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Aging Associated Loss Of Vegfa Impairs Muscle Regeneration
Charles D. Hwang, MD, Yori Endo, PhD, Yuteng Zhang, PhD, Ronald L. Neppl, PhD, Shailesh Agarwal, MD, Indranil Sinha, MD
Harvard University, Boston, MA, USA.

Purpose: Global populations, particularly in developed countries, have continued to age. Notably, aging is intimately correlated with frailty, a parameter that has been previously demonstrated to correlate significantly with mortality and concomitantly correlate with loss of muscle mass. The molecular mechanisms behind aging associated impaired skeletal muscle regeneration remain incompletely understood. Our group previously demonstrated aging-associated loss of hypoxia signaling impairs skeletal muscle myogenic potential. Given these findings, we hypothesized vascular endothelial growth factor (VEGFA), a potent downstream signal well characterized in nascent vascular ingrowth and muscle progenitor differentiation, to critically regulate regeneration in young and aged skeletal muscle.

Methods: Young C57BL/6 mice (12-14 weeks old) and old C57BL/6 mice (24-25 months old) were subjected to a previously validated model of muscle cryoinjury (n=5-6/group) to induce regeneration. The tibialis anterior muscle was injured using direct contact with a metal probe cooled in dry ice. Quantifications of cross sectional area (CSA) of myofibers were performed across 10 photos/sample by blinded reviewer via ImageJ. Western blots were performed from whole TA muscle lysates for quantification of VEGFA with GAPDH for normalization. To evaluate the role of VEGF in muscle regeneration, we performed a similar and separate series of experiments using VEGFlo (Jackson Labs Stock No. 027315) mice, which exhibit a systemic, 75% decrease in VEGFA activity, and littermate controls. In addition, ML228, a hypoxia signaling activator known to increase VEGFA levels, was injected into both young and old mice, as well as VEGFlo and littermate controls, to determine whether VEGFA augmentation promotes muscle regeneration in aging.

Results: Old mice demonstrated markedly impaired ability of regenerating myofiber CSA on days post injury (DPI) 5 in comparison to young mice (Fig A. Young vs. Old: 526 vs. 373µm2 , p<.001) and DPI 10 (Y v. O: 1250 vs. 833µm2 , p<.001). VEGFA protein levels were markedly reduced in the gastrocnemius of old mice in comparison to young (Fig B). Similarly, VEGFlo mice exhibited significantly smaller CSA of regenerating fibers as compared to littermate controls (Fig C. WT vs. lo: 541 vs. 238µm2 , p=.0011). Pharmacologic augmentation of VEGFA, using ML228, improved skeletal muscle regeneration in both old mice and VEGFlo mice. (WT vs. lo: CSA 757 vs. 662µm2 , p=.387).

Conclusions: Muscle regeneration declines with with aging, in a fashion correlated to loss of VEGFA levels within skeletal muscle. Systemic loss of VEGFA is further correlated with poor muscle regeneration in genetically modified mice. Furthermore, exogenous activation of hypoxic signaling/VEGFA reverses this reduced capacity for muscle regeneration in old and VEGFlo mice. Supplementation of VEGFA could represent a therapeutic target in aging patients with skeletal muscle loss.

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