Diabetic Vasculature Research Articles

Long non-coding RNA PANDA drives diabetic vascular dysfunction by promoting endothelial senescence and oxidative damage

Abstract Funding Acknowledgements None. Background ROS and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. Long non-coding RNAs (lncRNAs) are emerging as important players in the pathogenesis of cardiovascular disease. Recent work has shown that PANDA, a newly identified lncRNA, is a key regulator of cellular senescence and apoptosis. Purpose To elucidate the role and molecular mechanism of lncRNA PANDA in diabetic vascular disease. Methods Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5 mmol/L) and high glucose concentrations (HG, 25 mmol/L). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection. Expression of PANDA was assessed by real time PCR. RNAs sequencing (RNA-seq) and bioinformatic analysis (network perturbation amplitude, NPA) were leveraged to unveil transcriptional changes upon PANDA depletion. PANDA RNA immuno-precipitation (RIP) was performed to check its binding to relevant transcriptional factor. Western Blots was used to investigate on NRF2 protein and its products. Beta-galactosidase staining was used to detect endothelial senescence while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. ROS production and NRF2 localization were investigated by fluorescence staining. The ex vivo effect of PANDA siRNA on endothelial function was assessed in aortic rings from diabetic mice. Results PANDA expression was significantly increased in HAECs exposed to HG as compared to NG. Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 as the top-ranking transcript in HG-treated cells (Fig). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation. Under HG conditions PANDA perturbed the pathway of the transcription factor NRF2, thus inhibiting the expression of NRF2-dependent pro-survival and anti-aging gene. Indeed, silencing of PANDA in HG-treated HAECs reduced the apotosis and senescence (Figure 1). PANDA depletion in HG improved endothelial migration and tube formation, while ROS production was reduced. In HG, there was an increasing of PANDA-NRF2 binding (Fig). Interesting, in HG NRF2 was more present in the cytosol, while PANDA siRNA allowed its translocation into the where it can promote the transcription of HMOX1. Of interest, PANDA levels were increased in aortas from diabetic mice while depletion of PANDA rescued endothelial dysfunction (Figure 2). Conclusions The hyperglycemia induced the upregulation of PANDA driving endothelial senescence and impairing angiogenic properties. PANDA depletion in HG-treated HAECs rescued maladaptive transcriptional changes enhancing NRF2 pathway. Of note, targeting PANDA in the diabetic vasculature was able to rescue endothelial dysfunction. These results indicate PANDA as a novel molecular target in the setting of diabetic vascular disease.

Extracellular glucose and dysfunctional insulin receptor signaling independently upregulate arterial smooth muscle TMEM16A expression.

Diabetes alters the function of ion channels responsible for regulating arterial smooth muscle membrane potential, resulting in vasoconstriction. Our prior research demonstrated an elevation of TMEM16A in diabetic arteries. Here, we explored the mechanisms involved in Transmembrane protein 16A (TMEM16A) gene expression. Our data indicate that a Snail-mediated repressor complex regulates arterial TMEM16A gene transcription. Snail expression was reduced in diabetic arteries while TMEM16A expression was upregulated. The TMEM16A promoter contained three canonical E-box sites. Electrophoretic mobility and super shift assays revealed that the -154 nt E-box was the binding site of the Snail repressor complex and binding of the repressor complex decreased in diabetic arteries. High glucose induced a biphasic contractile response in pressurized nondiabetic mouse hindlimb arteries incubated ex vivo. Hindlimb arteries incubated in high glucose also showed decreased phospho-protein kinase D1 and TMEM16A expression. In hindlimb arteries from nondiabetic mice, administration of a bolus dose of glucose activated protein kinase D1 signaling to induce Snail degradation. In both in vivo and ex vivo conditions, Snail expression exhibited an inverse relationship with the expression of protein kinase D1 and TMEM16A. In diabetic mouse arteries, phospho-protein kinase D1 increased while Akt2 and pGSK3β levels declined. These results indicate that in nondiabetic mice, high glucose triggers a transient deactivation of the Snail repressor complex to increase arterial TMEM16A expression independently of insulin signaling. Conversely, insulin resistance activates GSK3β signaling and enhances arterial TMEM16A channel expression. These data have uncovered the Snail-mediated regulation of arterial TMEM16A expression and its dysfunction during diabetes.NEW & NOTEWORTHY The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.

Ginsenoside Rk1 improves endothelial function in diabetes through activating peroxisome proliferator-activated receptors.

Ginsenoside Rk1, one kind of ginsenoside, is a minor ginsenoside found in Panax ginseng and used as traditional Chinese medicine for centuries. It exhibits anti-tumor and anti-aggregation effects. However, little research has been done on its effect on endothelial function. This study investigated whether ginsenoside Rk1 improved endothelial dysfunction in diabetes and the underlying mechanisms in vivo and in vitro. Male C57BL/6 mice were fed with a 12 week high-fat diet (60% kcal % fat), whereas treatment groups were orally administered with ginsenoside Rk1 (10 and 20 mg per kg per day) in the last 4 weeks. Aortas isolated from C57BL/6 mice were induced by high glucose (HG; 30 mM) and co-treated with or without ginsenoside Rk1 (1 and 10 μM) for 48 h ex vivo. Moreover, primary rat aortic endothelial cells (RAECs) were cultured and stimulated by HG (44 mM) to mimic hyperglycemia, with or without the co-treatment of ginsenoside Rk1 (10 μM) for 48 h. Endothelium-dependent relaxations of mouse aortas were damaged with elevated oxidative stress and downregulation of three isoforms of peroxisome proliferator-activated receptors (PPARs), PPAR-α, PPAR-β/δ, and PPAR-γ, as well as endothelial nitric oxide synthase (eNOS) phosphorylation due to HG or high-fat diet stimulation, which also existed in RAECs. However, after the treatment with ginsenoside Rk1, these impairments were all ameliorated significantly. Moreover, the vaso-protective and anti-oxidative effects of ginsenoside Rk1 were abolished by PPAR antagonists (GSK0660, GW9662 or GW6471). In conclusion, this study reveals that ginsenoside Rk1 ameliorates endothelial dysfunction and suppresses oxidative stress in diabetic vasculature through activating the PPAR/eNOS pathway.

Long non-coding RNA PANDA drives diabetic vascular dysfunction by promoting endothelial senescence and oxidative damage

Abstract Background Accumulation of reactive oxygen species and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. Long non-coding RNAs (lncRNAs) are emerging as important players in the pathogenesis of cardiovascular disease. Recent work has shown that PANDA, a newly identified lncRNA, is a key regulator of cellular senescence and apoptosis. Purpose To investigate the role of lncRNA PANDA in diabetic vascular disease. Methods Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5 mmol/L) and high glucose concentrations (HG, 25 mmol/L). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection while a scrambled RNA was used as a negative control. Expression of PANDA was assessed by real time PCR. PANDA RNA immuno-precipitation (RIP) was performed to check its binding to relevant transcriptional factors (NF-YA). Beta-galactosidase staining was used to detect endothelial senescence while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. Finally, RNAs sequencing (RNA-seq) and bioinformatic analysis (network perturbation amplitude, NPA) were leveraged to unveil transcriptional changes upon PANDA depletion. Finally, the ex vivo effect of PANDA siRNA on endothelial function was assessed in aortic rings from diabetic mice. Results PANDA expression was significantly increased in HAECs exposed to HG as compared to NG (Fig. 1A). We found that under HG conditions PANDA sequesters the transcription factor NF-YA, thus inhibiting the expression of NF-YA-dependent pro-survival and anti-aging genes (Fig. 1B). Indeed, silencing of PANDA in HG-treated HAECs restored the expression of anti-apoptotic genes Bcl-X(L) and Bcl-2 while blunting senescence-related genes such as p53 and p16INK (Fig. 1C). PANDA depletion also abolished HG-induced cellular apoptosis and senescence, as assessed by caspase-3 activity assay and b-gal staining (Fig. 1D-E), while improving endothelial migration and tube formation (Fig.1F-G). Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 (HMOX1) as the top-ranking transcript in HG-treated cells (Fig.1H). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation (Fig. 1I). Of interest, PANDA levels were increased in aortas from diabetic mice (Fig. 2A) while depletion of PANDA rescued endothelial dysfunction (Fig. 2B). Conclusions Hyperglycemia-induced upregulation of PANDA drives endothelial senescence while impairing angiogenic properties. PANDA depletion in HG-treated HAECs rescues maladaptive transcriptional changes by restoring expression of the antioxidant gene HMOX1. Of note, targeting PANDA in the diabetic vasculature was able to rescue endothelial dysfunction. Our results indicate PANDA as a novel molecular target in the setting of diabetic vascular disease.

Dose- and Time-Dependent Effect of Dietary Blueberries on Diabetic Vasculature Is Correlated with Gut Microbial Signature.

Evidence from our lab and others indicates the vascular effects of dietary blueberries. In the present study, we determined dietary blueberries' dose- and time-dependent effects on diabetic vasculature and their association with gut microbes. Seven-week-old db/db diabetic male mice were fed a diet supplemented with ± freeze-dried wild blueberry powder (FD-BB) for 4, 8, or 12 weeks (three cohorts). Diets contained 0%, 1.23%, 2.46%, and 3.7% of FD-BB, equivalent to 0, ½, 1, and 1.5 human servings of wild blueberries, respectively. The non-diabetic db/+ mice fed a standard diet served as controls. Metabolic parameters, vascular inflammation, and gut microbiome were assessed. Dietary supplementation of 3.7% FD-BB improved vascular inflammation in diabetic mice without improving systemic milieu in all three cohorts. Blueberries improved diabetes-induced gut dysbiosis depending on blueberry dosage and treatment duration. Spearman's correlation indicated that the opportunistic microbes and commensal microbes were positively and negatively associated with indices of vascular inflammation, respectively. Dietary blueberries reduced the opportunistic microbe that was positively associated with vascular inflammation (Desulfovibrio), and increased the commensal microbe that was negatively associated with vascular inflammation (Akkermansia). Dietary blueberries could be a potential adjunct strategy to beneficially modulate gut microbes and improve vascular complications in diabetes.

Apigenin alleviates diabetic endothelial dysfunction through activating AMPK/PI3K/Akt/eNOS and Nrf2/HO‐1 signaling pathways

AbstractApigenin (4′,5,7‐trihydroxyflavone) is a natural flavone reported to present anti‐diabetic and anti‐oxidative bioactivities. The effect of apigenin to mitigate endothelial dysfunction in diabetes remains to be investigated. In the present study, the vaso‐protective effect of apigenin as well as its underlying action mechanisms was investigated using in vitro‐ and in vivo‐based assays. Aortas were detached from C57BL/6J mice for ex vivo treatment, and primary rat aortic endothelial cells (RAECs) were cultured and further stimulated using high glucose (HG) with or without apigenin. In‐vivo diabetic model was established by feeding male C57BL/6J mice with a high‐fat diet (60% kcal% fat) for a total of 14 weeks, whereas the treatment group was orally administered apigenin (25 mg/kg/day) during the last 4 weeks. Exposure to HG (30 mM, 48 h) impaired acetylcholine‐induced endothelium‐dependent relaxation (EDR) in mouse aortas and induced oxidative stress with the downregulation of adenosine 5'‐monophosphate (AMP)‐activated protein kinase (AMPK)/phosphatidyl inositol 3‐kinase (PI3K)/protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) and nuclear factor erythroid 2‐related factor 2 (Nrf2)/heme oxygenase 1 (HO‐1) signaling pathways in aortas and RAECs. These impairments were reversed by apigenin treatments. However, the vaso‐protective effects of apigenin were abolished by Compound C (AMPK inhibitor) and wortmannin (PI3K inhibitor). Chronic oral administration with apigenin ameliorated EDR and reduced oxidative stress in the aortas of high‐fat diet (HFD)‐induced obese and diabetic mice. In conclusion, this study revealed that apigenin improved endothelial function and suppressed oxidative stress in the vasculature in diabetes through the activation of AMPK/PI3K/Akt/eNOS and Nrf2/HO‐1 pathways, suggesting its therapeutic potential as a natural dietary flavonoid against vascular diseases associated with diabetes.

Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes

AimsVascular dysfunction is a clinical hallmark of diabetes. While various pathways drive vascular alterations in diabetes, many gaps persist in understanding this process. Heat-shock protein 70 (HSP70) has a long-recognized role in diabetes, but the contributions of HSP70 to the diabetic vasculature remain largely unknown. Main methodsWe determined the systemic and local (aorta) levels of HSP70 in control (CTL) and streptozotocin (STZ)-induced diabetic rats. Functional studies were conducted in a wire myograph in the presence or absence of a pharmacological inhibitor for HSP70 (VER155008). Calcium (Ca2+) dynamics was indirectly evaluated as a function of change in force development in vehicle and VER-treated vessels, as well as in the presence of inhibitors for voltage-dependent and -independent plasmalemmal Ca2+ channels. Furthermore, mimicking the extracellular diabetic environment, we exposed aortic rings to serum from CTL and STZ-induced animals, which contains higher levels of HSP70, as well as to purified recombinant HSP70. Then, we performed functional studies following the modulation of Toll-like receptor 4 (TLR4) and its co-adaptor MD2, which interact with HSP70. Key findingsHSP70 plays a dual role in diabetes-induced vascular dysfunction: intracellular (i)HSP70 affects Ca2+ handling mechanisms, and extracellular (e)HSP70 modulates the TLR4-MD2 complex. SignificanceThese newly discovered roles of HSP70 push forward the field of vascular biology and open research avenues for other diseased states associated with altered vascular responses.

Dose and Time Dependent Effect of Dietary Blueberries on Diabetic Vasculature is Associated with Gut Microbes

BackgroundEvidence from our lab and others showed the vascular beneficial effects of dietary blueberries. However, dose‐ and time‐dependent effect of dietary blueberries on vascular complications in diabetes and the role of gut microbes in mediating this effect are unknown. We hypothesize that gut microbes are associated with the dose‐ and time‐dependent vascular beneficial effects of dietary blueberries.Methods7‐week‐old db/db male mice (Jackson Lab), a widely‐used type 2 diabetic animal model, were fed a diet with or without freeze‐dried wild blueberry powder (FD‐BB) for 4 or 8 or 12 weeks (D4, D8, and D12). Diets contained 0%, 1.23% (B1), 2.46% (B2) and 3.7% (B3) of FD‐BB, which is equivalent to 0, ½, 1, and 2 human servings of wild blueberries. Age‐matched, non‐diabetic db/+ mice fed a standard diet for 4 or 8 or 12 weeks (C4, C8, and C12) were used as controls. After the treatment period blood glucose, body composition, glucose tolerance, and insulin tolerance were assessed. Vascular inflammation was determined by the binding of fluorescent labelled mouse monocytic WEHI78/24 cells to vascular endothelium and mRNA expression of vascular inflammatory chemokines and adhesion molecules (IL8/KC, MCP1/JE, ICAM1, VCAM1 and E‐selectin). Microbial profiling of cecum was done by 16s rRNA amplification.ResultsDiabetic mice (D4, D8 and D12) had increased blood glucose, impaired glucose and insulin tolerance, increased body fat, decreased lean body mass, enhanced binding of monocytes to the aortic vessel, and increased expression of selected inflammatory molecules as compared to their respective control mice (C4, C8 and C12). Blueberry treatment at all levels didn’t improve the metabolic parameters in diabetic mice. However, 3.7% FD‐BB treatment reduced the binding of WEHI78/24 monocyte to vascular endothelium and suppressed the mRNA expression of selected inflammatory molecules in diabetic mice (D4B3 vs D4, D8B3 vs D8, and D12B3 vs D12). Microbial profiling showed an alteration in the relative abundance of gut microbes at different taxonomic levels in diabetic mice as compared to their respective control mice. At the genera level, 7, 9 and 10 genera were altered after 4‐, 8‐ and 12‐weeks treatment in diabetic vs control mice. Blueberry supplementation at different dosages improved the relative abundance of selected gut microbes in diabetic mice (>4 genera) at different time points. Specifically, blueberry treatment increased the abundance of commensal microbe Bifidobacterium and decreased the abundance of opportunistic Clostridium genera in diabetic mice.ConclusionThe beneficial effects of dietary blueberries on diabetic vasculature and gut microbiome varies with the doses and duration of treatment. In the present study, a dose of 3.7% FD‐BB in diet (equivalent to 1.5 human servings) was shown to exert potential beneficial effects on diabetic vasculature and is associated with specific gut microbes.

Hexokinase-2-Linked Glycolytic Overload and Unscheduled Glycolysis-Driver of Insulin Resistance and Development of Vascular Complications of Diabetes.

The recent discovery of the glucose-induced stabilization of hexokinase-2 (HK2) to proteolysis in cell dysfunction in model hyperglycemia has revealed a likely key initiating factor contributing to the development of insulin resistance and vascular complications in diabetes. Consequently, the increased flux of glucose metabolism without a change in the expression and activity of glycolytic enzymes produces a wave of increased glycolytic intermediates driving mitochondrial dysfunction and increased reactive oxygen species (ROS) formation, the activation of hexosamine and protein kinase C pathways, the increased formation of methylglyoxal-producing dicarbonyl stress, and the activation of the unfolded protein response. This is called HK2-linked glycolytic overload and unscheduled glycolysis. The conditions required to sustain this are GLUT1 and/or GLUT3 glucose uptake and the expression of HK2. A metabolic biomarker of its occurrence is the abnormally increased deposition of glycogen, which is produced by metabolic channeling when HK2 becomes detached from mitochondria. These conditions and metabolic consequences are found in the vasculature, kidneys, retina, peripheral nerves, and early-stage embryo development in diabetes and likely sustain the development of diabetic vascular complications and embryopathy. In insulin resistance, HK2-linked unscheduled glycolysis may also be established in skeletal muscle and adipose tissue. This may explain the increased glucose disposal by skeletal uptake in the fasting phase in patients with type 2 diabetes mellitus, compared to healthy controls, and the presence of insulin resistance in patients with type 1 diabetes mellitus. Importantly, glyoxalase 1 inducer—trans-resveratrol and hesperetin in combination (tRES-HESP)—corrected HK2-linked glycolytic overload and unscheduled glycolysis and reversed insulin resistance and improved vascular inflammation in overweight and obese subjects in clinical trial. Further studies are now required to evaluate tRES-HESP for the prevention and reversal of early-stage type 2 diabetes and for the treatment of the vascular complications of diabetes.

Quercetin Attenuated Myeloperoxidase-Dependent HOCl Generation and Endothelial Dysfunction in Diabetic Vasculature

Myeloperoxidase (MPO)-dependent hypochlorous acid (HOCl) generation plays crucial roles in diabetic vascular complications. As a natural polyphenol, quercetin has antioxidant properties in various diabetic models. Herein, we investigated the therapeutic mechanism for quercetin on MPO-mediated HOCl generation and endothelial dysfunction in diabetic vasculature. In vitro, the presence of MPO could amplify high glucose-induced endothelial dysfunction which was significantly inhibited by the NADPH oxidase inhibitor, HOCl or H2O2 scavengers, revealing the contribution of MPO/H2O2/HOCl to vascular endothelial injury. Furthermore, quercetin effectively inhibited MPO/high glucose-mediated HOCl generation and cytotoxicity to vascular endothelial cells. The inhibitive effect on MPO activity was related to the fact that quercetin reduced high glucose-induced H2O2 generation in endothelial cells and directly acted as a competitive substrate for MPO, thus limiting MPO/H2O2-dependent HOCl production. Moreover, quercetin could attenuate HOCl-caused endothelial dysfunction in endothelial cells and isolated aortas. In vivo, dietary quercetin significantly inhibited aortic endothelial dysfunction in diabetic mice, while this compound simultaneously suppressed vascular MPO expression and activity. Therefore, it was demonstrated herein that quercetin inhibited endothelial injury in diabetic vasculature via suppression of MPO/high glucose-dependent HOCl formation.

MiR145 Regulates the Proliferation and Apoptosis of Rat Vascular Endothelial Cells under Hyperglycemia by Targeting the ANGPT2 Gene and Involving the NFκB Signaling Pathway.

PurposeA majority of diabetes mellitus patients with disturbances of glucose metabolism present with vascular complications. This study aimed to explore regulatory mechanisms of miR145 and its potential target gene ANGPT2 on diabetic vasculopathy under hyperglycemia.MethodsBased on the fact that miR145 is detected in rat aortic endothelial cells (RAECs) under hyperglycemia, RAECs were transfected with miR145 mimics/inhibitor for further confirmation. RAEC proliferation was detected with CCK8 assays, and cell apoptosis and CD34+-cell population with annexinV–PI staining and anti-CD34FITC on flow cytometry, respectively. Then, qPCR and Western blot were applied to detect mRNA and protein expression of ANGPT2 and involved pathway factor NFκB p65. Subsequently, dual luciferase–reporter gene analysis was utilized to verify whether miR145 acted directly upon the 3ʹUTR of ANGPT2 mRNA.ResultsThe ANGPT2 gene was confirmed to be a direct target of miR145. miR145 mimics markedly downregulated the expression of ANGPT2 and NFκB p65, boosted the percentage of the CD34+ phenotype, and promoted proliferation and suppressed apoptosis of RAECs under hyperglycemia.ConclusionmiR145 might regulate the viability of RAECs via targeting ANGPT2 and involving NFκB signaling to exert a protective effect on diabetic vasculature.

Role of peroxisome proliferators-activated receptor-gamma in advanced glycation end product-mediated functional loss of voltage-gated potassium channel in rat coronary arteries

Abstract Background Voltage-gated K+ (Kv) channels in coronary artery smooth muscle cells (CSMCs), especially the major specific Kv1 subfamily, contribute to coronary artery vasodilation. Advanced glycation end products (AGEs) have been strongly implicated in diabetes-related cardiovascular complications. Our previous study showed AGEs can impair Kv channel-mediated coronary vasodilation by reducing Kv channel activity. However, its underlying mechanism remains unclear. Purpose Here, we used isolated rat small coronary arteries (RSCAs) and primary CSMCs to investigate the effect of AGEs on Kv channel-mediated coronary vasodilation and the possible involvement of peroxisome proliferators-activated receptor (PPAR)-γ pathway. Methods RSCAs and primary CSMCs were isolated, cultured and treated with bovine serum albumin (BSA), AGE-BSA, alagrebrium (ALA, AGE cross-linking breaker), pioglitazone (PIO) and/or GW9662, and then divided into the following groups: DMEM, BSA, AGE, AGE+ALA, AGE+PIO, and AGE+PIO+GW9662. Kv channel-mediated coronary vasodilation was analyzed using wire myograph. Histology and immunohistochemistry of RSCAs were performed. Western blot was used to detect the protein expression of RAGE, the major Kv1 channel subunits expressed in CSMCs (Kv1.2/1.5), PPAR-γ, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2). Results AGEs markedly reduced forskolin-induced Kv channel-mediated vasodilation of RSCAs by interacting with the receptor for AGEs (RAGE), and ALA or PIO significantly reversed this effect. In both RSCAs and primary CSMCs, AGEs decreased Kv1.2 and Kv1.5 channel protein expression, inhibited PPAR-γ expression, increased RAGE and NOX-2 expression. Treatment with ALA or PIO partially reversed the effects of AGEs on Kv1.2/Kv1.5 expression, accompanied by elevation of PPAR-γ level and diminished oxidative stress. Conclusion AGE/RAGE axis-induced inhibition of PPAR-γ pathway and enhancement of oxidative stress may contribute to AGEs-mediated Kv channel dysfunction and coronary vasodilation in RSCAs. Our results may provide new insights into developing therapeutic strategies to manage diabetic vasculature. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Natural Science Foundation of China; Natural Science Foundation of Beijing (7172059)

Carvacrol may alleviate vascular inflammation in diabetic db/db mice.

Type 2 diabetes mellitus (T2DM) is associated with chronic low-grade inflammation. Carvacrol has been confirmed to possess anti-inflammatory properties, but its effect on diabetic vasculature remains unknown. The aim of the present study was to investigate the possible protective effects of carvacrol against vascular endothelial inflammation. The mice were divided into four groups (n=15 per group) as follows: Non-diabetic control mice, db/db mice, db/db mice + carvacrol (low) and db/db mice + carvacrol (high) groups. The effects of carvacrol on the pathomorphism of the thoracoab-dominal aorta in db/db mice were evaluated using hematoxylin and eosin and Masson's trichrome staining. The serum levels of insulin signaling molecules, such as phosphorylated insulin receptor, phosphorylated insulin receptor substrate-1, insulin, triglyceride (TG) and inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-8] were measured by ELISA. Furthermore, the protein levels of the toll-like receptor (TLR)4/nuclear factor (NF)-κB inflammatory signaling pathway molecules were investigated in the thoracoabdominal aorta of db/db mice and in high glucose-induced endothelial cells. Vascular endothelial cell apoptosis and viability were assessed by using flow cytometry and Cell Counting Kit-8 assays, respectively. The results demonstrated that carva-crol alleviated vascular endothelial cell injury. Carvacrol reduced the expression levels of insulin signaling molecules, insulin, TG and inflammatory cytokines in the serum of db/db mice. Moreover, carvacrol reduced the activation of the TLR4/NF-κB signaling pathway in vivo and in vitro. In vitro, carvacrol inhibited high glucose-induced endothelial cell function by promoting vascular endothelial cell apoptosis and suppressing cell viability. These findings demonstrated that carvacrol could alleviate endothelial dysfunction and vascular inflammation in T2DM.

Diabetes Attenuates the Increase in Estrogen‐Mediated Endothelial Function

BackgroundApparently healthy premenopausal women are protected against cardiovascular disease (CVD) compared to men; and estrogen has been thought to play a key role. In the presence of diabetes; however, this overall sex‐dependent cardiovascular protection is lost. The increase in vascular function throughout the menstrual cycle in healthy premenopausal women has been established. The present investigation sought to test the hypothesis that the increase in vascular function from menses to the late follicular phase of the menstrual cycle is lost in premenopausal women with type 1 diabetes (T1D).MethodsPremenopausal women with a clinical diagnosis of T1D (n=22, HbA1c=8.2±1.7%, age=26±8 years, BMI=26.9±5.4 kg/m2) and seemingly healthy, age‐ and sex‐matched controls (n=10, HbA1c=5.2±0.3%, age=26±9 years, BMI=23.3±4.1 kg/m2) were recruited to participate. Flow mediated dilation (FMD), a measure of endothelial function, was assessed during the menses (cycle day=4±1) and late follicular phases (cycle day=13±1) of the menstrual cycle to represent periods of low and high concentrations of estrogen, respectively. Visit 1 (menses) was confirmed by a positive hemolytic urological multistix test and visit 2 (late follicular) was scheduled based on indications from an ovulation prediction kit. A small venous blood sample was collected at each visit to measure concentrations of estrogen. Endothelial independent vasodilation was determined by administration of sublingual nitroglycerin after each FMD test.ResultsThe increase in estrogen from menses to the follicular phase was similar (p=0.73) in both groups (Δ estrogen: T1D=78.3±92.6 vs Controls=85.6±30.0 pg/mL). Baseline brachial artery diameter was similar between T1D and controls (p=0.25) and was not different between visit 1 and visit 2 (p=0.76). Fasting blood glucose was higher (p<0.01) in women with T1D compared to controls at both visits; however, the change within groups was similar (p=0.49) across time. A near significant interaction (p=0.07) for endothelial function was observed such that FMD increased from the menses to follicular phase in controls (ΔFMD=1.151±1.603%), whereas no change was observed in patients with T1D (ΔFMD=−0.633±2.126%). Endothelial independent vasodilation was not different between groups (p=0.82). Additionally, the endothelial independent response was similar across the menstrual cycle (p=0.97).ConclusionThese data suggest that the presence of diabetes may attenuate the beneficial effects of estrogen on endothelial function in premenopausal women. The loss of estrogen’s cardiovascular protective effect on the vasculature in diabetes may explain the increased risk of CVD observed in women with T1D.Support or Funding Information1RO1HL137087‐01A1.

Supplementation of dietary nitrate attenuated oxidative stress and endothelial dysfunction in diabetic vasculature through inhibition of NADPH oxidase

The metabolic disorders in diabetes, which are usually accompanied by oxidative stress and impaired nitric oxide (NO) bioavailability, increase the risk of detrimental cardiovascular complications. Herein, we investigated the therapeutic potential of dietary nitrate, which is found in high content in green leafy vegetables, on vascular oxidative stress and endothelial dysfunction in diabetic mice induced by high-fat diet and streptozotocin injection. Dietary nitrate in drinking water fuelled a nitrate–nitrite–NO pathway, which inhibited vascular oxidative stress, endothelial dysfunction and many features of metabolic syndrome in diabetic mice. These beneficial effects of nitrate on diabetic mice were abolished by PTIO (NO scavenger) treatment and significantly prevented by febuxostat (xanthine oxidoreductase inhibitor), demonstrating the central importance of NO in bioactivation of nitrate. The favorable effects of nitrate were not further influenced by apocynin (NADPH oxidase inhibitor), suggesting NADPH oxidase as a possible target. In high glucose-incubated vascular endothelial cells, NO donor attenuated oxidative stress and endothelial dysfunction via the inhibition of NADPH oxidase, where a heme oxygenase-1 (HO-1)-dependent mechanism was demonstrated for the antioxidant abilities of NO. Altogether, boosting this nitrate–nitrite–NO signaling pathway resulted in the decreases of NADPH oxidase-derived oxidative stress, endothelial dysfunction and metabolic disorders in diabetic vasculature. These findings may have novel implications for the preventive strategy against diabetes-induced vascular dysfunction and associated complications.

Regulatory Effects of O-GlcNAcylation in Vascular Smooth Muscle Cells on Diabetic Vasculopathy

Vascular complications from uncontrolled hyperglycemia are the leading cause of death in patients with diabetes mellitus. Previous reports have shown a strong correlation between hyperglycemia and vascular calcification, which increases mortality and morbidity in individuals with diabetes. However, the precise underlying molecular mechanisms of hyperglycemia-induced vascular calcification remain largely unknown. Transdifferentiation of vascular smooth muscle cells (VSMC) into osteoblast-like cells is a known culprit underlying the development of vascular calcification in the diabetic vasculature. Pathological conditions such as high glucose levels and oxidative stress are linked to enhanced osteogenic differentiation of VSMC both in vivo and in vitro. It has been demonstrated that increased expression of runt-related transcription factor 2 (Runx2), a bone-related transcription factor, in VSMC is necessary and sufficient for the induction of VSMC calcification. Addition of a single O-linked β-N-acetylglucosamine (O-GlcNAc) moiety to the serine/threonine residues of target proteins (O-GlcNAcylation) has been observed in the arteries of diabetic patients, as well as in animal models in association with the enhanced expression of Runx2 and aggravated vascular calcification. O-GlcNAcylation is a dynamic and tightly regulated process, that is mediated by 2 enzymes, O-GlcNAc transferase and O-GlcNAcase. Glucose is metabolized into UDP-β-D-N-acetylglucosamine, an active sugar donor of O-GlcNAcylation via the hexosamine biosynthetic pathway. Overall increases in the O-GlcNAcylation of cellular proteins have been closely associated with cardiovascular complications of diabetes. In this review, the authors provide molecular insights into cardiovascular complications, including diabetic vasculopathy, that feature increased O-GlcNAcylation in people with diabetes.

Overexpression of Circulating Soluble Nogo-B Improves Diabetic Kidney Disease by Protecting the Vasculature.

Damage to the vasculature is the primary mechanism driving chronic diabetic microvascular complications such as diabetic nephropathy, which manifests as albuminuria. Therefore, treatments that protect the diabetic vasculature have significant therapeutic potential. Soluble neurite outgrowth inhibitor-B (sNogo-B) is a circulating N-terminus isoform of full-length Nogo-B, which plays a key role in vascular remodeling following injury. However, there is currently no information on the role of sNogo-B in the context of diabetic nephropathy. We demonstrate that overexpression of sNogo-B in the circulation ameliorates diabetic kidney disease by reducing albuminuria, hyperfiltration, and abnormal angiogenesis and protecting glomerular capillary structure. Systemic sNogo-B overexpression in diabetic mice also associates with dampening vascular endothelial growth factor-A signaling and reducing endothelial nitric oxide synthase, AKT, and GSK3β phosphorylation. Furthermore, sNogo-B prevented the impairment of tube formation, which occurred when human endothelial cells were exposed to sera from patients with diabetic kidney disease. Collectively, these studies provide the first evidence that sNogo-B protects the vasculature in diabetes and may represent a novel therapeutic target for diabetic vascular complications.

1392-P: The Potential Role of Placental α2-Adrenergic Receptors in the High Risk of Preeclampsia in Diabetes

Preeclampsia is a life-threatening complication of pregnancy that occurs four times more frequently in the presence of maternal diabetes. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt1), released from the placenta, plays a crucial role in PE development. Alpha 2-adrenoceptor (A2AR) dysfunction has been reported in diabetes, and is implicated in the development of vascular complications. Furthermore, A2AR agonist antihypertensive agents are frequently used to treat patients with preeclampsia, whether they have a disease-modifying role is unclear. We hypothesized that A2AR dysfunction could underlie up-regulation of placental sFlt1 expression, thus ‘driving’ preeclampsia in diabetes. We treated cultured human trophoblasts (HTR8/SVneo) with a stressor often present in the diabetic vasculature, ‘heavily oxidized, glycated’ low-density lipoproteins (HOG-LDL), vs. native LDL (at 100 µg protein/ml) with or without the A2AR agonist, clonidine, and antagonist, yohimbine (24h; both drugs at 10 μM and 100 μM). We determined α2A-, α2B- and α2C-AR mRNA expression (RT-PCR) and immunostaining; sFlt1 mRNA expression and protein release (ELISA); and cell viability (Cell Counting Kit-8 assay). Experiments were repeated three times independently. We found that all three A2AR subtypes were expressed in human trophoblasts. HOG- vs. N-LDL increased sFlt1 mRNA expression (2.0-fold, p<0.001) and protein release (2.5-fold, p<0.01), but neither clonidine nor yohimbine had any effect at either dose. Neither HOG- nor N-LDL altered A2AR expression or cell viability. Overall, the data confirm that modified lipoproteins may promote PE development in women with diabetes via upregulation and release of sFlt1, but do not provide clear evidence that A2ARs are implicated in this process. Further work will investigate whether A2ARs may be modified by other stressors of diabetes, and whether they may affect non-sFlt1 mechanisms for PE. Disclosure R.P. Chow: None. J. Zhao: None. T. Curtis: None. T. Lyons: None. J. Yu: None.

Circulating metabolites of strawberry mediate reductions in vascular inflammation and endothelial dysfunction in db/db mice

BackgroundCardiovascular disease is 2-4-fold more prevalent in patients with diabetes. Human studies support the cardiovascular benefits of strawberry consumption but the effects of strawberry on diabetic vasculature are unknown. We tested the hypothesis that dietary strawberry supplementation attenuates vascular inflammation and dysfunction in diabetic mice. MethodsSeven-week-old diabetic db/db mice that consumed standard diet (db/db) or diet supplemented with 2.35% freeze-dried strawberry (db/db + SB) for ten weeks were compared to non-diabetic control mice (db/+). Indices of vascular inflammation and dysfunction were measured. Endothelial cells (ECs) were isolated from the vasculature to determine the influence of strawberry on them. The effect of metabolites of strawberry on endothelial inflammation was determined by incubating mouse aortic ECs (MAECs) with ±5% serum, obtained from strawberry fed mice (metabolites serum) or standard diet fed mice (control serum) ± 25 mM glucose and 100 μM palmitate. Resultsdb/db mice exhibited an increased monocyte binding to vessel, elevated blood pressure, and reduced endothelial-dependent vasorelaxation compared with db/+ mice but each defect was attenuated in db/db + SB mice. The elevation of inflammatory molecules, NOX2 and inhibitor-κB kinase observed in ECs from db/db vs. db/+ mice was suppressed in db/db + SB mice. Glucose and palmitate increased endothelial inflammation in MAECs but were normalized by co-incubation with metabolites serum. ConclusionsDietary supplementation of strawberry attenuates indices of vascular inflammation and dysfunction in diabetic db/db mice. The effect of strawberry on vasculature is endothelial-dependent and possibly mediated through their circulating metabolites. Strawberry might complement conventional therapies to improve vascular complications in diabetics.

Increased global lysine acetylation in diabetic vasculature is associated with downregulation of SIRT‐1 in vascular smooth muscle cells.

Vascular complications are the main problems faced by patients with Type 2 Diabetes Mellitus (T2DM). In addition to endothelial dysfunction, vascular smooth muscle cell (VSMC) dysfunction is postulated to play a crucial role in the development of diabetic vascular complications. Recent studies from our laboratory have demonstrated that acetylation of lysine residues, a posttranslational modification, is increased in vascular tissue exposed to metabolic disorders, such as diabetes. We hypothesized that diabetic conditions would dysregulate lysine deacetylases and acetyltransferases in VSMC, consequently leading to increased global lysine acetylation in diabetic blood vessels. Using male adult Goto‐Kakizaki (GK) rats (16‐weeks‐old) as a model of non‐obese type 2 diabetes and their respective Wistar controls, we found that these diabetic rats exhibited high levels of fasting blood glucose (191 ± 12 vs. 98±7 mg/dL vs. controls, p<0.05), as well as hemoglobin A1C (6.82 ± 0.93 vs. 5.2 ±0.2 % controls, p<0.01). Through direct measurement of blood pressure via right carotid catheterization, we also found greater systolic blood pressure in the GK group (166.57 ±24.27 vs. 124.58 ±17.07 controls, p<0.01). This effect was accompanied by high levels of global lysine acetylation in GK blood vessels (3.3 fold of increase vs. control, p<0.001). To test our hypothesis that VSMC is a major cell type in blood vessels contributing to increased vascular lysine acetylation in diabetic conditions, we utilized isolated aortic VSMC from GK rats in primary cultures. After euthanasia with isoflurane (via nasal 5% in 100% O2), thoracic aortas from GK rats were dissected and VSMC were isolated by an enzymatic digestion method. Phenotypically, VSMC from GK rats exhibited an enhanced proliferative phenotype in comparison to VSMC from Wistar control rats (Figure 1). Strikingly, we found that VSMC from GK rats exhibited a marked decrease in SIRT‐1 deacetylase expression (3.5‐fold decrease vs. control, p<0.05). Moreover, expression of the acetyltransferase PCAF was increased in VSMC from GK rats (1.5‐fold increase vs. control, p<0.05). Taken together, our in vitro results suggest that downregulation of SIRT‐1 and upregulation of PCAF may mediate the increased levels of global lysine acetylation in diabetic blood vessels, confirming our hypothesis. To further this investigation, we will identify the proteins that are specifically acetylated in diabetic VSMC through mass spectrometry analysis, as well as the effects on global lysine acetylation when overexpressing SIRT‐1 in VSMC.Support or Funding InformationIn‐House Grant, NYITThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.