Protective effects of dapagliflozin against oxidative stress-induced cell injury in human proximal tubular cells

Elevated reactive oxygen species (ROS) in type 2 diabetes cause cellular damage in many organs. Recently, the new class of glucose-lowering agents, SGLT-2 inhibitors, have been shown to reduce the risk of developing diabetic complications; however, the mechanisms of such beneficial effect are largely unknown. Here we aimed to investigate the effects of dapagliflozin on cell proliferation and cell death under oxidative stress conditions and explore its underlying mechanisms. Human proximal tubular cells (HK-2) were used. Cell growth and death were monitored by cell counting, water-soluble tetrazolium-1 (WST-1) and lactate dehydrogenase (LDH) assays, and flow cytometry. The cytosolic and mitochondrial (ROS) production was measured using fluorescent probes (H2DCFDA and MitoSOX) under normal and oxidative stress conditions mimicked by addition of H2O2. Intracellular Ca2+ dynamics was monitored by FlexStation 3 using cell-permeable Ca2+ dye Fura-PE3/AM. Dapagliflozin (0.1–10 μM) had no effect on HK-2 cell proliferation under normal conditions, but an inhibitory effect was seen at an extreme high concentration (100 μM). However, dapagliflozin at 0.1 to 5 μM showed remarkable protective effects against H2O2-induced cell injury via increasing the viable cell number at phase G0/G1. The elevated cytosolic and mitochondrial ROS under oxidative stress was significantly decreased by dapagliflozin. Dapagliflozin increased the basal intracellular [Ca2+]i in proximal tubular cells, but did not affect calcium release from endoplasmic reticulum and store-operated Ca2+ entry. The H2O2-sensitive TRPM2 channel seemed to be involved in the Ca2+ dynamics regulated by dapagliflozin. However, dapagliflozin had no direct effects on ORAI1, ORAI3, TRPC4 and TRPC5 channels. Our results suggest that dapagliflozin shows anti-oxidative properties by reducing cytosolic and mitochondrial ROS production and altering Ca2+ dynamics, and thus exerts its protective effects against cell damage under oxidative stress environment

Mesenchymal stem cell-based Smad7 gene therapy for experimental liver cirrhosis

BackgroundBone mesenchymal stem cells (MSCs) can promote liver regeneration and inhibit inflammation and hepatic fibrosis. MSCs also can serve as a vehicle for gene therapy. Smad7 is an essential negative regulatory gene in the TGF-β1/Smad signalling pathway. Activation of TGF-β1/Smad signalling accelerates liver inflammation and fibrosis; we therefore hypothesized that MSCs overexpressing the Smad7 gene might be a new cell therapy approach for treating liver fibrosis via the inhibition of TGF-β1/Smad signalling.MethodsMSCs were isolated from 6-week-old Wistar rats and transduced with the Smad7 gene using a lentivirus vector. Liver cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl4) for 8 weeks. The rats with established liver cirrhosis were treated with Smad7-MSCs by direct injection of cells into the main lobes of the liver. The expression of Smad7, Smad2/3 and fibrosis biomarkers or extracellular matrix proteins and histopathological change were assessed by quantitative PCR, ELISA and Western blotting and staining.ResultsThe mRNA and protein level of Smad7 in the recipient liver and serum were increased after treating with Smad-MSCs for 7 and 21 days (P

Molecular Aspects of Cardiovascular Risk Factors

Cardiovascular diseases (CVDs) are the leading cause of death ..

TRPC Channels and Their Splice Variants are Essential for Promoting Human Ovarian Cancer Cell Proliferation and Tumorigenesis

TRPC channels are Ca2+-permeable cationic channels controlling Ca2+ influx response to the activation of G protein-coupled receptors and protein tyrosine kinase pathways or the depletion of Ca2+ stores. Here we aimed to investigate whether TRPC can act as the potential therapeutic targets for ovarian cancer. The mRNAs of TRPC1, TRPC3, TRPC4 and TRPC6 were detected in human ovarian adenocarcinoma. The spliced variants of TRPC1β, TRPC3a, TRPC4β, TRPC4γ, and TRPC6 with exon 3 and 4 deletion were highly expressed in the ovarian cancer cells, and a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1E9del) was identified. TRPC proteins were also detected by Western blotting and immunostaining. The expression of TRPC1, TRPC3, TRPC4 and TRPC6 was significantly lower in the undifferentiated ovarian cancer cells, but all-trans retinoic acid up-regulated the gene expression of TRPCs. The expression level was correlated to the cancer differentiation grade. The non-selective TRPC channel blockers, 2-APB and SKF-96365, significantly inhibited the cell proliferation, whilst the increase of TRPC channel activity by trypsin promoted the cell proliferation. Transfection with siRNA targeting TRPC1, TRPC3, TRPC4 and TRPC6 or application of specific blocking antibodies targeting to TRPC channels inhibited the cell proliferation. On the contrary, overexpression of TRPC1, TRPC1E9del, TRPC3, TRPC4, and TRPC6 increased the cancer cell colony growth. These results suggest that TRPCs and their spliced variants are important for human ovarian cancer development and alteration of the expression or activity of these channels could be a new strategy for anticancer therapy

Experimental investigation of a super performance dew point air cooler

This paper presents an experimental investigation of a super performance dew point air cooler which, by employing a super performance wet material layer, innovative heat and mass exchanger and intermittent water supply scheme, has achieved a significantly higher energy efficiency (i.e. Coefficient of Performance, COP) and a much lower electrical energy use compared to the existing air coolers of the same type. This involves the dedicated system design & construction, fully planned experimental testing under various simulated climatic conditions representing the climate of hot & dry, warm & dry, moderate, warm & humid and standard lab testing condition, testing results analysis and discussion, as well as the parallel comparison against the commercial dew point air cooler. Under the standard test condition, i.e. dry bulb temperature of 37.8 °C and coincident wet bulb temperature of 21.1 °C, the prototype cooler achieved the wet-bulb cooling effectiveness of 114% and dew-point cooling effectiveness of 75%, yielding a significantly high COP value of 52.5 at the optimal working air ratio of 0.364. The testing also indicated that the lower inlet air relative humidity led to a higher cooling efficiency, while the lower cooling output helped increase COP and cooling effectiveness (including the wet-bulb effectiveness and dew-point effectiveness) of the cooler

Involvement of TRPC Channels in Lung Cancer Cell Differentiation and the Correlation Analysis in Human Non-Small Cell Lung Cancer

The canonical transient receptor potential (TRPC) channels are Ca2+-permeable cationic channels controlling the Ca2+ influx evoked by G protein-coupled receptor activation and/or by Ca2+ store depletion. Here we investigate the involvement of TRPCs in the cell differentiation of lung cancer. The expression of TRPCs and the correlation to cancer differentiation grade in non-small cell lung cancer (NSCLC) were analyzed by real-time PCR and immunostaining using tissue microarrays from 28 patient lung cancer samples. The association of TRPCs with cell differentiation was also investigated in the lung cancer cell line A549 by PCR and Western blotting. The channel activity was monitored by Ca2+ imaging and patch recording after treatment with all-trans-retinoic acid (ATRA). The expression of TRPC1, 3, 4 and 6 was correlated to the differentiation grade of NSCLC in patients, but there was no correlation to age, sex, smoking history and lung cancer cell type. ATRA upregulated TRPC3, TRPC4 and TRPC6 expression and enhanced Ca2+ influx in A549 cells, however, ATRA showed no direct effect on TRPC channels. Inhibition of TRPC channels by pore-blocking antibodies decreased the cell mitosis, which was counteracted by chronic treatment with ATRA. Blockade of TRPC channels inhibited A549 cell proliferation, while overexpression of TRPCs increased the proliferation. We conclude that TRPC expression correlates to lung cancer differentiation. TRPCs mediate the pharmacological effect of ATRA and play important roles in regulating lung cancer cell differentiation and proliferation, which gives a new understanding of lung cancer biology and potential anti-cancer therapy. © 2013 Jiang et al

Ca2+ Influx through TRPC Channels Is Regulated by Homocysteine–Copper Complexes

An elevated level of circulating homocysteine (Hcy) has been regarded as an independent risk factor for cardiovascular disease; however, the clinical benefit of Hcy lowering-therapy is not satisfying. To explore potential unrevealed mechanisms, we investigated the roles of Ca2+ influx through TRPC channels and regulation by Hcy–copper complexes. Using primary cultured human aortic endothelial cells and HEK-293 T-REx cells with inducible TRPC gene expression, we found that Hcy increased the Ca2+ influx in vascular endothelial cells through the activation of TRPC4 and TRPC5. The activity of TRPC4 and TRPC5 was regulated by extracellular divalent copper (Cu2+) and Hcy. Hcy prevented channel activation by divalent copper, but monovalent copper (Cu+) had no effect on the TRPC channels. The glutamic acids (E542/E543) and the cysteine residue (C554) in the extracellular pore region of the TRPC4 channel mediated the effect of Hcy–copper complexes. The interaction of Hcy–copper significantly regulated endothelial proliferation, migration, and angiogenesis. Our results suggest that Hcy–copper complexes function as a new pair of endogenous regulators for TRPC channel activity. This finding gives a new understanding of the pathogenesis of hyperhomocysteinemia and may explain the unsatisfying clinical outcome of Hcy-lowering therapy and the potential benefit of copper-chelating therapy

High glucose enhances store-operated calcium entry by upregulating ORAI/STIM via calcineurin-NFAT signalling

© 2014, Springer-Verlag Berlin Heidelberg. Abstract: ORAI and stromal interaction molecule (STIM) are store-operated channel molecules that play essential roles in human physiology through a coupling mechanism of internal Ca 2+ store to Ca 2+ influx. However, the roles of ORAI and STIM in vascular endothelial cells under diabetic conditions remain unknown. Here, we investigated expression and signalling pathways of ORAI and STIM regulated by high glucose or hyperglycaemia using in vitro cell models, in vivo diabetic mice and tissues from patients. We found that ORAI1-3 and STIM1-2 were ubiquitously expressed in human vasculatures. Their expression was upregulated by chronic treatment with high glucose (HG, 25 mM d-glucose), which was accompanied by enhanced store-operated Ca 2+ influx in vascular endothelial cells. The increased expression was also observed in the aortae from genetically modified Akita diabetic mice (C57BL/6-Ins2 Akita /J) and streptozocin-induced diabetic mice, and aortae from diabetic patients. HG-induced upregulation of ORAI and STIM genes was prevented by the calcineurin inhibitor cyclosporin A and NFATc3 siRNA. Additionally, in vivo treatment with the nuclear factor of activated T cells (NFAT) inhibitor A-285222 prevented the gene upregulation in Akita mice. However, HG had no direct effects on ORAI1-3 currents and the channel activation process through cytosolic STIM1 movement in the cells co-expressing STIM1-EYFP/ORAIs. We concluded that upregulation of STIM/ORAI through Ca 2+ -calcineurin-NFAT pathway is a novel mechanism causing abnormal Ca 2+ homeostasis and endothelial dysfunction under hyperglycaemia. Key message: ORAI1-3 and STIM1-2 are ubiquitously expressed in vasculatures and upregulated by high glucose.Increased expression is confirmed in Akita (Ins2 Akita /J) and STZ diabetic mice and patients.Upregulation mechanism is mediated by Ca 2+ /calcineurin/NFATc3 signalling.High glucose has no direct effects on ORAI1-3 channel activity and channel activation process

Assessment of mitochondrial dysfunction and implications in cardiovascular disorders

Mitochondria play a pivotal role in cellular function, not only acting as the powerhouse of the cell, but also regulating ATP synthesis, reactive oxygen species (ROS) production, intracellular Ca2+ cycling, and apoptosis. During the past decade, extensive progress has been made in the technology to assess mitochondrial functions and accumulating evidences have shown that mitochondrial dysfunction is a key pathophysiological mechanism for many diseases including cardiovascular disorders, such as ischemic heart disease, cardiomyopathy, hypertension, atherosclerosis, and hemorrhagic shock. The advances in methodology have been accelerating our understanding of mitochondrial molecular structure and function, biogenesis and ROS and energy production, which facilitates new drug target identification and therapeutic strategy development for mitochondrial dysfunction-related disorders. This review will focus on the assessment of methodologies currently used for mitochondrial research and discuss their advantages, limitations and the implications of mitochondrial dysfunction in cardiovascular disorders

T-type Ca 2+ channel blocker mibefradil blocks ORAI channels via acting on extracellular surface

Background and purposeMibefradil (Mib), a T‐type Ca2+ channel blocker, has been investigated for treating solid tumours. However, its underlying mechanisms are still unclear. Here we aimed to investigate the pharmacological aspect of Mib on ORAI store‐operated Ca2+ channels.Experimental approachHuman ORAI1‐3 in tetracycline‐regulated pcDNA4/TO vectors was transfected into HEK293 T‐REx cells with STIM1 stable expression. The ORAI currents were recorded by whole‐cell and excised‐membrane patch clamp. Ca2+ influx or release was measured by Fura‐PE3/AM. Cell growth and death were monitored by WST‐1, LDH assays and flow cytometry.Key resultsMib inhibited ORAI1, ORAI2 and ORAI3 currents in a dose‐dependent manner. The IC50 for ORAI1, ORAI2 and ORAI3 was 52.6 μM, 14.1 μM and 3.8 μM, respectively. Outside‐out patch demonstrated that perfusion of 10 μM Mib to the extracellular surface completely blocked ORAI3 currents and single channel activity evoked by 2‐APB. Intracellular application of Mib did not alter ORAI3 channel activity. Mib at higher concentrations (>50 μM) inhibited Ca2+ release, but had no effect on cytosolic STIM1 translocation evoked by thapsigargin. The inhibition of Mib on ORAI channels is structure‐related, since other T‐type Ca2+ channel blockers with different structures, such as ethosuximide and ML218, had no or very small effect on ORAI channels. Moreover, Mib inhibited cell proliferation, induced apoptosis and arrested cell cycle progression.Conclusions and implicationsOur results suggest that Mib is a potent extracellular ORAI channel blocker, which provides a new pharmacological profile for the compound in regulating cell growth and death as an anti‐cancer drug