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doi:10.1378/chest.06-2243
(Chest. 2007; 132:1265-1274)
© 2007 American College of Chest Physicians
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A Nuclear Factor-{kappa}B Inhibitor Pyrrolidine Dithiocarbamate Ameliorates Pulmonary Hypertension in Rats*

Hirofumi Sawada, MD; Yoshihide Mitani, MD, PhD; Junko Maruyama, MD, PhD; Bao Hua Jiang, MD, PhD; Yukiko Ikeyama, MD; Francis A. Dida, MD; Hatsumi Yamamoto, MD, PhD; Kyoko Imanaka-Yoshida, MD, PhD; Hideto Shimpo, MD, PhD; Akira Mizoguchi, MD, PhD; Kazuo Maruyama, MD and Yoshihiro Komada, MD, PhD

* From the Departments of Pediatric and Developmental Science (Drs. Sawada, Mitani, Ikeyama, Dida, and Komada), Physiology (Dr. J. Maruyama), Anesthesiology (Drs. Jiang and K. Maruyama), Pathology and Matrix Biology (Dr. Imanaka-Yoshida), Thoracic and Cardiovascular Surgery (Dr. Shimpo), and Neural Regeneration and Cell Communication (Dr. Mizoguchi), Mie University Graduate School of Medicine, Tsu City, Japan; and Clinical Research Institute (Dr. Yamamoto), Mie-chuo Medical Center, National Hospital Organization, Tsu City, Mie, Japan.

Correspondence to: Yoshihide Mitani, MD, PhD, Department of Pediatric and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu city, Mie Prefecture, 514-8507, Japan; e-mail: ymitani{at}clin.medic.mie-u.ac.jp

Abstract

Background: Pulmonary hypertension (PH) is a fatal disorder that is associated with structural changes and inflammatory responses in the pulmonary vasculature. Nuclear factor (NF)-{kappa}B is a key transcription factor that is involved in the tissue remodeling mediated by inflammatory and fibroproliferative responses. However, the contribution of NF-{kappa}B-mediated inflammatory pathways to the development of PH is unknown.

Methods: We therefore investigated whether NF-{kappa}B activation and the expression of a downstream product vascular cell adhesion molecule (VCAM)-1 is associated with pulmonary vascular diseases in rats that have been injected with the toxin monocrotaline (MCT), and whether a NF-{kappa}B inhibitor, pyrrolidine dithiocarbamate (PDTC), ameliorates such diseases in rats.

Results: VCAM-1 expression and the nuclear localization of the p65 subunit of NF-{kappa}B, as analyzed immunohistochemically, was significantly up-regulated in the endothelium of diseased vessels on the days 8 to 22 (p < 0.05). Next, 39 rats were divided into three groups (rats injected with MCT and treated with saline solution or PDTC, and controls similarly treated with saline solution). Compared to controls, MCT treatment increased the mean (± SE) pulmonary artery pressure (31.2 ± 1.4 mm Hg [p < 0.05] vs 22.8 ± 0.9 mm Hg, respectively), which was reduced by PDTC treatment (24.3 ± 1.2 mm Hg; p < 0.05). Indexes of right ventricular hypertrophy and pulmonary vascular diseases induced by MCT were similarly inhibited (p < 0.05), which was associated with the suppression of VCAM-1 expression and macrophage infiltration.

Conclusions: We concluded that the NF-{kappa}B nuclear localization and VCAM-1 expression is temporally and spatially associated with the development of MCT-induced PH in rats, which was ameliorated by administering a NF-{kappa}B inhibitor, PDTC.

Key Words: cell adhesion molecules • inflammation • pulmonary hypertension • rats • transcription factors

Pulmonary hypertension (PH), which has been defined as a mean pulmonary artery (PA) pressure of > 25 mm Hg at rest,1 is histologically characterized by increased thickening of the walls of pulmonary arteries and the obstruction of small pulmonary arteries.2 Inflammation has been associated with some or all forms of severe PH. Pulmonary arterial hypertension (PAH) is a recognized complication of a number of systemic inflammatory conditions, which include scleroderma, systemic lupus erythematosus, HIV infection, and plasma cell dyscrasia with polyneuropathy, organomegaly, endocrinopathy, monoclonal protein and skin changes syndrome.3 A significant number of patients in whom idiopathic PAH has been diagnosed have evidence of the presence of antinuclear antibodies and increased serum levels of inflammatory cytokine such as interleukin (IL)-1 and IL-6.4 Lung histology revealed inflammatory infiltrates such as macrophages in plexiform lesion and neointima in patient with idiopathic PAH as well as in rats with PH induced by the toxin monocrotaline (MCT).567 However, the mechanisms by which inflammation is related to the pathogenesis of PH is poorly understood.

We focused on a transcription factor, nuclear factor (NF)-{kappa}B, that regulates the transcription of genes involved in inflammatory responses, cell growth control, and apoptosis, which include cytokines, cell adhesion molecules, chemoattractant proteins, and growth factors.8 NF-{kappa}B is a key regulator in vascular inflammatory processes, such as the induction of the expression of vascular cell adhesion molecule (VCAM)-1 and other gene products that mediate the interaction between the endothelium and circulating leukocytes.910 However, it is unknown whether NF-{kappa}B/VCAM-1-mediated inflammatory pathways are involved in the development of PH.

We therefore hypothesized that the activation of NF-{kappa}B and the subsequent VCAM-1 expression plays an important role in the development of PH in rats. To test this hypothesis, NF-{kappa}B nuclear localization and VCAM-1 expression were investigated by immunohistochemical analysis in MCT-treated rats. Furthermore, we examined the inhibitory effects of a NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC) on pulmonary vascular diseases in such animal models.

Materials and Methods

Expanded Materials and Methods are available in the online data supplement.

Animal Models
Protocols for all animal experiments were approved by the Animal Research Committee, Mie University. Seven-week-old male Sprague-Dawley rats (CLEA Japan; Osaka, Japan) were injected with MCT subcutaneously (SC),11 and were analyzed just before and 2, 4, 8, 16, and 22 days (n = 36) after the treatment. For the hemodynamic studies and the morphometric analysis of pulmonary arteries, 39 Sprague-Dawley rats were randomly assigned to one of the following three groups: MCT-injected rats treated with NF-{kappa}B inhibitor PDTC (Sigma; St. Louis, MO) [100 mg/kg once daily SC on days 3 to 16 (the MCT/PDTC group)] or with a similar volume of saline solution (once daily SC on days 3 to 16 [MCT/vehicle group]); or saline-injected rats were treated with a similar volume of saline solution (once daily SC on days-3 to 16 [control/vehicle group]).

Immunohistochemistry, Immunofluorescence, and Immunoblotting for I{kappa}B{alpha}
Lung sections were incubated with primary antibodies that recognize rat VCAM-1 (Berkeley; Richmond, CA), p65 active subunit of NF-{kappa}B12 (Chemicon; Temecula, CA), phosphorylated p65 subunit (Ser276) of NF-{kappa}B (Cell Signaling Technology; Beverly, MA), rat CD68 (clone ED-1; Serotec; Oxford, UK), and von Willebrand factor (vWF) [DAKO; Carpinteria, CA]. For double-labeling immunofluorescence, Alexa Fluor 488-conjugated or 594-conjugated secondary antibodies (Molecular Probes; Eugene, OR) were used. Quantification of the staining was performed according to previously established methods.1314 The antibodies used in immunoblotting for I{kappa}B{alpha} were anti-I{kappa}B{alpha} (diluted 1:1000) [Cell Signaling Technology]. The phosphorylation of the p65 subunit and the decrease in protein levels of I{kappa}B{alpha} have been reported1516 to be associated with the up-regulation of NF-{kappa}B activity.

Hemodynamic Studies and Morphometric Analysis of Pulmonary Arteries
PA catheterization, PA pressure measurement, and morphometric analysis of the vasculature were done, as has been described previously in detail.11

Statistical Analysis
The data are presented as the mean ± SE. Body weights (BWs), hemodynamic and morphologic parameters, and the numbers of ED-1-positive cells were compared between the treatment groups or time points by an one-way analysis of variance followed by Scheffé F test. The endothelial VCAM-1 expression scores were compared between the treatment groups or time points by the nonparametric Kruskal-Wallis analysis followed by Mann-Whitney U tests. A p value of < 0.05 was accepted as being statistically significant.

Results

Progression of PH and Macrophage Infiltration Into the Lung in MCT-Injected Rats
MCT treatment induced right ventricular (RV) hypertrophy on days 16 and 22 after injection, as evaluated by both the ratios of RV/left ventricle plus septum (LV + S) and of RV/BW (Fig 1 , top left, A, and bottom left, B) [RV/LV + S ratio: day 16, 0.45 ± 0.04; day 22, 0.69 ± 0.06; p < 0.01 vs 0.27 ± 0.01 on day 0, respectively; RV/BW: day 16, 0.63 ± 0.06; day 22, 0.93 ± 0.09; p < 0.01 vs 0.38 ± 0.01 on day 0, respectively). The number of infiltrated ED-1-positive macrophages was significantly higher on days 8, 16, and 22 than on day 0 (Fig 1, top right/top left, C, to bottom right, G) [day 8, 114.5 ± 9.3 (p < 0.05) vs day 0, 55.2 ± 5.8; day 16, 214 ± 7.1 vs day 22, 266 ± 23.8 (p < 0.01) on day 0].


Figure 1
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  		Figure 1.. Progression of RV hypertrophy and macrophage infiltration in lung tissues in MCT-injected rats. Top left, A: the ratio of the weight of the RV to that of the LV + S. Bottom left, B: the ratio of the RV to BW at each time point. Top right/top left, C, top right/top right, D: lung sections from MCT-treated rats were stained with anti-rat CD68 monoclonal antibody (ED-1). Top right/top left, C, days 0 to 4; top right/top right, D, day 8; top right/bottom left, E, days 16–22; top right/bottom right, F, days16 to 22 without primary antibody. Nuclei were counterstained with hematoxylin. Each scale bar represents 100 µm. Bottom right, G: the number of ED-1-positive monocytes/macrophages in the lungs was counted in 30 randomly chosen high-power fields. The data were presented as the mean ± SE (n = 6 at each time point). * = p < 0.01 compared with results on day 0; {dagger} = p < 0.05 compared with results on day 0.

 
NF-{kappa}B Nuclear Localization and VCAM-1 Expression in MCT-Treated Rats
The expression of VCAM-1 was rarely seen on days 0, 2, and 4 (Fig 2 , panels A, E, I, and M). The expression of VCAM-1 was observed in the endothelium of an hypertrophied muscular artery, newly muscularized small vessels, and capillaries on days 8, 16, and 22 (Fig 2, panels B, C, F, G, J, K, N, and O). In quantitative analysis of immunohistochemical findings, the endothelial expression of VCAM-1 was significantly up-regulated on days 8, 16, and 22 in the muscular arteries (Fig 2, panels D and H) and small pulmonary arteries (Fig 2, panel L), and on days 16 and 22 in the capillaries (Fig 2, panel P). VCAM-1 expression, as evaluated by double immunofluorescent staining with anti-vWF antibody, was localized to the inner surface of the endothelium in the hypertrophied arteries (Fig 2, panels Q to T) and newly muscularized arteries (Fig 2, panels U to X) in MCT-injected rats. Consistent with the VCAM-1 expression, the nuclear expression of active p65 subunit of NF-{kappa}B, which is unmasked in the absence of I{kappa}B, was not observed on days 0, 2, and 4 in any layers of the medium-sized muscular arteries and small peripheral arteries (Fig 3 , top left, A, and bottom left, D), but was strongly seen in the endothelium of hypertrophied arteries and newly muscularized small arteries on days 8, 16, and 22 (Fig 3, top middle, B, top right, C, bottom middle, E, and bottom right, F).


Figure 2
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  		Figure 2.. Expression of VCAM-1 in lung tissues in MCT-injected rats. Panels A to P: typical lung sections of VCAM-1 immunohistochemistry and quantitative analysis. Pictures were shown in varied vessel sizes at different time points. The brown color indicates VCAM-1 expression. Nuclei were counterstained with hematoxylin. The expression of VCAM-1 in each vessel was graded as follows: grade 0, no visible staining; grade 1, few cells with faint staining; grade 2, moderate intensity with multifocal staining; grade 3, intense staining of the cells. Panels Q to X: double immunofluorescent staining of pulmonary arteries on day 16 in rats treated with MCT using antibodies against vWF and VCAM-1. The first column, nuclei (blue); the second column, vWF (red); the third column, VCAM-1 (green); the last column, merged images. Upper row, muscular arteries larger than 200 µm in diameter (panels Q to T); lower row, the newly muscularized vessel 15 to 50 µm in diameter (panels V to X). Nuclei were counterstained with TO-PRO-3 iodide. The data were presented as the mean ± SE, n = 6 at each time point. * = p < 0.01 compared with results on day 0; each scale bar = 10 µm.

 

Figure 3
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  		Figure 3.. Expression of p65 subunit of NF-{kappa}B on the pulmonary arteries in MCT-injected rats. Top left, A, to bottom right, F: lung sections were stained with an anti-p65 subunit of NF-{kappa}B monoclonal antibody. The nuclear expression of the p65 subunit of NF-{kappa}B in the endothelium of the hypertrophied muscular arteries (top left, A, to top right, C), the newly muscularized smaller vessels (bottom left, D, to bottom right, F) on days 0 to 4 (top left, A, and bottom left, D), day 8 (top center, B, and bottom center, E) and days 16 to 22 (top right, C, and bottom right, F) in rats injected with MCT. Nuclei were counterstained with methylgreen. Each scale bar = 10 µm.

 
Effects of PDTC on BW, PH, RV Hypertrophy, and Pulmonary Vascular Remodeling
The initial BWs were similar in all of the three experimental groups (control/vehicle group, 311.4 ± 4.2 g [n = 11]; MCT/vehicle group, 316.9 ± 4.8 g [n = 13]; MCT/PDTC group, 308.6 ± 5.3 g [n = 15]). At the time of hemodynamic measurements, MCT-injected rats had a lower BW than control rats (343.2 ± 7.9 vs 374.5 ± 7.8 g, respectively; p < 0.05). PDTC treatment had no significant effect on the final BW in the MCT-injected groups of rats. The MCT-injected rats exhibited significant PH (31.2 ± 1.4 vs 22.8 ± 0.9 mm Hg; p < 0.05), which was reduced by PDTC treatment (24.3 ± 1.2 mm Hg; p < 0.05) [Fig 4 , top left, A]. The rats in the MCT/V group had significant RV hypertrophy, as indicated by a higher RV/LV + S ratio that that in the control/vehicle group (0.34 ± 0.01 vs 0.27 ± 0.01; p < 0.05), which was reduced by PDTC treatment (0.27 ± 0.01; p < 0.05) [Fig 4, bottom left, B]. MCT treatment significantly increased the percentage of muscularized arteries (at the alveolar duct level: MCT/vehicle group, 76.4 ± 4.5%; control/vehicle group, 23.8 ± 2.9% [p < 0.05]; at the alveolar wall level: MCT/vehicle group, 69.7 ± 4.0%; control/vehicle group, 18.0 ± 1.4% [p < 0.05]). PDTC treatment significantly reduced the degree of muscularization to 30.9 ± 3.1% at the alveolar duct level (p < 0.05) and 26.8 ± 1.9% at the alveolar wall level (p < 0.05) [Fig 4, top right/top left, C, to bottom right, I).


Figure 4
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  		Figure 4.. Effects of PDTC treatment on PH, RV hypertrophy, and pulmonary vascular disease induced by MCT. Top left, A: mean pulmonary artery pressure on day 16. Bottom left, B: ratio of the weight of the RV to that of the LV + S on day 16. Top right/top left, C, to top right/bottom right, H: typical photomicrographs of barium-filled pulmonary arteries in rats injected with saline solution or MCT. Top right/top right, C, and top right/bottom right, F: vehicle-treated rats injected with saline solution. Top right/top center, D, and top right/bottom center, G: vehicle-treated rats injected with MCT. Top right/top right, E, and top right/bottom right, H: PDTC-treated rats injected with MCT. Bottom right, I: the percentage of the muscularized arteries (% muscularization) in normally nonmuscular peripheral pulmonary arteries at the alveolar duct and alveolar wall levels in rats injected with MCT. Control/vehicle (C/V)-treated rats injected with saline; MCT/vehicle (MCT/V)-treated rats injected with MCT; MCT/PDTC-treated rats. Data represent the mean ± SE. Each scale bar: Top right/top left, C, to top right/top right, E, 100 µm; top right/bottom left, F, to top right/bottom right, H, 10 µm. The number of rats in each group was described in parentheses. * = p < 0.01 compared with C/V; {dagger} = p < 0.01 compared with MCT/V rats.

 
Effects of PDTC on VCAM-1 Expression, Monocyte/Macrophage Infiltration, NF-{kappa}B Phosphorylation (Ser 276 in p65), and I{kappa}B{alpha} Protein Levels
VCAM-1 expression was significantly induced in the endothelium of hypertrophied muscular arteries, muscularized small arteries, and capillaries in MCT-treated rats (p < 0.05), which was inhibited by PDTC treatment (p < 0.05) [Fig 5 ]. MCT treatment significantly increased the number of ED-1-positive cells in the adventitia of hypertrophied muscular arteries and newly muscularized small vessels (MCT/vehicle group, 203.2 ± 14.9 per 30 high-power fields; control/vehicle group, 64.4 ± 1.2 per 30 high-power fields; p < 0.05), which was inhibited by PDTC treatment (75.6 ± 5.5, p < 0.05) [Fig 6 ]. MCT treatment significantly increased the number of endothelial cells and infiltrated mononuclear cells, which express the phosphorylated p65 subunit of NF-{kappa}B in the nucleus (endothelial cells: MCT/vehicle group, 27.0 ± 2.5 per 30 high-power fields; control/vehicle group, 3.8 ± 1.2 per 30 high-power fields [p < 0.05]; mononuclear cells: MCT/vehicle group, 84.5 ± 8.3 per 30 high-power fields; control/vehicle group, 17.3 ± 3.1 per 30 high-power fields [p < 0.05]), which was inhibited by PDTC treatment (10.8 ± 2.7 and 26.8 ± 5.3 per 30 high-power fields, respectively; p < 0.05) [Fig 7 , top left, A, to middle right, F). MCT treatment significantly reduced the protein levels of I{kappa}B{alpha} in the lung tissues, which was restored by PDTC (Fig 7, bottom left, G, and middle bottom, H).


Figure 5
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  		Figure 5.. Effects of PDTC on VCAM-1 expression in MCT-injected rats. Panels A to I: representative photomicrographs of immunostaining for VCAM-1 in pulmonary arteries on day 16 in a rat injected with saline solution or MCT. Panels J to M: quantification of the VCAM-1 expression on the pulmonary endothelium in saline solution-injected or MCT-injected rats, treated or untreated with PDTC. See Figure 4 for abbreviations not used in the text. Data represent the mean ± SE. Each scale bar = 10 µm; * = p < 0.01 compared with C/V; {dagger} = p < 0.01 compared with MCT/V; n = 6 at each time point.

 

Figure 6
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  		Figure 6.. Effects of PDTC on monocyte/macrophage infiltration in MCT-injected rats. Panels A to K: representative photomicrographs of immunostaining for anti-rat CD68 (ED-1) in the lung tissues of saline-injected rats (top left, A, middle left, E, and bottom left, I) and MCT- injected rats in the presence of PDTC treatment (top center right, C, middle center right, G, and bottom center right, K) or the absence of PDTC treatment (top center left, B, middle center left, F, and bottom center left, J). Lung sections were stained with ED-1 monoclonal antibody 16 days after MCT injection. Bottom right, L: quantification of monocyte/macrophage infiltration in the lungs in saline solution-injected or MCT-injected rats, treated or untreated with PDTC. See Figure 4 for abbreviations not used in the text. The data represent the mean ± SE. Each scale bar: panels A to H, 10 µm; panels I to K, 100 µm. * = p < 0.01 compared with C/V; {dagger} = p < 0.01 compared with MCT/V; n = 6 at each time point.

 

Figure 7
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  		Figure 7.. Effects of PDTC on NF-{kappa}B phosphorylation (Ser 276 in p65) and I{kappa}B{alpha} protein levels in MCT-injected rats. Panels A to E: representative photomicrographs of immunostaining for the anti-phosphorylated p65 subunit of NF-{kappa}B in lung tissues of saline-injected rats (top left, A) and MCT-injected rats in the presence of PDTC treatment (top right, C) or the absence of PDTC treatment (top center, B, and middle center, E). Lung sections were stained with anti-phosphorylated p65 antibody 16 days after MCT injection. Middle left, D: isotype IgG control. Middle right, F: quantification of phosphorylated p65-positive cells in the lungs of saline solution-injected or MCT-injected rats, treated or untreated with PDTC. Bottom left, G, and bottom center, H: protein levels of I{kappa}B{alpha} were measured by Western blotting using anti-I{kappa}B{alpha} antibody. Quantitative analysis was performed by the densitometric scanning of blots. Values are expressed as the relative ratio of the control value. See Figure 4 for abbreviations not used in the text. The data represent the mean ± SE. Each scale bar: Top left, A, to middle left, D, 50 µm; middle center, E, 25 µm. * = p < 0.01 compared with C/V; {dagger} = p < 0.01 compared with MCT/V; n = 4 at each time point.

 
Discussion

This is the first study that has demonstrated the potential roles of NF-{kappa}B/VCAM-1-mediated inflammatory pathways in the development of PH in rats. Nuclear expression of an active subunit of NF-{kappa}B and VCAM-1 expression was temporally and spatially associated with the development of pulmonary vascular diseases in rats injected with MCT. Furthermore, PDTC, a potent and relatively specific inhibitor of NF-{kappa}B, ameliorates PH, RV hypertrophy, and vascular remodeling, in concert with the suppression of the nuclear expression of phosphorylated NF-{kappa}B and of VCAM-1 expression in the endothelium, as well as ED-1-positive cell infiltration in the perivascular space in MCT-treated rats. These effects of PDTC were correlated to the reversal of the MCT-induced decrease in the NF-{kappa}B inhibitory protein I{kappa}B{alpha}. These findings suggest that PDTC ameliorated PH and pulmonary vascular diseases in MCT-treated rats, at least in part by suppressing NF-{kappa}B/VCAM-1-mediated inflammatory pathways.

Temporal and Spatial Association of NF-{kappa}B Activation and VCAM-1 Expression With the Progression of PH Induced by MCT
The sequence of events associated with the development of pulmonary vascular diseases has been well described in MCT-treated rats.717 In a previous electron microscopic study,17 the evidence of endothelial injury occurred as early as 4 days after the injection of MCT followed by interstitial edema in the alveolar wall as well as in the perivascular tissue. These early electron microscopic changes are associated with the increased pulmonary vascular leakage in the physiologic studies.18 In the microscopic and hemodynamic study, the extension of smooth muscle into the normally nonmuscularized small vessel developed on day 8 without any increase in PA pressure.17 Thereafter, a further extension of muscularization as well as hypertrophy of the muscular arteries occurred on day 12, which was associated with an increase in PA pressure.17

In the present study, the nuclear localization of NF-{kappa}B, VCAM-1 expression, and macrophage infiltration was observed, not on day 2 or 4, but on days 8, 16, and 22, suggesting the MCT-induced electron microscopic endothelial changes precede the nuclear localization of NF-{kappa}B and VCAM-1 expression in the endothelium of the muscularized or hypertrophied vessels. These findings suggest MCT-induced early structural changes as well as pulmonary vascular leakage may occur independent of NF-{kappa}B and VCAM-1 expression in the endothelium.18 These early structural and functional changes after MCT injection may be caused by MCT pyrrole through alteration in the actin polymerization state and endothelial intercellular junction, not through mediation by NF-{kappa}B/VCAM-1-mediated inflammatory pathways.19 The presence of inflammatory cell infiltration in the interstitium of the alveolar walls as well as newly muscularized vessels on day 8 and later in the present study is consistent with the histologic findings in the study by Meyrick et al,7 in which inflammatory cells infiltrated into the interstitium of the alveolar walls and remodeled arteries on day 7. In addition, our results are consistent with monocyte chemoattractant protein (MCP)-1 expression in the lung.13 Since MCP-1 expression in the lung and increased MCP-1 concentrations in BAL fluid are obvious on day 14 and later, NF-{kappa}B/VCAM-1-mediated macrophage infiltration precedes MCP-1 expression in the lungs. In addition, since the nuclear expression of phosphorylated NF-{kappa}B was observed in infiltrating mononuclear cells, NF-{kappa}B in these cells may also contribute to the formation of vascular diseases by releasing NF-{kappa}B-regulated cytokines. Therefore, these lines of evidence suggest that NF-{kappa}B-mediated inflammatory responses may be involved in the development of MCT-induced PH upstream of MCP-1 expression in the lung.

However, the mechanisms by which NF-{kappa}B transactivation is induced are unknown. In MCT-injected rats, NF-{kappa}B-inducing cytokines, IL-1, and platelet-activating factor play a pivotal role in the development of PH and vascular remodeling.2021 Lung IL-1 levels peaked on day 3 and continued to be high throughout the experiment, in which an IL-1 receptor antagonist ameliorated the development of PH and vascular diseases in MCT rats.20 The administration of a platelet-activating factor antagonist prevented the development of these pulmonary vascular diseases as well as macrophage infiltrations in MCT models.21 Therefore, it is possible that these cytokines may work upstream of NF-{kappa}B/VCAM-1-mediated macrophage infiltration, culminating in the development of pulmonary vascular diseases in MCT models.

Effects of PDTC on MCT-Induced PH in Rats
To elucidate the cause-and-effect relationship between the up-regulation of NF-{kappa}B/VCAM-1-mediated inflammatory pathways and pulmonary vascular diseases, we examined the effect of a NF-{kappa}B inhibitor PDTC on MCT-induced PH in rats. In our study, PDTC markedly prevented PH and RV hypertrophy, which is associated with the suppression of the muscularization of small peripheral arteries. Furthermore, the protective effects of PDTC were associated with the suppression of the nuclear expression of phosphorylated NF-{kappa}B in the endothelium and mononuclear cells, and endothelial VCAM-1 expression and ED-1-positive cell infiltration in the perivascular space, in concert with the reversal of decreased levels of I{kappa}B{alpha} in the lungs. These findings indicate that NF-{kappa}B/VCAM-1-mediated inflammatory cascades may be causally related to MCT-induced PH. These findings are consistent with those of a previous study,22 which demonstrated that the gene transfer of the dominant negative form of MCP-1 ameliorated pulmonary vascular disease in MCT-injected rats by suppressing the inflammatory cell infiltration. Therefore, these lines of evidence suggest that MCP-1 may aggravate macrophage infiltration and VCAM-1 expression in the lungs, culminating in the inflammation-associated induction of pulmonary vascular disease in rats.

Limitations
Limitations should be considered in interpreting our results. Although the NF-{kappa}B inhibitor PDTC inhibited MCT-induced PH in rats, there is no genetic evidence, using mutant mice, demonstrating that NF-{kappa}B activation itself is necessary for the development of PH. Unfortunately, although mice with conditional mutations in the p65 and I{kappa}B genes using Cre recombinase-locus of crossing over in phage P1 (or Cre-LoxP) technology are possible, MCT-induced PH models cannot be reproduced in mice. Therefore, to further strengthen the role of NF-{kappa}B in the development of PH, we tried two other NF-{kappa}B inhibitors, diphenyleneiodonium sulfate, an nicotinamide adenine dinucleotide phosphate oxidase inhibitor, and N-acetylleucylleucylnorleucinal (calpain inhibitor I) in our studies. However, MCT rats were not tolerable to either compound. The specificity of PDTC was investigated in previous studies in which Schreck et al23 and Liu et al24 reported that PDTC inhibited NF-{kappa}B activation of various stimulants but had no effect on activator protein-1, cyclic adenosine monophosphate response element-binding protein, specificity protein-1, and octamer-binding proteins in several cell lines and in vivo. In addition to inhibiting NF-{kappa}B, PDTC might have some antioxidant effects as well.23 So we might not exclude the possibility that the beneficial effects of treatment were due in part to the property of PDTC as an antioxidant. Therefore, it would be appropriate to state that PDTC is a relatively selective NF-{kappa}B inhibitor, and that PDTC ameliorated PH and pulmonary vascular diseases in MCT-treated rats, at least in part by suppressing NF-{kappa}B/VCAM-1-mediated inflammatory pathways.

Acknowledgements

The authors thank Dr. Joji Morita (Department of Pathologic Oncology, Mie University Graduate School of Medicine) for his technical advice and skillful assistance.

Footnotes

Abbreviations: BW = body weight; IL = interleukin; LV + S = left ventricle plus septum; MCP = monocyte chemoattractant protein; MCT = monocrotaline; NF = nuclear factor; PA = pulmonary artery; PAH = pulmonary arterial hypertension; PDTC = pyrrolidine dithiocarbamate; PH = pulmonary hypertension; RV = right ventricle, ventricular; SC = subcutaneous, subcutaneously; VCAM = vascular cell adhesion molecule; vWF = von Willebrand factor

This work was supported in part by a grant-in-aid for scientific research (No. 17790701) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.

The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Received for publication September 10, 2006. Accepted for publication June 19, 2007.

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