Anti Human CX3CR1 VHH Molecule Attenuates Venous Neointimal Hyperplasia of Arteriovenous Fistula in Mouse Model.

Abstract

Fractalkine receptor 1 (CX3CR1) mediates macrophage infiltration and accumulation, causing venous neointimal hyperplasia (VNH)/venous stenosis (VS) in arteriovenous fistula (AVF). The effect of blocking CX3CR1 using an anti-human variable VHH molecule (hCX3CR1 VHH, BI 655088) on VNH/VS was determined using a humanized mouse in which the human <i>CX3CR1</i> (<i>hCX3CR1</i>) gene was knocked in (KI). Whole-transcriptomic RNA sequencing with bioinformatics analysis was used on human stenotic AVF samples, C57BL/6J, <i>hCX3CR1</i> KI mice with AVF and CKD, and in <i>in vitro</i> experiments to identify the pathways involved in preventing VNH/VS formation after hCX3CR1 VHH administration. Accumulation of CX3CR1 and CD68 was significantly increased in stenotic human AVFs. In C57BL/6J mice with AVF, there was increased <i>Cx3cr1, Cx3cl1, Cd68</i>, and <i>Tnf-α</i> gene expression, and increased immunostaining of CX3CR1 and CD68. In hCX3CR1-KI mice treated with hCX3CR1 VHH molecule (KI-A), compared with vehicle controls (KI-V), there was increased lumen vessel area and patency, and decreased neointima in the AVF outflow veins. RNA-seq analysis identified TNF-<i>α</i> and NF-<i>κ</i>B as potential targets of CX3CR1 inhibition. In KI-A-treated vessels compared with KI-V, there was decreased gene expression of <i>Tnf-</i> <i>α</i>, <i>Mcp-1</i>, and <i>Il-1</i> <i>β</i>; with reduction of <i>Cx3cl1</i>, NF-<i>κ</i>B, and <i>Cd68</i>; decreased M1, Ly6C, smooth muscle cells, fibroblast-activated protein, fibronectin, and proliferation; and increased TUNEL and M2 staining. In cell culture, monocytes stimulated with PMA and treated with hCX3CR1 VHH had decreased <i>TNF-</i> <i>α</i>, <i>CD68</i>, proliferation, and migration. CX3CR1 blockade reduces VNH/VS formation by decreasing proinflammatory cues.