The present study aimed to determine the isoform‐specific role of the NADPH oxidases (NOX) in the endothelium‐mediated vascular dysfunction associated with ageing. Endothelium‐dependent [intraluminal flow‐ and acetylcholine (ACh)‐induced] vasodilatation in human skeletal muscle feed arteries (SMFAs) of young (24 ± 1 years, n = 16), middle aged (45 ± 1 years, n = 18) and old (76 ± 2 years, n = 21) subjects was assessed in vitro with and without the inhibition of NOX1 (ML090), NOX2 (gp91) and NOX4 (plumbagin). To identify the role of nitric oxide (NO) bioavailability in these responses, NO synthase blockade (l‐NG‐monomethyl arginine citrate) was utilized. SMFA NOX1, NOX2 and NOX4 protein expression was determined by western blotting. Age related endothelium‐dependent vasodilatory dysfunction was evident in response to flow (young: 69 ± 3; middle aged: 51 ± 3; old: 27 ± 3%, P < 0.05) and ACh (young: 89 ± 2; middle aged: 72 ± 3; old: 45 ± 4%, P < 0.05). NOX1 inhibition had no effect on SMFA vasodilatation, whereas NOX2 inhibition restored flow‐ and ACh‐induced vasodilatation in the middle aged and the old SMFAs (middle aged + gp91: 69 ± 3; 86 ± 3, old + gp91: 65 ± 5; 83 ± 2%, P < 0.05) and NOX4 inhibition tended to restore these vasodilatory responses in these two groups, but neither achieved statistical significance (P ≈ 0.06). l‐NG‐monomethyl arginine citrate negated the restorative effects of NOX2 and NOX4 blockade. Only NOX2 and NOX4 protein expression was significantly greater in the two older groups and inversely related to vascular function (r = 0.48 to 0.93, P < 0.05). NOX2 and, to a lesser extent, NOX4 appear to play an important, probably NO‐mediated, role in age‐related endothelial dysfunction. Key points The present study aimed to determine the isoform‐specific role of the NADPH oxidases (NOX) in the endothelium‐mediated vascular dysfunction associated with ageing. Age related endothelium‐dependent vasodilatory dysfunction was evident in skeletal muscle feed arteries in response to both flow and acetylcholine. NOX2 inhibition (gp91) restored endothelium‐dependent vasodilatation in the middle aged and the old skeletal muscle feed arteries, and NOX4 inhibition (plumbagin) tended to restore these vasodilatory responses in these two groups. Nitric oxide synthase inhibition negated the restorative effects of NOX2 and NOX4 blockade. NOX2 and NOX4 protein expression was significantly greater in the two older groups and inversely related to vascular function. NOX2 and, to a lesser extent, NOX4 appear to play an important, probably nitric oxide‐mediated, role in age‐related endothelial dysfunction and could be important therapeutic targets to maintain vascular health with ageing.

BACKGROUND The accumulation of senescent cells is a hallmark of aging in the skeletal muscle which causes reactive oxygen species (ROS) and mitochondrial dysfunction. p21 is one of the major regulators and most recognized cellular markers for senescent cells. Recent evidence demonstrated that clearance of p21high cells enhances muscle function including grip strength, hanging endurance, and maximal walking speed in mice. However, it is still unclear how the muscle performance enhances through the clearance of p21high cells. One of the potential mechanisms is mitochondrial function and/or mitochondria‐derived ROS. Therefore, this study investigated the linkage between mitochondria and p21high cells in aging or high fat diet‐induced muscle dysfunction. METHODS Five p21‐Cre/+; +/+ (P) and p21‐Cre/+; DTA/+ (PD) obese mice fed by high fat diet were administrated with tamoxifen for twice. Five P lean mice fed by normal chow were adopted as normal control. We previously demonstrated that p21high cells accumulate in obese P mice, and can be eliminated in obese PD mice by tamoxifen treatment. Mitochondrial respiration was measured, by high‐resolution respirometry, in permeabilized muscle fibers from the soleus muscle. Mitochondria‐derive ROS production was determined using Amplex Red assays. One‐way ANOVAs with Bonferroni post‐hoc were used to determine differences between groups (P < 0.05). RESULTS Reductions in complex I + II state 3 respiration were observed in p21‐Cre mice with high fat diet but the clearance of p21high cells using tamoxifen enhanced complex I + II state 3 respiration (Lean P: 35.2 ± 3.13 pmol·s‐1·mg‐1; Obese P: 17.45 ± 2.85 pmol·s‐1·mg‐1; Obese PD: 30.04 ± 3.19 pmol·s‐1·mg‐1; P < 0.05). State 4 respiration did not differ between groups (P > 0.05). Respiratory Control Ratio (RCR), defined as respiration in state 3 divided by respiration in state 4, significantly decreased in high fat diet group but clearing p21high cells restored respiratory function (Lean P: 4.20 ± 1.18; Obese P: 2.21 ± 1.11; Obese PD: 3.85 ± 1.60; P<0.05). Also, high fat diet increased mitochondrial ROS production, but tamoxifen treatment attenuated ROS production (Lean P: 10.16 ± 0.41 pmol·s‐1·mg‐1; Obese P: 26.83 ± 0.54 pmol·s‐1·mg‐1; Obese PD: 12.23 ± 0.54 pmol·s‐1·mg‐1; P<0.05). CONCLUSION These results demonstrated that p21high cells may play a causal role in mitochondrial dysfunction and ROS emission in the skeletal muscle with obesity and other chronic diseases. Therefore, targeting p21high cells may enhance muscle performance through decreasing mitochondria‐derived ROS and enhancing mitochondrial function.

BACKGROUND: Cardiovascular diseases are the leading cause of death worldwide. Overweight and obesity are strongly associated with comorbidities such as hypertension and insulin resistance, which collectively contribute to the development of cardiovascular disease. Although high-fat diet shows increased vascular senescence and vascular dysfunction, it remains unclear how obesity could induce cellular senescence and whether vascular dysfunction is associated with senescence of cells in the vasculature. p21 is one of the major regulators and most recognized cellular markers for senescent cells. Recent evidence demonstrated that high fat diet induces the accumulation of p21 high cells in the endothelial cells. However, the relationship between p21 high cells and vascular function is not identified yet. Therefore, this study investigated the linkage between vasodilation and p21 high cells in aging or high-fat diet-induced vascular dysfunction. METHODS: Five p21-Cre/+; +/+ (P) and p21-Cre/+; DTA/+ (PD) obese mice fed by high fat diet were administrated with tamoxifen for twice. Five P lean mice fed by normal chow were adopted as normal control. We previously demonstrated that p21high cells accumulate in obese P mice, and can be eliminated in obese PD mice by tamoxifen treatment. Using pressure myography, vasodilation in femoral arteries was assessed in response to flow-induced shear stress, acetylcholine (ACh), and sodium nitroprusside (SNP). Free Radical Production were measured by Amplex Red Assays. One-way ANOVAs with Bonferroni post-hoc were used to determine differences between groups (P < 0.05). RESULTS: Endothelium-dependent vasodilation was significantly attenuated in the old, induced by both flow (Lean P: 62 ± 4, Obese P: 23 ± 3 %; p < 0.05) and ACh (Lean P: 77 ± 4, Obese P: 35 ± 4 %; p < 0.05). However, the clearance of p21 high cells using tamoxifen enhanced vasodilatory function response to both flow (Obese P: 23 ± 3 %, Obese PD: 58 ± 3 %; p < 0.05) and ACh (Obese P: 35 ± 4 %, Obese PD: 70 ± 3 %; p < 0.05) while endothelium-independent vasodilation was not altered by high fat diet. Also, high fat diet increased free radical production, but the clearance of p21 high cells using tamoxifen attenuated free radical production (Lean P: 10.16 ± 0.41 pmol·s-1·mg-1; Obese P: 26.83 ± 0.54 pmol·s-1·mg-1; Obese PD: 12.23 ± 0.54 pmol·s-1·mg-1; P<0.05). CONCLUSION: These results demonstrated that p21high cells may play a causal role in vascular dysfunction with obesity and other chronic disease. Therefore, targeting p21high cells may enhance vasodilatory function through decreasing oxidative stress. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.