He diagnosis of SScPAH, IPAH and PVOD was verified by reviewing the medical records. Only patients diagnosed with PAH upon right heart catheterization, with a mean resting pulmonary arterial pressure (mPpa) 25 mmHg and a pulmonary capillary wedge pressure 15 mmHg, were included. The diagnosis of SSc was established by a rheumatologist. SSc patients had to fulfil the preliminary ACR classification criteria for SSc and were classified according to LeRoy et al. [38,39]. Patients with restrictive disease as indicated by total lung capacity as a percentage of predicted (TLC ) <70 , vital capacity (VC ) <70 and/or PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29045898 severe fibrosis on HRCT scan were excluded. Lung tissue from five subjects who had died from extra-pulmonary trauma and who had no cardiorespiratory medical history, was used as a control. Histopathological diagnosis of pulmonary vascular disease was confirmed by independent reading by two pathologists (WJM, KG). PVOD was diagnosed based on the presence of a picture of patchy intense capillary congestion in the alveolar parenchyma, and obliterative intimal, loosely textured fibrosis of small veins and venules. PVOD cases did not have arterialised interlobular veins: this is indicative of congestive vasculopathy [40,41]. The cases were collected from the Departments of Pulmonary Diseases and Rheumatology of the VU University Medical Center, Amsterdam and from the Department of Rheumatology of the Radboud University Nijmegen Medical Center, Nijmegen, both in The Netherlands. The study, including the use of archived tissue was approved by the Institutional Review Board on Research Involving Human Subjects of the VU University Medical Center.Tissue preparation and immunohistochemistryImmunohistochemistry was performed on formalin-fixed paraffin-embedded 4 m sections of lung tissue. All sections were stained in one batch for each marker. Antibodies against PDGFR-b (Cell Signaling Technology, Danvers, MA, USA) and pPDGFR-b (Novus Biologicals, Littleton, CO, USA) were used at dilutions of 1:50 and 1:150, respectively. Active PDGF is built up by polypeptides that form hetero- and homodimers. An antibodyOverbeek et al. Arthritis Research Therapy 2011, 13:R61 http://arthritis-research.com/content/13/2/RPage 3 ofspecific for the PDGF-B form (Novus Biologicals) was used; it reacts with the PDGF (AB) and PDGF (BB) protein. The dilution used for this antibody was 1:400. For EGFR staining, a monoclonal antibody against EGFR (Novocastra, Newcastle upon Tyne, UK) was used. Immunostaining for the constitutively expressed endothelial marker CD31 (PECAM-1, clone JC70a, Dako, Glostrup, Denmark) served as a reference for the exact localization of PDGFR-b and EGFR staining, as well as for PDGFR-b- and EGFR staining intensity, as staining JNJ-26481585 chemical information intensity might be influenced by age of the blocks and duration of fixation. Isotype-matched control-staining was performed with rabbit anti-FITC IgG (Invitrogen, Camarillo, CA, USA). Additional detail on immunostaining is provided in an online data supplement (Additional file 1; figures of isotype-matched control staining: Additional file 2).Scoringimmunoreactivity, Fisher’s Exact test was used to compare non-parametric data between groups. A P-value < 0.05 was considered statistically significant. Other parameters were analysed descriptively due to lack of statistical power.Intensity of immunoreactivity was scored semi-quantitatively as absent, mild, moderate and strong on a 0 to 3 point scale. Immunoreactivity was a.
