Developmental endothelial locus-1 is a homeostatic factor in the central nervous system limiting neuroinflammation and demyelination.

Inflammation in the central nervous system (CNS) and disruption of its immune privilege are major contributors to the pathogenesis of multiple sclerosis (MS) and of its rodent counterpart, experimental autoimmune encephalomyelitis (EAE). We have previously identified developmental endothelial locus-1 (Del-1) as an endogenous anti-inflammatory factor, which inhibits integrin-dependent leukocyte adhesion. Here we show that Del-1 contributes to the immune privilege status of the CNS. Intriguingly, Del-1 expression decreased in chronic-active MS lesions and in the inflamed CNS in the course of EAE. Del-1-deficiency was associated with increased EAE severity, accompanied by increased demyelination and axonal loss. As compared with control mice, Del-1(-/-) mice displayed enhanced disruption of the blood-brain barrier and increased infiltration of neutrophil granulocytes in the spinal cord in the course of EAE, accompanied by elevated levels of inflammatory cytokines, including interleukin-17 (IL-17). The augmented levels of IL-17 in Del-1-deficiency derived predominantly from infiltrated CD8(+) T cells. Increased EAE severity and neutrophil infiltration because of Del-1-deficiency was reversed in mice lacking both Del-1 and IL-17 receptor, indicating a crucial role for the IL-17/neutrophil inflammatory axis in EAE pathogenesis in Del-1(-/-) mice. Strikingly, systemic administration of Del-1-Fc ameliorated clinical relapse in relapsing-remitting EAE. Therefore, Del-1 is an endogenous homeostatic factor in the CNS protecting from neuroinflammation and demyelination. Our findings provide mechanistic underpinnings for the previous implication of Del-1 as a candidate MS susceptibility gene and suggest that Del-1-centered therapeutic approaches may be beneficial in neuroinflammatory and demyelinating disorders.

Del-1 overexpression potentiates lung cancer cell proliferation and invasion.

Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild type of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial-mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma.

Association of cardio-ankle vascular index with diastolic heart function in hypertensive patients.

Arterial stiffness is an important risk factor of impaired left ventricular (LV) diastolic function as well as systolic dysfunction. The cardio-ankle vascular index (CAVI) and the ambulatory arterial stiffness index (AASI) can evaluate arteriosclerosis. We analyzed the relationship between arterial stiffness and diastolic function, and then compared the two methodologies to assess which method could serve as a more informative tool for diastology. In total, 136 patients with hypertension underwent 24-h ambulatory blood pressure monitoring (ABPM) and echocardiography including ventricular arterial coupling (VAC). Arterial stiffness was estimated using both CAVI and AASI derived from ABPM. Patients were classified into LV diastolic dysfunction and normal function groups. Those with diastolic dysfunction had a higher CAVI and AASI. Aside from LV torsion, mitral inflow parameters, tissue Doppler velocities and VAC showed a significantly greater association with CAVI, relative to AASI. The receiver operating characteristic curve analysis revealed that CAVI [area under the curve (AUC) = 0.869, p < 0.001] provided significantly more favorable accuracy for diastolic dysfunction compared with AASI (AUC = 0.672, p = 0.004). Multiple logistic regression analyses showed that CAVI [Odds ratio (OR) = 5.1, p = 0.009] had a greater association with diastolic dysfunction, relative to age, systolic blood pressure or AASI (OR = 1.4, p = 0.043). This study indicates that CAVI clinically provides diastolic functional information much better in hypertensive patients than AASI.

TNF-α polymorphisms and coronary artery disease: association study in the Korean population.

Coronary artery disease (CAD) results from atherosclerosis, a chronic inflammatory disease mediated in part by proinflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), which is expressed by atherosclerotic plaques. In this study, we investigated whether TNF-α gene promoter polymorphisms affect the incidence of CAD in Koreans by genotyping. 404 Control subjects and 197 patients who previously received a coronary artery stent for the G/A, C/T, and C/A polymorphisms at position -238, -857 and -863, respectively. The G/G, G/A and A/A genotypes at position -238 occurred in 85.8%, 14.2% and 0% CAD patients and 91.8%, 7.9% and 0.3% control subjects, respectively. The G/A polymorphisms at position -238 were significantly associated with CAD when assuming a dominant model of inheritance (OR = 1.87; 95% CI = 1.10-3.20; P = 0.02), and A allele carriers had a significantly increased risk of developing CAD relative to the G allele (OR = 1.74; 95% CI = 1.04-2.92; P = 0.03). However, the polymorphisms at positions -857 and -863 were not associated with CAD. Haplotype-based analysis revealed the CAD and control groups differed significantly in the frequencies of haplotype ACC at positions -238, -857 and -863 (OR = 1.77; 95% CI = 1.05-2.98; P = 0.03). This was confirmed by multivariate analysis after adjusting body mass index and the presence of diabetes and hypertension (OR = 2.06; 95% CI = 1.15-3.68; P = 0.015). Thus, the -238A allele of TNF-α is associated with an increased risk of CAD and could be used as predictor for CAD in Koreans. Further studies are needed to elucidate the clinical implications of these findings.

Clusterin attenuates the development of renal fibrosis.

Upregulation of clusterin occurs in several renal diseases and models of nephrotoxicity, but whether this promotes injury or is a protective reaction to injury is unknown. Here, in the mouse unilateral ureteral obstruction model, obstruction markedly increased the expression of clusterin, plasminogen activator inhibitor-1 (PAI-1), type I collagen, and fibronectin. Compared with wild-type mice, clusterin-deficient mice exhibited higher levels of PAI-1, type I collagen, and fibronectin and accelerated renal fibrosis in response to obstruction. In cultured rat tubular epithelium-like cells, adenovirus-mediated overexpression of clusterin inhibited the expression of TGF-ß-stimulated PAI-1, type I collagen, and fibronectin. Clusterin inhibited TGF-ß-stimulated Smad3 activity via inhibition of Smad3 phosphorylation and its nuclear translocation. Moreover, intrarenal delivery of adenovirus-expressing clusterin upregulated expression of clusterin in tubular epithelium-like cells and attenuated obstruction-induced renal fibrosis. In conclusion, clusterin attenuates renal fibrosis in obstructive nephropathy. These results suggest that upregulation of clusterin during renal injury is a protective response against the development of renal fibrosis.

Alpha-lipoic acid inhibits hepatic PAI-1 expression and fibrosis by inhibiting the TGF-beta signaling pathway.

Accumulating evidence suggests that plasminogen activator inhibitor (PAI)-1 plays an important role in the development of hepatic fibrosis via its involvement in extracellular matrix remodeling. We previously reported that alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, prevents hepatic steatosis by inhibiting the expression of sterol regulatory element binding protein-1c. The aim of the present study was to determine whether ALA prevents hepatic PAI-1 expression and fibrosis through the inhibition of multiple TGF-beta-mediated molecular mediators. We investigated whether ALA inhibited the development of hepatic fibrosis in mice following bile duct ligation (BDL), an established animal model of liver fibrosis. We found that ALA markedly inhibited BDL-induced hepatic fibrosis and PAI-1 expression. We also found that ALA attenuated TGF-beta-stimulated PAI-1 mRNA expression, and inhibited PAI-1 promoter activity in liver cells; this effect was mediated by Smads and the JNK and ERK pathways. The results of the present study indicate that ALA inhibits hepatic PAI-1 expression through inhibition of TGF-beta-mediated molecular mediators, including Smad3, AP1, and Sp1, and prevents the development of BDL-induced hepatic fibrosis. These findings suggest that ALA may have a clinical application in preventing the development and progression of hepatic fibrosis.

The orphan nuclear receptor SHP attenuates renal fibrosis.

The accumulation of extracellular matrix proteins is a common feature of fibrotic kidney diseases. Accumulating evidence suggests that TGF-beta and plasminogen activator inhibitor type 1 (PAI-1) promote the development of renal fibrosis by stimulating the generation and inhibiting the removal of matrix proteins. The small heterodimer partner (SHP) represses PAI-1 expression in the liver by inhibiting TGF-beta signaling, but whether SHP inhibits renal fibrosis is unknown. Here, unilateral ureteral obstruction (UUO) markedly increased the expression of PAI-1, type I collagen, and fibronectin but decreased SHP gene expression. Moreover, in kidneys of SHP-/- mice, the expression of PAI-1, type I collagen, fibronectin and alpha-smooth muscle actin (alpha-SMA) were higher compared with those in kidneys of wild-type mice. In addition, loss of SHP accelerated renal fibrosis after UUO. Adenovirus-mediated overexpression of SHP in cultured rat mesangial cells and renal tubular epithelial cells inhibited TGF-beta-stimulated expression of PAI-1, type I collagen, and fibronectin. SHP inhibited TGF-beta- and Smad3-stimulated PAI-1 promoter activities as well as TGF-beta-stimulated binding of Smad3 to its consensus response element on the PAI-1 promoter. Similarly, in vivo, adenovirus-mediated overexpression of SHP in the kidney inhibited the expression of UUO-induced PAI-1, type I collagen, fibronectin, and alpha-SMA. In summary, SHP attenuates renal fibrosis in obstructive nephropathy, making its pathway a possible therapeutic target for chronic kidney disease.

Forkhead transcription factor FoxO1 inhibits insulin- and transforming growth factor-beta-stimulated plasminogen activator inhibitor-1 expression.

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are considered a risk factor for chronic liver disease in patients with hyperinsulinemia. Insulin increases the expression of PAI-1, and inactivates the forkhead box-containing protein FoxO1. We were interested in whether the inactivation of FoxO1 is involved in the activation of PAI-1 expression under conditions of insulin stimulation. Here, we examined whether adenoviral-mediated expression of a constitutively active form of FoxO1 (Ad-CA-FoxO1) inhibited insulin-stimulated PAI-1 expression in human HepG2 hepatocellular liver carcinoma cells and mouse AML12 hepatocytes. Treatment of cells with insulin increased PAI-1 gene expression, and this effect was abolished by Ad-CA-FoxO1. Insulin also increased the transforming growth factor (TGF)-beta-induced expression of PAI-1 mRNA, and Ad-CA-FoxO1 inhibited this effect. Transient transfection assays using a reporter gene under the control of the PAI-1 promoter revealed that CA-FoxO1 inhibits Smad3-stimulated PAI-1 promoter activity. Taken together, our results indicate that FoxO1 inhibits PAI-1 expression through the inhibition of TGF-beta/Smad-mediated signaling pathways. Our data also suggest that in the hyperinsulinemic state, FoxO1 is inactivated by increased levels of insulin, and does not function as an inhibitor of TGF-beta-induced PAI-1 expression.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of glutamyl-tRNA synthetase (Xoo1504) from Xanthomonas oryzae pv. oryzae.

The gltX gene from Xanthomonas oryzae pv. oryzae (Xoo1504) encodes glutamyl-tRNA synthetase (GluRS), one of the most important enzymes involved in bacterial blight (BB), which causes huge production losses of rice worldwide. GluRS is a class I-type aminoacyl-tRNA synthetase (aaRS) that is primarily responsible for the glutamylation of tRNA(Glu). It plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. As it represents an important target for the development of new antibacterial drugs against BB, determination of the three-dimensional structure of GluRS is essential in order to understand its catalytic mechanism. In order to analyze its structure and function, the gltX gene was cloned and the GluRS enzyme was expressed, purified and then crystallized. A GluRS crystal belonging to the monoclinic space group C2 diffracted to 2.8 A resolution and had unit-cell parameters a = 186.8, b = 108.4, c = 166.1 A, beta = 96.3 degrees . The unit-cell volume of the crystal allowed the presence of six to eight monomers in the asymmetric unit, with a corresponding Matthews coefficient (V(M)) range of 2.70-2.02 A(3) Da(-1) and a solvent-content range of 54.5-39.3%.

Affective Factors That Contribute to the Quality of Life of Juvenile Inmates with Attention-Deficit/Hyperactivity Disorder: A Focus on Items from the Korean Youth Self Report.

OBJECTIVES: This study investigated quality of life in Korean juvenile inmates with attention-deficit/hyperactivity disorder (ADHD) and the impact of behavioral and emotional problems on quality of life. METHODS: In total, 200 inmates were evaluated using the Korean version of the Mini-International Neuropsychiatric Interview (K-MINI) and the Korean version of the Kiddie-Schedule for Affective Disorders and Schizophrenia-Present and Lifetime (K-SADS-PL-K). We extracted the inmates with ADHD and evaluated their quality of life, behavioral problems, and emotional problems with the Pediatric Quality of Life Inventory (PedsQL) and the Korean Youth Self Report (K-YSR) scale. Descriptive statistics, Pearson correlation analysis, and multiple regression analysis were conducted. RESULTS: Among the 200 total inmates, 68 were diagnosed with ADHD by the K-SADS-PL-K. Most of the correlations between PedsQL scores and K-YSR items were significant. Multiple regression analysis showed that PedsQL could be predicted by affective problems (among the DSM-oriented scales of the K-YSR) and attention problems (among the syndrome scales of the K-YSR). CONCLUSION: Our results demonstrate that, among juvenile inmates with ADHD, quality of life was negatively correlated with most behavioral and emotional problems. Meanwhile, the significant influence of affective and attention problems on inmates' quality of life suggests the necessity of comprehensive treatments for this group.

Endoplasmic reticulum stress-induced activation of activating transcription factor 6 decreases insulin gene expression via up-regulation of orphan nuclear receptor small heterodimer partner.

The highly developed endoplasmic reticulum (ER) structure of pancreatic beta-cells is a key factor in beta-cell function. Here we examined whether ER stress-induced activation of activating transcription factor (ATF)-6 impairs insulin gene expression via up-regulation of the orphan nuclear receptor small heterodimer partner (SHP; NR0B2), which has been shown to play a role in beta-cell dysfunction. We examined whether ER stress decreases insulin gene expression, and this process is mediated by ATF6. A small interfering RNA that targeted SHP was used to determine whether the effect of ATF6 on insulin gene expression is mediated by SHP. We also measured the expression level of ATF6 in pancreatic islets in Otsuka Long Evans Tokushima Fatty rats, a rodent model of type 2 diabetes. High glucose concentration (30 mmol/liter glucose) increased ER stress in INS-1 cells. ER stress induced by tunicamycin, thapsigargin, or dithiotreitol decreased insulin gene transcription. ATF6 inhibited insulin promoter activity, whereas X-box binding protein-1 and ATF4 did not. Adenovirus-mediated overexpression of active form of ATF6 in INS-1 cells impaired insulin gene expression and secretion. ATF6 also down-regulated pancreatic duodenal homeobox factor-1 and RIPE3b1/MafA gene expression and repressed the cooperative action of pancreatic duodenal homeobox factor-1, RIPE3b1/MafA, and beta-cell E box transactivator 2 in stimulating insulin transcription. The ATF6-induced suppression of insulin gene expression was associated with up-regulation of SHP gene expression. Finally, we found that expression of ATF6 was increased in the pancreatic islets of diabetic Otsuka Long Evans Tokushima Fatty rats, compared with their lean, nondiabetic counterparts, Long-Evans Tokushima Otsuka rats. Collectively, this study shows that ER stress-induced activation of ATF6 plays an important role in the development of beta-cell dysfunction.

Glucotoxicity in the INS-1 rat insulinoma cell line is mediated by the orphan nuclear receptor small heterodimer partner.

Prolonged elevations of glucose concentration have deleterious effects on beta-cell function. One of the hallmarks of such glucotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that inhibits nuclear receptor signaling in diverse metabolic pathways. In this study, we found that sustained culture of INS-1 cells at high glucose concentrations leads to an increase in SHP mRNA expression, followed by a decrease in insulin gene expression. Inhibition of endogenous SHP gene expression by small interfering RNA partially restored high-glucose-induced suppression of the insulin gene. Adenovirus-mediated overexpression of SHP in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. SHP downregulates insulin gene expression via two mechanisms: by downregulating PDX-1 and MafA gene expression and by inhibiting p300-mediated pancreatic duodenal homeobox factor 1-and BETA2-dependent transcriptional activity from the insulin promoter. Finally, the pancreatic islets of diabetic OLETF rats express SHP mRNA at higher levels than the islets from LETO rats. These results collectively suggest that SHP plays an important role in the development of beta-cell dysfunction induced by glucotoxicity.

Expression, crystallization and preliminary X-ray crystallographic analysis of XometC, a cystathionine gamma-lyase-like protein from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice (Oryza sativa L.), one of the most devastating diseases of rice in most rice-growing countries. XometC, a cystathionine gamma-lyase (CGL) like protein that is an antibacterial drug-target protein against Xoo, was cloned, expressed, purified and crystallized. CGL catalyzes the second step in the reverse-transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine. Crystals of two different shapes, plate-shaped and pyramid-shaped, diffracted to 2.9 and 3.2 A resolution and belonged to the primitive orthogonal space group P2(1)2(1)2(1) and the tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = 73.0, b = 144.9, c = 152.3 A and a = b = 78.2, c = 300.7 A, respectively. For the P2(1)2(1)2(1) crystals, three or four monomers exist in the asymmetric unit with a corresponding V(M) of 3.02 or 2.26 A(3) Da(-1) and a solvent content of 59.3 or 45.7%. For the P4(1) (or P4(3)) crystals, four or five monomers exist in the asymmetric unit with a corresponding V(M) of 2.59 or 2.09 A(3) Da(-1) and a solvent content of 52.5 or 40.6%.

Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo2316, a predicted 6-phosphogluconolactonase, from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is one of the most devastating diseases of rice (Oryza sativa L.) in many rice-growing countries. The coding sequence of Xoo2316 (a predicted 6-phosphogluconolactonase; 6PGL) from Xoo was cloned and expressed in Escherichia coli. 6PGL is an enzyme that is involved in the second step of the pentose phosphate pathway, which is essential for the synthesis of nucleotide sugars and NADPH, the main source of reducing power. The protein was purified and crystallized in order to elucidate the molecular basis for its enzymatic reaction. Native crystals diffracted to 2.4 A resolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 40.0, b = 65.1, c = 78.8 A. A monomer exists in the asymmetric unit with a corresponding V(M) of 1.93 A(3) Da(-1) and a solvent content of 36.5%.

Expression, crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Xanthomonas oryzae pv. oryzae.

Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides; this process is crucial for cell survival. As it is an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), PDF from Xoo was cloned, expressed, purified and crystallized. Native PDF crystals diffracted to 2.7 A resolution and belonged to the hexagonal space group P6(1)22, with unit-cell parameters a = b = 59.0, c = 266.3 A. One monomer is present in the asymmetric unit, with a corresponding crystal volume per protein weight of 3.50 A(3) Da(-1) and a solvent content of 64.9%.

Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo0352, D-alanine-D-alanine ligase A, from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. D-Alanine-D-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in D-alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of D-alanyl-D-alanine from two D-alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 A resolution and belonged to the primitive tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 83.0, c = 97.6 A. There is one molecule in the asymmetric unit, with a corresponding V(M) of 1.88 A(3) Da(-1) and a solvent content of 34.6%. The initial structure was determined by molecular replacement using D-alanine-D-alanine ligase from Staphylococcus aureus (PDB code 2i87) as a template model.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of 3-dehydroquinate synthase, Xoo1243, from Xanthomonas oryzae pv. oryzae.

The disease bacterial blight results in serious production losses of rice in Asian countries. The aroB gene encoding dehydroquinate synthase (DHQS), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo). DHQS plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. The aroB gene (Xoo1243) was cloned from Xoo and the corresponding DHQS protein was subsequently overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion method and yielded crystals that diffracted to 2.5 A resolution. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 118.2, c = 98.2 A. According to a Matthews coefficient calculation, the crystal contained two molecules in the asymmetric unit, with a corresponding V(M) of 2.06 A(3) Da(-1) and a solvent content of 40.4%.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of malonyl-CoA-acyl carrier protein transacylase (FabD) from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice, which is one of the most devastating diseases in rice-cultivating countries. The Xoo0880 (fabD) gene coding for a malonyl-CoA-acyl carrier protein transacylase (MCAT) from Xoo was cloned and expressed in Escherichia coli. MCAT is an essential enzyme that catalyzes a key reaction of fatty-acid synthesis in bacteria and plants: the conversion of malonyl-CoA to malonyl-acyl carrier protein. The FabD enzyme was purified and crystallized in order to elucidate its three-dimensional structure and to determine its enzymatic reaction mechanism and biological importance. The crystal obtained diffracted to 1.9 A resolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 41.4, b = 74.6, c = 98.5 A. According to Matthews coefficient calculations, the crystallographic structure contains only one monomeric unit in the asymmetric unit with a V(M) of 2.21 A(3) Da(-1) and a solvent content of 44.3%.

Effects of PGC-1alpha on TNF-alpha-induced MCP-1 and VCAM-1 expression and NF-kappaB activation in human aortic smooth muscle and endothelial cells.

Increased oxidative stress in vascular cells is implicated in the pathogenesis of atherosclerosis. Reactive oxygen species (ROS) induce vascular inflammation via the proinflammatory cytokine/NF-kappaB pathway. Several lines of evidence suggest that peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1alpha) is an important regulator of intracellular ROS levels. However, no studies have examined the effects of PGC-1alpha on this process. We investigated the effects of PGC-1alpha on inflammatory molecule expression and activity of the redox-sensitive transcription factor, NF-kappaB, in vascular cells. PGC-1alpha expressed in human aortic smooth (HASMCs) and endothelial cells (HAECs) is upregulated by AMP-activated protein kinase activators, including metformin, rosiglitazone and alpha-lipoic acid. Tumor necrosis factor-alpha (TNF-alpha), a major proinflammatory factor in the development of vascular inflammation, stimulates intracellular ROS production through an increase in both mitochondrial ROS and NAD(P)H oxidase activity. Adenovirus-mediated overexpression of the PGC-1alpha gene in HASMCs and HAECs leads to a significant reduction in intracellular and mitochondrial ROS production as well as NAD(P)H oxidase activity. Consequently, NF-kappaB activity and MCP-1 and VCAM-1 induced by TNF-alpha are suppressed. Our data support the possibility that agents stimulating PGC-1alpha expression in the vasculature aid in preventing the development of atherosclerosis.

Alpha-lipoic acid inhibits matrix metalloproteinase-9 expression by inhibiting NF-kappaB transcriptional activity.

The migration of vascular smooth muscle cells (VSMCs) into the intima, an important step in injury-induced neointimal hyperplasia, requires the activation of nuclear factor-kappaB (NF-kappaB) and the consequent up-regulation of matrix metalloproteinase-9 (MMP-9). This study was undertaken to test for a possible effect of alpha-lipoic acid (ALA), a potent inhibitor of NF-kappaB, on MMP-9 expression. ALA inhibited high-glucose- and TNF-alpha-stimulated VSMC migrations in vitro. It also inhibited high-glucose- and TNF-alpha-induced increases in MMP-9 expression. The activity of MMP-9-promoter constructs with mutations in the NF-kappaB binding site was not inhibited by ALA, indicating an involvement of the NF-kappaB signaling pathway in the ALA-specific inhibition of MMP-9. These data suggest the possibility that ALA may be useful for the prevention of neointimal hyperplasia after angioplasty, by inhibiting the NF-kappaB/MMP-9 pathway, especially with hyperglycemia.