A profibrotic polymorphism (of TGFbeta1) in systemic sclerosis.
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Humans↗
Biomedical subjects
E C LeRoy
Publications and source records attributed to E C LeRoy.
MCMV induces neointima in IFN-gammaR-/- mice: intimal cell apoptosis and persistent proliferation of myofibroblasts.
BACKGROUND: CMV infections have been linked to vasculopathies like atherosclerosis and Scleroderma. CMV infects vascular endothelium with intermittent shedding of the virus and the development of latency. METHODS: We adopted a model of arteritis, developed by Presti et al. (1998), triggered by murine cytomegalovirus (MCMV) infection. Our studies focused on neointima formation. Groups of mice include: 1) immunocompetent 129S, 2) immunocompetent 129S receiving whole body irradiation and MCMV, 3) IFN-gammaR-/- receiving MCMV, and 4) IFN-gammaR-/- receiving MCMV and whole body irradiation. RESULTS: Mice were inoculated with MCMV (5 x 10(4) or 1 x 10(5) PFU's) by i.p. injection; hearts and abdominal aortas were collected and histopathology evaluated. Infected immunocompetent animals exhibited widespread perivascular inflammation, which subsided by 8 weeks. Intimal pathology was not observed in any control group. Immunocompetent animals receiving MCMV and irradiation developed mild to moderate intimal lesions associated with medial and adventitial inflammation. IFN-gammaR-/- mice infected for 4 months and receiving whole body irradiation 2 months after infection developed pathology characterized by extensive adventitial and medial infiltrate and significant neointima, suggesting that infection and immunosuppression were co-requisites of neointima formation. Immunohistochemical analysis revealed myofibroblasts as a major component of neointima. The disease is characterized by up-regulation of growth factors (TGF-beta1, PDGF-A and B). Apoptosis was detected in the intimal layer of affected aortas. Active proliferation of myofibroblasts and infiltrating cells was also detected. CONCLUSION: These results indicate that CMV infections may lead to intimal injury that results in the formation of neointima characteristic of autoimmune vasculopathies.
Journal Article↗
Anti-fibrillin-1 autoantibodies in systemic sclerosis are GM and KM allotype-restricted.
GM and KM allotypes--genetic markers of immunoglobulin (Ig) gamma and kappa chains, respectively--have been shown to play an important role in genetic predisposition to some autoimmune diseases. To determine their role in susceptibility to systemic sclerosis (SSc; scleroderma) and in the generation of anti-fibrillin-1 antibodies, 148 SSc patients and 191 controls were typed for several GM and KM allotypes by a standard hemagglutination inhibition method. IgG and IgM antibodies to fibrillin-1 were measured by radioimmunoassay. GM and KM phenotypes were not significantly associated with SSc. However, these determinants significantly influenced the production of anti-fibrillin-1 antibodies in SSc patients. In Caucasians, GM1,3,17 23 5,13,21 and GM3 23 5,13 phenotypes were associated with the presence and absence of IgG autoantibodies, respectively. The production of these autoantibodies was also associated with KM allotypes, KM1,3 heterozygosity being associated with response and homozygosity for the KM3 allele with nonresponse to fibrillin-1. In African-Americans, the KM1 homozygotes were associated with the absence of anti-fibrillin-1 antibodies and the KM3 homozygotes with the presence of autoantibodies. In this ethnic group, the GM1,17 5,13 phenotype was associated with the absence of IgM autoantibodies. This represents the first description of genetic control of autoimmunity to fibrillin-1 in scleroderma.
Autoantibodies↗
Criteria for the classification of early systemic sclerosis.
We propose criteria for the early diagnosis and classification of systemic sclerosis that reflect the vascular and serological advances of the last 2 decades.
Humans↗
Autoimmunity and vascular involvement in systemic sclerosis (SSc).
Endothelial injury, obliterative microvascular lesions, and increased vascular wall thickness are present in all involved organs in scleroderma. The vascular pathology is associated with altered vascular function with increased vasospasm, reduced vasodilatory capacity and increased adhesiveness of the blood vessels to platelets and lymphocytes. The extent of injury and dysfunction is reflected by changes in the circulating levels of vascular markers. The initial triggers for the vascular pathology are not known. Possible viral triggers are visited here, including cytomegalovirus in view of increased levels of anti-CMV antibodies in scleroderma, and the remarkable similarities between CMV vasculopathies and scleroderma vascular disease. Endothelial apoptosis in scleroderma may be related to viral infection, immune reactions to viral or environmental factors, reperfusion injury or to anti-endothelial antibodies. The impact of the vascular pathology on the evolution of tissue fibrosis is not known; still, cytokines (TGFbeta, IL4), vascular factors (endothelin), and growth factors (PDGF) are possibly crucial signals that link the vascular disease to tissue fibrosis. Knowledge of the regulation of these and other factors will provide the opportunity to develop more rational therapeutic approaches to the disease.
Animals↗
Human cytomegalovirus and the vasculopathies of autoimmune diseases (especially scleroderma), allograft rejection, and coronary restenosis.
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Angioplasty, Balloon, Laser-Assisted↗
Diseases of the extracellular matrix.
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Connective Tissue Diseases↗
Pathogenesis of fibrosis: type 1 collagen and the skin.
This review on the pathogenesis of fibrosis emphasizes the similarities between tissue repair, a tightly regulated salutary biological response, and fibrosis, an unregulated pathological process. It focuses on the transcriptional regulation of type I collagen, the role of cytokines in fibroblast activation, integrins as examples of cell-matrix signaling pathways, and the heterogeneity of fibroblast populations as factors contributing to fibrosis. Tissue remodeling and the role of matrix metalloproteinases and metalloproteinase inhibitors are mentioned briefly. The capacity of extracellular matrix to modulate cellular function is a recurring theme.
Cell Adhesion↗
Identification of altered expression of ADP/ATP translocase during cellular senescence in vitro.
In this study, we have used the mRNA differential display technique to investigate the changes in gene expression that occur in the process of cellular aging. A number of cDNAs whose corresponding mRNAs are either increasingly or decreasingly expressed in senescent cells were thereby isolated. Through DNA sequencing, one of these differentially displayed mRNAs was identified as mitochondrial ADP/ATP translocase. The altered expression of ADP/ATP translocase in different stages of senescent fibroblasts was further confirmed by Northern blots and semiquantitative RT-PCR. Our results demonstrate that expression of ADP/ATP translocase is progressively decreased during the process of in vitro cellular senescence. Further analyses with MTT assays indicate that the decreased expression of ADP/ATP translocase in senescent cells is in parallel with the decline of mitochondrial functions, suggesting that altered expression of this important mitochondrial enzyme might play an active role in the process of cellular senescence.
Base Sequence↗
Oncostatin M stimulates transcription of the human alpha2(I) collagen gene via the Sp1/Sp3-binding site.
Oncostatin M (OSM), a member of the hematopoietic cytokine family, has been implicated in excessive bone growth and in the process of fibrosis. As part of an ongoing study of the molecular mechanisms of fibrosis, we have investigated the transcriptional regulation of the alpha2(I) collagen gene by OSM in human fibroblasts. An OSM response element was mapped by deletional analysis between base pairs (bp) -148 and -108 in the alpha2(I) collagen promoter. Further functional analysis of the alpha2(I) collagen promoter containing various substitution mutations revealed that both the basal activity and OSM stimulation of this promoter are mediated by a TCCTCC motif located between bp -128 and -123. Furthermore, three copies of the 12-bp synthetic alpha2(I) collagen promoter fragment containing the "TCC" motif conferred OSM inducibility to the otherwise unresponsive thymidine kinase promoter. Electrophoretic mobility shift assays demonstrated that the TCCTCC motif constitutes a novel binding site for the transcription factors Sp1 and Sp3. No differences have been observed in in vitro gel shift binding assays between unstimulated and OSM-stimulated fibroblasts. However, subtle conformational changes were detected in the region of the promoter surrounding TCC repeats after OSM stimulation using in vivo footprint analysis. In conclusion, this study characterized a dual-function response element that mediates the basal activity and OSM stimulation of the human alpha2(I) collagen promoter.
Binding Sites↗
Scleroderma and related conditions.
Systemic sclerosis is a generalized disorder characterized by fibrosis and microvascular injury in affected organs. Despite being recognized nearly 250 years ago, knowledge regarding pathogenesis remains limited, and treatment remains directed at symptomatic improvement. Early recognition of systemic sclerosis, however, is important in order to monitor for specific disease complications (i.e., fibrosing alveolitis, scleroderma renal crisis) as well as initiate manifestation specific therapies that improve quality of life.
Humans↗
Basic fibroblast growth factor inhibits basal and transforming growth factor-beta induced collagen alpha 2(I) gene expression in scleroderma and normal fibroblasts.
OBJECTIVE: Studies have shown that scleroderma (systemic sclerosis, SSc) and normal fibroblasts respond differently to basic fibroblast growth factor (bFGF), SSc fibroblasts being less responsive than normal fibroblasts in mitogenic assays in vitro, bFGF also stimulates the expression of platelet derived growth factor-alpha (PDGF-alpha) receptors in normal fibroblasts, but not in SSc fibroblasts. Conversely, transforming growth factor-beta (TGF-beta) stimulates PDGF-alpha receptor expression in SSc fibroblasts, but not in normal fibroblasts. Since bFGF has been shown to inhibit collagen gene expression in several cell types, we examined responses of SSc and normal fibroblasts to bFGF alone and in combination with TGF-beta with regard to collagen alpha 2(I) (COL1A2) expression. METHODS: Fibroblasts were obtained by skin biopsy from affected areas of patients with diffuse cutaneous SSc and from healthy donors and propagated in vitro. The effects of bFGF and TGF-beta on the COL1A2 mRNA expression levels in SSc and healthy fibroblasts were analyzed by Northern blot. The effects of bFGF on the COL1A2 promoter activities in both cell types were analyzed by transient transfection assays. The effects of bFGF and TGF-beta on collagen protein synthesis were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. RESULTS: While bFGF diminished COL1A2 mRNA in both SSc and normal cells, COL1A2 mRNA quantities in the SSc fibroblasts were not depressed to the levels expressed by normal controls. As anticipated, TGF-beta strongly induced COL1A2 mRNA levels in normal fibroblasts, and to a lesser degree in SSc fibroblasts. When cells were incubated with both TGF-beta and bFGF, the stimulatory effect of TGF-beta was completely suppressed in both cell types. bFGF decreased COL1A2 promoter activity in both cell types, suggesting that COL1A2 inhibition by bFGF occurs at least partially at the transcriptional level. The effects of bFGF and TGF-beta on the collagen protein synthesis correlated well with mRNA data, in that TGF-beta stimulated, while bFGF strongly inhibited, collagen synthesis. CONCLUSION: bFGF is a potent inhibitor of basal and TGF-beta stimulated collagen expression in human fibroblasts, and this effect is not different between SSc and healthy fibroblasts.
Collagen↗
Transcriptional regulation of the human alpha2(I) collagen gene. Combined action of upstream stimulatory and inhibitory cis-acting elements.
This study identifies three regions of the human alpha2(I) collagen promoter involved in the binding of nuclear factors. These regions include sequences from -173 to -155 (footprint I), -133 to -119 (footprint II), and -101 to -72 (footprint III). A novel positive cis-element containing a TCCTCC motif was identified within footprint II. In addition, we demonstrated that a pyrimidine-rich region within footprint I is a binding site for a transcriptional repressor, and a CCAAT motif within footprint III is a binding site for a transcriptional activator. Comparative functional analysis of the cis-acting elements within the proximal 350 base pairs of this promoter, including previously characterized Sp1 binding sites at -300, indicates that constitutive activity of this promoter is regulated equivalently by the three positive cis-acting elements at -300, -125, and -80. Mutations in the repressor site at -160 increase constitutive activity by 4-6-fold. However, simultaneous mutations of the repressor site and the cis-regulatory element at either the -300 or -125 sites result in no increase in constitutive transcription activity suggesting interaction between the activators and repressor elements. In contrast, simultaneous mutation of the CCAAT motif and the repressor site results in about a 4-fold increase, suggesting that activation via the CCAAT motif may be independent of this repressor.
Animals↗
Systemic sclerosis. A vascular perspective.
The horizon is bright for SSc in a vascular context. Surrogate markers can now be routinely used in the management of the active patient; new cytokines, such as VEGF, can be studied along with the known abnormalities of the cytokine cascade (TGF beta 1, PDGF) for a more integrated understanding of the vascular pathogenesis of SSc (Fig. 6); and combination therapies can be applied before vascular insufficiency leads to vital organ failure. Thus, despite reimbursement and research funding constraints, the future for both the SSc patient and the investigator of SSc is optimistic when based on a firm biologic foundation.
Arteries↗
IL-4 upregulates tenascin synthesis in scleroderma and healthy skin fibroblasts.
Tenascin (TN), a large extracellular matrix glycoprotein, is transiently expressed during embryonic development, but is absent from most normal adult tissues. TN is reexpressed, however, in healing wounds, in the stroma of some tumors, and in fibrotic diseases such as systemic sclerosis (SSc) and rheumatoid arthritis. To clarify the mechanisms regulating TN expression, we studied the effects of selected cytokines (PDGF, bFGF, TGF-beta, IL-1, IL-4, IL-6, IFN-gamma, and TNF-alpha) found in fibrotic tissue on TN expression by dermal fibroblasts. IL-4 strongly induced TN protein levels (up to 10-fold over the basal level), whereas PDGF and bFGF were less potent inducers of TN than IL-4. All other cytokines tested, including TGF-alpha1, did not stimulate TN synthesis. IL-4 also increased TN mRNA expression, and this effect was blocked by actinomycin D. Cycloheximide increased basal TN mRNA expression and induced TN mRNA in IL-4-treated fibroblasts, suggesting that repressor protein(s) may regulate transcription of the TN gene. Although no differences in constitutive TN expression or effects of cytokines on TN expression were observed between SSc and healthy fibroblasts, these data are consistent with the observations that high levels of both IL-4 and TN are present in the affected skin of patients with SSc. These results suggest that the high level of TN found in the affected tissue of patients with SSc results from the high level of IL-4 present.
Adult↗
Dot-like focal contacts in adherent eosinophils, their redistribution into peripheral belts, and correlated effects on cell migration and protected zone formation.
We have examined the organization of F-actin and focal contacts in eosinophils and the functional consequences correlated with their predistribution. In activated eosinophils adherent to laminin, F-actin is localized in large, uniformly distributed, dot-like structures. Co-localized with the F-actin are a variety of typical components of focal contacts including: the alpha 6 and beta 1 integrins subunits (which mediate eosinophil adhesion to laminin), vinculin, talin, pp125FAK, paxillin, and tyrosine phosphorylated proteins, but not the beta 2 integrin subunit which is not involved in eosinophil adhesion to laminin. In unactivated eosinophils on laminin (which do not adhere well) and in activated eosinophils on non-adhesive surfaces (human serum albumin, tenascin, or a mixture of tenascin and laminin), dot-like accumulations of F-actin do not form. When activated eosinophils are incubated on laminin in the presence of low concentrations of 1,1'-ethylidenebis(L-tryptophan), a remarkable rearrangement occurs. Both conventional and confocal microscopy suggest that the dot-like structures rearrange from a uniform distribution into thick, peripheral belts surrounding each cell with few dot-like structures internal to the belt. Functional consequences occur as a result of this rearrangement of focal contacts and the cytoskeleton. The belt of focal contacts acts as a size-dependent permeability barrier; fluorescent 8 kDa polyethylene glycol does not label the substrate underneath the cells whereas 3.5 kDa polyethylene glycol labels the substrate uniformly. The formation of belts of focal contacts also blocks the ability of eosinophils to migrate on laminin in response to a chemotactic gradient. While the mechanism of 1,1'-ethylidenebis(L-tryptophan) action remains to be determined, experiments indicate that the function of 1,1'-ethylidenebis(L-tryptophan) is distinct from the function of drugs that alter tyrosine kinase and serine/threonine kinase activities. Our observations reveal a novel mechanism for regulating cell behavior in invasive cells in which the rearrangement of focal contacts and the associated cytoskeleton may control cell migration and the sequestration of secreted cytotoxic and degradatory molecules. We propose that 1,1'-ethylidenebis(L-tryptophan) may mimic the function of an endogenous regulator of eosinophil morphology and behavior.
Actins↗
Paul Klee and scleroderma.
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Famous Persons↗
Characterization of a GC-rich region containing Sp1 binding site(s) as a constitutive responsive element of the alpha 2(I) collagen gene in human fibroblasts.
To analyze regulatory elements in the human alpha 2(I) collagen gene (COL1A2) promoter, a series of deletion mutants from -323 to -186 base pairs was tested in transient transfection assays in human fibroblasts. A strong positive responsive element was mapped to a GC-rich region located between base pairs -303 and -271. This region contains three binding sites (GC-boxes) resembling recognition sites for the transcription factor Sp1. Substitution mutations in the GC-boxes abolished binding to the GC-rich region in gel shift analyses and resulted in 90% reduction of promoter activity in transient transfection assays. We demonstrated that transcription factor Sp1 is essential for binding based on the following observations. 1) Sp1 consensus binding site alone competes by binding to the GC-rich region in the DNase I protection assay; 2) both Sp1 consensus binding site and Sp1 antibodies prevent the formation of a DNA-protein complex in the mobility shift assay; 3) anti-Sp1 antibodies recognize a component of the complex competed for by Sp1 consensus binding site.
Base Sequence↗