Circulating Heat Shock Protein 70 in Health, Aging and Disease

<p>Abstract</p> <p>Background</p> <p>Heat shock proteins (Hsp) are ubiquitously synthesised in virtually all species and it is hypothesised that they might have beneficial health effects. Recent studies have identified circulating Hsp as an important mediator in inflammation - the effects of low-grade inflammation in the aging process are overwhelming. While much is known about intracellular Hsp70, scant data exist on circulating Hsp70 in the aging context. Therefore, the objectives of this study were to investigate the effect of age and disease on circulating Hsp70 and, in particular, to evaluate the association between circulating Hsp70 and inflammatory parameters.</p> <p>Results</p> <p>Serum Hsp70, Interleukin (IL) -10, IL-6 and Tumor Necrosis Factor (TNF) alpha concentrations were determined in 90 hospitalised geriatric patients (aged 83 ± 6 years) and in 200 community-dwelling control subjects (100 elderly, aged 74 ± 5 years, and 100 young, aged 23 ± 3 years). In the community-dwelling elderly, serum Hsp70 and IL-10 concentrations were significantly lower and IL-6 was significantly higher when compared to healthy young control subjects. Elderly patients presenting inflammation (CRP serum levels ≥5 mg/L) showed significantly (p = 0.007) higher Hsp70 values; and Hsp70 correlated positively (p < 0.001) with IL-6 and CRP, but not with TNF-alpha or IL-10. A significant association was also noted between Hsp70 levels and the degree of dependency and cognitive decline in geriatric patients.</p> <p>Conclusions</p> <p>The present data provide new evidence that serum concentration of Hsp70 decreases with age in a normal population. Our study also shows that higher levels of Hsp70 are associated with inflammation and frailty in elderly patients.</p

Effect of vitamin E on monosodium glutamate induced hepatotoxicity and oxidative stress in rats

20-24Monosodium glutamate (MSG), administered to rats (by gavage) at a dose of 0.6 mg/g body weight for 10 days, significantly {P<0.05) induced lipid peroxidation (LPO), decreased reduced glutathione (GSH) level and increased the activities of glutathione-s-transferase (GST), catalase and superoxide dismutase (SOD) in the liver of the animals; these were observed 24 hr after 10 days of administration. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and ϒ glutamyl transferase (GGT) were also significantly increased in the serum, on MSG administration. Vitamin E (0.2 mg/g body wt) co-administered with MSG, significantly reduced the LPO, increased the GSH level and decreased the hepatic activities of GST, catalase and SOD. The activities of ALT, AST and GGT in the serum were also significantly reduced. The results showed that MSG at a dose of 0.6 mg/g body wt induced the oxidative stress and hepatotoxicity in rats and vitamin E ameliorated MSG-induced oxidative stress and hepatotoxicity

Class II HDAC inhibition hampers hepatic stellate cell activation by induction of microRNA-29.

BACKGROUND: The conversion of a quiescent vitamin A storing hepatic stellate cell (HSC) to a matrix producing, contractile myofibroblast-like activated HSC is a key event in the onset of liver disease following injury of any aetiology. Previous studies have shown that class I histone deacetylases (HDACs) are involved in the phenotypical changes occurring during stellate cell activation in liver and pancreas. AIMS: In the current study we investigate the role of class II HDACs during HSC activation. METHODS: We characterized the expression of the class II HDACs freshly isolated mouse HSCs. We inhibited HDAC activity by selective pharmacological inhibition with MC1568, and by repressing class II HDAC gene expression using specific siRNAs. RESULTS: Inhibition of HDAC activity leads to a strong reduction of HSC activation markers α-SMA, lysyl oxidase and collagens as well as an inhibition of cell proliferation. Knock down experiments showed that HDAC4 contributes to HSC activation by regulating lysyl oxidase expression. In addition, we observed a strong up regulation of miR-29, a well-known anti-fibrotic miR, upon treatment with MC1568. Our in vivo work suggests that a successful inhibition of class II HDACs could be promising for development of future anti-fibrotic compounds. CONCLUSIONS: In conclusion, the use of MC1568 has enabled us to identify a role for class II HDACs regulating miR-29 during HSC activation

Class II HDAC Inhibition Hampers Hepatic Stellate Cell Activation by Induction of MicroRNA-29

Background: The conversion of a quiescent vitamin A storing hepatic stellate cell (HSC) to a matrix producing, contractile myofibroblast-like activated HSC is a key event in the onset of liver disease following injury of any aetiology. Previous studies have shown that class I histone deacetylases (HDACs) are involved in the phenotypical changes occurring during stellate cell activation in liver and pancreas. Aims: In the current study we investigate the role of class II HDACs during HSC activation. Methods: We characterized the expression of the class II HDACs freshly isolated mouse HSCs. We inhibited HDAC activity by selective pharmacological inhibition with MC1568, and by repressing class II HDAC gene expression using specific siRNAs. Results: Inhibition of HDAC activity leads to a strong reduction of HSC activation markers alpha-SMA, lysyl oxidase and collagens as well as an inhibition of cell proliferation. Knock down experiments showed that HDAC4 contributes to HSC activation by regulating lysyl oxidase expression. In addition, we observed a strong up regulation of miR-29, a well-known anti-fibrotic miR, upon treatment with MC1568. Our in vivo work suggests that a successful inhibition of class II HDACs could be promising for development of future anti-fibrotic compounds. Conclusions: In conclusion, the use of MC1568 has enabled us to identify a role for class II HDACs regulating miR-29 during HSC activation

Effect of MC1568 treatment on fibrogenesis in a CCl₄ induced fibrosis mouse model.

<p>Mice were CCl₄ treated twice a week for a total period of 4 weeks. After the second week, mice also received intraperitoneal injections with MC1568 (50 mg/kg) every two days for two more weeks. The day after the final CCl₄ injection, mice were sacrificed and liver tissue was extracted for analysis. (A) Sirius Red staining was performed to visualize deposited collagens. Image J software was used for quantification of the red surface area. Scale bar = 100 µm. (B) Serum levels of ALT and AST. (C) HDAC activity was measured in protein lysates of livers at the end of treatments using HDAC-Glo. (D) A mathematical correlation between the HDAC activity and the red stained area, after Sirius staining was determined using Pearson correlation method. The a and b refer to HDAC-activity values of the corresponding liver lysates of the images given for CCl₄ + MC1568 in A).</p

Class IIa and class IIb HDACs are constantly expressed during <i>in vitro</i> mouse hepatic stellate cell activation.

<p>Mouse HSCs were isolated from healthy mice and cultured for the indicated time. (A) Shows morphological changes associated to the mHSC activation process <i>in vitro</i>. (B) mRNA levels of activation markers <i>Col1a1</i>, <i>Col3a1</i>, <i>Acta2 and Lox</i> were determined using qPCR to confirm <i>in vitro</i> activation. (C) mRNA expression of Class IIa and IIb <i>Hdac</i>s was measured by qPCR. Values represent 3 replicates, ***: p<0,001, **: p<0, 01, *: p<0, 05.</p

Effect of MC1568 treatment on stellate cell activation markers.

<p>(A) Freshly isolated HSCs were plated in presence or absence of 1 µM MC1568 for 10 days. The effect on HSC activation markers <i>Acta2</i>, <i>Lox</i>, <i>Col1a1</i> and <i>Col3a1</i> was evaluated by qPCR. (B) Mouse HSCs were cultured for 10 days in presence or absence of 1 µM MC1568. The effect on HSC activation markers Collagen I, Lysyl oxidase and α-Smooth muscle actin was investigated by western blot. β-actin was used as a loading control. (D) The effect of MC1568 treatment on HSC proliferation was investigated with an EdU incorporation assay. Cells were cultured for 48 hours in the presence or absence of MC1568. Nuclei were stained with 4′,6-Diamidino-2-phenylindole. The percentage of EdU-positive cells was determined from three independent experiments. (D) In order to test the reversibility of MC1568 treatment, freshly isolated mouse HSCs were treated for 7 days, after 7 days the inhibitor was washed out and cells were further cultured until day 10 (recovery). Then cells were collected and mRNA expression of HSC activation markers was determined. (E) Freshly isolated mouse HSCs were cultured for seven days. At day seven, cells were formalin fixed and stained with an antibody against the acetylated form of tubulin. Prior to fixation, cells were treated with 1 µM MC1568 for 24 hours. Nuclei were visualized with 4′,6-Diamidino-2-phenylindole. Scale bar = 100 µM. * p<0.05, ** p<0,01.</p

List of antibodies for immunocytochemistry and western blot.

<p>List of antibodies for immunocytochemistry and western blot.</p