Introduction: Autologous human cardiac stem/progenitor cell (hCPC) therapy is a novel treatment for patients with cardiomyopathy. However, the CPCs of elderly patients, who are most susceptible to myocardial disease, are senescent. Harsh in vitro modifications lead to decreased survival capacity of these senescent CPCs. Thus, it is vital to rejuvenate aged CPCs by reversing entry into senescence.
Hypothesis: Knockdown of p16INK4A, a cyclin dependent kinase inhibitor, will increase hCPC proliferation and survival, and rejuvenate aging hCPCs to a youthful phenotype.
Methods: Adult c-Kit+ hCPCs were sorted from atrial appendages of consenting patients during open-heart surgery at Albany Medical Center. Knockdown of p16INK4A was performed using shRNA lentiviral particles. Cell survival, proliferation, and reactive oxygen species (ROS) generation were examined by FACS analysis. QPCR and Western blot were performed to elucidate the molecular mechanisms underlying the effects of the knockdown.
Results: Data from qPCR and Western blots showed that upregulation of p16INK4A is associated with hCPC senescence. Both cell survival and proliferation were significantly increased in hCPCs infected with lentivirus expressing p16INK4A shRNA as compared to control hCPCs. The knockdown of p16INK4A also induced antioxidant properties. At basal cell metabolism, ROS generation was decreased by 50% in the knockdown hCPCs as compared to the controls. After exposure to oxidative stress, ROS generation was decreased by 25%. Anti-oxidant and cytoprotective genes such as HO-1, CYGB, phospho-p65, and total p65 were upregulated after the p16INK4A knockdown. A preponderance of pro-survival cytokines, such as VEGF, FGF family proteins, and interleukin family proteins, were upregulated upon knocking down p16INK4A.
Conclusions: Our data show that the p16INK4A knockdown induces a cytoprotective effect by lowering ROS production and increasing expression of anti-oxidant and pro-survival cytokine genes in aged hCPCs. This suggests that genetic knockdown of p16INK4A may play a significant role in inducing antioxidant effects and extending lifespan of aged hCPCs, which may enhance cell viability in the use of autologous hCPC therapy for repair of infarcted myocardium.