Reversal of Hyperglycemia-Induced Angiogenesis Deficit of Human Endothelial Cells by Overexpression of Glyoxalase 1 In Vitro (original) (raw)
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Annals of the New …
Dicarbonyl glycation of RGD and GFOGER sites in type IV collagen has been associated with decreased angiogenesis. In this study, we investigated whether overexpression of glyoxalase 1 to decrease dicarbonyl glycation would prevent the angiogenesis deficit induced by ...
Journal of Clinical Investigation, 1998
Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D -lactoylglutathione, which in turn is converted to D -lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo -transfected control cells (21.80 Ϯ 0.1 vs. 0.76 Ϯ 0.02 mol/min/mg protein, n ϭ 3, P Ͻ 0.001). In neo -transfected cells, 30 mM glucose incubation increased MG and D -lactate concentration approximately twofold above 5 M M (35.5 Ϯ 5.8 vs. 19.6 Ϯ 1.6, P Ͻ 0.02, n ϭ 3, and 21.0 Ϯ 1.3 vs. 10.0 Ϯ 1.2 pmol/ 10 6 cells, n ϭ 3, P Ͻ 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration at all, while increasing the enzymatic product D -lactate by Ͼ 10-fold (18.9 Ϯ 3.2 vs. 18.4 Ϯ 5.8, n ϭ 3, P ϭ NS, and 107.1 Ϯ 9.0 vs. 9.4 Ϯ 0 pmol/10 6 cells, n ϭ 3, P Ͻ 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58 Ϯ 0.15 vs. 0.19 Ϯ 0.03 total absorbance units, n ϭ 3, P Ͻ 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis. ( J. Clin.
Molecular and Cellular Biochemistry, 2010
Vascular endothelium is vulnerable to the attack of glucose-derived oxoaldehydes (glyoxal and methylglyoxal) during diabetes, through the formation of advanced glycation end products (AGEs). Although aminoguanidine (AG) has been shown to protect against the AGE-induced adverse effects, its protection against the glyoxal-induced alterations in vascular endothelial cells (ECs) such as cytotoxicity, barrier dysfunction, and inhibition of angiogenesis has not been reported and we investigated this in the bovine pulmonary artery ECs (BPAECs). The results showed that glyoxal (1-10 mM) significantly induced cytotoxicity and mitochondrial dysfunction in a dose-and time-dependent (4-12 h) fashion in ECs. Glyoxal was also observed to significantly inhibit EC proliferation. The study also revealed that glyoxal induced EC barrier dysfunction (loss of trans-endothelial electrical resistance), actin cytoskeletal rearrangement, and tight junction alterations in BPAECs. Furthermore, the results revealed that glyoxal significantly inhibited in vitro angiogenesis on the Matrigel. For the first time, this study demonstrated that AG significantly protected against the glyoxal-induced cytotoxicity, barrier dysfunction, cytoskeletal rearrangement, and inhibition of angiogenesis in BPAECs. Therefore, AG appears as a promising protective agent in the treatment of AGE-induced vascular endothelial alterations and dysfunction during diabetes, presumably by blocking the reactivity of the sugar-derived dicarbonyls such as glyoxal and preventing the formation of AGEs.
Scientific reports, 2016
Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of col...
The FASEB Journal, 2008
Introduction: Microalbuminuria is a clinical parameter of vascular dysfunction in patients with diabetes. The endothelial glycocalyx plays a role in regulation of vascular permeability. Therapeutic interventions using a mixture of glycosaminoglycans (GAGs) containing 80% heparin and 20% dermatan sulphate (sulodexide) have been shown to improve vascular barrier function by reducing microalbuminuria. Therefore we hypothesized that exogenous GAGs attenuate hyperglycemia-induced increases in endothelial permeability for albumin by restoring barrier properties of the endothelial glycocalyx. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured on semi-permeable inserts and exposed to normo-(5 mM) or hyperglycemia (25 mM) for 4 days, last 24 h in presence of the GAG mixture. Endothelial permeability was assessed by determining FITC-labeled albumin transfer over the monolayer (3 hours). Additionally, the glycocalyx glucosamine sugar residues on the endothelial cells were visualized with LEA-lectin staining. Results: Albumin permeability of endothelial cells under hyperglycemia was increased to 122 ± 8% (p < 0.01) compared to normoglycemia. Changes in albumin permeability under hyperglycemia normalized to normoglycemic control condition was-4 ± 3% (p < 0.05) in the presence of 0.06μg/mL sulodexide. Additionally, LEA-lectin revealed a 28 ± 1% (p < 0.05) increase in glucosamine staining in hyperglycemic cells in the presence of the GAG mix. Conclusion: GAG supplementation reverses the increased trans-endothelial albumin leakage under hyperglycemic conditions by restoring the barrier properties of the endothelial glycocalyx layer in vitro.
Proceedings of the National Academy of Sciences, 2003
We hypothesized that formation of advanced glycation end products (AGEs) associated with diabetes reduces matrix degradation by metalloproteinases (MMPs) and contributes to the impairment of ischemia-induced angiogenesis. Mice were treated or not with streptozotocin (40 mg͞kg) and streptozotocin plus aminoguanidine (AGEs formation blocker, 50 mg͞kg). After 8 weeks of treatment, hindlimb ischemia was induced by right femoral artery ligature. Plasma AGE levels were strongly elevated in diabetic mice when compared with control mice (579 ؎ 21 versus 47 ؎ 4 pmol͞ml, respectively; P < 0.01). Treatment with aminoguanidine reduced AGE plasma levels when compared with untreated diabetic mice (P < 0.001). After 28 days of ischemia, ischemic͞nonischemic leg angiographic score, capillary density, and laser Doppler skin-perfusion ratios were 1.4-, 1.5-, and 1.4-fold decreased in diabetic mice in reference to controls (P < 0.01). Treatment with aminoguanidine completely normalized ischemiainduced angiogenesis in diabetic mice. We next analyzed the role of proteolysis in AGE formation-induced hampered neovascularization process. After 3 days of ischemia, MMP-2 activity and MMP-3 and MMP-13 protein levels were increased in untreated and aminoguanidine-treated diabetic mice when compared with controls (P < 0.05). Despite this activation of the MMP pathway, collagenolysis was decreased in untreated diabetic mice. Conversely, treatment of diabetic mice with aminoguanidine restored collagenolysis toward levels found in control mice. In conclusion, blockade of AGE formation by aminoguanidine normalizes impaired ischemia-induced angiogenesis in diabetic mice. This effect is probably mediated by restoration of matrix degradation processes that are disturbed as a result of AGE accumulation.
Methylglyoxal-Glyoxalase 1 Balance: The Root of Vascular Damage
International Journal of Molecular Sciences, 2017
The highly reactive dicarbonyl methylglyoxal (MGO) is mainly formed as byproduct of glycolysis. Therefore, high blood glucose levels determine increased MGO accumulation. Nonetheless, MGO levels are also increased as consequence of the ineffective action of its main detoxification pathway, the glyoxalase system, of which glyoxalase 1 (Glo1) is the rate-limiting enzyme. Indeed, a physiological decrease of Glo1 transcription and activity occurs not only in chronic hyperglycaemia but also with ageing, during which MGO accumulation occurs. MGO and its advanced glycated end products (AGEs) are associated with age-related diseases including diabetes, vascular dysfunction and neurodegeneration. Endothelial dysfunction is the first step in the initiation, progression and clinical outcome of vascular complications, such as retinopathy, nephropathy, impaired wound healing and macroangiopathy. Because of these considerations, studies have been centered on understanding the molecular basis of endothelial dysfunction in diabetes, unveiling a central role of MGO-Glo1 imbalance in the onset of vascular complications. This review focuses on the current understanding of MGO accumulation and Glo1 activity in diabetes, and their contribution on the impairment of endothelial function leading to diabetes-associated vascular damage.
Diabetologia, 2014
Aims/hypothesis In diabetes, advanced glycation end-products (AGEs) and the AGE precursor methylglyoxal (MGO) are associated with endothelial dysfunction and the development of microvascular complications. In this study we used a rat model of diabetes, in which rats transgenically overexpressed the MGO-detoxifying enzyme glyoxalase-I (GLO-I), to determine the impact of intracellular glycation on vascular function and the development of early renal changes in diabetes. Methods Wild-type and Glo1-overexpressing rats were rendered diabetic for a period of 24 weeks by intravenous injection of streptozotocin. Mesenteric arteries were isolated to study ex vivo vascular reactivity with a wire myograph and kidneys were processed for histological examination. Glycation was determined by mass spectrometry and immunohistochemistry. Markers for inflammation, endothelium dysfunction and renal dysfunction were measured with ELISA-based techniques. Results Diabetes-induced formation of AGEs in mesenteric arteries and endothelial dysfunction were reduced by Glo1 overexpression. Despite the absence of advanced nephrotic lesions, early markers of renal dysfunction (i.e. increased Electronic supplementary material The online version of this article
Hyperglycemia reduces nitric oxide synthase and glycogen synthase activity in endothelial cells
Nitric Oxide, 2002
Hyperglycemia is considered a primary cause of diabetic vascular complications. A hallmark of vascular disease is endothelial cell dysfunction characterized by diminished nitric-oxide (NO)-dependent phenomena such as vasodilation, angiogenesis, and vascular maintenance. This study was designed to investigate the effects of a high level of D D-glucose on endothelial NO response, oxidative stress, and glucose metabolism. Bovine aortic endothelial cells (BAECs) were pretreated with a high concentration of glucose (HG) (22 mmol/L) for at least 2 weeks and compared with control cells exposed to 5 mmol/L glucose (NG). The effect of chronic hyperglycemia on endothelial NO-synthase (eNOS) activity and expression, glycogen synthase (GS) activity, extracellular-signalregulated kinase (ERK 1,2), p38, Akt expression, and Cu/Zn superoxide-dismutse (SOD-1) activity and expression were determined. Western blot analysis showed that eNOS protein expression decreased in HG cells and was accompanied by diminished eNOS activity. The activity of GS was also significantly lower in the HG cells than in NG cells, 25:0 AE 17:4 and 89 AE 22:5 nmol UDP-glucose Á mg protein À1 Â min À1 , respectively. Western blot analysis revealed a 40-60% decrease in ERK 1,2 and p38 protein levels, small modification of phosphorylated Akt expression, and a 30% increase in SOD-1 protein expression in HG cells. Although SOD expression was increased, no change was observed in SOD activity. These results support the findings that vascular dysfunction due to exposure to pathologically high D D-glucose concentrations may be caused by impairment of the NO pathway and increased oxidative stress accompanied by altered glucose metabolism.
Vascular hypertrophy in experimental diabetes. Role of advanced glycation end products
Journal of Clinical Investigation, 1997
The accelerated formation of advanced glycation end products (AGEs) and the overexpression of transforming growth factor beta (TGF-) have both been implicated in the pathogenesis of diabetic microvascular and macrovascular complications. Previous studies in our laboratory have demonstrated that the vascular changes in diabetes include hypertrophy of the mesenteric vasculature. To examine the role of AGEs in this process, streptozotocin-induced diabetic rats and control animals were randomized to receive aminoguanidine, an inhibitor of AGE formation, or no treatment. Animals were studied at 7 d, 3 wk, and 8 mo after induction of diabetes. When compared with control animals, diabetes was associated with an increase in mesenteric vascular weight and an increase in media wall/lumen area. By Northern analysis, TGF- 1 gene expression was increased 100-150% (P Ͻ 0.01) and ␣ 1 (IV) collagen gene expression was similarly elevated to 30-110% compared to controls (P Ͻ 0.05). AGEs and extracellular matrix were present in abundance in diabetic but not in control vessels. Treatment of diabetic rats with aminoguanidine resulted in significant amelioration of the described pathological changes including overexpression of TGF- 1 and ␣ 1 (IV) collagen. These data implicate the formation of AGEs in TGF- overexpression and tissue changes which accompany the diabetic state. (J.