肥胖影响缺血性血管再生新机制：CatK介导Notch1激活及其作用机制

Emerging evidence suggests that γ-secretase-independent Notch1 activation involves in pathological conditions. Cysteine protease cathepsin K (CatK) is one of the most potent proteolytic enzymes in the intracellular and extracellular spaces. Obesity induced ischemic vascular disease, but the effects and mechanisms of obesity on neovascularization remain unclear. Here, we investigated the hypothesis that Obesity-related decline in ischemia-induced neovascularization was associated with the reduction of CatK-mediated Notch1 activation in response to ischemic stress. Male CatK+/+ and CatK-/- mice fed on high-fat diet for 6 months received were underwent ischemic surgery, and were then processed for functional, morphological and biochemical studies to explore the possibility that obesity effects ischemia-induced angiogenesis associated with changed CatK activity and Notch1 activation. In vitro, we will investigate CatK inhibtion by genetic and pharmacological interventions could impair cellular angiogenic action via Notch-1 activation in endothelial cell under hypoxia. Furthermore, we used a short-interfering RNA against to siCatK and Ad-CystC to examine the role of CatK in Notch1 activation in several vascular cell lines. Finally, we will attempt to investigate the roles of bone-marrow (BM)-derived CatK by bone-marrow transplantation. This study will provide new evidences for prevention of obesity-induced ischemic vascular disease and proangiogenic therapy.

肥胖严重影响血管再生及下肢缺血性疾病，但其作用机制尚不清楚。组织蛋白酶K(CatK)在细胞内外参与修饰/分解多种蛋白质。Notch信号通路在血管再生过程中发挥重要作用，其中激活Notch是关键一步。本研究利用CatK和半胱氨酸蛋白酶抑制剂C（CystC）基因缺陷小鼠肥胖-下肢缺血性血管再生模型，应用免疫印迹、流式细胞术、骨髓移植及Proteome ProfilerTM Array等多种方法，探讨肥胖条件下CatK介导的Notch1活化及Notch1-VEGF/VEGFR2信号传导通路调控血管再生的作用机制；再利用培养内皮祖细胞和缺氧应激内皮细胞模型，通过基因沉默、基因转染等技术，进一步探索CatK调控Notch1激活及肥胖性血管再生机制。本研究阐明肥胖降低CatK活性、削弱Notch1激活导致血管再生能力降低的机制，将为预防、治疗LEAD奠定基础，为研究及开发新疗法、新药物提供新的思路。

随着经济社会发展和生活习惯的改变，全球肥胖人群呈逐年攀升之势。肥胖是代谢综合征、心血管疾病的主要危险因素，也是影响下肢动脉硬化性疾病(lower extremity atherosclerotic disease, LEAD)的主要原因之一。然而，肥胖等病理生理状态下血管再生能力减退的分子机制尚不清楚。组织蛋白酶K(cathepsin K, catK)是机体最强大的弹性蛋白酶和胶原酶，人们长期认为包括catK在内的组织蛋白酶仅在溶酶体内起作用，但最近研究表明其在细胞膜内外均显示蛋白酶活性，参与血管再生过程。本研究首次利用CatK基因敲除小鼠肥胖-下肢缺血性血管再生模型及缺氧应激暴露的内皮细胞模型，探讨CatK在肥胖影响缺血性血管再生中的作用及其机制。研究结果表明：高脂肪饲料显著增加小鼠体重及血浆CatK浓度；利用雄性野生型(CatK+/+)和CatK基因敲除型(CatK–/–)肥胖小鼠建立下肢缺血性血管再生模型，根据研究方案测定不同时间段缺血下肢血流恢复情况，发现CatK基因敲除明显改善缺血下肢血流灌流及毛细血管密度；腹腔注射CatK阻断剂方法也观察到类似的改善作用。同时观察到，CatK基因缺损减少了TLR-2、MCP-1、SDF-1的mRNA表达；cleaved Notch1、caspase 3、caspase 8、Bax等信号蛋白表达水平。细胞实验采用基因沉默(siRNA-CatK)和基因转染(Ad-CatK)方法，证实catK参与切割Notch1配体结合体、激活Notch1信号通路，调控内皮细胞的迁移、增殖等生物行为。以上结果提示，肥胖条件下CatK激活Notch1、活化caspase3/8调节机制，调节肥胖性缺血性血管再生过程，此研究将为合并肥胖等代谢异常LEAD的细胞治疗及预后提供重要的理论依据。