GPR124 Signaling Mediates Brain Pericyte Polarization And Migration

Stroke incidence and mortality correlated to stroke risk factors such as aging and the prevalence of cardiovascular risk factors in China,the mortality rate is much higher than Japan and European and American countries.Induced by a variety of factors in the brain vascular stenosis,occlusion or rupture caused by cerebral blood circulation disorders,including ischemic stroke accounted for 65%-80%,the clinical manifestations of a transient or permanent dysfunction symptoms and signs.Contrary to this,although scholars have made unremitting exploration,but so far in the field of drug treatment of cerebral ischemia has not yet been a substantive breakthrough and success,the lack of curative effect of new drugs in clinical practice.Therefore,to further explore the pathogenesis of cerebral ischemic disease,finding new drug targets,develop a safe and effective drug prevention and treatment strategies is an important scientific proposition.The further in-depth study of the cerebral vascular homeostasis and molecular mechanisms of neurovascular remodeling during brain ischemia is very important and urgent.As one of the important components of the neurovascular unit encircled pericytes and endothelial cells,which constitute the basic structure of the microvessels and maintain the stability of blood-brain barrier.The previously data reported that loss of pericytes can lead to neuronal degeneration and scarring induced phenomena.Stress can cause pericytes sustained contraction and lead to microcirculation disorders.In the acute phase of cerebral ischemia and late activation of endothelial cells and free radicals produced by hypoxia inducible pericyte recruitment,migration and rapid lumen ischemic area covering the newly formed capillaries in order to promote the maturity and stability,suggesting that pericytes may release some factors to maintain a stable microenvironment helping repair damaged nerve tissue.Currently,mechanism of pericytes during neurovascular unit rebuilding process after cerebral ischemia are far from clear,is a scientific problem to be solved.G protein-coupled receptors(Guanosine-binding Protein Coupled Receptor,GPCR)occupies an extremely important role in the pharmaceutical sector,by far the world market of small molecule drugs have been found in the total number of therapeutic drug targets as more than a third of the GPCRs.GPR124 is an important member of the family,an important cell signaling protein,its topological conformation seven transmembrane receptors.GPR124 has a long N-terminal initially be attributed to B family(also known as Secretin family)receptors,but accumulating data show that this type of GPCRs have long N-terminal family has a different function,GRP 124 as adhesion-type G protein coupled receptor family member,is a long-chain protein containing 1338 amino acids,containing the classic a-helix seven transmembrane(TM)domain,containing the characteristic comprises a plurality of long chain extracellular N-terminal conserved domain.Its N-terminal region includes multiple leucine-rich region(LRR),an immunoglobulin(1g)homology region,a hormone binding domain(HBD)and G protein coupled receptor proteolytic site(GPS).As a member of the GPCR family,GPR124 which signal transduction mediated by physiological and pathological function,whether it is a potential drug target is far from clear.Arouse our concern is:GPR124 plays a very important role in vascular development and angiogenesis process.GPR124 has been reported many types of normal tissue in the embryo during development of the nervous system,including the expression of the vascular system.Recent reports found that GPR124 knockout mouse died at embryonic phase,mainly due to reduced blood vessel extension,pathologic vascular plexus and bleeding,leading to developmental defects in the blood vessels of the central nervous system.In vitro experiments showed that endothelial cells,transforming growth factor-β1(TGF-β1)can induce up-regulation of expression of GPR124,indicate TGF-βl/GPR124 pathway with angiogenesis.We speculate that after GPR124 activation by binding to its ligand intracellular domain specific GAPs(GTPase-activating protein)active fragment downstream signaling,which lead to the biological effects of cell migration,immune response and angiogenesis.Nevertheless,so far as an important orphan receptor,function and regulation of GPR124 under pathophysiological conditions remain unknown.Based on its early development of cerebrovascular specifically expressed in mouse research foundation,were prompted GPR124 and cerebrovascular important developmental relevance.These also raise our interest on its role on the adult animal brain vascular function regulation,and a desire to know the brain microvascular homeostasis of GPR124 signal during brain ischemia.To better understand the role and mechanism of GPR124 in brain microvascular homeostasis regulation,rebuilding process,we constructed full-length GPR124,N terminal truncated and the seven transmembrane C-terminal truncated plasmid,which in combined with morphological,biochemical and neurological studies.First,the laser confocal immunofluorescence found specific GPR124 expression in human brain microvascularpericytes,partly located in Phalloidin positive staining structures.GPR124-CTF-EGFP displays membrane localization in pericytes after transfection.Stochastic optical reconstruction microscopy further helps us confirm GPR124 found in pericyte cells and the presence of F-actin co-localization,suggesting that GPR124 may be involved in pericyte polarization and migration process.Secondly,we found that cerebrovascular risk factors stimulate polarization change and migration and redistribution associated with GPR124.The quantitative results suggest that the surface area,the number of filopodia and other indicators of its expression after stimulation have changed significantly.The combination of these biochemical and morphological evidence,we believe pericytes GPR124 can regulate cellular and extracellular matrix(ECM)interactions.Finally,explore the unknown molecule GPR124 function,activation and signal transduction significant help us resolve cerebrovascular disease occurrence,development and outcome of pathological processes of brain microvascular homeostasis regulation,reconstruction and repair of the neurovascular unit,and then looking for potential drug targets.Confocal fluorescence staining showed that GPR124 and cell polarization of molecular markers PAR3 have a co-location,location in the movement of the front tip of the cell,suggesting that GPR124 is involved in cell migration process re-adhesion links.Furthermore,a lentivirusShRNA GPR124 transfection inhibited pericytes dynamic polarization and migration.More importantly,the microvascular pericytes was partially co-localizatied with Ki67 following microsphere embilism.GPR124 expression was also observed significantly increase in the pericytes of the damage zone,suggesting that it may be involved in angiogenesis process in vascular damage region during brain ischemia.Together,these results define the role of the GPR124 in linking pericyte polarization and migration to brain repair in vitro and in vivo and provide a rationale for targeting pericyte GPR124 for angiogenesis and neurovascular protection during brain ischemia.