Marie Wildt

Survival in patients with pulmonary arterial hypertension associated with systemic sclerosis from a Swedish single centre: prognosis still poor and prediction difficult

Objectives: To describe the survival rate in a cohort of systemic sclerosis (SSc) patients with pulmonary arterial hypertension (PAH) and to evaluate possible predictors for SSc-PAH in a cohort of SSc patients. Methods: Thirty patients with SSc-PAH and 150 SSc patients without PAH were included. Survival and survival on therapy were calculated. Clinical features at baseline were correlated to the risk for development of PAH during follow-up. Results: The 1-, 2-, 3-, and 4-year survival rates were 86, 59, 39, and 22%, respectively, from diagnosis of PAH. The hazard ratio for total mortality in the SSc-PAH group was 3.2 [95% confidence interval (CI) 1.8–5.7] compared to SSc without PAH (p < 0.001). Risk factors at baseline for the development of PAH were: limited skin involvement, low diffusing capacity of the lung for carbon monoxide (DLCO), high N-terminal pro-brain natriuretic peptide (NTProBNP), increased estimated systolic pulmonary arterial pressure (ESPAP), and the presence of teleangiectases. Severe peripheral vascular disease requiring iloprost treatment during follow-up was associated with an eightfold increased risk of PAH. Conclusion: Despite modern treatment and yearly screening by echocardiography, the survival in SSc-PAH is still low in our cohort. The identified risk factors should be assessed to select patients eligible for right heart catheterization (RHC) to make an earlier diagnosis.

Functional and phenotypical comparison of myofibroblasts derived from biopsies and bronchoalveolar lavage in mild asthma and scleroderma

BackgroundActivated fibroblasts, which have previously been obtained from bronchoalveolar lavage fluid (BALF), are proposed to be important cells in the fibrotic processes of asthma and scleroderma (SSc). We have studied the motility for BALF derived fibroblasts in patients with SSc that may explain the presence of these cells in the airway lumen. Furthermore, we have compared phenotypic alterations in activated fibroblasts from BALF and bronchial biopsies from patients with mild asthma and SSc that may account for the distinct fibrotic responses.MethodsFibroblasts were cultured from BALF and bronchial biopsies from patients with mild asthma and SSc. The motility was studied using a cell migration assay. Western Blotting was used to study the expression of alpha-smooth muscle actin (α-SMA), ED-A fibronectin, and serine arginine splicing factor 20 (SRp20). The protein expression pattern was analyzed to reveal potential biomarkers using two-dimensional electrophoresis (2-DE) and sequencing dual matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF). The Mann-Whitney method was used to calculate statistical significance.ResultsIncreased migration and levels of ED-A fibronectin were observed in BALF fibroblasts from both groups of patients, supported by increased expression of RhoA, Rac1, and the splicing factor SRp20. However, these observations were exclusively accompanied by increased expression of α-SMA in patients with mild asthma. Compared to BALF fibroblasts in mild asthma, fibroblasts in SSc displayed a differential protein expression pattern of cytoskeletal- and scavenger proteins. These identified proteins facilitate cell migration, oxidative stress, and the excessive deposition of extracellular matrix observed in patients with SSc.ConclusionThis study demonstrates a possible origin for fibroblasts in the airway lumen in patients with SSc and important differences between fibroblast phenotypes in mild asthma and SSc. The findings may explain the distinct fibrotic processes and highlight the motile BALF fibroblast as a potential target cell in these disorders.

Small intestinal manometry in patients with systemic sclerosis.

Objectives The study explores, by the use of manometry, the frequency and severity of small intestinal involvement in patients with systemic sclerosis, and relates the manometric findings to clinical symptoms, radiology, and some intestinal regulatory peptides. Methods Stationary antroduodeno-jejunal manometry was used to study small bowel involvement in 10 patients with systemic sclerosis and dysmotility of the oesophagus or signs of malabsorption. Measurements were made during fasting, after a meal, and after octreotide administration and were then compared with a sex-matched control group of healthy individuals. Plasma samples were taken in order to analyse levels of motilin, peptide YY, cholecystokinin, and somatostatin. Results Manometry was abnormal, with signs of intestinal pseudo-obstruction in eight out of 10 patients. In the control group, one individual had an abnormal manometry, as a result of burst activity. The mean contractile amplitudes during fasting and periods after food, spontaneous phase III periods, and octreotide-induced activity complexes were significantly reduced in the systemic sclerosis group when compared with controls. None of the patients, including two with advanced manometric intestinal disturbances, had small intestinal dilatation when examined by radiography. The plasma peptide levels did not differ significantly between the two groups. Conclusions In eight out of 10 patients the manometric criteria for intestinal pseudo-obstruction were fulfilled, with a motility pattern consistent with both neuropathy and myopathy. The release of motility-regulating peptides was unaffected.

A multicentre study on the reliability of qualitative and quantitative nail-fold videocapillaroscopy assessment

OBJECTIVE To investigate the inter- and intra-observer reliability of both qualitative and quantitative parameters used in the assessment of nail-fold capillaroscopy images. METHODS Fifty mosaic nail-fold images of healthy controls (n = 10), patients with primary RP (n = 10) and SSc (n = 30) were assessed in random order by two blinded observers on two occasions at centres in Sweden, UK and The Netherlands. Each image was therefore scored by six observers twice. RESULTS Inter- and intra-observer reliability of quantitative parameters showed substantial to almost perfect agreement [inter- and intra-observer weighted κs for the number of widened capillaries was 0.75 and 0.87 and giant capillaries was 0.84 and 0.92, intra-class correlation coefficients (ICCs) for capillary density was 0.87 and 0.92 and total loop width was 0.94 and 0.98, respectively]. Qualitative parameters including architecture, avascularity, haemorrhage, crossed, ramified and bushy capillaries showed moderate to substantial inter-observer reproducibility (weighted κ ranging from 0.47 to 0.73), and substantial intra-observer repeatability (weighted κ ranging from 0.71 to 0.80), whereas the scoring of tortuous and bizarre capillaries showed poor inter-observer and substantial intra-observer agreement (inter-observer weighted κs was 0.39 and 0.21 and intra-observer weighted κs was 0.68 and 0.76, respectively). CONCLUSION All quantitative and certain qualitative parameters are highly reliable in terms of inter- and intra-observer agreement. A combination of parameters with the highest reliability should be incorporated into future capillaroscopic scoring systems in studies of prediction and monitoring of SSc spectrum disorders.

Serum IL-15 in patients with early systemic sclerosis: a potential novel marker of lung disease

The pathogenesis of systemic sclerosis (SSc) is characterized by autoimmunity, vasculopathy and fibrosis. IL-15 is a pleiotropic cytokine that has impact on immune, vascular and connective tissue cells. We therefore investigated IL-15 in the circulation of patients with early SSc and explored possible associations of serum IL-15 with vasculopathy and fibrosis. Serum levels of IL-15 were analysed in 63 consecutive patients with SSc of disease duration less than 4 years and without disease-modifying treatment. Thirty-three age-matched healthy control individuals were enrolled. Serum IL-15 levels were increased in the sera of SSc patients compared with that of healthy control individuals (P < 0.01). Serum IL-15 levels correlated with impaired lung function, assessed both by the vital capacity (P < 0.05) and by the carbon monoxide diffusion capacity (P < 0.05). The association between IL-15 and the vital capacity remained after multiple linear regression analysis. Patients with intermediate serum IL-15 levels had a higher prevalence of increased systolic pulmonary pressure compared with patients with either low or high serum IL-15 levels (P < 0.05). Moreover, increased serum IL-15 levels were associated with a reduced nailfold capillary density in multivariable logistic regression analysis (P < 0.01). Serum IL-15 levels also correlated inversely with the systolic blood pressure (P < 0.01). We conclude that IL-15 is associated with fibrotic as well as vascular lung disease and vasculopathy in early SSc. IL-15 may contribute to the pathogenesis of SSc. IL-15 could also be a candidate biomarker for pulmonary involvement and a target for therapy in SSc.

Computer based quantitative analysis of capillary abnormalities in systemic sclerosis and its relation to plasma concentration of von Willebrand factor.

OBJECTIVES--To evaluate an objective and quantitative method for assessment of capillary abnormalities in systemic sclerosis (SSc). METHODS--Nailfold capillaries were investigated by capillary microscopy and photographed in 17 consecutive SSc patients (five with diffuse cutaneous systemic sclerosis (dSSc) and 12 with limited cutaneous systemic sclerosis (lSSc)) and in 17 healthy controls. Investigators having no access to clinical data made drawings from magnified projections of coded photographs and analysed them using a computer program. Capillary density (capillary loops/mm in the distal row) and median capillary loop area were calculated. Presence of functional or organic arterial changes was evaluated by measurement of finger pressure with finger cooling. Plasma concentration of von Willebrand factor (VWF) was analysed using an enzyme linked immunosorbent assay (ELISA). RESULTS--In 16 of 17 SSc patients and 13 of 17 controls the technical quality of the photographs was sufficient for computer analysis. Capillary density was decreased in dSSc (median 6.9 loops/mm) and in lSSc (median 3.8 loops/mm) compared with healthy controls (8.9 loops/mm) and median capillary loop area was increased in dSSc (7.3 x 10(-3) mm2) and in lSSc (8.5 x 10(-3) mm2) compared with healthy controls (5.0 x 10(-3) mm2). An inverse relation was found between capillary density and median capillary loop area in SSc patients. Plasma VWF was increased in patients (median 401 IE/l in dSSc and 409 IE/l in lSSc) compared with controls matched for age and sex (median 276 IE/l). Computer based analysis showed capillary density below the control range and median capillary loop area above the control range in 14 of 16 SSc patients. Measurement of finger pressure with finger cooling showed organic vascular changes in nine of 13 SSc patients. CONCLUSION--Computer based quantitative analysis has low interobserver variability and is a quantitative and sensitive method of assessing capillary abnormalities in SSc.

BAL fluid derived fibroblasts differ from biopsy derived fibroblasts in systemic sclerosis.

Growth of fibroblasts from bronchoalveolar lavage fluid (BALF) in patients with systemic sclerosis (SSc) has previously been described. The purpose of the present study was to characterise fibroblasts from BALF and bronchial biopsies from SSc patients with alveolitis and from controls, to analyse fibroblast proliferation, migration, stress fibres and proteoglycan production. BALF and bronchial biopsies were collected from 10 patients with SSc and alveolitis and from 15 controls. Outgrowth of fibroblasts was observed from the BALF of four patients, particularly in those with a markedly increased percentage of eosinophils in BALF, but not in any member of the control group. Increased levels of granulocyte-macrophage colony-stimulating factor, correlating with the percentage of eosinophils in BALF, were found in patients when compared with controls. Fibroblasts from BALF showed an elongated, mobile phenotype and increased proteoglycan production compared to the corresponding biopsy fibroblasts. In conclusion, outgrowth of fibroblasts with an altered phenotype is reported from bronchoalveolar lavage fluid in systemic sclerosis patients with alveolitis and an increased percentage of eosinophils in the bronchoalveolar lavage fluid. These findings indicate a possible role for eosinophil–fibroblast interaction in pulmonary fibrosis in systemic sclerosis.

Specific autoantibody profiles and disease subgroups correlate with circulating micro-RNA in systemic sclerosis

OBJECTIVE To evaluate the expression profiles of cell-free plasma miRNAs in SSc and to characterize their correlation with disease subgroups (lcSSc and dcSSc) and with autoantibody profiles. METHODS Using quantitative RT-PCR, the abundance of 45 mature miRNAs in plasma was determined in 95 patients (lcSSc = 63; dcSSc = 32), representing the following autoantibody subgroups: ACA, anti-DNA topoisomerase I, anti-RNA polymerase III and anti-U1-ribonucleoprotein. MiRNA data were correlated with clinical and paraclinical data. Multiple regression was used to model membership of the lcSSc, dcSSc and autoantibody subgroups, based on miRNA expression profiles. RESULTS Thirty-six miRNAs were measurable in all samples. Four (miRNA-223, -181b, -342-3p and -184) were differently expressed in lcSSc and dcSSc (false discovery rate < 0.05). Ten miRNAs exhibited statistically significantly different levels in one or more autoantibody groups, and five (miRNA-409, -184, -92a, -29a and -101) remained significant after correction for multiple comparisons. Multiple regression models accurately predicted ACA and anti-DNA topoisomerase I antibody-positive patients (area under the curve (AUC) = 0.97 and 0.93, respectively) as well as membership of the dcSSc and lcSSc groups (AUC = 0.88). CONCLUSION Circulating miRNA profiles differ between lcSSc and dcSSc patients and between patients with different autoantibodies. This is the first time autoantibody profiles, disease phenotypes and plasma miRNA profiles have been shown to correlate in an autoimmune disease. The data support a pathobiological role of miRNAs because specific miRNAs associate with autoantibody profiles of known diagnostic and prognostic value.

Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.

Splicosomal and serine and arginine-rich splicing factors as targets for TGF-β

BackgroundTransforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins.Methodology/Principal findingsThe expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/− 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence.ConclusionsThe results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.