IRS-1/KLF-4-Mediated Vascular Smooth Muscle Cells Differentiation Is Critical for Arteriovenous Fistula Maturation in a Mouse Model

Background: We previously reported that insulin receptor substrate-1 (IRS-1) is required to maintain differentiation of vascular smooth muscle cells (VSMCs) via regulating KLF-4/P53 complex formation. High glucose or IRS-1 knockout (KO) decreased p53 levels resulting in decreased association of p53 with KLF-4. However, these studies were performed in aortic VSMCs only. In this study, we generated IRS-1 smooth muscle (SM) specific KO mice and created arteriovenous fistula (AVF) to study the functions of IRS-1 in AVF maturation in a mouse model. Methods: Aged 14-16 weeks of IRS-1 SM KO mice were used to create AVF following an established protocol. An end-to-side anastomosis was created between the jugular vein and carotid artery. High-resolution micro-computed tomography (micro-CT) was performed in vivo on days 21 post-surgery. After imaging, animals were sacrificed and AVF tissues were collected for histological analysis and IHF staining. Results: CT images of AVF section showed severe vessel narrow down in both venous segment and arterial segment of AVF (highlighted) (Figure 1 right panel), compared to the same location in wild type (WT) animal (left panel). HE staining results clearly showed the severe blockage caused by neointimal hyperplasia and/or thrombus in venous segments from anastomosis to around 800 um distal position. In addition, IHF staining confirmed the KO IRS-1 signal in SM only but not in endothelial cells and adventitia. Further, like observations obtained from diabetic animals, KLF-4 expression in VSMCs was dramatically increased when IRS-1 was knockout in SM. Conclusion: Eliminating IRS-1 in SM significantly enhanced neointimal hyperplasia in venous segments of AVF. Our results support the hypothesis that the previously described inhibition of IRS-1 in the setting of diabetes or uremia could be the pathway responsible for the more aggressive AVF stenosis that likely occurs in these clinical settings. Funding: NIDDK SupportFigure 1. CT scan images of AVF in wild type (WT) mouse and IRS-1 smooth muscle (SM) knockout (KO) mouse