help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on August 18, 2004, doi:10.1164/rccm.200312-1668OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200312-1668OCv1
170/10/1101    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kunichika, N.
Right arrow Articles by Yuan, J. X.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kunichika, N.
Right arrow Articles by Yuan, J. X.-J.
American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 1101-1107, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200312-1668OC

Original Article

Bosentan Inhibits Transient Receptor Potential Channel Expression in Pulmonary Vascular Myocytes

Naomi Kunichika, Judd W. Landsberg, Ying Yu, Hideki Kunichika, Patricia A. Thistlethwaite, Lewis J. Rubin and Jason X.-J. Yuan

Department of Medicine and Department of Surgery, University of California, San Diego, La Jolla, California

Correspondence and requests for reprints should be addressed to Jason X.-J. Yuan, M.D., Ph.D., Division of Pulmonary and Critical Care Medicine, University of California, San Diego, Medical Teaching Facility, Room 252, 9500 Gilman Drive, La Jolla, CA 92093-0725. E-mail: xiyuan{at}ucsd.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bosentan, a dual endothelin receptor blocker, has been used clinically to treat idiopathic pulmonary arterial hypertension (IPAH). However, the mechanism of its antiproliferative effect on pulmonary artery smooth muscle cells (PASMCs) remains unclear. A rise in cytoplasmic Ca2+ stimulates PASMC proliferation and the canonical transient receptor potential (TRPC) channels are an important pathway for Ca2+ entry during PASMC proliferation. Bosentan (20–50 µM) significantly inhibited endothelin-1– or platelet-derived growth factor (PDGF)–mediated PASMC growth and [3H]thymidine uptake. In PASMCs, endothelin-1 (1 µM) and PDGF (10 ng/ml) both upregulated protein expression of TRPC6, whereas bosentan markedly downregulated TRPC6 protein levels. Furthermore, TRPC6 expression in PASMCs from patients with IPAH was greater than in normal PASMCs, and the antiproliferative effect of bosentan was significantly enhanced in IPAH-PASMCs in comparison with normal PASMCs. These observations demonstrate that the antiproliferative effect of bosentan on PASMCs involves the downregulation of TRPC6 channels via a mechanism possibly independent of endothelin receptor blockade. The greater effect of bosentan on IPAH-PASMCs than on normal PASMCs suggests that increased TRPC6 expression and function may be involved in the overgrowth of PASMCs in patients with IPAH.

Key Words: Ca2+ channels • endothelin-1 • transient receptor potential cation channel

Idiopathic pulmonary arterial hypertension (IPAH), formerly referred to as primary pulmonary hypertension, is a progressive and fatal disease that predominantly affects women. Although the pathogenesis and etiology of IPAH remain unclear, pulmonary vascular remodeling (characterized by vascular intimal and medial hypertrophy) is the major cause for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with IPAH (1, 2). Pulmonary vascular medial hypertrophy is caused mainly by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). The search for the cause of the inappropriate PASMC proliferation in IPAH has yielded a heterogeneous spectrum of derangements including elevation of circulating mitogens (3–6), dysfunction of growth factor receptors (7–9), and abnormality of ion channels (10).

A rise in cytoplasmic Ca2+ concentration ([Ca2+]cyt) in PASMCs is an important stimulus for PASMC proliferation (11, 12). [Ca2+]cyt can be increased in PASMCs by Ca2+ release from the sarcoplasmic or endoplasmic reticulum and by Ca2+ influx from extracellular sites to the cytosol via Ca2+ channels in the plasma membrane. In human PASMCs, there are three classes of Ca2+ channels: voltage-dependent, receptor-operated, and store-operated Ca2+ channels (13–17). Receptor- and store-operated Ca2+ channels are important Ca2+ entry pathways in PASMCs, especially on activation of membrane receptors for growth factors and mitogenic agonists (14–18). The transient receptor potential (TRP) channel genes have been demonstrated to encode subunits that form functional receptor- and store-operated Ca2+ channels (17). In animal and human PASMCs, canonical TRP (TRPC) channels are involved in agonist-mediated pulmonary vasoconstriction (14) and mitogen-mediated cell proliferation (11, 12).

Endothelin-1 is a potent pulmonary vasoconstrictor and PASMC mitogen (3–6, 18–22). Its mechanism of action involves binding to endothelin receptors with the subsequent activation of phospholipase C, which causes hydrolysis of phosphatidylinositol and generation of cytosolic inositol trisphosphate and membrane-bound diacylglycerol. Inositol trisphosphate causes a rapid Ca2+ increase followed by a sustained Ca2+ influx to induce contraction. Diacylglycerol activates protein kinase C and triggers intracellular mechanisms that promote cell proliferation and migration through the mitogen-activated protein kinase cascade (21). Enhanced local production of endothelin-1 by pulmonary vascular endothelial cells correlates with the severity of pulmonary vascular remodeling, suggesting a causal role for endothelin-1 in the development of pulmonary arterial hypertension (3, 5, 22).

Bosentan, a potent antagonist of endothelin receptors, is an orally administered drug approved by the Food and Drug Administration for the treatment of pulmonary arterial hypertension. Randomized clinical trial studies show that treatment with bosentan significantly increased cardiac index, reduced pulmonary vascular resistance, decreased mean pulmonary arterial pressure, and increased exercise capacity in patients with severe pulmonary arterial hypertension (23–25). However, little is known about its in vitro mechanism of action. This study was designed to test the hypothesis that endothelin-1–mediated PASMC proliferation involves the upregulation of TRPC channel expression, and the therapeutic effect of bosentan is due partially to its downregulatory effect on TRPC channels. To test the specificity of the antiproliferative effects of bosentan, we also examined whether bosentan affected PASMC proliferation mediated by platelet-derived growth factor (PDGF), a mitogen that works independent of endothelin receptors. Some of the results of these studies have been previously reported in the form of an abstract (26).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Preparation and Culture
Human PASMCs from normal subjects were purchased from Cambrex Bio Science (Walkersville, MD) and used at Passage 4–6. The cells were cultured at 37°C in smooth muscle growth medium (SMGM), which is composed of smooth muscle basal medium (SMBM), 5% fetal bovine serum (FBS), human epidermal growth factor (0.5 ng/ml), human fibroblast growth factor (2 ng/ml), and insulin (5 µg/ml). PASMCs from transplant patients were used in some experiments (10). There were two patients with IPAH from whom lung tissues were obtained to prepare PASMCs for the study. The diagnosis of IPAH was established clinically in these patients on the basis of the criteria used in the National Institutes of Health Registry on Primary Pulmonary Hypertension and was confirmed histopathologically. The mean pulmonary arterial pressure of the two patients with IPAH (a 57-year-old white woman and a 32-year-old white man) was 66 and 53 mm Hg, respectively. Both of the patients with IPAH had been treated with epoprostenol (Flolan), warfarin, digoxin, and furosemide before lung transplantation. The patients from whom we obtained lung tissues for preparing PASMCs gave written informed consent: all protocols were approved by the Institutional Review Board of the University of California, San Diego (La Jolla, CA). Lung tissues, removed from patients in the operating room, were immediately placed in cold (4°C) saline and taken to the laboratory for dissection. Muscular pulmonary arteries were incubated in Hanks' balanced salt solution containing collagenase (2 mg/ml; Worthington Biochemical, Lakewood, NJ) for 20 minutes. The adventitia was stripped, and the endothelium was removed. The remaining smooth muscle was digested with collagenase (2.25 mg/ml), elastase (0.5 mg/ml), and albumin (1 mg/ml) (Sigma, St. Louis, MO) at 37°C to make a cell suspension of PASMCs. The cells were resuspended and then incubated in SMGM at 37°C.

Rat PASMCs were isolated from intrapulmonary arteries (Division 3 or 4) of male Sprague-Dawley rats (125–250 g) and cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing FBS (10%), penicillin (100 U/ml) and streptomycin (100 mg/ml). The cells were passaged by trypsinization with 0.05% trypsin–EDTA and used for experiments at passages 3–6 (12, 27).

Determination of Cell Growth
Cell number was determined with a hemocytometer. Cell counts in the four 1-mm3 corner squares of the hemocytometer were averaged to calculate total cell number per milliliter of cell suspension. Cell number, normalized by the size of the petri dishes (cells per centimeter squared), was used to compare cell growth rate. Cell viability was determined with 0.45% trypan blue (Sigma). [3H]Thymidine incorporation was determined to evaluate DNA synthesis. Briefly, PASMCs were seeded in 24-well microplates (at about 2 x 104 cells per well) and cultured in 10% FBS–DMEM (for rat PASMCs) or SMGM (for human PASMCs) for 24 hours before growth was arrested by incubating the cells in FBS-free DMEM or SMBM for 24 hours. Cells were then incubated in 0.2% FBS–DMEM or SMBM with or without PDGF (10 ng/ml) or endothelin-1 (1–10 µM) for 48 hours, with 1 µCi of [3H]thymidine added to the cells for the last 16 hours. Incorporation of radioactivity into trichloroacetic acid-insoluble material was measured with a liquid scintillation counter.

Western Blot Analysis
Cells were gently washed twice in cold phosphate-buffered saline, scraped into 0.3 ml of lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, phenylmethylsulfonyl fluoride [100 µg/ml], and aprotinin [30 µl/ml]), and incubated for 30 minutes on ice. The cell lysates were then sonicated and centrifuged at 12,000 rpm for 10 minutes, and the insoluble fraction was discarded. The protein concentration in the supernatant was determined by the bicinchoninic acid protein assay, using bovine serum albumin as a standard. Ten to 25 µg of proteins was mixed and boiled in sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer for 5 minutes. The protein samples separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis were then transferred to nitrocellulose membranes by electroblotting in a Mini Trans-Blot cell transfer apparatus according to the manufacturer's instructions (Bio-Rad, Hercules, CA). After incubation overnight at 4°C in a blocking buffer (0.1% Tween 20 in phosphate-buffered saline) containing 5% nonfat dry milk powder, the membranes were incubated with anti-actin monoclonal antibody (Sigma) and anti-TRPC6 polyclonal antibody (Alomone Labs, Jerusalem, Israel). The membranes were then washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG for 90 minutes at room temperature. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ).

Statistical Analysis
Data are expressed as means ± SD. Statistical analysis was performed by paired or unpaired Student t test, or by analysis of variance and post hoc tests (Student–Newman–Keuls) as indicated. Differences are considered to be significant when p < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Bosentan Inhibits Endothelin-1–mediated Human PASMC Proliferation
The mitogenic effect of endothelin was first examined in human PASMCs cultured in low-serum (1% FBS) medium. Endothelin-1 at concentrations of 10–7 and 10–6 M (for 48 hours) significantly increased cell number (by 22 and 31%, respectively) and [3H]thymidine uptake (by 36 and 41%, respectively) compared with a vehicle control (Figure 1A). Blockade of endothelin receptors with bosentan significantly inhibited endothelin-1–mediated PASMC proliferation (Figure 1B). In the presence of endothelin-1 (10–6 M), bosentan markedly inhibited endothelin-1–mediated increases in PASMC number and [3H]thymidine incorporation. The inhibitory effect of bosentan was dose dependent; 50 µM bosentan caused 60% inhibition of endothelin-1–mediated cell number increase and almost abolished endothelin-1–induced increase in [3H]thymidine uptake (Figure 1B).



View larger version (56K):
[in this window]
[in a new window]
  		Figure 1. Bosentan inhibits endothelin-1-mediated PASMC proliferation. (A) Summarized data (means ± SD) showing cell numbers (a, n = 9 experiments) before (Basal) and 48 hours after treatment of normal human PASMCs with 0 (solid column), 10–8, 10–7, and 10–6 M endothelin-1 (ET-1) (shaded columns), as well as [3H]thymidine incorporation (b, n = 6 experiments) 48 hours after treatment of cells with 0 (solid column), 10–8, 10–7, and 10–6 M endothelin-1. (B) Summarized data (means ± SD) showing cell numbers (a, n = 9 experiments) and [3H]thymidine uptake (b, n = 6 experiments) before (Basal) and 48 hours after incubation of human PASMCs in 1 µM endothelin-1-containing medium with 0 (solid column), 10, 20, and 50 µM bosentan (shaded columns). **p < 0.01, ***p < 0.001 versus solid columns.

 
To examine whether bosentan has any toxic effect on human PASMCs, we first incubated PASMCs in medium with or without 50 µM bosentan for 48 hours. We then measured the number of cells that were positively stained with 0.45% trypan blue. Of PASMCs incubated in medium without bosentan, the percentage of trypan blue-positive cells was 3.27%, whereas of cells cultured in medium containing 50 µM bosentan, the percentage of necrotic cells was 6.05% (p > 0.1). These results indicate that bosentan has little toxic effect on human PASMCs.

Bosentan Inhibits PDGF-mediated Human PASMC Proliferation
Increased levels of endothelin-1 and PDGF in lung tissues and blood plasma have been observed in patients and animals with pulmonary arterial hypertension (3–6, 28–30). To investigate whether bosentan interferes with other proliferative mechanisms (e.g., PDGF signaling pathway) in PASMCs, we examined the effect of bosentan on PDGF-mediated PASMC proliferation.

In rat PASMCs, PDGF (10 ng/ml in 0.2% FBS–DMEM, for 48 hours) caused an approximately 3-fold increase in cell number (p < 0.001). Interestingly, bosentan caused a dose-dependent inhibition of PDGF-mediated rat PASMC growth (Figure 2A). The PDGF-induced increase in cell number was reduced by 33, 39, and 73%, respectively, in PASMCs treated with 10, 20, and 50 µM bosentan. Furthermore, bosentan also significantly inhibited the PDGF-mediated increase in [3H]thymidine uptake (Figure 2A). The inhibitory effect of bosentan on PASMC growth was also replicated in normal human PASMCs (Figure 2B).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2. Bosentan inhibits platelet-derived growth factor (PDGF)-mediated pulmonary artery smooth muscle cell (PASMC) proliferation. Summarized data (means ± SD) from rat (A, panel a; n = 9 experiments) and human (B, n = 9 experiments) PASMCs showing cell numbers before (Basal) and 48 hours after incubation in PDGF (10 ng/ml)-containing medium with 0 (solid column), 10, 20, and 50 µM bosentan (shaded columns). [3H]thymidine uptake (A, panel b; n = 6 experiments) was measured before (Basal, open column) and 48 hours after incubation of cells in PDGF-containing medium in the absence or presence of bosentan (10 µM). **p < 0.01, ***p < 0.001 versus solid columns.

 
Many mechanisms are involved in vascular smooth muscle cell proliferation when cells are stimulated with mitogenic agonists (2, 31). In PASMCs, we previously reported that inhibition of both TRPC channel expression and function inhibited PASMC proliferation, suggesting an important role for Ca2+ influx through TRPC channels in mediating PASMC growth (11, 12, 27). The next set of experiments was designed to test whether the antiproliferative effect of bosentan is due to attenuation of TRPC channel expression.

Bosentan Inhibits Endothelin-1–mediated and PDGF-mediated Upregulation of TRPC6 in PASMCs
TRPC6 is a canonical TRP channel isoform highly expressed in lung tissues and vasculature (14–16, 32–34). TRPC6 participates in forming receptor- and store-operated Ca2+ channels that are activated by vasoconstrictive and mitogenic agonists (16). In human PASMCs, endothelin-1 (10–6 M, for 48 hours) significantly increased the protein expression level of TRPC6 (Figure 3A), whereas bosentan caused a dose-dependent inhibition of endothelin-1–mediated TRPC6 upregulation (Figure 3A). Similar to endothelin-1, PDGF also upregulated the protein expression of TRPC6 in human and rat PASMCs, whereas bosentan markedly downregulated TRPC6 expression. Bosentan not only abolished the PDGF-mediated increase in TRPC6 protein expression, but also decreased the protein level of TRPC6 to a level lower than the level in cells treated with vehicle (Figures 3B and 3C). These results suggest that (1) endothelin-1–mediated and PDGF-mediated PASMC proliferation is associated with upregulation of TRPC6, and (2) bosentan-induced TRPC6 downregulation may be an important mechanism by which it inhibits PASMC proliferation.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 3. Endothelin-1 and PDGF upregulate TRPC6 (transient receptor potential channel) protein expression and bosentan inhibits TRPC6 expression in PASMCs. (A) Western blot analysis (top) of TRPC6 and {alpha}-actin in human PASMCs before and 48 hours after treatment with 1 µM endothelin-1 (ET-1) in the absence (0 µM) or presence of 10, 20, or 50 µM bosentan. Summarized data (means ± SD, n = 3 experiments, bottom) showing the averaged protein level of TRPC6 (normalized to the protein level of {alpha}-actin) corresponding to the bands shown at the top. (B and C) Western blot analysis of TRPC6 and {alpha}-actin in human (B) and rat (C) PASMCs before and 48 hours after treatment with PDGF (10 ng/ml) in the absence (0 µM) or presence of 10, 20, or 50 µM bosentan. Summarized data (means ± SD, bar graphs) show the averaged protein level of TRPC6 (normalized to the protein level of {alpha}-actin) corresponding to the bands shown at the top (n = 4 experiments for human and rat PASMCs, respectively). *p < 0.05, ***p < 0.001 versus solid columns.

 
Effects of Ca2+ Channel Blockers on PDGF-mediated TRPC6 Upregulation and PASMC Proliferation
To investigate whether PDGF-mediated TRPC6 upregulation and PASMC proliferation depend on Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) or through receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs), we examined the effects of nifedipine (a specific blocker of VDCC) and SKF 96365 (SKF, a selective blocker of ROCs and SOCs) on TRPC6 expression and PASMC growth. As shown in Figure 4A, blockade of VDCCs with nifedipine or of ROCs and SOCs with SKF negligibly affected PDGF-induced TRPC6 upregulation. However, blockade of ROCs and SOCs with SKF markedly inhibited PDGF-induced PASMC proliferation, whereas blockade of VDCCs with nifedipine had little effect (Figure 4B). These data indicate that (1) PDGF-mediated TRPC6 upregulation is not dependent on Ca2+ influx through either VDCCs or ROCs and SOCs; and (2) PDGF-mediated PASMC proliferation is partially dependent on Ca2+ influx through the SKF-sensitive ROCs and SOCs. Because TRPC6 is an important subunit that participates in forming functional ROCs and SOCs, bosentan, by downregulating TRPC6 expression, had a similar inhibitory effect on PASMC proliferation in response to SKF (Figure 4B).



View larger version (49K):
[in this window]
[in a new window]
  		Figure 4. Effects of nifedipine and SKF 96365 on PDGF-mediated TRPC6 upregulation and PASMC proliferation. (A) Western blot analysis of TRPC6 and {alpha}-actin in human PASMCs before (Control) and 48 hours after treatment with PDGF (10 ng/ml) in the absence or presence of nifedipine (Nif, 1 µM) or SKF 96365 (SKF, 10 µM). (B) Normalized increase in cell numbers (means ± SE) by PDGF (10 ng/ml) in the absence (Cont) or in the presence of bosentan (50 µM), Nif (1 µM), SKF (10 µM), or bosentan plus Nif (n = 16 experiments). ***p < 0.001 versus Cont (solid column).

 
Bosentan Inhibits PDGF-mediated Phosphorylation of Signal Transducer and Activator of Transcription
On activation of PDGF receptor, a receptor tyrosine kinase, phosphorylation of signal transducer and activator of transcription-3 (STAT3) is an immediate downstream signal transduction step in the cascade of PDGF-mediated cell proliferation. Indeed, PDGF (10 ng/ml) increased the protein level of phosphorylated STAT3 (pSTAT3) in human PASMCs. Bosentan (50 µM), however, significantly inhibited PDGF-mediated STAT3 phosphorylation (Figure 5). These results indicate that bosentan, in addition to blockade of endothelin receptors, may also interfere with the kinase activity of PDGF receptors and thus inhibit PDGF-mediated STAT3 phosphorylation.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 5. Bosentan inhibits PDGF-mediated phosphorylation of STAT3. Western blot analysis of phosphorylated STAT3 (pSTAT) and {alpha}-actin in human PASMCs before (Control) and 48 hours after treatment with PDGF (10 ng/ml) in the absence or presence of bosentan (50 µM).

 
Bosentan-mediated Antiproliferative Effect Is Enhanced in IPAH-PASMCs
To examine whether the bosentan-mediated antiproliferative effect on PASMCs is different in patients with IPAH, we performed the same experiments using PASMCs from patients with IPAH. As shown in Figure 6, the inhibitory effect of bosentan on PDGF-mediated PASMC growth was much greater in cells from patients with IPAH than in cells from normal subjects. Furthermore, the protein level of TRPC6 was much higher in IPAH-PASMCs than in normal human PASMCs (Figure 7A). Incubation of IPAH-PASMCs with bosentan not only decreased the basal level of TRPC6 proteins, but also inhibited PDGF-mediated TRPC6 upregulation (Figures 7B and 7C). These results indicate that (1) upregulation of TRPC channels may play an essential role in PASMC overgrowth in patients with IPAH; (2) downregulation of TRPC6 channels is an effective mechanism for bosentan to attenuate overgrowth of IPAH-PASMCs; and (3) the TRPC6 channel is an important target in developing therapeutic approaches for IPAH.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 6. Bosentan-mediated antiproliferative effect on PASMCs is enhanced in IPAH. Dose–response curves of bosentan-induced changes in cell numbers in normal PASMCs and PASMCs from patients with IPAH. Growth-arrested cells were cultured in SMGM containing various concentrations of bosentan (0, 10, 20, and 50 µM) for 48 hours. Normalized increase in cell numbers (means ± SD) by PDGF (10 ng/ml) after treatment with bosentan shows that bosentan-induced inhibition on cell growth is significantly greater than in IPAH-PASMCs (n = 9 experiments) than in normal PASMCs (n = 9 experiments). **p < 0.01 versus normal PASMCs.

 


View larger version (38K):
[in this window]
[in a new window]
  		Figure 7. TRPC6 protein expression in PASMCs is upregulated in patients with IPAH. (A) Western blot analysis of TRPC6 and {alpha}-actin in PASMCs from normal subjects and PASMCs from patients with IPAH. (B) Western blot analysis of TRPC6 and {alpha}-actin in IPAH-PASMCs treated with or without bosentan (50 µM for 48 hours). (C) Western blot analysis (top) of TRPC6 in IPAH-PASMCs cultured in PDGF-containing medium in the absence (0 µM) or presence of 20 and 50 µM bosentan. Summarized data (means ± SD, n = 4 experiments, bar graph) showing the averaged protein level of TRPC6 (normalized to the protein level of {alpha}-actin) in IPAH-PASMCs with or without treatment of bosentan. **p < 0.01 versus solid columns.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pulmonary vascular wall thickening due to inappropriate PASMC proliferation is a central cause of the elevated pulmonary vascular resistance and arterial pressure in patients with IPAH (1, 2). Increased production of mitogenic agonists, such as endothelin-1 and PDGF in the blood plasma and lung tissues, has been linked to the development of pulmonary vascular remodeling regardless of the etiology (1–6, 21). The medical treatment of IPAH and severe pulmonary arterial hypertension has been revolutionized by the availability of new therapeutic agents. Bosentan, an orally administered endothelin receptor antagonist, has been shown to produce sustained improvements in pulmonary hemodynamics, 6-minute walk, and other measures of clinical outcome in patients with severe pulmonary arterial hypertension (23–25).

A rise in [Ca2+]cyt serves as a critical signal to promote gene expression and propel cells to go through the cell cycle. Mitogenic stimulation of PASMCs leads to elevated [Ca2+]cyt by triggering Ca2+ release from the sarcoplasmic reticulum and Ca2+ influx through receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs) (14–18, 32–38). Removal of extracellular Ca2+ and chelation of intracellular Ca2+ significantly inhibit PASMC proliferation in the presence of serum and growth factors (12, 27), suggesting that a constant Ca2+ influx or a sufficient level of [Ca2+] in the cytosol and intracellular organelles is an essential requisite for mitogen-mediated PASMC proliferation.

The canonical transient receptor potential (TRPC) channels are believed to participate in forming functional ROCs and SOCs in many cell types including PASMCs (11, 12, 14–17, 32–34). Indeed, downregulation of TRPC channels (e.g., TRPC1 and TRPC6) with antisense oligonucleotides inhibits receptor-mediated Ca2+ influx (16) and efficiently attenuates PASMC proliferation (11, 12, 27). It has been demonstrated that endothelin receptors are actually colocalized with TRPC channels in caveolae (15); therefore, TRPC channels serve as a critical effecter for the endothelin receptor in endothelin-mediated vasoconstrictive and mitogenic effect on vascular smooth muscle cells.

The data from this study show that bosentan, as a nonselective blocker of endothelin receptors (ETA and ETB), not only inhibits endothelin-1–mediated PASMC proliferation, but also attenuates PDGF-mediated PASMC proliferation (Figure 8). The antiproliferative effect of bosentan results, at least in part, from its downregulating effect on the TRPC6 channel, a TRPC channel subunit that forms function ROCs (16) and SOCs (12). Taken together, these data suggest that, in addition to blockade of endothelin receptors, downregulation of TRPC6 channel expression is another important mechanism by which bosentan inhibits PASMC proliferation. How bosentan decreases TRPC6 protein expression in PASMCs remains unclear; interference with STAT3 phosphorylation seems to be a potential mechanism by which bosentan inhibits PDGF-mediated TRPC6 upregulation. Given the fact that bosentan also inhibits inflammatory reactions and prevents development of fibrosis in animals with pulmonary inflammation (39) as well as other diseases (21), it is fair to speculate that the therapeutic action of bosentan may involve multiple targets and downstream signaling pathways that lead to the progression of pulmonary vascular remodeling in patients with IPAH.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 8. Potential mechanisms involved in the antiproliferative effect of bosentan on PASMCs. PDGF-mediated activation of tyrosine kinase receptors (TKRs) and endothelin-1-mediated activation of endothelin receptors (ETA and ETB) mediate upregulation of TRPC6 expression. The resultant increase in the activity of receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs) would increase [Ca2+]cyt or enhance agonist-mediated increase in [Ca2+]cyt, and stimulate cell proliferation. In addition to blockade of endothelin receptors and their downstream signal transduction pathway, bosentan may also directly downregulate TRPC6 expression by repressing the gene transcription or translation and/or interfering with the signaling mechanism (e.g., signal transducer and activator of transcription-3 [STAT3] phosphorylation) required for growth factor-mediated TRPC6 expression. DAG = diacylglycerol; PKC = protein kinase C.

 
In PASMCs from patients with IPAH, our data show that the protein expression level of TRPC6 channels is much higher than in PASMCs from normal subjects. Interestingly, the antiproliferative effect of bosentan is also greater in IPAH-PASMCs than in normal PASMCs; this may be due to the abnormally upregulated TRPC6 in PASMCs from patients with IPAH. It must be emphasized that this study is limited to PASMCs from a limited number of patients, with no data allowing for pathological, hemodynamic, or clinical correlations.

TRPC6 is highly expressed in the lung and vascular smooth muscles relative to other organs (e.g., heart and kidney) and tissue types (32, 33), suggesting that TRPC6 is a critical Ca2+ channel involved in regulating [Ca2+]cyt in pulmonary vascular smooth muscle and endothelial cells (40, 41). Indeed, inhibition of TRPC6 channel expression with antisense oligonucleotides attenuates, whereas overexpression of TRPC6 channels enhances, agonist- or mitogen-mediated increases in [Ca2+]cyt (16) as well as store depletion-mediated increases in [Ca2+]cyt (12). These data suggest that whether the TRPC6 channel is a ROC or SOC may depend on its distribution (i.e., cell or tissue specific), and on the type of TRPC isoforms with which it forms heterotetramers (17, 42). Regardless of whether it forms either a ROC or SOC, TRPC6 is a critical Ca2+-permeable channel in regulating [Ca2+]cyt; its function and expression may play a critical role in PASMC proliferation. Overexpression of TRPC6 channels may contribute to the development or progression of pulmonary vascular remodeling in patients with IPAH. Drugs that specifically inhibit TRPC6 channel function and expression would enhance the efficacy of treatment of these patients (especially those who do not respond to conventional Ca2+ channel blockers and vasodilators).

Pulmonary vascular remodeling is caused by a heterogeneous constellation of genetic derangements, which ultimately give rise to a common pathologic event, PASMC overgrowth (1). These derangements can be broadly classified on the basis of the mechanisms of action. For example, mutations of the bone morphogenetic protein receptor Type II gene (BMPR2) are ultimately linked to increased PASMC proliferation and decreased PASMC apoptosis (7–9), whereas enhanced angiopoietin-1 expression is associated, via pulmonary vascular endothelial cells, with elevated levels of serotonin, a potent vasoconstrictor and mitogen (43, 44). At present, we do not know whether the proliferative effect of angiopoietin-1 and the proapoptotic or antiproliferative effect of bone morphogenetic proteins directly involve regulation of TRPC6 channel expression and function. Nevertheless, it is plausible that TRPC6 or other TRPC channels serve as a downstream effecter in mediating PASMC proliferation in patients with IPAH who have mutant BMPR2 and overexpressed angiopoietin-1.

In summary, bosentan potently inhibits mitogen-mediated PASMC growth. The bosentan-induced antiproliferative effect on normal and IPAH-PASMCs is, at least in part, due to downregulation of TRPC6 protein expression. These observations raise the hypothesis that the antiproliferative effect of bosentan on PASMCs involves the downregulation of TRPC6 channels, possibly independent of endothelin receptor blockade. These findings suggest that inhibition of TRPC protein expression in PASMCs is an efficacious downstream target in the development of new therapeutic approaches for the treatment of pulmonary arterial hypertension.


   Acknowledgments
 
The authors are grateful to Ann Nicholson, M.S., for excellent technical assistance; Donna Tigno, M.D., for contributing to the collection of demographic and hemodynamic data from patients; Timothy Morris, M.D., for providing bosentan; and Gordon Yung, M.D., and Jolene Kriett, M.D., for obtaining lung tissue samples from patients undergoing transplantation.


   FOOTNOTES
 
Supported by the National Heart, Lung, and Blood Institute (National Institutes of Health grants HL 66012, HL 64945, HL 54043, and HL 66941).

Conflict of Interest Statement: N.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.W.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; Y.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; P.A.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; L.J.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.X.-J.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 8, 2003; accepted in final form August 16, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Runo JR, Loyd JE. Primary pulmonary hypertension. Lancet 2003;361:1533–1544.[CrossRef][Medline]
  2. Giaid A, Yanagisawa M, Langleben D, Michel RP, Levy R, Shennib H, Kimura S, Masaki T, Duguid WP, Stewart DJ. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 1993;328:1732–1739.[Abstract/Free Full Text]
  3. Stenmark KR, Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 1997;59:89–144.[CrossRef][Medline]
  4. Berg JT, Breen EC, Fu Z, Mathieu-Costello O, West JB. Alveolar hypoxia increases gene expression of extracellular matrix proteins and platelet-derived growth factor-B in lung parenchyma. Am J Respir Crit Care Med 1998;158:1920–1928.[Abstract/Free Full Text]
  5. Kourembanas S, Marsden PA, McQuillan LP, Faller DV. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J Clin Invest 1991;88:1054–1057.
  6. Santos S, Peinado VI, Ramirez J, Morales-Blanhir J, Bastos R, Roca J, Rodriguez-Roisin R, Barbera J. Enhanced expression of vascular endothelial growth factor in pulmonary arteries of smokers and patients with moderate chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2002;167:1250–1256.
  7. Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, et al. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet 2000;67:737–744.[CrossRef][Medline]
  8. Runo JR, Vnencak-Jones CL, Prince M, Loyd JE, Wheeler L, Robins IM, Lane KB, Newman JH, Johnson J, Nichols WC, et al. Pulmonary veno-occlusive disease caused by an inherited mutation in bone morphogenetic protein receptor II. Am J Respir Crit Care Med 2002;167:889–894.
  9. Atkinson C, Stewart S, Upton PD, Machado R, Thomson JR, Trembath RC, Morrell NW. Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation 2002;105:1672–1678.[Abstract/Free Full Text]
  10. Yuan JX-J, Aldinger AM, Juhaszova M, Wang J, Conte JV Jr, Gaine SP, Orens JB, Rubin LJ. Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation 1998;98:1400–1406.[Abstract/Free Full Text]
  11. Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ, Yuan JX-J. Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol 2001;280:H746–H755.[Abstract/Free Full Text]
  12. Yu Y, Sweeney M, Zhang S, Platoshyn O, Landsberg J, Rothman A, Yuan JX-J. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol 2003;284:C316–C330.[Abstract/Free Full Text]
  13. Yuan X-J. Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary artery myocytes. Circ Res 1995;77:370–378.[Abstract/Free Full Text]
  14. Ng LC, Gurney AM. Store operated channels mediate Ca2+ influx and contraction in rat pulmonary artery. Circ Res 2001;89:923–929.[Abstract/Free Full Text]
  15. Bergdahl A, Gomez MF, Dreja K, Xu S-Z, Adner M, Beech DJ, Broman J, Hellstrand P, Sward K. Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TRPC1. Circ Res 2003;93:839–847.[Abstract/Free Full Text]
  16. Inoue R, Okada T. Onoue H, Hara Y, Shimizu S, Naitoh S, Ito Y, Mori Y. The transient receptor potential protein homologue TRP6 is the essential component of vascular {alpha}1-adrenoceptor-activated Ca2+-permeable cation channel. Circ Res 2001;88:325–332.[Abstract/Free Full Text]
  17. Clapham DE, Runnels LW, Strubing C. The TRP ion channel family. Nat Rev Neurosci 2001;2:387–396.[Medline]
  18. Hyvelin JM, Guibert C, Marthan R, Savineau JP. Cellular mechanisms and role of endothelin-1-induced calcium oscillations in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 1998;275:L269–L282.[Abstract/Free Full Text]
  19. Davie N, Haleen SJ, Upton PD, Polak JM, Yacoub MH, Morrell NW, Wharton J. ETA and ETB receptors modulate the proliferation of human pulmonary artery smooth muscle cells. Am J Respir Crit Care Med 2002;165:398–405.[Abstract/Free Full Text]
  20. Yang Z, Krasnici N, Luscher TF. Endothelin-1 potentiates human smooth muscle cell growth to PDGF: effects of ETA and ETB receptor blockade. Circulation 1999;100:5–8.[Abstract/Free Full Text]
  21. Remuzzi G, Perico N, Benigni A. New therapeutics that antagonize endothelin: promises and frustrations. Nat Rev Drug Discov 2002;1:986–1001.[CrossRef][Medline]
  22. Wort SJ, Woods M, Warner TD, Evans TW, Mitchell JA. Endogenously released endothelin-1 from human pulmonary artery smooth muscle promotes cellular proliferation: relevance to pathogenesis of pulmonary hypertension and vascular remodeling. Am J Respir Cell Mol Biol 2001;25:104–110.[Abstract/Free Full Text]
  23. Channick RN, Simonneau G, Sitbon O, Robbins IM, Front A, Tapson VF, Badesch DB, Roux S, Rainisio M, Bodin F, et al. Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hyperyension: a randomized placebo controlled study. Lancet 2001;358:1119–1123.[CrossRef][Medline]
  24. Sitbon O, Badesch DB, Channick RN, Front A, Robbins IM, Simonneau G, Tapson VF, Rubin LJ. Effects of the dual endothelin receptor antagonist bosentan in patients with pulmonary arterial hypertension: a 1-year follow-up study. Chest 2003;124:247–254.[Abstract/Free Full Text]
  25. Rubin LJ, Badesch DB, Barst RJ, Galie N, Black CM, Keogh A, Pulido T, Frost A, Roux S, Leconte I, et al. Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 2002;336:111–117.
  26. Kunichika N, Yu Y, Thistlethwaite PA, Rubin LJ, Yuan JX-J. Bosentan inhibits human pulmonary vascular smooth muscle cell proliferation by downregulating TRPC6 expression [abstract]. Am J Respir Crit Care Med 2003;167:A1003.
  27. Sweeney M, Yu Y, Platoshyn O, Zhang S, McDaniel SS, Yuan JX-J. Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol 2002;283:L144–L155.[Abstract/Free Full Text]
  28. Cacoub P, Dorent R, Maistre G, Nataf P, Carayon A, Piette C, Godeau P, Cabrol C, Gandjbakhch I. Endothelin-1 in primary pulmonary hypertension and the Eisenmenger syndrome. Am J Cardiol 1993;71:448–450.[CrossRef][Medline]
  29. Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, Dewit D, Yanagisawa M. ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 1994;78:473–485.[CrossRef][Medline]
  30. Humber M, Monti G, Fartoukh M, Magnan A, Brenot F, Rain B, Capron F, Galanaud P, Duroux P, Simonneau G, et al. Platelet-derived growth factor expression in primary pulmonary hypertension: comparison of HIV seropositive and HIV seronegative patients. Eur Respir J 1998;11:554–559.[Abstract]
  31. Berk BC. Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 2001;81:999–1030.[Abstract/Free Full Text]
  32. Walker RL, Hume JR, Horowitz B. Differential expression and alternative splicing of TRP channel genes in smooth muscles. Am J Physiol Cell Physiol 2001;280:C1184–C1192.[Abstract/Free Full Text]
  33. Boulay G, Zhu X, Peyton M, Jian M, Hurst R, Stefani E, Birnbaumer L. Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J Biol Chem 1997;272:29672–29680.[Abstract/Free Full Text]
  34. Doi S, Damron DS, Horibe M, Murray PA. Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2000;278:L118–L130.[Abstract/Free Full Text]
  35. Hardingham GE, Chawla S, Johnson CM, Bading H. Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression. Nature 1997;385:260–265.[CrossRef][Medline]
  36. Berridge MJ. Calcium signaling and cell proliferation. Bioessays 1995;17:491–500.[CrossRef][Medline]
  37. Zhang WM, Yip KP, Lin MJ, Shimoda LA, Li WH, Sham JS. ET-1 activates Ca2+ sparks in PASMC: local Ca2+ signaling between inositol trisphosphate and ryanodine receptors. Am J Physiol Lung Cell Mol Physiol 2003;285:L680–L690.[Abstract/Free Full Text]
  38. Shimoda LA, Sylvester JT, Sham JSK. Mobilization of intracellular Ca2+ by endothelin-1 in rat intrapulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2000;278:L148–L156.[Abstract/Free Full Text]
  39. Muller DN, Mervaala EM, Schmidt F, Park JK, Dechend R, Genersch E, Breu V, Loffler BM, Ganten D, Schneider W, et al. Effect of bosentan on NF-{kappa}B, inflammation, and tissue factor in angiotensin II-induced end-organ damage. Hypertension 2000;36:282–290.[Abstract/Free Full Text]
  40. Fantozzi I, Zhang S, Platoshyn O, Remillard CV, Cowling RT, Yuan JX-J. Hypoxia increases AP-1 binding activity by enhancing capacitative Ca2+ entry in human pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 2003;285:L1233–L1245.[Abstract/Free Full Text]
  41. Cioffi DL, Wu S, Stevens T. On the endothelial cell ISOC. Cell Calcium 2003;33:323–336.[CrossRef][Medline]
  42. Hofmann T, Schaefer M, Schultz G, Gudermann T. Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci USA 2002;99:7461–7466.[Abstract/Free Full Text]
  43. Du LL, Sullivan CC, Chu D, Cho AJ, Kido M, Wolf PL, Yuan JX-J, Deutsch R, Jamieson SW, Thistlethwaite PA. Signaling molecules in nonfamilial pulmonary hypertension. N Engl J Med 2003;348:500–509.[Abstract/Free Full Text]
  44. Sullivan CC, Du L, Chu D, Cho AJ, Kido M, Wolf PL, Jamieson SW, Thistlethwaite PA. Induction of pulmonary hypertension by an angiopoietin1/TIE2/serotonin pathway. Proc Natl Acad Sci USA 2003;100:12331–12336.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
W. Lu, J. Wang, L. A. Shimoda, and J. T. Sylvester
Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia
Am J Physiol Lung Cell Mol Physiol, July 1, 2008; 295(1): L104 - L113.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Nishida, N. Onohara, Y. Sato, R. Suda, M. Ogushi, S. Tanabe, R. Inoue, Y. Mori, and H. Kurose
G{alpha}12/13-mediated Up-regulation of TRPC6 Negatively Regulates Endothelin-1-induced Cardiac Myofibroblast Formation and Collagen Synthesis through Nuclear Factor of Activated T Cells Activation
J. Biol. Chem., August 10, 2007; 282(32): 23117 - 23128.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
B. Nilius, G. Owsianik, T. Voets, and J. A. Peters
Transient Receptor Potential Cation Channels in Disease
Physiol Rev, January 1, 2007; 87(1): 165 - 217.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. W. Bush, D. B. Hood, P. J. Papst, J. A. Chapo, W. Minobe, M. R. Bristow, E. N. Olson, and T. A. McKinsey
Canonical Transient Receptor Potential Channels Promote Cardiomyocyte Hypertrophy through Activation of Calcineurin Signaling
J. Biol. Chem., November 3, 2006; 281(44): 33487 - 33496.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. Inoue, L. J. Jensen, J. Shi, H. Morita, M. Nishida, A. Honda, and Y. Ito
Transient Receptor Potential Channels in Cardiovascular Function and Disease
Circ. Res., July 21, 2006; 99(2): 119 - 131.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
D. F. Alvarez, J. A. King, and M. I. Townsley
Resistance to Store Depletion-induced Endothelial Injury in Rat Lung after Chronic Heart Failure
Am. J. Respir. Crit. Care Med., November 1, 2005; 172(9): 1153 - 1160.
[Abstract] [Full Text] [PDF]

Home page
 Sci SignalHome page
B. Nilius, T. Voets, and J. Peters
TRP Channels in Disease
Sci. Signal., August 2, 2005; 2005(295): re8 - re8.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
T. D. Bradley, Y. E. Miller, F. J. Martinez, D. C. Angus, W. MacNee, and E. Abraham
Interstitial Lung Disease, Lung Cancer, Lung Transplantation, Pulmonary Vascular Disorders, and Sleep-disordered Breathing in AJRCCM in 2004
Am. J. Respir. Crit. Care Med., April 1, 2005; 171(7): 675 - 685.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200312-1668OCv1
170/10/1101    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kunichika, N.
Right arrow Articles by Yuan, J. X.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kunichika, N.
Right arrow Articles by Yuan, J. X.-J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2004 American Thoracic Society