Abstract Body: Background: Angiogenesis is driven by endothelial cell (EC) proliferation, migration and network formation, which is central for vascular regeneration and tissue perfusion in peripheral artery disease. Leucine-Rich Repeat-Containing 8 (LRRC8) family is a heterohexameric protein complex coding the mechanoresponsive volume-regulated anion channel (VRAC). We previously determined the endothelial VRAC to be composed of LRRC8A/B/C heteromer where LRRC8A functions as a core subunit and LRRC8C plays a non-redundant role in regulating vasoreactivity and blood pressure. This study is aimed at identifying a novel LRRC8-mediated hypoxia-sensing pathway in angiogenesis.
Methods: EC migration, tube formation and ex vivo angiogenesis were assessed by collagen invasion assay and wound scratch assay in human umbilical vein endothelial cells (HUVECs), and aortic ring sprouting from endothelial specific Lrrc8a knockout (eLrrc8a KO), global Lrrc8b KO and global Lrrc8c KO mice. Acute hindlimb ischemia in mice was established by femoral artery ligation. Blood flow recovery was monitored by laser doppler perfusion imaging and collateral vascular formation was assessed by 3D mapping of hindlimb vasculature after fluorescent-labeled lectin perfusion and tissue clearing.
Results: Lrrc8c knockdown, but not Lrrc8a or Lrrc8b, in HUVECs enhanced tube formation and wound closure as compared to control siRNA. Similarly, Lrrc8c but not Lrrc8a or Lrrc8b KO mice exhibited increased aortic ring sprouting, and accelerated perfusion recovery with collateral vascular formation in the ischemic limb, compared to their wildtype control mice. H&E staining of gastrocnemius muscle revealed more centrally located myonuclei in gLrrc8c KO mice, indicating increased muscular regeneration secondary to improved angiogenesis. Mechanistically, LRRC8C expression increased in cultured ECs exposed to 24 hours of 0.2% hypoxia, and contributes to both endothelial VRAC current and ATP conduction. Lrrc8c KD in HUVECs reduced LRRC8A expression, increased LRRC8E expression, and enhanced hypoxia-stimulated ERK1/2 phosphorylation.
Conclusion: LRRC8C negatively regulates EC migration, tube formation and enhances perfusion recovery and collateral vascular formation following acute hindlimb ischemia. Further studies are warranted to delineate the precise mechanisms underlying the LRRC8C-dependent and hypoxia-triggered shift in LRRC8 complex composition and activation of pro-angiogenic signaling in vascular regeneration.