Microvesicles derived from hypoxia/reoxYgenation-treated human umbilical vein endothellal cells impair relaxation of rat thoracic aortic rings

Objective To investigate the effects of microvesicles(MVs) derived from hypoxia/reoxygenation(H/R)-treated human umbilical vein endothelial cells(HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings.Methods H/R injury model was established to induce HUVECs to release H/R-EMVs.H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium.H/R-EMVs were characterized using 1 urn latex beads and anti-PE-CD144 by flow cytometry.Thoracic aortic rings of rats were incubated with 2.5,5,10,20 μg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours,and their endothelium-dependent relaxation in response to acetylcholine(ACh) or endothelium-independent relaxation in response to sodium nitroprusside(SNP) was recorded in vitro.The nitric oxide(NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent.The expression of endothelial NO synthase(eNOS) and phosphorylated eNOS(p-eNOS,Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting.Furthermore,the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit.Results H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation.The membrane vesicles(< 1 urn) induced by H/R were CD144 positive.ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner(P<0.05,P<0.01).The expression of total eNOS(t-eNOS)was not affected by H/R-EMVs.However,the expression of p-eNOS decreased after treated with H/R-EMVs.The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings(P<0.01).Conclusion ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner.The mechanisms included a decrease in NO production,p-eNOS expression and an increase in oxidative stress.

Flow cytometric analysis of circulating microvesicles derived from myocardial ischemic preconditioning and cardioprotection of ischemia/reperfusion injury in rats

Objective: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles(MVs) from myocardial ischemic preconditioning(IPC) treated rats(IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion(I/R) injury in rats. Methods: Myocardial IPC was elicited by three cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending(LAD) coronary artery. Platelet-free plasma(PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFP. PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 μm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure(MAP), heart rate(HR) and ST-segment of electrocardiogram(ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin(HE) staining. The activity of plasma lactate dehydrogenase(LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. Results: Total IPC-MVs and different phenotypes, including platelet-derived MVs(PMVs), endothelial cell-derived MVs(EMVs), leucocyte-derived MVs(LMVs) and erythrocyte-derived MVs(RMVs) were all isolated which were identified membrane vesicles(<1 μm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats(P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR(P<0.01), decreased ST-segment and LDH activity(P<0.05, P<0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment(P<0.01). Conclusion: The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.

Effects of endothelial microvesicles induced by A23187 on H9c2 cardiomyocytes

Objective To investigate the effects of endothelial microvesicles(EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes. Methods Human umbilical vein endothelial cells(HUVECs) were treated with 10 μmol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 μm latex beads and antiPE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/PI double staining. Results EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles(< 1 μm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner(P<0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry. Conclusion Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.

Vitamin D receptor(VDR)contributes to the development of hypercalciuria by sensitizing VDR target genes to vitamin D in a genetic hypercalciuric stone-forming(GHS)rat model

Human idiopathic hypercalciuria(IH)is the most common cause of calcium oxalate nephrolithiasis with perturbed calcium metabolism with increased bone resorption and decreased renal calcium reabsorption,which can be phenotype-copied in the genetic hypercalciuric stone-forming(GHS)rat model.We previously demonstrated that high VDR expression plays important roles in the development of hypercalciuria in the GHS rats.However,the underlying mechanism through which VDR impact hypercalciuria development remains to be fully understood.Here,we sought to determine how VDR regulated its target genes that are implicated in calcium homeostasis and potentially hypercalciuria.We found that VDR expression in the GHS rats was elevated in the calcium transporting tissues,as well as in the thymus and prostate,but not in lung,brain,heart,liver and spleen,when compared with control SD rats.Snail expression in the GHS rats was significantly downregulated in kidney,intestine,thymus and testis.Intraperitoneal injection of 1,25(OH)2D3 significantly upregulated the expression of renal calcium sensing receptor(CaSR),intestinal calcium transporters transient receptor potential vanilloid type 6(TRPV6),and VDR in GHS rats,compared with that in control SD rats.ChIP assays revealed that VDR specifically bound to the proximal promoters of target genes,followed by histone H3 hyperacetylation or hypermethylation.Collectively,our results suggest that elevated VDR expressi on may con tribute to the development of hypercalciuria by sensi・tizing VDR target genes to 1,25(OH)2D3 through histone modifications at their promoter regions in a genetic hypercalciuric stone-forming(GHS)rat model.

Adherence to Lifestyle Advice and Treatments in Pakistani Patients with Type 2 Diabetes Mellitus

Background: Type 2 diabetes mellitus (T2DM) is a chronic disease that has become a major health care concern, especially in developing countries like Pakistan. Lifestyle modification and appropriate pharmacotherapy are shown to improve blood glucose levels, lipid abnormalities and blood pressure. It is not known how many patients adhere to advice and drugs prescribed. This study aimed to determine adherence to lifestyle and therapeutic advice. Methods: A cross sectional hospital based study was conducted among patients attending the diabetic clinic at the Aga Khan University Hospital, using a structured questionnaire. Adult patients with T2DM and with at least one year duration of diabetes were included in the study. Results: Participants were aged between 32 and 92 years old with a mean age of 55.7 years old (SD ± 10.7). Mean duration of diabetes was 10.7 years old (SD ± 7.7). Majority (94%) of the patients were literate. Around half (47.3%) of the patients have had achieved glycemic target (HbA1c < 7%). Above target glycemic control was more common among patients with ischemic heart disease (68.1%), neuropathy (64.8%) and those on insulin (62.5%). Self-reported non-adherence for blood sugar monitoring (9.5%), physical activity (61.7%), tobacco use (43.4%) and foot care (43.9%) were noted. About 25% of the participants were not fully adherent to dietary advice. None of the patients from our study reported non-adherence to medications. Good adherence to physical activity was found in males with college degree. The highest percentage of tobacco use (33.3%) was reported among businessmen. Conclusion: We noted low adherence to advice for physical activity, tobacco use and SMBG, but a high adherence to prescribed medications and insulin. This was a selected group visiting a teaching hospital. This will need to be studied further in the community and efforts are required to motivate patients.

Saudi Consensus for GLP-1 RAs Switching Guidance: Consensus Report

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) provide adequate glycemic control, weight reduction, low risk of hypoglycemia, and CV risk reduction. Their usage for type 2 DM (T2DM) is recommended mainly when hypoglycemia or weight gain should be considered, also, whenever initial therapy is failed. There are many recent updates in the treatment paradigm of T2DM. There are many types of GLP-1RAs, with a knowledge gap regarding switching between the different types. A Saudi task force gathered to develop an explicit, evidence-based consensus for switching between GLP-1RAs, when, why, and how? This article contains the expert panel’s recommendations as a contribution to complement the knowledge gap in this area from the national perspective. As an alternative to intensifying therapy, switching from one GLP-1RA to another has various advantages. Improvements in glycemic control, weight loss, adherence, and medications with established cardiovascular benefits are among them. Also, switching needs to be individualized upon many discussed factors like the dose of the previous GLP1-RA and gastrointestinal adverse effects. Discussion with patients about the why and how to switch is critical.