Resistin-like molecule-beta in scleroderma-associated pulmonary hypertension

Abstract

Scleroderma is a systemic, mixed connective tissue disease that can impact the lungs through pulmonary fibrosis, vascular remodeling, and the development of pulmonary hypertension and right heart failure. Currently, little is known about the molecular mechanisms that drive this condition, but we have recently identified a novel gene product that is up-regulated in a murine model of hypoxia-induced pulmonary hypertension. This molecule, known as hypoxia-induced mitogenic factor (HIMF), is a member of the newly described resistin gene family. We have demonstrated that HIMF has mitogenic, angiogenic, vasoconstrictive, inflammatory, and chemokine-like properties, all of which are associated with vascular remodeling in the lung. Here, we demonstrate that the human homolog of HIMF, resistin-like molecule (RELM)-beta, is expressed in the lung tissue of patients with scleroderma-associated pulmonary hypertension and is up-regulated compared with normal control subjects. Immunofluorescence colocalization revealed that RELM-beta is expressed in the endothelium and vascular smooth muscle of remodeled vessels, as well as in plexiform lesions, macrophages, T cells, and myofibroblast-like cells. We also show that addition of recombinant RELM-beta induces proliferation and activation of ERK1/2 in primary cultured human pulmonary endothelial and smooth muscle cells. These results suggest that RELM-beta may be involved in the development of scleroderma-associated pulmonary hypertension.

Figure 1.

Immunohistochemical localization of resistin-like molecule (RELM)-β in human lungs. Paraffin-embedded sections from normal human lung (A, F, K, and P) and from lungs of patients with scleroderma-associated pulmonary hypertension (B–D, G–I, L–N, and Q–S) were stained with a rabbit anti–human RELM-β polyclonal antibody. In negative control samples, no primary antibody was added to serial sections (E, J, O, and T). A, airway; P, plexiform lesion; S, septum; SSc, scleroderma; V, vasculature. Scale bars, 25 μm.

Figure 2.

Immunohistochemical localization of RELM-β in lungs from patients with idiopathic pulmonary hypertension (IPH). Paraffin-embedded sections from normal human lung (A) and from lungs of patients with IPH (B–D) were stained with a rabbit anti-human RELM-β polyclonal antibody. Scale bars, 25 μm.

Figure 3.

Quantification of RELM-β in lungs from patients with scleroderma-associated pulmonary hypertension (SSc) and IPH. (A and C) Frozen human colon (control) and lung tissue samples (SSc and IPH) were homogenized, resolved by 4–20% SDS-PAGE, and transferred to nitrocellulose membranes. The blots were then probed with mouse monoclonal RELM-β antibodies, followed by horseradish peroxidase (HRP)–conjugated anti-mouse IgG antibodies, and developed with enhanced chemiluminescence (ECL). To ensure equal loading and transfer, blots were stripped and reprobed with anti–β-actin antibodies. IB, immunoblot; IB*, immunoblot after stripping. (B and D) Laser densitometry was used to quantify RELM-β levels in the lung samples. Data are normalized to β-actin expression and expressed as relative intensity (mean ± SEM). *P < 0.01 compared with the normal lung.

Figure 4.

(A–H) Colocalization of RELM-β with pulmonary vascular endothelium in patients with scleroderma-associated pulmonary hypertension. (A and E) Light micrograph of fluorescence images to show structure. Paraffin-embedded lung sections were dual stained with a rabbit anti-human RELM-β polyclonal antibody that was visualized by an Alexa Fluor 488–conjugated goat anti-rabbit IgG antibody (B and F) and a mouse anti–von Willebrand (vWF) factor monoclonal antibody visualized by a Cy3-conjugated donkey anti-mouse IgG antibody (C and G). The arrows in the merged images demonstrate colocalization of RELM-β with the pulmonary vascular endothelium (D and H). (I–T) Colocalization of RELM-β with pulmonary vascular smooth muscle in patients with scleroderma-associated pulmonary hypertension. (I, M, and Q) Light micrograph of fluorescence images to show structure. Paraffin-embedded lung sections were dual stained with a rabbit anti–human RELM-β polyclonal antibody that was visualized by an Alexa Fluor 488–conjugated goat anti-rabbit IgG antibody (J, N, and R), and with a mouse anti–α-smooth muscle actin monoclonal antibody that was visualized by a Cy3-conjugated donkey anti-mouse IgG antibody (K, O, and S). The arrows in the merged images demonstrate colocalization of RELM-β with pulmonary vascular smooth muscle (L and P) and with myofibroblasts (T). Scale bar, 25 μm.

Figure 5.

(A–H) Colocalization of RELM-β with macrophages in patients with scleroderma-associated pulmonary hypertension. (A and E) Light micrograph of fluorescence images to show structure. Paraffin-embedded lung sections were dual stained with a rabbit anti-human RELM-β polyclonal antibody that was visualized by an Alexa Fluor 488–conjugated goat anti-rabbit IgG antibody (B and F) and with a mouse anti-CD68 monoclonal antibody that was visualized by a Cy3-conjugated donkey anti-mouse IgG antibody (C and G). The arrows in the merged images demonstrate colocalization of RELM-β with the pulmonary macrophages (D and H). (I–P) Colocalization of RELM-β with T cells in patients with scleroderma-associated pulmonary hypertension. (I and M) Light micrograph of fluorescence images to show structure. Paraffin-embedded lung sections were dual stained with a rabbit anti-human RELM-β polyclonal antibody that was visualized by an Alexa Fluor 488–conjugated goat anti-rabbit IgG antibody (J and N) and with a mouse anti-CD3 monoclonal antibody that was visualized by a Cy3-conjugated donkey anti-mouse IgG antibody (K and O). The arrows in the merged images demonstrate colocalization of RELM-β with T cells (L and P). Scale bar, 25 μm.

Figure 6.

Recombinant human (rh) RELM-β has mitogenic effects on human lung microvascular endothelial cells (HMVEC-Ls) and human pulmonary artery smooth muscle cells (HPASMCs) and activates ERK1/2. HMVEC-Ls (A) and HPASMCs (B) were serum and growth factor starved for 24 hours before being treated with various concentrations of rhRELM-β (0 [control], 25, 50, 100 ng/ml) with or without U0126 (10 μM) for 48 hours. After treatment, the cells were washed, trypsinized, and counted with a hemacytometer. Results are reported as mean (±SEM) of fold increase compared with control. *P < 0.01, **P < 0.001 compared with control; ^†P < 0.05, ^††P < 0.01 compared with rhRELM-β (100 ng/ml). HMVEC-Ls (C) and HPASMCs (D) were cultured to approximately 50% confluence, serum and growth factor starved overnight, and then exposed to rhRELM-β (100 ng/ml) or vehicle (0.1% BSA/PBS) for up to 60 minutes. Cells were lysed and resolved with 4–20% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were probed with rabbit anti–phospho-p44/42 mitogen-activated protein kinase (MAPK; thr202/tyr204), followed by HRP-conjugated anti-rabbit IgG antibodies, and developed with ECL. To ensure equal loading and transfer, blots were stripped and reprobed with anti-p44/42 MAPK antibodies. IB, immunoblot; IB*, immunoblot after stripping.