Adventitial delivery of nanoparticles encapsulated with 1α, 25-dihydroxyvitamin D ₃ attenuates restenosis in a murine angioplasty model

Abstract

Percutaneous transluminal angioplasty (PTA) of stenotic arteriovenous fistulas (AVFs) is performed to maintain optimal function and patency. The one-year patency rate is 60% because of venous neointimal hyperplasia (VNH) and venous stenosis (VS) formation. Immediate early response gene X-1 ( Iex-1) also known as Ier3 increases in response to wall shear stress (WSS), and can cause VNH/VS formation in murine AVF. In human stenotic samples from AVFs, we demonstrated increased gene expression of Ier3. We hypothesized that 1α, 25-dihydroxyvitamin D ₃, an inhibitor of IER3 delivered as 1α, 25-dihydroxyvitamin D ₃ encapsulated in poly lactic- co-glycolic acid (PLGA) nanoparticles loaded in Pluronic F127 hydrogel (1,25 NP) to the adventitia of the stenotic outflow vein after PTA would decrease VNH/VS formation by reducing Ier3 and chemokine (C–C motif) ligand 2 ( Ccl2) expression. In our murine model of AVF stenosis treated with PTA, increased expression of Ier3 and Ccl2 was observed. Using this model, PTA was performed and 10-μL of 1,25 NP or control vehicle (PLGA in hydrogel) was administered by adventitial delivery. Animals were sacrificed at day 3 for unbiased whole genome transcriptomic analysis and at day 21 for immunohistochemical analysis. Doppler US was performed weekly after AVF creation. At day 3, significantly lower gene expression of Ier3 and Ccl2 was noted in 1,25 NP treated vessels. Twenty-one days after PTA, 1,25 NP treated vessels had increased lumen vessel area, with decreased neointima area/media area ratio and cell density compared to vehicle controls. There was a significant increase in apoptosis, with a reduction in CD68, F4/80, CD45, pro-inflammatory macrophages, fibroblasts, Picrosirius red, Masson’s trichrome, collagen IV, and proliferation accompanied with higher wall shear stress (WSS) and average peak velocity. IER3 staining was localized to CD68 and FSP-1 (+) cells. After 1,25 NP delivery, there was a decrease in the proliferation of α-SMA (+) and CD68 (+) cells with increase in the apoptosis of FSP-1 (+) and CD68 (+) cells compared to vehicle controls. RNA sequencing revealed a decrease in inflammatory and apoptosis pathways following 1,25 NP delivery. These data suggest that adventitial delivery of 1,25 NP reduces VNH and venous stenosis formation after PTA.