Although the incidence of pulmonary hypertension is higher in female patients, numerous experimental studies have demonstrated better outcome in female animals, exacerbation of the disease after ovariectomy, and a strong protective effect of estrogen: a phenomenon known as the “estrogen paradox” of pulmonary hypertension. On the other hand, some clinical studies have indirectly linked estrogen to increased risk of portopulmonary hypertension, whereas others implicate increased estrogen metabolism and high levels of certain estrogen metabolites in promoting pulmonary vascular remodeling in familial pulmonary arterial hypertension. In this review we investigate the estrogen paradox through highlighting the differential receptor-mediated effects of estrogen. Although estrogen and estrogen receptor–based therapies have shown promise in rescuing preexisting pulmonary hypertension in animals, their role is yet to be defined in humans.
estrogen paradox; pulmonary hypertension; estrogen receptors; estrogen metabolites
Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.
Reduced vascular expression of bone morphogenetic type IA receptor (Bmpr1a) has been found in patients with pulmonary arterial hypertension (PAH). Our previous studies in mice with patchy deletion of Bmpr1a in vascular smooth muscle cells and cardiac myocytes showed decreased distal vascular remodeling despite a similar severity of hypoxic pulmonary hypertension (HPH). We speculate increased stiffness from ectopic deposition of collagen in proximal pulmonary arteries (PAs) might account for HPH. Pulsatile pressure-flow relationships were measured in isolated, ventilated, perfused lungs of SM22α;TRE-Cre;R26R;Bmpr1aflox/flox (KO) mice and wild-type (WT) littermates following 21 days (hypoxia) and 0 days (control) of chronic hypoxia. Pulmonary vascular impedance, which yields insight into proximal and distal arterial remodeling, was calculated. Reduced Bmpr1a expression had no effect on input impedance Z0 (P=0.52) or characteristic impedance ZC (P=0.18) under control conditions; it also had no effect on the decrease in Z0 via acute rho kinase inhibition. However, following chronic hypoxia, reduced Bmpr1a expression increased ZC (P<0.001) without affecting Z0 (P=0.72). These results demonstrate that Bmpr1a deficiency does not significantly alter the hemodynamic function of the distal vasculature or its response to chronic hypoxia but larger, more proximal arteries are affected. In particular, reduced Bmpr1a expression likely decreased dilatation and increased stiffening in response to hypoxia, probably by collagen accumulation. Increased PA stiffness can have a significant impact on right ventricular function. This study illustrates for the first time how proximal pulmonary artery changes in the absence of distal pulmonary artery changes contribute to pulmonary hypertension.
Characteristic impedance; pulsatile pressure-flow relationships; arterial stiffness; pulmonary hypertension; knockout mouse; pulmonary hemodynamics
Recent clinical and experimental studies are redefining the cellular and molecular bases of pulmonary arterial hypertension (PAH). The genetic abnormalities first identified in association with the idiopathic form of PAH — together with a vast increase in our understanding of cell signaling, cell transformation, and cell-cell interactions; gene expression; microRNA processing; and mitochondrial and ion channel function — have helped explain the abnormal response of vascular cells to injury. Experimental and clinical studies now converge on the intersection and interactions between a genetic predisposition involving the BMPR2 signaling pathway and an impaired metabolic and chronic inflammatory state in the vessel wall. These deranged processes culminate in an exuberant proliferative response that occludes the pulmonary arterial (PA) lumen and obliterates the most distal intraacinar vessels. Here, we describe emerging therapies based on preclinical studies that address these converging pathways.
Pulmonary arterial hypertension (PAH) remains a life-limiting condition with a major impact on the ability to lead a normal life. Although existing therapies may improve the outlook in some patients there remains a major unmet need to develop more effective therapies in this condition. There have been significant advances in our understanding of the genetic, cell and molecular basis of PAH over the last few years. This research has identified important new targets that could be explored as potential therapies for PAH. In this review we discuss whether further exploitation of vasoactive agents could bring additional benefits over existing approaches. Approaches to enhance smooth muscle cell apotosis and the potential of receptor tyrosine kinase inhibition are summarised. We evaluate the role of inflammation, epigenetic changes and altered glycolytic metabolism as potential targets for therapy, and whether inherited genetic mutations in PAH have revealed druggable targets. The potential of cell based therapies and gene therapy are also discussed. Potential candidate pathways that could be explored in the context of experimental medicine are identified.
pulmonary arterial hypertension; cellular mechanisms; inflammation; metabolism; tyrosine kinase inhibition; genetics; epigenetics; apoptosis
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.
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+CD25hi or CD4+CD25- T cell subsets prior to vascular injury attenuated the development of PAH. Immune reconstitution 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.
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.
pulmonary arterial hypertension; inflammation; regulatory T cell; bone morphogenetic protein receptor type 2
Rationale: Mechanical ventilation with O2-rich gas (MV-O2) offers life-saving treatment for respiratory failure, but also promotes lung injury. We previously reported that MV-O2 of newborn mice increased lung elastase activity, causing elastin degradation and redistribution of elastic fibers from septal tips to alveolar walls. These changes were associated with transforming growth factor (TGF)-β activation and increased apoptosis leading to defective alveolarization and lung growth arrest, as seen in neonatal chronic lung disease.
Objectives: To determine if intratracheal treatment of newborn mice with the serine elastase inhibitor elafin would prevent MV-O2–induced lung elastin degradation and the ensuing cascade of events causing lung growth arrest.
Methods: Five-day-old mice were treated via tracheotomy with recombinant human elafin or vehicle (lactated-Ringer solution), followed by MV with 40% O2 for 8–24 hours; control animals breathed 40% O2 without MV. At study's end, lungs were harvested to assess key variables noted below.
Measurements and Main Results: MV-O2 of vehicle-treated pups increased lung elastase and matrix metalloproteinase-9 activity when compared with unventilated control animals, causing elastin degradation (urine desmosine doubled), TGF-β activation (pSmad-2 tripled), and apoptosis (cleaved-caspase-3 increased 10-fold). Quantitative lung histology showed larger and fewer alveoli, greater inflammation, and scattered elastic fibers. Elafin blocked these MV-O2–induced changes.
Conclusions: Intratracheal elafin, by blocking lung protease activity, prevented MV-O2–induced elastin degradation, TGF-β activation, apoptosis, and dispersion of matrix elastin, and attenuated lung structural abnormalities noted in vehicle-treated mice after 24 hours of MV-O2. These findings suggest that elastin breakdown contributes to defective lung growth in response to MV-O2 and might be targeted therapeutically to prevent MV-O2–induced lung injury.
elafin; elastin degradation; neonatal chronic lung disease; ventilator-induced lung injury; bronchopulmonary dysplasia
Rationale: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown.
Objectives: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH.
Methods: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B−/−), or early growth response-1 (Egr-1−/−), were evaluated for vascular morphology and hemodynamics.
Measurements and Main Results: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B−/− or Egr-1−/−, but not Beclin 1+/−, mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia.
Conclusions: The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B−/− and Egr-1−/− mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.
autophagy; hypoxia; hypertension, pulmonary
Idiopathic and familial forms of pulmonary arterial hypertension (PAH) occur more frequently in women than men. However, the reason for this remains unknown. Both the calcium binding protein S100A4/Mts1 (Mts1) and its endogenous receptor (receptor for advanced glycosylation end products; RAGE) have been implicated in the development of PAH. We wished to investigate if the Mts1/RAGE pathway may play a role in the gender bias associated with PAH.
We investigated the effects of gender on development of PAH in mice over-expressing Mts1 (Mts1+ mice) via measurement of pulmonary arterial remodeling, systolic right ventricular pressure (sRVP) and right ventricular hypertrophy (RVH). Gender differences in pulmonary arterial Mts1 and RAGE expression were assessed by qRT-PCR and immunohistochemistry. Western blotting and cell counts were used to investigate interactions between 17β-estradiol, Mts1 and RAGE on proliferation of human pulmonary artery smooth muscle cells (hPASMCs). Statistical analysis was by one-way analysis of variance with Dunnetts post test or two-way analysis of variance with Bonferronis post test, as appropriate.
Female Mts1+ mice developed increased sRVP and pulmonary vascular remodeling, whereas male Mts1+ mice remained unaffected. The development of plexiform-like lesions in Mts1+ mice was specific to females. These lesions stained positive for both Mts1 and RAGE in the endothelial and adventitial layers. Expression of pulmonary arterial Mts1 was greater in female than male Mts1+ mice, and was localised to the medial and adventitial layers in non plexiform-like pulmonary arteries. RAGE gene expression and immunoreactivity were similar between male and female Mts1+ mice and RAGE staining was localised to the endothelial layer in non plexiform-like pulmonary arteries adjacent to airways. In non-plexiform like pulmonary arteries not associated with airways RAGE staining was present in the medial and adventitial layers. Physiological concentrations of 17β-estradiol increased Mts1 expression in hPASMCs. 17β-estradiol-induced hPASMC proliferation was inhibited by soluble RAGE, which antagonises the membrane bound form of RAGE.
Mts1 over-expression combined with female gender is permissive to the development of experimental PAH in mice. Up-regulation of Mts1 and subsequent activation of RAGE may contribute to 17β-estradiol-induced proliferation of hPASMCs.
The Division of Lung Diseases of the National Heart, Lung, and Blood Institute, with the Office of Rare Diseases Research, held a workshop to identify priority areas and strategic goals to enhance and accelerate research that will result in improved understanding of the lung vasculature, translational research needs, and ultimately the care of patients with pulmonary vascular diseases. Multidisciplinary experts with diverse experience in laboratory, translational, and clinical studies identified seven priority areas and discussed limitations in our current knowledge, technologies, and approaches. The focus for future research efforts include the following: (1) better characterizing vascular genotype–phenotype relationships and incorporating systems biology approaches when appropriate; (2) advancing our understanding of pulmonary vascular metabolic regulatory signaling in health and disease; (3) expanding our knowledge of the biologic relationships between the lung circulation and circulating elements, systemic vascular function, and right heart function and disease; (4) improving translational research for identifying disease-modifying therapies for the pulmonary hypertensive diseases; (5) establishing an appropriate and effective platform for advancing translational findings into clinical studies testing; and (6) developing the specific technologies and tools that will be enabling for these goals, such as question-guided imaging techniques and lung vascular investigator training programs. Recommendations from this workshop will be used within the Lung Vascular Biology and Disease Extramural Research Program for planning and strategic implementation purposes.
right ventricle; pulmonary hypertension; metabolism; genomics; phenotyping
Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. We demonstrate that depletion of autophagic proteins microtubule associated protein-1 light chain 3B (LC3B) and Beclin 1 enhances caspase-1 activation and secretion of interleukin-1β and interleukin-18. Autophagic protein depletion promoted accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial ROS. Cytosolic mtDNA contributed to IL-1β and IL-18 secretion in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.
Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPARγ and β-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPARγ/β-catenin–dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPARγ in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction.
Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except α2, α4, γ and δ in four different types of ECs. Using siRNA methodology, we found that the α7 nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration and tube formation). The α9 and α7 nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the α7 nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis.
Vascular smooth muscle cell motility relies on interdependent activation of canonical and noncanonical Wnt signal transduction pathways; fibronectin, produced in response to BMP-2–mediated activation of β-catenin, promotes motility by activating an integrin-linked kinase via α4-integrin.
We present a novel cell-signaling paradigm in which bone morphogenetic protein 2 (BMP-2) consecutively and interdependently activates the wingless (Wnt)–β-catenin (βC) and Wnt–planar cell polarity (PCP) signaling pathways to facilitate vascular smooth muscle motility while simultaneously suppressing growth. We show that BMP-2, in a phospho-Akt–dependent manner, induces βC transcriptional activity to produce fibronectin, which then activates integrin-linked kinase 1 (ILK-1) via α4-integrins. ILK-1 then induces the Wnt–PCP pathway by binding a proline-rich motif in disheveled (Dvl) and consequently activating RhoA-Rac1–mediated motility. Transfection of a Dvl mutant that binds βC without activating RhoA-Rac1 not only prevents BMP-2–mediated vascular smooth muscle cell motility but promotes proliferation in association with persistent βC activity. Interfering with the Dvl-dependent Wnt–PCP activation in a murine stented aortic graft injury model promotes extensive neointima formation, as shown by optical coherence tomography and histopathology. We speculate that, in response to injury, factors that subvert BMP-2–mediated tandem activation of Wnt–βC and Wnt–PCP pathways contribute to obliterative vascular disease in both the systemic and pulmonary circulations.
Previously we related fibronectin (FN) mRNA translation to an interaction between an AU-rich element (ARE) in the FN-3′ UTR and Light Chain 3 (LC3) of microtubule associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce both little FN and LC3, we used these cells to investigate how LC3-mediated FN mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced FN mRNA translation that depended on an interaction between a triple arginine motif in LC3 and the ARE in FN mRNA, as determined by relating FN synthesis to mRNA levels, by polysome analysis of the FN mRNA transcript, and by RNA binding assays. Wild type, (WT) but not mutant LC3, accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of LC3-WT with vector-transfected HT1080 cells revealed increased FN-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in LC3-WT vs. vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show by qRT-PCR that enhanced expression of CTGF is FN- dependent and that LC3 mediated adhesion, invasion and proliferation are CTGF-dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types.
mRNA translation; microtubule associated protein; tumor invasiveness; LC3; fibronectin; connective tissue growth factor
S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMC), through an interaction with the receptor for advanced glycation end products (RAGE).
We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (R) II, observed in pulmonary arterial hypertension (PAH).
Methods and Results
Both S100A4/Mts1 (500ng/ml) and BMP-2 (10ng/ml) induce migration of hPASMCS in a novel co-dependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII siRNA. Phosphorylation of ERK is induced by both ligands and is required for motility by inducing MMP2 activity, but phosphoERK1/2 is blocked by anti-RAGE and not by BMPRII siRNA. In contrast, BMPRII siRNA, but not anti-RAGE, reduces expression of intracellular chloride channel 4 (CLIC4), 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 (MHCIIA), but does alter alignment of MHCIIA 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 lamellipodiae in motile cells.
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, i.e., cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.
Bone morphogenetic protein; S100 protein; Vascular smooth muscle cells; Intracellular chloride channel; migration
Although obesity, dyslipidemia, and insulin resistance (IR) are well known risk factors for systemic cardiovascular disease, their impact on pulmonary arterial hypertension (PAH) is unknown. Our previous studies indicate that IR may be a risk factor for PAH. We now investigate the prevalence of IR in PAH and explore its relationship to disease severity.
Clinical data and fasting blood samples were evaluated in 81 non-diabetic PAH females. National Health and Nutrition Examination Surveys (NHANES) females (n=967) served as controls. Fasting triglyceride to high-density lipoprotein cholesterol ratio (TG/HDL-C) was used as a surrogate of insulin sensitivity.
While BMI was similar in NHANES vs PAH females (28.6 vs. 28.7 kg/m2), PAH females were more likely to be IR (45.7% vs. 21.5%) and less likely to be IS (43.2% vs. 57.8%, p<0.0001). PAH females mostly had NYHA class II and III symptoms (82.7%). Etiology, NYHA class, 6-minute-walk-distance, and hemodynamics did not differ between IR and IS PAH groups. However, the presence of IR and a higher NYHA class were associated with poorer 6-months event-free survival (58% vs. 79%, p<0.05).
Insulin Resistance appears to be more common in PAH females than in the general population, and may be a novel risk factor or disease modifier which might impact survival.
Insulin Resistance; Obesity; Pulmonary Arterial Hypertension
Mutations in bone morphogenetic protein (BMP) receptor II (BMPRII) are associated with pulmonary artery endothelial cell (PAEC) apoptosis and the loss of small vessels seen in idiopathic pulmonary arterial hypertension. Given the low penetrance of BMPRII mutations, abnormalities in other converging signaling pathways may be necessary for disease development. We hypothesized that BMPRII supports normal PAEC function by recruiting Wingless (Wnt) signaling pathways to promote proliferation, survival, and motility. In this study, we report that BMP-2, via BMPRII-mediated inhibition of GSK3-β, induces β-catenin (β-C) accumulation and transcriptional activity necessary for PAEC survival and proliferation. At the same time, BMP-2 mediates phosphorylated Smad1 (pSmad1) or, with loss of BMPRII, pSmad3-dependent recruitment of Disheveled (Dvl) to promote RhoA–Rac1 signaling necessary for motility. Finally, using an angiogenesis assay in severe combined immunodeficient mice, we demonstrate that both β-C– and Dvl-mediated RhoA–Rac1 activation are necessary for vascular growth in vivo. These findings suggest that the recruitment of both canonical and noncanonical Wnt pathways is required in BMP-2–mediated angiogenesis.
Expression of bone morphogenetic protein receptor 1a (Bmpr1a) is attenuated in lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We therefore ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMC) by breeding mice with 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 cell proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMC 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 matrix metalloproteinase (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 human pulmonary artery SMC reduced MMP-2 and MMP-9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP-2 or MMP-9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of Bmpr1a reduced MMP-2 activity and knock-down of either Bmpr1a or MMP-2 caused resistance to apoptosis. Thus loss of Bmpr1a, by decreasing MMP-2 and/or MMP-9 activity, can account for vascular dilatation and persistence of brain microvessels leading to impaired organogenesis documented in the brain.
Bmpr1a (Alk3); vasculogenesis; heart development; craniofacial development; matrix metalloproteinase-2 and -9; smooth muscle cell proliferation; pericyte apoptosis
Vascular expression of bone morphogenetic type IA receptor (Bmpr1a) is reduced in lungs of patients with pulmonary arterial hypertension (PAH), but the significance of this observation is poorly understood. To elucidate the role of Bmpr1a in the vascular pathology of PAH and associated right ventricular dysfunction, we deleted Bmpr1a in vascular smooth muscle cells (SMC) and in cardiac myocytes in mice using the SM22α;TRE-Cre/LoxP;R26R system. The LacZ distribution reflected patchy deletion of Bmpr1a in the lung vessels, aorta, and heart of SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutants. This reduction in BMPR-IA expression was confirmed by western immunoblot and immunohistochemistry in the flox/flox group. This did not affect pulmonary vasoreactivity to acute hypoxia (10% O2) or the increase in right ventricular (RV) systolic pressure (RVSP) and RV hypertrophy (RVH) following three weeks in chronic hypoxia. However, both SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutant mice had fewer muscularized distal pulmonary arteries (PAs) and attenuated loss of peripheral PAs compared to age-matched control littermates in hypoxia. When Bmpr1a expression was reduced by siRNA in cultured PASMC, serum-induced proliferation was attenuated explaining decreased hypoxia-mediated muscularization of distal vessels. When Bmpr1a was reduced in cultured microvascular pericytes by siRNA, resistance to apoptosis was observed and this could account for protection against hypoxia-mediated vessel loss. The similar elevation in RVSP and RVH, despite the attenuated remodeling with chronic hypoxia in SM22α;TRE-Cre;R26R;Bmpr1aflox/flox mutants vs. controls, was not a function of elevated LV end diastolic pressure but was associated with increased periadventitial deposition of elastin and collagen, potentially enhancing vascular stiffness.
Bmpr1a; Pulmonary hypertension; Hypoxia; Vascular remodeling; Smooth muscle cell; Pericytes; Proliferation; Apoptosis; Transgenic mice
Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4+ T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule α, but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH.
Recent investigations have suggested that it might be possible to reverse the pathology of pulmonary arterial hypertension (PAH), a disorder that can be rapidly progressive and fatal despite current treatments including i.v. prostacyclin. This review will address the cellular and molecular processes implicated in clinical, genetic, and experimental studies as underlying the pulmonary vascular abnormalities associated with PAH. Emerging treatments are aimed at inducing apoptosis of abnormal vascular cells that obstruct blood flow and at promoting regeneration of “lost” distal vasculature.
Loss-of-function mutations in bone morphogenetic protein receptor II (BMP-RII) are linked to pulmonary arterial hypertension (PAH); the ligand for BMP-RII, BMP-2, is a negative regulator of SMC growth. Here, we report an interplay between PPARγ and its transcriptional target apoE downstream of BMP-2 signaling. BMP-2/BMP-RII signaling prevented PDGF-BB–induced proliferation of human and murine pulmonary artery SMCs (PASMCs) by decreasing nuclear phospho-ERK and inducing DNA binding of PPARγ that is independent of Smad1/5/8 phosphorylation. Both BMP-2 and a PPARγ agonist stimulated production and secretion of apoE by SMCs. Using a variety of methods, including short hairpin RNAi in human PASMCs, PAH patient–derived BMP-RII mutant PASMCs, a PPARγ antagonist, and PASMCs isolated from PPARγ- and apoE-deficient mice, we demonstrated that the antiproliferative effect of BMP-2 was BMP-RII, PPARγ, and apoE dependent. Furthermore, we created mice with targeted deletion of PPARγ in SMCs and showed that they spontaneously developed PAH, as indicated by elevated RV systolic pressure, RV hypertrophy, and increased muscularization of the distal pulmonary arteries. Thus, PPARγ-mediated events could protect against PAH, and PPARγ agonists may reverse PAH in patients with or without BMP-RII dysfunction.
Rationale: Injurious agents often cause less severe injury in neonates as compared with adults.
Objective: We hypothesized that maturational differences in lung inflammation induced by lipopolysaccharide (LPS) may be related to the nature of the nuclear factor (NF)-κB complex activated, and the profile of target genes expressed.
Methods: Neonatal and adult mice were injected with intraperitoneal LPS. Lung inflammation was assessed by histology, and apoptosis was determined by TUNEL (terminal deoxynucleotidyl transferase UTP nick-end labeling). The expression of candidate inflammatory and apoptotic mediators was evaluated by quantitative real-time polymerase chain reaction and Western immunoblot.
Results: Neonates demonstrated reduced inflammation and apoptosis, 24 hours after LPS exposure, as compared with adults. This difference was associated with persistent activation of NF-κB p65p50 heterodimers in the neonates in contrast to early, transient activation of p65p50 followed by sustained activation of p50p50 in the adults. Adults had increased expression of a panel of inflammatory and proapoptotic genes, and repression of antiapoptotic targets, whereas no significant changes in these mediators were observed in the neonates. Inhibition of NF-κB activity in the neonates decreased apoptosis, but heightened inflammation, with increased expression of the same inflammatory genes elevated in the adults. In contrast, inhibition of NF-κB in the adults resulted in partial suppression of the inflammatory response.
Conclusions: NF-κB activation in the neonatal lung is antiinflammatory, protecting against LPS-mediated lung inflammation by repressing similar inflammatory genes induced in the adult.
acute lung injury; apoptosis; gene expression regulation