Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells

Shen Zhang,1,* Ivana Fantozzi,1,* Donna D. Tigno,1 Eunhee S. Yi,2 Oleksandr Platoshyn,1 Patricia A. Thistlethwaite,3 Jolene M. Kriett,3 Gordon Yung,1 Lewis J. Rubin,1 and Jason X.-J. Yuan1

1Division of Pulmonary and Critical Care Medicine, Department of Medicine, 2Department of Pathology, and 3Division of Cardiothoracic Surgery, Department of Surgery, University of California, San Diego, California 92103-8382

Submitted 16 August 2002 ; accepted in final form 28 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Pulmonary vascular medial hypertrophy in primary pulmonary hypertension (PPH) is mainly caused by increased proliferation and decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Mutations of the bone morphogenetic protein (BMP) receptor type II (BMP-RII) gene have been implicated in patients with familial and sporadic PPH. The objective of this study was to elucidate the apoptotic effects of BMPs on normal human PASMCs and to examine whether BMP-induced effects are altered in PASMCs from PPH patients. Using RT-PCR, we detected six isoforms of BMPs (BMP-1 through -6) and three subunits of BMP receptors (BMP-RIa, -RIb, and -RII) in PASMCs. Treatment of normal PASMCs with BMP-2 or -7 (100-200 nM, 24-48 h) markedly increased the percentage of cells undergoing apoptosis. The BMP-2-mediated apoptosis in normal PASMCs was associated with a transient activation or phosphorylation of Smad1 and a marked downregulation of the antiapoptotic protein Bcl-2. In PASMCs from PPH patients, the BMP-2- or BMP-7-induced apoptosis was significantly inhibited compared with PASMCs from patients with secondary pulmonary hypertension. These results suggest that the antiproliferative effect of BMPs is partially due to induction of PASMC apoptosis, which serves as a critical mechanism to maintain normal cell number in the pulmonary vasculature. Inhibition of BMP-induced PASMC apoptosis in PPH patients may play an important role in the development of pulmonary vascular medial hypertrophy in these patients.

hypertension; arteries; Smad; Bcl; transforming growth factor-{beta}


PULMONARY VASOCONSTRICTION, vascular wall remodeling, and in situ thrombosis are major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure (PAP) found in patients with primary pulmonary hypertension (PPH). The pulmonary vascular remodeling in PPH is characterized by changes in pulmonary vascular structure associated with medial hypertrophy, which is mainly caused by imbalanced proliferation and apoptosis in pulmonary artery smooth muscle cells (PASMCs; Refs. 3, 9, 43, 45, 50, 55). Increased PASMC proliferation and decreased PASMC apoptosis can concurrently mediate thickening of the pulmonary vasculature, which subsequently reduces the inner-lumen diameter of pulmonary arteries, increases pulmonary vascular resistance, and raises PAP (45). In animal experiments, it has been demonstrated that hypertrophied PASMCs in the intact pulmonary arteries can be made to undergo apoptosis, and the induction of PASMC apoptosis results in progressive regression of pulmonary vascular medial hypertrophy (7, 8, 43). These observations suggest a novel strategy to treat pulmonary vascular disease by inducing regression of pulmonary vascular medial thickening, which is a pathological feature in patients with PPH and other types of severe pulmonary hypertension (7, 8, 43).

Bone morphogenetic proteins (BMPs) are signaling molecules that belong to the transforming growth factor-{beta} (TGF-{beta}) superfamily (22, 35-37, 53). BMPs are synthesized and secreted from a variety of cell types including pulmonary vascular smooth muscle and endothelial cells and play an important role in regulating cell proliferation, apoptosis, and differentiation (22, 35-39, 56-61). Signal transduction of BMP-mediated effects, similar to TGF-{beta}, involves two types of transmembrane serine-threonin kinase receptors: BMP receptor types I and II (BMP-RI and -RII, respectively; Refs. 22, 35-37, 41, 44, 53). In the absence of ligand (i.e., BMPs), both homomeric and heterogeneous BMP receptors (BMP-RIa, -RIb, and -RII) are found in the surface membrane of living cells (17, 42). Binding of BMPs to BMP-RI (BMP-RIa or -RIb) augments heterooligomerization of BMP-RI and -RII, which is required to activate the downstream signaling elements [mainly the receptor-activated "mothers against decapentaplegic" (Smad) proteins; e.g., Smad1, Smad5, and Smad8; Refs. 22, 35-39, 41, 44, 53]. Among the Smad-responsive genes, many encode proteins that are required to arrest cell growth and induce apoptosis (35-37, 41, 48, 53, 56).

Mutations of the BMP-RII gene have been demonstrated to be the genetic basis for familial PPH. The BMP-RII gene mutations have also been implicated in 15-25% of patients with sporadic PPH, which suggests a common genetic defect in the BMP-RII gene for the pathogenesis of both familial and sporadic PPH (9, 10, 32, 34, 41). However, how mutations in the BMP-RII gene (located at chromosome 2q33) cause PPH is unclear. To reveal the pathological mechanisms by which a mutant BMP-RII gene mediates vascular abnormalities in PPH patients, it is important to understand the functional role of BMP-mediated activation of BMP receptors in PASMCs from normal subjects. This study was designed to characterize the molecular identities of BMPs and BMP receptors in normal human PASMCs and to examine the effects of BMPs on PASMCs isolated from normal subjects and patients with secondary pulmonary hypertension (SPH) and PPH.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Demographic, clinical, and hemodynamic characteristics of patients. There were five subjects from whom lung tissue was obtained to prepare PASMCs for the study. The diagnosis of PPH was established clinically in two patients on the basis of the criteria used in the National Registry on Primary Pulmonary Hypertension and was confirmed histopathologically. The mean PAP of the two PPH patients (a 57-yr-old woman and a 31-yr-old man) were 51 and 53 mmHg, respectively. Both of the PPH patients had been treated with Flolan, warfarin, digoxin, and furosenmide before lung transplantation. Three subjects had pulmonary hypertension (SPH) resulting from known causes: a 69-yr-old male patient with idiopathic pulmonary fibrosis (mean PAP, 26 mmHg), a 58-yr-old female patient with emphysema (mean PAP, 33 mmHg), and a 37-yr-old female patient with lymphangioleiomyomatosis (LAM; mean PAP, 50 mmHg). The three SPH patients were treated with aspirin, citalopram, estrogen, or narcotic pain medicines before transplantation. All patients were Caucasian except the LAM patient, who was Hispanic.

Cell preparation and culture. Primary cultured PASMCs from transplant patients were used in this study (63, 64). Lung tissue removed from patients in the operating room was immediately placed in cold (4°C) saline and taken to the laboratory for dissection. Peripheral muscular pulmonary arteries (diameter range, 300-500 µm) isolated from the explanted lung tissues within 5 h after transplantation were incubated in Hanks' balanced salt solution that contained 2 mg/ml collagenase (Worthington Biochemical) for 20 min. The adventitia was stripped, and endothelium was removed. The remaining smooth muscle was digested with (in mg/ml) 2.25 collagenase, 0.5 elastase, and 1 albumin (Sigma) at 37°C to make a cell suspension of PASMCs. The cells were resuspended, plated onto 25-mm coverslips or 10-cm petri dishes, and incubated in a humidified atmosphere of 5% CO2-95% air at 37°C in the smooth muscle growth medium (SMGM, Clonetics). Human PASMCs from normal subjects (who had no implication of pulmonary hypertension) purchased from Clonetics were also used in some experiments. The cells were seeded in flasks at a density of 2,500-3,500 cells/cm2 and were incubated in SMGM. The medium was changed after 24 h and every 48 h thereafter. The SMGM was composed of smooth muscle basal medium supplemented with 5% FBS, 0.5 ng/ml human epidermal growth factor (hEGF), 2 ng/ml human fibroblast growth factor (hFGF), and 5 µg/ml insulin. Cells were subcultured or plated onto 25-mm coverslips using trypsin-EDTA buffer (Clonetics) when 70-90% confluence was achieved. The morphology of the cells was examined by using an inverted phase-contrast microscope attached to a digital camera.

Measurement of cell death. The cells, grown on 25-mm coverslips, were first washed with PBS and then fixed in 95% ethanol (for 20 min at 0°C) and stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI, Sigma). DAPI (5 µM) was dissolved in an antibody buffer that contained 500 mM NaCl, 20 µM NaN3, 10 µM MgCl2, and 20 µM Tris·HCl (pH 7.4). The blue fluorescence emitted at 461 nm was used to visualize the cell nuclei. The DAPI-stained cells were examined by using a Nikon TE 300 fluorescence microscope, and the cell (nuclear) images were acquired by using a high-resolution Solamere fluorescence imaging system (Solamere Technology Group, Salt Lake City, UT). DAPI staining reveals nuclear morphological changes of cells undergoing apoptosis. For each coverslip, seven fields (with 20-25 cells/field) were randomly selected to determine the percentage of apoptotic cells in the total cells based on the morphological characteristics of apoptosis. The cells with clearly defined nuclear breakage, remarkably condensed nuclear fluorescence, and significantly shrunken cell nuclei were defined as apoptotic cells (30). To quantify apoptosis, terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) assay was also performed with an in situ cell death detection kit (Roche; Ref. 30).

The annexin V-FITC Apoptosis Detection Kit (BD Phar-Mingen) was used for flow cytometry experiments to detect apoptotic cells (40). Human PASMCs cultured in SMGM with or without apoptosis inducers were washed twice with cold PBS and then resuspended in 1x binding buffer at a concentration of 1 x 106 cells/ml. The cell suspension (100 µl, ~1 x 105 cells) was transferred to a 5-ml culture tube and mixed with 5 µl of annexin V-FITC and 10 µl of propidium iodide (PI). The cells were gently vortexed and incubated for 15 min at room temperature (20-25°C) in the dark. Then 400 µl of 1x binding buffer was added to each tube and analyzed by fluorescence-activated cell sorting (with FACS Calibur) by using CellQuest software (Becton Dickinson, Mountain View, CA).

RT-PCR. Total RNA was extracted from human PASMCs by using the RNeasy Mini Kit (Qiagen). Total RNA from human brain and lung tissues was purchased from GIBCO (2.5 µg/µl). Genomic DNA was removed with RNase-free DNase according to the manufacturer's instruction. Super-Script reverse transcriptase (Invitrogen) was used to synthesize cDNA. RNA (2 µg) was first incubated with oligo(dT) (1 µl at 0.5 µg/µl) at 70°C for 10 min. Then 8 µl of a solution that contained 10x buffer, 10 mM dNTP, 20 mM MgCl2, 0.1 M DTT, 40 U/µl RNaseOUT, and 50 U/µl SuperScript II reverse transcriptase were added to the samples and incubated for 10 min at 30°C, 60 min at 42°C, and 5 min at 95°C. RNase-H (1 µlat2U/µl; GIBCO) was added to each reaction, and the samples were incubated for 20 min at 37°C. The sense and antisense primers were specifically designed from the coding regions of each gene (Table 1). Bax and Bcl-2 mRNA expression was detected by using specific primers (EZ41 for Bax and EZ42 for Bcl-2) purchased from Oxford Biomedical Research (Rochester Hills, MI). The sense and antisense primers are 5'-TTCTGACGGCAACTTCAACTGG-3' (sense) and 5'-AGGAAGTCCAATGTCCAGCC-3' (antisense; 135 bp) for Bax and 5'-TGTGGTATGAAGCCAGACCTCC-3' (sense) and 5'-CAGGATAGCAGCACAGGATTGG (antisense; 153 bp) for Bcl-2 (47). The sense and antisense primers for GAPDH are 5'-GAGCCAAAAGGGTCATCATCTC-3' (sense) and 5'-AGGGTCTCTCTCTTCCTCTT-3' (antisense; 719 bp). The fidelity and specificity of the sense and antisense oligonucleotides were examined by using the BLAST program.


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Table 1. Oligonucleotide sequences of the primers used for RT-PCR

 

PCR was performed by using the GeneAmp PCR System (Perkin-Elmer, Norwalk, CT) with platinum PCR supermix (GIBCO). The first-strand cDNA reaction mixture (1 µl) was used in a 50-µl PCR reaction that consisted of 1 µl of each primer (10 µM), 10 mM Tris·HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 200 µM of each dNTP, and 2 U Taq DNA polymerase. The cDNA samples were amplified in a DNA thermal cycler under the following conditions: the mixture was annealed at 55°C (30 s), extended at 72°C (30 s), and denatured at 94°C (30 s) for 32 cycles. This was followed by a final extension at 72°C (10 min) to ensure complete product extension. The PCR products were electrophoresed through a 1.5% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining. To semiquantify the RT-PCR products, an invariant mRNA of GAPDH was used as an internal control to normalize the mRNA levels of BMP ligands, BMP receptors, and Smad proteins as well as Bax and Bcl-2. The net intensity values of cDNA bands (e.g., for Bax and Bcl-2 transcripts) measured by a Kodak Electrophoresis Documentation System were normalized to the net intensity values of the GAPDH signals; the ratios are expressed as arbitrary units (AU) for quantitative comparison.

Western blot analysis. PASMCs were gently washed twice in cold PBS, scraped into 0.3 ml of radioimmunoprecipitation assay buffer [1x PBS, 1% Nonidet P-40 (Amaresco), 0.5% sodium deoxycholate, and 0.1% SDS], and incubated on ice for 45 min during which the cell mixture was shaken for 30 s by vortex three times. Resulting cell lysates were sonicated and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant was collected, and protein concentrations were determined by Coomassie Plus protein assay reagent (Pierce Biotechnology) by using BSA as a standard. Protein (20 µg) was mixed and boiled in 2x sample buffer (0.25 M Tris·HCl, pH 6.8, 20% glycerol, 8% SDS, and 0.02% bromophenol blue). Protein suspensions were electrophoretically separated on a 10% acrylamide gel, and protein bands were transferred to nitrocellulose membranes by electroblot in a Mini Trans-Blot cell transfer apparatus (Bio-Rad) under conditions recommended by the manufacturer. After 1 h of incubation in a blocking buffer (0.1% Tween 20 and 5% nonfat dry milk powder), the membranes were incubated with the affinitypurified rabbit polyclonal antibody against the phosphorylated Smad1 (Upstate Biotechnology, Lake Placid, NY) and with the mouse monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Finally, the membranes were washed and exposed to antirabbit or anti-mouse horseradish peroxidase-conjugated IgG for 90 min at room temperature. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham). The monoclonal anti-{alpha}-actin antibody (Upstate) was used as a control.

Immunofluorescence labeling. The PASMCs from SPH and PPH patients were first stained with the membrane-permeant nucleic acid stain DAPI (5 µM, Sigma), and the blue fluorescence emitted at 461 nm was used to estimate total cell numbers in the culture. A specific monoclonal antibody raised against smooth muscle {alpha}-actin (Upstate) was then used to evaluate cellular purity of culture, and a secondary antibody (Affinipute donkey anti-mouse IgG) conjugated with FITC (Jackson ImmunoResearch) was used to display the fluorescent image (emitted at 529 nm). The cells were mounted in 10% of 1 M Tris·HCl/90% glycerol (pH 8.5) that contained 1 mg/ml p-phenylenediamine. The cell images were processed by the Solamere fluorescence-imaging system (Solamere Technology Group); the FITC fluorescence was colored red and DAPI fluorescence was colored green to display images with red-green overlay. The DAPI-stained cells that also cross-reacted with the smooth muscle cell {alpha}-actin antibody were defined as smooth muscle cells.

Histological preparation. The explanted lungs were grossly examined and fixed overnight in 10% neutral buffered formalin. The formalin-fixed lungs were serially cut, and representative sections were sampled from each lobe (typically 2-3 samples/lobe) for microscopic examination. The tissues were routinely processed and embedded in paraffin blocks in an automatic tissue processor (Sakura Tissue-Tek VIP, Torrance, CA). The paraffin-embedded tissues were cut into 5-µm-thick sections for standard hematoxylin eosin staining. Selected sections were also stained with trichrome and elastin.

Chemicals. Staurosporine (ST, Sigma) was prepared as a 1-mM stock solution in DMSO; aliquots of the stock solution were then diluted 1:10,000-500,000 times to the culture media or physiological salt solution for experimentation. Human TGF-{beta}1 (R&D Systems) was prepared as a stock solution in sterile HCl (4 mM) that contained 0.1% BSA. Recombinant human BMP-2 and -7 (R&D Systems) and Fas ligand (Sigma) were prepared as stock solutions in sterile PBS that contained 0.1% BSA. Cells were synchronized or growth-arrested in smooth muscle basal medium for 48 h before each experiment.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed by using the paired or unpaired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) as indicated. Differences were considered to be significant when P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Molecular identity of BMPs and BMP receptors in human PASMCs. Using RT-PCR analysis, we detected transcripts of BMP-1, -2, -3, -4, -5, and -6 in normal human PASMCs from subjects who had no implication of pulmonary hypertension (Fig. 1A). However, the BMP-7 transcript was not detectable in PASMCs (Fig. 1A, top), although it was highly expressed in human lung tissues (Fig. 1A, bottom). In human PASMCs, mRNA expression levels of all BMP receptors (BMPRIa, -RIb, and -RII) were similar to those in human lung tissues (Fig. 1B), which suggests that BMP receptors are highly expressed in human PASMCs. These results indicate that BMP-1, -2, -3, -4, -5, and -6 are the endogenously expressed BMPs in human PASMCs, which can be synthesized and secreted to the intercellular space and can activate BMP receptors on adjacent PASMCs. Although BMP-7 seems to be an exogenous BMP for PASMCs, it may be synthesized in other types of pulmonary cells such as vascular endothelial cells and alveolar epithelial cells and subsequently secreted to the intercellular space to affect pulmonary vascular smooth muscle cells.



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Fig. 1. RT-PCR analysis of bone morphogenetic proteins (BMPs), BMP receptors, and "mothers against decapentaplegic" (Smads) in human pulmonary artery smooth muscle cells (PASMCs) and lung tissues. A: PCR-amplified products are displayed in agarose gels for BMP-1 (448 bp), BMP-2 (671 bp), BMP-3 (395 bp), BMP-4 (400 bp), BMP-5 (406 bp), BMP-6 (529 bp), and BMP-7 (574 bp). B: PCR-amplified products for BMP receptors BMP-RIa (232 bp), -Ib (630 bp), and -RII (694 bp). C: PCR-amplified products for Smad1 (353 bp), Smad2 (268 bp), Smad3 (283 bp), Smad4 (264 bp), Smad5 (395 bp), Smad6 (297 bp), Smad7 (294 bp), and GAPDH (719 bp). M, 1 kb plus DNA ladder. mRNA from human lung tissues was purchased from GIBCO and used as positive control. Primer sequences are shown in Table 1.

 

When two types of transmembrane serine-threonine kinase receptors, BMP-R1 and -RII, are activated by binding to ligands (i.e., BMPs), the type II (BMP-RII) and type I (BMP-RI) receptors form a heterotetrameric complex (22, 35-37, 53, 56). The phosphorylated or activated BMP-RI directly phosphorylates the receptor-activated Smads (R-Smad; e.g., Smad1, Smad5, or Smad8), which then form a heteromeric complex with the co-Smad, Smad4, and enter the nucleus for transcriptional regulation. The R-Smad/Smad-4 complex binds to the specific Smad binding site (5'-CGCA-3') in DNA (located in the gene promoter), and activates or represses Smad-responsive genes (22, 35-37, 48, 53, 56). In human PASMCs, we detected seven isoforms of Smad transcripts, Smad1 to Smad7 (Fig. 1C). The mRNA levels of all Smads in human PASMCs were comparable to those in lung tissues, which suggests that the proteins of the Smad family may serve as important downstream-signaling proteins in PASMCs when BMP receptors are activated by their ligands, BMPs.

In addition to BMPs and BMP receptors, human PASMCs also express three isoforms of TGF-{beta} (TGF-{beta}1, -{beta}2, and -{beta}3) and two types of TGF-{beta} receptors (TGF-{beta}-RI and -RII; Fig. 2). The mRNA expression levels of TGF-{beta} and TGF-{beta} receptors in human PASMCs were also comparable to those in human lung tissues.



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Fig. 2. RT-PCR analysis of transforming growth factor-{beta} (TGF-{beta}) and TGF-{beta} receptors (TGF-{beta}-R) in human PASMCs and lung tissues. A: PCR-amplified products displayed in agarose gels for TGF-{beta}1 (405 bp), TGF-{beta}2 (565 bp), and TGF-{beta}3 (522 bp). B: PCR-amplified products for TGF-{beta}-RI (250 bp), TGF-{beta}-RII (137 bp), and GAPDH (719 bp) in human PASMCs. M, 1 kb plus DNA ladder. Primer sequences are shown in Table 1.

 

It must be emphasized that the mRNA expression of BMPs, BMP receptors, and the downstream signal transduction proteins (Smads) does not directly represent the protein expression of these ligands, receptors, and signaling proteins. Further immunoblot or immunocytochemistry experiments are needed to demonstrate the protein expression and distribution of BMPs, BMP receptors, and Smads in human PASMCs as well as their functional linkages to the effects of BMPs on cell growth and apoptosis.

Apoptotic effects of BMP-2, TGF-{beta}1, and Fas ligand on human PASMCs. It has been demonstrated that low doses (10-100 nM) of BMPs and TGF-{beta}s inhibited human PASMC proliferation in the presence of low concentrations of serum (38). To test whether the antiproliferative effects of BMPs and TGF-{beta}1 also involve mediating apoptosis in human PASMCs, we determined and compared the percentage of cells undergoing apoptosis in cells cultured in media with or without BMP-2 or TGF-{beta}1.

Under baseline control conditions, the percentage of apoptotic cells (as determined by morphological changes of nuclei by using DAPI staining and TUNEL assay) ranged from 2 to 7% in normal human PASMCs. After 48 h of treatment with 200 nM BMP-2, ~22.9 ± 1.9% of the cells contained apoptotic nuclei as identified by DAPI staining (nuclear condensation and breakage) and TUNEL assay (Fig. 3, A and Ca). Treatment of PASMCs with 50 nM of TGF-{beta}1 or 200 nM of Fas ligand (anti-Fas) also increased the percentage of the cells undergoing apoptosis (Fig. 3, A and Ca). The EC50 for BMP-2-induced apoptosis in human PASMCs was ~75 nM (Fig. 3Cb) and for Fas ligand was ~97 nM (Fig. 3Cc). Furthermore, incubation of human PASMCs in media that contained 20 nM ST increased the percentage of apoptotic cells from 3.0 ± 0.7 to 44.5 ± 4.2% (Fig. 3, B and Ca). The time course of the apoptotic effect of ST (20 nM) on PASMCs indicated that the ST-mediated apoptosis in human PASMCs maximized ~20-24 h after treatment (Fig. 3Bb).



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Fig. 3. Apoptotic effects of BMP-2, TGF-{beta}1, Fas ligand (FasL), and staurosporine (ST) on human PASMCs. A: representative 4',6'-diamidino-2-phenylindole (DAPI; top)- and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL; middle)-stained nuclei in control cells (Control) and cells treated with BMP-2 (200 nM), TGF-{beta}1 (50 nM), and Fas ligand (200 nM) for 48 h. Apoptotic cell nuclei were positively stained with TUNEL reagent and are shown in green (middle) and yellow (bottom). Ba: representative DAPI- and TUNEL-stained nuclei in control (left) and ST-treated (20 nM for 24 h; right) cells. TUNEL-positive cells are defined as apoptotic cells and are shown in green (middle) and yellow (bottom). Bb: time course of the ST-induced apoptosis in PASMCs indicates that 24 h of treatment with 20 nM ST increased the percentage of cells undergoing apoptosis from 4 to 60%. Ca: summarized data (means ± SE) show the percentage of apoptotic nuclei in control PASMCs (Cont) and cells treated for 48 h with BMP-2, TGF-{beta}1, Fas ligand, and ST (20 nM); ***P < 0.001 vs. Cont. Cb and Cc: dose-response curve of BMP-2-mediated apoptotic effects. [BMP2], concentration of BMP-2. **P < 0.01. C, c: dose-response curve of Fas ligand-mediated apoptotic effects. [FasL], concentration of Fas ligand. Data are means ± SE; n = 21-28 fields of cells from 4 coverslips for each data point. EC50 values for BMP-2- and FasL-mediated apoptosis in normal human PASMCs (vertical lines) and baseline control level of apoptosis before treatment with BMP-2 or Fas ligand (horizontal dashed lines) are shown.

 

BMP-2 activates Smad1 and downregulates Bcl-2 expression. The basic signaling system of BMP is composed of two BMP receptors and the Smad proteins that are phosphorylated and translocated into the nucleus on activation of BMP receptors (23, 35-37, 53, 56). In mammalian cells, the ligand-activated BMP receptors recognize and phosphorylate the R-Smads such as Smad1. As shown in Fig. 4A, treatment of human PASMCs with 200 nM BMP-2 rapidly increased the protein level of the phosphorylated Smad1 (pSmad); the maximal activation (or phosphorylation) of Smad1 took place 8 h after treatment. These results suggest that the phosphorylation or activation of Smad1 is an important step involved in downstream signal transduction in the BMP-2-mediated apoptotic effect on human PASMCs.



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Fig. 4. BMP-2-induced activation of Smad1 and downregulation of Bcl-2 expression in human PASMCs. Aa: Western blot analysis of the phosphorylated Smad1 (pSmad1) and {alpha}-actin in PASMCs before (Cont) and after treatment with 200 nM BMP-2 for 2, 4, 8, 24, and 36 h. Ab: summarized data (means ± SE) show the protein level of pSmad1 before (open bar) and after (solid bars) treatment with BMP-2 for 2, 4, 8, 24, and 36 h; *P < 0.05; **P < 0.01 vs. open bar (0 h in BMP-2 treatment). Ba: PCR-amplified products displayed in agarose gels for Bcl-2 (153 bp) and Bax (131 bp) in control PASMCs (-) and cells treated with 200 nM BMP2 (+) for 12 h. M, 1 kb plus DNA ladder. Bb: ratio of Bcl-2 to Bax mRNA levels in PASMC treated with (+) or without (-) BMP-2; **P < 0.01 vs. open bar. Ca: Western blot analysis of Bcl-2 and {alpha}-actin in PASMCs before (Cont) and after treatment with 200 nM BMP-2 for 12, 24, and 48 h. Cb: summarized data show the protein level of Bcl-2 (normalized to protein level of {alpha}-actin) before (Cont, open bar) and after (solid bars) treatment with BMP-2 for 12, 24, and 48 h; ***P < 0.001 vs. open bar (Cont).

 

It has been well documented that Bcl-2 is an antiapoptotic protein of the Bcl-2 protein family (1, 6) that inhibits apoptosis by preventing the release of cytochrome c (28, 62) and an apoptogenic protease (52) from mitochondria to the cytosol. To test whether BMP-2-induced apoptosis is related to Bcl-2, we examined the effects of BMP-2 on mRNA and protein expression levels of Bcl-2 in human PASMCs.

Incubation of PASMCs with 200 nM BMP-2 for 12 h significantly downregulated the mRNA expression of Bcl-2, but negligibly affected the mRNA expression of Bax (Fig. 4Ba), a proapoptotic protein of the Bcl-2 protein family (1). The ratio of Bcl-2 to Bax mRNA levels (Bcl-2/Bax) was reduced by ~20% (n = 8; P < 0.01) in human PASMCs treated with BMP-2 (Fig. 4Bb). Consistent with the inhibitory effect on mRNA expression, BMP-2 also downregulated the protein expression of Bcl-2 in human PASMCs (Fig. 4C). These results provide evidence that downregulated Bcl-2 expression may serve as a critical mechanism involved in BMP-2-mediated apoptosis in human PASMCs. The precise sequences of events involved in the BMP-2-mediated downregulation of Bcl-2 expression and how the phosphorylated Smad1 leads to inhibition of Bcl-2 gene expression are unclear.

Apoptotic effects of BMP-7 on human PASMCs. As shown in Fig. 1, BMP-7 transcript was not detectable in human PASMCs, whereas it was highly expressed in the lung tissues. This suggests that human PASMCs may not express BMP-7 endogenously (or the expression level is extremely low). Using annexin V staining (annexin V binds phosphatidylserine, a phosphoaminolipid that is externalized during apoptosis) that detects apoptosis earlier in the process than DNA-based assays (e.g., TUNEL; Ref. 40), we examined whether BMP-7, an exogenous BMP for human PASMCs, also induces apoptosis in these cells.

Under baseline conditions, 5.35 ± 1.34% of PASMCs were stained positive for annexin V. After treatment with 200 nM BMP-7 for 48 h, there was a significant increase in annexin V staining to 23.57 ± 4.35% (Fig. 5, A and B). The annexin V-positive cells exhibited the decreased cell size that is indicative of apoptotic cell shrinkage. Under baseline conditions, 7.98 ± 0.53% of the cells were in a size range of 50-260 AU; after treatment of the cells with BMP-7, 25.12 ± 8.79% of the cells were in this size range (Fig. 5C). Based on the dose-response curve shown in Fig. 5D (left), BMP-7 (200 nM for 48 h) induced apoptosis in human PASMCs with an EC50 of ~50 nM. The time course indicated that the minimal time required for BMP-7 (200 nM) to induce apoptosis in human PASMCs was between 12 and 24 h, and the time required to cause 50% of the maximal apoptotic effect was ~35 h (Fig. 5D, right). These results demonstrate that BMP-7, although not expressed in PASMCs, also induces apoptosis in human PASMCs.



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Fig. 5. Apoptotic effect of BMP-7 on human PASMCs. A: representative cell nuclei stained with DAPI and annexin-V in a control cell and a cell treated with 200 nM of BMP-7. Apoptotic cell was stained positively with annexin V (green). B: PASMCs stained with annexin V-FITC and propidium iodide (PI) were analyzed by flow cytometry. A scatter plot of PI (y-axis) vs. annexin V fluorescein (x-axis) indicates the alive cells (bottom-left quadrant), necrotic cells (top-left quadrant), end-stage apoptotic cells (top-right quadrant), and early apoptotic cells (bottom-right quadrant). Number of annexin V-positive apoptotic cells (top- and bottom-right quadrants) was increased from 7.44% in control cells to 23.57% in BMP-7-treated cells. C: treatment with BMP-7 (200 nM for 48 h) displays a shift of the cell population to lower values in the forward scatter scale (M1), which is indicative of cell shrinkage. D: dose-response curve (left) and time course (right) of BMP-7-induced PASMC apoptosis; n = 21 fields of cells from 3 coverslips for each data point; *P < 0.05; ***P < 0.001 vs. control cells (0 nM BMP-7 or 0 h in BMP-7 treatment). Baseline control levels of apoptosis in control cells are indicated (horizontal dashed lines).

 

BMP- and ST-induced apoptosis is inhibited in PASMCs from PPH patients. Intrinsic abnormalities of pulmonary vascular smooth muscle are present in PPH and may be important in its pathogenesis and etiology (3, 9, 15, 45, 55). In patients with pulmonary hypertension secondary to cardiopulmonary disease such as chronic obstructive pulmonary disease and interstitial pulmonary fibrosis, the elevated PAP may arise from different cellular and molecular mechanisms. To elucidate whether the programmed cell death in PASMCs is altered uniquely in PPH, we compared the apoptotic effects of BMPs and ST between PASMCs isolated from two patients with PPH and three patients with SPH, pulmonary hypertension secondary to idiopathic pulmonary fibrosis, emphysema, and lymphangioleiomyomatosis. The averaged mean PAP of the two PPH patients was 52.0 ± 1.4 mmHg, whereas the mean PAP of the three SPH patients was 36.5 ± 12.3 mmHg. Unlike the SPH patients, the PPH patients were treated with Flolan (prostacyclin) intravenously before lung transplantation.

As shown in Fig. 6A, both SPH and PPH patients exhibited significant pulmonary vascular wall thickening, and the overall thickness of pulmonary arterial wall was similar between SPH and PPH patients (Fig. 6B). The PASMCs prepared from these hypertrophied peripheral pulmonary arteries, which had been cultured and passaged for the same period of time before experimentation, had similar morphologies in SPH and PPH patients (Fig. 7). However, the growth rate (data not shown) and cytoplasmic free Ca2+ concentration (63) were significantly increased, whereas K+ channel activity was markedly reduced (63, 64), in PASMCs from PPH patients compared with SPH patients.



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Fig. 6. Histological examination of pulmonary arteries in lung tissues isolated from patients with secondary and primary pulmonary hypertension (SPH and PPH, respectively). A: muscular arteries seen in two SPH (left) and two PPH (right) patients demonstrate a similar degree of medial thickening as shown (hematoxylin eosin stain, original magnification x200). B: summarized data show the averaged thickness of pulmonary artery (PA) wall determined by arbitrary measurement of PA wall thickness on the histological slices in SPH and PPH patients.

 


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Fig. 7. Morphology and purity of human PASMCs in culture. A: phase-contrast photomicrographs show cultured PASMCs prepared from SPH and PPH patients. B: cultures that were stained with the smooth muscle {alpha}-actin antibody (red) and the nucleic acid dye, DAPI (green) show that all DAPI-positive cells cross-react with the {alpha}-actin antibody in both SPH- and PPH-PASMCs. Horizontal bars denote 20 µm.

 

To determine whether basal apoptosis and BMP-mediated apoptosis are different in PASMCs from SPH and PPH patients, we isolated PASMCs from each of the patients' lung tissues and plated the cells on coverslips in six-well petri dishes. In each of the following experiments, the percentage of the cells undergoing apoptosis either under baseline control conditions or after treatment with BMPs (BMP-2 and -7) was measured in at least 12 coverslips of PASMCs from each of the patients. To compare the basal apoptosis rate and BMP-mediated apoptosis between SPH and PPH patients, the percentage of the cells undergoing apoptosis was averaged, respectively, among all the PASMCs tested from the two PPH patients (PPH-PASMCs) and all the PASMCs tested from the three SPH patients (SPH-PASMCs).

In PASMCs isolated from these patients, the apoptotic nuclei under baseline conditions were found in 7.2 ± 0.8% (n = 32) of SPH-PASMCs and 4.3 ± 0.5% of PPH-PASMCs (n = 32; P < 0.01). Treatment of the cells with BMP-2 and -7 induced apoptosis in PASMCs from both SPH and PPH patients. In SPH-PASMCs, BMP-2 and -7 increased the percentage of apoptotic cells from 7.35 ± 1.08 to 27.85 ± 2.47% (a 2.79-fold increase) and from 9.11 ± 1.25 to 29.29 ± 2.28% (a 2.21-fold increase), respectively (Fig. 8, A and B, left). In PPH-PASMCs, BMP-2 and -7 increased the percentage of apoptotic cells from 4.73 ± 0.88 to 12.56 ± 1.29% (a 1.65-fold increase) and from 5.52 ± 0.73 to 14.03 ± 1.15% (a 1.54-fold increase), respectively (Fig. 8, A and B, left).



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Fig. 8. Comparison of apoptotic effects of BMP-2, BMP-7, and ST in PASMCs isolated from patients with SPH (gray bars) and PPH (solid bars). Cells were cultured in media that included vehicle (Cont). A: 200 nM BMP-2 with vehicle. B: 200 nM BMP-7 with vehicle. C: 20 nM ST with vehicle. Percentage (left) of cells undergoing apoptosis in control cells and cells treated with BMP-2, BMP-7, and ST were summarized from 14-15 experiments for each treatment. Normalized apoptotic effects of BMP-2, -7, and ST (to baseline control levels of apoptosis before treatment) are shown (right); **P < 0.01; ***P < 0.001 vs. Cont or SPH.

 

To test whether PASMC apoptosis is inhibited in PPH, we compared the BMP-2- and -7-induced apoptosis between SPH- and PPH-PASMCs by determining the net increase of apoptotic nuclei after treatment (i.e., the difference between the percentage of cells undergoing apoptosis before and after treatment). As shown in Fig. 8, A and B (right), BMP-2-mediated PASMC apoptosis was inhibited by 68% in PPH patients (24.49 ± 2.07 in SPH-PASMCs vs. 7.83 ± 1.51% in PPH-PASMCs; P < 0.001), and BMP-7-mediated apoptosis was inhibited by 58% in PPH-PASMCs (20.18 ± 2.99 in SPH-PASMCs vs. 8.50 ± 2.02% in PPH-PASMCs; P < 0.001). These results suggest that PASMCs isolated from PPH patients are more resistant to BMP-2 and -7 than cells isolated from SPH patients.

Furthermore, the ST-mediated apoptosis was also inhibited in PPH patients by 30.7% compared with cells from SPH patients (Fig. 8C). These results suggest that there are other apoptotic pathways that may be abnormal in PPH-PASMCs in addition to the BMP-mediated apoptotic pathway.

Downregulated BMP-RII in PASMCs from PPH patients. Mutations in the BMP-RII gene have been found in 20-25% patients with sporadic PPH (9, 10, 32, 34, 41). Using immunohistological approaches, Atkinson et al. (4) reported that protein expression of BMP-RII was markedly decreased in the peripheral lung of PPH patients. The downregulation of BMP-RII not only existed in the PPH patients that harbored BMP-RII gene mutations but also was found in the PPH patients without BMP-RII gene mutations (4). Consistent with these results, we also observed that mRNA expression of BMP-RII in PASMCs from PPH patients was much lower than that in PASMCs from SPH patients (Fig. 9, A and B). However, mRNA levels of BMP-RIa and -RIb appeared to be comparable in PASMCs from PPH and SPH patients (Fig. 9, A and B). These results suggest that reduced expression of BMP-RII protein in PPH patients (4) may be due to inhibited transcription of the BMP-RII gene in PASMCs, and that the downregulated or mutated BMP-RII may lead to dysfunction of BMP signaling (9, 46). Indeed, BMP-2-mediated activation or phosphorylation of Smad1 was significantly reduced in PASMCs from PPH patients (22.82 ± 9.05 AU) compared with cells from SPH patients (37.53 ± 6.51 AU; n = 5 experiments; P < 0.01 by paired t-test; Fig. 9C). The 39% reduction of BMP-2-mediated phosphorylation or activation of Smad1 in PPH-PASMCs suggests an inhibited BMP-RII function in PPHPASMCs. It is unclear, however, whether the inhibited BMP-RII function is due to mutation or downregulation of BMP-RII in these cells (4, 9, 38, 46).



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Fig. 9. mRNA expression of BMP-RII and the BMP-2-mediated Smad1 activation are inhibited in PASMCs from PPH patients. A: PCR-amplified products displayed in agarose gels for BMP receptors BMP-RIa (232 bp), -Ib (630 bp), and -II (694 bp) in PASMCs isolated from two PPH (left) and two SPH (right) patients. As control, mRNA levels of GAPDH in PASMCs from two SPH (1, 2) and two PPH (1, 2) patients were measured (bottom). M, 1 kb plus DNA ladder. B: summarized data normalized to the mRNA levels of GAPDH show the mRNA expression levels of BMP-RIa, -Ib, and -II in PASMCs isolated from PPH and SPH patients (n = 6 experiments); ***P < 0.001 vs. open bars. C: Western blot analysis (top) of the pSmad1 in PPH- and SPH-PASMCs before (C) and after (B) treatment with 200 nM BMP-2. Summarized data (bottom) show the BMP-2-mediated increase in pSmad1 protein level in PPH- and SPH-PASMCs; **P < 0.01 vs. open bar (SPH) determined by paired t-test.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Apoptosis is crucial for normal development and homeostasis of multicellular organisms. The programmed cell death plays an important role in cell number control in various tissues and organs by balancing cell growth and multiplication and by eliminatsistance (3, 43, 45, 50, 55). It has been demonstrated that increased PASMC proliferation and/or inhibited PASMC apoptosis both contribute to induce pulmonary vascular medial hypertrophy (3, 7, 8, 43, 50, 55). However, the precise mechanisms involved in the regulation of PASMC proliferation and apoptosis in PPH are still incompletely understood.

PASMC proliferation and apoptosis are regulated by many vasoactive agonists, growth factors, and cytokines (3, 9, 43, 50, 55). The TGF-{beta} family includes multifunctional peptides that are involved in the regulation of embryonic development and tissue homeostasis. The TGF-{beta} family members, which include TGF-{beta}s, activins, and BMPs, are secreted intercellular signaling molecules that regulate cell proliferation, differentiation, migration, and apoptosis (9, 22, 35-39, 41, 53, 56-61). Because mutations of the BMP-RII gene have been found in familial and sporadic PPH patients (10, 32, 34, 41), it is important to elucidate the physiological role of the BMP signaling system in the regulation of normal PASMC growth and apoptosis, and to define the difference of BMP-mediated effects on PASMCs from SPH and PPH patients.

Molecular identification of BMP signaling system in human PASMCs. BMPs, which belong to the large superfamily of TGF-{beta}, initiate several distinct signaling cascades by binding to two types of transmembrane serine-threonin kinase receptors, BMP-RI and -RII (29, 35-37, 44). The BMP signaling cascades play an important role in embryonal development and adult tissue homeostasis (5, 27). The well-recognized roles of TGF-{beta} superfamily members in regulating cell proliferation, differentiation, and apoptosis suggest that BMPs must be involved in regulating vascular smooth muscle cell proliferation and apoptosis under both physiological and pathological conditions (9, 22, 35-39, 41, 53, 56-61).

The results from this study show that 1) six types of BMP transcripts (BMP-1 through -6), three types of BMP receptor transcripts (BMP-RIa, -RIb, and -RII), and seven types of Smad transcripts (Smad1 through -7) were detected by using RT-PCR analysis in PASMCs from normal subjects; 2) BMP-7 was not detectable in PASMCs (prepared from either normal subjects or patients with SPH and PPH) but was highly expressed in lung tissues; and 3) three isoforms of TGF-{beta} transcripts (TGF-{beta}1, -{beta}2, and -{beta}3) and two types of TGF-{beta} receptor transcripts (TGF-{beta}-RI and -RII) were found in PASMCs. These results suggest that the BMP/TGF-{beta} signaling system may play an important role in the regulation of human PASMC growth and apoptosis.

Antiproliferative effect of BMPs involves induction of apoptosis. In human aortic smooth muscle cells, BMP-2 and -7 inhibit cell proliferation (11, 57) and increase cell differentiation (11), whereas BMP-2 inhibits rat vascular smooth muscle cell proliferation (39). Recently, Morrell and colleagues (38) demonstrated that TGF-{beta}1 and BMP-2, -4, and -7 at doses of 1-100 ng/ml inhibited [3H]thymidine incorporation in PASMCs from normotensive and SPH patients cultured in media that contained FBS. The antiproliferative effect of TGF-{beta}1 and BMP-2, -4, and -7 (which appeared to be independent of the doses ranging from 0.1 to 100 ng/ml) was significantly attenuated and even reversed to be proliferative in PASMCs from PPH patients (38).

Furthermore, compared with normotensive and SPH patients, the protein expression of BMP-RII in lung tissues (e.g., in pulmonary vascular endothelium and arterial smooth muscle) was markedly reduced in PPH patients with and without mutations of the BMP-RII gene (4). These results provide compelling evidence that BMPs and their receptors and downstream signal transduction are required for preventing normal PASMCs from overgrowth (i.e., hypertrophy and hyperplasia), which is important in maintaining the thin pulmonary vascular wall and low pulmonary vascular resistance under normal conditions. Mutation and/or downregulation of the BMP ligands and receptors as well as defects in the downstream signaling pathway would therefore enhance pulmonary vascular remodeling and increase pulmonary vascular resistance and PAP in patients with PPH (4, 9, 10, 32, 34, 41, 46).

Role of inhibited PASMC apoptosis in development of pulmonary vascular remodeling. The results from this study demonstrate that, in PASMCs isolated from normal subjects and SPH patients, 1) BMP-2 (which is highly expressed in PASMCs) and BMP-7 (which is not expressed or the expression level is extremely low in PASMCs) both significantly increased the percentage of the cells undergoing apoptosis at doses ranging from 100 to 200 nM; and 2) the BMP-2-mediated apoptosis in PASMCs was associated with activation or phosphorylation of Smad1 and with a marked inhibition of Bcl-2 expression. In PASMCs isolated from PPH patients, the BMP-2- and -7-mediated apoptosis as well as the ST-induced apoptosis were all significantly inhibited (by 30-68%) compared with PASMCs isolated from normal subjects and SPH patients. Furthermore, the mRNA expression of BMP-RII and the BMP-2-mediated phosphorylation or activation of Smad1 were markedly inhibited in PASMCs from PPH patients compared with cells from SPH patients. These results suggest that 1) an additional mechanism involved in the antiproliferative effect of BMPs is to induce apoptosis in normal human PASMCs when the cells are exposed to high doses of BMPs, and 2) the inhibited apoptosis in PASMCs from PPH patients may contribute to the initiation and/or progression of pulmonary vascular medial hypertrophy in these patients. It is also important to note that the SPH-PASMCs and PPH-PASMCs were derived from similarly sized vessels (the peripheral resistance pulmonary arteries with the diameter ranges from 300 to 500 µm) and yet responded differently to the apoptotic inducers (BMP-2, -7, and ST).

Apoptosis is a highly regulated process that eliminates unnecessary cells (14, 20, 26, 54) such as cells migrated into the vascular lumen and hypertrophied cells accumulated in the pulmonary vasculature (8, 43). Thus timing and location of cell death as well as cell growth and division must be precisely controlled to maintain the normal pulmonary vascular structure. Compared with PASMCs from normal subjects and patients with SPH, PASMCs from PPH patients exhibited a significant resistance to apoptotic inducers such as BMP-2, -7, and ST. The inhibited apoptosis in PPHPASMCs would thus at least in part contribute to the thickening of the pulmonary vascular wall that is observed in patients with PPH. Our histological data indicate that SPH and PPH patients both exhibited significant pulmonary vascular remodeling characterized by medial hypertrophy or vascular wall thickening in small pulmonary vessels. However, the BMP- and ST-mediated apoptosis was only inhibited in PPHPASMCs but not in SPH-PASMCs. These results suggest that pulmonary arterial medial hypertrophy is caused by different mechanisms in patients with SPH and PPH. A unique mechanism involving the BMP signaling pathway responsible for normal apoptosis in PASMCs is inhibited in PPH patients but not in SPH patients.

The PPH patients from whom we isolated PASMCs for this study were treated with Flolan (prostacyclin) before lung transplantation, whereas the SPH patients were not treated with Flolan but were treated with other drugs such as aspirin, citalopram, estrogen, or narcotic pain medicines. We cannot exclude the possibility that the difference of BMP-mediated apoptosis between SPH-PASMCs and PPH-PASMCs was related to the different treatment for SPH and PPH patients. It is, however, unlikely that the inhibited apoptosis in PPH-PASMCs was due to continuous treatment of the PPH patients with Flolan, since prostacyclin has been demonstrated to inhibit PASMC proliferation and cause regression of pulmonary arterial medial hypertrophy. If the decreased apoptotic effect of BMPs on PPH-PASMCs compared with SPH-PASMCs was due to the Flolan treatment, one would have to assume that prostacyclin inhibits PASMC apoptosis and/or attenuates BMP-mediated apoptosis. To our knowledge, prostacyclin has not been demonstrated to have antiapoptotic effects on vascular smooth muscle cells and to interfere with the BMP signaling system.

Whether the apoptosis rate is inhibited in vivo in pulmonary arteries from the PPH patients is unknown. Rabinovitch and colleagues have reported that the induction of PASMC apoptosis by suppression of metalloproteinase and tenascin-C expression results in regression of pulmonary medial hypertrophy and reduction of PAP (7, 8, 43). Taken together with our results shown in this study, we speculate that inhibition of apoptosis in PASMCs is involved in the development and progression of pulmonary arterial medial hypertrophy, whereas induction or enhancement of PASMC apoptosis may be targeted to develop therapeutic approaches for pulmonary vascular remodeling in patients with PPH. As shown in Fig. 8, an apoptotic response to BMP-2 or -7 (~15% or two- to threefold above the baseline) still existed in PPH-PASMCs. It is unclear whether this "residual" apoptosis induced by BMP-2 or -7 in PPH-PASMCs would be sufficient to remove unnecessary or "misguided" PASMCs in vivo and to prevent pulmonary medial hypertrophy in these patients.

Regulation of gene transcription by BMP signaling system. Binding of BMP ligands (e.g., BMP-2 and -7) to either of the receptors (BMP-RI or -RII) encourages the two types of receptors to dimerize with one another and to form a ligand-receptor complex. The activated BMP-RI phosphorylates the R-Smad proteins (e.g., Smad3, -5, and -8), which then dimerize with co-Smad (e.g., Smad4). The R-Smad and co-Smad complex translocates into the nucleus and controls transcription of the target genes that contain the Smad binding sequence (5'-CAGAC-3' and 5'-GTCTG-3') in their promoter (22, 35-37, 44, 48, 56). In addition to the R-Smads and co-Smads, vertebrates and humans also express the antagonistic Smads including Smad6 and -7 (18, 19, 23-25), which mediate negative feedback within TGF-{beta}/BMP signaling pathways and regulatory inputs from other pathways. The antagonistic Smads serve as an RSmad decoy to compete for the activated tyrosine kinase and therefore to inhibit activation of R-Smads (35). Smad6 is an antagonistic Smad that preferentially inhibits BMP signaling by blocking activation of Smad1, -5, or -8 (18, 23, 24).

In addition to activating gene transcription by binding onto the Smad-binding sequence in the promoter, increased Smads in the nucleus can form heterogeneous polymers with corepressors such as the homeodomain protein TGIF (59) and the two related proteins c-Ski and SnoN (2, 33, 51) to induce repression of target gene transcription (35, 48, 56). An additional inhibitor of Smads is the Smad-interacting protein-1 (35), a zinc-finger/homeodomain protein that interacts with Smad1 and -5 in mammalian cells and inhibits BMP-mediated effects.

Increased Smads in the nucleus can also form heterogeneous polymers with other transcription factors (e.g., activator protein-1) to mediate apoptosis (60). Furthermore, Smad proteins have been reported to interact with calmodulin (65), a Ca2+-sensitive protein in the cytosol. Overexpression of calmodulin inhibits Smad activation and attenuates the response of TGF-{beta}/BMP signal transduction (65), which suggests that an increase in cytoplasmic Ca2+ concentration may exert an inhibitory effect on the TGF-{beta}/BMP signaling pathway by activating calmodulin (13) and thereby attenuate TGF-{beta}/BMP-mediated apoptosis in human PASMCs.

Cellular mechanisms involved in BMP-induced apoptosis. It has been demonstrated that BMPs inhibit proliferation in rat (39) and human (11, 57) aortic smooth muscle cells and human PASMCs (38) and induce apoptosis in human PASMCs (this study), normal human lung epithelial cells (61), and HepB3 cell lines (60). The precise mechanisms by which BMP-2 and -7 induce apoptosis in human PASMCs are still unknown. Our study indicates that treatment of PASMCs from normal subjects with BMP-2 decreased Bcl-2 mRNA and protein expression. It is unknown whether and which of the nuclear corepressors are involved in the BMP-mediated downregulation of Bcl-2 gene transcription in PASMCs.

Bcl-2 is an antiapoptotic protein that attenuates apoptosis by 1) inhibiting cytochrome c and apoptogenic protease release (28, 52, 62); 2) blocking K+ channels (12); 3) maintaining Ca2+ in the sarcoplasmic and endoplasmic reticulum (21, 31); and 4) regulating proton flux in mitochondria (49). In lung tissues from sporadic and familial PPH patients, Geraci et al. (16) showed that the mRNA expression of Bcl-2 was up-regulated. The upregulated Bcl-2 gene transcription may be related to the mutations in the BMP-RII gene and/or dysfunction of BMP signaling. These results imply that modulation of Bcl-2 gene expression is a critical mechanism in directing human PASMCs to undergo proliferation or apoptosis (Fig. 10).



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Fig. 10. Schematic diagram depicts the proposed mechanism involved in BMP-induced apoptosis in human PASMCs. On ligand (BMPs) binding with BMP receptors, the two types of receptors (BMP-RI and -II) dimerize with one another and form a ligand-receptor complex. Type I receptor (BMP-RI) is phosphorylated and activated by the type II receptor (BMP-RII). Activated BMP-RI then phosphorylates and activates the receptor-activated Smads (R-Smads) such as Smad1, Smad5, and Smad8. On the way to the nucleus, the activated R-Smads associate with the co-Smads such as Smad4. In addition to R-Smads and co-Smads, there are antagonistic Smads such as Smad6 and Smad7, which inhibit ligand-induced activation of R-Smads and mediate negative feedback within BMP signaling pathways. In the nucleus, R-Smad/co-Smad complexes interact with DNA and are involved in transcriptional regulation of various target genes whose primer contains the Smad-binding element (5'-AGAC-3'). Smad1/Smad4 complex, in association with a different corepressor, may be involved in downregulating expression of Bcl-2, an antiapoptotic protein that blocks cytochrome c release from the mitochondrial intermembrane space to the cytosol and activates K+ channels in the plasma membrane. Downregulated Bcl-2 via the BMP/BMP-R/Smad1 signaling pathway may lead to an increase in cytosolic cytochrome c, which subsequently activates caspase-3 and leads to cell apoptosis. Downregulated Bcl-2 would also increase K+ efflux and cause the apoptotic cell shrinkage that is an early hallmark of apoptosis. [K+]cyt, cytosolic K+ concentration; AVD, apoptotic volume decrease; Cyt-c, cytochrome c; [Cyt-c]cyt, cytoplasmic cytochrome c content.

 

Taken together with the data from this study and from other investigators, we speculate that BMP-mediated apoptosis in PASMCs from normal subjects is at least in part due to downregulation of Bcl-2 expression, whereas decreased BMP-receptor signaling (e.g., in PPH patients who have BMP-RII mutations) may lead to the upregulation of Bcl-2 (16) and inhibition of cytochrome c- or caspase-9-dependent apoptosis in PASMCs. The subsequent decrease in the ratio of cell apoptosis to proliferation favors PASMC growth, thereby mediating pulmonary vascular medial hypertrophy (Fig. 10). It is unknown whether the PPH patients from whom we obtained PASMCs contain any mutations of BMP-RII as reported by Machado et al. (34). The resistance to BMP-induced apoptosis in PASMCs from the PPH patients suggests that reduced BMP-RII signaling and/or BMP-RII/Smad-mediated gene transcription play an important role in the pathogenesis of PPH in patients with or without mutations of the BMP-RII gene (4, 9, 10, 32, 34, 41).

Summary. In normal PASMCs, the antiproliferative effects of BMPs (e.g., BMP-2 and -7) involve the induction of apoptosis. The cellular mechanisms by which BMPs induce PASMC apoptosis involve phosphorylation of Smad1 and downregulation of Bcl-2 expression. In PASMCs from PPH patients, the basal apoptosis and the BMP-mediated apoptosis rates are both inhibited compared with PASMCs from SPH patients. Although they need to be verified by in vivo study, the results from these in vitro experiments suggest that inhibition of apoptosis in PASMCs may play a role in the development of pulmonary vascular remodeling in patients with PPH.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043, HL-64945, HL-66012, HL-69758, and HL-66941.


    ACKNOWLEDGMENTS
 
The authors thank Bethany R. Lapp and Nicole Elliot for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. X.-J. Yuan, Dept. of Medicine, UCSD Medical Center, 200 West Arbor Dr., San Diego, CA 92103-8382 (E-mail: xiyuan{at}ucsd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S. Zhang and I. Fantozzi contributed equally to this work. Back


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCLOSURES
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