Faye N. Hant

Endothelial Fli1 Deficiency Impairs Vascular Homeostasis : A Role in Scleroderma Vasculopathy

Systemic sclerosis or scleroderma (SSc) is a complex autoimmune connective tissue disease characterized by obliterative vasculopathy and tissue fibrosis. The molecular mechanisms underlying SSc vasculopathy are largely unknown. Friend leukemia integration factor 1 (Fli1), an important regulator of immune function and collagen fibrillogenesis, is expressed at reduced levels in endothelial cells in affected skin of patients with SSc. To develop a disease model and to investigate the function of Fli1 in the vasculature, we generated mice with a conditional deletion of Fli1 in endothelial cells (Fli1 CKO). Fli1 CKO mice showed a disorganized dermal vascular network with greatly compromised vessel integrity and markedly increased vessel permeability. We show that Fli1 regulates expression of genes involved in maintaining vascular homeostasis including VE-cadherin, platelet endothelial cell adhesion molecule 1, type IV collagen, matrix metalloproteinase 9, platelet-derived growth factor B, and S1P(1) receptor. Accordingly, Fli1 CKO mice are characterized by down-regulation of VE-cadherin and platelet endothelial cell adhesion molecule 1, impaired development of basement membrane, and a decreased presence of alpha-smooth muscle actin-positive cells in dermal microvessels. This phenotype is consistent with a role of Fli1 as a regulator of vessel maturation and stabilization. Importantly, vascular characteristics of Fli1 CKO mice are recapitulated by SSc microvasculature. Thus, persistently reduced levels of Fli1 in endothelial cells may play a critical role in the development of SSc vasculopathy.

Surfactant Protein D and KL-6 as Serum Biomarkers of Interstitial Lung Disease in Patients with Scleroderma

Objective. To assess whether serum concentrations of surfactant protein D (SP-D) and Krebs von den Lungen-6 (KL-6), glycoproteins expressed by type II pneumocytes, correlate with the presence of “alveolitis” and measures of lung function in patients enrolled in the Scleroderma Lung Study (SLS). Methods. Serum obtained at baseline screening of patients with systemic sclerosis (SSc, scleroderma) in the SLS was assayed. “Alveolitis” was defined by either bronchoalveolar lavage or thoracic high-resolution computed tomography (HRCT) by SLS criteria. SP-D and KL-6 levels were measured by ELISA in 66 SSc patients (44 with “alveolitis,” 22 without “alveolitis”) and in 10 healthy controls. These were compared to clinical measures of lung disease and “alveolitis” in the SLS patients. Results. SP-D levels were 300 ± 214 ng/ml (mean ± SD) in the SSc patients compared to 40 ± 51 ng/ml in controls (p < 0.0001). KL-6 levels were 1225 ± 984 U/ml in the SSc patients and 333 ± 294 U/ml in controls (p < 0.0001). SSc patients with “alveolitis” had higher levels of both SP-D and KL-6 than those without “alveolitis.” The level of SP-D was 353 ± 219 ng/ml in patients with “alveolitis” and 161 ± 143 ng/ml without “alveolitis” (p = 0.0002). The level of KL-6 was 1458 ± 1070 U/ml in patients with “alveolitis” and 640 ± 487 U/ml without “alveolitis” (p = 0.0001). Receiver operator characteristic curve analysis demonstrated high sensitivity and specificity of both SP-D and KL-6 for the determination of “alveolitis.” KL-6 and SP-D were positively correlated with maximum fibrosis scores, but not with maximum ground-glass opacities, on HRCT. Conclusion. Serum levels of SP-D and KL-6 appear to be indicative of “alveolitis” in SSc patients as defined by the SLS, and are significantly higher than in SSc patients without “alveolitis.” Serum SP-D and KL-6 may serve as noninvasive serological means of assessing interstitial lung disease in patients with SSc.

Endoglin promotes TGF-β/Smad1 signaling in scleroderma fibroblasts.

TGF‐β is the primary inducer of extracellular matrix proteins in scleroderma (systemic sclerosis, SSc). Previous studies indicate that in a subset of SSc fibroblasts TGF‐β signaling is activated via elevated levels of activin receptor‐like kinase (ALK) 1 and phosphorylated Smad1 (pSmad1). The goal of this study was to determine the role of endoglin/ALK1 in TGF‐β/Smad1 signaling in SSc fibroblasts. In SSc fibroblasts, increased levels of endoglin correlated with high levels of pSmad1, collagen, and connective tissue growth factor (CCN2). Endoglin depletion via siRNA in SSc fibroblasts inhibited pSmad1 but did not affect pSmad2/3. Following endoglin depletion mRNA and protein levels of collagen and CCN2 were significantly decreased in SSc fibroblasts but remained unchanged in normal fibroblasts. ALK1 was expressed at similar levels in SSc and normal fibroblasts. Depletion of ALK1 resulted in inhibition of pSmad1 and a moderate but significant reduction of mRNA and protein levels of collagen and CCN2 in SSc fibroblasts. Furthermore, constitutively high levels of endoglin were found in complexes with ALK1 in SSc fibroblasts. Overexpression of constitutively active ALK1 (caALK1) in normal and SSc fibroblasts led to a moderate increase of collagen and CCN2. However, caALK1 potently induced endothelin 1 (ET‐1) mRNA and protein levels in SSc fibroblasts. Additional experiments demonstrated that endoglin and ALK1 mediate TGF‐β induction of ET‐1 in SSc and normal fibroblasts. In conclusion, this study has revealed an important profibrotic role of endoglin in SSc fibroblasts. The endoglin/ALK1/Smad1 pathway could be a therapeutic target in patients with SSc if appropriately blocked. J. Cell. Physiol. 226: 3340–3348, 2011.

Dihydrosphingosine 1-phosphate has a potent antifibrotic effect in scleroderma fibroblasts via normalization of phosphatase and tensin homolog levels

OBJECTIVE Previous studies have revealed a phosphatase and tensin homolog (PTEN)-dependent interaction between the sphingolipid agonist dihydrosphingosine 1-phosphate (dhS1P) and the transforming growth factor beta/Smad3 signaling pathway. This study was undertaken to examine responses of systemic sclerosis (SSc) fibroblasts to sphingosine 1-phosphate (S1P) and dhS1P and to gain further insight into the regulation of the S1P/dhS1P/PTEN pathway in SSc fibrosis. METHODS Fibroblast cultures were established from skin biopsy samples obtained from patients with SSc and matched healthy controls. Western blotting and quantitative polymerase chain reaction were used to measure protein and messenger RNA levels, respectively. PTEN protein was examined in skin biopsy samples by immunohistochemistry. RESULTS PTEN protein levels were low in SSc fibroblasts and correlated with elevated levels of collagen and phospho-Smad3 and reduced levels of matrix metalloproteinase 1 (MMP-1). Treatment with dhS1P restored PTEN levels and normalized collagen and MMP-1 expression, as well as Smad3 phosphorylation status in SSc fibroblasts. S1P was strongly profibrotic in SSc and control fibroblasts. Distribution of S1P receptor isoforms was altered in SSc fibroblasts, which had reduced levels of S1P receptor 1 and S1P receptor 2 and elevated levels of S1P receptor 3. Only depletion of S1P receptor 1 abrogated the effects of dhS1P and S1P in control dermal fibroblasts. In contrast, depletion of either S1P receptor 1 or S1P receptor 2 prevented the effects of S1P and dhS1P in SSc fibroblasts. CONCLUSION Our findings demonstrate that PTEN deficiency is a critical determinant of the profibrotic phenotype of SSc fibroblasts. The antifibrotic effect of dhS1P is mediated through normalization of PTEN expression, suggesting that dhS1P or its derivatives may be effective as therapeutic antifibrotic agents. The distribution and function of S1P receptors differ in SSc and healthy fibroblasts, suggesting that alteration in the sphingolipid signaling pathway may contribute to SSc fibrosis.

Pulmonary manifestations of scleroderma and mixed connective tissue disease.

Pulmonary manifestations are common in connective tissue diseases, and are associated with significant morbidity and mortality in this patient population. Systemic sclerosis (SSc) and mixed connective tissue disease (MCTD) are clinical entities for which the detection of lung involvement is essential to improve patient care and outcomes. This article discusses the pathogenesis, clinical presentation, and evaluation of the patient with pulmonary disease related to SSc and MCTD, with an emphasis on interstitial lung disease and pulmonary hypertension.

Autocrine Transforming Growth Factor β Signaling Regulates Extracellular Signal-regulated Kinase 1/2 Phosphorylation via Modulation of Protein Phosphatase 2A Expression in Scleroderma Fibroblasts

BackgroundDuring scleroderma (SSc) pathogenesis, fibroblasts acquire an activated phenotype characterized by enhanced production of extracellular matrix (ECM) and constitutive activation of several major signaling pathways including extracellular signal-related kinase (ERK1/2). Several studies have addressed the role of ERK1/2 in SSc fibrosis however the mechanism of its prolonged activation in SSc fibroblasts is still unknown. Protein phosphatase 2A (PP2A) is a key serine threonine phosphatase responsible for dephosphorylation of a wide array of signaling molecules. Recently published microarray data from cultured SSc fibroblasts suggests that the catalytic subunit (C-subunit) of PP2A is downregulated in SSc. In this study we examined the role and regulation of PP2A in SSc fibroblasts in the context of ERK1/2 phosphorylation and matrix production.ResultsWe show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression. Furthermore, transforming growth factor β (TGFβ), a major profibrotic cytokine implicated in SSc fibrosis, downregulates PP2A expression in healthy fibroblasts. PP2A-specific small interfering RNA (siRNA) was utilized to confirm the role of PP2A in ERK1/2 dephosphorylation in dermal fibroblasts. Accordingly, blockade of autocrine TGFβ signaling in SSc fibroblasts using soluble recombinant TGFβ receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression. In addition, we observed that inhibition of ERK1/2 in SSc fibroblasts increased PP2A expression suggesting that ERK1/2 phosphorylation also contributes to maintaining low levels of PP2A, leading to an even further amplification of ERK1/2 phosphorylation.ConclusionsTaken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFβ signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts.

Akt inhibition up-regulates MMP1 through a CCN2-dependent pathway in human dermal fibroblasts.

Please cite this article as: Akt inhibition up‐regulates MMP1 through a CCN2‐dependent pathway in human dermal fibroblasts. Experimental Dermatology 2010.

Biomarkers of Scleroderma Lung Disease: Recent Progress

This article reviews the clinical background and significance of selected biomarkers that have been studied in relation to systemic sclerosis, or scleroderma, a devastating connective tissue disease whose morbidity and mortality are often related to pulmonary complications. Interstitial lung disease is the most common pulmonary manifestation in systemic sclerosis, and the search for a noninvasive biomarker to assess and monitor patients and their lung disease is a nascent and expending field of study. In this article, we examine the background and significance of a variety of selected biomarkers and assess their role in relation to systemic sclerosis–related interstitial lung disease.

Imaging of scleroderma.

Systemic sclerosis is a rare autoimmune condition that affects a variety of organ systems. Knowledge of the imaging features in this patient population is essential in facilitating accurate diagnosis and guiding treatment. Common and rare imaging features of systemic sclerosis are reviewed in this article. Skin, musculoskeletal, pulmonary, cardiac, gastrointestinal, renal, and oral imaging are discussed. Conventional radiography, computed tomography of the chest, echocardiography, enterography, scintigraphy, and panorex dental imaging are reviewed. In addition, the evolving applications of ultrasonography and magnetic resonance imaging to evaluate the musculoskeletal and cardiac features of systemic sclerosis are discussed.

Elevated expression of cav-1 in a subset of SSc fibroblasts contributes to constitutive Alk1/Smad1 activation.

Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin‐1 (cav‐1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin‐1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up‐regulated cav‐1 expression in vitro, and that cav‐1 up‐regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho‐Smad1 were down‐regulated after inhibition of cav‐1 in SSc dermal fibroblasts. Caveolin‐1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav‐1 and adenoviral cav‐1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav‐1 and that cav‐1 is sufficient for Smad‐1 phosphorylation. We also show that cav‐1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ‐induced CCN2 levels. In conclusion, this study has revealed an important role of cav‐1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.