The Safety and Efficacy of Sodium-Glucose Cotransporter-2 Inhibitors for Patients with Sarcopenia or Frailty: Double Edged Sword?

Sodium-glucose cotransporter-2 inhibitors (SGLT-2is) show cardiovascular protective effects, regardless of the patient's history of diabetes mellitus (DM). SGLT2is suppressed cardiovascular adverse events in patients with type 2 DM, and furthermore, SGLT-2is reduced the risk of worsening heart failure (HF) events or cardiovascular death in patients with HF. Along with these research findings, SGLT-2is are recommended for patients with HF in the latest guidelines. Despite these benefits, the concern surrounding the increasing risk of body weight loss and other adverse events has not yet been resolved, especially for patients with sarcopenia or frailty. The DAPA-HF and DELIVER trials consistently showed the efficacy and safety of SGLT-2i for HF patients with frailty. However, the Rockwood frailty index that derived from a cumulative deficit model was employed for frailty assessment in these trials, which might not be suitable for the evaluation of physical frailty or sarcopenia alone. There is no fixed consensus on which evaluation tool to use or its cutoff value for the diagnosis and assessment of frailty in HF patients, or which patients can receive SGLT-2i safely. In this review, we summarize the methodology of frailty assessment and discuss the efficacy and safety of SGLT-2i for HF patients with sarcopenia or frailty.

ß1 Adrenergic Receptor Autoantibodies and IgG Subclasses: Current Status and Unsolved Issues.

A wide range of anti-myocardial autoantibodies have been reported since the 1970s. Among them, autoantibodies against the ß1-adrenergic receptor (ß1AR-AAb) have been the most thoroughly investigated, especially in dilated cardiomyopathy (DCM). Β1AR-Aabs have agonist effects inducing desensitization of ß1AR, cardiomyocyte apoptosis, and sustained calcium influx which lead to cardiac dysfunction and arrhythmias. Β1AR-Aab has been reported to be detected in approximately 40% of patients with DCM, and the presence of the antibody has been associated with worse clinical outcomes. The removal of anti-myocardial autoantibodies including ß1AR-AAb by immunoadsorption is beneficial for the improvement of cardiac function for DCM patients. However, several studies have suggested that its efficacy depended on the removal of AAbs belonging to the IgG3 subclass, not total IgG. IgG subclasses differ in the structure of the Fc region, suggesting that the mechanism of action of ß1AR-AAb differs depending on the IgG subclasses. Our previous clinical research demonstrated that the patients with ß1AR-AAb better responded to ß-blocker therapy, but the following studies found that its response also differed among IgG subclasses. Further studies are needed to elucidate the possible pathogenic role of IgG subclasses of ß1AR-AAbs in DCM, and the broad spectrum of cardiovascular diseases including HF with preserved ejection fraction.

Reactive Oxygen Species in the Aorta and Perivascular Adipose Tissue Precedes Endothelial Dysfunction in the Aorta of Mice with a High-Fat High-Sucrose Diet and Additional Factors.

Metabolic syndrome (Mets) is the major contributor to the onset of metabolic complications, such as hypertension, type 2 diabetes mellitus (DM), dyslipidemia, and non-alcoholic fatty liver disease, resulting in cardiovascular diseases. C57BL/6 mice on a high-fat and high-sucrose diet (HFHSD) are a well-established model of Mets but have minor endothelial dysfunction in isolated aortas without perivascular adipose tissue (PVAT). The purpose of this study was to evaluate the effects of additional factors such as DM, dyslipidemia, and steatohepatitis on endothelial dysfunction in aortas without PVAT. Here, we employed eight-week-old male C57BL/6 mice fed with a normal diet (ND), HFHSD, steatohepatitis choline-deficient HFHSD (HFHSD-SH), and HFHSD containing 1% cholesterol and 0.1% deoxycholic acid (HFHSD-Chol) for 16 weeks. At week 20, some HFHSD-fed mice were treated with streptozocin to develop diabetes (HFHSD-DM). In PVAT-free aortas, the endothelial-dependent relaxation (EDR) did not differ between ND and HFHSD (p = 0.25), but in aortas with PVAT, the EDR of HFHSD-fed mice was impaired compared with ND-fed mice (p = 0.005). HFHSD-DM, HFHSD-SH, and HFHSD-Chol impaired the EDR in aortas without PVAT (p < 0.001, p = 0.019, and p = 0.009 vs. ND, respectively). Furthermore, tempol rescued the EDR in those models. In the Mets model, the EDR is compromised by PVAT, but with the addition of DM, dyslipidemia, and SH, the vessels themselves may result in impaired EDR.

A Low Arginine/Ornithine Ratio is Associated with Long-Term Cardiovascular Mortality.

AIMS: The long-term prognostic value of the bioavailability of L-arginine, an important source of nitric oxide for the maintenance of vascular endothelial function, has not been investigated fully. We therefore investigated the relationship between amino acid profile and long-term prognosis in patients with a history of standby coronary angiography. METHODS: We measured the serum concentrations of L-arginine, L-citrulline, and L-ornithine by high-speed liquid chromatography. We examined the relationship between the L-arginine/L-ornithine ratio and the incidence of all-cause death, cardiovascular death, and major adverse cardiovascular events (MACEs) in 262 patients (202 men and 60 women, age 65±13 years) who underwent coronary angiography over a period of ≤ 10 years. RESULTS: During the observation period of 5.5±3.2 years, 31 (12%) patients died, including 20 (8%) of cardiovascular death, while 32 (12%) had MACEs. Cox regression analysis revealed that L-arginine/L-ornithine ratio was associated with an increased risk for all-cause death (unadjusted hazard ratio, 95% confidence interval) (0.940, 0.888-0.995) and cardiovascular death (0.895, 0.821-0.965) (p＜0.05 for all). In a model adjusted for age, sex, hypertension, hyperlipidemia, diabetes, current smoking, renal function, and log10-transformed brain natriuretic peptide level, cardiovascular death (0.911, 0.839-0.990, p=0.028) retained an association with a low L-arginine/ L-ornithine ratio. When the patients were grouped according to an L-arginine/L-ornithine ratio of 1.16, the lower L-arginine/L-ornithine ratio group had significantly higher incidence of all-cause death, cardiovascular death, and MACEs. CONCLUSION: A low L-arginine/L-ornithine ratio may be associated with increased 10-year cardiac mortality.

Short-Chain Fatty Acids in Gut-Heart Axis: Their Role in the Pathology of Heart Failure.

Heart failure (HF) is a syndrome with global clinical and socioeconomic burden worldwide owing to its poor prognosis. Accumulating evidence has implicated the possible contribution of gut microbiota-derived metabolites, short-chain fatty acids (SCFAs), on the pathology of a variety of diseases. The changes of SCFA concentration were reported to be observed in various cardiovascular diseases including HF in experimental animals and humans. HF causes hypoperfusion and/or congestion in the gut, which may lead to lowered production of SCFAs, possibly through the pathological changes of the gut microenvironment including microbiota composition. Recent studies suggest that SCFAs may play a significant role in the pathology of HF, possibly through an agonistic effect on G-protein-coupled receptors, histone deacetylases (HDACs) inhibition, restoration of mitochondrial function, amelioration of cardiac inflammatory response, its utilization as an energy source, and remote effect attributable to a protective effect on the other organs. Collectively, in the pathology of HF, SCFAs might play a significant role as a key mediator in the gut-heart axis. However, these possible mechanisms have not been entirely clarified and need further investigation.

Endothelial Extracellular Signal-Regulated Kinase/Thromboxane A2/Prostanoid Receptor Pathway Aggravates Endothelial Dysfunction and Insulin Resistance in a Mouse Model of Metabolic Syndrome.

Background Metabolic syndrome is characterized by insulin resistance, which impairs intracellular signaling pathways and endothelial NO bioactivity, leading to cardiovascular complications. Extracellular signal-regulated kinase (ERK) is a major component of insulin signaling cascades that can be activated by many vasoactive peptides, hormones, and cytokines that are elevated in metabolic syndrome. The aim of this study was to clarify the role of endothelial ERK2 in vivo on NO bioactivity and insulin resistance in a mouse model of metabolic syndrome. Methods and Results Control and endothelial-specific ERK2 knockout mice were fed a high-fat/high-sucrose diet (HFHSD) for 24 weeks. Systolic blood pressure, endothelial function, and glucose metabolism were investigated. Systolic blood pressure was lowered with increased NO products and decreased thromboxane A2/prostanoid (TP) products in HFHSD-fed ERK2 knockout mice, and Nω-nitro-l-arginine methyl ester (L-NAME) increased it to the levels observed in HFHSD-fed controls. Acetylcholine-induced relaxation of aortic rings was increased, and aortic superoxide level was lowered in HFHSD-fed ERK2 knockout mice. S18886, an antagonist of the TP receptor, improved endothelial function and decreased superoxide level only in the rings from HFHSD-fed controls. Glucose intolerance and the impaired insulin sensitivity were blunted in HFHSD-fed ERK2 knockout mice without changes in body weight. In vivo, S18886 improved endothelial dysfunction, systolic blood pressure, fasting serum glucose and insulin levels, and suppressed nonalcoholic fatty liver disease scores only in HFHSD-fed controls. Conclusions Endothelial ERK2 increased superoxide level and decreased NO bioactivity, resulting in the deterioration of endothelial function, insulin resistance, and steatohepatitis, which were improved by a TP receptor antagonist, in a mouse model of metabolic syndrome.

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.

Effectiveness of the d-ROMs oxidative stress test to predict long-term cardiovascular mortality.

BACKGROUND: The long-term prognostic value of the derivatives of reactive oxidative metabolites (d-ROMs) oxidative stress test, which measures hydroperoxide in blood, has not been fully investigated. METHODS AND RESULTS: We administered the d-ROMs test to 265 patients with cardiovascular disease (204 men, 61 women; age, 65 ± 13 years) and followed these patients for up to 10 years. During the observational period of 5.82 (2.47-8.34) years, 31 (12%) patients died, including 20 (8%) of cardiovascular death, and 33 (12%) had major adverse cardiovascular events (MACEs). Cox regression analysis revealed that patients with a d-ROMs value ≥395 U.CARR had a greater risk for all-cause mortality [unadjusted hazard ratio (95% confidence interval), 3.586 (1.772-7.257)], cardiovascular death [7.034 (2.805-17.640)], and MACEs [4.440 (2.237-8.814)] (p < 0.001 for all). In a model adjusted for age, sex, estimated glomerular filtration rate, C-reactive protein, diabetes, hypertension, hyperlipidemia, coronary artery diseases, current smoking, and log-transformed brain natriuretic peptide, all-cause death [2.311 (1.059-5.135), p = 0.036], cardiovascular death [4.398 (1.599-12.099), p = 0.004], MACEs [2.696 (1.266-5.739), p = 0.010] were still significant in patients with high d-ROMS values. CONCLUSION: A high d-ROMs value is an independent predictor of the long-term risk of cardiovascular mortality. A d-ROMs value of 395 U.CARR was considered to be an appropriate threshold for distinguishing prognosis.

Artificially Created Reentry Circuit by Laser Irradiation Causes Atrial Tachycardia to Persist in Murine Atria.

BACKGROUND: There is a gradual progression from paroxysmal to persistent atrial fibrillation (AF) in humans. To elucidate the mechanism involved, the creation of an artificial atrial substrate to persist AF in mice was attempted.Methods and Results:This study used wild type (WT) mice, but it is difficult to induce AF in them. A novel antegrade perfusion method from the left ventricle (LV) to enlarge both atria for artificial atrial modification was proposed in this study. Short duration AF was induced by burst pacing under this method. Optical mapping analysis revealed non-sustained focal type and meandering spiral reentrants after short duration AF. A tiny artificial substrate (~1.2 mm in diameter) was added in by laser irradiation to create a critical atrial arrhythmogenic substrate. Burst pacing was performed in a non-laser group (n=8), a circular-shape laser group (n=8), and a wedge-shaped dent laser group (n=8). We defined AF and atrial tachycardia (AT) as atrial arrhythmia (AA). Long-lasting AA was defined as lasting for ≥30 min. Long-lasting AA was observed in 0/8, 0/8, and 6/8 (75%) mice in each group. Optical mapping analysis revealed that the mechanism was AT with a stationary rotor around the irradiated margin. CONCLUSIONS: Regrettably, this study failed to reproduce persistent AF, but succeeded in creating an arrhythmic substrate that causes sustained AT in WT mice.

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.

Activation of LKB1 rescues 3T3-L1 adipocytes from senescence induced by Sirt1 knock-down: a pivotal role of LKB1 in cellular aging.

Previous reports have shown that excess calorie intake promotes p53 dependent senescence in mouse adipose tissues. The objective of the current study was to address the mechanism underlying this observation, i.e. adipocyte aging. Using cultured 3T3-L1 cells, we investigated the involvement of energy regulators Sirt1, AMPK, and LKB1 in senescence. Fifteen days post differentiation, Sirt1 knock-down increased senescence-associated beta-galactosidase (SA-ß-Gal) staining by 20-40% (p<0.05, n=12) and both cyclin kinase inhibitor p21Cip and chemokine receptor IL8Rb expression by 2-4 fold. ATP and expression of mitochondria Complex 1 were also reduced by 30% and 50%, respectively (p<0.05, n=4). Such energy depletion may have caused the observed increase in AMPK activity, despite LKB1 activity downregulation. This association between Sirt1 and LKB1 activity was confirmed in vivo in mouse adipose tissue. Upregulation of LKB1 activity by expression of the Sirt1-insensitive LKB1-K48R mutant in 3T3-L1 cells completely prevented the senescence-associated changes of Sirt1 knock-down. In addition, cellular senescence, which also occurs in cultured primary human aortic endothelial cells, was largely prevented by ectopic expression of LKB1. These results suggest that LKB1 plays a pivotal role in cellular senescence occurring in adipocytes and other cell types.

Impaired nicotinamide adenine dinucleotide (NAD+ ) metabolism in diabetes and diabetic tissues: Implications for nicotinamide-related compound treatment.

One of the biochemical abnormalities found in diabetic tissues is a decrease in the cytosolic oxidized to reduced forms of the nicotinamide adenine dinucleotide ratio (NAD+ /NADH also known as pseudohypoxia) caused by oxidation of excessive substrates (glucose through the polyol pathway, free fatty acids and lactate). Subsequently, a decline in NAD+ levels as a result of the activation of poly adenine nucleotide diphosphate-ribose polymerase (mainly in type 1 diabetes) or the inhibition of adenine nucleotide monophosphate-activated protein kinase (in type 2 diabetes). Thus, replenishment of NAD+ levels by nicotinamide-related compounds could be beneficial. However, these compounds also increase nicotinamide catabolites that cause oxidative stress. This is particularly troublesome for patients with diabetes, because they have impaired nicotinamide salvage pathway reactions at the level of nicotinamide phosphoribosyl transferase and phosphoribosyl pyrophosphate, which occurs by the following mechanisms. First, phosphoribosyl pyrophosphate synthesis from pentose phosphate pathway is compromised by a decrease in plasma thiamine and transketolase activity. Second, nicotinamide phosphoribosyl transferase expression is decreased because of reduced adenosine monophosphate-activated protein kinase activity, which occurs in type 2 diabetes. The adenosine monophosphate-activated protein kinase inhibition is caused by an activation of protein kinase C and D1 as a result of enhanced diacylglycerol synthesis caused by pseudohypoxia and increased fatty acids levels. In this regard, nicotinamide-related compounds should be given with caution to treat diabetes. To minimize the risk and maximize the benefit, nicotinamide-related compounds should be taken with insulin sensitizers (for type 2 diabetes), polyphenols, benfotiamine, acetyl-L-carnitine and aldose reductase inhibitors. The efficacy of these regimens can be monitored by measuring serum NAD+ and urinary nicotinamide catabolites.

Impact of oxidative posttranslational modifications of SERCA2 on heart failure exacerbation in young patients with non-ischemic cardiomyopathy: A pilot study.

BACKGROUND: Oxidative posttranslational modifications (OPTM) impair the function of Sarcoplasmic/endoplasmic reticulum (SR) calcium (Ca2+) ATPase (SERCA) 2 and trigger cytosolic Ca2+ dysregulation. We investigated the extent of OPTM of SERCA2 in patients with non-ischemic cardiomyopathy (NICM). METHODS AND RESULTS: Endomyocardial biopsy (EMB) was obtained in 40 consecutive patients with NICM. Total expression and OPTM of SERCA2, including sulfonylation at cysteine-674 (S-SERCA2) and nitration at tyrosine-294/295 (N-SERCA2), were examined by immunohistochemical analysis. S-SERCA2 increased in the presence of late gadolinium enhancement on cardiac magnetic resonance imaging. S-SERCA2/SERCA2 and N-SERCA2/SERCA2 correlated with cardiac fibrosis evaluated by Masson's trichrome staining of EMB. SERCA2 expression modestly increased in parallel with an upward trend in OPTM of SERCA2 with aging. This tendency became prominent only in patients aged >65 years. OPTM of SERCA2 positively correlated with brain natriuretic peptide (BNP) values only in patients aged ≤65 years. Composite major adverse cardiac events (MACE) increased more in the high OPTM group of younger patients; however, MACE-free survival was similar irrespective of the extent of OPTM in older patients. CONCLUSIONS: OPTM of SERCA2 correlate with myocardial fibrosis in NICM. In younger patients, OPTM of SERCA2 correlate with elevated BNP and increased composite MACE.

Production of adeno-associated virus vectors for in vitro and in vivo applications.

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010-1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

Endothelial Insulin Resistance of Freshly Isolated Arterial Endothelial Cells From Radial Sheaths in Patients With Suspected Coronary Artery Disease.

Background Endothelial insulin resistance is insulin-insensitivity in the vascular endothelium and can be observed in experimental models. This study aimed to investigate endothelial insulin resistance in patients with suspected coronary artery disease. To this end, a novel method of obtaining freshly isolated arterial endothelial cells from a radial catheter sheath was developed. Methods and Results Freshly isolated arterial endothelial cells were retrieved from catheter sheaths placed in radial arteries for coronary angiography (n=69, patient age 64±12 years). The endothelial cells were divided into groups for incubation with or without insulin, vascular endothelial growth factor, or acetylcholine. The intensity of phosphorylated endothelial nitric oxide synthase at Ser1177 (p- eNOS ) was quantified by immunofluorescence microscopy. The percentage increase of insulin-induced phosphorylated endothelial nitric oxide synthase correlated negatively with derivatives of reactive oxygen metabolites, an oxidative stress test ( r=-0.348, n=53, P=0.011), E/E', an index of left ventricular diastolic dysfunction in Doppler echocardiography (ρ=-0.374, n=49, P=0.008), and log-transformed brain natriuretic peptide ( r=-0.266, n=62, P=0.037). Furthermore, percentage increase of insulin-induced p- eNOS was an independent factor for the cardio-ankle vascular index (standardized coefficient ß=-0.293, n=42, P=0.021) in the multivariate regression analysis of adaptive least absolute shrinkage and selection operator. Conclusions Our results suggested that endothelial insulin resistance is associated with oxidative stress, left ventricular diastolic dysfunction, heart failure, and arterial stiffness.

Computed tomography-measured pulmonary artery to aorta ratio and EUTOS score for detecting dasatinib-induced pulmonary arterial hypertension.

BACKGROUND: Periodic echo-based screening to detect early stages of a rare complication of dasatinib, pulmonary arterial hypertension (PAH), is inefficient and weakens the potential benefit of dasatinib as a potent drug for chronic myelogenous leukemia (CML). This study aimed to identify the predisposing factors of DASA-PAH to stratify high-risk patients for dasatinib-induced PAH (DASA-PAH). METHODS: Sixty consecutive adult patients who received dasatinib were enrolled in this case-control study. We defined DASA-PAH when at least one of the following four criteria was met: (1) recent electrocardiographic changes indicating right ventricular pressure overload, (2) estimated systolic pulmonary arterial pressure > 40 mmHg measured by Doppler echocardiography; (3) computed tomography (CT)-measured pulmonary artery to aorta diameter (PaD/AoD) ratio > 1; and (4) mean pulmonary arterial pressure > 25 mmHg and pulmonary artery wedge pressure < 15 mmHg measured by right heart catheterization. RESULTS: We identified 13 patients with DASA-PAH among 59 patients analyzed. Baseline PaD/AoD ratios of patients who developed DASA-PAH (PH group) were significantly larger than those who did not (NPH group). A dramatic rise in PaD/AoD ratio after dasatinib treatment was observed. Interestingly, the EUTOS score and spleen size were significantly smaller in the PH than in the NPH group. CONCLUSION: High baseline PaD/AoD ratio and low EUTOS score were associated with DASA-PAH development. The spleen might play a protective role against DASA-PAH.

Preserved Vasoconstriction and Relaxation of Saphenous Vein Grafts Obtained by a No-Touch Technique for Coronary Artery Bypass Grafting.

BACKGROUND: To obtain a saphenous vein graft (SVG) for coronary artery bypass grafting (CABG), the benefit of using a no-touch (NT) technique in vascular function has not been fully investigated. Methods and Results: The pathological and physiological functions of human SVGs with a NT technique to preserve the perivascular adipose tissue (PVAT) and ones obtained by using a conventional (CON) technique removing PVAT, were examined. Immunohistochemistry of the section of SVGs showed that the phosphorylation of endothelial nitric oxide synthase in the endothelium of the NT group was more responsive to vascular endothelial growth factor. A myograph of SVGs showed greater contraction with phenylephrine in the NT group. However, the strong contraction was eliminated in SVGs taken by electrocautery. In the 10 patients whose SVGs were taken without electrocautery, endothelial-dependent relaxation with bradykinin was apparently increased in the CON group more than in the NT group. Smooth muscle relaxation with nitroprusside was higher in the CON group at the lower concentrations; however, the relaxation became greater in the NT group at the high concentrations. Therefore, the effect of neutralizing PVAT-released factors in the both groups was further examined. After medium of NT and CON were exchanged in half, relaxation of SVGs was immediately restored in the NT group. CONCLUSIONS: The results suggest that the NT technique preserves the functions of vasoconstriction and relaxation. Also, the presence of PVAT-released vasoconstrictive factors was suspected.

Resveratrol-Induced AMP-Activated Protein Kinase Activation Is Cell-Type Dependent: Lessons from Basic Research for Clinical Application.

Despite the promising effects of resveratrol, its efficacy in the clinic remains controversial. We were the first group to report that the SIRT1 activator resveratrol activates AMP-activated protein kinase (AMPK) (Diabetes 2005; 54: A383), and we think that the variability of this cascade may be responsible for the inconsistency of resveratrol's effects. Our current studies suggest that the effect of SIRT1 activators such as resveratrol may not be solely through activation of SIRT1, but also through an integrated effect of SIRT1-liver kinase B1 (LKB1)-AMPK. In this context, resveratrol activates SIRT1 (1) by directly binding to SIRT1; and (2) by increasing NAD⁺ levels by upregulating the salvage pathway through Nampt activation, an effect mediated by AMPK. The first mechanism promotes deacetylation of a limited number of SIRT1 substrate proteins (e.g., PGC-1). The second mechanism (which may be more important than the first) activates other sirtuins in addition to SIRT1, which affects a broad spectrum of substrates. Despite these findings, detailed mechanisms of how resveratrol activates AMPK have not been reported. Here, we show that (1) resveratrol-induced activation of AMPK requires the presence of functional LKB1; (2) Resveratrol increases LKB1 activity, which involves translocation and phosphorylation at T336 and S428; (3) Activation of LKB1 causes proteasomal degradation of LKB1; (4) At high concentrations (50-100 µM), resveratrol also activates AMPK through increasing AMP levels; and (5) The above-mentioned activation mechanisms vary among cell types, and in some cell types, resveratrol fails to activate AMPK. These results suggest that resveratrol-induced activation of AMPK is not a ubiquitous phenomenon. In addition, AMPK-mediated increases in NAD⁺ in the second mechanism require several ATPs, which may not be available in many pathological conditions. These phenomena may explain why resveratrol is not always consistently beneficial in a clinical setting.