OBJECTIVE Aberrant transforming growth factor beta (TGFbeta) signaling has been implicated in the pathogenesis of scleroderma (systemic sclerosis [SSc]), but the contribution of specific components in this pathway to SSc fibroblast phenotype remains unclear. This study was undertaken to delineate the role of TGFbeta receptor type I (TGFbetaRI) and TGFbetaRII in collagen overexpression by SSc fibroblasts. METHODS Primary dermal fibroblasts from SSc patients and healthy adults were studied (n = 10 matched pairs). Adenoviral vectors were generated for TGFbetaRI (AdTGFbetaRI), TGFbetaRII (AdTGFbetaRII), and kinase-deficient TGFbetaRII (AdDeltakRII). TGFbetaRI basal protein levels were analyzed by (35)S-methionine labeling/immunoprecipitation and immunohistochemistry. Type I collagen and TGFbetaRII basal protein levels were analyzed by Western blot and newly secreted collagen by (3)H-proline incorporation assay. RESULTS Analysis of endogenous TGFbetaRI and TGFbetaRII protein levels revealed that SSc TGFbetaRI levels were increased 1.7-fold (P = 0.008; n = 7) compared with levels in healthy controls, while TGFbetaRII levels were decreased by 30% (P = 0.03; n = 7). This increased TGFbetaRI:TGFbetaRII ratio correlated with SSc collagen overexpression. To determine the consequences of altered TGFbetaRI:TGFbetaRII ratio on collagen expression, healthy fibroblasts were transduced with AdTGFbetaRI or AdTGFbetaRII. Forced expression of TGFbetaRI in the range corresponding to elevated SSc TGFbetaRI levels increased basal collagen expression in a dose-dependent manner, while similar TGFbetaRII overexpression had no effect, although transduction of fibroblasts at higher multiplicities of infection led to a marked reduction of basal collagen levels. Blockade of TGFbeta signaling via AdDeltakRII resulted in approximately 50% inhibition of basal collagen levels in healthy fibroblasts and in 5 of 9 SSc cell lines. A subset of SSc fibroblasts (4 of 9 cell lines) was resistant to this treatment. SSc fibroblasts with the highest levels of TGFbetaRI were the least responsive to collagen inhibition via DeltakRII. CONCLUSION This study indicates that an increased TGFbetaRI:TGFbetaRII ratio may underlie aberrant TGFbeta signaling in SSc and contribute to elevated basal collagen production, which is insensitive to TGFbeta signaling blockade via DeltakRII.

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.

OBJECTIVE:To examine the mechanism of collagen induction by connective tissue growth factor (CTGF), a profibrotic cytokine overexpressed in the skin of patients with systemic sclerosis (SSc).METHODS:Dermal fibroblasts from 7 SSc patients and 7 matched healthy adult donors were stimulated with CTGF in the presence or absence of the culture-medium supplement, insulin-transferrin-selenium (ITS). Expression of collagen protein was analyzed by a (3)H-proline incorporation assay. To identify the signaling pathways mediating CTGF induction of collagen, pharmacologic inhibitors were used, including rottlerin, a protein kinase C delta (PKC delta) inhibitor.RESULTS:Collagen levels in both SSc and normal fibroblasts were increased after treatment with transforming growth factor beta in serum-free medium, whereas no stimulation was observed following addition of CTGF. In the presence of ITS, CTGF (2.5 ng/ml) potently stimulated collagenous protein levels in SSc cell lines (n = 5); however, CTGF was not stimulatory in the majority of normal fibroblasts (n = 6). ITS alone induced collagen levels in normal fibroblasts to the levels observed in SSc skin fibroblasts, thereby diminishing the hallmark difference in basal collagen levels in these cell types. Insulin was the ITS component responsible for promoting the basal and CTGF stimulation of collagenous proteins. Rottlerin, the PKC delta inhibitor, down-regulated collagen synthesis in normal and SSc fibroblasts cultured in ITS, and inhibited the stimulatory effects of CTGF in cooperation with insulin or of insulin (500 ng/ml) alone.CONCLUSION:Increased responsiveness of SSc fibroblasts to CTGF-mediated collagen synthesis requires the costimulatory activation of insulin signaling pathways to induce matrix production. Blockade of this effect via rottlerin may suggest that PKC delta is a downstream signaling molecule necessary for CTGF stimulation of collagen synthesis.

Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.

The main manifestation of systemic sclerosis (SSc) is the overproduction of extracellular matrix, predominantly type I collagen. This study was undertaken to evaluate the effects of noncytotoxic doses of the topoisomerase I inhibitor camptothecin (CPT) on collagen production in the activated dermal fibroblasts from patients with SSc and healthy donors. The fibroblasts were cultured in the presence or absence of CPT. Production of collagenous proteins by fibroblasts was determined in cell and matrix layers by ELISA and in conditioned media by [3H]proline incorporation, gel electrophoresis, and autoradiography. Expression of α2(I) collagen (COL1A2) mRNA was measured by northern blot, and the activity of COL1A2 promoter was determined by a chloramphenicol acetyltransferase assay. CPT (10-7 M) decreased the deposition of type I collagen by 68%, of type III by 38%, and of type VI by 21% in SSc fibroblasts and to a lesser degree in healthy controls. Similarly, CPT (10-8 M to 10-6 M) significantly inhibited secretion of newly synthesized collagenous proteins into conditioned media by 50%. CPT (10-8 M to 10-6 M) caused a significant dose-dependent inhibition of COL1A2 mRNA levels and COL1A2 promoter activity, both by as much as 60%. The inhibitory effect of CPT on collagen production by fibroblasts from patients with SSc suggests that topoisomerase I inhibitors may be effective in limiting fibrosis in such patients.

Background Elevated expression of platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta have been observed in a number of fibrotic diseases, including systemic sclerosis (SSc). This suggests a possible interaction between these factors in establishing a profibrotic programme in dermal fibroblasts.Objectives To examine the effects of PDGF isoforms on the expression of TGF-beta receptors in human dermal fibroblasts.Methods Steady-state mRNA levels of TGF-beta receptor I and II (T betaR-I and T betaR-II) were analysed by northern blot. T betaR-I protein levels were analysed by immunoprecipitation of S-35 metabolically labelled cells. T betaR-II protein levels were analysed by western blot.Results Steady-state mRNA levels of T betaR-I and T betaR-II were induced in response to PDGF isoforms. PDGF-AA and PDGF-AB stimulated both receptors with similar potency, whereas PDGF-BB was less potent. The MEK1 (mitogen-activated protein kinase [MAPK] or extracellular signal regulated kinase) inhibitor, PD98059, abrogated the stimulatory effect of PDGF-AB. In contrast to mRNA levels, only TPR-II protein levels were elevated in response to PDGF.Conclusions These data suggest that PDGF receptor alpha and MAPK mediate stimulation of TGF-beta receptors by PDGF. Furthermore, TGF-beta receptor protein levels are discordantly regulated by PDGF.

Background: The efficacy and safety of sustained-release nifedipine for the treatment of primary Raynaud phenomenon (RP) has not previously been demonstrated by a randomized, controlled trial. Temperature biofeedback has been studied in patients with primary RP but not in a large multicenter controlled trial or compared with nifedipine therapy.Objective: To evaluate and compare the effectiveness of sustained-release nifedipine and temperature biofeedback for the treatment of primary RP.Participants and Methods: This is a randomized, controlled clinical trial, double-masked for drug and placebo but not masked for temperature and control biofeedback. It included 313 persons with primary RP as defined by medical history, physical examination findings, normal nailfold capillaries, and a history of 2 or more attacks per day during the previous cold season. Participants were randomized to 1 of 4 treatment groups: (1) sustained-release nifedipine, (2) pill placebo, (3) temperature biofeedback, or (4) control (electromyographic) self-reported, color chart-verified RP attacks during 1 winter month approximately 1 year after initiation of treatment. Secondary outcome measures included verified attacks at 2 months, all attacks at 2 months and 1 year, and quality of life.Results: Nifedipine-treated participants showed a 66% reduction in verified attacks compared with placebo recipients (P<.001); temperature biofeedback training did not reduce attacks significantly compared with control biofeedback (P = .37). Comparison of nifedipine and temperature biofeedback treatments favored nifedipine use (P = .08); similar results were obtained for the secondary end points. Adverse effects resulted in discontinuation of nifedipine treatment in 15% of participants.Conclusions: Temperature biofeedback is not better than its control treatment and is inferior to sustained-release nifedipine for treating primary RP, whereas sustained-release nifedipine is a safe and effective treatment for this disease.

Our previous studies have suggested that digital blood pressure response to cooling could provide a measure of the efficacy of treatments that are administered to patients with Raynaud phenomenon (RP). This method was used on 158 primary RP patients participating in a multicenter, randomized clinical trial that compared the efficacy of sustained-release nifedi-pine with temperature biofeedback in the treatment of RP. A pill placebo and electromyography served as controls. The response to local finger cooling was measured at 30°,20°,15° and 10°C in a temperature-controlled room under standardized conditions. The results showed that, at the 15°C and 10°C local cooling temperatures, the patients in the nifedipine group had a higher mean digital systolic blood pressure, a higher relative digital systolic blood pressure (RDSP), a smaller proportion of subjects with RDSP, 70% and a smaller proportion of subjects with a zero reopening pressure than the patients in the three other treatment groups. These results were statistically significant at 10°C, the nifedipine group being significantly different from all others (p, 0.05); no significant difference was found between the three other treatment groups.

Two patients with diffuse cutaneous systemic sclerosis and spinal calcification, involving the lumbar spine in one and the cervical spine in the other, are described. Computed tomography-guided aspiration of the calcific masses was performed, and material aspirated from one patient was shown to be apatite, Ca5(PO4)3OH. One patient showed improvement following lumbar laminotomy, hemilaminectomy, and diskectomy.

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.

OBJECTIVE:Systemic sclerosis (scleroderma, SSc) frequently affects the lungs, and interstitial pulmonary fibrosis is one of its major complications. The pathophysiology of SSc lung disease s poorly understood, but recent studies document an inflammatory process resembling that of idiopathic pulmonary fibrosis with increased numbers of activated alveolar macrophages and granulocytes in bronchoalveolar lavage (BAL) fluid). We determined levels of 2 potentially important mediators of fibroproliferative repair in BAL fluid from patients with SSc.METHODS:Using Western blot and ELISA techniques we measured levels of platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta in BAL fluid from patients with SSc and healthy controls. The mitogenic effect of these cytokines on SSc lung myofibroblasts was determined by [3H]thymidine incorporation.RESULTS:SSc BAL fluid contains significantly elevated levels of PDGF-AA and PDGF-BB. Where TGF-beta 1 was significantly elevated in SSc lavage fluid, the amount of TGF-beta 2 was significantly less than that observed in normal lavage fluid. Myofibroblasts cultured from SSc lavage fluid exhibited enhanced [3H]thymidine incorporation upon exposure to the growth factors present in SSc BAL fluid: PDGF and TGF-beta 1. SSc lung myofibroblasts pretreated with TGF-beta 1 exhibited an enhanced mitogenic effect upon stimulation by PDGF, due in part to the induction of the PDGF alpha receptor.CONCLUSION:Our studies support a role for PDGF and TGF-beta 1 in the pathogenesis of SSc lung disease.

Mitogen-activated protein kinase (MAPK) is activated in many cell types in response to growth factors during the G0-G1 transition in the cell cycle. We investigated the effects of platelet-derived growth factor (PDGF) AA and PDGF BB on activation of MAPK in human dermal fibroblasts, and asked whether its activation correlates with proliferative responses of human fibroblasts to PDGF AA and PDGF BB. Treatment with either PDGF isoform for 20 min resulted in equal phosphorylation of MAPK as visualized by gel shifts in Western blotting with anti-MAPK polyclonal antibody. This finding was confirmed by measurements of MAPK activity in response to increasing doses (2-20 ng/ml) of PDGF AA and PDGF BB in in vitro assays with myelin basic protein as a substrate. PDGF AA was slightly less potent than PDGF BB, but both growth factors induced similar maximal activations. Kinetics of activation were also similar for both isoforms, with maximal induction of MAPK at 10-20 min after growth factor addition followed by a gradual decline to control levels at 1 h. Activation of MAPK by both PDGF isoforms was also confirmed by measuring myelin basis protein phosphorylation in MAPK immunoprecipitates. Thus both PDGF AA and PDGF BB are potent activators of MAPK in human dermal fibroblasts. In contrast, PDGF BB elicited a strong mitogenic response, while PDGF AA had no significant effect on DNA synthesis in human dermal fibroblasts. These data indicate that acute activation of MAPK is not sufficient to stimulate cells to progress through cell cycle. PDGF AA may have other biologic functions or may play a co-stimulatory role in proliferation of human dermal fibroblasts.

Differences in the responses to growth factors of normal fibroblasts and scleroderma fibroblasts have been demonstrated previously. Because human dermal fibroblasts are heterogeneous populations, whether known differences between papillary and reticular dermal fibroblasts could account for the noted differences between normal and scleroderma fibroblasts was investigated. Papillary dermal fibroblasts were grown from a dermatome section of normal skin from an adult donor. Reticular dermal fibroblasts were cultured from punch biopsy specimens taken from the same location. In vitro, papillary dermal fibroblasts proliferated more rapidly, had a higher mitotic index and reached greater density at confluence, confirming previous observations. The reticular dermal fibroblasts were more dendritic. Reticular dermal fibroblasts had higher rates of tritiated thymidine uptake and larger increases in mitotic index in response to isoforms of platelet-derived growth factor (PDGF). The characteristic response of scleroderma fibroblasts, potentiation of the mitogenicity of PDGF AA by transforming growth factor-beta (TGF-beta), was not observed in either cell type. Therefore, the phenotypic characteristics of scleroderma fibroblasts cannot be explained by an unusual admixture of papillary and reticular fibroblasts.

Scleroderma fibrotic lesions demonstrate vascular disease, mononuclear cell infiltrates, and increased collagen. Fibroblasts in these lesions are activated to synthesize increased extracellular matrix substances, a phenotype that continues when these cells are removed and grown in tissue culture. Levels of messenger RNA for connective-tissue substances, measured directly in biopsies of scleroderma skin, show increased message for type I collagen, but not type III collagen or fibronectin. Increased procollagen type I in scleroderma skin occurs in the papillary dermis, perivascular areas, and deep interstitium, even in skin areas that are not yet fibrotic. Scleroderma fibroblasts express more intercellular adhesion molecule 1 on their surfaces than do normal cells, and this molecule is increased in endothelial cells, mononuclear cells, and fibroblasts. In vitro scleroderma fibroblasts adhere more frequently to extracellular matrix substances and retract collagen lattices to a greater extent. Peripheral blood lymphocytes from scleroderma patients produce excessive amounts of interleukin-2 when incubated with type I collagen, and circulating basophils release more histamine than do normal cells. There is evidence for activated eosinophils both in the dermis and pulmonary lesions in scleroderma, which may play a role in fibrosis. Transforming growth factor-beta is overexpressed by alveolar macrophages from patients with fibrotic pulmonary disease. Scleroderma fibroblasts, when exposed to transforming growth factor-beta, overexpress the alpha-type receptor for platelet-derived growth factor. Scleroderma sera more frequently contain measurable quantities of interleukin-4, interleukin-6, and interleukin-2. Interleukin-4 causes adult dermal fibroblasts to proliferate and to make interleukin-6. Interleukin-6 has been shown to stimulate fibroblast synthesis of collagen and glycosaminoglycans.(ABSTRACT TRUNCATED AT 250 WORDS)