Platelet-derived growth factor receptor-β and epidermal growth factor receptor in pulmonary vasculature of systemic sclerosis-associated pulmonary arterial hypertension versus idiopathic pulmonary arterial hypertension and pulmonary veno-occlusive disease: a case-control study

Abstract

Introduction: Systemic sclerosis (SSc) complicated by pulmonary arterial hypertension (PAH) carries a poor prognosis, despite pulmonary vascular dilating therapy. Platelet-derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR) are potential therapeutic targets for PAH because of their proliferative effects on vessel remodelling. To explore their role in SScPAH, we compared PDGFR- and EGFR-mmunoreactivity in lung tissue specimens from SScPAH. We compared staining patterns with idiopathic PAH (IPAH) and pulmonary veno-occlusive disease (PVOD), as SScPAH vasculopathy differs from IPAH and sometimes displays features of PVOD. Immunoreactivity patterns of phosphorylated PDGFR-β (pPDGFR-β) and the ligand PDGF-B were evaluated to provide more insight into the patterns of PDGFR-b activation.

Methods: Lung tissue specimens from five SScPAH, nine IPAH, six PVOD patients and five controls were examined. Immunoreactivity was scored for presence, distribution and intensity.

Results: All SScPAH and three of nine IPAH cases (P = 0.03) showed PDGFR-β-immunoreactivity in small vessels (arterioles/venules); of five SScPAH vs. two of nine IPAH cases (P = 0.02) showed venous immunoreactivity. In small vessels, intensity was stronger in SScPAH vs. IPAH. No differences were found between SScPAH and PVOD. One of five normal controls demonstrated focally mild immunoreactivity. There were no differences in PDGF-ligand and pPDGFR-b-immunoreactivity between patient groups; however, pPDGFR-b-immunoreactivity tended to be more prevalent in SScPAH small vasculature compared to IPAH. Vascular EGFR-immunoreactivity was limited to arterial and arteriolar walls, without differences between groups. No immunoreactivity was observed in vasculature of normals.

Conclusions: PDGFR-β-immunoreactivity in SScPAH is more common and intense in small- and post-capillary vessels than in IPAH and does not differ from PVOD, fitting in with histomorphological distribution of vasculopathy. PDGFR-β immunoreactivity pattern is not paralleled by pPDGFR-β or PDGF-B patterns. PDGFR-β- and EGFR-immunoreactivity of pulmonary vessels distinguishes PAH patients from controls.

Figure 1

Plateled-derived growth factor receptor (PDGFR)-β immunoreactivity in pulmonary vessels in SScPAH, IPAH and PVOD. (Original magnification 200×). A) PDGFR-β immunoreactivity in the endothelial layer of a small corner vessel without fibrosis in SScPAH. Note the epithelial immunoreactivity at the alveolar attachments of the corner vessel, serving as an internal positive control. B) PDGFR-β immunoreactivity in the endothelium of a small corner vessel with fibrosis in SScPAH, and in the (congested) capillary endothelium. C) Absence of PDGFR-β immunoreactivity in the endothelial layer of a small corner vessel with intimal fibrosis, in an area of capillary congestion, in PVOD. There is epithelial immunoreactivity at the alveolar attachments of the corner vessel. D) PDGFR-β immunoreactivity in endothelial cells of capillaries in a SScPAH patient. E) Intense PDGFR-β endothelial immunoreactivity in an area of congestion in a PVOD patient. F) PDGFR-β endothelial immunoreactivity in a vein in a SScPAH patient. G) PDGFR-β immunoreactivity in the basal side of the endothelium of an arteriole with intimal fibrosis and media hyperplasia in an IPAH patient. H) PDGFR-β immunoreactivity in a plexiform lesion in an IPAH patient; immunoreactivity of basal side of the endothelial cells (insert) in the glomeruloid lesion, surrounded by vein-like branches (dilatation lesions).

Figure 2

Number of cases with plateled-derived growth factor receptor (PDGFR)-β-immunoreactivity in the intima of pulmonary vessels in SScPAH, IPAH, PVOD, controls. Small vessels: those arterioles and/or venules that cannot be distinguished based on their anatomical localisation. * P < 0.05

Figure 3

Intensity of plateled-derived growth factor-receptor (PDGFR)-β-immunostaining in the intima of pulmonary vessels in SScPAH, IPAH, PVOD and controls. A: arterioles. B: small vessels.For the pooled arterioles and small vessels, intensity in SScPAH was stronger than in IPAH (P = 0.02). Each image represents one case. Small vessels: those arterioles and/or venules that cannot be distinguished as such based on their anatomical localisation.

Figure 4

Phosphorylated plateled-derived growth factor receptor (pPDGFR)-β immunoreactivity in pulmonary vessels in SScPAH, IPAH and PVOD. (Original magnification 200×) A) pPDGFR-β immunoreactivity in endothelial cell nuclei in a small vessel with intimal fibrosis in a patient with SScPAH. B) pPDGFR-β immunoreactivity in cell nuclei in the intima of an arteriole with concentric laminar intimal fibrosis in a patient with SScPAH (see also 6B). C) pPDGFR-β immunoreactivity in cell nuclei of a small vessel and capillaries in a PVOD patient. D) pPDGFR-β immunoreactivity in the basal side of the endothelium, the thickened intima and in smooth muscle cells of the hyperplastic media of an arteriole of an IPAH patient (see also Figures 1G and 6D). E) pPDGFR-β immunoreactivity in stroma and endothelium of a plexiform lesion in an IPAH patient. Lower right quadrant parent artery. In the center a glomeruloid lesion surrounded by vein-like branches (dilatation lesions).

Figure 5

Amount of phosphorylated plateled-derived growth factor receptor (pPDGFR)-β-positively immunostained cells in intima of the small vessels in SScPAH, IPAH, PVOD and controls. With a cut off of 25% cell staining, a trend was shown (P = 0.09) in favor of more positive cell immunoreactivity in small vasculature in SScPAH patients vs. IPAH patients. Each image represents one case.

Figure 6

Plateled-derived growth factor (PDGF) B immunoreactivity in pulmonary vessels in SScPAH, IPAH and PVOD. (Original magnification 200×). A) PDGF-B immunoreactivity in small vessel and capillaries in a patient with SScPAH. B) PDGF-B immunoreactivity in the intima layers of an arteriole with concentric laminar intimal fibrosis in a patient with SScPAH (see also 4B). C) PDGF-B immunoreactivity in a small vessel and (congested) capillaries in a PVOD patient. D) PDGF-B immunoreactivity in the basal side of the endothelium, the thickened intima and in smooth muscle cells in the hyperplastic media of an arteriole in a IPAH patient. E) PDGF-B immunoreactivity in stroma and endothelium of a plexiform lesion in an IPAH patient. In the center a glomeroid lesiom surrounded by vein-like branches (dilatation lesions).

Figure 7

Epidermal growth factor receptor (EGFR) immunoreactivity in pulmonary vessels in SScPAH, IPAH and PVOD. (Original magnification 200×). A) EGFR expression of alveolar epithelium surrounding a parenchymal arteriole (corner vessel) in a SScPAH patient, showing weak immunoreactivity of the media and endothelium. B) Weak EGFR immunoreactivity of endothelial cells in a SScPAH patient. C) EGFR expression in media and fibrotic intimal layer of an axial artery of a SScPAH patient. Left lower quadrant: parent artery. Center: glomeruloid lesion, surrounded by dilatation lesions (original magnification 100×). D) Plexiform lesion in an IPAH patient showing weak stromal EFGR expression.

Figure 8

Number of cases with positive immunostaining for epidermal growth factor receptor (EGFR) in the intima of pulmonary vessels. A: arterioles, B: Small vessels, C: veins) in SScPAH, IPAH, PVOD and normal controls. No staining was observed in the capillaries. Small vessels: those arterioles and/or venules that cannot be distinguished as such by their anatomical localisation