Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

Abstract

Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia inducible factor-1 alpha at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.

New Insights into Dialysis Vascular Access: Molecular Targets in Arteriovenous Fistula and Arteriovenous Graft Failure and Their Potential to Improve Vascular Access Outcomes

“…These can be divided into their mechanisms of action based on inflammation, oxidative stress, fibrosis, and endothelial dysfunction (several potential targets are discussed in preceding sections and below) (Figure 3). Recently, experimental studies have demonstrated that anti-VEGF-A therapy using bevacizumab treatment before the placement of experimental AVF is protective for formation of venous stenosis (Figure 4) (73). In a small group of patients who received intravitreal treatment of bevacizumab for macular degeneration before the placement of a hemodialysis vascular access, the Misra laboratory showed that the median patency was improved more than two-fold compared with control group (paper under review).…”

“…In addition, in AVGs there is an active macrophage cell layer lining on the polytetrafluoroethylene graft itself (28,30). Several publications have demonstrated that neointimal hyperplasia is related to endothelial cell activation, activation, and infiltration of circulating inflammatory cells, along with production of chemokines and growth factors that regulate migration of smooth muscle cells and myofibroblasts into the intimal layer of the vessel (23,31). Moreover, several perivascularly delivered antiproliferative therapies have been evaluated in AVG (32,33), but none of these studies have advanced past early phases in clinical trials.…”

c-Kit signaling determines neointimal hyperplasia in arteriovenous fistulae

“…These include vein configuration (22), hemodynamics stress (39), inflammation (20), and preexisting diseases like diabetes (40) and kidney failure (26). The above conditions induce de novo accumulation of growth factors such as VEGF (48), plateletderived growth factor (41), fibroblast growth factor (42), and transforming growth factor-␤ (18) and their receptor tyrosine kinases (RTKs) in the outflow vein, which ultimately increase the proliferation, migration, and survival of neointimal cells and the subsequent thickening of the vessel wall. Recently, vascular expression of stem cell factor (SCF) and its receptor c-Kit have also been implicated in A-V fistula NIH (8).…”

Hyperglycemia-Induced Modulation of the Physiognomy and Angiogenic Potential of Fibroblasts Mediated by Matrix Metalloproteinase-2: Implications for Venous Stenosis Formation Associated with Hemodialysis Vascular Access in Diabetic Milieu

“…To assess the efficacy of glycemic changes upon the invasiveness of fibroblasts and myofibroblasts, the expression of the constitutive matrix regulatory and signaling moiety of Mmp-2 was silenced with lentivirus (LV)-mediated shRNA transduction [4,17]. Briefly, AKR-2B cells were seeded into 10-cm dishes and grown to about 50-60% of confluency.…”

“…AVFs have been used for more than 50 years and are prone to venous stenosis formation [2]. Although the exact mechanism for venous stenosis is not yet clear, several major factors have been hypothesized including hypoxia, shear stress, oxidative stress and inflammation [3,4]. As a consequence, these factors lead to an accumulation of macrophages, fibroblasts and smooth-muscle cells in the vessel wall, that lead to stenosis formation [5].…”