Abstract 3750: Endothelial NO Synthase Uncoupling Impairs Formation and Induces Senescence of Endothelial Progenitor Cells in Mild Hyperhomocysteinemic Mice
Mild hyperhomocysteinemia (H-Hcy) is associated with increased risk of cardiovascular disease; however, the exact mechanism whereby H-Hcy leads to vascular damage is unclear. Using a mouse model heterozygous for the gene deletion of methylene tetrahydrofolate reductase (Mthfr +/−) mimicking mild H-Hcy in humans, we previously showed that the heterozygous mice present endothelial dysfunction as demonstrated by reduced endothelium-dependent vasodilatation. Recent studies have shown that cardiovascular risk factors such as hypertension are inversely correlated with endothelial progenitor cell (EPC) number and function. We hypothesized that EPC formation would be impaired in Mthfr +/− mice and would be correlated with the endothelial dysfunction. Bone marrow-derived mononuclear cells (BM-MNCs) were obtained by flushing tibia and femur of wild-type or Mthfr +/− mice. BM-MNCs (1.5x106) per ml were plated on cell culture dishes for 7 days. EPCs were characterized by dual-positive staining for both AcLDL-Dil and BS-1. We found that mild H-Hcy reduced the ability of adherent BM-MNCs to differentiate into EPCs (p<0.001 vs wild-type mice) which was associated with an increase by 30% of the phosphorylation of p38MAPK in Mthfr +/− (p<0.05 vs wild-type mice). Reduced EPCs numbers and activity were recently associated with EPCs senescence. After ex vivo culture, EPCs became senescent as determined by acidic β-galactosidase staining. Mild H-Hcy in Mthfr +/− increased EPC senescence by 80% (p<0.005) and decreased Akt activation by 44% (p<0.05 vs wild-type). When BM-MNC, during their differentiation, were treated with sepiapterin, which is a precursor of tetrahydrobiopterin that is a co-factor for nitric oxide (NO) synthesis, EPC senescence was significantly reduced in Mthfr +/− compared to the untreated mice (p<0.05). Taken together, these results show that mild H-Hcy attributable to heterozygous Mthfr deficiency impairs EPC formation and increases EPC senescence by uncoupling endothelial NO synthase, leading to endothelial cell dysfunction. These results explain in part the endothelial dysfunction of resistance arteries in this mouse model of human disease.