87 results on '"Christopher D. Kontos"'
Search Results
2. Data from Loss of Phosphatase and Tensin Homologue Increases Transforming Growth Factor β–Mediated Invasion with Enhanced SMAD3 Transcriptional Activity
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Jeremy N. Rich, Christopher D. Kontos, Xiao-Fan Wang, Darell D. Bigner, Janet L. Hart, Qing Shi, Mark D. Hjelmeland, and Anita B. Hjelmeland
- Abstract
In normal epithelial tissues, the multifunctional cytokine transforming growth factor-β (TGF-β) acts as a tumor suppressor through growth inhibition and induction of differentiation whereas in advanced cancers, TGF-β promotes tumor progression through induction of tumor invasion, neoangiogenesis, and immunosuppression. The molecular mechanisms through which TGF-β 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-β. The TGF-β effector SMAD3 inducibly interacts with PTEN on TGF-β treatment under endogenous conditions. RNA interference (RNAi) suppression of PTEN expression enhances SMAD3 transcriptional activity and TGF-β–mediated induction of SMAD3 target genes whereas reconstitution of PTEN in a null cancer cell line represses the expression of TGF-β–regulated target genes. Targeting PTEN expression through RNAi in a PTEN wild-type cell line increases TGF-β–mediated invasion but does not affect TGF-β–mediated growth inhibition. Reconstitution of PTEN expression in a PTEN-null cell line blocks TGF-β–induced invasion but does not modulate TGF-β–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-β receptors and phosphatidylinositol 3-kinase signaling downstream from PTEN cooperate to block TGF-β–mediated invasion. Thus, the loss of PTEN expression in human cancers may contribute to a role for TGF-β as a tumor enhancer with specific effects on cellular motility and invasion. (Cancer Res 2005; 65(24): 11276-81)
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- 2023
3. Data from Systemic Overexpression of Angiopoietin-2 Promotes Tumor Microvessel Regression and Inhibits Angiogenesis and Tumor Growth
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Mark W. Dewhirst, Christopher D. Kontos, Chuan-Yuan Li, Bryan M. Clary, Jing Mi, Yulin Zhao, Shanling Liu, Pierre Sonveaux, and Yiting Cao
- Abstract
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. [Cancer Res 2007;67(8):3835–44]
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- 2023
4. Muscle progenitor cells are required for skeletal muscle regeneration and prevention of adipogenesis after limb ischemia
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Hasan Abbas, Lindsey A. Olivere, Michael E. Padgett, Cameron A. Schmidt, Brian F. Gilmore, Timothy J. McCord, Kevin W. Southerland, Joseph M. McClung, and Christopher D. Kontos
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Cardiology and Cardiovascular Medicine - Abstract
Skeletal muscle injury in peripheral artery disease (PAD) has been attributed to vascular insufficiency, however evidence has demonstrated that muscle cell responses play a role in determining outcomes in limb ischemia. Here, we demonstrate that genetic ablation of Pax7+ muscle progenitor cells (MPCs) in a model of hindlimb ischemia (HLI) inhibited muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to control mice (Pax7WT), the ischemic limb of Pax7-deficient mice (Pax7Δ) was unable to generate significant force 7 or 28 days after HLI. A significant increase in adipose was observed in the ischemic limb 28 days after HLI in Pax7Δ mice, which replaced functional muscle. Adipogenesis in Pax7Δ mice corresponded with a significant increase in PDGFRα+ fibro/adipogenic progenitors (FAPs). Inhibition of FAPs with batimastat decreased muscle adipose but increased fibrosis. In vitro, Pax7Δ MPCs failed to form myotubes but displayed increased adipogenesis. Skeletal muscle from patients with critical limb threatening ischemia displayed increased adipose in more ischemic regions of muscle, which corresponded with fewer satellite cells. Collectively, these data demonstrate that Pax7+ MPCs are required for muscle regeneration after ischemia and suggest that muscle regeneration may be an important therapeutic target in PAD.
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- 2023
5. Coupled myovascular expansion directs cardiac growth and regeneration
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Paige DeBenedittis, Anish Karpurapu, Albert Henry, Michael C. Thomas, Timothy J. McCord, Kyla Brezitski, Anil Prasad, Caroline E. Baker, Yoshihiko Kobayashi, Svati H. Shah, Christopher D. Kontos, Purushothama Rao Tata, R. Thomas Lumbers, and Ravi Karra
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Vascular Endothelial Growth Factor A ,Mice ,Infant, Newborn ,Animals ,Endothelial Cells ,Humans ,Heart ,Myocytes, Cardiac ,Molecular Biology ,Cell Proliferation ,Signal Transduction ,Developmental Biology - Abstract
Heart regeneration requires multiple cell types to enable cardiomyocyte (CM) proliferation. How these cells interact to create growth niches is unclear. Here, we profile proliferation kinetics of cardiac endothelial cells (CECs) and CMs in the neonatal mouse heart and find that they are spatiotemporally coupled. We show that coupled myovascular expansion during cardiac growth or regeneration is dependent upon VEGF-VEGFR2 signaling, as genetic deletion of Vegfr2 from CECs or inhibition of VEGFA abrogates both CEC and CM proliferation. Repair of cryoinjury displays poor spatial coupling of CEC and CM proliferation. Boosting CEC density after cryoinjury with virus encoding Vegfa enhances regeneration. Using Mendelian randomization, we demonstrate that circulating VEGFA levels are positively linked with human myocardial mass, suggesting that Vegfa can stimulate human cardiac growth. Our work demonstrates the importance of coupled CEC and CM expansion and reveals a myovascular niche that may be therapeutically targeted for heart regeneration.
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- 2022
6. VEGF-C and VE-cadherin: balancing sinusoidal and lymphatic angiogenesis
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Timothy J. McCord and Christopher D. Kontos
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- 2022
7. Endothelial Regulation of Microvascular Growth and Stability by Ang‐Tie and VEGF Signaling Pathways: A Mechanistic Computational Systems Biology Model
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Yu Zhang, Christopher D. Kontos, Brian H. Annex, and Aleksander S. Popel
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Genetics ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2022
8. Using genetically encoded fluorescent biosensors to interrogate ovarian cancer metabolism
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Shree Bose, Haipei Yao, Qiang Huang, Regina Whitaker, Christopher D. Kontos, Rebecca A. Previs, and Xiling Shen
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Ovarian Neoplasms ,Obstetrics and Gynecology ,Biosensing Techniques ,Carcinoma, Ovarian Epithelial ,NAD ,Carboplatin ,Adenosine Diphosphate ,Adenosine Triphosphate ,Oncology ,Culture Media, Conditioned ,Tumor Microenvironment ,Humans ,Female ,NADP - Abstract
Background Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy and patients present with significant metastatic burden, particularly to the adipose-rich microenvironment of the omentum. Recent evidence has highlighted the importance of metabolic adaptations in enabling this metastasis, leading to significant interest in evolving the arsenal of tools used to study OC metabolism. In this study, we demonstrate the capability of genetically encoded fluorescent biosensors to study OC, with a focus on 3D organoid models that better recapitulate in vivo tumor microenvironments. Materials and methods Plasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval were transfected into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n = 8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin. Results Exposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients. Conclusions Our study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC.
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- 2022
9. Pearls of wisdom for aspiring physician-scientist residency applicants and program directors
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Emily J. Gallagher, Don C. Rockey, Christopher D. Kontos, Jatin M. Vyas, Lawrence F. Brass, Patrick J. Hu, Carlos M. Isales, Olujimi A. Ajijola, W. Kimryn Rathmell, Paul R. Conlin, Robert A. Baiocchi, Barbara I. Kazmierczak, Myles H. Akabas, and Christopher S. Williams
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Physicians ,Surveys and Questionnaires ,Humans ,Internship and Residency ,General Medicine ,Fellowships and Scholarships ,Child ,Research Personnel - Abstract
Postgraduate physician-scientist training programs (PSTPs) enhance the experiences of physician-scientist trainees following medical school graduation. PSTPs usually span residency and fellowship training, but this varies widely by institution. Applicant competitiveness for these programs would be enhanced, and unnecessary trainee anxiety relieved, by a clear understanding of what factors define a successful PSTP matriculant. Such information would also be invaluable to PSTP directors and would allow benchmarking of their admissions processes with peer programs. We conducted a survey of PSTP directors across the US to understand the importance they placed on components of PSTP applications. Of 41 survey respondents, most were from internal medicine and pediatrics residency programs. Of all components in the application, two elements were considered very important by a majority of PSTP directors: (a) having one or more first-author publications and (b) the thesis advisor's letter. Less weight was consistently placed on factors often considered more relevant for non-physician-scientist postgraduate applicants - such as US Medical Licensing Examination scores, awards, and leadership activities. The data presented here highlight important metrics for PSTP applicants and directors and suggest that indicators of scientific productivity and commitment to research outweigh traditional quantitative measures of medical school performance.
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- 2022
10. Addressing the physician-scientist pipeline: strategies to integrate research into clinical training programs
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Christopher D. Kontos, Patrick J. Hu, Sallie R. Permar, Jatin M. Vyas, Rebecca A Ward, Katherine E Hartmann, Rasheed Gbadegesin, Katherine J. Barrett, Christopher S. Williams, and Stephanie A. Freel
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Medical education ,Biomedical Research ,Career Choice ,Education, Medical ,Extramural ,MEDLINE ,General Medicine ,Pipeline (software) ,Research Personnel ,Nobel Prize ,Viewpoint ,Physicians ,Clinical training ,Humans ,Psychology ,Career choice - Published
- 2020
11. G6PD inhibition sensitizes ovarian cancer cells to oxidative stress in the metastatic omental microenvironment
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Shree Bose, Qiang Huang, Yunhan Ma, Lihua Wang, Grecia O. Rivera, Yunxin Ouyang, Regina Whitaker, Rebecca A. Gibson, Christopher D. Kontos, Andrew Berchuck, Rebecca Previs, and Xiling Shen
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Ovarian Neoplasms ,Pentose Phosphate Pathway ,Oxidative Stress ,Tumor Microenvironment ,Humans ,Female ,Carcinoma, Ovarian Epithelial ,Glucosephosphate Dehydrogenase ,Omentum ,General Biochemistry, Genetics and Molecular Biology - Abstract
SummaryOvarian cancer (OC) is the most lethal gynecological malignancy, with aggressive metastatic disease responsible for the majority of ovarian cancer related deaths. In particular, OC tumors preferentially metastasize to and proliferate rapidly in the omentum. Here, we show metastatic OC cells experience increased oxidative stress in the omental microenvironment. Metabolic reprogramming, including upregulation of the pentose phosphate pathway (PPP), a key cellular redox homeostasis mechanism, allows OC cells to compensate for this challenge. Inhibition of G6PD, the rate-limiting enzyme of the PPP, reduces tumor burden in pre-clinical models of OC, suggesting this adaptive metabolic dependency is important for OC omental metastasis.HighlightsThe omental microenvironment poses a high oxidative stress metastatic niche for ovarian cancer cells.G6PD, a key enzyme involved in redox homeostasis and the rate-limiting enzyme of the pentose phosphate pathway (PPP) is upregulated in omental metastasis.Inhibition of G6PD increases oxidative stress and cytotoxicity in the omental microenvironment.Pharmacological G6PD inhibition reduces omental metastases in vivo.
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- 2021
12. Computational Systems Biology Modeling of the Angiopoietin‐Tie Signaling Pathway and its Crosstalk with α5β1 Integrin in Endothelial Cells
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Christopher D. Kontos, Aleksander S. Popel, Yu Zhang, and Brian H. Annex
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Angiopoietin ,Crosstalk (biology) ,Chemistry ,Modelling biological systems ,Genetics ,Tie signaling pathway ,Molecular Biology ,Biochemistry ,α5β1 integrin ,Biotechnology ,Cell biology - Published
- 2021
13. A systems biology model of junctional localization and downstream signaling of the Ang-Tie signaling pathway
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Yu Zhang, Brian H. Annex, Aleksander S. Popel, and Christopher D. Kontos
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Cell biology ,Chemistry ,QH301-705.5 ,Applied Mathematics ,Systems biology ,Systems Biology ,Endothelial Cells ,Vascular permeability ,Receptor, TIE-2 ,General Biochemistry, Genetics and Molecular Biology ,TIE1 ,Article ,Computer Science Applications ,Endothelial stem cell ,Modeling and Simulation ,Drug Discovery ,Computer modelling ,Extracellular ,Tie signaling pathway ,Signal transduction ,Biology (General) ,Receptor ,Biological Phenomena ,Signal Transduction - Abstract
The Ang–Tie signaling pathway is an important vascular signaling pathway regulating vascular growth and stability. Dysregulation in the pathway is associated with vascular dysfunction and numerous diseases that involve abnormal vascular permeability and endothelial cell inflammation. The understanding of the molecular mechanisms of the Ang–Tie pathway has been limited due to the complex reaction network formed by the ligands, receptors, and molecular regulatory mechanisms. In this study, we developed a mechanistic computational model of the Ang–Tie signaling pathway validated against experimental data. The model captures and reproduces the experimentally observed junctional localization and downstream signaling of the Ang–Tie signaling axis, as well as the time-dependent role of receptor Tie1. The model predicts that Tie1 modulates Tie2’s response to the context-dependent agonist Ang2 by junctional interactions. Furthermore, modulation of Tie1’s junctional localization, inhibition of Tie2 extracellular domain cleavage, and inhibition of VE-PTP are identified as potential molecular strategies for potentiating Ang2’s agonistic activity and rescuing Tie2 signaling in inflammatory endothelial cells.
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- 2021
14. Coupled myovascular expansion directs cardiac growth and regeneration
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Christopher D. Kontos, Prasad A, Anish Karpurapu, Paige DeBenedittis, Svati H. Shah, Kyla D Brezitski, Timothy J. McCord, Purushothama Rao Tata, Michael C Thomas, Ravi Karra, Albert Henry, Lumbers Rt, and Yoshihiko Kobayashi
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Vascular endothelial growth factor A ,Cell type ,Incomplete regeneration ,Regeneration (biology) ,cardiovascular system ,Neonatal mouse ,Proliferation kinetics ,Biology ,Border zone ,biology.organism_classification ,Zebrafish ,Cell biology - Abstract
Innate heart regeneration in zebrafish and neonatal mammals requires multiple cell types, such as epicardial cells, nerves, and macrophages, to enable proliferation of spared cardiomyocytes (CMs). How these cells interact to create growth niches is unclear. Here we profile proliferation kinetics of cardiac endothelial cells (CECs) and CMs in the neonatal mouse heart and find that CM and CEC expansion is spatiotemporally coupled. We show that coupled myovascular expansion during cardiac growth or regeneration is dependent upon VEGF-VEGFR2 signaling, as genetic deletion of Vegfr2 from CECs or inhibition of VEGFA abrogates both CEC and CM proliferation. Repair of cryoinjury, a model of incomplete regeneration, displays poor spatial coupling of CEC and CM proliferation. Boosting CEC density in the border zone by injection of virus encoding Vegfa enhances CM proliferation and the efficacy of heart regeneration, suggesting that revascularization strategies to increase CEC numbers may be an important adjunct for approaches designed to promote CM proliferation after injury. Finally, we use a human Mendelian randomization study to demonstrate that circulating VEGFA levels are positively associated with higher myocardial mass among healthy individuals, suggesting similar effects on human cardiac growth. Our work demonstrates the importance of coupled CEC and CM expansion for cardiomyogenesis and reveals the presence of a myovascular niche that underlies cardiac growth and regeneration.
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- 2021
15. Muscle progenitor cells are required for the regenerative response and prevention of adipogenesis after limb ischemia
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Joseph M. McClung, Brian F. Gilmore, Christopher D. Kontos, Kevin W. Southerland, Cameron A. Schmidt, Hasan Abbas, Michael E. Padgett, and Lindsey A. Olivere
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0303 health sciences ,medicine.medical_specialty ,business.industry ,Myogenesis ,Ischemia ,Adipose tissue ,Skeletal muscle ,Critical limb ischemia ,030204 cardiovascular system & hematology ,medicine.disease ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,medicine.anatomical_structure ,Adipogenesis ,Internal medicine ,medicine ,Progenitor cell ,medicine.symptom ,business ,Ex vivo ,030304 developmental biology - Abstract
Peripheral artery disease (PAD) is nearly as common as coronary artery disease, but few effective treatments exist, and it is associated with significant morbidity and mortality. Although PAD studies have focused on the vascular response to ischemia, skeletal muscle cells play a critically important role in determining the phenotypic manifestation of PAD. Here, we demonstrate that genetic ablation of Pax7+muscle progenitor cells (MPCs, or satellite cells) in a murine model of hind limb ischemia (HLI) resulted in a complete absence of normal muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to ischemic muscle of control mice (Pax7WT), the ischemic limb of Pax7-deficient mice (Pax7Δ) was unable to generate significant force 7- or 28-days after HLI inex vivoforce measurement studies. A dramatic increase in adipose infiltration was observed 28 days after HLI in Pax7Δmice, which replaced functional muscle. To investigate the mechanism of this adipogenic change, mice with inhibition of fibro/adipogenic precursors (FAPs), another pool of MPCs, were subjected to HLI. Inhibition of FAPs decreased muscle adipose fat but increased fibrosis. MPCs cultured from mouse muscle tissue failed to form myotubesin vitrofollowing depletion of satellite cellsin vivo, and they displayed an increased propensity to differentiate into fat in adipogenic medium. Importantly, this phenotype was recapitulated in patients with critical limb ischemia (CLI), the most severe form of PAD. Skeletal muscle samples from CLI patients demonstrated an increase in adipose deposition in more ischemic regions of muscle, which corresponded with a decrease in the number of satellite cells in those regions. Collectively, these data demonstrate that Pax7+MPCs are required for normal muscle regeneration after ischemic injury, and they suggest that targeting muscle regeneration may be an important therapeutic approach to prevent muscle degeneration in PAD.
- Published
- 2020
16. Mitochondrial dysfunction in human immunodeficiency virus‐1 transgenic mouse cardiac myocytes
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Fabian Jana Prado, Nana Merabova, Manish K. Gupta, Joseph M. McClung, Joseph Y. Cheung, Jennifer Gordon, Jianliang Song, Xue-Qian Zhang, Santhanam Shanmughapriya, Sudarsan Rajan, Kamel Khalili, Christopher D. Kontos, Tijana Knezevic, Muniswamy Madesh, Dhanendra Tomar, Arthur M. Feldman, Farzaneh G. Tahrir, Paul E. Klotman, and JuFang Wang
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0301 basic medicine ,Membrane potential ,chemistry.chemical_classification ,Genetically modified mouse ,Reactive oxygen species ,Contraction (grammar) ,Physiology ,Chemistry ,Protein subunit ,Transgene ,Clinical Biochemistry ,Wild type ,Cell Biology ,Molecular biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Myocyte - Abstract
The pathophysiology of human immunodeficiency virus (HIV)-associated cardiomyopathy remains uncertain. We used HIV-1 transgenic (Tg26) mice to explore mechanisms by which HIV-related proteins impacted on myocyte function. Compared to adult ventricular myocytes isolated from nontransgenic (wild type [WT]) littermates, Tg26 myocytes had similar mitochondrial membrane potential (ΔΨ m ) under normoxic conditions but lower Δ Ψ m after hypoxia/reoxygenation (H/R). In addition, Δ Ψ m in Tg26 myocytes failed to recover after Ca 2+ challenge. Functionally, mitochondrial Ca 2+ uptake was severely impaired in Tg26 myocytes. Basal and maximal oxygen consumption rates (OCR) were lower in normoxic Tg26 myocytes, and further reduced after H/R. Complex I subunit and ATP levels were lower in Tg26 hearts. Post-H/R, mitochondrial superoxide (O 2 •- ) levels were higher in Tg26 compared to WT myocytes. Overexpression of B-cell lymphoma 2-associated athanogene 3 (BAG3) reduced O 2 •- levels in hypoxic WT and Tg26 myocytes back to normal. Under normoxic conditions, single myocyte contraction dynamics were similar between WT and Tg26 myocytes. Post-H/R and in the presence of isoproterenol, myocyte contraction amplitudes were lower in Tg26 myocytes. BAG3 overexpression restored Tg26 myocyte contraction amplitudes to those measured in WT myocytes post-H/R. Coimmunoprecipitation experiments demonstrated physical association of BAG3 and the HIV protein Tat. We conclude: (a) Under basal conditions, mitochondrial Ca 2+ uptake, OCR, and ATP levels were lower in Tg26 myocytes; (b) post-H/R, Δ Ψ m was lower, mitochondrial O 2 •- levels were higher, and contraction amplitudes were reduced in Tg26 myocytes; and (c) BAG3 overexpression decreased O 2 •- levels and restored contraction amplitudes to normal in Tg26 myocytes post-H/R in the presence of isoproterenol.
- Published
- 2018
17. BAG3 (Bcl-2–Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy
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Douglas A. Marchuk, Espen E. Spangenburg, Thomas D. Green, Sehoon Keum, Ayotunde O. Dokun, Joseph M. McClung, Christopher D. Kontos, Timothy J. McCord, Jessica L. Reinardy, Christopher D. Lascola, Brian H. Annex, Cameron A. Schmidt, Sarah B. Mueller, Terence E. Ryan, Talaignair N. Venkatraman, and Kevin W. Southerland
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0301 basic medicine ,Pathology ,medicine.medical_specialty ,Necrosis ,business.industry ,Ischemia ,Skeletal muscle ,Critical limb ischemia ,Hindlimb ,Anatomy ,medicine.disease ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Physiology (medical) ,Heat shock protein ,medicine ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,Myopathy ,business ,Perfusion - Abstract
Background: Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1 , that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. Methods: We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2–associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. Results: We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6– Lsq1-3 ). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein– (n=29) expressing animals. BAG3 Ile81 , but not BAG3 Met81 , improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus–BAG3 Ile81 (n=9), but not BAG3 Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3 Met81 , BAG3 Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. Conclusions: Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.
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- 2017
18. Precision Medicine for Heart Failure
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Arthur M. Feldman, Glenn S. Gerhard, Joseph M. McClung, Christopher D. Kontos, Kamel Khalili, and Joseph Y. Cheung
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medicine.medical_specialty ,Pathology ,Alternative medicine ,Genomics ,Disease ,030204 cardiovascular system & hematology ,Medical Oncology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Medicine ,Medical physics ,Tumor biopsy ,Precision Medicine ,Adaptor Proteins, Signal Transducing ,Heart Failure ,business.industry ,Cancer ,medicine.disease ,Precision medicine ,United States ,Molecular analysis ,Clinical trial ,030220 oncology & carcinogenesis ,Apoptosis Regulatory Proteins ,Cardiology and Cardiovascular Medicine ,business - Abstract
In his State of the Union Address in January 2015, President Barack Obama launched the Precision Medicine Initiative “to bring us closer to curing diseases like cancer and diabetes.” Francis Collins, the Director of the National Institutes of Health, noted that advances in molecular biology, genomics, and bioinformatics and converging trends of increased connectivity through social media and mobile devices had set the stage for the President’s visionary initiative.1 The initiative would start by focusing on cancer. Although cardiovascular disease remains the leading cause of death in the United States, the decisions to focus initially on cancer highlighted the advances in precision medicine for cancer that have clearly outpaced that for any other field of medicine. In fact, cardiology in general and heart failure (HF) specifically have made little progress toward precision medicine. Nonetheless, important lessons can be learned from both the success and failures of precision oncology, which will potentially provide a template for advances toward a precise approach to the therapy of HF and other cardiovascular diseases. The fundamental principle that underlies cancer precision medicine is that molecular analysis of an individual patient’s tumor can enable the identification of the appropriate drug for that tumor which would in turn lead to improved efficacy. Molecular analysis of cancer has been facilitated by the confluence of technological and analytic advances, including the completion of the Human Genome Project,2 the introduction of high-throughput and relatively inexpensive next-generation sequencing,3 and the development of sufficient data storage and computational analytics.4 The Precision Medicine Initiative at the National Cancer Institute has also been facilitated by the Exceptional Responders Initiative and new constructs for clinical trials. In the Molecular Analysis for Therapy Choice (MATCH) program, patients undergo a tumor biopsy followed by next-generation sequencing supplemented by immunohistochemistry or fluorescence in situ …
- Published
- 2017
19. Dysregulation of mitochondrial bioenergetics and quality control by HIV-1 Tat in cardiomyocytes
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Tijana Knezevic, Christopher D. Kontos, Muniswamy Madesh, Manish K. Gupta, Kamel Khalili, Santhanam Shanmughapriya, Taha Mohseni Ahooyi, Joseph M. McClung, Jennifer Gordon, Arthur M. Feldman, Farzaneh G. Tahrir, and Joseph Y. Cheung
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0301 basic medicine ,Time Factors ,Bioenergetics ,Physiology ,Clinical Biochemistry ,Primary Cell Culture ,Apoptosis ,Oxidative phosphorylation ,Mitochondrion ,Mitochondria, Heart ,Oxidative Phosphorylation ,Article ,Membrane Potentials ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Adenosine Triphosphate ,Ubiquitin ,Sequestosome-1 Protein ,Autophagy ,Animals ,Myocytes, Cardiac ,Cells, Cultured ,chemistry.chemical_classification ,Reactive oxygen species ,biology ,Mitophagy ,Cell Biology ,Cell Hypoxia ,Cell biology ,030104 developmental biology ,chemistry ,Cell culture ,Host-Pathogen Interactions ,biology.protein ,HIV-1 ,Calcium ,tat Gene Products, Human Immunodeficiency Virus ,Calcium Channels ,Energy Metabolism ,Reactive Oxygen Species ,Microtubule-Associated Proteins ,030217 neurology & neurosurgery ,Homeostasis ,Signal Transduction - Abstract
Cardiovascular disease remains a leading cause of morbidity and mortality in HIV positive patients, even in those whose viral loads are well controlled with antiretroviral therapy. However, the underlying molecular events responsible for the development of cardiac disease in the setting of HIV remain unknown. The HIV encoded Tat protein plays a critical role in the activation of HIV gene expression and profoundly impacts homeostasis in both HIV infected cells and uninfected cells that have taken up released Tat via a bystander effect. Since cardiomyocyte function, including excitation-contraction coupling, greatly depends on energy provided by the mitochondria, in this study we performed a series of experiments to assess the impact of Tat on mitochondrial function and bioenergetics pathways in a primary cell culture model derived from neonatal rat ventricular cardiomyocytes (NRVCs). Our results show that the presence of Tat in cardiomyocytes is accompanied by a decrease in oxidative phosphorylation, a decline in the levels of ATP, and an accumulation of reactive oxygen species (ROS). Tat impairs the uptake of mitochondrial Ca2+ ([Ca2+]m) and the electrophysiological activity of cardiomyocytes. Tat also affects the protein clearance pathway and autophagy in cardiomyocytes under stress due to hypoxia-reoxygenation conditions. A reduction in the level of ubiquitin along with dysregulated degradation of autophagy proteins including SQSTM1/p62 and a reduction of LC3 II were detected in cardiomyocytes harboring Tat. These results suggest that, by targeting mitochondria and protein quality control, Tat significantly impacts bioenergetics and autophagy resulting in dysregulation of cardiomyocyte health and homeostasis. This article is protected by copyright. All rights reserved
- Published
- 2017
20. More than skin deep: connecting melanocyte pigmentation and angiogenic diseases
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Christopher D. Kontos
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Angiogenesis ,Neovascularization, Physiologic ,Skin Pigmentation ,Melanocyte ,Biology ,Transforming Growth Factor beta1 ,Extracellular matrix ,Neovascularization ,Mice ,Cell Movement ,medicine ,Animals ,Humans ,Cells, Cultured ,Cell Proliferation ,Extracellular Matrix Proteins ,Genetic variants ,Endothelial Cells ,General Medicine ,eye diseases ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Immunology ,Commentary ,Melanocytes ,Proteoglycans ,Racial differences ,Endothelium, Vascular ,sense organs ,medicine.symptom ,Fibromodulin ,Research Article - Abstract
Studies have established that pigmentation can provide strong, protective effects against certain human diseases. For example, angiogenesis-dependent diseases such as wet age-related macular degeneration and infantile hemangioma are more common in light-skinned individuals of mixed European descent than in African-Americans. Here we found that melanocytes from light-skinned humans and albino mice secrete high levels of fibromodulin (FMOD), which we determined to be a potent angiogenic factor. FMOD treatment stimulated angiogenesis in numerous in vivo systems, including laser-induced choroidal neovascularization, growth factor-induced corneal neovascularization, wound healing, and Matrigel plug assays. Additionally, FMOD enhanced vascular sprouting during normal retinal development. Deletion of Fmod in albino mice resulted in a marked reduction in the amount of neovascularization induced by retinal vein occlusion, corneal growth factor pellets, and Matrigel plugs. Our data implicate the melanocyte-secreted factor FMOD as a key regulator of angiogenesis and suggest an underlying mechanism for epidemiological differences between light-skinned individuals of mixed European descent and African-Americans. Furthermore, inhibition of FMOD in humans has potential as a therapeutic strategy for treating angiogenesis-dependent diseases.
- Published
- 2013
21. Gene therapy for the prevention of vein graft disease
- Author
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Dawn E. Bowles, Kevin W. Southerland, Christopher D. Kontos, Sarah B. Frazier, and Carmelo A. Milano
- Subjects
medicine.medical_specialty ,Intimal hyperplasia ,Genetic Vectors ,Gene delivery ,Inferior vena cava ,Article ,Veins ,Coronary artery disease ,Blood vessel prosthesis ,Physiology (medical) ,medicine ,Humans ,Vein ,Clinical Trials as Topic ,business.industry ,Biochemistry (medical) ,Gene Transfer Techniques ,Public Health, Environmental and Occupational Health ,Genetic Therapy ,General Medicine ,medicine.disease ,Blood Vessel Prosthesis ,Surgery ,medicine.anatomical_structure ,medicine.vein ,Bypass surgery ,business ,Vein graft disease - Abstract
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.
- Published
- 2013
22. Methods for Acute and Subacute Murine Hindlimb Ischemia
- Author
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Christopher D. Kontos, Joseph M. McClung, Timothy J. McCord, and Michael E. Padgett
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Necrosis ,General Chemical Engineering ,Ischemia ,Neovascularization, Physiologic ,Hindlimb ,Femoral artery ,030204 cardiovascular system & hematology ,General Biochemistry, Genetics and Molecular Biology ,Neovascularization ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,medicine.artery ,medicine ,Animals ,Muscle, Skeletal ,General Immunology and Microbiology ,business.industry ,General Neuroscience ,Vascular surgery ,medicine.disease ,Surgery ,Femoral Artery ,Disease Models, Animal ,030104 developmental biology ,medicine.anatomical_structure ,Regional Blood Flow ,Cardiology ,Medicine ,medicine.symptom ,business ,Ligation ,Artery - Abstract
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.
- Published
- 2016
23. Angiopoietin-1 promotes atherosclerosis by increasing the proportion of circulating Gr1+ monocytes
- Author
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Takeshi, Fujisawa, Keqing, Wang, Xi-Lin, Niu, Stuart, Egginton, Shakil, Ahmad, Peter, Hewett, Christopher D, Kontos, and Asif, Ahmed
- Subjects
Male ,Vascular Endothelial Growth Factor A ,Genetic Vectors ,Aortic Diseases ,Aorta, Thoracic ,Diet, High-Fat ,Monocytes ,Adenoviridae ,Tissue Culture Techniques ,Apolipoproteins E ,Vascular Biology ,Angiopoietin-1 ,Animals ,Antigens, Ly ,Humans ,Genetic Predisposition to Disease ,Chemokine CCL2 ,Mice, Knockout ,CD11b Antigen ,Original Articles ,Atherosclerosis ,Plaque, Atherosclerotic ,Mice, Inbred C57BL ,Disease Models, Animal ,Phenotype ,Signal Transduction - Abstract
Aims 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. Methods and results 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. Conclusions Ang-1 specifically increases circulating Gr1+ inflammatory monocytes and increases monocyte/macrophage retention in atherosclerotic plaques, thereby contributing to development of atherosclerosis.
- Published
- 2016
24. In vivo tumor targeting by a NGR-decorated micelle of a recombinant diblock copolypeptide
- Author
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Ashutosh Chilkoti, Miriam Amiram, Gabi Hanna, Wenge Liu, Mark W. Dewhirst, Christopher D. Kontos, and Andrew J. Simnick
- Subjects
Light ,Mice, Nude ,Pharmaceutical Science ,Angiogenesis Inhibitors ,CD13 Antigens ,Ligands ,Micelle ,Article ,Mice ,In vivo ,Cell Line, Tumor ,Amphiphile ,Escherichia coli ,Animals ,Humans ,Scattering, Radiation ,Receptor ,Micelles ,Drug Carriers ,Oligopeptide ,Microscopy, Confocal ,Neovascularization, Pathologic ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,Temperature ,Xenograft Model Antitumor Assays ,Elastin ,Microscopy, Fluorescence ,Biochemistry ,Cell culture ,Drug delivery ,Biophysics ,Electrophoresis, Polyacrylamide Gel ,Female ,Peptides ,Drug carrier ,Hydrophobic and Hydrophilic Interactions ,Oligopeptides - Abstract
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.
- Published
- 2011
25. Targeting the TIE2 pathway with a novel small molecule vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor in high-grade serous ovarian cancer
- Author
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Zhiqing Huang, Christopher D. Kontos, Joseph A. Herbert, Gabi Hanna, K. Peters, Lauren Patterson Cobb, Gregory M. Palmer, Andrew Berchuck, Ashlyn G. Rickard, Angeles Alvarez Secord, and Sharareh Siamakpour-Reihani
- Subjects
VASCULAR ENDOTHELIAL PROTEIN-TYROSINE PHOSPHATASE ,Oncology ,biology ,business.industry ,Serous ovarian cancer ,biology.protein ,Cancer research ,Obstetrics and Gynecology ,Medicine ,business ,Angiopoietin receptor ,Small molecule - Published
- 2018
26. VEGF and soluble VEGF receptor-1 (sFlt-1) distributions in peripheral arterial disease: an in silico model
- Author
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Christopher D. Kontos, Florence T. H. Wu, Brian H. Annex, Marianne O. Stefanini, Feilim Mac Gabhann, and Aleksander S. Popel
- Subjects
Vascular Endothelial Growth Factor A ,Physiology ,Angiogenesis ,Regulator ,Biology ,Models, Biological ,chemistry.chemical_compound ,Physiology (medical) ,Neuropilin 1 ,Humans ,Therapeutic angiogenesis ,Muscle, Skeletal ,Peripheral Vascular Diseases ,Leg ,Vascular Endothelial Growth Factor Receptor-1 ,Neovascularization, Pathologic ,Kinase insert domain receptor ,Articles ,Vascular Endothelial Growth Factor Receptor-2 ,Neuropilin-1 ,Capillaries ,Vascular endothelial growth factor ,Vascular endothelial growth factor A ,chemistry ,Regional Blood Flow ,Immunology ,Cancer research ,Signal transduction ,Cardiology and Cardiovascular Medicine ,Signal Transduction - Abstract
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.
- Published
- 2010
27. Inactivation of the tumour suppressor, PTEN, in smooth muscle promotes a pro-inflammatory phenotype and enhances neointima formation
- Author
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Insun Park, Christopher D. Kontos, Vicki VanPutten, Raphael A. Nemenoff, Seth B. Furgeson, Henrick Horita, Mary C.M. Weiser-Evans, and Peter A. Simpson
- Subjects
Neointima ,Vascular smooth muscle ,Physiology ,Myocytes, Smooth Muscle ,Calponin ,Protein Serine-Threonine Kinases ,Biology ,Muscle, Smooth, Vascular ,Mice ,Phosphatidylinositol 3-Kinases ,Physiology (medical) ,Animals ,Tensin ,PTEN ,Protein kinase B ,Cells, Cultured ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Inflammation ,Mice, Knockout ,TOR Serine-Threonine Kinases ,Intracellular Signaling Peptides and Proteins ,NF-kappa B ,PTEN Phosphohydrolase ,Original Articles ,Cell Dedifferentiation ,musculoskeletal system ,Rats ,CXCL1 ,Disease Models, Animal ,Phenotype ,cardiovascular system ,biology.protein ,Cancer research ,Cytokines ,RNA Interference ,Inflammation Mediators ,Carotid Artery Injuries ,Tunica Intima ,Cardiology and Cardiovascular Medicine ,Proto-Oncogene Proteins c-akt ,Signal Transduction - Abstract
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-κB) activity, reduced expression of SM markers (SM-α-actin and calponin), and increased production of stromal cell-derived factor-1α (SDF-1α), 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-κB 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-α-actin and calponin expression, and increased NF-κB 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.
- Published
- 2010
28. Phosphatase and Tensin Homologue on Chromosome 10 (PTEN) Directs Prostaglandin E2-mediated Fibroblast Responses via Regulation of E Prostanoid 2 Receptor Expression
- Author
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Jessica Tsui, Christopher D. Kontos, Janet Melonakos, Steven K. Huang, Megan N. Ballinger, Mei Yan, David Alan Stephenson, David M. Aronoff, Rommel Sagana, Ashley M. Cornett, Eric S. White, Marc Peters-Golden, and Bethany B. Moore
- Subjects
Receptor expression ,Prostaglandin E2 receptor ,Down-Regulation ,Mice, Transgenic ,Lipids and Lipoproteins: Metabolism, Regulation, and Signaling ,Biochemistry ,Dinoprostone ,Receptors, G-Protein-Coupled ,Mice ,Phosphatidylinositol 3-Kinases ,Cyclic AMP ,medicine ,Animals ,Humans ,Receptors, Prostaglandin E ,Tensin ,PTEN ,Receptor ,Fibroblast ,Molecular Biology ,PI3K/AKT/mTOR pathway ,G protein-coupled receptor ,biology ,PTEN Phosphohydrolase ,Muscle, Smooth ,Cell Biology ,Fibroblasts ,Actins ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Cancer research ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Collagen - Abstract
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.
- Published
- 2009
29. Computational kinetic model of VEGF trapping by soluble VEGF receptor-1: effects of transendothelial and lymphatic macromolecular transport
- Author
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Christopher D. Kontos, Marianne O. Stefanini, Feilim Mac Gabhann, Florence T. H. Wu, Brian H. Annex, and Aleksander S. Popel
- Subjects
Vascular Endothelial Growth Factor A ,Call for Papers: Computational Modeling of Physiological Genomics ,Physiology ,Angiogenesis ,media_common.quotation_subject ,Biology ,chemistry.chemical_compound ,Cell surface receptor ,Neuropilin 1 ,Genetics ,Humans ,Internalization ,Lymphatic Vessels ,media_common ,Vascular Endothelial Growth Factor Receptor-1 ,Models, Theoretical ,Vascular endothelial growth factor ,Kinetics ,Protein Transport ,Vascular endothelial growth factor A ,Solubility ,Biochemistry ,chemistry ,Biophysics ,Signal transduction ,Soluble fms-like tyrosine kinase-1 - Abstract
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.
- Published
- 2009
30. A VEGF Trap Inhibits the Beneficial Effect of bFGF on Vasoreactivity in Corporal Tissues of Hypercholesterolemic Rabbits
- Author
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Christopher D. Kontos, Clarence M. Findley, Brian H. Annex, Craig F. Donatucci, Donghua Xie, Jason M. Greenfield, and Anne M. Pippen
- Subjects
Male ,Vascular Endothelial Growth Factor A ,medicine.medical_specialty ,Nitric Oxide Synthase Type III ,Endothelium ,Urology ,Endocrinology, Diabetes and Metabolism ,Hypercholesterolemia ,Basic fibroblast growth factor ,Injections, Intramuscular ,Nitric oxide ,Impotence, Vasculogenic ,chemistry.chemical_compound ,Endocrinology ,Western blot ,Internal medicine ,medicine ,Animals ,Protein kinase B ,medicine.diagnostic_test ,business.industry ,Gene Transfer Techniques ,Effective dose (pharmacology) ,Recombinant Proteins ,Vasodilation ,Vascular endothelial growth factor ,Disease Models, Animal ,Psychiatry and Mental health ,Receptors, Vascular Endothelial Growth Factor ,medicine.anatomical_structure ,Reproductive Medicine ,chemistry ,Phosphorylation ,Fibroblast Growth Factor 2 ,Endothelium, Vascular ,Rabbits ,business ,Proto-Oncogene Proteins c-akt ,Penis - Abstract
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 µg bFGF ICI and 2.5 × 10 11 viral particle unit (vpu) of adenovirus encoding β-galactosidase (Adβ-gal) ICI, 10 days later. Group 2 received a 2.5 µg bFGF ICI and 2.5 × 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 × 10 11 vpu Adβ-gal ICI, 10 days later. Group 4 received PBS ICI and 2.5 × 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 phoshorylated 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. 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 2008;5:2069–2078.
- Published
- 2008
31. VEGF Induces Tie2 Shedding via a Phosphoinositide 3-Kinase/Akt–Dependent Pathway to Modulate Tie2 Signaling
- Author
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Clarence M. Findley, Christopher D. Kontos, Asif Ahmed, and Melissa J. Cudmore
- Subjects
Vascular Endothelial Growth Factor A ,Pyridines ,Angiogenesis ,Apoptosis ,p38 Mitogen-Activated Protein Kinases ,Mice ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,Phosphorylation ,Cells, Cultured ,Phosphoinositide-3 Kinase Inhibitors ,biology ,Kinase ,Imidazoles ,Dipeptides ,Receptor, TIE-2 ,Cell biology ,Vascular endothelial growth factor ,embryonic structures ,cardiovascular system ,Tetradecanoylphorbol Acetate ,Cardiology and Cardiovascular Medicine ,Signal Transduction ,medicine.medical_specialty ,Morpholines ,Neovascularization, Physiologic ,Matrix Metalloproteinase Inhibitors ,Transfection ,Angiopoietin-2 ,Internal medicine ,Angiopoietin-1 ,medicine ,Animals ,Humans ,PTEN ,Protein Kinase Inhibitors ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Phosphoinositide 3-kinase ,PTEN Phosphohydrolase ,Endothelial Cells ,Fibroblasts ,Matrix Metalloproteinases ,Peptide Fragments ,Endocrinology ,chemistry ,Chromones ,NIH 3T3 Cells ,biology.protein ,Proto-Oncogene Proteins c-akt ,Peptide Hydrolases - Abstract
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.
- Published
- 2007
32. 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
- Author
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Surovi Hazarika, Brian H. Annex, Christopher D. Kontos, Anne M. Pippen, Yongjun Li, and Donghua Xie
- Subjects
Male ,Vascular Endothelial Growth Factor A ,medicine.medical_specialty ,Transcription, Genetic ,Angiogenesis ,Endocrinology, Diabetes and Metabolism ,Genetic Vectors ,Neovascularization, Physiologic ,Type 2 diabetes ,Biology ,Protein Engineering ,Neovascularization ,Mice ,chemistry.chemical_compound ,Ischemia ,Diabetes mellitus ,Internal medicine ,Internal Medicine ,medicine ,Animals ,RNA, Messenger ,Therapeutic angiogenesis ,Muscle, Skeletal ,Protein kinase B ,Gene Transfer Techniques ,Zinc Fingers ,Genetic Therapy ,medicine.disease ,Hindlimb ,Mice, Inbred C57BL ,Vascular endothelial growth factor ,Endocrinology ,Diabetes Mellitus, Type 2 ,Gene Expression Regulation ,chemistry ,Angiogenesis Inducing Agents ,medicine.symptom ,Ligation ,Signal Transduction ,Transcription Factors - Abstract
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 μg 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.
- Published
- 2007
33. Abstract 066: The Undescribed Protein Caskin2 Is a Novel Regulator of eNOS Phosphorylation and Systemic Blood Pressure
- Author
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Christopher D. Kontos, Susan B. Gurley, and Sarah B. Mueller
- Subjects
medicine.medical_specialty ,biology ,Phosphatase ,Wild type ,Protein phosphatase 1 ,biology.organism_classification ,Nitric oxide synthase ,Endocrinology ,Enos ,Internal medicine ,Knockout mouse ,Internal Medicine ,biology.protein ,medicine ,Phosphorylation ,Signal transduction - Abstract
Disruptions in the function of the quiescent endothelial cells (ECs) that line mature vessels can both result in and contribute to the progression of numerous cardiovascular diseases including hypertension, atherosclerosis, and disorders of vascular permeability. Despite recent attention, the signaling pathways that are active in quiescent ECs remain poorly characterized relative to those that regulate EC activation. In an effort to provide mechanistic insight into these pathways, we have characterized the previously undescribed protein Caskin2, which we hypothesize is a novel regulator of EC quiescence. Caskin2 is expressed in ECs throughout the vasculature, including the aorta, coronary arteries, and renal glomeruli. In vitro, Caskin2 promotes a quiescent EC phenotype characterized by decreased proliferation and increased resistance to apoptosis-inducing factors. Caskin2 knockout mice are viable and fertile. However, preliminary radiotelemetry measurements indicate that Caskin2 knockout (KO) mice have mildly elevated systemic blood pressure (BP). Compared to wild type (WT) littermates (n=8), Caskin2 KO mice (n=7) had increased mean arterial pressure (119+/-1 vs. 113+/-1, p=0.012), systolic BP (138+/-2 vs. 132+/-2, p=0.023), and diastolic BP (99+/-1 vs. 93+/-1, p=0.014) at baseline. To explore the molecular mechanisms of Caskin2’s effects, we used mass spectrometry to identify interacting proteins. Among the 67 proteins identified were the Ser/Thr phosphatase protein phosphatase 1 (PP1) and eNOS. Using standard in vitro biochemical techniques, we demonstrated that Caskin2 acts as a PP1 regulatory subunit. Interestingly, homologous expression of Caskin2 in vitro resulted in a marked increase in phosphorylation of eNOS on S1177, which is known to promote eNOS activity, and a decrease in phosphorylation on T495, which is associated with eNOS inhibition. Finally, PP1 has been shown to dephosphorylate eNOS T495 in vitro, suggesting a molecular mechanism for our in vivo findings. Ongoing work aims to determine if the interaction of Caskin2 and PP1 is required for the Caskin2-induced increase in activating phosphorylation of eNOS and to characterize the physiological mechanisms responsible for Caskin2’s effects on BP in more detail.
- Published
- 2015
34. Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment
- Author
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David Brown, Terence E. Ryan, Sabah N. A. Hussain, Sarah B. Mueller, Christopher D. Kontos, Cameron A. Schmidt, Joseph M. McClung, Thomas D. Green, Jessica L. Reinardy, and Timothy J. McCord
- Subjects
Tube formation ,Myoblast proliferation ,paracrine ,lcsh:QP1-981 ,progenitor cell ,Physiology ,Angiogenesis ,Myogenesis ,vascular disease ,Biology ,MyoD ,lcsh:Physiology ,Cell biology ,Ischemia ,Physiology (medical) ,Immunology ,Myosin ,cardiovascular system ,Regeneration ,Muscle ,Myocyte ,Myogenin ,Original Research - Abstract
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.
- Published
- 2015
35. Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway
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Jessica L. Reinardy, Daniel M. Corey, Christelle Golzio, Nicholas Katsanis, Christopher D. Kontos, and Sarah B. Mueller
- Subjects
rac1 GTP-Binding Protein ,Angiogenesis ,lcsh:Medicine ,Neovascularization, Physiologic ,Receptor tyrosine kinase ,chemistry.chemical_compound ,PAK1 ,Protein Interaction Mapping ,Angiopoietin-1 ,Human Umbilical Vein Endothelial Cells ,Morphogenesis ,Animals ,Humans ,Protein phosphorylation ,Phosphorylation ,lcsh:Science ,Protein kinase B ,Zebrafish ,Multidisciplinary ,biology ,lcsh:R ,Tyrosine phosphorylation ,Receptor, TIE-1 ,Zebrafish Proteins ,Threonine Phosphorylation Site ,Cell biology ,Protein Structure, Tertiary ,Enzyme Activation ,Drug Combinations ,Phosphothreonine ,chemistry ,Biochemistry ,p21-Activated Kinases ,biology.protein ,Mutagenesis, Site-Directed ,Blood Vessels ,lcsh:Q ,Proteoglycans ,Collagen ,Endothelium, Vascular ,Laminin ,Protein Processing, Post-Translational ,Proto-Oncogene Proteins c-akt ,Signal Transduction ,Research Article - Abstract
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.
- Published
- 2015
36. Angiopoietin‐1 Enhances Skeletal Muscle Regeneration in Mice
- Author
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Sabah N. A. Hussain, Christopher D. Kontos, Joseph M. McClung, Amy E. Pickett, Gawiyou Danialou, Bassman Tappuni, Laurent Huck, Sharon Harel, Mahroo Mofarrahi, Elaine C. Davis, and Nicolay Moroz
- Subjects
Male ,Time Factors ,Physiology ,Angiogenesis ,MyoD ,Muscle Development ,Biochemistry ,Myoblasts ,0302 clinical medicine ,Cell Movement ,Myocyte ,Cells, Cultured ,0303 health sciences ,Myogenesis ,Chemistry ,Cell Differentiation ,Cell biology ,Endothelial stem cell ,medicine.anatomical_structure ,Angiopoietin-1 ,030220 oncology & carcinogenesis ,cardiovascular system ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction ,Biotechnology ,Adult ,medicine.medical_specialty ,Cell Survival ,Genetic Vectors ,Biology ,Cardiotoxins ,Adenoviridae ,Angiopoietin-2 ,Necrosis ,03 medical and health sciences ,Cardiotoxin ,Muscular Diseases ,Physiology (medical) ,Internal medicine ,Genetics ,medicine ,Animals ,Humans ,Regeneration ,RNA, Messenger ,Progenitor cell ,Muscle, Skeletal ,Molecular Biology ,Myogenin ,030304 developmental biology ,Translational Physiology ,Regeneration (biology) ,Skeletal muscle ,Genetic Therapy ,Mice, Inbred C57BL ,Disease Models, Animal ,Endocrinology ,Gene Expression Regulation ,Myogenic regulatory factors - Abstract
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.
- Published
- 2015
37. Contributors
- Author
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Mathew G. Angelos, Jacopo Baglieri, Carmen Bertoni, Laura Breda, Hildegard Büning, Lawrence Chan, Wenhao Chen, Laurence J.N. Cooper, Alisa Dong, Christopher H. Evans, Charles A. Gersbach, Steven C. Ghivizzani, Saar Gill, Joseph C. Glorioso, William F. Goins, Perry B. Hackett, H. Kirk Hammond, Manu Jain, Michael Kalos, Dan S. Kaufman, Fahad Kidwai, Christopher D. Kontos, Robert A. Kratzke, Robert E. MacLaren, Michelle E. McClements, Federico Mingozzi, Sarah B. Mueller, Jianfang Ning, Manish R. Patel, Michelle Prickett, Samuel D. Rabkin, Stefano Rivella, Paul D. Robbins, Michele Simonato, Timothy K. Starr, Tong Tang, Pratiksha I. Thakore, Jakub Tolar, Lars U. Wahlberg, Christopher E. Walsh, Jie Wu, Aini Xie, and Yisheng Yang
- Published
- 2015
38. Gene Therapy for the Prevention of Vein Graft Disease
- Author
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Christopher D. Kontos and Sarah B. Mueller
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medicine.medical_specialty ,Intimal hyperplasia ,business.industry ,Genetic enhancement ,Disease ,Gene delivery ,medicine.disease ,Surgery ,Coronary artery disease ,surgical procedures, operative ,medicine.anatomical_structure ,Bypass surgery ,cardiovascular system ,Medicine ,business ,Vein graft disease ,Vein - Abstract
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.
- Published
- 2015
39. A Comparison of Antiangiogenic Therapies for the Prevention of Liver Metastases
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Yiting Cao, Shiva Sarraf-Yazdi, Mark W. Dewhirst, Jing Mi, Christopher D. Kontos, Chuan-Yuan Li, Bryan M. Clary, and Xiuwu Zhang
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Pathology ,medicine.medical_specialty ,Angiogenesis ,Angiogenesis Inhibitors ,Transfection ,Umbilical vein ,Viral vector ,Mice ,chemistry.chemical_compound ,In vivo ,Tumor Cells, Cultured ,Animals ,Medicine ,Angiostatins ,Cell Proliferation ,Mice, Inbred BALB C ,Angiostatin ,Neovascularization, Pathologic ,business.industry ,Liver Neoplasms ,Endothelial Cells ,Neoplasms, Experimental ,Receptor, TIE-2 ,Vascular Endothelial Growth Factor Receptor-2 ,Endostatins ,Vascular endothelial growth factor ,chemistry ,Colonic Neoplasms ,Cancer research ,Female ,Surgery ,Endostatin ,business - Abstract
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.
- Published
- 2006
40. Cholesterol Feeding Reduces Vascular Endothelial Growth Factor Signaling in Rabbit Corporal Tissues
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Brian H. Annex, Craig F. Donatucci, Christopher D. Kontos, and Donghua Xie
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Male ,Vascular Endothelial Growth Factor A ,medicine.medical_specialty ,Endothelium ,Angiogenesis ,Urology ,Endocrinology, Diabetes and Metabolism ,Blotting, Western ,Hypercholesterolemia ,Enzyme-Linked Immunosorbent Assay ,chemistry.chemical_compound ,Endocrinology ,Erectile Dysfunction ,Enos ,Internal medicine ,medicine ,Animals ,RNA, Messenger ,Phosphorylation ,Neurons ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,Muscle, Smooth ,biology.organism_classification ,Vascular endothelial growth factor ,Vascular endothelial growth factor B ,Nitric oxide synthase ,Psychiatry and Mental health ,Vascular endothelial growth factor A ,Cholesterol ,medicine.anatomical_structure ,Reproductive Medicine ,Vascular endothelial growth factor C ,biology.protein ,Rabbits ,Nitric Oxide Synthase ,Energy Intake ,Proto-Oncogene Proteins c-akt ,Penis ,Signal Transduction - Abstract
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).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.Cholesterol feeding was associated with a significant decrease in p-Akt/Akt 2.16-fold (P0.05), 3.28-fold (P0.02), and 3.42-fold (P0.02) 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.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.
- Published
- 2005
41. A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension
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Shoukang Zhu, Christopher D. Kontos, Don C. Rockey, Richard T. Premont, and Songling Liu
- Subjects
Male ,medicine.medical_specialty ,G-Protein-Coupled Receptor Kinase 2 ,Nitric Oxide Synthase Type III ,Portal venous pressure ,Protein Serine-Threonine Kinases ,Nitric Oxide ,General Biochemistry, Genetics and Molecular Biology ,Nitric oxide ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Enos ,Proto-Oncogene Proteins ,Internal medicine ,Hypertension, Portal ,medicine ,Animals ,Protein kinase B ,Cells, Cultured ,biology ,business.industry ,Endothelial Cells ,General Medicine ,medicine.disease ,biology.organism_classification ,Cyclic AMP-Dependent Protein Kinases ,Rats ,Isoenzymes ,Nitric oxide synthase ,Endothelial stem cell ,Endocrinology ,chemistry ,beta-Adrenergic Receptor Kinases ,biology.protein ,Portal hypertension ,Nitric Oxide Synthase ,business ,Proto-Oncogene Proteins c-akt - Abstract
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.
- Published
- 2005
42. Systemic soluble Tie2 expression inhibits and regresses corneal neovascularization
- Author
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Saadia Rashid, Eric Higgins, E. Macnamara, Balamurali K. Ambati, Christopher D. Kontos, Nirbhai Singh, and Jayakrishna Ambati
- Subjects
Vascular Endothelial Growth Factor A ,genetic structures ,Angiogenesis ,Biophysics ,In Vitro Techniques ,Transfection ,medicine.disease_cause ,Biochemistry ,Adenoviridae ,Neovascularization ,Andrology ,Angiopoietin ,Mice ,Cornea ,Animals ,Medicine ,Corneal Neovascularization ,Molecular Biology ,Mice, Inbred BALB C ,business.industry ,Men ,Cell Biology ,Anatomy ,medicine.disease ,Receptor, TIE-2 ,eye diseases ,Vascular endothelial growth factor A ,medicine.anatomical_structure ,Solubility ,Corneal neovascularization ,sense organs ,medicine.symptom ,business - Abstract
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.
- Published
- 2005
43. Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts
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Jason A. Petrofski, Christopher D. Kontos, Jonathan A. Hata, Sarah H. Timberlake, Thomas R. Gehrig, Michael T. Corwin, Carmelo A. Milano, Anne M. Pippen, Amy Solan, Andre Jakoi, Matthew L. Williams, and Jacob N. Schroder
- Subjects
Pulmonary and Respiratory Medicine ,medicine.medical_specialty ,Pathology ,Vascular smooth muscle ,Intimal hyperplasia ,Muscle, Smooth, Vascular ,Phosphatidylinositol 3-Kinases ,Dogs ,medicine ,PTEN ,Tensin ,Animals ,Saphenous Vein ,Coronary Artery Bypass ,Vein ,Hyperplasia ,biology ,business.industry ,Graft Occlusion, Vascular ,medicine.disease ,Surgery ,medicine.anatomical_structure ,biology.protein ,Phosphatidylinositol 3-kinase signaling ,business ,Tunica Intima ,Cardiology and Cardiovascular Medicine ,Cell Division ,Artery ,Signal Transduction - Abstract
ObjectivesFifty 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.MethodsAdult 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 (β-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.ResultsSaphenous 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 mm2, 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.ConclusionThis 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.
- Published
- 2005
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44. Engineered Zinc Finger–Activating Vascular Endothelial Growth Factor Transcription Factor Plasmid DNA Induces Therapeutic Angiogenesis in Rabbits With Hindlimb Ischemia
- Author
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Edward J. Rebar, Pascal J. Goldschmidt-Clermont, Chunming Dong, S. Kaye Spratt, Bruce Klitzman, Christopher D. Kontos, Casey C. Case, Brian H. Johnstone, Qunsheng Dai, Keith L. March, Jianhua Huang, Brian H. Annex, and Joseph Rokovich
- Subjects
Vascular Endothelial Growth Factor A ,Endothelium ,Angiogenesis ,medicine.medical_treatment ,Genetic Vectors ,DNA, Recombinant ,Ischemia ,Cytomegalovirus ,Neovascularization, Physiologic ,Apoptosis ,Simian virus 40 ,Protein Engineering ,Injections, Intramuscular ,Andrology ,chemistry.chemical_compound ,Physiology (medical) ,Genes, Synthetic ,medicine ,Animals ,Protein Isoforms ,RNA, Messenger ,Therapeutic angiogenesis ,Antigens, Viral, Tumor ,Promoter Regions, Genetic ,Binding Sites ,business.industry ,Growth factor ,NF-kappa B ,Transcription Factor RelA ,Zinc Fingers ,DNA ,Genetic Therapy ,medicine.disease ,Capillaries ,Hindlimb ,Femoral Artery ,Vascular endothelial growth factor ,Vascular endothelial growth factor A ,medicine.anatomical_structure ,Gene Expression Regulation ,chemistry ,Immunology ,Female ,Rabbits ,Cardiology and Cardiovascular Medicine ,business ,Blood vessel - Abstract
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 μg ZFP-VEGF plasmid) or no ZFP treatment (β-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 VEGF 121 , VEGF 165 , VEGF 189 , 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 P P Conclusions— This engineered ZFP-VEGF–activating transcription factor may provide a novel approach to treat peripheral arterial disease.
- Published
- 2004
45. Endothelin-1 Activates Endothelial Cell Nitric-oxide Synthase via Heterotrimeric G-protein βγ Subunit Signaling to Protein Kinase B/Akt
- Author
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Christopher D. Kontos, Richard T. Premont, Jianhua Huang, Don C. Rockey, and Songling Liu
- Subjects
Male ,Time Factors ,Protein Serine-Threonine Kinases ,Nitric Oxide ,Models, Biological ,Biochemistry ,Culture Media, Serum-Free ,Rats, Sprague-Dawley ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,GTP-Binding Protein gamma Subunits ,Proto-Oncogene Proteins ,Heterotrimeric G protein ,Animals ,Phosphorylation ,Molecular Biology ,Protein kinase B ,Endothelin-1 ,Receptors, Endothelin ,Chemistry ,Akt/PKB signaling pathway ,GTP-Binding Protein beta Subunits ,Endothelial Cells ,Tyrosine phosphorylation ,Cell Biology ,Molecular biology ,Protein Structure, Tertiary ,Rats ,Cell biology ,Enzyme Activation ,Endothelial stem cell ,Vascular endothelial growth factor A ,cardiovascular system ,Tyrosine ,Nitric Oxide Synthase ,Signal transduction ,Proto-Oncogene Proteins c-akt ,Protein Binding ,Signal Transduction - Abstract
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.
- Published
- 2003
46. Activation of Vascular Endothelial Growth Factor Receptor-1 Sustains Angiogenesis and Bcl-2 Expression Via the Phosphatidylinositol 3-Kinase Pathway in Endothelial Cells
- Author
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Asif Ahmed, Shakil Ahmad, Wen Guo Jiang, Christopher D. Kontos, Jianhua Huang, Jun Cai, and Michael E. Boulton
- Subjects
Vascular Endothelial Growth Factor A ,medicine.medical_specialty ,Platelet-derived growth factor ,Cell Survival ,Angiogenesis ,Endocrinology, Diabetes and Metabolism ,Apoptosis ,Pregnancy Proteins ,Biology ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,Internal medicine ,Internal Medicine ,medicine ,Animals ,Humans ,Cells, Cultured ,PI3K/AKT/mTOR pathway ,Placenta Growth Factor ,Vascular Endothelial Growth Factor Receptor-1 ,Neovascularization, Pathologic ,Microcirculation ,Retinal Vessels ,Cell biology ,Enzyme Activation ,Endothelial stem cell ,Vascular endothelial growth factor ,Vascular endothelial growth factor B ,Vascular endothelial growth factor A ,Endocrinology ,Proto-Oncogene Proteins c-bcl-2 ,chemistry ,Vascular endothelial growth factor C ,Cattle ,Endothelium, Vascular - Abstract
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.
- Published
- 2003
47. Deletion of the Carboxyl Terminus of Tie2 Enhances Kinase Activity, Signaling, and Function
- Author
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Christopher D. Kontos, Kevin G. Peters, and Xi-Lin Niu
- Subjects
Kinase ,C-terminus ,Wild type ,Tyrosine phosphorylation ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,Receptor tyrosine kinase ,chemistry.chemical_compound ,surgical procedures, operative ,chemistry ,embryonic structures ,cardiovascular system ,biology.protein ,sense organs ,Kinase activity ,Receptor ,tissues ,Molecular Biology ,Protein kinase B - Abstract
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 (ΔCT). Compared to wild type Tie2, in vitroautophosphorylation and kinase activity were significantly enhanced by the ΔCT 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-ΔCT. Moreover, the ΔCT 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.
- Published
- 2002
48. PTEN Modulates Vascular Endothelial Growth Factor-Mediated Signaling and Angiogenic Effects
- Author
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Christopher D. Kontos and Jianhua Huang
- Subjects
Male ,Vascular Endothelial Growth Factor A ,Angiogenesis ,Neovascularization, Physiologic ,Apoptosis ,Endothelial Growth Factors ,In Vitro Techniques ,Biochemistry ,Rats, Sprague-Dawley ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,Cell Movement ,Animals ,Humans ,PTEN ,Phosphatidylinositol ,Molecular Biology ,Cells, Cultured ,Tube formation ,Lymphokines ,biology ,Vascular Endothelial Growth Factors ,Tumor Suppressor Proteins ,PTEN Phosphohydrolase ,Cell Biology ,Phosphoric Monoester Hydrolases ,Rats ,Cell biology ,Vascular endothelial growth factor B ,Endothelial stem cell ,Vascular endothelial growth factor ,chemistry ,Cancer research ,biology.protein ,Tumor necrosis factor alpha ,Endothelium, Vascular ,Signal Transduction - Abstract
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.
- Published
- 2002
49. Abstract 14: Caskin2 is a Novel Regulator of Endothelial Cell Quiescence
- Author
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Sarah B Mueller, Shubhangi Arora, Natalie Mattocks, Susan B Gurley, and Christopher D Kontos
- Subjects
Cardiology and Cardiovascular Medicine - Abstract
Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. Morpholino knockdown of Caskin2 in zebrafish results in abnormal vascular development characterized by overly branched intersegmental vessels and failure to form the dorsal longitudinal vessels. Interestingly, Caskin2 knockout mice are viable and fertile. However, compared to wild-type mice, adult Caskin2 knockout mice have significantly more abdominal adipose and higher fasting blood glucose levels even when fed a standard chow diet. In vitro, Caskin2 overexpression inhibits serum-induced EC proliferation and DNA synthesis but promotes EC survival during serum starvation. Caskin2 localizes to the nucleus as well as the cytoplasm and promotes the downregulation of genes associated with EC activation (e.g., IL-8, VEGFR2, ANG2) and the upregulation of genes associated with EC quiescence (e.g., Notch1, KLF2/4, ANG1). More broadly, pathway analysis of microarray data demonstrates that Caskin2 primarily regulates cell cycle and metabolic genes. Additional data suggest that these effects result from the interaction of Caskin2 with the Ser/Thr phosphatase PP1alpha, which is mediated by a consensus PP1 binding motif at the Caskin2 N-terminus. Taken together, our data demonstrate that Caskin2 is sufficient to suppress EC proliferation in vitro and necessary to prevent dysregulated angiogenesis in at least one in vivo model. These findings indicate that Caskin2 is a novel regulator of EC quiescence and suggest a role for Caskin2 in the prevention of EC dysfunction in vivo. Understanding Caskin2’s function on the molecular level may lead to the development of novel pharmacological approaches to prevent inappropriate angiogenesis, normalize dysfunctional vessels, and improve vascular function in a variety of cardiovascular diseases.
- Published
- 2014
50. HCPTPA, a Protein Tyrosine Phosphatase That Regulates Vascular Endothelial Growth Factor Receptor-mediated Signal Transduction and Biological Activity
- Author
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Sabita Sankar, Liwen Huang, Shu-Mang Feng, Eugene H. Cha, Michael A. Blanar, Christopher D. Kontos, Zhiming Yu, Kevin G. Peters, Charles Lin, Robert L. Van Etten, Alfred D. Schroff, and Su-Feng Li
- Subjects
Vascular Endothelial Growth Factor A ,Phosphotyrosine binding ,Angiogenesis ,Neovascularization, Physiologic ,Endothelial Growth Factors ,Protein tyrosine phosphatase ,Biochemistry ,Receptor tyrosine kinase ,Substrate Specificity ,Two-Hybrid System Techniques ,Animals ,Humans ,Receptors, Growth Factor ,Phosphorylation ,Molecular Biology ,Aorta ,Cells, Cultured ,Lymphokines ,biology ,Vascular Endothelial Growth Factors ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Molecular biology ,Recombinant Proteins ,Rats ,Cell biology ,Vascular endothelial growth factor B ,Vascular endothelial growth factor A ,Receptors, Vascular Endothelial Growth Factor ,cardiovascular system ,biology.protein ,Protein Tyrosine Phosphatases ,Signal transduction ,Platelet-derived growth factor receptor ,Signal Transduction - Abstract
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.
- Published
- 1999
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