Diabetes-associated Vascular Disease Research Articles

Abstract 13700: LncRNA MERRICAL Controls Macrophage Chemotaxis and Vascular Inflammation by In-cis Regulation of CCL3/4 via H3k4me3 in Diabetes-Associated Atherosclerosis

Introduction: Diabetes-associated atherosclerosis is characterized by excessive vascular wall inflammation, including impaired lipid deposition, increased immune cell infiltration and plaque formation in the vessel wall. Macrophages are instrumental for lesion formation, however the mechanisms underlying their impaired functional capacity remain poorly understood in diabetes. Herein, we identify a unique role of a macrophage-enriched lncRNA MERRICAL (Macrophage Enriched lncRNA Regulates Inflammation, Chemotaxis, and AtheroscLerosis) in the development of diabetes-associated atherosclerosis. Methods: Transcriptomic analysis of the aortic intima from Ldlr -/- mice was performed at 4 time points along the continuum of lesion progression and regression under a high fat, high sucrose-containing (HFSC) or chow diet, respectively, to identify differentially expressed lncRNAs. Gain- and loss-of-function cell-based assays, RNA-protein interactions, and in vivo knockdown were performed to uncover the role of a top lncRNA candidate. Results: LncRNA MERRICAL is nuclear localized and increased by 249% in intimal lesions during progression. Transcriptomic analysis of MERRICAL -deficient bone marrow-derived macrophages showed decreased chemokine and inflammatory gene expression for Ccl3, Ccl4, and Il1β, with reduced chemotaxis and inflammatory-associated gene ontology pathways. Gain- and loss-of-function studies demonstrated the role of lncRNA MERRICAL in cis -regulation of its neighboring genes Ccl3 and Ccl4, an effect that is mediated in part via interaction with the histone modification enzyme H3K4me3. Remarkably, systemic knockdown of MERRICAL in HFSC-fed mice strongly reduced lesion formation by 73% in the aortic sinus by inhibiting the pro-inflammatory and Ccl3 - and Ccl4 -associated chemotactic response and decreasing the recruitment of leukocytes into the aortic wall. Conclusions: These findings establish a cis -regulatory mechanism by which a macrophage-specific lncRNA, MERRICAL , potently inhibits pro-inflammatory and chemotactic responses to alleviate lesion progression in diabetes, with implications for a broad range of diabetes-associated vascular disease states.

Hypoglycemia, Vascular Disease and Cognitive Dysfunction in Diabetes: Insights from Text Mining-Based Reconstruction and Bioinformatics Analysis of the Gene Networks.

Hypoglycemia has been recognized as a risk factor for diabetic vascular complications and cognitive decline, but the molecular mechanisms of the effect of hypoglycemia on target organs are not fully understood. In this work, gene networks of hypoglycemia and cardiovascular disease, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, cognitive decline, and Alzheimer’s disease were reconstructed using ANDSystem, a text-mining-based tool. The gene network of hypoglycemia included 141 genes and 2467 interactions. Enrichment analysis of Gene Ontology (GO) biological processes showed that the regulation of insulin secretion, glucose homeostasis, apoptosis, nitric oxide biosynthesis, and cell signaling are significantly enriched for hypoglycemia. Among the network hubs, INS, IL6, LEP, TNF, IL1B, EGFR, and FOS had the highest betweenness centrality, while GPR142, MBOAT4, SLC5A4, IGFBP6, PPY, G6PC1, SLC2A2, GYS2, GCGR, and AQP7 demonstrated the highest cross-talk specificity. Hypoglycemia-related genes were overrepresented in the gene networks of diabetic complications and comorbidity; moreover, 14 genes were mutual for all studied disorders. Eleven GO biological processes (glucose homeostasis, nitric oxide biosynthesis, smooth muscle cell proliferation, ERK1 and ERK2 cascade, etc.) were overrepresented in all reconstructed networks. The obtained results expand our understanding of the molecular mechanisms underlying the deteriorating effects of hypoglycemia in diabetes-associated vascular disease and cognitive dysfunction.

Diabetic Complications and Oxidative Stress: A 20-Year Voyage Back in Time and Back to the Future.

Twenty years have passed since Brownlee and colleagues proposed a single unifying mechanism for diabetic complications, introducing a turning point in this field of research. For the first time, reactive oxygen species (ROS) were identified as the causal link between hyperglycemia and four seemingly independent pathways that are involved in the pathogenesis of diabetes-associated vascular disease. Before and after this milestone in diabetes research, hundreds of articles describe a role for ROS, but the failure of clinical trials to demonstrate antioxidant benefits and some recent experimental studies showing that ROS are dispensable for the pathogenesis of diabetic complications call for time to reflect. This twenty-year journey focuses on the most relevant literature regarding the main sources of ROS generation in diabetes and their role in the pathogenesis of cell dysfunction and diabetic complications. To identify future research directions, this review discusses the evidence in favor and against oxidative stress as an initial event in the cellular biochemical abnormalities induced by hyperglycemia. It also explores possible alternative mechanisms, including carbonyl stress and the Warburg effect, linking glucose and lipid excess, mitochondrial dysfunction, and the activation of alternative pathways of glucose metabolism leading to vascular cell injury and inflammation.

Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus

BackgroundEndothelial progenitor cell (EPC) dysfunction contributes to vascular disease in diabetes mellitus. However, the molecular mechanism underlying EPC dysfunction and its contribution to delayed reendothelialization in diabetes mellitus remain unclear. Our study aimed to illustrate the potential molecular mechanism underlying diabetic EPC dysfunction in vivo and in vitro. Furthermore, we assessed the effect of EPC transplantation on endothelial regeneration in diabetic rats.MethodsLate outgrowth EPCs were isolated from the bone marrow of rats for in vivo and in vitro studies. In vitro functional assays and Western blotting were conducted to reveal the association between C-X-C chemokine receptor type 7 (CXCR7) expression and diabetic EPC dysfunction. To confirm the association between cellular CXCR7 levels and EPC function, CXCR7 expression in EPCs was upregulated and downregulated via lentiviral transduction and RNA interference, respectively. Western blotting was used to reveal the potential molecular mechanism by which the Stromal-Derived Factor-1 (SDF-1)/CXCR7 axis regulates EPC function. To elucidate the role of the SDF-1/CXCR7 axis in EPC-mediated endothelial regeneration, a carotid artery injury model was established in diabetic rats. After the model was established, saline-treated, diabetic, normal, or CXCR7-primed EPCs were injected via the tail vein.ResultsDiabetic EPC dysfunction was associated with decreased CXCR7 expression. Furthermore, EPC dysfunction was mimicked by knockdown of CXCR7 in normal EPCs. However, upregulating CXCR7 expression reversed the dysfunction of diabetic EPCs. The SDF-1/CXCR7 axis positively regulated EPC function by activating the AKT-associated Kelch-like ECH-associated protein 1 (keap-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis, which was reversed by blockade of AKT and Nrf2. Transplantation of CXCR7-EPCs accelerated endothelial repair and attenuated neointimal hyperplasia in diabetes mellitus more significantly than transplantation of diabetic or normal EPCs. However, the therapeutic effect of CXCR7-EPC transplantation on endothelial regeneration was reversed by knockdown of Nrf2 expression.ConclusionsDysfunction of diabetic EPCs is associated with decreased CXCR7 expression. Furthermore, the SDF-1/CXCR7 axis positively regulates EPC function by activating the AKT/keap-1/Nrf2 axis. CXCR7-primed EPCs might be useful for endothelial regeneration in diabetes-associated vascular disease.

Specific NLRP3 Inhibition Protects Against Diabetes-Associated Atherosclerosis.

Low-grade persistent inflammation is a feature of diabetes-driven vascular complications, in particular activation of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome to trigger the maturation and release of the inflammatory cytokine interleukin-1β (IL-1β). We investigated whether inhibiting the NLRP3 inflammasome, through the use of the specific small-molecule NLRP3 inhibitor MCC950, could reduce inflammation, improve vascular function, and protect against diabetes-associated atherosclerosis in the streptozotocin-induced diabetic apolipoprotein E-knockout mouse. Diabetes led to an approximately fourfold increase in atherosclerotic lesions throughout the aorta, which were significantly attenuated with MCC950 (P < 0.001). This reduction in lesions was associated with decreased monocyte-macrophage content, reduced necrotic core, attenuated inflammatory gene expression (IL-1β, tumor necrosis factor-α, intracellular adhesion molecule 1, and MCP-1; P < 0.05), and reduced oxidative stress, while maintaining fibrous cap thickness. Additionally, vascular function was improved in diabetic vessels of mice treated with MCC950 (P < 0.05). In a range of cell lines (murine bone marrow-derived macrophages, human monocytic THP-1 cells, phorbol 12-myristate 13-acetate-differentiated human macrophages, and aortic smooth muscle cells from humans with diabetes), MCC950 significantly reduced IL-1β and/or caspase-1 secretion and attenuated leukocyte-smooth muscle cell interactions under high glucose or lipopolysaccharide conditions. In summary, MCC950 reduces plaque development, promotes plaque stability, and improves vascular function, suggesting that targeting NLRP3-mediated inflammation is a novel therapeutic strategy to improve diabetes-associated vascular disease.

Analysis of long non-coding RNA expression profiles in high-glucose treated vascular endothelial cells

BackgroundDiabetes mellitus is often associated with microvascular and macrovascular lesions, and hyperglycemia-induced vascular endothelial cell damage is a key factor.MethodsWe investigated long non-coding RNAs (lncRNAs) and mRNAs that are affected by hyperglycemia-induced damage using human umbilical vein endothelial cells (HUVECs) as a model. HUVECs were cultured under high (25 mmol/L) or normal (5 mmol/L) glucose conditions for 6 d, and then lncRNAs and protein-coding transcripts were profiled by RNA-seq.ResultAmong 40,379 lncRNAs screened, 214 were upregulated (log2 [fold-change] > 1, FDR < 0.05) and 197 were downregulated (log2 [fold-change] < − 1, FDR < 0.05) in response to high-glucose. Furthermore, among 28,431 protein-coding genes screened, 778 were upregulated and 998 were downregulated. A total of 945 lncRNA/mRNA pairs were identified, including 126 differentially expressed lncRNAs predicted to target 201 mRNAs, among which 26 were cis-regulatory interactions. The corresponding lncRNA-mRNA network was composed of 354 lncRNA nodes, 1167 mRNA nodes and 9735 edges. Dozens of lncRNAs with high degree may play important roles in high-glucose-induced HUVEC damage, including ENST00000600527, NONHSAT037576.2, NONHSAT135706.2, ENST00000602127, NONHSAT200243.1, NONHSAT217282.1, NONHSAT176260.1, NONHSAT199075.1, NONHSAT067063.2, NONHSAT058417.2.ConclusionThese observations may provide novel insights into the regulatory molecules and pathways of hyperglycemia-related endothelial dysfunction in diabetes-associated vascular disease.

Why the endothelium? The endothelium as a target to reduce diabetes-associated vascular disease.

Over the past 66 years, our knowledge of the role of the endothelium in the regulation of cardiovascular function and dysfunction has advanced from the assumption that it is a single layer of cells that serves as a barrier between the blood stream and vascular smooth muscle to an understanding of its role as an essential endocrine-like organ. In terms of historical contributions, we pay particular credit to (1) the Canadian scientist Dr. Rudolf Altschul who, based on pathological changes in the appearance of the endothelium, advanced the argument in 1954 that "one is only as old as one's endothelium" and (2) the American scientist Dr. Robert Furchgott, a 1998 Nobel Prize winner in Physiology or Medicine, who identified the importance of the endothelium in the regulation of blood flow. This review provides a brief history of how our knowledge of endothelial function has advanced and now recognize that the endothelium produces a plethora of signaling molecules possessing paracrine, autocrine, and, arguably, systemic hormone functions. In addition, the endothelium is a therapeutic target for the anti-diabetic drugs metformin, glucagon-like peptide I (GLP-1) receptor agonists, and inhibitors of the sodium-glucose cotransporter 2 (SGLT2) that offset the vascular disease associated with diabetes.

The protective effects of orexin a against high glucose-induced activation of NLRP3 inflammasome in human vascular endothelial cells

Vascular disease is one of the most significant threats to the lives of patients suffering from diabetes, and chronic exposure of vascular endothelial cells to high glucose has been shown to significantly contribute to the process of endothelial cell dysfunction, one of the earliest events in diabetes-associated vascular disease. Nucleotide oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in initiating the inflammatory process by facilitating the production of interleukin-1β (IL-1β) and IL-18. ASC and caspase 1 are also implicated in NLRP3 inflammasome-mediated chronic inflammation. While under normal conditions, a balance exists between oxidants and antioxidants, exposure to high glucose significantly increases the production of ROS, which is enhanced by NOX4 expression. In the present study, we explored the role of orexin A, an endogenous peptide produced in the hypothalamus, in high glucose-induced activation of the NLRP3 inflammasome, oxidative stress, and expression of several key cytokines. Our findings demonstrate that orexin A exerts potent antioxidant effects in human aortic endothelial cells exposed to high glucose by inhibiting mitochondrial ROS and expression of NOX4 at both the mRNA and protein levels as revealed by MitoSOX staining, real-time PCR, and Western blot analysis. We also show that orexin A inhibits high glucose-induced expression of TxNIP, which is crucial to the activation of the NLRP3 inflammasome, as well as that of HMGB1. We confirmed via real-time PCR and Western blot analysis that orexin A suppressed the production of the inflammatory cytokines IL-1β and IL-18. Additionally, through SIRT1 knockdown siRNA experimentation, we confirmed that SIRT1 knockdown abolishes the effects of orexin A described above, thereby indicating a critical role of SIRT in the capacity of orexin A to ameliorate high glucose-induced oxidative stress and activation of NLRP3 inflammasome.

Canagliflozin Inhibits Human Endothelial Cell Proliferation and Tube Formation.

Recent clinical trials revealed that sodium-glucose co-transporter 2 (SGLT2) inhibitors significantly reduce cardiovascular events in type 2 diabetic patients, however, canagliflozin increased limb amputations, an effect not seen with other SGLT2 inhibitors. Since endothelial cell (EC) dysfunction promotes diabetes-associated vascular disease and limb ischemia, we hypothesized that canagliflozin, but not other SGLT2 inhibitors, impairs EC proliferation, migration, and angiogenesis. Treatment of human umbilical vein ECs (HUVECs) with clinically relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin, inhibited cell proliferation. In particular, 10 μM canagliflozin reduced EC proliferation by approximately 45%. The inhibition of EC growth by canagliflozin occurred in the absence of cell death and was associated with diminished DNA synthesis, cell cycle arrest, and a striking decrease in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer partially rescued the proliferative response of HUVECs treated with canagliflozin. A high concentration of canagliflozin (50 μM) modestly inhibited HUVEC migration by 20%, but markedly attenuated their tube formation by 65% and EC sprouting from mouse aortas by 80%. A moderate 20% reduction in HUVEC migration was also observed with a high concentration of empagliflozin (50 μM), while neither empagliflozin nor dapagliflozin affected tube formation by HUVECs. The present study identified canagliflozin as a robust inhibitor of human EC proliferation and tube formation. The anti-proliferative action of canagliflozin occurs in the absence of cell death and is due, in part, to the blockade of cyclin A expression. Notably, these actions are not seen with empagliflozin or dapagliflozin. The ability of canagliflozin to exert these pleiotropic effects on ECs may contribute to the clinical actions of this drug.

Canagliflozin Inhibits Human Endothelial Cell Inflammation via the Induction of Heme Oxygenase-1

Vascular disease is the major cause of mortality in diabetes. Although many factors contribute to diabetes-associated vascular disease, inflammation of endothelial cells (ECs) plays a critical role. Sodium-glucose transporter 2 (SGLT2) inhibitors are recently approved anti-hyperglycemic drugs that cause a significant reduction in cardiovascular events in type 2 diabetic patients, but the mechanisms underlying this protection are not fully known. In the present study, we investigated the effect of SGLT2 inhibitors on the expression of the vasoprotective protein heme oxygenase-1 (HO-1) in human umbilical vein ECs. Treatment of ECs with canagliflozin, dapagliflozin, or empagliflozin stimulated the expression of HO-1 mRNA and protein; however, only clinically achievable concentrations of canagliflozin (3-10µM) increased HO-1 levels. The induction of HO-1 by canagliflozin was associated with an increase in Nrf2 activity and reactive oxygen species (ROS) formation. Canagliflozin also stimulated HO-1 promoter activity, and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the canagliflozin-mediated induction of HO-1 was abolished by N-acetyl-L-cysteine or rotenone. Significantly, prior treatment of ECs with canagliflozin inhibited the induction of the adhesion molecules ICAM-1 and VCAM-1 in response to high concentrations of glucose (25mM) and oxidized low-density lipoprotein (50mg/L) that mimic the diabetic milieu. Significantly, the anti-inflammatory action of canagliflozin was abrogated by blocking HO-1 activity or expression. In conclusion, this study demonstrates that canagliflozin stimulates HO-1 gene expression in the vascular endothelium via the ROS-Nrf2 pathway, and that canagliflozin mitigates inflammation in human ECs exposed to a diabetic environment via the induction of HO-1. Thus, canagliflozin may exert some of it cardiovascular benefit in diabetes by targeting HO-1. Disclosure K.J. Peyton: None. G. Behnammanesh: None. W. Durante: None.

232 Endothelial ship2 knockout causes nox2 nadph oxidase-dependent oxidative stress and endothelial dysfunction

IntroductionInsulin-resistant type 2 diabetes mellitus (DM) leads to premature death and disability, primarily as a consequence of accelerated vascular disease. Shc homology 2-containing inositol 5 phosphatase-2 (SHIP2) is a lipid...

The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activator dh404 protects against diabetes-induced endothelial dysfunction

BackgroundVascular dysfunction is a pivotal event in the development of diabetes-associated vascular disease. Increased inflammation and oxidative stress are major contributors to vascular dysfunction. Nrf2, a master regulator of several anti-oxidant genes and a suppressor of inflammatory NF-κB, has potential as a target to combat oxidative stress and inflammation. The aim of this study was to investigate the effects of a novel Nrf2 activator, the bardoxolone methyl derivative dh404, on endothelial function in vitro and in vivo.Methodsdh404 at 3 mg/kg was administered to male Akita mice, an established diabetic mouse model of insulin insufficiency and hyperglycemia, from 6 weeks of age. At 26 weeks of age, vascular reactivity was assessed by wire myography, pro-inflammatory expression was assessed in the aortas by qRT-PCR and immunohistochemistry, and systemic and vascular oxidative stress measurements were determined. Additionally, studies in human aortic endothelial cells (HAECs) derived from normal and diabetic patients in the presence or absence of dh404 included assessment of pro-inflammatory genes by qRT-PCR and western blotting. Oxidative stress was assessed by three methods; L-012, DCFDA and amplex red. Static adhesion assays were performed to determine the leukocyte–endothelial interaction in the presence or absence of dh404.ResultsDh404 significantly attenuated endothelial dysfunction in diabetic Akita mice characterized by reduced contraction in response to phenylephrine and the downregulation of inflammatory genes (VCAM-1, ICAM-1, p65, IL-1β) and pro-oxidant genes (Nox1 and Nox2). Furthermore, reduced systemic and vascular oxidative stress levels were observed in diabetic Akita mice. dh404 exhibited cytoprotective effects in diabetic HAECs in vitro, reflected by significant upregulation of Nrf2-responsive genes, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), reduction of oxidative stress markers (O2·− and H2O2), inhibition of inflammatory genes (VCAM-1 and the p65 subunit of NF-κB) and attenuation of leukocyte–endothelial interactions (P < 0.05 for all in vitro and in vivo parameters; one or two-way ANOVA as appropriate with post hoc testing).ConclusionThese studies demonstrate that upregulation of Nrf2 by dh404 represents a novel therapeutic strategy to limit diabetes-associated vascular injury.

A global profile of glucose-sensitive endothelial-expressed long non-coding RNAs.

Hyperglycemia-related endothelial dysfunction is believed to be the crux of diabetes-associated micro- and macro-vascular complications. We conducted a systematic transcriptional survey to screen for human endothelial long non-coding RNAs (lncRNAs) regulated by elevated glucose levels. lncRNAs and protein-coding transcripts from human umbilical vein endothelial cells (HUVECs) cultured under high (25 mmol/L) or normal (5 mmol/L) glucose conditions for 24 h were profiled with the Arraystar Human LncRNA Expression Microarray V3.0. Of the 30 586 lncRNAs screened, 100 were significantly upregulated and 186 appreciably downregulated (P < 0.05) in response to high-glucose exposure. In the same HUVEC samples, 133 of the 26 109 mRNAs screened were upregulated and 166 downregulated. Of these 299 differentially expressed mRNAs, 26 were significantly associated with 28 differentially expressed long intergenic non-coding RNAs (P < 0.05). Bioinformatics analyses indicated that the mRNAs most upregulated are primarily enriched in axon guidance signaling pathways; those most downregulated are notably involved in pathways targeting vascular smooth muscle cell contraction, dopaminergic signaling, ubiquitin-mediated proteolysis, and adrenergic signaling. This is the first lncRNA and mRNA transcriptome profile of high-glucose-mediated changes in human endothelial cells. These observations may prove novel insights into novel regulatory molecules and pathways of hyperglycemia-related endothelial dysfunction and, accordingly, diabetes-associated vascular disease.

Subcellular proteomic approach for identifying the signaling effectors of protein kinase C-β₂ under high glucose conditions in human umbilical vein endothelial cells.

The high glucose-induced activation of protein kinase C-β2 (PKC-β2) has an essential role in the pathophysiology of diabetes-associated vascular disease. In the present study, human umbilical vein endothelial cells (HUVECs) were cultured in high and normal glucose conditions prior to being infected with a recombinant adenovirus to induce the overexpression of PKC-β2. The activity of PKC-β2 was also decreased using a selective PKC-β2 inhibitor. A series of two-dimensional electrophoresis images detected ~800 spots in the nuclei, and ~600 spots in the cytosol. Following intra- and inter-group cross-matching, 38 significantly altered spots were identified as high glucose-induced and PKC-β2-associated nuclear proteins. In addition to the observation that the regulation of key proteins involved in the nuclear factor (NF)-κB signaling cascade occurred in the cytosol, various transcription factors, including peroxisome proliferator-activated receptor δ (PPAR-δ), were also altered in the nuclei. A human protein-protein interaction network of potential connections of PKC-β2-associated proteins was constructed in the proteomics investigation using Biological General Repository for Interaction Datasets. The results indicated that PKC-β2 may be involved in high glucose-induced glucose and lipid crosstalk by regulating PPAR-δ. In addition, NF-κB inhibitor-interacting Ras-like protein 1 may be important in the PKC-β2-NF-κB inhibitor-NF-κB signaling pathway in HUVECs under high-glucose conditions.

Bilirubin: striking gold in diabetic vasculopathy?

Vascular complications are the primary cause of morbidity and mortality and a major contributor to health care costs in patients with diabetes (1). While the etiology of vascular disease is multifactorial, endothelial dysfunction characterized by a decrease in nitric oxide (NO) synthesis from endothelial NO synthase (eNOS) and blunted endothelium-dependent vasodilation is a seminal feature in diabetes-associated vascular disease. Given the dramatic increase in the incidence of diabetes worldwide, there is an urgent need for new therapies that targets this cellular defect in diabetes. Although long considered a toxic waste product of heme metabolism, the yellow pigment bilirubin is now recognized as an important vasoprotective molecule. Bilirubin is formed by the action of inducible heme oxygenase-1 (HO-1) or constitutively expressed HO-2, which degrades heme into biliverdin, carbon monoxide (CO), and iron (Fig. 1). Biliverdin is rapidly reduced to bilirubin by biliverdin reductase (BVR). Bilirubin is further processed in the liver, where it is conjugated with glucuronic acid by uridine diphosphate-glucuronosyltransferase A1A (UGT1A1) to a water-soluble form for elimination. While early work identified the potent antioxidant properties of bilirubin, more recent studies found that bilirubin elicits anti-inflammatory and antiapoptotic effects in endothelial cells and improves endothelium-dependent vasodilation in murine hypercholesterolemic blood vessels (2–5). Clinical studies also support a protective role, as serum bilirubin concentrations show an inverse relationship with risk for a constellation of cardiovascular disorders (6). Interestingly, serum bilirubin levels are lower …

Apocynin decreases AGEs-induced stimulation of NF-κB protein expression in vascular smooth muscle cells from GK rats

Context: Elevated oxidative stress plays a key role in diabetes-associated vascular disease. Excessive production of reactive oxygen species via advanced glycation end products (AGEs) activates peroxisome proliferator-activated receptor gamma (PPARγ) and the transcription factor nuclear factor-kB (NF-κB) in aortic vascular smooth muscle cells (VSMCs). Apocynin, a drug with an antioxidant effect, has also been proposed as a therapeutic agent for atherosclerotic disease.Objectives: This work investigates the effects of apocynin on the PPARγ and NF-κB protein expression evoked by AGEs in cultured VSMCs from Goto–Kakisaki (GK) rats, a non-obese insulin model of both insulin resistance and type 2 diabetes.Materials and methods: VSMCs, isolated from aortas of GK and non-diabetic rats, were cultured. The expression of proteins was evaluated by Western blot. The blood glucose concentration was measured with a blood glucose test meter. The diabetes of GK rats was controlled by blood glucose and insulin determinations (non-fasting values). The serum insulin concentration was determined by radioimmunoassay.Results: In VSMCs from non-diabetic and GK rats, apocynin (1 and 10 µM) abolished the protein overexpression of NF-κB induced by glycated bovine serum albumin (AGEs-BSA) incubation. However, apocynin (1 and 10 µM) enhanced the expression of PPARγ protein in the presence of AGEs-BSA (100 μg/mL) in VSMCs from non-diabetic, but not from GK rats.Conclusion: These findings suggest that apocynin decreases the incidence of alterations in VSMCs induced by AGEs through the reduction of NF-κB and may represent an attractive therapeutic approach to treat diabetes mellitus.

Insulin Induces Production of New Elastin in Cultures of Human Aortic Smooth Muscle Cells

Diabetes mellitus accelerates atherosclerotic progression, peripheral angiopathy development, and arterial hypertension, all of which are associated with elastic fiber disease. However, the potential mechanistic links between insulin deficiency and impaired elastogenesis in diabetes have not been explored. Results of the present study reveal that insulin administered in therapeutically relevant concentrations (0.5 to 10 nmol/L) selectively stimulates formation of new elastic fibers in cultures of human aortic smooth muscle cells. These concentrations of insulin neither up-regulate collagen type I and fibronectin deposition nor stimulate cellular proliferation. Further, the elastogenic effect of insulin occurs after insulin receptor activation, which triggers the PI3K downstream signaling pathway and activates elastin gene transcription. In addition, the promoter region of the human elastin gene contains the CAAATAA sequence, consistent with the FoxO-recognized element, and the genomic effects of insulin occur after removal of the FoxO1 transcriptional inhibitor from the FoxO-recognized element in the elastin gene promoter. In addition, insulin signaling facilitates the association of tropoelastin with its specific 67-kDa elastin-binding protein/spliced form of β-galactosidase chaperone, enhancing secretion. These results are crucial to understanding of the molecular and cellular mechanisms of diabetes-associated vascular disease, and, in particular, endorse use of insulin therapy for treatment of atherosclerotic lesions in patients with type 1 diabetes, in which induction of new elastic fibers would mechanically stabilize the developing plaques and prevent arterial occlusions.

Diabetes-Related Microvascular and Macrovascular Diseases in the Physical Therapy Setting

Physical therapists commonly treat people with diabetes for a wide variety of diabetes-associated impairments, including those from diabetes-related vascular disease. Diabetes is associated with both microvascular and macrovascular diseases affecting several organs, including muscle, skin, heart, brain, and kidneys. A common etiology links the different types of diabetes-associated vascular disease. Common risk factors for vascular disease in people with diabetes, specifically type 2 diabetes, include hyperglycemia, insulin resistance, dyslipidemia, hypertension, tobacco use, and obesity. Mechanisms for vascular disease in diabetes include the pathologic effects of advanced glycation end product accumulation, impaired vasodilatory response attributable to nitric oxide inhibition, smooth muscle cell dysfunction, overproduction of endothelial growth factors, chronic inflammation, hemodynamic dysregulation, impaired fibrinolytic ability, and enhanced platelet aggregation. It is becoming increasingly important for physical therapists to be aware of diabetes-related vascular complications as more patients present with insulin resistance and diabetes. The opportunities for effective physical therapy interventions (such as exercise) are significant.

Oxidative stress and increased eNOS and NADPH oxidase expression in mouse microvessel endothelial cells

Elevated oxidative stress plays a key role in diabetes-associated vascular disease. In this study, we tested the hypothesis that high glucose-induced oxidative stress was associated with changes in the expression of NADPH oxidase, superoxide dismutase (SOD) and endothelial nitric oxide synthase (eNOS). Oxidative stress was assessed in cell cultures of mouse microvessel endothelial cells (MMECs) by fluorescence labelling with dihydroethidium, lucigenin-enhanced chemiluminescence and determining NADPH oxidase subunit and eNOS expression with real-time polymerase chain reaction protocol and Western blotting. Oxidative stress and expression of the NADPH oxidase subunit, p22phox, were both increased, SOD1 and 3 expression lowered and eNOS significantly elevated in MMECs treated with 40 mM glucose for 72 h compared to low glucose medium. Oxidative stress, p22phox mRNA, eNOS mRNA, and protein were lowered by concurrent incubation with sepiapterin. When eNOS protein expression in endothelial cells was significantly decreased by eNOS siRNA treatment, superoxide generation was significantly higher in the MMECs grown in low glucose, but reduced in those grown in high glucose for 72 h. Thus, exposure of MMECs to high glucose results in increased oxidative stress that is associated with increased eNOS and NADPH oxidase subunit expression, notably p22phox, and decreased expression of SOD1 and 3.

Increased oxidative stress in the streptozotocin-induced diabetic apoE-deficient mouse: Changes in expression of NADPH oxidase subunits and eNOS

Elevated oxidative stress plays a key role in the development of atherosclerosis and endothelial dysfunction in diabetes-associated vascular disease. Glucose-induced changes in the activity of NADPH oxidase and endothelial nitric oxide synthase (eNOS) may result in vascular endothelial cell dysfunction via dysregulation of eNOS and/or changes in the expression of the subunits of NADPH oxidase. In this study, we have investigated whether changes in the expression of the subunits of NADPH oxidase, or eNOS mRNA, can be associated with oxidative stress in the streptozotocin-induced type 1 diabetic apolipoprotein E-deficient (apoE −/−) diabetic mouse. Oxidative stress was assessed in aorta and mesenteric arteries by immunofluorescence labelling with dihydroethidium and levels of NADPH oxidase subunits and eNOS were determined by a real-time polymerase chain reaction protocol. Blood glucose levels and oxidative stress were significantly increased following 4, 8 and 16 weeks after treatment with streptozotocin in both streptozotocin-apoE −/− aorta and mesenteric arteries compared to the time- and age-matched vehicle (citrate buffer)-treated non-diabetic apoE −/−. In the mesenteric arteries the expression of nox4 (4 weeks) and gp91phox (nox2) (8 weeks) subunits of NADPH oxidase from streptozotocin-apoE −/− were enhanced as were eNOS mRNA and protein ( P < 0.05). However, only eNOS mRNA and protein remained increased at 16 weeks. These data indicate that increased oxidative stress in the vasculature of streptozotocin-apoE −/− mice is linked to changes in eNOS, superoxide dismutase (SOD) and NADPH oxidase expression.