Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells (original) (raw)
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Hypoxia induces myocyte-dependent COX-2 regulation in endothelial cells: role of VEGF
American Journal of …, 2003
There is increasing evidence that cyclooxygenase (COX)-2 possess both angiogenic and cardioprotective properties. We examined the effects of hypoxic cardiac myocytes (H9c2 cells) on COX-2 expression in human umbilical vein endothelial cells (HUVECs) to determine the pathway involved in COX-2 regulation. The medium from hypoxic (Ͻ1% O 2) cardiac myocytes (HMCM) or normoxic cardiac myocytes (21% O 2) was added to HUVEC cultures. HMCM induced a transient increase of COX-2 mRNA expression at 1 and 3 h without affecting the COX-1 mRNA level. A similar effect also observed in HMCM from cultured primary cardiac myocytes (rat neonatal cardiac myocytes). The increased COX-2 mRNA was associated with a time-dependent increase in COX-2 protein expression. COX-2 was significantly induced by VEGF (4.86 Ϯ 1.03-fold) and IL-1 (3.93 Ϯ 0.89fold) and slightly increased by TNF-␣ but not by FGF2, IGF-1, or PDGFs. Analysis of proteins secreted in HMCM showed increased levels of VEGF but not IL-1 or TNF-␣. The HMCM-induced COX-2 expression was inhibited by the addition of an anti-VEGF neutralizing antibody. VEGF induced endothelial cell COX-2 expression by both increasing COX-2 transcription and prolonging the COX-2 mRNA halflife. Furthermore, staurosporine, a nonselective PKC inhibitor, prevented the induction of VEGF by hypoxia. Both a selective PKC-␣ and- inhibitor and an inducible nitric oxide synthase (NOS) inhibitor decreased the induction of COX-2 by HMCM and VEGF. Finally, HMCM-induced upregulation of COX-2 was accompanied by upregulation of PGI 2 and PGE2. These results suggest that VEGF is one of the principal factors produced by hypoxic myocytes that is responsible for the induction of endothelial cell COX-2 expression. This process likely involves both PKC and NOS pathways. Our findings have important implications regarding the cardiac protection of COX-2 in ischemic heart disease. cyclooxygenase-2; vascular endothelial growth factor; cardiac myocytes CYCLOOXYGENASE (COX) is a key regulatory enzyme in eicosanoid metabolism, converting free arachidonic
Toxicology, 2000
Introduction: Oxidative stress, as exerted by free radicals within biological systems, is known to exert numerous physiological and pathological effects on the cardiovascular system. Short-term exposure to environmental conditions such as low oxygen tension can cause such oxidative stress in vivo through inhalational hypoxia/reoxygenation. In this report the effects of different durations of hypoxia were investigated on myocardial protein expression of vascular endothelial growth factor (VEGF), a major angiogenic growth factor, and also explore the possible modulatory role of transcription factor NFkB on such expression. Methods: Forty eight male Sprague -Dawley rats (300 g b.w.) were randomly divided into four groups and subjected to either 1, 2 or 4 h of systemic normobaric hypoxemic hypoxia (10 90.4% O 2 ) in an anesthesia chamber, or to 4 h of normoxia (ambient 20.9 9 0.4% O 2 ) to time-match the maximal hypoxic duration. All rats were then kept under normoxic conditions. Rats were sacrificed and hearts harvested either after 2 h for later electrophoretic mobility gel shift assay for NFkB, or after 24 h for later Western blot analysis for VEGF. Results: Western blot analysis for VEGF revealed significantly elevated protein expression (2.4-fold compared to baseline control) in the 1 h group. This elevated level persisted in the 2 and 4 h groups as well. Two hours post-hypoxia gel shift assay for NFkB indicated significant nuclear translocation and DNA binding of this transcription factor in the 1 and 2 h groups, with moderate decrease in the 4 h group. Conclusion: In vivo oxidative stress caused by systemic inhalational hypoxemic hypoxia increases cardiac VEGF protein expression and may trigger myocardial angiogenesis. The results suggest that NFkB modulates such an effect.
Molecular and cellular biochemistry, 2000
Tissue hypoxia has been identified as being a particularly important stimulus for triggering angiogenesis. Here we report early effects of hypoxia/reoxygenation (H/R) on the protein expression profiles and localization patterns of the VEGF and Angiopoietin-Tie systems in adult rat myocardium. Western blot as well as immunohistochemical analyses were performed on hearts obtained from rats exposed to various durations of in vivo systemic hypoxemic hypoxia followed by 24 h reoxygenation. The relative time course of protein expression in response to increasing durations of hypoxia, as indicated from our experiments, seems to suggest the involvement of the VEGF system and the Ang-Tie system in early angiogenesis. An apparent relationship between the expression profiles of Flk-1 and Ang-2 was observed. The most significant and interesting relationship which came to light was the surprisingly coincident yet opposite temporal trends between Ang-1 and Ang-2 protein levels. In the 1 h hypoxia...
Hypoxia Increases Nitric Oxide-Dependent Inhibition of Angiogenic Growth
International Journal of Translational Medicine, 2021
Nitric oxide (NO) is a proangiogenic factor acting through the soluble guanylate cyclase (sGC) pathway. However, angiogenic growth increases energy demand, which may be hampered by NO inhibition of cytochrome c oxidase (CcO). Then, NO activity would be the balanced result of sGC activation (pro-angiogenic) and CcO inhibition (anti-angiogenic). NO activity in a rat and eNOS−/− mice aortic ring angiogenic model and in a tube formation assay (human aortic endothelial cells) were analyzed in parallel with mitochondrial O2 consumption. Studies were performed with NO donor (DETA-NO), sGC inhibitor (ODQ), and NOS or nNOS inhibitors (L-NAME or SMTC, respectively). Experiments were performed under different O2 concentrations (0–21%). Key findings were: (i) eNOS-derived NO inhibits angiogenic growth by a mechanism independent on sGC pathway and related to inhibition of mitochondrial O2 consumption; (ii) NO inhibition of the angiogenic growth is more evident in hypoxic vessels; (iii) in the ab...
Differential activation of vascular genes by hypoxia in primary endothelial cells
Experimental Cell Research, 2004
Changes in the local environment, such as reduced oxygen tension (hypoxia), elicit transcriptional activation of a variety of genes in mammalian cells. Here we have analyzed the effect of hypoxia in different vascular endothelial cells (ECs) with emphasis on hypoxiaregulated transcription factors and genes of importance for blood vessel dynamics. While hypoxia induced the transcription factor hypoxiainducible factor-1a (HIF-1a) in all endothelial cells tested, the closely related HIF-2a protein was markedly induced in microvascular/ capillary endothelial cells, but only weakly or not at all in artery and vein endothelial cells. Furthermore, microvascular/capillary endothelial cells responded to hypoxia with increased number of transcripts encoding vascular endothelial growth factor-A (VEGF-A), VEGF receptor-2, the angiopoietin receptor Tie2, platelet-derived growth factor-B (PDGF-B), and inducible nitric oxide synthase (iNOS). In vein endothelial cells, hypoxia instead increased transcripts encoding lymphatic vascular components VEGF-C,-D, and VEGF receptor-3. Finally, reduced VEGF receptor levels and phosphorylation indicated establishment of a functional autocrine VEGF-A loop in hypoxic endothelial cells. Our results show that endothelial cells, derived from different vascular beds, mount different transcriptional responses to changes in oxygen tension.
Cancer research, 1996
Hypoxia can select for cells that have lost their apoptotic potential, thereby making them resistant to adverse conditions. However, long-term survival of transformed cells which have diminished apoptotic sensitivity when exposed to low oxygen conditions would require the activation of their angiogenic program to compensate for an insufficient oxygen supply. In this report, we show that the activity (of oncogenic Ha-ras, either constitutively or transiently, enhances the induction of the angiogenic mitogen, vascular endothelial growth factor (VEGF), by hypoxia. Analysis of the 5' flanking region of the VEGF promoter indicates that a HIF-1-like sequence is to promote a 15-fold increase in reporter gene activity in Ha-ras-transformed cells when exposed to hypoxia, whereas mutations in the same site totally inhibited VEGF induction. Under low oxygen conditions, VEGF induction is inhibited in cells expressing a mutant inhibitory allele of Ha-ras (RasN17), indicating a direct role fo...
FEBS Letters, 1995
Vessel growth is often associated with ischemia. VEGF, a potent angiogenic factor, has been shown to be induced by low oxygen concentrations. These studies were conducted to investigate the molecular basis of the hypoxia-induced increase in VEGF mRNA. Run-on analysis of VEGF revealed a minimal increase in the rate of gene transcription in a human retinal epithelial cell line grown under hypoxic conditions. Examination of VEGF mRNA stability revealed that the half-life of VEGF transcripts under normoxia was short, 30-45 min, but was dramatically increased to 6-8 h in cells grown under hypoxia. Cobalt chloride, which elevates VEGF and has been suggested to be similar to hypoxia in its mechanism of action, had only a slight effect on decay rate. We postulate that hypoxia-induced increases in mRNA stability provide the sustained increases in VEGF mRNA levels necessary to support a neovascular response.
Interindividual Heterogeneity in the Hypoxic Regulation of VEGF
Circulation, 1999
Background —The coronary artery collateral circulation may be beneficial in protecting against myocardial ischemia and necrosis. However, there is a tremendous interindividual variability in the degree of new collateral formation in patients with coronary artery disease. The basis for this interindividual heterogeneity is not understood. In this study we test the hypothesis that failure to generate collateral vessels is associated with a failure to appropriately induce with hypoxia or ischemia the angiogenic factor, vascular endothelial growth factor (VEGF). Methods and Results —We correlated the VEGF response to hypoxia in the monocytes harvested from patients with coronary artery disease with the presence of collaterals visualized during routine angiography. We found that there was a highly significant difference in the hypoxic induction of VEGF in patients with no collaterals compared with patients with some collaterals (mean fold induction 1.9±0.2 versus 3.2±0.3 , P <0.0001)....
Journal of Molecular and Cellular Cardiology, 2001
H. S, P. S. R, L. Z, H. O, T. A N. M. Hypoxia/Reoxygenation Promotes Myocardial Angiogenesis via an NF B-dependent Mechanism in a Rat Model of Chronic Myocardial Infarction. Journal of Molecular and Cellular Cardiology (2001) 33, 283-294. Therapeutic angiogenesis achieved either through the use of discreet angiogenic proteins or by gene therapy is fast emerging as a highly attractive treatment modality for ischemic heart disease. Herein we examine a novel method of stimulating myocardial angiogenesis by hypoxic preconditioning at both capillary and arteriolar levels, and the potential role of NF B in mediating such a response. We also investigate the functional relevance of such treatment by assessing whether the induced neovascularization can help preserve left ventricular contractile functional reserve in the setting of developing heart failure secondary to myocardial infarction. Male Sprague-Dawley rats were randomly divided into eight groups: normoxia+sham surgery (NS), normoxia+permanent left anterior descending coronary artery (LAD) occlusion (NMI), hypoxic preconditioning+sham surgery (HS), hypoxic preconditioning+permanent LAD occlusion (HMI), PDTC (NF B inhibitor)+hypoxic preconditioning+LAD occlusion (PHMI), PDTC+normoxia+LAD occlusion (PNMI), PDTC+hypoxic preconditioning+sham surgery (PHS) and PDTC+normoxia+sham surgery (PNS). Rats in the preconditioned groups were subjected to systemic hypoxemic hypoxic exposure (10±0.4% O 2 ) for 4 h followed by a 24-h period of normoxic reoxygenation prior to undergoing LAD occlusion. Rats in the normoxia groups were time matched with the preconditioned group and maintained under normoxic conditions for the 28-h period prior to LAD occlusion. The HMI group displayed significant increases in capillary as well as arteriolar density after 2, 4 and 7 days post-operation compared to the NMI. Prior PDTC administration prevented such increases in the PHMI group and effectively abolished the pro-angiogenic effect of hypoxic preconditioning (HP). One week after sham surgery or LAD occlusion, rats underwent a pharmacological stress test with dobutamine in progressively increasing doses which revealed significantly elevated values of dp/dt max at each dose point in the HMI group compared to the NMI or PHMI groups. Hypoxic preconditioning also decreases endothelial cell injury as determined by the extent of endothelial cell apoptosis using anti-VWF factor labelling and TUNEL assay. The results suggest that HP stimulates myocardial angiogenesis via redox-regulated transcription factor, NF B-dependent pathway to an extent sufficient to exert significant preservation of contractile functional reserve in a rat model of myocardial infarction progressing to heart failure.