Inhibition of NF-κB prevents high glucose-induced proliferation and plasminogen activator inhibitor-1 expression in vascular smooth muscle cells

Recent epidemiologic studies clearly showed that early intensive glucose control has a legacy effect for preventing diabetic macrovascular complications. However, the cellular and molecular processes by which high glucose leads to macrovascular complications are poorly understood. Vascular smooth muscle cell (VSMC) dysfunction due to high glucose is a characteristic of diabetic vascular complications. Activation of nuclear factor-κB (NF-κB) may play a key role in the regulation of inflammation and proliferation of VSMCs. We examined whether VSMC proliferation and plasminogen activator inhibitor-1 (PAI-1) expression induced by high glucose were mediated by NF-κB activation. Also, we determined whether selective inhibition of NF-κB would inhibit proliferation and PAI-1 expression in VSMCs. VSMCs of the aorta of male SD rats were treated with various concentrations of glucose (5.6, 11.1, 16.7, and 22.2 mM) with or without an inhibitor of NF-κB or expression of a recombinant adenovirus vector encoding an IκB-α mutant (Ad-IκBαM). VSMC proliferation was examined using an MTT assay. PAI-1 expression was assayed by real-time PCR and PAI-1 protein in the media was measured by ELISA. NF-κB activation was determined by immunohistochemical staining, NF-κB reporter assay, and immunoblotting. We found that glucose stimulated VSMC proliferation and PAI-1 expression in a dose-dependent manner up to 22.2 mM. High glucose (22.2 mM) alone induced an increase in NF-κB activity. Treatment with inhibitors of NF-κB such as MG132, PDTC or expression of Ad-IκB-αM in VSMCs prevented VSMC proliferation and PAI-1 expression induced by high glucose. In conclusion, inhibition of NF-κB activity prevented high glucose-induced VSMC proliferation and PAI-1 expression.     

Preeclampsia serum-induced collagen I expression and intracellular calcium levels in arterial smooth muscle cells are mediated by the PLC-γ1 pathway

In women with preeclampsia (PE), endothelial cell (EC) dysfunction can lead to altered secretion of paracrine factors that induce peripheral vasoconstriction and proteinuria. This study examined the hypothesis that PE sera may directly or indirectly, through human umbilical vein ECs (HUVECs), stimulate phospholipase C-γ1-1,4,5-trisphosphate (PLC-γ1-IP₃) signaling, thereby increasing protein kinase C-α (PKC-α) activity, collagen I expression and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in human umbilical artery smooth muscle cells (HUASMCs). HUASMCs and HUVECs were cocultured with normal or PE sera before PLC-γ1 silencing. Increased PLC-γ1 and IP₃ receptor (IP₃R) phosphorylation was observed in cocultured HUASMCs stimulated with PE sera (P<0.05). In addition, PE serum significantly increased HUASMC viability and reduced their apoptosis (P<0.05); these effects were abrogated with PLC-γ1 silencing. Compared with normal sera, PE sera increased [Ca²⁺]_i in cocultured HUASMCs (P<0.05), which was inhibited by PLC-γ1 and IP₃R silencing. Finally, PE sera-induced PKC-α activity and collagen I expression was inhibited by PLC-γ1 small interfering RNA (siRNA) (P<0.05). These results suggest that vasoactive substances in the PE serum may induce deposition in the extracellular matrix through the activation of PLC-γ1, which may in turn result in thickening and hardening of the placental vascular wall, placental blood supply shortage, fetal hypoxia–ischemia and intrauterine growth retardation or intrauterine fetal death. PE sera increased [Ca²⁺]_i and induced PKC-α activation and collagen I expression in cocultured HUASMCs via the PLC-γ1 pathway.     

Extracellular HIV-1 Tat up-regulates expression of matrix metalloproteinase-9 via a MAPK-NF-��B dependent pathway in human astrocytes

The infiltration of monocytes into the CNS represents one of the early steps to inflammatory events in AIDS-related encephalitis and dementia. Increased activity of selected matrix metalloproteinases (MMPs) such as MMP-9 impairs the integrity of blood-brain barrier leading to enhanced monocyte infiltration into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of MMP-9 in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein levels of MMP-9, as measured by Western blot analysis, zymography and an ELISA. Treatment of CRT-MG cells with HIV-1 Tat protein markedly increased mRNA levels of MMP-9, as analyzed by RT-PCR. Pretreatment of CRT-MG cells with NF-κB inhibitors led to decrease in Tat-induced protein and mRNA expression of MMP-9. Pretreatment of CRT-MG cells with MAPK inhibitors suppressed Tat-induced MMP-9 expression. Furthermore, HIV-1 Tat-induced expression of MMP-9 was significantly inhibited by neutralization of TNF-α, but not IL-1β and IL-6. Taken together, our results indicate that HIV-1 Tat can up-regulate expression of MMP-9 via MAPK-NF-κB-dependent mechanisms as well as Tat-induced TNF-α production in astrocytes.     

Synovial fluid of patients with rheumatoid arthritis induces ��-smooth muscle actin in human adipose tissue-derived mesenchymal stem cells through a TGF-��1-dependent mechanism

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. Transforming growth factor-β1 (TGF-β1) is a secreted protein that promotes differentiation of synovial fibroblasts to α-smooth muscle actin (α-SMA)-positive myofibroblasts to repair the damaged joints. Synovial fluid from patients with RA (RA-SF) induced expression of α-SMA in human adipose tissue-derived mesenchymal stem cells (hASCs). RA-SF-induced α-SMA expression was abrogated by immunodepletion of TGF-β1 from RA-SF with anti-TGF-β1 antibody. Furthermore, pretreatment of hASCs with the TGF-β type I receptor inhibitor SB431542 or lentiviral small hairpin RNA-mediated silencing of TGF-β type I receptor expression in hASCs blocked RA-SF-induced α-SMA expression. Small interfering RNA-mediated silencing of Smad2 or adenoviral overexpression of Smad7 (an inhibitory Smad isoform) completely inhibited RA-SF-stimulated α-SMA expression. These results suggest that TGF-β1 plays a pivotal role in RA-SF-induced differentiation of hASCs to α-SMA-positive cells.     

Toll-like receptor 4 on islet β cells senses expression changes in high-mobility group box 1 and contributes to the initiation of type 1 diabetes

Type 1 diabetes mellitus is caused by the autoimmune destruction of β cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were β cells; few α cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on β cells and that HMGB1 may signal via TLR4 to selectively damage β cells rather than α cells during the development of type 1 diabetes mellitus.     

Quantitative evaluation of the biosynthetic pathways leading to δ-aminolevulinic acid from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus by analysis of 13C-labeled coproporphyrinogen III biosynthesized from [2-13C]glycine, [1-13C]acetate, and [2-13C]acetate using 13C NMR spectroscopy

The biosynthetic pathways leading to δ-aminolevulinic acid (ALA) from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus were quantitatively evaluated by means of feeding experiments with [2-¹³C]glycine, sodium [1-¹³C]acetate, and sodium [2-¹³C]acetate, followed by analysis of the labeling patterns of coproporphyrinogen III (Copro’gen III) (biosynthesized from ALA) using ¹³C NMR spectroscopy. Two biosynthetic pathways leading to ALA from glycine via the C5 pathway were identified: i.e., transformation of glycine to l-serine catalyzed by glycine hydroxymethyltransferase, and glycine synthase-catalyzed catabolism of glycine to N ⁵,N ¹⁰-methylene-tetrahydrofolic acid (THF), which reacts with another molecule of glycine to afford l-serine. l-Serine is transformed to acetyl-CoA via pyruvic acid. Acetyl-CoA enters the tricarboxylic acid cycle, affording 2-oxoglutaric acid, which in turn is transformed to l-glutamic acid. The l-glutamic acid enters the C5 pathway, affording ALA in A. hyalinus. A ¹³C NMR spectroscopic comparison of the labeling patterns of Copro’gen III obtained after feeding of [2-¹³C]glycine, sodium [1-¹³C]acetate, and sodium [2-¹³C]acetate showed that [2-¹³C]glycine transformation and [2-¹³C]glycine catabolism in A. hyalinus proceed in the ratio of 52 and 48 %. The reaction of [2-¹³C]glycine and N ⁵,N ¹⁰-methylene-THF, that of glycine and N ⁵,N ¹⁰-[methylene-¹³C]methylene-THF generated from the [2-¹³C]glycine catabolism, and that of [2-¹³C]glycine and N ⁵,N ¹⁰-[methylene-¹³C]methylene-THF transformed the fed [2-¹³C]glycine to [1-¹³C]acetyl-CoA, [2-¹³C]acetyl-CoA, and [1,2-¹³C₂]acetyl-CoA in the ratios of 42, 37, and 21 %, respectively. These labeled acetyl-CoAs were then incorporated into ALA. Our results provide a quantitative picture of the pathways of biosynthetic transformation to ALA from glycine in A. hyalinus.