Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells.
(17/13538)
Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu. (+info)
Paracrine changes in the peritoneal environment of women with endometriosis.
(18/13538)
During the past decade, macrophage-derived substances such as prostanoids, cytokines, growth factors and angiogenic factors have been detected in the peritoneal fluid of women with endometriosis. In particular, growth-promoting and angiogenic factors are considered to be substantially involved in the pathogenesis of endometriosis. In this study, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and intercellular adhesion molecule 1 (ICAM-1), substances recently detected in the peritoneal fluid of women with endometriosis, were assessed with regard to their concentrations in different stages of endometriosis and changes of the peritoneal paracrine activity after medical treatment with a gonadotrophin releasing hormone agonist (GnRHa). Peritoneal fluid was obtained from patients with endometriosis during laparoscopy before and after a 4-month treatment with a GnRHa. VEGF, TGF-beta and ICAM-1 could be detected in all women presenting with various stages of active endometriosis. After GnRHa therapy, all patients showed significant decreases in mean concentrations of VEGF (194+/-77 pg/ml), TGF-beta (902+/-273 pg/ml) and ICAM-1 (157+/-52 ng/ml). Patients with stage III and IV endometriosis (according to the rAFS score) had much higher concentrations of VEGF and TGF-beta before treatment compared with those patients with mild endometriosis (rAFS stages I and II). The most striking decrease in concentration was for TGF-beta, from 902 pg/ml before to 273 pg/ml after therapy. These results indicate an important role for paracrine activity in the establishment and maintenance of endometriosis. Indeed, treatment with a GnRHa may reduce paracrine activity in the peritoneal cavity via hypo-oestrogenism and provide proof of successful therapy. (+info)
Recovery following relief of unilateral ureteral obstruction in the neonatal rat.
(19/13538)
BACKGROUND: Obstructive nephropathy is a primary cause of renal insufficiency in infants and children. This study was designed to distinguish the reversible and irreversible cellular consequences of temporary unilateral ureteral obstruction (UUO) on the developing kidney. METHODS: Rats were subjected to UUO or sham operation in the first 48 hours of life, and the obstruction was removed five days later (or was left in place). Kidneys were removed for study 14 or 28 days later. In additional groups, kidneys were removed at the end of five days of obstruction. Immunoreactive distribution of renin was determined in arterioles, and the distribution of epidermal growth factor, transforming growth factor-beta1, clusterin, vimentin, and alpha-smooth muscle actin was determined in tubules and/or interstitium. The number of glomeruli, glomerular maturation, tubular atrophy, and interstitial collagen deposition was determined by morphometry. Renal cellular proliferation and apoptosis were measured by proliferating cell nuclear antigen and the TdT uridine-nick-end-label technique, respectively. The glomerular filtration rate was measured by inulin clearance. RESULTS: Renal microvascular renin maintained a fetal distribution with persistent UUO; this was partially reversed by the relief of obstruction. Although glomerular maturation was also delayed and glomerular volume was reduced by UUO, the relief of obstruction prevented the reduction in glomerular volume. Although relief of obstruction did not reverse a 40% reduction in the number of nephrons, the glomerular filtration rate of the postobstructed kidney was normal. The relief of obstruction did not improve tubular cell proliferation and only partially reduced apoptosis induced by UUO. This was associated with a persistent reduction in the tubular epidermal growth factor. In addition, the relief of obstruction reduced but did not normalize tubular expression of transforming growth factor-beta1, clusterin, and vimentin, all of which are evidence of persistent tubular injury. The relief of obstruction significantly reduced interstitial fibrosis and expression of alpha-smooth muscle actin by interstitial fibroblasts, but not to normal levels. CONCLUSIONS: The relief of obstruction in the neonatal rat attenuates, but does not reverse, renal vascular, glomerular, tubular, and interstitial injury resulting from five days of UUO. Hyperfiltration by remaining nephrons and residual tubulointerstitial injury in the postobstructed kidney are likely to lead to deterioration of renal function later in life. (+info)
Up-regulation of glomerular extracellular matrix and transforming growth factor-beta expression in RF/J mice.
(20/13538)
BACKGROUND: RF/J mice were first reported as a murine model of spontaneous glomerulosclerosis by Gude and Lupton in 1960, but the precise histologic characteristics and immunopathological background of this mouse have not been investigated further. METHODS: Measurements of serum levels of immunoglobulins, anti-single strand DNA (anti-ss-DNA) antibody, complement (C3), and circulating immune complex (IC) were performed. Analyses of glomerular histological and immunopathological lesions in association with the detection of mRNA expression of collagen IV, TGF-beta, matrix protein turnover related enzymes, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2) and platelet-derived growth factor (PDGF) were also performed in young (10-week-old) and elderly (60-week-old) RF/J mice with age-matched BALB/C mice as the controls. RESULTS: High levels of serum IgA and IgG from as early as 20 weeks of age were noted in the RF/J mice. Serum anti-ss-DNA antibody of aged RF/J mice increased up to 23% of that of aged MRL-lpr/lpr mice, and serum C3 concentration significantly decreased with age, reaching lower levels than that of BALB/c mice. IgA-IC levels were significantly high compared to BALB/C mice both in the early and late stages of life, whereas IgG-IC levels were high only in mice younger than 20 weeks. Semiquantitative and quantitative analyzes of renal histopathological findings revealed significantly marked and age-related mesangial matrix expansion in RF/J mice, with increasing frequency of global glomerular sclerosis and tubulointerstitial damage. On the other hand, although precise measurements of glomerular cell numbers also showed an apparent augmentation in both young and old RF/J mice compared to BALB/C mice, glomerular cellularity decreased with age in RF/J mice. Immunohistochemical study revealed massive immunoglobulin deposition from a young age in association with significantly higher accumulation of matrix proteins, such as types I and IV collagen and laminin from the early stage of life. In addition, in these glomeruli, transforming growth factor-beta1 (TGF-beta1) was highly expressed both in young and old mice. The mRNA expression of MMP-2 was up-regulated only in the early stage of life. Although PDGF mRNA of RF/J mice was significantly up-regulated in the early stage of life, the differences between the mice disappeared in the late stage of life. CONCLUSIONS: These findings suggest that in RF/J mice, an immunopathological background inducing high serum immunoglobulin and IC levels from the early stage of life is closely related to mesangioproliferative glomerular lesions mediated by PDGF, and that development of massive extracellular matrix accumulation in glomeruli was induced by up-regulated expression of TGF-beta with inappropriate regulation of protein turnover-related enzyme production. (+info)
Blocking angiotensin II ameliorates proteinuria and glomerular lesions in progressive mesangioproliferative glomerulonephritis.
(21/13538)
BACKGROUND: The renin-angiotensin system is thought to be involved in the progression of glomerulonephritis (GN) into end-stage renal failure (ESRF) because of the observed renoprotective effects of angiotensin-converting enzyme inhibitors (ACEIs). However, ACEIs have pharmacological effects other than ACE inhibition that may help lower blood pressure and preserve glomerular structure. We previously reported a new animal model of progressive glomerulosclerosis induced by a single intravenous injection of an anti-Thy-1 monoclonal antibody, MoAb 1-22-3, in uninephrectomized rats. Using this new model of progressive GN, we examined the hypothesis that ACEIs prevent the progression to ESRF by modulating the effects of angiotensin II (Ang II) on the production of transforming growth factor-beta (TGF-beta) and extracellular matrix components. METHODS: We studied the effect of an ACEI (cilazapril) and an Ang II type 1 receptor antagonist (candesartan) on the clinical features and morphological lesions in the rat model previously reported. After 10 weeks of treatment with equihypotensive doses of cilazapril, cilazapril plus Hoe 140 (a bradykinin receptor B2 antagonist), candesartan, and hydralazine, we examined systolic blood pressure, urinary protein excretion, creatinine clearance, the glomerulosclerosis index, and the tubulointerstitial lesion index. We performed a semiquantitative evaluation of glomerular immunostaining for TGF-beta and collagen types I and III by immunofluorescence study and of these cortical mRNA levels by Northern blot analysis. RESULTS: Untreated rats developed massive proteinuria, renal dysfunction, and severe glomerular and tubulointerstitial injury, whereas uninephrectomized control rats did not. There was a significant increase in the levels of glomerular protein and cortical mRNA for TGF-beta and collagen types I and III in untreated rats. Cilazapril and candesartan prevented massive proteinuria, increased creatinine clearance, and ameliorated glomerular and tubulointerstitial injury. These drugs also reduced levels of glomerular protein and cortical mRNA for TGF-beta and collagen types I and III. Hoe 140 failed to blunt the renoprotective effect of cilazapril. Hydralazine did not exhibit a renoprotective effect. CONCLUSION: These results indicate that ACEIs prevent the progression to ESRF by modulating the effects of Ang II via Ang II type 1 receptor on the production of TGF-beta and collagen types I and III, as well as on intrarenal hemodynamics, but not by either increasing bradykinin activity or reducing blood pressure in this rat model of mesangial proliferative GN. (+info)
Vitamin E succinate (VES) induces Fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis.
(22/13538)
Fas (CD95/APO-1) is an important mediator of apoptosis. We show that Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 human breast cancer cells become responsive to anti-Fas (CD95) agonistic antibody-triggered apoptosis after pretreatment or cotreatment with vitamin E succinate (VES; RRR-alpha-tocopheryl succinate). In contrast, no enhancement of anti-Fas agonistic antibody-triggered apoptosis was observed following VES pretreatment or cotreatment with Fas-sensitive primary cultures of human mammary epithelial cells, immortalized MCF-10A cells, or T47D human breast cancer cells. Although VES is itself a potent apoptotic triggering agent, the 6-h pretreatment procedure for Fas sensitization did not initiate VES-mediated apoptosis. The combination of VES plus anti-Fas in pretreatment protocols was synergistic, inducing 2.8-, 3.0-, and 6.3-fold enhanced apoptosis in Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively. Likewise, cotreatment of Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells with VES plus anti-Fas enhanced apoptosis 1.9-, 2.0-, and 2.6-fold, respectively. Functional knockout of Fas-mediated signaling with either Fas-neutralizing antibody (MCF-7-, MDA-MB-231-, and MDA-MB-435-treated cells) or Fas antisense oligomers (MDA-MB-435-treated cells only), reduced VES-triggered apoptosis by approximately 50%. Analyses of whole cell extracts from Fas-sensitive cells revealed high constitutive expression of Mr 43,000 Fas, whereas Fas-resistant cells expressed low levels that were confined to the cytosolic fraction. VES treatment of the Fas-resistant cells caused a depletion of cytosolic Mr 43,000 Fas with a concomitant increase in Mr 43,000 membrane Fas. These data show that VES can convert Fas-resistant human breast cancer cells to a Fas-sensitive phenotype, perhaps by translocation of cytosolic Mr 43,000 Fas to the membrane and show that VES-mediated apoptosis involves Mr 43,000 Fas signaling. (+info)
Downregulation of interleukin-12 (IL-12) responsiveness in human T cells by transforming growth factor-beta: relationship with IL-12 signaling.
(23/13538)
Interleukin-12 (IL-12) is a cytokine that plays a central role in the control of cell-mediated immunity. We have previously shown that transforming growth factor-beta1 (TGF-beta) inhibitory effects on human primary allogeneic cytotoxicity and proliferative responses interfere with IL-12 pathway. The present study was undertaken to further elucidate the biochemical basis of the functional interaction between these two cytokines and to define the site of TGF-beta action on the signaling pathway activated by IL-12. Our data indicate that TGF-beta induced an inhibition of interferon-gamma (IFN-gamma) production without affecting the IL-12Rbeta1 and IL-12Rbeta2 subunits mRNA expression by activated T cells. We further show that TGF-beta has a significant inhibitory effect on the early signal transduction events following interaction of IL-12 with its receptor on activated T cells, resulting in the inhibition of both JAK2 and Tyk2 phosphorylation. In addition, TGF-beta was found to significantly inhibit IL-12-induced phosphorylation of the STAT4 transcription factor. Electrophoretic mobility shift assay indicated that TGF-beta induced a decrease in IL-12-induced STAT4 DNA binding activity in T lymphocytes. This study suggests that TGF-beta influences IL-12 responsiveness at least in part by inhibiting early signaling events essential to gene induction in IL-12-activated T cells. (+info)
Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators.
(24/13538)
Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta (TGF-beta) and the liphophilic hormone vitamin D. TGF-beta causes activation of SMAD proteins acting as coactivators or transcription factors in the nucleus. Vitamin D controls transcription of target genes through the vitamin D receptor (VDR). Smad3, one of the SMAD proteins downstream in the TGF-beta signaling pathway, was found in mammalian cells to act as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus. Thus, Smad3 may mediate cross-talk between vitamin D and TGF-beta signaling pathways. (+info)