Expression Of Robo4 In Mouse Cardiac Microvascular Endothelial Cells And Impact Of Slit2/robo4 Signaling On Proliferation And Migration Of Cardiac Microvascular Endothelial Cells

Neural axon guidance signaling caused by the combination of Roboprotein and ligand Slit can guide axon growth and control migration ofneurocytes. Recently, reserches found this signaling could efftct thepromotion or inhibition of tumors angiogenesis, organ development, andinhibit chemotaxis of leukocyte and retinal neovascularization, it plays animportant role in endothelial cell migration of angiogenesis as well. The Robofamily consists of Robo1, Robo2, Robo3, Robo4, mainly were expressed inthe nervous system, but were detectable in other systems and tissues, such asvascular tissues, smooth muscles, kidneys, uterus, retina, tumor tissues and soon. Robo4, vascular-specific receptor, was expressed in human umbilical veinendothelial cells, human aortic endothelial cells, human microvascularendothelial cells. Robo1 was expressed in human umbilical vein endothelialcells, but the expression of Robo1 in microvascular endothelial cells wascontroversial. Robo2、Robo3 do not express in endothelial cells. At present,there is no evidences to prove that expressions of Robo in cardiacmicrovascular endothelial cells could be detected. In the experiment, RT-PCRwas used to detect whether Robo1gene and Robo4gene were expressed inmouse ventricular muscle tissue. Results showed that Robo4 gene wasexpressed in mouse ventricle muscle tissue, but Robo1gene was not expressed in mouse ventricle muscle tissue. It can be seen that mouse ventricular muscleblood vessel and cardiac microvascular endothelial cells were identified toexpress Robo4 by using immunohistochemistry。Slit is the ligand of Robo. There are three family members Slit1, Slit2and Slit3. Slit1 only presented in nervous system, Slit2, Slit3 also expressedin other systems, such as vascular endothelial, smooth muscles ,some solidtumors and so on. The researches have shown that expressions of Robo4 inhuman umbilical vein endothelial cells and human microvascular endothelialcells ,interacted with Slit2 to inhibit VEGF-,bFGF-induced endothelial cellsmigration. How does Slit2/Robo4 signaling pathway effect mouse cardiacmicrovascular endothelial cells and what is the impact on cardiacmicrovascular endothelial cells proliferation and migration are not clear. Inthe course of CCK-8 proliferation experiment, exogenous Slit2 was used indifferent concentrations (50 ng/mLSlit2, 75 ng/mL Slit2, 100 ng/mLSlit2, 125ng/mLSlit2, 150 ng/mL Slit2) , results indicated that when compared with ODof the negative control group(p>0.05), there was no efftec of Slit2/Robo4signaling on proliferation of mouse cardiac microvascular endothelial cells.Results of transwell assay implied that exogenous 100 ng/mLSlit2 couldinhibit VEGF-induced mouse cardiac microvascular endothelial cellmigration ,so Slit2/Robo4 signaling inhibits VEGF-induce endothelial cellmigration. Conclusion: Robo4 was expressed in mouse ventricular muscleblood vessels and cardiac microvascular endothelial cells, Robo1 genes wasnot in ventricular muscle. Different concentrations of exogenous Slit2 didn’teffect the proliferation of mouse cardiac microvascular endothelial cells.Exogenous 100ng/mL Slit2 could inhibit VEGF- induced mouse cardiacmicrovascular endothelial cell migration. The Slit2/Robo4 signaling which can inhibit VEGF-induced mouse cardiac microvascular endothelial cellmigration, introduced new ways to explore ischemic myocardialangiogenesis ,that is to say, by blocking the interaction of Robo4 with Slit2 ordistracting Slit2 downstream signaling to possibly promote endothelial cellmigration, thus to lay the foundation of angiogenesis of ischemic myocardial.