This Article Abstract Full Text (PDF) All Versions of this Article: 110/11/1499    most recent 01.CIR.0000141576.39579.23v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mathew, R. Articles by Sehgal, P. B. Search for Related Content PubMed PubMed Citation Articles by Mathew, R. Articles by Sehgal, P. B. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB MONOCROTALINE Medline Plus Health Information Pulmonary Hypertension Related Collections Pulmonary circulation and disease

(Circulation. 2004;110:1499-1506.)
© 2004 American Heart Association, Inc.

Original Articles

Disruption of Endothelial-Cell Caveolin-1/Raft Scaffolding During Development of Monocrotaline-Induced Pulmonary Hypertension

Rajamma Mathew, MD; Jing Huang, MD; Mehul Shah, MD; Kirit Patel, BS; Michael Gewitz, MD; Pravin B. Sehgal, MD PhD

From the Departments of Pediatrics (R.M., J.H., M.G.), Cell Biology & Anatomy (M.S., K.P., P.B.S.), and Medicine (P.B.S.), New York Medical College, Valhalla, NY.

Correspondence to Pravin B. Sehgal, MD, PhD, Basic Sciences Bldg, Department of Cell Biology & Anatomy, New York Medical College, Valhalla, NY 10595. E-mail pravin_sehgal{at}nymc.edu

Received February 13, 2004; de novo received April 13, 2004; revision received May 24, 2004; accepted May 25, 2004.

	Abstract

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Background— In the monocrotaline (MCT)-treated rat, there is marked stimulation of DNA synthesis and megalocytosis of pulmonary arterial endothelial cells (PAECs) within 3 to 4 days, followed by pulmonary hypertension (PH) 10 to 14 days later. Growing evidence implicates caveolin-1 (cav-1) in plasma membrane rafts as a negative regulator of promitogenic signaling. We have investigated the integrity and function of endothelial cell–selective cav-1/raft signaling in MCT-induced PH.

Methods and Results— Although PH and right ventricular hypertrophy developed by 2 weeks after MCT, a reduction in cav-1 levels in the lung was apparent within 48 hours, declining to 30% by 2 weeks, accompanied by an increase in activation of the promitogenic transcription factor STAT3 (PY-STAT3). Immunofluorescence studies showed a selective loss of cav-1 and platelet endothelial cell adhesion molecule-1 in the PAEC layer within 48 hours after MCT but an increase in PY-STAT3. PAECs with cav-1 loss displayed high PY-STAT3 and nuclear immunostaining for proliferating cell nuclear antigen (PCNA). Biochemical studies showed a loss of cav-1 from the detergent-resistant lipid raft fraction concomitant with hyperactivation of STAT3. Moreover, cultured PAECs treated with MCT-pyrrole for 48 hours developed megalocytosis associated with hypo-oligomerization and reduction of cav-1, hyperactivation of STAT3 and ERK1/2 signaling, and stimulation of DNA synthesis.

Conclusions— MCT-induced disruption of cav-1 chaperone and scaffolding function in PAECs likely accounts for diverse alterations in endothelial cell signaling in this model of PH.

Key Words: endothelium • hypertension, pulmonary • interleukins • signal transduction • transcription factors

	Introduction

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Pulmonary hypertension (PH) is a progressive disease with high morbidity and mortality.¹ Because the clinical manifestations of PH occur long after the initial injury, experimental models provide an opportunity to investigate the initiating mechanisms of this disease process. The monocrotaline (MCT)-treated rat is a well-established experimental model of PH.^2–4 This plant alkaloid is bioactivated in the liver to pyrrolic metabolites.⁵ The bioactive derivatives produced in the liver have a half-life of 3 seconds and thus significantly affect mainly the pulmonary arterial endothelium.⁵ Within 24 to 48 hours, there is evidence of endothelial cell damage⁶ and a pulmonary vascular leak.² This is followed within 3 to 4 days by a 3- to 5-fold stimulation of pulmonary arterial endothelial cell (PAEC) DNA synthesis and megalocytosis resulting from abortive cytokinesis^7–12. These hypertrophic endothelial cells are thought to secrete growth- and motility-promoting factors such as platelet-derived growth factor-B, transforming growth factor-ß, and interleukin-6 (IL-6), which contribute to the migration and megalocytosis of underlying smooth muscle cells.^10–14 On its abluminal side, increased deposition of insoluble elastin and collagen¹⁰ and migration and megalocytosis of smooth muscle cells are observed.^3,7–10 On the luminal side, there is an increase in adhesiveness of the endothelial cell surface to cellular elements in the bloodstream.¹⁵

The initial in vivo phenotypic changes in PAECs in lungs of MCT-treated rats have been extensively studied in cell culture. Briefly, within 48 hours, monocrotaline pyrrole (MCTP)-treated PAECs show a marked increase in cell size, enlarged nuclei, and increased and continuing DNA synthesis, leading to hypertetraploidy.^5,16,17 Vascular smooth muscle cells in culture show minimal changes.⁸

The molecular mechanism that initiates PAEC DNA synthesis and megalocytosis in the MCT-treated rat is largely unclear. Recent data have emphasized the role of IL-6 in the development of PH in this model.⁴ An increase in IL-6 in the rat lung is observed within 48 hours of MCT.⁴ IL-6 activates promitogenic signaling, including activation of the transcription factor STAT3 and the ras-raf-MAPK pathway.^18–20 IL-6 signaling involves specialized cholesterol-rich detergent-resistant plasma membrane microdomains called rafts.^19,20 Raft microdomains represent preassembled complexes of cytokine and growth factor receptors, their associated signaling molecules with scaffolding proteins such as the caveolins (cav).^19–23 Cav-1, a major protein component in rafts/caveolae, is largely a negative regulator of mitogenic signaling.^21,22 Interestingly, a 25% reduction in the number of caveolae per unit endothelial cell volume has been reported in MCT-treated rat lungs (see Reference ¹¹, Table IV). In mouse and rat lungs, the cav-1 isoform but not the N-terminal truncated cav-1ß isoform is selectively found in endothelial cells, whereas the reverse is true for the alveolar epithelium.²⁴ Compared with the endothelium, vascular smooth muscle cells express minimal cav-1.²⁴

In the present study, we investigated whether the MCT-induced disruption of cav-1/raft integrity and consequent hyperactivation of promitogenic signaling results in early endothelial dysfunction followed by PH. While this study was in progress, a causal role for loss of cav-1 in the development of PH in the mouse was established by the observation that homozygous deletion of the murine cav-1 gene spontaneously produced pulmonary endothelial cell hyperplasia and PH.²⁵ The present study extends the mechanism of cav-1 loss in initiating PH to the MCT-rat model.

	Methods

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Monocrotaline Administration and Hemodynamic Function
Male Sprague-Dawley rats (age, 6 to 7 weeks) weighing 125 to 150 g (Charles River) were given 1 subcutaneous injection of MCT (60 mg/kg), and pulmonary artery pressure and right ventricular hypertrophy were assessed at 48 hours, 1 week, and 2 weeks after MCT and in controls as described previously.⁴ Experimental protocols were approved by the institutional Animal Care and Use Committee.

Western Blot Analyses
Protein aliquots (50 µg) of respective lung extracts were Western blotted, and cav-1 (sc-894),²⁴ STAT3, tyrosine-phosphorylated STAT3 (PY-STAT3), and glucose-regulated protein 58 (GRP58, also called ER-60 and ERp57) antigens were detected as described previously.^19,20 Protein bands were quantified using a Hoefer GS300 densitometer.^19,20

Double-Label Immunofluorescence Studies
Formaldehyde-fixed, paraffin-embedded sections (6 microns thick) of lung tissue were probed in double-label immunofluorescence assays for endothelial-selective cav-1 and von Willebrand Factor (vWF), cav-1 and platelet endothelial cell adhesion molecule-1 (PECAM-1), cav-1 and PY-STAT3, cav-1 and proliferating cell nuclear antigen (PCNA), and PY-STAT3 and PCNA using respective rabbit and goat polyclonal antibodies or murine monoclonal antibodies and corresponding AlexaFluor 488-, AlexaFluor 594-, CY3-, or rhodamine-tagged secondary antibodies. Images were collected with a MRC 1024 ES (Bio-Rad) confocal microscopy system at a magnification of x180 (Figures 2 and 3), x270 (Figure 4A), or x450 (Figure 4B and 4C). Immunostaining intensities were quantified (10 to 12 arteries per section and 3 to 4 animals in each group) by use of a pixel histogram procedure (Adobe Photoshop 7.0) and expressed in arbitrary luminosity units.

View larger version (89K): [in this window] [in a new window]   Figure 2. Rapid and sustained loss of cav-1 but not vWF from PAECs in lungs of MCT-treated rats. Double-label immunofluorescence analyses of lung sections were carried out as described in Methods. Magnification of recorded images was x180; arterial vascular segments illustrated are 50 to 150 µm in diameter. For quantification, 2 to 3 individual animals and 2 to 3 different sections of each lung were evaluated at each time point. Within each group, relative cav-1 and vWF staining intensities were quantified in 6 to 16 different arteries per section. Luminosity data, expressed in arbitrary units (mean±SEM), are summarized in B. *P<0.001.

View larger version (91K): [in this window] [in a new window]   Figure 3. Rapid and sustained loss of cav-1 and PECAM-1 from PAECs in lungs of MCT-treated rats. Double-label immunofluorescence analyses of lung sections were carried out as described in Methods. Magnification of recorded images was x180; arterial vascular segments illustrated are 50 to 150 µm in diameter. Luminosity data, expressed in arbitrary units (mean±SEM), are summarized in B. *P<0.001.

View larger version (67K): [in this window] [in a new window]   Figure 4. Cav-1 loss and reciprocal activation of PY-STAT3 and DNA synthesis in PAECs. A, Selective loss of cav-1 from pulmonary arterial segments. Comparison of cav-1 immunofluorescence in arterial (juxtabronchiolar) and venous (long thin-walled channels) segments in same section of lungs from either control or MCT-treated (2 weeks) rats. Magnification of recorded images was x270. B, C, Relationships between immunostaining for cav-1 and PY-STAT3 (B) and PY-STAT3 and PCNA (C) in PAEC layer in lungs of rats treated with MCT for 48 hours. Double-label immunofluorescence analyses of lung sections were carried out as described in Methods. Magnification of recorded images was x450; arterial vascular segments illustrated are 50 to 150 µm in diameter. Arrowheads in B point to endothelial cells with high PY-STAT3; in C, to cells with high PY-STAT3 and nuclear PCNA immunostaining.

Membrane Fractionation and Isolation of Rafts
Triton X-100–resistant (0.05%) membrane rafts were isolated from sonicated and homogenized lung tissue²¹ by equilibrium sucrose-density flotation by use of the method of Lafont and Simons²³ as modified.^19,20 Seven fractions were collected from each gradient^22,23 corresponding to the visible band at the very top (fraction 1), just above the 13/43% sucrose interface (fraction 2), at the 13/43% interface (fraction 3), just below the 13/43% interface (fraction 4), at the 43/60% interface (fraction 5), an aliquot from the 60% loading region (fraction 6), and the pellet (fraction 7). Fractions 2, 3, 4, 5, and 7 were diluted in cold Triton-solubilizing buffer,²³ resedimented at 15 000g for 20 minutes, and resuspended in 200 µL solubilizing buffer. Aliquots were assayed for the plasma membrane marker 5' nucleotidase (5' ND) (Sigma) and for various proteins by SDS-PAGE and Western blotting.^20,21,23 Fraction 3 is the detergent-resistant lipid raft fraction.^20,21,23

PAEC Cell Culture, MCTP Treatment, and Cell Fractionation
Growth and fractionation of primary bovine PAECs (BPAECs) into the "P15" membrane fraction, the cytosolic "S100" fraction, and the "NE" nuclear extract were carried out as described earlier.²⁰ MCTP was prepared by use of the procedure of Mattocks et al.²⁶ Protein-matched aliquots of the respective cellular fractions were Western blotted for cav-1, STAT3, STAT1, PY-STAT3, ERK1/2, and P-ERK1/2. DNA-shift assays for STAT-specific binding in nuclear extracts were carried out with the SIE oligonucleotide probe,^19,20 and the SIF-A band observed (as in Figure 6A) was verified to be due to PY-STAT3 homodimers through the use of an antibody supershift assay (data not shown). Phase contrast confirmed megalocytosis of MCTP-treated BPAECs; respective cultures in 6-well plates were double-immunostained for cav-1 and PCNA after methanol-acetone fixation.

View larger version (45K): [in this window] [in a new window]   Figure 6. MCTP-induced hyperactivation of promitogenic signaling in cultured BPAECs. A, BPAEC cultures were treated with MCTP (100 µg/mL) for 48 hours and then IL-6 (10 ng/mL) for another 30 minutes. Equally packed cell volumes of control and treated cultures were fractionated into P15 membrane, S100 cytosolic, and NE extract fractions. Protein-matched aliquots of each were Western blotted with respective antibodies. PY-STAT3 DNA binding activity was assayed in NE fraction by use of SIE oligonucleotide (which gives SIF-A gel shift band with homodimeric PY-STAT3). B, Multimeric status of cav-1 in detergent-resistant 13/43% sucrose interface fraction (similar to fraction 3 in Figure 5) was evaluated by treatment with n-octylglucoside (50 mmol/L) and velocity sedimentation.27 Estimate of cav-1 oligomer state: n 20 in control and n <8 on MCTP treatment. C, Inverse relationship between cav-1 content and nuclear PCNA immunofluorescence in BPAEC treated with MCTP for 48 hours. Single arrowheads point to cell nuclei without PCNA staining (not in DNA synthesis); double arrowheads, to nuclear PCNA immunostaining (cells in DNA synthesis).

View larger version (35K): [in this window] [in a new window]   Figure 5. Loss of cav-1 from Triton X-100–resistant lipid rafts from lungs of MCT-treated rats and reciprocal activation of STAT3. Representative sets of protein content–matched lung extracts from untreated and MCT-treated rats were subjected to detergent treatment and equilibrium flotation. Western blots were probed for cav-1 (A), PY-STAT3 (B), STAT3 (C), gp130 (D), STAT1 (not shown), and GRP58 (not shown). Fraction 3 corresponds to low-density raft/caveolar fraction at 13/43% interface. Additionally, each fraction was assayed for 5' ND activity. Association of respective proteins with raft fraction was quantified by densitometry; these data, together with 5' ND levels, are summarized (E) as percent of that in the raft fraction from untreated control lung.

Cav-1 Oligomerization Status
This status was assessed by treating respective BPAEC cellular fractions with n-octylglucoside (50 mmol/L) and subjecting the sample to velocity sedimentation as described earlier.²⁷

Statistical Analyses
Statistical analyses were carried out with Student’s t test (for paired samples, 2 tailed; each variable group was compared pairwise with controls). Additionally, we used 1-Way ANOVA and the Tukey-Kramer multiple comparison test.

	Results

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Progressive Loss of Cav-1 and Increased Tyr-Phosphorylation of STAT3 in Lungs
Figure 1A and 1B show the occurrence of significant PH and right ventricular hypertrophy by 2 weeks in rats administered MCT. Remarkably, Western blotting data showed a reduction in the endothelial cell–selective cav-1 isoform beginning as early as 48 hours after MCT and declining to 30% of control levels by 2 weeks (Figure 1C and 1E). During this time, there was reciprocal activation of STAT3 as indicated by increased levels of PY-STAT3 (Figure 1D and 1E). MCT had little effect on the levels of total STAT3 or of GRP58, a protein known to be derivatized by MCT pyrrole in cell culture²⁸ (Figure 1E). Consistent with previous reports,²⁹ little cav-2 or cav-3 protein was detected (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 1. Hemodynamic and Western blotting data from rats treated with MCT. A, B, Significant PH and right ventricular hypertrophy in rats at 2 weeks after MCT. C, D, Reciprocal downregulation of cav-1 and activation of STAT3 (PY-STAT3). E, Representative Western blots first probed with anti–cav-1 pAb (sc-894) and then reprobed with additional antibodies as indicated. Numerical data are summarized as mean±SEM. PAP indicates pulmonary artery pressure; RVH, right ventricular hypertrophy. *P<0.001.

Immunofluorescence Studies of Cav-1, vWF, and PECAM-1, in PAECs
As expected, the most intense cellular immunostaining for cav-1 colocalized with vWF, a marker for endothelial cells³⁰ (Figure 2A). There was a progressive loss of cav-1 beginning as early as 48 hour post-MCT in the intimal layer (Figure 2). In contrast, there was little alteration in vWF (Figure 2). The lack of alteration in vWF suggests that the integrity of PAEC per se was maintained during development of PH.

Figure 3 confirms the early and sustained loss of cav-1 in PAEC, and also shows the concomitant loss of PECAM-1, another plasma membrane-spanning scaffolding protein which negatively regulates diverse Tyr-kinase signaling pathways,^31,32 including STAT3.³²

Compared with the marked loss of cav-1 in arterial segments, there was little alteration of cav-1 in the endothelium of pulmonary venous segments (Figure 4A).

Disruption of Cav-1/Raft Association and Modulation of cav-1/Raft/STAT3 Signaling
Figure 5A (control) confirms a major distribution of cav-1 in the low-density raft fraction at the 13/43% sucrose interface (fraction 3). Figure 5A (MCT groups) and Figure 5E taken together show a selective and marked loss (>90%) of cav-1 but not of the 5' ND plasma membrane marker from the raft fraction (fraction 3) during development of PH.

Figure 5B and 5E shows that activated Tyr-phosphorylated STAT3 (PY-STAT3) was associated with the raft fraction as early as 48 hours after MCT, with sustained and increasing PY-STAT3/raft association throughout the 2-week period. Additionally, Figure 5C and 5E shows a rapid and sustained recruitment of the transcription factor STAT3 per se to the raft fraction beginning within 48 hours (see fraction 3 at 48 hours after MCT) and that of STAT1 (Figure 5E and data not shown). In contrast, the gp130 signal-transducing chain of the IL-6 receptor, which was found in both raft (fraction 3) and nonraft (fraction 5) membrane fractions, was progressively downregulated (Figure 5D and 5E). The association of GRP58 with the raft fraction was largely unchanged (Figure 5E and data not shown).

Inverse Relationship Between Cav-1 Loss and PY-STAT3 and Nuclear PCNA at the Single Endothelial Cell Level In Vivo
Figure 4B replicates the loss of cav-1 in the PAEC layer 48 hours after MCT, with a concomitant increase in PY-STAT3 at the cellular level. Importantly, Figure 4C shows that endothelial cells with high PY-STAT3 showed nuclear immunostaining for PCNA, indicative of active DNA synthesis. In additional controls, we have confirmed the absence of PCNA staining in high–cav-1-containing PAECs from untreated rats and the presence of PCNA immunostaining in low–cav-1-containing PAECs from rats 48 hours after MCT (data not shown).

Effects of MCTP on PAEC in Culture
Treatment of BPAECs in culture with MCTP (100 mmol/L) for 48 hours produced megalocytosis (not shown). Figure 6A shows that MCTP-treated BPAECs displayed (1) a reduction in cav-1 in the P15 membrane fraction, (2) cytosolic activation of phospho-ERK1/2, and (3) increased nuclear translocation of STAT3 and STAT1. Additionally, IL-6 stimulation of MCTP-treated cells led to hyperactivation of STAT3 signaling (increased Western-blottable nuclear PY-STAT3 and "SIF-A" DNA binding activity in nuclear extracts) and further activation of phospho-ERK1/2. Figure 6B shows that cav-1 in the detergent-resistant fraction derived from MCTP-treated cells banding at the 13/43% sucrose interface was hypo-oligomeric (n <8-mer compared with n20-mer in control). This low-density membrane fraction from MCTP-treated cells but not from untreated cells was enriched in the Golgi marker GM130 (data not shown), indicating that MCTP blocks cav-1 trafficking and assembly in the Golgi compartment.^27,33 Figure 6C illustrates the inverse relationship between cav-1 and DNA synthesis (nuclear accumulation of PCNA indicative of cells in S phase) in MCTP-treated cells. Overall, >90% of the cells showing nuclear PCNA immunostaining were low–cav-1-expressing cells.

	Discussion

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Our studies show that administration of MCT results in a rapid reduction of cav-1 expression in rat lungs with a reciprocal activation of PY-STAT3 and of DNA synthesis before the development of PH. The loss of cav-1 was most marked in the detergent-resistant raft fractions. The loss of cav-1 was also associated with a parallel loss of PECAM-1, another scaffolding protein and an endothelial cell marker. Interestingly, PECAM-1 also negatively regulates STAT3.³² However, vWF, another marker for the endothelial cells which is contained in Weibel-Palade bodies,³⁰ was not altered, indicating that MCT-induced injury is confined mainly to the raft/scaffolding component of the endothelial cells. The inverse relationship between cav-1 and DNA synthesis and activation of promitogenic endothelial cell signaling, including hyperactivation of PY-STAT3, P-ERK1/2, and PY-STAT3 DNA binding activity, was confirmed in MCTP-treated PAECs in culture. The low-density membrane fraction from these cells showed hypo-oligomerization of cav-1 (Figure 6B), and this fraction was enriched in the Golgi scaffolding protein GM130 (data not shown). Mechanistically, the hypo-oligomerized state of cav-1 in detergent-resistant membranes from MCTP-treated PAECs suggests a block in trafficking of cav-1 through the Golgi compartment.^27,34 An MCTP-induced Golgi-block would shunt cav-1 away from the plasma membrane, resulting in enlarged Golgi and enlarged intracellular membranes as has been observed in vivo.^10,11 Thus, an MCT-induced Golgi block may be the subcellular mechanism leading to cav-1 loss, hyperactivation of promitogenic signaling, and DNA synthesis, yet a block at the Golgi fragmentation control point at the G2+M border³³ results in hyperploidy and the megalocytotic cell phenotype^8,9,16,17 and endothelial cell dysfunction.

Cav-1, the major protein constituent of caveolae, has emerged as a "master regulator" of signaling in endothelial cells.³⁵ Cav-1 multimers in raft/caveolae provide a scaffold for colocalization of cytokine and growth factor receptors, together with their signaling molecules.^19–23,34 Cav-1 serves both a positive chaperone function in ferrying receptors to plasma membrane raft domains and a negative function attenuating signaling by sequestering activated signaling intermediates.^19–23,34 Loss of cav-1 resulting in hyperactivation of STAT3 in the MCT model is significant because STAT3 has been shown to be involved in cell proliferation.³⁶ Furthermore, the upregulation of cav-1 is reported to attenuate epidermal growth factor signaling.²⁹ Cav-1 also negatively regulates endothelial nitric oxide synthase (eNOS) activity, but cav-1 chaperone function is needed to deliver eNOS to the plasma membrane caveolae.³⁵ Thus, a reduction in cav-1 secondary to a trafficking block at the Golgi could adversely affect the eNOS activity, resulting in low bioavailability of NO, an underlying feature of MCT-induced PH.³⁷ Interestingly, the endothelial cell angiopoietin-1 receptor Tie2, which is associated with cav-1,³⁸ is also reported to be decreased in MCT-induced PH.³⁹ Thus, the cav-1 loss is associated not only with the activation of promitogenic pathways such as PY-STAT3, ERK1/2, and DNA synthesis but also with negative regulation of eNOS and Tie2 pathways. All these factors individually or in concert are capable of participating in the pathogenesis of PH.

To summarize, the MCT-induced loss of cav-1 chaperone and scaffolding function in PAECs may provide an underlying mechanism for diverse alterations in endothelial cell signaling in this model of PH.

	Note Added in Proof

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Historically, endothelial cell proliferation induced by pyrrolizidine plant alkaloids was reported by Davidson,⁴⁰ the marked enlargement of target cell types was reported by Harris et al,⁴¹ and the term "megalocytosis" was introduced to describe this phenotype by Bull.⁴²

	Acknowledgments

 
This work was supported in part by research grant HL–73301 from the National Heart Lung and Blood Institute, National Institutes of Health. We thank Dr Susan Olson for BPAEC cell stocks and Joanne Abrahams for expert help in preparing the figures.

	References

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
1. Mathew R, Gewitz MH. Pulmonary hypertension in infancy and childhood. Heart Dis. 2000; 2: 362–368.[Medline] [Order article via Infotrieve]

2. Valdivia E, Sonnad J, Hayashi Y, et al. Experimental interstitial pulmonary edema. Angiology. 1967; 18: 378–383.[Free Full Text]

3. Meyrick B, Gamble W, Reid L. Development of Crotolaria pulmonary hypertension. Am J Physiol. 1981; 239: H692–H702.

4. Bhargava A, Kumar A, Yuan N, et al. Monocrotaline induces interleukin-6 mRNA expression in rat lungs. Heart Dis. 1999; 1: 126–132.[Medline] [Order article via Infotrieve]

5. Roth RA, Reindel JF. Lung vascular injury from monocrotaline pyrrole, a putative hepatic metabolite. Adv Exp Med Biol. 1991; 283: 477–487.[Medline] [Order article via Infotrieve]

6. Schultze AE, Gunaga KP, Wagner JG, et al. Lactate dehydrogenase activity and isozyme patterns in tissues and bronchoalveolar lavage fluid from rats treated with monocrotaline pyrrole. Toxicol Appl Pharmacol. 1994; 126: 301–310.[CrossRef][Medline] [Order article via Infotrieve]

7. Meyrick BO, Reid LM. Crotalaria-induced pulmonary hypertension: uptake of ³H-thymidine by the cells of the pulmonary circulation and alveolar walls. Am J Pathol. 1982; 106: 84–94.[Abstract]

8. Reindel JF, Roth RA. The effects of monocrotaline pyrrole on cultured bovine pulmonary artery endothelial and smooth muscle cells. Am J Pathol. 1991; 138: 707–719.[Abstract]

9. Lappin PB, Ross KL, King LE, et al. The response of pulmonary vascular endothelial cells to monocrotaline pyrrole: cell proliferation and DNA synthesis in vitro and in vivo. Toxicol Appl Pharmacol. 1998; 150: 37–48.[CrossRef][Medline] [Order article via Infotrieve]

10. Todorovich-Hunter L, Johnson DJ, Ranger P, et al. Altered elastin and collagen synthesis associated with progressive pulmonary hypertension induced by monocrotaline: a biochemical and ultrastructural study. Lab Invest. 1988; 58: 184–195.[Medline] [Order article via Infotrieve]

11. Rosenberg HC, Rabinovitch M. Endothelial injury and vascular reactivity in monocrotaline pulmonary hypertension. Am J Physiol. 1988; 255: H1484–H1491.[Medline] [Order article via Infotrieve]

12. Lappin PB, Roth RA. Hypertrophy and prolonged DNA synthesis in smooth muscle cells characterize pulmonary arterial wall thickening after monocrotaline pyrrole administration to rats. Toxicol Pathol. 1997; 25: 372–380.[Abstract/Free Full Text]

13. Wang Z, Newman WH. Smooth muscle cell migration stimulated by interleukin-6 is associated with cytoskeleton reorganization. J Surg Res. 2003; 111: 261–266.[CrossRef][Medline] [Order article via Infotrieve]

14. Arcot SS, Lipke DW, Gillespie MN, et al. Alterations of growth factor transcripts in rat lungs during development of monocrotaline-induced pulmonary hypertension.

15. Kato S, Kishiro I, Sugimura H, et al. Changes of sequestered leukocytes and platelets by inhalation prostaglandin E₁ in the pulmonary microvasculature of rats with monocrotaline-induced pulmonary hypertension. J Vasc Res. 2002; 39: 83–87.[CrossRef][Medline] [Order article via Infotrieve]

16. Thomas HC, Lamé MW, Wilson DW, et al. Cell cycle alterations associated with covalent binding of monocrotaline pyrrole to pulmonary artery endothelial cell DNA. Toxicol Appl Pharmacol. 1996; 141: 319–329.[Medline] [Order article via Infotrieve]

17. Wilson DW, Lamé MW, Dunston SK, et al. DNA damage cell checkpoint activities are altered in monocrotaline pyrrole-induced cell cycle arrest in human pulmonary endothelial cells. Toxicol App Pharmacol. 2000; 166: 69–80.[CrossRef][Medline] [Order article via Infotrieve]

18. Mackiewicz A, Koj A, Sehgal PB, eds. Interleukin-6-type cytokines. Ann N Y Acad Sci. 1995; 762: 1–522.[Medline] [Order article via Infotrieve]

19. Sehgal PB, Guo GG, Shah M, et al. Cytokine signaling: STATs in plasma membrane rafts. J Biol Chem. 2002; 277: 12067–12074.[Abstract/Free Full Text]

20. Shah M, Patel K, Fried VA, et al. Interaction of STAT3 with caveolin-1 and heat shock protein 90 in plasma membrane raft and cytosolic complexes. J Biol Chem. 2002; 277: 45662–45669.[Abstract/Free Full Text]

21. Lisanti MP, Scherer PE, Vidugiriene J, et al. Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J Cell Biol. 1994; 126: 111–126.[Abstract/Free Full Text]

22. Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin Invest. 2002; 110: 597–603.[CrossRef][Medline] [Order article via Infotrieve]

23. Lafont F, Simons K. Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling. Proc Natl Acad Sci U S A. 2001; 98: 3180–3184.[Abstract/Free Full Text]

24. Kogo H, Aiba T, Fujimoto T. Cell type-specific occurrence of caveolin-1 and -1ß in the lung caused by expression of distinct mRNAs. J Biol Chem. 2004; 279: 25574–25581.[Abstract/Free Full Text]

25. Zhao YY, Liu Y, Stan RV, et al. Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice. Proc Natl Acad Sci U S A. 2002; 99: 11375–11380.[Abstract/Free Full Text]

26. Mattocks AR, Jukes R, Brown J. Simple procedures for preparing putative toxic metabolites of pyrrolizidine alkaloids. Toxicon. 1989; 27: 561–567.[Medline] [Order article via Infotrieve]

27. Lee H, Park DS, Razani B, et al. Caveolin-1 mutations (P132L and null) and the pathogenesis of breast cancer. Am J Pathol. 2002; 161: 1357–1369.[Abstract/Free Full Text]

28. Lamé MW, Jones AD, Wilson DW, et al. Protein targets of monocrotaline pyrrole in pulmonary endothelial cells. J Biol Chem. 2000; 275: 29091–29099.[Abstract/Free Full Text]

29. Park W-Y, Park J-S, Cho K-A, et al. Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells. J Biol Chem. 2003; 275: 20847–20852.[CrossRef]

30. Müller AM, Skrzynski C, Skipka G, et al. Expression of von Willebrand factor by human pulmonary endothelial cells in vivo. Respiration. 2002; 69: 526–533.[CrossRef][Medline] [Order article via Infotrieve]

31. Newman PJ, Newman DK. Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular biology. Arterioscler Thromb Vasc Biol. 2003; 23: 953–964.[Abstract/Free Full Text]

32. Ilan N, Cheung S, Miller S, et al. PECAM-1 is a modulator of STAT family member phosphorylation and localization: lessons from a transgenic mouse. Dev Biol. 2001; 232: 219–232.[CrossRef][Medline] [Order article via Infotrieve]

33. Sutterlin C, Hsu P, Mallabiabarrena A, et al. Fragmentation and dispersal of the pericentriolar Golgi complex is required for entry into mitosis in mammalian cells. Cell. 2002; 109: 359–369.[CrossRef][Medline] [Order article via Infotrieve]

34. Pol A, Martin S, Fernandez MA, et al. Dynamic and regulated association of caveolin with lipid bodies: modulation of lipid body motility and function by a dominant negative mutant. Mol Biol Cell. 2004; 15: 99–110.[Abstract/Free Full Text]

35. Minshall RD, Sessa WC, Stan RV, et al. Caveolin regulation of endothelial function. Am J Physiol Lung Cell Mol Physiol. 2003; 285: L1179–L1183.[Abstract/Free Full Text]

36. Levy DE, Lee CK. What does STAT3 do? J Clin Invest. 2002; 109: 1143–1148.[CrossRef][Medline] [Order article via Infotrieve]

37. Mathew R, Yuan N, Rosenfeld L, et al. Effects of monocrotaline on endothelial nitric oxide synthase expression and sulfhydryl levels in rat lungs. Heart Dis. 2002; 4: 152–158.[CrossRef][Medline] [Order article via Infotrieve]

38. Yoon MJ, Cho CH, Lee CS, et al. Localization of Tie2 and phospholipase D in endothelial caveolae is involved in angiopoietin-1-induced MEK/ERK phosphorylation and migration in endothelial cells. Biochem Biophys Res Commun. 2003; 308: 101–105.[CrossRef][Medline] [Order article via Infotrieve]

39. Zhao YD, Campbell AIM, Robb M, et al. Protective role of angiopoietin-1 in experimental pulmonary hypertension. Circ Res. 2003; 92: 984–991.[Abstract/Free Full Text]

40. Davidson J. The action of retrorsine on rat’s liver. J Path Bact. 1935; 40: 285–295.[CrossRef]

41. Harris PN, Anderson RC, Chen KK. The action of senecionine, integerrimine, jacobine, longilobine, and spartioidine, especially on the liver. J Pharmacol Exptl Ther. 1942; 75: 60–77.

42. Bull LA. The histological evidence of liver damage from pyrrolizidine alkaloids. Aust Vet J. 1955; 31: 33–40.[CrossRef]

This article has been cited by other articles:

				J. Huang, P. M. Kaminski, J. G. Edwards, A. Yeh, M. S. Wolin, W. H. Frishman, M. H. Gewitz, and R. Mathew Pyrrolidine dithiocarbamate restores endothelial cell membrane integrity and attenuates monocrotaline-induced pulmonary artery hypertension Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1250 - L1259. [Abstract] [Full Text] [PDF]

				N. A. Maniatis, V. Shinin, D. E. Schraufnagel, S. Okada, S. M. Vogel, A. B. Malik, and R. D. Minshall Increased pulmonary vascular resistance and defective pulmonary artery filling in caveolin-1-/- mice Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L865 - L873. [Abstract] [Full Text] [PDF]

				S. Mukhopadhyay, M. Shah, F. Xu, K. Patel, R. M. Tuder, and P. B. Sehgal Cytoplasmic provenance of STAT3 and PY-STAT3 in the endolysosomal compartments in pulmonary arterial endothelial and smooth muscle cells: implications in pulmonary arterial hypertension Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L449 - L468. [Abstract] [Full Text] [PDF]

				F. Xu, S. Mukhopadhyay, and P. B. Sehgal Live cell imaging of interleukin-6-induced targeting of "transcription factor" STAT3 to sequestering endosomes in the cytoplasm Am J Physiol Cell Physiol, October 1, 2007; 293(4): C1374 - C1382. [Abstract] [Full Text] [PDF]

				H. H. Patel, S. Zhang, F. Murray, R. Y. S. Suda, B. P. Head, U. Yokoyama, J. S. Swaney, I. R. Niesman, R. T. Schermuly, S. S. Pullamsetti, et al. Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension FASEB J, September 1, 2007; 21(11): 2970 - 2979. [Abstract] [Full Text] [PDF]

				P. B. Sehgal and S. Mukhopadhyay Dysfunctional Intracellular Trafficking in the Pathobiology of Pulmonary Arterial Hypertension Am. J. Respir. Cell Mol. Biol., July 1, 2007; 37(1): 31 - 37. [Abstract] [Full Text] [PDF]

				P. B. Sehgal and S. Mukhopadhyay Pulmonary arterial hypertension: a disease of tethers, SNAREs and SNAPs? Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H77 - H85. [Abstract] [Full Text] [PDF]

				P. B. Sehgal, S. Mukhopadhyay, F. Xu, K. Patel, and M. Shah Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, June 1, 2007; 292(6): L1526 - L1542. [Abstract] [Full Text] [PDF]

				J.-F. Jasmin, I. Mercier, J. Dupuis, H. B. Tanowitz, and M. P. Lisanti Short-Term Administration of a Cell-Permeable Caveolin-1 Peptide Prevents the Development of Monocrotaline-Induced Pulmonary Hypertension and Right Ventricular Hypertrophy Circulation, August 29, 2006; 114(9): 912 - 920. [Abstract] [Full Text] [PDF]

				S. Mukhopadhyay and P. B. Sehgal Discordant regulatory changes in monocrotaline-induced megalocytosis of lung arterial endothelial and alveolar epithelial cells Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1216 - L1226. [Abstract] [Full Text] [PDF]

				R. O. D. Achcar, Y. Demura, P. R. Rai, L. Taraseviciene-Stewart, M. Kasper, N. F. Voelkel, and C. D. Cool Loss of Caveolin and Heme Oxygenase Expression in Severe Pulmonary Hypertension Chest, March 1, 2006; 129(3): 696 - 705. [Abstract] [Full Text] [PDF]

				P. A. Singleton, S. M. Dudek, E. T. Chiang, and J. G. N. Garcia Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and {alpha}-actinin FASEB J, October 1, 2005; 19(12): 1646 - 1656. [Abstract] [Full Text] [PDF]

				M. Shah, K. Patel, and P. B. Sehgal Monocrotaline pyrrole-induced endothelial cell megalocytosis involves a Golgi blockade mechanism Am J Physiol Cell Physiol, April 1, 2005; 288(4): C850 - C862. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 110/11/1499    most recent 01.CIR.0000141576.39579.23v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mathew, R. Articles by Sehgal, P. B. Search for Related Content PubMed PubMed Citation Articles by Mathew, R. Articles by Sehgal, P. B. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB MONOCROTALINE Medline Plus Health Information Pulmonary Hypertension Related Collections Pulmonary circulation and disease