Endothelial RhoA GTPase is essential for in vitro endothelial functions but dispensable for physiological in vivo angiogenesis

Abstract

Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro , RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo , through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting.

Low Dose of Penfluridol Inhibits VEGF-Induced Angiogenesis

“…Cell migration assay was performed using a 48-well Boyden chamber, as described previously [16,34]. Briefly, the upper chamber and lower chamber were separated by an 8-µm pore size polyvinyl pyrrolidone-free polycarbonate membrane (NeuroProbe) coated with collagen.…”

“…Tube formation assay was performed with serum-starved HUVECs using growth factor-reduced matrigel, as previously described [16,32,35]. Wells of a 96-well culture plate were carefully coated to avoid bubble formation, with 40 µL/well RGF-Basement membrane extract.…”

“…VEGF is the main target of anti-angiogenic therapy. It is secreted at high levels from tumor cells and induces endothelial cell proliferation, migration, and tube formation [11,15,16]. VEGF binds to three receptor tyrosine kinases, VEGFR1, VEGFR2 and VEGFR3, which have overlapping but distinct expression patterns: VEGFR1 is highly expressed in monocytes and macrophages, VEGFR2 in vascular endothelial cells, and VEGFR3 in lymphatic endothelial cells.…”

Regulation of lung endothelial permeability by NEK kinases

“…In NEK9 immunoprecipitates, it was shown that NEK9 interacts with both Rho GTPase‐activating protein 11A, as well as myosin phosphatase Rho‐interacting protein. Both proteins are regulators of endothelial permeability . It was suggested that NEK9 forms two separate signaling modules: The first one is composed of NEK6 and Mklp2, and the second one is consisted of NEK7 and the Kinesin‐like protein KIF20A.…”

“…Both proteins are regulators of endothelial permeability. 25 It was suggested that NEK9 forms two separate signaling modules: The first one is composed of NEK6 and Mklp2, and the second one is consisted of NEK7 and the Kinesin-like protein KIF20A. Indeed, NEK9 activates NEK6 and NEK7 by releasing autoinhibitory conformations through direct binding, as well as by phosphorylating the corresponding activation sites.…”