Impaired hydrogen sulfide protein expression in patients with peripheral artery disease

INTRODUCTION: Hydrogen sulfide (H2S) is a gaseous signaling molecule that serves various roles in the vasculature, such as upregulating angiogenesis, vascular smooth muscle relaxation, protecting endothelial function, and regulating redox balance. Despite H2S’s positive impacts on vascular homeostasis, it is important to note that its actions depend on its concentrations. At high concentrations, H2S has been reported to increase oxidative stress damage, such as oxidation of cysteine residues and lipid peroxidation. This may indicate that H2S may act as a ‘double-edged sword’ in the field of vascular physiology. Peripheral artery disease (PAD) is an atherosclerotic disease which manifested by claudication (leg pain during walking). Growing evidence suggests that abnormal H2S level may present with vascular diseases, however, only a few animal studies investigated the H2S and H2S -mediated oxidative stress damage in vascular disease models, and there are currently no available studies for human vascular disease patients, such as patients with PAD. Therefore, the purpose of this study was to examine the H2S and oxidative stress damage in peripheral blood mononuclear cells (PBMCs) and skeletal muscle tissues from patients with PAD. METHODS: Western blot was performed using skeletal muscle tissues and PBMCs to examine protein expression of cystathionase (CTH), which catalyzes production of H2S, and glutathione peroxidase-4 (GPx-4) and catalase (CAT), which are antioxidant markers, from healthy adults (CON) and patients with PAD (PAD). RESULTS: Patients with PAD show a lower expression of CTH compared to CON (P \u3c 0.01, PAD: 1.61 ± 0.44, CON: 8.53 ± 0.46). However, CAT expression was not different between groups (P = 0.429, PAD: 0.03 ± 0.02, CON: 0.01 ± 0.01). In addition, CAT and GPx-4 expression was assessed in CON PBMCs (CAT: 5.07 ± 1.14, GPx-4: 0.63 ± 0.3). CONCLUSION: CTH protein expression in the skeletal muscle is attenuated in PAD compared to CON. However, CAT protein expression in the skeletal muscle is not different between groups. These data suggest an impairment is present in the H2S signaling system in the skeletal muscle of patients with PAD

Endothelial cell Nrf2-KO attenuates endothelial function and skeletal muscle antioxidant capacity

INTRODUCTION: Endothelial cells line the inner surface of blood vessels and play a major role in modulating blood flow and gas exchange. Endothelial dysfunction is thought to be a contributor to cardiovascular disease development, and it is well-accepted that excessive reactive oxygen species (harmful molecules) likely contribute to endothelial dysfunction. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered the master regulator of cellular protection in response to elevated reactive oxygen species. Therefore, Nrf2 may be a potential therapeutic target to protect against endothelial dysfunction. However, the roles of endothelial cell-specific Nrf2 on endothelial function are not known. The purpose of this study was to investigate the impacts of endothelial cell-specific Nrf2 deletion on vascular function (endothelium-dependent and endothelium-independent vasodilation) and skeletal muscle antioxidant status. METHODS: Leg arteries were harvested from 6-mo old C57BL/6 mice (WT, n = 6) and endothelial cell-specific Nrf2-knockout mice (Tie2-Cre-Nrf2 floxed-KO, n = 6). Endothelium-dependent vasodilation was assessed in response to flow (30 uL·min-1) and acetylcholine (ACh, 10-7-10-3 M) with and without Nω-Nitro-L-arginine methyl ester (L-NAME), and endothelium-independent vasodilation was assessed with sodium nitroprusside (SNP, 10-9-10-4 M) using videomicroscopy. Skeletal muscle antioxidant protein expression for glutathione peroxidase-1 (GPX-1) and catalase (CAT) was assessed by immunoblotting. RESULTS: Endothelium-dependent vasodilation was lower in Nrf2-KO compared to WT induced by flow (WT: 34.8±2.9%, Nrf2-KO: 20.7±3.7%, P-3M, WT: 68.3±8.2%, Nrf2-KO: 44.5±7.1%, PP-3 M, 19.1±4.4%, PP=0.28) or ACh (10-3 M, 37.7±7.0%, P = 0.16). Endothelium-independent vasodilation was not different (SNP 10-4 M, WT: 92.7±3.6%, Nrf2-KO: 81.9± 0.2%, P=0.157). In addition, GPX-1 was lower in Nrf2-KO mice (WT: 0.47±0.06, Nrf2-KO: 0.001±0.003, PP=0.08). CONCLUSIONS: Endothelial cell Nrf2 may play a key role in endothelial-mediated vasodilatory function. The nitric oxide synthase inhibitor L-NAME attenuated endothelial-mediated vasodilation in WT but not in endothelial cell Nrf2-KO. Furthermore, endothelial cell Nrf2 may play a role in skeletal muscle antioxidant homeostasis, which suggests potential systemic implications of endothelial cell Nrf2 deletion. These results collectively suggest that the endothelial cell Nrf2 system is linked to endothelial dysfunction and changes in the skeletal muscle redox environment, likely through nitric oxide- and oxidative stress-related mechanisms

Effects of dietary nitrate on skeletal muscle microvascular and autonomic function in peripheral artery disease

Peripheral artery disease (PAD) is an atherosclerotic disease characterized by compromised lower-extremity blood flow that impairs walking capacity. We recently showed that a moderate body mass-normalized dose of dietary nitrate in the form of beetroot juice (BRJ, 0.11 mmol/kg) can improve macrovascular function and maximal walking distance in patients with PAD. However, its impacts on the skeletal muscle microcirculation and autonomic nervous system have not been investigated. PURPOSE: To investigate the effects of a moderate dose of dietary nitrate on skeletal muscle microvascular function and autonomic nervous system function and to further relate these measurements to walking distance and symptom onset in patients with PAD. METHODS: Patients with PAD (n = 10) ingested either BRJ or placebo in a randomized crossover design. Autonomic nervous system function, skeletal muscle microvascular function, and 6-min walking distance were assessed by heart rate variability, reactive hyperemia with near-infrared spectroscopy (tissue oxygenation index (TOI) recovery rate), and the 6-min walk test, respectively, pre- and post-BRJ and placebo intake. RESULTS: There were significant group x time interactions (P \u3c 0.05) for skeletal muscle microvascular function and 6-min walking distance whereas no changes (P \u3e 0.05) occurred in heart rate variability or pain-free walking distance. The BRJ group demonstrated improved skeletal muscle microvascular function (Δ 22.1 ± 7.5%·min-1) and a longer 6-min walking distance (Δ 37.5 ± 9.1m). Furthermore, TOI recovery rate was positively associated with 6-min walking distance (r = 0.5) and pain-free walking distance (r = 0.6). CONCLUSIONS: These results suggest that a moderate dose of dietary nitrate may support skeletal muscle microvascular function, which is related to improvements in 6-min and pain-free walking distance in patients with PAD