Published online before print December 13, 2007, doi: 10.1161/CIRCRESAHA.107.161059
© 2008 American Heart Association, Inc.
Integrative Physiology |
Smooth Muscle Protein 22
–Mediated Patchy Deletion of Bmpr1a Impairs Cardiac Contractility but Protects Against Pulmonary Vascular Remodeling
From the Cardiopulmonary Research Program (N.E.-B., L.W., S.L.M., C.G., M.R.), Vera Moulton Wall Center for Pulmonary Vascular Disease; Departments of Pediatrics (N.E.-B., L.W., S.L.M., C.G., T.U. M.R.), Biochemistry (T.D.), Pulmonary and Critical Care Medicine (T.D.), and Cardiothoracic Surgery (A.Y.S.), Stanford University School of Medicine, Calif; Department of Cell Biology (R.H.K., R.P.M.), Washington University School of Medicine, St Louis, Mo; and Molecular Developmental Biology Group (Y.M.), Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC.
Correspondence to Marlene Rabinovitch, MD, Stanford University School of Medicine, CCSR-2245B, 269 Campus Dr, Stanford, CA 94305-5162. E-mail marlener{at}stanford.edu
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Abstract |
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Vascular expression of bone morphogenetic type IA receptor (Bmpr1a) is reduced in lungs of patients with pulmonary arterial hypertension, but the significance of this observation is poorly understood. To elucidate the role of Bmpr1a in the vascular pathology of pulmonary arterial hypertension and associated right ventricular (RV) dysfunction, we deleted Bmpr1a in vascular smooth muscle cells and in cardiac myocytes in mice using the SM22
;TRE-Cre/LoxP;R26R system. The LacZ distribution reflected patchy deletion of Bmpr1a in the lung vessels, aorta, and heart of SM22;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 RV systolic pressure and RV hypertrophy following 3 weeks in chronic hypoxia. However, both SM22;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutant mice had fewer muscularized distal pulmonary arteries and attenuated loss of peripheral pulmonary arteries compared with age-matched control littermates in hypoxia. When Bmpr1a expression was reduced by short interference RNA in cultured pulmonary arterial smooth muscle cells, serum-induced proliferation was attenuated explaining decreased hypoxia-mediated muscularization of distal vessels. When Bmpr1a was reduced in cultured microvascular pericytes by short interference RNA, resistance to apoptosis was observed and this could account for protection against hypoxia-mediated vessel loss. The similar elevation in RV systolic pressure and RV hypertrophy, despite the attenuated remodeling with chronic hypoxia in the flox/flox mutants versus controls, was not a function of elevated left ventricular end diastolic pressure but was associated with increased periadventitial deposition of elastin and collagen, potentially influencing vascular stiffness.
Key Words: Bmpr1a (Alk3) • pulmonary hypertension • hypoxia • smooth muscle cell • pericyte • transgenic mice
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Introduction |
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Idiopathic pulmonary arterial hypertension is a rare and potentially fatal condition,1 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 flow.3 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 pulmonary arterial hypertension (PAH).4
BMPR-II gene mutations confer a reduction in BMPR-II signaling activity,5 resulting from a dose-dependent modulation of BMPR-II oligomerization with its coreceptor, most commonly, BMPR-IA.6 A marked reduction in BMPR-II expression has also been documented in patients with idiopathic and secondary PAH without a mutation, as well as in experimental animal models of pulmonary hypertension (PH).7 Steady-state levels of BMPR-IA are also reduced in the pulmonary vasculature of patients with nonfamilial idiopathic PAH or secondary PAH,8 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 Bmpr29 or Bmpr1a10 die in utero because of defective mesodermal formation. Their phenotypes also resembled those of BMP411 or Smad412 homozygous null mice. Compared with wild-type (WT) littermates, mice heterozygous for Bmpr2 deletion develop mild PH with resistance to vascular remodeling in hypoxia13 but enhanced PH following stimulation with either lipoxygenase14 or serotonin plus hypoxia.15 Mice expressing smooth muscle protein (SM)22-specific dominant negative Bmpr2 exhibit more severe PH, albeit with relatively modest vascular remodeling.16
To elucidate how reduced BMPR-IA might contribute to the pathogenesis of PAH and the associated right ventricular (RV) dysfunction, and to avoid the embryonic lethality that we observed in SM22-Cre;R26R;Bmpr1aflox/flox mice,17 we created a mouse that would allow deletion of Bmpr1a in vascular smooth muscle cells (VSMCs) and in cardiac myocytes on tetracycline withdrawal. Embryonic lethality did not occur in the SM22;TRE-Cre;R26R;Bmpr1aflox/flox mutant mouse so produced without using tetracycline, owing to the patchy Bmpr1a deletion. SM22;TRE-Cre;R26R;Bmpr1aflox/+ and SM22;TRE-Cre;R26R;Bmpr1aflox/flox knockout (KO) and control mice showed similar RV systolic pressure (RVSP) in room air, similar acute hypoxic vasoconstrictive response, and similar rise in RVSP and RV hypertrophy (RVH) following chronic hypoxia. However, despite PAH, increased muscularization of distal vessels and reduced arterial density were not observed. Our investigations of the mechanisms involved revealed that knockdown of Bmpr1a in human pulmonary arterial smooth muscle cells (PASMCs) resulted in reduced proliferation, perhaps explaining the decreased hypoxia-induced neomuscularization. Knockdown of Bmpr1a in vascular pericytes resulted in resistance to apoptosis, possibly explaining protection against hypoxia-mediated loss of vessels. Ectopic deposition of elastin and collagen in proximal pulmonary arteries (PAs) in the mutants may have influenced the chronic hypoxia-induced rise in PA pressure despite the reduced distal remodeling.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
SM22;TRE-Cre;Bmpr1a Conditional KO Mice
See the online data supplement for the breeding strategy used.
Genotyping
DNA extracted from yolk sacs of embryonic day (E)11.5 embryos and tail biopsies of adult mice was used for genotyping. PCRs were used to amplify tTA,18 Cre,19 and R26R20 genes, the floxed Bmpr1a gene,21 and the Bmpr1a gene with exon 2 deletion.21
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% paraformaldehyde with 0.2% gluteraldehyde and stained with 0.75 or 1 mg/mL 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, respectively, following a modified protocol22 described in the online data supplement. Frozen sections of embedded heart and lungs were counterstained with Nuclear Fast Red (Vector Labs, Burlingame, Calif).
Hemodynamic Assessments
Balanced numbers of littermate mice matched for gender and genotype were used in each group. To measure acute vasoreactivity,23 12- to 14-week-old mice were anesthetized with 1.5% to 2% isoflurane and continuous RVSP measurements were obtained using a 1.4F Millar catheter (Millar Instruments Inc, Houston, Tex)24 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 were obtained. The mean systemic arterial pressure was assessed by direct catheterization of the carotid artery. Left ventricular (LV) fractional shortening and cardiac output were evaluated by echocardiography using the Acuson Sequoia 256 ultrasound system (Siemens Medical, Mountain View, Calif). For hematocrit measurement, blood was collected by direct cardiac puncture. RVH was assessed as described previously.24
The above measurements were also made in 11- to 14-week-old mice after a 3-week period in chronic hypoxia (10% O2).23 To further evaluate LV function after chronic hypoxia, we also assessed pressure–volume relationships25 to obtain LV elastance (Emax) and LV end-diastolic pressure. The LV function studies were performed in mice 22 to 29 weeks of age, older than our initial cohort, but with similar levels of RVSP.
PA Compliance
Compliance measurements of the left branch PA of 18- to 26-week-old mice following exposure to chronic hypoxia were made with assessments of external diameter of the vessel for each level of intravascular pressure applied as described previously.26
Pulmonary Vascular Morphometry
Isolated lungs were prepared27 and embedded as described previously.23 Muscularity of distal PAs was assessed as in our previous studies,28 and the number of peripheral arteries per millimeter squared was calculated. One blinded observer performed all of the morphometric analyses.
Lung Tissue Preparation for Histological Analyses
Sections of whole-mount LacZ-stained lungs were used to assess medial wall thickness (WT) and external diameter (ED) in large preacinar PAs using the Java image-processing program ImageJ. The internal lumen 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 were stained with Hart’s stain.29 To detect changes in collagen, sections of paraformaldehyde-fixed paraffin-embedded lungs were stained with Masson’s trichrome stain. To quantify differences, the surface area of collagen relative to the area of the muscular media of the vessel wall was assessed in 2 fields at x20 magnification from each lung section using the ImageJ program described above.
Immunofluorescence and Western Immunoblotting
To determine whether LacZ reflected loss of Bmpr1a, we performed whole-mount immunohistochemistry on the aorta. A modified protocol30 was used in which tissues were incubated with rabbit anti–BMPR-IA (Orbigen Inc, San Diego, Calif; 1:200) primary antibody, followed by goat anti–rabbit-A555 (Invitrogen, Carlsbad, Calif; 1:250) secondary antibody and nuclear stain 4'-6-diamidino-2-phenylindole (DAPI) (Invitrogen; 1:1000). An aorta stained in the absence of primary antibody was used as a negative control.
Detection and quantification of BMPR-IA protein levels were assessed by Western immunoblot using heart homogenates. Detection of BMPR-IA was performed using a rabbit polyclonal BMPR-IA primary antibody (Orbigen Inc; 1:100), followed by a goat anti-rabbit IgG–horseradish peroxidase secondary antibody (Santa Cruz Biotechnology Inc, Santa Cruz, Calif; 1:5000). BMPR-IA protein expression was quantified by densitometric analysis and normalized to the level of S6 ribosomal protein (Cell Signaling Technology Inc, Danvers, Ma; 1:1000).
Primary Cell Cultures and RNA Interference
Adult human PASMCs (HPASMCs) (Cascade Biologics, Portland, Ore) and human brain vascular pericytes (HBVPs) (ScienCell Research Laboratories, San Diego, Calif) were transiently transfected with control short interference (si)RNA or human Bmpr1a siRNA (Dharmacon, Lafayatte, Colo; D-001206-13-05 and M-004933-03, respectively) using Lipofectamine 2000 (Invitrogen) under serum starvation (0.1% FBS) for 48 hours. Extracted total RNA was reverse-transcribed using the QuantiTect reverse-transcription kit (Qiagen, Valencia, Calif). Bmpr1a gene expression levels were quantified using a preverified Assay on-Demand TaqMan primer/probe sets (Applied Biosystems, Foster City, Calif) and normalized to β2 microglobulin (β2M) using the comparative 
–CT method.
Cell Proliferation and Apoptosis
Forty-eight hours following transfection in serum starvation, HPASMCs were exposed to 10% FBS (Gibco, Invitrogen) for 72 hours, and cell growth was assessed by the 3-(4,5-dimethylthiazol-yl-2)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay (American Type Culture Collection, Manassas, Va) and by cell counts using a hemocytometer. Transfected HPASMCs were exposed to 7.7 nmol/L (200 ng/mL) of BMP2 (Sigma, St Louis, Mo) for 24 hours, and apoptosis was then assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay using the In Situ Cell Death Detection kit, TMR Red (Roche Applied Science, Indianapolis, Ind). Forty-eight hours after transfection in serum starvation, HBVPs kept under serum-free conditions for an additional 24 hours, after which apoptosis was assessed by TUNEL assay.
Statistical Analysis
Values for each determination are expressed as means±SEM. For multiple group comparisons, 1-way ANOVA, followed by Bonferroni test, was performed. ANOVA followed by Dunnett’s test was used to compare the 3 genotypes under the same condition. For comparisons made between normoxia and chronic hypoxia for a given genotype, or for comparison of only 2 groups, the unpaired 2-tailed t test was used. A probability value of <0.05 was considered significant. The number of mice or samples used in each determination is indicated in the figure legends.
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Results |
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Bmpr1a Conditional Deletion in Embryos and Adult Mice
Mice were genotyped by PCR (Figure I in the online data 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 (Figure 1). No background staining was noted in the SM22
;R26R control mouse embryos (Figure 1A and 1C). Patchy blue staining was observed in the dorsal aorta, heart, and lungs of SM22;TRE-Cre;R26R control and SM22;TRE-Cre;R26R; Bmpr1aflox/+ (Figure 1B and 1D through 1F) and SM22;TRE-Cre;R26R;Bmpr1aflox/flox mutant embryos. This patchy distribution persisted in cardiovascular tissues of adult mutants and control littermates (SM22;TRE-Cre;R26R) (Figure 1G through 1L). Whole-mount LacZ staining performed on lungs and heart isolated en bloc from adult mice revealed LacZ staining in large intrapulmonary arteries (Figure 1G and 1H) and in small distal pulmonary vessels in pericytes (Figure 1I). A higher proportion of LacZ staining was apparent in the unsectioned main PAs (Figure 1J) than the preacinar large PAs, which was not the result of penetration. Similarly, unsectioned adult hearts showed patchy β-galactosidase activity throughout the ventricles (Figure 1K and 1L). To assess the pattern of Bmpr1a deletion in SM22;TRE-Cre;R26R;Bmpr1aflox/flox compared with WT adult mice, we performed whole-mount immunofluorescence studies using aortae as representative vascular tissues. Compared with WT (Figure 1M), diffuse BMPR-IA immunoreactivity was appreciably reduced in the flox/flox aortae (Figure 1N) and absent in the negative control (Figure 1O). In Western immunoblots from adult mice hearts, we confirmed a 20% reduction in protein expression of BMPR-IA in the flox/flox versus WT group (P<0.05) (Figure 1P).
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Pulmonary Vasoreactivity
To study the effect of reduced Bmpr1a in PAs on pulmonary vasoreactivity, RVSP was measured in SM22;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox KO and WT mice maintained in 40% oxygen (baseline), after inhalation of 10% O2 (acute hypoxia) for 15 minutes, and following return to baseline O2 (40%) for 10 minutes. The 3 groups of mice had similar baseline RVSP values, comparable hypoxia-mediated acute vasoconstriction (10 mm Hg rise), and return to baseline values on "recovery" in 40% O2 (Figure 2).
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P<0.05 and
RVSP and Cardiac Function in Chronic Hypoxia
SM22;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox KO and control mice exposed to chronic hypoxia had a comparable sustained increase in RVSP (Figure 3A). SM22;TRE-Cre;R26R;Bmpr1aflox/+ mice showed depressed RV maximal dP/dt, and the flox/flox group exhibited reduced maximal as well as minimal dP/dt compared with controls but only in normoxia (Figure 3B). These values were similar in all 3 groups following chronic hypoxia. Echocardiography revealed a depression in the percentage of LV fractional shortening in the flox/flox mutant mice but only under chronic hypoxia (Figure 3C). This was confirmed in another group of mice in which a depression in Emax was observed with no change in LV end-diastolic pressure measurements (supplemental Table I). No significant differences in measurements of heart rate, cardiac output, hematocrit (supplemental Table I), or mean systemic arterial pressure (data not shown) were noted among the 3 groups of mice under either normoxia or chronic hypoxia.
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RV Hypertrophy and Pulmonary Vascular Changes
The development of RVH judged by an increase in the RV weight (RV) over body weight (BW) (data not shown) or over the weight of LV plus septum (RV/LV+S) (Figure 4A) was similar in SM22;TRE-Cre;R26R;Bmpr1aflox/+, 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 remodeling31 was observed in the WT mice, as evidenced by an increase in the proportion of muscularized arteries at the alveolar duct and wall levels (Figure 4B). However, both SM22;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mice were resistant to neomuscularization, as no significant increase in the percentage of partially and fully muscularized peripheral PAs was noted. Similarly, whereas a significant 33% loss of distal PAs at the alveolar duct and wall level was detected in WT mice under chronic hypoxia, no significant decrease in the number of vessels per millimeter squared was noted in the KO groups (Figure 4C).
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PA Elastin and Collagen Deposition
To explain the hypoxia-induced rise in RVSP observed in the SM22;TRE-Cre;R26R;Bmpr1aflox/flox mice with lack of distal pulmonary vascular remodeling, we evaluated whether structural changes were present in the more proximal PAs that might reduce vascular compliance. Medial wall thickness (Figure 5A) and external diameter (Figure 5B) of LacZ-stained PAs at terminal and respiratory bronchiolus level (
200 µm of external diameter) were increased in the WT group in hypoxia versus normoxia (P<0.05) without a significant increase in internal lumen (Figure 5C). There were no significant hypoxia-induced changes in these features in the SM22;TRE-Cre;R26R;Bmpr1aflox/flox group, although room air values tended to be higher than in the WT groups. However, in the SM22;TRE-Cre;R26R;Bmpr1aflox/flox group, where there was evidence of Cre activity (green color in Figure 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 SM22;TRE-Cre;R26R;Bmpr1aflox/flox (Figure 5K) compared with control mice (Figure 5J) following chronic hypoxia, in terminal and respiratory bronchiolus vessels, but this was not evident in smaller vessels that were either nonmuscular or where muscle was abnormally differentiated from pericytes. The difference in collagen deposition was confirmed by quantitative assessments (Figure 5L) (P<0.05). We therefore determined whether this feature might be reflected in reduced compliance of the isolated left branch PA, but the response of this major vessel was similar in the 2 groups (Figure 5M).
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Loss of Bmpr1a Reduces Proliferation and Enhances Apoptosis in PASMCs but Causes Resistance to Apoptosis in Pericytes
To determine whether the reduced hypoxia-related neomuscularization in the SM22;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mice could be attributed to attenuated VSMC proliferation, we knocked down Bmpr1a in HPASMCs using RNA interference under serum deprivation. Forty-eight hours later, the mRNA transcript level of Bmpr1a relative to β2M was decreased by 66% (Figure 6A). There was a 42% reduction in cell proliferation assessed by MTT assay, 72 hours after stimulation with 10% FBS, in cells treated with siBmpr1a compared with siControl (Figure 6B). Similar results were obtained with cell counts (data not shown). Because hypoxia increases BMP2 expression in intrapulmonary arteries concomitant with enhanced apoptosis,7 we reasoned that loss of BMPR-IA might negate this effect. However, cell apoptosis assessed by TUNEL assay was 2-fold increased in siBmpr1a HPASMCs in response to a high concentration of BMP2 at 7.7 nmol/L (200 ng/mL) versus siControl cells (Figure 6C). Thus loss of BMPR-IA leads to reduced proliferation and enhanced apoptosis, even in response to BMP2.
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We next determined whether lack of appreciable hypoxia-induced reduction in the number of distal PAs in the SM22;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mice was attributable to resistance of pericytes to apoptosis, thus protecting against endothelial cell apoptosis. We knocked down Bmpr1a in HBVPs by 53% using RNA interference in serum starvation (0.1% FBS) for 48 hours (data not shown). By TUNEL assay, we noted a 68% decrease in apoptosis of siBmpr1a versus siControl pericytes (Figure 6D) when cells were serum-starved for an additional 24 hours.
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Discussion |
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The aim of the present study was to assess how reduced expression of Bmpr1a might contribute to the vascular pathology of PAH and associated RV dysfunction. The incomplete deletion of Bmpr1a likely reflects different levels of expression of Cre,32 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 explanation suggested was "microheterogeneity" of smooth muscle cells (SMCs).33 In our case, the patchy deletion was likely a function of introducing the tTA/TRE genetic components because our recent data17 show homogeneous LacZ staining in vascular SMC and cardiac myocytes of murine embryos produced using the SM22
-Cre34/LoxP-R26R system lacking these components. We pursued this model of mosaic deletion of Bmpr1a because it could be useful in assessing features that might be specifically related to these LacZ-marked cells during hypoxia-induced PH.35 The similar phenotypes in the SM22;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 SMCs rendering them haploinsufficient rather than nulls.
Although 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 PAH patients.36 It was unexpected that in neither SM22;TRE-Cre;R26R;Bmpr1aflox/+ or flox/flox mutant group was there impaired RV contractility following chronic hypoxia, but it is possible that the RVH 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% and 26% reduction in fractional shortening by echocardiography and in LV elastance by pressure–volume loops, respectively. Selective deletion of Bmpr1a in cardiac myocytes results in embryonic lethality associated with cardiac defects, including myocardial thinning.37 Therefore, it is not surprising that even "patchy" deletion is associated with increased ventricular vulnerability.
Although previous discrepancies between RVSP and the extent of vascular remodeling are documented in the SM22-driven dominant negative16 and the haploinsufficient13 Bmpr2 and in the BMP4 heterozygous nulls,38 the SM22;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutant mice actually show complete lack of distal vascular remodeling.
Aberrant muscularization of small, normally nonmuscular PAs in chronic hypoxia has been attributed to differentiation of pericytes or fibroblasts to SMCs and their subsequent proliferation,39 accompanied perhaps by resistance to apoptosis.7 We used HPASMCs derived from large arteries as a surrogate to show that loss of Bmpr1a by RNA interference was associated with reduced proliferation in response to serum and enhanced apoptosis in response to a high concentration of BMP2. Thus loss of Bmpr1a could protect against proliferation and increase susceptibility to apoptosis in response to chronic hypoxia, assuming that SMCs in small vessels have properties similar to the SMCs harvested from the proximal PA and used in our culture system.
Hypoxia-induced loss of peripheral vessels has been attributed both to endothelial cell40 and pericyte apoptosis.41 We therefore used HBVPs, in which the purity of the population was shown previously, as a surrogate for distal PA pericytes and determined that loss of Bmpr1a conferred resistance to apoptosis.
To further explain the discrepancy between the chronic hypoxia-induced elevation in RVSP, despite the lack of vascular remodeling in the SM22;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutants, we directed our attention to assessing differences in the distribution of extracellular matrix molecules known to influence vascular tone.42 Enhanced proteolytic activities causing increased extracellular matrix turnover results in net increases in deposition of structural proteins such as elastin and collagen as part of the vascular remodeling associated with PAH.42,43 We noted ectopic deposition of elastin as well as periadventitial and interstitial collagen accumulation in large preacinar arteries of the lungs of the SM22;TRE-Cre;R26R;Bmpr1aflox/flox mutant mice following chronic hypoxia. Although we could not document a reduction in compliance in the branch PA of SM22;TRE-Cre;R26R;Bmpr1aflox/flox versus control mice, it is possible that other methods that incorporate intrapulmonary vessels in assessing compliance44 would reveal a "stiffer" vasculature in these mice.
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. It is likely that modifier genes need to be expressed to derepress what appears to be a protective effect on SMC proliferation or pericyte apoptosis that is observed with BMPR-IA loss of function.
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Acknowledgments |
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We thank Dr Michal Roof for assistance in the preparation of the figures.
Sources of Funding
This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (to Y.M.) and by NIH grant R01 HL074186 (to M.R.). N.E.-B. was supported by a fellowship from the American Heart Association/Pulmonary Hypertension Association, and M.R. was supported by the Dunlevie Professorship.
Disclosures
None.
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Footnotes |
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Original received August 1, 2007; revision received October 30, 2007; accepted November 29, 2007.
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