microRNA-21-3p调控NOX4在脑海绵状血管畸形发生中的作用及机制

Cerebral cavernous malformations (CCMs) are relatively common vascular malformations that arise predominantly in the central nervous system, resulting in cerebral hemorrhage and seizure with a disastrous clinical outcome. Current treatment for CCMs consists of palliative therapies, neurosurgical resection or radiotherapy. However, no effective precision treatment yet exists. CCMs can be sporadic or familial in humans with similar pathogenesis. CCMs arise from loss of function mutations in one of three genes: KRIT1 (also known as Ccm1), Ccm2 or PDCD10 (also known as Ccm3), that encode components of a heterotrimeric, intracellular adaptor protein complex (the “CCM complex”). However, how loss of Ccm gene affecting CCM complex causes the subsequent deficit in brain microvascular endothelial cells (BMVECs) and CCM formation is not yet fully elucidated. . Recent studies with numerous signaling mechanisms as causal for CCM formation have implicated that oxidative stress may play an important role in the pathogenesis of CCMs. NADPH oxidase (NOX)-4 is a major source of reactive oxygen species (ROS) in BMVECs. A recent study has shown that loss of KRIT1 gene increases endothelial ROS production via NOX signaling, up-regulates NOX4 expression, and exacerbates vascular permeability. Our previous studies have shown that multiple microRNAs via downregulating their direct target gene NOX4 protect against cerebral ischemia reperfusion injury. However, the contribution of increased ROS via microRNAs to the phenotype of Ccm2-depleted BMVECs and the pathophysiology of CCM remains unknown. . Our preliminary data indicate that microRNA regulation of NOX4 and vascular endothelial (VE)-cadherin contributes to CCM formation. We found the increased expression of NOX4 and decreased expression of VE-cadherin in the abnormal endothelial cells from human CCM lesions. CCM animal model was generated in Ccm2ECKO mice with endothelial-specific deletion of Ccm2 gene. Increased expression of NOX4 and decreased expression of VE-cadherin were found in the BMVECs from Ccm2ECKO mice. We also found over-expression of miR-27a-3p and down-expression of miR-21-3p in these BMVECs. VE-cadherin is the direct target gene for miR-27a-3p. In Ccm2ECKO mice model, inhibitor of miR-27-3p increased the expression of VE-cadherin and maintained the integrity of endothelial cell, and inhibited CCM formation and development. On the other hand, increased expression of NOX4 was found in the Ccm2-depleted BMVECs. Inhibition of NOX4 using siRNA reduced ROS production, alleviated oxidative stress injury and inhibited vascular permeability in these Ccm2-depleted BMVECs. NOX4 is the direct target gene for miR-21-3p. Therefore, we hypothesized that ROS-generating NOX4 oxidative stress contributes to BMVECs dysfunction and CCM formation through miR-21-3p pathway. . To test the hypothesis, human CCM lesions, CCM animal model, and BMVECs from Ccm2ECKO mice will be used. The expression of miR-21-3p, NOX4, VE-cadherin and ROS in these samples will be analyzed by immunofluorescent histochemistry, qRT-PCR, Western blot, and in situ hybridization. Then, the effect of miR-21-3p/NOX4/ROS signaling responsible for endothelial cells dysfunction in the Ccm2-depleted BMVECs will be studied by regulation of miR-21-3p and NOX4. The mechanisms of oxidative stress, apoptosis and inflammation will be assessed. Finally, a therapeutic intervention study will be carried out in Ccm2ECKO mice model using miR-21-3p mimic, siRNA NOX4 and NOX4 inhibitor. This study will test the efficacy of regulation of miR-21-3p/NOX4/ROS pathway to maintain the integrity of BMVECs and inhibit CCM formation and developement. . This study will help to elucidate the mechanisms of miR-21-3p/NOX4/ROS pathway in CCM formation using clinical samples, in vivo and in vitro models, and to develop the potential therapeutic interventions to attenuate neurological dysfunction from CCMs disease.

脑海绵状血管畸形(CCM)临床上容易引起脑出血和癫痫，目前缺乏精准治疗方法。血管内皮细胞3个Ccm致病基因功能缺失后可导致疾病发生和进展，但是microRNA参与CCM疾病的机制尚不明确。我们前期在内皮细胞特异性Ccm2基因敲除小鼠疾病模型，发现下调miR-27维护了内皮细胞粘附连接功能，明显减小CCM病变。在CCM患者手术切除组织和Ccm2基因敲除小鼠的脑微血管内皮细胞，初步发现miR-21-3p下调和靶基因NOX4上调，抑制脑血管内皮细胞NOX4后，降低了活性氧的产生和内皮细胞通透性的增加。据此我们提出假设：Ccm2基因缺失后通过miR-21-3p/NOX4/活性氧通路，引起脑微血管内皮细胞功能失调，参与CCM形成。本项目拟通过临床、动物模型和体外细胞研究，进一步阐明miR-21-3p调控NOX4在CCM发生和进展中的作用机制，为明确miR-21-3p作为CCM精准治疗新靶点提供依据。

脑海绵状血管畸形是一种脑血管病，临床上容易引起脑出血和癫痫，治疗方法以显微外科手术切除为主，目前缺乏精准治疗方法。血管内皮细胞3个Ccm致病基因功能缺失后可导致疾病发生和进展，但是微小核糖核酸（microRNA）参与脑海绵状血管畸形疾病的机制尚不明确。. 本项目以揭示脑海绵状血管畸形的发病机制为目标，通过临床样本、体外细胞模型和动物模型研究，阐明miR-21-3p/NOX4/ROS通路在脑海绵状血管畸形发生和发展中的作用及机制。在人脑海绵状血管畸形病变组织和脑微血管内皮细胞中，发现miR-21-3p和Ccm2基因表达降低，NADPH氧化酶4（NOX4）、Toll样受体4（TLR4）、细胞间粘附分子（ICAM）-1表达升高，血管内皮钙粘蛋白（VE-cadherin）表达降低。在脑海绵状血管畸形病变中miR-21-3p、NOX4、TLR4、ICAM-1和VE-cadherin的表达水平与头颅MR影像学脑出血表现有关。在Ccm2基因缺失的人脑微血管内皮细胞体外模型中，发现miR-21-3p表达显著降低，其直接靶基因NOX4表达显著升高。采用miR-21-3p激动剂、miR-21-3p抑制剂、siRNA NOX4进行干预，发现miR-21-3p激动剂干预后抑制NOX4的上调表达，降低脑微血管内皮细胞活性氧（ROS）的过度产生，减轻血管内皮细胞通透性的升高，改善脑微血管内皮细胞功能障碍，并降低脑微血管内皮细胞的增殖、迁移和管腔形成能力，起到抑制血管生成的作用。在Ccm2基因下调的斑马鱼活体模型中，miR-21-3p激动剂显著降低斑马鱼模型的脑出血发生。. 研究结果表明microRNA与脑海绵状血管畸形疾病的形成和发展密切相关，miR-21-3p/NOX4/ROS通路在脑海绵状血管畸形的形成发展和脑出血中发挥关键作用，miR-21-3p是脑海绵状血管畸形的一个有效治疗靶点。取得的研究成果在国内学术会议交流，部分研究结果发表在PLoS Biology杂志，培养多名研究生，建立了一支脑血管病临床和转化医学研究团队。