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Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells.

ABSTRACT: Diabetes mellitus leads to a high incidence of several so-called complications, sharing similar pathophysiological features in several territories. Previous reports points at early nonenzymatic glycosylation products (Amadori adducts) as mediators of diabetic vascular complications. In the present study, we analysed a possible role for Amadori adducts as stimulators of proinflammatory pathways in human peritoneal mesothelial cells (HPMCs). Cultured HPMCs isolated from 13 different patients (mean age 38.7+/-16 years) were exposed to different Amadori adducts, that is, highly glycated haemoglobin (10 nM) and glycated bovine serum albumin (0.25 mg ml(-1)), as well as to their respective low glycosylation controls. Amadori adducts, but not their respective controls, elicited a marked increase of NF-kappaB activation, as determined by electromobility shift assays and transient transfection experiments. Additionally, Amadori adducts significantly increased the production of NF-kappaB-related proinflammatory molecules, including cytokines, such as TNF-alpha, IL-1beta or IL-6, and enzymes, such as cyclooxygenase-2 and inducible nitric oxide (NO) synthase, this latter leading to the release of NO by HPMCs. The effects of Amadori adducts were mediated by different reactive oxygen and nitrosative species (e.g. superoxide anions, hydroxyl radicals, and peroxynitrite), as they were blunted by coincubation with the appropriate scavengers. Furthermore, NO generated upon exposure to Amadori adducts further stimulated NF-kappaB activation, either directly or after combination with superoxide anions to form peroxynitrite. We conclude that Amadori adducts can favour peritoneal inflammation by exacerbating changes in NO synthesis pathway and triggering NF-kappaB-related proinflammatory signals in human mesothelial cells.

SUBMITTER: Nevado J

PROVIDER: S-EPMC1576262 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells.

Nevado Julián J Peiró Concepción C Vallejo Susana S El-Assar Mariam M Lafuente Nuria N Matesanz Nuria N Azcutia Verónica V Cercas Elena E Sánchez-Ferrer Carlos F CF Rodríguez-Mañas Leocadio L

British journal of pharmacology 20050901 2

Diabetes mellitus leads to a high incidence of several so-called complications, sharing similar pathophysiological features in several territories. Previous reports points at early nonenzymatic glycosylation products (Amadori adducts) as mediators of diabetic vascular complications. In the present study, we analysed a possible role for Amadori adducts as stimulators of proinflammatory pathways in human peritoneal mesothelial cells (HPMCs). Cultured HPMCs isolated from 13 different patients (mean ...[more]

PMID: 15997235

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Project description:Patients undergoing continuous ambulatory peritoneal dialysis are classified according to their peritoneal permeability as low transporter (low solute permeability) or High transporter (high solute permeability). Factors that determine the differences in permeability between them have not been fully disclosed. We investigated morphological features of cultured human peritoneal mesothelial cells from low or high transporter patients and its response to All trans retinoic Acid (ATRA, vitamin A active metabolite), as compared to non-uremic human peritoneal mesothelial cells. Control cells were isolated from human omentum. High or low transporter cells were obtained from dialysis effluents. Cells were cultured in media containing ATRA (0, 50, 100 or 200 nM). We studied length and distribution of microvilli and cilia (scanning electron microscopy), epithelial (cytokeratin, claudin-1, ZO-1 and occludin) and mesenchymal (vimentin and ?-smooth muscle actin) transition markers by immunofluorescence and Western blot, and transforming growth factor ?1 expression by Western blot. Low and high transporter exhibited hypertrophic cells, reduction in claudin-1, occludin and ZO-1 expression, cytokeratin and vimentin disorganization and positive ?-smooth muscle actin label. Vimentin, ?-smooth muscle actin and transforming growth factor-?1 were overexpressed in low transporter. Ciliated cells were diminished in low and high transporters. Microvilli number and length were severely reduced in high transporter. ATRA reduced hypertrophic cells number in low transporter. It also improved cytokeratin and vimentin organization, decreased vimentin and ?-smooth muscle actin expression, and increased claudin 1, occludin and ZO-1 expression, in low and high transporter. In low transporter, ATRA reduced transforming growth factor-?1 expression. ATRA augmented percentage of ciliated cells in low and high transporter. It also augmented cilia length in high transporter. Alterations in structure, epithelial mesenchymal markers and transforming growth factor-?1 expression were differential between low and high transporter. Beneficial effects of ATRA were improved human peritoneal mesothelial cells morphology tending to normalize structures.

Project description:Peritoneal mesothelial cells are uniquely located to regulate cellular events in the peritoneal cavity and are an important source for various cytokines and growth factors. This study was conducted to analyze the capacity of human peritoneal mesothelial cells (HPMCs) to synthesize and release basic fibroblast growth factor (bFGF) and to characterize its regulation by inflammatory cytokines. HPMCs constitutively synthesized and released considerable amounts of bFGF as detected by a specific immunoassay. Almost 80% of bFGF (1547 +/- 173 pg/10(5) cells) was localized intracellularly. Approximately 20% of the bFGF (357 +/- 27 pg/10(5) cells) was associated with extracellular matrix components on the HPMC surface. Small amounts of bFGF (<1%) were detectable in tissue culture supernatants (8.4 +/- 1.4 pg/10(5) cells). Treatment of HPMCs with interleukin-1beta (IL-1beta; 1 ng/ml) resulted in a significant increase in bFGF production. The intracellular bFGF content showed a rapid but only transient increase, which was significant above background levels after 24 hours (41% increase; P < 0.05). This increase in intracellular bFGF concentration was associated with an induction of the release of bFGF. Within 96 hours, the release of bFGF to the cell surface and into the supernatant increased by 58% (564 +/- 52.4 pg/10(5) cells; P < 0.01) and by 214% (26.4 +/- 3.2 pg/10(5) cells; P < 0.001), respectively. Neither tumor necrosis factor-alpha nor interferon-gamma affected bFGF synthesis by HPMCs. Stimulation of HPMCs with IL-1beta increased steady-state levels of bFGF-specific mRNA. Immunohistochemical analyses of peritoneal tissue revealed constitutive expression of bFGF by HPMCs. This in situ expression proved to be most pronounced in areas of serosal inflammation in activated HPMCs. Our study demonstrates that HPMCs synthesize and release significant amounts of bFGF and that the expression of this growth factor is significantly up-regulated by the proinflammatory cytokine IL-1beta. The data support the view that HPMCs are key regulators of abdominal disease processes such as peritonitis, peritoneal fibrosis, or peritoneal tumor metastasis.

Dataset Information

Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells.

Publications

Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells.

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