The Long Noncoding RNA Hotair Regulates Oxidative Stress and Cardiac Myocyte Apoptosis during Ischemia-Reperfusion Injury.

Oxidative stress and subsequent cardiac myocyte apoptosis play central roles in the initiation and progression of myocardial ischemia-reperfusion (I/R) injury. Homeobox transcript antisense intergenic RNA (Hotair) was previously implicated in various heart diseases, yet its role in myocardial I/R injury has not been clearly demonstrated. Mice with cardiac-restricted knockdown or overexpression of Hotair were exposed to I/R surgery. H9c2 cells were cultured and subjected to hypoxia/reoxygenation (H/R) stimulation to further verify the role and underlying mechanisms of Hotair in vitro. Histological examination, molecular detection, and functional parameters were determined in vivo and in vitro. In response to I/R or H/R treatment, Hotair expression was increased in a bromodomain-containing protein 4-dependent manner. Cardiac-restricted knockdown of Hotair exacerbated, whereas Hotair overexpression prevented I/R-induced oxidative stress, cardiac myocyte apoptosis, and cardiac dysfunction. Mechanistically, we observed that Hotair exerted its beneficial effects via activating AMP-activated protein kinase alpha (AMPKα). Further detection revealed that Hotair activated AMPKα through regulating the enhancer of zeste homolog 2/microRNA-451/calcium-binding protein 39 (EZH2/miR-451/Cab39) axis. We provide the evidence that endogenous lncRNA Hotair is an essential negative regulator for oxidative stress and cardiac myocyte apoptosis in myocardial I/R injury, which is dependent on AMPKα activation via the EZH2/miR-451/Cab39 axis.

Circulating MiR-19b-3p, MiR-134-5p and MiR-186-5p are Promising Novel Biomarkers for Early Diagnosis of Acute Myocardial Infarction.

BACKGROUND/AIMS: Recent studies have shown that circulating microRNAs (miRNAs) are emerging as promising biomarkers for cardiovascular diseases. This study aimed to determine whether miR-19b-3p, miR-134-5p and miR-186-5p can be used as novel indicators for acute myocardial infarction (AMI). METHODS: To investigate the kinetic expression of the three selected miRNAs, we enrolled 18 patients with AMI and 20 matched controls. Plasma samples were collected from each participant, and total RNA was extracted. Quantitative real-time PCR and ELISA assays were used to investigate the expression of circulating miRNAs and cardiac troponin I (cTnI), respectively. Plasma samples from another age- and gender-matched cohort were collected to investigate the impact of medications for AMI on the expression of the selected miRNAs. RESULTS: Levels of plasma miR-19b-3p, miR-134-5p and miR-186-5p were significantly increased in early stage of AMI. Plasma miR-19b-3p and miR-134-5p levels reached peak expression immediately after admission (T0), whereas miR-186-5p achieved peak expression at 4 h after T0. All of these times were earlier than the peak for cTnI (8 h after T0). In addition, all three miRNAs were positively correlated with cTnI. Receiver Operating Characteristic (ROC) analysis indicated that each single miRNA showed considerable diagnostic efficiency for predicting AMI. Furthermore, combining all three miRNAs in a panel increased the efficiency of distinguishing between patients with AMI and controls. Moreover, we found that heparin and medications for AMI did not impact the expression of these circulating miRNAs. CONCLUSION: Circulating miR-19b-3p, miR-134-5p and miR-186-5p could be considered promising novel diagnostic biomarkers for the early phase of AMI.

Human umbilical vein endothelial cells promote the inhibitory activation of CD4(+)CD25(+)Foxp3(+) regulatory T cells via PD-L1.

BACKGROUND: Atherosclerosis (AS) is a chronic inflammation characterized by massive infiltration of inflammatory cells in arterial wall plaques. Programmed death ligand-1 (PD-L1), a co-stimulatory molecule, plays a vital role in regulating immune responses. We investigated the role and mechanisms of PD-L1 expressed on oxidized low-density lipoprotein (ox-LDL)-impaired human umbilical vein endothelial cells (HUVECs) in promoting activation and cytokine production of CD4(+)CD25(+) forkhead box P3 (FoxP3) regulatory T cells (Tregs). METHODS AND RESULTS: Tregs were incubated alone, with HUVECs or HUVECs pre-stimulated with ox-LDL in the presence of anti-CD3 monoclonal antibodies (mAbs) for 48 h. HUVECs were shown to upregulate the immune phenotypic markers of Tregs, such as glucocorticoid-induced TNF receptor (GITR), cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 protein (PD-1). Moreover, HUVECs modulated cytokine production of Tregs (e.g., interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1)). HUVECs treated with anti-PD-L1 mAbs were unable to regulate the surface expression and cytokine production of Tregs. The Transwell culture system suggested that interaction between HUVECs and Tregs via PD-L1 requires cell-to-cell contact. CONCLUSION: Expression of the negative co-stimulatory molecule PD-L1 on HUVECs may upregulate the inhibitory activation and cytokine production of CD4(+)CD25(+)Foxp3(+) regulatory T cells in AS.

Activated regulatory T-cells attenuate myocardial ischaemia/reperfusion injury through a CD39-dependent mechanism.

Regulatory T-cells (Tregs) are generally regarded as key immunomodulators that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. However, its role in myocardial ischaemia/reperfusion injury (MIRI) remains unknown. The purpose of the present study was to determine whether Tregs exert a beneficial effect on mouse MIRI. We examined the role of Tregs in murine MIRI by depletion using 'depletion of regulatory T-cell' (DEREG) mice and adoptive transfer using Forkhead box P3 (Foxp3)-GFP knockin mice and the mechanisms of cardio protection were further studied in vivo and in vitro. Tregs rapidly accumulated in murine hearts following MIRI. Selective depletion of Tregs in the DEREG mouse model resulted in aggravated MIRI. In contrast, the adoptive transfer of in vitro-activated Tregs suppressed MIRI, whereas freshly isolated Tregs had no effect. Mechanistically, activated Treg-mediated protection against MIRI was not abrogated by interleukin (IL)-10 or transforming growth factor (TGF)-ß1 inhibition but was impaired by the genetic deletion of cluster of differentiation 39 (CD39). Moreover, adoptive transfer of in vitro-activated Tregs attenuated cardiomyocyte apoptosis, activated a pro-survival pathway involving Akt and extracellular-signal-regulated kinase (ERK) and inhibited neutrophil infiltration, which was compromised by CD39 deficiency. Finally, the peripheral blood mononuclear cells of acute myocardial infarction (AMI) patients after primary percutaneous coronary intervention (PCI) revealed a decrease in CD4+CD25+CD127low Tregs and a relative increase in CD39+ cells within the Treg population. In conclusion, our data validated a protective role for Tregs in MIRI. Moreover, in vitro-activated Tregs ameliorated MIRI via a CD39-dependent mechanism, representing a putative therapeutic strategy.

Impaired circulating CD4+ LAP+ regulatory T cells in patients with acute coronary syndrome and its mechanistic study.

OBJECTIVE: CD4(+) latency-associated peptide (LAP)(+) regulatory T cells (Tregs) are a newly discovered T cell subset in humans and the role of these cells in patients with acute coronary syndrome (ACS) has not been explored. We designed to investigate whether circulating frequency and function of CD4(+)LAP(+) Tregs are defective in ACS. METHODS: One hundred eleven ACS patients (acute myocardial infarction and unstable angina) and 117 control patients were enrolled in the study. The control patients consisted of chronic stable angina (CSA) and chest pain syndrome (CPS). The frequencies of circulating CD4(+)LAP(+) Tregs and the expression of the transmembrane protein glycoprotein-A repetitions predominant (GARP) on CD4(+) T cells were determined by flow cytometry. The function of CD4(+)LAP(+) Tregs was detected using thymidine uptake. Serum interleukin-10 (IL-10) and transforming growth factor-ß protein (TGF-ß) levels were detected using ELISA and expression of GARP mRNA in peripheral blood mononuclear cells (PBMCs) was measured by real time-polymerase chain reaction. RESULTS: We found ACS patients had a significantly lower frequency of circulating CD4(+)LAP(+) Tregs, and the function of these cells was reduced compared to controls. The expression of GARP in CD4(+) T cells and the serum levels of TGF-ß in ACS patients were lower than those of control patients. The serum levels of IL-10 were similar between the two cohorts. CONCLUSIONS: A novel regulatory T cell subset, defined as CD4(+)LAP(+) T cells is defective in ACS patients.

Wnt1 from cochlear schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model.

Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted NSCs.

[Effects of rhubarb on the intestinal barrier function of patients with acute myocardial infarction-heart].

OBJECTIVE: To clarify the intestinal barrier function (IBF) state of patients with acute myocardial infarction-heart failure (AMI-HF), and to compare the therapeutic effects of rhubarb and Pantoprazole (proton pump inhibitor). METHODS: Enrolled were 107 AMI patients from ICU, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from May 2008 to April 2010. Of them, 47 AMI patients without HF were recruited as the control group, while 60 AMI-HF patients were randomly assigned to the rhubarb group (30 cases, treated by rhubarb + Pantoprazole) or the Pantoprazole group (30 cases, treated by Pantoprazole + routine treatment). All patients were treated till the 14th day of the onset. The fecal occult blood (FOB) test was performed daily. The occurrence of the digestive tract hemorrhage on the 14th day after onset was compared. The N-terminal pro-brain natriuretic peptide (NT-proBNP), serum D-lactic acid, plasma glutamine (Gln), endotoxin and cytokines [high sensitive C reaction protein (hsCRP), tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10)], and heart function were compared among the three groups before and after treatment. RESULTS: There was no statistical difference in the case number of using aspirin, clopidogel, low molecular weight heparin, ACEI/ARB, statins, insulin, and antibiotics among the 3 groups. The case number of using beta-blocker was obviously lower in the two medication groups than in the control group (P < 0.05). The case number of using diuretics was obviously higher in the two medication groups than in the control group (P < 0.05). There was no statistical difference in the incidence of digestive tract hemorrhage (P = 0.413). Compared with the control group before treatment, Gln and ejection fraction (EF) were both lowered, NT-proBNP, D-lactic acid, endotoxin, hsCRP, TNF-alpha, and IL-10 increased in the two medication groups (P < 0.01). There was no statistical difference in each index between the two medication groups (P > 0.05). Compared with before treatment, NT-proBNP, D-lactic acid, endotoxin, hsCRP, TNF-alpha, and IL-10 decreased in the Pantoprazole group (P < 0.01), and no obvious change in Gin or EF was found (P > 0.05). Gin and EF increased in the rhubarb group after treatment, and they were higher than those of the control group. Blood NT-proBNP, D-lactic acid, endotoxin, hsCRP, TNF-alpha, and IL-10 decreased in the rhubarb group after treatment, showing statistical difference when compared with the control group (P < 0.01, P < 0.05). CONCLUSIONS: Impaired IBF and endotoxemia existed in AMI-HF patients. Rhubarb not only could prevent the digestive tract hemorrhage, but also could reduce endotoxemia, inhibit inflammatory reactions, and improve the heart function through ameliorating the IBF.

Protective role of hydrogen sulfide against noise-induced cochlear damage: a chronic intracochlear infusion model.

BACKGROUND: A reduction in cochlear blood flow plays an essential role in noise-induced hearing loss (NIHL). The timely regulation of cochlear perfusion determines the progression and prognosis of NIHL. Hydrogen sulfide (H(2)S) has attracted increasing interest as a vasodilator in cardiovascular systems. This study identified the role of H(2)S in cochlear blood flow regulation and noise protection. METHODOLOGY/PRINCIPAL FINDINGS: The gene and protein expression of the H(2)S synthetase cystathionine-γ-lyase (CSE) in the rat cochlea was examined using immunofluorescence and real-time PCR. Cochlear CSE mRNA levels varied according to the duration of noise exposure. A chronic intracochlear infusion model was built and artificial perilymph (AP), NaHS or DL-propargylglycine (PPG) were locally administered. Local sodium hydrosulfide (NaHS) significantly increased cochlear perfusion post-noise exposure. Cochlear morphological damage and hearing loss were alleviated in the NaHS group as measured by conventional auditory brainstem response (ABR), cochlear scanning electron microscope (SEM) and outer hair cell (OHC) count. The highest percentage of OHC loss occurred in the PPG group. CONCLUSIONS/SIGNIFICANCE: Our results suggest that H(2)S plays an important role in the regulation of cochlear blood flow and the protection against noise. Further studies may identify a new preventive and therapeutic perspective on NIHL and other blood supply-related inner ear diseases.

Beta structure motifs of islet amyloid polypeptides identified through surface-mediated assemblies.

We report here the identification of the key sites for the beta structure motifs of the islet amyloid polypeptide (IAPP) analogs by using scanning tunneling microscopy (STM). Duplex folding structures in human IAPP(8-37) (hIAPP(8-37)) assembly were observed featuring a hairpin structure. The multiplicity in rIAPP assembly structures indicates the polydispersity of the rat IAPP(8-37) (rIAPP(8-37)) beta-like motifs. The bimodal length distribution of beta structure motifs for rIAPP(8-37) R18H indicates the multiple beta segments linked by turns. The IAPP(8-37) analogs share common structure motifs of IAPP(8-17) and IAPP(26-37) with the most probable key sites at positions around Ser(19)/Ser(20) and Gly(24). These observations reveal the similar amyloid formation tendency in the C and N terminus segments because of the sequence similarity, while the differences in specific amino acids at each key site manifest the effect of sequence variations. The results could be beneficial for studying structural polymorphism of amyloidal peptides with multiple beta structure motifs.

[Therapeutic effect of transplantation of bone marrow mesenchymal stem cells over-expressing Cx43 on heart failure in post-infarction rats].

OBJECTIVE: To explore the therapeutic effect of transplantation of bone marrow mesenchymal stem cells (BMSCs) over-expressing Cx43 on heart failure in post-infarction rats. METHODS: Sixty SD rats were randomly divided equally into 4 groups: sham group, DMEM/F12 group injected with DMEM/F12, EGFP group with transplanted EGFP transfected BMSCs and Cx43 group with transplanted Cx43 transfected BMSCs. Myocardial infarction model was established by ligating anterior descending branch and the cells were transplanted after 30 minutes. At Week 4 post-infarction, the heart functions of rats were evaluated by echocardiography. After the rats were sacrificed, their tissue samples were collected. The areas of myocardial infarction and the levels of collagen fiber content were measured. And the expressions of EGFR and Cx43 were observed under laser confocal microscopy. The level of Cx43 mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: As compared with the DMEM/F12 group, cardiac function was improved significantly, myocardial infarct area shrunk and collagen fiber content decreased significantly in the EGFP and group in Cx43 groups the. Survival of BMSCs and the formation of gap junction between BMSCs and the host myocardium could be observed under laser confocal microscopy both in EGFP group and Cx43 groups. And the post-infarction, expression of Cx43 mRNA in myocardial tissue decreased significantly in the group DMEM/F12, when compared with sham group (0.18 ± 0.05 vs 0.78 ± 0.14, P < 0.01). There was no significant difference on expression of Cx43 mRNA between DMEM/F12 group and EGFP group (0.18 ± 0.05 vs 0.20 ± 0.09, P > 0.05). The lever of Cx43 mRNA was higher in group Cx43 than in group DMEM/F12 and group EGFP(0.39 ± 0.14 vs 0.18 ± 0.05, P < 0.01; 0.39 ± 0.14 vs 0.20 ± 0.09, P < 0.05). CONCLUSION: Transplantation of BMSCs attenuates ventricular remodeling and improves cardiac functions. It may result from the over-expression of Cx43 gene through its effects of improving gap junction remodeling and increasing electro-mechanical coupling between myocardial cells in peri-infarct area.

Overexpression of CXCL16 promotes a vulnerable plaque phenotype in Apolipoprotein E-Knockout Mice.

BACKGROUND: CXCL16 has been shown to be involved in atherosclerotic lesion development, but its role in preexisting lesions is still unclear. This study aims to assess the effect of CXCL16 on the stability of preexisting lesions. METHODS: We firstly measured plasma CXCL16 level in Apolipoprotein E-Knockout (ApoE KO) mice with either high-cholesterol diet (HCD) or normal diet (ND) by enzyme-linked immunosorbent assay (ELISA). Then, silastic collars were placed around the carotid arteries in HCD-ApoE KO mice to accelerate atherosclerotic lesions. Five weeks later, CXCL16 was overexpressed by intravenous injection of lentivirus carrying CXCL16 transgene. Two weeks after infection, lesions were stained with hematoxylin and eosin (HE) and with oil red O. Biomarkers in the lesions, such as MMPs, CCL2, VCAM-1 and TNF-α were measured by real-time polymerase chain reaction (RT-PCR), which indicate the instability of plaques. RESULTS: The level of CXCL16 in plasma was higher in HCD-ApoE KO mice as compared to ND-ApoE KO mice. Circulating CXCL16 overexpression does not affect the size of preexisting plaques, but it leads to vulnerable plaque morphology and increases the expression of markers of plaque destabilization. CONCLUSION: Systemic CXCL16 becomes much higher in atherosclerosis, and it could be a potential atherogenic biomarker. Overexpression of CXCL16 promotes the evolution of preexisting lesions to vulnerable plaques in ApoE KO mice.

Significantly improved expression and biochemical properties of recombinant Serratia marcescens lipase as robust biocatalyst for kinetic resolution of chiral ester.

A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 x 105 U/l, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 °C and within the pH range of 5.0-6.8, giving temperature and pH optima at 40 °C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca²+, Cu²+, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C12). Using racemic trans-3-(4'-methoxy-phenyl)-glycidyl methyl ester [(±)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene-water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 °C under shaking condition (160 rpm), affording (-)-MPGM in nearly 100% ee. The K(m) and V(max) values of the lipase for (±)-MPGM were 222 mM and 1.24 mmol min⁻¹ mg⁻¹, respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.

The role of RANTES as a crucial downstream cytokine in calcineurin-dependent VSMC apoptosis stimulated by INFgamma and CD40L.

Many studies have suggested that VSMC (vascular smooth muscle cell) apoptosis plays a key role in destabilization and rupture of atherosclerotic plaques. Therefore protection for VSMCs from apoptosis is a promising approach to stabilize 'vulnerable' lesions. However, the mechanisms as to why VSMCs in the fibrous cap often appear as profilerated in early stages, but turn apoptotic in advanced stages, are still unknown. In the present study, using RNAi (RNA interference) technology and a CaN (calcineurin) antagonist, the correlation between CaN and RANTES (regulated upon activation, normal T-cell expressed and secreted) in cultured rat apoptotic VSMCs stimulated by IFNgamma (interferon gamma; 20 ng/ml) and CD40L (CD40 ligand; 100 ng/ml) was investigated. RANTES released from VSMCs in each group was measured by ELISA and its mRNA in VSMCs was determined by RT (reverse transcription)-PCR. The total activity and expression of CaN in VSMCs were detected by the zymochemistry method and Western blot analysis respectively. From the results of the present study it can be hypothesized that an elevated CaN concentration in endochylema, by the CD40-CD40L signal pathway, induces VSMC apoptosis accomplished by the overexpression of RANTES. Therefore RANTES is a potential target for treating vulnerable atherosclerotic plaques owing to its crucial downstream regulating role in CaN-dependent VSMC apoptosis.

Downregulation of T helper cell type 3 in patients with acute coronary syndrome.

BACKGROUND AND AIMS: There is an imbalance between Th1 and Th2 in the development and progression of atherosclerosis and in patients with acute coronary syndrome (ACS) including acute myocardial infarction (AMI) and unstable angina. T helper cell type 3 (Th3), which primarily secretes transforming growth factor beta-1 (TGF-beta1), has been shown to inhibit both Th1 and Th2 cells. The present study was designed to investigate whether Th3 cells are involved in plaque destabilization and the onset of ACS. METHODS: Ninety one patients who underwent diagnostic catheterization were classified into four groups (AMI group, unstable angina group, stable angina group and chest pain syndrome group). The cell frequencies of Th1, Th2 and Th3 were detected using flow cytometry, and the concentrations of their related cytokines IFN-gamma, IL-4 and TGF-beta1 were studied by ELISA. RESULTS: Apart from the imbalance between Th1 and Th2, results revealed a significant decrease in peripheral Th3 number and levels of TGF-beta1 in patients with ACS as compared with those in patients with stable angina and chest pain syndrome (p<0.01). CONCLUSIONS: Downregulation of Th3 cells in patients with ACS may play a potential role in plaque destabilization and the onset of ACS.

Metformin inhibits nuclear factor kappaB activation and decreases serum high-sensitivity C-reactive protein level in experimental atherogenesis of rabbits.

Previous studies demonstrated that metformin has obvious antiatherogenic properties, but the exact mechanism remains unclear. Therefore, we established an atherosclerotic rabbit model in order to investigate the potential effects of metformin on transcription factor nuclear factor kappaB (NF-kappaB) and serum high-sensitivity C-reactive protein (hs-CRP) level, which had been regarded as proatherogenic factors. New Zealand rabbits were randomly divided into three groups: a control group (n = 8), an atherosclerotic group (AS group, n = 8), and a metformin treatment group (Met group, n = 8). The experimental atherosclerotic rabbit model was successfully established at the end of the 8th week. From the 9th week, rabbits in the Met group were administered with 150 mg/kg metformin daily by gavage. Blood samples were collected at days 0 and 8, and at 16 weeks to detect the level of blood lipid and serum glucose. At the end of the experiment, blood samples were withdrawn for determining serum hs-CRP. Aortic samples were harvested for histomorphometric analysis. Immunohistochemistry and Western blotting were used to detect the expression of NF-kappaB subunit p65 in nuclear extracts and phosphorylation of inhibitor of nuclear factor kappaB (IkappaB) in cytoplasmic extracts. An experimental atherosclerotic rabbit model was successfully established. The expression of nuclear NF-kappaB subunit p65 and cytoplasmic phosphorylation of IkappaB protein in the vessel wall was enhanced (P < 0.01, respectively) in the AS group, and serum hs-CRP level was significantly increased in the AS group compared with the control group (3.90 +/- 0.25 mg/l versus 1.36 +/- 0.14 mg/l, P < 0.01). Treatment with metformin significantly attenuated the progression of aortic atherosclerosis. In the Met group, there was a marked reduction in nuclear NF-kappaB subunit p65 and cytoplasmic phosphorylation of IkappaB protein expression (P < 0.01). Serum hs-CRP concentration was also significantly decreased (3.20 +/- 0.20 mg/l versus 3.90 +/- 0.25 mg/l, P < 0.05). Metformin inhibits the phosphorylation of IkappaB and the activation of NF-kappaB in the vessel wall of experimental atherogenesis of rabbits, as well as decreasing the serum level of hs-CRP, thus suggesting that metformin has vascular anti-inflammatory properties, which may be one of its antiatherogenic mechanisms.

Antiapoptosis and mitochondrial effect of pioglitazone preconditioning in the ischemic/reperfused heart of rat.

PURPOSE: Pioglitazone, used clinically in the treatment of type 2 diabetes mellitus, has been implicated as a regulator of cellular inflammatory and ischemic responses. The present study examined whether pioglitazone could inhibit cardiomyocyte apoptosis and reduce mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of the rat. Furthermore, we investigated whether the protective effect of pioglitazone was related to opening of the mitochondrialATP-sensitive potassium channels. METHODS: Adult male Sprague-Dawley rats were subjected to 30 min of ischemia followed by 4 h of reperfusion. At 24 h before ischemia, rats were randomized to receive 0.9% saline, 5-hydroxydecanoate (5-HD, 10 mg kg(-1), i.v.) plus pioglitazone (3 mg kg(-1), i.v.) or pioglitazone (3 mg kg(-1), i.v.). One group served as sham control. We investigated mitochondrial structure, apoptosis rate and Bcl-2, Bax and Caspase-3 proteins by immunohistochemistry staining. RT-PCR was used to determine the expression of P38MAPKmRNA and JNKmRNA. Western blotting was used to measure the expression of P38MAPK, JNK and NFkappaB P65. A second group of rats were randomly divided into sham-operated, ischemia/reperfusion (I/R), pioglitazone treatment, 5-HD + pioglitazone and 5-HD groups and the size of myocardial infarction was determined. Primary cultured cardiomyocytes of neonatal Sprague-Dawley rats were divided into control, hypoxia reoxygenation, different concentrations of pioglitazone and 5-HD + pioglitazone groups. JC-1 staining flowcytometry was used to examine mitochondrial membrane potential (DeltaPsim). RESULTS: Pioglitazone decreased mitochondrial ultrastructural damage compared to I/R, and reduced infarct size from 34.93 +/- 5.55% (I/R) to 20.24 +/- 3.93% (P < 0.05). Compared with the I/R group, the apoptosis rate and positive cell index (PCI) of Bax and Caspase-3 proteins in the pioglitazone group were significantly decreased (P < 0.05), while the PCI of Bcl-2 protein was increased (P < 0.05). There was no significant difference between the I/R and 5-HD + pioglitazone groups. Compared with the sham-operated group, the expression of P38MAPK mRNA, JNK mRNA and protein of P38MAPK, JNK and NFkappaB P65 in I/R was increased (P < 0.05). Pioglitazone did inhibit the increase in expressions vs I/R (P < 0.05). The rate of loss DeltaPsim cells in the pioglitazone group was significantly lower than in the hypoxia reoxygenation group, while the addition of 5-HD inhibited the effect of pioglitazone. CONCLUSION: Pioglitazone inhibited cardiomyocyte apoptosis and reduced mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of rat. These protective effects of pioglitazone may be related to opening mitochondrial(ATP)-sensitive potassium channels.

[Effect of human bone marrow mesenchymal stem cells on allogeneic regulatory T cells and its possible mechanism].

The study was purposed to investigate the immune regulatory effects of human bone marrow mesenchymal stem cells (hMSCs) on Foxp3 expressing CD4(+)CD25(+) regulatory T cells and to explore the mechanism of immune modulation by hMSCs. Human MSCs were isolated and expanded from bone marrow cells, and identified with cell morphology, and the phenotypes were assessed by immunohistochemistry. Human peripheral blood mononuclear cells (hPBMNCs) were prepared by centrifugation on a Ficoll Hypaque density gradient. The hMSCs (1 x 10(3), 1 x 10(4), 1 x 10(5)) were added into wells containing hPBMNCs (1 x 10(6)) from an unrelated donor in the presence of rhIL-2. After 5 days of co-culture, the percentage of CD4(+)CD25(+) T cells was detected by flow cytometry. T cell proliferation was assessed by [(3)H] thymidine incorporation using a liquid scintillation counter. The expression of Foxp3 in CD4(+)CD25(+) T cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Cytokines (TGF-beta, IL-12, IFN-gamma, IL-10) concertrations of cultured supernatants were measured with ELISA. The results indicated that in all the experiments, the presence of hMSCs with hPBMNCs resulted in a statistically significant decrease in T cell proliferation, in dose-dependent manner. The increase of percentage of CD4(+)CD25(+) T cells in the peripheral CD4(+) T cell was observed after coculturing lymphocytes with hMSCs (p < 0.01). The expression of Foxp3-mRNA (Foxp3/beta-actin) in hMSCs groups was significantly higher than that in the control and was negatively associated with the value of CPM representing T proliferation. The levels of TGF-beta and IL-10 were higher in hMSCs groups than that in the control, and the levels of TGF-beta and IL-10 correlated positively with Foxp3-mRNA expression and the percentage of CD4(+)CD25(+) T cells. However, the secretion of IL-12 and IFN-gamma was significantly attenuated by hMSCs coculture, and there was no correlation with Foxp3-mRNA expression and the percentage of CD4(+)CD25(+) T cells. It is concluded that the Foxp3 expressing regulatory T cells may play an important role in the immune regulatory by hMSCs. Its mechanism is related to that the hMSCs-mediated TGF-beta and IL-10 convert CD4(+)CD25(-) T cells into CD4(+)CD25(+) T regulatory T cells, which specifically inhibits the proliferation of T cells.

[Mechanism of the angiogenic effect of bone marrow stromal cells implantation on acute myocardial infarction].

AIM: To study the mechanism of the therapeutic angiogenesis effect of bone marrow stromal cells (BMSCs) implantation on rat acute myocardial infarction models. METHODS: The rat acute myocardial infarction models were made by coronary artery ligation and divided into 2 groups at random. In the experiment group, twice passaged BMSCs were labeled with BrdU and then implanted into the infarction region of the recipients in 4 weeks. The control group was the model rats received only DMEM injection. In control group, the hearts were harvested on the day 3, 7, 14, 28, 42 and 56. The infarction regions were examined to identify the angiogenesis and the expression of the VEGF and bFGF. In experiment group, the hearts were examined on the day 42 and 56 after AMI (the day 14 and 28 after cells implantation). RESULTS: Viable cells labeled with BrdU could be identified in host hearts. Histologic examination found most donor cells within the infarction region expressed fibroblastic and endothelial phenotype. The transplantation regions had a greater capillary density than the control regions did (14 +/- 4.7/HPF vs 6 +/- 2.4/HPF, P < 0.05). In the control group, the expression of VEGF and bFGF within the infarction regions peaked on day 7, and then decreased over time. In the experiment groups, the expression of bFGF and VEGF on the day 42 and 56 had a higher level than the control group did. CONCLUSION: The expression of VEGF and bFGF is significantly increased after cells therapy during the late phase of AMI. It indicates that BMSCs implantation promoted the angiogenesis is mediated by its differentiation into endothelium and the increased release of VEGF and bFGF.