Genetic Deficiency of Mas Receptor Impairs Vascular Repair by Bone Marrow Progenitor Cells
Authors: Vasam Goutham , Joshi Shrinidh , Jarajapu Yagna PR 
Published: November 2023

Bone marrow progenitor cells (BMPCs) are mobilized into the systemic circulation and maintain cardiovascular health. We have recently showed that Mas receptor (MasR) is a promising approach for reversing the impaired mobilization of BMPCs, in conditions such as diabetes. This study investigated the role of endogenous MasR expression in BMPC mobilization. Age‐matched male wild type (WT) or MasR knockout (MasR‐KO) mice in C57BL/6 background were used. Circulating BMPCs were enumerated by flow cytometry of lineage‐negative, Sca‐1+ and c‐Kit+ (LSK) cells or by Colony Forming Unit (CFU) assay. Effects of clinically used mobilizers, plerixafor (5 mg/kg, s.c.) or granulocyte‐colony stimulating factor (G‐CSF, 125 μg/kg, s.c., twice a day for four days) were evaluated. Hind‐limb ischemia (HLI) was induced by femoral artery ligation. Mobilization of BMPCs was monitored and blood flow recovery was determined by Laser Doppler imaging system for 21 days post‐HLI. Neovascularization of ischemic areas was assessed by quantification of capillary density by immunohistochemistry. In vitro migration and proliferation were evaluated in response to stromal cell derived factor (SDF) or vascular endothelial growth factor (VEGF). Fluorescent probes, CellROX, DAF‐FM and MitoSOX were used to estimate the intracellular levels of reactive oxygen species (ROS), nitric oxide (NO) and mitochondrial ROS, respectively, and fluorescence intensity was evaluated by using flow cytometry. Mitochondrial membrane potential/Annexin V apoptosis kit was used to measure the mitochondrial apoptosis. MasR deficiency decreased BMPCs mobilization by plerixafor (LSK, P<0.05; CFUs, P<0.05; n=6) or G‐CSF (LSK, P<0.01; CFUs, P<0.001, n=6). HLI‐induced mobilization of BMPCs in WT mice showed peak response at day‐2 (5‐fold) following the injury and returned to normal by day 10 (n=6). In MasR‐KO mice ischemia failed to induce mobilization of BMPCs (P<0.001, n=6). Along similar lines, blood flow recovery in ischemic limbs was 85±5% of contralateral non‐ischemic limb by day 21 in the WT, which was reduced to 64±2% (P<0.001, n=6) in MasR‐KO mice. In agreement with this, capillary density in the ischemic regions observed in the MasR‐KO mice is lower compared to WT mice (P<0.05, n=3). The total number of BMPCs residing in the bone marrow were similar in WT and KO mice. At the age of 12‐months, even the partial deficiency of MasR (heterozygotes) resulted in severe ischemia, which lead to amputation by day‐3 post‐HLI. Proliferation and migration of BMPCs were decreased in partial MasR‐KO compared to WT mice in basal or in response to SDF or VEGF (P<0.05, n=7). In MasR‐KO LSK cells, cellular and mitochondrial ROS levels were elevated (P<0.05, n=5), mitochondrial membrane potential was decreased with increased apoptosis (P<0.05, n=4) in comparison with WT cells. This study provides compelling evidence for an indispensable role of MasR in the mobilization of BMPCs in response to pharmacological mobilizers or to an ischemic insult, and ischemic vascular repair, most likely by increasing the sensitivity of BMPCs to ischemia‐regulated factors and by maintaining mitochondrial homeostasis.

Support or Funding Information

MasR‐KO mice were provided by Prof. Lisa Cassis of University of Kentucky, Lexington, KY.