Toll-like receptors in the functional orientation of cardiac progenitor cells.

Cardiac progenitor cells (CPCs) have the potential to differentiate into several cell lineages with the ability to restore in cardiac tissue. Multipotency and self-renewal activity are the crucial characteristics of CPCs. Also, CPCs have promising therapeutic roles in cardiac diseases such as valvular disease, thrombosis, atherosclerosis, congestive heart failure, and cardiac remodeling. Toll-like receptors (TLRs), as the main part of the innate immunity, have a key role in the development and differentiation of immune cells. Some reports are found regarding the effect of TLRs in the maturation of stem cells. This article tried to find the potential role of TLRs in the dynamics of CPCs. By showing possible crosstalk between the TLR signaling pathways and CPCs dynamics, we could achieve a better conception related to TLRs in the regeneration of cardiac tissue.

Cytoprotective and cytofunctional effect of polyanionic polysaccharide alginate and gelatin microspheres on rat cardiac cells.

This study investigated the cyto-functional effect of Alginate-Gelatin microspheres on rat cardiomyoblasts after 7 days. Rat cardiomyoblasts were encapsulated inside Alginate-Gelatin microspheres via application of high voltage rate and dropping in a stirring CaCl2 solution. The swelling rate, biodegradation, and mechanical features were measured. Cell viability was assessed using MTT. Cell membrane integrity was monitored via calculation supernatant SGOT, SGPT, CPK, and LDH. We also measured SOD, GPx, and anti-oxidant capacity. Protein levels of Nrf-2 and PCCG-1α were detected via western blotting. The cyto-functional activity of encapsulated cells was monitored using real-time PCR assay targeting the expression of Connexin-43, α-actinin, and myosin light chain. Data showed suitable biodegradation and swelling rate in Alginate-gelatin microspheres by time. 7-day incubation of rat cells inside microspheres did not exert cytotoxicity compared to control cells (p > 0.05). The release of SGPT, SGOT, CPK, and LDH in encapsulated cells was significantly decreased compared to the control group (p < 0.05). We also found enhanced anti-oxidant capacity and SOD and GPx activity in cells after being-encapsulated inside Alginate-Gelatin microspheres (p < 0.05) coincided with increased Nrf-2 synthesis (p < 0.05) compared to control cells. The expression of Connexin-43, α-actinin, and myosin light chain was significantly up-regulated, showing cyto-functional effect of Alginate-Gelatin microspheres after 7-days.

Protective effect of bacterial lipase on lipopolysaccharide-induced toxicity in rat cardiomyocytes; H9C2 cell line.

Introduction: Cardiovascular system is highly sensitive to LPS-induced oxidative damage. This study aimed to show the inhibitory effect of bacterial Lipase on LPS-induced cardiomyoblasts toxicity. Methods: Rat cardiomyoblasts H9C2 were classified into Control, LPS (cells received 0.1, 1 and 10 µg/mL LPS) and LPS+ Lipase groups. In LPS+Lipase group, different concentrations of lipopolysaccharide were pre-incubated with 5 mg/mL bacterial lipase at 37˚C overnight prior to cell treatment. After 72 hours, cell viability was assessed by MTT assay. The expression of key genes related to toll-like receptor signaling pathways was assessed by real-time PCR assay. Percentage of fatty acids was evaluated in each group using gas chromatography assay. The levels of NO was also measured using the Griess reaction. Results: Data showed H9C2 cells viability was decreased after exposure to LPS in a dose-dependent manner (P < 0.05). Incubation of LPS with lipase increased cell survival rate and closed to near-to-control levels (P < 0.05). Lipase had the potential to blunt the increased expression of IRAK and NF-κB in cells after exposure to the LPS. Compared to the LPS group, lipase attenuated the increased level of NO-induced by LPS (P < 0.05). Gas chromatography analysis showed the reduction of saturated fatty acids in cells from LPS group while the activity of lipase prohibited impact of LPS on cell fatty acid composition. LPS decreased the ability of cardiomyoblasts to form colonies. Incubation of LPS with lipase enhanced clonogenic capacity. Conclusion: Reduction in lipopolysaccharide-induced cytotoxicity is possibly related to lipase activity and reduction of modified lipopolysaccharide with toll-like receptor.

Encapsulation of rat cardiomyoblasts with alginate-gelatin microspheres preserves stemness feature in vitro.

INTRODUCTION: The emergence of numerous tissue engineering and regenerative medicine techniques cell encapsulation paves a way to heal and restore the function of various injured tissues mainly cardiovascular system. Here, we aimed to investigate the role of alginate-gelatin encapsulation on the dynamic of rat cardiomyoblasts in vitro. MATERIALS AND METHODS: Rat cardiomyoblasts cell line H9C2 were enclosed by using alginate-gelatin microspheres and incubated for 7 days. MTT method was used to examine cell viability. The level of genes associated with cardiomyoblasts maturation MYL7, NPPA, NKX2-5, and GATA4 real-time PCR. ELISA was used to measure the protein levels of Bcl-2 and Bax factor post-encapsulation. The level of SOD, GPx, and TAC was detected by biochemical analyses. Western blotting was performed to measure the content of AMP-activated protein kinase. RESULTS: We found that encapsulation was able to increase the viability of rat cardiomyocytes after 7 days. The decreased level of Bcl-2 (p < 0.001) coincided with non-significant differences in the level of Bax (p > 0.05). The transcription level of all genes MYL7, NPPA, NKX2-5, and GATA4 were found to down-regulate compared to the control non-treated cells (p < 0.05). No significant differences were found regarding the level of SOD, GPx, and TAC compared to the control (p>0.05). According to western blotting, revealed a reduced level of AMPK following 7-day incubation of rat cardiomyoblasts (p < 0.05). CONCLUSION: Data confirmed that the encapsulation of rat cardiomyoblasts with alginate-gelatin microspheres maintained the cells multipotentiality.

Nrf2 activation and down-regulation of HMGB1 and MyD88 expression by amnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cells.

BACKGROUND: human Amniotic Membrane (hAM) extracts contain bioactive molecules such as growth factors and cytokines. Studies have confirmed the ability of hAM in reduction of post-operative dysfunction in patients with cardiac surgery. However, the function of Amniotic Membrane Proteins (AMPs), extracted from hAM, against hypoxia-induced H9c2 cells injury have never been investigated. In this study, we aimed to appraise the protective impact of AMPs on H9c2 cells under hypoxia condition. METHODS: Cardiomyocyte cells were pre-incubated with AMPs and subjected to 24 h hypoxia to elucidate its effects on expression of Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1). Furthermore, the high mobility group box-1 (HMGB1) and Myeloid differentiation primary response 88 (MyD88) expressions were detected by qPCR and western-blotting. The mitochondrial membrane potential (ΔΨm) was estimated by JC-1 using fluorescent microscopy and fluorimetry. Moreover, the cell apoptosis and intracellular calcium levels were measured by flow cytometry. RESULTS: Pre-treatment of AMPs resulted in significant induction in cell viability and decreased the LDH release under hypoxic condition in H9c2 cells. Accordingly, these protective effects of AMPs were associated with a reduction in apoptosis rates and intracellular Ca2+, meanwhile, ΔΨm was increased. Pre-treatment with AMPs resulted in degradation of HMGB1 and MyD88 levels and depicted pro-survival efficacy of AMPs against hypoxia-induced cell damage through induction of HO-1 and Nrf2. CONCLUSION: The data indicated that AMPs mediated HO-1 regulation by Nrf2 activation and plays critical protective effects in hypoxia-induced H9c2 injury in vitro by the inhibition of myocardial HMGB1 and MyD88 inflammatory cascade.

Soluble Expression of Humanized Anti-CD20 Single Chain Antibody in Escherichia coli by Cytoplasmic Chaperones Co-expression.

BACKGROUND: CD20 is an important cell surface receptor that is used for target therapy of B cell lymphoma and some related blood diseases due to vital function of CD20. In previous studies, a Rituximab based humanized single chain variable fragment (scFv) antibody showed good reactivity against B cell related cancer cells. But this recombinant protein produced Inclusion Bodies (IBs) in Escherichia coli (E. coli) cytoplasm. The aim of this study was to investigate the effect of coexpression with cytoplasmic chaperones on expression and solubility of humanized anti-CD20 scFv in E. coli. METHODS: For this purpose, the fragment coding for anti-CD20 huscFv subcloned into the pET22b (+) and transformed into the E. coli BL21 (DE3) was evaluated. In order to inhibit the production of IBs, the effects of co-expression with cytoplasmic chaperones GroEL, DnaK, GroES, Tig, DnaJ and GrpE were investigated. RESULT: Coexpression with cytoplasmic chaperones led to increased soluble expression of anti-CD20 recombinant protein. Among investigated chaperones, pKJE7 chaperone plasmid containing DnaJ, GrpE, DnaK chaperone genes had significant effects with an expression yield of 325 µg/ml soluble anti-CD20 scFv. CONCLUSION: The result of this study demonstrated remarkable effect of pKJE7 chaperone on enhancement of soluble expression of anti-CD20 huscFv antibody in E. coli.

Decreasing serum homocysteine and hypocholesterolemic effects of Bovine lactoferrin in male rat fed with high-cholesterol diet.

Introduction: Lipid metabolism disorder or hyperlipidemia is known as a risk factor for cardiovascular disease, the increase in serum homocysteine and leptin are associated with atherosclerotic disease. The purpose of the present study was to examine the effects of bovine lactoferrin (bLF) on serum homocysteine (Hcy), apolipoproteinA-I (ApoA-I) and B (ApoB), leptin and lipid profile changes in high-cholesterol-diet (HCD) fed rats. Methods: The Healthy Adult Sprague-Dawley (SD) male rats were randomly assigned into three experimental groups. Each group consisted of eleven male rats including control group, HCD rats and hypercholesterolemic rats, which were treated with bLF (HCD+bLF). bLF was given by gavage (200 mg/kg/d). After 4 weeks of feeding and overnight fasting, total blood samples were collected. Results: The results showed the elevated level of Hcy, leptin, total cholesterol, low density lipoprotein cholesterol (LDL-C), ApoB and decrease in ApoA-I in non-treated HCD group compared to the control rats. Administration of bLF significantly ameliorated the Hcy and leptin levels with decrease in LDL-C and total cholesterol in rats fed with a high-cholesterol diet. bLF also tended to increase low serum concentration of ApoA-I and high density lipoprotein cholesterol (HDL-C) in HCD rats. Meanwhile, upon bLF-treated rats, there was a significant decrease in ApoB in HCD group. Conclusion: The findings indicated that bLF can improve the alteration of serum Hcy, leptin, apolipproteins and lipid changes in male rats fed with high-cholesterol diet. So, bLF can counteract with HCD elicited hyper-homocysteinemia and hyper-leptinemia, suggesting it to have the useful therapeutic potential in patients with atherosclerosis and lipid disorder.

An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation.