Association between polymorphic variation in VDR and RXRA and circulating levels of vitamin D metabolites.

The vitamin D metabolite 1,25(OH)2D is the bioactive ligand of the vitamin D receptor (VDR). VDR forms a heterodimer with the retinoid X receptors (RXRs) that when bound to ligand influences the transcriptional control of genes that regulate circulating levels of vitamin D metabolites. Whether genetic variation in VDR or RXRA affects circulating levels of 1,25(OH)2D or 25(OH)D has not been established. We used a single nucleotide polymorphism (SNP) tagging approach to evaluate the association between SNPs in VDR and RXRA and serum levels of 1,25(OH)2D and 25(OH)D. A total of 42 tagSNPs in VDR and 32 in RXRA were analyzed in a sample of 415 participants. Principal components analyses revealed a gene-level association between RXRA and serum 1,25(OH)2D concentrations (P=0.01), but not 25(OH)D. No gene-level association was found for VDR with either serum biomarker. At the single-SNP level, a significant positive trend was observed for increasing 1,25(OH)2D levels with each additional copy of the A allele for RXRA SNP rs9409929 (P-trend=0.003). After a multiple comparisons adjustment, no individual SNP in VDR or RXRA was significantly associated with either outcome. These results demonstrate an association between genetic variation in RXRA and 1,25(OH)2D serum concentrations.

The 72-kilodalton IE-1 protein of human cytomegalovirus (HCMV) is a potent inducer of connective tissue growth factor (CTGF) in human dermal fibroblasts.

Latent human cytomegalovirus (HCMV) infection has been implicated in diseases characterized by tissue remodeling. Because of recent evidence indicating the possibility of a partial HCMV reactivation, the purpose of this study was to examine the role of the HCMV immediate early (IE) genes in the regulation of extracellular matrix (ECM) related host genes. Adenoviral vector expressing IE1 was generated to allow efficient gene delivery into human fibroblasts. IE1 stimulated the prolonged expression of connective tissue growth factor (CTGF) and TIMP1. IE1-dependent stimulation of CTGF was partially mediated by TGF-beta. Moreover, whereas collagenous proteins and collagen type 1 mRNA were only transiently induced by IE1 in the majority of fibroblasts, in selected fibroblast strains IE1 induced persistent ECM upregulation for up to 120 hours. This study suggests that transient or limited HCMV reactivation may play a direct role in abnormal matrix remodeling in GVHD, scleroderma, atherosclerosis and other HCMV-linked diseases.

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.

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.

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.

A disease severity scale for systemic sclerosis: development and testing.

OBJECTIVE: To develop and test a severity scale for individual organ involvements in systemic sclerosis (SSc, scleroderma). METHODS: An international study group completed the following tasks: (1) developed a glossary of terms including all pertinent variables for 9 potentially affected organ systems; (2) collected prospective data to determine the feasibility and practicality of each proposed variable; (3) revised the initial list of variables; (4) determined the association of each variable with mortality (a proxy for morbidity) using 579 patients in an existing comprehensive longitudinal scleroderma databank; (5) developed a severity grading scale for each organ system by discussion and consensus; and (6) externally validated the scale using an independent group of 680 patients from the same databank. RESULTS: Nine organ-specific severity scales were developed from 0 (no documented involvement) to 4 (endstage disease). The data required for scale completion are relatively easy and practical for all physicians to obtain. CONCLUSION: This preliminary severity scale will be useful for assessing disease severity status in individual patients both at one point in time and longitudinally. The severity scale will assist in the design and conduct of clinical trials and the comparison of study populations with one another. The scale will serve as a framework for developing a scleroderma disease activity index.

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.

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.

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.

Thrombospondin 1 is expressed by mesenchymal cells in mouse post-natal skin and hair follicle development.

Thrombospondin 1 is an extracellular matrix glycoprotein with multiple functions. In the skin, it has been immunolocalized to basement membrane, and its expression increases during embryogenesis and wound healing. Its normal cellular source in the skin is not known, except during wound healing, where macrophages and keratinocytes seem to be the primary source. We have analysed the expression of thrombospondin 1 mRNA in normal mouse skin at different ages by in situ hybridization. It was found that the mRNA is expressed by dermal mesenchymal cells and mature fibroblasts and developmentally regulated during post-natal skin growth and morphogenesis. In adult mouse skin, expression of the thrombospondin is restricted to the mesenchymal cells of hair follicle papilla. These results suggest that the regulation of thrombospondin 1 transcription in mesenchymal cells can play an important role in post-natal skin development. Its mRNA expression is a characteristic of adult dermal papilla cells with a potential role in hair development.

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.

Quinolinic acid accumulation in injured spinal cord: time course, distribution, and species differences between rat and guinea pig.

Experimental compression injury of the spinal cord in guinea pigs results in delayed neurologic deficits that continue to increase in severity for several days following trauma, coincident with inflammatory responses, including invasion of the lesion by mononuclear phagocytes and increased levels of the neurotoxin quinolinic acid (QUIN). Inflammatory responses and QUIN elevation also occur following spinal cord contusion in rats, but maximal neurologic deficits develop immediately. In this study, somatosensory evoked potentials (SEP) and tissue, serum, and cerebrospinal fluid levels of QUIN were measured in guinea pigs and rats following similar compression injuries of the thoracic spinal cord. SEP changes differed between the species, consistent with other neurological changes. In guinea pigs, increases in QUIN levels at the lesion site began at 1 day postinjury, achieved maximal elevation (100-fold) by 12 days, then declined, but remained above serum levels at 25 days postinjury. A similar increase occurred in adjacent areas of the spinal cord, with lower peak levels. In rats, tissue QUIN at the center of the lesion remained below serum levels at all times, increasing moderately (<10-fold) up to 7 days, then decreasing between 7 and 25 days. These data demonstrate differences in the time course and magnitude of QUIN accumulation and neurological deficit between guinea pig and rat, which may relate to differences in secondary pathological mechanisms. Such profound differences may affect the use of these species for evaluation of experimental therapy in this and other inflammatory conditions of the central nervous system.

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.

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.

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.

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.