Abstract 17966: The Roles of S-Phase Kinase-Associated Protein-2 and Mitochondrial Dysfunction in the Aging-Related Endothelial Progenitor Cell-Cycle Retardation

Abstract

Background Deceleration of cell-cycle progression is a prominent feature of cellular senescence. S-phase kinase-associated protein-2 (Skp2) is a subunit of SCF Skp2 ubiquitin ligase responsible for ubiquitination and subsequent degradation of many cell-cycle regulators, such as cyclin-dependent kinase inhibitors p27 Kip1 and p21 Cip1 , and therefore is a key mediator for G1-S progression. However, it remains poorly understood whether Skp2 regulation and mitochondrial dysfunction are associated with endothelial progenitor cell (EPC) senescence. Materials and Methods In vitro EPC senescence was induced by serial passages. Bone marrow mononuclear cells from bone marrows (BM-MNC) of young (3 months) and old (24 months) rats were served as in vivo model of aging. Results Senescent EPC had a significantly longer cell doubling time, along with a typical increase of senescence-associated β-galactosidase (SA-βGal) activity and the enhanced expression of aging-related proteins, such as p21 Cip1 and p16 INK4a . In line with a slow-down of S-phase entry, aging EPC demonstrated a graded downregulation of Skp2 expression. Compared with young EPC, old EPC demonstrated a more elongated mitochondrial network along with a reduction of ATP production. Mitochondrial fission protein Fis-1 was found to be remarkably reduced in old EPC and silencing Fis-1 in young EPC resulted in an elongated mitochondrial network and a reduced ATP level similar to those of old EPC. However, Skp2 levels were not affected by Fis-1 silence, suggesting a mitochondria-independent regulation. Surprisingly, adenovirus-mediated Skp2 expression significantly reversed SA-βGal activity and almost completely rescued old EPC proliferation back to the level of young EPC, though Fis-1 levels and ATP production were not affected. Bone marrow blood from old rats had significantly reduced CD34 + /KDR + cells. Importantly, Skp2 expression in BM-MNC from old rats was also able to reverse SA-βGal activity and cell proliferation. Conclusion Skp2 downregulation and mitochondrial dysfunction develop with EPC senescence. Skp2 expression reverses SA-βGal and cell proliferation in old EPC and in old rat BM-MNC. Skp2 is at least one of the important factors that may rejuvenate senescent EPC.