Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Mar 9.
Published in final edited form as: Circ Res. 2007 Dec 13;102(3):380–388. doi: 10.1161/CIRCRESAHA.107.161059

SM22α-Mediated Patchy Deletion of Bmpr1a Impairs Cardiac Contractility but Protects Against Pulmonary Vascular Remodeling

Nesrine El-Bizri 1,2, Lingli Wang 1,2, Sandra L Merklinger 1,2, Christophe Guignabert 1,2, Tushar Desai 3,4, Takashi Urashima 2, Ahmad Y Sheikh 5, Yuji Mishina 6, Marlene Rabinovitch 1,2
PMCID: PMC2652676  NIHMSID: NIHMS76611  PMID: 18079409

Abstract

Vascular expression of bone morphogenetic type IA receptor (Bmpr1a) is reduced in lungs of patients with pulmonary arterial hypertension (PAH), but the significance of this observation is poorly understood. To elucidate the role of Bmpr1a in the vascular pathology of PAH and associated right ventricular dysfunction, we deleted Bmpr1a in vascular smooth muscle cells (SMC) and in cardiac myocytes in mice using the SM22α;TRE-Cre/LoxP;R26R system. The LacZ distribution reflected patchy deletion of Bmpr1a in the lung vessels, aorta, and heart of SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutants. This reduction in BMPR-IA expression was confirmed by western immunoblot and immunohistochemistry in the flox/flox group. This did not affect pulmonary vasoreactivity to acute hypoxia (10% O2) or the increase in right ventricular (RV) systolic pressure (RVSP) and RV hypertrophy (RVH) following three weeks in chronic hypoxia. However, both SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutant mice had fewer muscularized distal pulmonary arteries (PAs) and attenuated loss of peripheral PAs compared to age-matched control littermates in hypoxia. When Bmpr1a expression was reduced by siRNA in cultured PASMC, serum-induced proliferation was attenuated explaining decreased hypoxia-mediated muscularization of distal vessels. When Bmpr1a was reduced in cultured microvascular pericytes by siRNA, resistance to apoptosis was observed and this could account for protection against hypoxia-mediated vessel loss. The similar elevation in RVSP and RVH, despite the attenuated remodeling with chronic hypoxia in SM22α;TRE-Cre;R26R;Bmpr1aflox/flox mutants vs. controls, was not a function of elevated LV end diastolic pressure but was associated with increased periadventitial deposition of elastin and collagen, potentially enhancing vascular stiffness.

Keywords: Bmpr1a, Pulmonary hypertension, Hypoxia, Vascular remodeling, Smooth muscle cell, Pericytes, Proliferation, Apoptosis, Transgenic mice

Introduction

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and potentially fatal condition1, in which loss of distal arteries and obliterative changes in larger proximal intra-acinar arteries owing to vascular cell proliferation and migration2 result in progressive increased resistance to flow3. Different heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor (BMPR-II), Bmpr2, have been identified in familial and idiopathic forms of PAH4.

BMPR-II is a member of the transforming growth factor (TGF) ß superfamily of receptors5. There are two classes of transmembrane receptors, type I receptors (ACVR-I, BMPR-IA, and BMPR-IB), and the type II receptors (BMPR-II, ActR-IIA, and ActR-IIB). Ligand binding induces phosphorylation of type I receptors by type II receptors, leading to activation of downstream signaling pathways5. BMPR-II gene mutations confer a reduction in the BMPR-II signaling activity6 resulting from a dose-dependent modulation of BMPR-II oligomerization with its co-receptor, most commonly, BMPR-IA7. A marked reduction in BMPR-II expression has also been documented in patients with idiopathic and secondary PAH (SPAH) without a mutation, as well as in experimental animal models of chronic hypoxia-induced PH8. Steady-state levels of BMPR-IA are also reduced in the pulmonary vasculature of patients with non-familial IPAH or SPAH9, suggesting that disrupted BMP signaling, whether by BMPR-II and/or BMPR-IA dysfunction, could contribute to the pathogenesis of PAH.

Mice homozygous null for Bmpr210 or Bmpr1a11 die in utero due to defective mesodermal formation. Their phenotypes also resembled those of mice that are homozygous null for BMP412 or Smad413. Compared to wild type (WT) littermates, mice heterozygous for Bmpr2 deletion develop mild PH that is resistant to vascular remodeling in hypoxia14, but enhanced PH following stimulation with lipoxygenase15 or serotonin infusion during hypoxia16. Mice expressing a SMC-specific (SM22α) dominant negative Bmpr2 exhibit more severe PH, albeit with relatively modest vascular remodeling17.

To elucidate how reduced BMPR-IA might contribute to the pathogenesis of PAH and the associated right ventricular dysfunction, and to avoid the embryonic lethality that we observed in SM22α;R26R;Bmpr1aflox/flox mice18, we created a mouse that would allow deletion of Bmpr1a in vascular smooth muscle cells (VSMC) and in cardiac myocytes upon tetracycline withdrawal. Embryonic lethality did not occur in the SM22α;TRE-Cre;R26R; Bmpr1aflox/flox mutant mouse so produced without using tetracycline, owing to the patchy Bmpr1a deletion, as reflected by LacZ staining in the heart and arteries and confirmed by western immunoblot and immunohistochemistry. SM22α;TRE-Cre;Bmpr1aflox/+ and SM22α;TRE-Cre; Bmpr1aflox/flox knock-out (KO) mice showed normal RVSP in room air, a similar acute hypoxic vasoconstrictive response, and a similar rise in RVSP and RVH in chronic hypoxia. However, despite PAH, increased muscularization of distal vessels and reduced arterial density were not observed. Despite less severe hypoxia induced distal vascular remodeling in the KO mice, the similar level of PAH to WT mice may be related to ectopic deposition of elastin and accumulation of collagen in the adventitia of more proximal muscularized arteries causing increased vascular stiffness. Our investigations of the mechanisms involved, revealed that knockdown of Bmpr1a using siRNA in human PASMC resulted in reduced proliferation in response to serum, perhaps explaining the decreased muscularization of distal vessels during hypoxia. In contrast, knockdown of Bmpr1a in vascular pericytes, resulted in resistance to apoptosis, perhaps accounting for protection against hypoxia-mediated loss of vessels.

Materials and Methods: (Expanded Version in Online Supplement)

SM22α;TRE-Cre;Bmpr1a Conditional Knock-Out Mice

See Supplement for breeding strategy used.

Genotyping

DNA extracted from yolk sacs of E11.5 embryos and tail biopsies of adult mice was used for genotyping. Polymerase chain reactions (PCR) were used to amplify tTA19, Cre20, and R26R21 genes, the floxed Bmpr1a gene22, and the Bmpr1a gene with exon2 deletion22.

Whole-mount LacZ Staining in Embryos and Adult Tissues

Isolated E11.5 mouse embryos or heart and lungs (en bloc) from 11 to 16 week-old mice were fixed with a mixture of 2 or 1% paraforlmaldehyde (PFA) with 0.2% gluteraldehyde (GTA), and stained with 0.75 or 1 mg/ml X-gal, respectively, following a modified version of an earlier protocol23 as described in the online Supplement. Stained PAs were then cleared in benzyl alcohol and benzyl benzoate before microscopic evaluation. Frozen sections of embedded heart and lungs were counterstained with standard procedures using the Nuclear Fast Red (Vector Labs, Burlingame, California).

Hemodynamic Assessments

Balanced numbers of adult littermate male and female mice matched for gender and genotype were used in each group. To measure acute vasoreactivity24, 12 to 14 week-old mice were anesthetized with 1.5 to 2% isofluorane and continuous RVSP measurements were obtained using a 1.4F Millar catheter (Millar Instruments Inc, Houston, Texas)25 at baseline (40% O2), during acute hypoxia (10% O2 for 15 minutes), and with return back to baseline 40% O2 for 10 minutes. Simultaneous measurements of RV pressure first derivative (dP/dt) and heart rate (HR) were obtained. The mean systemic arterial pressure (MAP) was obtained by direct catheterization of the carotid artery. Left ventricular (LV) fractional shortening (FS) and cardiac output were evaluated by echocardiography using the Acuson Sequoia 256 ultrasound system (Siemens Medical, Mountain View, California). For measurement of hematocrit, blood was collected by direct cardiac puncture. Right ventricular hypertrophy (RVH) was assessed as described earlier25.

The above measurements were also made in 11 to 14 week-old mice after chronic hypoxia for three weeks as previously described24. To further evaluate LV function after chronic hypoxia, we also assessed pressure-volume (PV) relationships performed as previously described26 to obtain LV elastance (Emax) and LV end diastolic pressure (LVEDP). These studies were carried out in mice 22 to 29 weeks of age, older than our initial cohort, but with similar levels of RVSP.

Pulmonary Vascular Morphometry

Isolated lungs were prepared27 and embedded as previously described24. Muscularity of distal PAs was assessed as in our previous studies28 and the number of peripheral arteries per mm2 was calculated. One blinded observer performed all the morphometric analyses.

Lung Tissue Preparation for Histological Analyses

Sections of whole-mount LacZ stained lungs prepared as described above was used to assess medial wall thickness (WT) and external diameter (ED) in large pre-acinar PAs using the Java image processing program, ImageJ. The internal lumen (IL) was calculated using the formula, ED-2XWT.

To relate deletion of Bmpr1a to regional changes in elastin deposition, the whole-mount LacZ stained frozen sections described above were stained with Hart’s stain29. To detect changes in collagen, sections of PFA-fixed paraffin-embedded lungs were stained with Masson’s trichrome stain.

Immunofluorescence and Western Immunoblotting

To determine whether LacZ reflected loss of Bmpr1a, we carried out whole-mount immunohistochemistry on the aorta. Isolated vessels were immersion-fixed in Dent’s fixative and bleached in Dent’s bleach. A modified version of an earlier protocol30 was used for immunostaining. Tissues were incubated in blocking solution containing rabbit anti-BMPR-IA (Orbigen Inc., San Diego, California; 1:200) primary antibody, washed, and incubated with goat anti-rabbit-A555 (Invitrogen, Carlsbad, California; 1:250) secondary antibody and nuclear stain 4’-6-Diamidino-2-phenylindole, DAPI (Invitrogen; 1:1000). An aorta stained following the above procedure with absence of primary antibody was used as a negative control. Specimens were then stored in Vectashield (Vector Labs) at 4°C for later microscopic assessment.

Detection and quantification of BMPR-IA protein expression in total heart tissue homogenates were carried out by western immunoblot. Total protein (50 mg) from each heart sample was separated by SDS-PAGE then transferred onto a PVDF membrane (Amersham Biosciences, Piscataway, New Jersey). The membranes were incubated with a rabbit polyclonal BMPR-IA primary antibody (Orbigen Inc.; 1:100) followed by a goat anti-rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, California; 1:5000). In each sample BMPR-IA protein expression was quantified by densitometric analysis and normalized to the level of S6 ribosomal protein (Cell Signaling Technology Inc., Danvers, Massachusetts; 1:1000).

Primary Cell Cultures and RNA Interference

Adult human pulmonary artery smooth muscle cells (HPASMC; Cascade Biologics, Portland, Oregon) and human brain vascular pericytes (HBVP; ScienCell Research Laboratories, San Diego, California) at 60 or 70% confluence were transiently transfected with control short interference RNA (siRNA) or human Bmpr1a siRNA (Dharmacon, Lafayatte, Colorado; D-001206-13-05 and M-004933-03, respectively) using Lipofectamine™ 2000 (Invitrogen, Carlsbad, California) under serum starvation conditions for 48 hours (hr). Total RNA was extracted using Trizol (Invitrogen) and RNeasy Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using QuantiTect Reverse Transcription Kit (Qiagen). Bmpr1a gene expression levels were quantified using a pre-verified Assay on-Demand TaqMan primer/probe sets (Applied Biosystems, Foster City, California) and normalized to β2M using the comparative delta-CT method.

Cell Proliferation (MTT) and Apoptosis (TUNEL)

Forty-eight hr following transfection in serum starvation (0.1% FBS), HPASMC were exposed to 10% fetal bovine serum (FBS; Gibco™, Invitrogen) for 72 hr, and cell growth was assessed in 96- and 24-well plates by the MTT cell proliferation assay (American Type Culture Collection (ATCC), Manassas, Virginia) and by cell counts using a hemocytometer, respectively. Transfected HPASMC grown in 4-well slides were exposed to 7.7 nmol/L (200 ng/ml) of BMP2 (Sigma, Saint Louis, Missouri) for 24 hr and apoptosis was then assessed by the TUNEL (Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assay using the In Situ Cell Death Detection Kit, TMR Red (Roche Applied Science, Indianapolis, Indiana), performed as per manufacturer’s instructions. Transfected HBVP grown in poly-L-Lysine (ScienCell Research Laboratories) coated 4-well slides for 48 hr in serum starvation were kept under serum-free conditions for an additional 24 hr after which apoptosis was assessed by TUNEL assay.

Statistical Analysis

Values for each determination are expressed as mean±SEM. For comparisons made to assess multiple groups, one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparison test was carried out. ANOVA followed by Dunnett’s test was used to compare three genotypes (WT, flox/+, and flox/flox KO) under the same condition. For comparisons made between normoxia and chronic hypoxia for a given genotype, or for comparison of only two genotypes (WT vs. flox/flox KO) or two groups, statistical significance was determined using the unpaired two-tailed t-Test. A P value <0.05 was considered significant. The number of mice or samples used in each determination is indicated in the Figure legends.

Results

Bmpr1a Conditional Deletion in Embryos and Adult Mice

Mice were genotyped by PCR as shown in Fig. 1 of the Supplement. To assess the profile of Cre expression reflecting areas of Bmpr1a deletion early in life, whole-mount LacZ staining was performed on mouse embryos at E11.5 (Fig. 1). No background staining was noted in the SM22α;R26R control mouse embryos (Fig. 1A, 1C). Patchy blue staining was observed in the dorsal aorta, heart, and lungs of control embryos expressing tTA, Cre, and R26R (SM22α;TRE-Cre;R26R) and in SM22α;TRE-Cre;R26R;Bmpr1aflox/+ (Fig. 1B, 1D-F) and SM22α;TRE-Cre;R26R; Bmpr1aflox/flox mutant embryos. This patchy distribution persisted in cardiovascular tissues of mutants and adult control littermates (SM22α;TRE-Cre;R26R) (Fig. 1G-L). Whole-mount LacZ staining performed on lungs and heart isolated en bloc from adult mice revealed LacZ staining in large intrapulmonary arteries (Fig. 1G, 1H), and in small distal pulmonary vessels in pericytes (Fig, 1I). A higher proportion of LacZ staining was apparent in the unsectioned main PAs (Fig. 1J) than the pre-acinar large PAs, which we determined was not the result of penetration. Similarly, unsectioned adult hearts showed patchy β-galactosidase activity distributed throughout the ventricles (Fig. 1K, 1L). To assess the pattern of BMPR-IA deletion in SM22α;TRE-Cre;R26R; Bmpr1aflox/flox compared to WT adult mice, we performed whole-mount immunofluorescence studies using aortae as representative vascular tissues. BMPR-IA immunoreactivity was diffuse in both the WT (Fig. 1M) and the flox/flox aortae (Fig. 1N), but there was appreciable reduction of immunostatining in the flox/flox aorta. No immunoreactivity was seen in the WT aorta treated with secondary antibody only and used as a negative control (Fig. 1O). We also performed western immunoblot of extracted proteins from adult mice hearts and we observed a 20% reduction in protein expression of BMPR-IA in the flox/flox group (P<0.05) (Fig. 1P).

Figure 1. Whole-mount LacZ staining of SM22α;TRE-Cre;R26R; Bmpr1aflox/+ and flox/flox KO embryos and adult mice and their controls.

Figure 1

Open in a new tab

Patchy LacZ staining shown in SM22α;TRE-Cre;R26R;Bmpr1aflox/+ KO E11.5 embryonic tissues (B, D, E, F) as indicated by red arrows as compared to control littermates not expressing R26R with absence of staining (A, C). RA: right atrium. Patchy staining in large (G and H) and distal PAs (I) in sections of whole-mount LacZ stained lungs of an adult control mouse and in the main PA (J) and heart (K, L) of whole-mount LacZ stained lungs and heart of SM22α;TRE-Cre;R26R;Bmpr1aflox/flox adult KO mouse. H is an enlargement of the outlined area in G and * identifies the lung airway. L represents a section of the heart at the level of the line indicated in K. Whole-mount immunofluorescence revealed a global reduction in BMPR-IA expression (red) in the aorta of SM22α;TRE-Cre; Bmpr1aflox/flox adult KO mouse (N) as compared to WT adult mouse (N). Note the clustered BMPR-IA expression in both WT and KO aortae. No staining was seen in the negative control (O). The blue color in M and N represents DAPI nuclear staining. Bars (I and L)= 50 μm, (G and H)= 100 μm. P. On the left is a western immunoblot of whole heart tissue showing BMPR-IA relative to S6 protein expression in two representative WT and flox/flox adult mice. Densitometric quantification of values from three WT and four flox/flox adult mice is depicted graphically on the right. Bars represent mean±SEM. *P<0.05.

Pulmonary Vasoreactivity

To study the effect of reduced Bmpr1a in PAs on pulmonary vasoreactivity, RVSP was measured in SM22α;TRE-Cre;R26R; Bmpr1aflox/+ and flox/flox adult KO and WT mice by closed-chest technique. The mice were maintained in 40% oxygen to obtain baseline measurements, then challenged by inhalation of 10% O2 to induce acute hypoxia for 15 min, followed by baseline O2 (40%) for 10 min. Figure 2 shows that the three groups of mice had similar RVSP values at baseline and exhibited a similar hypoxia-mediated acute vasoconstriction, reflected in an approximate 10 mmHg rise in RVSP from baseline, and a similar return to normal baseline values upon ‘recovery’ in 40% O2.

Figure 2. Comparable pulmonary vasoreactivity in SM22α;TRE-Cre;R26R; Bmpr1aflox/+ and flox/flox KO adult mice and their controls under acute hypoxia.

Figure 2

Open in a new tab

RVSP was measured in anesthetized SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox adult KO mice and age-matched controls under baseline 40% O2, in response to hypoxia (10% O2) for 15 min, and upon return to 40% O2 for 10 min. Bars represent mean±SEM (n=6-8). **P<0.01 and ***P<0.001 compared to normoxia; †P<0.05 and †††P<0.001, recovery compared to hypoxia.

RVSP and Cardiac Function in Chronic Hypoxia

We next determined the consequences of reduced Bmpr1a in PAs and heart, on pulmonary hemodynamic and cardiac function in response to chronic hypoxia. SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox KO mice and control littermates were exposed to hypoxia (10% O2) for 3 weeks. All three groups exhibited a comparable sustained increase in RVSP (Fig. 3A). To study the impact of reduced Bmpr1a on myocardial function, we assessed the RV rate of contraction (maximal dP/dt) and rate of relaxation (minimal dP/dt), and the LV FS at baseline and following chronic hypoxia. While SM22α;TRE-Cre;R26R;Bmpr1aflox/+ mice showed depressed RV maximal dP/dt at baseline, the flox/flox group exhibited reduced maximal as well as minimal dP/dt (Fig. 3B). Echocardiography revealed a depression in the % of LV FS in the flox/flox mutant mice, but only under chronic hypoxia (Fig. 3C). This was confirmed in another group of mice where a depression in Emax was observed with no change in LVEDP measurements (Supplement Table 1). No significant differences in measurements of HR, CO, hematocrit (Supplement Table 1), or systemic MAP (not shown) were noted among the three groups of mice under either normoxia or chronic hypoxia.

Figure 3. Right ventricular systolic pressure (RVSP) and cardiac function in adult mice following 3 weeks of normoxia or chronic hypoxia.

Figure 3

Open in a new tab

RVSP (A), max and min dP/dt (B), and fractional shortening (FS; C) were measured in anesthetized SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox adult KO mice and age-matched control littermates in normoxia and following three weeks of chronic hypoxia (10% O2). Bars represent mean±SEM (A and B, n=5-11; C, n=3-5). *P<0.05, **P<0.01, and ***P<0.001 between hypoxia and normoxia for each genotype; † P<0.05 and †† P<0.01 between the mutants and WT in chronic hypoxia.

RV Hypertrophy and Pulmonary Vascular Changes

The development of RVH judged by an increase in the RV weight (RVW) over total body weight (BW) (Fig. 4A) or over the weight of LV+septum (not shown) was similar in SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutant mice and WT littermates subjected to chronic hypoxia. In keeping with the elevation in RVSP and RVH during chronic hypoxia, pulmonary vascular remodeling previously described31 was observed in the WT mice as evidenced by an increase in the proportion of partially and fully muscularized arteries at the alveolar duct and wall levels (Fig. 4B). However, both SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mice were resistant to neomuscularization as no significant increase in the % of partially and fully muscularized pulmonary distal vessels was noted. Similarly, while a significant 33% loss of distal PAs at alveolar duct and wall level was detected in WT mice under chronic hypoxia, no significant decrease in the number of vessels per mm2 was noted in the KO groups (Fig. 4C).

Figure 4. Comparable right ventricular hypertrophy (RVH) but depressed vascular remodeling of SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox adult KO mice compared to controls following chronic hypoxia.

Figure 4

Open in a new tab

RVH (A), % of partially and fully muscularized per total number of distal arteries (B), and number of distal arteries per mm2 (C) in SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox KO mice in normoxia and following three weeks of chronic hypoxia (10% O2). Panels in B show EVG-stained paraffin sections of barium-injected lungs where arteries were identified by barium filling (grey color). Note the thick vessel walls in controls compared to the thin vessel walls seen in the KO (red arrows). Bars represent mean±SEM (A, n=5-13; B, n=5-9; C, n=5-9). *P<0.05, **P<0.01, and ***P<0.001 between hypoxia and normoxia for each genotype; †P<0.05 between the mutants and WT in chronic hypoxia.

Pulmonary Artery Elastin and Collagen Deposition

To explain the rise in RVSP observed in the SM22α;TRE-Cre;R26R; Bmpr1aflox/flox mice with lack of distal pulmonary vascular remodeling in response to chronic hypoxia, we evaluated whether structural changes were present in the more proximal PAs that might reduce vascular compliance. Medial wall thickness (WT; Fig 5A) and external diameter (ED; Fig. 5B) of LacZ stained PAs at terminal and respiratory bronchiolus level (≤200 μm of ED) were increased in the WT group in hypoxia versus normoxia (P<0.05) without a significant increase in internal lumen (IL; Fig. 5C). There were no significant hypoxia induced changes in these features in the SM22α;TRE-Cre;R26R;Bmpr1aflox/flox group although room air values tended to be higher than in the WT groups. However, in the SM22α;TRE-Cre;R26R;Bmpr1aflox/flox group, where there was evidence of Cre activity (green color in Fig. 5G), we noted striking hypoxia induced irregularities and thinning of the elastic laminae associated with periadventitial ectopic deposition of elastin fibers. Collagen, judged by trichrome staining, appeared to be ubiquitously deposited more densely in the adventitia of the SM22α;TRE-Cre;R26R;Bmpr1aflox/flox mice (Fig. 5K) compared to control mice (Fig. 5J) following chronic hypoxia.

Figure 5. Changes in pulmonary vessel wall structure in chronic hypoxia.

Figure 5

Open in a new tab

Quantitative measurements of wall thickness (A), external diameter (B), and internal lumen (C) in pre-acinar large PAs (≤200 μm) in lung sections of SM22α;TRE-Cre;R26R;Bmpr1aflox/flox KO mice and age-matched controls following 3 weeks of hypoxia versus normoxia. Bars represent mean±SEM (n=5-9). *P<0.05. D-K Lung sections of flox/flox KO and control mice in normoxia (upper panels) and chronic hypoxia (lower panels). D-G Hart’s elastin staining of sections of whole-mount LacZ stained lungs. H-K Masson’s trichrome (collagen) staining. Note the ectopic elastin deposition in brown color neighboring areas of Cre activity (green color, G) as well as compact collagen deposition in proximal large vessels of SM22α;TRE-Cre;R26R;Bmpr1a flox/flox KO lungs under chronic hypoxia (K). Panels D-G and H-K were acquired with the same magnification as D and H respectively; Bars in D and H are 100 μm.

Loss of Bmpr1a Reduces Proliferation and Enhances Apoptosis in PASMC, but Leads to Resistance to Apoptosis in Pericytes

To determine whether we could attribute the reduced hypoxia-related neo-muscularization in the SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mice to attenuated proliferation of VSMC, we knocked-down Bmpr1a in HPASMC using RNAi under serum deprivation. Forty-eight hr later, the mRNA transcript level of Bmpr1a normalized to β2M was decreased by 66% (Fig. 6A). Cell proliferation assessed by MTT assay 72 hr after stimulation with 10% FBS showed a 42% reduction in proliferation of siBmpr1a cells as compared to siControl cells (Fig. 6B). Similar results were obtained with cell counts (data not shown). Since hypoxia increases BMP2 expression in intrapulmonary arteries concomitant with enhanced apoptosis8 we reasoned that loss of BMPR-IA, might negate this effect. However, cell apoptosis assessed by TUNEL assay was 2-fold increased in siBmpr1a HPASMC in response to a high concentration of BMP2 at 7.7 nmol/L (200 ng/ml) when compared to siControl cells (Fig. 6C). Thus loss of BMPR-IA leads to reduced proliferation and enhanced apoptosis, even in response to BMP2.

Figure 6. Knock-down of Bmpr1a attenuates proliferation and survival of HPASMC and promotes survival of HBVP.

Figure 6

Open in a new tab

A. HPASMC were transfected with siControl (SiCtrl) and siBmpr1a and the relative message transcript levels of Bmpr1a normalized to β2M. Bars represent mean±SEM (n=3 experiments run in quadruplicates). **P<0.01. B-D. Bmpr1a in HPASMC or HBVP was knocked-down using siBmpr1a for 48 hr. B. Proliferation of SiControl and SiBmpr1a HPASMC in presence of 10% FBS for 72 hr by MTT assay. Bars represent mean±SEM of fold increase in OD570nm readings relative to baseline SiControl cells (n=14-16). ***P<0.001. C. Apoptosis of SiControl and SiBmpr1a HPASMC in response to 7.7 nmol/L (200 ng/ml) BMP2 by TUNEL assay. D. Apoptosis of SiControl and SiBmpr1a HBVP in response to a total of 72 hr of serum deprivation by TUNEL assay. Bars in C and D represent mean±SEM of percentages of TUNEL positive cells over total number of cells labeled with nuclei-staining DAPI (stained in red and blue in their corresponding microscopic views, respectively) (n=4 for both). *P<0.05; **P<0.01.

We next determined whether lack of appreciable reduction in the number of distal PAs in the SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mice exposed to chronic hypoxia was due to resistance pericytes to apoptosis thus protecting against endothelial cell (EC) apoptosis. We knocked-down Bmpr1a in HBVP by 53% using RNAi in serum starved conditions for 48 hr (data not shown). Using the TUNEL assay, we noted a 68% decrease in apoptosis of pericytes that were transfected with siBmpr1a compared to siControl pericytes (Fig. 6D) when maintained under serum starvation (0.1% FBS) for an additional 24 hr (a total of 72 hr).

Discussion

The aim of the present study was to assess how reduced expression of Bmpr1a might contribute to the vascular pathology of PAH and associated right ventricular dysfunction. The incomplete deletion of Bmpr1a likely reflects different levels of expression of Cre32 as observed by the patchy ‘striated’ appearance of LacZ. This patchy pattern was also seen in other models of SM-specific Cre;R26R mice and the explanations suggested were either “microheterogeneity” of SMC that require further regulatory elements to activate the promoter, or periodicity of transgene expression33. The former explanation seems unlikely in the light of other findings34 and our recent data18, showing homogeneous LacZ staining in VSMC and cardiac myocytes of murine embryos produced using the SM22α-Cre35/LoxP-R26R system lacking the tTA/TRE genetic components18. We, therefore, pursued this model of mosaic deletion of Bmpr1a as it could be particularly useful in assessing features that might be specifically related to these LacZ marked cells during the course of hypoxia-induced PH36. The similar phenotypes in the SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and the flox/flox mice may be the result of variable expression of Cre with incomplete deletion of Bmpr1a in some flox/flox SMC rendering them haploinsufficient rather than nulls.

While incomplete loss of Bmpr1a in cardiac myocytes did not influence the development of RVH, it did result in depressed RV myocardial function in room air and in LV function following chronic hypoxia. These studies underscore the need, in genetic mouse models of PAH, to assess changes in ventricular function that might relate to the decompensation observed in patients with PAH37. While a reduced rate of RV contractility was observed in SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and especially in flox/flox mutants in room air, it was unexpected that in neither group was there impaired contractility following chronic hypoxia. It is possible that the RV hypertrophy compensated for the dysfunction observed under room air conditions. In contrast, the LV that does not hypertrophy in response to chronic hypoxia may be more vulnerable, and impaired LV contractility was manifest by a 30% reduction in FS by echocardiography and a 26% decrease in LV elastance by PV loops, respectively. Selective deletion of Bmpr1a in cardiac myocytes when Cre is driven by the ventricular myosin light chain promoter results in embryonic lethality in association with both endocardial cushion defects and myocardial thinning38. Targeted deletion of Bmpr1a to the myocardium of atrio-ventricular (AV) canal disrupted the development of AV valves and the annulus fibrosus in mice39. So it is not surprising that even ‘patchy’ deletion is associated with increased ventricular vulnerability.

The discrepancy between the rise in RVSP and the lack of vascular remodeling was observed both in the SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutant mice. A similar discrepancy has been noted in the SM22α-driven dominant negative (DN)17 and the haploinsufficient14 Bmpr2. In the SM22α-DN-Bmpr2, the severe elevation in RVSP was associated with very mild vascular disease17. With the halploinsufficient Bmpr214, there was actually a decrease in medial hypertrophy with chronic hypoxia, despite the higher pressures observed relative to WT controls. This discrepancy between RVSP and vascular remodeling has also been shown in mice that are heterozygous null for BMP440.

Aberrant muscularization of small, normally non-muscular PAs, in chronic hypoxia has been attributed to differentiation of pericytes or fibroblasts to SMC and their subsequent proliferation41 accompanied possibly by reduced SMC apoptosis8. We used HPASMC derived from large arteries as a surrogate to show that loss of Bmpr1a by RNAi was associated with reduced proliferation in response to serum and enhanced apoptosis in response to high concentration of BMP2. This makes the assumption that SMC that have abnormally differentiated in small vessels have properties similar to the SMC in the proximal PA that we studied in our culture system. Our studies inducing apoptosis in response to BMP2 imply possible signaling through BMPR-II and a different co-receptor, likely BMPR-IB. Hypoxia-induced loss of peripheral vessels has been attributed both to EC42 and pericyte apoptosis43. We therefore used HBVP in which the purity of the population was previously shown, and determined that loss of Bmpr1a conferred resistance to apoptosis. Although the data are consistent with our observations, differences in pericyte morphology and distribution among vascular beds are known to occur44. It is also possible that there is an EC non-autonomous effect resulting from knock-down of Bmpr1a in pericytes. The preservation of peripheral vessel density could also explain the lack of muscularization of distal vessels.

To further explain the discrepancy between the chronic hypoxia- induced elevation in RVSP, despite the attenuated vascular remodeling in the SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutants, we directed our attention to assessing differences in the distribution of extracellular matrix (ECM) molecules known to influence vascular tone45. Enhanced proteolytic and elastolytic activities along with increased ECM turnover results in increases in deposition of structural matrix proteins such as elastin and collagen as part of the vascular remodeling associated with PAH45,46. We noted ectopic deposition of elastin in areas neighboring Bmpr1a deletion as well as periadventitial and interstitial collagen accumulation in large pre-acinar arteries of the lungs of the SM22α;TRE-Cre;R26R;Bmpr1aflox/flox mutant mice following chronic hypoxia. This could result in reduced compliance or increased stiffness of large pulmonary vessels as was noted in hypobaric hypoxic model of PH in mice47. These studies underscore the need to consider patterns of altered gene expression in the pathobiology of PAH as they relate both to proximal and distal vasculature, as well as to cardiac structure and function.

In view of our findings, it is possible that reduced Bmpr1a seen in patients with PAH actually protects against vascular disease, specifically distal muscularization and loss of small vessels in the pulmonary circulation. This protective effect might be due to partnering of BMPR-II receptor with a type I receptor other than BMPR-IA, namely IB, preserving the BMP signaling as observed in our preliminary studies and as seen in PASMC with loss of BMPR-II where recruitment of ActR-IIA receptor by type I receptors was documented48. Therefore, it could be that a corresponding reduction in expression or function of Bmpr2, may be required for the pathology of PAH to be expressed. Alternatively, other modifier genes may be expressed to de-repress what appears to be a protective effect on SMC proliferation or pericytes apoptosis observed in BMPR-IA loss of function.

Supplementary Material

Online supplem

Acknowledgments

We thank Dr Michal Roof for her assistance in the preparation of the manuscript figures.

Sources of funding: This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences to YM, and by the NIH Grant R01 HL074186 to MR. NB was supported by a fellowship from the American Heart Association/Pulmonary Hypertension Association and MR by the Dunlevie Professorship.

Footnotes

Disclosures: None of the authors had any disclosure.

Subject Codes:

[18] Pulmonary circulation and disease

[115] Remodeling

[131] Apoptosis

[145] Genetically altered mice

[156] Pulmonary biology and circulation

[162] Smooth muscle cell proliferation and differentiation

References

  • 1.Abenhaim L, Moride Y, Brenot F, Rich S, Benichou J, Kurz X, Higenbottam T, Oakley C, Wouters E, Aubier M, Simonneau G, Begaud B. Appetite-suppressant drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary Hypertension Study Group. N Engl J Med. 1996;335:609–616. doi: 10.1056/NEJM199608293350901. [DOI] [PubMed] [Google Scholar]
  • 2.Jeffery TK, Morrell NW. Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension. Prog Cardiovasc Dis. 2002;45:173–202. doi: 10.1053/pcad.2002.130041. [DOI] [PubMed] [Google Scholar]
  • 3.Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM, Christman BW, Weir EK, Eickelberg O, Voelkel NF, Rabinovitch M. Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol. 2004;43:13S–24S. doi: 10.1016/j.jacc.2004.02.029. [DOI] [PubMed] [Google Scholar]
  • 4.Thomson J, Machado R, Pauciulo M, Morgan N, Yacoub M, Corris P, McNeil K, Loyd J, Nichols W, Trembath R. Familial and sporadic primary pulmonary hypertension is caused by BMPR2 gene mutations resulting in haploinsufficiency of the bone morphogenetic protein tuype II receptor. J Heart Lung Transplant. 2001;20:149. doi: 10.1016/s1053-2498(01)00259-5. [DOI] [PubMed] [Google Scholar]
  • 5.de Caestecker M. The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev. 2004;15:1–11. doi: 10.1016/j.cytogfr.2003.10.004. [DOI] [PubMed] [Google Scholar]
  • 6.Foletta VC, Lim MA, Soosairajah J, Kelly AP, Stanley EG, Shannon M, He W, Das S, Massague J, Bernard O. Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J Cell Biol. 2003;162:1089–1098. doi: 10.1083/jcb.200212060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Gilboa L, Nohe A, Geissendorfer T, Sebald W, Henis YI, Knaus P. Bone morphogenetic protein receptor complexes on the surface of live cells: a new oligomerization mode for serine/threonine kinase receptors. Mol Biol Cell. 2000;11:1023–1035. doi: 10.1091/mbc.11.3.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Takahashi H, Goto N, Kojima Y, Tsuda Y, Morio Y, Muramatsu M, Fukuchi Y. Downregulation of type II bone morphogenetic protein receptor in hypoxic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol. 2006;290:L450–458. doi: 10.1152/ajplung.00206.2005. [DOI] [PubMed] [Google Scholar]
  • 9.Du L, Sullivan CC, Chu D, Cho AJ, Kido M, Wolf PL, Yuan JX, Deutsch R, Jamieson SW, Thistlethwaite PA. Signaling molecules in nonfamilial pulmonary hypertension. N Engl J Med. 2003;348:500–509. doi: 10.1056/NEJMoa021650. [DOI] [PubMed] [Google Scholar]
  • 10.Beppu H, Kawabata M, Hamamoto T, Chytil A, Minowa O, Noda T, Miyazono K. BMP type II receptor is required for gastrulation and early development of mouse embryos. Dev Biol. 2000;221:249–258. doi: 10.1006/dbio.2000.9670. [DOI] [PubMed] [Google Scholar]
  • 11.Mishina Y, Suzuki A, Ueno N, Behringer RR. Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 1995;9:3027–3037. doi: 10.1101/gad.9.24.3027. [DOI] [PubMed] [Google Scholar]
  • 12.Winnier G, Blessing M, Labosky PA, Hogan BL. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 1995;9:2105–2116. doi: 10.1101/gad.9.17.2105. [DOI] [PubMed] [Google Scholar]
  • 13.Sirard C, de la Pompa JL, Elia A, Itie A, Mirtsos C, Cheung A, Hahn S, Wakeham A, Schwartz L, Kern SE, Rossant J, Mak TW. The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo. Genes Dev. 1998;12:107–119. doi: 10.1101/gad.12.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Beppu H, Ichinose F, Kawai N, Jones RC, Yu PB, Zapol WM, Miyazono K, Li E, Bloch KD. BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia. Am J Physiol Lung Cell Mol Physiol. 2004;287:L1241–1247. doi: 10.1152/ajplung.00239.2004. [DOI] [PubMed] [Google Scholar]
  • 15.Song Y, Jones JE, Beppu H, Keaney JF, Jr, Loscalzo J, Zhang YY. Increased susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice. Circulation. 2005;112:553–562. doi: 10.1161/CIRCULATIONAHA.104.492488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Long L, MacLean MR, Jeffery TK, Morecroft I, Yang X, Rudarakanchana N, Southwood M, James V, Trembath RC, Morrell NW. Serotonin increases susceptibility to pulmonary hypertension in BMPR2-deficient mice. Circ Res. 2006;98:818–827. doi: 10.1161/01.RES.0000215809.47923.fd. [DOI] [PubMed] [Google Scholar]
  • 17.West J, Fagan K, Steudel W, Fouty B, Lane K, Harral J, Hoedt-Miller M, Tada Y, Ozimek J, Tuder R, Rodman DM. Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle. Circ Res. 2004;94:1109–1114. doi: 10.1161/01.RES.0000126047.82846.20. [DOI] [PubMed] [Google Scholar]
  • 18.El-Bizri N, Wang L, Chang CP, Helms JA, Mishina Y, Rabinovitch M. Vascular, Cardiac, and Craniofacial Defects in Mice with Smooth Muscle Cell-Specific Deletion of Bone Morphogenetic Protein Type IA Receptor (BMPR-IA) Circ Suppl. 2006;114:II–141. Abstract. [Google Scholar]
  • 19.Ju H, Gros R, You X, Tsang S, Husain M, Rabinovitch M. Conditional and targeted overexpression of vascular chymase causes hypertension in transgenic mice. Proc Natl Acad Sci U S A. 2001;98:7469–7474. doi: 10.1073/pnas.131147598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Saam JR, Gordon JI. Inducible gene knockouts in the small intestinal and colonic epithelium. J Biol Chem. 1999;274:38071–38082. doi: 10.1074/jbc.274.53.38071. [DOI] [PubMed] [Google Scholar]
  • 21.Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–71. doi: 10.1038/5007. [DOI] [PubMed] [Google Scholar]
  • 22.Mishina Y, Hanks MC, Miura S, Tallquist MD, Behringer RR. Generation of Bmpr/Alk3 conditional knockout mice. Genesis. 2002;32:69–72. doi: 10.1002/gene.10038. [DOI] [PubMed] [Google Scholar]
  • 23.Koop EA, Lopes SM, Feiken E, Bluyssen HA, van der Valk M, Voest EE, Mummery CL, Moolenaar WH, Gebbink MF. Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice. Int J Dev Biol. 2003;47:345–354. [PubMed] [Google Scholar]
  • 24.Merklinger SL, Wagner RA, Spiekerkoetter E, Hinek A, Knutsen RH, Kabir MG, Desai K, Hacker S, Wang L, Cann GM, Ambartsumian NS, Lukanidin E, Bernstein D, Husain M, Mecham RP, Starcher B, Yanagisawa H, Rabinovitch M. Increased fibulin-5 and elastin in S100A4/Mts1 mice with pulmonary hypertension. Circ Res. 2005;97:596–604. doi: 10.1161/01.RES.0000182425.49768.8a. [DOI] [PubMed] [Google Scholar]
  • 25.Zaidi SH, You XM, Ciura S, Husain M, Rabinovitch M. Overexpression of the serine elastase inhibitor elafin protects transgenic mice from hypoxic pulmonary hypertension. Circulation. 2002;105:516–521. doi: 10.1161/hc0402.102866. [DOI] [PubMed] [Google Scholar]
  • 26.Georgakopoulos D, Mitzner WA, Chen CH, Byrne BJ, Millar HD, Hare JM, Kass DA. In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry. Am J Physiol. 1998;274:H1416–1422. doi: 10.1152/ajpheart.1998.274.4.H1416. [DOI] [PubMed] [Google Scholar]
  • 27.Ye CL, Rabinovitch M. Inhibition of elastolysis by SC-37698 reduces development and progression of monocrotaline pulmonary hypertension. Am J Physiol. 1991;261:H1255–1267. doi: 10.1152/ajpheart.1991.261.4.H1255. [DOI] [PubMed] [Google Scholar]
  • 28.Todd L, Mullen M, Olley PM, Rabinovitch M. Pulmonary toxicity of monocrotaline differs at critical periods of lung development. Pediatr Res. 1985;19:731–737. doi: 10.1203/00006450-198507000-00019. [DOI] [PubMed] [Google Scholar]
  • 29.Pierce RA, Albertine KH, Starcher BC, Bohnsack JF, Carlton DP, Bland RD. Chronic lung injury in preterm lambs: disordered pulmonary elastin deposition. Am J Physiol. 1997;272:L452–460. doi: 10.1152/ajplung.1997.272.3.L452. [DOI] [PubMed] [Google Scholar]
  • 30.Sillitoe RV, Hawkes R. Whole-mount immunohistochemistry: a high-throughput screen for patterning defects in the mouse cerebellum. J Histochem Cytochem. 2002;50:235–244. doi: 10.1177/002215540205000211. [DOI] [PubMed] [Google Scholar]
  • 31.Rabinovitch M, Gamble W, Nadas AS, Miettinen OS, Reid L. Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am J Physiol. 1979;236:H818–827. doi: 10.1152/ajpheart.1979.236.6.H818. [DOI] [PubMed] [Google Scholar]
  • 32.Topouzis S, Majesky MW. Smooth muscle lineage diversity in the chick embryo. Two types of aortic smooth muscle cell differ in growth and receptor-mediated transcriptional responses to transforming growth factor-beta. Dev Biol. 1996;178:430–445. doi: 10.1006/dbio.1996.0229. [DOI] [PubMed] [Google Scholar]
  • 33.Madsen CS, Regan CP, Hungerford JE, White SL, Manabe I, Owens GK. Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5’-flanking and first intronic DNA sequence. Circ Res. 1998;82:908–917. doi: 10.1161/01.res.82.8.908. [DOI] [PubMed] [Google Scholar]
  • 34.Regan CP, Manabe I, Owens GK. Development of a smooth muscle-targeted cre recombinase mouse reveals novel insights regarding smooth muscle myosin heavy chain promoter regulation. Circ Res. 2000;87:363–369. doi: 10.1161/01.res.87.5.363. [DOI] [PubMed] [Google Scholar]
  • 35.Holtwick R, Gotthardt M, Skryabin B, Steinmetz M, Potthast R, Zetsche B, Hammer RE, Herz J, Kuhn M. Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure. Proc Natl Acad Sci U S A. 2002;99:7142–7147. doi: 10.1073/pnas.102650499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 1995;75:487–517. doi: 10.1152/physrev.1995.75.3.487. [DOI] [PubMed] [Google Scholar]
  • 37.Marcus JT, Vonk Noordegraaf A, Roeleveld RJ, Postmus PE, Heethaar RM, Van Rossum AC, Boonstra A. Impaired left ventricular filling due to right ventricular pressure overload in primary pulmonary hypertension: noninvasive monitoring using MRI. Chest. 2001;119:1761–1765. doi: 10.1378/chest.119.6.1761. [DOI] [PubMed] [Google Scholar]
  • 38.Gaussin V, Van de Putte T, Mishina Y, Hanks MC, Zwijsen A, Huylebroeck D, Behringer RR, Schneider MD. Endocardial cushion and myocardial defects after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK3. Proc Natl Acad Sci U S A. 2002;99:2878–2883. doi: 10.1073/pnas.042390499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Gaussin V, Morley GE, Cox L, Zwijsen A, Vance KM, Emile L, Tian Y, Liu J, Hong C, Myers D, Conway SJ, Depre C, Mishina Y, Behringer RR, Hanks MC, Schneider MD, Huylebroeck D, Fishman GI, Burch JB, Vatner SF. Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus. Circ Res. 2005;97:219–226. doi: 10.1161/01.RES.0000177862.85474.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Frank DB, Abtahi A, Yamaguchi DJ, Manning S, Shyr Y, Pozzi A, Baldwin HS, Johnson JE, de Caestecker MP. Bone morphogenetic protein 4 promotes pulmonary vascular remodeling in hypoxic pulmonary hypertension. Circ Res. 2005;97:496–504. doi: 10.1161/01.RES.0000181152.65534.07. [DOI] [PubMed] [Google Scholar]
  • 41.Yang X, Sheares KK, Davie N, Upton PD, Taylor GW, Horsley J, Wharton J, Morrell NW. Hypoxic induction of cox-2 regulates proliferation of human pulmonary artery smooth muscle cells. Am J Respir Cell Mol Biol. 2002;27:688–696. doi: 10.1165/rcmb.2002-0067OC. [DOI] [PubMed] [Google Scholar]
  • 42.Campbell AI, Zhao Y, Sandhu R, Stewart DJ. Cell-based gene transfer of vascular endothelial growth factor attenuates monocrotaline-induced pulmonary hypertension. Circulation. 2001;104:2242–2248. doi: 10.1161/hc4201.097838. [DOI] [PubMed] [Google Scholar]
  • 43.Zhao YD, Campbell AI, Robb M, Ng D, Stewart DJ. Protective role of angiopoietin-1 in experimental pulmonary hypertension. Circ Res. 2003;92:984–991. doi: 10.1161/01.RES.0000070587.79937.F0. [DOI] [PubMed] [Google Scholar]
  • 44.Hirschi KK, D’Amore PA. Pericytes in the microvasculature. Cardiovasc Res. 1996;32:687–698. [PubMed] [Google Scholar]
  • 45.Hassoun PM. Deciphering the “matrix” in pulmonary vascular remodelling. Eur Respir J. 2005;25:778–779. doi: 10.1183/09031936.05.00027305. [DOI] [PubMed] [Google Scholar]
  • 46.Maruyama K, Ye CL, Woo M, Venkatacharya H, Lines LD, Silver MM, Rabinovitch M. Chronic hypoxic pulmonary hypertension in rats and increased elastolytic activity. Am J Physiol. 1991;261:H1716–1726. doi: 10.1152/ajpheart.1991.261.6.H1716. [DOI] [PubMed] [Google Scholar]
  • 47.Kobs RW, Muvarak NE, Eickhoff JC, Chesler NC. Linked mechanical and biological aspects of remodeling in mouse pulmonary arteries with hypoxia-induced hypertension. Am J Physiol Heart Circ Physiol. 2005;288:H1209–1217. doi: 10.1152/ajpheart.01129.2003. [DOI] [PubMed] [Google Scholar]
  • 48.Yu PB, Beppu H, Kawai N, Li E, Bloch KD. Bone morphogenetic protein (BMP) type II receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells. J Biol Chem. 2005;280:24443–24450. doi: 10.1074/jbc.M502825200. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Online supplem
NIHMS76611-supplement-Online_supplem.pdf (577.9KB, pdf)

RESOURCES