Molecular Mechnism Of Bradykinin On Proliferation,Migration And Apoptosis In Human Cardiac C-Kit~+ Progenitor Cells

It is believed that human cardiac c-Kit⁺ progenitor cells?or stem cells?are an ideal cell source for myocardial regeneration in patients with ischemic cardiomyopathy.Although the promising results of improving heart function are observed in patients with transplantation of human cardiac c-Kit⁺ progenitor cells,there are several challenges in cell transplantation for myocardial repair using cardiac progenitor cells or stem cells,including the very low survival rate,limited cardiomyocyte differentiation,etc.Understanding the regulation of cellular biological functions in cardiac c-Kit⁺ progenitor cells may be important to find out the potential strategy to increase survival rate,cardiomyocyte differentiation,etc in cell therapy for myocardial repair.The aims of my PhD project were to 1)investigate the regulation of cell proliferation and migration by bradykinin?an endogenous cardiac protection factor?,2)clarify the molecular mechanisms of bradykinin,and 3)explore whether bradykinin has anti-oxidation and anti-apoptosis in cultured human cardiac c-Kit⁺progenitor cells using multiple cell biology and molecular biological technologies.We found that gene and protein of B2Rs,but not B1Rs,are expressed in human cardiac c-Kit⁺ progenitor cells.Bradykinin?1-10 nM?stimulated cell growth and migration in a concentration-dependent manner.Bradykinin significantly promoted G0/G1 phase into G2 and S phase.Western blot analysis showed that bradykinin increased pAkt and pERK1/2 as well as cyclin Dl,which were countered by the B2Rs antagonist HOE140 or silencing B2Rs,or by the PI3K inhibitor LY294002,the PLC inhibitors U73122 and neomycin,and/or the PKC inhibitor chelerythrine and the MAPK inhibitor PD98059.Further experiments demonstrated that bradykinin increased intracellular free Ca²⁺(Ca²⁺i)level by activating PLC with two components,a transient component mediated by IPR3,which is prevented by IP3R3 blocker araguspongin B or silencing IP3R3,and a sustained component mediated by SOCE channels,which is prevented by SOCE channel blockers La3+ and SKF-96365 or silencing the SOCE components TRPC1,Stim1 or Orai1.It is interesting that bradykinin-induced increase of cell proliferation and migration were partially decreased in cells with silencing IP3R3,TRPC1,Stiml or Orail,and bradykinin-induced increase of pAkt and pERK1/2 as well as cyclin D1 was also reduced by silencing these genes,suggesting that increase of Ca²⁺i is related to the cell proliferation and migration stimulated by bradykinin.Moreover,bradykinin reduced ROS production induced by H2O2,and therefore showed anti-apoptosis by increasing the anti-apoptotic protein Bcl-2 and decreasing the apoptotic proteins Bax in human cardiac c-Kit⁺ progenitor cells.Collectively,the results of my PhD project demonstrate for the first time that bradykinin stimulates cell proliferation and migration by increasing Ca²⁺i levels via activating IP3R3 and SOCE channels and activating pAkt,pERK1/2 and/or cyclin D1 in human cardiac c-Kit⁺ progenitor cells.It remains further study whether bradykinin can be used to make the preconditioning treatment of human cardiac c-Kit⁺ progenitor cells for myocardial repair in patients with ischemic cardiomyopathy.