Release of endothelial cell associated VEGFR2 during TGF-β modulated angiogenesis in vitro

Abstract

BackgroundSprouting angiogenesis requires vascular endothelial proliferation, migration and morphogenesis. The process is regulated by soluble factors, principally vascular endothelial growth factor (VEGF), and via bidirectional signaling through the Jagged/Notch system, leading to assignment of tip cell and stalk cell identity. The cytokine transforming growth factor beta (TGF-β) can either stimulate or inhibit angiogenesis via its differential surface receptor signaling. Here we evaluate changes in expression of angiogenic signaling receptors when bovine aortic endothelial cells were exposed to TGF-β1 under low serum conditions.ResultsTGF-β1 induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on Matrigel, maximal at 5.0 ng/ml. This occurred via ALK5-dependent pathways and was accompanied by significant upregulation of the TGF-β co-receptor endoglin, and SMAD2 phosphorylation, but no alteration in Smad1/5 activation. TGF-β1 also induced ALK5-dependent downregulation of Notch1 but not of its ligand delta-like ligand 4. Cell associated VEGFR2 (but not VEGFR1) was significantly downregulated and accompanied by reciprocal upregulation of VEGFR2 in conditioned medium. Quantitative polymerase chain reaction analysis revealed that this soluble VEGFR2 was not generated by a selective shift in mRNA isoform transcription. This VEGFR2 in conditioned medium was full-length protein and was associated with increased soluble HSP-90, consistent with a possible shedding of microvesicles/exosomes.ConclusionsTaken together, our results suggest that endothelial cells exposed to TGF-β1 lose both tip and stalk cell identity, possibly mediated by loss of VEGFR2 signaling. The role of these events in physiological and pathological angiogenesis requires further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-017-0127-y) contains supplementary material, which is available to authorized users.

Furin inhibition prevents hypoxic and TGFβ-mediated blood-brain barrier disruption

“…It seems likely that a balance between activation of ALK1 versus ALK5 determines whether ECs remain quiescent or initiate an angiogenic response, an observation previously described by Goumans et al, in 2002 [11]. In HUVEC and bovine aortic EC ALK5 majorly contributed to angiogenic processes [31,43]. Similarly ALK5 blockade prevented TGFβ-induced activation of Smad2 (without affecting pSmad1/5 levels) and suppressed brain EC migration in agreement with those findings.…”

“…In ECs, canonical binding of TGFβ to ALK5 causes downstream activation and phosphorylation of Smad2 resulting in stimulation of angiogenesis [31], whereas signaling via ALK1 induces phosphorylation of Smad1, 5 and 8 and subsequent endothelial quiescence [32]. Exposure of brain ECs to TGFβ but not hypoxia increased phosphorylated Smad2 levels whereas the opposite effect was observed on phospho-Smad1/5 levels (Supp.…”

It Takes Two: Endothelial-Perivascular Cell Cross-Talk in Vascular Development and Disease

“…These two receptors have opposing effects on the response of the cell to TGF-β. ALK-5 stimulation causes down regulation of proangiogenic VEGFR-2 and upregulation of the antiangiogenic VEGFR-1 leading to reduced proliferation and increased differentiation of endothelial cells ( 45 , 46 ). Similarly, ALK-5 activation causes differentiation of the perivascular cells and promotes release of ECM proteins such as fibronectin from both cell types ( 40 ).…”

Exosomes: A Rising Star in Failing Hearts

“…Although exosomes are relatively stable compartments, under certain conditions they may liberate their components, such as interleukin-1β (Qu et al, 2007) and vascular endothelial growth factor receptor 2 (VEGFR2) (Jarad et al, 2017). Also, those exosomes that maintain structural integrity can selectively bind to the cell surface via ligand–receptor recognition.…”