The Effects Of Cathepsin K On Ischemia Induced Neovascularization In Mice Of Advanced Age

Objective:As the aging of society continues to deepen,the incidence of ischemic diseases such as lower extremity atherosclerotic disease(LEAD)increases year by year.Cathepsin K(CatK)is a member of the family of cysteine protease,widely distributed in cardiovascular cells and inflammatory cells.CatK is the main elastase and collagenase in the body.It is involved in extracellular matrix degradation,cell migration,proliferation,apoptosis and other biological function.It is well known that the Notch signaling pathway plays an important role in neovascularization.Aging limits angiogenesis and LEAD prognosis,but the mechanisms remain largely undefined.We use aged CatK+/+ and CatK-/mice were underwent hindlimb ischemic surgery,proved CatK-mediated Notch1 activation-based mechanism that plays an essential role in angiogenic actions.Our findings will provide new therapeutic target that improve ischemia-induced neovascularization in aging.Methods:Mouse model of revascularization is that wild-type(CatK+/+)and Cathepsin K gene knockout(CatK-/-)aged mice(96weeks)were subjected to unilateral hindlimb ischemic surgery.Hindlimb blood flow was determined by using a laser doppler blood flow(LDBF)analyzer.The number and density of new capillaries in ischemia was determined by using immunohistochemical staining.Two groups of mouse aortic vessels were cultured in vitro,and observed condition aortic-ring microvessel sprouting.Flow cytometry observed the distribution of EPCs in bone marrow and peripheral blood fluid,and analyzed EPCs mobilization and homing ability.Western blot(Western blotting,WB)to detect the expression of signaling proteins such as Notchl,c-Notchl(cleaved Notchl),vascular endothelial growth factor(vascular endothelial growth factor,VEGF)and Akt in ischemia tissue and EPCs.Result:1.On day 4,the blood flow of ischemic lower limbs was beganing gradually recovered in two groups,and CatK-/-group had markedly weaker blood flow recovery than wild-type group(P<0.05).2.Immunohistochemical staining observed that CatK-/-group exhibited significantly low of number and capillary density compared wild-type group.(P<0.05).3.The area of aortic-ring microvessel sprouting in CatK-/-group was significantly less than the wild-type(P<0.05).4.Western blot analysis revealed that the levels of c-Notchl,p-Akt and VEGFwere markedly declined in CatK deficient mice(P<0.05).5.The numbers of CD31+/c-Kit+EPC-like cells were observed by flow cytometry.The numbers of CD31+/c-Kit+EPC-like cells in peripheral blood were significantly lower in CatK-/-group than in wild-type group(P<0.05),however,there was no significant difference between in bone marrow two groups.6.The expression of c-Notchl and Notchl in EPC was observed,and the level of c-Notchl was significantly reduced in CatK-/-group compared with wild-type group(P<0.05).Conclusion:Cathepsin K may activate Notchl and downstream signaling pathway,regulate the function of endothelial cells and endothelial progenitor cells,and affect the ischemia-induced neovascularization in mice of advanced age.