Development of a high-yield, high-quality purification process for adeno-associated virus vectors that can be used in vivo without ultracentrifugation: Application to a lung endothelial cell-targeted adeno-associated virus.

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 × 1013 ± 3.58 × 1012 vector genome from one φ150 mm dish (mean ± SEM).

Metabolic Remodeling with Hepatosteatosis Induced Vascular Oxidative Stress in Hepatic ERK2 Deficiency Mice with High Fat Diets.

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-1ß 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.

Resistance to Obesity in SOD1 Deficient Mice with a High-Fat/High-Sucrose Diet.

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.

Substitution of the SERCA2 Cys674 reactive thiol accelerates atherosclerosis by inducing endoplasmic reticulum stress and inflammation.

BACKGROUND AND PURPOSE: The cysteine674 (C674) thiol of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 is easily and irreversibly oxidized under atherosclerotic conditions. However, the contribution of the C674 thiol redox status in the development of atherosclerosis remains unclear. Our goal was to elucidate the possible mechanism involved. EXPERIMENTAL APPROACH: Heterozygous SERCA2 C674S knock-in mice in which half of the C674 was substituted by serine (S674) were used to mimic the removal of the reactive C674 thiol, which occurs under pathological conditions. Bone marrow-derived macrophages (BMDMs) and cardiac endothelial cells (ECs) were used for intracellular Ca2+ , macrophage adhesion, and protein expression analysis. The whole aorta and aortic root were isolated for histological analysis. KEY RESULTS: Cell culture studies suggest the partial substitution of SERCA2 C674 increased intracellular Ca2+ levels and induced ER stress in both BMDMs and ECs. The release of proinflammatory factors and macrophage adhesion increased in SKI BMDMs. In ECs, overexpression of S674 induced endothelial inflammation and promoted macrophage recruitment. SKI mice developed more severe atherosclerotic plaque and macrophage accumulation. Additionally, 4-phenyl butyric acid, an ER stress inhibitor, suppressed ER stress and inflammatory responses in BMDMs and ECs, and alleviated atherosclerosis in SKI mice. CONCLUSIONS AND IMPLICATIONS: The substitution of SERCA2 C674 thiol accelerates the development of atherosclerosis by inducing ER stress and inflammation. Our findings highlight the importance of SERCA2 C674 redox state in the context of atherosclerosis and open up a novel therapeutic strategy to combat atherosclerosis.

Deletion of Superoxide Dismutase 1 Blunted Inflammatory Aortic Remodeling in Hypertensive Mice under Angiotensin II Infusion.

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.

Redox-dependent PPARγ/Tnpo1 complex formation enhances PPARγ nuclear localization and signaling.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ has been implicated in the pathogenesis of various human diseases including fatty liver. Although nuclear translocation of PPARγ plays an important role in PPARγ signaling, details of the translocation mechanisms have not been elucidated. Here we demonstrate that PPARγ2 translocates to the nucleus and activates signal transduction through H2O2-dependent formation of a PPARγ2 and transportin (Tnpo)1 complex via redox-sensitive disulfide bonds between cysteine (Cys)176 and Cys180 of the former and Cys512 of the latter. Using hepatocyte cultures and mouse models, we show that cytosolic H2O2/Tnpo1-dependent nuclear translocation enhances the amount of DNA-bound PPARγ and downstream signaling, leading to triglyceride accumulation in hepatocytes and liver. These findings expand our understanding of the mechanism underlying the nuclear translocation of PPARγ, and suggest that the PPARγ and Tnpo1 complex and surrounding redox environment are potential therapeutic targets in the treatment of PPARγ-related diseases.

Elevation of Derivatives of Reactive Oxygen Metabolites Elevated in Young "Disaster Responders" in Hypertension due to Great East Japan Earthquake.

There have been very few studies on serum biomarkers associated with hypertension in disaster situations. We assessed biomarkers associated with disaster-related hypertension (DRH) due to the Great East Japan Earthquake of March 2011.We collected blood samples from members of the Japan Self Defense Forces (JSDF) (n = 77) after completing disaster relief operations. We divided them into two groups based on systolic blood pressure. We defined DRH as either systolic blood pressure greater than 140 mmHg or diastolic blood pressure greater than 90 mmHg at the time of completing missions.In subjects with DRH, the mean blood pressure was 143.5 ± 5.0/99.5 ± 2.4 mmHg. Height and body weight measurements were slightly greater in the DRH group but the differences were not significant, and age was significantly higher in the DRH group. There were no differences in serum biochemical tests including metabolic markers, sulfur-containing amino acids, and cytokines. Among nitric oxide-related amino acids, asymmetric dimethylarginine (ADMA) was lower in the DRH group than in the normotension group (0.40 ± 0.02 versus 0.31 ± 0.02 µmol/L P = 0.04). The serum oxidative stress metabolite levels (d-ROMs; indicators of active oxygen metabolite products) were significantly higher in the DRH group (273.6 ± 6.08 versus 313.5 ± 13.7 U.CARR P = 0.016). Using multivariable regression analysis, d-ROMs levels were particularly predictive for DRH.Oxidative stress is associated with DRH in responders to the disaster of the Great East Japan Earthquake.

Vascular Smooth Muscle Sirtuin-1 Protects Against Aortic Dissection During Angiotensin II-Induced Hypertension.

BACKGROUND: Sirtuin-1 (SirT1), a nicotinamide adenine dinucleotide(+)-dependent deacetylase, is a key enzyme in the cellular response to metabolic, inflammatory, and oxidative stresses; however, the role of endogenous SirT1 in the vasculature has not been fully elucidated. Our goal was to evaluate the role of vascular smooth muscle SirT1 in the physiological response of the aortic wall to angiotensin II, a potent hypertrophic, oxidant, and inflammatory stimulus. METHODS AND RESULTS: Mice lacking SirT1 in vascular smooth muscle (ie, smooth muscle SirT1 knockout) had drastically high mortality (70%) caused by aortic dissection after angiotensin II infusion (1 mg/kg per day) but not after an equipotent dose of norepinephrine, despite comparable blood pressure increases. Smooth muscle SirT1 knockout mice did not show any abnormal aortic morphology or blood pressure compared with wild-type littermates. Nonetheless, in response to angiotensin II, aortas from smooth muscle SirT1 knockout mice had severely disorganized elastic lamellae with frequent elastin breaks, increased oxidant production, and aortic stiffness compared with angiotensin II-treated wild-type mice. Matrix metalloproteinase expression and activity were increased in the aortas of angiotensin II-treated smooth muscle SirT1 knockout mice and were prevented in mice overexpressing SirT1 in vascular smooth muscle or with use of the oxidant scavenger tempol. CONCLUSIONS: Endogenous SirT1 in aortic smooth muscle is required to maintain the structural integrity of the aortic wall in response to oxidant and inflammatory stimuli, at least in part, by suppressing oxidant-induced matrix metalloproteinase activity. SirT1 activators could potentially be a novel therapeutic approach to prevent aortic dissection and rupture in patients at risk, such as those with hypertension or genetic disorders, such as Marfan's syndrome.

Association between brachial-ankle pulse wave velocity and the ratio of l-arginine to asymmetric dimethylarginine in patients undergoing coronary angiography.

BACKGROUND: Endothelial dysfunction causes vasomotor dysregulation and vascular stiffening in addition to structural changes. By influencing NO synthesis, deficiency of l-arginine relative to asymmetric dimethylarginine (ADMA), which is an l-arginine derivative that acts as a competitive NO synthase inhibitor, may lead to the promotion of arterial stiffness. This study investigated the relationship between the l-arginine/ADMA ratio and brachial-ankle pulse wave velocity (baPWV), an indicator of arterial stiffness. METHODS AND RESULTS: This cross-sectional study enrolled 74 patients (62 men, 12 women; mean age, 67±10 years) undergoing elective coronary angiography. A total of 54 (73%) patients had coronary artery disease. Serum l-arginine and ADMA were measured by high-performance liquid chromatography with fluorescence detection. The ratio of l-arginine to ADMA and the serum l-arginine level was associated with baPWV in univariate regression analysis (l-arginine/ADMA ratio: ß=-0.323, p=0.005; l-arginine: ß=-0.247, p=0.034). In addition, baPWV was related to blood hemoglobin concentration, hematocrit, brain natriuretic peptide level, symmetric dimethylarginine, renal function, blood pressure, and heart rate. In multivariate analysis, the l-arginine/ADMA ratio was a significant predictor of baPWV (ß=-0.310, p<0.001). In subgroup analyses, the l-arginine/ADMA ratio was associated with baPWV in elderly patients (n=46, ß=-0.359, p=0.004), and in younger patients (n=28, ß=-0.412, p=0.006). CONCLUSION: A low l-arginine/ADMA ratio may be associated with high baPWV in patients undergoing coronary angiography.

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase C674 promotes ischemia- and hypoxia-induced angiogenesis via coordinated endothelial cell and macrophage function.

Ischemia is a complex phenomenon modulated by the concerted action of several cell types. We have identified that sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2 (SERCA 2) cysteine 674 (C674) S-glutathiolation is essential for ischemic angiogenesis, vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) migration and network formation. A heterozygote SERCA 2 C674S knockin (SKI) mouse shows impaired ischemic blood flow recovery after femoral artery ligation, and its ECs show depleted endoplasmic reticulum (ER) Ca(2+) stores and impaired angiogenic behavior. Here we studied the role of SERCA 2 C674 in the interaction between ECs and macrophages in the context of ischemia and discovered the involvement of the ER stress response protein, ER oxidoreductin-1α (ERO1). In wild type (WT) mice, expression of ERO1 was increased in the ischemic hind limb in vivo, as well as in ECs and macrophages exposed to hypoxia in vitro. The increase in ERO1 to ischemia/hypoxia was less in SKI mice. In WT ECs, both vascular cell adhesion molecule 1 (VCAM1) expression and bone marrow-derived macrophage adhesion to ECs were increased by hypoxia, and both were attenuated in SKI ECs. In WT ECs, ERO1 siRNA blocked hypoxia-induced VCAM1 expression and macrophage adhesion. In WT macrophages, hypoxia also stimulated both ERO1 and VEGF expression, and both were less in SKI macrophages. Compared with conditioned media of hypoxic SKI macrophages, conditioned media from WT macrophages had a greater effect on EC angiogenic behavior, and were blocked by VEGF neutralizing antibody. Taken together, under hypoxic conditions, SERCA 2 C674 and ERO1 enable increased VCAM1 expression and macrophage adhesion to ECs, as well as macrophage VEGF production that, in turn, promote angiogenesis. This study highlights the hitherto unrecognized interaction of two ER proteins, SERCA 2 C674 and ERO1, which mediate the EC and macrophage angiogenic response to ischemia/hypoxia.

Arginase inhibition augments nitric oxide production and facilitates left ventricular systolic function in doxorubicin-induced cardiomyopathy in mice.

A metabolizing enzyme arginase can decrease nitric oxide (NO) production by competing with NO synthase for arginine as a substrate, but its pathophysiological role in heart failure remains unknown. We aimed to investigate the effect of pharmacological inhibition of arginase on left ventricular function in doxorubicin-induced cardiomyopathy in mice. Doxorubicin administration for 5 weeks significantly increased protein expression levels or activity of arginase in the lungs and liver, and caused moderate increase in arginase 2 expression in the aorta. In the lungs, accumulated interstitial cells strongly expressed both arginase 1 and arginase 2 by doxorubicin administration. Echocardiography revealed that administration of a potent, reversible arginase inhibitor N-omega-hydroxy-nor-l-arginine completely reversed doxorubicin-induced decrease in the ejection fraction, in parallel with expression levels of BNP mRNA, without affecting apoptosis, hypertrophy, fibrosis, or macrophage infiltration in the left ventricle. Arginase inhibition reversibly lowered systolic blood pressure, and importantly, it recovered doxorubicin-induced decline in NO concentration in the serum, lungs, and aorta. Furthermore, arginase inhibition stimulated NO secretion from aortic endothelial cells and peritoneal macrophages in vitro. In conclusion, pharmacological inhibition of arginase augmented NO concentration in the serum, lungs, and aorta, promoted NO-mediated decrease in afterload for left ventricle, and facilitated left ventricular systolic function in doxorubicin-induced cardiomyopathy in mice.

Hepatic extracellular signal-regulated kinase 2 suppresses endoplasmic reticulum stress and protects from oxidative stress and endothelial dysfunction.

BACKGROUND: Insulin signaling comprises 2 major cascades: the insulin receptor substrate/phosphatidylinositol 3'-kinase/protein kinase B and Ras/Raf/mitogen-activated protein kinase/kinase/ERK pathways. While many studies on the tissue-specific effects of the insulin receptor substrate/phosphatidylinositol 3' -kinase/protein kinase B pathway have been conducted, the role of the other cascade in tissue-specific insulin resistance has not been investigated. High glucose/fatty acid toxicity, inflammation, and oxidative stress, all of which are associated with insulin resistance, can activate ERK. The liver plays a central role in metabolism, and hepatosteatosis is associated with vascular diseases. The aim of study was to elucidate the role of hepatic ERK2 in hepatosteatosis, metabolic remodeling, and endothelial dysfunction. METHODS AND RESULTS: We created liver-specific ERK2 knockout mice and fed them with a high-fat/high-sucrose diet for 20 weeks. The high-fat/high-sucrose diet-fed liver-specific ERK2 knockout mice exhibited a marked deterioration in hepatosteatosis and metabolic remodeling represented by impairment of glucose tolerance and decreased insulin sensitivity without changes in body weight, blood pressure, and serum cholesterol/triglyceride levels. In the mice, endoplasmic reticulum stress was induced together with decreased mRNA and protein expressions of hepatic sarco/endoplasmic reticulum Ca(2+)-ATPase 2. In a hepatoma cell line, inhibition of ERK activation- induced endoplasmic reticulum stress only in the presence of palmitate. Vascular reactive oxygen species were elevated with upregulation of nicotinamide adenine dinucleotide phosphate oxidase1 (Nox1) and Nox4 and decreased phosphorylation of endothelial nitric oxide synthase, which resulted in the remarkable endothelial dysfunction in high-fat/high-sucrose diet-fed liver-specific ERK2 knockout mice. CONCLUSIONS: Hepatic ERK2 suppresses endoplasmic reticulum stress and hepatosteatosis in vivo, which results in protection from vascular oxidative stress and endothelial dysfunction. These findings demonstrate a novel role of hepatic ERK2 in obese-induced insulin resistance in the protection from hepatovascular metabolic remodeling and vascular diseases.