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Objective: To study the correlation of Wnt/β-catenin pathway changes with cytokines and apoptosis genes in diabetic foot wound. Methods: diabetic foot patients admitted to our hospital between February 2015 and October 2017 were selected as the diabetic foot group, and non-diabetic patients admitted to our hospital during the same period were selected as the control group. Wound tissues of the diabetic foot group and normal skin tissues of the control group were collected to determine the mRNA expression levels of Wnt/β-catenin pathway molecules and apoptosis genes as well as the contents of cytokines. Results: The mRNA expression levels of Wnt1, β-catenin and B-cell lymphoma-2 (Bcl-2) as well as the contents of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) in the wounds of the diabetic foot group were significantly lower than those of the control group whereas the mRNA expression levels of glycogen synthase kinase-3 (GSK-3β), Bcl-2-associated X protein (Bax) and cysteinyl aspartate specific proteinase-3 (Caspase-3) as well as the contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly higher than those of the control group; Wnt1 and β-catenin were positively correlated with VEGF, IGF-1, bFGF and Bcl-2, and negatively correlated with TNF-α, IL-6, Bax and Caspase-3; GSK-3β was negatively correlated with VEGF, IGF-1, bFGF, and Bcl-2, and positively correlated with TNF-α, IL-6, Bax and Caspase-3. Conclusion: Wnt/β-catenin pathway is inhibited in diabetic foot wound and it is related to the changes in cytokine secretion and apoptosis gene expression.

Objective: To study the correlation of miR-29b expression level with oxidative stress mediators and inflammatory cytokines in diabetic foot (DF) wound. Methods: The DF patients admitted to our hospital between February 2015 and October 2017 were selected as the DF group and wound tissues were collected; non-diabetic patients admitted to our hospital due to trauma during the same period were selected as the control group and normal skin tissues were collected. The expression level of miR-29b as well as the contents of oxidative stress mediators and inflammatory cytokines in the tissues was measured. Results: miR-29b expression as well as malondialdehyde (MDA), interleukin-1β (IL-1β), interleukin-18 (IL-18), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) contents in the wounds of DF group was significantly higher than those of control group while superoxide dismutase (SOD) and glutathione peroxidase (GPX) contents were significantly lower than those of control group; miR-29b expression was positively correlated with MDA, IL-1β, IL- 18, TNF-α and VCAM-1 contents, and negatively correlated with GPX and SOD contents. Conclusion: The high expression of miR-29b in diabetic foot wound is related to the overactivation of oxidative stress response and inflammatory response.

Aim: The aim of this study was to investigate the relationship between CXCL-6 levels in wound exudates and healing of diabetic foot ulcers (DFU). Materials & methods: One hundred patients with neuropathic DFU were recruited. Wound exudate CXCL-6 levels were measured by enzyme-linked immunosorbent assay. Patients were followed for 24 weeks and divided into rapidly healing and nonhealing groups. Results: Compared with the NH group, the mean CXCL-6 levels in the wound exudates of the rapidly healing group were significantly higher. After adjusting for traditional risk factors, wound exudate CXCL-6 levels were still significantly associated with wound healing. Conclusion: CXCL6 is an independent predictor of wound healing in DFU patients and may be a potentially novel therapeutic target for the treatment of DFU.

Background: We recently reported that transplantation of autologous bone marrow mononuclear cells (BM-MNCs) may be an effective and promising therapy to treat refractory diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes mellitus (T2DM). This study was designed to investigate the potential mechanisms of BM-MNCs therapy.Methods: This clinical study recruited 60 patients with DPN, 30 T2DM patients without complications, 30 healthy control participants. All clinical parameters, the levels of inflammatory markers and growth factors in three groups were compared. All patients with DPN received one intramuscular injection of BM-MNCs and clinical follow-ups after the therapy for 2 days, 1, 4, 12, 24, and 48 weeks. Then they were divided into the responder and non-responder groups based on the improvement of nerve conduction velocity. Binary logistic regression was performed to evaluate the corresponding prognostic factors for BM-MNCs treatment.Results: Patients in DPN group had higher level of tumor necrosis factor-α (TNF-α) and lower level of vascular endothelial growth factor (VEGF) than those in control group. DPN group had the highest level of soluble intercellular adhesion molecule-1 (sICAM-1) among three groups. The level of nerve growth factor (NGF) in DPN group was slightly lower than that in DM group. Neuropathic symptoms were significantly improved after BM-MNCs injection. Thirty five of 54 patients with DPN (64.8%) reached the primary endpoint, and were regarded as the responders. Compared to non-responders, responders were younger, had a longer history of diabetes, and had higher numbers of mobilized CD34+ cells and BM-MNCs. The levels of TNF-α and sICAM-1 decreased just after BM-MNCs injection in both groups and slowly reverted to baseline levels. The durations of downtrend of TNF-α and sICAM-1 in responder group lasted longer than that in non-responder group. Serum level of VEGF in responder group increased immediately after BM-MNC therapy, and reached the highest point after the injection for 12 weeks. On the other hand, VEGF levels in the non-responder group only increased slightly. The number of applied CD34+ cells (OR=1.567, 95% CI 1.106-2.222, p=0.012) and duration of diabetes (OR=0.760, 95% CI 0.597-0.967, p=0.025) were the independent predictors of responding to BM-MNCs therapy. No adverse event associated with the treatment was observed during follow-up observations.Conclusions: BM-MNCs transplantation is an effective and promising therapeutic strategy to treat refractory DPN. The immune regulation and paracrine function of BM-MNCs may contribute to the improvement of DPN.

Expanded hexanucleotide GGGGCC repeat in a noncoding region of C9ORF72 is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). However, its molecular pathogenesis remains unclear. In our previous study, the expanded GGGGCC repeats have been shown to be sufficient to cause neurodegeneration. In order to investigate the further role of expanded GGGGCC repeats in the neuron, the normal r(GGGGCC)3 and mutant-type expanded r(GGGGCC)30 expression vectors were transfected into Neuro-2a cells. Cell proliferation, dendrite development, and the proteins’ levels of microtubule-associated protein-2 (MAP2) and cyclin-dependent kinase-5 (CDK5) were used to evaluate the cell toxicity of GGGGCC repeats on Neuro-2a cells. The results were shown that expression of expanded GGGGCC repeats caused neuronal cell toxicity in Neuro-2a cells, enhanced the expression of pMAP2 and pCDK5. Moreover, overexpression of Purα repaired expanded GGGGCC repeat-inducing neuronal toxicity in Neuro-2a cells and reduced the expression of pMAP2 and pCDK5. In all, our findings suggested that the expanded GGGGCC repeats might cause neurodegeneration through destroyed neuron cells. And the GGGGCC repeat-induced neuronal cell toxicity was inhibited by upregulation of Purα. We inferred that Purα inhibits expanded GGGGCC repeat-inducing neurodegeneration, which might reveal a novel mechanism of neurodegenerative diseases ALS and FTD.

Objective: To study the correlation of substance P (SP) expression in diabetic foot ulcer tissue with oxidative stress and apoptosis. Methods: Patients with diabetic foot ulcer who were treated in the Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology between January 2016 and March 2017 were selected as the diabetic foot ulcer (DFU) group of the research, the diabetic foot ulcer tissue samples were collected, patients who received debridement surgery due to trauma in the Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology during the same period were selected as the control group of the research, and normal wound tissue samples were collected. The contents of SP, oxidative stress molecules and apoptosis molecules in the wound specimens were detected. Results: SP, Prdx6, GSHPX and SOD contents in diabetic foot ulcer wound specimens of DFU group were significantly lower than those in normal wound specimens of control group while MDA, AOPP, Cyto-C, Caspase-9, Fas, FasL, Caspase-8 and Caspase-3 contents were significantly higher than those in normal wound specimens of control group; Prdx6, GSHPX and SOD contents in wound of patients with high SP were significantly higher than those of patients with low SP while MDA, AOPP, Cyto-C, Caspase-9, Fas, FasL, Caspase-8 and Caspase-3 contents were significantly lower than those of patients with low SP. Conclusion: The low expression of SP in diabetic foot ulcer tissue can activate oxidative stress response and apoptosis to participate in the formation of ulcer wound.

Critical Limb Ischemia (CLI) is common but uncommonly diagnosed. Improved recognition and early diagnostic markers for CLI are needed. Therefore, the aim of our study was to identify plasma biomarkers of CLI in patients with type 2 diabetes mellitus (T2DM). In this study, antibody-coated glass slide arrays were used to determine the plasma levels of 274 human cytokines in four matched cases of diabetes with and without CLI. Potential biomarkers were confirmed in an independent cohort by ELISA. After adjusting for confounding risk factors, only plasma level of Siglec-5 remained significantly associated with an increased odds ratio (OR) for diabetes with CLI by binary logistic regression analysis. Receiver operating characteristic (ROC) curve analysis revealed the optimal cut-off points for Siglec-5 was 153.1 ng/ml. After entering Siglec-5, the AUC was 0.99, which was higher than that of confounding risk factors only (AUC = 0.97, P

Supplement_materials_0703 for Autologous Bone Marrow Mononuclear Cell Transplantation Therapy Improved Symptoms in Patients with Refractory Diabetic Sensorimotor Polyneuropathy via the Mechanisms of Paracrine and Immunomodulation: A Controlled Study by Wei Wei, Li Li, Lin Deng, Zhong-Jing Wang, Jing-Jian Dong, Xiao-Yu Lyu, Ting Jia, Li Wang, Hong-Xiang Wang, Hong Mao and Shi Zhao in Cell Transplantation

The purpose of this study was to investigate the effect of glycaemic control on subclinical hypothyroidism (SCH) in Chinese type 2 diabetic patients.The study included 476 diabetic patients with SCH admitted for treatment of type 2 diabetes. The controls were selected euthyroid patients with similar characteristics regarding [age, body mass index (BMI), gender, previous hypertension or duration of diabetes, and smoke]. Total cholesterol (TC), LDL cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), the homeostatic model assessment of insulin resistance ratio (HOMA-IR), fasting plasma glucose, hemoglobin AThe LDL-C level and HOMA-IR value were significantly higher in the SCH group. Glycaemic control reduced HOMA-IR, HbAOur study shows that glycaemic control may bring some benefits to type 2 diabetic patients with SCH.

Objectives. Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The aim of our study was to identify novel biomarkers of impaired wound healing in diabetic foot ulcers. Methods. 109 patients with neuropathic diabetic foot ulcers and 30 burn victims otherwise healthy participated. Antibody-coated glass slide arrays were used to determine the levels of 80 human cytokines in pooled plasma or pooled wound exudate of diabetic foot ulcers with rapidly healing (RH, n=12) and matched nonhealing (NH, n=12) patients. Potential biomarkers were confirmed in an independent cohort by enzyme-linked immunosorbent assay (ELISA). Results. Protein array profiling identified 27 proteins or 15 proteins significantly altered in protein profiling of pooled plasma or pooled wound exudate of 12 RH patients compared with 12 matched NH patients, respectively. In an independent cohort, quantitative ELISA validation confirmed a decrease in MCP-2 and ENA-78 levels in NH patients versus RH patients or burn victims. After adjusting for the traditional risk factors (sex, age, body mass index, fasting plasma glucose, ulcer area, HbA1C, diabetes duration, hyperlipidemia, and antibiotic therapy), only wound exudate level of ENA-78 remained having a significant association with an increased odds ratio (OR) for wound healing by binary logistic regression analysis (P<0.05). Conclusion. Decreased wound exudate ENA-78 was independently associated with wound healing of patients with diabetic foot. Exudate ENA-78 level is implicated as a novel predictor of wound healing in patients with diabetic foot ulcers.

The study aimed to determine the relationships between the basal metabolic rate (BMR) and body composition of overweight and obese Chinese adults with type 2 diabetes mellitus (DM). This cross-sectional clinical study enrolled 193 Chinese adults with type 2 DM who were overweight (24 kg/m(2)=BMI≤28 kg/m(2), n=99), or obese (BMI ≥28 kg/m(2), n=94). Ninety-seven adults with normal BMIs, including 50 DM patients and 47 healthy adults, were recruited as a control group. BMR was measured by indirect calorimetry; predicted BMR was calculated according to the Schofield equation; and the relationships between BMR, body composition, and biochemical results were determined by the Pearson correlation. The results showed that obese DM patients had significantly higher BMRs than both overweight patients (P

Purpose : To investigate the effect of recombinant adenovirus-mediated human β-nerve growth factor (Ad-EGFP-hβ-NGF) on the differentiation of endothelial progenitor cells (EPCs) in rats. Methods: The successfully constructed Ad-EGFP-hβ-NGF and its negative control Ad-EGFP were infected into the isolated and purified rat EPCs to observe their morphological changes. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the levels of vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and basic fibroblast growth factor (bFGF) in different rat EPC culture solutions. Western blot was performed to determine the expression of tyrosine kinase receptor A (TrKA) protein in different groups of EPCs. Results : Primary fibrous EPCs were converted into epithelium-like cells. After infection with Ad-EGFPhβ- NGF for 1 week, some EPCs became round and exhibited neural stem cell-like changes. The expression levels of VEGF, vWF and bFGF in the Ad-EGFP-hβ-NGF infection group were significantly higher than those in the control group (p < 0.01). TrKA protein in Ad-EGFP-hβ-NGF infection was also significantly up-regulated compared with that in the negative control and blank control groups (p

The aim of this study was to investigate whether osteopontin(OPN)variants are associated with susceptibility to ankylosing spondylitis (AS) in a Chinese population. Polymorphisms at the 9175th position in exon 7 ofOPNand rs17524488 were genotyped using direct sequencing in 186 unrelated AS patients and 188 ethnically matched healthy controls. Serum concentration of OPN was measured by enzyme-linked immunosorbent assay (ELISA) in all participants. AS patients displayed significantly higher OPN serum levels than the controls (P<001). A heterozygous, novel 9175 T>A in exon 7 of theOPNgene was found in this study. In healthy controls, subjects carrying the rs17524488 G/G genotype of theOPNdisplay significantly higher OPN serum levels than the GG/GG genotype (P<0.05). Plasma OPN level is implicated as an early diagnostic marker of AS. The novel 9175th- (exon 7) position polymorphism ofOPNand rs17524488 were related to susceptibility to AS in a Chinese population, the rs17524488 G/G genotype may be involved in the pathogenesis of AS, and the precise molecular mechanism underlying the influence ofOPNpolymorphisms on the development of AS remains to be determined in the further prospective studies.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease, which is common but rarely diagnosed. Noninvasive biomarkers are urgently required to assist in the diagnosis of CLI. Accumulating evidence indicates that miRNAs play an important role in the development of various diseases. In this study, microarray profiling revealed 11 miRNAs with significantly altered expression in four T2DM patients with CLI compared with that in four sex- and age-matched T2DM patients without CLI. In independent cohorts, qRT-PCR validation confirmed the increased miRNA-4739 level in patients with CLI versus patients without CLI. miRNA-4739 levels increased with FPG and HbA1c (all P < 0.05). After adjusting for the risk factors, miRNA-4739 levels were found to be associated with an increased odds ratio (OR) of T2DM with CLI (OR =12.818, 95% confidence intervals (CI) 1.148 to 143.143, P = 0.038). ROC curve analysis revealed that the area under the curve (AUC) of miR-4739+confounding risk factors was 0.94 (95% CI 0.891 to 0.998, P < 0.001), which was higher than that of confounding risk factors (AUC 0.94 vs. 0.91, 95% CI -0.122 to 0.060, P > 0.05) and of miR-4739 (AUC 0.94 vs. 0.69, 95% CI -0.399 to -0.101, P < 0.001), respectively. We conclude that elevated plasma miRNA-4739 levels are independently associated with CLI in T2DM patients. miRNA-4739 is implicated as a novel diagnostic marker and a potential therapeutic target for CLI in diabetes.

Objective To investigate whether height is associated with peripheral arterial disease (PAD) in patients with type 2 diabetes. Research design and methods This was an observational study performed in 4,528 Chinese patients with type 2 diabetes. Anthropometric measures and the ankle-brachial index (ABI) were performed on each subject. PAD was defined as those patients with a history of revascularization or amputation due to ischemia, or an ABI

The role of hydrogen sulfide (H2S) in myocardial infarction (MI) has not been previously studied. We therefore investigated the effect of H2S in a rat model of MI in vivo. Animals were randomly divided into three groups ( n = 80) and received either vehicle, 14 μmol/kg of sodium hydrosulfide (NaHS), or 50 mg/kg propargylglycine (PAG) everyday for 1 wk before surgery, and the treatment was continued for a further 2 days after MI when the animals were killed. The mortality was 35% in vehicle-treated, 40% in PAG-treated, and 27.5% in NaHS-treated ( P < 0.05 vs. vehicle) groups. Infarct size was 52.9 ± 3.5% in vehicle-treated, 62.9 ± 7.6% in PAG-treated, and 43.4 ± 2.8% in NaHS-treated ( P < 0.05 vs. vehicle) groups. Plasma H2S concentration was significantly increased after MI (59.2 ± 7.16 μM) compared with the baseline concentration (i.e., 38.2 ± 2.07 μM before MI; P < 0.05). Elevated plasma H2S after MI was abolished by treatment of animals with PAG (39.2 ± 5.02 μM). We further showed for the first time cystathionine-gamma-lyase protein localization in the myocardium of the infarct area by using immunohistochemical staining. In the hypoxic vascular smooth muscle cells, we found that cell death was increased under the stimuli of hypoxia but that the increased cell death was attenuated by the pretreatment of NaHS (71 ± 1.2% cell viability in hypoxic vehicle vs. 95 ± 2.3% in nonhypoxic control; P < 0.05). In conclusion, endogenous H2S was cardioprotective in the rat model of MI. PAG reduced endogenous H2S production after MI by inhibiting cystathionine-gamma-lyase. The results suggest that H2S might provide a novel approach to the treatment of MI.

The purpose of the current study is to evaluate the cardioprotective effects of purified Salvia miltiorrhiza extract (PSME) on myocardial ischemia/reperfusion injury in isolated rat hearts. Hearts were excised and perfused at constant flow (7 – 9 ml min−1) via the aorta. Non-recirculating perfusion with Krebs-Henseleit (KH) solution was maintained at 37°C and continuously gassed with 95% O2 and 5% CO2. KH solution with or without PSME (100 mg per liter solution) was used after 30-min zero-flow ischemia for the PSME and control group, respectively. Left ventricular (LV) developed pressure; its derivatives, diastolic pressure, and so on were continuously recorded via a pressure transducer attached to a polyvinylchloride balloon that was placed in the left ventricle through an incision in the left atrium. PSME treated hearts showed significant postischemic contractile function recovery (developed pressure recovered to 44.2 ± 4.9% versus 17.1 ± 5.7%, P

Hepatocyte transplantation is considered to be an alternative to orthotopic liver transplantation. Cells can be used to bridge patients waiting for a donor organ, decrease mortality in acute liver failure, and support metabolic liver diseases. The limited availability of primary human hepatocytes for such applications has led to the generation of alternative hepatocyte-like cells from various adult stem or precursor cells. The aim of this study was to generate hepatocyte-like cells from adipose-derived mesenchymal stem cells (Ad-MSCs) for clinical applications, which are available “off the shelf.” Epigenetic changes in hepatocyte-like cells were induced by 5-azacytidine, which, in combination with other supplements, leads to significantly improved metabolic and enzymatic activities compared to nontreated cells. Cells with sufficient hepatic features were generated with a four-step protocol: 5-azacytidine (step 1); epidermal growth factor (step 2); fibroblast growth factor-4, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 3); and hepatocyte growth factor, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 4). Generated differentiated cells had higher phase I (CYP1A1/2, CYP2E1, CYP2B6, CYP3A4) and phase II activities compared to the undifferentiated cells. A strong expression of CYP3A7 and a weak expression of 3A4, as well as the important detoxification markers α-fetoprotein and albumin, could also be detected at the mRNA level. Importantly, urea metabolism (basal, NH4-stimulated, NH4- and ornithine-stimulated) was comparable to freshly isolated human hepatocytes, and unlike cryopreserved human hepatocytes, this activity was maintained after 6 months of cryopreservation. These findings suggest that these cells may be suitable for clinical application, especially for treatment of urea cycle disorders.

Autologous peripheral stem cell transplantation was first reported in 2007 to treat type 1 diabetes mellitus (DM) and achieved encouraging effect, but whether similar outcome can be achieved in type 2 DM is not well demonstrated. The objective of this study was to determine the effect of combination of autologous bone marrow stem cell transplantation (BMT) and hyperbaric oxygen treatment on type 2 DM.The study involved 31 patients with type 2 DM (aged 33 to 62 years) from January 2009 to January 2011 in the Central Hospital of Wuhan, China. Clinical variables (body mass index, duration of DM, insulin requirement, oral hypoglycemic drugs, time free from insulin, time free from oral drugs) and laboratory variables (hemoglobin A1c (HbA1c)), mononuclear cells infused, and C-peptide in four time points) were assessed. Purified bone marrow stem cells were infused into major pancreatic arteries. Follow-up was performed at the 30, 90, 180, 360, 540 and 720 days (mean 321 days) after BMT.Mean HbA1c values showed a significant reduction during follow-up in all patients after BMT. It decreased by more than 1.5% (from 8.7% to 7.1%) as quickly as at 30 days after BMT. Afterwards mean HbA1c fluctuated between plus or minus 0.5% until 24 months rather than declined continuously. At 90 days after the combined therapy C-peptide increased significantly compared with baseline (P0.0001). But in other time points C-peptide was similar with baseline data (P0.3). All patients had insulin and/or oral hypoglycemic drugs reduced to different levels. The dose of insulin of 7 patients (7/26, 27%) reduced for a period of time after BMT.Combined therapy of intrapancreatic BMT and hyperbaric oxygen treatment can improve glucose control and reduce the dose of insulin and/or oral hypoglycemic drugs in type 2 DM patients, but it only improve pancreatic β-cell function transiently. Further randomized controlled clinical trials involved more patients will be required to confirm these findings and the mechanism needs to be illustrated deeply.

Cytochrome P450 (CYP) epoxygenases CYP2C8 and CYP2J2 generate epoxyeicosatrienoic acids (EETs) from arachidonic acid. Mice with expression of CYP2J2 in cardiomyocytes (αMHC-CYP2J2 Tr) or treated with synthetic EETs have increased functional recovery after ischemia/reperfusion (I/R); however, no studies have examined the role of cardiomyocyte- vs. endothelial-derived EETs or compared the effects of different CYP epoxygenase isoforms in the ischemic heart. We generated transgenic mice with increased endothelial EET biosynthesis (Tie2-CYP2C8 Tr and Tie2-CYP2J2 Tr) or EET hydrolysis (Tie2-sEH Tr). Compared to wild-type (WT), αMHC-CYP2J2 Tr hearts showed increased recovery of left ventricular developed pressure (LVDP) and decreased infarct size after I/R. In contrast, LVDP recovery and infarct size were unchanged in Tie2-CYP2J2 Tr and Tie2-sEH Tr hearts. Surprisingly, compared to WT, Tie2-CYP2C8 Tr hearts had significantly reduced LVDP recovery (from 21 to 14%) and increased infarct size after I/R (from 51 to 61%). Tie2-CYP2C8 Tr hearts also exhibited increased reactive oxygen species (ROS) generation, dihydroxyoctadecenoic acid (DiHOME) formation, and coronary resistance after I/R. ROS scavengers and CYP2C8 inhibition reversed the detrimental effects of CYP2C8 expression in Tie2-CYP2C8 Tr hearts. Treatment of WT hearts with 250 nM 9,10-DiHOME decreased LVDP recovery compared to vehicle (16 vs. 31%, respectively) and increased coronary resistance after I/R. These data demonstrate that increased ROS generation and enhanced DiHOME synthesis by endothelial CYP2C8 impair functional recovery and mask the beneficial effects of increased EET production following I/R.—Edin, M. L., Wang, Z. J., Bradbury, J. A., Graves, J. P., Lih, F. B., DeGraff, L. M., Foley, J. F., Torphy, R., Ronnekleiv, O. K., Tomer, K. B., Lee, C. R., Zeldin, D. C. Endothelial expression of human cytochrome P450 epoxygenase CYP2C8 increases susceptibility to ischemia-reperfusion injury in isolated mouse heart.

In this study, the cardioprotective effects of nitric oxide (NO)-aspirin, the nitroderivative of aspirin, were compared with those of aspirin in an anesthetized rat model of myocardial ischemia-reperfusion. Rats were given aspirin or NO-aspirin orally for 7 consecutive days preceding 25 min of myocardial ischemia followed by 48 h of reperfusion (MI/R). Treatment groups included vehicle (Tween 80), aspirin (30 mg·kg−1·day−1), and NO-aspirin (56 mg·kg−1·day−1). NO-aspirin, compared with aspirin, displayed remarkable cardioprotection in rats subjected to MI/R as determined by the mortality rate and infarct size. Mortality rates for vehicle ( n = 23), aspirin ( n = 22), and NO-aspirin groups ( n = 22) were 34.8, 27.3, and 18.2%, respectively. Infarct size of the vehicle group was 44.5 ± 2.7% of the left ventricle (LV). In contrast, infarct size of the LV decreased in the aspirin- and NO-aspirin-pretreated groups, 36.7 ± 1.8 and 22.9 ± 4.3%, respectively (both P < 0.05 compared with vehicle group; P < 0.05, NO-aspirin vs. aspirin ). Moreover, NO-aspirin also improved ischemiareperfusion-induced myocardial contractile dysfunction on postischemic LV developed pressure. In addition, NO-aspirin downregulated inducible NO synthase (iNOS; 0.37-fold, P < 0.01) and cyclooxygenase-2 (COX-2; 0.61-fold, P < 0.05) gene expression compared with the vehicle group after 48 h of reperfusion. Treatment with NG-nitro-l-arginine methyl ester (l-NAME; 20 mg/kg), a nonselective NOS inhibitor, aggravated myocardial damage in terms of mortality and infarct size but attenuated effects when coadministered with NO-aspirin. l-NAME administration did not alter the increase in iNOS and COX-2 expression but did reverse the NO-aspirin-induced inhibition of expression of the two genes. The beneficial effects of NO-aspirin appeared to be derived largely from the NO moiety, which attenuated myocardial injury to limit infarct size and better recovery of LV function following ischemia and reperfusion.

Hydrogen sulfide (H2S) is synthesized in the body from L-cysteine by several enzymes including cystathionine-gamma-lyase (CSE). To date, there is little information about the potential role of H2S in inflammation. We have now investigated the part played by H2S in endotoxin-induced inflammation in the mouse. E. coli lipopolysaccharide (LPS) administration produced a dose (10 and 20 mg/kg ip)- and time (6 and 24 h)-dependent increase in plasma H2S concentration. LPS (10 mg/kg ip, 6 h) increased plasma H2S concentration from 34.1 +/- 0.7 microM to 40.9 +/- 0.6 microM (n=6, P

We investigated the apoptotic development and apoptotic-related gene expression after myocardial infarction (MI) at different time points in the current study. Bax gene expression was increased at 12 h after MI and peaked at 24 h. Fas gene started to over-express at 12 h after MI as well but it reached maximum at 72 h. In the MI groups, strongest staining of apoptosis was detected in rats 3 days post operation. Our results demonstrate that apoptotic development after MI is time dependent in the ischemic area and there could be some linkage with the over expression of angiotensin II receptors post MI.

Introduction: Angiotensin II (Ang II) exerts its biological effects by binding to Ang II receptors. Two major subtypes of Ang II receptors, AT1R and AT2R, have been recognized by ligand binding studies. AT1R mediates most of the Ang II effects in the cardiovascular system. Usually, the AT2R only occurs in the growing animal but disappears in adults. The function of the AT2R so far has not been elucidated clearly. Recent studies have suggested that the AT2R is coupled to an antigrowth process that counteracts the growth-promoting program initiated by AT1R activation. We previously reported that gene expression of AT1R and AT2R were regulated differently in a time-dependent manner after myocardial infarction (MI). However, it is still not clear how the transcriptional levels of AT1R and AT2R are regulated as well as their localization after MI. Therefore, the present study is to investigate Ang II receptor expression in rat heart after using specific Ang II receptor antibodies. Materials and Methods: MI was surgically induced in Wistar rats weighing around 200g by permanent ligation of the left anterior descending coronary artery. After 3, 7, 14 and 21 days of surgery, these rats were sacrificed and rat heart was collected. H&E staining was used to evaluate the morphological changes and immunohistochemistry was used to investigate the receptor expression and localization of AT1R and AT2R. Results: H&E Staining: Degraded myocardium and fibrosis were seen in the infarcted area of left ventricle. Immunohistochemistry: Degraded myocytes of infarcted area in the left ventricle demonstrated the strongest AT1R staining. Positive immunostaining for AT1R was also detected in the endocardium, myocardium, and epicardium. Occasionally, positivity was also observed in the coronary vessels, mainly coronary vascular endothelium, but not smooth muscle cells. In the left ventricular wall, AT1R expression increased mainly on day 3 but decreased thereafter as a result of degrading myocytes. On the other hand, in the non-infarcted area, AT1R expression was detected but weaker than the infarcted area. On the contrary, AT2R positive immunostaining was less intense than AT1R. It was mainly expressed in the degraded myocardium of infarcted area. Some positive staining was also in the endocardium and epicardium though very weakly. Occasionally, the endothelium of large coronary arteries was positively stained but not in the small vascular endothelium or vascular smooth muscle. The expression of AT2R was increased over time. The strongest expression of AT2R was found on day 21, post-surgery. There was no AT2 expression detected in the non-infarct area. Conclusion: (1) Ang II expression was changed in the heart after MI. (2) Both AT1R and AT2R expressions are increased after remodeling. (3) Up and down-regulation of AT1R and AT2R respectively in the different time points suggested that there is some interaction between each other but its mechanism needs to be further elucidated.

Myocardial infarction (MI) remains the leading cause of death from cardiovascular diseases over the past decades. Apoptosis is ‘programmed’ cell death which leads to clearance of ‘unwanted’ cells without disruption of tissue structure or function. Recently, we reported that angiotensin receptor subtype AT1 and AT2 mRNA levels were time dependently regulated after MI. AT1 and AT2 receptor mRNA levels markedly increased at 30 min and peaked 24 h post-MI. The time-dependent increase of AT1 and AT2 receptor mRNA is associated with the early remodeling process of the non-infarcted myocardium post MI. There is a further question raised whether the up-regulation of AT2 receptor is linked to apoptosis. Therefore, we investigated the apoptotic development and apoptotic related gene expression after MI at different time points in the current study. MI was induced in Wistar rats by ligation of the left anterior descending coronary artery. Bax gene expression was found to be increased at 12 h after MI and peaked at 24 h (4.3-fold). It declined at 72 h after MI. Fas gene started to over-express at 12 h after MI as well but it reached maximum at 72 h (4.7-fold). Protein levels of Bax and Fas expression were detected in the necrotic area and area at risk (surrounding area) at different time points after MI. Apoptosis was detected in the infarcted areas. No apoptosis was detected in the sham operated rats. In the MI groups, strongest staining of apoptosis was detected in rats 3 days post operation. Weak staining was found 1 day, 7 day post MI. Very fewer apoptotic cells were detected in the rats 2 weeks after MI. Our results demonstrate that apoptotic development after MI is time dependent in the ischemic area and there could be some linkage with the over expression of AT2 receptor post MI.