Prostacyclin is a powerful vasodilator and inhibits platelet adhesion and cell growth. We hypothesized that a decrease in expression of the critical enzyme PGI2 synthase (PGI2-S) in the lung may represent an important manifestation of pulmonary endothelial dysfunction in severe pulmonary hypertension (PH). Immunohistochemistry and Western blot analysis were used to assess lung PGI2-S protein expression, and in situ hybridization was used to assess PGI2-S mRNA expression. In the normal pulmonary circulation (n = 7), PGI2-S was expressed in 48% of small, 67% of medium, and 76% of large pulmonary arteries as assessed by immunohistochemistry. PPH (n = 12), cirrhosis-associated (n = 4) and HIV-associated PH (n = 2) lungs exhibited a marked reduction in PGI2-S expression, involving all size ranges of pulmonary arteries. Vessels with concentric lesions showed complete lack of PGI2-S expression. Congenital heart (n = 4) and CREST (n = 2) cases exhibited a more variable immunohistological pattern of PGI2-S expression. These results were complemented by in situ hybridization and Western blots of representative lung samples. We conclude that the different sizes of the pulmonary arteries express PGI2-S differently and that the loss of expression of PGI2-S represents one of the phenotypic alterations present in the pulmonary endothelial cells in severe PH.
In severe chronic pulmonary hypertension (PH), pulmonary blood vessel remodeling involves a wide range of morphological alterations that span the entire vessel wall. Medial hypertrophy occurs in forms of PH caused by hypoxic vasoconstriction and associated with interstitial lung disease (1, 2). In primary pulmonary hypertension (PPH) or in severe pulmonary hypertension associated with the CREST syndrome (calcinosis cutis, Raynaud's phenomenon, esophageal motility disorder[s], sclerodactyly, and telangiectasia), hepatic cirrhosis, human immunodeficiency virus (HIV) infection, or cardiac malformations (secondary PH), altered endothelial cell function likely determines the progression and irreversibility of the vascular remodeling of the vessels (3, 4). We postulate that this dysfunctional pulmonary hypertensive endothelial cell phenotype is characterized by uncontrolled proliferation (5, 6), increased production of vasoconstrictor mediators such as endothelin (7), expression of 5-lipoxygenase (8, 9), and decreased synthesis of prostacyclin (10).
Prostacyclin, the principal arachidonic acid metabolite of vascular endothelial and smooth muscle cells (11, 12), is a powerful vasodilator (13), and inhibits platelet adhesion and cell growth (14, 15). While a decrease in urinary excretion of the 2,3-dinor-6-keto prostaglandin F1α (PGF1α) metabolite occurs in patients with PPH (16), there is no direct histologic documentation of the prostacyclin-producing cell sites in normal lungs or in the lungs of patients with severe PH. A loss or reduction of pulmonary prostacyclin synthesis might contribute to the pathogenesis of severe PH (3, 16).
In the present study, we describe the pattern of prostaglandin I2 synthase (PGI2-S) expression by immunohistochemistry, Western blot assay, and in situ hybridization in lungs of patients with severe primary and secondary PH. The reduction in the number of pulmonary arteries positive for PGI2-S expression and the lack of PGI2-S in concentric and plexiform lesions are consistent with the concept of endothelial cell dysfunction in severe PH.
Paraffin-embedded lung tissue was obtained from 12 patients with PPH, 4 with cirrhosis-associated PH, 4 with PH associated with cardiac congenital abnormalities, and 2 with AIDS-related PH (Table 1). Normal lung tissue was obtained from seven patients undergoing open lung biopsy (owing to localized inflammation [n = 2] or owing to primary or metastatic malignancies [n = 5]; six males, one female; 50 ± 3.5 [SEM] yr). Patients with normal lung tissue had no clinical evidence of PH. Ice-cold isopentane-frozen fresh lung tissue was available from 7 of the 24 patients with severe PH (3 PPH, 3 CREST, and 1 congenital) and from 5 normal lungs.
|Patient||Sex/age||PAP (s/d/m)||CO||Underlying Condition||Final Diagnosis||PGI2 †||Procedure|
|15||F/44||101/44/64||5.1||Primary biliary cirrhosis||PH||No||Transplant|
|20||F/21||130/50/78||NA||CHD (SV, TGV)||PH||No||Transplant|
Frozen tissue. All lung tissue samples were obtained during surgery except for one (case 21), which was harvested within 24 h of death. Whole lobes or lungs were maintained on ice for approximately 15 to 45 min, and then expanded and cryoprotected as previously described (17). Lung fragments were frozen in Tissue Tek embedding medium (Sakura, Torrance, CA) and maintained at −70° C until sectioning. Five- or 7-μm sections were mounted onto Superfrost slides (Fisher Scientific, Pittsburgh, PA). At least two slides from each lung were processed for in situ hybridization.
Paraffin-embedded tissue. Fragments of lungs were fixed overnight (autopsy) or for approximately 2 h (lung biopsy) in 10% buffered formalin before paraffin embedding. Five-micrometer sections were mounted onto Superfrost slides. One slide per case was selected for immunohistochemistry studies.
The diagnosis of PH was established on the basis of structural pulmonary vascular lesions described previously (1, 5, 6). The normal lungs were confirmed as such by assessment of hematoxylin and eosin (H&E)-stained slides.
A polyclonal anti-PGI2-S antibody produced against the peptide corresponding to amino acids 115–124 [(C)NFNPSEEKAR] of the rat PGI2-S cDNA was used for the immunohistochemistry and Western blot studies. Antibody specificity was confirmed by enzyme-linked immunosorbent assay (ELISA) using solid-phase immobilized antigen (0.1 μg/100 μl per microwell of peptide Cys-GGG) to capture the peptide-specific antibody.
To optimize immunostaining, PGI2-S was retrieved from formalin-fixed, paraffin-embedded sections by microwave pressure cooker heating in a sodium citrate solution for 18 min. After endogenous peroxidase blocking with 3% H2O2, the slides were automatically stained using the Ventana (Tucson, AZ) ES automatic immunostaining device. The primary antibody against PGI2-S was used at 1:50 dilution, and the immunodetection was performed using the Ventana avidin- biotin peroxidase system.
In each case serial sections were incubated with preimmune serum (1:50 dilution) from the same rabbit used to generate the anti-PGI2 antibody, as negative controls for immunohistochemistry.
Frozen sections of seven lungs with severe PH and five normal lungs were subjected to in situ hybridization studies. The procedure for in situ hybridization for the detection of PGI2-S mRNA was as described previously (18). The PGI2-S probe consisted of a 1500-base pair (bp) rat cDNA inserted into pGEM T vector (Promega, Madison, WI) (19). Development of the hybridization signal was allowed to proceed for 2 h, and then stopped by immersion of the slides in phosphate-buffered saline (PBS) (8).
The mean histological area (mm2 ± SEM) examined in normal, PPH, cirrhosis–PH, congenital PH, CREST–PH, and HIV–PH samples was 2.99 ± 0.32, 2.97 ± 0.48, 3.45 ± 0.55, 2.9 ± 0.6, 0.6 ± 0.4, and 3.2 ± 0.5 mm2, respectively. Each section was reviewed independently by two investigators (R.M.T. and C.C.), and for each vessel it was determined whether PGI2-S expression was present or absent (patchy staining was considered negative) (Figure 1). The pulmonary arteries were categorized as small (less than 100 μm in diameter), medium (between 100 and 500 μm in diameter), and large (more than 500 μm in diameter). PGI2-S staining was considered negative when the signal matched the background intensity of the parallel sections incubated with preimmune rabbit serum.
Lung tissue was homogenized in a 0.1 M Tris, pH 7.4 buffer containing 0.1 mM flurbiprofen, using a Tissuemizer (Tekmar, Cincinnati, OH) at 4° C (20, 21). The homogenate was initially centrifuged at 10,000 × g for 10 min at 4° C to remove tissue fragments. The resulting supernatant was centrifuged at 200,000 × g for 1 h at 4° C to collect the microsomal pellets. The microsomes were then suspended in a buffer of 10 mM potassium phosphate, pH 7.4, containing 0.1 mM EDTA, 0.1 mM dithiothreitol, and 10% glycerol. Solubilization of the microsomes was performed by stirring the microsomal suspension at 4° C for 30 min with 0.5% cholic acid and 0.2% Lubrol PX. Subsequently, the suspension was centrifuged at 200,000 × g for 1 h and the supernatant collected. Direct protein quantitation was by the DC protein assay kit (Bio-Rad, Hercules, CA). To assure equal protein loading of all gels, each sample was quantified, and 20 μg of protein was loaded in each lane of a 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Transfer of proteins to Optitran nitrocellulose (Schleicher & Schuell, Keene, NH) was performed by electrophoresis at 100 V for 1 h in a Bio-Rad Western blot apparatus. Membranes were then blocked for 1 h in a 2% membrane-blocking buffer (Rad-Free calorimetric detection system; Schleicher & Schuell). The anti-PGI2-S polyclonal antibody was used as the primary antibody at 1:50 dilution. After the primary antibody incubation, the membranes were exposed to three brief washes in distilled water followed by a 5-min rinse in Rad-Free (Schleicher & Schuell) wash buffer; this wash cycle was repeated three times before the addition of alkaline phosphatase-conjugated goat anti-rabbit IgG for 30 min at room temperature, followed by three more wash cycles and the addition of 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT) color substrate.
To control for protein quality of normal and PH lung samples, Western blot analysis of endothelial nitric oxide synthase (eNOS) was performed as described (22). Twenty-five micrograms of total cellular protein was loaded onto an SDS-polyacrylomide gel. eNOS was detected with a monoclonal antibody (Transduction Laboratories, Lexington, KY) at a dilution of 1:1000 and visualized by enhanced chemiluminescence (ECL kit; Amersham, Arlington Heights, IL).
The morphological alterations of the lung vessels present in the cases of PPH and secondary PH were extensive and included medial and intimal thickening, and plexiform and concentric lesions (5, 6). Positive factor FVIII-related antigen and negative smooth muscle actin immunostaining confirmed the endothelial cell nature of the plexiform lesions and concentric lesions in PPH and secondary PH.
PPH. PGI2-S immunolocalization was performed with a polyclonal antibody directed against a peptide designed on the basis of the primary sequence of rat PGI2-S. Only the pulmonary artery endothelial cells (not the medial smooth muscle cells or perivascular fibroblasts) exhibited immunoreactivity for PGI2-S (Figures 1 and 2). PGI2-S staining was stronger in large pulmonary arteries (> 500 μm in diameter) than in small pulmonary vessels (< 100 μm in diameter) (Figures 1-3). In the normal lungs, PGI2-S expression was detectable in 48% (170 of 316) of small, 67% (119 of 191) of medium, and 76% (6 of 8) of large pulmonary arteries sampled (Figure 3). Pulmonary veins located within interlobular septa expressed PGI2-S in an intensity pattern between that of small and medium-sized pulmonary arteries. On the other hand, PPH lungs exhibited a lower number of PGI2-S-positive endothelial cells within small (60 of 285; 21%) and medium-sized (73 of 208; 35%) pulmonary arteries when compared with normal vessels (Figures 2 and 3). PGI2-S expression in large vessels in PPH lungs (20 of 41; 49%) was not significantly different from that in normal lungs. The reduction in PGI2-S expression in PPH lungs did not alter the pattern of PGI2-S expression, that is, medium-sized and large arteries in PPH lungs were more frequently positive for PGI2-S expression than were small pulmonary arteries, similar to the normal lungs (Figure 3). Although there was a trend toward a total loss of PGI2-S expression in the more severely remodeled pulmonary vessels in PPH, some vessels with mild remodeling also showed no PGI2-S immunoreactivity (Figure 2). There was a smaller (but statistically insignificant) number of pulmonary arteries per square millimeter of lung section in the PPH lungs when compared with normal lungs (small, 10.5 ± 2.7 [SEM] versus 13.7 ± 2.3; medium, 7.31 ± 1.4 [SEM] versus 7.8 ± 1.7 [SEM], respectively).
In 12 patients with PPH, we detected a total of 48 plexiform and 33 concentric lesions (mean per case: 1.39 ± 0.6 plexiform and 1.44 ± 0.5 [SEM] concentric lesions per square centimeter of lung section). Forty-three percent of the plexiform lesions and 97% of the concentric lesions exhibited no PGI2-S expression at all (Figure 2). We have previously demonstrated that the plexiform and concentric lesions are composed predominantly of endothelial cells and we consider these lesions the result of an abnormal process of angiogenesis (5). Although most of the endothelial cells within the lesions lacked PGI2-S, there was some immunoreactivity in the cells lining the slit-like lumenal areas. These areas may represent abnormal early blood vessel formation (Figure 2). Low-intensity immunostaining of PGI2-S was seen in alveolar septa (Figures 1 and 2).
Prostacyclin treatment (Flolan infusion) likely had no influence on the pattern of PGI2-S expression because four patients with PPH (patients 6–8 and 12) chronically treated with prostacyclin exhibited a lung PGI2-S staining pattern similar to that observed in the eight untreated patients with PPH. It is possible that the advanced stage of pulmonary blood vessel remodeling may account for the lack of a prostacyclin effect on the overall pulmonary vessel PGI2-S expression among patients with PPH.
Secondary PH. The lungs of patients with cirrhosis-related PH (n = 4) exhibited a pattern of PGI2-S expression similar to that observed in the patients with PPH (percentage positive arteries ± SEM in cirrhosis–PH: small, 15.7 ± 8.6; medium, 45 ± 21; large, 58 ± 25) (Table 2). The two patients with HIV–PH also had a similar frequency of positive PGI2-S: small (9%), medium (57%), and large (41%) pulmonary arteries (Table 2). Patients with congenital PH (n = 4) and CREST–PH (n = 2) exhibited a wider range of PGI2-S expression; two of the patients with congenital PH and one with CREST–PH showed preserved immunoreactivity comparable to that of normal lungs, while the other three presented a pattern similar to that of PPH lungs (Table 2). Only 3 of 32 plexiform and no concentric lesions (n = 36) in the secondary group expressed PGI2-S.
|Patient*||Pulmonary Arteries†||Plexiform Lesions‡||Concentric Lesions‡|
|13||5/16 (31)||7/8 (87)||2/2 (100)||3/3||0/3|
|14||1/55 (1.8)||0/20 (0)||1/3 (33)||0/0||0/6|
|15||15/50 (30)||18/24 (30)||2/2 (100)||0/9||0/2|
|16||0/8 (0)||3/15 (20)||0/3 (0)||0/1||0/7|
|17||6/20 (30)||4/11 (36)||0/0||0/3||0/1|
|18||0/4 (0)||2/7 (28)||1/1 (100)||0/5||0/1|
|19||25/46 (54)||10/24 (42)||5/6 (83)||0/1||0/5|
|20||20/22 (91)||18/21 (86)||7/8 (87)||0/2||0/3|
|21||6/32 (19)||8/13 (62)||1/1 (100)||0/0||0/3|
|22||9/10 (90)||8/8 (100)||0/0||0/0||0/0|
|23||16/63 (25)||7/10 (70)||3/3 (100)||0/0||0/0|
|24||0/4 (0)||5/10 (50)||1/5 (20)||0/5||0/5|
A similar PGI2-S immunostaining pattern was obtained in frozen sections with the polyclonal PGI2-S antiserum (kindly provided by D. Dewitt, Michigan State University, East Lansing, MI) (12) and the polyclonal antibody K376 (provided by V. Ulrich, University of Konstanz, Germany) (23). The following monoclonal anti-PGI2-S antibodies did not react in paraffin-embedded and frozen sections of lung and brain tissue (positive control): anti-PGI2-S (Oxford Biomedical Research, Oxford, MI), ISN-1 (kindly provided by W. Smith, Michigan State University, East Lansing, MI) and anti-PGI2-S (Cayman Chemicals, Ann Arbor, MI) (dilutions ranging from 1:20 to 1:500).
PGI2-S transcripts were detected in the normal and partially remodeled pulmonary arteries and in the alveolar septa (Figure 4). The intensity of the specific hybridization signal in the pulmonary vessels paralleled the intensity of their immunohistochemical staining. The strongest signal was present in the larger vessels followed by small vessels and alveolar septa from patients with secondary pH and in normal lungs. Plexiform and concentric lesions and pulmonary vessels with marked intimal fibrosis exhibited low or absent hybridization signals for PGI2-S mRNA (Figure 4). The partial correlation between the expression of PGI2-S mRNA and protein in the alveolar septa might have occurred owing to decreased protein stability or to the presence of low levels of proteins in the alveolar walls.
We detected a faint or absent PGI2-S protein signal in the lungs of patients with PPH (n = 2), CREST (n = 1), or PH associated with congenital heart disease (n = 1) in comparison with the strong signal demonstrated in lung samples from control patients (n = 2). These same normal and PH lung samples exhibited a strong eNOS signal by Western blot (Figure 5).
This study demonstrated that, in normal lungs, larger (more proximal) pulmonary arteries expressed more PGI2-S than did smaller arteries, and that the number of pulmonary arteries expressing PGI2-S was reduced in PPH lungs when compared with normal lungs. The reduction in PGI2-S expression in PPH lungs was supported by in situ hybridization for PGI2-S mRNA and by Western blot analysis.
In both the normal and the diseased human pulmonary circulation, the endothelium was clearly the major site of lung vascular PGI2-S expression. In contrast, little or no PGI2-S expression was found in the subintimal and medial smooth muscle cells. The lack of PGI2-S expression in human pulmonary vascular smooth muscle cells is remarkable because lung tissue sections from a wide range of species express PGI2-S by immunofluorescence in the pulmonary arterial smooth muscle and endothelial cells (12). Although endothelial cells are the major sites of PGI2-S protein expression in the human pulmonary blood vessels, we cannot rule out that the vascular smooth muscle cells express PGI2-S below the detection level of our immunohistochemical and molecular techniques. The regional distribution pattern of PGI2-S in the normal circulation indicated that a more uniform expression was present in larger pulmonary arteries and that at least 50% of the normal small pulmonary arteries had no detectable PGI2-S.
The reduction in PGI2-S protein and mRNA expression in severe pulmonary hypertension was observed in arteries ranging from 1 mm to less than 100 μm in diameter. These changes were significant in the small and medium-sized but not large-sized arteries. This may have occurred because the present study was conducted retrospectively, using lung tissue obtained for diagnosis, with limited sampling of large pulmonary arteries. PGI2-S was absent in severely remodeled vessels containing plexiform and concentric lesions and in minimally remodeled small pulmonary arteries. The marked reduction of the PGI2-S signal by Western blot analysis in the lungs from patients with PPH and secondary PH may reflect the severity of the pulmonary arterial disease and the endothelial cell dysfunction of severe PH (3, 4). Because the overall surface area of the smaller pulmonary arteries is significantly larger than that of the larger vessels, a reduction of small vessel PGI2-S expression could have accounted for the Western blot results. We believe that the decreased PGI2 synthesis by the hypertensive lungs can explain the earlier data of reduced urinary excretion of PGI2 metabolites in patients with PPH (16). These results in the aggregate suggest that the reduction of prostacyclin synthesis in otherwise morphologically normal to minimally remodeled vessels may play a role in the early stages of pathogenesis. Alternatively, endothelial cells of pulmonary small arteries may become dysfunctional, as the disease progresses and the pulmonary artery pressure or shear stress progressively rise. The decrease in PGI2 production by pulmonary endothelial cells, inferred by the decreased PGI2-S staining intensity and by the lack of lung tissue PGI2-S protein, could predispose the lung vessels to additional vasoconstriction and/ or in situ thrombosis due to enhanced platelet adhesion.
Giaid and coworkers noted a reduction in expression of nitric oxide synthase, which paralleled the extent of blood vessel remodeling and the severity of PH (24). However, the presence of eNOS expression in PPH (our Western blot analysis) agreed with reports by Xue and Johns (25) and Mason and co-workers (26) indicating that the loss of eNOS expression is not part of the pulmonary hypertensive endothelial cell dysfunction in partially remodeled vessels in PPH. In addition, there is evidence of increased eNOS levels in experiment models of pulmonary hypertension (27, 28).
In addition to the alteration of the phenotypic properties of the vascular cells in the pulmonary circulation, there is growing evidence of a genetic basis for the development of PPH. A region on the long arm of chromosome 2 has been associated with familial cases of PPH (29). The finding that, in PPH but not in secondary PH, the plexiform and concentric lesions are composed of a monoclonal endothelial cell population suggests that a somatic genetic event has caused an endothelial cell growth advantage in PPH (30). Because there is a comparable reduction of PGI2-S expression in secondary PH, it is unlikely that a reduction of PGI2-S expression in PPH could account for a genetic event creating a predisposition to monoclonal endothelial cell growth in PPH.
The expression of PGI2-S in cirrhosis- and HIV-associated PH was similar to that exhibited by PPH lungs. However, lungs with CREST-associated and congenital heart-associated PH showed a spectrum of vascular PGI2-S expression, and we wonder whether the extent of the phenotypic alterations of the endothelial cells in some forms of secondary PH is less pronounced than in PPH.
In summary, our study demonstrates that small and medium-sized hypertensive lung vessels, with mild and severe remodeling, have a decreased expression of PGI2-S when compared with normal pulmonary vessels. Decreased or absent PGI2-S expression may be an important tissue marker for pulmonary endothelial cell dysfunction in severe PH.
The authors thank Jenny Allard and Marilee Horan for performing Western blot analyses, and Velma Parker and Ginger Woodward for secretarial work.
Supported by an NIH Vascular Center Award, R01 Grant HL43180-01A1, and a PPH Cure Foundation grant to R.M.T., M.W.G., and N.F.V.
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