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(Chest. 2006;129:696-705.)
© 2006 American College of Chest Physicians

Loss of Caveolin and Heme Oxygenase Expression in Severe Pulmonary Hypertension*

Rosane O. D. Achcar, MD; Yoshiki Demura, MD; Pradeep R. Rai, MD; Laima Taraseviciene-Stewart, PhD; Michael Kasper, MD; Norbert F. Voelkel, MD and Carlyne D. Cool, MD

* From the Pulmonary Hypertension Center and Departments of Pathology (Dr. Rai) and Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, CO; and Institute of Anatomy (Dr. Kasper), Technical University Dresden, Medical Faculty Carl Gustav Carus, Dresden, Germany.

Correspondence to: Norbert F. Voelkel, MD, Division of Pulmonary Sciences and Critical Care Medicine, 4200 E. Ninth Ave, C272, Denver, CO 80262; e-mail: norbert.voelkel{at}uchsc.edu

Abstract

Caveolae are cell plasma membrane microdomains implicated in organizing and concentrating many signaling molecules. In the lung, caveolae are in endothelium, smooth muscle, fibroblasts, and pneumocytes. Caveolin is the main structural protein of caveolae. Caveolin 1 is down-regulated in transformed cells and may be a tumor suppressor protein. Caveolin 2 is coexpressed and hetero-oligomerizes with caveolin 1. Because the cells of the plexiform lesions in severe pulmonary hypertension (PH) are phenotypically altered, we wondered whether these cells lack caveolin. We now demonstrate by immunolocalization that while caveolin is expressed in lung endothelial, smooth-muscle, and alveolar septal cells, its expression is absent or decreased in plexiform lesions and in some muscularized precapillary arterioles. In contrast, Western blot analysis of total lung extracts from patients with severe PH shows no significant reduction in caveolin. Similar to the human lung tissue, a rat model of severe PH demonstrates absent-to-decreased caveolin expression in the complex vascular lesions. Additionally, it appears that caveolin and heme oxygenase 1 (HO-1) [a heat shock protein] are co-expressed since HO-1 expression parallels caveolin expression in vascular lesions. We propose that loss of caveolin expression in the cells of the complex vascular lesions in severe PH reflects the proliferating and apoptosis-resistant nature of these cells.

Key Words: caveolin • heme oxygenase 1 • pulmonary hypertension

Caveolae are flask or {omega}-shaped cell membrane invaginations of cholesterol-rich cell surface microdomains that have been implicated in assembling many signaling molecules and facilitating and integrating their functions.12 Caveolae may also play an important role in sequestering inactive signaling molecules and down-regulating receptor activities. Additionally, caveolae may provide an environment for the regulated activation of these molecules and explain the cross-talk between different signaling pathways. Caveolae have been the object of intense research since the discovery of a biochemical marker protein, caveolin, in the early 1990s.34 Currently, the caveolin gene family includes caveolin 1, caveolin 2, and caveolin 3.56 They are highly homologous but different in their tissue distribution and probably their physiologic roles in signal transduction. In the lung, caveolae are present in the endothelial cells, smooth-muscle cells, fibroblasts, and type 1 pneumocytes.2 Caveolin 1 constitutes the main structural protein of caveolae. Caveolin 2 is co-expressed and hetero-oligomerizes with caveolin 2. A region has been defined within caveolin that mediates its interaction with other proteins. This protein domain is termed the caveolin-scaffolding domain.789 Lipid rafts are lipid microenvironments on the cell surface10 and, like caveolae, are thought to be rich in cholesterol and sphingolipids; however, they are flat and do not contain caveolin.11 These lipid rafts may represent precursors of caveolae and facilitate the cholesterol-dependent insertion of caveolins into membranes. Therefore, most researchers now believe that caveolae are a subset of lipid rafts, formed by the acquisition of caveolin.11

Since caveolin interactions with signaling molecules lead to altered cell signaling, and because caveolin-1 expression is lost or reduced during cell transformation by activated oncogenes as well as in several mammary carcinoma cell lines,1213 it has been suggested that caveolin may possess tumor suppressor activity.3914 Of importance, knockout mice deficient in caveolin 1, although fertile, show severe abnormalities in their lungs characterized by thick alveolar walls and hyperproliferation of angioblastic cells, indicating strongly that caveolin plays a role in the control of proper lung vascular development1516 and in lung endothelial cell biology. Caveolin 2-deficient mice have lung phenotypes identical to those reported in caveolin 1-deficient mice.17

We have proposed that the endothelial cells present in the plexiform lesions of lungs from patients with severe pulmonary hypertension (PH) to some degree resemble a malignant cell phenotype.18192021 Because caveolin may function as a tumor suppressor protein,3 and because plexiform lesion endothelial cells grow abnormally,18 we hypothesized that plexiform lesions and remodeled small arteries in severe PH may demonstrate a loss of caveolin expression. This hypothesis is based in part on results of gene expression profiling in randomly sampled lung tissue from patients with severe PH vs that of normal lung tissue. In that study, we found a decreased expression of caveolin 1 messenger RNA in lung tissues from patients with severe PH.22 In the present study, we demonstrate by immunohistochemistry that caveolin 1 and 2 are decreased in the plexiform lesions of patients with severe PH, while the endothelial and smooth-muscle cells of patent, normal-appearing vessels retain their caveolin expression. The caveolins are also decreased in some of the muscularized precapillary arterioles. A decrease in caveolin 1 and 2 expression by immunohistochemistry was also found in two rat models of severe PH.2324

Jung et al25 demonstrated the association of caveolin 1 and 2 with heme oxygenase-1 (HO-1) in mouse mesangial cells. More recently, Kim et al26 report the localization of HO-1 to caveolae in rat pulmonary artery endothelial cells as well as providing evidence that caveolin-1 interacts with and modulates heme oxygenase activity. Because of the growing evidence for an association of HO-1 with caveolin and the fact that enhancement of endogenous HO-1 has been shown to prevent hypoxia-induced PH,27 we also hypothesized that HO-1 expression would be absent or decreased in plexiform lesions, in parallel to the caveolin 1 and 2 expression pattern.

Materials and Methods

Tissue Samples
Lung tissue was obtained from 14 patients with plexiform pulmonary arteriopathy (clinical diagnosis of severe PH [Table 1 ]). Ten of the lung specimens were obtained at autopsy, 3 at lung transplant, and 1 at open-lung biopsy. One of the specimens was obtained from lung tissue from a patient with severe PH associated with the use of an anorexigen (dexfenfluramine) and one from a patient with AIDS. "Normal" tissue from four patients with no history of PH was used as a control. Lobectomy was the tissue source for these four patients, each with a diagnosis of carcinoma preoperatively. Lung tissue from a rat model of severe PH and liver tissue from a patient with a liver hemangioma (positive control for neoangiogenesis) were also examined.


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  		Table 1.. Clinical Data*

 
Immunohistochemistry
The tissue was fixed in 10% formalin, processed, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. All of the severe PH cases demonstrated plexiform arteriopathy. Plexiform lesions were defined as intravascular networks of abnormal cells, highlighted by the endothelial cell marker, FVIII-related antigen (FVIII-r.ag).20 All cases, including the rat lung tissue and the liver hemangioma, were stained for caveolin 1 and 2 (monoclonal antibodies, 1:400 dilution; Transduction Laboratories; Lexington, KY), and five PH blocks were selected for serial sectioning and examination with immunohistochemical staining for endothelial cells, smooth-muscle cells, and caveolin-1.

In the serially sectioned cases, the first slide was immunostained for caveolin 1 (clone 2297). The 4-µm paraffin sections were deparaffinized, dried overnight, and microwaved in 0.01 mol/L sodium citrate buffer. After washing in phosphate-buffered saline solution (pH 7.4), conventional immunohistochemical procedure was followed using a commercially available detection system (Vectastain Elite Kit; Vector Laboratories; Burlingame, CA). The peroxidase activity was developed with 3'-3'-diaminobenzidine, and counterstaining with hematoxylin was performed. For control, the primary antibody was replaced by phosphate-buffered saline solution or by nonspecific mouse IgG.

The second slide of each serial sectioned case was stained with FVIII-r.ag (polyclonal antibody, 1:500 dilution; Dako Corporation; Carpinteria, CA),28 and the third slide of each serial sectioned case was stained with {alpha}-smooth-muscle actin (SMA) stain (monoclonal antibody, 1:10000 dilution; Sigma; St. Louis, MO).29 Heat-induced antigen retrieval using pressure cooker heating in a sodium citrate solution was used to optimize immunostaining. An automatic immunostaining device (Ventana ES; Ventana Medical Systems; Tucson, AZ) was employed for the immunohistochemical staining. Incubations with unrelated antibodies were used as controls for the above methods. Appendix was used as the positive control tissue for {alpha}-SMA and FVIII-r.ag. In addition, each lung section provided its own internal control for {alpha}-SMA (bronchial smooth-muscle cells) and FVIII-r.ag (alveolar septal capillaries).

Additional slides were stained using HO-1 antibody (SPA-895, rabbit polyclonal, 1:1500 dilution; Stressgen; Victoria, BC, Canada). Heat-induced antigen retrieval in a sodium citrate solution was used. The slides were incubated with the primary antibody overnight at 4°C.

Immunostaining Analysis
Lumen-occluding FVIII-r.ag.-positive cells were used to identify plexiform lesions. Some of the cells in the plexiform lesion also stained positive for {alpha}-SMA. Twenty-eight plexiform lesions were examined for caveolin expression. All FVIII-r.ag and {alpha}-SMA-positive cells in the plexiform lesions were counted (total of 5,479 cells) and graded as either positive or negative for caveolin 1 expression. Images were obtained with a digital camera (Axiocam-HRC; Zeiss; Thornwood, NY) connected to a personal computer (TEC; Knoxville, TN). Image analysis was performed using software (KS 300 3.0 Image; Zeiss). Plexiform lesions in lung tissue sections from the same 14 patients with severe PH were also examined for the expression of HO-1.

Caveolin Western Blot
Frozen human lung tissue was homogenized in homogenization buffer containing 50 mM hydroxyethyl piperazine-ethanesulfonic acid, pH 7.55, 1 mM dithiothreitol, 10% glycerol, and 0.1% Triton X-100 using homogenizer (model PT10/35; Polytron; Westbury, NY). The lysates were centrifuged twice in a centrifuge (Eppendorf, Hamburg, Germany) at 14,000 revolutions per minute for 10 min. The protein concentration in the supernatant was measured using the Bradford assay. Equal amount of proteins were loaded on NuPAGE 4 to 12% Bis-Tris gels (Novex; Invitrogen Corporation; Carlsbad, CA) and transferred to PolyScreen PVDF transfer membrane (NEN Life Science Products; Foster City, CA) in NuPAGE transfer buffer containing 10% methanol. Prestained molecular mass marker proteins (Bio-Rad Laboratories; Hercules, CA) were used as standards. Rabbit polyclonal anti-caveolin antibodies (BD Transduction Laboratories, San Diego, CA) were diluted 1:1000. Western blots were visualized using a reagent (Renaissance Western Blot Chemiluminescence Reagent; NEN Life Science Product, Piscataway, NJ). Intensity of experimental bands was measured by the ImageQuant program (Molecular Dynamics). Results are expressed as mean ± SEM. Statistical analysis was performed using Student t test to determine the significance of change in the densitometric measurements. A significant difference was considered at p ≤ 0.05.

Rat Lung Tissue Samples
Rat lung tissues were obtained from five adult male Sprague-Dawley rats weighing 200 g. The rats were injected subcutaneously with the synthetic vascular endothelial growth factor (VEGF) receptor 2 inhibitor 3-[(2,4 dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one (SU5416).30 After a single injection (200 mg/kg) of SU5416, the animals were exposed to 3 weeks of chronic hypoxia (simulated altitude of 5,000 m in a hypobaric chamber, with an inspired partial oxygen pressure of approximately 76 mm Hg).23 The animals were then returned to Denver altitude. Immunohistochemistry for caveolin 1 and 2 expression was performed as described above.

Results

Decreased Expression of Caveolin 1 and 2 in the Vascular Lesions of Patients With Severe PH
Caveolin staining was ubiquitous throughout the lung tissue of all patients with primary and secondary PH and in the liver hemangioma. However, the complex vascular structures of severe PH—the plexiform lesions—frequently demonstrated a striking decrease or absence of caveolin 1 and 2 staining (Fig 1, 2 ), although some of the cells lining the residual lumens stained positive for caveolin (Fig 2). When serial sections were stained using antibodies against Factor VIII-r.ag, {alpha}-SMA, and caveolin 1, some of the plexiform lesions demonstrated composite endothelial and smooth-muscle cell phenotypes. In these lesions, both the endothelial and smooth-muscle cells lost their caveolin expression (Fig 3 ).


Figure 1
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  		Figure 1.. Top, A: Immunostaining for caveolin 1 demonstrates decreased-to-absent staining in the cells of the plexiform lesion. The endothelial cells lining the multichanneled lumens are positive (arrows) [original x 400]. Bottom, B: The same lesion as in top, A immunostained for caveolin 2. The caveolin 2 immunostaining mirrors the caveolin 1 immunostaining pattern (original x 400).

 

Figure 2
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  		Figure 2.. Top, A: Plexiform lesion immunostained for caveolin 1. Note that the majority of the cells that make up this complex vascular lesion lack caveolin1 staining and only the endothelial cells that line the multiple lumens are positive (arrows). The surrounding alveolar septal structures express caveolin 1 (arrowheads) [original x 200]. Bottom, B: Plexiform lesion from another patient with PPH demonstrating markedly decreased expression of caveolin 1 (original x 400).

 

Figure 3
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  		Figure 3.. Serial section analysis of a plexiform lesion from a patient with primary PH. The lesion occurs just distal to the bifurcation point of a pulmonary artery. Top, A: The intravascular lesion stains positive for the endothelial cell marker FVIII-r.ag. (arrows) [original x 200]. Center, B: The same lesion as shown in top, A immunostained for {alpha}-SMA (arrowhead). The endothelial cells of the plexiform lesion are negative for this smooth-muscle stain (arrows) [original x 200]. Bottom, C: The same lesion as in top, A and center, B immunostained for caveolin 1, showing that both the endothelial as well as the smooth-muscle cells of the plexiform lesion (identified in top, A and center, B above) have decreased or absent caveolin 1 staining (original x 200).

 
Altogether, 26 of 29 vascular lesions in the patients with severe (primary and secondary) PH were found to be markedly abnormal, ie, they showed clearly decreased or absent staining for caveolin 1. In the 26 lesions, approximately 70% (range, 52 to 85%) of the cells comprising each plexiform lesion demonstrated lack of caveolin 1 expression (as assessed by Zeiss image analysis). Caveolin 2 expression mirrored the caveolin 1 expression pattern. In contrast, remodeled pulmonary arteries with medial hypertrophy only (Fig 4 ) and the endothelium of the hemangioma (Fig 5 ) demonstrated abundant caveolin 1 and 2 expression. The Western blot of the whole lung extract did not demonstrate a significant difference in the expression of caveolin 1 protein (Fig 6 ), illustrating the importance of immunohistochemical studies for the documentation of localized (ie, lesion) abnormalities. With p < 0.3, we found no significant difference in this comparison.


Figure 4
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  		Figure 4.. Caveolin 1 expression in alveolar septal cells (arrows), smooth-muscle cells (arrowhead), and endothelial cells (dashed arrow) of remodeled pulmonary hypertensive arteries (original x 200). P = plexiform lesion.

 

Figure 5
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  		Figure 5.. Liver hemangioma showing the high expression of caveolin 1 (arrows) in the endothelial cells. Fibroblast staining is nearly absent (original x 200).

 

Figure 6
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  		Figure 6.. Western blot analysis depicting whole lung extracts from normal lungs (N) as well as primary PH lungs (PPH). No significant difference in caveolin 1 was detected.

 
Decreased Expression of HO-1 in the Vascular Lesions of Patients With Severe PH
Sections of lung tissue samples from patients with severe PH demonstrated co-expression of caveolin 1 and HO-1; lesions that lacked caveolin 1 expression also lacked HO-1 expression (Fig 7 ).


Figure 7
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  		Figure 7.. Top left, A: Normal lung demonstrating expression of HO-1 in endothelial cells of pulmonary arteries (arrow) [original x 400]. Top right, B: Normal lung demonstrating HO-1 expression in the vessel endothelium as well as the lung parenchyma (original x 400). Bottom left, C and bottom right, D: Plexiform lesions from patients with severe PH. Bottom left, C: Absent HO-1 expression in all of the cells of plexiform lesion; bottom right, D: Continued presence of HO-1 in the alveolar septae surrounding the lesion (original x 400). See Figure 4 legend for expansion of abbreviation.

 
Decreased Expression of Caveolin 1 and 2 in the Vascular Lesions of a Rat Model of Severe PH
In the rat model of severe PH, which is based on the single subcutaneous injection of the VEGF receptor antagonist SU 5416 and subsequent exposure of the animals to 3 weeks of chronic hypoxia, a striking proliferative pulmonary vasculopathy develops.23 Staining of lung tissue sections obtained from five animals treated with SU 5416 and exposed to 3 weeks of hypoxia showed many complex cellular arterial lesions demonstrating striking reduction or absence of caveolin 1 and 2 staining (Fig 8 ). Some of the lesions demonstrate a rim of caveolin-positive cells lining the residual lumen, similar to the immunohistochemical appearance of the human plexiform lesions.


Figure 8
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  		Figure 8.. Top, A: Rat model of severe PH showing decreased expression of caveolin 1 (arrows) in the vascular lesions. Many of the cells that comprise these proliferative lesions lack caveolin 1 staining, while residual luminal endothelial cell staining remains (original x 100). Bottom, B: Caveolin 2 immunostaining from the rat model of severe PH. There is a parallel lack of caveolin 2 expression in the lesions (arrow) [original x 200].

 
Discussion

Exuberant endothelial cell growth has been described in the lungs from patients with severe PH.18 The elucidation of the mechanisms involved in the control of endothelial cell proliferation is fundamentally important in the pathogenesis of severe PH31 and has recently received increased attention.22313233

Endothelial cells are a major cell type in the lung tissue and normally express high levels of caveolin 1.34 Caveolin 1 and 2 null animal lungs are markedly abnormal with thickened alveolar walls and hypercellularity due primarily to endothelial cells.151617 We previously demonstrated reduced caveolin-1 gene expression in the lungs from patients with PPH.22 In the present study, we examine caveolin expression in the lung tissue obtained from patients with severe PH and show that there is a strong caveolin 1 and 2 expression in normal endothelial, smooth muscle, and alveolar septal cells, but markedly decreased expression of caveolin 1 and 2 in the plexiform lesions. A majority of the cells comprising the plexiform lesions show greatly reduced expression of the caveolins. However, protein expression for caveolin 1 is not clearly diminished in Western blots of whole lung tissue extracts from patients with plexiform arteriopathy, indicating the importance of immunolocalization of proteins in lung tissue. In this study, we also show that a rat model of severe PH, which is characterized by endothelial cell growth and partial lumen obliteration,23 demonstrates diminished expression of both caveolin 1 and 2 in the vascular lesions.

The loss or decrease in caveolin expression in the abnormally structured pulmonary vascular lesions appears to be specific for the vascular lesions since the lung tissue surrounding the lesions express caveolin normally, which likely explains the results of the Western blot protein data. It is also of interest that angiogenic growth per se does not necessarily proceed via cells that have an impaired caveolin expression, as is illustrated by the example of the liver hemangioma. However, the vascular lesions in the rat model of severe PH, which are composed of proliferating endothelial cells, also show loss or reduction in expression of caveolin 1 and 2, indicating that there are similarities between the cells involved in the pathogenesis of severe human PH and this animal model.

It has been previously stated that proliferating endothelial cells form the cellular basis of the plexiform lesions in severe PH.1831 These dysfunctional endothelial cells have a central and critical role in the initiation and progression of severe PH.32 The loss of important cell growth control mechanisms allows for the expansion of endothelial cells, which may have acquired a selective growth advantage. Because caveolin may function as a tumor suppressor protein,3 and because plexiform lesion endothelial cells grow abnormally,1832 we consider that loss of caveolin expression may participate in the development and/or maintenance of plexiform lesions in severe PH. In cell culture experiments, capillary-like tubule formation dramatically increased with increasing caveolin 1 levels and, conversely, decreased with down-regulation of caveolin 1 expression.35 The levels of endogenous caveolin 1 expression were at maximum just prior to formation of the capillary-like tubules, further supporting a critical role for caveolin 1 in endothelial cell differentiation and growth.

Not only do the endothelial cells of the plexiform lesion lack caveolin expression, but the occasional smooth-muscle cell of the plexiform lesion also demonstrates absent or decreased immunostaining for caveolin 1 and 2. Previous work36 has shown that differentiated contractile smooth-muscle cells found in the normal arterial media express plasma membrane caveolae in a several-fold-higher density than the synthetic smooth-muscle cells that appear in the neointima after vascular injury. In addition, the handling of lipoprotein-derived cholesterol is also changed, since cholesterol is preferentially associated with caveolae on the surface of contractile smooth-muscle cells and is more randomly dispersed over the surface of noncontractile synthetic smooth cells. Therefore, one consequence of loss of caveolae and presumably decreased caveolin expression is an impaired uptake and transport of cholesterol.37 Via H-Ras, a critical connection has been established between caveolin in cholesterol trafficking and cell signaling.38

Knockout mice deficient in caveolin display uncontrolled proliferation of lung endothelial cells and fibroblasts.151617 Although the etiology of the caveolin 1 null phenotype is unclear, an assessment of endothelial cell function in aortic ring experiments151639 shows decreased vasoconstriction due to uninhibited nitric oxide production (a result which is consistent with the known inhibitory effect of caveolin 1 on endothelial nitric oxide synthase). The previously described strong endothelial nitric oxide synthase expression in plexiform lesions of severe PH40 may be due to decreased caveolin 1 expression. Caveolin 2 null mice, while demonstrating the lung parenchymal septal thickening and endothelial cell hyperplasia of the caveolin 1 null mice, do not show the abnormal vascular responses or altered lipid metabolism of the caveolin 1 null mice, indicating a possible specific pulmonary role for caveolin 2.17 Caveolin 2 expression is also decreased in the caveolin 1 null mice, further support for a pulmonary-specific role.17

At present, the functional importance of decreased caveolin expression in severe PH remains unclear, but the markedly reduced caveolin 1 and 2 expression in plexiform lesions provides another marker of the altered endothelial and vascular smooth-muscle cell phenotypes in the lungs of patients with severe PH. It is also of interest that the complex vascular lesions that characterize the SU5416/hypoxia rat model of severe PH23 also have diminished or absent expression of caveolin 1 and 2. Further, it appears that in the lung there is a co-expression of caveolin1 and HO-1 protein, similar to that described in mouse mesangial cells.25

HO-1 has antioxidant functions,41 and enhancement of endogenous HO-1 prevents hypoxia-induced PH.27 Thus, loss of expression of HO-1 in the complex pulmonary hypertensive lesions could contribute to an oxidant stress milieu in the lungs from patients with severe PH.42 Not only might HO-1 and caveolin 1 coexist in caveolae, but there may also be coexistence of peroxisome proliferator-activated receptor (PPAR)-{gamma} and caveolin 1, since it has been shown that PPAR-{gamma} participates in the regulation of caveolin gene expression in cancer cells.43 However, VEGF receptor 2 (KDR) activity is regulated by caveolin 1,4445 and incubation of endothelial cells with VEGF leads to a marked down-regulation of caveolin 1.46

In this context, we have reported marked overexpression of both VEGF and VEGF receptor 2 proteins in the plexiform lesions31 as well as loss of expression of PPAR-{gamma} protein,47 and we can now characterize these vascular lesion cells as apoptosis resistant and angiogenic. We suggest that both the overexpression of VEGF ligand and the loss of PPAR-{gamma} protein may contribute to the decrease in cellular caveolin expression and that the decreased caveolin expression may, in turn, contribute to enhanced VEGF receptor-induced signaling and endothelial cell proliferation18 (Fig 9 ). Whether VEGF ligand and PPAR-{gamma} can also determine HO-1 expression is unknown.


Figure 9
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  		Figure 9.. Schematic illustrating the possible relationships between VEGF, PPAR-{gamma}, caveolin, and angiogenesis. VEGFRII = VEGF receptor 2.

 
Lastly, there is emerging evidence that caveolin expression is also tied to the fate of vascular smooth-muscle cells (VSMCs); caveolin expression is decreased in secretory VSMC phenotypes as compared to contractile phenotypes or proliferating VSMCs.4849 Loss or decreased expression of the caveolins in the pulmonary hypertensive plexiform lesion smooth-muscle cells and the smooth-muscle cells of the hypertrophied pulmonary arteries may be a marker of a proliferating, apoptosis-resistant VSMC phenotype.50 It remains unclear, however, whether loss of caveolin expression precedes cell proliferation. We propose that the multiple phenotypical alterations of the plexiform lesion cells2021334348 are part of a quasimalignant cell growth program, ie, growth-inhibiting genes are being switched off and growth-promoting genes are being turned on.

Footnotes

Abbreviations: FVIII-r.ag = FVIII-related antigen; HO-1 = heme oxygenase 1; PH = pulmonary hypertension; PPAR = peroxisome proliferator-activated receptor; SMA = smooth-muscle actin; VEGF = vascular endothelial growth factor; VSMC = vascular smooth-muscle cell

This work is supported by RO1 HL60913–01 (Dr. Voelkel) and K08 HL03911–04 (Dr. Cool) from the Heart, Lung, and Blood Institute, National Institutes of Health.

Received for publication February 2, 2005. Accepted for publication July 11, 2005.

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N. F. Voelkel, I. S. Douglas, and M. Nicolls
Angiogenesis in Chronic Lung Disease
Chest, March 1, 2007; 131(3): 874 - 879.
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