DEVELOPMENT AND DISEASE RESEARCH ARTICLE 2981

Development 135, 2981-2991 (2008) doi:10.1242/dev.017863 SM22α-targeted deletion of bone morphogenetic receptor 1A in mice impairs cardiac and vascular development, and influences organogenesis Nesrine El-Bizri1,2, Christophe Guignabert1,2, Lingli Wang1,2, Alexander Cheng1,2, Kryn Stankunas3, Ching-Pin Chang3, Yuji Mishina4 and Marlene Rabinovitch1,2,*

Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMCs) by breeding mice possessing a loxP allele of Bmpr1a (Bmpr1aflox) expressing R26R with SM22α- Cre mice. SM22α-Cre;R26R;Bmpr1aflox/flox mice died soon after embryonic day 11 (E11) with massive vascular and pericardial hemorrhage and impaired brain development. At E10.5, SM22α-Cre;R26R;Bmpr1aflox/flox embryos showed thinning of the myocardium associated with reduced proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMCs that was also related to reduced proliferation. SM22α-Cre;R26R;Bmpr1aflox/flox mice showed collapsed telencephalon in association with impaired clearing of brain microvessels in areas where reduced apoptosis was observed. Transcript and protein levels of (MMP) 2 and 9 were reduced in E9.5 and E10.5 SM22α- Cre;R26R;Bmpr1aflox/flox embryos, respectively. Knock-down of BMPR1A by RNA interference in pulmonary artery SMCs reduced MMP2 and MMP9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP2 or MMP9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of BMPR1A reduced MMP2 activity and knock-down of either BMPR1A or MMP2 caused resistance to apoptosis. Thus, loss of Bmpr1a, by decreasing MMP2 and/or MMP9 activity, can account for vascular dilatation and persistence of brain microvessels, leading to the impaired organogenesis documented in the brain.

KEY WORDS: Bmpr1a (Alk3), Vasculogenesis, Heart development, Craniofacial development, Matrix metalloproteinase (metallopeptidase), MMP2, MMP9, Smooth muscle cell proliferation, Pericyte apoptosis, SM22α (transgelin, Tagln), Mouse

INTRODUCTION account for the progressive increase in pulmonary vascular Bone morphogenetic protein receptors (BMPRs) are members of resistance culminating in right-side heart failure (Humbert et al., the transforming growth factor β superfamily of receptors (de 2004; Rubin, 1997). Caestecker, 2004; Mehra and Wrana, 2002). Heteromeric Mice homozygous null for Bmpr2 (Beppu et al., 2000), Bmpr1a complexes form between BMPR1 and BMPR2 (Gilboa et al., (Mishina et al., 1995), the ligand Bmp4 (Winnier et al., 1995) and 2000). Aberrant BMP signaling has been linked to pulmonary the effector Smad4 (Sirard et al., 1998) die early in embryonic life arterial hypertension (PAH). Various germline mutations in BMPR2 owing to a lack of mesodermal induction. In mice with Flk1-targeted have been identified in familial and even sporadic forms of the deletion of Bmpr1a (Flk1-Cre;Bmpr1a flox/flox) (Flk1 is also known disease (Deng et al., 2000; Lane et al., 2000; Thomson et al., 2001). Kdr – Mouse Genome Informatics) (Park et al., 2006), lethality Moreover, independent of a mutation, expression of BMPR2 occurs between E10.5 and E11.5, in association with massive (Atkinson et al., 2002) and BMPR1A (Du et al., 2003) is reduced in abdominal hemorrhage. These mice exhibit dilatation of large lungs of PAH patients. vessels owing to poor recruitment of VSMCs around the EC layer, PAH is a potentially fatal disease (Abenhaim et al., 1996) but it is not clear whether the vascular phenotype is due to Bmpr1a- characterized by both obliteration of proximal pulmonary arteries deficient ECs or SMCs (Park et al., 2006). resulting from vascular smooth muscle cell (VSMC) proliferation In this study, we determined whether VSMC deletion of Bmpr1a and migration (Jeffery and Morrell, 2002), and loss of distal arteries could cause abnormalities in vasculogenesis that might explain a associated with endothelial cell (EC) (Campbell et al., 2001) and propensity to PAH. We bred mice expressing floxed Bmpr1a and pericyte apoptosis (Zhao et al., 2003). These pathological features ROSA26 with SM22α-Cre mice [SM22α is also known as transgelin (Tagln) – Mouse Genome Informatics]. Progeny homozygous for deletion of Bmpr1a, SM22α-Cre;R26R;Bmpr1a flox/flox, died soon 1Cardiopulmonary Research Program, Vera Moulton Wall Center for Pulmonary after E11 with massive vascular and pericardial hemorrhage. These Vascular Disease, Departments of 2Pediatrics and 3Medicine, Stanford University mice had a thin ventricular wall and aneurysmal dilatation of large 4 School of Medicine, Stanford, California, CA XXXXX?, USA. Molecular vessels associated with reduced myocyte proliferation related to Developmental Biology Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle decreased MMP9 and MMP2 activities. Defective brain Park, North Carolina, NC XXXXX?, USA. development documented in the SM22α-Cre;R26R;Bmpr1a flox/flox mice was associated with impaired clearing of brain microvessels *Author for correspondence (e-mail: [email protected]) related to a resistance of pericytes to apoptosis and decreased levels

Accepted 2 July 2008 of MMP2. DEVELOPMENT 2982 RESEARCH ARTICLE Development 135 (17)

MATERIALS AND METHODS IgG (1:500, Jackson ImmunoResearch). Embryos were then subjected to Experimental model: SM22α-Cre;R26R;Bmpr1aflox/flox mice DAB substrate (Vector Labs), cleared (benzyl alcohol/benzyl benzoate) for We crossed SM22α-Cre mice with mice homozygous for the floxed Bmpr1a better visualization of the vascular tree and then assessed under the (Mishina et al., 2002) and the Cre reporter gene ROSA26 (R26R) microscope or sectioned and counterstained with Methyl Green (Vector (Soriano, 1999) (Bmpr1a flox/flox;R26R+/+). F2 breeding was then realized by Labs) for histological analysis. backcrossing F1 mice (SM22α-Cre;R26R+/–;Bmpr1a flox/+) with the Bmpr1a flox/flox;R26R+/+ mice to produce mice that were SM22α- Primary cell cultures and RNA interference (RNAi) Cre;R26R;Bmpr1a flox/flox (flox/flox). All studies were performed under a Adult human pulmonary artery smooth muscle cells (HPASMCs) and protocol approved by the Animal Care Committee at Stanford University in human brain vascular pericytes (HBVPs) were cultured as previously accordance with the guidelines of the American Physiological Society. described (El-Bizri et al., 2008). Cells were transiently transfected with control, human BMPR1A, MMP9 or MMP2 siRNA (Dharmacon, Lafayatte, Genotyping CO) in Opti-MEM I (Gibco, Invitrogen) using Lipofectamine 2000 PCR was used to amplify Cre (Saam and Gordon, 1999), R26R (Soriano, (Invitrogen). ‘Starvation media’ (media supplemented with 0.1% FBS) were 1999), the floxed Bmpr1a gene (Mishina et al., 2002), and the Bmpr1a gene added 6 hours later for a total of 48 hours. with an exon 2 deletion (Mishina et al., 2002), using DNA extracted from mouse embryonic yolk sacs. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from whole E9.5 mouse embryos or from HPASMCs Preparation of embryos for histological analyses or HBVPs and then reverse transcribed as previously described (El-Bizri et al., Isolated E9.5-11 mouse embryos were fixed with formalin or 4% 2008). levels were quantified using preverified Assays-on- paraformaldehyde (PFA) in phosphate-buffered saline (PBS), embedded in Demand TaqMan primer/probe sets (Applied Biosystems, Foster City, CA) paraffin and cut transversely (7 μm). and normalized to 18S RNA and B2M for murine and human samples, respectively, using the comparative delta-CT method. Histology and immunostaining Paraffin sections of brains, hearts and dorsal aortae of formalin-fixed embryos Cell proliferation (MTT) and apoptosis (caspase 3 and 7 activity) were stained with Hematoxylin and Eosin (H&E) to assess the phenotype Forty-eight hours following transfection in 0.1% FBS, HPASMCs were resulting from deletion of Bmpr1a. To assess apoptosis, we performed the exposed to 10% FBS for 72 hours, and cell growth was assessed by the MTT TUNEL assay using the ApopTag Peroxidase In Situ Oligo Ligation Cell Proliferation Assay (American Type Culture Collection, Manassas, VA) Apoptosis Detection Kit (Chemicon International, Temecula, CA). Sections and by cell counts. Transfected HBVPs were kept under serum-free were counterstained with Methyl Green (Vector Labs, Burlingame, CA). conditions for an additional 24 hours, after which apoptosis was assessed by To assess alpha smooth muscle (αSM-actin) or the proliferating cell measuring caspase 3 and 7 activity using the Caspase 3/Caspase 7 nuclear antigen (PCNA), formalin-fixed tissue sections were incubated with Luminescent Assay Kit (Caspase-Glo, Promega, Madison, WI), and either mouse anti-αSM-actin antibody (1:200, Sigma-Aldrich, St Louis, MO) proliferation was assessed by the MTT assay. or with biotinylated mouse anti-PCNA antibody (1:100, Zymed, South San α Cell migration assay (Boyden Chamber) Francisco, CA). For SM-actin staining, sections were then incubated with Migration was assessed using a modified Boyden Chamber (BD Falcon, BD goat anti-mouse-biotinylated antibody (1:500, Jackson ImmunoResearch, α Biosciences) as previously described (Leung et al., 2004). SiControl- and West Grove, PA). For both SM-actin and PCNA staining, sections were SiBMPR1A-transfected HPASMCs were stimulated to migrate for 6 hours incubated with streptavidin-horseradish peroxidase (HRP)-conjugated in 0.1% FBS for baseline measurements and in response to 10% FBS or antibody (1:500, Jackson ImmunoResearch). Brown immunoreactivity was PDGF-BB (20 ng/ml) (R&D Systems, Minneapolis, MN) as chemo- observed by subjecting the sections to diaminobenzidine substrate (DAB; attractants in the lower compartments of the chambers. Vector Labs). Sections stained with antibodies to αSM-actin and PCNA were counterstained with Hematoxylin and Methyl Green, respectively. Gelatin zymography To assess apoptosis in brain pericytes, TUNEL assay using the ApopTag Conditioned media collected from the upper compartments of the Boyden Red In Situ Apoptosis Detection Kit (Chemicon) was followed by Chambers to evaluate production of MMPs in HPASMCs migrating in immunostaining for the pericyte marker NG2 (CSPG4 – Mouse Genome response to 0.1% FBS, 10% FBS or PDGF-BB (20 ng/ml) and from HBVPs Informatics) (primary antibody, 1:100, Chemicon) on formalin fixed-head after 48 hours of serum starvation as well as extracts of individual mouse sections. embryos were used for gelatin zymography. The supernatants were Expression of MMP2 and MMP9 in aortic walls and heart was analyzed subjected to electrophoresis in an 8% SDS-PAGE gel co-polymerized with in tissue sections of PFA-fixed embryos incubated with either an anti-MMP2 gelatin (1 mg/ml, Sigma-Aldrich) (Cann et al., 2008). The gelatinolytic (Ab-4) mouse mAb (75-7F7) or an anti-MMP9 (Ab-3) mouse mAb (56- activities were detected as transparent bands against the background of 2A4) (1:100, Calbiochem, EMD Biosciences, San Diego, CA) followed by Coomassie Brilliant Blue-stained gelatin and quantified using ImageJ. Alexa Fluor 488 goat anti-mouse IgG (H+L, 1:200, Molecular Probes, Invitrogen, Carlsbad, CA). Statistical analysis To assess BMP10 signaling in embryo hearts, PFA-fixed tissue sections Values for each determination are expressed as mean±s.e.m. For were incubated with a p57KIP2 (CDKN1C – Mouse Genome Informatics) comparisons between two groups, statistical significance was determined primary antibody (clone 57P06, 1:100, Neomarkers, Fremont, CA) followed using the unpaired two-tailed t-test. For comparisons of multiple groups, by a biotinylated rabbit anti-mouse secondary antibody (1:250, BMK-2202, one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple- MOM Kit, Vector Labs) and ABC Reagent (PK6100, ABC Elite Kit, Vector comparison test was carried out. The number of mouse embryos or samples Labs). Sections were then subjected to DAB+ (DAKO, Carpinteria, CA) and used in each experiment is provided in the figure legends. counterstained with Hematoxylin.

Whole-mount lacZ staining RESULTS E8.5-10.5 PFA-fixed mouse embryos were stained with 0.7 mg/ml X-Gal Embryonic lethality in transgenic mice with for assessment under the microscope or were sectioned and counterstained SM22α-targeted deletion of Bmpr1a with Nuclear Fast Red (Vector Labs). Mouse embryos were genotyped as described under Materials and Whole-mount PECAM staining methods and are illustrated in Fig. 1A. Ubiquitous expression of PFA-fixed E10.5 mouse embryos were incubated with PECAM antibody Bmpr1a is observed early in mouse development, starting at E6.5 [1:100, rat anti-mouse CD31 (PECAM1), clone MEC13.3, BD Pharmingen, (Mishina et al., 1995; Roelen et al., 1997). To assess the profile of

BD Biosciences, San Jose, CA] followed by HRP-conjugated goat anti-rat Cre activity reflecting areas of Bmpr1a deletion, we performed DEVELOPMENT SM22α-driven Bmpr1a loss in mice RESEARCH ARTICLE 2983

Fig. 1. Genotyping and phenotyping SM22α-Cre;Bmpr1aflox/flox mouse embryos. (A) Genotyping of mouse embryos. Targeted deletion of exon 2 of Bmpr1a occurs only in mice expressing Cre and the floxed Bmpr1a gene. SM22α-Cre;Bmpr1aflox/+ (f/+) and flox/flox (f/f) represent mice heterozygous and homozygous for the floxed gene, respectively. Mice expressing either Cre or the floxed Bmpr1a gene represent the WT group. Any mutant (f/fR26R) or WT (WTR26R) mouse expressing Cre and the R26R gene, expresses β-galactosidase and can be used for lacZ staining. (B) Cre activity in SM22α-Cre;R26R;Bmpr1aflox/flox embryos. Whole-mount lacZ staining of SM22α-Cre;R26R;Bmpr1aflox/+ embryos showing blue staining (a) in the heart (asterisk) and dorsal aorta (arrow) at E9.25, and (b) in smaller vessels as well as somatic myotomes (arrows) at E10.5. (C) Phenotype of SM22α-Cre;R26R;Bmpr1aflox/flox (b,d,f) compared with WT (a,c,e) embryos. SM22α-Cre;R26R;Bmpr1aflox/flox mutants appear normal at E9.5 (b versus a) and relatively reduced in size at E10.5 (d versus c); at E11, areas of hemorrhage are noted in the heart (asterisk) and abdomen (asterisk), as well as near the mouth (arrow) and brain (arrowhead) (f versus e). (D) Percentage of total mice of SM22α-Cre;R26R;Bmpr1aflox/flox genotype. Numbers of genotyped mice of each age are depicted in parentheses. whole-mount lacZ staining on SM22α-Cre;R26R;Bmpr1a flox/+ matched littermate control embryos (WT) was carried out to show the mouse embryos at E8.5-10.5 (Fig. 1B). Cre activity was evident in four chambers and outflow tract anatomy at multiple levels. We noted the heart from E8.5 by blue lacZ staining (data not shown), and in thinning of the ventricular wall in the flox/flox versus WT hearts (Fig. the heart and vasculature at E9.25 (Fig. 1Ba). At E10.5, smaller 2, compare D with C), quantified as a ~35% reduction in the number intersomitic vascular branches and somitic myotomes showed of ventricular cells per heart section (P<0.05) (Fig. 2E). The cardiac positive lacZ staining (Fig. 1Bb). phenotype was not due to enhanced apoptosis as only the occasional SM22α-Cre;R26R;Bmpr1a flox/flox mice appeared normal until TUNEL-positive cell was seen (Fig. 2F,G), but rather was associated E9.5 (Fig. 1C, compare b with a). By E10.5, they were somewhat with attenuated cell proliferation. There was a reduction in the smaller than wild-type (WT) (Fig. 1C, compare d with c), and at E11 percentage of PCNA-positive cells over the total number of the mice showed massive perivascular and pericardial hemorrhage ventricular cells in heart sections of the flox/flox (Fig. 2I) versus WT (Fig. 1C, compare f with e) and died soon after. Following the (Fig. 2H) (P<0.05) at E9.5 that persisted at E10.5-11 (Fig. 2J). As breeding strategy described in the Materials and methods, the Bmp10-deficient embryos show thinning of the myocardium expected frequency of the flox/flox genotype was 25%. This associated with decreased cell proliferation and ectopic expression of frequency was observed by genotyping embryos at different ages up p57KIP2 (Chen et al., 2004), we assessed the expression of p57KIP2 to E11-11.5; the frequency was 10% at E12.5, and there were no by immunofluorescence to address the possibility that a deletion of fetuses with this genotype by E18.0 or in postnatal mice assessed Bmpr1a in the heart might lead to a defect in BMP10 signaling. Our after weaning (Fig. 1D). results showed no difference in p57KIP2 immunoreactivity between mutant (Fig. 2L) and WT hearts (Fig. 2K). Cardiac defect in SM22α-Cre;R26R;Bmpr1aflox/flox embryos: thinning of the myocardium associated Vascular defect in SM22α-Cre;R26R;Bmpr1aflox/flox with reduced proliferation embryos: dilatation of large vessels associated Cre activity was confined to atrial and ventricular myocytes, with no with reduced proliferation of vascular smooth expression in the endocardium of E10.5 flox/flox embryos as muscle cells assessed by whole-mount lacZ staining (Fig. 2A,B). Myocardial To characterize the vascular defect resulting in perivascular deletion by SM22α-Cre is consistent with transient expression of hemorrhage and lethality in flox/flox embryos, we performed whole- SM22α in the developing heart (Li et al., 1996; Umans et al., 2007). mount PECAM staining on embryos at E10.5. Gross morphological To assess the sequelae of loss of Bmpr1a in cardiomyocytes on examination revealed massive dilatation of the large vessels embryonic cardiac development, histological analysis of heart appreciated in the dorsal aortae, mesenteric (Fig. 3Ab,d,f) and α flox/flox sections of viable E10.5-11 SM22 -Cre;R26R;Bmpr1a and age- cranial vessels (not shown) of the mutants versus WT littermates DEVELOPMENT 2984 RESEARCH ARTICLE Development 135 (17)

Fig. 2. Cardiac defect in SM22α-Cre;R26R;Bmpr1aflox/flox mouse embryos. (A) Blue staining (Cre activity) in the atrial (am) and ventricular (vm) myocytes but not in endocardial cells (ec) by whole-mount lacZ staining in E10.5 SM22α-Cre;R26R;Bmpr1aflox/flox embryos. (B) An enlargement of A. (C,D,F,G,H,I) Consecutive transverse sections of wild-type (WT) (C,F,H) and SM22α-Cre;R26R;Bmpr1aflox/flox (D,G,I) hearts taken at the same level from viable E11 embryos. H&E staining shows thinning of the ventricular wall (arrowheads in high-magnification insets) in the flox/flox mutant heart (D) versus WT (C). Apoptosis was infrequent in ventricular sections of E11 SM22α-Cre;R26R;Bmpr1aflox/flox mutant (G) and WT (F) by TUNEL immunostaining. However, fewer PCNA-positive cells (brown) were observed in the mutant ventricles (I, high-magnification inset, arrows) compared with the WT (H, inset, arrows). (E) A numerical assessment of hematoxylin-stained nuclei per ventricular section of E10.5-11 WT and SM22α- Cre;R26R;Bmpr1aflox/flox mutants. Bars indicate mean±s.e.m. (n=3). *P<0.05. (J) A numerical assessment of percentage of PCNA-positive cells over the total number of ventricular cells in heart sections of WT and flox/flox (f/f) mutant embryos at E9.5 and E10.5-11. Bars indicate mean±s.e.m. (n=3-4). *P<0.05. (K,L) p57KIP2 immunostaining in E10.5 WT (K) and flox/flox mutant (L) heart sections. RA, right atrium; RV and LV, right and left ventricle, respectively; EC, endocardial cushions; IVS, interventricular septum. Panels depicting WT and their corresponding mutants have the same magnification. Scale bars: 100 μm.

(Fig. 3Aa,c,e). There were more ramifications or interconnections of embryos incubated with ethidium bromide under UV light. in the interlimb vessels of the flox/flox (Fig. 3Af) versus WT (Fig. Compared with WT (Fig. 4Ba,c), flox/flox mutant embryos (Fig. 3Ae) embryos. H&E-stained transverse sections of E10.5 embryos 4Bb,d) showed brain compression and collapse of telencephalic showed dilated aortae (Fig. 3Ah) in flox/flox embryos relative to WT vesicles. These defects were apparent in H&E-stained transverse controls (Fig. 3Ag). sections of the heads at multiple levels (Fig. 4Bf,h,j,l). To determine Whole-mount staining revealed poor investment of SM22α-Cre- how loss of Bmpr1a in SM22α-expressing cells could impair brain expressing lacZ-positive cells in the dilated aortic wall of the development, we performed whole-mount PECAM staining on SM22α-Cre;R26R;Bmpr1a flox/flox mutants (Fig. 3Aj) as compared embryos at E9.5 and E10.5. We observed similar brain vessel with WT (Fig. 3Ai), where strong lacZ staining was evident. The distribution in the WT and flox/flox mutants at E9.5 (data not lacZ-positive cells were identified as being of smooth muscle shown); however, at E10.5, we noted evidence of clearing of lineage by immunoperoxidase staining using an antibody for αSM- telancephalic vessels in the WT (Fig. 4Ca,c) but not the mutants actin. There were also fewer surrounding mesenchymal cells (Fig. 4Cb,d). expressing αSM-actin in SM22α-Cre;R26R;Bmpr1a flox/flox (Fig. Transverse sections of the brains stained for PECAM at the level 3Bb) versus WT (Fig. 3Ba) embryos. TUNEL staining on sections of the nasal-mandibular processes showed histologic evidence of of aorta revealed only occasional positive mesenchymal cells (Fig. clearing of vessels in the WT heads (Fig. 4Ce), whereas flox/flox 3Bc,d, arrows). Instead, the decreased number of αSM-actin- mutant heads (Fig. 4Cf) showed persistent vessels (brown). To positive perivascular cells was consistent with reduced proliferation determine whether the clearing of vessels is related to apoptosis, the as assessed by PCNA staining (Fig. 3B, compare f with e, arrows). TUNEL assay was performed on brain sections. TUNEL-positive Quantitative analysis revealed a ~53% reduction in the percentage cells were plentiful in the WT (Fig. 4Cg) but were almost absent of PCNA-positive SMCs forming the vessel wall (Fig. 3Bg) from the flox/flox mutant heads (Fig. 4Ch). Quantitative analysis (P<0.05). PECAM staining of sections did not reveal a difference in showed a ~62% reduction in the percentage of TUNEL-positive the number of ECs surrounding the dilated vessels, but the cells cells over the total number of cells in the flox/flox group (Fig. 4Ck) appeared ‘stretched’ (data not shown). (P<0.05). Since pericytes express SM22α (Ding et al., 2004), we speculated Defective brain development of SM22α- that loss of Bmpr1a in these cells led to resistance to apoptosis and Cre;R26R;Bmpr1aflox/flox embryos associated with reduced clearing of brain microvessels. We therefore performed a impaired clearing of small vessels fluorescent TUNEL assay followed by fluorescent immunostaining Cre activity was seen in the forebrain of an E10.5 WT embryo by for NG2, a pericyte marker. Reduced apoptosis was associated with whole-mount lacZ staining (Fig. 4A). To characterize and better an increased number of pericytes in the mutant (Fig. 4Cj) versus WT

visualize any brain development abnormality, we examined heads (Fig. 4Ci) brains. Because co-localization of the TUNEL and NG2 DEVELOPMENT SM22α-driven Bmpr1a loss in mice RESEARCH ARTICLE 2985

Fig. 3. Dilated large vessels with reduced VSMCs in SM22α-Cre;R26R;Bmpr1aflox/flox mouse embryos. (A) Whole-mount PECAM staining of E10.5 embryos (a-f) shows a dilated aorta in the SM22α-Cre;R26R;Bmpr1aflox/flox compared with WT (d versus c, arrowheads). Panels c and d are high magnifications of the framed areas in a and b, respectively. Dilatation of large abdominal vessels with frequent interconnections are seen in the flox/flox mutants versus WT (f versus e, arrowheads). H&E-stained transverse sections show dilatation of the mutant aortae (h) compared with WT (g). Poor investment of lacZ staining (blue) SM22α-expressing VSMCs around the mutant aortae in sections of whole-mount mutant embyros (j) versus WT (i). Sections h and j are at the same level as g and i, respectively. (B) αSM-actin staining (brown) shows poor investment of SMCs in the dilated aortic wall of SM22α-Cre;R26R;Bmpr1aflox/flox (b) versus WT (a). TUNEL staining (arrows) revealed similar occasional apoptotic cells surrounding the dilated aorta in the flox/flox mutant (d) and WT (c). However, reduced PCNA staining (arrows) was seen in VSMCs of the dilated aorta and in the neighboring mesenchymal cells in the flox/flox (f) versus WT (e). Panels a, c and e are similar consecutive sections in the WT embryo as b, d and f in the flox/flox mutant embryo. (g) Quantification of percentage of PCNA-positive cells over total number of SMCs in the vessel walls in dorsal aortae of E10.5-11 WT and SM22α-Cre;R26R;Bmpr1aflox/flox mutants. Bars indicate mean±s.e.m. (n=4). *P<0.05. Panels depicting WT and mutants are of the same magnification. Scale bars: 100 μm in Ah,j; 50 μm in Bf. staining was not observed in the WT brain (Fig. 4Ci), we could not levels of implicated in vasculo/angiogenesis – vascular confirm ongoing apoptosis of pericytes, suggesting that this endothelial growth factor (Vegf; also known as Vegfa) and the occurred before E10.5. Persistence of brain microvessels was not angiopoietins (Angpt1, Angpt2) – and of the phosphatase and due to enhanced cell growth, as PCNA immunoreactivity showed no tensin homolog gene (Pten), a known gene downstream of BMP difference between E10.5 WT and flox/flox mutants (Fig. 4Cl). signaling implicated in juvenile polyposis (He et al., 2004) that might also impact cell growth (Beck and Carethers, 2007). RNA Reduced MMP9 and MMP2 in embryos with was extracted from E9.5 embryos, preceding the appearance of the SM22α-targeted deletion of Bmpr1a phenotype in SM22α-Cre;R26R;Bmpr1a flox/flox mice. We found a We investigated the level of expression of candidate genes significant decrease in the mRNA expression of Mmp9 (P<0.05) dysregulated by loss of Bmpr1a that could account both for and Mmp2 (P<0.05), and trends toward reduced expression of resistance to apoptosis in pericytes and repression of proliferation tenascin C (Tnc), fibronectin, connective tissue growth factor in VSMCs. For example, BMPs increase MMP activity and (Ctgf) and urokinase plasminogen activator (uPA; Plau – Mouse mRNA expression (Mishina et al., 2004; Palosaari et al., 2003) and Genome Informatics) were observed (Fig. 5A). No differences in MMPs can regulate cell survival (Jones et al., 1997) and induce tissue plasminogen activator (tPA; Plat) (Fig. 5A), Angpt1, Angpt2, proliferation of VSMCs (Zempo et al., 1994). Quantitative RT- Vegf (data not shown) and Pten (Fig. 5A) mRNA levels were noted PCR was applied to embryonic extracts to assess differential between the WT and mutants. expression of MMPs and other genes that This decrease in Mmp2 and Mmp9 transcripts was associated with could be modulated by loss of Bmpr1a and might account for these a decrease, although not statistically significant, in the pro (40%) and altered vascular cell phenotypes. Many of the genes that we active (30%) forms of MMP2 in SM22α-Cre;R26R;Bmpr1a flox/flox assessed are modified in other embryonic mouse models of mutants versus WT, as assessed by gelatin zymography on mouse

vascular dilatation (Oh et al., 2000). We also assessed the transcript embryos (data not shown). DEVELOPMENT 2986 RESEARCH ARTICLE Development 135 (17)

To determine whether the decrease in mRNA levels of Mmp9 and siRNA (SiBMPR1A), and observed, by gelatin zymography, that Mmp2 in whole E9.5 mutant mouse embryos is translated into SiBMPR1A-transfected cells had decreased levels of the pro and reduced protein expression at a later age, we performed active forms of MMP9 (P<0.05 for both) and of MMP2 (P<0.01 and immunostaining of MMP9 and MMP2 at E10.5. We found abundant P<0.05, respectively) versus SiControl-transfected cells (Fig. 6A). MMP9 and, to a greater extent, MMP2, in the aortic walls of WT Consistent with our hypothesis and our findings in the mouse embryos (Fig. 5Ba,c) and only weak immunoreactivity in the embryo, we showed that RNAi-mediated reduction in mRNA of mutants (Fig. 5Bb,d). However, a low and diffuse immunostaining BMPR1A (by 66%), MMP9 (to undetectable levels) or MMP2 (by was noted in the heart and brains of WT and mutants (data not >80%), resulted in a 35-40% reduction in HPASMC proliferation in shown). response to 10% FBS as assessed by the MTT assay (P<0.001, Fig. 6B) and cell counts (data not shown). Since MMP9 and MMP2 Loss of BMPR1A attenuates proliferation and levels increase in migrating SMCs (Bendeck et al., 2002; Franco et directed migration of vascular smooth muscle al., 2006; Kuzuya et al., 2003; Mason et al., 1999), we determined cells and induces pericyte resistance to apoptosis whether the chemotactic migratory behavior of SMCs was impaired via reduced MMP9 and MMP2 by loss of BMPR1A, in association with reduced MMP9 and/or Subsequent studies were carried out using cultured human MMP2. A deficiency in SMC migration could also account for the pulmonary artery smooth muscle cells (HPASMCs) and human lack of SMC investment of the aneurysmally dilated vessels in brain (micro)vascular pericytes (HBVPs) to determine (1) whether flox/flox embryos. We serum starved HPASMCs in 0.1% FBS for 48 reducing levels of BMPR1A by RNAi would result in suppression hours and then assessed their response to a 6-hour treatment with of MMP9 and/or MMP2 activities and (2) whether reducing PDGF-BB (20 ng/ml) using a modified Boyden Chamber assay. The BMPR1A, MMP9 and/or MMP2, represses proliferation of VSMCs MMP9 and MMP2 activities in SiControl HPASMCs, as assessed and induces resistance to apoptosis in pericytes. We reduced the by gelatin zymography, were repressed in SiBMPR1A-treated cells mRNA level of BMPR1A by 66% by transfecting HPASMCs with (P<0.01 for MMP9 and P<0.001 for proMMP2 and MMP2) (Fig.

Fig. 4. Brain distortion in SM22α-Cre;R26R;Bmpr1aflox/flox mouse embryos. (A) Whole-mount lacZ staining of an E10.5 WT embryo brain showing blue staining in the forebrain. (B) Collapse of telencephalic vesicles (b arrows) and compressed brains (d) of SM22α- Cre;R26R;Bmpr1aflox/flox versus WT (a,c) embryos at E10.5. H&E-stained cross-sections of WT (e,g,i,k) and mutant (f,h,j,l) head at the levels of the dashed lines in c and d, respectively. Sections 1, 2 and 3 are at levels of the frontonasal processes and section 4 is at the nasal-mandibular level. Panels depicting WT and mutants are of the same magnification. (C) Whole-mount PECAM staining shows unilateral clearing of telancephalic vessels in the WT (a,c) but not the flox/flox (b,d) head at E10.5. The clearing is evident at nasal-mandibular areas of the WT head (e) as opposed to resistance to clearing (arrows) in the mutant (f). TUNEL staining (arrows) on brain sections show less apoptosis in the mutants (h) than WT (g). By TUNEL assay (red) combined with NG2 immunofluorescence (green), no apoptosis was seen in NG2-positive areas in the WT brain sections (i); however, more immunoreactivity was seen in the mutants (j). Panels depicting WT are of the same magnification as their corresponding mutants. Percentage TUNEL-positive (k) and PCNA-positive (l) over total number of cells in nasal-mandibular areas (examined using a 20ϫ objective) of E10.5 WT and SM22α-Cre;R26R; Bmpr1aflox/flox (f/f) mutants. Bars indicate mean±s.e.m. (n=3 in k and n=3-4 in l). *P<0.05. Scale bars: 100 μm in Cf,h,j; μ

200 m in Bl. DEVELOPMENT SM22α-driven Bmpr1a loss in mice RESEARCH ARTICLE 2987

Fig. 5. Attenuated MMP9 and MMP2 expression in SM22α-Cre;R26R;Bmpr1aflox/flox mouse embryos. (A) Profiling by qRT-PCR the expression of genes that might be modified by the SM22α-targeted deletion of Bmpr1a. Values are shown relative to 18S RNA. n=3 where each sample combines 3-4 embryos. *P<0.05. Eln, elastin; Colla1, collagen Ia1. (B) Reduction of MMP9 (b versus WT in a) and MMP2 (d versus WT in c) protein expression in aortic walls of flox/flox embryos as assessed by immunofluorescence. Note the absence of immunoreactivity in an aortic section of a WT incubated with only the secondary antibody as a negative control (e). Panels depict WT and mutants at the same magnification. Scale bar: 50 μm.

7A). Although basal levels of migration were increased in starvation (0.1% FBS) for 48 hours, we showed a 53% reduction in SiBMPR1A-transfected HPASMCs (P<0.001), these cells did not BMPR1A transcript levels in HBVPs. To induce apoptosis, cells significantly migrate in response to PDGF-BB when compared with were serum deprived for an additional 24 hours, after which a SiControl HPASMCs (P<0.05) (Fig. 7B). reduction in both pro and active forms of MMP2 was demonstrated We then investigated whether pericytes with loss of BMPR1A by gelatin zymography (P<0.05) (Fig. 8A). We then used RNAi to would be resistant to apoptosis owing to a reduction in MMP2 reduce mRNA levels of MMP2 in HBVPs and confirmed the activity, as MMP2 activity is proapoptogenic in pericytes of diabetic decrease in pro and active forms of MMP2 by gelatin zymography patients (Yang et al., 2007). Using RNAi under conditions of serum (P<0.001) (Fig. 8B). A control experiment showing the sensitivity

Fig. 6. RNAi-induced loss of BMPR1A, by reducing MMP9 and MMP2 expression, attenuates proliferation of human vascular smooth muscle cells (HPASMCs). (A) Gelatin zymography using conditioned media of HPASMCs. Gelatin zymograms performed on conditioned media (left) and densitometric analysis (right) show MMP9 and MMP2 activities in SiBMPR1A-treated (SiBR1A) versus SiControl-treated (SiC) cells in response to serum (10% FBS) for 6 hours. Bars indicate mean±s.e.m. of densitometric values of SiBR1A proMMP9, MMP9, proMMP2 and MMP2 normalized to their corresponding SiC values (n=3-4). *P<0.05 and **P<0.01 between SiBR1A and SiC. (B) Proliferation of HPASMCs in response to serum using the MTT assay. HPASMCs transfected with SiC (white bars), SiBR1A, SiMMP9, SiMMP2 and combined SiMMP9 and SiMMP2 were subjected to 0.1% FBS (all white bars) or stimulated with 10% FBS (all black bars) for 72 hours, after which proliferation was assessed by the MTT assay. Bars indicate mean±s.e.m. of arbitrary OD570 values normalized to values of SiC under 0.1% FBS. n=12 for siMMP2 and/or siMMP9 and n=26 for SiC and SiBMPR1A from three independent experiments. †††P<0.001 for serum-stimulated versus unstimulated comparisons, and ***P<0.001

for comparisons with SiC at 10% FBS. DEVELOPMENT 2988 RESEARCH ARTICLE Development 135 (17)

Fig. 7. RNAi-induced loss of BMPR1A, by reducing MMP9 and MMP2 expression, impairs directed migration of human vascular smooth muscle cells. (A) Gelatin zymography in response to PDGF-BB. Gelatin zymography performed on conditioned media (left) and densitometric analysis (right) show MMP9 and pro and active MMP2 activities in SiBMPR1A- treated (SiBR1A) versus SiControl-treated (SiC) cells in response to PDGF-BB (20 ng/ml) stimulation for 6 hours. Bars indicate mean±s.e.m. of densitometric values of SiBR1A MMP forms normalized to the corresponding SiC values. (n=4). **P<0.01, ***P<0.001. (B) Migration in response to PDGF-BB as assessed by modified Boyden Chamber assay. SiC- or SiBR1A-transfected HPASMCs were stimulated with 20 ng/ml of PDGF-BB for 6 hours. Bars represent mean±s.e.m. of migrating cells in 5-6 different microscopic fields. (n=4). †P<0.05 for stimulated versus unstimulated comparisons for each Si; ***P<0.001 for comparisons between SiBR1A and SiC. Representative micrographs show the number of migrating cells under each condition. Scale bar: 100 μm. of gel zymography to detect gelatinase activity in a dose-dependent 2004). However, our results showed that BMP10 signaling in E10.5 manner is provided in Fig. S1 (see supplementary material). SM22α-Cre;R26R;Bmpr1a flox/flox heart sections was not affected by Transfecting HBVPs with SiBMPR1A, or with SiMMP2, induced Bmpr1a deletion. resistance to apoptosis when compared with SiControl HBVPs It is interesting that in the mouse in which Bmpr1a was deleted (P<0.001), as assessed by caspase 3 and 7 activities (Fig. 8C), following activation of the cardiac myocyte-specific promoter alpha without affecting cell proliferation as assessed by the MTT assay myosin heavy chain (αMHC-Cre;Bmpr1a flox/flox) (Gaussin et al., (data not shown). Therefore, lack of MMP2 in pericytes could 2002), the ventricular thinning that took place at a later time point account for the resistance to apoptosis seen in highly vascularized (E11.5-12.5) was attributed to enhanced apoptosis. Ventricular areas of flox/flox mutant brains. thinning was also seen at E11.5-12.5 in mice lacking Bmpr1a in cardiac progenitors (Islet1-Cre;Bmpr1a nulls) (Yang et al., 2006), DISCUSSION or at E11.5 with cardiac-specific ablation of Smad4 (Song et al., Bmpr1a in cardiac development 2007), and both were associated with attenuated proliferation and The cardiac phenotype in E10.5 SM22α-Cre;R26R;Bmpr1a flox/flox enhanced apoptosis of the ventricular septal myocytes. This suggests mouse embryos was characterized by thinning of the ventricular that differences in the timing of promoter activation and Bmpr1a wall and was attributed to reduced cell proliferation evident at E9.5. deletion in cardiomyocytes might dictate whether the thinning of the Bmp10-null mice also develop hearts with hypoplastic walls owing ventricular wall will be the result of apoptosis and/or reduced to reduced proliferation of cardiac myocytes at E9.0-9.5 (Chen et al., proliferation.

Fig. 8. RNAi-induced loss of BMPR1A, by reducing MMP2 expression, attenuates apoptosis of human pericytes (HBVPs). (A) Gelatin zymography using conditioned media of HBVPs in response to serum deprivation. Gelatin zymograms of conditioned media (top) with densitometric analysis (beneath) to assess MMP2 activities in SiBMPR1A-transfected (SiBR1A) as compared with SiControl-transfected (SiC) cells. Bars represent mean±s.e.m. of densitometric values of SiBR1A MMP2 forms normalized to the corresponding SiC value. n=7-8 for pro and n=3-4 for active MMP2. *P<0.05 versus SiC. (B) Reduced MMP2 activity in HBVPs transfected with MMP2 RNAi. Gelatin zymogram of conditioned media (top) and densitometric values (beneath) of MMP2 in HBVPs transfected with SiMMP2. Bars represent mean±s.e.m. of densitometric values of SiBR1A MMP2 forms normalized to SiC value (n=4). ***P<0.001. (C) Apoptosis of HBVPs in response to serum deprivation using caspase 3/7 assay. Apoptosis in HBVPs transfected with SiC, SiBR1A or SiMMP2 was induced by serum-deprivation for 24 hours and assessed by a caspase 3/7 luminescent assay. Bars represent mean±s.e.m. of arbitrary luminescent values (n=6-9). ***P<0.001

versus SiC. DEVELOPMENT SM22α-driven Bmpr1a loss in mice RESEARCH ARTICLE 2989

Bmpr1a and vasculogenesis We cannot exclude the possibility that the myocardial thinning is secondary to a hemodynamic abnormality caused by the vascular phenotype observed in the SM22α-Cre;R26R;Bmpr1a flox/flox embryos, and characterized by aneurysmal dilatation of the dorsal aorta and other large vessels. Dilatation of the aorta was observed in embryos with Flk1-targeted deletion of Bmpr1a (Flk1- Cre;Bmpr1a flox/flox) (Park et al., 2006) and in embryos null for Alk1 (Acvrl1 – Mouse Genome Informatics) (Oh et al., 2000) or Smad5 (Yang et al., 1999). In those models, the dilatation was attributed to a paracrine effect of Bmpr1a-deficient ECs repressing the recruitment of VSMCs or pericytes, as observed in mice lacking PDGF-BB or PDFG-Rβ (Hellstrom et al., 1999; Lindahl et al., 1997). Other possibilities suggested include poor transdifferentiation of ECs into SMCs, or a defect in SMC growth affecting vessel maturation and integrity (Park et al., 2006). The Fig. 9. Schematic of defective BMPR1A signaling in vascular smooth muscle cells and pericytes. Deletion of BMPR1A results in third explanation fits best with the further delineation of the decreased MMP9 and MMP2 activities in VSMCs, and decreased MMP2 α flox/flox phenotype of SM22 -Cre;R26R; Bmpr1a embryos that we activity in pericytes, culminating in reduced proliferation and apoptosis, carried out. respectively. We were able to assess the impact of Bmpr1a deletion in reducing VSMC proliferation in the tissue as well as in cultured cells, in which we also observed impaired PDGF-BB-directed VSMC observed in abdominal aortic aneurysm (Goodall et al., 2001; migration. No defect in the EC layer was noted in the SM22α- Thompson et al., 1995). Moreover, reduction of MMP9 activity by Cre;R26R;Bmpr1a flox/flox embryos by whole-mount PECAM Doxycycline protects against experimentally induced aortic immunostaining (data not shown) that might explain the vascular aneurysm (Kaito et al., 2003), as does local expression of TIMP1, defect through a non-cell-autonomous contribution. In addition, we an inhibitor of MMP9 activity (Allaire et al., 1998; McMillan et al., did not observe upregulation of angiogenic factors, such as of 1995). In addition, mice that are null for Mmp9 are resistant to Angpt1 and Angpt2 as described in Alk1-null embryos (Oh et al., elastase-induced aortic aneurysms (Pyo et al., 2000). It therefore 2000), or of Vegf as observed in both Flk1-Cre;Bmpr1a flox/flox (Park appears that during vascular development, a reduction in both et al., 2006) and Alk1 nulls (Oh et al., 2000). It follows that MMP9 and MMP2 in SMCs is required to produce aneurysmal there was no concomitant angiogenic defect in SM22α- dilatation, as a result of reduced proliferation and perhaps migration Cre;R26R;Bmpr1a flox/flox mutant embryos, such as the impaired yolk of SMCs. It is interesting that the Mmp2/9 double nulls (Lambert et sac vascular remodeling seen in the Flk1-Cre;Bmpr1a flox/flox (Park al., 2003) do not recapitulate our phenotype. This could reflect et al., 2006) and the Alk1-null (Oh et al., 2000) embryos. Since Flk1 compensatory induction of other MMPs in response to a global, is a mesodermal marker and Alk1 is mostly expressed in ECs, the rather than a tissue-specific, deletion. Alternatively, the mixed angiogenic defect in the yolk sac is likely to be due to the loss of background of the flox/flox mutants compared with the C57BL/6J Bmpr1a in ECs, a feature we reproduced by ablating Bmpr1a using background of the Mmp2/9 double nulls might account for the Tie2-Cre (our unpublished observations) (Tie2 is also known as Tek). difference in the phenotype. In contrast to other mice models of aneurysmal vascular dilatation, the SM22α-Cre;R26R;Bmpr1a flox/flox mice did not exhibit an Bmpr1a expression in pericytes mediates vessel increase in expression of proteases such as uPA and tPA (Oh et al., regression during brain development 2000; Park et al., 2006). In contrast to the Smad5-null embryos with SM22α-Cre;R26R;Bmpr1a flox/flox mutants showed severe brain dilated aorta, the SM22α-Cre;R26R; Bmpr1a flox/flox embryos did not asymmetry and collapse of telencephalic vesicles. A vascular defect show apoptosis in VSMCs or in neighboring mesenchymal cells produced by impaired BMPR1A signaling that has not previously (Yang et al., 1999). been described might explain these abnormalities. When we assessed gene expression of extracellular matrix Regression of vessels is crucial in triggering mesenchymal and proteinases previously implicated in VSMC condensation culminating in chondrogenesis and skeletogenesis (Yin proliferation and migration, a consistent reduction in the expression and Pacifici, 2001). As MMP2 activity is linked to retinal pericyte of Mmp9 and Mmp2, genes downstream of BMP signaling in other apoptosis in diabetic retinopathy (Yang et al., 2007), we reasoned that cell types (Mishina et al., 2004; Palosaari et al., 2003), was observed. suppression of MMP2 resulting from lack of BMPR1A signaling A direct association between reduced BMPR1A and impaired might make pericytes resistant to apoptosis, preventing EC apoptosis production of MMP9 and MMP2 was then demonstrated in cultured and microvessel clearing, and subsequently leading to defective brain human VSMCs, in which knock-down of BMPR1A by RNAi development. Indeed, we showed that lack of BMPR1A or MMP2 by attenuated MMP9 and MMP2 activities. The role of both MMP9 RNAi renders pericytes in culture resistant to apoptosis. and MMP2 in VSMC proliferation and migration is well The same phenomenon might explain the enhanced ramification documented (Bendeck et al., 2002; Franco et al., 2006; Kuzuya et of the interlimb vessels seen in the flox/flox mutants, suggesting that al., 2003; Mason et al., 1999). Expression of Pten, downstream of vascular deletion of Bmpr1a might impair organogenesis of other Bmpr1a and implicated in juvenile polyposis and affecting cell tissues not investigated here. In the rat aortic model of growth, was not modified in the mutants. angiogenesis, MMP9 and MMP2 expression and activity not only Our observations linking reduced MMP9 and MMP2 to increased during the angiogenic growth phase of microvessels, but aneurysmal dilatation might seem at odds with clinical studies in also remained elevated and were necessary for microvessel

human tissue in which increased MMP2 and especially MMP9 are regression (Zhu et al., 2000). Consistent with this, maximal MMP2 DEVELOPMENT 2990 RESEARCH ARTICLE Development 135 (17) activity is observed in the late corpus luteum concomitant with Cann, G. M., Guignabert, C., Ying, L., Deshpande, N., Bekker, J. M., Wang, vessel regression (Duncan et al., 1998). The deletion of Bmpr1a in L., Zhou, B. and Rabinovitch, M. (2008). Developmental expression of LC3alpha and beta: absence of fibronectin or autophagy phenotype in LC3beta brain cells (Hebert et al., 2002) did not recapitulate the phenotype, knockout mice. Dev. Dyn. 237, 187-195. further indicating the importance of the vasculature in this cell- Chen, H., Shi, S., Acosta, L., Li, W., Lu, J., Bao, S., Chen, Z., Yang, Z., autonomous mechanism. Schneider, M. D., Chien, K. R. et al. (2004). BMP10 is essential for maintaining The discrepancy between the phenotypes resulting from loss of cardiac growth during murine cardiogenesis. Development 131, 2219-2231. de Caestecker, M. (2004). The transforming growth factor-beta superfamily of BMPR1A in VSMCs and pericytes might be related to the fact that receptors. Cytokine Growth Factor Rev. 15, 1-11. they have different basement membranes (Meyrick and Reid, 1979) Deng, Z., Morse, J. H., Slager, S. L., Cuervo, N., Moore, K. J., Venetos, G., and hence could exhibit different effects resulting from reduced Kalachikov, S., Cayanis, E., Fischer, S. G., Barst, R. J. et al. (2000). Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the MMPs (Fig. 9). bone morphogenetic protein receptor-II gene. Am. J. Hum. Genet. 67, 737-744. It is worth mentioning that our findings did not recapitulate any Ding, R., Darland, D. C., Parmacek, M. S. and D’Amore, P. A. (2004). aspects of juvenile polyposis (JP), a condition associated with Endothelial-mesenchymal interactions in vitro reveal molecular mechanisms of smooth muscle/pericyte differentiation. Stem Cells Dev. 13, 509-520. mutations in BMPR1A. However, the site of pathology in JP, Du, L., Sullivan, C. C., Chu, D., Cho, A. J., Kido, M., Wolf, P. L., Yuan, J. X., the villus, forms after E15.5 (Batts et al., 2006) and SM22α- Deutsch, R., Jamieson, S. W. and Thistlethwaite, P. A. (2003). Signaling Cre;R26R;Bmpr1a flox/flox embryos die several days earlier. molecules in nonfamilial pulmonary hypertension. New Engl. J. Med. 348, 500- 509. Duncan, W. C., McNeilly, A. S. and Illingworth, P. J. (1998). The effect of luteal Clinical significance “rescue” on the expression and localization of matrix metalloproteinases and Our study is the first to show that both MMP9 and MMP2 are their tissue inhibitors in the human corpus luteum. J. Clin. Endocrinol. Metab. developmentally regulated by expression of Bmpr1a and that 83, 2470-2478. El-Bizri, N., Wang, L., Merklinger, S. L., Guignabert, C., Desai, T., Urashima, attenuation in their levels could reduce the proliferation of SMCs T., Sheikh, A. Y., Knutsen, R. H., Mecham, R. P., Mishina, Y. et al. (2008). leading to aneurysmal dilatation of large vessels. These observations Smooth muscle protein 22alpha-mediated patchy deletion of Bmpr1a impairs could also explain the reduced cell proliferation that leads to cardiac contractility but protects against pulmonary vascular remodeling. Circ. thinning of the ventricular wall. Our findings linking repression of Res. 102, 380-388. Franco, C., Ho, B., Mulholland, D., Hou, G., Islam, M., Donaldson, K. and Bmpr1a-mediated MMP2 activity to reduced apoptosis of pericytes, Bendeck, M. P. (2006). Doxycycline alters vascular smooth muscle cell adhesion, point to a feature not only of developmental importance in clearing migration, and reorganization of fibrillar collagen matrices. Am. J. Pathol. 168, of microvessels, but potentially to a mechanism that might help in 1697-1709. Gaussin, V., Van de Putte, T., Mishina, Y., Hanks, M. C., Zwijsen, A., preserving or regenerating microvessels in disease. In our recent Huylebroeck, D., Behringer, R. R. and Schneider, M. D. (2002). Endocardial studies (El-Bizri et al., 2008), in which patchy deletion of Bmpr1a cushion and myocardial defects after cardiac myocyte-specific conditional was induced in VSMCs, mice were actually protected against both deletion of the bone morphogenetic protein receptor ALK3. Proc. Natl. Acad. Sci. USA 99, 2878-2883. the excessive muscularization and loss of distal vessels associated Gilboa, L., Nohe, A., Geissendorfer, T., Sebald, W., Henis, Y. I. and Knaus, P. with chronic hypoxia-induced PAH. (2000). Bone morphogenetic protein receptor complexes on the surface of live cells: a new oligomerization mode for serine/threonine kinase receptors. Mol. This research is supported by the Intramural Research Program of the NIH, Biol. Cell 11, 1023-1035. NIEHS to Y.M., and by NIH Grant R01 HL074186 to M.R. N.E. is supported by a Goodall, S., Crowther, M., Hemingway, D. M., Bell, P. R. and Thompson, M. fellowship from the American Heart Association (AHA)/Pulmonary M. (2001). Ubiquitous elevation of matrix metalloproteinase-2 expression in the Hypertension Association and M.R. by the Dunlevie Professorship. C.-P.C. is vasculature of patients with abdominal aneurysms. Circulation 104, 304-309. supported by funds from the National Heart Lung and Blood Institute He, X. C., Zhang, J., Tong, W. G., Tawfik, O., Ross, J., Scoville, D. H., Tian, Q., (HL085345), AHA, Children Heart Foundation, March of Dimes Foundation Zeng, X., He, X., Wiedemann, L. M. et al. (2004). BMP signaling inhibits and Baxter Foundation. K.S. is supported by an AHA postdoctoral fellowship. intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nat. Genet. 36, 1117-1121. Supplementary material Hebert, J. M., Mishina, Y. and McConnell, S. K. (2002). BMP signaling is required locally to pattern the dorsal telencephalic midline. Neuron 35, 1029-1041. Supplementary material for this article is available at Hellstrom, M., Kalen, M., Lindahl, P., Abramsson, A. and Betsholtz, C. (1999). http://dev.biologists.org/cgi/content/full/135/17/2981/DC1 Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. References Development 126, 3047-3055. Abenhaim, L., Moride, Y., Brenot, F., Rich, S., Benichou, J., Kurz, X., Humbert, M., Morrell, N. W., Archer, S. L., Stenmark, K. R., MacLean, M. R., Higenbottam, T., Oakley, C., Wouters, E., Aubier, M. et al. (1996). Lang, I. M., Christman, B. W., Weir, E. K., Eickelberg, O., Voelkel, N. F. et Appetite-suppressant drugs and the risk of primary pulmonary hypertension. al. (2004). Cellular and molecular pathobiology of pulmonary arterial International Primary Pulmonary Hypertension Study Group. New Engl. J. Med. hypertension. J. Am. Coll. Cardiol. 43, 13S-24S. 335, 609-616. Jeffery, T. K. and Morrell, N. W. (2002). Molecular and cellular basis of Allaire, E., Forough, R., Clowes, M., Starcher, B. and Clowes, A. W. (1998). pulmonary vascular remodeling in pulmonary hypertension. Prog. Cardiovasc. Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and Dis. 45, 173-202. rupture in a rat model. J. Clin. Invest. 102, 1413-1420. Jones, P. L., Crack, J. and Rabinovitch, M. (1997). Regulation of tenascin-C, a Atkinson, C., Stewart, S., Upton, P. D., Machado, R., Thomson, J. R., vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 Trembath, R. C. and Morrell, N. W. (2002). Primary pulmonary hypertension is integrin to promote epidermal growth factor receptor phosphorylation and associated with reduced pulmonary vascular expression of type II bone growth. J. Cell Biol. 139, 279-293. morphogenetic protein receptor. Circulation 105, 1672-1678. Kaito, K., Urayama, H. and Watanabe, G. (2003). Doxycycline treatment in a Batts, L. E., Polk, D. B., Dubois, R. N. and Kulessa, H. (2006). Bmp signaling is model of early abdominal aortic aneurysm. Surg. Today 33, 426-433. required for intestinal growth and morphogenesis. Dev. Dyn. 235, 1563-1570. Kuzuya, M., Kanda, S., Sasaki, T., Tamaya-Mori, N., Cheng, X. W., Itoh, T., Beck, S. E. and Carethers, J. M. (2007). BMP suppresses PTEN expression via Itohara, S. and Iguchi, A. (2003). Deficiency of gelatinase a suppresses smooth RAS/ERK signaling. Cancer Biol. Ther. 6, 1313-1317. muscle cell invasion and development of experimental intimal hyperplasia. Bendeck, M. P., Conte, M., Zhang, M., Nili, N., Strauss, B. H. and Farwell, S. Circulation 108, 1375-1381. M. (2002). Doxycycline modulates smooth muscle cell growth, migration, and Lambert, V., Wielockx, B., Munaut, C., Galopin, C., Jost, M., Itoh, T., Werb, matrix remodeling after arterial injury. Am. J. Pathol. 160, 1089-1095. Z., Baker, A., Libert, C., Krell, H. W. et al. (2003). MMP-2 and MMP-9 Beppu, H., Kawabata, M., Hamamoto, T., Chytil, A., Minowa, O., Noda, T. synergize in promoting choroidal neovascularization. FASEB J. 17, 2290-2292. and Miyazono, K. (2000). BMP type II receptor is required for gastrulation and Lane, K. B., Machado, R. D., Pauciulo, M. W., Thomson, J. R., Phillips, J. A., early development of mouse embryos. Dev. Biol. 221, 249-258. 3rd, Loyd, J. E., Nichols, W. C. and Trembath, R. C. (2000). Heterozygous Campbell, A. I., Zhao, Y., Sandhu, R. and Stewart, D. J. (2001). Cell-based germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial gene transfer of vascular endothelial growth factor attenuates monocrotaline- primary pulmonary hypertension. The International PPH Consortium. Nat. Genet.

induced pulmonary hypertension. Circulation 104, 2242-2248. 26, 81-84. DEVELOPMENT SM22α-driven Bmpr1a loss in mice RESEARCH ARTICLE 2991

Leung, W. C., Lawrie, A., Demaries, S., Massaeli, H., Burry, A., Yablonsky, S., Rubin, L. J. (1997). Primary pulmonary hypertension. New Engl. J. Med. 336, 111- Sarjeant, J. M., Fera, E., Rassart, E., Pickering, J. G. et al. (2004). 117. Apolipoprotein D and platelet-derived growth factor-BB synergism mediates Saam, J. R. and Gordon, J. I. (1999). Inducible gene knockouts in the small vascular smooth muscle cell migration. Circ. Res. 95, 179-186. intestinal and colonic epithelium. J. Biol. Chem. 274, 38071-38082. Li, L., Miano, J. M., Cserjesi, P. and Olson, E. N. (1996). SM22 alpha, a marker Sirard, C., de la Pompa, J. L., Elia, A., Itie, A., Mirtsos, C., Cheung, A., Hahn, of adult smooth muscle, is expressed in multiple myogenic lineages during S., Wakeham, A., Schwartz, L., Kern, S. E. et al. (1998). The tumor embryogenesis. Circ. Res. 78, 188-195. suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior Lindahl, P., Johansson, B. R., Leveen, P. and Betsholtz, C. (1997). Pericyte loss development of the mouse embryo. Genes Dev. 12, 107-119. and microaneurysm formation in PDGF-B-deficient mice. Science 277, 242-245. Song, L., Yan, W., Chen, X., Deng, C. X., Wang, Q. and Jiao, K. (2007). Mason, D. P., Kenagy, R. D., Hasenstab, D., Bowen-Pope, D. F., Seifert, R. A., Myocardial smad4 is essential for cardiogenesis in mouse embryos. Circ. Res. Coats, S., Hawkins, S. M. and Clowes, A. W. (1999). Matrix 101, 277-285. metalloproteinase-9 overexpression enhances vascular smooth muscle cell Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter migration and alters remodeling in the injured rat carotid artery. Circ. Res. 85, strain. Nat. Genet. 21, 70-71. 1179-1185. Thompson, R. W., Holmes, D. R., Mertens, R. A., Liao, S., Botney, M. D., McMillan, W. D., Patterson, B. K., Keen, R. R., Shively, V. P., Cipollone, M. Mecham, R. P., Welgus, H. G. and Parks, W. C. (1995). Production and and Pearce, W. H. (1995). In situ localization and quantification of mRNA for localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An 92-kD type IV collagenase and its inhibitor in aneurysmal, occlusive, and normal elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. aorta. Arterioscler. Thromb. Vasc. Biol. 15, 1139-1144. J. Clin. Invest. 96, 318-326. Mehra, A. and Wrana, J. L. (2002). TGF-beta and the Smad signal transduction Thomson, J., Machado, R., Pauciulo, M., Morgan, N., Yacoub, M., Corris, P., pathway. Biochem. Cell Biol. 80, 605-622. McNeil, K., Loyd, J., Nichols, W. and Trembath, R. (2001). Familial and Meyrick, B. and Reid, L. (1979). Ultrastructural features of the distended sporadic primary pulmonary hypertension is caused by BMPR2 gene mutations pulmonary arteries of the normal rat. Anat. Rec. 193, 71-97. resulting in haploinsufficiency of the bone morphogenetic protein type II Mishina, Y., Suzuki, A., Ueno, N. and Behringer, R. R. (1995). Bmpr encodes a receptor. J. Heart Lung Transplant. 20, 149. type I bone morphogenetic protein receptor that is essential for gastrulation Umans, L., Cox, L., Tjwa, M., Bito, V., Vermeire, L., Laperre, K., Sipido, K., during mouse embryogenesis. Genes Dev. 9, 3027-3037. Moons, L., Huylebroeck, D. and Zwijsen, A. (2007). Inactivation of Smad5 in Mishina, Y., Hanks, M. C., Miura, S., Tallquist, M. D. and Behringer, R. R. endothelial cells and smooth muscle cells demonstrates that Smad5 is required (2002). Generation of Bmpr/Alk3 conditional knockout mice. Genesis 32, 69- for cardiac homeostasis. Am. J. Pathol. 170, 1460-1472. 72. Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. (1995). Bone Mishina, Y., Starbuck, M. W., Gentile, M. A., Fukuda, T., Kasparcova, V., morphogenetic protein-4 is required for mesoderm formation and patterning in Seedor, J. G., Hanks, M. C., Amling, M., Pinero, G. J., Harada, S. et al. the mouse. Genes Dev. 9, 2105-2116. (2004). Bone morphogenetic protein type IA receptor signaling regulates Yang, L., Cai, C. L., Lin, L., Qyang, Y., Chung, C., Monteiro, R. M., Mummery, postnatal osteoblast function and bone remodeling. J. Biol. Chem. 279, 27560- C. L., Fishman, G. I., Cogen, A. and Evans, S. (2006). Isl1Cre reveals a 27566. common Bmp pathway in heart and limb development. Development 133, Oh, S. P., Seki, T., Goss, K. A., Imamura, T., Yi, Y., Donahoe, P. K., Li, L., 1575-1585. Miyazono, K., ten Dijke, P., Kim, S. et al. (2000). Activin receptor-like kinase 1 Yang, R., Liu, H., Williams, I. and Chaqour, B. (2007). Matrix metalloproteinase- modulates transforming growth factor-beta 1 signaling in the regulation of 2 expression and apoptogenic activity in retinal pericytes: implications in diabetic angiogenesis. Proc. Natl. Acad. Sci. USA 97, 2626-2631. retinopathy. Ann. N. Y. Acad. Sci. 1103, 196-201. Palosaari, H., Pennington, C. J., Larmas, M., Edwards, D. R., Tjaderhane, L. Yang, X., Castilla, L. H., Xu, X., Li, C., Gotay, J., Weinstein, M., Liu, P. P. and and Salo, T. (2003). Expression profile of matrix metalloproteinases (MMPs) and Deng, C. X. (1999). Angiogenesis defects and mesenchymal apoptosis in mice tissue inhibitors of MMPs in mature human odontoblasts and pulp tissue. Eur. J. lacking SMAD5. Development 126, 1571-1580. Oral Sci. 111, 117-127. Yin, M. and Pacifici, M. (2001). Vascular regression is required for mesenchymal Park, C., Lavine, K., Mishina, Y., Deng, C. X., Ornitz, D. M. and Choi, K. condensation and chondrogenesis in the developing limb. Dev. Dyn. 222, 522- (2006). Bone morphogenetic protein receptor 1A signaling is dispensable for 533. hematopoietic development but essential for vessel and atrioventricular Zempo, N., Kenagy, R. D., Au, Y. P., Bendeck, M., Clowes, M. M., Reidy, M. A. endocardial cushion formation. Development 133, 3473-3484. and Clowes, A. W. (1994). Matrix metalloproteinases of vascular wall cells are Pyo, R., Lee, J. K., Shipley, J. M., Curci, J. A., Mao, D., Ziporin, S. J., Ennis, T. increased in balloon-injured rat carotid artery. J. Vasc. Surg. 20, 209-217. L., Shapiro, S. D., Senior, R. M. and Thompson, R. W. (2000). Targeted gene Zhao, Y. D., Campbell, A. I., Robb, M., Ng, D. and Stewart, D. J. (2003). disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development Protective role of angiopoietin-1 in experimental pulmonary hypertension. Circ. of experimental abdominal aortic aneurysms. J. Clin. Invest. 105, 1641-1649. Res. 92, 984-991. Roelen, B. A., Goumans, M. J., van Rooijen, M. A. and Mummery, C. L. Zhu, W. H., Guo, X., Villaschi, S. and Francesco Nicosia, R. (2000). Regulation (1997). Differential expression of BMP receptors in early mouse development. of vascular growth and regression by matrix metalloproteinases in the rat aorta Int. J. Dev. Biol. 41, 541-549. model of angiogenesis. Lab. Invest. 80, 545-555. DEVELOPMENT