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Published as doi: 10.1096/fj.07-8424com.
 (The FASEB Journal. 2007;21:2970-2979.)
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Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension

Hemal H. Patel*,1, Shen Zhang{dagger},1, Fiona Murray*,1, Ryan Y. S. Suda*, Brian P. Head*, Utako Yokoyama*, James S. Swaney*, Ingrid R. Niesman{ddagger}, Ralph T. Schermuly§, Soni Savai Pullamsetti§, Patricia A. Thistlethwaite||, Atsushi Miyanohara, Marilyn G. Farquhar{ddagger},{dagger}{dagger}, Jason X.-J. Yuan{dagger} and Paul A. Insel*,{ddagger},2

Departments of
* Pharmacology,

{dagger} Medicine,

{ddagger} Cellular and Molecular Medicine,

|| Surgery, and

Gene Therapy Program,

{dagger}{dagger} Pathology, University of California, San Diego, La Jolla, California, USA; and

§ University of Giessen Lung Center, Medical Clinic II/V, Giessen, Germany

2Correspondence: University of California, San Diego, Department of Pharmacology, 9500 Gilman Dr., BSB 3076, La Jolla, CA 92093-0636, USA. E-mail: pinsel{at}ucsd.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vasoconstriction and vascular medial hypertrophy, resulting from increased intracellular [Ca2+] in pulmonary artery smooth muscle cells (PASMC), contribute to elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Caveolae, microdomains within the plasma membrane, contain the protein caveolin, which binds certain signaling molecules. We tested the hypothesis that PASMC from IPAH patients express more caveolin-1 (Cav-1) and caveolae, which contribute to increased capacitative Ca2+ entry (CCE) and DNA synthesis. Immunohistochemistry showed increased expression of Cav-1 in smooth muscle cells but not endothelial cells of pulmonary arteries from patients with IPAH. Subcellular fractionation and electron microscopy confirmed the increase in Cav-1 and caveolae expression in IPAH-PASMC. Treatment of IPAH-PASMC with agents that deplete membrane cholesterol (methyl-ß-cyclodextrin or lovastatin) disrupted caveolae, attenuated CCE, and inhibited DNA synthesis of IPAH-PASMC. Increasing Cav-1 expression of normal PASMC with a Cav-1-encoding adenovirus increased caveolae formation, CCE, and DNA synthesis; treatment of IPAH-PASMC with siRNA targeted to Cav-1 produced the opposite effects. Treatments that down-regulate caveolin/caveolae expression, including cholesterol-lowering drugs, reversed the increased CCE and DNA synthesis in IPAH-PASMC. Increased caveolin and caveolae expression thus contribute to IPAH-PASMC pathophysiology. The close relationship between caveolin/caveolae expression and altered cell physiology in IPAH contrast with previous results obtained in various animal models, including caveolin-knockout mice, thus emphasizing unique features of the human disease. The results imply that disruption of caveolae in PASMC may provide a novel therapeutic approach to attenuate disease manifestations of IPAH.—Patel, H. H., Zhang, S., Murray, F., Suda, R. Y. S., Head, B. P., Yokoyama, U., Swaney, J. S., Niesman, I. R., Schermuly, R. T., Savai Pullamsetti, S., Thistlethwaite, P. A., Miyanohara, A., Farquhar, M. G., Yuan, J. X.-J., Insel, P. A. Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension.


Key Words: statins • calcium • primary pulmonary hypertension • secondary pulmonary hypertension


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PULMONARY ARTERIAL HYPERTENSION (PAH) is a disorder with multiple etiologies. Idiopathic PAH (IPAH), previously termed primary pulmonary hypertension (PPH), is a form of the disease associated with a mutation in bone morphogenetic protein type II receptor (BMPR2) in some patients (1 , 2) . Three predominant factors contribute to the pathophysiology of IPAH: vasoconstriction, cell proliferation resulting in vascular wall thickening, and thrombosis; however, the dominant factor in the pathophysiology of IPAH is vascular remodeling (e.g., proliferation, hypertrophy, migration, transformation, inflammatory mediator recruitment, matrix changes, and neovascularization) (3 4 5) . The prognosis of IPAH is poor, with untreated PAH leading to heart failure and death in 2–8 years (6) . Although new treatments have yielded some success in targeting cellular and molecular processes in IPAH (7) , the precise mechanisms that mediate the vascular remodeling characterizing this disorder remain poorly understood.

An emerging idea in signal transduction posits the existence of spatially organized complexes of signaling molecules in microdomains of the plasma membrane (8 , 9) . One such domain, lipid rafts, are cholesterol- and sphingolipid-enriched regions, a subset of which, caveolae, contains the protein caveolin (Cav), of which three isoforms have been identified (10 , 11) . Caveolins have scaffolding domains that anchor and regulate proteins (12 , 13) . Caveolin-1 and -2 (Cav-1 and -2) are expressed in multiple cell types, while caveolin-3 (Cav-3) is found primarily in striated (skeletal and cardiac) muscle and certain smooth muscle cells (14) . Caveolins are involved in multiple cellular processes including vesicular transport, cholesterol and calcium homeostasis (15 16 17) , and signal transduction (9 , 18) . Caveolins function as chaperones and scaffolds recruiting signaling molecules to caveolae to provide direct temporal and spatial regulation of signal transduction (9 , 19) . Caveolins can inhibit activity of signaling proteins by interaction of the caveolin scaffolding domain, with a caveolin binding motif present in many proteins found in caveolae, including eNOS and ERK1/2 (20 21 22) . Alternatively, caveolins can promote signaling via enhanced receptor-effector coupling or enhanced receptor affinity when caveolins are up-regulated or overexpressed (12 , 23) . This has led to the concept of a "caveolar paradox" in which caveolins may produce direct allosteric inhibition of molecules such as eNOS under basal conditions but facilitate increased signaling on agonist stimulation through compartmentation (12 , 24) .

Although mice with knockout of caveolin-1 (Cav-1) have PAH (25) and animal models of PAH show decreased caveolin-1 expression (26 , 27) , caveolin expression has not been assessed in individual pulmonary vascular cell types in patients with PAH. Since Cav-1 knockout mice used in previous studies involve animals with total body and lifetime elimination of Cav-1, it is dubious whether such animals mirror a tissue-selective disease such as IPAH (28) . It was recently suggested that Cav-1 contributes to the expression and localization of canonical-like transient receptor potential (TRPC) channels, which form store- and receptor-operated Ca2+ channels (SOC and ROC) (29) . IPAH-PASMC have up-regulated expression in TRPC channels (30) . Influx of Ca2+ through SOC (i.e., capacitative Ca2+ entry, or CCE) contributes to PASMC proliferation, hypertrophy of the pulmonary vascular wall, and increased pulmonary vascular resistance (31) . Thus, the elevated TRPC expression in human PASMC may be related to Cav-1 expression. In the current study we sought to 1) characterize the expression of caveolins and caveolae in PASMC of normal subjects and patients with IPAH and 2) determine the role of caveolae formation on morphological and functional activity of PASMC, in particular on CCE and cell proliferation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
A detailed description of the methods is presented in the Supplemental Materials section.

Demographic, clinical, and hemodynamic characteristics of patients
Tissue for immunoblot analysis was obtained from seven non-IPAH donors and seven IPAH patients undergoing lung transplantation. Formalin-fixed sections of lung were obtained from four donors and three IPAH patients in a study protocol approved by the Ethk-Kommission am Fachbereich Humanmedizin der Justus-Liebig-Universitaet Giessen of the University Hospital Giessen (Giessen, Germany) in accordance with national law and with Good Clinical Practice/International Conference on Harmonisation guidelines. Written informed consent was obtained from each individual patient or the patient's next of kin.

PASMC were isolated and cultured from PAH patients, all Caucasians, who underwent lung/heart transplantation. Studies were approved by the Institutional Review Board and the patients gave informed consent. The diagnosis of IPAH was based on the criteria of the National Registry on Primary Pulmonary Hypertension and was confirmed histologically. The mean pulmonary arterial pressure (PAP) of two IPAH patients (a 57-year-old woman and a 31-year-old man), from whom PASMCs were prepared and cultured, was 51 and 53 mmHg, respectively. Both patients had been treated with beraprost, warfarin, digoxin, and furosemide before transplantation.

Cell preparation and culture
PASMC from healthy individuals were purchased from Clonetics (San Diego, CA, USA) and used at passages 4–6 in parallel with primary cultured PASMCs studied at a similar passage number (detailed isolation protocol in Supplemental Material).

Immunoblot analysis
Proteins in whole cell lysates (5 µg) or cellular fractions were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted as described (32) .

Immunofluorescent microscopy and deconvolution image analysis
Whole lung tissue sections (3 µm) and PASMC from normal and IPAH patients were fixed in 4% formalin and assessed by immunofluorescent microscopy and deconvolution image analysis, as described (32) .

Electron microscopy (EM)
Cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at room temperature, postfixed in 1% OsO4 in 0.1 M cacodylate buffer (1 h) at room temperature, and embedded as monolayers as described previously (33) . Sections were stained in uranyl acetate and lead citrate and observed by EM. Cells for EM analysis were either untreated or treated with methyl-ß-cyclodextrin (MßCD, 5 mM, 2 h), cholesterol-loaded MßCD (5 mM, 2 h), lovastatin (10 µM, 24 h), adenovirus Cav-1 (72 h), or siRNA for Cav-1 (72 h). Random EM fields were taken from different platings of cells or after drug, adenovirus, or siRNA treatments (n=3). An average of 15 fields was studied for each treatment (by a person blinded to the treatment or cell type) to determine caveolae/µm membrane.

Measurement of [Ca2+]i
Capacitative calcium entry (CCE) was assessed in untreated or treated [MßCD (5 mM, 2 h), cholesterol plus MßCD (5 mM, 2 h), or lovastatin (10 µM, 24 h)] cells. Multiple cells were imaged in a single field; a peripheral cytosolic area from each cell was spatially averaged.

Cell proliferation assay
[3H]Thymidine incorporation was used to evaluate DNA synthesis as a measure of cell proliferation. PASMC were seeded (1x104 cells/well) in 24-well microplates, cultured in smooth muscle growth media (SMGM) for 48–72 h, then incubated in 5% FBS-SMGM in the absence or presence of lovastatin (24 h) and MßCD (2 h) with 1 µCi [3H]thymidine for at least 24 h. Liquid scintillation counting was used to quantitate incorporation of radioactivity into trichloroacetic acid-insoluble material.

Adenoviral Cav-1 overexpression and siRNA
An adenoviral construct for human Cav-1 (a gift from Dr. E. Rodriguez-Boulan, Cornell University, Ithaca, NY, USA) was used to overexpress Cav-1 in control PASMC. Cells were treated with the Cav-1-containing adenovirus or LacZ control adenovirus for 24 h (MOI=100). The expression of Cav-1 was suppressed in IPAH-PASMC using targeted siRNA. Cells were treated with 1.5 µg of siRNA using Gene Porter as the transfection reagent for 72 h. Transfection reagent and negative siRNA (scrambled sequence of similar length) served as controls.

Statistical analysis
Data are expressed as mean ± SE. Statistical analysis was performed using unpaired Student's t tests or ANOVA as indicated. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Caveolin expression in normal and IPAH-PASMC and formation of caveolae: impact of cholesterol-depleting agents
Immunoblotting revealed decreased expression of Cav-1 in whole lung prepared from IPAH patients, results consistent with those in animal models of PAH (Fig. 1 A) (26 , 27) . By contrast, we observed different results when we used immunohistochemistry to evaluate Cav-1 expression in formalin-fixed sections of lungs from non-IPAH and IPAH patients and, in parallel, assessed the expression of Cav-1 with {alpha}-smooth muscle actin ({alpha}SMA, a smooth muscle cell marker) or von Willebrand factor (vWF, an endothelial cell marker). Sections of lungs from IPAH patients revealed the expected hypertrophy of smooth muscle cells and constriction of the pulmonary arterial lumen; in addition, we found a prominent increase in Cav-1 expression in the smooth muscle cell layer, but not endothelial cell layer, of sections from IPAH patients compared with those from controls (Fig. 1B-D ). In tissue from controls, Cav-1 staining was localized almost exclusively to the endothelium, whereas in IPAH Cav-1 localized to the pulmonary vascular smooth muscle (Fig. 1D ). In subsequent studies we focused our efforts on Cav-1 expression in isolated PASMC.


Figure 1
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  		Figure 1. Expression of Cav-1 in normal and IPAH lungs and isolated PASMC. A) Immunoblot for Cav-1 expression of whole lung lysates. GAPDH served as a loading control. B) Representative immunohistochemical images of sections (3 µm) of lungs from normal and IPAH patients stained with {alpha}-smooth muscle actin ({alpha}-SMA) and Cav-1; colocalization is represented by yellow in the merged images. C) Bar graph shows percent colocalization of {alpha}-SMA and Cav-1 in images taken from normal (n=4) and IPAH patients (n=3). Cav-1 expression is elevated in smooth muscle cells from IPAH patients. D) Representative immunohistochemical images of sections of lungs from normal and IPAH patients stained for von Willebrand factor (vWF) and Cav-1; colocalization is represented by yellow in the merged images. *P < 0.01; L represents lumen.

Using mRNA analysis, we found that PASMC isolated from IPAH patients had much higher expression of Cav-1 mRNA compared with PASMC obtained from control patients (data not shown). Immunoblot analysis revealed an increase in the expression of Cav-1 protein (Fig. 2 A) in IPAH-PASMC compared with normal cells. To assess the impact of increased Cav-1 expression in IPAH-PASMC, we used sucrose density fractionation and EM analysis. Immunoblotting of cells subjected to fractionation documented enrichment of Cav-1 in fractions 4–5 (in which buoyant caveolae localize) of IPAH-, but not normal-PASMC (Fig. 2B ). EM revealed a 3-fold increased (per µm of membrane) expression of structures characteristic of caveolae, flask-like invaginations of the plasma membrane in IPAH-PASMC compared with normal PASMC (Fig. 2C ).


Figure 2
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  		Figure 2. Sucrose density gradient fractionation and electron microscopy analysis of normal and cholesterol depleted PASMC. A) Immunoblot analysis of Cav-1 protein expression in isolated PASMC. Cav-1 is increased in IPAH-PASMC compared with control PASMC. B) Sucrose density gradient fractionation, followed by immunoblot analysis for Cav-1. Buoyant caveolin-containing fractions are found at the 5–35% sucrose interface (fraction 4–5). IPAH-PASMC show enrichment of Cav-1 and higher expression than did control PASMC. C) Ultrastructure of the plasma membrane was assessed by EM. {blacktriangledown} indicates individual caveolae and * regions with multiple caveolae and increased vesicle formation. IPAH-PASMC have increased flask-like invaginations of the plasma membrane consistent with the morphology of caveolae. D) Membrane morphology was assessed in IPAH-PASMC after treatment with drugs that deplete cholesterol. MßCD and lovastatin disrupt membrane morphology and diminish the expression of caveolae. Lovastatin causes distortion and blebbing of the plasma membrane. No apparent changes in membrane structure were observed after treatment with cholesterol-loaded MßCD. The right panel is an enlarged inset of the area outlined in black on the left panel. White bar on left panel represents 1 µm and on right panel, 50 nm.

MßCD and lovastatin treatment results in altered Ca2+ influx and reduced cell proliferation
Caveolae possess caveolins and are enriched in cholesterol and sphingolipids (8) . To test whether depletion of cholesterol content would modify morphology (i.e., expression of caveolae) and cell function, we treated IPAH-PASMC with MßCD, which depletes membrane cholesterol (34 35 36) , or with the HMG-CoA reductase inhibitor lovastatin, which inhibits the rate-limiting step in cholesterol synthesis. Both treatments altered the morphology of IPAH-PASMC plasma membranes. MßCD treatment produced a loss of caveolae, a response not observed after treatment with cholesterol-loaded MßCD, a cyclodextrin not capable of removing cellular cholesterol (36 , 37) ; cells treated with lovastatin showed bleb formation and a decreased number of caveolae (Fig. 2D ).

As a measure of smooth muscle function, we assessed CCE, which we induced by passive depletion of intracellular Ca2+ stores with cyclopiazonic acid (CPA), and found that CCE was elevated in IPAH-PASMC compared with control PASMC. MßCD treatment decreased CCE in IPAH-PASMC, but treatment with cholesterol-loaded MßCD as a control for the effect of MßCD yielded CCE values similar to those of untreated cells (Fig. 3 A, B). Treatment with lovastatin had no effect on CCE in control PASMC (data not shown) but reduced CCE ~ 33% in IPAH-PASMC (Fig. 3A, B ). In addition, incubation with MßCD or lovastatin decreased proliferation of IPAH-PASMC, but this response was not observed in cells treated with cholesterol-loaded MßCD (Fig. 3C ).


Figure 3
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  		Figure 3. Modulation of capacitative Ca2+ entry and cell proliferation by MßCD and lovastatin. Capacitative Ca2+ entry (CCE) was induced in IPAH-PASMC by passive depletion of Ca2+ from the SR using cyclopiazonic acid (CPA) in Ca2+-free media, followed by restoration of extracellular Ca2+. Cells were treated with MßCD (5 mM, for 1 h), cholesterol-loaded MßCD (5 mM, 1 h), or lovastatin (10 µM, 24 h) before measuring CCE. A) Representative traces of [Ca2+]i change over time. The initial peak represents leakage of Ca2+ from SR stores and the second peak represents CCE. CCE is elevated in IPAH-PASMC compared with control PASMC. MßCD and lovastatin, but not cholesterol-loaded MßCD, have reduced CCE. B) Bar graph shows average results (mean±SE) for CCE. *P < 0.05 vs. IPAH-PASMC (untreated). C) DNA synthesis, assessed by [3H]-thymidine incorporation, was reduced by treatment with MßCD or lovastatin but not by cholesterol-loaded MßCD. Data are mean ± SE; *P < 0.05 vs. control.

Overexpression or inhibition of Cav-1 expression alters caveolae formation, CCE, and proliferation
Treatment of control PASMC with an adenoviral construct containing Cav-1 increased expression of Cav-1 (Fig. 4 A), expression of buoyant caveolin (Fig. 4B ), and increased formation of caveolae (4-fold increase/µm membrane vs. control) to levels found in IPAH-PASMC (Fig. 4C, D ). The increase in Cav-1 expression and formation of caveolae was associated with increased CCE (Fig. 4E, F ). Treatment with adenoviral Cav-1 also increased sarcoplasmic reticulum (SR) Ca2+content, as evidenced by increased Ca2+ release upon addition of CPA. No increase in CCE or release from SR occurred in adenoviral LacZ-transfected (control) PASMC. In addition, DNA synthesis ([3H]-thymidine incorporation) was increased in PASMC treated with the adenoviral Cav-1 construct (Fig. 4G ).


Figure 4
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  		Figure 4. Overexpression of Cav-1 in control PASMC increases caveolae, CCE, and DNA synthesis. Cav-1 was overexpressed using an adenoviral construct. A) Immunoblot analysis of Cav-1 expression in adenoviral LacZ (control) or Cav-1-treated PASMC after 24 h. B) Sucrose density gradient fractionation of Cav-1 expression. Overexpressed Cav-1 partitioned in buoyant fractions. C) Representative EM images of control (untreated), adenoviral LacZ-, or adenoviral Cav-1-treated PASMC reveal that adenoviral Cav-1 treatment increases formation of caveolae. White bar = 50 nm. D) Caveolae per µm membrane. Adenoviral Cav-1 treatment increased the number of caveolae/per µm of membrane to a level similar to that of IPAH-PASMC. *P < 0.01 vs. control and LacZ. E) Representative traces of time-dependent changes in [Ca2+]i. The initial peak represents leakage of Ca2+ from internal stores and the second peak represents CCE. Adenoviral Cav-1 treatment, but not the adenoviral LacZ control, increases CCE. F) Bar graph shows average results (mean±SE) for CCE in PASMC treated with adenoviral LacZ or adenoviral Cav-1 constructs. *P < 0.05 vs. LacZ. G) Cell proliferation ([3H]-thymidine incorporation) was increased in control PASMC treated with the adenoviral Cav-1 construct but not with adenoviral LacZ. *P < 0.05 vs. LacZ.

Treatment of IPAH-PASMC with three different siRNA constructs targeted to Cav-1 reduced expression of Cav-1 protein (Fig. 5 A); the greatest reduction was observed with siRNA-1, which also reduced the amount of buoyant Cav-1 (Fig. 5B ). Cells treated with siRNA-1 had a 3-fold decrease in the number of caveolae/µm membrane (Fig. 5C, D ) as well as reduced CCE (Fig. 5E, F ) and DNA synthesis (Fig. 5G ), results not observed with a transfection reagent control or with control siRNA. Thus, inhibition of expression of Cav-1 produced changes in cell morphology and function that largely normalized abnormalities observed in IPAH-PASMC.


Figure 5
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  		Figure 5. Knockdown of Cav-1 in IPAH PASMC decreases Cav-1 expression, caveolae formation, CCE, and DNA synthesis. A) Representative immunoblots and densitometric analysis of Cav-1 protein expression in IPAH-PASMC incubated 72 h with siRNA constructs. All three siRNA constructs reduced Cav-1 protein expression, with the largest reduction by siRNA-1, n = 5 independent treatments. B) Sucrose density gradient fractionation shows that treatment with siRNA for Cav-1 decreased Cav-1 expression in buoyant fractions. C) Representative EM images of untreated IPAH-PASMC, cells treated with negative siRNA or with Cav-1 siRNA-1. Treatment with Cav-1 siRNA-1 decreased expression of caveolae. White bar = 50 nm. D) Caveolae per µm membrane. Treatment with Cav-1 siRNA-1 decreased the number of caveolae/per µm of membrane. *P < 0.01 vs. control. E) Representative traces of time-dependent changes in [Ca2+]i. Knockdown of Cav-1 decreased CCE. No effects were observed in cells treated with control siRNA. F) Bar graph shows average results (mean±SE) for CCE in IPAH-PASMC treated with Cav-1 siRNA. *P < 0.05 vs. control. G) Cell proliferation ([3H]-thymidine incorporation) was decreased in IPAH-PASMC treated with Cav-1 siRNA compared with cells treated with control siRNA. *P < 0.05 vs. Neg siRNA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent efforts to understand the pathogenesis and to develop treatments for IPAH have emphasized increased proliferation of PASMC leading to vascular wall hypertrophy and increased vascular tone as possible therapeutic targets (38) . This "hyperproliferative" state has been linked to increased [Ca2+]i levels in PASMC (31) , but little is known regarding the precise molecular and cellular determinants. The data shown here involving assessment of mRNA, protein, ultrastructure, function, and molecular manipulation are all consistent with the idea that increased [Ca2+]i in PASMC from IPAH patients is linked to overexpression of Cav-1 and caveolae. We found overexpression of Cav-1 in tissue sections and in PASMC cultured from patients with IPAH. In addition, two different interventions (MßCD and lovastatin) that deplete cholesterol and perturb the structure of caveolae decreased CCE and proliferation of IPAH-PASMC. We observed similar effects in IPAH-PASMC treated with siRNA directed against Cav-1. By contrast, overexpression of Cav-1 in normal PASMC increased formation of caveolae (to levels comparable to those found in IPAH) and recapitulated the enhanced CCE observed in IPAH-PASMC. Taken together, these multiple complementary pieces of evidence strongly implicate the expression of Cav-1 and caveolae in the regulation of [Ca2+]i in IPAH-PASMC. The results suggest that statins (or perhaps other cholesterol-lowering agents) or therapies that reduce Cav-1 expression in PASMC may have therapeutic benefit for IPAH. However, such caveolae-targeted therapeutics must take into account the possibility of effects on other cell types; further data are needed to develop such a therapeutic approach.

How might the overexpression of Cav-1 and caveolae contribute to the hyperproliferative phenotype of IPAH? Certain neoplasms [i.e., prostate cancer (39 , 40) , bladder cancer (41) , adenocarcinomas (42) , esophageal squamous cell carcinomas (43) , and both benign and malignant smooth muscle tumors (44) ] have elevated Cav-1 expression. Timme et al. have shown an interplay between Cav-1 and c-Myc-induced apoptosis: the Cav-1 gene can be down-regulated by c-myc, and maintaining high levels of Cav-1 suppresses c-myc-induced apoptosis (45) . Cav-1 has also been shown to maintain active Akt in prostate cancer cells by inhibiting protein phosphatase activity, an additional mechanism that may contribute to cellular resistance to apoptosis (46) . Such findings imply that, in IPAH, Cav-1 overexpression may be antiapoptotic and/or block proapoptotic pathways. Mutations in Cav-3 have been linked to a pathological process (i.e., muscular dystrophy) (47 , 48) . The current results indicate that another caveolin, Cav-1, contributes to pathology and may serve as both a marker and therapeutic target in IPAH.

Cav-1-deficient mice, which lack Cav-1 and -2 expression and caveolae formation, develop pulmonary hypertension, right ventricular hypertrophy, as well as structural remodeling, vasculopathies, and hyperproliferation in the lung (25 , 49 , 50) . Recent data suggest that administration to rats of a cell-permeable Cav-1 peptide prevents monocrotaline-induced pulmonary hypertension (51) . Such findings contrast with our findings in human IPAH, implying that the latter disease is not easily approximated in animal models. It is likely that the Cav-1-knockout mice develop pulmonary hypertension secondary to other pathologies (such as cardiac or other effects; see ref. 28 , 52 ). Expression of Cav-1 and Cav-2 is decreased in the lungs of rats with SPH, including monocrotaline- and myocardial infarction-induced pulmonary hypertension (53 , 54) . Such data contrast with our results shown here for IPAH-PASMC, further demonstrating the difficulty of modeling the human disorder in studies with experimental animals. Limited data for patients with IPAH have utilized whole lung tissue and shown a reduction in caveolin mRNA (27) and protein (26) . When we analyzed tissue from whole lung, we, too, found a decrease in Cav-1 expression; however, assessment of specific cell types revealed a dramatic elevation of Cav-1 expression in vascular smooth muscle of IPAH patients with decreased expression in endothelial cells, the latter of which are likely the predominant contributors to analyses of caveolin expression in whole lung preparations. Our other data show that increased caveolin expression contributes to the altered smooth muscle cell function in patients with IPAH. One must therefore be cautious in extrapolating animal data (55) to a complex human disease such as IPAH, which may show cell-specific changes such as those we observe in the expression of caveolins.

In the current study we show that increased caveolar formation increases CCE and [Ca2+]i. [Ca2+]i is tightly regulated: a rise in [Ca2+]i is a trigger for vasoconstriction, and a stimulant of cell proliferation. [Ca2+]i also regulates numerous enzymes (56 , 57) . Depletion of Ca2+ from the SR leads to the opening of plasma membrane TRPC channels, thereby increasing Ca2+ influx (CCE), to refill the SR stores and allowing for sustained increase in cytoplasmic [Ca2+]. PASMC isolated from IPAH patients have higher [Ca2+]i levels than do normal cells (58) , likely attributable to up-regulation of TRPC that comprise the SOC. Increased expression of Cav-1 and caveolae in IPAH may contribute to this increased [Ca2+]i via up-regulation of TRPC expression and localization in caveolae (29) . Disruption of caveolae via MßCD and lovastatin, as used in our study, may displace components of the Ca2+ signaling machinery from close association with SR whose proximity is necessary for activation of SOC in a local microenvironment. Since Cav-1 also localizes with BMPR2 and nitric oxide synthase (59 60 61 62) , such interactions may also influence IPAH. The current findings provide complementary evidence from upward and downward manipulation of expression of Cav-1 to establish a molecular proof-of-principle for the role of enhanced Cav-1 expression and caveolae formation in altered Ca2+ handling and cellular responses in IPAH.

The therapeutic role of statins in PAH has been assessed in animals with hypoxia- and monocrotaline-induced pulmonary hypertension (63 , 64) , but such studies do not reveal the cellular and molecular mechanisms for beneficial responses. Although statins have pleiotropic effects (65) , the findings we obtained with PASMC imply that statins may act via cholesterol depletion and resultant decreased expression of caveolae; this mechanism will require further studies in animals that are administered such drugs. Our data showing that statins modulate CCE in IPAH-PASMC in parallel with altered membrane morphology and disruption of caveolae suggest that statins modify IPAH-PASMC physiology toward a nonproliferative phenotype linked to a decrease in [Ca2+]i.

Our results should be interpreted with certain limitations. Although the current data emphasize the increase in expression of Cav-1 in IPAH-PASMC, other data (not shown) indicate that expression of Cav-2 mRNA and protein are increased in IPAH-PASMC and that siRNA for Cav-1 also results in a down-regulation of Cav-2. The latter finding is consistent with reports in the literature indicating that Cav-1 is the dominant caveolin isoform that regulates cell physiology and that expression and localization of Cav-2 depend on the expression and localization of Cav-1 (49 , 66) . Such data suggest that Cav-1 is the more important isoform and are consistent with findings we obtained in siRNA and overexpression studies. In addition, we observed no expression of Cav-3 in either normal or IPAH-PASMC.

It is theoretically possible that the treatments administered to the IPAH patients we studied may have altered the expression of Cav-1 and caveolae; samples from untreated patients were not available to us. As noted above, it is possible that the beneficial effects we observed with statins are mediated independent of cholesterol reduction and, in turn, are independent of caveolae expression. Nevertheless, the current findings provide evidence for a previously unappreciated role of increased expression of Cav-1 and caveolae in the pathophysiology of IPAH and suggest that modifying their expression may have therapeutic benefit (Fig. 6 ).


Figure 6
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  		Figure 6. Schematic diagram depicting the role of Cav-1 in IPAH-PASMC. The diagram depicts the pathophysiology of IPAH as related to increased Cav-1 and caveolae in PASMC. Treatment with MßCD, Cav-1 siRNA, or statins shifts the balance toward a normal phenotype whereas overexpression of Cav-1 in normal PASMC leads to a hypertensive phenotype. TRPC, transient receptor potential channel.


   ACKNOWLEDGMENTS
 
This study was supported in part by grants from the National Institutes of Health (HL74625 to HHP; CA10076 to M.G.F.; HL66012 and HL64945 to J.X-J.Y.; HL66941 to P.A.I.), the Ellison Medical Foundation (to P.A.I.), American Heart Association (0630039N to H.H.P.), and American Lung Association (RT-9094-N to F.M.).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication March 13, 2007. Accepted for publication March 29, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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