Abstract-Bone morphogenetic peptides (BMPs), a family of cytokines critical to normal development, were recently implicated in the pathogenesis of familial pulmonary arterial hypertension. The type-II receptor (BMPRII) is required for recognition of all BMPs, and targeted deletion of BMPRII in mice results in fetal lethality before gastrulation. To overcome this limitation and study the role of BMP signaling in postnatal vascular disease, we constructed a smooth muscle-specific transgenic mouse expressing a dominant-negative BMPRII under control of the tetracycline gene switch (SM22-tet-BMPRII delx4ϩ mice). When the mutation was activated after birth, mice developed increased pulmonary artery pressure, RV/LVϩS ratio, and pulmonary arterial muscularization with no increase in systemic arterial pressure. Studies with SM22-tet-BMPRII delx4ϩ mice support the hypothesis that loss of BMPRII signaling in smooth muscle is sufficient to produce the pulmonary hypertensive phenotype. Key Words: artery Ⅲ bone morphogenetic peptide Ⅲ hypertension Ⅲ smooth muscle Ⅲ vascular A ssociation between abnormal bone morphogenetic peptide (BMP) signaling and pulmonary vascular disease was suggested by genetic studies of familial pulmonary arterial hypertension (PAH), a disorder characterized by the pathological development of increased pressure and structural remodeling in the pulmonary circulation later in life. 1,2 Fifteen to 25% of cases of PAH occur in families, with autosomal-dominant inheritance, and in the year 2000, two groups independently identified the presence of mutations in the BMRII gene in familial PAH. 3,4 Subsequently, approximately 25% of sporadic cases of PAH were also found to be associated with mutations in BMPRII. 5 Dissecting the function of BMPII using transgenic mice has identified a critical role for BMP signaling in development. 6 Although there is redundancy at the level of ligands, type I receptors, intra-and extracellular inhibitors and downstream signaling via SMADs, functional BMPRII is an absolute requirement for BMP signaling. Consequently, BM-PRII Ϫ/Ϫ mice die early in development, before gastrulation, whereas BMPRII ϩ/Ϫ mice develop normally and have no apparent phenotype. 7 To overcome the developmental lethality identified by traditional transgenic approaches, we constructed a conditional, tissue-specific BMPRII transgenic mouse, using a smooth muscle cell-specific promotor and a dominant-negative BMPRII identified in a family with PAH. 8 Using these mice, we asked two questions: (1) is expression of a strong loss-of-function mutation in BMPRII sufficient to produce pulmonary hypertension, and (2) is loss of BMPRII function in smooth muscle alone sufficient to produce pulmonary hypertension? Materials and Methods Construction of SM22-tet-BMPRII delx4؉ Transgenic MiceThe mice were generated at the University of Cincinnati Transgenic Mouse Science from plasmids we provided. The murine SM22 promotor (488 bp starting 340 bp before the start site and extending 148 bp into the 5Ј UTR) was used to drive...

Familial pulmonary arterial hypertension (PAH) is associated with mutations in bone morphogenetic protein type II receptor (BMPR2). Many of these mutations occur in the BMPR2 tail domain, leaving the SMAD functions intact. To determine the in vivo consequences of BMPR2 tail domain mutation, we created a smooth muscle-specific doxycycline-inducible BMPR2 mutation with an arginine to termination mutation at amino acid 899. When these SM22-rtTA x TetO(7)-BMPR2(R899X) mice had transgene induced for 9 wk, starting at 4 wk of age, they universally developed pulmonary vascular pruning as assessed by fluorescent microangiography. Approximately one-third of the time, the induced animals developed elevated right ventricular systolic pressures (RVSP), associated with extensive pruning, muscularization of small pulmonary vessels, and development of large structural pulmonary vascular changes. These lesions included large numbers of macrophages and T cells in their adventitial compartment as well as CD133-positive cells in the lumen. Small vessels filled with CD45-positive and sometimes CD3-positive cells were a common feature in all SM22-rtTA x TetO(7)-BMPR2(R899X) mice. Gene array experiments show changes in stress response, muscle organization and function, proliferation, and apoptosis and developmental pathways before RVSP increases. Our results show that the primary phenotypic result of BMPR2 tail domain mutation in smooth muscle is pulmonary vascular pruning leading to elevated RVSP, associated with early dysregulation in multiple pathways with clear relevance to PAH. This model should be useful to the research community in examining early molecular and physical events in the development of PAH and as a platform to validate potential treatments.

Objective: This study was performed to assess the utility of selective small-molecule inhibitors of class I HDACs in a preclinical model of pulmonary hypertension. Methods and Results:Rats were exposed to hypobaric hypoxia for 3 weeks in the absence or presence of a benzamide HDAC inhibitor, MGCD0103, which selectively inhibits class I HDACs 1, 2, and 3. The compound reduced pulmonary arterial pressure more dramatically than tadalafil, a standard-of-care therapy for human pulmonary hypertension that functions as a vasodilator. MGCD0103 improved pulmonary artery acceleration time and reduced systolic notching of the pulmonary artery flow envelope, which suggests a positive impact of the HDAC inhibitor on pulmonary vascular remodeling and stiffening. Similar results were obtained with an independent class I HDAC-selective inhibitor, MS-275. Reduced pulmonary arterial pressure in MGCD0103-treated animals was associated with blunted pulmonary arterial wall thickening because of suppression of smooth muscle cell proliferation. Right ventricular function was maintained in MGCD0103-treated animals. Although the class I HDAC inhibitor only modestly reduced right ventricular hypertrophy, it had multiple beneficial effects on the right ventricle, which included suppression of pathological gene expression, inhibition of proapoptotic caspase activity, and repression of proinflammatory protein expression. Key Words: histone deacetylase Ⅲ pulmonary hypertension Ⅲ proliferation Ⅲ gene expression Ⅲ signaling pathways I n patients with pulmonary hypertension (PH), restricted blood flow through the pulmonary arterial circulation due to increased pulmonary vascular resistance often results in right-sided heart failure. Despite recent advances in the treatment of PH, the 5-year mortality rate for individuals with this disease still approaches 50%, which highlights an urgent need for novel therapeutics. 1 Current standards of care for patients with PH typically involve the use of vasoactive drugs, including endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, and prostacyclins. More effective therapeutic strategies will likely be based on the combined use of vasodilators and agents that target distinct pathogenic mechanisms in PH, such as pulmonary vascular inflammation and fibrosis, as well as aberrant proliferation of smooth muscle cells, endothelial cells, and fibroblasts in the lung vasculature. 2 Additionally, because right ventricular (RV) failure is the cause of death in the majority of PH patients, 3,4 and it is unclear whether standards of care for left ventricular Original received October 8, 2011; revision received January 11, 2012; accepted January 18, 2012. In December 2011 HDACs control cell proliferation, inflammation, and fibrosis by catalyzing removal of acetyl groups from lysine residues in a variety of proteins. The 18 mammalian HDACs are encoded by distinct genes and are grouped into 4 classes. 6 Two broad-spectrum HDAC inhibitors are approved for the treatment of cancer. One of these compounds,...

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.

Abstract-While Ca 2ϩ influx is essential for activation of the cell cycle machinery, the processes that regulate Ca 2ϩ influx in this context have not been fully elucidated. Electrophysiological and molecular studies have identified multiple Ca 2ϩ

Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45(pos)/Flk-1(pos) and CD45(neg)/Flk-1(pos) bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.

Idiopathic pulmonary arterial hypertension (IPAH) in human patients is associated with mutations in type 2 receptor for the bone morphogenic protein pathway (BMPR2). Mice expressing an inducible dominant negative form of BMPR2 in smooth muscle develop elevated right ventricular pressures when the transgene is activated. We hypothesized that transcriptional changes in these mice may allow insight into the early molecular events leading to IPAH. Microarray analysis was used to examine the transcriptional changes induced in whole lung by loss of normal smooth muscle cell (SMC) BMPR2 signaling in adult male or female mice (12 wk at time of death) expressing the transgene for either 1 or 8 wk. Our key results include a decrease in markers of smooth muscle differentiation, an increase in cytokines and markers of immune response, particularly in female mice, and a decrease in angiogenesis-related genes. These broad patterns of gene expression appear as early as 1 wk and are well established by 8 wk. Results were confirmed by quantitative RT-PCR to RNA from individual mice. Primary pulmonary artery SMC cultures transfected with small interfering RNA to BMPR2 also show loss of SMC markers myosin heavy chain 11 and calponin by quantitative RT-PCR and Western blot. These studies show classes of genes differentially regulated in response to loss of BMPR2 in SMC in vivo with clear relevance to the IPAH disease process, suggesting that the relevance of BMPR2 dysregulation may extend beyond proliferation.

BackgroundPulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations. However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown.MethodsIn vivo experiments were performed on adult mice with conditional endothelial-specific expression of the truncation mutation Bmpr2delx4+, with age-matched transactivator-only mice as controls. Phenotype was assessed by RVSP, counts of muscularized vessels and proliferating cells, and staining for thromboses, inflammatory cells, and apoptotic cells. The effects of BMPR2 knockdown in PMVEC by siRNA on rates of apoptosis were assessed. Affymetrix expression arrays were performed on PMVEC isolated and cultured from triple transgenic mice carrying the immortomouse gene, a transactivator, and either control, Bmpr2delx4+ or Bmpr2R899X mutation.ResultsTransgenic mice showed increased RVSP and corresponding muscularization of small vessels, with histologic alterations including thrombosis, increased inflammatory cells, increased proliferating cells, and a moderate increase in apoptotic cells. Expression arrays showed alterations in specific pathways consistent with the histologic changes. Bmpr2delx4+ and Bmpr2R899X mutations resulted in very similar alterations in proliferation, apoptosis, metabolism, and adhesion; Bmpr2delx4+ cells showed upregulation of platelet adhesion genes and cytokines not seen in Bmpr2R899X PMVEC. Bmpr2 mutation in PMVEC does not cause a loss of differentiation markers as was seen with Bmpr2 mutation in smooth muscle cells.ConclusionsBmpr2 mutation in PMVEC in vivo may drive PAH through multiple, potentially independent, downstream mechanisms, including proliferation, apoptosis, inflammation, and thrombosis.