目的：高血压是一种由环境和遗传因素共同决定的复杂疾病。近年来有证据表明表观遗传参与高血压病理过程，但其机制尚未阐明。DNA甲基化是表观遗传学调控的方式之一，本课题组前期发现高血压人群中输卵管糖蛋白1（Oviductal glycoprotein 1，OVGP1）启动子区cg20823859位点甲基化程度降低，在高血压人群血浆和大鼠球囊损伤血管重塑模型中OVGP1表达上调。然而，OVGP1参与血压调节和血管重塑的具体机制尚不清楚。本研究主要通过构建转基因和敲除小鼠模型，观察过表达和敲除OVGP1后血压和血管重塑的变化及分子机制，旨在探讨OVGP1参与血压调节和血管重塑的具体分子机制，为高血压新药的开发及血管重塑性疾病的治疗提供理论依据。方法与结果：通过构建全身性Ovgp1转基因小鼠（Transgenic，TG），TG和同窝对照小鼠（Wild type, WT）采用尾套法和电子监测血压发现TG小鼠的收缩压和舒张压均高于WT同窝小鼠；血管紧张素II（Angiotensin II，Ang II）（490ng/kg/min）诱导后，与WT小鼠相比，TG组小鼠的血压显著升高。采用CRISPR/CAS9的技术构建了Ovgp1全身性敲除小鼠（Knockout, KO）。通过尾套法和电子监测血压发现，与对照组（WT）相比，KO组小鼠的血压在基础水平和Ang II诱导后在均低于其同窝WT小鼠。为了探讨OVGP1引起血压升高的可能机制，本研究采用免疫组织化学染色和免疫荧光染色的方法检测了小鼠不同组织中OVGP1的表达，结果显示OVGP1在全身组织广泛性表达，在血管组织中表达较高，主要在血管中膜平滑肌层表达。在重塑血管中表达升高，且主要在新生的内膜中表达，主要是增生的平滑肌细胞。小鼠血尿生化指标检测发现过表达OVGP1对小鼠肾功能无显著影响。通过小动物超声观察到过表达OVGP1后可增加主动脉直径，但对心脏结构和射血功能均无明显影响，因此推测OVGP1可能通过影响血管重塑发挥作用。采用Ang II诱导的血管重塑模型，经超声、HE、Masson染色显示TG小鼠主动脉壁增厚，胶原沉积增加；实时定量聚合酶链反应（RT-PCR）显示TG组小鼠炎症因子IL-6、MCP-1表达升高，纤维化因子Collagen I表达升高，α-Smooth Muscle Actin (α-SMA)表达降低；免疫荧光染色表明TG组Collagen I表达升高，α-SMA表达降低；DHE染色发现与WT相比，TG组血管活性氧（Reactive oxygen species, ROS）产生增加，RT-PCR检测发现TG组NADPH、NOX2表达水平上调。进一步病理染色结果显示敲除OVGP1减轻了Ang II诱导的血管壁增厚，胶原沉积和氧化应激；RT-PCR检测发现敲除OVGP1减轻了Ang II诱导的炎症因子IL-6、MCP-1表达升高。进一步构建了小鼠股动脉损伤模型，分别在第14天和第28天取材进行HE染色和新生内膜厚度、内膜与中膜比（I/M）统计。结果发现，过表达OVGP1促进血管损伤后内膜增生，敲除OVGP1抑制血管损伤后内膜增生。免疫荧光染色发现过表达OVGP1促进新生内膜中PCNA和MMP9表达升高。血管张力实验发现，与WT相比，TG组小鼠苯肾上腺素（Phenylephrine，PHE）诱导的肠系膜上动脉血管收缩张力增加，内皮依赖性舒张剂乙酰胆碱（Acetylcholine，ACH）和非内皮依赖性舒张剂硝普纳（Sodium Nitroprusside，SNP）诱导的肠系膜上动脉血管舒张功能降低。在Ang II诱导后，进一步加重了上述的血管功能障碍。KO小鼠血管张力实验发现，与WT相比，敲除OVGP1减轻了Ang II诱导的肠系膜上动脉血管功能障碍。为探讨OVGP1在平滑肌细胞中的功能机制，本研究在人主动脉平滑肌细胞中过表达OVGP1后采用转录组测序，共检测到781个差异基因，与敲低OVGP1的表达谱芯片结果取交集，共得到155个共同差异表达的基因。GO和KEGG分析发现，差异基因主要参与胞外基质重塑，调控细胞增殖，磷酸化信号转导，参与细胞迁移，炎症反应等过程，参与Wnt信号通路、PI3K-AKT等信号通路。这些通路与血管重塑密切相关，选取在多条通路中均有参与的基因进行RT-PCR和Western blot验证，发现WNT2和IGFBP5在过表达OVGP1后显著上调，可能是OVGP1的靶基因之一。采用CCK8和Transwell小室迁移实验，发现过表达OVGP1引起平滑肌增殖、迁移和ROS产生，在PDGF-BB诱导后加重了这一反应，敲低OVGP1后抑制了PDGF-BB引起的增殖和迁移。PI3K-AKT信号通路在上述结果中被显著富集，且在心血管疾病中具有重要的作用。检测过表达和敲低OVGP1后AKT通路变化，结果显示，过表达OVGP1后AKT的磷酸化水平升高，PDGF-BB诱导后进一步升高；敲低OVGP1抑制了AKT磷酸化水平，表明AKT是OVGP1的下游通路。为进一步明确OVGP1的作用机制，本研究采用质谱检测的方法筛选OVGP1相互作用蛋白并采用CoIP进行验证，结果发现OVGP1与MYH9在平滑肌细胞中相互作用，并在平滑肌细胞和主动脉血管中存在共定位。敲低MYH9抑制了过表达OVGP1引起的平滑肌细胞氧化应激和肥大，抑制了OVGP1下游通路AKT的磷酸化水平。在动物水平上给予MYH9抑制剂布雷他汀（Blebbistatin，BLEB）后，观察TG小鼠血压水平和血管重塑情况，结果显示，与对照组（Saline）相比，MYH9抑制剂BLEB显著改善了过表达OVGP1引起的血管重塑和血压升高；改善了过表达OVGP1引起的血管功能障碍。结论：OVGP1通过与MYH9相互作用，激活下游AKT信号通路，ROS产生增加，引起血管氧化应激、炎症和胶原沉积，导致血管重塑及功能障碍，引起血压升高。

目的：环状RNA（circRNA）由于在外周血中较为稳定，可作为疾病诊断较有潜力的生物标志物，circRNA参与缺血性脑卒中的病理生理过程，然而在出血性脑卒中的表达特征和功能机制尚不清楚。本研究旨在探讨高血压脑出血（ICH）后circRNA的表达特征和潜在的诊断价值。方法与结果：本研究采用转录组测序（RNA-seq）的方法在发现人群（44例ICH患者，42例高血压（HTN）对照和43例脑梗死（CI）患者）和独立的验证人群（20例ICH患者，18例HTN对照和16例CI患者）中检测circRNA的表达谱。通过在两个人群中进行ICH vs HTN 和CI vs HTN比较，发现15个circRNA包括5个上调和10个下调circRNA仅在ICH中一致性表达。进一步采用实时定量聚合酶链反应在所有样本中对候选circRNA进行验证，结果发现，与HTN和CI相比，ICH中的hsa_circ_0001240和hsa_circ_0001947表达上调，hsa_circ_0001386表达下调。采用逻辑回归分析发现，与HTN相比，3个circRNA组合诊断ICH的曲线面积为0.92，敏感性为86％，特异性为88％。结合ICH危险因素，3个circRNA组合诊断ICH的曲线面积为0.97。Spearman相关性分析表明，hsa_circ_0001240，hsa_circ_0001947和hsa_circ_0001386的表达水平与ICH危险因素相关。circRNA–miRNA–mRNA网络分析表明，circRNA的靶标miRNA和mRNA的功能与ICH的病理过程相关，包括赖氨酸降解，脂肪酸代谢与生物合成，整联蛋白细胞表面相互作用和免疫系统功能。结论：这是首次报道ICH外周血中circRNA的表达谱，并提出三种circRNA可作为ICH的潜在生物标志物。

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 methylation 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 Ovgp1, 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 Ovgp1 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 II (Ang II) (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 after Ang II 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 OVGP1 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 an Ang II-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 α-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 II-induced vascular wall thickening, collagen deposition and oxidative stress; RT-PCR detection of inflammatory factors found that knocking out OVGP1 alleviated Ang II-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 OVGP1 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 II, it further aggravated vascular dysfunction. The vascular tension experiments of KO mice found that compared with WT, knocking out OVGP1 alleviated Ang II-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_circ_0001240 and hsa_circ_0001947 were upregulated and that hsa_circ_0001386 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_circ_0001240, hsa_circ_0001947 and hsa_circ_0001386 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.