[The Role of DNA Double-strain Damage Repairing Mechanisms in Diabetic Atheroscolersis].

OBJECTIVES: To identify the role of DNA double-strain damage repairing pathway in the development of diabetics atherosclerosis. METHODS: Wistar male rats were randomly divided into three groups: control group (group A), balloon injury group (group B) and diabetes + balloon injury group (group C). Streptozotocin (STZ) was injected into rat abdomen to induce diabetes. After stabilizing high glucose, rats in group B and group C were both under aortic balloon injury technique and fed high lipid forage post-operatively. Glucose levels and weight were observed weekly. Segments of aortoa of three groups were taken at 2, 4, 6 and 8 weeks, staining of senescent ß-galactosidase (SA-ß-gal) staining, HE and changes of aorta under light microscope were observed. The area of tunica intima (I) and tunica media (M) in aorta was measured, and their ratio (I/M) were analyzed. Expressions of gamma-histong family 2A variant (γ-H2AX), phosphorylated ataxia telangiectasia mutated (ATM), phosphorylated checkpoint kinasen 2 (CHK2) and phosphorylated P53 were detected by immunohistochemical staining. RESULTS: SA-ß-gal staining positive areas were dotted around in group B and group C [CM(155.3mm]but not in group A at two weeks.At the same time, a slight hyperlasia of aortic neointima was observed in HE staining of group B and group C. SA-ß-gal staining was positive scattered within the tunica intima of aorta of group B and group C at four weeks, and HE staining promted a significantly greater of aortic neointima in the group C than that in the other two group (P<0.05). Positive regions of SA-ß-gal staining were more in group C than group B at six weeks. Typical atherosclerotic plaques were formed, vascular smooth muscle cells were disordered arranged and foam cells were aggregated in the plaques of group C at six weeks post-operatively, and intimal membrane areas increased than group A and group B (P <0.05). At 8 weeks, SA-ß-gal positive areas in group C were greater than in group B. The arteriolar wall was markedly thickened and the lumen was narrowed. The area of intimal membrane and the I/M radio were significantly greater in group C than those in group A and group B (P <0.05). Positive expressed of γ-H2AX, phosphorylated ATM, phosphorylated CHK2 and phosphorylated P53 were observed in typical atherosclerotic foci of group C, and weaker expressed in group B. CONCLUSION: Cellular senescence of vascular edothelium is triggered and DNA double-strain damage is increased in diabetes. The DNA double-strain damage repairing machines may participate in the development of diabetic atherosclerosis.

Vascular endothelial injury and apoptosis in rats with severe acute pancreatitis.

We explored mechanisms of vascular endothelial injury that lead to systemic multiple organ failure by detecting the soluble endothelial protein C receptor (sEPCR), von Willebrand factor (vWF), serum nitric oxide (NO), and tumor necrosis factor alpha (TNF-α) and Bcl-2 mRNA and Bax mRNA expression in a severe acute pancreatitis (SAP) rat model. Compared to controls, the levels of TNF-α, vWF, and sEPCR were significantly increased in the experimental group at 12 hours and 24 hours and the NO level was significantly decreased. After 12 hours, the aortic endothelial apoptosis index and Bax mRNA expression in aortic endothelial cells had increased in the experimental group, but Bcl-2 mRNA levels had decreased. All these changes appeared at both 12 h and 24 hours. The results indicated that vascular endothelial injury and apoptosis markers were elevated in SAP. Endothelial injury and increased apoptosis in the experimental group were related to the increased expression of TNF-α.

Importance of high-density lipoprotein cholesterol levels in elderly diabetic individuals with type IIb dyslipidemia: A 2-year survey of cardiovascular events.

AIM: The risk factors for ischemic heart disease (IHD) or cerebrovascular accident (CVA) in elderly diabetic individuals with type IIb dyslipidemia are not fully known. Therefore, we investigated the relationship between lipid levels and IHD and CVA in diabetic individuals with type IIb dyslipidemia. METHOD: The Japan Cholesterol and Diabetes Mellitus Study is a prospective cohort study of 4014 type 2 diabetic patients (1936 women; age 67.4 ± 9.5 years). The primary end-points were the onset of IHD or CVA. Lipid and glucose levels, and other factors were investigated in relation to the occurrence of IHD or CVA. A total of 462 participants were included in the group of patients with type IIb dyslipidemia. RESULTS: The 462 diabetic participants with type IIb dyslipidemia were divided into those who were aged <65 years, 65-74 years and >75 years (n=168, 190 and 104, respectively). High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol/HDL-C were significantly associated with the risk of cardiovascular events in diabetic individuals with type IIb dyslipidemia who were aged <65 years, and HDL-C and diastolic blood pressure was significantly associated with cardiovascular events in patients aged 65-74 years. Non-HDL-C was not significantly associated with the risk of cardiovascular events. Multiple regression analysis showed that lower HDL-C was significantly associated with the risk of cardiovascular events in diabetic individuals with type IIb dyslipidemia who were aged <65 years and 65-74 years. CONCLUSIONS: Lower HDL-C was an important risk factor for cardiovascular events in diabetic individuals with type IIb dyslipidemia who were aged <75 years.

[Effect of hyaluronic acid on promoting bone marrow-derived mesenchymal stem cells to differentiate into neural cells in vitro].

OBJECTIVE: To determine the effect of hyaluronic acid (HA) on promoting bone marrow-derived mesenchymal stem cells (BMSCs) to differentiate into neural cells. METHODS: BMSCs were purified and monitored. NSE+/NF+ cells and nestin+ cells were detected by immunocytochemistry. The mRNA levels of NSE, NF and nestin were determined by RT-PCR. RESULTS: The BMSCs adhered to HA hydrogel, with many surface projections, branches or crossing (Group A). Those cultured in brain tissue culture medium grew projections with fewer branches (Group B). Group A had more NSE+ cells (92.58 +/- 15.84) than Group B (80.26 +/- 16.47), and more NF+ cells (71.25 +/- 17.44) than Group B (52.37 +/- 14.75) (P < 0.05). At day 2 post-stimulation, more nestin+ cells were found in Group A (48.3 +/- 7.7) compared with Group B (34.6 +/- 5.2) (P < 0.05). At day 7, the mRNA levels of NSE and NF increased in both groups, but more in Group A. Nestin mRNA increased at day 2 post-stimulation and dropped at day 7 in both groups, where more significant in Group A. CONCLUSION: HA hydrogel provides structural support and proper microenvironment for the growth, proliferation and differentiation of BMSCs, and promotes BMSCs to differentiate into neuron cells.

[The role of DNA double strains damage repairing mechanisms in high glucose-induced endothelial senescence].

OBJECTIVE: To investigate the roles of DNA double stains damage repairing mechanisms in high glucose-induced cellular senescence. METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated with different concentrations of glucose (5.5 mmol/L, 11 mmol/L, 22 mmol/L and 33 mmol/L) for 72 hrs before the assay of senescence-associated beta-galactosidase staining. The superoxides were detected by flow cytometry. The levels of NO were detected by enzyme assay. Gamma-H2AX and phosphorylated P53 protein were measured by Western blot. Changes after co-incubation with KU55993 (an inhibitor of ATM) were examined with methods mentioned above. RESULTS: Compared with control group, percentage of positive cells of senescence-associated beta-galactosidase staining increased significantly in high glucose groups. The corresponding levels of reactive oxygen increased and NO decreased in a concentration-dependent manner. Intra-cellular levels of gamma-H2AX and phosphorylated P53 protein were significantly increased in high glucose groups. Statistical significances were revealed between high-glucose group and control group, as well as among different high-glucose groups, but no significant difference was observed between mannitol and control group. KU55993, an inhibitor of ATM, significantly reduced the levels of gamma-H2AX, phosphorylated P53 protein, and positive rate of senescence-associated beta-galactosidase staining. CONCLUSION: High glucose may promote DNA double strains damage by enhancing oxidative stress and decreasing NO, and thus accelerate cellular senescence. ATM-P53 pathway, the key proteins related to DNA double strain damage repairing mechanisms, may play an important role in high glucose induced cellular senescence and atherosclerosis.

[Effects of testosterone substitution on metabolic syndrome-related factors in hypogonadal males: a meta-analysis].

OBJECTIVE: To conduct a meta-analysis on the effects of testosterone on the related factors of metabolic syndrome in hypogonadal males. METHODS: Based on the principles and methods of Cochrane systematic reviews, we searched the PubMed (1980 to August 2009), Embase (1980 to August 2009), the Cochrane Central Register of Controlled Trials and CNKI (1995 to August 2009) , and handsearched some relevant journals and conference proceedings as well. We also identified randomized controlled trials addressing the use of testosterone for the treatment of hypogonadism, screened the retrieved studies according to the predefined inclusion and exclusion criteria, evaluated the quality of the included studies, and performed a meta-analysis on the results of homogeneous studies using the Cochrane Collaboration's RevMan 5.0 software. RESULTS: Six randomized controlled trials were included. The results of analysis indicated that testosterone substitution could significantly ameliorate fasting blood glucose, total cholesterol and insulin resistance in hypogonadism patients, and it could also reduce LDL, HDL, triglyceride and systolic blood pressure, though with no significant difference from the controls. However, there was insufficient evidence to show the effects of testosterone on waist circumference, waist-hip ratio and diastolic blood pressure. CONCLUSION: Existing clinical evidence has demonstrated the positive effects of testosterone substitution on the improvement of insulin resistance, blood glucose and lipids, but due to the heterogeneity and high risk of bias in the included studies, the evidence might be insufficient to give full support to the demonstration. Further large-scale trials are required to define the metabolic effects of testosterone in the treatment of hypogonadism.

[Study on the accuracy of dual source computed tomography coronary angiography in detection of coronary artery stenoses in old patients].

OBJECTIVE: To investigate the accuracy of dual source computed tomography (DSCT) coronary angiography in detecton of coronary artery stenoses in patients older than 60 years. METHODS: The study was performed in 102 patients older than 60 years who were suspected of coronary artery stenoses, and without any contraindications to CT Scan and iodnated contrast agents. These patients underwent DSCT, as well as selective coronary angiography (SCA). All the data of DSCT were compared with the results of SCA which were regarded as gold standard to assess the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of DSCT in classifying coronary artery stenoses in old patients. RESULTS: In these 102 patients, DSCT indicated 56 patients with coronary artery stenoses and SCA indicated 52 patients. DSCT correctly classified 51 patients out of 52 patients who had significant coronary artery stenoses. The sensitivity of DSCT was 98.1%, specificity was 90.0%, positive predictive value was 91.1%, negative predictive value was 97.8%, and accuracy was 94.1%. DSCT also assessed and measured 408 lesions in all the patients. The sensitivity of DSCT was 95.8%, specificity was 96.2%, positive predictive value was 88.5%, negative predictive value was 98.7%, and accuracy was 96.1%. CONCLUSION: DSCT appears to be a useful method for the detection of coronary artery stenoses with a high accuracy in elder patients more than 60-year.

[Effect of total flavonoids of Hippophae rhamnoides L. on the expression of MCP-1 in aorta of spontaneously hypertensive rats].

OBJECTIVE: To study the effects of total flavonoids of Hippophae rhamnoides L. (TFH) on the expression of monocyte chemoattractant protein (MCP-1) in aorta of Spontaneously Hypertensive Rats (SHR) and to study the relationship of expression of MCP-1 in aorta and intimal medial thickness (IMT) of aorta. METHODS: Twelve-week-old male SHR (n=12) were randomly devided into three groups to receive TFH [30 mg/(kg x d), n=4], Enalapril [10 mg/(kg x d), n= 4] and Hydrochlorothiazide [25 mg/(kg x d), n=4] for 12 weeks, respectively. Another 4 SHR and 4 WKY which receive placebo served as positive control group and negative control group. The systolic blood pressure (SBP), intimal medial thickness and inside diameter of aorta of the rats were measured. The expression of MCP-1 in aorta was examined by real-time PCR and immunohistochemistry and the concentration of serum MCP-1 protein by ELSA. RESULTS: Twelve weeks later, systolic blood pressure in TFH was significantly decreased, compared with that in Enalapril and Hydrochlorothiazide without statistical differences. TFH markedly reduced the intimal medial thickness of aorta and expression of MCP-1 in aorta, similar with Enalapril, stronger than Hydrochlorothiazide. CONCLUSION: TFH can markedly decrease SBP of SHR and the decrease value of the TFH group was similar with that of the Enalapril and Hydrochlorothiazide group. TFH may inhibit the expression of MCP-1 in aorta and intimal medial thickness of aorta beyond BP Lowering effect. The effect of THF on the expression of MCP-1 and intimal medial thickness of aorta was similar with Enalapril. TFH may through inhibite the expression of MCP-1 in aorta to reduce the intimal medial thickness of aorta and protect hypertensive injury in target organs.

Clinical factors such as B-type natriuretic peptide link to factor VII, endothelial NO synthase and estrogen receptor alpha polymorphism in elderly women.

AIMS: This study evaluated the presence of genetic mutations in relation to thrombosis or atherosclerosis in elderly women. MAIN METHODS: This is an observational study of 93 Japanese women with a mean age of 80.9 years recruited from outpatient clinics of Nagoya University and its related hospitals. Ten single nucleotide polymorphisms (SNPs) were studied. Each gene studied acts in or is related to either blood coagulation (factor V Leiden, prothrombin G20210A, factor XIII Val34Leu, factor VII Arg353Gln, MTHFR C677T, beta-fibrinogen G-455A, PAI-1 4G/5G), metabolic syndrome-related pathways (PPARalpha Leu162Val), or endothelium/estrogen system (eNOS Glu298Asp, ERalpha IVS1-401). SNPs were analyzed for their relation to clinical values including lipids, B-type natriuretic peptide (BNP), fasting plasma glucose, tumor necrosis factor-alpha, interleukin-6, cyclic GMP, and nitric oxide metabolites. KEY FINDINGS: Comparisons between the distributions of different genotypes and clinical values showed three relationships. First, factor VII Arg353Gln and HDL-cholesterol (HDL-C) were linked to Arg/Arg carriers at higher levels (P=.049). The HDL-C to LDL-cholesterol ratio supported this link (P=.027). Second, eNOS Glu298Asp and triglycerides were linked to Glu/Glu carriers at higher levels (P=.031). Third, ERalpha IVS1-401 and BNP were related to CC genotype at lower levels (P=.031). Additionally, the last two relations showed that genotype does not influence the demarcation line of biomarkers, but the plasma/serum levels of biomarkers instead. SIGNIFICANCE: Correlations of factor VII Arg353Gln with HDL-C and eNOS Glu298Asp with triglycerides are new findings. Polymorphisms in the endothelium/estrogen system and the heart failure marker BNP are also correlated, with ERalpha IVS1-401 being the first identified marker. SNPs may be helpful for understanding the pathophysiology of atherosclerotic diseases in elderly women.

Risks of CHD identified by different criteria of metabolic syndrome and related changes of adipocytokines in elderly postmenopausal women.

The objective of this study was to assess the capacity of different criteria of metabolic syndrome (MetS) to identify risks of coronary heart diseases (CHDs) and related changes of adipocytokines in postmenopausal women. A cross-sectional study was carried out in 225 community-dwelling, elderly postmenopausal Chinese women (age, 66.77+/-5.09 years) without hormone replacement therapy (HRT). Baseline data such as blood pressure, body mass index (BMI), serum lipid profiles, and fasting glucose were analyzed, and insulin sensitivity was estimated via the homeostasis model assessment for insulin resistance. Serum tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), and adiponectin were measured simultaneously. The prevalence of MetS identified by the Third Report of the National Cholesterol Education Programme Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, the International Diabetes Federation (IDF), the Chinese Diabetes Society (CDS), and the Japanese Society of Internal Medicine (JPN) were 27.31%, 37.34%, 23.29%, and 13.65%, respectively. No significant differences of baseline data were found among different MetS groups, except for a significant higher waist circumference in the JPN-MetS group as compared with other MetS groups. The prevalence of confirmed CHD in the four MetS groups were 26.2%, 18.6%, 26.9%, and 32%, respectively. Odds ratios for CHD were 1.905 (95% CI=1.273-2.851), 1.208 (95% CI=0.778-1.876), 1.997 (95% CI=1.238-3.221), and 2.336 (95% CI=1.119-4.876), respectively. The JPN-MetS group had higher levels of TNFalpha and IL-6, whereas the CDS-MetS group correlated better with lower adiponectin levels. The IDF definition for MetS is the most sensitive one with regard to metabolic disorders, whereas JPN and CDS definitions correlate better with CHD and changes of adipocytokines among the four criteria studied.

17beta-estradiol antagonizes the down-regulation of endothelial nitric-oxide synthase and GTP cyclohydrolase I by high glucose: relevance to postmenopausal diabetic cardiovascular disease.

In postmenopausal women, the risk of diabetic cardiovascular disease drastically increases compared with that of men or premenopausal women. However, the mechanism of this phenomenon has not yet been clarified. We hypothesized that the beneficial effects of estrogen on endothelial function may be relevant to protection against hyperglycemia-induced vascular derangement. Bovine aortic endothelial cells were incubated for 72 h in the presence and absence of the physiological concentration of 17beta-estradiol (17beta-E2) under normal and high-glucose conditions. The presence of 17beta-E2 significantly counteracted the reduction in basal nitric oxide production under high-glucose conditions. This finding was associated with the recovery of endothelial nitric-oxide synthase (eNOS) protein expression, tetrahydrobiopterin (BH4) levels, and the activity and gene expression of GTP cyclohydrolase I (GTPCH-I), a rate-limiting enzyme for BH4 synthesis. Both the gene transfer of estrogen receptor alpha using adenovirus and treatment with the protein kinase C inhibitor bisindolylmaleimide I significantly enhanced the effects of 17beta-E2 treatment under high-glucose conditions, whereas these effects were abolished by the estrogen receptor antagonist ICI 182,780 (faslodex). Transfection of small-interfering RNA targeting eNOS resulted in a marked reduction in GTPCH-I mRNA under both normal and high-glucose conditions, but this reduction was strongly reversed by 17beta-E2. These results suggest that the activation of ERalpha with 17beta-E2 can counteract high-glucose-induced down-regulation of eNOS and GTPCH-I in endothelial cells. Therefore, estrogen deficiency may result in an exaggeration of hyperglycemia-induced endothelial dysfunction, leading to the development of cardiovascular disease in postmenopausal diabetic women.

Endothelial cellular senescence is inhibited by nitric oxide: implications in atherosclerosis associated with menopause and diabetes.

Senescence may contribute to the pathogenesis of atherosclerosis. Although the bioavailability of nitric oxide (NO) is limited in senescence, the effect of NO on senescence and its relationship to cardiovascular risk factors have not been investigated fully. We studied these factors by investigating senescence-associated beta-galactosidase (SA-beta-gal) and human telomerase activity in human umbilical venous endothelial cells (HUVECs). Treatment with NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) and transfection with endothelial NO synthase (eNOS) into HUVECs each decreased the number of SA-beta-gal positive cells and increased telomerase activity. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) abolished the effect of eNOS transfection. The physiological concentration of 17beta-estradiol activated hTERT, decreased SA-beta-gal-positive cells, and caused cell proliferation. However, ICI 182780, an estrogen receptor-specific antagonist, and L-NAME each inhibited these effects. Finally, we investigated the effect of NO bioavailability on high glucose-promoted cellular senescence of HUVECs. Inhibition by eNOS transfection of this cellular senescence under high glucose conditions was less pronounced. Treatment with L-arginine or L-citrulline of eNOS-transfected cells partially inhibited, and combination of L-arginine and L-citrulline with antioxidants strongly prevented, high glucose-induced cellular senescence. These data demonstrate that NO can prevent endothelial senescence, thereby contributing to the anti-senile action of estrogen. The ingestion of NO-boosting substances, including L-arginine, L-citrulline, and antioxidants, can delay endothelial senescence under high glucose. We suggest that the delay in endothelial senescence through NO and/or eNOS activation may have clinical utility in the treatment of atherosclerosis in the elderly.

The effect of high glucose on NO and O2- through endothelial GTPCH1 and NADPH oxidase.

Although endothelial dysfunction deteriorates diabetic angiopathy, the mechanisms are obscure. We revealed that high glucose augmented eNOS through stimulation of eNOS mRNA in cultured BAECs. NO was decreased and O2- was increased simultaneously. NOS inhibitor, inhibited O2- release, so did NADPH oxidase inhibitor. The effects were synergistic. Both intracellular BH4 level and GTPCH1 activity were decreased by high glucose, in line with decrease of GTPCH1 mRNA. HMG-CoA reductase inhibitor, atorvastatin increased GTPCH1 mRNA and activity, and BH4 level. Conclusively, high glucose leads to eNOS dysfunction by inhibiting BH4 synthesis and atorvastatin stimulate BH4 synthesis directly, and it may work as atherogenic process.