Study On The Mechanism Of OVGP1 Increasing Blood Pressure And CircRNA Markers In Hypertensive Intracerebral Hemorrhage

Objective:Hypertension is a complex disease determined by both environmental and genetic factors.In recent years,there is evidence that epigenetics is involved in the pathological process of hypertension,but its mechanism has not been elucidated.DNA methy lation is one of the ways of epigenetic regulation.Previous study found that the level of methylation at cg20823859 in the promoter region of OVGP1 was decreased in hypertensive people,and OVGP1 was up-regulated in the plasma of hypertensive people and vascular remodeling model.However,the specific mechanism of OVGP1 in blood pressure and vascular remodeling is still unclear.This study mainly used the construction of transgenic and knockout mice models to observe the changes and molecular mechanisms of blood pressure and vascular remodeling after overexpression and knockout of Ovgpl,aiming to explore the specific molecular mechanisms of OVGP1 involved in blood pressure regulation and vascular remodeling,to provide theoretical basis for the development of new hypertension drugs and the treatment of vascular remodeling diseases.Methods and Results:Global Ovgpl transgenic mice(TG)were constructed.Using the tail-cuff method and electronic monitoring blood pressure,the results showed that the systolic and diastolic blood pressures of TG mice were higher than those littermates wild-type mice(WT).After angiotensin ?(Ang ?)(490ng/kg/min)induction,the blood pressure of the TG group mice was significantly higher than that of the WT mice.CRISPR/CAS9 technology was used to construct Ovgp1 global knockout mice(KO).Through the tail-cuff method and electronic monitoring of blood pressure,it was found that compared with the control group(WT),the blood pressure of the KO group mice was lower at the basal level and afterAng? induction.In order to explore the possible mechanism of the increase of blood pressure caused by OVGP1,immunohistochemistry and immunofluorescence staining methods were used to detect the expression of OVGP1 in the different tissues of mice.The results showed that OVGP1 is widely expressed in whole body tissues,particularly in vascular tissues and in the middle smooth muscle layer.The expression was increased in remodeling vascular,mainly in the neointima where smooth muscle cells proliferated.The detection of blood and urine biochemical indicators showed that overexpression of OVGP1 had no significant effect on the renal function of mice.It has been observed by ultrasound that overexpression of OVGPI can increase the diameter of the aorta,but has no significant effect on the structure of the heart and ejection function.Therefore,it is speculated that OVGP1 may play a role by affecting vascular remodeling.Using anAng?-induced vascular remodeling model,results of ultrasound,HE,and Masson staining showed that the thickness of the aortic wall and the collagen components of TG mice were increased;real-time quantitative polymerase chain reaction(RT-PCR)showed that the expression of inflammatory factors IL-6 and MCP-1 in TG group mice were increased.The expression of collagen I was increased and the expression of ?-SMA was decreased by both RT-PCR and immunofluorescence staining.DHE staining showed that the TG group vascular reactive oxygen species(ROS)production was increased compared with WT group,RT-PCR detection showed that the expression levels of NADPH and NOX2 were up-regulated.Further pathological staining results showed that knocking out OVGP1 alleviated Ang?-induced vascular wall thickening,collagen deposition and oxidative stress;RT-PCR detection of inflammatory factors found that knocking out OVGP1 alleviated Ang?-induced inflammatory factors IL-6 and MCP-1 expression.This study furtherly constructed a wide-induce femoral artery injury model.The arteries were collected on the 14th day and the 28th day for HE staining,and calculating neointima area and intima-to-media ratio(I/M).The results showed that the overexpression of OVGP1 promoted neointima information after vascular injury,and knocking out OVGPI inhibited neointima information.Immunofluorescence staining found that overexpression of OVGP1 promoted the expression of PCNA and MMP9 that were the representative markers of proliferation and migration in the neointima.The vascular tension experiment found that compared with WT,TG mice increased vasoconstriction tension induced by phenylephrine(PHE),and vasodilation induced by the endothelium-dependent relaxant acetylcholine(ACH)and the non-endothelial-dependent sodium nitroprusside(SNP)was decreased.After the infusion of Ang?,it further aggravated vascular dysfunction.The vascular tension experiments of KO mice found that compared with WT,knocking out OVGP1 alleviated Ang?-induced vascular dysfunction.RNA sequencing was used in OVGP1 overexpressed smooth muscle cells,a total of 781 differentially expressed genes were detected.Overlapped with the expression profile of OVGP1 knockdown and 155 commonly differentially expressed genes were identified.GO and KEGG analysis found that the differentially expressed genes were mainly involved in extracellular matrix remodeling,regulation of cell proliferation,phosphorylation signal transduction,cell migration,inflammation and Wnt signaling pathway,PI3K-AKT signaling pathways.These pathways are closely related to vascular remodeling.The genes involved in multiple pathways were selected for RT-PCR and western blot validation.It was found that WNT2 and IGFBP5 were significantly up-regulated after overexpression of OVGP1,which may be one of the target genes of OVGP1.Using CCK8 and Transwell chamber migration experiments,it was found that overexpression of OVGP1 caused smooth muscle proliferation,migration and ROS production,which aggravated after PDGF-BB induction,and knocking down OVGP1 inhibited the proliferation and migration caused by PDGF-BB.The PI3K-AKT signaling pathway is enriched in the above results and has an important role in cardiovascular disease.Western blot showed that overexpressed OVGP1 increased the phosphorylation of AKT,which aggravated after the PDGF-BB induction.Knockdown of OVGP1 inhibited the level of AKT phosphorylation,indicating that AKT is a downstream pathway of OVGP1.To further clarify the mechanism of OVGP1,mass spectrometry and co-immunoprecipitation was used to screen and verify OVGP1 binding proteins.The results found that OVGP1 binds with MYH9 in smooth muscle cells and co-localizes in smooth muscle cells and aortic vessels.Knockdown of MYH9 inhibits the oxidative stress and hypertrophy of smooth muscle cells caused by overexpression of OVGP1.Knockdown of MYH9 inhibits the phosphorylation level of AKT in the downstream pathway of OVGP1.After further administration of MYH9 inhibitor Blebbistatin(BLEB)at the animal level,the blood pressure level and vascular remodeling of TG mice were observed.The results showed that compared with the control group(saline),after giving the inhibitor BLEB,TG mice blood pressure was significantly reduced;the pathological staining results showed that BLEB significantly reversed the vascular remodeling caused by overexpression of OVGP1;the vascular function showed that BLEB improved the vascular dysfunction caused by overexpression of OVGP1.Conclusion:OVGP1 binds with MYH9 to activate the AKT signaling pathway and increase ROS production,causing vascular oxidative stress,inflammation and collagen deposition,leading to vascular remodeling and dysfunction,and elevating blood pressure.Objective:Circular RNAs(circRNAs)have shown promise as biomarkers because of their stability in peripheral blood,and they participate in various pathological processes of ischemic stroke;however,their expression profiles and the potential diagnostic value for intracerebral hemorrhage stroke remain unclear.This study aims to investigate the expression profiles of circRNAs after hypertensive intracerebral hemorrhage(ICH).Methods and Results:RNA sequencing was used to investigate the expression profiles of circRNA in a discovery sample of 129 subjects(44 patients with ICH,42 hypertension(HTN)controls,and 43 patients with cerebral infarction(CI)),and an independent validation sample of 54 subjects(20 patients with ICH,18 HTN controls,and 16 patients with CI).By comparing ICH and hypertension(HTN)controls,cerebral infarction(CI)and HTN in two samples,we found that 15 circRNAs including 5 upregulated circRNAs and 10 downregulated circRNAs,were consistently altered only after ICH(fold change>1.5 and FDR<0.05).We further applied quantitative real-time polymerase chain reaction to validate the circRNA expression levels in all samples and found that hsa_circ₀001240 and hsa_circ₀001947 were upregulated and that hsa_circ₀001386 was downregulated in ICH compared with HTN and CI.Logistic regression analysis of the combination of 3 circRNAs showed an area under the curve of 0.92 with a sensitivity of 86%and a specificity of 88%for diagnosing ICH compared to HTN controls.Together with ICH risk factors,the circRNAs showed an area under the curve of 0.97 in diagnosing ICH.Spearman's correlation analysis suggested that the hsa_circ₀001240,hsa_circ₀001947 and hsa_circ₀001386 expression levels correlated with ICH risk factors.The circRNA-miRNA-mRNA functional analysis indicated that the target miRNAs and mRNAs were involved in were associated with ICH-related pathways,including lysine degradation,fatty acid metabolism and biosynthesis,integrin cell surface interactions and immune system function.Conclusion:This is the first study to report expression profiles of circRNAs after ICH and to propose that three circRNAs are potential biomarkers for ICH.