Aging Impairs VEGF-A mRNA Stability and Consequent Inflammatory ArteriogenesisdissertationsCaballero, OlivyacreatorMorrison, AlanAdvisorMantsounga, ChrisReaderOancea, ElenaReaderBrown University. Biology and Medicine: Biotechnologysponsor2024, 40 p.born digitalThesis (Sc. M.)--Brown University, 2024thesesAbstract: Aging impairs VEGF-A mRNA stability and consequent inflammatory arteriogenesis, by Olivya Caballero, Sc.M, Brown University, May, 2024. Objective: Aging is associated with impaired inflammatory arteriogenesis in response to vascular injury. Following inflammation and ischemia, macrophage recruitment and early macrophage pro-angiogenic VEGF-A isoform (VEGF-A165a) expression contribute to setting the stage for post developmental arteriogenesis. Preliminary data demonstrated reduced expression of proangiogenic VEGF-A levels by both mRNA and protein by bone marrow-derived macrophages (BMDMs) from aged (52-week-old) mice when compared to young (12-week-old) mice. We sought to determine the age-related mechanisms which contribute to the reduction of pro-angiogenic VEGF-A expression and consequent impaired inflammatory-mediated arteriogenesis. Methods: We used aged (52-week-old) and young (12-week-old) C57BL/6 mice as well as mouse strains with tamoxifen-inducible, myeloid-specific knockouts and knock-ins of Dicer1. A hind limb ischemia procedure was used to mimic the effects of lost blood flow due to peripheral artery disease. Subsequent blood flow recovery was measured using laser Doppler imaging of the rear paws in addition to microCT angiography. These findings were validated with RT-qPCR of lysed muscle tissue on day 3 post HLI. In addition, BMDMs were collected and analyzed using immunoblot, RT-qPCR, and ELISA. Results: In an experimental model of hindlimb ischemia, we found decreased blood flow recovery in aged mice and significant reductions in new small arterial development by microCT angiography consistent with decreased arteriogenesis. Reduced arteriogenesis was associated with decreased muscle tissue VEGF-A and VEGF-A165a levels during early inflammation 3 days after induction of hindlimb ischemia despite adequate macrophage recruitment to the muscle tissue. BMDMs from aged mice demonstrated increased levels of DNA methylation. BMDMs from aged mice also revealed decreased VEGF-A165a mRNA half-life along with decreased association of VEGF-A with the RNA-stabilizing protein, HuR. Treatment of BMDMs with a DNA methyltransferase inhibitor led to improved association of HuR with VEGF-A mRNA and improved blood flow recovery during hindlimb ischemia, indicating that age associated increases in DNA methylation impact HuR-mediated VEGF-A mRNA stability and consequent inflammation-driven arteriogenesis. Conclusions: Understanding and reversing the mechanisms driving age-related impairments in vascular healing may pave the way for new therapeutic strategies to improve vascular healing in the elderly.InflammationAgingAngiogenesisEnglishRPB20240505