Expression of endothelin-converting enzyme, endothelin-1 and endothelin receptors at the site of percutaneous coronary intervention in humans

OBJECTIVE: The repair process at the site of injury after percutaneous coronary intervention (PCI) is dominated by neointimal formation composed mainly of smooth muscle cells (SMC). Endothelin-1 (ET-1) is a powerful vasoconstrictor and SMC mitogen. Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin processing. The effects of ET-1 are mediated by binding to endothelin type A (ETA) and endothelin type B (ETB) receptors. The ligand/receptor/ligand-producing system (ET system) could be involved in the pathogenesis of neointimal formation in humans. METHODS: Fifteen post-PCI sites obtained at autopsy and eight atherectomy specimens obtained from restenotic sites were investigated using immunohistochemical single and double staining techniques. Frozen sections were stained with antibodies against ECE, ET-1, ETA and ETB receptors, SMC, macrophages and endothelial cells. RESULTS: At the early stage, less than 3 months after PCI, neointimal SMC were positive for ECE, ET-1, ETA and ETB receptors. The expression of ECE, ET-1, ETA and ETB receptors in these neointimal SMC decreased markedly from 6 months onwards. The ECE, ET-1, ETA and ETB receptor-positive cell areas were significantly (P < 0.005) greater in the first 3 months after PCI compared with 6 months or more after PCI. Atherectomy specimens also showed similar positivity. CONCLUSIONS: These observations strongly suggest that the expression of ECE, ET-1, ETA and ETB receptors is enhanced in neointimal SMC at early stages after PCI injury in human coronary arteries. The increased expression of the ET system may contribute to SMC proliferation/migration and vasoconstriction in human post-PCI coronary lesions.     

Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1.

The endothelins (ETs) form a group of three vasoactive peptides (ET-1, ET-2, and ET-3) for which two types of cellular receptors have been identified, types A and B ET receptors (ETA and ETB receptors, respectively). To address possible targets for ETs within the liver, we isolated the four principal liver cell populations and placed them in short-term primary culture. By ligand-binding assay and mRNA levels, expression of ET receptors was greatest on hepatic lipocytes (Ito cells or fat-storing cells), which are perisinusoidal cells exhibiting features of smooth muscle cells. Moreover, lipocytes expressed both ETA and ETB receptors. The mRNA for ETB receptor, but not for ETA receptor, was detectable in sinusoidal endothelial cells and Kupffer cells; neither mRNA was detectable in hepatocytes. Both ET-1 and ET-3 elicited contraction of activated lipocytes cultured on collagen lattices; the EC50 value for ET-1 was 3 +/- 1 nM and for ET-3 was 17 +/- 12 nM. In cell isolates from injured liver (after administration of carbon tetrachloride), expression of ET receptors was unchanged. However, mRNA for ET-1 was significantly increased in activated lipocytes, suggesting an autocrine loop for the initiation of lipocyte contraction. The findings imply that ET-1 may play a role in regulating sinusoidal perfusion through its effect on lipocytes, particularly in injury states.     

Role of endothelin-1/endothelin receptor system in endotoxic shock rats.

Endothelin (ET)-1, a potent vasoconstrictor peptide derived from the endothelium, is markedly increased in endotoxic shock, although the pathophysiological role of ET-1 under septic conditions remains obscure. To delineate the role of ET-1 and its receptor subtype in endotoxic shock, we here attempted to determine the changes of circulating levels of ET-1 and its biosynthetic intermediate big ET-1 in endotoxic shock rats, to evaluate the gene expression of ET-1 as well as the ET-1 receptor subtypes (ETA and ETB) in the heart, lung and liver, and to study the effects of ET receptor antagonists on systemic arterial blood pressure, heart rate and survival rate. Administration of bacterial lipopolysaccharide (LPS) caused profound hypotension, increased heart rate and death, and these effects were blocked by a nonselective ETA/ETB receptor antagonist (TAK044), but not by an ETA selective antagonist (BQ123). Administration of exogenous ET-1 caused a profound pressor response in control rats, but not in the LPS-pretreated rats. Injection of LPS caused marked elevation of plasma levels of both ET-1 and big ET-1, which were not affected by treatment with either ET receptor antagonist. Administration of LPS caused up-regulation of ET-1 and ETB receptor mRNA in the heart, whereas ETA receptor mRNA was markedly down-regulated in the heart, lung and liver. These data suggest differential gene regulation of ET-1 and its receptor subtypes in various organs from endotoxic shock rats, and that nonselective ETA/ETB receptor antagonist, but not ETA receptor antagonist, ameliorates endotoxin-induced hypotension and death.     

Regulation of the endothelin/endothelin receptor system by interleukin-1{beta} in human myometrial cells

Proinflammatory cytokines produced at the fetomaternal interface, such as IL-1beta, have been implicated in preterm and term labor. The present study was performed to evaluate the influence of IL-1beta on the endothelin (ET)/ET receptor system in human myometrial cells. We report that myometrial cells under basal conditions not only respond to but also secrete ET-1, one of the main regulators of uterine contractions. Prolonged exposure of the cells to IL-1beta led to a decrease in prepro-ET-1 and ET-3 mRNA correlated with a decrease in immunoreactive ET-1 and ET-3 levels in the culture medium. Whereas ETA receptor expression at both protein and mRNA levels was not affected by IL-1beta treatment, we demonstrated an unexpected predominance of the ETB receptor subtype under this inflammatory condition. Whereas the physiological function of ETB remains unclear, we confirmed that only ETA receptors mediate ET-1-induced myometrial cell contractions under basal conditions. By contrast, prolonged exposure of the cells to IL-1beta abolished the contractile effect induced by ET-1. Such a regulation of IL-1beta on the ET release and the balance of ETA to ETB receptors leading to a loss of ET-1-induced myometrial cell contractions suggest that complex regulatory mechanisms take place to constraint the onset of infection-induced premature contractions.     

Tumor necrosis factor-alpha induces vascular endothelial growth factor-C expression in rheumatoid synoviocytes

OBJECTIVE: To determine the expression of vascular endothelial growth factor-C (VEGF-C) in the synovial fluid of patients with rheumatoid arthritis (RA) and to investigate the regulation of VEGF-C production by major proinflammatory cytokines in fibroblast-like synoviocytes (FLS). METHODS: The concentrations of VEGF-C, tumor necrosis factor-alpha (TNF-alpha), and interleukin 1beta (IL-1beta) were measured using an ELISA method in synovial fluids obtained from 20 patients with RA and 20 with osteoarthritis (OA). Primary cultured RA FLS were stimulated with TNF-alpha or IL-1beta, and the expression levels of VEGF-C mRNA and protein were assessed by quantitative real-time polymerase chain reaction and ELISA. RESULTS: Significantly higher levels of VEGF-C were found in RA synovial fluids compared to OA synovial fluids. VEGF-C levels showed a highly significant correlation with the levels of both TNF-alpha and IL-1beta in the synovial fluid of patients with RA. TNF-alpha stimulation significantly increased VEGF-C mRNA and protein expression in RA FLS in a dose-dependent manner. A tendency to increased expression of VEGF-C was also observed after IL-1beta stimulation in FLS. CONCLUSION: Overexpression of VEGF-C in FLS by stimulation with TNF-alpha may play an important role in the progression of synovial inflammation and hyperplasia in RA by contributing to local lymphangiogenesis and angiogenesis.     

Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor alpha

OBJECTIVES: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis. METHODS: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha. RESULTS: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha. CONCLUSIONS: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.     

Influence of hypoxia on the expression of matrix metalloproteinase-1, -3 and tissue inhibitor of metalloproteinase-1 in rheumatoid synovial fibroblasts

OBJECTIVE: The rheumatoid synovium is a hypoxic environment, and hypoxia has been implicated as a factor in the pathogenesis of rheumatoid arthritis (RA). The purpose of this study was to investigate the effect of hypoxia on the expression of matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of metalloproteinase (TIMP)-1 in rheumatoid synovial fibroblasts. METHODS: Synovial fibroblasts obtained from RA patients were cultured for 48 h under normoxic or hypoxic conditions. Assays included western blot analysis and enzyme-linked immunosorbent assay (ELISA) for MMP-1, -3 and TIMP-1, and northern blot analysis to measure TIMP-1 mRNA levels. RESULTS: Compared with normoxic culture, hypoxia increased MMP-1 and MMP-3 expression in rheumatoid synovial fibroblasts. Hypoxia decreased TIMP-1 expression in rheumatoid synovial fibroblasts, as measured by both protein and mRNA levels. CONCLUSION: These results suggest that microenvironmental conditions, such as hypoxia, may directly contribute to joint destruction in RA by increasing the ratio of MMP-1, -3 to TIMP-1 production in synovial fibroblasts.     

Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll-like receptor signaling

OBJECTIVE: To test the hypothesis that bacterial products acting as adjuvants, such as CpG oligodeoxynucleotides (ODNs) and peptidoglycans (PGs), are able to activate synoviocytes, and to determine the involvement of Toll-like receptors (TLRs) in this activation process. METHODS: Cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were stimulated with CpG ODNs or PGs. The expression of various integrins was determined by fluorescence-activated cell sorting. TLR and matrix metalloproteinase (MMP) messenger RNA (mRNA) was measured by real-time polymerase chain reaction. Additionally, levels of interleukin-6 (IL-6) and IL-8 in the culture supernatants were assessed by enzyme-linked immunosorbent assay. Blocking experiments were performed by adding anti-TLR-2 and anti-TLR-4 monoclonal antibodies to cultures stimulated with bacterial PGs. RESULTS: Incubation of synovial fibroblasts with CpG ODNs resulted in neither up-regulation of the expression of integrins on the cell surface, up-regulation of MMP mRNA expression, nor IL-6 and IL-8 production. However, incubation of RA synovial fibroblasts as well as OA synovial fibroblasts with staphylococcal PGs led to an up-regulation of CD54 (ICAM-1) surface expression and to increased expression of MMP-1, MMP-3, and MMP-13 mRNA. Furthermore, production of the proinflammatory cytokines IL-6 and IL-8 was increased by treatment with PGs. We demonstrated that cultured synovial fibroblasts express low levels of TLR-2 and TLR-9 mRNA. TLR-2 was up-regulated after stimulation with PGs, whereas TLR-9 mRNA remained at baseline levels after stimulation with CpG ODNs. Anti-TLR-2 monoclonal antibodies significantly inhibited production of IL-6 and IL-8 induced by stimulation with PGs. CONCLUSION: We demonstrate that bacterial PGs activate synovial fibroblasts, at least partially via TLR-2, to express integrins, MMPs, and proinflammatory cytokines. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction.     

The TNF superfamily member LIGHT contributes to survival and activation of synovial fibroblasts in rheumatoid arthritis

OBJECTIVES: The TNF superfamily member LIGHT has a T-cell co-stimulatory role and has previously been associated with inflammation and autoimmunity. To investigate its role in rheumatoid arthritis (RA), a disease where activated T cells contribute in a prominent way, we have analysed the expression of LIGHT and its receptors in RA and analysed its effects on synovial fibroblasts in vitro. METHODS: The expression of LIGHT was measured in synovial tissues and fluids and the receptors of LIGHT were detected on synovial fibroblasts derived from patients with RA and osteoarthritis (OA). The effects of recombinant LIGHT on the production of proinflammatory cytokines and proteases and on the apoptosis of synovial fibroblasts was assessed. RESULTS: LIGHT mRNA was present in synovial tissues of patients with RA but not with OA. Correspondingly, soluble LIGHT protein could be detected in RA synovial fluid samples at much higher levels than in synovial fluid from patients with OA. Immunohistochemical detection of LIGHT and analysis of synovial fluid cells by flow cytometry revealed CD4 T cells as the major source of LIGHT in the rheumatoid joint. Synovial fibroblasts from RA patients were found to express the LIGHT receptors HVEM and LTbetaR. Recombinant LIGHT induced RA synovial fibroblasts to upregulate MMP-9 mRNA, CD54 and IL-6 in an NF-kappaB-dependent fashion. In vitro, exposure of cultured synovial fibroblasts to LIGHT reduced FAS-mediated apoptosis significantly, without affecting the rate of spontaneous apoptosis. CONCLUSIONS: The results provide evidence for a novel T-cell-dependent activation of synovial fibroblasts by LIGHT in joints of patients with RA, contributing to an inflammatory and destructive phenotype.     

Endothelin-1[1-31]: a novel autocrine-paracrine regulator of human adrenal cortex secretion and growth.

Endothelin (ET)-1[1-21] stimulates steroid secretion and zona glomerulosa growth and is expressed in the human and rat adrenal cortex together with its receptor subtypes A and B (ETA and ETB). Although ET-1[1-21] is generated from bigET-1 by an ET-converting enzyme (ECE-1), there is evidence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide, the role of which in adrenal pathophysiology is largely unknown. Gene expression and immunohistochemical studies allowed localization of chymase in the normal human adrenal cortex. Sizable amounts, not only of ET-1[1-21] but also of ET-1[1-31], were found in the adrenal vein plasma of three patients. ET-1[1-21] and ET-1[1-31] elicited a clear-cut secretory response by dispersed human adrenocortical cells, ET-1[1-31] being significantly less potent than ET-1[1-21]. The secretagogue effect of ET-1[1-31] was abolished by the ETA receptor antagonist BQ-123 and was unaffected by the ETB receptor antagonist BQ-788. Because, in humans, the secretagogue effect of ET-1[1-21] involves both ETA and ETB receptors, the weaker action of ET-1[1-31] could be attributable to a selective ETA receptor activation. Two lines of evidence support this contention: 1) ET-1[1-31] was more effective than ET-1[1-21] in stimulating ETA-mediated cell proliferation of human adrenocortical cells cultured in vitro; and 2) autoradiography showed that a) ET-1[1-31] displaced in vitro [(125)I]ET-1[1-21] binding to the ETA, but not ETB receptors, in human internal thoracic artery rings; and b) BQ-123, but not BQ-788, eliminated [(125)I]ET-1[1-31] binding in the rat adrenal cortex.     

Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis

OBJECTIVE: Sentrin, a novel antiapoptotic molecule, has been shown to interact with the signal-competent form of Fas/APO-1 and tumor necrosis factor receptor I (TNFRI), and thereby, to protect cells against anti-Fas/APO-1- and TNF-induced cell death. Since reduced apoptosis in the synovial lining is supposed to contribute to synovial hyperplasia in rheumatoid arthritis (RA), we searched for the expression of sentrin-1 messenger RNA (mRNA) in synovium from patients with RA. METHODS: The expression of sentrin-1 mRNA was examined by in situ hybridization on snap-frozen sections of normal and RA synovial tissues as well as on paraffin-embedded RA synovial specimens, including the interface of cartilage-bone and invading synovium. Immunohistochemical double labeling after in situ hybridization was performed to further characterize sentrin-1 mRNA-expressing cells. In addition, quantitative analysis of sentrin-1 mRNA expression in RA synovial fibroblasts (RASF), osteoarthritis synovial fibroblasts (OASF), and normal fibroblasts was performed by quantitative real-time polymerase chain reaction. Expression levels were standardized to the expression of GAPDH. The in vivo maintenance of sentrin expression in RASF aggressively invading human cartilage was explored in the SCID mouse model of RA. RESULTS: A marked expression of sentrin-1 mRNA could be seen in all RA synovial specimens, predominantly in SF of the lining layer and at sites of invasion of RA synovium into cartilage. In normal synovial tissues, no sentrin-1 mRNA was detectable. RASF showed a maximum 32.5-fold (mean +/- SD 14.9 +/- 11.6) increase of sentrin-1 mRNA expression compared with normal fibroblasts and a maximum 31.4-fold (mean +/-SD 14.3 +/- 10.9) increase compared with OASF. When coimplanted with normal human cartilage in the SCID mouse model, invading RASF maintained their sentrin-1 mRNA expression for at least 60 days in vivo. CONCLUSION: The marked expression of sentrin in rheumatoid synovial tissue, but not in normal or OA synovial tissue, may contribute to the modulation of Fas- and TNFR-mediated apoptosis in RA synovium, and thereby extend the lifespan of invasive, cartilage-destructive SF.     

Constitutive expression of angiopoietin-1 and -2 and modulation of their expression by inflammatory cytokines in rheumatoid arthritis synovial fibroblasts

OBJECTIVE: Angiopoietin- I (Ang-1) and Ang-2 are ligands for the receptor tyrosine kinase, Tie-2. Ang-1, a Tie-2 agonist, may have a vascular stabilizing role in angiogenesis, while Ang-2, an endogenous antagonist of Tie-2, may have an early role in angiogenesis, destabilizing existing vasculature. We show that these ligands are expressed by rheumatoid synovial fibroblasts (RSF) and investigate whether their expression was modulated by proinflammatory cytokines present in the joint in rheumatoid arthritis (RA). METHODS: Using quantitative PCR we determined the level of expression of these 2 ligands in RSF and chronic inflamed synovial tissue. The level of expression of these ligands after treatment with proinflammatory cytokines and hypoxia was also determined. RESULTS: We observed constitutive expression of Ang-1 and Ang-2 in RSF and chronic inflamed synovial tissue. Ang-1 was the most highly expressed ligand in late stage RA synovial fibroblasts; however, in chronic inflamed synovial tissue, Ang-2 was predominant and was expressed at strikingly high levels (70 to 120-fold increase). We observed that tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), but not interleukin 1beta or hypoxia, stimulated Ang-1 gene expression in RSE This was confirmed at the protein level as media from TNF-alpha treated RSF resulted in increased autophosphorylation of Tie-2. In contrast, TNF-alpha and TGF-beta had no effect on Ang-2 expression in RSF, but augmented expression of Ang-2 in normal synovial fibroblasts. CONCLUSION: The angiopoietins are important angiogenic factors constitutively present in RA, and their expression is modulated by certain cytokines. Ang-2 may have an important role in rheumatoid tissue where vigorous angiogenesis is occurring.