Diabetic Endothelial Cell Dysfunction Research Articles

AMPK-Dependent YAP Inhibition Mediates the Protective Effect of Metformin against Obesity-Associated Endothelial Dysfunction and Inflammation.

Hyperglycemia is a crucial risk factor for cardiovascular diseases. Chronic inflammation is a central characteristic of obesity, leading to many of its complications. Recent studies have shown that high glucose activates Yes-associated protein 1 (YAP) by suppressing AMPK activity in breast cancer cells. Metformin is a commonly prescribed anti-diabetic drug best known for its AMPK-activating effect. However, the role of YAP in the vasoprotective effect of metformin in diabetic endothelial cell dysfunction is still unknown. The present study aimed to investigate whether YAP activation plays a role in obesity-associated endothelial dysfunction and inflammation and examine whether the vasoprotective effect of metformin is related to YAP inhibition. Reanalysis of the clinical sequencing data revealed YAP signaling, and the YAP target genes CTGF and CYR61 were upregulated in aortic endothelial cells and retinal fibrovascular membranes from diabetic patients. YAP overexpression impaired endothelium-dependent relaxations (EDRs) in isolated mouse aortas and increased the expression of YAP target genes and inflammatory markers in human umbilical vein endothelial cells (HUVECs). High glucose-activated YAP in HUVECs and aortas was accompanied by increased production of oxygen-reactive species. AMPK inhibition was found to induce YAP activation, resulting in increased JNK activity. Metformin activated AMPK and promoted YAP phosphorylation, ultimately improving EDRs and suppressing the JNK activity. Targeting the AMPK-YAP-JNK axis could become a therapeutic strategy for alleviating vascular dysfunction in obesity and diabetes.

Comprehensive analysis of m6A modification lncRNAs in high glucose and TNF-α induced human umbilical vein endothelial cells.

N6-methyladenosine (m6A) RNA methylation, as a reversible epigenetic modification of mammalian mRNA, holds a critical role in multiple biological processes. m6A modification in Long non-coding RNAs (lncRNAs) has increasingly attracted more attention in recent years, especially in diabetics, with or without metabolic syndrome. We investigated via m6A-sequencing and RNA-sequencing the differentially expressed m6A modification lncRNAs by high glucose and TNF-α induced endothelial cell dysfunction in human umbilical vein endothelial cells. Additionally, gene ontology and kyoto encyclopedia of genes and genomes analyses were performed to analyze the biological functions and pathways for the target of mRNAs. Lastly, a competing endogenous RNA network was established to further reveal a regulatory relationship between lncRNAs, miRNAs and mRNAs. A total of 754 differentially m6A-methylated lncRNAs were identified, including 168 up-regulated lncRNAs and 266 down-regulated lncRNAs. Then, 119 significantly different lncRNAs were screened out, of which 60 hypermethylated lncRNAs and 59 hypomethylated lncRNAs. Moreover, 122 differentially expressed lncRNAs were filtered, containing 14 up-regulated mRNAs and 18 down-regulated lncRNAs. Gene ontology and kyoto encyclopedia of genes and genomes analyses analyses revealed these targets were mainly associated with metabolic process, HIF-1 signaling pathway, and other biological processes. The competing endogenous RNA network revealed the regulatory relationship between lncRNAs, miRNAs and mRNAs, providing potential targets for the treatment and prevention of diabetic endothelial cell dysfunction. This comprehensive analysis for lncRNAs m6A modification in high glucose and TNF-α-induced human umbilical vein endothelial cells not only demonstrated the understanding of characteristics of endothelial cell dysfunction, but also provided the new targets for the clinical treatment of diabetes. Private information from individuals will not be published. This systematic review also does not involve endangering participant rights. Ethical approval will not be required. The results may be published in a peer-reviewed journal or disseminated at relevant conferences.

BASP1 promotes high glucose-induced endothelial apoptosis in diabetes via activation of EGFR signaling.

Diabetes mellitus is a common chronic disease of glucose metabolism. Endothelial dysfunction is an early event in diabetes complicated by cardiovascular disease. This study aimed to reveal the expression of BASP1 and its biological roles in endothelial cell dysfunction in diabetes complicated by cardiovascular disease. By analyzing the databases related to diabetes complicated with coronary heart disease, BASP1 was screened out as an upregulated gene. Human umbilical vein endothelial cells (HUVECs) and primary mouse aortic endothelial cells were treated with high glucose to establish cell models of diabetes-related endothelial dysfunction, and the expression changes of BASP1 were verified by RT-qPCR, western blot, and immunofluorescence. BASP1 was silenced or overexpressed by siRNA or overexpression plasmid, and its effects on cell migration, apoptosis, tube formation, inflammatory response, and ROS were detected. The possible signaling pathway of BASP1 was found and the mechanism of BASP1 on promoting the progression of endothelial dysfunction was explored using the EGFR inhibitor, gefitinib. Bioinformatics analysis indicated that the expression of BASP1 in patients with diabetes mellitus and concomitant coronary heart disease was increased. High glucose induced the upregulation of BASP1 expression in endothelial cells, and showed a time-dependent relationship. Silencing of BASP1 alleviated the damage of high glucose to endothelial cells. BASP1 regulated EGFR positively. The promoting effect of BASP1 on endothelial cell apoptosis may be achieved by regulating the EGFR pathway. BASP1 promotes endothelial cell injury induced by high glucose in patients with diabetes, which may be activated by activating the EGFR pathway.

Endothelial Dysfunction in Diabetes Is Aggravated by Glycated Lipoproteins; Novel Molecular Therapies.

Diabetes and its vascular complications affect an increasing number of people. This disease of epidemic proportion nowadays involves abnormalities of large and small blood vessels, all commencing with alterations of the endothelial cell (EC) functions. Cardiovascular diseases are a major cause of death and disability among diabetic patients. In diabetes, EC dysfunction (ECD) is induced by the pathological increase of glucose and by the appearance of advanced glycation end products (AGE) attached to the plasma proteins, including lipoproteins. AGE proteins interact with their specific receptors on EC plasma membrane promoting activation of signaling pathways, resulting in decreased nitric oxide bioavailability, increased intracellular oxidative and inflammatory stress, causing dysfunction and finally apoptosis of EC. Irreversibly glycated lipoproteins (AGE-Lp) were proven to have an important role in accelerating atherosclerosis in diabetes. The aim of the present review is to present up-to-date information connecting hyperglycemia, ECD and two classes of glycated Lp, glycated low-density lipoproteins and glycated high-density lipoproteins, which contribute to the aggravation of diabetes complications. We will highlight the role of dyslipidemia, oxidative and inflammatory stress and epigenetic risk factors, along with the specific mechanisms connecting them, as well as the new promising therapies to alleviate ECD in diabetes.

MicroRNA-139-5p upregulation is associated with diabetic endothelial cell dysfunction by targeting c-jun.

Dysfunction of endothelial cells (ECs) and their progenitor cells is an important feature of diabetic vascular disease. MicroRNA (miR)-139-5p is involved in inhibiting the metastasis and progression of diverse malignancies. However, the role of miR-139-5p in ECs still remains unclarified. Here we demonstrated that miR-139-5p expression was elevated in endothelial colony-forming cells (ECFCs) isolated from patients with diabetes, ECs derived from the aorta of diabetic rodents, and human umbilical vein endothelial cells (HUVECs) cultured in high glucose media. MiR-139-5p mimics inhibited tube formation, migration, proliferation, and down-regulated expression of c-jun, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF)-B, in ECFCs and HUVECs, respectively; moreover, miR-139-5p inhibitors reversed the tendency. Further, gain- and-loss function experiments and ChIP assay indicated that miR-139-5p regulate functions of ECFCs by targeting c-jun-VEGF/PDGF-B pathway. In vivo experiments (Matrigel plug assay and hindlimb ischemia model) showed that miR-139-5p downregulation further promoted ECFC-mediated angiogenesis and blood perfusion. In conclusion, diabetes-mediated high miR-139-5p expression inhibits the c-jun-VEGF/PDGF-B pathway, thus decreasing ECFCs migration, tube formation and proliferation, which subsequently reduces ECs survival. Therefore, miR-139-5p might be an important therapeutic target in the treatment of diabetic vasculopathy in the future.

The Effects of Known Cardioprotective Drugs on Proinflammatory Cytokine Secretion From Human Coronary Artery Endothelial Cells.

Endothelial cell dysfunction in diabetes is involved in the pathogenesis and progression of premature atherosclerosis. High-dextrose has been shown to induce both oxidative stress and endoplasmic reticulum stress in cultured human coronary artery endothelial cells (HCAEC). To determine whether or not several classes of cardioprotective drugs inhibit proinflammatory cytokine expression by HCAEC. To determine the effects of high dextrose on expression of proinflammatory cytokines by HCAEC, cells were treated with either 5.5 mM or 27.5 mM dextrose for 24 hours and interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor α were measured by enzyme immunoassay in the presence or absence of known cardioprotective drugs, including select β-blockers, statins, and renin-angiotensin system inhibitors. IL-1β levels increased significantly in cells treated with high dextrose; however, IL-6 and IL-8 levels did not change. Treatment of cells with carvedilol, atenolol, and propranolol decreased levels of all 3 cytokines in cells exposed to either 5.5 or 27.5 mM dextrose. Similar effects on IL-1β, IL-6, and IL-8 levels were observed when cells were treated with simvastatin, pravastatin, and the renin-angiotensin system inhibitors spironolactone, captopril, lisinopril, candesartan, and losartan. No Il-2 or tumor necrosis factor α expression was observed in any of the experiments indicating that HCAEC do not express these cytokines. We conclude that each of the classes of drugs tested possess pleiotropic anti-inflammatory activities and are effective in both low- and high-dextrose-treated cells.

MicroRNA-106 attenuates hyperglycemia-induced vascular endothelial cell dysfunction by targeting HMGB1

Chronic wounds are a common surgical problem exacerbated by diabetes. A hyperglycemic microenvironment induces inflammation and apoptosis, and plays an important role in vascular endothelial cell dysfunction in diabetes. Increasing evidence shows that high mobility group box 1 (HMGB1) expression is related to inflammation and apoptosis. The aim of this study was to determine the function of HMGB1 in hyperglycemia-induced vascular endothelial cell dysfunction. The results showed that the expression of HMGB1 was increased in human umbilical vein endothelial cells (HUVECs) after exposure to high glucose (25 mM). Downregulation of HMGB1 attenuated the high glucose-induced antiangiogenesis of HUVECs, and the decrease expression of HMGB1 inhibiting HUVEC apoptosis and inflammatory factor expression. In addition, miR-106 expression in HUVECs was decreased under high glucose conditions. Increased miR-106 significantly reversed the high glucose-induced vascular endothelial cell dysfunction by inhibition of HUVEC apoptosis and inflammatory factor expression. However, HMGB1 overexpression attenuated the protective effect of miR-106 on HUVECs in high glucose conditions. This suggested that miR-106 suppressed hyperglycemia-induced vascular endothelial cell dysfunction by targeting HMGB1. Double fluorescent reporter assays confirmed that miR-106 interacted with the 3′-UTR of HMGB1 and inhibited HMGB1 expression. Taken together, these data collectively suggested that miR-106 was a potential molecular target for inhibiting high glucose-induced inflammation and apoptosis by targeting HMGB1.

The AGP-PPARγ axis promotes oxidative stress and diabetic endothelial cell dysfunction.

Alkyl-glycerophosphate (AGP) accumulates in atherogenic oxidized-LDL and human atherosclerotic plaques and is a potent agonist of peroxisome-proliferator-activated receptor-gamma (PPARγ). Recent studies suggest a potential regulatory role for PPARγ in endothelial nitric oxide synthase (eNOS) expression/activation and nitrogen oxide (NO) generation in the vascular endothelium. Importantly, eNOS-induced NO and advanced glycation end-products (AGEs) are involved in blood-vessel damage, and diabetic patients exhibit high serum NO and AGE levels; however, the effect of AGP on NO- and AGE-mediated endothelium dysfunction remains unknown. Investigation of the AGP-specific effects on NO- and AGE-mediated dysfunction and the underlying molecular mechanisms revealed that AGP upregulated eNOS expression and NO production, and that eNOS silencing and PPARγ antagonism inhibited AGP-mediated eNOS upregulation and NO production. Moreover, AGP-PPARγ-axis-mediated NO production promoted the generation of reactive oxygen species and AGE formation. These results suggested that AGP plays a significant role in the initiation/progression of diabetes-related atherosclerosis through PPARγ activation.

Why Wounds Won’t Heal for Diabetics

Activation of Notch signaling by the transcriptional coactivator PGC-1α contributes to endothelial cell dysfunction in diabetes.

The oligomerized polyphenol oligonol ameliorates high glucose‐ and TNF‐α‐induced NADPH oxidase activation and insulin signaling down‐regulation in vascular endothelial cells

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease. We investigated to explore the possible mechanism(s) by which the oligomerized polyphenol oligonol, used as an efficacious healthy aging dietary supplement, may be beneficial to endothelial cell dysfunction in diabetes. Porcine aortic endothelial cells were cultured in the presence of high glucose and TNF‐α with or without oligonol. In the presence of high glucose and TNF‐α, treatment of cells with oligonol prevented the increased expression level of the NADPH oxidase (Nox) subunit p67Phox and the enhanced production of radical oxygen. Furthermore, the impaired phosphorylated levels of Akt and GSK‐3β and the down‐regulated expression level of insulin receptors were improved by oligonol treatment. These results indicate that oligonol can decrease the oxidative stress through inhibition of Nox activity and amend the insulin signaling pathway in the endothelial cells under conditions that mimic diabetes. This mechanism may offer an important standpoint for the beneficial cardiovascular health effects of oligonol.

Unique Responses of Stem Cell-Derived Vascular Endothelial and Mesenchymal Cells to High Levels of Glucose

Diabetes leads to complications in selected organ systems, and vascular endothelial cell (EC) dysfunction and loss is the key initiating and perpetuating step in the development of these complications. Experimental and clinical studies have shown that hyperglycemia leads to EC dysfunction in diabetes. Vascular stem cells that give rise to endothelial progenitor cells (EPCs) and mesenchymal progenitor cells (MPCs) represent an attractive target for cell therapy for diabetic patients. Whether these vascular stem/progenitor cells succumb to the adverse effects of high glucose remains unknown. We sought to determine whether adult vascular stem/progenitor cells display cellular activation and dysfunction upon exposure to high levels of glucose as seen in diabetic complications. Mononuclear cell fraction was prepared from adult blood and bone marrow. EPCs and MPCs were derived, characterized, and exposed to either normal glucose (5 mmol/L) or high glucose levels (25 mmol/L). We then assayed for cell activity and molecular changes following both acute and chronic exposure to high glucose. Our results show that high levels of glucose do not alter the derivation of either EPCs or MPCs. The adult blood-derived EPCs were also resistant to the effects of glucose in terms of growth. Acute exposure to high glucose levels increased caspase-3 activity in EPCs (1.4x increase) and mature ECs (2.3x increase). Interestingly, MPCs showed a transient reduction in growth upon glucose challenge. Our results also show that glucose skews the differentiation of MPCs towards the adipocyte lineage while suppressing other mesenchymal lineages. In summary, our studies show that EPCs are resistant to the effects of high levels of glucose, even following chronic exposure. The findings further show that hyperglycemia may have detrimental effects on the MPCs, causing reduced growth and altering the differentiation potential.

Abstract 9849: Aerobic Exercise Improves Endothelial Dysfunction in Pre-Diabetes Patients

Current literature confirms presence of endothelial cell dysfunction in diabetes compared to non-diabetes state. However there is lack of data on endothelial function in Pre-diabetes (defined as per Diabetes Care, Clinical Guideline, Jan'11 ). Methods: We selected exercise-naive patients, 20-70 yrs, BMI 25-34.9, n=20 in each group and studied their endothelial function after 150min/week of aerobic exercise (similar to Diabetes Prevention Program) and after non exercise phase (6wks phase each), in a cross over design with a 4 wk wash-out period between the 2 groups. For endothelial function assessment we looked at, 1. flow mediated dilatation (FMD of brachial artery), 2. CD34/KDR +ve cell count from peripheral blood derived mononuclear cell fraction at day0 by Flow cytometry (FACS) 3. endothelial progenitor cell (EPC) associated colony-forming units (Hill-CFU) at day5 of culture, 4.endothelial bio-inflammatory markers and BP, fasting lipids, insulin, glucose. We also looked at gene expression profile of critical endothelial function genes in CD34/KDR+ve cells. Results: FMD studies (undertaken by 3 observers, blinded to glycemic status) showed, mean FMD in non-exercise group of 5.7+0.6% and post exercise it improved to 11.2±0.9%. Both CFU estimation, at day5 and CD34/KDR +ve cells estimation at day0 showed significant increase in numbers following exercise. There was no weight loss noted between 2 groups, however, there was statistically significant reduction in leptin, IL-6, TNFα, fasting Triglyceride, LDL and ApoB levels. Insulin sensitivity (HOMA) improved in the exercise group. Gene expression analysis of CD34+ve cells showed decrease of eNOS and increase of PECAM-1 in non-exercise which improved significantly following exercise, with no change in von-Willebrand's factor. Summary: Prediabetic patients have significant endothelial dysfunction, similar to patients with diabetes. In this population, FMD measurements and CD34/KDR+ve cell number and gene profile assays along with usual biochemical markers can serve as robust bio-markers for monitoring endothelial cell dysfunction. Conclusion: Pre-diabetic state is associated with significant endothelial dysfunction which can be improved, by undertaking regular aerobic exercise.

Inhibition of JAK/STAT signaling pathway prevents high-glucose-induced increase in endothelin-1 synthesis in human endothelial cells

Emerging evidence demonstrates the involvement of endothelin-1 (ET-1) in the pathophysiology of cardiovascular disorders associated with diabetes mellitus. The molecular mechanisms accountable for the increased production of ET-1 are not completely defined. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is an essential pathogenic mechanism leading to endothelial cell dysfunction. Our aim has been to investigate the role of JAK/STAT in the regulation of ET-1 synthesis in human endothelial cells (EAhy926 cells line). EAhy926 cells were exposed to normal (5 mM) or high (25 mM) glucose concentrations in the presence/absence of various JAK/STAT inhibitors. Using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gene reporter assay, we found that JAK/STAT inhibitors (STAT1 decoy oligodeoxynucleotides, AG490, S3I201, WP1066) significantly diminished the high-glucose-dependent up-regulation of ET-1 mRNA, peptide synthesis, and promoter activity. In silico analysis of the human ET-1 promoter revealed the presence of typical STAT1-gamma-activated sequence (STAT1-GAS) elements. Transient overexpression of STAT1 indicated an up-regulation of ET-1 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1 proteins with the predicted GAS sites. Regulation of ET-1 synthesis by the JAK/STAT pathway thus represents a novel mechanism by which high glucose induces endothelial cell dysfunction in diabetes. Since the JAK/STAT system is an important regulator of the response of endothelial cells to injury, the modulation of this system and the subsequent decrease in ET-1 level may represent a key pharmacological target in diabetes-associated cardiovascular disorders.

Delayed autoregulation of glucose transport in vascular endothelial cells

We aimed to characterise the development of autoregulation of glucose transport in vascular endothelial cells and its relationship to 12-lipoxygenase (12-LO) expression. Bovine aortic endothelial cells were exposed to 5.5 and 23.0 mmol/l glucose for up to 48 h. The rates of glucose transport, GLUT-1 and 12-LO expression and of 12-hydroxyeicosatetraenoic acid (12-HETE) production were determined. We showed high glucose-dependent downregulation of glucose transport and transporter in vascular endothelial cells within 36-48 h. A similar time-dependent increase in the expression of 12-LO and the generation of its product 12-HETE was also observed. This downregulatory process was prevented when lipoxygenase activity was inhibited. Vascular endothelial cells, which were previously thought to be "glucose-blind", do in fact downregulate GLUT-1 expression and the rate of glucose transport in response to extended exposure to high glucose concentrations. This slow development of glucose-induced downregulation in vascular endothelial cells is related to the slower basal rate of glucose transport in these cells and the slow induction of 12-LO. These data are interesting in view of current hypotheses that attribute vascular endothelial cell dysfunction in diabetes to the lack of a glucose-induced autoregulatory response.

Chronic exposure to high glucose impairs bradykinin-stimulated nitric oxide production by interfering with the phospholipase-C-implicated signalling pathway in endothelial cells: evidence for the involvement of protein kinase C

Overwhelming evidence indicates that endothelial cell dysfunction in diabetes is characterised by diminished endothelium-dependent relaxation, but the matter of the underlying molecular mechanism remains unclear. As nitric oxide (NO) production from the endothelium is the major player in endothelium-mediated vascular relaxation, we investigated the effects of high glucose on NO production, and the possible alterations of signalling pathways implicated in this scenario. NO production and intracellular Ca(2+) levels ([Ca(2+)](i)) were assessed using the fluorescent probes 4,5-diaminofluorescein diacetate and fura-2 respectively. Exposure of cultured bovine aortic endothelial cells to high glucose for 5 or 10 days significantly reduced NO production induced by bradykinin (but not by Ca(2+) ionophore) in a time- and dose-dependent manner. This was probably due to an attenuation in bradykinin-induced elevations of [Ca(2+)](i) under these conditions, since a close correlation between [Ca(2+)](i) increases and NO generation was observed in intact bovine aortic endothelial cells. Both bradykinin-promoted intracellular Ca(2+) mobilisation and extracellular Ca(2+) entry were affected. Moreover, bradykinin-induced formation of Ins(1,4,5)P(3), a phospholipase C product leading to increases in [Ca(2+)](i), was also inhibited following high glucose culture. This abnormality was not attributable to a decrease in inositol phospholipids, but possibly to a reduction in the number of bradykinin receptors. The alterations in NO production, the increases in [Ca(2+)](i), and the bradykinin receptor number due to high glucose could be largely reversed by protein kinase C inhibitors and D: -alpha-tocopherol (antioxidant). Chronic exposure to high glucose reduces NO generation in endothelial cells, probably by impairing phospholipase-C-mediated Ca(2+) signalling due to excess protein kinase C activation. This defect in NO release may contribute to the diminished endothelium-dependent relaxation and thus to the development of cardiovascular diseases in diabetes.

Profilin acts downstream of LDL to mediate diabetic endothelial cell dysfunction

The changes occurring at the luminal surface of endothelial cells in diabetes and their relevance to endothelial dysfunction are poorly characterized in vivo. In this study, we developed an integrated strategy to discover cell surface proteins associated with diabetes and to test their role in endothelial dysfunction. First, a peptide phage display library was screened over the endothelial surface of the intact aorta or in retinal endothelial cells from diabetic and control rats. Then, we purified profilin-1 as a binding partner for one of the diabetic aorta-specific phage. Profilin was increased in the aortic endothelium of human diabetic individuals and streptozotocin-diabetic rats. Furthermore, overexpressing profilin in rat aortic endothelial cells triggered 3 indicators of endothelial dysfunction: increased apoptosis, elevated expression of ICAM-1, and decreased phosphorylation of the vasodilator-stimulated phosphoprotein, a marker for nitric oxide signaling. The changes in ICAM-1 and vasodilator-stimulated phosphoprotein were recapitulated in the diabetic aorta in vivo. LDL and oxysterols elevated profilin in cultured aortic endothelial cells. Interference with the de novo synthesis of profilin abrogated the LDL-mediated increase in ICAM-1 expression. Finally, profilin expression was markedly elevated in atherosclerotic plaques. These data indicate that profilin contributes to endothelial dysfunction in a pathway that is downstream of LDL.

Vascular action of the hypoglycaemic agent gliclazide in diabetic rabbits.

ATP-dependent potassium channel blockers used as hypoglycaemic agents may have effects on vascular disease in diabetes mellitus beyond their effect on blood glucose control. This study was designed to determine the effects of treatment with gliclazide on the isolated abdominal aorta of diabetic rabbits in which endothelium-dependent relaxation is impaired by a mechanism involving oxygen-derived free radicals. After induction of diabetes with alloxan, there was no effect of gliclazide (10 mg x kg(-1) day(-1) orally) on blood glucose or insulin levels over a 6 week period. Hence, this permitted an examination of the vascular effects of gliclazide in diabetic rabbits exclusive of metabolic effects. Acetylcholine- and nitric oxide-induced relaxation in aortae from rabbits treated with or without gliclazide were measured in the absence or presence of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine (L-NAME). Diabetes was associated with significant impairment of acetylcholine-induced endothelium-dependent relaxation of the abdominal aorta which was not significant in diabetic rabbits treated with gliclazide in vivo. Aortae from diabetic rabbits studied in the presence of L-NAME showed an exaggerated contraction to acetylcholine which was prevented in rabbits treated with gliclazide. Gliclazide treatment did not affect the response to acetylcholine of normal rabbit aorta, and gliclazide when added in vitro had no effect on the response of diabetic rabbit aorta, suggesting that the effect of gliclazide was specific to the abnormality arising with diabetes and was not due to an acute effect of the drug. These data indicate that gliclazide, aside from either a direct antioxidant action or an effect on insulin or glucose levels, may ameliorate diabetic endothelial cell dysfunction.

High glucose induces antioxidant enzymes in human endothelial cells in culture. Evidence linking hyperglycemia and oxidative stress.

It has been suggested that oxidative stress may play an important role in the pathogenesis of diabetic complications. Hyperglycemia may cause increased production of free radicals, and evidence supports a prominent role for these reactive molecules as mediators of endothelial cell dysfunction in diabetes. It has been demonstrated that active oxygen species induce antioxidant enzyme expression in some tissues, and this phenomenon is considered proof of an existing oxygen-dependent toxicity. In this study, human endothelial cells from umbilical vein, immortalized human endothelial cells, and immortalized human endothelial cells transfected to express high glutathione peroxidase levels were grown in normal and high-glucose conditions. High glucose delayed replication after 7 and 14 days of culture of human endothelial cells, both from umbilical vein and immortalized, while transfected cells were not affected. The activity and the mRNA expression of the antioxidant enzymes CuZn-superoxide-dismutase, Mn-superoxide-dismutase, catalase, and glutathione peroxidase were evaluated after 2, 7, and 14 days of culture. High glucose at days 7 and 14 induced an overexpression of CuZn-superoxide-dismutase, catalase, and glutathione peroxidase in both human endothelial cells from umbilical vein and immortalized human endothelial cells, while in transfected cells it did not. This study demonstrates that high glucose induces an increase in antioxidant enzyme levels in human endothelial cells, suggesting that elevated glucose levels may produce an oxidative stress in the cells.

Plasma free N‐terminal fibronectin 30‐kDa domain as a marker of endothelial dysfunction in type 1 diabetes mellitus

The plasma free N-terminal fibronectin 30-kDa domain was measured in 44 type 1 diabetic patients and in 20 healthy subjects. A significantly raised mean concentration of a free N-terminal fibronectin 30-kDa domain was found in plasma of diabetic patients with proliferative retinopathy as compared with healthy persons (P less than 0.001). A positive correlation was observed between free N-terminal fibronectin 30-kDa domain and von Willebrand factor in plasma of all examined subjects (r = 0.62, P less than 0.01). A similar correlation was present between 30-kDa domain and albuminuria (r = 0.56, P less than 0.01). However, no relationship was found between fibronectin 30-kDa domain and control of diabetes as assessed by fructosamine concentration. The free N-terminal fibronectin 30-kDa domain may be used as a marker of actual endothelial cell dysfunction in diabetes.