The Role of Hypoxia In Venous Neointimal Hyperplasia In Hemodialysis Grafts

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PROJECT SUMMARY/ABSTRACT: Nearly 600,000 patients in the US have end stage renal disease (ESRD), a population expected to double in the next decade. The long-term goal of this current proposal and research program is to improve the care of patients with ESRD, the vast majority of who use long-term hemodialysis as their mode of renal replacement therapy. These patients require highly functioning vascular access for optimal therapeutic adequacy. Hemodialysis vascular access failure is frequently from venous stenosis secondary to neointimal hyperplasia (VNH). Our preliminary data demonstrate that outflow veins treated with AMSCs plus bevacuzimab have a significant increase in lumen vessel area and a decrease in the ratio of neointima/media + adventitia compared to controls. This effect is associated with a reduction in PPAR-γ gene expression. Furthermore, we show that bevacuzimab localizes to the endothelium and not the adventitia where AMSCs are transplanted and thus will not disrupt AMSC function. VEGF-A staining of this tissue demonstrates a reduction in sub-endothelial VEGF- A where bevacuzimab is localized. We used THP-1 monocytes (cell line from acute monocytic leukemia patients) to simulate the in vivo findings of monocyte to macrophage differentiation under hypoxia, which shows that THP-1 monocytes differentiate to macrophages via an increase in PPAR-γ. When monocytes are co-cultured with AMSCs plus bevacuzimab and subjected to hypoxic stress, there is a decrease in differentiation to macrophages with a reduction in PPAR-γ gene expression compared to controls. In aggregate, these data support the following CENTRAL Hypothesis, which will be tested in the present revised competitive renewal: Adventitial transplantation of AMSCs to the outflow vein of AVF combined with bevacuzimab therapy reduces PPAR-γ gene expression in monocytes leading to decreased macrophage differentiation and subsequent VNH formation. To test our hypothesis, we have developed three specific aims: SPECIFIC AIM 1: Evaluate the efficacy of AMSCs combined with bevacizumab on reducing VNH using murine and porcine models. Optimal bevacizumab combination with AMSCs will be determined in a murine model and next validated in a preclinical porcine model of AVF with CKD. The pathological and radiological evaluation will be carried out in the tissue samples to assess long-term patency and vascular remodeling. SPECIFIC AIM 2: Determine in vitro role(s) of AMSCs combined with bevacuzimab on reducing hypoxia-induced up-regulation of PPAR-γ in monocytes leading to a reduction in macrophage differentiation. Initially, the effect of hypoxia on expression of PPAR-γ in monocytes will be examined. Next, experiments will be conducted using co-culture with AMSCs to override the negative effect of PPAR-γ caused by hypoxia followed by administering bevacuzimab. The outcomes will include the expression of genes responsible for inflammation and vascular remodeling as well as cellular function. These results will be validated in tissue samples from Aim 1. SPECIFIC AIM 3: Assess in vivo role(s) of PPAR-γ using genetic approaches on VNH formation. We will use monocyte specific conditional knockout of PPAR-γ in mice with AVF and CKD and examine the effect on venous stenosis formation with gene and protein expression of pro-inflammatory cytokines. Next, we will determine the role(s) of bevacuzimab plus monocyte specific conditional knockout of PPAR-γ mice on VNH.