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
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ABSTRACT |
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hypertension; arteries; Smad; Bcl; transforming growth factor-
Bone morphogenetic proteins (BMPs) are signaling molecules that belong to
the transforming growth factor- (TGF-
) 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-
, 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.
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MATERIALS AND METHODS |
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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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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--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 -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
-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-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.
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RESULTS |
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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- (TGF-
1, -
2, and
-
3) and two types of TGF-
receptors (TGF-
-RI and
-RII; Fig. 2). The mRNA
expression levels of TGF-
and TGF-
receptors in human PASMCs were
also comparable to those in human lung tissues.
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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-1,
and Fas ligand on human PASMCs. It has been demonstrated that low doses
(10-100 nM) of BMPs and TGF-
s inhibited human PASMC proliferation in the
presence of low concentrations of serum
(38). To test whether the
antiproliferative effects of BMPs and TGF-
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-
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-
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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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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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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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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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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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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DISCUSSION |
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PASMC proliferation and apoptosis are regulated by many vasoactive
agonists, growth factors, and cytokines
(3,
9,
43,
50,
55). The TGF- family
includes multifunctional peptides that are involved in the regulation of
embryonic development and tissue homeostasis. The TGF-
family members,
which include TGF-
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-, 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-
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- transcripts
(TGF-
1, -
2, and -
3) and two
types of TGF-
receptor transcripts (TGF-
-RI and -RII) were found
in PASMCs. These results suggest that the BMP/TGF-
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-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-
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-/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-/BMP signal transduction
(65), which suggests that an
increase in cytoplasmic Ca2+ concentration may exert an
inhibitory effect on the TGF-
/BMP signaling pathway by activating
calmodulin (13) and thereby
attenuate TGF-
/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).
|
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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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