Supplemental Oxygen Reverses Hypoxia-induced Smooth Muscle Cell Proliferation by Modulating HIF-alpha and VEGF Levels in a Rabbit Arteriovenous Fistula Model

Abstract

Numerous mechanisms for the formation of intimal hyperplasia have been proposed but none have been proven or accepted. Our research focuses on the potential role of hypoxia-inducible factors (HIFs), vascular endothelial growth factor (VEGF), and platelet-derived growth factors as well as the extracellular signal-regulated kinase (ERK), phosphatidylinositide 3-kinase /protein Kinase B (PI3-K/AKT) pathway in hypoxia-mediated intimal hyperplasia processes. We hypothesize that HIF and VEGF will be downregulated with supplemental oxygen in our arteriovenous fistula rabbit model. Rabbits were randomized into different experimental groups with varying oxygen exposure (21% O2 or 30% O2) and receipt of surgery (surgery with fistula formation, no surgery, or sham operation with skin incision only). Plasma samples were collected at designated intervals in which cytokines and smooth muscle cell proliferation were measured. In addition, cell specimens were exposed to hyperoxic, normoxic, and hypoxic environments with cytokines measured at various time points. Placement of an arteriovenous fistula resulted in hypoxia-induced HIF stabilization with a concurrent increase in VEGF levels. There was a 4.2-fold induction in HIF-1α levels in animals that were placed in normal air after surgery when compared with animals that were exposed to hyperoxic air. Also, VEGF level significantly increased after surgery in the normoxic group, reaching a maximum of 959 pg/mL. Plasma VEGF levels in the surgery and supplemental oxygen group were significantly lower than the normoxic surgery group with almost a 45% reduction in plasma VEGF levels (524 pg/mL). Activation of VEGF receptors on smooth muscle cells through ERK1 and AKT pathways resulted in significant smooth muscle cell proliferation and migration. These effects are dramatically reduced in animals that are exposed to a hyperoxic environment of 30% oxygen. Our results suggest that short-term administration of supplemental oxygen inhibits HIFs and VEGF signaling to reduce smooth muscle proliferation in the local blood vessel. These results provide strong support for the therapeutic use of supplemental oxygen after arterial surgery to reduce intimal hyperplasia. These findings also provide a nidus for future clinical trials to determine whether this is clinically applicable in humans.