Search

Objective: Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH., Methods: SMC-specific Bmpr2 -/- mice ( BKO SMC ) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKO SMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism., Results: BKO SMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (β-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (β-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension., Conclusions: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.

Background: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC., Methods: RNA sequencing was applied to pulmonary arterial SMC (PASMC) from PAH patients with and without a BMPR2 (bone morphogenetic receptor 2) mutation versus control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 ( ALDH1A3) , an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC., Results: ALDH1A3 mRNA and protein were increased in PAH versus control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced pulmonary arterial muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl coenzyme A to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at NFYA (nuclear transcription factor Y subunit α) binding sites via the acetyltransferase KAT2B (lysine acetyltransferase 2B) and permitted NFY-mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was β-catenin dependent., Conclusions: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not endothelial cells, is an important therapeutic consideration.

Rationale: In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of BMPR2 (bone morphogenetic protein type 2 receptor) via two downstream transcription factors, PPARγ (peroxisome proliferator-activated receptor gamma), and p53., Objective: We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ-p53 transcription factor complex in the pulmonary endothelium., Methods and Results: In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial and lung MV (microvascular) endothelial cells in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation sequencing and RNA-sequencing established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung endothelial cell survival, angiogenesis and DNA repair including, EPHA2 ( ephrin type-A receptor 2 ), FHL2 ( four and a half LIM domains protein 2 ), JAG1 ( jagged 1 ), SULF2 ( extracellular sulfatase Sulf-2 ), and TIGAR ( TP53-inducible glycolysis and apoptosis regulator ). Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in pulmonary artery endothelial cells (PAECs) subjected to oxidative stress. In endothelial cell-specific Bmpr2 -knockout mice unable to stabilize p53 in endothelial cells under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN ( apelin ) and JAG1 , to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAECs from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis., Conclusions: We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAECs downstream of dysfunctional BMPR2 to rehabilitate PAH PAECs, regenerate pulmonary microvessels, and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.

Background: Bmpr2 (bone morphogenetic protein receptor 2) mutations are critical risk factors for hereditary pulmonary arterial hypertension (PAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-LO (5-lipoxygenase) provokes lung inflammation and transient PAH in Bmpr2 +/ - mice. Accordingly, 5-LO and its metabolite, leukotriene B 4 , are candidates for the second hit. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats., Methods: Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to 1 year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus, monocrotaline, SU5416, SU5416 with chronic hypoxia, or chronic hypoxia alone. Bmpr2 -mutant hereditary PAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells with impaired BMPR2 signaling were exposed to increased 5-LO-mediated inflammation and were assessed for phenotypic and transcriptomic changes., Results: Lung inflammation, induced by intratracheal delivery of 5-LO-expressing adenovirus, elicited severe PAH with intimal remodeling in Bmpr2 +/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2 +/- rats gained endogenous 5-LO expression associated with elevated leukotriene B 4 biosynthesis. Bmpr2 -mutant hereditary PAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant and proliferative pulmonary artery endothelial cells with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced leukotriene B 4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated nuclear factor Kappa B subunit (NF-κB), interleukin-6, and transforming growth factor beta (TGF-β) signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-β antagonism suggests that TGF-β is critical for neointimal transformation., Conclusions: In a new 2-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2 +/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-β signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced leukotriene B 4 production. This study offers an explanation of how an environmental injury unleashes the destructive potential of an otherwise silent genetic mutation.

Rationale: Accumulating evidence implicates inflammation in pulmonary arterial hypertension (PAH) and therapies targeting immunity are under investigation, although it remains unknown if distinct immune phenotypes exist., Objective: Identify PAH immune phenotypes based on unsupervised analysis of blood proteomic profiles., Methods and Results: In a prospective observational study of group 1 PAH patients evaluated at Stanford University (discovery cohort; n=281) and University of Sheffield (validation cohort; n=104) between 2008 and 2014, we measured a circulating proteomic panel of 48 cytokines, chemokines, and factors using multiplex immunoassay. Unsupervised machine learning (consensus clustering) was applied in both cohorts independently to classify patients into proteomic immune clusters, without guidance from clinical features. To identify central proteins in each cluster, we performed partial correlation network analysis. Clinical characteristics and outcomes were subsequently compared across clusters. Four PAH clusters with distinct proteomic immune profiles were identified in the discovery cohort. Cluster 2 (n=109) had low cytokine levels similar to controls. Other clusters had unique sets of upregulated proteins central to immune networks-cluster 1 (n=58; TRAIL [tumor necrosis factor-related apoptosis-inducing ligand], CCL5 [C-C motif chemokine ligand 5], CCL7, CCL4, MIF [macrophage migration inhibitory factor]), cluster 3 (n=77; IL [interleukin]-12, IL-17, IL-10, IL-7, VEGF [vascular endothelial growth factor]), and cluster 4 (n=37; IL-8, IL-4, PDGF-β [platelet-derived growth factor beta], IL-6, CCL11). Demographics, PAH clinical subtypes, comorbidities, and medications were similar across clusters. Noninvasive and hemodynamic surrogates of clinical risk identified cluster 1 as high-risk and cluster 3 as low-risk groups. Five-year transplant-free survival rates were unfavorable for cluster 1 (47.6%; 95% CI, 35.4%-64.1%) and favorable for cluster 3 (82.4%; 95% CI, 72.0%-94.3%; across-cluster P<0.001). Findings were replicated in the validation cohort, where machine learning classified 4 immune clusters with comparable proteomic, clinical, and prognostic features., Conclusions: Blood cytokine profiles distinguish PAH immune phenotypes with differing clinical risk that are independent of World Health Organization group 1 subtypes. These phenotypes could inform mechanistic studies of disease pathobiology and provide a framework to examine patient responses to emerging therapies targeting immunity.

Rationale: Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as atherosclerosis and pulmonary arterial hypertension., Objective: We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity., Methods and Results: We found that in SMC and EC contact cocultures, BMPR2 (bone morphogenetic protein receptor 2) is required by both cell types to produce collagen IV to activate ILK (integrin-linked kinase). This enzyme directs p-JNK (phospho-c-Jun N-terminal kinase) to the EC membrane, where it stabilizes presenilin1 and releases N1ICD (Notch1 intracellular domain) to promote EC proliferation. This response is necessary for EC regeneration after carotid artery injury. It is deficient in EC-SMC Bmpr2 double heterozygous mice in association with reduced collagen IV production, decreased N1ICD, and attenuated EC proliferation, but can be rescued by targeting N1ICD to EC. Deletion of EC- Notch1 in transgenic mice worsens hypoxia-induced pulmonary hypertension, in association with impaired EC regenerative function associated with loss of precapillary arteries. We further determined that N1ICD maintains EC proliferative capacity by increasing mitochondrial mass and by inducing the phosphofructokinase PFKFB3 (fructose-2,6-bisphosphatase 3). Chromatin immunoprecipitation sequencing analyses showed that PFKFB3 is required for citrate-dependent H3K27 acetylation at enhancer sites of genes regulated by the acetyl transferase p300 and by N1ICD or the N1ICD target MYC and necessary for EC proliferation and homeostasis., Conclusions: Thus, SMC-EC contact is required for activation of Notch1 by BMPR2, to coordinate metabolism with chromatin remodeling of genes that enable EC regeneration, and to maintain monolayer integrity and vascular homeostasis in response to injury.

Rationale: Pulmonary arterial hypertension (PH) is a life-threatening condition associated with immune dysregulation and abnormal regulatory T cell (Treg) activity, but it is currently unknown whether and how abnormal Treg function differentially affects males and females., Objective: To evaluate whether and how Treg deficiency differentially affects male and female rats in experimental PH., Methods and Results: Male and female athymic rnu/rnu rats, lacking Tregs, were treated with the VEGFR2 (vascular endothelial growth factor receptor 2) inhibitor SU5416 or chronic hypoxia and evaluated for PH; some animals underwent Treg immune reconstitution before SU5416 administration. Plasma PGI 2 (prostacyclin) levels were measured. Lung and right ventricles were assessed for the expression of the vasoprotective proteins COX-2 (cyclooxygenase 2), PTGIS (prostacyclin synthase), PDL-1 (programmed death ligand 1), and HO-1 (heme oxygenase 1). Inhibitors of these pathways were administered to athymic rats undergoing Treg immune reconstitution. Finally, human cardiac microvascular endothelial cells cocultured with Tregs were evaluated for COX-2, PDL-1, HO-1, and ER (estrogen receptor) expression, and culture supernatants were assayed for PGI 2 and IL (interleukin)-10. SU5416-treatment and chronic hypoxia produced more severe PH in female than male athymic rats. Females were distinguished by greater pulmonary inflammation, augmented right ventricular fibrosis, lower plasma PGI 2 levels, decreased lung COX-2, PTGIS, HO-1, and PDL-1 expression and reduced right ventricular PDL-1 levels. In both sexes, Treg immune reconstitution protected against PH development and raised levels of plasma PGI 2 and cardiopulmonary COX-2, PTGIS, PDL-1, and HO-1. Inhibiting COX-2, HO-1, and PD-1 (programmed death 1)/PDL-1 pathways abrogated Treg protection. In vitro, human Tregs directly upregulated endothelial COX-2, PDL-1, HO-1, ERs and increased supernatant levels of PGI 2 and IL-10., Conclusions: In 2 animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on the normal Treg function to counteract the effects of pulmonary vascular injury leading to PH., (© 2018 American Heart Association, Inc.)

Background: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies., Methods: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat., Results: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1β, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6., Conclusions: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH., (© 2017 American Heart Association, Inc.)

Objective: We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells previously reported, PA elastic fibers are susceptible to degradation because of their abnormal assembly., Approach and Results: Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β1 (TGFβ1). Thus, we considered whether BMPs like TGFβ1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with Bmpr2/1a compound heterozygosity, elastic fibers are susceptible to degradation. In PA smooth muscle cells and adventitial fibroblasts, TGFβ1 increased elastin mRNA, but the elevation in elastin protein was dependent on BMPR2; TGFβ1 and BMP4, via BMPR2, increased extracellular accumulation of fibrillin-1. Both BMP4- and TGFβ1-stimulated elastic fiber assembly was impaired in idiopathic (I) PAH-PA adventitial fibroblast versus control cells, particularly those with hereditary (H) PAH and a BMPR2 mutation. This was related to profound reductions in elastin and fibrillin-1 mRNA. Elastin protein was increased in IPAH PA adventitial fibroblast by TGFβ1 but only minimally so in BMPR2 mutant cells. Fibrillin-1 protein increased only modestly in IPAH or HPAH PA adventitial fibroblasts stimulated with BMP4 or TGFβ1. In Bmpr2/1a heterozygote mice, reduced PA fibrillin-1 was associated with elastic fiber susceptibility to degradation and more severe pulmonary hypertension., Conclusions: Disrupting BMPR2 impairs TGFβ1- and BMP4-mediated elastic fiber assembly and is of pathophysiologic significance in PAH., (© 2017 American Heart Association, Inc.)

Background: Right ventricular (RV) end-systolic dimensions provide information on both size and function. We investigated whether an internally scaled index of end-systolic dimension is incremental to well-validated prognostic scores in pulmonary arterial hypertension., Methods and Results: From 2005 to 2014, 228 patients with pulmonary arterial hypertension were prospectively enrolled. RV end-systolic remodeling index (RVESRI) was defined by lateral length divided by septal height. The incremental values of RV free wall longitudinal strain and RVESRI to risk scores were determined. Mean age was 49±14 years, 78% were female, 33% had connective tissue disease, 52% were in New York Heart Association class ≥III, and mean pulmonary vascular resistance was 11.2±6.4 WU. RVESRI and right atrial area were strongly connected to the other right heart metrics. Three zones of adaptation (adapted, maladapted, and severely maladapted) were identified based on the RVESRI to RV systolic pressure relationship. During a mean follow-up of 3.9±2.4 years, the primary end point of death, transplant, or admission for heart failure was reached in 88 patients. RVESRI was incremental to risk prediction scores in pulmonary arterial hypertension, including the Registry to Evaluate Early and Long-Term PAH Disease Management score, the Pulmonary Hypertension Connection equation, and the Mayo Clinic model. Using multivariable analysis, New York Heart Association class III/IV, RVESRI, and log NT-proBNP (N-Terminal Pro-B-Type Natriuretic Peptide) were retained (χ 2 , 62.2; P <0.0001). Changes in RVESRI at 1 year (n=203) were predictive of outcome; patients initiated on prostanoid therapy showed the greatest improvement in RVESRI. Among right heart metrics, RVESRI demonstrated the best test-retest characteristics., Conclusions: RVESRI is a simple reproducible prognostic marker in patients with pulmonary arterial hypertension., (© 2017 American Heart Association, Inc.)

Background: We previously reported high-throughput RNA sequencing analyses that identified heightened expression of the chromatin architectural factor High Mobility Group AT-hook 1 (HMGA1) in pulmonary arterial endothelial cells (PAECs) from patients who had idiopathic pulmonary arterial hypertension (PAH) in comparison with controls. Because HMGA1 promotes epithelial-to-mesenchymal transition in cancer, we hypothesized that increased HMGA1 could induce transition of PAECs to a smooth muscle (SM)-like mesenchymal phenotype (endothelial-to-mesenchymal transition), explaining both dysregulation of PAEC function and possible cellular contribution to the occlusive remodeling that characterizes advanced idiopathic PAH., Methods and Results: We documented increased HMGA1 in PAECs cultured from idiopathic PAH versus donor control lungs. Confocal microscopy of lung explants localized the increase in HMGA1 consistently to pulmonary arterial endothelium, and identified many cells double-positive for HMGA1 and SM22α in occlusive and plexogenic lesions. Because decreased expression and function of bone morphogenetic protein receptor 2 (BMPR2) is observed in PAH, we reduced BMPR2 by small interfering RNA in control PAECs and documented an increase in HMGA1 protein. Consistent with transition of PAECs by HMGA1, we detected reduced platelet endothelial cell adhesion molecule 1 (CD31) and increased endothelial-to-mesenchymal transition markers, αSM actin, SM22α, calponin, phospho-vimentin, and Slug. The transition was associated with spindle SM-like morphology, and the increase in αSM actin was largely reversed by joint knockdown of BMPR2 and HMGA1 or Slug. Pulmonary endothelial cells from mice with endothelial cell-specific loss of Bmpr2 showed similar gene and protein changes., Conclusions: Increased HMGA1 in PAECs resulting from dysfunctional BMPR2 signaling can transition endothelium to SM-like cells associated with PAH., (© 2016 American Heart Association, Inc.)

Objective: Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome., Approach and Results: Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs., Conclusions: Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome., (© 2014 American Heart Association, Inc.)

This review summarizes an expanding body of knowledge indicating that failure to resolve inflammation and altered immune processes underlie the development of pulmonary arterial hypertension. The chemokines and cytokines implicated in pulmonary arterial hypertension that could form a biomarker platform are discussed. Pre-clinical studies that provide the basis for dysregulated immunity in animal models of the disease are reviewed. In addition, we present therapies that target inflammatory/immune mechanisms that are currently enrolling patients, and discuss others in development. We show how genetic and metabolic abnormalities are inextricably linked to dysregulated immunity and adverse remodeling in the pulmonary arteries., (© 2014 American Heart Association, Inc.)

Rationale: Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins promote pulmonary angiogenesis by recruiting the Wnt/β-catenin pathway, we proposed that β-catenin activation could reduce loss and induce regeneration of small pulmonary arteries (PAs) and attenuate PH., Objective: This study aims to establish the role of β-catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis after injury., Methods and Results: To assess the impact of β-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (Apc(Min/+)) mouse, where reduced APC causes constitutive β-catenin elevation. Surprisingly, hypoxic Apc(Min/+) mice displayed greater PH and small PA loss compared with control C57Bl6J littermates. PA endothelial cells isolated from Apc(Min/+) demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase-1 and phospho Akt. We found that PA endothelial cells from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin, and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC., Conclusions: We show that APC is integral to PA endothelial cells adhesion and survival and is reduced in PA endothelial cells from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.

Rationale: Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood., Objective: To determine whether immune dysregulation due to absent T-cell populations directly contributes to the development of PAH., Methods and Results: Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell-deficient rats but not in immune-reconstituted (IR) rats. T cell-lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH, whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4(+)CD25(hi) or CD4(+)CD25(-) T cell subsets prior to vascular injury attenuated the development of PAH. IR limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3(+), IL-10- and TGF-β-expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)-expressing cells, a receptor that activates endothelial cell survival pathways., Conclusions: PAH may arise when regulatory T-cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.

Rationale: S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMCs), through an interaction with the RAGE (receptor for advanced glycation end products)., Objective: We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (BMPR)II, observed in pulmonary arterial hypertension., Methods and Results: Both S100A4/Mts1 (500 ng/mL) and BMP-2 (10 ng/mL) induce migration of hPASMCs in a novel codependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII short interference (si)RNA. Phosphorylation of extracellular signal-regulated kinase is induced by both ligands and is required for motility by inducing matrix metalloproteinase 2 activity, but phospho-extracellular signal-regulated kinase 1/2 is blocked by anti-RAGE and not by BMPRII short interference RNA. In contrast, BMPRII short interference RNA, but not anti-RAGE, reduces expression of intracellular chloride channel (CLIC)4, a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA, but does alter alignment of myosin heavy chain IIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodia in motile cells., Conclusions: Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, ie, cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.

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 SM22alpha;TRE-Cre/LoxP;R26R system. The LacZ distribution reflected patchy deletion of Bmpr1a in the lung vessels, aorta, and heart of SM22alpha;TRE-Cre;R26R;Bmpr1a(flox/+) 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 SM22alpha;TRE-Cre;R26R;Bmpr1a(flox/+) 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.

Transgenic mice overexpressing the calcium binding protein, S100A4/Mts1, occasionally develop severe pulmonary vascular obstructive disease. To understand what underlies this propensity, we compared the pulmonary vascular hemodynamic and structural features of S100A4/Mts1 with control C57Bl/6 mice at baseline, following a 2-week exposure to chronic hypoxia, and after 1 and 3 months "recovery" in room air. S100A4/Mts1 mice had greater right ventricular systolic pressure and right ventricular hypertrophy at baseline, which increased further with chronic hypoxia and was sustained after 3 months "recovery" in room air. These findings correlated with a heightened response to acute hypoxia and failure to vasodilate with nitric oxide or oxygen. S100A4/Mts1 mice, when compared with C57Bl/6 mice, also had impaired cardiac function judged by reduced ventricular elastance and decreased cardiac output. Despite higher right ventricular systolic pressures with chronic hypoxia, S100A4/Mts1 mice did not develop more severe PVD, but in contrast to C57Bl/6 mice, these features did not regress on return to room air. Microarray analysis of lung tissue identified a number of genes differentially upregulated in S100A4/Mts1 versus control mice. One of these, fibulin-5, is a matrix component necessary for normal elastin fiber assembly. Fibulin-5 was localized to pulmonary arteries and associated with thickened elastic laminae. This feature could underlie attenuation of pulmonary vascular changes in response to elevated pressure, as well as impaired reversibility.

Heightened expression of the S100 calcium-binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100beta, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT1B), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT-induced S100A4/Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal-regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4-mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT1B receptor and SERT are codependent in regulating S100A4/Mts1.

We identified apolipoprotein (apo)D in a search for proteins upregulated in a posttranscriptional manner similar to fibronectin in motile smooth muscle cells (SMCs). To address the function of apoD in SMCs, we cloned a partial apoD cDNA from ovine aortic (Ao) SMCs using RT-PCR. We documented a 2.5-fold increase in apoD protein but no increase in apoD mRNA in Ao SMCs 48 hours after a multiwound migration assay (P<0.01). Confocal microscopy revealed prominent perinuclear and trailing edge expression of apoD in migrating SMCs but not in the confluent monolayer. Stimulation of Ao SMCs with 10 ng/mL platelet-derived growth factor (PDGF)-BB increased apoD protein expression (P<0.05). Moreover, PDGF-BB-stimulated migration of human pulmonary artery SMCs was suppressed by knock-down of apoD using RNAi. Stable overexpression of apoD in Ao SMCs cultured in 10% fetal bovine serum promoted random migration by 62% compared with vector-transfected cells (P<0.01). Overexpression of apoD or addition of exogenous apoD to a rat aortic SMC line (A10) stimulated their migration in response to a subthreshold dose of PDGF-BB (P<0.05). This was unrelated to increased phosphorylation of ERK1/2 or of phospholipase C-gamma1, but correlated with enhanced Rac1 activation. This study shows that apoD can be expressed or taken up by SMCs and can regulate their motility in response to growth factors.

Objective: Elevated apolipoprotein D (apoD) levels are associated with reduced proliferation of cancer cells. We therefore investigated whether apoD, which occurs free or associated with HDL, suppresses vascular smooth muscle cell (VSMC) proliferation, which is related to the pathobiology of disease., Methods and Results: Intense immunoreactivity for apoD was observed in human atherosclerotic plaque but not in normal coronary artery. However, an increase in apoD mRNA was seen in quiescent relative to proliferating fetal lamb aortic VSMCs, and in the rat aortic VSMC line (A10), we demonstrated uptake of apoD from serum. Stable transfection of apoD in A10 cells in the absence of serum did not influence VSMC proliferation assessed by [3H]-thymidine incorporation. ApoD, administered at a dose of 100 ng/mL, completely inhibited basal as well as platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation (P<0.01) but had no effect on fibroblast growth factor-induced VSMC proliferation. ApoD did not suppress PDGF-BB or fibroblast growth factor-2-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 but selectively inhibited PDGF-BB-mediated ERK1/2 nuclear translocation., Conclusions: Our data suggest that apoD selectively modulates the proliferative response of VSMC to growth factors by a mechanism related to nuclear translocation of ERK1/2.

Background: Creation of a bi-directional cavopulmonary shunt after the Norwood procedure for hypoplastic left heart syndrome is delayed to allow pulmonary vascular resistance to fall with maturation of the pulmonary vascular bed. We hypothesized that unfractionated heparin (UFH) and low molecular weight heparin (LMWH), which promote angiogenesis and inhibit smooth muscle cell growth, could accelerate this process., Methods and Results: Fifty-six newborn rabbits were randomly selected to receive UFH 225U/kg (n=12), LMWH 1 mg/kg (n=14), LMWH 10 mg/kg (n=16), or saline (n=14) by subcutaneous injection every 12 hours for 14 days. Treatment with heparin reduced mean pulmonary artery (PA) pressure by 12% to 16% relative to controls [9.0+/-0.2 (UFH), 9.4+/-0.1 (LMWH 1 mg/kg), 9.2+/-0.2 (LMWH 10 mg/kg) versus 10.7+/-0.2 mm Hg (saline), P=0.0001]. Lower PA pressures were associated with reduced alveolar:arterial ratio consistent with enhanced pulmonary angiogenesis in heparin treated animals [8+/-1 (UFH), 13+/-2 (LMWH 1 mg/kg), 12+/-2 (LMWH 10 mg/kg) versus 23+/-5 (saline), P<0.03]. Reduced PA medial wall thickness and muscularization, two additional features of pulmonary vascular maturation, were also more evident in heparin treated animals. Mean PA pressures in 14-day-old rabbits treated with heparin were lower than those measured in control rabbits less than 7 weeks of age suggesting that heparin shortens the pulmonary vascular maturation process by over 60%., Conclusions: These results indicate that both UFH and LMWH are effective at accelerating pulmonary vascular maturation in newborn rabbits. This raises the possibility that administration of heparin to children after the Norwood procedure might allow for earlier conversion to a bi-directional cavopulmonary shunt.

Background: Integrins are involved in structural remodeling and tissue repair. This study aimed to elucidate the role of the beta-integrins in cardiac remodeling after myocardial infarction (MI)., Methods and Results: The MI model was created by ligation of the left anterior descending coronary artery in rats. We detected cardiac integrins beta1 and beta3 gene expression (quantitative in situ hybridization) and protein production (Western blot and immunohistochemistry) and potential regulation by tumor necrosis factor (TNF) using neonatal ventricular myocytes and TNF-/- knockout mice. Integrins beta1 and beta3 gene expression and protein production were low in sham-operated hearts. After MI, the beta1 and beta3 mRNA and proteins were significantly increased at the site of MI at day 3, reached a peak at day 7, and gradually declined thereafter. Integrin beta1A localized primarily in fibroblasts and inflammatory cells, beta1D localized in myocytes, and integrin beta3 was associated primarily with endothelial and smooth muscle cells in peri-infarct vessels. In cultured myocytes, there was isoform transition from the adult beta1D to the fetal beta1A on exposure to TNF-alpha. This was confirmed in vivo in the peri-infarct myocytes, but the transition was voided in TNF-/--knockout mice., Conclusions: Integrins beta1 and beta3 are significantly activated in the infarcted myocardium. Integrin beta1 is active particularly at sites of inflammation and fibrosis, whereas integrin beta3 localizes to vessels in the peri-infarct zone in a temporally coordinated manner. Integrin beta1D to beta1A isoform transition in myocytes is regulated by TNF-alpha.

Background: Increased serine elastase activity has been implicated in the vascular remodeling associated with chronic hypoxia-related pulmonary hypertension in rats., Methods and Results: In this study we determined the time course of hypoxia-induced serine elastase activity in the murine lung and related this to initiation of a proteolytic cascade characterized by an increase in matrix metalloproteinases (MMPs). We then used transgenic mice in which overexpression of the selective serine elastase inhibitor elafin was targeted to the cardiovascular system to determine whether upregulation of a naturally occurring serine elastase inhibitor suppresses MMPs and the hemodynamic and structural response to chronic hypoxia (air at 380 mm Hg). In nontransgenic but not in elafin-transgenic mice, we documented a transient increase in serine elastase activity after 12 hours of hypoxic exposure attributed to a 30-kDa protein as determined by elastin zymography and fluorophosphonate/fluorophosphate-biotin labeling. Two days after hypoxia, the pro-forms of MMP-2 and MMP-9 were induced in the nontransgenic mice, but MMP-9 was suppressed in elafin-transgenic mice. Acute hypoxic vasoconstriction was similar in nontransgenic and elafin-transgenic littermates. Chronic hypoxia for 26 days resulted in >1-fold increase in right ventricular pressure (P<0.004) in nontransgenic compared with control or elafin-transgenic littermates. In the latter mice, normalization of the right ventricular pressure was associated with reduced muscularization and preservation of the number of distal vessels (P<0.04 for both comparisons)., Conclusions: Modulation of the severity of chronic hypoxia-induced pulmonary vascular disease could be a function of endogenously expressed serine elastase inhibitors.

Increased proteolytic activity may be a factor in intimal hyperplasia after balloon angioplasty (BA). The objectives of this study were to assess elastase activity after BA in a rabbit arterial double-injury model and the effects of elastase inhibition. Elastase activity increased immediately after BA, reached an 8-fold peak at 1 week, and declined to baseline levels by 4 weeks. Elastin zymography showed that the elastase activity was associated predominantly with a molecular mass of 25 kDa. Elastase activity was significantly inhibited in vitro by elafin and phenylmethylsulfonyl fluoride, selective inhibitors of serine elastases. A second group of animals was transfected after BA with a plasmid containing the cDNA for either elafin or a control (chloramphenicol acetyltransferase, CAT) construct by using a hemagglutinating virus of Japan-liposome transfection technique. Arterial segments were obtained at 48 hours, 1 week, and 4 weeks to assess transgene expression, arterial wall elastase activity, and intimal cross-sectional area, respectively. Elafin transgene expression was evident at 48 hours and resulted in a significant (80%) inhibition of elastase activity compared with chloramphenicol acetyltransferase-transfected arteries. There was a 43% reduction in intimal cross-sectional area in elafin-transfected arteries (0.28+/-0.22 versus 0.16+/-0.07 mm(2) for CAT-transfected versus elafin-transfected arteries, respectively; P<0.05). These data suggest that an early increase in serine elastase activity after BA contributes to intimal hyperplasia. Serine elastase inhibition may be a potential therapeutic approach to inhibit intimal hyperplasia.

Postcardiac transplant coronary arteriopathy is associated with tumor necrosis factor-alpha (TNF-alpha) induction of fibronectin-dependent smooth muscle cell (SMC) migration into the subendothelium, resulting in occlusive neointimal formation. Because expression of inducible nitric oxide synthase (iNOS) is elevated in neointimal formation after transplantation and upregulated in vascular SMCs by TNF-alpha, we investigated whether TNF-alpha induction of fibronectin synthesis in coronary artery (CA) SMCs is mediated by nitric oxide (NO). TNF-alpha caused a dose-dependent increase in reactive oxygen and nitrogen intermediates in CA SMCs (P<0.05). This correlated with increased NO production (P<0.05) and fibronectin synthesis (P<0.05). TNF-alpha induction of fibronectin synthesis was abrogated by the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) (P<0.05) or the flavonoid-containing enzyme inhibitor diphenyleneiodonium (DPI) (P<0.05) and reproduced with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (P<0.05). Northern blotting showed no effect of TNF-alpha on steady-state fibronectin mRNA levels. TNF-alpha increased expression of light chain 3 (LC-3), a protein shown previously to facilitate fibronectin mRNA translation through its interaction with an adenosine-uracil rich element (ARE) in the 3'-untranslated region of fibronectin mRNA. RNA gel mobility shift and UV cross-linking assays using CA SMC lysates revealed protein binding complexes with radiolabeled oligonucleotide containing the ARE, similar to those generated with recombinant LC-3. One of these complexes increased after TNF-alpha treatment, an effect inhibited with L-NMMA or DPI. These data demonstrate a novel paradigm whereby cytokines regulate mRNA translation of extracellular matrix proteins through NO-dependent modulation of RNA binding protein interaction with mRNA.

Background: Leukocyte infiltration and serine elastase activity lead to smooth muscle cell proliferation in association with posttransplant coronary arteriopathy and may also be involved in vein graft neointimal formation. A number of therapies have targeted cellular proliferation, but the inhibition of serine elastase-mediated extracellular matrix remodeling has not been investigated as a potential strategy to prevent neointimal formation and subsequent atherosclerotic degeneration in vein grafts., Methods and Results: We studied jugular vein grafts 48 hours after interposition into the carotid arteries of rabbits and demonstrated inflammatory cell infiltration and elevated serine elastase activity, a stimulus for matrix remodeling and deposition of elastin. Therefore, elastolytic activity in vein grafts was targeted through transient expression of the selective serine elastase inhibitor elafin with hemagglutinating virus of Japan liposome-mediated gene transfer. Elafin transfection reduced inflammation by 60% at 48 hours and neointimal formation by approximately 50% at 4 weeks after implantation. At 3 months, a 74% decrease in neointimal elastin deposition correlated with protection against cholesterol-induced macrophage infiltration and lipid accumulation, which were both reduced by approximately 50% in elafin-transfected grafts relative to controls., Conclusions: Gene transfer of the selective serine elastase inhibitor elafin in vein grafts is effective in reducing the early inflammatory response. Although transient expression of elafin delays neointimal formation, it is also sufficient to cause an alteration in elastin content of the extracellular matrix, making it relatively resistant to atherosclerotic degeneration.

Extracellular matrix (ECM) regulates vascular smooth muscle cell proliferation. The role of ECM in myocardial growth is unexplored. We sought to determine whether human fetal ventricular myocytes (HFVMs) produce ECM and whether synthesis and attachment to ECM are necessary for their epidermal growth factor (EGF)-dependent and -independent proliferation. Cultured HFVMs proliferate in the presence but not absence of serum and EGF, as determined by increase in cell number and [(3)H]thymidine and [(14)C]leucine incorporation (measures of DNA and protein synthesis, respectively). Using a cyanogen bromide digestion technique to measure collagen and elastin and using affinity chromatography for fibronectin, we found that HFVMs synthesized collagen and fibronectin but not elastin. HFVMs grown on exogenous ECM (including fibronectin and type I collagen and laminin) demonstrated no change in proliferation or DNA and protein synthesis with or without EGF. However, inhibition of collagen synthesis using cis-4-hydroxyproline resulted in a decrease in EGF-related HFVM proliferation and DNA and protein synthesis, which was reversed by exposure to L-proline but not by growth on type I collagen. Use of beta(1) but not beta(3) integrin antibody to inhibit cell interaction with ECM resulted in a decrease in HFVM proliferation and DNA and protein synthesis in response to EGF. Furthermore, EGF-dependent proliferation was enhanced by alpha(1)beta(1) and alpha(5)beta(1) antibodies that act as functional ligands, but not alpha(3)beta(1), the only beta(1) subtype expressed in adult myocytes. In conclusion, proliferating HFVMs synthesize collagen and fibronectin. The proliferative response of HFVMs to EGF requires the synthesis of collagen as well as attachment to specific alpha/beta(1) integrin heterodimers.

Increased elastase activity and deposition of the matrix glycoprotein tenascin-C (TN), codistributing with proliferating smooth muscle cells (SMCs), are features of pulmonary vascular disease. In pulmonary artery (PA) SMC cultures, TN is regulated by matrix metalloproteinases (MMPs) and mechanical stress. On attached collagen gels, MMPs upregulate TN, leading to SMC proliferation, whereas on floating collagen, reduced MMPs suppress TN and induce SMC apoptosis. We now investigate the response of SMCs in the whole vessel by comparing attached and floating conditions using either normal PAs derived from juvenile pigs or normal or hypertrophied rat PAs that were embedded in collagen gels for 8 days. Normal porcine PAs in attached collagen gels were characterized by increasing activity of MMP-2 and MMP-9 assessed by zymography and TN deposition detected by Western immunoblotting and densitometric analysis of immunoreactivity. PAs on floating collagen showed reduced activity of both MMPs and deposition of TN. Tenascin-rich foci were associated with proliferating cell nuclear antigen immunoreactivity, and TN-poor areas with apoptosis, by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, but no difference in wall thickness was observed. Although normal rat PAs were similar to piglet vessels, hypertrophied rat PAs showed an amplified response. Increased elastase, MMP-2, TN, and elastin deposition, as well as SMC proliferating cell nuclear antigen positivity, correlated with progressive medial thickening on attached collagen, whereas reduced MMP-2, elastase, TN, and induction of SMC apoptosis accompanied regression of the thickened media on floating collagen. In showing that hypertrophied SMCs in the intact vessel can be made to apoptose and that resorption of extracellular matrix can be achieved by inhibition of elastase and MMPs, our study suggests novel strategies to reverse vascular disease.

Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule-associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.

In previous studies, we showed that induction of pulmonary artery (PA) smooth muscle cell (SMC) elastase activity by serum-treated elastin (STE) requires DNA transcription. We therefore used differential mRNA display to identify transcripts expressed coincident with elastase induction. Twenty-four individual transcripts were differentially expressed from a screen of approximately 2000 mRNA sequences. An mRNA with sequence homology to the human transcription factor AML1 was identified and subsequently cloned from ovine PA SMCs. Since AML1 binds to a consensus sequence in the promoter of neutrophil elastase, we pursued the possibility that AML1 is a candidate transcription factor for SMC elastase. We documented by immunohistochemistry that serum stimulation induces increased expression of AML1 in the nucleus of PA SMCs. We also showed that STE induction of elastase activity is associated with early expression of AML1 mRNA and protein and that AML1 consensus sequence DNA binding activity is increased in nuclear extracts of STE-treated cells. In addition, AML1 antisense oligonucleotides reduced serum induction of elastase activity. Our study thus provides the first functional evidence of AML1 transcriptional activity related to elastase genes and offers novel insights into the broader biological significance of AML1 in nonmyeloid cells.

Tenascin-C, an extracellular matrix glycoprotein prominent during tissue remodeling, has been linked to cell migration, proliferation, and apoptosis. To determine its potential role in the pathobiology of pulmonary hypertension, we compared tenascin expression in adult and infant rat pulmonary arteries (PAs) after injection of the toxin monocrotaline. Immunohistochemistry, in situ hybridization, and Northern blot analysis demonstrated induction of tenascin in adult rat central and peripheral PA. Tenascin was not, however, detected in infant vessels, which show spontaneous regression of vascular lesions. To determine a function for tenascin, we correlated its expression with evidence of apoptosis and cell proliferation using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and 5-bromo-2'-deoxyuridine labeling, respectively. Apoptosis was observed only in the adult rat PA endothelial cell layer, preceding the induction of tenascin, which colocalized both temporally and spatially with proliferating smooth muscle cells (SMCs). A cause-and-effect relationship was documented in cultured rat PA SMCs, where tenascin promoted growth in response to basic fibroblast growth factor and was a prerequisite for epidermal growth factor-induced proliferation. These data provide novel functional information suggesting that endothelial cell apoptosis precedes progressive pulmonary hypertension and that induction of tenascin may be critical to growth factor-dependent SMC proliferation.

We investigated the association between tissue elastolytic activity and the development of neointimal formation using a previously described porcine aortic organ culture. Neointimal formation is associated with the presence of intact endothelium (nondenuded cultures) but is markedly reduced if endothelial cells are removed (denuded cultures). In nondenuded organ cultures, elastolytic activity assessed by using [3H]elastin increased sixfold at day 3 after initiation of the culture (P < .01), a time earlier than the previously published increase in intimal smooth muscle cells (ISMCs). Elastolytic activity did not increase from day 3 to day 7 despite doubling of ISMCs but did double by day 14 (P < .01) and remained elevated to day 28, correlating with increases in ISMCs. In denuded organ cultures, elastolytic activity was much lower than in nondenuded organ cultures at day 3 (P < .05) but increased fivefold in the presence of nondenuded organ culture conditioned medium (P < .01). Addition of alpha 1-proteinase inhibitor for 14 days caused a 60% decrease in elastolytic activity in nondenuded organ cultures and a 27% reduction in ISMCs compared with untreated controls (P < .05 for both). The elastolytic activity, resolved as lytic bands on an elastin substrate gel, reflected candidate enzymes, one at 76 kD and perhaps a doublet at 43 and 50 kD. Our study suggests that endothelial cells release a soluble agent that enhances elastin-degrading activity in the aorta and may at least partially account for the initiation of neointimal formation.

Background: We previously identified in piglet cardiac allografts an immunoinflammatory response in coronary arteries in which increased fibronectin regulated by interleukin-1 beta was associated with early evidence of intimal thickening. In the present study, we used rabbits to assess whether acute neointimal formation after cardiac transplantation was reduced by blockade of tumor necrosis factor (TNF)-alpha, which modulates interleukin-1 beta, or by cyclosporine A., Methods and Results: Sixteen rabbits underwent heterotopic cardiac transplantation and were given saline, TNF-soluble receptor (sr), or cyclosporine A. In host hearts from saline- or TNFsr-treated groups, few coronary arteries (approximately 13% to 16%) had intimal thickening, whereas values were higher in the cyclosporine A-treated group (approximately 30%). In donor hearts from the saline-treated group, however, approximately 68% of vessels had intimal thickening versus approximately 32% in TNFsr- and approximately 30% in cyclosporine A-treated groups (P < .01 for both). Severity of intimal thickening assessed quantitatively as percent vessel area was approximately 38% in the saline-treated group but reduced in TNFsr- and cyclosporine A-treated groups to approximately 22% and 18%, respectively (P < .01 for each). Immunohistochemistry revealed increased staining for major histocompatibility complex II, T cells, interleukin-1 beta, TNF-alpha, and fibronectin in donor coronary arteries from saline-treated animals when compared with TNFsr- and cyclosporine A-treated animals. Grade 3 myocardial rejection was observed in both saline- and TNFsr-treated groups, but only grade 1 was apparent in the cyclosporine A-treated group., Conclusions: In vivo blockade of TNF-alpha suppresses the acute development of neointimal formation by selectively reducing the vascular immunoinflammatory reaction and accumulation of fibronectin, whereas cyclosporine A suppresses both the myocardial and the vascular immune reaction.

Background: Graft coronary arteriopathy has become the major complication observed in the late follow-up of cardiac transplant patients. We investigated this process experimentally in piglets after a heterotopic cardiac transplant and observed early changes in donor coronary arteries compatible with an immune-inflammatory process, ie, there is increased expression of interleukin-1 beta (IL-1 beta), fibronectin, and activated lymphocytes associated with intimal thickening., Methods and Results: In this study, we cultured porcine coronary artery endothelial cells from host and donor hearts and found similarities in morphology and uptake of acetylated low-density lipoprotein (LDL). As well, host and donor cells showed similar patterns of growth, protein, and glycosaminoglycan synthesis. Endothelial cell fibronectin synthesis was determined after radiolabeling with [35S]-methionine in serum-free medium, gelatin-sepharose extraction of the culture medium and resolution on 5% SDS-PAGE. Donor coronary artery endothelial cell fibronectin synthesis was up to fivefold higher than that of host but was not associated with comparable increased levels of fibronectin mRNA. IL-1 beta appeared to mediate this enhanced fibronectin production, since the IL-1 receptor antagonist caused a 50% decrease in this feature, a change not observed in host cells. Furthermore, donor endothelial cells produced twice the amount of IL-1 beta compared with host cells as judged by immunoprecipitation., Conclusions: Increased donor coronary artery endothelial cell fibronectin appears to be regulated at least partly by an autocrine mechanism involving IL-1 beta, and fibronectin may mediate lymphocyte trafficking and smooth muscle cell migration related to graft arteriopathy.