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Published ahead of print on September 10, 2004, doi:10.1164/rccm.200311-1602OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 1340-1348, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200311-1602OC

Original Article

Impaired Transforming Growth Factor-ß Signaling in Idiopathic Pulmonary Arterial Hypertension

Amy Richter, Michael E. Yeager, Ari Zaiman, Carlyne D. Cool, Norbert F. Voelkel and Rubin M. Tuder

Division of Cardiopulmonary Pathology, Department of Pathology, and Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and Graduate Program in Experimental Pathology, Department of Pathology, Pulmonary Hypertension Center, and Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado School of Medicine, Denver, Colorado

Correspondence and requests for reprints should be addressed to Rubin M. Tuder, M.D., Division of Cardiopulmonary Pathology, Department of Pathology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 519, Baltimore, MD 21205. E-mail: rtuder{at}jhmi.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in transforming growth factor-ß family receptor-II, bone morphogenetic protein receptor-2, and activin-like kinase-1 have been associated with pulmonary hypertension. In the present study, we determined that pulmonary arteries in normal lungs and in lungs of patients with emphysema and idiopathic pulmonary arterial hypertension comparably expressed transforming growth factor-ß receptors I and II, Smad(1, 5, 8), Smad2, Smad3, Smad4, phosphorylated Smad(1, 5, 8), and phosphorylated Smad2 (the latter two both indicative of active in vivo signaling) in endothelial cells, as assessed by immunohistochemistry and quantitative morphometry. Medial or intimal smooth muscle cells had weak or absent expression of these molecules. In clear contrast to endothelial cell expression in pulmonary arteries and in endothelial cells lining incipient vessels within plexiform lesions of hypertensive lungs, endothelial cells present in the core of the lesions lacked expression of all examined members of the signaling molecules. These findings were made irrespective of the mutation status of bone morphogenetic protein receptor-2 in hypertensive patients. Our findings suggest that pulmonary artery endothelial cells in both normal and severely hypertensive lungs have active transforming growth factor-ß family signaling, and that loss of signaling might contribute to the abnormal growth of endothelial cells in plexiform lesions in idiopathic pulmonary arterial hypertension.

Key Words: endothelial cells • pulmonary hypertension • Smad • smooth muscle cells • transforming growth factor-ß

There is growing evidence that abnormalities of transforming growth factor (TGF)-ß signaling are linked to the pathogenesis of severe pulmonary arterial hypertension (severe PAH) (1). First, about 50% of members of families with PAH (familial PAH) and 2 to 10% of sporadic idiopathic PAH (IPAH [2], previously known as primary pulmonary hypertension) harbor heterozygous germline mutations of bone morphogenetic protein receptor (BMPR)-2 (3, 4). Second, endothelial cells of patients with IPAH contain somatic mutations of the transforming growth factor-ß receptor (TßR)-I (5), and IPAH has been reported in patients with hereditary hemorrhagic telangiectasia due to mutations of activin-like kinase (Alk)-1 (6). Finally, a decrease in BMPR-1 levels in idiopathic PAH and PAH with associated conditions has been proposed as a common link between the genetic and acquired basis of PAH (7).

The prototypic members of the TGF-ß family, TGF-ß1, TGF-ß2, and TGF-ß3, are stored after synthesis in an inactive form in the extracellular matrix. Known mediators of TGF-ß stromal release and subsequent activation include plasmin, thombospondin-1, integrins ({alpha}5ß6), and ionizing irradiation. On TGF-ß binding to the extracellular domain of TGF-ß receptor Type II (TßRII), heterodimeric complexes are formed with recruitment, phosphorylation, and activation of Type I receptors (TßRI) Alk-5 and Alk-1. The accessory binding proteins betaglycan and endoglin facilitate clustering and activation of TGF-ß receptors. Subsequently, recruitment, phosphorylation, and activation of receptor Smad2 or Smad3 (if TßRI is activated) or of Smad1, Smad5, or Smad8 (if Alk-1 is activated) lead to receptor Smad(s) partnering with Smad4, followed by nuclear translocation and activation of TGF-ß-dependent gene transcription. Smad1, Smad5, or Smad8 also participates in cell signaling after binding of the TGF-ß family member bone morphogenetic protein to BMPR-2 and BMPR-1. Complex interactions of receptor and partner Smad(s) with inhibitory Smad(s), coactivators or repressors, and regulatory proteins ultimately determine the wide range of activation or inhibition of TGF-ß-dependent gene expression (8). The Smad–TGF-ß family signaling interacts with other signaling circuits such as Ras or the mitogen-activated protein kinase kinase (MKK)4–Jun kinase and MKK3–p38 (9). The role of the TGF-ß superfamily in angiogenesis and endothelial cell growth is best illustrated by the finding that TGF-ß1 knockout mice die in utero of multiple abnormalities including impaired vasculogenesis, hematopoiesis, and cardiac malformations (10).

Despite intense efforts to define the mechanisms underlying the defects in TGF-ß or BMP signaling in cell culture systems and animal models of pulmonary hypertension, there are few data on the pattern of expression of TGF-ß signaling molecules in normal and remodeled pulmonary arteries in humans (11). TßRII and BMPR-2 have been localized to endothelial cells in IPAH lungs, and we have reported the lack of TßRII expression in plexiform lesion endothelial cells (5).

In the present study, we asked whether there is evidence of an abnormal cellular pattern and expression intensity of TGF-ß signaling molecules TßRI and TßRII; Smad1, Smad5, or Smad8; Smad2; Smad3; and Smad4 in pulmonary artery endothelial and smooth muscle cells in IPAH pulmonary arteries as compared with remodeled pulmonary arteries in patients with emphysema and in normal pulmonary arteries. To assess in vivo TGF-ß signaling in normal lungs and lungs with remodeled pulmonary arteries in chronic cigarette smokers or in patients with IPAH, we performed immunohistochemical detection of phosphorylated active Smad1, Smad5, and Smad8 [phosphoSmad(1, 5, 8)], using an antibody that detects an epitope common to all three proteins, and immunohistochemical detection of phosphorylated Smad2 (phosphoSmad2).

Our findings revealed that endothelial cells lining normal or remodeled pulmonary arteries with varying degrees of intimal thickening or medial hypertrophy concordantly express all TGF-ß signaling molecules, in normal, emphysema, and IPAH lungs. In contrast, core endothelial cells in plexiform lesions in IPAH failed to express several members that relay TGF-ß signal transduction.

Some of the results of these studies have been previously reported in the form of an abstract (12).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Case Selection
The Joint Committee on Clinical Investigation of the Johns Hopkins University School of Medicine (Baltimore, MD) and the Human Subjects Committee of the University of Colorado Health Sciences Center (Denver, CO) approved this study. A formalin-fixed, paraffin-embedded block of autopsy or explanted lung (obtained at the time of lung transplantation) was selected as representative of IPAH from the pathology archives at Johns Hopkins Hospital (n = 5) and the University of Colorado Health Sciences Center (n = 7 cases) (these cases were part of the case population used in a study described in Reference 13), in addition to emphysematous lungs due to long-term cigarette smoking (n = 5), and histologically normal lungs (n = 5) for immunohistochemical localization of TGF-ß receptors and Smad(s). IPAH cases from the University of Colorado Health Sciences Center were screened for human herpesvirus-8 status on the basis of lung tissue latency-associated nuclear antigen-1 expression and BMPR-2 mutations as described previously (13). Relevant clinical information was obtained through chart review, and if not available, through contacts with referring health care professionals (see Table E1 in the online supplement).

Antibodies
The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): goat IgG (N-18, sc-6031) that recognizes a common epitope on Smad1, Smad5, and Smad8 [as the antibody cannot discriminate the selective expression of Smad1, Smad5, or Smad8, the results are described as pertinent to Smad(1, 5, 8)], used at 1:50 dilution; goat IgG anti-Smad2 (S-20, sc-6200), used at 1:100 dilution; goat IgG anti-Smad3 (I-20, sc-6202), used at 1:100 dilution; goat IgG anti-Smad4 (N-16, sc-1908), used at 1:100 dilution; mouse monoclonal anti-Smad4 (B-8, sc-7966), used at 1:50 dilution; rabbit IgG anti-TßRI (V22, sc-398) (Alk-5), used at 1:200 dilution; and rabbit IgG anti-TßRII (L-21, sc-400), used at 1:100 dilution. Rabbit anti-phosphorylated Smad1, Smad5, and Smad8 [called hereafter phosphoSmad(1, 5, 8)] and anti-phosphorylated Smad2 (phosphoSmad2) were kindly provided by P. ten Dijke and C.-H. Heldin (14, 15) and were used at 1:50 and 1:1500 dilution, respectively; rabbit anti-factor VIII-related antigen (DakoCytomation, Carpinteria, CA) was used at 1:50 dilution; and rabbit anti-vascular endothelial growth factor (DakoCytomation) was used at 1:500 dilution (13).

Immunohistochemistry
See the online supplement for a detailed outline of the immunohistochemical procedures. Immunohistochemical detection of TßRI, TßRII, Smad(1, 5, 8), phosphoSmad(1, 5, 8), Smad2, phosphoSmad2, Smad3, and Smad4 was performed in paraffin-embedded, formalin-fixed lung tissue. Negative controls consisted of primary antibody absorbed to its corresponding immunizing peptide [for all antibodies except phosphoSmad(1, 5, 8) and phosphoSmad2], provided by the vendor, or of normal rabbit serum at a similar dilution as the phosphoSmad(1, 5, 8) or phosphoSmad2 antibody. The peptide was incubated at a ratio of 10:1 with the cognate antibody, at 4°C, overnight.

Assessment of TGF-ß Family Signaling Protein Expression
The expression of TGF-ß family signaling proteins was quantified with Image-Pro (MediaCybernetics, Silver Spring, MD) by an observer blinded to the identity of the slides (R.M.T.) (see METHODS in the online supplement). Pulmonary vascular remodeling was assessed by observation of the accumulation of smooth muscle cells in the intima (intima thickening) and smooth muscle medial hypertrophy (medial hypertrophy), which can be seen in emphysematous lungs (16) and IPAH (17). A single layer (monolayer) of endothelial cells characteristically lines these vessels. Complex vascular lesions occur exclusively in severe PAH (17, 18) and include plexiform lesions. Plexiform lesions are intravascular glomeruloid projections, which represent a form of abnormal angiogenesis. These lesions are composed of endothelial cells lining incipient blood vessels (called herein lumenal endothelial cells), often in proximity to the residual vascular lumen, and a solid cellular core embedded in a matrix containing a variable population of myofibroblast-like cells often extending in continuity with the vascular wall (18). The extent of vascular wall thickening was determined by means of assessment of percent medial plus intimal (named percent vascular wall thickness) thickening (outside diameter – inside diameter/outside diameter) and vascular wall area/outside perimeter. See Table E2 in the online supplement for the detailed number of vascular profiles examined per lung of each TGF-ß signaling molecule immunostaining. Overall, 211, 189, and 239 lung fields containing one or two pulmonary arteries, with an average of 5.6, 5.2, and 2.7 lung fields per lung, were analyzed in normal (n = 5 lungs), emphysema (n = 5 lungs), and IPAH (n = 12 lungs) lungs, respectively. When possible, the vascular remodeling was also classified histologically as medial hypertrophy or intimal thickening by a pulmonary pathologist (R.M.T.) during the quantification of TGF-ß signaling molecule expression. Plexiform and concentric lesions were seen exclusively in IPAH lungs (total of 33 lesions; n = 12 lungs) (Table E2). Overall, 107 lung fields containing plexiform lesions were analyzed for expression TGF-ß signaling molecules.

The mean expression of each TGF-ß signaling molecule in endothelial cells, vascular smooth muscle cells, alveolar macrophages, and alveolar septum in each lung was determined by a multitier averaging process, which involved (1) averaging the expression intensity measured along a line drawn over endothelial and smooth muscle cells, macrophages, and alveolar septum, using the Image-Pro line profile measurement tool (see METHODS in the online supplement); (2) averaging the expression intensity of two or three replicate measurements of endothelial and medial smooth muscle cells, alveolar macrophages, and alveolar septum per lung field; (3) individual lung expression levels determined by averaging the individual lung field measurements (which ranged between 3 and 10 per lung); and (4) final mean values of expression intensity in normal, emphysema, or IPAH groups obtained by averaging individual lung values within each particular group.

Statistical Analysis
Mean expression intensities of each TGF-ß signaling molecule in endothelial cells, medial smooth muscle cells, alveolar macrophages, and alveolar septal cells were compared among normal, emphysema, and IPAH lungs and within each group by analysis of variance (ANOVA), and of endothelial cell versus smooth muscle cell expression differences in each group by t test. The vascular size range of pulmonary arteries sampled in each group was determined by measuring the outer vascular perimeter and by mathematically determining the outer diameter. Differences in the extent of pulmonary vascular remodeling among the lung tissues of the three study groups were assessed by ANOVA comparison of the percent vascular wall thickness or medial area/outer perimeter (both indicative of pulmonary vascular remodeling) (see METHODS in the online supplement). The correlation between endothelial or vascular smooth muscle cell expression of each TGF-ß signaling molecule and pulmonary arterial size or wall thickness was assessed by Spearman correlation coefficient. Statistical analyses were performed with the program SigmaStat (SPSS, Chicago, IL) and statistical significance was set at a p value of less than 0.05. Figures 3 and 5 and Figures E1, E2, and E4 in the online supplement show normally distributed data as bar graphs with means ± standard error, whereas nonnormally distributed data are shown with box plots in which the boxes define the 25th and 75th percentiles, with a line at a median, and error bars defining the 10th and 90th percentiles.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Population and Pulmonary Artery Morphology
Patient clinical data are displayed in Table E1. Normal lung tissue obtained during surgical removal of metastases or primary lung cancers showed normal pulmonary arteries and no significant pathological alterations. Pulmonary emphysema was confirmed histologically and graded as mild (n = 2), moderate (n = 1), or severe (n = 2) emphysema on the basis of pathological and clinical data.

Pulmonary arteries of patients with emphysema showed intimal and medial remodeling as described (16). Lungs of patients with IPAH had complex vascular lesions (i.e., plexiform and concentric vascular lesions) and extensive intimal and medial thickening (17). Muscularization of intraalveolar pulmonary arteries was evident in the lungs of patients with emphysema and patients with IPAH. The sizes of resistance pulmonary arteries examined for immunohistochemical expression of TGF-ß signaling molecules ranged from 56 to 165 µm (outer diameter) in normal, emphysema, and IPAH lungs (Figure E1). There were no significant differences in sampled pulmonary vessel sizes between emphysema and IPAH lungs, but normal pulmonary arteries were slightly larger than emphysematous or IPAH vessels (median values: normal, 157 µm; emphysematous, 145 µm; IPAH, 135 µm; ANOVA, p < 0.05, Dunn post hoc test). This size range of pulmonary arteries is the site of major remodeling in IPAH and includes normally nonmuscularized vessels of less than 70 µm in diameter, which undergo muscularization in emphysema and IPAH. Pulmonary arteries in emphysema lungs had some evidence of enhanced pulmonary vascular remodeling as assessed by percent vascular wall thickness or medial area/outside perimeter, when compared with normal pulmonary arteries. These differences were, however, not significant (ANOVA, p > 0.05, Dunn post hoc test) (Figure E1A). IPAH pulmonary arteries had the most pronounced vascular wall thickening among the three groups (ANOVA, p < 0.01 versus normal and emphysema lungs, Dunn post hoc test) (Figure E1B).

Endothelial and Smooth Muscle Cell Expression of TGF-ß Signaling Molecules
Endothelial cells were the main pulmonary vascular site of TßR and Smad expression in all (normal, emphysema, and IPAH) pulmonary arteries, with levels of expression that were about two- to fourfold higher than those of vascular smooth muscle cells (Figures 1– 3) (ANOVA, p < 0.01, t test). There were no significant differences in endothelial expression among the three groups for all TGF-ß signaling molecules, except for phosphoSmad2, which showed a trend toward increased expression in IPAH pulmonary arteries (Figure 3). We also confirmed our Smad4 expression data with a second antibody (Figure E2). IPAH cases had similar levels of expression of TGF-ß signaling molecules in BMPR-2 wild-type IPAH lungs (n = 4) when compared with heterozygous mutant IPAH cases (n = 3) or with cases with (n = 5) or without (n = 2) evidence of human herpesvirus-8 infection (13).



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  		Figure 1. Expression of TßRI (A and B), TßRII (C and D), Smad(1, 5, 8) (E and F), Smad2 (G and H), Smad3 (I and J), and Smad4 (K and L) in normal pulmonary arteries from a normal lung (A, C, E, G, I, and K) or after absorption with specific peptide (negative controls; B, D, F, H, J, and L). Note the predominance of endothelial cell expression (arrows) as compared with the less intense and more irregular expression by medial smooth muscle cells (arrowheads). Magnification bars (A–L): 100 µm.

 


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  		Figure 2. Expression of TßRI (A and B), TßRII (C and D), Smad(1, 5, 8) (E and F), Smad2 (G and H), Smad 3 (I and J), and Smad4 (K and L) in pulmonary arteries with intimal thickening in an emphysema lung (A, C, E, G, I, and K) or an IPAH lung (B, D, F, H, J, and L). The intimal thickening in IPAH lungs is more pronounced than in emphysema lungs, yet the expression pattern of each receptor and Smad(s) is similar in both pulmonary arteries in that endothelial cells express each member of TGF-ß signaling member more intensively than smooth muscle cells (arrows). Magnification bars (A–L): 100 µm.

 


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  		Figure 3. Endothelial cell (black columns) and vascular smooth muscle cell (gray columns) expression levels of TßRI (A), TßRII (B), Smad(1, 5, 8) (C), Smad2 (D), Smad3 (E), and Smad4 (F) in normal (N), emphysematous (E), and IPAH (P) lungs (columns indicate mean, bars indicate SE; endothelial cell versus smooth muscle cell expression, ANOVA, p < 0.05).

 
Subintimal and medial vascular cells had absent or faint expression of TGF-ß signaling molecules (Figures 1–3) in normal, emphysema, and IPAH lungs. Low-level expression was restricted to scattered cells, with no distinct pattern or preferential site of localization (i.e., subintimal, medial, or subadventitial).

The correlation between pulmonary artery size and vascular wall remodeling and expression intensity of each TGF-ß signaling molecule in endothelial or smooth muscle cells in normal, emphysematous, and IPAH lungs is shown in Table E3. In IPAH, endothelial cell expression of TßRI, Smad2, phosphoSmad2, Smad3, and Smad4 and smooth muscle cell expression of TßRI, Smad2, and Smad4 correlated inversely with pulmonary artery size (Table E3).

Alveolar macrophages expressed all members of the TGF-ß signaling intensively and uniformly in all three groups, whereas alveolar septal cells (composed of Type I and II epithelial and endothelial cells) expressed TGF-ß signaling molecules at levels comparable to those detected in pulmonary artery endothelial cells (Figures E3 and E4).

Plexiform Lesion Expression of TGF-ß Signaling Molecules
Plexiform lesions occurred exclusively in lungs of patients with IPAH. We studied the expression of TGF-ß receptor and Smad proteins in 33 lesions of a total of 12 IPAH lungs, both in lumenal endothelial cells (endothelial cells lining incipient vessels) and in core endothelial cells (19) (Table E2). Our most important finding was that core endothelial cells did not express TGF-ß receptors or Smad(s) (ANOVA, p < 0.05, Tukey post hoc test), whereas their expression was preserved in lumenal endothelial cells and cells lining the proximal pulmonary artery (Figure 4), at levels similar to those present in endothelial cells of IPAH pulmonary arteries (ANOVA, p > 0.05, Tukey post hoc test; Figures 3 and 5). Many of these proximal arteries showed concentric intimal or marked eccentric intimal thickening. Core and lumenal plexiform lesion endothelial cells expressed the endothelial cell marker factor VIII-related antigen and the angiogenic vascular endothelial growth factor as previously described (20) (Figure E5).



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  		Figure 4. Lack of expression of TßRI (A), TßRII (B), Smad(1, 5, 8) (C), Smad2 (D), Smad3 (E), and Smad4 (F) in core (arrows) endothelial cells in a plexiform lesion. Expression is preserved in endothelial cells in the segment that feeds the lesion and in vascular slits within the lesion (arrowheads). Magnification bar (A–F): 100 µm.

 


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  		Figure 5. Expression intensities of TßRI (TBR1), TßRII (TBR2), Smad(1, 5, 8) [S(1,5,8)], phosphoSmad(1, 5, 8) [pSmad(1,5,8)], Smad2 (S2), phosphoSmad2 (pS2), Smad3 (S3), and Smad4 (S4) in endothelial cells, which line pulmonary arteries as a monolayer (black columns), in lumenal endothelial cells in plexiform lesions (light gray columns), and in core endothelial cells (dark gray columns). Lumenal endothelial cell expression of TßRI, TßRII, and Smad(s) in plexiform lesions is similar to expression by endothelial cells in IPAH pulmonary artery endothelial cells. Lesional core endothelial cells have significantly lower expression of all TGF-ß signaling molecules as compared with endothelial cells of IPAH pulmonary arteries or lumenal endothelial cells lining vascular slits in plexiform lesions (ANOVA, p < 0.05, Tukey post hoc test).

 
Expression of Phosphorylated Smad2 and Phosphorylated Smad(1, 5, 8)
Because expression of phosphoSmad(1, 5, 8) and phosphoSmad2 is highly indicative of active TGF-ß family signaling in vivo (14, 15), we assessed smooth muscle or endothelial cell expression of these signaling molecules in normal lungs (n = 2), emphysema lungs (n = 2), and IPAH lungs (up to six lungs). Bronchiolar epithelial cells, cartilage cells, and alveolar macrophages expressed phosphoSmad(1, 5, 8) and phosphoSmad2 intensely, in both a cytoplasmic and intranuclear pattern. Pulmonary artery endothelial cells, but not medial smooth muscle cells, expressed phosphoSmad(1, 5, 8) and phosphoSmad2 in all three groups examined (Figures 6–8) (ANOVA, p < 0.01, except for endothelial versus smooth muscle cell expression of phosphoSmad2, which was not significantly different in normal and in emphysema lungs) in the cytoplasm and in the nucleus (Figures 6 and 7). Endothelial cell expression levels of phoshoSmad2 were lower in normal lungs (mean = 11.65 ± 7.3), intermediate in emphysema lungs (mean, 28.4 ± 1.6), and most intense in IPAH lungs (mean, 81 ± 14.6). These differences had a trend toward statistical significance (ANOVA, p = 0.063, Tukey post hoc test) (Figure 8). Levels of endothelial cell expression of phosphoSmad2 correlated inversely with pulmonary artery size and, less prominently, with the extent of vascular wall remodeling (Figure 9 and Table E3).



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  		Figure 6. PhosphoSmad2 immunolocalization in normal (A), emphysema (B and H), and IPAH lungs (C–G and I). PhosphoSmad2 is predominantly expressed by endothelial cells in all pulmonary arteries, including those in normal arteries (A), mild medial thickening (B), or marked medial and intimal thickening (IT) (D and E). Endothelial cell expression pattern is predominantly cytoplasmic but also frequently nuclear [arrows in (A–E)], as apparent in an IPAH pulmonary artery without hematoxylin counterstain (E). Smooth muscle cell expression is weak and focal, or absent (E, arrowhead). Plexiform lesion (PL) endothelial cells do not express PhosphoSmad2 (F). As internal positive controls, Type I cells (arrowhead) and alveolar macrophages (arrow) (IPAH lung, G), bronchiolar cells (emphysema and IPAH lungs, highlighted by the letter b, in B–D and F), and bronchial cartilaginous cells (emphysema lung, H) express phosphoSmad2 consistently in the cytoplasmic and nucleus in normal, emphysema and IPAH lungs. (I) Pulmonary artery of an IPAH lung stained with normal rabbit serum as negative control. Magnification bars: (A–C and I) 100 µm; (D–H) 50 µm. PA = pulmonary artery.

 


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  		Figure 7. PhosphoSmad(1, 5, 8) immunolocalization in normal (A), emphysema (B), and IPAH lungs (C–F). Note endothelial cell expression of phosphoSmad(1, 5, 8) (arrows) in representative pulmonary arteries in all three groups, whereas low or absent expression is present in vascular smooth muscle cells (A–C). PhosphoSmad(1, 5, 8) is expressed in lumenal endothelial cells in plexiform lesions (PL, arrows in D-E) and in bronchiolar epithelial cells [thin arrow (D)], but not present in core endothelial cells (E, arrowhead) (amplified boxed area in D). Rabbit serum control is shown in (F). Magnification bars: (A–C, E, and F) 25 µm; (D) 50 µm.

 


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  		Figure 8. Endothelial cell (black columns) and vascular smooth muscle cell (gray columns) expression levels of phosphoSmad2 (A) and phosphoSmad(1, 5, 8) (B) in normal (N), emphysematous (E) and IPAH (P) lungs (columns indicate mean, bars indicate SE).

 


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  		Figure 9. Inverse correlation between pulmonary artery size (outer perimeter) (A) or pulmonary artery wall remodeling (percent vascular wall thickness) (B) in lungs of patients with IPAH and expression levels of phosphoSmad2, providing evidence of TGF-ß signaling in IPAH pulmonary arteries. CC = Spearman's correlation coefficient.

 
Expression of phosphoSmad2 (Figure 6) and phosphoSmad(1, 5, 8) (Figure 7) was present in endothelial cells lining the proximal artery and incipient vascular channels in plexiform lesions, whereas no positivity was detected in core endothelial cells (nine lesions). PhosphoSmad(1, 5, 8) expression was more intense in pulmonary artery endothelial cells in all three groups when compared with phosphoSmad2 expression intensity (Figure 8). On the other hand, macrophages and alveolar septum expressed similar levels of phosphoSmad(1, 5, 8) and phosphoSmad2 in all three groups examined (Figures E3 and E4).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that pulmonary artery endothelial cells intensely expressed TGF-ß receptors and Smad proteins. Comparatively, medial smooth muscle cell expression was less marked, and, when present, expressed by fewer pulmonary arteries than those with positively staining endothelial cells. This overall difference in expression pattern occurred irrespective of whether the vessels were from normal, emphysema, or IPAH lungs. The important finding of our study is that endothelial cells present in the core of plexiform lesions lacked or had markedly reduced expression of all members of the examined TGF-ß family signaling proteins, regardless of the BMPR-2 mutation status of patients with IPAH. Our results are of interest because, given the evidence linking abnormal TGF-ß family signaling and IPAH, one might have expected that the pulmonary arteries in IPAH would show uniform reduction or lack of expression of TGF-ß family signaling molecules (11).

Studies based on lung paraffin sections reflect the state of lung cells at the time of organ removal or patient death and cannot reliably determine cause-and-effect relationships or changes of gene expression over time. Notwithstanding these limitations, the evidence of abnormal TGF-ß signaling in IPAH lungs and differences between endothelial and vascular smooth muscle cells in TGF-ß signaling protein levels were validated by the expression pattern of phosphoSmad2 and phosphoSmad(1, 5, 8). Expression of phosphorylated Smad(s) correlates with TGF-ß family signaling in vivo (14, 15), because phosphoSmad2 and phosphoSmad(1, 5, 8) detection likely reflects activation of TGF-ß family receptors and nuclear accumulation of partners of phosphorylated receptor Smad(s) and Smad4 (21). However, the in vivo functional implications of Smad2 or Smad(1, 5, 8) phosphorylation are unknown.

Findings have highlighted the role of dysregulated growth of lung vascular cells in the pathogenesis of the disease (22). These discoveries were followed by immunohistochemical studies, which demonstrated preferential expression of BMPR-2 and TGF-ß receptors in endothelial cells rather than in medial smooth muscle cells in normal pulmonary arteries and in pulmonary arteries of patients with IPAH and PAH with associated conditions (secondary PAH) (11). Our findings revealed a similar expression pattern regarding the expression of TGF-ß receptors as that observed with BMPR-2 expression in IPAH pulmonary artery endothelial cells and in lumenal endothelial cells in plexiform lesions (11). Our studies extend these observations further because we also found consistent and concordant expression of several members of the TGF-ß family signaling system when compared with TßRI and TßRII, and evidence of active TGF-ß family signaling in vivo. In contrast to the studies by Atkinson and coworkers, we did not find a reduction in expression of TGF-ß family signaling proteins or lack of signaling in alveolar septal cells as suggested by the report of reduced alveolar expression of BMPR-2 in IPAH lungs (11). Moreover, we did not observe differences in the expression pattern of TGF-ß signaling molecules between sporadic IPAH cases with and without BMPR-2 mutations. Finally, our findings also indicate that there is evidence of active Smad(1, 5, 8)-dependent signaling in IPAH pulmonary arteries. These results argue against the conclusions of Du and coworkers that reduced BMPR-1 expression levels in IPAH and PAH with associated conditions might lead to decreased Smad(1, 5, 8)-dependent cell signaling in all forms of PAH (7).

We found a consistently high level of expression and potential active cell signaling by phosphoSmad(1, 5, 8) and phosphoSmad2 in endothelial cells in pulmonary arteries of IPH lungs as compared with pulmonary arteries of normal and emphysema lungs. Furthermore, phosphoSmad2 expression was higher in the smaller size pulmonary vessels when compared with larger diameter resistance pulmonary arteries in IPAH lungs. Interestingly, normal and hypertensive pulmonary arteries express predominantly TGF-ß2 and TGF-ß3, and less TGF-ß1, in medial layers and, in an irregular pattern, in the endothelium (23). These findings suggest that paracrine or autocrine TGF-ß family signaling may significantly contribute to the maintenance of quiescent pulmonary artery endothelial cells, because TGF-ß decreases cyclin-dependent kinase inhibitors (such as p27 and p57), induces apoptosis (8, 24), and may inhibit endothelial cell migration and proliferation by means of decreasing vascular endothelial growth factor receptor-2 expression (25).

Importantly, our finding of discrepant TGF-ß signaling molecule expression in lumenal versus core endothelial cells in plexiform lesions is consistent with our prior report of phenotypic heterogeneity of endothelial cells in these lesions (19). The lack of expression of TGF-ß receptors and signaling Smad(s) in the core region of plexiform lesions suggests that a loss of TGF-ß signaling might contribute to the disorganized growth of endothelial cells in plexiform lesions. Impaired TGF-ß cell signaling may either cause or be the result of a lack of endothelial cell differentiation or endothelial smooth muscle cell transdifferentiation in plexiform lesions (26, 27). The lack of expression of multiple members of the TGF-ß signaling cascade downstream of TGF-ß receptors (i.e., TßRI and/or TßRII) is likely to cause a generalized loss of TGF-ß–dependent cell responses. Smad(s) can also be activated by TßRI-TßRII-independent signaling (21) and each Smad may play independent and possibly complementary roles in suppressing lung endothelial cell growth, as shown by the proangiogenesis afforded by loss of Smad4 in pancreatic cancers (28). Different pathobiological events, such as impaired TGF-ß signaling, microsatellite instability (29), overexpression of angiogenic factors (20), or infection with the vasculotropic and angiogenic human herpesvirus-8 alone or in combination, might ultimately contribute to uncontrolled pulmonary artery remodeling in IPAH. Defective BMPR-2/BMPR-1/Smad(1, 5, 8)/Smad4 or TßRII/Alk-1/Smad(1, 5, 8)/Smad4 signaling might facilitate endothelial cell apoptosis (30) early in the development of the disease, whereas defective Alk5 signaling may afford growth advantages to abnormal endothelial cells, as shown in in vitro studies with endothelial cells (31).

Our study did not address modifiers of TGF-ß family signaling and gene expression control, such the coactivator CBP/p300, SARA, c-Ski, SnoN, Smurf, the homeodomain protein TGIF (transforming growth factor inhibitory factor), or inhibitory Smad6 and Smad7 (8, 32–35), among others, which may also control TGF-ß family signaling and endothelial cell growth. Although we focused our study on TßRI (Alk-5)/TßRII and Smad lung expression, it will be necessary to extend our observations to Alk-1, Alk-2, Alk-3, and Alk-6 and endoglin lung expression because of their role in vascular morphogenesis and function (8).

In summary, our studies indicate that pulmonary artery endothelial cells are the major lung vascular site of TGF-ß family member expression and cell signaling. Furthermore, we did not find evidence of a loss or reduction of expression of TGF-ß family signaling molecules in remodeled pulmonary arteries in IPAH lungs. However, core endothelial cells in plexiform lesions in IPAH and vascular smooth muscle cells in normal, emphysema, and IPAH pulmonary arteries lacked expression of TßR(s) and signaling Smad(s).


   Acknowledgments
 
The authors thank Mrs. Teresa Smith for secretarial work, Dr. Irina Petrache for critical review of the manuscript, Mr. Rai Pradeep for histological assistance, the personnel of the Hopkins Lung Transplant Program for clinical information, and Drs. Carl-Henrik Heldin and Peter ten Dijke for providing anti-phosphoSmad(1, 5, 8) and phosphoSmad2.


   FOOTNOTES
 
Supported by American Heart Association Grant-in-Aid 0150595N to R.M.T. and by NIH P01-HL66254 to N.F.V. and R.M.T.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: A.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.E.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.D.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; N.F.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.M.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 24, 2003; accepted in final form September 7, 2004


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 RESULTS
 DISCUSSION
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