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Mentions: Adenoviral-mediated overexpression of sFlt-1 in HUVEC inhibited endothelial cell proliferation (Figure 3a) and MAP kinase ERK-1/-2 phosphorylation (Figure 3a insert). Subsequently, to test whether knockdown of sFlt-1 would promote endothelial cell proliferation, HUVEC were transfected with two synthetic siRNA sequences targeted to the unique carboxyl-terminus region of sFlt-1. sFlt-1 siRNA transfection resulted in a substantial reduction in the release of sFlt-1 from HUVEC after 24 hours (Figure 3b). Endothelial cell proliferation was significantly increased (Figure 3c) and interestingly, sFlt-1 knockdown also led to a concomitant increase in VEGFR-2 phosphorylation at tyrosine 951 (Y951) (Figure 3d). In addition, sFlt-1 siRNA increased both basal and VEGF-A-mediated endothelial cell migration (Figure 4a) and tube formation on Matrigel (Figure 4b and 4c). VEGF stimulates eNOS activity and NO release [23,36] to mediate angiogenesis [33,37], thus we predicted that loss of sFlt-1 would increase eNOS phosphorylation in HUVEC. Phosphorylation of eNOS (ser1177) was significantly increased in cells lacking sFlt-1 (Figure 4d). These data provide direct evidence that sFlt-1 is itself a negative regulator of endothelial function. It is likely that sFlt-1 sequesters VEGF and PlGF to maintain a physiological steady state until angiogenesis is required, at which point this system must be overridden.
Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis
Bottom Line: In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation.Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta.These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.
Affiliation: University/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK. firstname.lastname@example.org.
Background: The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors.
Methods and results: Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta.
Conclusion: These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.