The Role and Mechanisms of RhoA/ROCK Signaling in Angiogenic Effects of Endothelial Progenitor Cells

Neovascularization is involved in many physiological and pathological processes. It plays an important role in initiation and progression (such as the formation of vasa vasorum in the pathological change of coronary atherosclerosis) and treatment (such as neovascularization in ischemia tissue in myocardial infarction) of cardiovascular disease. Angiotensin II (Ang Ⅱ) is the main biologically active peptide of the important humoral regulation system—renin-angiotensin system (RAS). It was found that Ang Ⅱ could promote neovascularization. The Rho proteins, whose prototypical member is RhoA controls a diverse array of cellular processes. RhoA and its downstream effector Rho associated kinase (ROCK) which could be efficiently activated by Ang Ⅱ is an essential part of intracellular signal transduction pathway. Recent studies found that RhoA/ROCK and its downstream effectors mitogen-activated protein kinases (MAPK) were involved in the regulation of neovascularization. Endothelial progenitor cells (EPCs) comprise a cell population that has the capacity to circulate, proliferate and differentiate into mature endothelial cells but that have not yet acquired characteristic mature endothelial markers nor formed a lumen. There is a growing body of data that these precursor cells not only participate in angiogenesis, but also contribute to postnatal vasculogenesis. There is no research on the role of RhoA/ROCK-MAPK signaling in Ang11-mediated angiogenic effects of EPCs. This study explore the role of RhoA/ROCK-MAPK signaling in Ang Ⅱ-mediated angiogenic effects of EPCs, thus provide an important therapeutic target in neovascularization-related disease. Part1Role of RhoA/ROCK signaling in angiotensin Ⅱ-mediated angiogenic effects of endothelial progenitor cellsAim:We undertook our research to explore whether Ang Ⅱ affects EPC proliferation, migration, adhesion, and in vitro vasculogenesis, and whether it involves RhoA/ROCK-MAPK signaling.Methods:(1) EPCs were cultured in serum-free EBM-2medium for24h before incubation with various concentrations of Ang Ⅱ (0,0.1,1,10,100μM) for48h. EPC proliferation, migration, adhesion, and in vitro vasculogenesis capacities were measured.(2) EPCs were cultured in serum-free EBM-2medium for24h. Then EPCs were pre-incubated with C3exoenzyme, GGTI-286, or Y-27632in the addition of Ang Ⅱ (1μM) for48h. EPC proliferation, migration, adhesion, and in vitro vasculogenesis capacities were measured.(3) EPCs were pre-incubated with C3exoenzyme for48h, before incubation with Ang Ⅱ (1μM) for15min, Pull down assay and western blot analysis for RhoA activity and expression were performed.(4) EPCs were pre-incubated with C3exoenzyme, GGTI-286or Y-27632for48h, before incubation with Ang Ⅱ (1μM) for15min. Besides, the activity of ERK1/2, p-38and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) was performed by western blot analysis.Results:(1) Ang Ⅱ improves EPC migration, adhesion, and in vitro vasculogenesis capacities, especially between1-10μM, but has no effect on proliferation ability. (2) The influence of Ang Ⅱ on EPC migration, adhesion, and in vitro vasculogenesis capacities can be abolished by GGTI-286. a geranylgeranyltransferase-I inhibitor. C3exoenzyme, an inhibitor of Rho or Y-27632, an inhibitor of ROCK.(3) Ang Ⅱ enhance RhoA activity, and this effect can be abolished by C3exoenzyme, an inhibitor of Rho.(4) Increased-phosphorylation of P-38and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) by Ang Ⅱ were attenuated by C3exoenzyme, GGTI-286, and Y-27632.Conlusions:In conclusion, Ang Ⅱ improves EPC migration, adhesion, and in vitro vasculogenesis capacities, through activating RhoA/ROCK and MAPK pathway down stream. Part2The role and mechanisms of farnesylpyrophosphate synthase inhibitor Zoledronate in endothelial progenitor cells angiogenic propertiesAim:We undertook our research to explore whether farnesylpyrophosphate synthase inhibitor Zoledronate and Ang II affects EPC proliferation, migration, adhesion, and in vitro vasculogenesis, and whether the RhoA/ROCK-MAPK signaling is involved.Methods:(1) EPCs were cultured in serum-free EBM-2medium for24h before incubation with various concentrations of Zol (0,25,50,75,100μM) and GGOH (50μM) for48h. EPC proliferation, migration, adhesion, paracrine, in vitro vasculogenesis capacities and apoptosis were measured.(2) EPCs were pre-incubated with Zol (50μM) and GGOH (50μM) for48h before incubation with Ang Ⅱ (1μM) for15min. Pull down assay and western blot analysis for RhoA, ERK1/2, JNK and p38MAPK activity were performed.Results:(1) Zol dose-dependently impaires the promotion of EPC proliferation, migration, adhesion, and in vitro vasculogenesis abilities induced by Ang II. GGOH of the same amount can partly reverse the effects of Zol on EPCs.(2) Zol suppresses the activation of RhoA, ERK1/2and JNK stimulated by Ang II. These effects can be partly reversed by GGOH of the same amount.Conlusions:In conclusion, Zol dose-dependently impaires Ang Ⅱ induced neovascularization by suppressing EPC proliferation, migration, adhesion, and in vitro vasculogenesis abilities, through suppressing RhoA/ROCK and MAPK downstream.