MMP-9介导的血脑屏障损伤在CADASIL发病中的作用机制研究

Cerebral autosomal dominant arteriopathy with Subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited small-vessel disease, is associated with vascular aggregation of mutant Notch3 protein, dysfunction of cerebral vessels, and dementia. Notch3 extracellular domain (Notch3ECD) gathering in the cerebral vascular wall is expected to result in smooth muscle cell (SMC) injury and death which are considered the mechanism of CADASIL. SMC activity in CADASIL mutant mice is not affected, while the pericyte is injury. Therefore, we put forward the hypothesis: the brain pericyte injury is induced by Notch3ECD, and pericyte secrets matrix metalloproteinase -9 (MMP-9), damaging endothelial cell tight junction and disruption of the blood-brain barrier which causes white matter lesions in CADASIL. To test this hypothesis, we use CADASIL mutant mice, separate and identify brain microvascular pericytes, and detect Notch3 expression by RT-PCR; CADASIL mutant mice and wild-type controls were examined regarding Notch3 aggregation in pericytes, the coverage of cerebral vessels by pericytes, pericyte numbers, capillary density, blood–brain barrier (BBB) integrity and the expression of endothelial adherens junction protein using immunostaining and Western blot analysis. We explore that the effect of matrix metalloproteinase-9 mediated blood-brain barrier injury is involved in the pathogenesis of CADASIL and provide a new way of interfering with pericytes for the treatment of CADASIL.

伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病（CADASIL）是由Notch3基因突变引起，突变Notch3的胞外段聚集在脑血管管壁导致平滑肌细胞（SMC）损伤被认为CADASIL发病机制，但在CADASIL小鼠模型中SMC并未受影响，而观察到周细胞损伤。为此，我们提出假说：Notch3ECD致脑周细胞损伤，周细胞分泌基质金属蛋白酶-9（MMP-9），导致内皮细胞紧密连接损伤，破坏血脑屏障引起白质病变，致CADASIL发生。为验证该假说，我们利用CADASIL小鼠模型，荧光共聚焦显微镜观察大脑皮质突变Notch3聚集于周细胞、周细胞缺失及毛细血管上覆盖周细胞数目减少；Western Blot检测缝隙连接、黏着连接及紧密连接蛋白表达减少，提示血脑屏障的完整性破坏，从而探讨MMP-9介导的血脑屏障损伤在CADASIL发病中的作用机制，为周细胞可能成为治疗CADASIL新靶点提供依据。

脑小血管病（CSVD）白质高信号的病理生理机制包括血脑屏障（BBB）受损，通透性增加。神经炎症可能参与CSVD患者BBB的破坏。其中基质金属蛋白9（MMP-9）参与神经炎症性疾病BBB的损伤。此外，慢性脑灌注不足（CCH）与 CSVD 中的BBB功能障碍密切相关。因此，了解血脑屏障损伤的分子机制，对预防CSVD血脑屏障破坏具有重要意义， .研究发现白细胞介素-1β（IL-1β）诱导周细胞分泌MMP-9，通过破坏VE-cadherin、occludin、claudin-5和zonula-occluddin-1的破坏，增加BBB对荧光素钠（Na-F）的通透性。褪黑素降低BBB对Na-F的通透性，抑制粘附蛋白和紧密连接蛋白的破坏。褪黑素还下调MMP-9表达并上调TIMP-1基因表达，降低MMP-9/TIMP-1比值。此外，IL-1β诱导周细胞NF-κB/p65核移位上调MMP-9表达，NF-κB抑制剂PDTC抑制MMP-9表达。然而，NOTCH3抑制剂DAPT明显抑制NF-κB/p65向细胞核的移位，而褪黑素联合DAPT明显抑制NF-κB/p65向细胞核的移位。此外，我们通过永久性双侧颈总动脉闭塞（2VO）建立了CSVD的大鼠模型，以模拟CSVD的慢性低充气状态。结果发现CCH后胼胝体（CC）中BBB的短暂但较严重的破坏。BBB早在术后1天就严重受损，术后3天受损最严重。此外，周细胞丢失与BBB损伤有关，血清蛋白的积累是由CC中内皮转吞作用增加介导的。 RNA测序也显示胞吞基因表达增加。BBB功能障碍通过调节TGF-β/Smad2信号传导导致脑损伤。此外，伊马替尼治疗改善了血清蛋白渗漏、少突胶质细胞祖细胞（OPC）活化、内皮胞吞作用、小胶质细胞活化和异常TGF-β/Smad2信号激活。.我们得出结论褪黑素降低了MMP-9诱导的BBB通透性。褪黑素通过调节周细胞NOTCH3/NF-κB信号通路降低IL-1β诱导的MMP-9的表达和活性，提示周细胞参与BBB功能完整性的调节。周细胞覆盖率降低导致BBB通透性通过内皮胞吞作用增加。伊马替尼通过抑制内皮胞吞作用对BBB完整性起保护作用。维持BBB完整性通过调节CCH后的TGF-β / Smad2信号来改善脑损伤。因此，逆转BBB功能障碍可能是CSVD治疗的一种有效的治疗策略。