Adipose Stromal Vascular Fraction Reverses Mitochondrial Hyperfission in Coronary Microvessels in Aging

Abstract

IntroductionIn aging post‐menopausal women, Coronary Microvascular Disease (CMD) leads to hyperconstricted tone, reduced perfusion and chronic micro‐ischemia with angina. We previously showed an age‐related increase in coronary microvascular ROS alongside increased prooxidant gene and protein expression associated with blunted vasodilation. Adipose Stromal Vascular Fraction (SVF) is a heterogenous cell population that reduces vascular ROS to improve vasodilation. Oxidative stress with aging may be mediated by mitochondrial dysfunction, including fission/fusion imbalance. Therefore, we hypothesize aging leads to mitochondrial hyperfission and reduced fusion gene and protein expression, reversed by SVF therapy.MethodsCoronary microvessels from young (YC, 4 months), old (OC, 24 months), or old + SVF (OSVF, tail vein injection 4 weeks prior to sacrifice) from female Fischer 344 rats were assessed via RNA sequencing (n=3) and immunofluorescence imaging of fission/fusion proteins Dynamin Related Protein 1 (DRP‐1) and Mitofusin 1 & 2 (MFN1 & 2). Morphometric analysis was accomplished using ImageJ and the MiNA Image‐J tool to analyze 60x confocal images of microvessels stained with antibodies against mitochondrial TOM20. Statistics were preformed using One‐Way ANOVA with Bonferroni post hoc analysis.ResultsGene expression of DRP‐1 was significantly reduced, and MFN‐1 significantly enhanced in OSVF vs. OC (p = 0.035, 0.019). There was no significant difference in gene expression of fission or fusion mediators Fis‐1, MFN‐2 or Opa‐1. Protein expression of DRP‐1 was enhanced with aging, reduced to youthful levels by SVF (p = 0.007, 0.033) (Fig.1a). There were no differences in MFN1 or MFN2 protein expression with aging, however, protein expression of MFN‐1 was significantly increased after SVF (YC vs. OSVF p = 0.003, OC vs. OSVF p < .001). Mitochondrial aspect ratio and form factor were reduced in aging (p = 0.024, < 0.001), restored to youthful levels by SVF (p = < 0.001, < 0.001) (Fig. 1b). Mitochondrial fission factor was elevated with aging (p = < 0.001), reversed by SVF therapy (p = < 0.001) (Fig. 1c). Mitochondrial area was reduced with aging (p = 0.002). Mitochondrial branch length and numbers of branches within a network were greatest in youth, significantly attenuated with aging (p < 0.001, < 0.001), and significantly increased (albeit not to youthful levels) with SVF therapy (OC vs. OSVF: p = 0.017, 0.011, YC vs. OSVF: p = 0.004, 0.003) (Fig. 1d).ConclusionsDuring youth, mitochondrial morphology is more rod‐like and interconnected, vs. punctate and isolated in aging. Injection of SVF reversed mitochondrial circularity and partially restored mitochondrial network density in aging. These findings were complimented by enhanced expression of DRP‐1 with aging with reduced DRP‐1 and enhanced MFN‐1 expression with SVF. SVF is a plausible therapy for CMD by reversing mitochondrial dysfunction including alleviating oxidative stress, attenuating mitochondrial fission, and rejuvenating mitochondrial fusion.