A Color-Based Image Segmentation Pipeline Depicts Collagen, Versican, And Secretory Vascular Smooth Muscles Constituting The Wall Of Human Explantedarteriovenous Fistulas

Background: Arteriovenous Fistula (AVF) is the lifeline of patients with End-Stage Kidney Disease (ESKD) on hemodialysis. AVF undergoes profound remodeling to accommodate arterial pressure and flow, a process called AVF maturation or arterialization of the vein. In the US, more than 50% of AVF fail to mature. Despite the central importance of this process in functional AVF, there is a dearth of studies characterizing the components of walls of the mature AVFs in humans. Methods: The AVF explants were probed with Masson Trichrome, Sirius Red, and Versican, a predominant proteoglycan in the vessel. This was followed by a color-based segmentation pipeline with a machine-learning approach to quantify the components of AVF explants. The state of vascular smooth muscle cells (VSMCs) was characterized using specific markers. Results: Twenty-one patients had AVFs explanted for aneurysm (71.42%), pseudoaneurysm, stenosis, or aesthetic reasons (each in 9.52%). Neointimal hyperplasia characterized by intimal thickening was present in all AVFs. Other features included calcification, inflammatory infiltrate, needle track injury, and thrombus. Collagen was deposited concentrically in the explanted AVFs with aneurysms and in an eccentric manner in the AVFs with pseudoaneurysms (Fisher’s exact test, P= 0.025). Quantitative histological analysis showed that in the AVF wall, collagen, proteoglycan, and VSMCs, constituted 27.78%, 31.45%, and 19.80% of the wall, respectively. Compared to VSMC, the collagen and versican were in the ECM higher by 1.40-fold and 2.76-fold, respectively (p <0.001). Compared to Ki67 expression, VSMCs showed prominent expression of MYH11 (P= 0.0016), α-smooth muscle actin (P= 0.0023), and Calponin (P= 0.006), all markers of differentiated VSMCS with secretory phenotype. Conclusion: This is the first study elucidating the composition of mature AVF in humans and shows a preponderance of collagen and versican as components of AVF walls. The VSMCs demonstrated secretory phenotype in the wall of the explanted AVFs. This study supports future investigation in regulating collagen and versican deposition and secretory VSMCs in the maturation of the outflow vein of AVF.