Senescence in the Arteriovenous Fistula (AVF): Early Development and the Prosenescent Effects of Heme Treatment In Vivo and In Vitro

Background: Our prior studies demonstrated a senescent phenotype in the rat AVF at 1 and 2 weeks after creation, as measured by elevated p16 and p21 mRNA levels, increased senescence-associated-β-galactosidase (SA-β-Gal) activity, and a prominent senescence associated secretory phenotype (SASP). We also showed that chronic treatment with hemin reduced AVF flow, the latter hypothesized to be a pro-senescent effect of this treatment. We now examine whether senescence occurs at an earlier timepoint in the AVF, and if heme treatment induces markers of senescence in the vasculature of rats with AVFs, and in human umbilical vein endothelial cells (HUVECs). Methods: Subtotal nephrectomy was performed in rats after which an AVF was created by anastomosing the femoral artery and vein. At 3 days after AVF creation, AVF veins were assessed for expression of cell cycle inhibitors (p16 and p21 mRNA), and SASP factor expression. In additional studies, the effects of heme (hemin) treatment were assessed in the aortas of rats with AVFs and in HUVECs using established measurements. Results: 3 days after AVF creation, p16 and p21 mRNA levels were markedly elevated in AVF veins compared with sham veins and was accompanied by the appearance of a robust SASP (SASP factors such as TNF-a, IL-6, CCL2, IL-1β, CINC-1, and PAI-1 mRNA were all induced). Three-week heme treatment in rats with AVFs induced SASP factor mRNA expression (TNF-a, IL-6, CCL2, IL-1b, SDF-1, TF, CCL5) in the aorta. In HUVECs, 16-hour heme treatment increased p16 protein expression, decreased lamin-B1 protein expression, and significantly increased SA-β-Gal activity and SASP factor mRNA expression - all effects consistent with a senescent phenotype. Conclusion: We demonstrate that the rat AVF in the presence of CKD exhibits a senescent phenotype as early as 3 days after AVF creation. We also identify heme as a novel inducer of senescence in vitro and in vivo. Intravascular hemorrhage is known to be a driver of atherosclerosis, putatively through the effects of free heme. Neoangiogenesis is also known to occur in both rodent and human AVFs, and this may predispose to intramural hemorrhage. We speculate that, in this manner, heme may contribute to a senescence phenotype in the AVF and, in turn, neointimal hyperplasia and AVF dysfunction. Funding: NIDDK Support