Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of two superoxide anions (O₂^·−) into hydrogen peroxide (H₂O₂) and oxygen (O₂) and is generally known to protect against oxidative stress. Angiotensin II (AngII) causes vascular hypertrophic remodeling which is associated with H₂O₂ generation. The aim of this study is to investigate the role of cytosolic SOD (SOD1) in AngII-induced vascular hypertrophy. We employed C57/BL6 mice (WT) and SOD1 deficient mice (SOD1^−/−) with the same background. They received a continuous infusion of saline or AngII (3.2 mg/kg/day) for seven days. The blood pressures were equally elevated at 1.5 times with AngII, however, vascular hypertrophy was blunted in SOD1^−/− mice compared to WT mice (WT mice 91.9 ± 1.13 µm versus SOD1^−/− mice 68.4 ± 1.41 µm p < 0.001). The elevation of aortic interleukin 6 (IL-6) and phosphorylation of pro-inflammatory STAT3 due to AngII were also blunted in SOD1^−/− mice’s aortas. In cultured rat vascular smooth muscle cells (VSMCs), reducing expression of SOD1 with siRNA decreased AngII induced IL-6 release as well as phosphorylation of STAT3. Pre-incubation with polyethylene glycol (PEG)-catalase also attenuated phosphorylation of STAT3 due to AngII. These results indicate that SOD1 in VSMCs plays a role in vascular hypertrophy due to increased inflammation caused by AngII, probably via the production of cytosolic H₂O₂.

We previously demonstrated the marked hepatosteatosis and endothelial dysfunction in hepatocyte-specific ERK2 knockout mice (LE2KO) with a high-fat/high-sucrose diet (HFHSD), but detailed metabolic changes and the characteristics in insulin-sensitive organs were not tested. This study aimed to characterize metabolic remodeling with changes in insulin-sensitive organs, which could induce endothelial dysfunction in HFHSD-LE2KO. The serum glucose and fatty acid (FA) were modestly higher in HFHSD-LE2KO than HFHSD-Control. FA synthesis genes were up-regulated, which was associated with the decreased phosphorylation of AMPK and ACC, and with the up-regulation of SREBP-1 in the liver from HFHSD-LE2KO. In FA and amino acids fraction analysis, arachidonic acid/eicosapentaenoic acid ratio, L-ornithine/arginine ratio, asymmetric dimethylarginine and homocysteine levels were elevated in HFHSD-LE2KO. Insulin-induced phosphorylation of AKT was blunted in skeletal muscle. Serum leptin and IL-1β were elevated, and serum adiponectin was decreased with the enlargement of epididymal adipocytes. Finally, the enhanced superoxide levels in the aorta, which were blunted with CCCP, apocynin, and tempol, were observed in HFHSD-LE2KO. A pre-incubation of aortic rings with tempol improved endothelial dysfunction in HFHSD-LE2KO. HFHSD-LE2KO revealed an acceleration of FA synthesis in the liver leading to insulin resistance in skeletal muscle and the enlargement of visceral adipocytes. Global metabolic remodeling such as changes in arginine metabolism, ω3/ω6 ratio, and adipocytokines, could affect the vascular oxidative stress and endothelial dysfunction in HFHSD-LE2KO.

Metabolic syndrome (Mets) is an important condition because it may cause stroke and heart disease in the future. Reactive oxygen species (ROSs) influence the pathogenesis of Mets; however, the types of ROSs and their localization remain largely unknown. In this study, we investigated the effects of SOD1, which localize to the cytoplasm and mitochondrial intermembrane space and metabolize superoxide anion, on Mets using SOD1 deficient mice (SOD1^−/−). SOD1^−/− fed on a high-fat/high-sucrose diet (HFHSD) for 24 weeks showed reduced body weight gain and adipose tissue size compared to wild-type mice (WT). Insulin secretion was dramatically decreased in SOD1^−/− fed on HFHSD even though blood glucose levels were similar to WT. Ambulatory oxygen consumption was accelerated in SOD1^−/− with HFHSD; however, ATP levels of skeletal muscle were somewhat reduced compared to WT. Reflecting the reduced ATP, the expression of phosphorylated AMPK (Thr 172) was more robust in SOD1^−/−. SOD1 is involved in the ATP production mechanism in mitochondria and may contribute to visceral fat accumulation by causing insulin secretion and insulin resistance.

Recombinant adeno‐associated viruses (rAAVs) are useful vectors for expressing genes of interest in vivo because of their low immunogenicity and long‐term gene expression. Various mutations have been introduced in recent years and have enabled high‐efficacy, stabilized, and organ‐oriented transduction. Our purpose for using rAAV is to express our target gene in the mouse lung to investigate pulmonary artery hypertension. We constructed a self‐complementary AAV having mutant capsids with the ESGHGYF insert, which directs the vectors to lung endothelial cells. However, when this mutant virus was purified from the producing cells by the conventional method using an ultracentrifuge, it resulted in a low yield. In addition, the purification method using an ultracentrifuge is tedious and labor‐intensive. Therefore, we aimed to develop a simple, high‐quality method for obtaining enough lung‐targeted rAAV. First, we modified amino acids (T491V and Y730F) of the capsid to stabilize the rAAV from degradation, and we optimized culture conditions. Next, we noticed that many rAAVs were released from the cells into the culture medium. We, therefore, improved our purification method by purifying from the culture medium without the ultracentrifugation step. Purification without ultracentrifugation had the problem that impurities were mixed in, causing inflammation. However, by performing PEG precipitation and chloroform extraction twice, we were able to purify rAAV that caused only as little inflammation as that obtained by the ultracentrifuge method. Sufficient rAAV was obtained and can now be administered to a rat as well as mice from a single dish: 1.50 × 10 ¹³ ± 3.58 × 10 ¹² vector genome from one φ150 mm dish (mean ± SEM).

Background: Perivascular adipose tissue (PVAT) releases factors that regulate vascular tone (adipocyte-derived factors (ADFs) ). Mitogen-activated protein kinases (MAPKs) in blood vessels have been implicated in the development of hypertension. This study aimed to determine whether MAPKs are involved in impaired ADF-induced relaxation in hypertension. Methods and Results: Aortic rings of Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) with or without PVAT (+PVAT rings and −PVAT rings, respectively) were placed in an organ bath filled with 5 ml of physiological salt solution (PSS). In the aortic rings of SHRs, 5-hydroxytryptamine (5-HT) -induced contraction of the −PVAT ring was increased compared with that in WKY rats. The 5-HT-induced contraction decreased in WKY rats and SHRs when PVAT was retained (+PVAT ring). After contraction with 5-HT (10^−6.5−10^−5.5 M), PSS (2 ml) was removed from the other chambers. The PSS from the +PVAT rings of WKY rats slowly relaxed the −PVAT rings; however, the PSS from the −PVAT rings of WKY rats did not. The PSS collected from the chamber with the +PVAT ring failed to relax the pre-constructed −PVAT rings of the SHR. After pretreatment of the −PVAT rings of SHR with PD98052 (20 μM: MAPK/ERK Kinase (MEK) inhibitor), the PSS from the +PVAT rings relaxed to similar degrees as in WKY rats (WKY 44%±4%, SHR −15%±8%; P<0.001 vs WKY, SHR+PD98052 45%±7%). Conclusion: PVAT releases ADFs; however, the aorta from SHR resists relaxation with ADFs. The impairment of ADF-induced relaxation and hyperconstriction by 5-HT can be mediated by MAPKs, potentially contributing to the pathogenesis of hypertension.