Abstract 1159: Harnessing Limb Revascularization With Synthetic Probiotics

Abstract

Peripheral arterial disease (PAD) is a critical vascular condition that results in impaired blood flow leading to chronic limb threatening ischemia (CLTI), tissue necrosis and oftentimes to lower-limb amputation. This is largely due to suboptimal revascularization and inflammatory programs, that fail to instigate neovascularization at sites of vessel stenosis. The incidence of CLTI is on the rise but yet there is a paucity of effective treatment options for patients. We hypothesized that the coordinated action of regenerative angiogenesis and immunosurveillance would likely stimulate effective revascularization in the ischemic leg. The neuroimmune cue, Netrin-1 has been shown to possess such properties. We have shown that Netrin-1 can assist atherosclerosis onset by modulating macrophage chemotaxis and shown its critical role in modulating vascular remodeling. Here we show that the conditional knock-down of Netrin-1 in myeloid cells (Ntn1 flox/flox LysM cre+ ), protects mice from revascularization following femoral artery ligation compared to WT littermates. The deletion of Netrin-1 in endothelial cells nor vascular smooth muscle cells was insufficient to produce such effects. Notably, in human amputated tissue sections, the expression of Netrin-1 was repressed at sites proximal to amputated sites compared to distal regions with preserved blood flow. Tissue macrophages were packed with Netrin-1 and it exerted its effects in an autocrine/paracrine manner via its receptor Unc5b. The use of synthetic biology to engineer bacteria with tailored functionalities presents a cutting-edge avenue for developing novel therapeutic tools. We therefore employed a synthetic biology approach to deliver exogenous Netrin-1 to the ischemic leg. The Nissle probiotic was genetically engineered to overexpress murine Netrin-1. The local administration of this probiotic to ischemic limb of Ntn1 flox/flox LysM cre+ mice was sufficient to recapitulate blood flow and reperfusion in the leg comparable to WT mice (and sham probiotic) in 2 weeks. Our results show the therapeutic potential of the use of synthetic microorganisms and Netrin-1 for innovative and adaptable solutions in limb regenerative medicine.