Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation

<p>Abstract</p> <p>Background</p> <p>Agonist stimulation of airway smooth muscle (ASM) results in IP₃mediated Ca²⁺release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na⁺influx. This localised increase in Na⁺levels can potentially switch the Na⁺/Ca²⁺exchanger into reverse mode and so result in a further influx of Ca²⁺. The aim of this study was to characterise the expression and physiological function of the Na⁺/Ca²⁺exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca²⁺influx into these cells.</p> <p>Methods</p> <p>The expression profile of NCX (which encodes the Na⁺/Ca²⁺exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca²⁺measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.</p> <p>Results</p> <p>NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na⁺loading and addition of extracellular Ca²⁺induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca²⁺and induced relaxation of porcine peripheral airways.</p> <p>Conclusions</p> <p>Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca²⁺homeostasis and link store depletion via STIM1 directly with NCX activation.</p

Cardiac diastolic dysfunction in angiotensin II treated hypertensive rats is associate with endothelial upregulation of SGLT1/2 in the macro and microcirculation: Protective effect of empagliflozin

Background: /Introduction: The angiotensin II (Ang II)/AT1R/NADPH oxidase-prooxidant pathway has been shown to upregulate the expression of SGLT1 and 2 and to induce premature endothelial dysfunction in coronary endothelial cells. Since all these effects are prevented by the dual SGLT1/2 inhibitor sotagliflozin (sota) and the SGLT2 inhibitor empagliflozin (empa), SGLT1 and 2 are involved in the induction of endothelial dysfunction. Purpose: The aim of the study was to evaluate the role of SGLT1 and 2 in the Ang II-induced hypertensive response leading to cardiac and vascular endothelial dysfunction. Methods: Male Wistar rats received empa (30 mg/kg/day) provided in the diet for 5 weeks. After 1 week, rats underwent sham surgery (sham rats) or surgery with implantation of an osmotic mini-pump infusing Ang II (0.4 mg/kg/d) for 4 weeks. Systolic blood pressure (SBP) was assessed by plethysmography, the cardiac function using echocardiography, the expression level of target proteins by immunofluorescence staining, fibrosis by Sirius red staining, the function of SGLT1 and 2 indirectly by the uptake of glucose-conjugated anthocyanins, and the level of oxidative stress using dihydroethidium staining. Results: The administration of Ang II to rats caused an increased systolic blood pressure from about 140 to 180 mmHg, which was not affected by empa. In the heart, the hypertensive response resulted in an increased left ventricle mass and a diastolic dysfunction as indicated by an elevated E/e' and a decreased IVRT, fibrosis and increased collagen I and ANP expression levels. In the secondary branch of the mesenteric artery, it resulted in an increased level of oxidative stress, AT1R, collagen I and of endothelial ACE, VCAM-1, MCP-1, MMP-2, MMP-9 associated with a down-regulation of eNOS. It was also associated with an increased endothelial expression level of SGLT1 in the aorta, mesenteric resistance vessel and coronary microcirculation. Moreover, an increased uptake of anthocyanins is observed in the aorta predominantly in the endothelium and this effect is markedly reduced by sota and empa. All of the above parameters were markedly reduced in the Ang II plus empa group whereas the empa treatment alone had little effect compared to the control group. Conclusion: The findings indicate that Ang II-induced hypertension promoting left ventricle cardiac dysfunction, and both macro and microvascular endothelial dysfunction are associated with an up-regulation of SGLT1 and 2 in endothelial cells. They further indicate that empa prevents the noxious impact of Ang II on the heart and vasculature despite persistent hypertension

Empagliflozin treatment does not affect the hypertensive response to Ang II administration to rats but decreases oxidative stress in the arterial wall, and endothelial and cardiac dysfunction

Background Selective sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular protection in type 2 diabetes patients with established cardiovascular disease independently of glycemic control. Angiotensin II (Ang II) and H2O2 have been shown to be strong inducers of the expression of SGLT2 and 1 in endothelial cells promoting oxidative stress and endothelial dysfunction. Purpose This study examined the cardiovascular protective effect of empagliflozin (empa) in a normoglycemic experimental model of hypertension in the rat. Methods Male Wistar rats received empa (30 mg/kg/day) provided in the diet for 5 weeks. After 1 week, rats underwent sham surgery (sham rats) or surgery with implantation of an osmotic mini-pump infusing Ang II (0.4 mg/kg/d) for 4 weeks. Systolic blood pressure (SBP) was assessed by sphygmomanometry, the cardiac function using echocardiography, the expression level of target proteins by immunofluorescence staining, and the level of oxidative stress using dihydroethidium staining. Results Angiotensin II administration increased systolic blood pressure from about 130 to 180 mmHg, which was not affected by the empa treatment. The 4-week Ang II treatment did not significantly affect the systolic cardiac function (cardiac output, left ventricle ejection fraction) but impaired the diastolic function as indicated by a reduced E' and IVRT values, and an increased E/E' value. The Ang II treatment increased significantly the heart and right ventricle weight whereas the left ventricle + septum weight was slightly but not significantly increased. No such functional and structural changes were observed in the Ang II + empa treatment group. An increased immunofluorescence eNOS signal in the endothelium, and a higher level of ROS throughout the aorta wall were observed in the Ang II-treated group, both of which were significantly reduced in the empa + Ang II-treated group. In the Ang II-treated group, the high level of oxidative stress in the aorta was significantly reduced by the AT1 receptor antagonist losartan, the NADPH oxidase inhibitor VAS-2871, the eNOS inhibitor NG-nitro-L-arginine and also to a greater extent by the selective SGLT2 inhibitor empa compared to the dual SGLT1/2 inhibitor sotagliflozin. Conclusion(s) The present findings indicate that although the empa treatment did not affect the hypertensive response of rats to Ang II, the SGLT2 inhibitor prevented the deleterious impact of Ang II on the diastolic cardiac function and remodeling, and the upregulation of eNOS expression and oxidative stress in the aorta wall. Thus, these findings highlight the protective potential of empa on the cardiovascular system in a normoglycemic hypertensive experimental model. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Boehringer Ingelheim Pharma GmbH &amp; Co KG (Biberach an der Riss, Germany

Sodium-glucose co-transporter 1 and 2 expression in the mammary artery of patients with bypass surgery: role of the pro-inflammatory response and contribution to oxidative stress

Abstract Background Selective sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular protection independently of glycemic control. Angiotensin II (Ang II) and H2O2 induced the expression of SGLT1 and 2 in cultured endothelial cells and isolated arteries to promote oxidative stress and endothelial dysfunction. However, the expression level and role of SGLT1 and 2 in human arteries remain poorly studied. Purpose This study examined the expression level of SGLT1 and 2 in the human internal mammary artery (IMA) obtained from bypass surgery patients, and, if so, determined the underlying mechanism and function. Methods IMAs were obtained from 40 bypass surgery patients (age 45 to 82). The expression level of target factors was assessed by Western blot analysis, immunofluorescence staining and RT-PCR, and the level of oxidative stress using dihydroethidium staining. Human kidney was used as a control tissue known to express SGLT1 and 2. Porcine coronary artery endothelial cells (CAEC) were cultured and studied at passage 1. Results Western blot analysis of 40 IMA samples indicated a high level of both SGLT1 and 2 in 16 and 17 IMAs, an intermediate level in 8 and 6 IMAs, and a low one in 16 and 17 IMAs, respectively. Immunofluorescence staining of IMA sections indicated that SGLT1 and 2 immunofluorescence signals were observed predominantly in the intima thickening and the media. The expression levels of SGLT1 and 2 were associated with p-p65 NF-kB signals but not angiotensin-converting enzyme (ACE), AT1R, MCP-1, VCAM-1. IMAs with a high expression level of SGLT1 and 2 had a high level of ROS throughout the arterial wall including the intima thickening and endothelium, which was inhibited by the antioxidant N-acetylcysteine, the ACE inhibitor perindoprilat, the AT1R antagonist losartan, and also by the dual SGLT1 and 2 inhibitor sotagliflozin and the selective SGLT2 inhibitor empagliflozin. Pro-inflammatory cytokines mRNA levels of IL-1β, TNF-α and IL-6 were detected in IMAs. Exposure of CAEC to either TNF-α, IL-1β or IL-6 caused a concentration-dependent upregulation of SGLT1 and 2. Conclusion The present findings indicate that SGLT1 and 2 expression is observed in some but not all IMAs of bypass surgery patients predominantly in the media, the intima thickening and the endothelium. High expression levels of SGLT1 and 2 are associated with NF-kB activation and oxidative stress that is prevented by a selective SGLT2 inhibitor and by a dual SGLT1/2 inhibitor. Since pro-inflammatory cytokines triggered SGLT1 and 2 expression in endothelial cells, the inflammatory burden of patients appears to be an important trigger regulating SGLT1/2 expression and the subsequent pro-oxidant response prompting pro-inflammatory and pro-thrombotic responses. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This work was supported by an unrestricted research grant from Boehringer Ingelheim Pharma GmbH &amp; Co. KG, Biberach, Germany

Sodium-glucose co-transporter 1 and 2 expression in the mammary artery of patients with bypass surgery: role of the pro-inflammatory response and contribution to oxidative stress

Abstract Background Selective sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular protection independently of glycemic control. Angiotensin II (Ang II) and H2O2 induced the expression of SGLT1 and 2 in cultured endothelial cells and isolated arteries to promote oxidative stress and endothelial dysfunction. However, the expression level and role of SGLT1 and 2 in human arteries remain poorly studied. Purpose This study examined the expression level of SGLT1 and 2 in the human internal mammary artery (IMA) obtained from bypass surgery patients, and, if so, determined the underlying mechanism and function. Methods IMAs were obtained from 40 bypass surgery patients (age 45 to 82). The expression level of target factors was assessed by Western blot analysis, immunofluorescence staining and RT-PCR, and the level of oxidative stress using dihydroethidium staining. Human kidney was used as a control tissue known to express SGLT1 and 2. Porcine coronary artery endothelial cells (CAEC) were cultured and studied at passage 1. Results Western blot analysis of 40 IMA samples indicated a high level of both SGLT1 and 2 in 16 and 17 IMAs, an intermediate level in 8 and 6 IMAs, and a low one in 16 and 17 IMAs, respectively. Immunofluorescence staining of IMA sections indicated that SGLT1 and 2 immunofluorescence signals were observed predominantly in the intima thickening and the media. The expression levels of SGLT1 and 2 were associated with p-p65 NF-kB signals but not angiotensin-converting enzyme (ACE), AT1R, MCP-1, VCAM-1. IMAs with a high expression level of SGLT1 and 2 had a high level of ROS throughout the arterial wall including the intima thickening and endothelium, which was inhibited by the antioxidant N-acetylcysteine, the ACE inhibitor perindoprilat, the AT1R antagonist losartan, and also by the dual SGLT1 and 2 inhibitor sotagliflozin and the selective SGLT2 inhibitor empagliflozin. Pro-inflammatory cytokines mRNA levels of IL-1β, TNF-α and IL-6 were detected in IMAs. Exposure of CAEC to either TNF-α, IL-1β or IL-6 caused a concentration-dependent upregulation of SGLT1 and 2. Conclusion The present findings indicate that SGLT1 and 2 expression is observed in some but not all IMAs of bypass surgery patients predominantly in the media, the intima thickening and the endothelium. High expression levels of SGLT1 and 2 are associated with NF-kB activation and oxidative stress that is prevented by a selective SGLT2 inhibitor and by a dual SGLT1/2 inhibitor. Since pro-inflammatory cytokines triggered SGLT1 and 2 expression in endothelial cells, the inflammatory burden of patients appears to be an important trigger regulating SGLT1/2 expression and the subsequent pro-oxidant response prompting pro-inflammatory and pro-thrombotic responses. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This work was supported by an unrestricted research grant from Boehringer Ingelheim Pharma GmbH &amp; Co. KG, Biberach, Germany

In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifylline: Involvement of caspase cascades and I?B? phosphorylation

The aim of this work was to investigate whether in vivo and in vitro pentoxifylline (PTX) sensitizes hematological tumor cells to adriamycin (ADM)-induced apoptosis, and to investigate the involvement of caspase cascades and phosphorylated forms of I?B?. Balb/c mice inoculated intraperitoneally with L5178-Y murine lymphoma cells were used for in vivo experiments and for survival studies. The U937 human monocytic cell line was used for in vitro experiments. Both cell lines were treated under similar experimental conditions with PTX and/or ADM to assess their effects on apoptosis. Apoptosis was evaluated by fluorescence microscopy with ethidium bromide and acridine orange staining and confirmed by electrophoretic DNA analysis. Caspase inhibitors Z-VAD-fmk, Z-DEVD-fmk, and Z-LEHD-fmk were used to investigate the involvement of caspase cascades. C-terminally and Ser32 phosphorylated forms of I?B? were evaluated in cytoplasmic extracts in the absence or presence of TNF?. Results: In vivo, PTX (50 mg/kg) with ADM (5 mg/kg) increased the apoptotic index relative to PTX or ADM administered alone, time- and dose-dependently. DNA laddering appeared in lymphoma cells treated with PTX + ADM at 24 h, whereas neither untreated control, PTX-, nor ADM-treated cells showed DNA fragmentation. All (100%) tumor-bearing mice treated with PTX (25 mg/kg) + ADM (2.5 mg/kg) survived for 1 year, whereas the mortality rates of mice treated with either PTX or ADM alone at the same doses were similar to that of untreated tumor-bearing mice (28 � 3 days). Caspase inhibitors inhibited apoptosis more efficiently in PTX- or ADM-treated cultures than in PTX + ADM-treated cultures. Pretreatment with TNF? (10 ng/mL) increased apoptosis in PTX- or ADM-treated U937 cells. However, the apoptotic index of PTX + ADM-treated cultures was significantly reduced and the expression of C-terminally and Ser32 phosphorylated I?B? was reduced. PTX sensitizes hematological malignancies to ADR-induced apoptosis. An independent caspase pathway is involved in PTX + ADM-induced apoptosis. The phosphorylation status of I?B? is closely related via TNF? to the possible mechanisms of drug resistance. � 2005 Elsevier B.V. All rights reserved

P6278- Ageing is associated with increased endothelial sodium-glucose cotransporter 1 expression at arterial sites at risk promoting enhanced anthocyanin accumulation and improved vascular oxidative stress

Introduction Ageing is characterized by endothelial dysfunction and vascular oxidative stress affecting initially arterial sites at risk. Anthocyanin-rich products are potent stimulators of the endothelial formation of nitric oxide. Sodium-glucose co-transporter 1 (SGLT1) expression has been shown to be increased by oxidative stress and mediate anthocyanin uptake in endothelial cells. Purpose The study determined whether ageing is associated with an upregulation of SGLT1 in arterio-susceptible (aortic arch) and resistant (aorta) sites, and evaluated the vascular SGLT1-mediated anthocyanin uptake. In addition, the impact of a 2-week ingestion of an anthocyanin-rich blackcurrant concentrate (ARBC) by old rats on vascular anthocyanin uptake and oxidative stress, and systolic blood pressure (SBP) was assessed. Methods Male Wistar rats (22-month old) were either untreated or treated with ARBC (60 and 120 mg/kg/day) in the drinking water for 2 weeks. SGLT1 expression was assessed by immunofluorescence, anthocyanin accumulation by Neu A reagent using a purified extract (BCE) prepared from ARBC, oxidative stress by dihydroethidium using confocal microscopy, and SBP by tail-cuff sphingomanometry. Results SGLT1 immunofluorescence was observed predominantly in the endothelium and was higher in the aortic arch than the aorta in old rats whereas only low levels were observed in young rats (12-week old). Exposure of vascular sections to BCE resulted in anthocyanin uptake exclusively in the endothelium, which was higher in the aortic arch than the aorta, and more pronounced in old than young rats. Anthocyanin uptake induced by BCE in the aorta was markedly reduced by LX4211 (a SGLT1/2 inhibitor) both in old and young rats. A high level of oxidative stress was observed throughout the aortic wall of old compared to young rats, which was inhibited by LX4211. Ingestion of ARBC by old rats resulted in a dose-dependent accumulation of anthocyanins throughout the aorta wall and the aortic arch. The tissue accumulation of anthocyanins was associated with a reduced level of oxidative stress. Ageing was associated with increased SBP by about 8 mmHg, which was reduced by ARBC 60 and 120 mg/kg/day treatment by about 5 and 7 mmHg, respectively. Conclusion The present findings indicate that ageing is associated with an upregulation of SGLT1 predominantly in the endothelium and that this effect is more pronounced at the aortic arch than the aorta. The increased endothelial expression level of SGLT1 promoted a greater accumulation of anthocyanins sensitive to LX4211. In addition, a 2-week ingestion of ARBC by old rats resulted in the accumulation of anthocyanins throughout the arterial wall of the aortic arch and aorta, and resulted in a reduced level of oxidative stress and systolic blood pressure. Thus, SGLT1 may be an attractive target to restore vascular protection at arterial sites at risk by promoting endothelial and vascular uptake of anthocyanins

Anthocyanin-rich blackcurrant intake by old rats improves blood pressure, vascular oxidative stress and endothelial dysfunction associated with SGLT1- and 2-mediated vascular uptake of anthocyanin

Introduction Aging-related endothelial dysfunction and vascular oxidative stress affect early arterial sites at risk. Anthocyanins uptake via sodium-glucose co-transporter 1 (SGLT1) are potent inducers of endothelial formation of nitric oxide (NO). Aims This study examined if anthocyanin-rich blackcurrant (ARB) improves the endothelial function in old rats. Method Male Wistar rats (22-month old) received ARB (60 and 120 mg/kg/d) orally for 2 weeks. Systolic blood pressure (SBP) was assessed by tail-cuff sphygmomanometry, vascular reactivity using organ chambers, protein expression by immunofluorescence, oxidative stress using dihydroethidium, and anthocyanin uptake by Neu reagent. Results Old rats showed increased SBP, abolished endothelium-dependent hyperpolarization-mediated relaxation and increased contractile response to phenylephrine in the mesenteric artery, which were improved by ARB. Old aorta showed increased oxidative stress and expression levels of eNOS, which were improved by ARB. SGLT1 immunofluorescence predominantly in the endothelium was more pronounced in the aortic arch than the aorta and higher in old than young rats, whereas the SGLT2 signal was low. The ARB treatment induced a dose-dependent accumulation of anthocyanins in the aorta and aortic arch. An ARB purified extract promoted ex vivo greater anthocyanins uptake mostly in the endothelium in the aortic arch than aorta, and in old compared to young rats. The anthocyanins uptake was inhibited to a greater extent by a dual SGLT1/2 inhibitor than by a selective SGLT2 inhibitor in the aorta of young and old rats. Both SGLT inhibitors reduced also ex vivo the age-related vascular oxidative stress. Conclusion The upregulation of SGLT1, and the greater SGLT1 and SGLT2-mediated uptake of anthocyanins predominantly in the endothelium at arterial sites at risk in old rats suggest that anthocyanins appear as interesting natural products to protect the endothelial function with increasing age

P6278 - Ageing is associated with increased endothelial sodium-glucose cotransporter 1 expression at arterial sites at risk promoting enhanced anthocyanin accumulation and improved vascular oxidative stress

Introduction Ageing is characterized by endothelial dysfunction and vascular oxidative stress affecting initially arterial sites at risk. Anthocyanin-rich products are potent stimulators of the endothelial formation of nitric oxide. Sodium-glucose co-transporter 1 (SGLT1) expression has been shown to be increased by oxidative stress and mediate anthocyanin uptake in endothelial cells. Purpose The study determined whether ageing is associated with an upregulation of SGLT1 in arterio-susceptible (aortic arch) and resistant (aorta) sites, and evaluated the vascular SGLT1-mediated anthocyanin uptake. In addition, the impact of a 2-week ingestion of an anthocyanin-rich blackcurrant concentrate (ARBC) by old rats on vascular anthocyanin uptake and oxidative stress, and systolic blood pressure (SBP) was assessed. Methods Male Wistar rats (22-month old) were either untreated or treated with ARBC (60 and 120 mg/kg/day) in the drinking water for 2 weeks. SGLT1 expression was assessed by immunofluorescence, anthocyanin accumulation by Neu A reagent using a purified extract (BCE) prepared from ARBC, oxidative stress by dihydroethidium using confocal microscopy, and SBP by tail-cuff sphingomanometry. Results SGLT1 immunofluorescence was observed predominantly in the endothelium and was higher in the aortic arch than the aorta in old rats whereas only low levels were observed in young rats (12-week old). Exposure of vascular sections to BCE resulted in anthocyanin uptake exclusively in the endothelium, which was higher in the aortic arch than the aorta, and more pronounced in old than young rats. Anthocyanin uptake induced by BCE in the aorta was markedly reduced by LX4211 (a SGLT1/2 inhibitor) both in old and young rats. A high level of oxidative stress was observed throughout the aortic wall of old compared to young rats, which was inhibited by LX4211. Ingestion of ARBC by old rats resulted in a dose-dependent accumulation of anthocyanins throughout the aorta wall and the aortic arch. The tissue accumulation of anthocyanins was associated with a reduced level of oxidative stress. Ageing was associated with increased SBP by about 8 mmHg, which was reduced by ARBC 60 and 120 mg/kg/day treatment by about 5 and 7 mmHg, respectively. Conclusion The present findings indicate that ageing is associated with an upregulation of SGLT1 predominantly in the endothelium and that this effect is more pronounced at the aortic arch than the aorta. The increased endothelial expression level of SGLT1 promoted a greater accumulation of anthocyanins sensitive to LX4211. In addition, a 2-week ingestion of ARBC by old rats resulted in the accumulation of anthocyanins throughout the arterial wall of the aortic arch and aorta, and resulted in a reduced level of oxidative stress and systolic blood pressure. Thus, SGLT1 may be an attractive target to restore vascular protection at arterial sites at risk by promoting endothelial and vascular uptake of anthocyanins