Regulation Of ARHGEF7 On Vascular Endothelial Cell Function And Association Between Variants Of ARHGEF7 And Stroke

BackgroundSurvivors of stroke are often disability,required long-term care,and are at high risk of mortality.Vascular remodeling and endothelial integrity play critical roles in the pathogenesis of stroke.ARHGEF7(Rho Guanine Nucleotide Exchange Factor 7,ARHGEF7)are highly expressed in brain tissues and vascular.ARHGEF7 promote the proliferation,migration,extracellular matrix reconstruction and angiogenesis in vascular endothelial cells.And ARHGEF7 are important for regulating the stability and permeability of blood vessel walls,and play an important regulatory role in the process development,remodeling and maturation for vascular.The genetic variation of ARHGEF7 may affect expression of ARHGEF7,and then affect the stability and permeability of vascular,which make people susceptible to the risk factor for stroke.However,the gene variant profile in ARHGEF7 has not been studied in patients with stroke.Objectives1.In order to determine whether polymorphisms of ARHGEF7 are genetic risk factors for stroke and post-stroke,the multi-center case-control and prospective study was performed.2.To examine the regulatory effects of a single nucleotide polymorphism of ARHGEF7 in 5'UTR and 3'UTR.3.To study the effects of ARHGEF7 on the function of vascular endothelial cells.Subjects and methods1.The multi-center case-control study for assessment the association between variations of ARHGEF7 and stroke.This study included 1669 patients with initial stroke and 1758 controls.Stroke patients were followed-up for 4.5 years.2.In order to investigate the reasons for gender differences in the correlation between variants of ARHGEF7 and stroke,we test the effects of estrogen and its receptor antagonist on the expression of ARHGEF7.3.ARHGEF7 promoter/luciferase reporter vectors and 3'UTR/luciferase reporter vectors were constructed respectively,which contains wild allele or mutant allele of SNPs in ARHGEF7 5 'UTR or 3' UTR respectively.4.To detect the effects of transcription factors NFI and Oct-2 on ARHGEF7 expression,expression vectors of transcription factors NFI and Oct-2 were constructed.In addition,to detect the effect of microRNAs on the expression of ARHGEF7,endothelial cells were transcribed with different microRNA.5.The effects of ARHGEF7 on the proliferation,injury repair,adhesion and vascular formation ability of vascular endothelial cells were tested by CCK-8,scraching experiment,adhesion experiment and tubule formation experiment respectively.Results1.The allele A of rs3 809340 significantly increased the risk of ICH(cerebral hemorrhage,ICH)in women(OR=1.70,95%CI:1.22-2.36,P=0.002,Bonferroni correction P=0.02).Subjects carrying rs3809340 A(AA or TA genotype)significantly increased the risk of ICH in women(OR=1.89;95%CI,1.22 to 2.93,P=0.004,Bonferroni correction P=0.04).2.Subjects carrying rs3809340 A allele(AA or TA genotype)significantly increased risk of cardiovascular mortality after stroke(HR=1.36,95%CI 1.06-1.89,P=0.03)in women.Furthermore,stroke patients carrying with rs4773327 A genotype(AA or GA genotype)significantly increased the risk of cardiovascular death after stroke(HR=1.42,95%CI 1.061.99,P=0.04).3.Estrogen inhibited the expression ARHGEF7 both in mRNA and protein level.However,the estrogen receptor antagonists promoted expression of ARHGEF7.4.Transcription factor NFI inhibited expression of ARHGEF7 both in mRNA and protein level.The variant rs3 809340 A might change the binding ability of transcription factor NFI and then inhibits the transcription activity of ARHGEF7 and results the lower expression of ARHGEF7.5.Variations of ARHGEF7 gene in 3 'UTR altered the binding sites of microRNAs.However,these microRNAs have no significant effects on the expression of ARHGEF7.6.ARHGEF7 promoted the ability of proliferation,injury repair,adhesion and angiogenesis in vascular endothelial cell.Conclusion1.The variants rs3809340 and rs4773327 of ARHGEF7 in 5'UTR are associated with the risk of ICH and post-stroke cardiovascular mortality.Gender difference of association between variants of ARHGEF7 and risk of stroke might because of estrogen affects.2.The variant rs3809340 A might change the binding ability of transcription factor NFI and then inhibits the transcription activity of ARHGEF7 and results the lower expression of ARHGEF7.This might the reason of the correlation between rs3809340 and ICH.3.ARHGEF7 regulated function of vascular endothelial cells and then effected the function of vascular.This is might be the mechanism of the ARHGEF7 on the effects of stroke.4.This is the first time to study the association between variants of ARHGEF7 and stroke.Furthermore,we studied the regulatory of ARHGEF7 on vascular endothelial cells.