25mM D-glucose Research Articles

ERM Complex, A Therapeutic Target for Vascular Leakage Induced by Diabetes.

Ezrin, radixin, and moesin (the ERM complex) interact directly with membrane proteins regulating their attachment to actin filaments. ERM protein activation modifies cytoskeleton organization and alters the endothelial barrier function, thus favoring vascular leakage. However, little is known regarding the role of ERM proteins in diabetic retinopathy (DR). This study aimed to examine whether overexpression of the ERM complex exists in db/db mice and its main regulating factors. 9 male db/db mice and 9 male db/+ aged 14 weeks were analyzed. ERM proteins were assessed by western blot and by immunohistochemistry. Vascular leakage was determined by the Evans blue method. To assess ERM regulation, HRECs were cultured in a medium containing 5.5 mM D-glucose (mimicking physiological conditions) and 25 mM D-glucose (mimicking hyperglycemia that occurs in diabetic patients). Moreover, treatment with TNF-α, IL-1β, or VEGF was added to a high glucose condition. The expression of ERM proteins was quantified by RT-PCR. Cell permeability was evaluated by measuring movements of FITC-dextran. A significant increase of ERM in diabetic mice in comparison with non-diabetic mice was observed. A high glucose condition alone did not have any effect on ERM expression. However, TNF-α and IL-1β induced a significant increase in ERM proteins. The increase of ERM proteins induced by diabetes could be one of the mechanisms involved in vascular leakage and could be considered as a therapeutic target. Moreover, the upregulation of the ERM complex by diabetes is induced by inflammatory mediators rather than by high glucose itself.

Glucose‐Derived Extracellular Endothelial Microvesicles Induce a Pathologic Endothelial Phenotype

Hyperglycemia is associated with an increased risk and prevalence of cardiovascular disease (CVD), due in large part to the adverse effects of glucose on endothelial cells. Indeed, hyperglycemic conditions can damage endothelial cells, in turn, impairing endothelial vasomotor and fibrinolytic function, exacerbating inflammatory and oxidative processes and propagating a proapoptotic phenotype. In addition to these primary effects, glucose also stimulates the release of extracellular microvesicles from endothelial cells. Extracellular endothelial microvesicles (EMVs) have emerged as causative agents in vascular pathology owing to their numerical increase in disease states, including diabetes, and cellular effects. The effects of glucose-derived EMVs on endothelial cell function is not clear. The experimental aim of this study was to determine, in vitro, the effect of glucose-derived EMVs on endothelial cell inflammation, oxidative stress, and nitric oxide (NO) production. Human umbilical vein endothelial cells (HUVECs) were cultured (3rdpassage) and incubated with RPMI 1640 media containing 25mM D-glucose (concentration representing a diabetic glycemic state) or 5mM D-glucose (control, normoglycemic condition) for 48 h to generate EMVs. EMVs (CD144-PE) were counted and isolated by flow cytometry. Thereafter, HUVECs (2 x106cells/condition) were treated with EMVs (2:1 EMV:cell ratio) generated from either the high (hgEMV) or normal (ngEMV) glucose condition for 24 h. EMV release was markedly higher (~280%; P<0.05) in cells treated with high vs normal glucose (134.7±15.6 vs. 35.4±3.2 EMV/µL). hgEMVs induced significantly higher release of cytokines IL-6 (26.6±1.5 vs. 20.7±1.6 pg/mL) and IL-8 (40.5±4.1 vs. 26.4±2.9 pg/mL); in addition, expression of active NF-κB p65 (Ser-536) was higher in the hgEMV treated cells (7.6±1.0 vs. 3.4±0.5 AU; P<0.05). Intracellular ROS production was higher(~26%) in the cells treated with hgEMVs (72.5±3.4 %) vs ngEMVs (57.7±1.8%). Active endothelial nitric oxide synthase (p-eNOS Ser1177) (10.6±1.6 vs 31.7±5.2 AU) and NO production (7.7±0.2 vs. 11.6±1.1 umol/L) were both significantly lower (~30% and 20%, respectively) in hgEMV treated cells. Blocking NF-kB activation using IKK-2 inhibitor IV abolished the increase in IL-6 and IL-8 release in response to hgEMVs, verifying hgEMV induced NF-kB-mediated inflammation. Inhibiting endocytosis prevented the proinflammatory, pro-oxidative and anti-NO effects of hgEMVs; whereas, RNase-inactivation (RNase A/T1) of hgEMVs eliminated their negative effects on endothelial proteins regulating inflammation, oxidative stress and NO production. These data demonstrate that: 1)high glucose-derived EMVs induce a proinflammatory, pro-oxidative, proatherogenic endothelial phenotype; and 2) EMV internalization and intracellular delivery of RNA regulatory cargo are central mechanisms underlying these deleterious cellular effects.

Activation of Histone Deacetylase 6 (HDAC6) in the diabetic retina and in retinal endothelial cells exposed to glucidic stress, promotes oxidative stress through suppression of the thioredoxin system

Retinal microvascular dysfunction characterizes the pathophysiology of diabetic retinopathy (DR), a common complication of diabetes and a leading cause of blindness in the World. Oxidative stress and inflammation have been implicated as major pathogenic hubs for the induction and progression of DR. Reduced endogenous antioxidant activities within the retinal microvasculature can significantly contribute to oxidative stress in the diabetic retina. We have previously shown that diabetes inhibits the thioredoxin system (TrxS) and others have shown that the cytosolic histone deacetylase 6 (HDAC6) can promote oxidative stress in a not yet understood mechanism. Here we have investigated the role of HDAC6 in regulating the TrxS in the diabetic retinal microvasculature. HDAC6 expression and activity were assessed by immunoblotting and fluorimetric assay (respectively) in retinas of streptozotocin‐induced diabetic rats (STZ‐rats, 8 weeks of diabetes) and in primary culture of human retinal endothelial cells (HuREC) exposed to disease‐relevant glucose levels (HG=25mM D‐glucose) for 48 hours. Changes in TrxS were monitored by assessing the expression and activity of its functional unit, thioredoxin‐1 (Trx‐1), by immunoblotting and a commercially available fluorimetric assay, in STZ‐rats retinas and in HuREC exposed to HG. Some experiments in HuREC were conducted also in presence of 5μM of the specific HDAC6 inhibitor tubastatin A. CellROX assay and dot‐blot assessing 5‐nitrotyrosine (5‐NT) and 4 hydroxynonenal (4‐HNE) were used to measure oxidative stress in our experimental groups. HDAC6 expression and activity were found significantly elevated in retinas of STZ‐rats compared to normal age‐matched controls and in HuREC exposed to HG, but not to normal glucose (5mM D‐glucose) or the osmotic control L‐glucose (25mM). At the same time Trx‐1 expression was increased in diabetic retinas and in HuREC exposed to HG, whereas its activity was significantly decreased. Parallel to loss of Trx‐1 activity and increased HDAC6 expression and activity, we also found that oxidative stress parameters were also elevated. In HuREC, tubastatin A was able to block HG‐induced up‐regulation of oxidative stress parameters while up‐regulating Trx‐1 activity and normalizing its expression. In summary, our data confirm previous observations on the role of HDAC6 in promoting oxidative stress and disclose HDAC6 role in suppressing Trx‐1 activity. Therefore, our results, support HDAC6 pathogenic role in promoting oxidative stress in the diabetic retinal microvasculature and its implication in the pathogenesis of diabetic retinopathy.Support or Funding InformationEY022416This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GW29-e0204 Bradykinin Inhibits High-Glucose-Induced Senescence of Cardiac Progenitor Cells through the B2R/PI3K/AKT/mTOR/P53 Signal Pathways

The aim of this study is to determine the mechanism of bradykinin (BK) inhibit D-glucose induced premature senescence of c-kit positive cardiac progenitor cells (CPCs). C-kit positive CPCs were cultured from C57BL/6J mouses. CPCs were exposed to 25mM D-glucose (D-Glu) with or without treatment with

T-Resveratrol Protects against Acute High Glucose Damage in Endothelial Cells.

Postprandial hyperglycemia in diabetic and nondiabetic subjects is associated with endothelial dysfunction. Evidence shows that high glucose generates oxidative stress and a pro-inflammatory state promoting the development of cardiovascular diseases. trans-Resveratrol (t-RV) has been shown to reduce cardiovascular risk. To determine whether t-RV acts as a protector against acute high glucose (AHG)-induced damage, two in vitro models, rat aortic rings (RAR) and human umbilical vein endothelial cells (HUVEC) were used. RAR pretreated with AHG (25mM D-glucose) for 3h dramatically decreased the endothelium-dependent relaxation (EDR) induced by acetylcholine in phenylephrine (PE)-precontracted vessels. However, coincubation with t-RV significantly mitigated the damage induced by AHG on EDR. Pretreatment with AHG did not affect the vasodilation induced by sodium nitroprusside. HUVEC treated with t-RV decreased cytotoxicity and reduced radical oxygen species production induced by AHG. Taken together, these results suggest that t-RV can mitigate the AHG-induced EDR damage through a mechanism involving ROS scavenging and probably an increase in the bioavailability of NO.

Silymarin prevents diabetes-induced hyperpermeability in human retinal endothelial cells

IntroductionVascular endothelial growth factor (VEGF) plays an essential role in development of diabetic macular edema (DME). While there is evidence suggesting that silymarin, a flavonoid extracted from Silybum marianum, could be useful for prevention and treatment of diabetic nephropathy, no studies have been conducted in diabetic retinopathy (DR). The aim of this study was to assess the effect of silymarin on disruption of inner blood retinal barrier (BRB), the primary cause of DME. Materials and methodsHuman retinal endothelial cells (HRECs) were cultured under standard (5.5mM D-glucose) and diabetogenic conditions (25mM D-glucose and 25mM D-glucose + recombinant vascular endothelial growth factor [rVEGF, 25mg/mL]). To assess cell viability, three concentrations of silymarin were tested (2, 4 and 10μg/mL). The effect of silymarin on HREC disruption was determined using a dextran (70kD) permeability asssay. ResultsNo differences were found in the viability of HRECs treated with 2 or 4μg/mL of silymarin as compared to untreated cells, but viability significantly decreased after using 10μg/mL. The concentration of 4 μg/mL was therefore selected. Silymarin (4μg/mL) caused a significant decrease in VEGF-induced permeability in both media with 5.5nM (422±58 vs. 600±72 ng/mL/cm2; p<0.03) and 25nM of D-glucose (354 ± 28 vs. 567 ± 102 ng/mL/cm2; p<0.04). DiscussionOur results show that silymarin is effective for preventing hyperpermeability induced by diabetic conditions in HRECs. Further studies are needed to assess whether silymarin could be useful to treat DME.

Silymarin prevents diabetes-induced hyperpermeability in human retinal endothelial cells

IntroductionVascular endothelial growth factor (VEGF) plays an essential role in development of diabetic macular edema (DME). While there is evidence suggesting that silymarin, a flavonoid extracted from Silybum marianum, could be useful for prevention and treatment of diabetic nephropathy, no studies have been conducted in diabetic retinopathy (DR). The aim of this study was to assess the effect of silymarin on disruption of inner blood retinal barrier (BRB), the primary cause of DME. Materials and methodsHuman retinal endothelial cells (HRECs) were cultured under standard (5.5mM D-glucose) and diabetogenic conditions (25mM D-glucose and 25mM D-glucose + recombinant vascular endothelial growth factor [rVEGF, 25mg/mL]). To assess cell viability, three concentrations of silymarin were tested (2, 4 and 10μg/mL). The effect of silymarin on HREC disruption was determined using a dextran (70kD) permeability asssay. ResultsNo differences were found in the viability of HRECs treated with 2 or 4μg/mL of silymarin as compared to untreated cells, but viability significantly decreased after using 10μg/mL. The concentration of 4 μg/mL was therefore selected. Silymarin (4μg/mL) caused a significant decrease in VEGF-induced permeability in both media with 5.5nM (422±58 vs. 600±72 ng/mL/cm2; p<0.03) and 25nM of D-glucose (354 ± 28 vs. 567 ± 102 ng/mL/cm2; p<0.04). DiscussionOur results show that silymarin is effective for preventing hyperpermeability induced by diabetic conditions in HRECs. Further studies are needed to assess whether silymarin could be useful to treat DME.

Abstract 483: Effect of Glucose on Endothelial Cell Expression of Inflammation-Related miRNAs

Hyperglycemia is an independent risk factor for cardiovascular disease (CVD). A key factor underlying hyperglycemia-related CVD risk is endovascular inflammation. The mechanisms underlying hyperglycemia induced endovascular inflammation are not clear. It is now recognized that microRNAs (miRs) play a pivotal role in regulating cellular inflammation pathways, vascular health and, in-turn, cardiovascular disease (CVD). It is currently unknown whether high glucose concentrations adversely affect the expression of inflammation-related miRs in endothelial cells; if so, miR dysregulation may provide novel insight into the proinflammatory endothelial phenotype associated with hyperglycemia. The aim of this study was to determine the effect of high glucose concentrations on the expression of inflammation-related miRs (miR-34a, -92a, -126, -146a, -150, -155 and -181b). Cultured human umbilical vein endothelial cells were harvested on the 3rd passage and plated in 6-well plates at a density of 5.0x10 5 cells/condition. Cells were incubated with RPMI 1640 media containing 25mM D-glucose (concentration representing a hyperglycemic state) or 5mM D-glucose (control condition) for 48 hours. Thereafter, RNA was extracted from 1.0x10 5 cells and miRs were reverse transcribed and expression was determined by RT-PCR. Cellular expression was normalized to RNU6 and calculated as fold change in ΔΔCt from control (N=6, experimental units). There was no effect of high glucose concentrations on the expression of proinflammatory miRs: miR-34a (0.93±0.18 fold), -92a (0.93±0.07 fold) and -155 (0.85±0.10 fold). However, cellular expression of anti-inflammatory miRs: miR-146a (0.64±0.06 fold), -150 (0.69±0.12 fold), and -181b (0.73±0.07 fold), were significantly reduced (~60%, ~45%, and ~40% respectively; P&lt;0.05) in response to the high glucose condition. Lower cellular expression of miR-146a, miR-150 and miR-181b is consistent with a pro-inflammatory endothelial phenotype. These miRs directly target and suppress NF-kB activation and immune cell activation, primary mediators of cellular inflammation. The seminal and novel finding of this study is that high glucose concentration compromises endothelial cell expression of key anti-inflammatory miRs.

Abstract 203: Glucose Stimulated Endothelial Microparticles Increase Endothelial Cell Apoptotic Susceptibility

Clinical interest in endothelial cell-derived microparticles (EMPs) has increased due to their role in the pathogenesis of vascular disease. Although released by the endothelium, EMPs have autocrine properties that can significantly impact endovascular health. Hyperglycemic conditions, such as diabetes, are known to stimulate EMP release; however, the effects of these glucose-related microparticles on endothelial cell function are not well understood. High glucose concentrations induce endothelial cell apoptosis through a caspase-3-dependent mechanism. The aim of this study was to determine the effect of EMPs derived from a hyperglycemic condition on endothelial cell susceptibility to apoptosis. Human umbilical vein endothelial cells (HUVECs) were cultured (3 rd passage) and plated in 6-well plates at a density of 5.0 x 10 5 cell/condition. Cells were incubated with RPMI 1640 media containing 25mM D-glucose (concentration representing a diabetic glycemic state) or 5mM D-glucose (control, normoglycemic, condition) for 48 h to generate EMPs. EMPs derived from both conditions were pelleted by centrifugation and resuspended in culture media. EMP identification (CD144 + expression) and number was determined by flow cytometry. HUVECs (2 x10 6 cells/condition) were treated with EMPs (2:1 ratio) generated from either the hyperglycemic or normoglycemic conditions for 24 h. Thereafter, cells were treated with staurosporine (1μmol/L) for 3 h at 37°C and biotin-ZVKD-fmk inhibitor for 1 h at 37°C. Intracellular concentration of active caspase-3 was determined by enzyme immune assay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. Hyperglycemic EMPs resulted in significant increase in basal (1.5 + 0.1 vs 1.0 + 0.1 ng/mL) and staurosporine-stimulated (2.2 + 0.2 vs 1.4 + 0.1 ng/mL) caspase-3 activity compared with normoglycemic EMPs. Additional, the expression of miR-Let7a was markedly reduced (~140%) in response to hyperglycemic EMPs (0.43 + 0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell apoptotic susceptibility. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let7a expression.

Influence of Clinically Relevant Mechanical Forces on Vascular Smooth Muscle Cells Under Chronic High Glucose: An In Vitro Dynamic Disease Model

In this study, we subjected vascular smooth muscle cells (VSMC) to acute and chronic high glucose conditions under physiologically relevant levels of cyclic strain and low wall shear forces to compare phenotypic modulation and thus conceptualize a dynamic-disease test model which captures cellular response more accurately in comparison with static cultures. P2-P6 rat aortic smooth muscle cells were seeded on type I collagen-coated silicone membranes and subjected to 0-7% cyclic strain at 1Hz and 0.3dynes/cm(2) shear stress from flow for 24hr under acute (25mM d-glucose, 84hr) and chronic high glucose conditions (25mM d-glucose, 3-4weeks). Samples were analyzed for cell proliferation, percent apoptosis, cellular hypertrophy, and expression levels of smooth muscle contractile state-associated markers with 0.05 level of significance. Concomitant application of cyclic strain and flow shear resulted in an overall increase in proliferation of VSMCs under both acute and chronic high glucose conditions as compared with normal glucose control (P<0.0001). Application of both cyclic strain and cyclic strain shear resulted in a significant increase in percent apoptosis with chronic high glucose treatment in comparison with both normal glucose controls (P<0.0001) and acute high glucose (P<0.0001). Cellular hypertrophy as estimated by measuring cell area and aspect ratio revealed a significantly altered morphology due to concomitant loading under chronic high glucose conditions with significantly higher cell area (P<0.0001) and lower aspect ratio (P<0.0001) indicative of a relatively rounded morphology as compared with normal glucose controls. Western blot analysis demonstrated reduced expression of SM α-actin (P<0.0001), calponin (P<0.0001), and SM22α (P=0.0008) for concomitant loading under chronic high glucose treatment as compared with normal glucose controls. Concomitant application of cyclic strain and low wall shear stress resulted in greater phenotypic modulation of VSMCs due to chronic high glucose treatment as compared with normal glucose controls, thus implicating cellular-response differences which may impact progression of in-stent restenosis in diabetic patients with poorly controlled hyperglycemia. Similarity of VSMC response from our study to existing preclinical models of diabetes and reports of altered phenotype of VSMCs isolated from diabetic patients substantiate the relevance of our dynamic disease test model.

Myelinated 3D neural culture platform as a physiological model of diabetic peripheral neuropathy

Event Abstract Back to Event Myelinated 3D neural culture platform as a physiological model of diabetic peripheral neuropathy Ashwin Sivakumar1, Reed Gioe1 and Michael Moore1, 2 1 Tulane University, Department of Biomedical Engineering, United States 2 Tulane University, Department of Neuroscience, United States Introduction: We present a microengineered 3D hydrogel platform for culture of myelinated embryonic peripheral neural tissue as shown in Figure 1. This novel in-vitro platform enables morphological and physiological metrics analogous to clinical nerve fiber density and nerve conduction tests. We demonstrate the feasibility of this model system for studying peripheral neuropathy caused by glucose-induced neurotoxicity, the most common form of neuropathy. Figure 1: Microengineered myelinated neural fiber tract 3D architecture resembling native peripheral nerve anatomy. Materials and Methods: Dorsal root ganglia (DRG) from embryonic day 15 rats were grown over a period of 4 weeks in a dual hydrogel system consisting of a methacrylated heparin gel in a polyethylene glycol micro-mold. The dual hydrogel system served to constrain the axon growth to be polarized with high density, fasciculation and myelination. The DRG’s were subject to a three-stage media regimen including ascorbic acid (AA) in the final stage to induce Schwann Cell myelination. The myelinated cultures were subjected to 60mM concentrations of the D-glucose in media over a period of 48 hours to simulate diabetic neuropathy. The control cultures were exposed to a 25mM D-glucose concentration. Electrophysiological field recordings were taken with and without the exposure of high glucose, and changes in the compound action potentials in the field recording were observed and compared to the control. Results and Discussion: Compound action potentials show little or no synaptic input, increased delay and decreased amplitude with higher glucose concentration exposure as shown in Figure 2. The higher glucose concentration is thought to trigger the sorbitol pathway and induce oxidative stress in the neurons of the DRG, leading to apoptosis and axonal degeneration. Figure 2: Average traces representing compound action potentials recorded with and without high glucose treatment. The change in amplitude and latency are also quantified as bar plots. (n=10, * = p<0.05 and ***=p<0.001) Conclusion: Microengineered myelinated neural fiber tracts have morphology, physiology and, toxicity response analogous to a peripheral nerve. The platform could be used as an in-vitro model for neuropathy studies and drug development. Funding provided by Tulane University

Abstract 255: Hur/ppar-gamma Mediates Posttranscriptional Regulation of Hyperglycemia-induced Pro-inflammatory Response

Metabolic disorders including diabetes and obesity are associated with persistent activation of the innate immune response, which leads to low-grade inflammation and increased cardiovascular risk. The RNA-binding protein HuR binds to and post-transcriptionally regulates inflammatory cytokines, mRNA stability and gene expression, although it’s exact role in the diabetic condition remains unclear. Bone marrow-derived macrophages (BMM) exposed to high glucose (HG, 25mM D-glucose) displayed elevated expression of HuR and reduced expression of nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) as compared to low glucose (LG, 5mM D-glucose)-treated cells. The increase in HuR expression was associated with an increase in the gene expression of various inflammatory cytokines including TNF-alpha, MCP-1, IL-1beta and IL-6. At the molecular level, macrophages activated with LPS showed enhancements in expression of inflammatory mRNAs and also promoted their stability. Conversely, knockdown of HuR induced post-transcriptional silencing, reduced the mRNA stability and expression of inflammatory cytokines. Macrophages treated with selective antagonists of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma), GW9662 displayed increases in HuR expression and of various inflammatory cytokines. In an in vivo model of myocardial infarction, mice receiving intramyocardial injection of HuR-specific shRNA displayed a reduced inflammatory cytokine profile in the myocardium as compared to control shRNA treated mice. Taken together, these data highlight the role of HuR as a homeostatic coordinator of cytokine mRNA that regulates innate inflammatory effects and demonstrates the potential of modulating the effects of HuR for clinical benefit against pathologic inflammation in diabetes/obesity.

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

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, 25mM 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. 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.

Abstract 11059: Role of Sirtuin 6 in Macrophage Polarization and Cardiac Repair in Diabetes

Clinical and experimental studies provide evidence that metabolic and inflammatory pathways are functionally interconnected to cardiovascular diseases. Dynamic changes in macrophage activation [classical M1 activation (promote inflammation) or alternative M2 activation (promote wound healing)], in response to various stress signals, modulate cardiac physiopathology in diabetes. Sirtuin 6 (SIRT6), a NAD-dependent nuclear deacetylase plays an important role in genomic stability, cellular metabolism, stress response and aging. However, the mechanism by which SIRT6 activity affects macrophage phenotype and cardiac function in diabetes is still unexplored. Mouse bone marrow-derived macrophages (BMM) exposed to high glucose (HG, 25mM D-glucose) showed reduced expression of SIRT6 as compared to low glucose (LG, 5mM D-glucose)- and osmotic control (OC, 5mM D-glucose+20mM D-mannitol)-treated cells, associated with increased expression of proinflammatory cytokine and transcription factors (NFkb, c-JUN, FOXO, SP1 and STAT1). In addition, SIRT6 level was reduced in peritoneal macrophages of both diabetic models (streptozotocin-induced and db/db mice) as compared to non-diabetic mice. SIRT6 knockdown in RAW 264.7 cells exaggerated inflammatory response when exposed to HG. In contrast, IL-4-induced increase in mRNA expression of macrophage M2 phenotype markers like Arg1, Chi4l4, Retnla and IRS-2, but not IRS-1 expression was repressed suggesting that alternative macrophage (M2) phenotype was defective in SIRT6 deficient BM-macrophages under HG condition. SIRT6 protein expression was low in myocardial infarction-induced (MI) and diabetes-affected hearts. Interestingly, mice receiving intramyocardial injection of SIRT6-deficient macrophages showed further deterioration in left ventricular function, post-MI. Taken together, these data highlight a role for SIRT6 in regulating the balance of M1/M2 polarization, therefore, modulate macrophage mediated cardiac repair and regeneration in numerous inflammatory disease states including diabetes

Abstract 214: Role Of Sirtuin 6 In Macrophage Polarization In Diabetes

Clinical and experimental studies provide evidence that metabolic and inflammatory pathways are functionally interconnected to cardiovascular diseases. Dynamic changes in macrophage activation [classical M1 activation (promote inflammation) or alternative M2 activation (promote wound healing)], in response to various signals, modulate tissue pathology in diabetes. Sirtuin 6 (SIRT6), a NAD-dependent nuclear deacetylase plays an important role in genomic stability, cellular metabolism, stress response and aging. However, the mechanism by which SIRT6 activity affects macrophage function in diabetes is still unclear. Mouse bone marrow-derived macrophages (BMM) exposed to high glucose (HG, 25mM D-glucose) showed reduced expression of SIRT6 as compared to low glucose (LG, 5mM D-glucose)- and osmotic control (OC, 5mM D-glucose+20mM D-mannitol)-treated cells. SIRT6 knockdown in RAW 264.7 cells exaggerated inflammatory response in macrophages exposed to high glucose (HG) and in contrast, IL4-induced alternative macrophage (M2) phenotype was defective in SIRT6 deficient BM-macrophages under high glucose condition. SIRT6 protein expression was low in failing (MI-induced) and diabetes-affected hearts. Interestingly, mice receiving intramyocardial injection of SIRT6-deficient macrophages showed further deterioration in LV function, post-MI. Taken together, these data highlighting a role for SIRT6 in regulating both M1 and M2 polarization might have broad implications for numerous inflammatory disease states including insulin resistance and wound healing.

Molecular Mechanisms of Oxidative Stress‐induced STAT3 Activation in Endothelial Cells

We and others have established that STAT3 is a critical mediator of VEGF function and expression in microvascular endothelial cells (EC). In inflammatory diseases, including diabetic microangiopathies, enhanced STAT3 activation has been correlated with increased oxidative stress and VEGF expression. Here we wanted to determine whether and how elevation of reactive oxygen species (ROS) formation affects the activation of STAT3 in endothelial cells. EC were cultured in the presence of 25mM D-glucose for 24 hours and then challenged with 20ng/ml VEGF for different times. This treatment resulted in enhanced reactive oxygen species (ROS) formation, as demonstrated by hydroethidine staining. Furthermore, western blotting and immunoprecipitation analyses revealed that EC cultured in the presence of HG showed sustained VEGF-dependent STAT3 tyrosine phosphorylation which correlated with decreased phosphorylation of the tyrosine phosphatase SHP-1 and blockade of STAT3/SHP-1 interaction. These HG/ oxidative stress effects were blocked by concomitant treatment of EC with the antioxidants superoxide dismutase and catalase. In addition, antisense-mediated inhibition of SHP-1 expression resulted in constitutive STAT3 tyrosine phosphorylation. In conclusion, these results demonstrate a key role for SHP-1 in the regulation of VEGF-dependent STAT3 activation in EC and suggest that oxidative stress-mediated STAT3 activation occurs through inhibition of SHP-1.