组织蛋白酶K介导的Notch1信号通路在老年缺血性血管再生中的作用及分子机制研究

Cysteine protease cathepsin K (CatK) is one of the most potent mammalian elastases. We have previously shown increased expression of CatK in atherosclerotic and neointimal lesions. However, the roles of CatK in pathophysiological angiogenesis are poorly understood in young and aged humans and animals. Young (8-weeks) male CatK+/+ and CatK?/? mice were underwent hindlimb ischemic surgery, and were then processed for functional (blood-flow recovery), morphological and biochemical studies at specific time points to explore the possibility that CatK deficiency impaired ischemia-induced angiogenesis associated with the reduction in Notch1 activation and downstream signaling pathway activity. To explore the mechanisms of aging-related impaired angiogenesis, both old (96-weeks) genotype mice were also applied to examinations. In vitro, we will investigate whether genetic and pharmacological interventions of CatK impair cellular angiogenic action via Notch-1 activation in cultured endothelial cell under hypoxia. We will further investigate the role of CatK in Notch1 activation in several vascular cell lines by a short-interfering RNA against to siCatK and Ad-CystC. Finally, we will investigate the roles of bone-marrow (BM)-derived CatK using bone-marrow transplantation. Our findings will help understanding molecular mechanisms of the impairment of aging-related angiogenesis, and provide new therapeutic approach to improve ischemia-induced neovascularization in aged humans.

治疗老年缺血性疾病成为医学专家亟需解决的难题。Notch信号通路在血管再生过程中发挥关键作用，其激活Notch1是非常重要的一步。CatK在细胞内外参与修饰、分解多种蛋白质。本研究利用CatK和半胱氨酸蛋白酶抑制剂C（CystC）基因缺陷小鼠的下肢缺血性血管再生模型和缺氧应激暴露的内皮细胞模型，探讨CatK介导下Notch1激活以及Notch1-VEGF/VEGFR2信号传导通路调节血管再生的机制。通过基因沉默（siCatK）、基因转染（ad-CystC）等进一步探索CatK调控Notch1激活以及血管再生机制；再通过不同年龄小鼠下肢缺血性血管再生模型，探讨衰老对CatK活性、Notch1激活及Notch1-VEGF/VEGFR2信号传导通路的影响。阐明衰老降低CatK活性、削弱Notch1激活导致血管再生能力降低的机制，为预防和治疗LEAD奠定基础，为研究、开发新疗法和新药物提供新思路。

治疗老年缺血性疾病成为医学专家亟需解决的难题。Notch信号通路在血管再生过程中发挥关键作用，其激活Notch1是非常重要的一步。CatK在细胞内外参与修饰、分解多种蛋白质。本研究利用CatK和半胱氨酸蛋白酶抑制剂C（CystC）基因缺陷小鼠的下肢缺血性血管再生模型和缺氧应激暴露的内皮细胞模型，探讨CatK介导下Notch1激活以及Notch1-VEGF/VEGFR2信号传导通路调节血管再生的机制。通过基因沉默（siCatK）、基因转染（ad-CystC）等进一步探索CatK调控Notch1激活以及血管再生机制；再通过不同年龄小鼠下肢缺血性血管再生模型，探讨衰老对CatK活性、Notch1激活及Notch1-VEGF/VEGFR2信号传导通路的影响。.小鼠制备L-FAL下肢缺血性血管再生模型14天后，应用激光多普勒血流测定仪（laser dopplar blood flowmetry, LDBF）扫描测定双下肢血流，发现不同月龄catk-/-小鼠缺血下肢血流恢复明显弱于野生型组。免疫组织化学染色法发现8周龄catk-/-组缺血下肢毛细血管数量明显少于同龄野生型组。96周龄小鼠中观察到部分catk-/-组小鼠缺血下肢完全萎缩，测定不到血流，说明老化减弱缺血性血管再生能力，CatK基因缺失明显影响缺血性血管再生过程。利用免疫印迹杂交技术检测缺血组织中Notch1、cleaved Notch1、VEGF、Flk1、Akt等磷酸化及蛋白表达。观察到8周龄catk-/-小鼠缺血组织cleaved Notch1、VEGF、Flt1、p-Akt等蛋白表达明显减少；同样在96周龄小鼠缺血组织中也观察到，catk-/-组cleaved Notch1表达明显减少，与野生型组相比具有显著性差异。Notch1表达两组之间无明显差异。说明CatK激活Notch1信号传导通路参与血管再生过程。老化减弱小鼠缺血性血管再生能力，CatK基因缺失明显影响缺血性血管再生过程，Notch1-VEGF/VEGFR2信号通路可能参与此过程，提示catK可能介导的Notch1激活。分析Notch1活化的整个过程不难发现Notch1发挥作用的关键环节在Notch1蛋白水解被激活步骤。.本研究阐明了衰老降低CatK活性、削弱Notch1激活导致血管再生能力降低的机制，为预防和治疗下肢缺血奠定基础，为研究、开发新疗