Adipose Stromal Vascular Fraction Restores Coronary Microvascular Flow‐Mediated Dilation in Aging Female Rats via Enhanced Peroxynitrite Signaling

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Introduction Coronary Microvascular Disease (CMD) presents in a majority of aging post-menopausal women and is defined by hyperconstricted tone. CMD can lead to chronic ischemia exacerbated by reduced perfusion with aging. Flow-mediated dilation (FMD) primarily regulates vascular patency via endothelial shear stress, but the signaling mechanism of FMD alters throughout the lifespan from vasoprotective nitric oxide (NO) in youth to the less efficacious hydrogen peroxide (H2O2) in advancing age. Reactive Oxygen Species (ROS), including H2O2, suppress NO, cause cellular damage and are pro-atherogenic. Adipose Stromal Vascular Fraction (SVF) is a heterogenous cell population that has antioxidant and regenerative properties. We hypothesize that coronary microvascular FMD is reduced with age, but aged rats treated with SVF (OSVF) will exhibit restored FMD via increased NO and decreased ROS at baseline and with flow. Methods Isolated coronary microvessels from young (4 months), old (24 months), or OSVF (tail vein injection 4 weeks prior to sacrifice at 24 months) from female Fischer 344 rats were assessed via pressure myography. FMD was assessed at flow rates of 5, 10, 15, 20, and 25 μL/min at physiologic pressure (45 mmHg) and temperature (37°C). Mean Fluorescence Intensity (MFI) of NO, H2O2, superoxide (O2-), peroxynitrite (ONOO-) and antioxidant glutathione (GSH) were assessed via intraluminally-infused fluorescence dyes (2,3-diaminonapthalene, mitopy1, mitosox, biotracker 515, and SEMKUR-IM, respectively) at baseline and during flow at 10 and 25 μL/min. Results Percent relaxation to intraluminal flow (FMD) is reduced with aging and restored to youthful levels by SVF (Figure 1a). At baseline, H2O2 and ONOO- MFI are significantly increased with aging compared to young and OSVF (p = < .001, .01 vs. young, < .001, .024 vs. OSVF). O2- MFI is reduced at baseline in OSVF compared to aging alone (p = .004). NO and GSH MFI are significantly reduced at baseline with aging (p = .01, < .001) and increased slightly (non-significant) with OSVF. Intraluminal flow preferentially increases MFI of NO in youth, H2O2 in aging, and ONOO- in OSVF. These data correspond functionally as FMD was significantly attenuated in youth via L-NAME inhibition of NO production, inhibited in OSVF via uric acid scavenging of ONOO-, but was unaffected by reducing H2O2 signaling via catalase in aging controls (Figure 1b-d). Conclusions FMD is abrogated with aging, contributing to CMD hyperconstriction associated with increased ROS and loss of NO and GSH. FMD can be restored with therapeutic SVF associated with increased ONOO- signaling in response to intraluminal flow. Although ONOO- is considered a ROS, it could be considered a NO reservoir inducing FMD as a NO donor since O2- and NO combine to form ONOO-. Alternatively, ONOO- can elicit FMD as an Endothelium-Derived Hyperpolarizing Factor. Future directions will differentiate between these alternative mechanisms. SVF represents a plausible future clinical direction to alleviate vascular oxidative stress and restore microvascular patency and perfusion.