The aim of this study was to investigate hypoxia effects on vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in human umbilical vein endothelial cells (HUVEC) and to determine their modulation by the peptide somatostatin (SRIF) and its analogues. The involvement of signal transducer and activator of transcription (STAT) 3 and hypoxia inducible factor (HIF)-1 was also investigated. Quantitative real-time PCR, Western blot and ELISA were used. Hypoxia upregulated VEGF expression and release, whereas it downregulated VEGFR-1 and VEGFR-2. In contrast, neither the expression nor the phosphorylation of the platelet-derived growth factor receptor (PDGFR) β was affected by hypoxia. SU1498 at 1 μM did not affect pVEGFR-2 and pPDGFRβ, whereas at 20 μM it inhibited pVEGFR-2, but not pPDGFRβ. Upregulated VEGF expression and release were prevented by SU1498, which also inhibited the hypoxia-induced pSTAT3 and HIF-1α. Blocking pSTAT3 with S3I-201 inhibited HIF-1α and VEGF upregulation, suggesting the existence of an autocrine loop involving STAT3, HIF-1, VEGF and VEGFR-2. Endothelial cells express somatostatin (SRIF) receptors (sst1–5) although less is known in HUVEC. We found that sst1 and sst4 were expressed by HUVEC with sst1 more expressed than sst4 mRNA. Hypoxia downregulated sst1, whereas it upregulated sst4. The sst1 downregulation, but not the sst4 upregulation, was prevented by SU1498, S3I-201 or YC-1, an inhibitor of HIF-1α. SRIF and the sst1 agonist CH-275, but not the sst4 agonist L803,087 and the sst2/sst3/sst5 agonist octreotide, prevented hypoxia effects on VEGF and its receptors. In addition, SRIF and CH-275 inhibited the hypoxia-induced pSTAT3 and HIF-1α accumulation. Our results suggest that SRIF acting at sst1 limits upregulated VEGF expression and release through a control on the activity of STAT3 and HIF-1, supporting the possible use of sst1 agonists in antiangiogenic therapies.

Hypoxia is a trigger of VEGF expression, the primary cause of retinal pathologies characterized by neovascularization. During hypoxia, transcription factors such as STAT3 and HIF‐1 promote the increase in VEGF expression. Octreotide, a somatostatin receptor 2 (sst 2 )‐preferring agonist, reduces retinal VEGF expression and neovascularization. To investigate the intracellular pathways linking sst 2 activation to the inhibition of hypoxia‐induced VEGF up‐regulation, we used pharmacological approaches and siRNA in mouse retinal explants cultured in normoxia or hypoxia. In hypoxic explants in which STAT3 or HIF‐1 was inhibited, we observed the existence of reciprocal interactions between STAT3 and HIF‐1, which synergistically induced VEGF expression. Octreotide prevented hypoxia‐induced activation of STAT3 and HIF‐1, and the downstream increase in VEGF expression, as evaluated in hypoxic explants treated with pharmacological inhibitors of STAT3 or HIF‐1 and in normoxic explants in which pharmacological activators of STAT3 or HIF‐1 were used to mimic a hypoxia‐like response. The effect of octreotide on STAT3 activation is in part indirect, through the blockade of VEGFR‐2 phosphorylation. The effect of octreotide on STAT3, HIF‐1, VEGFR‐2, and VEGF required Src homology region 2 domain‐containing phosphatase 1 (SHP‐1). In hypoxic extracts, octreotide induced SHP‐1 phosphorylation and activation, and inhibiting SHP‐1 abolished the octreotide effect on STAT3, HIF‐1, VEGFR‐2, and VEGF. The central role of SHP‐1 in the modulation of STAT3 and HIF‐1 was confirmed in normoxic explants in which pharmacologically activated SHP‐1 prevented the effect of STAT3 or HIF‐1 activation. Immunohistochemical studies showed that under hypoxia sst 2 and VEGF are expressed by retinal vessels, thus indicating a possible direct effect of octreotide on VEGF‐containing endothelial cells. These data clarify the mechanism by which octreotide prevents hypoxia‐induced VEGF up‐regulation and support the effectiveness of octreotide in treatment of oxygen‐induced retinopathies. These results may have implications in designing therapies targeting STAT3 and/or HIF‐1 aimed at preventing retinal neovascularization. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.