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Membrane raft-lysosome redox signalling platforms in coronary endothelial dysfunction induced by adipokine visfatin.

ABSTRACT: The adipokine visfatin, produced during obesity, has been reported to participate in the development of cardiovascular disease associated with metabolic syndrome. The present study was designed to test a hypothesis that visfatin causes coronary endothelial dysfunction through lysosome trafficking and fusion to cell membranes, membrane raft (MR) clustering, and formation of redox signalosomes.By using confocal microscopy, it was found that visfatin, but not adiponectin, stimulated NADPH oxidase (NOX) subunits, gp91(phox) aggregation in MR clusters and p47(phox) translocation to these MR clusters in bovine coronary arterial endothelial cells (CAECs), leading to activation of NOX with a 2.5-fold increase in O(2)(·-) production. A signalling lipid, ceramide, was found to be enriched in such membrane MR-NOX complexes of CAECs. Lysosomal fluorescent dye (FM1-43) quenching and de-quenching revealed that visfatin induced the fusion of lysosomes to cell membranes and incorporation of acid sphingomyelinase and its product, ceramide, in such MR-NOX signalling platforms. Functionally, visfatin significantly attenuated endothelium-dependent vasodilation in small coronary arteries (by 80%), which was blocked by lysosomal function inhibitor and MR disruptors.These results suggest that lysosome-associated molecular trafficking and consequent ceramide accumulation in cell membrane may mediate the assembly of NOX subunits and their activation in response to adipokine visfatin in CAECs, thereby producing endothelial dysfunction in coronary circulation.

SUBMITTER: Xia M

PROVIDER: S-EPMC3020127 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Membrane raft-lysosome redox signalling platforms in coronary endothelial dysfunction induced by adipokine visfatin.

Xia Min M Zhang Chun C Boini Krishna M KM Thacker Audrey M AM Li Pin-Lan PL

Cardiovascular research 20100907 2

<h4>Aims</h4>The adipokine visfatin, produced during obesity, has been reported to participate in the development of cardiovascular disease associated with metabolic syndrome. The present study was designed to test a hypothesis that visfatin causes coronary endothelial dysfunction through lysosome trafficking and fusion to cell membranes, membrane raft (MR) clustering, and formation of redox signalosomes.<h4>Methods and results</h4>By using confocal microscopy, it was found that visfatin, but no ...[more]

PMID: 20823276

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Project description:Recent studies have indicated that the inflammasome plays a critical role in the pathogenesis of vascular diseases. However, the pathological relevance of this inflammasome activation, particularly in vascular cells, remains largely unknown. Here, we investigated the role of endothelial (Nucleotide-binding Oligomerization Domain) NOD-like receptor family pyrin domain containing three (Nlrp3) inflammasomes in modulating inter-endothelial junction proteins, which are associated with endothelial barrier dysfunction, an early onset of obesity-associated endothelial injury. Our findings demonstrate that the activation of Nlrp3 inflammasome by visfatin markedly decreased the expression of inter-endothelial junction proteins including tight junction proteins ZO-1, ZO-2 and occludin, and adherens junction protein VE-cadherin in cultured mouse vascular endothelial (VE) cell monolayers. Such visfatin-induced down-regulation of junction proteins in endothelial cells was attributed to high mobility group box protein 1 (HMGB1) release derived from endothelial inflammasome-dependent caspase-1 activity. Similarly, in the coronary arteries of wild-type mice, high-fat diet (HFD) treatment caused a down-regulation of inter-endothelial junction proteins ZO-1, ZO-2, occludin and VE-cadherin, which was accompanied with enhanced inflammasome activation and HMGB1 expression in the endothelium as well as transmigration of CD43(+) T cells into the coronary arterial wall. In contrast, all these HFD-induced alterations in coronary arteries were prevented in mice with Nlrp3 gene deletion. Taken together, these data strongly suggest that the activation of endothelial Nlrp3 inflammasomes as a result of the increased actions of injurious adipokines such as visfatin produces HMGB1, which act in paracrine or autocrine fashion to disrupt inter-endothelial junctions and increase paracellular permeability of the endothelium contributing to the early onset of endothelial injury during metabolic disorders such as obesity or high-fat/cholesterol diet.

Project description:Visfatin/extracellular-nicotinamide-phosphoribosyltranferase-(eNampt) is a multifaceted adipokine enhanced in type-2-diabetes and obesity. Visfatin/eNampt cause in vitro endothelial dysfunction and vascular inflammation, although whether the same effects are achieved in vivo is unknown. Toll-like receptor-4 (TLR4), a main surface pattern recognition receptor of innate immune system is a potential target for visfatin/eNampt. We studied its capacity to generate vascular dysfunction in vivo, focusing on TLR4 role and downstream activation of nod-like-receptor-protein-3 (NLRP3)-inflammasome. 4 month-old C57BL/6 mice were exposed to 7 days infusion of visfatin/eNampt, alone or together with FK 866 (Nampt enzymatic inhibitor), CLI 095 (TLR4 blocker), MCC 950 (NLRP3-inflammasome inhibitor), or anakinra (interleukin(IL)-1-receptor antagonist). Endothelial dysfunction was tested in isolated microvessels. In human umbilical endothelial cells (HUVEC), proteins related to the NLRP3-inflammasome phosphorylated p-65, NLRP3, caspase-1, pro-IL-1?, and mature IL-1? were determined by Western blot, while the inflammasome related apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC-specks) was studied by immunofluorescence. Impaired endothelium-dependent relaxations were observed in isolated mesenteric microvessels from visfatin/eNampt-infused mice. This effect was attenuated by co-treatment with FK 866 or CLI 095, supporting a role for Nampt enzymatic activity and TLR4 activation. Moreover, cultured HUVEC exposed to visfatin/eNampt showed higher expression and activation of NLRP3-inflammasome. Again, this effect relied on Nampt enzymatic activity and TLR4 activation, and it was abrogated by the inflammasome assembly blockade with MCC 950. The endothelial dysfunction evoked by visfatin/eNampt infusion in vivo was also sensitive to both MCC 950 and anakinra treatments, suggesting that the NLRP3-inflammasome-driven tissular release of IL-1? is the final mediator of endothelial damage. We conclude that Visfatin/eNampt produces in vivo vascular dysfunction in mice by a Nampt-dependent TLR4-mediated pathway, involving NLRP3-inflammasome and paracrine IL-1?. Thus, those targets may become therapeutic strategies for attenuating the adipokine-mediated vascular dysfunction associated to obesity and/or type-2-diabetes.

Project description:INTRODUCTION: Visfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA). With a homodimeric conformation, it exerts nicotinamide phosphoribosyltransferase (Nampt) enzymatic activity, essential for nicotinamide adenine dinucleotide biosynthesis. We examined the tissular origin and conformation of visfatin/Nampt in human OA joints and investigated the role of visfatin/Nampt in chondrocytes and osteoblasts by studying Nampt enzymatic activity. METHODS: Synovium, cartilage and subchondral bone from human OA joints were used for protein extraction or incubated for 24 hours in serum-free media (conditioned media), and synovial fluid was obtained from OA patients. Visfatin/Nampt expression in tissular extracts and conditioned media was evaluated by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Nampt activity was assessed in OA synovium by colorimetric assay. Primary cultures of murine chondrocytes and osteoblasts were stimulated with visfatin/Nampt and pretreated or not with APO866, a pharmacologic inhibitor of Nampt activity. The effect on cytokines, chemokines, growth factors and hypertrophic markers expression was examined by quantitative reverse transcriptase polymerase chain reaction and/or ELISA. RESULTS: In tissular explants, conditioned media and synovial fluid, visfatin/Nampt was found as a homodimer, corresponding to the enzymatically active conformation. All human OA joint tissues released visfatin/Nampt (synovium: 628?±?106 ng/g tissue; subchondral bone: 195?±?26 ng/g tissue; cartilage: 152?±?46 ng/g tissue), with significantly higher level for synovium (P <0.0005). Nampt activity was identified ex vivo in synovium. In vitro, visfatin/Nampt significantly induced the expression of interleukin 6, keratinocyte chemoattractant and monocyte chemoattractant protein 1 in chondrocytes and osteoblasts. APO866 decreased the mRNA and protein levels of these pro-inflammatory cytokines in the two cell types (up to 94% and 63% inhibition, respectively). Levels of growth factors (vascular endothelial growth factor, transforming growth factor ?) and hypertrophic genes were unchanged with treatment. CONCLUSION: Visfatin/Nampt is released by all human OA tissues in a dimeric enzymatically active conformation and mostly by the synovium, which displays Nampt activity. The Nampt activity of visfatin is involved in chondrocyte and osteoblast activation, so targeting this enzymatic activity to disrupt joint tissue interactions may be novel in OA therapy.

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Membrane raft-lysosome redox signalling platforms in coronary endothelial dysfunction induced by adipokine visfatin.

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Membrane raft-lysosome redox signalling platforms in coronary endothelial dysfunction induced by adipokine visfatin.

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