STAT5 and Prolactin Participate in a Positive Autocrine Feedback Loop That Promotes Angiogenesis

We have shown previously that the murine prolactin/growth hormone family member proliferin plays a pivotal role in angiogenesis induced by the FGF2/STAT5 signaling cascade. To delineate the signaling pathway downstream of STAT5 in the human system, where proliferin does not exist, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in hCMEC/D3 human brain endothelial cells. We found that conditioned medium from CA-STAT5A- but not from DN-STAT5A-overexpressing endothelial cells (EC) is sufficient to induce EC migration and tube formation but not proliferation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. We identified prolactin (PRL) as a candidate autocrine factor. CA-STAT5A expression stimulates PRL production at the RNA and protein level, and STAT5A binds to the PRL promoter region, suggesting direct transcriptional regulation. Medium conditioned by CA-STAT5A-overexpressing EC induces phosphorylation of the PRL receptor and activates MAPK. Knockdown of PRL expression by shRNA or blocking of PRL activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. The addition of recombinant PRL restores this activity. STAT5A-induced PRL in the conditioned medium can activate STAT5, STAT1, and to a lesser extent STAT3 in hCMEC/D3 cells, suggesting the existence of a positive feedback loop between STAT5 and PRL that promotes angiogenesis. Furthermore, we find that VEGF, a potent proangiogenic factor, is induced by activation of STAT5A, and VEGF induction depends on PRL expression. These observations demonstrate a STAT5/PRL/VEGF signaling cascade in human brain EC and implicate PRL and VEGF as autocrine regulators of EC migration, invasion, and tube formation.

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The Inhibitor of VEGF-Receptors Tyrosine Kinase PTK-787 Blocks the Invasive Potential of Adult T-Cell Leukemia Cells through the Endothelium.

Blood ◽

10.1182/blood.v104.11.84.84 ◽

2004 ◽

Vol 104 (11) ◽

pp. 84-84

Author(s):

Ali Bazarbachi ◽

Roa Harb ◽

Hilda Khoury ◽

Lara Haddad ◽

Youmna Kfoury ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Conditioned Medium ◽

Tube Formation ◽

Adult T Cell Leukemia ◽

Vegf Signaling ◽

Anti Vegf ◽

Endothelial Cell Retraction ◽

Cell Retraction

Abstract Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis. HTLV-I associated adult T-cell leukemia/lymphoma (ATL) is an aggressive disease characterized by multiple organ invasion. We have shown that ATL-derived cells induce angiogenesis and communicate with endothelial cells through gap junctions. This results in induction of endothelial-derived metalloproteinases, sub-endothelial basement membrane degradation followed by endothelial cell retraction allowing neoplastic lymphocytes extravasation. In this study we have used the specific tyrosine kinase inhibitor of VEGF receptors PTK787, functional blocking antibodies against VEGF and the gap junction communication inhibitor18-a-G to dissect the respective role of VEGF signaling pathway and cell-to-cell communication in endothelial cell retraction and tumor cell extravasation. PTK787 has mild growth inhibitory and cytotoxic effects on both endothelial and tumor cells at a concentration of 50μM and significantly inhibited cell proliferation at 100μM. Hence, 20μM PTK787 which did not exhibit any cytotoxic or antiproliferative effects on both cell types was used in experiments that last 24 hours or longer (in vitro tube formation assay and invasion assay). Endothelial cells monolayers incubated with either ATL derived cells or their cell-free conditioned medium for 48 hours induced in vitro tube formation in Matrigel angiogenesis assay. This was inhibited by both anti-VEGF antibody and 18-a-G and to a greater extent and in a dose dependent manner by PTK787. Importantly, PTK787, and to a lesser extent anti-VEGF antibody and 18-alpha-G significantly attenuated the ability of ATL-derived cells to traverse endothelial cell monolayers in double compartment invasion assays. Signaling cascade that is activated upon VEGF stimulation of endothelial cells was investigated in confluent endothelial cells cocultured with ATL derived cells or cell-free conditioned medium in the absence or presence of PTK787 or anti-VEGF antibody, using phospho-specific antibodies against FAK Tyr 861, FAK Tyr 397, Src, ERK, PLCgamma, and Akt/PKB, known to be signal mediators in the Ras-Raf-MEKK-ERK signaling cascade. The addition of ATL derived cells or cell-free conditioned medium resulted in a transient activation of the above signal mediators with a peak increase at 15 minutes and gradual return to baseline after 60 minutes. This activation was down regulated in the presence of anti-VEGF antibody and to a greater extent in the presence of PTK787. This data establishes a novel role of VEGF signaling in tumor cell invasion and supports the potential use of PTK787 in the treatment of ATL and other hematological malignancies with angiogenic and invasive properties.

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Adenosine triphosphate is a critical determinant for VEGFR signal during hypoxia

AJP Cell Physiology ◽

10.1152/ajpcell.00145.2016 ◽

2016 ◽

Vol 311 (6) ◽

pp. C985-C995 ◽

Cited By ~ 2

Author(s):

Abdullah Al Mamun ◽

Hisaki Hayashi ◽

Miho Sakima ◽

Motohiko Sato

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Signal Propagation ◽

Tube Formation ◽

Activation Process ◽

Vegf Receptor ◽

Vegf Mrna ◽

Critical Determinant ◽

Hypoxia Exposure ◽

Vegf Signaling

Hypoxia induces angiogenesis through the VEGF signaling pathway; however, signal propagation of VEGF in hypoxia is not fully understood. In this study, we examined alterations in VEGF signaling during hypoxia conditions and its determinant in endothelial cells. To analyze VEGF signaling during hypoxia, human umbilical vein endothelial cells (HUVECs) were exposed to 3 h of hypoxia (1% O2) followed by 3 h of reoxygenation or 12 h of hypoxia. Hypoxia induced expression of VEGF mRNA, but it was not associated with an increase in tube formation by HUVECs. During 3 h of hypoxia, VEGF-induced phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream molecules were significantly inhibited without a change in VEGFR-2 expression, but it was completely restored after reoxygenation. VEGF-mediated VEGFR-2 phosphorylation is associated with a reduction in cellular ATP in hypoxia conditions (65.93 ± 8.32% of normoxia, means ± SE, P < 0.01). Interestingly, attenuation of VEGFR-2 phosphorylation was restored by addition of ATP to prepared membranes from cells that underwent 3 h of hypoxia. In contrast to 3 h of hypoxia, exposure of cells to 12 h of hypoxia decreased VEGFR-2 expression and VEGF-mediated VEGFR-2 phosphorylation. The magnitude of VEGFR-2 phosphorylation was not fully restored by addition of ATP to prepared membranes from cells exposed to 12 h of hypoxia. These data indicate that ATP is an important determinant of VEGF signaling in hypoxia and suggest that the activation process of VEGFR-2 was modified by sustained hypoxia. These observations contribute to our understanding of signal alterations in VEGF in endothelial cells during hypoxia.

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Cardiac Extracellular Vesicles (EVs) Released in the Presence or Absence of Inflammatory Cues Support Angiogenesis in Different Manners

International Journal of Molecular Sciences ◽

10.3390/ijms20246363 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6363 ◽

Cited By ~ 1

Author(s):

Christien Madlen Beez ◽

Maria Schneider ◽

Marion Haag ◽

Kathleen Pappritz ◽

Sophie Van Linthout ◽

...

Keyword(s):

Endothelial Cells ◽

Conditioned Medium ◽

Extracellular Vesicles ◽

Quantitative Polymerase Chain Reaction ◽

Cell Interaction ◽

Tube Formation ◽

Vascular Endothelial ◽

Human Proteins ◽

Intracellular Detection ◽

Endothelial Growth Factor

Cells release extracellular vesicles (EVs) to communicate in a paracrine manner with other cells, and thereby influence processes, such as angiogenesis. The conditioned medium of human cardiac-derived adherent proliferating (CardAP) cells was recently shown to enhance angiogenesis. To elucidate whether their released EVs are involved, we isolated them by differential centrifugation from the conditioned medium derived either in the presence or absence of a pro-inflammatory cytokine cocktail. Murine recipient cells internalized CardAP-EVs as determined by an intracellular detection of human proteins, such as CD63, by a novel flow cytometry method for studying EV–cell interaction. Moreover, endothelial cells treated for 24 h with either unstimulated or cytokine stimulated CardAP-EVs exhibited a higher tube formation capability on Matrigel. Interestingly, unstimulated CardAP-EVs caused endothelial cells to release significantly more vascular endothelial growth factor and interleukin (IL)-6, while cytokine stimulated CardAP-EVs significantly enhanced the release of IL-6 and IL-8. By nCounter® miRNA expression assay (NanoString Technologies) we identified microRNA 302d-3p to be enhanced in unstimulated CardAP-EVs compared to their cytokine stimulated counterparts, which was verified by quantitative polymerase chain reaction. This study demonstrates that both CardAP-EVs are pro-angiogenic by inducing different factors from endothelial cells. This would allow to select potent targets for a safe and efficient therapeutic application.

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Roundabout4 Suppresses Glioma-Induced Endothelial Cell Proliferation, Migration and Tube Formation in Vitro by Inhibiting VEGR2-Mediated PI3K/AKT and FAK Signaling Pathways

Cellular Physiology and Biochemistry ◽

10.1159/000373982 ◽

2015 ◽

Vol 35 (5) ◽

pp. 1689-1705 ◽

Cited By ~ 27

Author(s):

Heng Cai ◽

Yixue Xue ◽

Zhen Li ◽

Yi Hu ◽

Zhenhua Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Signaling Pathways ◽

Conditioned Medium ◽

Tube Formation ◽

Endothelial Cell Proliferation ◽

Akt Inhibitor ◽

Over Expression

Background and Aims: Endothelial cell (EC) proliferation, migration, and tube formation are the critical steps for tumor angiogenesis, which is involved in the formation of new tumor blood vessels. Roundabout4 (Robo4), a new member of Robo proteins family, is specifically expressed in endothelial cells. This study aimed to investigate the effects of Robo4 on glioma-induced endothelial cell proliferation, migration and tube formation in vitro. Methods and Results: We found that Robo4 was endogenously expressed in Human Brain Microvascular Endothelial Cells (HBMECs), while Robo4 was significantly down-regulated in endothelial cells cultured in glioma conditioned medium. Robo4 over-expression remarkably suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro. In addition, Robo4 influenced the glioma-induced angiogenesis via binding to its ligand Slit2. Further studies demonstrated that the knockdown of Robo4 up-regulated the phosphorylation of VEGFR2, PI3K, AKT and FAK in EC cultured in glioma conditioned medium. VEGFR2 inhibitor SU-1498, AKT inhibitor LY294002 and FAK inhibitor 14 (FAK inhibitor) blocked the Robo4 knockdown-mediated alteration in glioma angiogenesis in vitro. Conclusion: Our results proved that Robo4 suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro by inhibiting VEGR2-mediated activation of PI3K/AKT and FAK signaling pathways.

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A Disintegrin and Metalloprotease 15 is Expressed on Rheumatoid Arthritis Synovial Tissue Endothelial Cells and may Mediate Angiogenesis

Cells ◽

10.3390/cells8010032 ◽

2019 ◽

Vol 8 (1) ◽

pp. 32 ◽

Cited By ~ 2

Author(s):

Shinichiro Nishimi ◽

Takeo Isozaki ◽

Kuninobu Wakabayashi ◽

Hiroko Takeuchi ◽

Tsuyoshi Kasama

Keyword(s):

Rheumatoid Arthritis ◽

Endothelial Cells ◽

Conditioned Medium ◽

Inflammatory Diseases ◽

Umbilical Vein ◽

Tube Formation ◽

Tnf Α ◽

Adhesion Index

A disintegrin and metalloprotease 15 (ADAM15) is involved in several malignancies. In this study, we investigated the role of ADAM15 in rheumatoid arthritis (RA) angiogenesis. Soluble ADAM15 (s-ADAM15) in serum from RA and normal (NL) subjects was measured using ELISA. To determine membrane-anchored ADAM15 (ADAM15) expression in RA synovial tissues, immunohistochemistry was performed. To examine the role of ADAM15 in angiogenesis, we performed in vitro Matrigel assays and monocyte adhesion assays using human umbilical vein endothelial cells (HUVECs) transfected with ADAM15 siRNA. Finally, to investigate whether angiogenic mediators were affected by ADAM15, cytokines in ADAM15 siRNA-transfected HUVEC-conditioned medium were measured. ADAM15 was significantly higher in RA serum than in NL serum. ADAM15 was also expressed on RAST endothelial cells. ADAM15 siRNA-treated HUVECs had decreased EC tube formation in response to RA synovial fluids compared with non-treated HUVECs. The adhesion index of ADAM15 siRNA-transfected HUVECs was significantly lower than the adhesion index of control siRNA-transfected HUVECs. ENA-78/CXCL5 and ICAM-1 were decreased in tumor necrosis factor (TNF)-α-stimulated ADAM15 siRNA-transfected HUVEC-conditioned medium compared with TNF-α-stimulated control siRNA-transfected HUVEC-conditioned medium. These data show that ADAM15 plays a role in RA angiogenesis, suggesting that ADAM15 might be a potential target in inflammatory diseases such as RA.

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Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

AJP Cell Physiology ◽

10.1152/ajpcell.00230.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. C1292-C1301 ◽

Cited By ~ 74

Author(s):

Anke C. Webler ◽

U. Ruth Michaelis ◽

Rüdiger Popp ◽

Eduardo Barbosa-Sicard ◽

Andiappan Murugan ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Protein Expression ◽

Second Messengers ◽

Tube Formation ◽

Dominant Negative ◽

Endothelial Cell Proliferation ◽

Angiogenic Response

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5( Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

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Prostate-Specific Membrane Antigen (PSMA) Promotes Angiogenesis of Glioblastoma Through Interacting With ITGB4 and Regulating NF-κB Signaling Pathway

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.598377 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yang Gao ◽

Hui Zheng ◽

Liangdong Li ◽

Mingtao Feng ◽

Xin Chen ◽

...

Keyword(s):

Endothelial Cells ◽

Conditioned Medium ◽

Poor Prognosis ◽

Umbilical Vein ◽

Tube Formation ◽

Prostate Specific Membrane Antigen ◽

Umbilical Vein Endothelial Cells ◽

Specific Membrane

BackgroundGlioblastoma multiforme (GBM) is the most common primary malignant tumor in the central nervous system (CNS), causing the extremely poor prognosis. Combining the role of angiogenesis in tumor progression and the role of prostate-specific membrane antigen (PSMA) in angiogenesis, this study aims to explore the functions of PSMA in GBM.MethodsClinical GBM specimens were collected from 60 patients who accepted surgical treatment in Fudan University Shanghai Cancer Center between January 2018 and June 2019. Immunohistochemical staining was used to detect PSMA and CD31 expression in GBM tissues. Prognostic significance of PSMA was evaluated by bioinformatics. Human umbilical vein endothelial cells (HUVECs) transfected with PSMA overexpression plasmids or cultured with conditioned medium collected based on GBM cells, were used for CCK8, Transwell and tube formation assays. High-throughput sequencing and immunoprecipitation were used to explore the underlying mechanism. Furthermore, the in vivo experiment had been also conducted.ResultsWe demonstrated that PSMA was abundantly expressed in endothelium of vessels of GBM tissues but not in vessels of normal tissues, which was significantly correlated with poor prognosis. Overexpression of PSMA could promotes proliferation, invasion and tube formation ability of human umbilical vein endothelial cells (HUVECs). Moreover, U87 or U251 conditioned medium could upregulated PSMA expression and induce similar effects on phenotypes of HUVECs, all of which could be partially attenuated by 2-PMPA treatment. The mechanistic study revealed that PSMA might promote angiogenesis of GBM through interacting with Integrin β4 (ITGB4) and activating NF-κB signaling pathway. The in vivo growth of GBM could be alleviated by the treatment of 2-PMPA.ConclusionThis study identified PSMA as a critical regulator in angiogenesis and progression of GBM, which might be a promising therapeutic target for GBM treatment.

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MicroRNA-183 inhibition exerts suppressive effects on diabetic retinopathy by inactivating BTG1-mediated PI3K/Akt/VEGF signaling pathway

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00444.2018 ◽

2019 ◽

Vol 316 (6) ◽

pp. E1050-E1060 ◽

Cited By ~ 4

Author(s):

Zhen-Zhen Zhang ◽

Xiu-Hong Qin ◽

Jing Zhang

Keyword(s):

Endothelial Cells ◽

Diabetic Retinopathy ◽

Cell Growth ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Vascular Endothelial Cells ◽

Tube Formation ◽

Vascular Endothelial ◽

Vegf Signaling ◽

Vegf Signaling Pathway

Diabetic retinopathy (DR) is a serious diabetic complication caused by both environmental and genetic factors. Molecular mechanisms of DR may lead to the discovery of reliable prognostic indicators. The current study aimed to clarify the mechanism of microRNA-183 (miR-183) in DR in relation to the PI3K/Akt/VEGF signaling pathway. Microarray-based gene expression profiling of DR was used to identify the differentially expressed genes. Sprague-Dawley rats were used for the establishment of DR models, and then miR-183 was altered by mimic or inhibitor or BTG1 was downregulated by siRNA to explore the regulatory mechanism of miR-183 in DR. Furthermore, the expression of miR-183, CD34, endothelial nitric oxide synthase (eNOS), BTG1 and the PI3K/Akt/VEGF signaling pathway-related genes as well as reactive oxygen species (ROS) level was determined, and the relationship between miR-183 and BTG1 was also verified. Cell growth, cell apoptosis, and angiogenesis were determined. Microarray analysis revealed the involvement of miR-183 in DR via the PI3K/Akt/VEGF signaling pathway by targeting BTG1. Upregulated miR-183 and downregulated BTG1 were observed in retinal tissues of DR rats. miR-183 overexpression activated the PI3K/Akt/VEGF signaling pathway, upregulated CD34, eNOS, and ROS, and inhibited BTG1. BTG1 was confirmed as a target gene of miR-183. miR-183 overexpression or BTG1 knockdown promoted cell growth and tube formation while it suppressed cell apoptosis of vascular endothelial cells in DR rats. In this study, we demonstrated that miR-183 silencing inhibiting cell growth and tube formation in vascular endothelial cells of DR rats via the PI3K/Akt/VEGF signaling pathway by upregulating BTG1.

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Celastrol inhibits the proliferation and angiogenesis of high glucose-induced human retinal endothelial cells

BioMedical Engineering OnLine ◽

10.1186/s12938-021-00904-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Jian Fang ◽

Xiaoke Chang

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

High Glucose ◽

Microvascular Complications ◽

Tube Formation ◽

Immunofluorescence Assay ◽

Retinal Endothelial Cells ◽

Vegf Signaling ◽

Qpcr Analysis ◽

Vegf Signaling Pathway

Abstract Background Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. Celastrol plays a certain role in the improvement of various diabetes complications. Therefore, this study aimed to explore whether celastrol inhibited the proliferation and angiogenesis of high glucose (HG)-induced human retinal endothelial cells (hRECs) by down-regulating the HIF1/VEGF signaling pathway. Methods The viability and proliferation of hRECs treated with glucose, celastrol or dimethyloxallyl glycine (DMOG) were analyzed by MTT assay. The invasion and tube formation ability of hRECs treated with glucose, celastrol or DMOG were in turn detected by transwell assay and tube formation assay. The expression of HIF1α and VEGF in hRECs after indicated treatment was analyzed by Western blot analysis and RT-qPCR analysis and ICAM-1 expression in hRECs after indicated treatment was detected by immunofluorescence assay Results HG induction promoted the proliferation, invasion and tube formation ability and increased the expression of HIF-1α and VEGF of hRECs, which were gradually suppressed by celastrol changing from 0.5 to 2.0 μM. DMOG was regarded as a HIF1α agonist, which attenuated the effect of celastrol on HG-induced hRECs. Conclusion Celastrol inhibited the proliferation and angiogenesis of HG-induced hRECs by down-regulating the HIF1α/VEGF signaling pathway.

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Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00533.2008 ◽

2009 ◽

Vol 296 (5) ◽

pp. C1162-C1170 ◽

Cited By ~ 56

Author(s):

Barbara Olszewska-Pazdrak ◽

Travis W. Hein ◽

Paulina Olszewska ◽

Darrell H. Carney

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Coronary Artery ◽

Chronic Hypoxia ◽

Tube Formation ◽

Endothelial Cell Migration ◽

No Production ◽

Vegf Receptor ◽

Human Coronary Artery ◽

Vegf Signaling

Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O2) for 24 h decreased VEGF-stimulated endothelial cell migration (∼82%), proliferation (∼30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) (∼72%) and reduced NO production in VEGF-stimulated cells from 237 ± 38.8 to 61.3 ± 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by ∼50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 ± 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 ± 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.

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