Methylglyoxal modification of mSin3A links glycolysis to angiopoietin-2 transcription

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here, we report that in retinal Müller cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. A similar mechanism involving methylglyoxal-modification of other coregulator proteins may play a role in the pathobiology of a variety of conditions associated with changes in methylglyoxal concentration, including cancer and diabetic vascular disease.     

Angiogenic activity of endothelial progenitor cells through angiopoietin-1 and angiopoietin-2

Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of different cell types particularly mature endothelial cells and recently discovered progenitor cells, named as endothelial progenitor cells (EPCs). Up to now, many attempts have been made to understand the dynamic balance of pro- and anti-angiogenic factors on EPCs on different milieu. It has been accepted that Ang-1, -2 and Tie-1, -2 signaling play a key role on angiogenesis pathways in endothelial lineage cells. In the current experiment, the angiogenic/angio-modulatory potency of Ang-1 and -2 was investigated on isolated EPCs. Freshly isolated EPCs were exposed to different concentrations of Ang-1 and -2 (25 and 50?ng/ml) over a course of 7 and 14 days. Corroborating to our results, a superior effect of Ang-1 on angiogenic properties, including an increased concentration of vascular endothelial growth factor, in vitro tubulogenesis, EPC migratory, Tie-2 expression and clonogenicity, was determined. A large amount of positive mature endothelium markers was achieved in EPCs being-exposed to Ang-1 peptide. Nonetheless, the number of CD133 positive cells increased in the presence of Ang-2. Collectively, we conclude that Ang-1 potentially induces functional and mature vascular-like behavior in EPCs more than Ang-2.     

High glucose increases B1-kinin receptor expression and signaling in endothelial cells

The loss of endothelial function is the initiating factor in the development of diabetic vascular disease. Kinins control endothelial function by the activation of two receptors: the B2 which is constitutively expressed, and the B1 which is highly induced in pathological conditions. In the present study, we observed that the levels of B1-receptor mRNA and protein are induced in endothelial cells incubated in high glucose. An increase in B1-receptor was also observed in the endothelial layer of aortas, from 4-week diabetic rats. When cells were grown in high glucose, the B1 agonist des-Arg9-BK increased nitrite levels, whereas in normal glucose nitrite levels were unchanged. Nitrite increase was blocked by L-NAME and 1400W indicating the participation of the inducible Nitric Oxide Synthase (iNOS). iNOS protein levels were also increased in high glucose. These results demonstrate the participation of the B1 receptor in the signaling pathways mediated by kinins in high glucose.     

Antiproliferative effect of elevated glucose in human microvascular endothelial cells

Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P < 0.05) HMEC-1 proliferation after 7, 10, and 14 days. This effect was not mimicked by 20 mM mannitol. The antiproliferative effect was more pronounced with longer exposure (1–14 days) to elevated glucose and was irreversible 4 days after a 10-day exposure. The antiproliferative effect was partially reversed in the presence of a PKA inhibitor, Rp-cAMP (10–50 μM), and/or a PKC inhibitor, Calphostin C (10 nM). HMEC-1 exposed to elevated glucose (20 mM) for 14 days caused an increase in cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular pathophysiology associated with diabetic microangiopathy. J. Cell. Biochem. 71:491–501, 1998. © 1998 Wiley-Liss, Inc.     

Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells

The atherogenic serum lysophosphatidylcholine (LPC) is known to mediate vascular endothelial responses ranging from upregulation of adhesion molecules and growth factors to secretion of chemokines and superoxide anion. We investigated whether endothelial cells express receptors for LPC, which may account for their actions. Human brain microvascular (HBMEC) and dermal microvascular endothelial cells (HMEC) were prepared for RT-PCR analysis for possible expression of the G protein-coupled receptors, GPR4 and G2A, which are believed to be specific LPC receptors. Results indicated that HBMEC expressed low basal GPR4 mRNA, but stimulation with tumor necrosis factor-alpha (TNF-alpha) (100 U/ml) or H2O2 (50 micromol/l) for 2 h or overnight upregulated expression severalfold. In contrast, HMEC expressed high basal GPR4 mRNA, which was not further increased by either TNF-alpha or H2O2 stimulation. Another LPC receptor, G2A, was not detected in either endothelial cell type. Competition binding studies were made to evaluate specific binding of [3H]LPC to the intact endothelial cell monolayer. Basal specific [3H]LPC binding in HBMEC was approximately eight times lower than in HMEC; however, TNF-alpha or H2O2 stimulation increased [3H]LPC binding on HMBEC but not HMEC. The results indicated that GPR4 expression was consistent with specific [3H]LPC binding. Overall, we report that endothelial cells selectively expressed GPR4, a specific LPC receptor. Furthermore, GPR4 expression by HBMEC, but not HMEC, was increased by inflammatory stresses. We conclude that endogenous GPR4 in endothelial cells may be a potential G protein-coupled receptor by which LPC signals proinflammatory activities.     

High glucose increases expression of cyclooxygenase‐2, increases oxidative stress and decreases the generation of nitric oxide in mouse microvessel endothelial cells

Hyperglycaemia is a key factor that contributes to the development of diabetes‐related microvascular disease. Both cyclooxygenase I and cyclooxygenase II are expressed in endothelial cells and play key roles in the regulation of cardiovascular function. In the current study we tested the hypothesis that hyperglycaemia‐induced increased expression of cyclooxygenase II is a contributing factor both to the increased oxidative stress and to the reduction in the generation of nitric oxide in microvessel endothelial cells following their exposure to high glucose. We demonstrated that the exposure of mouse microvascular endothelial cells to high glucose for 3 days decreased the generation of nitric oxide and enhanced production of superoxide. Western blots illustrated that exposure to high glucose also increased endothelial nitric oxide synthase and cyclooxygenase II protein expression levels and decreased the dimer/monomer ratio of endothelial nitric oxide synthase protein. All the changes induced by the high glucose culture media could be reversed by either the cyclooxygenase II inhibitor CAY10404, the non‐selective cyclooxygenase inhibitor indomethacin or the protein kinase C inhibitor chelerythrine, but not solely by preincubation with the antioxidant and putative NADPH oxidase inhibitor, apocynin. Our data indicate that high glucose induced oxidative stress is linked to an increase in the expression of cyclooxygenase II and a reduced generation of nitric oxide that is associated with an uncoupled endothelial nitric oxide synthase, possibly due to decreased dimer/monomer ratio. J. Cell. Physiol. 222: 669–675, 2010. © 2009 Wiley‐Liss, Inc.     

High level glucose increases mutagenesis in human lymphoblastoid cells

Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was also investigated by using human lymphoblastoid cell lines derived from same donor but differs in p53 statuses; TK6 has wild-type p53, NH32 has null p53, and WTK1 has mutant p53 (ile237). In addition, we studied the influence of antioxidant treatment on HG-induced mutagenesis. Mutation fractions at both loci increased significantly in all three lines at 21 and 28 days after HG treatments. At tk locus, the increase of a class of mutants with normal growth rate is mainly responsible for the overall increased mutant fraction. Compared to TK6 cells, both NH32 and WTK1 cells showed an early onset of mutagenesis. Treatment of cells with antioxidant N-acetyl-L-cysteine partially reduced HG induced mutagenesis. This study is the first to indicate that HG is able to induce gene mutation which may be one of the important mechanisms of diabetes-associated carcinogenesis.     

A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro

Although tight-junctions (TJs) at the blood-brain barrier (BBB) are important to prevent non-specific entry of compounds into the CNS, molecular mechanisms regulating TJ maintenance remain still unclear. The purpose of this study was therefore to identify molecules, which regulate occludin expression, derived from astrocytes and pericytes that ensheathe brain microvessels by using conditionally immortalized adult rat brain capillary endothelial (TR-BBB13), type II astrocyte (TR-AST4) and brain pericyte (TR-PCT1) cell lines. Transfilter co-culture with TR-AST4 cells, and exposure to conditioned medium of TR-AST4 cells (AST-CM) or TR-PCT1 cells (PCT-CM) increased occludin mRNA in TR-BBB13 cells. PCT-CM-induced occludin up-regulation was significantly inhibited by an angiopoietin-1-neutralizing antibody, whereas the up-regulation by AST-CM was not. Immunoprecipitation and western blot analyses confirmed that multimeric angiopoietin-1 is secreted from TR-PCT1 cells, and induces occludin mRNA, acting through tyrosine phosphorylation of Tie-2 in TR-BBB13 cells. A fractionated AST-CM study revealed that factors in the molecular weight range of 30-100 kDa led to occludin induction. Conversely, occludin mRNA was reduced by transforming growth factor beta 1, the mRNA of which was up-regulated in TR-AST4 cells following hypoxic treatment. In conclusion, in vitro BBB model studies revealed that the pericyte-derived multimeric angiopoietin-1/Tie-2 pathway induces occludin expression.     

Sodium butyrate inhibits angiogenesis of human intestinal microvascular endothelial cells through COX-2 inhibition

We examined the effect of sodium butyrate on in vitro angiogenesis and cyclooxygenase (COX) expression using primary cultures of human intestinal microvascular endothelial cells (HIMEC). Butyrate inhibited VEGF-induced cellular proliferation, transmigration and tube formation of HIMEC. Butyrate also inhibited COX-2 expression as well as prostaglandin (PG)E2 and PGI2 production, and administration of PGI2 analog partially reversed the effect of butyrate on HIMEC angiogenesis. These results indicate that sodium butyrate inhibits HIMEC angiogenesis through down-regulation of COX-2 expression and PG production, and suggest that anti-angiogenic mechanisms may also be involved in the inhibitory effect of sodium butyrate on tumor growth.     

Endotoxin increases intercellular resistance in microvascular endothelial cells by a tyrosine kinase pathway

Gap junction communication between microvascular endothelial cells has been proposed to contribute to the coordination of microvascular function. Septic shock may attenuate microvascular cell-to-cell communication. We hypothesized that lipopolysaccharide (LPS) attenuates communication between microvascular endothelial cells derived from rat hindlimb skeletal muscle. Endothelial cells grown in monolayers expressed mRNA for connexin 37, 40, and 43. The expression of connexin 43 protein was confirmed, but connexin 40 protein was not detected by immunocytochemistry or immunoblot analysis. Intercellular resistance between cells of the monolayer, calculated using a Bessel function model, was increased from 3.3 to 5.3 MOmega by LPS. The effect was seen after 1 h of exposure and required a minimum concentration of 10 ng/ml. Intercellular resistance returned to normal 1 h following removal of LPS. Neither the response to LPS, nor its reversal, was blocked by the protein synthesis inhibitor cycloheximide (10 microg/ml). Pretreatment of monolayers with the tyrosine kinase inhibitors PP-2 (10 nM), lavendustin-C (1 microM), and geldanamycin (200 nM) prevented this LPS response; geldanamycin was also able to reverse the response. Inhibitors of MAP kinases, PD 98059 (5 microM) and SB 202190 (5 microM), and PKC (500 nM bisindolylmaleimide I) were unable to block the LPS response. We propose that LPS attenuates cell-to-cell communication through a signaling pathway that is tyrosine kinase dependent.