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Kontos, Christopher Dale

Overview:

The Kontos Lab studies the molecular mechanisms of angiogenesis and vascular remodeling. Studies are directed toward understanding how signal transduction by endothelial receptor tyrosine kinase (RTKs) regulates vascular growth in both physiological and pathological processes, including exercise, peripheral artery disease (PAD), atherosclerosis, and cancer.

Current projects include:

1. Signal transduction, mechanisms of activation, and downregulation of endothelial RTKs (mainly Tie1, Tie2, VEGFR-1, and VEGFR-2)

2. Differential effects of the Angiopoietins

3. Role of the inositol phosphatase PTEN in the regulation of vascular cell growth and remodeling

4. PTEN gene therapy for prevention of vein graft disease

5. Angiogenic signaling in skeletal muscle, including mechanisms of exercise-induced angiogenesis

6. Proteolytic cleavage and shedding of endothelial RTKs

7. Angiogenic proteins as biomarkers in peripheral artery disease

Positions:

Associate Professor of Medicine

Medicine, Cardiology
School of Medicine

Associate Professor of Pharmacology & Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

M.D. 1989

M.D. — Virginia Commonwealth University

News:

Grants:

Genetic dissection of angiogenesis in the tuberculous granuloma

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
February 15, 2017
End Date
January 31, 2022

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

The role of muscle progenitor cells in muscle and vascular regeneration in PAD

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 13, 2017
End Date
March 12, 2020

Pharmacology Industry Internships for Ph.D. Students

Administered By
Pharmacology & Cancer Biology
AwardedBy
American Society for Pharmacology and Experimental Therapeutics
Role
Participating Faculty Member
Start Date
January 01, 2017
End Date
December 31, 2019

Macrophages and Treatment-Resistant NVAMD

Administered By
Ophthalmology
AwardedBy
National Institutes of Health
Role
Advisor
Start Date
May 01, 2015
End Date
April 30, 2019

Skeletal Muscle and Vascular Remodeling in Peripheral Artery Disease

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2015
End Date
August 31, 2018

A Novel Molecular Regulator of Vascular Homeostasis

Administered By
Medicine, Cardiology
AwardedBy
American Heart Association
Role
Principal Investigator
Start Date
July 01, 2016
End Date
June 30, 2018

Enhancing dendritic cell migration to drive potent anti-tumor immune responses

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 2013
End Date
June 30, 2017

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1997
End Date
June 30, 2017

Duke Training Grant in Nephrology

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Preceptor
Start Date
September 20, 1995
End Date
June 30, 2017

Skeletal Muscle and Vascular Remodeling in Peripheral Artery Disease

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2014
End Date
August 31, 2016

Tie2 Activation for the Treatment of Chemical-Induced Acute Lung Injury

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 26, 2013
End Date
August 31, 2016

Muscle-Resident Stem Cells for Angiogenesis and Vascular Maturation in PAD

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2013
End Date
July 31, 2016

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Cancer Institute
Role
Mentor
Start Date
July 01, 1993
End Date
March 31, 2016

Institutional Training Grant in Pediatric Cardiology

Administered By
Pediatrics, Cardiology
AwardedBy
National Institutes of Health
Role
Faculty Member
Start Date
April 01, 2009
End Date
June 30, 2015

Novel pathways modulating Raf-mediated cardiac hypertrophy

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
February 15, 2013
End Date
May 31, 2015

The role of satellite cells in adult skeletal muscle growth and maintenance

Administered By
Medicine, Cardiology
AwardedBy
University of Kentucky
Role
Principal Investigator
Start Date
September 01, 2013
End Date
August 31, 2014

Peripheral endothelial and muscle cell pathology in cardiovascular disease

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
August 20, 2010
End Date
June 30, 2013

PTEN Gene Therapy for Vein Graft Disease

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 15, 2009
End Date
April 30, 2012

The Role of PTEN in Endothelial Biology

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 15, 2008
End Date
January 31, 2012

The Role of ADAM15 in sTie@ Shedding and Ischemic Angiogenesis

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 30, 2009
End Date
June 29, 2010

Systems Biology of Angiogenesis

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
September 27, 2006
End Date
August 31, 2009

Endothelial Cell Molecular Alterations in Cancer

Administered By
Pathology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
August 23, 2002
End Date
July 31, 2008

Therapeutic Angiogenesis for Erectile Dysfunction

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
January 01, 2004
End Date
June 30, 2008

The Role of Tie2 and the Angiopoietins in EPC Biology

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2005
End Date
July 17, 2007

Downregulation of Tie2

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 2004
End Date
June 30, 2007

The Angiopoietins in Vascular Maintenance and Remodeling

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2002
End Date
March 31, 2007

TIE2-Associated Molecular Targets in Tumor Vasculature

Administered By
Pathology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
July 01, 2002
End Date
June 30, 2005

Endothelial signal transduction by tek and tie

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1997
End Date
August 31, 2002

Role of TEK signaling in endothelial biology

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 1996
End Date
February 28, 2002

Endothelial Signal Transduction By Tek And Tie

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1997
End Date
August 31, 1999

Tek Signaling And Endothelial Biology

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 1996
End Date
February 28, 1999
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Publications:

Angiopoietin-1 promotes atherosclerosis by increasing the proportion of circulating Gr1+ monocytes.

Atherosclerosis is a chronic inflammatory disease occurring within the artery wall. A crucial step in atherogenesis is the infiltration and retention of monocytes into the subendothelial space of large arteries induced by chemokines and growth factors. Angiopoietin-1 (Ang-1) regulates angiogenesis and reduces vascular permeability and has also been reported to promote monocyte migration in vitro. We investigated the role of Ang-1 in atherosclerosis-prone apolipoprotein-E (Apo-E) knockout mouse.Apo-E knockout (Apo-E-/-) mice fed a western or normal chow diet received a single iv injection of adenovirus encoding Ang-1 or control vector. Adenovirus-mediated systemic expression of Ang-1 induced a significant increase in early atherosclerotic lesion size and monocyte/macrophage accumulation compared with control animals receiving empty vector. Ang-1 significantly increased plasma MCP-1 and VEGF levels as measured by ELISA. FACS analysis showed that Ang-1 selectively increased inflammatory Gr1+ monocytes in the circulation, while the cell-surface expression of CD11b, which mediates monocyte emigration, was significantly reduced.Ang-1 specifically increases circulating Gr1+ inflammatory monocytes and increases monocyte/macrophage retention in atherosclerotic plaques, thereby contributing to development of atherosclerosis.

Authors
Fujisawa, T; Wang, K; Niu, X-L; Egginton, S; Ahmad, S; Hewett, P; Kontos, CD; Ahmed, A
MLA Citation
Fujisawa, T, Wang, K, Niu, X-L, Egginton, S, Ahmad, S, Hewett, P, Kontos, CD, and Ahmed, A. "Angiopoietin-1 promotes atherosclerosis by increasing the proportion of circulating Gr1+ monocytes." Cardiovascular research 113.1 (January 2017): 81-89.
PMID
28069704
Source
epmc
Published In
Cardiovascular Research
Volume
113
Issue
1
Publish Date
2017
Start Page
81
End Page
89
DOI
10.1093/cvr/cvw223

Subacute limb ischemia induces skeletal muscle injury in genetically susceptible mice independent of vascular density.

The primary preclinical model of peripheral artery disease, which involves acute limb ischemia (ALI), can result in appreciable muscle injury that is attributed to the acuity of the ischemic injury. A less acute model of murine limb ischemia using ameroid constrictors (ACs) has been developed in an attempt to mimic the chronic nature of human disease. However, there is currently little understanding of how genetics influence muscle injury following subacute arterial occlusion in the mouse.We investigated the influence of mouse genetics on skeletal muscle tissue survival, blood flow, and vascular density by subjecting two different mouse strains, C57BL/6 (BL6) and BALB/c, to ALI or subacute limb ischemia using single (1AC) or double (2AC) AC placement on the femoral artery.Similar to ALI, the 2AC model resulted in significant tissue necrosis and limb perfusion deficits in genetically susceptible BALB/c but not BL6 mice. In the 1AC model, no outward evidence of tissue necrosis was observed, and there were no differences in limb blood flow between BL6 and BALB/c. However, BALB/c mice displayed significantly greater muscle injury, as evidenced by increased inflammation and myofiber atrophy, despite having no differences in CD31(+) and SMA(+) vascular density and area. BALB/c mice also displayed significantly greater centralized myonuclei, indicating increased muscle regeneration.The susceptibility of skeletal muscle to ischemia-induced injury is at least partly independent of muscle blood flow and vascular density, consistent with a muscle cell autonomous response that is genetically determined. Further development of preclinical models of peripheral artery disease that more accurately reflect the nature of the human disease may allow more accurate identification of genetic targets for therapeutic intervention.

Authors
McClung, JM; McCord, TJ; Southerland, K; Schmidt, CA; Padgett, ME; Ryan, TE; Kontos, CD
MLA Citation
McClung, JM, McCord, TJ, Southerland, K, Schmidt, CA, Padgett, ME, Ryan, TE, and Kontos, CD. "Subacute limb ischemia induces skeletal muscle injury in genetically susceptible mice independent of vascular density." Journal of vascular surgery 64.4 (October 2016): 1101-1111.e2.
PMID
26254821
Source
epmc
Published In
Journal of Vascular Surgery
Volume
64
Issue
4
Publish Date
2016
Start Page
1101
End Page
1111.e2
DOI
10.1016/j.jvs.2015.06.139

Tie1: an orphan receptor provides context for angiopoietin-2/Tie2 signaling.

Angiopoietin-1/Tie2 (ANG1/Tie2) signaling is well documented as regulating angiogenesis and vessel maturation. This pathway is complicated by involvement of the orphan receptor Tie1, which has been implicated as both a positive and negative regulator of ANG1/Tie2 signaling, and ANG2, which can serve as both a Tie2 agonist and antagonist, depending on the context. Two papers in this issue of the JCI provide new insight into this complicated pathway. Korhonen et al. reveal that Tie1 acts to modulate the effects of ANG1 and ANG2 on Tie2 in vitro and in vivo. Kim et al. demonstrate that ANG2 acts as a Tie2 agonist in non-pathological conditions, whereas in the setting of inflammation, ANG2 functions as a Tie2 antagonist and promotes vascular dysfunction. Both studies indicate that inflammation promotes cleavage of the ectodomain of Tie1 and that this cleavage event corresponds with the switch of ANG2 from a Tie2 agonist to an antagonist. The results of these studies lay the groundwork for future strategies to therapeutically exploit this pathway in diseases characterized by adverse vascular remodeling and increased permeability.

Authors
Mueller, SB; Kontos, CD
MLA Citation
Mueller, SB, and Kontos, CD. "Tie1: an orphan receptor provides context for angiopoietin-2/Tie2 signaling." The Journal of clinical investigation 126.9 (September 2016): 3188-3191.
PMID
27548526
Source
epmc
Published In
Journal of Clinical Investigation
Volume
126
Issue
9
Publish Date
2016
Start Page
3188
End Page
3191
DOI
10.1172/jci89963

Methods for Acute and Subacute Murine Hindlimb Ischemia.

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators' ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Authors
Padgett, ME; McCord, TJ; McClung, JM; Kontos, CD
MLA Citation
Padgett, ME, McCord, TJ, McClung, JM, and Kontos, CD. "Methods for Acute and Subacute Murine Hindlimb Ischemia." Journal of visualized experiments : JoVE 112 (June 21, 2016).
PMID
27403963
Source
epmc
Published In
Journal of Visualized Experiments
Issue
112
Publish Date
2016
DOI
10.3791/54166

Angiopoietin-1 enhances skeletal muscle regeneration in mice.

Activation of muscle progenitor cell myogenesis and endothelial cell angiogenesis is critical for the recovery of skeletal muscle from injury. Angiopoietin-1 (Ang-1), a ligand of Tie-2 receptors, enhances angiogenesis and skeletal muscle satellite cell survival; however, its role in skeletal muscle regeneration after injury is unknown. We assessed the effects of Ang-1 on fiber regeneration, myogenesis, and angiogenesis in injured skeletal muscle (tibialis anterior, TA) in mice. We also assessed endogenous Ang-1 levels and localization in intact and injured TA muscles. TA fiber injury was triggered by cardiotoxin injection. Endogenous Ang-1 mRNA levels immediately decreased in response to cardiotoxin then increased during the 2 wk. Ang-1 protein was expressed in satellite cells, both in noninjured and recovering TA muscles. Positive Ang-1 staining was present in blood vessels but not in nerve fibers. Four days after the initiation of injury, injection of adenoviral Ang-1 into injured muscles resulted in significant increases in in situ TA muscle contractility, muscle fiber regeneration, and capillary density. In cultured human skeletal myoblasts, recombinant Ang-1 protein increased survival, proliferation, migration, and differentiation into myotubes. The latter effect was associated with significant upregulation of the expression of the myogenic regulatory factors MyoD and Myogenin and certain genes involved in cell cycle regulation. We conclude that Ang-1 strongly enhances skeletal muscle regeneration in response to fiber injury and that this effect is mediated through induction of the myogenesis program in muscle progenitor cells and the angiogenesis program in endothelial cells.

Authors
Mofarrahi, M; McClung, JM; Kontos, CD; Davis, EC; Tappuni, B; Moroz, N; Pickett, AE; Huck, L; Harel, S; Danialou, G; Hussain, SNA
MLA Citation
Mofarrahi, M, McClung, JM, Kontos, CD, Davis, EC, Tappuni, B, Moroz, N, Pickett, AE, Huck, L, Harel, S, Danialou, G, and Hussain, SNA. "Angiopoietin-1 enhances skeletal muscle regeneration in mice." American journal of physiology. Regulatory, integrative and comparative physiology 308.7 (April 2015): R576-R589.
PMID
25608750
Source
epmc
Published In
American journal of physiology. Regulatory, integrative and comparative physiology
Volume
308
Issue
7
Publish Date
2015
Start Page
R576
End Page
R589
DOI
10.1152/ajpregu.00267.2014

Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment.

Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy.

Authors
McClung, JM; Reinardy, JL; Mueller, SB; McCord, TJ; Kontos, CD; Brown, DA; Hussain, SNA; Schmidt, CA; Ryan, TE; Green, TD
MLA Citation
McClung, JM, Reinardy, JL, Mueller, SB, McCord, TJ, Kontos, CD, Brown, DA, Hussain, SNA, Schmidt, CA, Ryan, TE, and Green, TD. "Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment." Frontiers in physiology 6 (January 2015): 161-.
PMID
26042050
Source
epmc
Published In
Frontiers in Physiology
Volume
6
Publish Date
2015
Start Page
161
DOI
10.3389/fphys.2015.00161

Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway.

The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1's role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.

Authors
Reinardy, JL; Corey, DM; Golzio, C; Mueller, SB; Katsanis, N; Kontos, CD
MLA Citation
Reinardy, JL, Corey, DM, Golzio, C, Mueller, SB, Katsanis, N, and Kontos, CD. "Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway." PloS one 10.10 (January 2015): e0139614-.
PMID
26436659
Source
epmc
Published In
PloS one
Volume
10
Issue
10
Publish Date
2015
Start Page
e0139614
DOI
10.1371/journal.pone.0139614

Gene Therapy for the Prevention of Vein Graft Disease

© 2015 Elsevier Inc. All rights reserved..Despite advances in the medical management of atherosclerosis, surgical revascularization using autologous veins remains a mainstay of therapy for both coronary and peripheral artery disease. However, long-term outcomes following bypass surgery are limited by relatively high failure rates due to vein graft disease. Vein graft failure, together with the limited supply of native veins, has led to interest in gene therapy to prevent vein graft disease. Bypass grafting presents an ideal opportunity for gene therapy because vein grafts can be treated with gene delivery vectors ex vivo to maximize gene delivery, minimize systemic toxicity, and target the pathogenesis of vein graft disease at its onset. Here we discuss the pathogenesis of vein graft disease, vector delivery strategies, and potential molecular targets for preventing vein graft disease. We summarize the preclinical and clinical literature on gene therapy in vein grafting and discuss considerations for future therapies to prevent vein graft disease.

Authors
Mueller, SB; Kontos, CD
MLA Citation
Mueller, SB, and Kontos, CD. "Gene Therapy for the Prevention of Vein Graft Disease." Translating Gene Therapy to the Clinic: Techniques and Approaches. November 17, 2014. 227-246.
Source
scopus
Publish Date
2014
Start Page
227
End Page
246
DOI
10.1016/B978-0-12-800563-7.00015-4

Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature.

Retinal and choroidal neovascularization (NV) and vascular leakage contribute to visual impairment in several common ocular diseases. The angiopoietin/TIE2 (ANG/TIE2) pathway maintains vascular integrity, and negative regulators of this pathway are potential therapeutic targets for these diseases. Here, we demonstrated that vascular endothelial-protein tyrosine phosphatase (VE-PTP), which negatively regulates TIE2 activation, is upregulated in hypoxic vascular endothelial cells, particularly in retinal NV. Intraocular injection of an anti-VE-PTP antibody previously shown to activate TIE2 suppressed ocular NV. Furthermore, a small-molecule inhibitor of VE-PTP catalytic activity (AKB-9778) activated TIE2, enhanced ANG1-induced TIE2 activation, and stimulated phosphorylation of signaling molecules in the TIE2 pathway, including AKT, eNOS, and ERK. In mouse models of neovascular age-related macular degeneration, AKB-9778 induced phosphorylation of TIE2 and strongly suppressed NV. Ischemia-induced retinal NV, which is relevant to diabetic retinopathy, was accentuated by the induction of ANG2 but inhibited by AKB-9778, even in the presence of high levels of ANG2. AKB-9778 also blocked VEGF-induced leakage from dermal and retinal vessels and prevented exudative retinal detachments in double-transgenic mice with high expression of VEGF in photoreceptors. These data support targeting VE-PTP to stabilize retinal and choroidal blood vessels and suggest that this strategy has potential for patients with a wide variety of retinal and choroidal vascular diseases.

Authors
Shen, J; Frye, M; Lee, BL; Reinardy, JL; McClung, JM; Ding, K; Kojima, M; Xia, H; Seidel, C; Lima e Silva, R; Dong, A; Hackett, SF; Wang, J; Howard, BW; Vestweber, D; Kontos, CD; Peters, KG; Campochiaro, PA
MLA Citation
Shen, J, Frye, M, Lee, BL, Reinardy, JL, McClung, JM, Ding, K, Kojima, M, Xia, H, Seidel, C, Lima e Silva, R, Dong, A, Hackett, SF, Wang, J, Howard, BW, Vestweber, D, Kontos, CD, Peters, KG, and Campochiaro, PA. "Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature." The Journal of clinical investigation 124.10 (October 2014): 4564-4576.
PMID
25180601
Source
epmc
Published In
Journal of Clinical Investigation
Volume
124
Issue
10
Publish Date
2014
Start Page
4564
End Page
4576
DOI
10.1172/jci74527

More than skin deep: connecting melanocyte pigmentation and angiogenic diseases.

Epidemiological studies have identified racial differences in susceptibility to numerous diseases, including several ocular and skin diseases characterized by increased vascular growth. In most cases, the specific mechanisms and genetic variants responsible for these differences have remained elusive. In this issue of the JCI, Adini et al. explore a direct connection between skin pigmentation and susceptibility to angiogenic diseases and identify an extracellular matrix protein that is regulated by melanogenesis and potently modulates angiogenesis.

Authors
Kontos, CD
MLA Citation
Kontos, CD. "More than skin deep: connecting melanocyte pigmentation and angiogenic diseases." J Clin Invest 124.1 (January 2014): 76-79.
PMID
24355914
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
124
Issue
1
Publish Date
2014
Start Page
76
End Page
79
DOI
10.1172/JCI73559

Angiogenesis in peripheral artery disease: An emerging therapy targeting skeletal muscle

Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities causing claudication, exercise intolerance and a decreased quality of life. Despite the fact that stenosis of conduit vessels are largely responsible for PAD diagnosis, and re-vascularization of these arteries are routinely performed as a treatment strategy, hemodynamics of conduit vessels do not entirely explain the functional limitation observed in PAD patients. Due to the inherent purpose the microvasculature plays in blood delivery and oxygen exchange to skeletal muscle, angiogenesis of the microvasculature may play a prominent role in PAD and has become the focus of both basic science and a therapeutic target for PAD clinical trials. This review will discuss what is currently known about skeletal muscle capillary density in PAD patients compared to normal subjects, how capillary density relates to exercise intolerance and how exercise training may be the best therapeutic intervention for initiating angiogenesis to improve exercise tolerance. Last, we will discuss the mechanisms of angiogenesis in skeletal muscle and the use of growth factors in therapeutic clinical trials. © 2013 by Nova Science Publishers, Inc. All rights reserved.

Authors
Duscha, BD; Robbins, JL; Kontos, CD; Kraus, WE; Annex, BH
MLA Citation
Duscha, BD, Robbins, JL, Kontos, CD, Kraus, WE, and Annex, BH. "Angiogenesis in peripheral artery disease: An emerging therapy targeting skeletal muscle." (December 1, 2013): 99-134. (Chapter)
Source
scopus
Publish Date
2013
Start Page
99
End Page
134

Inhibiting the inhibitor: targeting vascular endothelial protein tyrosine phosphatase to promote tumor vascular maturation.

Authors
Kontos, CD; Willett, CG
MLA Citation
Kontos, CD, and Willett, CG. "Inhibiting the inhibitor: targeting vascular endothelial protein tyrosine phosphatase to promote tumor vascular maturation." J Natl Cancer Inst 105.16 (August 21, 2013): 1163-1165.
PMID
23899554
Source
pubmed
Published In
Journal of the National Cancer Institute
Volume
105
Issue
16
Publish Date
2013
Start Page
1163
End Page
1165
DOI
10.1093/jnci/djt199

Gene therapy for the prevention of vein graft disease.

Ischemic cardiovascular disease remains the leading cause of death worldwide. Despite advances in the medical management of atherosclerosis over the past several decades, many patients require arterial revascularization to reduce mortality and alleviate ischemic symptoms. Technological advancements have led to dramatic increases in the use of percutaneous and endovascular approaches, yet surgical revascularization (bypass surgery) with autologous vein grafts remains a mainstay of therapy for both coronary and peripheral artery disease. Although bypass surgery is highly efficacious in the short term, long-term outcomes are limited by relatively high failure rates as a result of intimal hyperplasia, which is a common feature of vein graft disease. The supply of native veins is limited, and many individuals require multiple grafts and repeat procedures. The need to prevent vein graft failure has led to great interest in gene therapy approaches to this problem. Bypass grafting presents an ideal opportunity for gene therapy, as surgically harvested vein grafts can be treated with gene delivery vectors ex vivo, thereby maximizing gene delivery while minimizing the potential for systemic toxicity and targeting the pathogenesis of vein graft disease at its onset. Here we will review the pathogenesis of vein graft disease and discuss vector delivery strategies and potential molecular targets for its prevention. We will summarize the preclinical and clinical literature on gene therapy in vein grafting and discuss additional considerations for future therapies to prevent vein graft disease.

Authors
Southerland, KW; Frazier, SB; Bowles, DE; Milano, CA; Kontos, CD
MLA Citation
Southerland, KW, Frazier, SB, Bowles, DE, Milano, CA, and Kontos, CD. "Gene therapy for the prevention of vein graft disease." Transl Res 161.4 (April 2013): 321-338. (Review)
PMID
23274305
Source
pubmed
Published In
Translational Research, The Journal of Laboratory and Clinical Medicine
Volume
161
Issue
4
Publish Date
2013
Start Page
321
End Page
338
DOI
10.1016/j.trsl.2012.12.003

p53 functions in endothelial cells to prevent radiation-induced myocardial injury in mice.

Radiation therapy, which is used for the treatment of some cancers, can cause delayed heart damage. In the heart, p53 influences myocardial injury that occurs after multiple types of stress. Here, we demonstrated that p53 functioned in endothelial cells to protect mice from myocardial injury after whole-heart irradiation. Mice with an endothelial cell-specific deletion of p53 succumbed to heart failure after whole-heart irradiation as a result of myocardial necrosis, systolic dysfunction, and cardiac hypertrophy. Moreover, the onset of cardiac dysfunction was preceded by alterations in myocardial vascular permeability and density, which resulted in cardiac ischemia and myocardial hypoxia. Mechanistic studies with primary cardiac endothelial cells irradiated in vitro indicated that p53 signaling caused mitotic arrest and protected cardiac endothelial cells from cell death resulting from abnormal mitosis or mitotic catastrophe. Furthermore, mice lacking the cyclin-dependent kinase inhibitor p21, which is a transcriptional target of p53, were also sensitized to myocardial injury after whole-heart irradiation. Together, our results demonstrate that the p53-p21 axis functions to prevent radiation-induced myocardial injury in mice.

Authors
Lee, C-L; Moding, EJ; Cuneo, KC; Li, Y; Sullivan, JM; Mao, L; Washington, I; Jeffords, LB; Rodrigues, RC; Ma, Y; Das, S; Kontos, CD; Kim, Y; Rockman, HA; Kirsch, DG
MLA Citation
Lee, C-L, Moding, EJ, Cuneo, KC, Li, Y, Sullivan, JM, Mao, L, Washington, I, Jeffords, LB, Rodrigues, RC, Ma, Y, Das, S, Kontos, CD, Kim, Y, Rockman, HA, and Kirsch, DG. "p53 functions in endothelial cells to prevent radiation-induced myocardial injury in mice. (Published online)" Sci Signal 5.234 (July 24, 2012): ra52-.
PMID
22827996
Source
pubmed
Published In
Science Signaling
Volume
5
Issue
234
Publish Date
2012
Start Page
ra52
DOI
10.1126/scisignal.2002918

Skeletal muscle-specific genetic determinants contribute to the differential strain-dependent effects of hindlimb ischemia in mice.

Genetics plays an important role in determining peripheral arterial disease (PAD) pathology, which causes a spectrum of clinical disorders that range from clinically silent reductions in blood flow to limb-threatening ischemia. The cell-type specificity of PAD pathology, however, has received little attention. To determine whether strain-dependent differences in skeletal muscle cells might account for the differential responses to ischemia observed in C57BL/6 and BALB/c mice, endothelial and skeletal muscle cells were subjected to hypoxia and nutrient deprivation (HND) in vitro, to mimic ischemia. Muscle cells were more susceptible to HND than were endothelial cells. In vivo, C57BL/6 and BALB/c mice displayed strain-specific differences in myofiber responses after hindlimb ischemia, with significantly greater myofiber atrophy, greater apoptosis, and attenuated myogenic regulatory gene expression and stress-responsive signaling in BALB/c mice. Strain-specific deficits were recapitulated in vitro in primary muscle cells from both strains after HND. Muscle cells from BALB/c mice congenic for the C57BL/6 Lsq-1 quantitative trait locus were protected from HND-induced atrophy, and gene expression of vascular growth factors and their receptors was significantly greater in C57BL/6 primary muscle cells. Our results indicate that the previously identified specific genetic locus regulating strain-dependent collateral vessel density has a nonvascular or muscle cell-autonomous role involving both the myogenic program and traditional vascular growth factor receptor expression.

Authors
McClung, JM; McCord, TJ; Keum, S; Johnson, S; Annex, BH; Marchuk, DA; Kontos, CD
MLA Citation
McClung, JM, McCord, TJ, Keum, S, Johnson, S, Annex, BH, Marchuk, DA, and Kontos, CD. "Skeletal muscle-specific genetic determinants contribute to the differential strain-dependent effects of hindlimb ischemia in mice." Am J Pathol 180.5 (May 2012): 2156-2169.
PMID
22445571
Source
pubmed
Published In
The American journal of pathology
Volume
180
Issue
5
Publish Date
2012
Start Page
2156
End Page
2169
DOI
10.1016/j.ajpath.2012.01.032

In vivo tumor targeting by a NGR-decorated micelle of a recombinant diblock copolypeptide.

Antivascular targeting is a promising strategy for tumor therapy. This strategy has the potential to overcome many of the transport barriers associated with targeting tumor cells in solid tumors, because the tumor vasculature is directly accessible to targeting vehicles in systemic circulation. We report a novel nanoscale delivery system consisting of multivalent polymer micelles to target receptors that are preferentially upregulated in the tumor vasculature and perivascular cells, specifically CD13. To this end we utilized amphiphilic block copolymers, composed of a genetically engineered elastin-like polypeptide (ELP) that self-assemble into monodisperse spherical micelles. These polymer micelles were functionalized by incorporating the NGR tripeptide ligand, which targets the CD13 receptor, on their corona. We examined the self-assembly and in vivo tumor targeting by these NGR-functionalized nanoparticles and show that multivalent presentation of NGR by micelle self-assembly selectively targets the tumor vasculature by targeting CD13. Furthermore, we show greater vascular retention and extravascular accumulation of nanoparticles in tumor tissue compared to normal tissue, although the enhancement is modest. These results suggest that enhanced delivery to solid tumors can be achieved by targeting upregulated receptors in the tumor vasculature with multivalent ligand-presenting nanoparticles, but additional work is required to optimize such systems for multivalent targeting.

Authors
Simnick, AJ; Amiram, M; Liu, W; Hanna, G; Dewhirst, MW; Kontos, CD; Chilkoti, A
MLA Citation
Simnick, AJ, Amiram, M, Liu, W, Hanna, G, Dewhirst, MW, Kontos, CD, and Chilkoti, A. "In vivo tumor targeting by a NGR-decorated micelle of a recombinant diblock copolypeptide." J Control Release 155.2 (October 30, 2011): 144-151.
PMID
21763734
Source
pubmed
Published In
Journal of Controlled Release
Volume
155
Issue
2
Publish Date
2011
Start Page
144
End Page
151
DOI
10.1016/j.jconrel.2011.06.044

Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death.

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd induces neuronal apoptosis in part through activation of the mammalian target of rapamycin (mTOR) pathway. However, the underlying mechanism is unknown. Here we show that Cd induces the generation of reactive oxygen species (ROS) by upregulating the expression of NADPH oxidase 2 and its regulatory proteins (p22(phox), p67(phox), p40(phox), p47(phox), and Rac1) in PC12 and SH-SY5Y cells. Cd induction of ROS contributed to the activation of mTOR signaling, as pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, prevented this event. Further studies reveal that Cd induction of ROS increased phosphorylation of the type I insulin-like growth factor receptor (IGFR) β subunit, which was abrogated by NAC. Wortmannin, a phosphoinositide 3'-kinase (PI3K) inhibitor, partially attenuated Cd-induced phosphorylation of Akt, p70 S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1, as well as apoptosis of the neuronal cells. In addition, overexpression of wild-type phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or pretreatment with aminoimidazole carboxamide ribonucleotide, an AMP-activated protein kinase (AMPK) activator, partially prevented Cd-induced ROS and activation of the mTOR pathway, as well as cell death. The results indicate that Cd induction of ROS activates mTOR signaling, leading to neuronal cell death, in part by activating the positive regulators IGFR/PI3K and by inhibiting the negative regulators PTEN/AMPK. The findings suggest that inhibitors of PI3K and mTOR, activators of AMPK, or antioxidants may be exploited for the prevention of Cd-induced neurodegenerative diseases.

Authors
Chen, L; Xu, B; Liu, L; Luo, Y; Zhou, H; Chen, W; Shen, T; Han, X; Kontos, CD; Huang, S
MLA Citation
Chen, L, Xu, B, Liu, L, Luo, Y, Zhou, H, Chen, W, Shen, T, Han, X, Kontos, CD, and Huang, S. "Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death." Free Radic Biol Med 50.5 (March 1, 2011): 624-632.
PMID
21195169
Source
pubmed
Published In
Free Radical Biology and Medicine
Volume
50
Issue
5
Publish Date
2011
Start Page
624
End Page
632
DOI
10.1016/j.freeradbiomed.2010.12.032

VEGF and soluble VEGF receptor-1 (sFlt-1) distributions in peripheral arterial disease: an in silico model.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, the growth of new capillaries from existing microvasculature. In peripheral arterial disease (PAD), lower extremity muscle ischemia develops downstream of atherosclerotic obstruction. A working hypothesis proposed that the maladaptive overexpression of soluble VEGF receptor 1 (sVEGFR1) in ischemic muscle tissues, and its subsequent antagonism of VEGF bioactivity, may contribute to the deficient angiogenic response in PAD, as well as the limited success of therapeutic angiogenesis strategies where exogenous VEGF genes/proteins are delivered. The objectives of this study were to develop a computational framework for simulating the systemic distributions of VEGF and sVEGFR1 (e.g., intramuscular vs. circulating, free vs. complexed) as observed in human PAD patients and to serve as a platform for the systematic optimization of diagnostic tools and therapeutic strategies. A three-compartment model was constructed, dividing the human body into the ischemic calf muscle, blood, and the rest of the body, connected through macromolecular biotransport processes. Detailed molecular interactions between VEGF, sVEGFR1, endothelial surface receptors (VEGFR1, VEGFR2, NRP1), and interstitial matrix sites were modeled. Our simulation results did not support a simultaneous decrease in plasma sVEGFR1 during PAD-associated elevations in plasma VEGF reported in literature. Furthermore, despite the overexpression in sVEGFR1, our PAD control demonstrated increased proangiogenic signaling complex formation, relative to our previous healthy control, due to sizeable upregulations in VEGFR2 and VEGF expression, thus leaving open the possibility that impaired angiogenesis in PAD may be rooted in signaling pathway disruptions downstream of ligand-receptor binding.

Authors
Wu, FTH; Stefanini, MO; Mac Gabhann, F; Kontos, CD; Annex, BH; Popel, AS
MLA Citation
Wu, FTH, Stefanini, MO, Mac Gabhann, F, Kontos, CD, Annex, BH, and Popel, AS. "VEGF and soluble VEGF receptor-1 (sFlt-1) distributions in peripheral arterial disease: an in silico model." Am J Physiol Heart Circ Physiol 298.6 (June 2010): H2174-H2191.
PMID
20382861
Source
pubmed
Published In
American journal of physiology. Heart and circulatory physiology
Volume
298
Issue
6
Publish Date
2010
Start Page
H2174
End Page
H2191
DOI
10.1152/ajpheart.00365.2009

Inactivation of the tumour suppressor, PTEN, in smooth muscle promotes a pro-inflammatory phenotype and enhances neointima formation.

AIMS: Phosphatase and tensin homolog (PTEN) is implicated as a negative regulator of vascular smooth muscle cell (SMC) proliferation and injury-induced vascular remodelling. We tested if selective depletion of PTEN only in SMC is sufficient to promote SMC phenotypic modulation, cytokine production, and enhanced neointima formation. METHODS AND RESULTS: Smooth muscle marker expression and induction of pro-inflammatory cytokines were compared in cultured SMC expressing control or PTEN-specific shRNA. Compared with controls, PTEN-deficient SMC exhibited increased phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signalling and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) activity, reduced expression of SM markers (SM-alpha-actin and calponin), and increased production of stromal cell-derived factor-1alpha (SDF-1alpha), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and chemokine (C-X-C motif) ligand 1 (KC/CXCL1) under basal conditions. PI3K/Akt or mTOR inhibition reversed repression of SM marker expression, whereas PI3K/Akt or NF-kappaB inhibition blocked cytokine induction mediated by PTEN depletion. Carotid ligation in mice with genetic reduction of PTEN specifically in SMC (SMC-specific PTEN heterozygotes) resulted in enhanced neointima formation, increased SMC hyperplasia, reduced SM-alpha-actin and calponin expression, and increased NF-kappaB and cytokine expression compared with wild-types. Lesion formation in SMC-specific heterozygotes was similar to lesion formation in global PTEN heterozygotes, indicating that inactivation of PTEN exclusively in SMC is sufficient to induce considerable increases in neointima formation. CONCLUSION: PTEN activation specifically in SMC is a common upstream regulator of multiple downstream events involved in pathological vascular remodelling, including proliferation, de-differentiation, and production of multiple cytokines.

Authors
Furgeson, SB; Simpson, PA; Park, I; Vanputten, V; Horita, H; Kontos, CD; Nemenoff, RA; Weiser-Evans, MCM
MLA Citation
Furgeson, SB, Simpson, PA, Park, I, Vanputten, V, Horita, H, Kontos, CD, Nemenoff, RA, and Weiser-Evans, MCM. "Inactivation of the tumour suppressor, PTEN, in smooth muscle promotes a pro-inflammatory phenotype and enhances neointima formation." Cardiovasc Res 86.2 (May 1, 2010): 274-282.
PMID
20051384
Source
pubmed
Published In
Cardiovascular Research
Volume
86
Issue
2
Publish Date
2010
Start Page
274
End Page
282
DOI
10.1093/cvr/cvp425

A systems biology perspective on sVEGFR1: its biological function, pathogenic role and therapeutic use.

Angiogenesis is the growth of new capillaries from pre-existent microvasculature. A wide range of pathological conditions, from atherosclerosis to cancer, can be attributed to either excessive or deficient angiogenesis. Central to the physiological regulation of angiogenesis is the vascular endothelial growth factor (VEGF) system--its ligands and receptors (VEGFRs) are thus prime molecular targets of pro-angiogenic and anti-angiogenic therapies. Of growing interest as a prognostic marker and therapeutic target in angiogenesis-dependent diseases is soluble VEGF receptor-1 (sVEGFR1, also known as sFlt-1)--a truncated version of the cell membrane-spanning VEGFR1. For instance, it is known that sVEGFR1 is involved in the endothelial dysfunction characterizing the pregnancy disorder of pre-eclampsia, and sVEGFR1's therapeutic potential as an anti-angiogenic agent is being evaluated in pre-clinical models of cancer. This mini review begins with an examination of the protein domain structure and biomolecular interactions of sVEGFR1 in relation to the full-length VEGFR1. A synopsis of known and inferred physiological and pathological roles of sVEGFR1 is then given, with emphasis on the utility of computational systems biology models in deciphering the molecular mechanisms by which sVEGFR1's purported biological functions occur. Finally, we present the need for a systems biology perspective in interpreting circulating VEGF and sVEGFR1 concentrations as surrogate markers of angiogenic status in angiogenesis-dependent diseases.

Authors
Wu, FTH; Stefanini, MO; Mac Gabhann, F; Kontos, CD; Annex, BH; Popel, AS
MLA Citation
Wu, FTH, Stefanini, MO, Mac Gabhann, F, Kontos, CD, Annex, BH, and Popel, AS. "A systems biology perspective on sVEGFR1: its biological function, pathogenic role and therapeutic use." J Cell Mol Med 14.3 (March 2010): 528-552. (Review)
PMID
19840194
Source
pubmed
Published In
Journal of Cellular and Molecular Medicine
Volume
14
Issue
3
Publish Date
2010
Start Page
528
End Page
552
DOI
10.1111/j.1582-4934.2009.00941.x

Phosphatase and tensin homologue on chromosome 10 (PTEN) directs prostaglandin E2-mediated fibroblast responses via regulation of E prostanoid 2 receptor expression.

Prostaglandin E(2) (PGE(2)) is an arachidonic acid metabolite that counters transforming growth factor-beta-induced fibroblast activation via E prostanoid 2 (EP2) receptor binding. Phosphatase and tensin homologue on chromosome 10 (PTEN) is a lipid phosphatase that, by antagonizing the phosphoinositol 3-kinase (PI3K) pathway, also inhibits fibroblast activation. Here, we show that PTEN directly regulates PGE(2) inhibition of fibroblast activation by augmenting EP2 receptor expression. The increase in collagen production and alpha-smooth muscle actin expression observed in fibroblasts in which PTEN is deficient was resistant to the usual suppressive effects of PGE(2). This was due to marked down-regulation of EP2, a G(s) protein-coupled receptor (GPCR) that mediates the inhibitory actions of this prostanoid via cAMP. pten(-/-) or PTEN-inhibited fibroblasts in which the PI3K pathway was blocked demonstrated a restoration of EP2 receptor expression, due to augmented gene transcription and mRNA instability. Importantly, restoration of the balance between PI3K and PTEN reestablished the inhibitory effect of PGE(2) on fibroblast activation. No such influence of PTEN was observed on alternative E prostanoid GPCRs. Moreover, our studies identified a positive feedback loop in which cAMP signaling enhanced EP2 receptor expression, independent of PTEN. Therefore, our findings indicate that PTEN regulates the antifibrotic effects of PGE(2) by a specific and permissive effect on EP2 receptor expression. Further, our data imply that cAMP signaling circumvents EP2 down-regulation in pten-deficient cells to restore EP2 receptor expression. This is the first description, to our knowledge, of PI3K/PTEN balance directing GPCR expression, and provides a novel mechanism for cellular effects of PTEN.

Authors
Sagana, RL; Yan, M; Cornett, AM; Tsui, JL; Stephenson, DA; Huang, SK; Moore, BB; Ballinger, MN; Melonakos, J; Kontos, CD; Aronoff, DM; Peters-Golden, M; White, ES
MLA Citation
Sagana, RL, Yan, M, Cornett, AM, Tsui, JL, Stephenson, DA, Huang, SK, Moore, BB, Ballinger, MN, Melonakos, J, Kontos, CD, Aronoff, DM, Peters-Golden, M, and White, ES. "Phosphatase and tensin homologue on chromosome 10 (PTEN) directs prostaglandin E2-mediated fibroblast responses via regulation of E prostanoid 2 receptor expression." J Biol Chem 284.47 (November 20, 2009): 32264-32271.
PMID
19808686
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
47
Publish Date
2009
Start Page
32264
End Page
32271
DOI
10.1074/jbc.M109.004796

Angiopoietin-2 confers Atheroprotection in apoE-/- mice by inhibiting LDL oxidation via nitric oxide.

Atherosclerosis is promoted by a combination of hypercholesterolemia and vascular inflammation. The function of Angiopoietin (Ang)-2, a key regulator of angiogenesis, in the maintenance of large vessels is unknown. A single systemic administration of Ang-2 adenovirus (AdAng-2) to apoE(-/-) mice fed a Western diet significantly reduced atherosclerotic lesion size ( approximately 40%) and oxidized LDL and macrophage content of the plaques. These beneficial effects were abolished by the inhibition of nitric oxide synthase (NOS). In endothelial cells, endothelial NOS activation per se inhibited LDL oxidation and Ang-2 stimulated NO release in a Tie2-dependent manner to decrease LDL oxidation. These findings demonstrate a novel atheroprotective role for Ang-2 when endothelial cell function is compromised and suggest that growth factors, which stimulate NO release without inducing inflammation, could offer atheroprotection.

Authors
Ahmed, A; Fujisawa, T; Niu, X-L; Ahmad, S; Al-Ani, B; Chudasama, K; Abbas, A; Potluri, R; Bhandari, V; Findley, CM; Lam, GKW; Huang, J; Hewett, PW; Cudmore, M; Kontos, CD
MLA Citation
Ahmed, A, Fujisawa, T, Niu, X-L, Ahmad, S, Al-Ani, B, Chudasama, K, Abbas, A, Potluri, R, Bhandari, V, Findley, CM, Lam, GKW, Huang, J, Hewett, PW, Cudmore, M, and Kontos, CD. "Angiopoietin-2 confers Atheroprotection in apoE-/- mice by inhibiting LDL oxidation via nitric oxide." Circ Res 104.12 (June 19, 2009): 1333-1336.
PMID
19461044
Source
pubmed
Published In
Circulation Research
Volume
104
Issue
12
Publish Date
2009
Start Page
1333
End Page
1336
DOI
10.1161/CIRCRESAHA.109.196154

Computational kinetic model of VEGF trapping by soluble VEGF receptor-1: effects of transendothelial and lymphatic macromolecular transport.

Vascular endothelial growth factor (VEGF) signal transduction through the cell surface receptors VEGFR1 and VEGFR2 regulates angiogenesis-the growth of new capillaries from preexistent microvasculature. Soluble VEGF receptor-1 (sVEGFR1), a nonsignaling truncated variant of VEGFR1, has been postulated to inhibit angiogenic signaling via direct sequestration of VEGF ligands or dominant-negative heterodimerization with surface VEGFRs. The relative contributions of these two mechanisms to sVEGFR1's purported antiangiogenic effects in vivo are currently unknown. We previously developed a computational model for predicting the compartmental distributions of VEGF and sVEGFR1 throughout the healthy human body by simulating the molecular interaction networks of the VEGF ligand-receptor system as well as intercompartmental macromolecular biotransport processes. In this study, we decipher the dynamic processes that led to our prior prediction that sVEGFR1, through its ligand trapping mechanism alone, does not demonstrate significant steady-state antiangiogenic effects. We show that sVEGFR1-facilitated tissue-to-blood shuttling of VEGF accounts for a counterintuitive and drastic elevation in plasma free VEGF concentrations after both intramuscular and intravascular sVEGFR1 infusion. While increasing intramuscular VEGF production reduces free sVEGFR1 levels through increased VEGF-sVEGFR1 complex formation, we demonstrate a competing and opposite effect in which increased VEGF occupancy of neuropilin-1 (NRP1) and the corresponding reduction in NRP1 availability for internalization of sVEGFR1 unexpectedly increases free sVEGFR1 levels. In conclusion, dynamic intercompartmental transport processes give rise to our surprising prediction that VEGF trapping alone does not account for sVEGFR1's antiangiogenic potential. sVEGFR1's interactions with cell surface receptors such as NRP1 are also expected to affect its molecular interplay with VEGF.

Authors
Wu, FTH; Stefanini, MO; Mac Gabhann, F; Kontos, CD; Annex, BH; Popel, AS
MLA Citation
Wu, FTH, Stefanini, MO, Mac Gabhann, F, Kontos, CD, Annex, BH, and Popel, AS. "Computational kinetic model of VEGF trapping by soluble VEGF receptor-1: effects of transendothelial and lymphatic macromolecular transport." Physiol Genomics 38.1 (June 10, 2009): 29-41.
PMID
19351908
Source
pubmed
Published In
Physiological genomics
Volume
38
Issue
1
Publish Date
2009
Start Page
29
End Page
41
DOI
10.1152/physiolgenomics.00031.2009

Endogenous S-nitrosothiols protect against myocardial injury.

Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR(-/-)) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) under normoxic conditions. We further show that S-nitrosylated HIF-1alpha binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function.

Authors
Lima, B; Lam, GKW; Xie, L; Diesen, DL; Villamizar, N; Nienaber, J; Messina, E; Bowles, D; Kontos, CD; Hare, JM; Stamler, JS; Rockman, HA
MLA Citation
Lima, B, Lam, GKW, Xie, L, Diesen, DL, Villamizar, N, Nienaber, J, Messina, E, Bowles, D, Kontos, CD, Hare, JM, Stamler, JS, and Rockman, HA. "Endogenous S-nitrosothiols protect against myocardial injury." Proceedings of the National Academy of Sciences of the United States of America 106.15 (April 2009): 6297-6302.
PMID
19325130
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
15
Publish Date
2009
Start Page
6297
End Page
6302
DOI
10.1073/pnas.0901043106

Phosphatase and tensin homolog (PTEN) regulates hepatic lipogenesis, microsomal triglyceride transfer protein, and the secretion of apolipoprotein B-containing lipoproteins.

Hepatic apolipoprotein B (apoB) lipoprotein production is metabolically regulated via the phosphoinositide 3-kinase cascade; however, the role of the key negative regulator of this pathway, the tumor suppressor phosphatase with tensin homology (PTEN), is unknown. Here, we demonstrate that hepatic protein levels of apoB100 and microsomal triglyceride transfer protein (MTP) are significantly down-regulated (73% and 36%, respectively) in the liver of PTEN liver-specific knockout (KO) mice, and this is accompanied by increased triglyceride (TG) accumulation and lipogenic gene expression, and reduced hepatic apoB secretion in freshly isolated hepatocytes. MTP protein mass and lipid transfer activity were also significantly reduced in liver of PTEN KO mice. Overexpression of the dominant negative mutant PTEN C/S124 (adenovirus expressing PTEN C/S mutant [AdPTENC/S]) possessing constitutive phospoinositide 3-kinase activity in HepG2 cells led to significant reductions in both secreted apoB100 and cellular MTP mass (76% and 34%, respectively), and increased messenger RNA (mRNA) levels of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Reduced apoB100 secretion induced by AdPTENC/S was associated with increased degradation of newly-synthesized cellular apoB100, in a lactacystin-sensitive manner, suggesting enhanced proteasomal degradation. AdPTENC/S also reduced apoB-lipoprotein production in McA-RH7777 and primary hamster hepatocytes. Our findings suggest a link between PTEN expression and hepatic production of apoB-containing lipoproteins. We postulate that perturbations in PTEN not only may influence hepatic insulin signaling and hepatic lipogenesis, but also may alter hepatic apoB-lipoprotein production and the MTP stability. On loss of PTEN activity, increased lipid substrate availability in the face of reduced hepatic lipoprotein production capacity can rapidly lead to hepatosteatosis and fatty liver.

Authors
Qiu, W; Federico, L; Naples, M; Avramoglu, RK; Meshkani, R; Zhang, J; Tsai, J; Hussain, M; Dai, K; Iqbal, J; Kontos, CD; Horie, Y; Suzuki, A; Adeli, K
MLA Citation
Qiu, W, Federico, L, Naples, M, Avramoglu, RK, Meshkani, R, Zhang, J, Tsai, J, Hussain, M, Dai, K, Iqbal, J, Kontos, CD, Horie, Y, Suzuki, A, and Adeli, K. "Phosphatase and tensin homolog (PTEN) regulates hepatic lipogenesis, microsomal triglyceride transfer protein, and the secretion of apolipoprotein B-containing lipoproteins." Hepatology 48.6 (December 2008): 1799-1809.
PMID
19026012
Source
pubmed
Published In
Hepatology
Volume
48
Issue
6
Publish Date
2008
Start Page
1799
End Page
1809
DOI
10.1002/hep.22565

Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells.

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P(1-3) receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P(1) and S1P(2) was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P(2) receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P(2) to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P(2)-mediated activation of the lipid phosphatase PTEN.

Authors
Estrada, R; Zeng, Q; Lu, H; Sarojini, H; Lee, J-F; Mathis, SP; Sanchez, T; Wang, E; Kontos, CD; Lin, C-Y; Hla, T; Haribabu, B; Lee, M-J
MLA Citation
Estrada, R, Zeng, Q, Lu, H, Sarojini, H, Lee, J-F, Mathis, SP, Sanchez, T, Wang, E, Kontos, CD, Lin, C-Y, Hla, T, Haribabu, B, and Lee, M-J. "Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells." J Biol Chem 283.44 (October 31, 2008): 30363-30375.
PMID
18765664
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
44
Publish Date
2008
Start Page
30363
End Page
30375
DOI
10.1074/jbc.M804392200

A VEGF trap inhibits the beneficial effect of bFGF on vasoreactivity in corporal tissues of hypercholesterolemic rabbits.

INTRODUCTION: Hypercholesterolemia causes a decrease in normal corporal tissue vasoreactivity in a preclinical model of erectile dysfunction. Previous studies have shown that intracorporal injection (ICI) of basic fibroblast growth factor (bFGF) reverses some of the detrimental vasoreactivity effects of hypercholesterolemia and increases vascular endothelial growth factor (VEGF) expression. AIM: We sought to determine whether the beneficial effects of bFGF are VEGF-mediated. METHODS: A total of 32 New Zealand white rabbits were fed a 1% cholesterol diet for 6 weeks and randomly divided into four groups (N = 8/group). Group 1 received a 2.5 microg bFGF ICI and 2.5 x 10(11) viral particle unit (vpu) of adenovirus encoding beta-galactosidase (Ad beta-gal) ICI, 10 days later. Group 2 received a 2.5 microg bFGF ICI and 2.5 x 10(11) vpu of adenovirus encoding soluble VEGF receptor (VEGFR) (AdsVEGFR, a VEGF trap) ICI, 10 days later. Group 3 received phosphate buffered saline solution (PBS) ICI and 2.5 x 10(11) vpu Ad beta-gal ICI, 10 days later. Group 4 received PBS ICI and 2.5 x 10(11) vpu AdsVEGFR ICI, 10 days later. MAIN OUTCOME MEASURES: The corpus cavernosum was harvested for vasoreactivity studies 10 days post viral injection. The effective dose of 50% maximum relaxation was determined. VEGF levels were assessed by enzyme-linked immunosorbent assay. Total and phosphorylated Akt and endothelial nitric oxide were analyzed by Western blot. RESULTS: Endothelium-dependent vasoreactivity was significantly greater in Group 1 vs. all other groups. The VEGF trap eliminated the beneficial effects of bFGF on endothelium-dependent vasoreactivity and decreased Akt and nitric oxide phosphorylation. CONCLUSIONS: These data demonstrate that VEGF activity contributes much of the therapeutic modulation of bFGF-mediated vasoreactivity in corporal tissue.

Authors
Xie, D; Findley, CM; Greenfield, JM; Pippen, AM; Kontos, CD; Donatucci, CF; Annex, BH
MLA Citation
Xie, D, Findley, CM, Greenfield, JM, Pippen, AM, Kontos, CD, Donatucci, CF, and Annex, BH. "A VEGF trap inhibits the beneficial effect of bFGF on vasoreactivity in corporal tissues of hypercholesterolemic rabbits." J Sex Med 5.9 (September 2008): 2069-2078.
PMID
18637998
Source
pubmed
Published In
The Journal of Sexual Medicine
Volume
5
Issue
9
Publish Date
2008
Start Page
2069
End Page
2078
DOI
10.1111/j.1743-6109.2008.00933.x

Plasma levels of soluble Tie2 and vascular endothelial growth factor distinguish critical limb ischemia from intermittent claudication in patients with peripheral arterial disease.

OBJECTIVES: Our purpose was to determine whether factors that regulate angiogenesis are altered in peripheral arterial disease (PAD) and whether these factors are associated with the severity of PAD. BACKGROUND: Alterations in angiogenic growth factors occur in cardiovascular disease (CVD), but whether these factors are altered in PAD or correlate with disease severity is unknown. METHODS: Plasma was collected from patients with PAD (n = 46) and healthy control subjects (n = 23). Peripheral arterial disease patients included those with intermittent claudication (IC) (n = 23) and critical limb ischemia (CLI) (n = 23). Plasma angiopoietin-2 (Ang2), soluble Tie2 (sTie2), vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR-1), and placenta growth factor (PlGF) were measured by enzyme-linked immunoadsorbent assay. In vitro, endothelial cells (ECs) were treated with recombinant VEGF to investigate effects on sTie2 production. RESULTS: Plasma concentrations of sTie2 (p < 0.01), Ang2 (p < 0.001), and VEGF (p < 0.01), but not PlGF or sVEGFR-1, were significantly greater in PAD patients compared with control subjects. Plasma Ang2 was significantly increased in both IC and CLI compared with control subjects (p < 0.0001), but there was no difference between IC and CLI. Plasma VEGF and sTie2 were similar in control subjects and IC but were significantly increased in CLI (p < 0.001 vs. control or IC). Increased sTie2 and VEGF were independent of CVD risk factors or the ankle-brachial index, and VEGF treatment of ECs in vitro significantly increased sTie2 shedding. CONCLUSIONS: Levels of VEGF and sTie2 are significantly increased in CLI, and sTie2 production is induced by VEGF. These proteins may provide novel biomarkers for CLI, and sTie2 may be both a marker and a cause of CLI.

Authors
Findley, CM; Mitchell, RG; Duscha, BD; Annex, BH; Kontos, CD
MLA Citation
Findley, CM, Mitchell, RG, Duscha, BD, Annex, BH, and Kontos, CD. "Plasma levels of soluble Tie2 and vascular endothelial growth factor distinguish critical limb ischemia from intermittent claudication in patients with peripheral arterial disease." J Am Coll Cardiol 52.5 (July 29, 2008): 387-393.
PMID
18652948
Source
pubmed
Published In
Journal of the American College of Cardiology
Volume
52
Issue
5
Publish Date
2008
Start Page
387
End Page
393
DOI
10.1016/j.jacc.2008.02.045

Inhibition of in vivo tumor angiogenesis and growth via systemic delivery of an angiopoietin 2-specific RNA aptamer.

BACKGROUND: Cellular events mediated by the Tie2 receptor are important to tumor neovascularization. Despite the complex interplay of the best-characterized Tie2 ligands, angiopoietins 1 and 2, Ang2 is purportedly "proangiogenic" in the presence of vascular endothelial growth factor. We examined whether in vivo administration of an RNA aptamer that specifically blocks Ang 2 would inhibit tumor angiogenesis and growth. METHODS: Ang2-mediated Tie2 receptor phosphorylation was assessed in vitro in the absence and presence of aptamer coupled to polyethylene glycol. IN VIVO ANGIOGENESIS ASSAY: CT26 murine colon carcinoma cells expressing green fluorescent protein were delivered into mouse dorsal skinfold window chambers. Animals received daily intraperitoneal injections of phosphate-buffered saline, low-dose (Ang2 aptamer-LD; 1 mg/kg/d), or high-dose aptamer (Ang2 aptamer-HD; 10 mg/kg/d). Vascular length density was measured under fluorescence microscopy. PRIMARY TUMOR GROWTH: CT26 cells expressing luciferase were injected into flanks of BALB/c mice to allow tumor growth monitoring by bioluminescence imaging. Animals received continuous phosphate-buffered saline or aptamer (1 mg/kg/d) via ALZET pumps. Tumors were assessed for CD31/PECAM-1 immunostaining and Hoechst dye uptake. RESULTS: Pegylated aptamer inhibited Tie2 phosphorylation. Systemic aptamer administration reduced vascular length density (P < or = 0.03) and decreased bioluminescence emission (P < 0.04), corresponding to 50% decrease in tumor volume (P = 0.04). Control tumors displayed abundant vascular marker staining, in contrast to tumors from aptamer-treated animals. CONCLUSIONS: in vivo administration of a clinically relevant, pegylated RNA aptamer specifically designed against Ang2 inhibited tumor angiogenesis and growth. These findings support targeted Ang2 inhibition as a relevant anti-angiogenic, anti-neoplastic strategy.

Authors
Sarraf-Yazdi, S; Mi, J; Moeller, BJ; Niu, X; White, RR; Kontos, CD; Sullenger, BA; Dewhirst, MW; Clary, BM
MLA Citation
Sarraf-Yazdi, S, Mi, J, Moeller, BJ, Niu, X, White, RR, Kontos, CD, Sullenger, BA, Dewhirst, MW, and Clary, BM. "Inhibition of in vivo tumor angiogenesis and growth via systemic delivery of an angiopoietin 2-specific RNA aptamer." J Surg Res 146.1 (May 1, 2008): 16-23.
PMID
17950331
Source
pubmed
Published In
Journal of Surgical Research
Volume
146
Issue
1
Publish Date
2008
Start Page
16
End Page
23
DOI
10.1016/j.jss.2007.04.028

High cholesterol feeding in C57/Blc6 mice alters expression within the VEGF receptor-ligand family in corporal tissue.

INTRODUCTION: Angiogenesis, the growth and proliferation of blood vessels from existing vascular structures, is mediated by many cytokine growth factors and receptors, among the most important are the vascular endothelial growth factor (VEGF) family. AIM: Decreases in VEGF receptor signaling have been linked to abnormalities in vasoreactivity in corporal tissue, but it is unknown if alterations in the VEGF ligands and/or receptors contribute to this process. MAIN OUTCOME MEASURES: We sought to determine changes in vasoreactivity and the expression of the family of VEGF ligands and receptors in corporal tissue with cholesterol feeding in C57BL6 mice. Methods. Twenty-four mice (N = 8/group) were fed a normal diet (Group 1) or a 1.25% high cholesterol diet for 4 (Group 2) or 12 (Group 3) weeks. Isometric tension studies were performed on corporal strips and dose response curves were generated to evaluate endothelium-dependent and endothelium-independent vasoreactivities. Levels of VEGF-A, B, C, D, VEGF receptors (VEGFRs) were detected by PCR (polymerase chain reaction) and/or western blot/enzyme-linked immunosorbent assay (ELISA). Endothelial and smooth muscle cell contents were determined by immunohistochemistry. RESULTS: At 4 weeks there was a small but significant decrease in endothelium-dependent vasoreactivity. Both mRNA and protein levels of VEGFR-1 were decreased, while VEGF-B was increased in Group 2 vs. Group 1, with no change in VEGF-A or endothelial cell content. By 12 weeks, decreases in both endothelium-dependent and endothelium-independent vasoreactivity were evident with decrease in most VEGF ligands (except VEGF-B), receptors, and receptor signaling. CONCLUSIONS: Cholesterol feeding in C57BL6 mice results in alterations in the VEGF receptor-ligand family that may initially serve to limit the degree of vascular injury but these adaptations fail with the continuation of cholesterol feeding.

Authors
Xie, D; Hazarika, S; Andrich, AJ; Padgett, ME; Kontos, CD; Donatucci, CF; Annex, BH
MLA Citation
Xie, D, Hazarika, S, Andrich, AJ, Padgett, ME, Kontos, CD, Donatucci, CF, and Annex, BH. "High cholesterol feeding in C57/Blc6 mice alters expression within the VEGF receptor-ligand family in corporal tissue." J Sex Med 5.5 (May 2008): 1137-1148.
PMID
18439153
Source
pubmed
Published In
The Journal of Sexual Medicine
Volume
5
Issue
5
Publish Date
2008
Start Page
1137
End Page
1148
DOI
10.1111/j.1743-6109.2008.00801.x

RNA aptamer-targeted inhibition of NF-kappa B suppresses non-small cell lung cancer resistance to doxorubicin.

Due to the prevalence of tumor chemoresistance, the clinical response of advanced non-small cell lung cancer (NSCLC) to chemotherapy is poor. We suppressed tumor resistance to doxorubicin (Dox) in A549 cells, a human NSCLC cell line, both in vitro and in vivo in a lung tumor xenograft model, using a novel adenoviral expression system to deliver an RNA aptamer (A-p50) that specifically inhibits nuclear factor-kappaB (NF-kappaB) activation. By achieving selective, targeted, and early inhibition of NF-kappaB activity, we demonstrate that NF-kappaB plays a critical role in Dox-induced chemoresistance by regulating genes involved in proliferation (Ki-67), response to DNA damage (GADD153), antiapoptosis (Bcl-XL), and pH regulation (CA9). This Dox-induced NF-kappaB activation and subsequent chemoresistance is dependent on expression of p53. We also demonstrate that NF-kappaB promotes angiogenesis in the presence of Dox via the hypoxia-inducible factor-1alpha/vascular endothelial growth factor (HIF-1alpha/VEGF) pathway, revealing a previously unknown mechanism of NSCLC resistance to Dox. These studies provide important insights into the mechanisms of Dox-induced chemoresistance, and they demonstrate a novel, effective, and clinically practical strategy for interfering with these processes.

Authors
Mi, J; Zhang, X; Rabbani, ZN; Liu, Y; Reddy, SK; Su, Z; Salahuddin, FK; Viles, K; Giangrande, PH; Dewhirst, MW; Sullenger, BA; Kontos, CD; Clary, BM
MLA Citation
Mi, J, Zhang, X, Rabbani, ZN, Liu, Y, Reddy, SK, Su, Z, Salahuddin, FK, Viles, K, Giangrande, PH, Dewhirst, MW, Sullenger, BA, Kontos, CD, and Clary, BM. "RNA aptamer-targeted inhibition of NF-kappa B suppresses non-small cell lung cancer resistance to doxorubicin." Mol Ther 16.1 (January 2008): 66-73.
PMID
17912235
Source
pubmed
Published In
Molecular Therapy
Volume
16
Issue
1
Publish Date
2008
Start Page
66
End Page
73
DOI
10.1038/sj.mt.6300320

A nuclease-resistant RNA aptamer specifically inhibits angiopoietin-1-mediated Tie2 activation and function.

Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2'-fluoro-modified ribonucleotides for Ang1-binding aptamers. After nine rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 (K ( d ) 2.8 nM) but not Ang2 (K ( d ) > 1 microM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases.

Authors
White, RR; Roy, JA; Viles, KD; Sullenger, BA; Kontos, CD
MLA Citation
White, RR, Roy, JA, Viles, KD, Sullenger, BA, and Kontos, CD. "A nuclease-resistant RNA aptamer specifically inhibits angiopoietin-1-mediated Tie2 activation and function." Angiogenesis 11.4 (2008): 395-401.
PMID
19037734
Source
pubmed
Published In
Angiogenesis
Volume
11
Issue
4
Publish Date
2008
Start Page
395
End Page
401
DOI
10.1007/s10456-008-9122-4

VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling.

OBJECTIVE: Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. METHODS AND RESULTS: sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt. CONCLUSIONS: These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling.

Authors
Findley, CM; Cudmore, MJ; Ahmed, A; Kontos, CD
MLA Citation
Findley, CM, Cudmore, MJ, Ahmed, A, and Kontos, CD. "VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling." Arterioscler Thromb Vasc Biol 27.12 (December 2007): 2619-2626.
PMID
17901375
Source
pubmed
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
27
Issue
12
Publish Date
2007
Start Page
2619
End Page
2626
DOI
10.1161/ATVBAHA.107.150482

Impaired angiogenesis after hindlimb ischemia in type 2 diabetes mellitus: differential regulation of vascular endothelial growth factor receptor 1 and soluble vascular endothelial growth factor receptor 1.

Deficient angiogenesis after ischemia may contribute to worse outcomes of peripheral arterial disease in patients with diabetes mellitus (DM). Vascular endothelial growth factor (VEGF) and its receptors promote angiogenesis. We hypothesized that in peripheral arterial disease, maladaptive changes in VEGF ligand/receptor expression could account for impaired angiogenesis in DM. Skeletal muscle from diet-induced, type 2 diabetic (DM) and age-matched normal chow (NC)-fed mice was collected at baseline and 3 and 10 days after hindlimb ischemia and analyzed for expression of VEGF (n=10 per group), full-length VEGF receptor (VEGFR)-1, soluble VEGFR-1, and markers of downstream VEGF signaling (n=20 per group) using ELISA, reverse transcriptase-polymerase chain reaction, and Western blots. In the absence of ischemia, DM mice had increased VEGF (NC versus DM: 26.6+/-2.6 versus 53.5+/-8.8 pg/mg protein; P<0.05), decreased soluble and membrane-bound VEGFR-1 (NC versus DM: 1.44+/-0.30 versus 0.85+/-0.08 and 1.03+/-0.10 versus 0.72+/-0.10, respectively; P<0.05), decreased phospho-AKT/AKT and phospho-endothelial NO synthase/endothelial NO synthase (NC versus DM: 0.76+/-0.2 versus 0.38+/-0.1 and 0.36+/-0.06 versus 0.25+/-0.04, respectively; P<0.05), and no change in VEGFR-2. After ischemia, both DM and NC had comparable increases in VEGF-A. VEGFR-1 and soluble VEGFR-1 expression increased in both groups, but the fold increase was significantly greater in DM. These data demonstrate that soluble VEGFR-1, an angiogenesis inhibitor, is regulated in skeletal muscle by type 2 DM and ischemia. In the absence of ischemia, despite reductions in both soluble VEGFR-1 and VEGFR-1, VEGF ligand signaling is lower in DM compared with controls. After ischemia, maladaptive upregulation of these receptors further reduces the capacity of VEGF to induce an angiogenic response, which may provide a novel target for therapy.

Authors
Hazarika, S; Dokun, AO; Li, Y; Popel, AS; Kontos, CD; Annex, BH
MLA Citation
Hazarika, S, Dokun, AO, Li, Y, Popel, AS, Kontos, CD, and Annex, BH. "Impaired angiogenesis after hindlimb ischemia in type 2 diabetes mellitus: differential regulation of vascular endothelial growth factor receptor 1 and soluble vascular endothelial growth factor receptor 1." Circ Res 101.9 (October 26, 2007): 948-956.
PMID
17823371
Source
pubmed
Published In
Circulation Research
Volume
101
Issue
9
Publish Date
2007
Start Page
948
End Page
956
DOI
10.1161/CIRCRESAHA.107.160630

Systemic overexpression of angiopoietin-2 promotes tumor microvessel regression and inhibits angiogenesis and tumor growth.

Angiopoietin-2 (Ang-2) is a conditional antagonist and agonist for the endothelium-specific Tie-2 receptor. Although endogenous Ang-2 cooperates with vascular endothelial growth factor (VEGF) to protect tumor endothelial cells, the effect on tumor vasculature of high levels of exogenous Ang-2 with different levels of VEGF has not been studied in detail. Here, we report that systemic overexpression of Ang-2 leads to unexpected massive tumor vessel regression within 24 h, even without concomitant inhibition of VEGF. By impairing pericyte coverage of the tumor vasculature, Ang-2 destabilizes the tumor vascular bed while improving perfusion in surviving tumor vessels. Ang-2 overexpression transiently exacerbates tumor hypoxia without affecting ATP levels. Although sustained systemic Ang-2 overexpression does not affect tumor hypoxia and proliferation, it significantly inhibits tumor angiogenesis, promotes tumor apoptosis, and suppresses tumor growth. The similar antitumoral, antiangiogenic efficacy of systemic overexpression of Ang-2, soluble VEGF receptor-1, and the combination of both suggests that concomitant VEGF inhibition is not required for Ang-2-induced tumor vessel regression and growth delay. This study shows the important roles of Ang-2-induced pericyte dropout during tumor vessel regression. It also reveals that elevated Ang-2 levels have profound pleiotropic effects on tumor vessel structure, perfusion, oxygenation, and apoptosis.

Authors
Cao, Y; Sonveaux, P; Liu, S; Zhao, Y; Mi, J; Clary, BM; Li, C-Y; Kontos, CD; Dewhirst, MW
MLA Citation
Cao, Y, Sonveaux, P, Liu, S, Zhao, Y, Mi, J, Clary, BM, Li, C-Y, Kontos, CD, and Dewhirst, MW. "Systemic overexpression of angiopoietin-2 promotes tumor microvessel regression and inhibits angiogenesis and tumor growth." Cancer Res 67.8 (April 15, 2007): 3835-3844.
PMID
17440098
Source
pubmed
Published In
Cancer Research
Volume
67
Issue
8
Publish Date
2007
Start Page
3835
End Page
3844
DOI
10.1158/0008-5472.CAN-06-4056

Engineered transcription factors for therapeutic angiogenesis.

Angiogenesis is defined as the growth and proliferation of blood vessels from existing vascular structures. Therapeutic angiogenesis seeks to harness the mechanisms of vascular growth to treat disorders of inadequate tissue perfusion. Early clinical trials of therapeutic angiogenesis met with limited success, in part due to the complex spatial and temporal regulation of angiogenesis, which requires the coordinate action of multiple growth factors and their receptors. Engineered transcription factors represent a novel and innovative approach to modulate this process as they offer the ability to induce expression of multiple angiogenic genes simultaneously. The rational design of these transcription factor agents, results of their use in preclinical models of ischemic disease and prospects for their use in human investigations are discussed in this review.

Authors
Kontos, CD; Annex, BH
MLA Citation
Kontos, CD, and Annex, BH. "Engineered transcription factors for therapeutic angiogenesis." Curr Opin Mol Ther 9.2 (April 2007): 145-152. (Review)
PMID
17458168
Source
pubmed
Published In
Current Opinion in Molecular Therapeutics
Volume
9
Issue
2
Publish Date
2007
Start Page
145
End Page
152

In mice with type 2 diabetes, a vascular endothelial growth factor (VEGF)-activating transcription factor modulates VEGF signaling and induces therapeutic angiogenesis after hindlimb ischemia.

Peripheral arterial disease is a major complication of diabetes. The ability to promote therapeutic angiogenesis may be limited in diabetes. Type 2 diabetes was induced by high-fat feeding C57BL/6 mice (n = 60). Normal chow-fed mice (n = 20) had no diabetes. Mice underwent unilateral femoral artery ligation and excision. A plasmid DNA encoded an engineered transcription factor designed to increase vascular endothelial growth factor expression (ZFP-VEGF). On day 10 after the operation, the ischemic limbs received 125 microg ZFP-VEGF plasmid or control. Mice were killed 3, 10, or 20 days after injection (n = 10/group, at each time point). Limb blood flow was measured by laser Doppler perfusion imaging. VEGF mRNA expression was examined by real-time PCR. VEGF, Akt, and phospho-Akt protein were measured by enzyme-linked immunosorbent assay. Capillary density, proliferation, and apoptosis were assessed histologically. Compared with normal mice, mice with diabetes had greater VEGF protein, reduced phospho-Akt-to-Akt ratio before ligation, and an impaired perfusion recovery after ligation. At 3 and 10 days after injection, in mice with diabetes, gene transfer increased VEGF expression and signaling. At later time points, gene transfer resulted in better perfusion recovery. Gene transfer with ZFP-VEGF was able to promote therapeutic angiogenesis mice with type 2 diabetes.

Authors
Li, Y; Hazarika, S; Xie, D; Pippen, AM; Kontos, CD; Annex, BH
MLA Citation
Li, Y, Hazarika, S, Xie, D, Pippen, AM, Kontos, CD, and Annex, BH. "In mice with type 2 diabetes, a vascular endothelial growth factor (VEGF)-activating transcription factor modulates VEGF signaling and induces therapeutic angiogenesis after hindlimb ischemia." Diabetes 56.3 (March 2007): 656-665.
PMID
17327433
Source
pubmed
Published In
Diabetes
Volume
56
Issue
3
Publish Date
2007
Start Page
656
End Page
665
DOI
10.2337/db06-0999

APOE4-VLDL inhibits the HDL-activated phosphatidylinositol 3-kinase/Akt Pathway via the phosphoinositol phosphatase SHIP2.

Endothelial cell dysfunction and apoptosis are critical in the pathogenesis of atherosclerotic cardiovascular disease (CVD). Both endothelial cell apoptosis and atherosclerosis are reduced by high-density lipoprotein (HDL). Low HDL levels increase the risk of CVD and are also a key characteristic of the metabolic syndrome. The apolipoprotein E4 (APOE4) allele also increases the risk of atherosclerosis and CVD. We previously demonstrated that the antiapoptotic activity of HDL is inhibited by APOE4 very-low-density lipoprotein (APOE4-VLDL) in endothelial cells, an effect similar to reducing the levels of HDL. Here we establish the intracellular mechanism by which APOE4-VLDL inhibits the antiapoptotic pathway activated by HDL. We show that APOE4-VLDL diminishes the phosphorylation of Akt by HDL but does not alter phosphorylation of c-Jun N-terminal kinase, p38, or Src family kinases by HDL. Furthermore APOE4-VLDL inhibits Akt phosphorylation by reducing the phosphatidylinositol 3-kinase product phosphatidylinositol-(3,4,5)-triphosphate (PI[3,4,5]P3). We further demonstrate that APOE4-VLDL reduces PI(3,4,5)P3, through the phosphoinositol phosphatase SHIP2, and not through PTEN. SHIP2 is already implicated as an independent risk factor for type II diabetes, hypertension and obesity, which are also all components of the metabolic syndrome and independent risk factors for CVD. Significantly, the association between CVD and type 2 diabetes or hypertension is further increased by the APOE4 allele. Therefore the activation of SHIP2 by APOE4-VLDL, with the subsequent inhibition of the HDL/Akt pathway, is a novel and significant biological mechanism and may be a critical intermediate by which APOE4 increases the risk of atherosclerotic CVD.

Authors
DeKroon, R; Robinette, JB; Hjelmeland, AB; Wiggins, E; Blackwell, M; Mihovilovic, M; Fujii, M; York, J; Hart, J; Kontos, C; Rich, J; Strittmatter, WJ
MLA Citation
DeKroon, R, Robinette, JB, Hjelmeland, AB, Wiggins, E, Blackwell, M, Mihovilovic, M, Fujii, M, York, J, Hart, J, Kontos, C, Rich, J, and Strittmatter, WJ. "APOE4-VLDL inhibits the HDL-activated phosphatidylinositol 3-kinase/Akt Pathway via the phosphoinositol phosphatase SHIP2." Circ Res 99.8 (October 13, 2006): 829-836.
PMID
16973905
Source
pubmed
Published In
Circulation Research
Volume
99
Issue
8
Publish Date
2006
Start Page
829
End Page
836
DOI
10.1161/01.RES.0000245479.03190.9f

An engineered vascular endothelial growth factor-activating transcription factor induces therapeutic angiogenesis in ApoE knockout mice with hindlimb ischemia.

OBJECTIVE: Angiogenesis is the growth and proliferation of blood vessels from existing vascular structures, and therapeutic angiogenesis seeks to promote blood vessel growth to improve tissue perfusion. Vascular endothelial growth factor (VEGF) is a prototypic angiogenic agent that exists in vivo in multiple isoforms, and studies with VEGF to date had used single isoform therapy with disappointing results. We tested plasmid and adenoviral vectors encoding a zinc-finger DNA-binding transcription factor (ZFP-32E) that was designed to increase the expression of all major VEGF isoforms in a preclinical model of peripheral arterial obstructive disease (PAOD) in hypercholesterolemic (ApoE knock-out) mice. METHODS: Unilateral femoral artery ligation/excision was performed in 117 mice. At 7 days postoperatively, the ischemic tibialis anterior (TA) and gastrocnemius (GAS) muscles received either ZFP-32E treatment (125 microg of plasmid, 2.5 x 10(11) viral particle units [vpu] of adenovirus; some mice received a second plasmid injection 3 days later) or no-ZFP treatment (125 microg of beta-galactosidase [beta-gal], a plasmid-lacking insert, or an equal dose of adenoviral encoding beta-gal; some mice received a second plasmid injection 3 days later). Group 1 mice (n = 31) were euthanized 3 days later, and VEGF messenger RNA (mRNA) and protein levels were measured. Group 2 mice (n = 38) were euthanized 7 days later, and measures of capillary density, cell proliferation, and apoptosis were quantified. Group 3 mice (n = 48) were euthanized 28 days later, and changes in lower limb blood flow perfusion were measured. RESULTS: In group 1, VEGF mRNA and protein levels were significantly higher in those with ZFP-32E treatment vs beta-gal. In group 2, capillary density and proliferating cells were significantly greater and apoptosis was significantly lower in those with ZFP-32E treatment vs beta-gal. Finally, in group 3, changes in the perfusion ratio (ischemic/nonischemic limb) at 21 days after injection were significantly greater in those with ZFP-32E treatment vs no-ZFP treatment. CONCLUSION: The ability of this engineered zinc-finger VEGF-activating transcription factor to induce therapeutic angiogenesis in hypercholesterolemic mice suggests this approach warrants investigation as a novel approach to treat PAOD.

Authors
Xie, D; Li, Y; Reed, EA; Odronic, SI; Kontos, CD; Annex, BH
MLA Citation
Xie, D, Li, Y, Reed, EA, Odronic, SI, Kontos, CD, and Annex, BH. "An engineered vascular endothelial growth factor-activating transcription factor induces therapeutic angiogenesis in ApoE knockout mice with hindlimb ischemia." J Vasc Surg 44.1 (July 2006): 166-175.
PMID
16753278
Source
pubmed
Published In
Journal of Vascular Surgery
Volume
44
Issue
1
Publish Date
2006
Start Page
166
End Page
175
DOI
10.1016/j.jvs.2006.03.024

Efficacy and mechanism of adenovirus-mediated VEGF-165 gene therapy for augmentation of skin flap viability.

Skin ischemic necrosis due to vasospasm and/or insufficient vascularity is the most common complication in the distal portion of the skin flap in reconstructive surgery. This project was designed to test our hypothesis that preoperative subdermal injection of adenoviral vectors encoding genes for vascular endothelial growth factor-165 (Ad.VEGF-165) or endothelial nitric oxide (NO) synthase (Ad.eNOS) effectively augments skin viability in skin flap surgery and that the mechanism of Ad.VEGF-165 gene therapy involves an increase in synthesis/release of the angiogenic and vasodilator factor NO. PBS (0.5 ml) or PBS containing Ad.VEGF-165, Ad.eNOS, or adenovirus (Ad.Null) was injected subdermally into the distal half of a mapped rat dorsal skin flap (4 x 10 cm) 7 days preoperatively, and skin flap viability was assessed 7 days postoperatively. Local subdermal gene therapy with 2 x 10(7)-2 x 10(10) plaque-forming units of VEGF-165 increased skin flap viability compared with PBS- or Ad.Null-injected control (P < 0.05). Subdermal Ad.VEGF-165 and Ad.eNOS gene therapies were equally effective in increasing skin flap viability at 5 x 10(8) plaque-forming units. Subdermal Ad.VEGF-165 therapy was associated with upregulation of eNOS protein expression, Ca2+ -dependent NOS activity, synthesis/release of NO, and increase in capillary density and blood flow in the distal portion of the skin flap. Injection of the NOS inhibitor Nomega-nitro-L-arginine (15 mg/kg im), but not the cyclooxygenase inhibitor indomethacin (5 mg/kg im), 45 min preoperatively completely abolished the increase in skin flap blood flow and viability induced by Ad.VEGF-165 injected subdermally into the mapped skin flap 7 days preoperatively. We have demonstrated for the first time that 1) Ad.VEGF-165 and Ad.eNOS mapped skin flap injected subdermally into the mapped skin flap 7 days preoperatively are equally effective in augmenting viability in the rat dorsal skin flap compared with control, 2) the mechanism of subdermal Ad.VEGF-165 gene therapy in augmenting skin flap viability involves an increase in NO synthesis/release downstream of upregulation of eNOS protein expression and Ca2+ -dependent NOS activity, and 3) the vasodilating effect of NO may predominantly mediate subdermal Ad.VEGF gene therapy in augmenting skin flap blood flow and viability.

Authors
Huang, N; Khan, A; Ashrafpour, H; Neligan, PC; Forrest, CR; Kontos, CD; Pang, CY
MLA Citation
Huang, N, Khan, A, Ashrafpour, H, Neligan, PC, Forrest, CR, Kontos, CD, and Pang, CY. "Efficacy and mechanism of adenovirus-mediated VEGF-165 gene therapy for augmentation of skin flap viability." Am J Physiol Heart Circ Physiol 291.1 (July 2006): H127-H137.
PMID
16461370
Source
pubmed
Published In
American journal of physiology. Heart and circulatory physiology
Volume
291
Issue
1
Publish Date
2006
Start Page
H127
End Page
H137
DOI
10.1152/ajpheart.01253.2005

A comparison of antiangiogenic therapies for the prevention of liver metastases.

Angiogenesis is essential for solid tumor growth. Although successful antiangiogenic therapies have been demonstrated in animal models, a systematic comparison of the efficacy of different antiangiogenic factors has not been described in the hepatic environment. To address this issue, CT26 murine colon carcinoma cells were transfected with retroviral vectors encoding murine endostatin (mEndostatin), human angiostatin (hAngiostatin), murine-soluble vascular endothelial growth factor receptor-2, (msFlk-1), or murine-soluble Tie2 (msTie2). The transfected cells were then subjected to another round of transfection with a luciferase cDNA-encoding retroviral vector. Expression of these putative antiangiogenic proteins inhibited the proliferation of human umbilical vein endothelial cells in vitro but not tumor cells. To examine effects on tumor growth in vivo, modified cells were delivered via intrasplenic injection into BALB/c mice to induce liver metastases. Tumor burden was measured weekly by bioluminescence. Growth of hepatic metastases in vivo was significantly reduced in mice that were administered cells expressing msTie2 (76% reduction compared with control cells 21 days after intrasplenic inoculation; P < 0.05). Similar results were observed with cells that expressed msFlk-1 and hAngiostatin. However, expression of mEndostatin had no significant effect on the growth of liver metastases compared with control animals. These findings indicate that multiple antiangiogenic pathways are necessary for the growth of hepatic metastases, and each of these pathways is a potential clinically relevant antiangiogenic target for the treatment of this disease.

Authors
Mi, J; Sarraf-Yazdi, S; Zhang, X; Cao, Y; Dewhirst, MW; Kontos, CD; Li, C-Y; Clary, BM
MLA Citation
Mi, J, Sarraf-Yazdi, S, Zhang, X, Cao, Y, Dewhirst, MW, Kontos, CD, Li, C-Y, and Clary, BM. "A comparison of antiangiogenic therapies for the prevention of liver metastases." J Surg Res 131.1 (March 2006): 97-104.
PMID
16242720
Source
pubmed
Published In
Journal of Surgical Research
Volume
131
Issue
1
Publish Date
2006
Start Page
97
End Page
104
DOI
10.1016/j.jss.2005.09.008

Negative regulation of myofibroblast differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on chromosome 10).

RATIONALE: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents. OBJECTIVE: To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs. METHODS: We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and alpha-smooth muscle actin (alpha-SMA). We used cell culture of pten(-/-) and wild-type fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits alpha-SMA, fibroblast proliferation, and collagen production. RESULTS: In human lung specimens of IPF, myofibroblasts within fibroblastic foci demonstrated diminished PTEN expression. Furthermore, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten(-/-) fibroblasts, and in normal fibroblasts in which PTEN was inhibited, alpha-SMA, proliferation, and collagen production was upregulated. Addition of transforming growth factor-beta to wild-type cells, but not pten(-/-) cells, resulted in increased alpha-SMA expression in a time-dependent fashion. In pten(-/-) cells, reconstitution of PTEN decreased alpha-SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibited transforming growth factor-beta-induced myofibroblast differentiation. It was observed that both the protein and lipid phosphatase actions of PTEN were capable of modulating the myofibroblast phenotype. CONCLUSIONS: The results indicate that in IPF, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.

Authors
White, ES; Atrasz, RG; Hu, B; Phan, SH; Stambolic, V; Mak, TW; Hogaboam, CM; Flaherty, KR; Martinez, FJ; Kontos, CD; Toews, GB
MLA Citation
White, ES, Atrasz, RG, Hu, B, Phan, SH, Stambolic, V, Mak, TW, Hogaboam, CM, Flaherty, KR, Martinez, FJ, Kontos, CD, and Toews, GB. "Negative regulation of myofibroblast differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on chromosome 10)." Am J Respir Crit Care Med 173.1 (January 1, 2006): 112-121.
PMID
16179636
Source
pubmed
Published In
American journal of respiratory and critical care medicine
Volume
173
Issue
1
Publish Date
2006
Start Page
112
End Page
121
DOI
10.1164/rccm.200507-1058OC

H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity.

Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers are limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation. In this report, we successfully expressed a 'pure' RNA aptamer specific for NF-kappaB p50 protein (A-p50) utilizing an adenoviral vector employing the H1 RNA polymerase III promoter. Binding of the expressed aptamer to its target and subsequent inhibition of NF-kappaB mediated intracellular events were demonstrated in human lung adenocarcinoma cells (A549), murine mammary carcinoma cells (4T1) as well as a human tumor xenograft model. This success highlights the promise of RNA aptamers to effectively target intracellular proteins for in vitro discovery and in vivo applications.

Authors
Mi, J; Zhang, X; Rabbani, ZN; Liu, Y; Su, Z; Vujaskovic, Z; Kontos, CD; Sullenger, BA; Clary, BM
MLA Citation
Mi, J, Zhang, X, Rabbani, ZN, Liu, Y, Su, Z, Vujaskovic, Z, Kontos, CD, Sullenger, BA, and Clary, BM. "H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity. (Published online)" Nucleic Acids Res 34.12 (2006): 3577-3584.
PMID
16855294
Source
pubmed
Published In
Nucleic Acids Research
Volume
34
Issue
12
Publish Date
2006
Start Page
3577
End Page
3584
DOI
10.1093/nar/gkl482

Loss of phosphatase and tensin homologue increases transforming growth factor beta-mediated invasion with enhanced SMAD3 transcriptional activity.

In normal epithelial tissues, the multifunctional cytokine transforming growth factor-beta (TGF-beta) acts as a tumor suppressor through growth inhibition and induction of differentiation whereas in advanced cancers, TGF-beta promotes tumor progression through induction of tumor invasion, neoangiogenesis, and immunosuppression. The molecular mechanisms through which TGF-beta shifts from a tumor suppressor to a tumor enhancer are poorly understood. We now show a role for the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in repressing the protumorigenic effects of TGF-beta. The TGF-beta effector SMAD3 inducibly interacts with PTEN on TGF-beta treatment under endogenous conditions. RNA interference (RNAi) suppression of PTEN expression enhances SMAD3 transcriptional activity and TGF-beta-mediated induction of SMAD3 target genes whereas reconstitution of PTEN in a null cancer cell line represses the expression of TGF-beta-regulated target genes. Targeting PTEN expression through RNAi in a PTEN wild-type cell line increases TGF-beta-mediated invasion but does not affect TGF-beta-mediated growth inhibition. Reconstitution of PTEN expression in a PTEN-null cell line blocks TGF-beta-induced invasion but does not modulate TGF-beta-mediated growth regulation. These effects are distinct from Akt and Forkhead family members that also interact with SMAD3 to regulate apoptosis or proliferation, respectively. Pharmacologic inhibitors targeting TGF-beta receptors and phosphatidylinositol 3-kinase signaling downstream from PTEN cooperate to block TGF-beta-mediated invasion. Thus, the loss of PTEN expression in human cancers may contribute to a role for TGF-beta as a tumor enhancer with specific effects on cellular motility and invasion.

Authors
Hjelmeland, AB; Hjelmeland, MD; Shi, Q; Hart, JL; Bigner, DD; Wang, X-F; Kontos, CD; Rich, JN
MLA Citation
Hjelmeland, AB, Hjelmeland, MD, Shi, Q, Hart, JL, Bigner, DD, Wang, X-F, Kontos, CD, and Rich, JN. "Loss of phosphatase and tensin homologue increases transforming growth factor beta-mediated invasion with enhanced SMAD3 transcriptional activity." Cancer Res 65.24 (December 15, 2005): 11276-11281.
PMID
16357132
Source
pubmed
Published In
Cancer Research
Volume
65
Issue
24
Publish Date
2005
Start Page
11276
End Page
11281
DOI
10.1158/0008-5472.CAN-05-3016

Cholesterol feeding reduces vascular endothelial growth factor signaling in rabbit corporal tissues.

PURPOSE: Hypercholesterolemia is a major risk factor for erectile dysfunction (ED), but the mechanisms are not completely understood. Vascular endothelial growth factor (VEGF) is reduced in rabbit corporal tissue with cholesterol feeding. VEGF signaling leads to the phosphorylation of Akt and endothelial nitric oxide synthase (p-Akt and p-eNOS). MATERIAL AND METHODS: New Zealand White rabbits (n = 50) were fed a 1% cholesterol (n = 8, 8, 8, 4) or normal (n = 6, 6, 6, 4) diet for 2, 4.5, 7.5, and 12 weeks. Akt, p-Akt, and p-Akt/Akt were measured by enzyme-linked immunosorbent assay. Levels of eNOS, p-eNOS, and neuronal and inducible nitric oxide synthase (nNOS and iNOS) mRNA and protein were assessed by polymerase chain reaction and Western analysis. RESULTS: Cholesterol feeding was associated with a significant decrease in p-Akt/Akt 2.16-fold (P < 0.05), 3.28-fold (P < 0.02), and 3.42-fold (P < 0.02) at 4.5, 7.5, and 12 weeks, respectively. The reduction in p-Akt/Akt with the cholesterol diet at 2 weeks was not significantly different, but the correlation between the duration of cholesterol feeding and the reduction in p-Akt/Akt was high (r( 2) = 0.858). eNOS protein or mRNA did not change with cholesterol feeding, but p-eNOS was significantly decreased at 4.5 weeks and all subsequent time points. nNOS mRNA and protein levels were decreased at 4.5 weeks and all subsequent time points, while iNOS was not different between groups. CONCLUSIONS: Hypercholesterolemia results in decreased VEGF signaling and decreased levels of the active form of eNOS in corporal tissue. Levels of nNOS were reduced by a different mechanism. VEGF signaling may provide a target to modulate ED.

Authors
Xie, D; Kontos, CD; Donatucci, CF; Annex, BH
MLA Citation
Xie, D, Kontos, CD, Donatucci, CF, and Annex, BH. "Cholesterol feeding reduces vascular endothelial growth factor signaling in rabbit corporal tissues." J Sex Med 2.5 (September 2005): 634-640.
PMID
16422820
Source
pubmed
Published In
The Journal of Sexual Medicine
Volume
2
Issue
5
Publish Date
2005
Start Page
634
End Page
640
DOI
10.1111/j.1743-6109.2005.00111.x

A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension.

Nitric oxide (NO) production by endothelial cell nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. We sought to understand the mechanism underlying defective eNOS function. Phosphorylation of the serine-threonine kinase Akt, which activates eNOS, was substantially reduced in sinusoidal endothelial cells from injured livers. Overexpression of Akt in vivo restored phosphorylation of Akt and production of NO and reduced portal pressure in portal hypertensive rats. We found that Akt physically interacts with G-protein-coupled receptor kinase-2 (GRK2), and that this interaction inhibits Akt activity. Furthermore, GRK2 expression increased in sinusoidal endothelial cells from portal hypertensive rats and knockdown of GRK2 restored Akt phosphorylation and NO production, and normalized portal pressure. Finally, after liver injury, GRK2-deficient mice developed less severe portal hypertension than control mice. Thus, an important mechanism underlying impaired activity of eNOS in injured sinusoidal endothelial cells is defective phosphorylation of Akt caused by overexpression of GRK2 after injury.

Authors
Liu, S; Premont, RT; Kontos, CD; Zhu, S; Rockey, DC
MLA Citation
Liu, S, Premont, RT, Kontos, CD, Zhu, S, and Rockey, DC. "A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension." Nat Med 11.9 (September 2005): 952-958.
PMID
16142243
Source
pubmed
Published In
Nature Medicine
Volume
11
Issue
9
Publish Date
2005
Start Page
952
End Page
958
DOI
10.1038/nm1289

Systemic soluble Tie2 expression inhibits and regresses corneal neovascularization.

This study was designed to determine if soluble Tie2 (sTie2) expression inhibits and regresses corneal neovascularization, and if VEGF contributes to its effect. The corneas of BALB/c mice were scraped and the mice were injected with either an adenovirus expressing soluble Tie2 (Ad.sTie2) or an empty adenoviral vector. When injected at the inhibition timepoint (one day prior to corneal injury), the mean percentage of neovascularized corneal area two weeks later in Ad.sTie2-treated mice vs. controls was 56.37+/-9.15% vs. 85.79+/-3.55% (p=0.04). At the regression timepoint (4 weeks after corneal scrape), the mean area of corneal neovascularization in Ad.sTie2-treated mice was 42.89+/-4.74% vs. 75.01+/-3.22% in the control group (p=0.007). VEGF expression was significantly higher in Ad.sTie2-treated mice at the inhibition timepoint and there was no significant difference at the regression timepoint. These findings suggest that sTie2 inhibits and regresses corneal neovascularization in a VEGF-independent manner.

Authors
Singh, N; Macnamara, E; Rashid, S; Ambati, J; Kontos, CD; Higgins, E; Ambati, BK
MLA Citation
Singh, N, Macnamara, E, Rashid, S, Ambati, J, Kontos, CD, Higgins, E, and Ambati, BK. "Systemic soluble Tie2 expression inhibits and regresses corneal neovascularization." Biochem Biophys Res Commun 332.1 (June 24, 2005): 194-199.
PMID
15896317
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
332
Issue
1
Publish Date
2005
Start Page
194
End Page
199
DOI
10.1016/j.bbrc.2005.04.108

Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts.

OBJECTIVES: Fifty percent of human aortocoronary saphenous vein grafts are occluded after 10 years. Intimal hyperplasia is an initial step in graft occlusion and consists of vascular smooth muscle cell proliferation. Phosphatidylinositol 3-kinase and its downstream regulator, the inositol 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), are important regulators of vascular smooth muscle cell proliferation, migration, and cell death. This study tests whether overexpression of PTEN in aortocoronary saphenous vein grafts can reduce intimal hyperplasia. METHODS: Adult dogs underwent aortocoronary bypass grafting to the left anterior descending artery by using the autologous saphenous vein. Saphenous vein grafts were treated with phosphate-buffered saline (n = 9), empty adenovirus (n = 8), or adenovirus encoding for PTEN (n = 8). Arteriography at 30 and 90 days assessed saphenous vein graft patency. A subset received saphenous vein grafts treated with a marker transgene (beta-galactosidase, n = 3), empty adenovirus (n = 4), or adenovirus encoding for PTEN (n = 4) and were killed on postoperative day 3 to confirm expression. Vascular smooth muscle cells were isolated from canine saphenous vein infected with adenovirus encoding for PTEN, and immunoblotting and proliferation assays were performed. RESULTS: Saphenous vein graft transgene expression was confirmed by means of immunohistochemistry, immunoblotting, and polymerase chain reaction. Arteriograms revealed all saphenous vein grafts to be patent. Saphenous vein grafts treated with adenovirus encoding for PTEN demonstrated reduced intimal area compared with those treated with empty adenovirus and phosphate-buffered saline (1.39 +/- 0.11 vs 2.35 +/- 0.3 and 2.57 +/- 0.4 mm 2 , P < .05), and the intima/media ratio was lower in saphenous vein grafts treated with adenovirus encoding for PTEN (0.50 +/- 0.05 vs 1.43 +/- 0.18 and 1.11 +/- 0.14, P < .005). PTEN overexpression in vascular smooth muscle cells inhibited platelet-derived growth factor-induced phosphorylation of Akt, a downstream effector of phosphatidylinositol 3-kinase. PTEN-treated vascular smooth muscle cells demonstrated decreased basal, platelet-derived growth factor-stimulated, and serum-stimulated proliferation. CONCLUSION: This study demonstrates that PTEN overexpression in aortocoronary saphenous vein grafts reduces intimal hyperplasia. The mechanism of this antiproliferative effect in vascular smooth muscle cells is likely due to inhibition of phosphatidylinositol 3-kinase signaling through Akt, with resultant decreases in vascular smooth muscle cell growth and survival. Therefore modulation of the phosphatidylinositol 3-kinase pathway through PTEN overexpression might represent a novel therapy to prevent saphenous vein graft intimal hyperplasia after coronary artery bypass grafting.

Authors
Hata, JA; Petrofski, JA; Schroder, JN; Williams, ML; Timberlake, SH; Pippen, A; Corwin, MT; Solan, AK; Jakoi, A; Gehrig, TR; Kontos, CD; Milano, CA
MLA Citation
Hata, JA, Petrofski, JA, Schroder, JN, Williams, ML, Timberlake, SH, Pippen, A, Corwin, MT, Solan, AK, Jakoi, A, Gehrig, TR, Kontos, CD, and Milano, CA. "Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts." J Thorac Cardiovasc Surg 129.6 (June 2005): 1405-1413.
PMID
15942585
Source
pubmed
Published In
Journal of Thoracic and Cardiovascular Surgery
Volume
129
Issue
6
Publish Date
2005
Start Page
1405
End Page
1413
DOI
10.1016/j.jtcvs.2004.11.048

PTEN as an effector in the signaling of antimigratory G protein-coupled receptor.

PTEN, a tumor suppressor phosphatase, is important in the regulation of cell migration and invasion. Physiological regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) by cell surface receptors has not been described. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P), which acts through the S1P2 receptor (S1P2R) G protein-coupled receptor (GPCR) to inhibit cell migration, utilizes PTEN as a signaling intermediate. S1P2R inhibition of cell migration is abrogated by dominant-negative PTEN expression. S1P was unable to efficiently inhibit the migration of Pten(DeltaloxP/DeltaloxP) mouse embryonic fibroblasts; however, the antimigratory effect was restored upon the expression of PTEN. S1P2R activation of Rho GTPase is not affected in Pten(DeltaloxP/DeltaloxP) cells, and dominant-negative Rho GTPase reversed S1P inhibition of cell migration in WT cells but not in Pten(DeltaloxP/DeltaloxP) cells, suggesting that PTEN acts downstream of the Rho GTPase. Ligand activation of the S1P2R receptor stimulated the coimmunoprecipitation of S1P2R and PTEN. Interestingly, S1P2R signaling increased PTEN phosphatase activity in membrane fractions. Furthermore, tyrosine phosphorylation of PTEN was stimulated by S1P2R signaling. These data suggest that the S1P2R receptor actively regulates the PTEN phosphatase by a Rho GTPase-dependent pathway to inhibit cell migration. GPCR regulation of PTEN maybe a general mechanism in signaling events of cell migration and invasion.

Authors
Sanchez, T; Thangada, S; Wu, M-T; Kontos, CD; Wu, D; Wu, H; Hla, T
MLA Citation
Sanchez, T, Thangada, S, Wu, M-T, Kontos, CD, Wu, D, Wu, H, and Hla, T. "PTEN as an effector in the signaling of antimigratory G protein-coupled receptor." Proc Natl Acad Sci U S A 102.12 (March 22, 2005): 4312-4317.
PMID
15764699
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
12
Publish Date
2005
Start Page
4312
End Page
4317
DOI
10.1073/pnas.0409784102

Adenovirus-mediated intraarterial delivery of PTEN inhibits neointimal hyperplasia.

OBJECTIVE: Phosphoinositide (PI) 3-kinase promotes vascular smooth muscle cell (VSMC) responses necessary for neointimal hyperplasia. We recently demonstrated that the inositol 3-phosphatase PTEN is expressed in VSMCs and that its overexpression inhibits these cellular responses. The purpose of this study was to determine the effects of adenovirus-mediated overexpression of PTEN on neointimal hyperplasia in vivo in the rat carotid injury model. METHODS AND RESULTS: Rat carotid arteries were balloon-injured and treated with a recombinant control adenovirus (AdEV) (n=6), an adenovirus encoding wild-type PTEN (AdPTEN) (n=8), or phosphate-buffered saline (sham) (n=5). Injured vessels demonstrated PTEN overexpression by Western blotting and immunohistochemistry after AdPTEN treatment. Neointimal hyperplasia was assessed 2 weeks after balloon injury and adenovirus administration. Compared with controls, AdPTEN treatment significantly decreased neointimal area and percent stenosis. To investigate the mechanisms of action of AdPTEN, vessels were harvested 3 days after balloon injury and virus infection. AdPTEN significantly increased medial cell apoptosis while decreasing proliferation of the remaining viable cells. CONCLUSIONS: PTEN overexpression potently inhibits neointimal hyperplasia through induction of apoptosis and inhibition of medial cell proliferation. These findings suggest that modulation of PTEN expression or activity may be a viable approach to treat neointimal hyperplasia. Phosphoinositide (PI) 3-kinase is a critical regulator of neointimal hyperplasia. The inositol 3-phosphatase PTEN modulates PI 3-kinase signaling by hydrolyzing the phospholipid products of PI 3-kinase, and overexpression of PTEN in vascular smooth muscle cells inhibits the cellular processes necessary for neointimal hyperplasia. The effects of adenovirus-mediated PTEN (AdPTEN) overexpression on neointimal hyperplasia were tested in the rat carotid injury model. Compared with control arteries, AdPTEN treatment significantly reduced neointimal area and percent stenosis by enhancing medial cell apoptosis and inhibiting proliferation of the remaining viable cells. Thus, PTEN provides a new target for the treatment of neointimal hyperplasia.

Authors
Huang, J; Niu, X-L; Pippen, AM; Annex, BH; Kontos, CD
MLA Citation
Huang, J, Niu, X-L, Pippen, AM, Annex, BH, and Kontos, CD. "Adenovirus-mediated intraarterial delivery of PTEN inhibits neointimal hyperplasia." Arterioscler Thromb Vasc Biol 25.2 (February 2005): 354-358.
PMID
15569824
Source
pubmed
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
25
Issue
2
Publish Date
2005
Start Page
354
End Page
358
DOI
10.1161/01.ATV.0000151619.54108.a5

Erratum: Cholesterol feeding reduces vascular endothelial growth factor signaling in rabbit corporal tissues (Journal of Sexual Medicine (2005) 2 (634-640))

Authors
Xie, D; Kontos, CD; Donatucci, CF; Annex, BH
MLA Citation
Xie, D, Kontos, CD, Donatucci, CF, and Annex, BH. "Erratum: Cholesterol feeding reduces vascular endothelial growth factor signaling in rabbit corporal tissues (Journal of Sexual Medicine (2005) 2 (634-640))." Journal of Sexual Medicine 2.6 (2005): 876--.
Source
scival
Published In
The Journal of Sexual Medicine
Volume
2
Issue
6
Publish Date
2005
Start Page
876-
DOI
10.1111/j.1743-6109.2005.00154.x

Acute local subcutaneous VEGF165 injection for augmentation of skin flap viability: efficacy and mechanism.

Distal skin ischemic necrosis is a common complication in skin flap surgery. The pathogenesis of skin flap ischemic necrosis is unclear, and there is no clinical treatment available. Here, we used the 4 x 10 cm rat dorsal skin flap model to test our hypothesis that subcutaneous injection of vascular endothelial growth factor 165 (VEGF165) in skin flaps at the time of surgery is effective in augmentation of skin flap viability, which is associated with an increase in nitric oxide (NO) production, and the mechanism involves 1) an increase in skin flap blood flow in the early stage after surgery and 2) enhanced angiogenesis subsequently to sustain increased skin flap blood flow and viability. We observed that subcutaneous injection of VEGF165 in skin flaps at the time of surgery increased skin flap viability in a dose-dependent manner. Subcutaneous injection of VEGF165 at the dose of 2 microg/flap increased skin flap viability by 28% (P < 0.05; n = 8). Over 80% of this effect was blocked by intramuscular injection of the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine (13 mg/kg) 45 min before surgery (P < 0.05; n = 8). The VEGF165 treatment also increased skin flap blood flow (2.68 +/- 0.63 ml x min(-1) x 100 g(-1)) compared with the control (1.26 +/- 0.10 ml x min(-1) x 100 g(-1); P < 0.05, n = 6) assessed 6 h postoperatively. There was no change in skin flap capillary density at this time point. VEGF165-induced increase in capillary density (32.2 +/- 1.1 capillaries/mm2; P < 0.05, n = 7) compared with control (24.6 +/- 1.4 capillaries/mm2) was seen 7 days postoperatively. There was also evidence to indicate that VEGF165-induced NO production in skin flaps was stimulated by activation of NOS activity followed by upregulation of NOS protein expression. These observations support our hypothesis and for the first time provide an important insight into the mechanism of acute local VEGF165 protein therapy in mitigation of skin flap ischemic necrosis.

Authors
Khan, A; Ashrafpour, H; Huang, N; Neligan, PC; Kontos, C; Zhong, A; Forrest, CR; Pang, CY
MLA Citation
Khan, A, Ashrafpour, H, Huang, N, Neligan, PC, Kontos, C, Zhong, A, Forrest, CR, and Pang, CY. "Acute local subcutaneous VEGF165 injection for augmentation of skin flap viability: efficacy and mechanism." Am J Physiol Regul Integr Comp Physiol 287.5 (November 2004): R1219-R1229.
PMID
15217788
Source
pubmed
Published In
American journal of physiology. Regulatory, integrative and comparative physiology
Volume
287
Issue
5
Publish Date
2004
Start Page
R1219
End Page
R1229
DOI
10.1152/ajpregu.00143.2004

Engineered zinc finger-activating vascular endothelial growth factor transcription factor plasmid DNA induces therapeutic angiogenesis in rabbits with hindlimb ischemia.

BACKGROUND: Therapeutic angiogenesis seeks to promote blood vessel growth to improve tissue perfusion. Vascular endothelial growth factor (VEGF) exists in multiple isoforms. We investigated an engineered zinc finger-containing transcription factor plasmid designed to activate the endogenous VEGF gene (ZFP-VEGF). METHODS AND RESULTS: New Zealand White rabbits (n=56) underwent unilateral femoral artery ligation and excision. At day 10 postoperatively, the ischemic muscle received ZFP treatment (500 microg ZFP-VEGF plasmid) or no ZFP treatment (beta-galactosidase, empty, or no plasmid). Group 1 (n=13) was harvested 3 days after injection to examine VEGF mRNA by real-time polymerase chain reaction and protein by ELISA. Groups 2 (n=13) and 3 (n=10) were harvested 11 days after injection. Group 2 was studied by histology and group 3, by histology and changes in blood flow. Groups 4 and 5 (n=10 each) were harvested 22 and 32 days after injection, respectively, and studied for changes in blood flow. In group 1, VEGF mRNA copy numbers were significantly higher for VEGF121, VEGF165, VEGF189, and protein in the ZFP-VEGF-treatment versus no-ZFP-treatment arms. In groups 2 and 3, capillary density and proliferating cells were significantly greater and apoptosis significantly lower in the treatment versus no-treatment arms. Changes in the blood flow ratio of the ischemic to the nonischemic limb were significantly greater in the treatment versus no-ZFP-treatment groups (6.57+/-1.52% versus 3.38+/-0.87%, P<0.005; 13.15+/-1.77% versus 6.13+/-1.55%, P<0.001; and 20.16+/-2.84% versus 13.88+/-3.14%, P<0.01, for groups 3, 4, and 5, respectively). CONCLUSIONS: This engineered ZFP-VEGF-activating transcription factor may provide a novel approach to treat peripheral arterial disease.

Authors
Dai, Q; Huang, J; Klitzman, B; Dong, C; Goldschmidt-Clermont, PJ; March, KL; Rokovich, J; Johnstone, B; Rebar, EJ; Spratt, SK; Case, CC; Kontos, CD; Annex, BH
MLA Citation
Dai, Q, Huang, J, Klitzman, B, Dong, C, Goldschmidt-Clermont, PJ, March, KL, Rokovich, J, Johnstone, B, Rebar, EJ, Spratt, SK, Case, CC, Kontos, CD, and Annex, BH. "Engineered zinc finger-activating vascular endothelial growth factor transcription factor plasmid DNA induces therapeutic angiogenesis in rabbits with hindlimb ischemia." Circulation 110.16 (October 19, 2004): 2467-2475.
PMID
15477407
Source
pubmed
Published In
Circulation
Volume
110
Issue
16
Publish Date
2004
Start Page
2467
End Page
2475
DOI
10.1161/01.CIR.0000145139.53840.49

IQGAP1, a novel vascular endothelial growth factor receptor binding protein, is involved in reactive oxygen species--dependent endothelial migration and proliferation.

Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)-mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels.

Authors
Yamaoka-Tojo, M; Ushio-Fukai, M; Hilenski, L; Dikalov, SI; Chen, YE; Tojo, T; Fukai, T; Fujimoto, M; Patrushev, NA; Wang, N; Kontos, CD; Bloom, GS; Alexander, RW
MLA Citation
Yamaoka-Tojo, M, Ushio-Fukai, M, Hilenski, L, Dikalov, SI, Chen, YE, Tojo, T, Fukai, T, Fujimoto, M, Patrushev, NA, Wang, N, Kontos, CD, Bloom, GS, and Alexander, RW. "IQGAP1, a novel vascular endothelial growth factor receptor binding protein, is involved in reactive oxygen species--dependent endothelial migration and proliferation." Circ Res 95.3 (August 6, 2004): 276-283.
PMID
15217908
Source
pubmed
Published In
Circulation Research
Volume
95
Issue
3
Publish Date
2004
Start Page
276
End Page
283
DOI
10.1161/01.RES.0000136522.58649.60

Responses of vascular endothelial cells to angiogenic signaling are important for tumor cell survival

Authors
Shan, S; Robson, ND; Cao, Y; Qiao, T; Li, CY; Kontos, CD; Garcia-Blanco, M; Dewhirst, MW
MLA Citation
Shan, S, Robson, ND, Cao, Y, Qiao, T, Li, CY, Kontos, CD, Garcia-Blanco, M, and Dewhirst, MW. "Responses of vascular endothelial cells to angiogenic signaling are important for tumor cell survival." FASEB JOURNAL 18.2 (February 2004): 326-328.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
18
Issue
2
Publish Date
2004
Start Page
326
End Page
328
DOI
10.1096/fj.03-0765fje

Targeting the Tie2/Tek receptor in astrocytomas.

Tie2 is an endothelial cell-specific receptor tyrosine kinase, whose activation is positively and negatively modulated by angiopoietin-1 and angiopoietin-2, respectively. Angiopoietin-mediated modulation of Tie2 activation contributes to normal vessel development and stability, however, its role in tumor angiogenesis is not well known. We investigated the role of Tie2 activation in malignant astrocytomas, a common and highly vascularized primary human brain tumor. We found that Tie2 expression and activation increases with increasing malignancy grade of astrocytomas. Inhibition of Tie2, using a kinase-deficient Tie2 construct, decreases growth of malignant human astrocytoma subcutaneous and intracranial xenografts. Tie2 inactivation disrupted the tumor vascularity, with a decrease in microvascular density, increased presence of abnormally dilated vessels, and loss of interaction between endothelial cells and surrounding smooth muscle cells, all collectively resulting in increased tumor cell apoptosis. Overall, these findings strongly suggest that Tie2 activation contributes significantly to astrocytoma tumor angiogenesis and growth. We postulate that targeting Tie2 activation, either independently or in conjunction with other anti-angiogenic therapies, such as against vascular endothelial growth factor, is of potential clinical interest.

Authors
Zadeh, G; Qian, B; Okhowat, A; Sabha, N; Kontos, CD; Guha, A
MLA Citation
Zadeh, G, Qian, B, Okhowat, A, Sabha, N, Kontos, CD, and Guha, A. "Targeting the Tie2/Tek receptor in astrocytomas." Am J Pathol 164.2 (February 2004): 467-476.
PMID
14742253
Source
pubmed
Published In
The American journal of pathology
Volume
164
Issue
2
Publish Date
2004
Start Page
467
End Page
476
DOI
10.1016/S0002-9440(10)63137-9

Responses of vascular endothelial cells to angiogenic signaling are important for tumor cell survival.

Neoplastic cells overexpress several angiogenic cytokines, which stimulate neovascularization. Whether the responses of the host endothelial cells to these signaling molecules affect tumor cells during early tumorigenesis has not been investigated. We investigated pre-angiogenic tumor cell survival and angiogenesis initiation by two murine tumor lines (4T1 mammary carcinoma and B16 melanoma), which constitutively expressed GFP, in dorsal skin-fold window chambers of mice treated with extracellular domain of Tie-2 (ExTek) or bFGF. ExTek reduced tumor cell survival, retarded tumor growth, and inhibited angiogenesis onset compared with controls. bFGF increased tumor cell survival and promoted earlier angiogenesis and tumor growth. Neither bFGF nor ExTek affected cell proliferation in vitro. RT-PCR showed mRNA expression of bFGF receptor 2 (FGFR2) IIIb, which does not bind bFGF efficiently, by 4T1 cells and B16 cells express FGFR1 but not FGFR2. B16 cells expressed angiopoietin (Ang) 2, but neither cell line expresses Ang1. Both tumor lines express VEGF. These findings suggested that effects of bFGF and ExTek on tumor cell survival and angiogenesis were not due to direct action but were instead a result of paracrine factors secreted by endothelial cells. These subsequent signals from endothelial cells promote early survival and proliferation of disseminated tumor cells before onset of angiogenesis.

Authors
Shan, S; Robson, ND; Cao, Y; Qiao, T; Li, CY; Kontos, CD; Garcia-Blanco, M; Dewhirst, MW
MLA Citation
Shan, S, Robson, ND, Cao, Y, Qiao, T, Li, CY, Kontos, CD, Garcia-Blanco, M, and Dewhirst, MW. "Responses of vascular endothelial cells to angiogenic signaling are important for tumor cell survival." FASEB J 18.2 (February 2004): 326-328.
PMID
14688196
Source
pubmed
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
18
Issue
2
Publish Date
2004
Start Page
326
End Page
328
DOI
10.1096/fj.03-0765fje

Functional significance of Tie2 signaling in the adult vasculature.

Abundant data now demonstrate that the growth of new blood vessels, termed angiogenesis, plays both pathological and beneficial roles in human disease. Based on these data, a tremendous effort has been undertaken to understand the molecular mechanisms that drive blood vessel growth in adult tissues. Tie2 recently was identified as a receptor tyrosine kinase expressed principally on vascular endothelium. Disrupting Tie2 function in mice resulted in embryonic lethality with defects in embryonic vasculature, suggesting a role in blood vessel maturation and maintenance. Based on these studies, we undertook a series of studies to probe the function of Tie2 in adult vasculature that will form the focus of this chapter. Consistent with a role in blood vessel growth in adult vasculature, Tie2 was upregulated and activated in the endothelium of rat ovary and in healing rat skin wounds, both areas of active angiogenesis. Moreover, Tie2 was upregulated in the endothelium of vascular "hot spots" in human breast cancer specimens. Surprisingly, Tie2 also was expressed and activated in the endothelium of all normal rat tissues examined, suggesting a role in maintenance of adult vasculature. To determine the functional role of Tie2 in tumor vasculature, a soluble Tie2 extracellular domain (ExTek) was designed that blocked the activation of Tie2 by its activating ligand, angiopoietin 1 (Ang1). Administration of recombinant ExTek protein or an ExTek adenovirus inhibited tumor growth and metastasis in rodent tumor models, demonstrating a functional role for Tie2 in pathological angiogenesis in adult tissues. To begin to understand the endothelial signaling pathways and cellular responses that mediate Tie2 function, we identified signaling molecules that are recruited to the activated, autophosphorylated Tie2 kinase domain. Two of these molecules, SHP2 and GRB2, are part of the pathway upstream of mitogen-activated protein kinase (MAPK) activation, a pathway that may be responsible for morphogenetic effects of Tie2 on endothelial cells. Another signaling molecule, p85, is responsible for recruitment of phosphatidylinositol 3 kinase (PI3-K) and activation of the Akt/PI3-K pathway. Akt/PI3-K has emerged as a critical pathway downstream of Tie2 that is necessary for cell survival effects as well as for chemotaxis, activation of endothelial nitric oxide synthase, and perhaps for anti-inflammatory effects of Tie2 activation. Taken together, these studies and many others demonstrate that the Tie2 pathway has important functions in adult tissues, in both quiescent vasculature and during angiogenesis, and help to validate the Tie2 pathway as a therapeutic target.

Authors
Peters, KG; Kontos, CD; Lin, PC; Wong, AL; Rao, P; Huang, L; Dewhirst, MW; Sankar, S
MLA Citation
Peters, KG, Kontos, CD, Lin, PC, Wong, AL, Rao, P, Huang, L, Dewhirst, MW, and Sankar, S. "Functional significance of Tie2 signaling in the adult vasculature." Recent Prog Horm Res 59 (2004): 51-71. (Review)
PMID
14749497
Source
pubmed
Published In
Recent progress in hormone research
Volume
59
Publish Date
2004
Start Page
51
End Page
71

Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt.

Endothelin-1 has dual vasoactive effects, mediating vasoconstriction via ETA receptor activation of vascular smooth muscle cells and vasorelaxation via ETB receptor activation of endothelial cells. Although it is commonly accepted that endothelin-1 binding to endothelial cell ETB receptors stimulates nitric oxide (NO) synthesis and subsequent smooth muscle relaxation, the signaling pathways downstream of ETB receptor activation are unknown. Here, using a model in which we have utilized isolated primary endothelial cells, we demonstrate that ET-1 binding to sinusoidal endothelial cell ETB receptors led to increased protein kinase B/Akt phosphorylation, endothelial cell nitric-oxide synthase (eNOS) phosphorylation, and NO synthesis. Furthermore, eNOS activation was not dependent on tyrosine phosphorylation, and pretreatment of endothelial cells with pertussis toxin as well as overexpression of a dominant negative G-protein-coupled receptor kinase construct that sequesters betagamma subunits inhibited Akt phosphorylation and NO synthesis. Taken together, the data elucidate a G-protein-coupled receptor signaling pathway for ETB receptor-mediated NO production and call attention to the absolute requirement for heterotrimeric G-protein betagamma subunits in this cascade.

Authors
Liu, S; Premont, RT; Kontos, CD; Huang, J; Rockey, DC
MLA Citation
Liu, S, Premont, RT, Kontos, CD, Huang, J, and Rockey, DC. "Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt." J Biol Chem 278.50 (December 12, 2003): 49929-49935.
PMID
14523027
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
50
Publish Date
2003
Start Page
49929
End Page
49935
DOI
10.1074/jbc.M306930200

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells.

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Authors
Cai, J; Ahmad, S; Jiang, WG; Huang, J; Kontos, CD; Boulton, M; Ahmed, A
MLA Citation
Cai, J, Ahmad, S, Jiang, WG, Huang, J, Kontos, CD, Boulton, M, and Ahmed, A. "Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells." Diabetes 52.12 (December 2003): 2959-2968.
PMID
14633857
Source
pubmed
Published In
Diabetes
Volume
52
Issue
12
Publish Date
2003
Start Page
2959
End Page
2968

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2.

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent.

Authors
White, RR; Shan, S; Rusconi, CP; Shetty, G; Dewhirst, MW; Kontos, CD; Sullenger, BA
MLA Citation
White, RR, Shan, S, Rusconi, CP, Shetty, G, Dewhirst, MW, Kontos, CD, and Sullenger, BA. "Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2." Proc Natl Acad Sci U S A 100.9 (April 29, 2003): 5028-5033.
PMID
12692304
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
9
Publish Date
2003
Start Page
5028
End Page
5033
DOI
10.1073/pnas.0831159100

Deletion of the carboxyl terminus of Tie2 enhances kinase activity, signaling, and function. Evidence for an autoinhibitory mechanism.

Tie2 is an endothelial receptor tyrosine kinase that is required for both embryonic vascular development and tumor angiogenesis. There is considerable interest in understanding the mechanisms of Tie2 activation for therapeutic purposes. The recent solution of the Tie2 crystal structure suggests that Tie2 activity is autoinhibited by its carboxyl terminus. Here we investigated the role of the C tail in Tie2 activation, signaling, and function both in vitro and in vivo by deleting the C terminus of Tie2 (Delta CT). Compared to wild type Tie2, in vitro autophosphorylation and kinase activity were significantly enhanced by the Delta CT mutation. In NIH 3T3 cells expressing chimeric Tie2 receptors, both basal and ligand-induced tyrosine phosphorylation were markedly enhanced compared to wild type in several independent clones of Tie2-Delta CT. Moreover, the Delta CT mutation enhanced basal and ligand-dependent activation of Akt and extracellular signal-regulated kinase. Enhanced Akt activation correlated with significant inhibition of staurosporine-induced apoptosis. These findings demonstrate that the Tie2 C tail performs a novel negative regulatory role in Tie2 signaling and function, and they provide important insights into the mechanisms by which the Tie2 kinase is activated.

Authors
Niu, X-L; Peters, KG; Kontos, CD
MLA Citation
Niu, X-L, Peters, KG, and Kontos, CD. "Deletion of the carboxyl terminus of Tie2 enhances kinase activity, signaling, and function. Evidence for an autoinhibitory mechanism." J Biol Chem 277.35 (August 30, 2002): 31768-31773.
PMID
12082108
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
35
Publish Date
2002
Start Page
31768
End Page
31773
DOI
10.1074/jbc.M203995200

Inhibition of vascular smooth muscle cell proliferation, migration, and survival by the tumor suppressor protein PTEN.

Phosphatidylinositol (PI) 3-kinase signaling regulates numerous cellular processes, including proliferation, migration, and survival, which are required for neointimal hyperplasia and restenosis. The effectors of PI 3-kinase are activated by the phospholipid products of PI 3-kinase. In this report, we investigated the hypothesis that overexpression of the tumor suppressor protein PTEN, an inositol phosphatase specific for the products of PI 3-kinase, would inhibit the vascular smooth muscle cell (VSMC) responses necessary for neointimal hyperplasia and restenosis. Effects of PTEN were assessed in primary rabbit VSMCs after overexpression with a recombinant adenovirus and compared with uninfected or control virus-infected cells. PTEN was expressed endogenously in VSMCs, and PTEN overexpression inhibited PDGF-induced phosphorylation of p70(s6k), Akt, and glycogen synthase kinase-3-alpha and -beta but not ERK1 or -2. Overexpression of PTEN significantly inhibited both basal and PDGF-mediated VSMC proliferation and migration, the latter possibly due in part to downregulation of focal adhesion kinase. Moreover, PTEN overexpression induced cleavage of caspase-3 and significantly increased apoptosis compared with control cells. Taken together, these results demonstrate that PTEN overexpression potently inhibits the VSMC responses required for neointimal hyperplasia and restenosis. Adenovirus-expressed PTEN may therefore provide a useful tool for the local treatment of these and other vascular proliferative disorders.

Authors
Huang, J; Kontos, CD
MLA Citation
Huang, J, and Kontos, CD. "Inhibition of vascular smooth muscle cell proliferation, migration, and survival by the tumor suppressor protein PTEN." Arterioscler Thromb Vasc Biol 22.5 (May 1, 2002): 745-751.
PMID
12006385
Source
pubmed
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
22
Issue
5
Publish Date
2002
Start Page
745
End Page
751

PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects.

Phosphatidylinositol 3-kinase is activated by vascular endothelial growth factor (VEGF), and many of the angiogenic cellular responses of VEGF are regulated by the lipid products of phosphatidylinositol 3-kinase. The tumor suppressor PTEN has been shown to down-regulate phosphatidylinositol 3-kinase signaling, yet the effects of PTEN on VEGF-mediated signaling and angiogenesis are unknown. Inhibition of endogenous PTEN in cultured endothelial cells by adenovirus-mediated overexpression of a dominant negative PTEN mutant (PTEN-C/S) enhanced VEGF-mediated Akt phosphorylation, and this effect correlated with decreases in caspase-3 cleavage, caspase-3 activity, and DNA degradation after induction of apoptosis with tumor necrosis factor-alpha. Overexpression of PTEN-C/S also enhanced VEGF-mediated endothelial cell proliferation and migration. In contrast, overexpression of wild-type PTEN inhibited the anti-apoptotic, proliferative, and chemotactic effects of VEGF. Moreover, PTEN-C/S increased the length of vascular sprouts in the rat aortic ring assay and modulated VEGF-mediated tube formation in an in vitro angiogenesis assay, whereas PTEN-wild type inhibited these effects. Taken together, these findings demonstrate that PTEN potently modulates VEGF-mediated signaling and function and that PTEN is a viable target in therapeutic approaches to promote or inhibit angiogenesis.

Authors
Huang, J; Kontos, CD
MLA Citation
Huang, J, and Kontos, CD. "PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects." J Biol Chem 277.13 (March 29, 2002): 10760-10766.
PMID
11784722
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
13
Publish Date
2002
Start Page
10760
End Page
10766
DOI
10.1074/jbc.M110219200

The endothelial receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt to inhibit apoptosis.

Tie1 is an orphan receptor tyrosine kinase that is expressed almost exclusively in endothelial cells and that is required for normal embryonic vascular development. Genetic studies suggest that Tie1 promotes endothelial cell survival, but other studies have suggested that the Tie1 kinase has little to no activity, and Tie1-mediated signaling pathways are unknown. To begin to study Tie1 signaling, a recombinant glutathione S-transferase (GST)-Tie1 kinase fusion protein was produced in insect cells and found to be autophosphorylated in vitro. GST-Tie1 but not a kinase-inactive mutant associated with a recombinant p85 SH2 domain protein in vitro, suggesting that Tie1 might signal through phosphatidylinositol (PI) 3-kinase. To study Tie1 signaling in a cellular context, a c-fms-Tie1 chimeric receptor (fTie1) was expressed in NIH 3T3 cells. Ligand stimulation of fTie1 resulted in Tie1 autophosphorylation and downstream activation of PI 3-kinase and Akt. Stimulation of fTie1-expressing cells potently inhibited UV irradiation-induced apoptosis in a PI 3-kinase-dependent manner. Moreover, both Akt phosphorylation and inhibition of apoptosis were abrogated by mutation of tyrosine 1113 to phenylalanine, suggesting that this residue is an important PI 3-kinase binding site. These findings are the first biochemical demonstration of a signal transduction pathway and corresponding cellular function for Tie1, and the antiapoptotic effect of Tie1 is consistent with the results of previous genetic studies.

Authors
Kontos, CD; Cha, EH; York, JD; Peters, KG
MLA Citation
Kontos, CD, Cha, EH, York, JD, and Peters, KG. "The endothelial receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt to inhibit apoptosis." Mol Cell Biol 22.6 (March 2002): 1704-1713.
PMID
11865050
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
22
Issue
6
Publish Date
2002
Start Page
1704
End Page
1713

Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide.

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.

Authors
Bussolati, B; Dunk, C; Grohman, M; Kontos, CD; Mason, J; Ahmed, A
MLA Citation
Bussolati, B, Dunk, C, Grohman, M, Kontos, CD, Mason, J, and Ahmed, A. "Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide." Am J Pathol 159.3 (September 2001): 993-1008.
PMID
11549592
Source
pubmed
Published In
The American journal of pathology
Volume
159
Issue
3
Publish Date
2001
Start Page
993
End Page
1008
DOI
10.1016/S0002-9440(10)61775-0

HCPTPA, a protein tyrosine phosphatase that regulates vascular endothelial growth factor receptor-mediated signal transduction and biological activity.

Angiogenesis is a tightly controlled process in which signaling by the receptors for vascular endothelial growth factor (VEGF) plays a key role. In order to define signaling pathways downstream of VEGF receptors (VEGFR), the kinase domain of VEGFR2 (Flk-1) was used as a bait to screen a human fetal heart library in the yeast two-hybrid system. One of the signaling molecules identified in this effort was HCPTPA, a low molecular weight, cytoplasmic protein tyrosine phosphatase. Although HCPTPA possesses no identifiable phosphotyrosine binding domains (i.e. SH2 or phosphotyrosine binding domains), it bound specifically to active, autophosphorylated VEGFR2 but not to a mutated, kinase-inactive VEGFR2. Recombinant VEGFR2 and endogenous VEGFR2 were substrates for recombinant HCPTPA, and HCPTPA was co-expressed with VEGFR2 in endothelial cell lines, suggesting that HCPTPA may be a negative regulator of VEGFR2 signal transduction. To pursue this possibility, an adenovirus directing the expression of HCPTPA was constructed. When used to infect cultured endothelial cells, this adenovirus directed high level expression of HCPTPA that resulted in impairment of VEGF-mediated VEGFR2 autophosphorylation and mitogen-activated protein kinase activation. Adenovirus-mediated overexpression of HCPTPA also inhibited VEGF-induced cellular responses (endothelial cell migration and proliferation) and inhibited angiogenesis in the rat aortic ring assay. Taken together, these findings indicate that HCPTPA may be an important regulator of VEGF-mediated signaling and biological activity. Potential interactions with other signaling pathways and possible therapeutic implications are discussed.

Authors
Huang, L; Sankar, S; Lin, C; Kontos, CD; Schroff, AD; Cha, EH; Feng, SM; Li, SF; Yu, Z; Van Etten, RL; Blanar, MA; Peters, KG
MLA Citation
Huang, L, Sankar, S, Lin, C, Kontos, CD, Schroff, AD, Cha, EH, Feng, SM, Li, SF, Yu, Z, Van Etten, RL, Blanar, MA, and Peters, KG. "HCPTPA, a protein tyrosine phosphatase that regulates vascular endothelial growth factor receptor-mediated signal transduction and biological activity." J Biol Chem 274.53 (December 31, 1999): 38183-38188.
PMID
10608891
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
53
Publish Date
1999
Start Page
38183
End Page
38188

Inhibition of rat vascular smooth muscle cell proliferation in vitro and in vivo by recombinant replication-competent herpes simplex virus - Editorial Comment

Authors
Kontos, CD
MLA Citation
Kontos, CD. "Inhibition of rat vascular smooth muscle cell proliferation in vitro and in vivo by recombinant replication-competent herpes simplex virus - Editorial Comment." STROKE 30.11 (November 1999): 2438-2439.
Source
wos-lite
Published In
Stroke
Volume
30
Issue
11
Publish Date
1999
Start Page
2438
End Page
2439

Angiogenesis.

Angiogenesis, the growth and proliferation of blood vessels from existing vascular structures, is tightly regulated in adult tissues, and abnormalities in angiogenesis are associated with a number of pathologic states. Strategies designed to promote angiogenesis to treat disorders of inadequate tissue perfusion, such as occurs in coronary artery and peripheral vascular disease, have led to the area of therapeutic angiogenesis. Approaches to block angiogenesis are actively being explored to treat diseases that range from arthritis to cancer. This article will review some of the basic concepts of vascular development and the mechanisms involved in angiogenesis. Particular attention will be paid to the growth factors and receptors that are known to mediate angiogenesis, and a description of some of the cell signaling mechanisms that are involved in the regulation of angiogenesis will be described. Finally, potential targets that may provide opportunities to enhance or block angiogenesis will be discussed.

Authors
Kontos, CD; Annex, BH
MLA Citation
Kontos, CD, and Annex, BH. "Angiogenesis." Curr Atheroscler Rep 1.2 (September 1999): 165-171. (Review)
PMID
11122706
Source
pubmed
Published In
Current Atherosclerosis Reports
Volume
1
Issue
2
Publish Date
1999
Start Page
165
End Page
171

Allelic and locus heterogeneity in inherited venous malformations.

Venous malformations are low-flow vascular lesions consisting of disorganized thin-walled vascular channels. These can occur sporadically but also as an autosomal dominant condition termed venous malformations, cutaneous and mucosal (VMCM; OMIM 600195). In two large unrelated kindreds mapping to chromosome 9, the identical R849W missense mutation was identified in the first kinase domain of Tie2, an endothelial cell-specific receptor tyrosine kinase. We report here the identification of four new kindreds with inherited venous malformations. Unlike the initial two families described, these four families demonstrate allelic and locus heterogeneity. In one of these families, the R849W mutation co-segregates with the disease phenotype. Three other families with venous malformations lack this mutation. One of these families is linked to markers near TIE2 on chromosome 9. In this family, we identified a novel mutation within the first kinase domain of Tie2 resulting in a Y897S change. Results from COS-1 cell transfections using expression constructs containing either the R849W or the Y897S mutation suggest that the receptors containing either mutation show ligand-independent hyperphosphorylation. These results suggest a gain-of-function mechanism for development of venous malformations in these families. Of the two remaining families, one excludes linkage to the TIE2 locus, establishing the existence of at least one additional locus for dominantly inherited venous malformations.

Authors
Calvert, JT; Riney, TJ; Kontos, CD; Cha, EH; Prieto, VG; Shea, CR; Berg, JN; Nevin, NC; Simpson, SA; Pasyk, KA; Speer, MC; Peters, KG; Marchuk, DA
MLA Citation
Calvert, JT, Riney, TJ, Kontos, CD, Cha, EH, Prieto, VG, Shea, CR, Berg, JN, Nevin, NC, Simpson, SA, Pasyk, KA, Speer, MC, Peters, KG, and Marchuk, DA. "Allelic and locus heterogeneity in inherited venous malformations." Hum Mol Genet 8.7 (July 1999): 1279-1289.
PMID
10369874
Source
pubmed
Published In
Human Molecular Genetics
Volume
8
Issue
7
Publish Date
1999
Start Page
1279
End Page
1289

Adenovirus-mediated gene transfer is augmented in basilar and carotid arteries of heritable hyperlipidemic rabbits - Comment

Authors
Kontos, CD
MLA Citation
Kontos, CD. "Adenovirus-mediated gene transfer is augmented in basilar and carotid arteries of heritable hyperlipidemic rabbits - Comment." STROKE 30.1 (January 1999): 125-125.
Source
wos-lite
Published In
Stroke
Volume
30
Issue
1
Publish Date
1999
Start Page
125
End Page
125

Expression of nerve growth factor and trkA after transient focal cerebral ischemia in rats - Comment

Authors
Kontos, CD
MLA Citation
Kontos, CD. "Expression of nerve growth factor and trkA after transient focal cerebral ischemia in rats - Comment." STROKE 29.8 (August 1998): 1697-1697.
Source
wos-lite
Published In
Stroke
Volume
29
Issue
8
Publish Date
1998
Start Page
1697
End Page
1697

Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt.

Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3, 4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

Authors
Kontos, CD; Stauffer, TP; Yang, WP; York, JD; Huang, L; Blanar, MA; Meyer, T; Peters, KG
MLA Citation
Kontos, CD, Stauffer, TP, Yang, WP, York, JD, Huang, L, Blanar, MA, Meyer, T, and Peters, KG. "Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt." Mol Cell Biol 18.7 (July 1998): 4131-4140.
PMID
9632797
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
18
Issue
7
Publish Date
1998
Start Page
4131
End Page
4140

Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumour angiogenesis.

Endothelial receptor tyrosine kinases may play important roles in pathological vascular growth, particularly in tumours. In this study, immunohistochemistry was used to evaluate the expression of a novel endothelial receptor tyrosine kinase, Tie2/Tek, in the endothelium of vascular 'hotspots' in normal breast tissue (n = 10), benign breast lesions (n = 10) and in breast tumours (n = 123). Tie2 expression was detected in the endothelium of all breast tissues examined. However, the strongest expression of Tie-2 was seen in vascular 'hot spots' within the inflammatory infiltrate at the periphery of invasive tumours. Moreover, the proportion of Tie2-positive vessels (Tie2 counts/CD31 counts) was significantly higher in breast tumours than the proportion of Tie2-positive vessels in either normal breast tissue or benign breast lesions (P = 0.004 and 0.0001 respectively). These data are consistent with a role for Tie2 in tumour angiogenesis and demonstrate the potential use of Tie2 expression as a novel marker of the tumour vasculature.

Authors
Peters, KG; Coogan, A; Berry, D; Marks, J; Iglehart, JD; Kontos, CD; Rao, P; Sankar, S; Trogan, E
MLA Citation
Peters, KG, Coogan, A, Berry, D, Marks, J, Iglehart, JD, Kontos, CD, Rao, P, Sankar, S, and Trogan, E. "Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumour angiogenesis." Br J Cancer 77.1 (1998): 51-56.
PMID
9459145
Source
pubmed
Published In
British Journal of Cancer
Volume
77
Issue
1
Publish Date
1998
Start Page
51
End Page
56

Intravenous basic fibroblast growth factor decreases brain injury resulting from focal ischemia in cats - Comment

Authors
Kontos, CD
MLA Citation
Kontos, CD. "Intravenous basic fibroblast growth factor decreases brain injury resulting from focal ischemia in cats - Comment." STROKE 28.3 (March 1997): 615-616.
Source
wos-lite
Published In
Stroke
Volume
28
Issue
3
Publish Date
1997
Start Page
615
End Page
616

Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo.

We used a cytochemical technique for the detection of superoxide in cerebral inflammation and ischemia-reperfusion in anesthetized cats. The technique is based on the oxidation of Mn2+ to Mn3+ by superoxide; Mn3+, in turn, oxidizes diaminobenzidine. The oxidized diaminobenzidine forms an osmiophilic electron-dense product that is detected by electron microscopy. The reagents, manganese chloride (2 mM) and diaminobenzidine (2 mg/ml), were placed topically on the brain surface of anesthetized cats equipped with cranial windows. Inflammation was induced by topical carrageenan with or without phorbol 12-myristate 13-acetate to activate leukocytes. In inflammation, superoxide was detected in the plasma membrane and in the phagocytic vacuoles of leukocytes. In ischemia-reperfusion, superoxide was identified in the meninges in association with blood vessels. It was located primarily in the extracellular space and occasionally in endothelial and vascular smooth muscle cells. In both inflammation and ischemia, the reaction product was eliminated by superoxide dismutase or by the omission of either manganese or diaminobenzidine. It was unaffected by sodium azide, which inhibits peroxidases. No superoxide was detected in the brain parenchyma. The findings confirm the generation of superoxide is cerebral ischemia-reperfusion and show that it is produced in cerebral vessels.

Authors
Kontos, CD; Wei, EP; Williams, JI; Kontos, HA; Povlishock, JT
MLA Citation
Kontos, CD, Wei, EP, Williams, JI, Kontos, HA, and Povlishock, JT. "Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo." Am J Physiol 263.4 Pt 2 (October 1992): H1234-H1242.
PMID
1329563
Source
pubmed
Published In
The American journal of physiology
Volume
263
Issue
4 Pt 2
Publish Date
1992
Start Page
H1234
End Page
H1242

Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo

We used a cytochemical technique for the detection of superoxide in cerebral inflammation and ischemia-reperfusion in anesthetized cats. The technique is based on the oxidation of Mn2+ to Mn3+ by superoxide; Mn3+, in turn, oxidizes diaminobenzidine. The oxidized diaminobenzidine forms an osmiophilic electron-dense product that is detected by electron microscopy. The reagents, manganese chloride (2 mM) and diaminobenzidine (2 mg/ml), were placed topically on the brain surface of anesthetized cats equipped with cranial windows. Inflammation was induced by topical carrageenan with or without phorbol 12-myristate 13-acetate to activate leukocytes. In inflammation, superoxide was detected in the plasma membrane and in the phagocytic vacuoles of leukocytes. In ischemia-reperfusion, superoxide was identified in the meninges in association with blood vessels. It was located primarily in the extracellular space and occasionally in endothelial and vascular smooth muscle cells. In both inflammation and ischemia, the reaction product was eliminated by superoxide dismutase or by the omission of either manganese or diaminobenzidine. It was unaffected by sodium azide; which inhibits peroxidases. No superoxide was detected in the brain parenchyma. The findings confirm the generation of superoxide is cerebral ischemia-reperfusion and show that it is produced in cerebral vessels.

Authors
Kontos, CD; Wei, EP; Williams, JI; Kontos, HA; Povlishock, JT
MLA Citation
Kontos, CD, Wei, EP, Williams, JI, Kontos, HA, and Povlishock, JT. "Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo." American Journal of Physiology - Heart and Circulatory Physiology 263.4 32-4 (1992): H1234-H1242.
Source
scival
Published In
American Journal of Physiology - Heart and Circulatory Physiology
Volume
263
Issue
4 32-4
Publish Date
1992
Start Page
H1234
End Page
H1242
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