Abstract 543: Leukadherine-1 Improves Neovascularization In A Mouse Model Of Hindlimb Ischemia

Objective: Critical limb ischemia (CLI) is the end stage of peripheral artery disease characterized by rest pain, gangrene, and amputations. CLI is secondary to severe stenosis/occlusion of arteries and to the failure of compensatory neovascularization; and immune cells play essential role on it. Mac-1 integrin (also known as CD11b/CD18) is a leukocyte surface receptor that contributes to monocytes adhesion and infiltration after vascular injury controlling to excessive inflammation. The goal of this study is to evaluate the pharmacological activation of Mac-1, by a compound called Leukadherin-1 (LA1), in neovascularization after limb ischemia. Methods: C57Bl6 mice were divided in three groups: control (vehicle), 1 dose LA1 (2 mg/kg), and 2 dose LA1 (2 doses of 2 mg/kg). Injections of LA1 or vehicle (ip) were given for 14 days starting at hindlimb ischemia surgery day. Blood flow recovery, angigogenesis, and arteriogenesis were measured on these groups 14 days after hindlimb ischemia. Single cell RNA sequencing of ischemic limb was performed on control and 2 dose LA1 groups 7 days after hindlimb ischemia. For proliferation, murine smooth muscle cells (SMCs) were cultured with murine monocytes exposed or not to LA1 and cell counting was performed 24h and 48h after treatment. Results: Mice who received LA1 treatment showed an improvement in blood flow recovery and remarkable angiogenesis and arteriogenesis. Interestingly, cell clustering findings from limb ischemic muscle single cell RNA-seq, demonstrated increased percentage of B cells in the ischemic muscle on 2 dose LA1 group compared to control group. Moreover, our in vitro data demonstrated that 48 hours after LA1 exposure SMCs proliferation is increased. Conclusions: Our data demonstrate that LA1 treatment improves neovascularization after hindlimb ischemia which might be associated with greater mobilization of B cells to the ischemic tissue and higher proliferation rate of SMCs.