Moderate- and high-intensity exhaustive exercise in the heat induce a similar increase in monocyte Hsp72

This study examined the relationship between exhaustive exercise in the heat at moderate and high intensities on the intracellular heat shock protein 72 (iHsp72) response. Twelve male subjects cycled to exhaustion at 60 and 75 % of maximal oxygen uptake in hot conditions (40 °C, 50 % RH). iHsp72 concentration was measured in monocytes before, at exhaustion and 24 h after exercise. Rectal temperature, heart rate and oxygen uptake were recorded during exercise. Volitional exhaustion occurred at 58.9 ± 12.1 and 27.3 ± 9.5 min (P < 0.001) and a rectal temperature of 39.8 ± 0.4 and 39.2 ± 0.6 °C (P = 0.002), respectively, for 60 and 75 %. The area under the curve above a rectal temperature of 38.5 °C was greater at 60 % (17.5 ± 6.6 °C min) than 75 % (3.4 ± 4.8 °C min; P < 0.001), whereas the rate of increase in rectal temperature was greater at 75 % (5.1 ± 1.7 vs. 2.2 ± 1.4 °C h⁻¹; P < 0.001). iHsp72 concentration increased similarly at exhaustion relative to pre-exercise (P = 0.044) and then increased further at 24 h (P < 0.001). Multiple regression analysis revealed no predictor variables associated with iHsp72 expression; however, a correlation was observed between exercise intensities for the increase in iHsp expression at exhaustion and 24 h (P < 0.05). These results suggest that iHsp72 expression increased in relation to the level of hyperthermia attained and sustained at 60 % and the higher metabolic rate and greater rate of increase in core temperature at 75 %, with the further increase in iHsp72 concentration 24 h after exercise reinforcing its role as a chaperone and cytoprotective agent.     

Mild eccentric exercise increases Hsp72 content in skeletal muscles from adult and late middle-aged rats

The loss of muscle mass with age or sarcopenia contributes to increased morbidity and mortality. Thus, preventing muscle loss with age is important for maintaining health. Hsp72, the inducible member of the Hsp70 family, is known to provide protection to skeletal muscle and can be increased by exercise. However, ability to increase Hsp72 by exercise is intensity-dependent and appears to diminish with advanced age. Thus, other exercise modalities capable of increasing HSP content and potentially preventing the age related loss of muscle need to be explored. The purpose of this study was to determine if the stress from one bout of mild eccentric exercise was sufficient to elicit an increase in Hsp72 content in the vastus intermedius (VI) and white gastrocnemius (WG) muscles, and if the Hsp72 response differed between adult and late middle-aged rats. To do this, 30 adult (6 months) and late middle-aged (24 months) F344BN rats were randomly divided into three groups (n = 6/group): control (C), level exercise (16 m^.min⁻¹) and eccentric exercise (16 m^.min⁻¹, 16 degree decline). Exercised animals were sacrificed immediately post-exercise or after 48 hours. Hematoxylin and Eosin staining was used to assess muscle damage, while Western Blotting was used to measure muscle Hsp72 content. A nested ANOVA with Tukey post hoc analysis was performed to determine significant difference (p < 0.05) between groups. Hsp72 content was increased in the VI for both adult and late middle-aged rats 48 hours after eccentric exercise when compared to level and control groups but no differences between age groups was observed. Hsp72 was not detected in the WG following any type of exercise. In conclusion, mild eccentric exercise can increase Hsp72 content in the rat VI muscle and this response is maintained into late middle-age.     

Extracellular Hsp72 concentration relates to a minimum endogenous criteria during acute exercise-heat exposure

Extracellular heat shock protein 72 (eHsp72) concentration increases during exercise-heat stress when conditions elicit physiological strain. Differences in severity of environmental and exercise stimuli have elicited varied response to stress. The present study aimed to quantify the extent of increased eHsp72 with increased exogenous heat stress, and determine related endogenous markers of strain in an exercise-heat model. Ten males cycled for 90 min at 50 % in three conditions (TEMP, 20 °C/63 % RH; HOT, 30.2 °C/51%RH; VHOT, 40.0 °C/37%RH). Plasma was analysed for eHsp72 pre, immediately post and 24-h post each trial utilising a commercially available ELISA. Increased eHsp72 concentration was observed post VHOT trial (+172.4 %) (p < 0.05), but not TEMP (−1.9 %) or HOT (+25.7 %) conditions. eHsp72 returned to baseline values within 24 h in all conditions. Changes were observed in rectal temperature (T_rec), rate of T_rec increase, area under the curve for T_rec of 38.5 and 39.0 °C, duration T_rec ≥ 38.5 and ≥39.0 °C, and change in muscle temperature, between VHOT, and TEMP and HOT, but not between TEMP and HOT. Each condition also elicited significantly increasing physiological strain, described by sweat rate, heart rate, physiological strain index, rating of perceived exertion and thermal sensation. Stepwise multiple regression reported rate of T_rec increase and change in T_rec to be predictors of increased eHsp72 concentration. Data suggests eHsp72 concentration increases once systemic temperature and sympathetic activity exceeds a minimum endogenous criteria elicited during VHOT conditions and is likely to be modulated by large, rapid changes in core temperature.     

Exercise reduces cellular stress related to skeletal muscle insulin resistance

This study sought to evaluate the effects of a single session of exercise on the expression of Hsp70, of c-jun N-terminal kinase (JNK), and insulin receptor substrate 1 serine 612 (IRS^ser612) phosphorylation in the skeletal muscle of obese and obese insulin-resistant patients. Twenty-seven volunteers were divided into three experimental groups (eutrophic insulin-sensitive, obese insulin-sensitive, and obese insulin-resistant) according to their body mass index and the presence of insulin resistance. The volunteers performed 60 min of aerobic exercise on a cycle ergometer at 60 % of peak oxygen consumption. M. vastus lateralis samples were obtained before and after exercise. The protein expressions were evaluated by Western blot. Our findings show that compared with paired eutrophic controls, obese subjects have higher basal levels of p-JNK (100 ± 23 % vs. 227 ± 67 %, p = 0.03) and p-IRS-1^ser612 (100 ± 23 % vs. 340 ± 67 %, p < 0.001) and reduced HSP70 (100 ± 16 % vs. 63 ± 12 %, p < 0.001). The presence of insulin resistance results in a further increase in p-JNK (460 ± 107 %, p < 0.001) and a decrease in Hsp70 (46 ± 5 %, p = 0.006), but p-IRS-1^ser612 levels did not differ from obese subjects (312 ± 73 %, p > 0.05). Exercise reduced p-JNK in obese insulin-resistant subjects (328 ± 33 %, p = 0.001), but not in controls or obese subjects. Furthermore, exercise reduced p-IRS-1^ser612 for both obese (122 ± 44 %) and obese insulin-resistant (185 ± 36 %) subjects. A main effect of exercise was observed in HSP70 (p = 0.007). We demonstrated that a single session of exercise promotes changes that characterize a reduction in cellular stress that may contribute to exercise-induced increase in insulin sensitivity.     

Treatment with atorvastatin is associated with a better prognosis in chronic heart failure with systolic dysfunction: results from The Daunia Heart Failure Registry

Background

Few works have evaluated the effect of statins on left ventricular dysfunction in patients with chronic heart failure (CHF), by using tissue Doppler imaging (TDI). We therefore aimed to investigate whether atorvastatin treatment may influence prognosis and myocardial performance evaluated by TDI in subjects with CHF.

Methods

Five hundred thirty-two consecutive CHF outpatients enrolled in a local registry, the Daunia Heart Failure Registry, were prospectively analysed. 195 patients with CHF and left ventricular ejection fraction (LVEF) ≤40 %, either in treatment with atorvastatin (N: 114) or without statins (N: 81), underwent TDI examination. Adverse events were evaluated during follow-up.

Results

The atorvastatin group showed a lower incidence of adverse events (cardiac death: 0 % vs 7 %, p < 0.01), and better TDI performance (E/E’ 15 ± 5.7 vs 18 ± 8.3, p < 001) than controls. Ischaemic CHF patients in treatment with atorvastatin also showed a lower incidence of adverse events (death: 10 % vs 26 %, p < 0.05; sustained ventricular arrhythmias: 5 % vs 19 %, p < 0.05, cardiac death: 0 vs 8 %, p < 0.05) and better TDI performance (E/E’ ratio: 15.00 ± 5.68 vs 19.72 ± 9.14, p < 0.01; St: 353.70 ± 48.96 vs 303.33 ± 68.52 msec, p < 0.01) than controls. The association between atorvastatin and lower rates of cardiac death remained statistically significant even after correction in a multivariable analysis (RR 0.83, 95 % CI 0.71–0.96, p < 0.05 in CHF with LVEF ≤40 %; RR 0.77, 95 % CI 0.62–0.95, p < 0.05 in ischaemic CHF with LVEF ≤40 %).

Conclusions

Treatment with atorvastatin in outpatients with systolic CHF is associated with fewer cardiac deaths, and a better left ventricular performance, as assessed by TDI.     

Cold stress induces antioxidants and Hsps in chicken immune organs

The aim of this study was to investigate the effects of cold stress on oxidative indexes, immune function, and the expression levels of heat shock protein (Hsp90, Hsp70, Hsp60, Hsp40, and Hsp27) in immune organs of chickens. Two hundred forty 15-day-old male chickens were randomly divided into 12 groups and kept under the temperature of (12 ± 1) °C for acute and chronic cold stress. There were one control group and five treatment groups for acute cold stress and three control groups and three treatment groups for chronic cold stress. The results showed that cold stress influence the activities of antioxidant enzymes in the immune organs. The activities of SOD and GSH-Px were first increased then decreased, and activity of total antioxidation capacity (T-AOC) was significantly decreased (P < 0.05) at the acute cold stress in chicks; however, T-AOC activities were significantly increased (P < 0.05) at the chronic cold stress in these tissues. Cold stress induced higher level of malondialdehyde (MDA) in chicken immune organs. In addition, the cytokine contents were increased in cold stress groups. As one protective factor, the expression levels of Hsps were increased significantly (P < 0.05) in both cold stress groups. These results suggested that cold stress induced the oxidative stress in the three tissues and influenced immune function of chicks. Higher expression of Hsps (Hsp90, Hsp70, Hsp60, Hsp40, and Hsp27) may play a role in protecting immune organs against cold stress.     

The lost correlation between heat shock protein 70 (HSPA1A) and plasminogen activator inhibitor-1 in patients with type 2 diabetes and albuminuria

We aimed to study the relation between plasma levels of stress-induced heat shock protein 70 (HSPA1A) with plasminogen activator inhibitor-1 (PAI-1) and high-density lipoprotein cholesterol (HDL-C), apolipoprotein A1 (Apo-A1), and HDL-C/Apo-A1 ratio. In a matched case-control study on patients with diabetes (40 patients with albuminuria and 40 without albuminuria matched for age, sex, and body mass index), we observed that plasma levels of HSPA1A and PAI-1 are increased in patients with albuminuria (0.55 ± 0.02 vs. 0.77 ± 0.04 ng/ml, p value <0.001 for HSPA1A; 25.9 ± 2 vs. 31.8 ± 2.4 ng/ml, p value <0.05 for PAI-1). There was a significant correlation between HSPA1A and PAI-1 in diabetic patients without albuminuria (r = 0.28; p value = 0.04), but not in those with albuminuria (r = 0.07; p value = 0.63). No association was found between HSPA1A and HDL-C, between HSPA1A and Apo-A1, or between HSPA1A and HDL-C/Apo-A1 ratio. We concluded that there is a direct correlation between plasma HSPA1A and PAI-1 levels in patients with diabetes, which is lost when they develop albuminuria.     

Hsp70 regulates the doxorubicin-mediated heart failure in Hsp70-transgenic mice

The aim of this study was to investigate the potential protective effect of the Hsp70 protein in the cardiac dysfunction induced by doxorubicin (DOX) and the mechanisms of its action. For this purpose, we used both wild-type mice (F1/F1) and Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70. Both types were subjected to chronic DOX administration (3 mg/kg intraperitoneally every week for 10 weeks, with an interval from weeks 4 to 6). Primary cell cultures isolated from embryos of these mice were also studied. During DOX administration, the mortality rate as well as weight reduction were lower in Tg/Tg compared to F1/F1 mice (P < 0.05). In vivo cardiac function assessment by transthoracic echocardiography showed that the reduction in left ventricular systolic function observed after DOX administration was lower in Tg/Tg mice (P < 0.05). The study in primary embryonic cell lines showed that the apoptosis after incubation with DOX was reduced in cells overexpressing Hsp70 (Tg/Tg), while the apoptotic pathway that was activated by DOX administration involved activated protein factors such as p53, Bax, caspase-9, caspase-3, and PARP-1. In myocardial protein extracts from identical mice with DOX-induced heart failure, the particular activated apoptotic pathway was confirmed, while the presence of Hsp70 appeared to inhibit the apoptotic pathway upstream of the p53 activation. Our results, in this DOX-induced heart failure model, indicate that Hsp70 overexpression in Tg/Tg transgenic mice provides protection from myocardial damage via an Hsp70-block in p53 activation, thus reducing the subsequent apoptotic mechanism.     

Salivary extracellular heat shock protein 70 (eHSP70) levels increase after 59 min of intense exercise and correlate with resting salivary secretory immunoglobulin A (SIgA) levels at rest

This study aimed to identify the response of a salivary stress protein, extracellular heat shock protein (eHSP70), to intense exercise and to investigate the relationship between salivary eHSP70 and salivary immunoglobulin A (SIgA) levels in response to exercise. Sixteen healthy sedentary young males (means ± SD 23.8 ± 1.5 years, 172.2 ± 6.4 cm, 68.3 ± 7.4 kg) performed 59 min of cycling exercise at 75 % VO2_max. Saliva and whole blood samples were collected before (Pre), immediately after (Post), and at 1, 2, 3, and 4 h after completion of the exercise (1, 2, 3, and 4 h). The salivary eHSP70 and SIgA levels were measured by enzyme-linked imunosorbent assay (ELISA), and the secretion rates were computed by multiplying the concentration by the saliva flow rate. White blood cells were analyzed using an automated cell counter with a direct-current detection system. The salivary eHSP70 secretion rates were 1.11 ± 0.86, 1.51 ± 1.47, 1.57 ± 1.32, 2.21 ± 2.04, 3.36 ± 2.72, and 6.89 ± 4.02 ng · min⁻¹ at Pre, Post, and 1, 2, 3, and 4 h, respectively. The salivary eHSP70 secretion rate was significantly higher at 4 h than that at Pre, Post, 1, and 3 h (p < 0.05). The SIgA secretion rates were 26.9 ± 12.6, 20.3 ± 10.4, 19.6 ± 11.0, 21.8 ± 12.8, 21.5 ± 11.9, and 21.9 ± 11.7 μg · min⁻¹ at Pre, Post, 1, 2, 3, and 4 h, respectively. The salivary SIgA secretion rate was significantly lower between 1 and 4 h than that at Pre (p < 0.05). There was a positive correlation between salivary eHSP70 and SIgA in both concentration and secretion rates before exercise (p < 0.05). The absolute number of white blood cells significantly increased after exercise, with a maximum at 2 h (p < 0.05). The neutrophil/lymphocyte ratio was significantly increased from 1 to 4 h when compared with that in the Pre samples (p < 0.05). The present study revealed that salivary eHSP70 significantly increased at 4 h after the 59 min of intense exercise in sedentary male subjects. Exercise stress can induce elevated salivary eHSP70 level and upregulate oral immune function partially.     

Heat shock protein 70 and albuminuria in patients with type 2 diabetes: a matched case control study

Here, we aimed to study serum heat shock protein (HSP) 70 levels in diabetic patients with and without albuminuria. We performed a 1:1 matched case control study on 40 diabetic patients with albuminuria as cases and 40 age, sex, body mass index matched diabetic patients without albuminuria (normoalbuminuria) as controls. Normoalbuminuria was defined as urinary albumin excretion rate <15 mg/12 h, and albuminuria was defined as urinary albumin excretion rate between 100–400 mg/12 h. Patients with albuminuria had a higher HSP70 than controls (0.83 ± 0.50 vs. 0.63 ± 0.06; p = 0.02), while they did not differ in any other studied variables. In ten of the studied pairs, the controls had higher HSP70 levels than cases (reverse relationship). Patients in the “direct relationship group” had higher HbA1c values than the patients in the “reverse relationship group” (8.9 ± 0.3 vs. 7.3 ± 0.6, p = 0.04). Cases in the reverse pairs had a lower low density lipoprotein cholesterol levels than their controls. The odds ratio of HSP70 in the prediction of albuminuria was (28.69 (3.2–250.1), p = 0.002). In conclusion, we have shown an increased HSP70 levels in diabetic patients with albuminuria.     

Impact of hyperthermic intraperitoneal chemotherapy on Hsp27 protein expression in serum of patients with peritoneal carcinomatosis

Despite the strong rationale for combining cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) in patients with peritoneal carcinomatosis, thermotolerance and chemoresistance might result from heat shock protein overexpression. The aim of the present study was thus to determine whether the heat shock protein 27 (Hsp27), a potential factor in resistance to treatment, could have a higher level in serum from patients under this combined therapy. Patients receiving CRS plus HIPEC for peritoneal carcinomatosis (group 1), patients with cancer or a history of cancer undergoing abdominal surgery (group 2), and patients without malignancies undergoing abdominal surgery (group 3) were included. Hsp27 serum levels were determined before and at different times following CRS and HIPEC using enzyme-linked immunosorbent assay. In group 1 (n = 25), the high Hsp27 levels, observed at the end of surgery compared with before (p < 0.0001), decreased during HIPEC, but remained significantly higher than before surgery (p < 0.0005). In groups 2 (n = 11) and 3 (n = 15), surgery did not significantly increase Hsp27 levels. A targeted molecular strategy, inhibiting Hsp27 expression in tumor tissue, could significantly reduce resistance to the combined CRS plus HIPEC treatment. This approach should be further assessed in a clinical phase I trial.     

Induction of the 72 kDa heat shock protein by glucose ingestion in black pregnant women

Obese Black women are at increased risk for development of gestational diabetes mellitus and have worse perinatal outcomes than do obese women of other ethnicities. Since hsp72 has been associated with the regulation of obesity-induced insulin resistance, we evaluated associations between glucose ingestion, hsp72 release and insulin production in Black pregnant women. Specifically, the effect of a 50-g glucose challenge test (GCT) on heat shock protein and insulin levels in the circulation 1 h later was evaluated. Hsp27 and hsp60 levels remained unchanged. In contrast, serum levels of hsp72 markedly increased after glucose ingestion (p = 0.0054). Further analysis revealed that this increase was limited to women who were not obese (body mass index <30). Insulin levels pre-GCT were positively correlated with body mass index (p = 0.0189). Median insulin concentrations also increased post GCT in non-obese women but remained almost unchanged in obese women. Post-GCT serum hsp72 concentrations were inversely correlated with post GCT insulin concentrations (p = 0.0111). These observations suggest that glucose intake during gestation in Black women rapidly leads to an elevation in circulating hsp72 only in non-obese Black women. The release of hsp72 may regulate the extent of insulin production in response to a glucose challenge and, thereby, protect the mother and/or fetus from development of hyperglycemia, hyperinsulinemia, and/or immune system alterations.     

A comparison of two commercially available ELISA methods for the quantification of human plasma heat shock protein 70 during rest and exercise stress

This study compared resting and exercise heat/hypoxic stress-induced levels of plasma extracellular heat shock protein 70 (eHSP70) in humans using two commercially available enzyme-linked immunosorbent assay (ELIS)A kits. EDTA plasma samples were collected from 21 males during two separate investigations. Participants in part A completed a 60-min treadmill run in the heat (HOT70; 33.0 ± 0.1 °C, 28.7 ± 0.8 %, n = 6) at 70 % V̇O_2max. Participants in part B completed 60 min of cycling exercise at 50 % V̇O_2max in either hot (HOT50; 40.5 °C, 25.4 relative humidity (RH)%, n = 7) or hypoxic (HYP50; fraction of inspired oxygen (F_IO₂) = 0.14, 21 °C, 35 % RH, n = 8) conditions. Samples were collected prior to and immediately upon termination of exercise and analysed for eHSP70 using EKS-715 high-sensitivity HSP70 ELISA and new ENZ-KIT-101 Amp’d™ HSP70 high-sensitivity ELISA. ENZ-KIT was superior in detecting resting eHSP70 (1.54 ± 3.27 ng·mL⁻¹; range 0.08 to 14.01 ng·mL⁻¹), with concentrations obtained from 100 % of samples compared to 19 % with EKS-715 assay. The ENZ-KIT requires optimisation prior to running samples in order to ensure participants fall within the standard curve, a step not required with EKS-715. Using ENZ-KIT, a 1:4 dilution allowed for quantification of resting HSP70 in 26/32 samples, with a 1:8 (n = 3) and 1:16 (n = 3) dilution required to determine the remaining samples. After exercise, eHSP70 was detected in 6/21 and 21/21 samples using EKS-715 and ENZ-KIT, respectively. eHSP70 was increased from rest after HOT70 (p < 0.05), but not HOT50 (p > 0.05) or HYP50 (p > 0.05) when analysed using ENZ-KIT. It is recommended that future studies requiring the precise determination of resting plasma eHSP70 use the ENZ-KIT (i.e. HSP70 Amp’d® ELISA) instead of the EKS-715 assay, despite additional assay development time and cost required.     

Cardiopulmonary exercise test predicts sustained ventricular arrhythmias in chronic heart failure

Background

The cardiopulmonary exercise test (CPX) is an affordable tool for risk prediction in patients with chronic heart failure (CHF). We aimed to determine the role of CPX parameters in predicting the risk of incidence of sustained ventricular arrhythmias (SVA) in CHF.

Methods

Sixty-one consecutive patients with CHF enrolled in the Daunia Heart Failure Registry underwent CPX and were followed for 327 ± 247 days. Clinical follow-up was performed every month and anticipated in case of re-hospitalisation for cardiac disease. Incidence of SVA was evaluated by direct clinical examination (ECG, ambulatory ECG).

Results

Patients with episodes of SVA (N 14) showed lower values of pVO2 and PetCO2, and higher values of VE/VCO2, VE/VCO2 slope, and VE%. After correction for age, gender, diabetes, ischaemic heart disease and left ventricular ejection fraction, peak VO2 (hazard ratio (HR) 0.68, 95 % confidence interval (CI) 0.51–0.91, p < 0.05), VE% (HR 1.38, 95 % CI 1.04–1.84, p < 0.05), VE/VCO2 (HR 1.38, 95 % CI 1.04–1.82, p < 0.05), VE/VCO2 slope (HR 1.77, 95 % CI 1.31–2.39, p < 0.01), PetCO2 (HR 0.66, 95 % CI 0.50–0.88, p < 0.01) were found as predictors of SVA. At Kaplan-Meier analysis, lower event-free rates were found in subjects with peak VO2 values below median (log rank p < 0.05), values of VE/VCO2 above mean (p < 0.05), higher VE/VCO2 slope tertiles (p <0.05), and values of PetCO2 below median (p < 0.05).

Conclusions

CPX provides prognostic independent information for risk of SVA in subjects with CHF.     

The role of heat shock proteins in inflammatory injury induced by cold stress in chicken hearts

The aim of this study was to investigate the effects of cold stress on the expression levels of heat shock proteins (Hsps90, 70, 60, 40, and 27) and inflammatory factors (iNOS, COX-2, NF-κB, TNF-α, and PTGEs) and oxidative indexes in hearts of chickens. Two hundred forty 15-day-old male chickens were randomly divided into 12 groups and kept at the temperature of 12 ± 1 °C for acute and chronic cold stress. There were one control group and five treatment groups for acute cold stress, three control groups, and three treatment groups for chronic cold stress. After cold stress, malondialdehyde level increased in chicken heart; the activity of superoxide dismutase and glutathione peroxidase in the heart first increased and then decreased. The inflammatory factors mRNA levels were increased in cold stress groups relative to control groups. The histopathological analysis showed that heart tissues were seriously injured in the cold stress group. Additionally, the mRNA levels of Hsps (70, 60, 40, and 27) increased significantly (P < 0.05) in the cold stress groups relative to the corresponding control group. Meanwhile, the mRNA level and protein expression of Hsp90 decreased significantly (P < 0.05) in the stress group, and showed a gradually decreasing tendency. These results suggested that the levels of inflammatory factors and Hsps expression levels in heart tissues can be influenced by cold stress. Hsps commonly played an important role in the protection of the heart after cold stress.     

Hsp70 regulation on Nox4/p22phox and cytoskeletal integrity as an effect of losartan in vascular smooth muscle cells

A series of signaling cascades are activated after angiotensin II binds to angiotensin II type I receptor (AT₁R), a peptide that is an important mediator of oxidative stress. Hsp70 regulates a diverse set of signaling pathways through interactions with proteins. Here, we tested the hypothesis of angiotensin II AT₁R inhibition effect on Hsp70 interaction with Nox4/p22phox complex and Hsp70 leading to actin cytoskeleton modulation in spontaneously hypertensive rats (SHR) vascular smooth muscle cells (VSMCs). SHR and Wistar–Kyotto rats (VSMCs from 8 to 10 weeks) were stimulated with angiotensin II (100 nmol/L) for 15 min (AII), treated with losartan (100 nmol/L) for 90 min (L), and with losartan for 90 min plus angiotensin in the last 15 min (L + AII). Whereas SHR VSMCs exposure to angiotensin II overexpressed AT₁R and Nox4 nicotinamide–adenine dinucleotide phosphate (NADPH) oxidase and slightly downregulated caveolin-1 expression, losartan decreased AT₁R protein levels and increased caveolin-1 and Hsp70 expression in SHR VSMC membranes. Immunoprecipitation and immunofluorescence confocal microscopy proved interaction and colocalization of membrane translocated Hsp70 and Nox4/p22phox. Increased levels of Hsp70 contrast with the decreased immunoprecipitation of Nox4/p22phox and RhoA in membranes from SHR VSMCs (L) vs SHR VSMCs (AII). Hsp72 depletion resulted in higher Nox4 expression and increased NADPH oxidase activity in VSMCs (L + AII) from SHR when contrasted with nontransfected VSMCs (L + AII). After Hsp72 knockdown in SHR VSMCs, losartan could not impair angiotensin II-enhanced stress fiber formation and focal adhesion assembly. In conclusion, our data showing a negative regulation of Hsp70 on Nox4/p22phox demonstrates a possible mechanism in explaining the antioxidative function joined to cytoskeletal integrity modulation within the effects of losartan in VSMCs from SHR.     

Relative Bioavailability of Chlorothiazide from Mucoadhesive Compacts in Pigs

The relative bioavailability of chlorothiazide from mucoadhesive polymeric compacts is compared to commercial oral suspension in pigs. A single-dose randomized study was conducted in 12 healthy pigs that are 9–10 weeks old. After overnight fasting, pigs were divided into two groups of six animals. To the first group, a reference product containing 50 mg of chlorothiazide suspension, and in the second group, test product (mucoadhesive compacts) chlorothiazide (50 mg) was administered with 75 mL of water via gastric tubes. Blood samples were collected between 0 to 24 h using catheters inserted into the jugular vein. Plasma was separated by protein precipitation, and chlorothiazide concentrations were determined using a high-performance liquid chromatography method. The mean T_max and the C_max of chlorothiazide following the administration of oral suspension and mucoadhesive compacts were 0.58 ± 0.20 h and 682.97 ± 415.69 ng/mL and 2.17 ± 0.98 h and 99.42 ± 124.08 ng/mL, respectively. The K_el and T_1/2 of chlorothiazide were found to be 1.06 ± 0.28 h⁻¹ and 0.70 ± 0.21 h from suspension and 0.95 ± 1.11 h⁻¹ and 2.05 ± 1.90 h from the compacts, respectively. The T_max of mucoadhesive compacts were significantly longer (p < 0.05; 2.17 h) than the reference products (0.58 h), whereas the C_max of compacts were significantly lower (99 ng/mL) than the reference product (683 ng/mL; p < 0.05). The area under the curve (AUC) of compacts accounts only 50.15% (404.32 ± 449.93 ng h/mL) of the reference product’s AUC (806.27 ± 395.97 ng h/mL). The relative bioavailability of the compacts was lower than that of the suspension, and this may be due to the narrow window of absorption for chlorothiazide.Key words: bioavailability, chlorothiazide, mucoadhesive compacts, pigs     

Novel links among peroxiredoxins,endothelial dysfunction,and severity of atherosclerosis in type 2 diabetic patients with peripheral atherosclerotic disease

Peroxiredoxins, a group of antioxidant protein enzymes (PRDX1 to 6), are reported as antiatherogenic factors in animals; however, human studies are lacking. The present work aims to provide baseline data regarding the phenotype of PRDX1, 2, 4, and 6 in diabetic patients with peripheral atherosclerosis disease (PAD) and their relation to endothelial dysfunction (ED) and disease severity. Plasma levels of PRDX1, 2, 4, and 6 and markers of endothelial dysfunction (ICAM-1 and VCAM-1) were measured using ELISA in 55 type 2 diabetic patients having PAD and 25 healthy subjects. Ankle–brachial index (ABI), body mass index (BMI), triglycerides (TG), total cholesterol, HbA1c, and insulin resistance (HOMA IR) were measured. PRDX1, 2, 4, and 6 levels were significantly higher in patients compared to controls (PRDX1 21.9 ± 5.71 vs 16.8 ± 3.9 ng/ml, P < 0.001, PRDX2 36.5 ± 14.83 vs 20.4 ± 8.61 ng/ml, P < 0.001, PRDX4 3,840 ± 1,440 vs 2,696 ± 1,972 pg/ml, P < 0.005, PRDX6 311 ± 110 vs 287.9 ± 114 pg/ml, P < 0.05). PRDX1 and PRDX4 correlated negatively with ABI (r = −0.273, P < 0.05 and r = −0.28, P < 0.05, respectively), while PRDX1 and PRDX2 correlated positively with HOMA/IR and TG (r = 0.276, P < 0.01 and r = 0.295, P < 0.01, respectively). ICAM-1 was associated with PRDX2 and log PRDX6 (r = 0.345, P = 0.0037 and r = 0.344, P = 0.0038). Our results provide strong links among PRDXs, ED, and severity of PAD in diabetic patients which warrants further evaluation to clarify whether high circulating levels of PRDXs are a consequence of chronic atherosclerotic disease or a predisposing factor for later cardiovascular events.     

Expression of heat shock proteins in premalignant and malignant urothelial lesions of bovine urinary bladder

Abnormal heat shock protein (HSP) levels have been observed in a number of human tumours, where they are involved in all hallmarks of cancer. Since bovine urothelial tumours share striking morphological and biochemical features with their human counterparts, the aim of this study was to evaluate the immunohistochemical levels of Hsp27, Hsp60, Hsp72, Hsp73 and Hsp90 in 28 normal bovine urinary bladders and 30 bovine papillomavirus-positive urothelial tumours (9 in situ carcinomas, 9 low-grade and 12 high-grade carcinomas) and adjacent premalignant lesions obtained from cows suffering from chronic enzootic haematuria, in order to investigate the role of these proteins in the process of urothelial carcinogenesis. A semi-quantitative method was used for the analysis of the results. Western blot analysis was also used to confirm HSP expression in normal controls. All investigated HSPs were expressed in normal bovine urothelium, showing characteristic patterns of immunolabelling throughout urothelial cell layers, which usually appeared to be conserved in urothelial hyperplasia and dysplasia. On the other hand, gradual loss of Hsp27 immunostaining resulted to be significantly associated with increasing histological grade of malignancy (P < 0.01). As well, a significantly reduced immunosignal of Hsp73 and Hsp90 was observed in high-grade and low-/high-grade carcinomas, respectively (P < 0.01). In contrast, Hsp60 (P < 0.01) and Hsp72 (P < 0.05) immunoreactivity appeared to be significantly increased both in premalignant and malignant lesions when compared to that observed in normal urothelium, thus suggesting an early involvement of these proteins in neoplastic transformation of urinary bladder mucosa.     

Neurocardiological differences between musicians and control subjects

Background

Exercise training is beneficial in health and disease. Part of the training effect materialises in the brainstem due to the exercise-associated somatosensory nerve traffic. Because active music making also involves somatosensory nerve traffic, we hypothesised that this will have training effects resembling those of physical exercise.

Methods

We compared two groups of healthy, young subjects between 18 and 30 years: 25 music students (13/12 male/female, group M) and 28 controls (12/16 male/female, group C), peers, who were non-musicians. Measurement sessions to determine resting heart rate, resting blood pressure and baroreflex sensitivity (BRS) were held during morning hours.

Results

Groups M and C did not differ significantly in age (21.4 ± 3.0 vs 21.2 ± 3.1 years), height (1.79 ± 0.11 vs 1.77 ± 0.10 m), weight (68.0 ± 9.1 vs 66.8 ± 10.4 kg), body mass index (21.2 ± 2.5 vs 21.3 ± 2.4 kg∙m⁻²) and physical exercise volume (39.3 ± 38.8 vs 36.6 ± 23.6 metabolic equivalent hours/week). Group M practised music daily for 1.8 ± 0.7 h. In group M heart rate (65.1 ± 10.6 vs 68.8 ± 8.3 beats/min, trend P =0.08), systolic blood pressure (114.2 ± 8.7 vs 120.3 ± 10.0 mmHg, P = 0.01), diastolic blood pressure (65.0 ± 6.1 vs 71.0 ± 6.2 mmHg, P < 0.01) and mean blood pressure (83.7 ± 6.4 vs 89.4 ± 7.1, P < 0.01) were lower than in group C. BRS in groups M and C was 12.9 ± 6.7 and 11.3 ± 5.8 ms/mmHg, respectively (P = 0.17).

Conclusions

The results of our study suggest that active music making has training effects resembling those of physical exercise training. Our study opens a new perspective, in which active music making, additionally to being an artistic activity, renders concrete health benefits for the musician.