Table 1.
Demographic and biochemical characteristics of study subjects.
Table 2.
Clinical characteristics of idiopathic PAH patients.
Figure 1.
Pulmonary arterial smooth muscle medial thickness.
Percentage medial thickness increased in pulmonary arterial hypertension (PAH) compared to controls from 20.5±1.5 to 34.0±1.2 (***p<0.001) in vessels 100–250 µm external diameter (ED) and from 18.9±1.5 to 25.6±1.08 (**p<0.01) in vessels 250–500 µm ED. Results expressed as mean ± SEM of 12 PAH and 14 control samples.
Figure 2.
Macrophage numbers in idiopathic pulmonary arterial hypertension.
Numbers of macrophages per 1002 of lung parenchyma were increased in pulmonary arterial hypertension (PAH) vs. controls (49.0±4.5 vs. 7.95±1.9/100 mm2, ***p<0.001). Additionally, p65+ macrophages were increased in PAH compared with controls (52.4±2.15% vs. 71.6±2.98%, ***p<0.001) and in the nucleus in PAH compared to controls (3.5±1.3% vs. 16.9±2.5%, ***p<0.001). Results are expressed as mean ± SEM for 12 PAH and 14 control subjects.
Figure 3.
NF-κB staining in perivascular macrophages in lung parenchyma a patient with idiopathic pulmonary arterial hypertension.
Macrophages (CD68+ cells, brown) are seen surrounding a diseased pulmonary artery in a post-transplantation lung specimen from a patient with idiopathic pulmonary arterial hypertension (PAH). Some of these cells exhibit nuclear p65-positive staining (pink staining, black arrow) i.e. show evidence of NF-κB activation; others demonstrate p65-negative staining (CD-68 brown only with no pink counter-staining, white arrow). In addition, small mononuclear cells (lymphocytes) stain avidly for p65. Magnification x200.
Figure 4.
NF-κB staining in a control subject and in patients with idiopathic pulmonary arterial hypertension.
(A) Control pulmonary artery showing p65- staining in sparse macrophages (arrows) and p65- vascular cells (x200). (B) Occluded pulmonary artery in a patient with idiopathic pulmonary arterial hypertension (PAH) showing intense p65 staining in pulmonary vascular cells and perivascular inflammatory cells, with further serial sections showing these cells also stain positively for B lymphocytes (CD20, Figure B1) and T lymphocytes (CD45, Figure B2). (C) High expression of p65 in endothelial cells (EC) and in PASMC nuclei in a pulmonary arteriole (x400). (D) Negative antibody-control in a non-PAH control (normal mouse IgG+normal rabbit IgG) (x200). Images representative of 12 iPAH and 14 control subjects.
Figure 5.
NF-κB p65 staining in pulmonary vascular cells in lung parenchyma a patient with idiopathic pulmonary arterial hypertension.
Light microscopy demonstrating immunohistochemical p65+ (pink)-staining in vascular cells in diseased pulmonary arteries in lung sections from pulmonary arterial hypertension (PAH) subjects. Pulmonary arterial endothelial cells show intense staining for p65+ as indicated with a black arrow (A and B). Pulmonary arterial smooth muscle cells (PASMC) stain positive for nuclear p65+ (black arrow) and p65-stain negative (unfilled arrow) (C). Results representative of those from 12 PAH subjects. Magnification x200.
Figure 6.
Quantification of NF-κB p65 staining in pulmonary vascular cells.
(A) Overall p65+ was increased in pulmonary arterial endothelial cells (EC) (62.3±2.9 vs. 14.4±3.8%, ***p<0.001), pulmonary arterial smooth muscle cells (PASMC) (22.6±2.3 vs. 11.2±2.0%, ***p<0.001) in PAH versus controls and (B) within pulmonary arterioles in both EC (86.4±3.28 vs. 4.68±2.29%, p<0.0001) and PASMC (39.2±5.29 vs. 6.39±2.61%, ***p<0.001). (C) In pulmonary endothelial cells, percentage p65+ was increased in pulmonary arterial EC in PAH versus control subjects following subdivision into cytoplasmic p65+ (23.7±1.6 vs. 8.0±2.2%, PAH, ***p<0.001) and nuclear p65+ (38.5±2.1 vs. 6.4±1.6%, ***p<0.001). Results represent mean ± SEM of 12 PAH and 14 control samples.
Figure 7.
Confocal immunofluorescence NF-κB p65 staining in pulmonary vascular cells.
Confocal immunofluorescence staining of acetone-fixed frozen lung specimens using NF-κB KAC310 AlexaFluor488 (cyan-green), and DAPI (blue) counterstaining, showed little positive nuclear staining in control lung specimens (A), and marked nuclear staining in idiopathic PAH (B and D). Antibody negative control staining of a control specimen is shown in C. Some green autofluorescence is seen with collagen fibers. Magnification x400 (insert x2400).
Figure 8.
Chemokine analysis in lung tissue.
qRT-PCR analysis performed on whole lung homogenates showed an increase in endothelin (ET)-1 mRNA (0.213±0.069 vs. 1.06±0.23, p<0.01) (A) and in CCL5 (RANTES) mRNA (0.16±0.045 vs. 0.26±0.039, p<0.05) (B) in PAH patients vs. controls. Data represent mean±SEM for n = 5 controls and n = 9 PAH patients, and were compared using the Student t test. * p<0.05 ** p<0.01.