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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)-kappaB 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-kappaB 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-kappaB 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-kappaB in the adults resulted in partial suppression of the inflammatory response.Conclusions: NF-kappaB activation in the neonatal lung is antiinflammatory, protecting against LPS-mediated lung inflammation by repressing similar inflammatory genes induced in the adult. [ABSTRACT FROM AUTHOR]

Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. The endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the artery-to-capillary-to-vein axis. Although our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site-specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease.

Rapid expansion of the pulmonary microvasculature through angiogenesis drives alveolarization, the final stage of lung development that occurs postnatally and dramatically increases lung gas-exchange surface area. Disruption of pulmonary angiogenesis induces long-term structural and physiologic lung abnormalities, including bronchopulmonary dysplasia, a disease characterized by compromised alveolarization. Although endothelial cells are primary determinants of pulmonary angiogenesis, mesenchymal cells (MC) play a critical and dual role in angiogenesis and alveolarization. Therefore, we performed single cell transcriptomics and in-situ imaging of the developing lung to profile mesenchymal cells during alveolarization and in the context of lung injury. Specific mesenchymal cell subtypes were present at birth with increasing diversity during alveolarization even while expressing a distinct transcriptomic profile from more mature correlates. Hyperoxia arrested the transcriptomic progression of the MC, revealed differential cell subtype vulnerability with pericytes and myofibroblasts most affected, altered cell to cell communication, and led to the emergence of Acta1 expressing cells. These insights hold the promise of targeted treatment for neonatal lung disease, which remains a major cause of infant morbidity and mortality across the world., (© 2024. The Author(s).)

Preterm birth affects ~10% of pregnancies in the US. Despite familial associations, identifying at-risk genetic loci has been challenging. We built deep learning and graphical models to score mutational effects at base resolution via integrating the pregnant myometrial epigenome and large-scale patient genomes with spontaneous preterm birth (sPTB) from European and African American cohorts. We uncovered previously unidentified sPTB genes that are involved in myometrial muscle relaxation and inflammatory responses and that are regulated by the progesterone receptor near labor onset. We studied genomic variants in these genes in our recruited pregnant women administered progestin prophylaxis. We observed that mutation burden in these genes was predictive of responses to progestin treatment for preterm birth. To advance therapeutic development, we screened ~4000 compounds, identified candidate molecules that affect our identified genes, and experimentally validated their therapeutic effects on regulating labor. Together, our integrative approach revealed the druggable genome in preterm birth and provided a generalizable framework for studying complex diseases.

Pulmonary arterial hypertension (PAH) is a disease characterized by increased vasoconstriction and vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) highly express the transcription factor hypoxia-inducible factor-1α (HIF-1α), yet the role of PASMC HIF-1α in the development of PAH remains controversial. To study the role of SMC HIF-1α in the pulmonary vascular response to acute and chronic hypoxia, we used a gain-of-function strategy to stabilize HIF-1α in PASMC by generating mice lacking prolyl hydroxylase domain (PHD) 1 and 2 in SM22α-expressing cells. This strategy increased HIF-1α expression and transcriptional activity under conditions of normoxia and hypoxia. Acute hypoxia increased right ventricular systolic pressure (RVSP) in control, but not in SM22α-PHD1/2 -/- mice. Chronic hypoxia increased RVSP and vascular remodeling more in control SM22α-PHD1/2 +/+ than in SM22α-PHD1/2 -/- mice. In vitro studies demonstrated increased contractility and myosin light chain phosphorylation in isolated PHD1/2 +/+ compared with PHD1/2 -/- PASMC under both normoxic and hypoxic conditions. After chronic hypoxia, there was more p27 and less vascular remodeling in SM22α-PHD1/2 -/- compared with SM22α-PHD1/2 +/+ mice. Hypoxia increased p27 in PASMC isolated from control patients, but not in cells from patients with idiopathic pulmonary arterial hypertension (IPAH). These findings highlight an SM22α-expressing cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling. Modulating HIF-1α expression in PASMC may represent a promising preventative and therapeutic strategy for patients with PAH. NEW & NOTEWORTHY In a mouse model wherein hypoxia-inducible factor 1 alpha (HIF-1α) is stabilized in vascular smooth muscle cells, we found that HIF-1α regulates vasoconstriction by limiting phosphorylation of myosin light chain and regulates vascular remodeling through p27 induction. These findings highlight a cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling.

Preterm infants with oxygen supplementation are at high risk for bronchopulmonary dysplasia (BPD), a neonatal chronic lung disease. Inflammation with macrophage activation is central to the pathogenesis of BPD. CXCL10, a chemotactic and pro-inflammatory chemokine, is elevated in the lungs of infants evolving BPD and in hyperoxia-based BPD in mice. Here, we tested if CXCL10 deficiency preserves lung growth after neonatal hyperoxia by preventing macrophage activation. To this end, we exposed Cxcl10 knockout (Cxcl10 -/- ) and wild-type mice to an experimental model of hyperoxia (85% O 2 )-induced neonatal lung injury and subsequent regeneration. In addition, cultured primary human macrophages and murine macrophages (J744A.1) were treated with CXCL10 and/or CXCR3 antagonist. Our transcriptomic analysis identified CXCL10 as a central hub in the inflammatory network of neonatal mouse lungs after hyperoxia. Quantitative histomorphometric analysis revealed that Cxcl10 -/-  mice are in part protected from reduced alveolar. These findings were related to the preserved spatial distribution of elastic fibers, reduced collagen deposition, and protection from macrophage recruitment/infiltration to the lungs in Cxcl10 -/-  mice during acute injury and regeneration. Complimentary, studies with cultured human and murine macrophages showed that hyperoxia induces Cxcl10 expression that in turn triggers M1-like activation and migration of macrophages through CXCR3. Finally, we demonstrated a temporal increase of macrophage-related CXCL10 in the lungs of infants with BPD. In conclusion, our data demonstrate macrophage-derived CXCL10 in experimental and clinical BPD that drives macrophage chemotaxis through CXCR3, causing pro-fibrotic lung remodeling and arrest of alveolarization. Thus, targeting the CXCL10-CXCR3 axis could offer a new therapeutic avenue for BPD., (© 2023. Japanese Society of Inflammation and Regeneration.)

Worldwide, the incidence of both preterm births and chronic lung disease of infancy, or bronchopulmonary dysplasia, remains high. Infants with bronchopulmonary dysplasia have larger and fewer alveoli, a lung pathology that can persist into adulthood. Although recent data point to a role for hypoxia-inducible factor-1α (HIF-1α) in mediating pulmonary angiogenesis and alveolarization, the cell-specific role of HIF-1α remains incompletely understood. Thus, we hypothesized that HIF-1α, in a distinct subset of mesenchymal cells, mediates postnatal alveolarization. To test the hypothesis, we generated mice with a cell-specific deletion of HIF-1α by crossing SM22α promoter-driven Cre mice with HIF-1α flox/flox mice (SM22α-HIF-1α -/- ), determined SM-22α-expressing cell identity using single-cell RNA sequencing, and interrogated samples from preterm infants. Deletion of HIF-1α in SM22α-expressing cells had no effect on lung structure at day 3 of life. However, at 8 days, there were fewer and larger alveoli, a difference that persisted into adulthood. Microvascular density, elastin organization, and peripheral branching of the lung vasculature were decreased in SM22α-HIF-1α -/- mice, compared with control mice. Single-cell RNA sequencing demonstrated that three mesenchymal cell subtypes express SM22α: myofibroblasts, airway smooth muscle cells, and vascular smooth muscle cells. Pulmonary vascular smooth muscle cells from SM22α-HIF-1α -/- mice had decreased angiopoietin-2 expression and, in coculture experiments, a diminished capacity to promote angiogenesis that was rescued by angiopoietin-2. Angiopoietin-2 expression in tracheal aspirates of preterm infants was inversely correlated with overall mechanical ventilation time, a marker of disease severity. We conclude that SM22α-specific HIF-1α expression drives peripheral angiogenesis and alveolarization in the lung, perhaps by promoting angiopoietin-2 expression.

Pulmonary angiogenesis drives alveolarization, but the transcriptional regulators directing pulmonary angiogenesis remain poorly defined. Global, pharmacological inhibition of nuclear factor-kappa B (NF-κB) impairs pulmonary angiogenesis and alveolarization. However, establishing a definitive role for NF-κB in pulmonary vascular development has been hindered by embryonic lethality induced by constitutive deletion of NF-κB family members. We created a mouse model allowing inducible deletion of the NF-κB activator, IKKβ, in endothelial cells (ECs) and assessed the effect on lung structure, endothelial angiogenic function, and the lung transcriptome. Embryonic deletion of IKKβ permitted lung vascular development but resulted in a disorganized vascular plexus, while postnatal deletion significantly decreased radial alveolar counts, vascular density, and proliferation of both endothelial and nonendothelial lung cells. Loss of IKKβ impaired survival, proliferation, migration, and angiogenesis in primary lung ECs in vitro, in association with decreased expression of VEGFR2 and activation of downstream effectors. Loss of endothelial IKKβ in vivo induced broad changes in the lung transcriptome with downregulation of genes related to mitotic cell cycle, extracellular matrix (ECM)-receptor interaction, and vascular development, and the upregulation of genes related to inflammation. Computational deconvolution suggested that loss of endothelial IKKβ decreased general capillary, aerocyte capillary, and alveolar type I cell abundance. Taken together, these data definitively establish an essential role for endogenous endothelial IKKβ signaling during alveolarization. A deeper understanding of the mechanisms directing this developmental, physiological activation of IKKβ in the lung vasculature may provide novel targets for the development of strategies to enhance beneficial proangiogenic signaling in lung development and disease. NEW & NOTEWORTHY This study highlights the cell-specific complexity of nuclear factor kappa B signaling in the developing lung by demonstrating that inducible loss of IKKβ in endothelial cells impairs alveolarization, disrupts EC angiogenic function, and broadly represses genes important for vascular development.

Introduction: Pulmonary arterial hypertension (PAH) is characterised by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. We hypothesised that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis, and its loss contributes to PAH., Methods: Lung tissue and PMVECs from healthy and PAH patients were screened for Wnt production. Global and endothelial-specific Wnt7a -/- mice were generated and exposed to chronic hypoxia and Sugen-hypoxia (SuHx)., Results: Healthy PMVECs demonstrated >6-fold Wnt7a expression during angiogenesis that was absent in PAH PMVECs and lungs. Wnt7a expression correlated with the formation of tip cells, a migratory endothelial phenotype critical for angiogenesis. PAH PMVECs demonstrated reduced vascular endothelial growth factor (VEGF)-induced tip cell formation as evidenced by reduced filopodia formation and motility, which was partially rescued by recombinant Wnt7a. We discovered that Wnt7a promotes VEGF signalling by facilitating Y1175 tyrosine phosphorylation in vascular endothelial growth factor receptor 2 (VEGFR2) through receptor tyrosine kinase-like orphan receptor 2 (ROR2), a Wnt-specific receptor. We found that ROR2 knockdown mimics Wnt7a insufficiency and prevents recovery of tip cell formation with Wnt7a stimulation. While there was no difference between wild-type and endothelial-specific Wnt7a -/- mice under either chronic hypoxia or SuHx, global Wnt7a +/- mice in hypoxia demonstrated higher pulmonary pressures and severe right ventricular and lung vascular remodelling. Similar to PAH, Wnt7a +/- PMVECs exhibited an insufficient angiogenic response to VEGF-A that improved with Wnt7a., Conclusions: Wnt7a promotes VEGF signalling in lung PMVECs and its loss is associated with an insufficient VEGF-A angiogenic response. We propose that Wnt7a deficiency contributes to progressive small vessel loss in PAH., Competing Interests: Conflict of interest: V.A. de Jesus Perez reports support for the present manuscript from the National Institutes of Health National Heart, Lung, and Blood Institute; and outside the submitted work, holds a leadership position as AHA Chair of Diversity subcommittee. All other authors have nothing to disclose., (Copyright ©The authors 2023.)

At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2) contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3 , a paternally imprinted transcription factor. Hyperoxia, an injury that impairs angiogenesis induced both common and unique endothelial gene signatures, dysregulated capillary EC crosstalk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Though survival rates for preterm infants are improving, the incidence of chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD), remains high. Histologically, BPD is characterized by larger and fewer alveoli. Hypoxia-inducible factors (HIFs) may be protective in the context of hyperoxia-induced lung injury, but the cell-specific effects of HIF expression in neonatal lung injury remain unknown. Thus, we sought to determine whether HIF stabilization in SM22α-expressing cells can limit hyperoxia-induced neonatal lung injury. We generated SM22α-specific HIF-1α-stabilized mice ( SM22α-PHD1/2 -/- mice) by cross-breeding SM22α-promotor-driven Cre recombinase mice with prolyl hydroxylase PHD1 flox/flox and PHD2 flox/flox mice. Neonatal mice were randomized to 21% O 2 (normoxia) or 80% O 2 (hyperoxia) exposure for 14 days. For the hyperoxia recovery studies, neonatal mice were recovered from normoxia for an additional 10 wk. SM22α-specific HIF-1α stabilization mitigated hyperoxia-induced lung injury and preserved microvessel density compared with control mice for both neonates and adults. In SM22α-PHD1/2 -/- mice, pulmonary artery endothelial cells (PAECs) were more proliferative and pulmonary arteries expressed more collagen IV compared with control mice, even under hyperoxic conditions. Angiopoietin-2 (Ang2) mRNA expression in pulmonary artery smooth muscle cells (PASMC) was greater in SM22α-PHD1/2 -/- compared with control mice in both normoxia and hyperoxia. Pulmonary endothelial cells (PECs) cocultured with PASMC isolated from SM22α-PHD1/2 -/- mice formed more tubes and branches with greater tube length compared with PEC cocultured with PASMC isolated from SM22α-PHD1/2 +/+ mice. Addition of Ang2 recombinant protein further augmented tube formation for both PHD1/2 +/+ and PHD1/2 -/- PASMC. Cell-specific deletion of PHD1 and 2 selectively increases HIF-1α expression in SM22α-expressing cells and protects neonatal lung development despite prolonged hyperoxia exposure. HIF stabilization in SM22α-expressing cells preserved endothelial cell proliferation, microvascular density, increased angiopoietin-2 expression, and lung structure, suggesting a role for cell-specific HIF-1α stabilization to prevent neonatal lung injury.

Rationale: Premature infants exposed to oxygen are at risk for bronchopulmonary dysplasia (BPD), which is characterised by lung growth arrest. Inflammation is important, but the mechanisms remain elusive. Here, we investigated inflammatory pathways and therapeutic targets in severe clinical and experimental BPD., Methods and Results: First, transcriptomic analysis with in silico cellular deconvolution identified a lung-intrinsic M1-like-driven cytokine pattern in newborn mice after hyperoxia. These findings were confirmed by gene expression of macrophage-regulating chemokines ( Ccl2 , Ccl7 , Cxcl5 ) and markers ( Il6 , Il17A , Mmp12 ). Secondly, hyperoxia-activated interleukin 6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signalling was measured in vivo and related to loss of alveolar epithelial type II cells (ATII) as well as increased mesenchymal marker. Il6 null mice exhibited preserved ATII survival, reduced myofibroblasts and improved elastic fibre assembly, thus enabling lung growth and protecting lung function. Pharmacological inhibition of global IL-6 signalling and IL-6 trans-signalling promoted alveolarisation and ATII survival after hyperoxia. Third, hyperoxia triggered M1-like polarisation, possibly via Krüppel-like factor 4; hyperoxia-conditioned medium of macrophages and IL-6-impaired ATII proliferation. Finally, clinical data demonstrated elevated macrophage-related plasma cytokines as potential biomarkers that identify infants receiving oxygen at increased risk of developing BPD. Moreover, macrophage-derived IL6 and active STAT3 were related to loss of epithelial cells in BPD lungs., Conclusion: We present a novel IL-6-mediated mechanism by which hyperoxia activates macrophages in immature lungs, impairs ATII homeostasis and disrupts elastic fibre formation, thereby inhibiting lung growth. The data provide evidence that IL-6 trans-signalling could offer an innovative pharmacological target to enable lung growth in severe neonatal chronic lung disease., Competing Interests: Conflict of interest: D. Hirani has nothing to disclose. Conflict of interest: C.M. Alvira has nothing to disclose. Conflict of interest: S. Danopoulos has nothing to disclose. Conflict of interest: C. Milla reports grants from Proteostasis Inc and Eloxx Pharmaceuticals, grants and personal fees for advisory board work from Vertex Pharmaceuticals, outside the submitted work. Conflict of interest: M. Donato has nothing to disclose. Conflict of interest: L. Tian has nothing to disclose. Conflict of interest: J. Mohr has nothing to disclose. Conflict of interest: K. Dinger has nothing to disclose. Conflict of interest: C. Vohlen has nothing to disclose. Conflict of interest: J. Selle has nothing to disclose. Conflict of interest: S. v. Koningsbruggen-Rietschel has nothing to disclose. Conflict of interest: V. Barbarino has nothing to disclose. Conflict of interest: C. Pallasch has nothing to disclose. Conflict of interest: S. Rose John has nothing to disclose. Conflict of interest: M. Odenthal has nothing to disclose. Conflict of interest: G.S. Pryhuber reports grants from NHLBI, during the conduct of the study. Conflict of interest: S. Mansouri has nothing to disclose. Conflict of interest: R. Savai has nothing to disclose. Conflict of interest: W. Seeger reports personal fees from Actelion, Abivax, Bayer AG, Vectura, Medspray, United Therapeutics, Liquidia and Pieris, outside the submitted work. Conflict of interest: P. Khatri has nothing to disclose. Conflict of interest: D. Al Alam has nothing to disclose. Conflict of interest: J. Dötsch has nothing to disclose. Conflict of interest: M.A. Alejandre Alcazar has nothing to disclose., (Copyright ©The authors 2022.)

Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that NF-κB promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF-κB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate proangiogenic NF-κB signaling in the developing pulmonary vasculature, proteomic analysis of the lung secretome was performed using two-dimensional difference gel electrophoresis. NF-κB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PECs) and TGFBI (transforming growth factor-β-induced protein)-regulated genes identified using RNA sequencing. Alveolarization and pulmonary angiogenesis was assessed in wild-type and Tgfbi null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Secreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PECs. Proteomic analysis identified TGFBI as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NF-κB via αvβ3 integrins. RNA sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PECs. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. Our studies identify TGFBI as a developmentally regulated protein that promotes NF-κB-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

At birth, the lungs rapidly transition from a pathogen-free, hypoxic environment to a pathogen-rich, rhythmically distended air-liquid interface. Although many studies have focused on the adult lung, the perinatal lung remains unexplored. Here, we present an atlas of the murine lung immune compartment during early postnatal development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed an orchestration of distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability., Competing Interests: RD, FZ, XC, ML, RJ, MS, SQ, DC, CA No competing interests declared, (© 2020, Domingo-Gonzalez et al.)

A wealth of evidence supports the broad therapeutic potential of NF-κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF-κB and the NF-κB interacting long non-coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non-canonical actions of EZH2 on the regulation of NF-κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non-transformed breast epithelial cells, triple negative MDA-MB-231 cells and hormone responsive MCF-7 cells, and measured changes in EZH2/NF-κB/NKILA levels to confirm their interdependence. We demonstrate cell line-specific fluctuations in these factors that functionally contribute to epithelial-to-mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA-MB-231 cell motility and CDK4-mediated MCF-7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non-transformed cells. Notably, these events are mediated by a cell-context dependent gain or loss of NKILA and NF-κB. Depletion of NF-κB in non-transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF-κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Intrauterine growth restriction (IUGR) in premature newborns increases the risk for bronchopulmonary dysplasia, a chronic lung disease characterized by disrupted pulmonary angiogenesis and alveolarization. We previously showed that experimental IUGR impairs angiogenesis; however, mechanisms that impair pulmonary artery endothelial cell (PAEC) function are uncertain. The NF-κB pathway promotes vascular growth in the developing mouse lung, and we hypothesized that IUGR disrupts NF-κB-regulated proangiogenic targets in fetal PAEC. PAECs were isolated from the lungs of control fetal sheep and sheep with experimental IUGR from an established model of chronic placental insufficiency. Microarray analysis identified suppression of NF-κB signaling and significant alterations in extracellular matrix (ECM) pathways in IUGR PAEC, including decreases in collagen 4α1 and laminin α4, components of the basement membrane and putative NF-κB targets. In comparison with controls, immunostaining of active NF-κB complexes, NF-κB-DNA binding, baseline expression of NF-κB subunits p65 and p50, and LPS-mediated inducible activation of NF-κB signaling were decreased in IUGR PAEC. Although pharmacological NF-κB inhibition did not affect angiogenic function in IUGR PAEC, angiogenic function of control PAEC was reduced to a similar degree as that observed in IUGR PAEC. These data identify reductions in endothelial NF-κB signaling as central to the disrupted angiogenesis observed in IUGR, likely by impairing both intrinsic PAEC angiogenic function and NF-κB-mediated regulation of ECM components necessary for vascular development. These data further suggest that strategies that preserve endothelial NF-κB activation may be useful in lung diseases marked by disrupted angiogenesis such as IUGR.

Pulmonary artery smooth muscle cells (PASMCs) express endothelin (ET-1), which modulates the pulmonary vascular response to hypoxia. Although cross-talk between hypoxia-inducible factor-1α (HIF-1α), an O 2 -sensitive transcription factor, and ET-1 is established, the cell-specific relationship between HIF-1α and ET-1 expression remains incompletely understood. We tested the hypotheses that in PASMCs 1) HIF-1α expression constrains ET-1 expression, and 2) a specific microRNA (miRNA) links HIF-1α and ET-1 expression. In human (h)PASMCs, depletion of HIF-1α with siRNA increased ET-1 expression at both the mRNA and protein levels ( P < 0.01). In HIF-1α -/- murine PASMCs, ET-1 gene and protein expression was increased ( P < 0.0001) compared with HIF-1α +/+ cells. miRNA profiles were screened in hPASMCs transfected with siRNA-HIF-1α, and RNA hybridization was performed on the Agilent (Santa Clara, CA) human miRNA microarray. With HIF-1α depletion, miRNA-543 increased 2.4-fold ( P < 0.01). In hPASMCs, miRNA-543 overexpression increased ET-1 gene ( P < 0.01) and protein ( P < 0.01) expression, decreased TWIST gene expression ( P < 0.05), and increased ET-1 gene and protein expression, compared with nontargeting controls ( P < 0.01). Moreover, we evaluated low passage hPASMCs from control and patients with idiopathic pulmonary arterial hypertension (IPAH). Compared with controls, protein expression of HIF-1α and Twist-related protein-1 (TWIST1) was decreased ( P < 0.05), and miRNA-543 and ET-1 expression increased ( P < 0.001) in hPASMCs from patients with IPAH. Thus, in PASMCs, loss of HIF-1α increases miRNA-543, which decreases Twist expression, leading to an increase in PASMC ET-1 expression. This previously undescribed link between HIF-1α and ET-1 via miRNA-543 mediated Twist suppression represents another layer of molecular regulation that might determine pulmonary vascular tone.

Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa-B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKβ, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKβ demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKβ induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKβ regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKβ regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKβ as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKβ activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Accessory subunits associated with the calcium-sensitive potassium channel (BK Ca ), a major determinant of vascular tone, confer functional and anatomical diversity. The β1 subunit increases Ca 2+ and voltagesensitivity of the BK Ca channel and is expressed exclusively in smooth muscle cells. Evidence supporting the physiological significance of the β1 subunit includes the observations that murine models with deletion of the β1 subunit are hypertensive and that humans with a gain-of-function β1 mutation are at a decreased risk of diastolic hypertension. However, whether the β1 subunit of the BK Ca channel contributes to the low tone that characterizes the normal pulmonary circulation or modulates the pulmonary vascular response to hypoxia remains unknown. To determine the role of the BK Ca channel β1 subunit in the regulation of pulmonary vascular tone and the response to acute and chronic hypoxia, mice with deletion of the Kcnmb1 gene that encodes for the β1 subunit ( Kcnmb1 -/- ) were placed in chronic hypoxia (10% O 2 ) for 21-24 days. In normoxia, right ventricular systolic pressure (RVSP) did not differ between Kcnmb1 +/+ (controls) and Kcnmb1 -/- mice. After exposure to either acute or chronic hypoxia, RVSP was higher in Kcnmb1 -/- mice compared with Kcnmb1 +/+ mice, without increased vascular remodeling. β1 subunit expression was predominantly confined to pulmonary artery smooth muscle cells (PASMCs) from vessels ≤ 150 µm. Peripheral PASMCs contracted collagen gels irrespective of β1 expression. Focal adhesion expression and Rho kinase activity were greater in Kcnmb1 -/- compared with Kcnmb1 +/+ PASMCs. Compromised PASMC β1 function may contribute to the heightened microvascular vasoconstriction that characterizes pulmonary hypertension.

Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared with adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown. To test the hypothesis that PEC barrier function is more well-preserved in neonatal lungs compared with adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase 1 (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and overexpression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult mice, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, whereas FAK overexpression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function compared with that of adults and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS.

In contrast to many other organs, a significant portion of lung development occurs after birth during alveolarization, thus rendering the lung highly susceptible to injuries that may disrupt this developmental process. Premature birth heightens this susceptibility, with many premature infants developing the chronic lung disease, bronchopulmonary dysplasia (BPD), a disease characterized by arrested alveolarization. Over the past decade, tremendous progress has been made in the elucidation of mechanisms that promote postnatal lung development, including extensive data suggesting that impaired pulmonary angiogenesis contributes to the pathogenesis of BPD. Moreover, in addition to impaired vascular growth, patients with BPD also frequently demonstrate alterations in pulmonary vascular remodeling and tone, increasing the risk for persistent hypoxemia and the development of pulmonary hypertension. In this review, an overview of normal lung development will be presented, and the pathologic features of arrested development observed in BPD will be described, with a specific emphasis on the pulmonary vascular abnormalities. Key pathways that promote normal pulmonary vascular development will be reviewed, and the experimental and clinical evidence demonstrating alterations of these essential pathways in BPD summarized.

Bronchopulmonary dysplasia (BPD), characterized by impaired alveolarization and vascularization in association with lung inflammation and apoptosis, often occurs after mechanical ventilation with oxygen-rich gas (MV-O2). As heightened expression of the proinflammatory cytokine TNF-α has been described in infants with BPD, we hypothesized that absence of TNF-α would reduce pulmonary inflammation, and attenuate structural changes in newborn mice undergoing MV-O2 Neonatal TNF-α null (TNF-α(-/-)) and wild type (TNF-α(+/+)) mice received MV-O2 for 8 h; controls spontaneously breathed 40% O2 Histologic, mRNA, and protein analysis in vivo were complemented by in vitro studies subjecting primary pulmonary myofibroblasts to mechanical stretch. Finally, TNF-α level in tracheal aspirates from preterm infants were determined by ELISA. Although MV-O2 induced larger and fewer alveoli in both, TNF-α(-/-) and TNF-α(+/+) mice, it caused enhanced lung apoptosis (TUNEL, caspase-3/-6/-8), infiltration of macrophages and neutrophils, and proinflammatory mediator expression (IL-1β, CXCL-1, MCP-1) in TNF-α(-/-) mice. These differences were associated with increased pulmonary transforming growth factor-β (TGF-β) signaling, decreased TGF-β inhibitor SMAD-7 expression, and reduced pulmonary NF-κB activity in ventilated TNF-α(-/-) mice. Preterm infants who went on to develop BPD showed significantly lower TNF-α levels at birth. Our results suggest a critical balance between TNF-α and TGF-β signaling in the developing lung, and underscore the critical importance of these key pathways in the pathogenesis of BPD. Future treatment strategies need to weigh the potential benefits of inhibiting pathologic cytokine expression against the potential of altering key developmental pathways., (Copyright © 2016 the American Physiological Society.)

The importance of gaining insight into the mechanisms underlying uterine quiescence and contractility is highlighted by the absence of an effective strategy to prevent or treat preterm labor, the greatest cause of perinatal mortality and morbidity worldwide. Although current evidence suggests that in myometrial smooth muscle cells (mSMCs) calcium homeostasis is modulated near term to promote uterine contractility, the efficacy of blocking voltage-operated calcium channels is limited by dose-related cardiovascular side effects. Thus, we considered whether uterine contractility might be modulated by calcium entry via transient receptor potential vanilloid 4 (TRPV4) channels. In mSMC, TRPV4 gene and protein expression increased with gestation, and TRPV4-mediated Ca(2+) entry and contractility were increased in mSMC from pregnant compared to nonpregnant rats. Cell membrane TRPV4 expression was specifically increased, whereas the expression of β-arrestin-1 and β-arrestin-2, molecules that can sequester TRPV4 in the cytoplasm, decreased. Physical interaction of β-arrestin-2 and TRPV4 was apparent in nonpregnant, but absent in pregnant, mouse uterus. Moreover, direct pharmacologic activation of TRPV4 increased uterine contraction, but oxytocin-induced myometrial contraction was blocked by pharmacologic inhibition of TRPV4 and decreased in mice with global deletion of TRPV4. Finally, TRPV4 channel blockade prolonged pregnancy in two distinct in vivo murine models of preterm labor, whereas the absence of either β-arrestin-1 or β-arrestin-2 increased susceptibility to preterm labor. These data suggest that TRPV4 channel activity modulates uterine contractility and might represent a therapeutic target to address preterm labor., (Copyright © 2015, American Association for the Advancement of Science.)

A significant portion of lung development is completed postnatally during alveolarization, rendering the immature lung vulnerable to inflammatory stimuli that can disrupt lung structure and function. Although the NF-κB pathway has well-recognized pro-inflammatory functions, novel anti-inflammatory and developmental roles for NF-κB have recently been described. Thus, to determine how NF-κB modulates alveolarization during inflammation, we exposed postnatal day 6 mice to vehicle (PBS), systemic lipopolysaccharide (LPS), or the combination of LPS and the global NF-κB pathway inhibitor BAY 11-7082 (LPS + BAY). LPS impaired alveolarization, decreased lung cell proliferation, and reduced epithelial growth factor expression. BAY exaggerated these detrimental effects of LPS, further suppressing proliferation and disrupting pulmonary angiogenesis, an essential component of alveolarization. The more severe pathology induced by LPS + BAY was associated with marked increases in lung and plasma levels of macrophage inflammatory protein-2 (MIP-2). Experiments using primary neonatal pulmonary endothelial cells (PEC) demonstrated that MIP-2 directly impaired neonatal PEC migration in vitro; and neutralization of MIP-2 in vivo preserved lung cell proliferation and pulmonary angiogenesis and prevented the more severe alveolar disruption induced by the combined treatment of LPS + BAY. Taken together, these studies demonstrate a key anti-inflammatory function of the NF-κB pathway in the early alveolar lung that functions to mitigate the detrimental effects of inflammation on pulmonary angiogenesis and alveolarization. Furthermore, these data suggest that neutralization of MIP-2 may represent a novel therapeutic target that could be beneficial in preserving lung growth in premature infants exposed to inflammatory stress., (Copyright © 2015 the American Physiological Society.)

Endothelin-1 (ET-1) increases pulmonary vascular tone through direct effects on pulmonary artery smooth muscle cells (PASMC) via membrane-bound ET-1 receptors. Circulating ET-1 contributes to vascular remodeling by promoting SMC proliferation and migration and inhibiting SMC apoptosis. Although endothelial cells (EC) are the primary source of ET-1, whether ET-1 produced by SMC modulates pulmonary vascular tone is unknown. Using transgenic mice created by crossbreeding SM22α-Cre mice with ET-1(flox/flox) mice to selectively delete ET-1 in SMC, we tested the hypothesis that PASMC ET-1 gene expression modulates the pulmonary vascular response to hypoxia. ET-1 gene deletion and selective activity of SM22α promoter-driven Cre recombinase were confirmed. Functional assays were performed under normoxic (21% O2) or hypoxic (5% O2) conditions using murine PASMC obtained from ET-1(+/+) and ET-1(-/-) mic and in human PASMC (hPASMC) after silencing of ET-1 using siRNA. Under baseline conditions, there was no difference in right ventricular systolic pressure (RVSP) between SM22α-ET-1(-/-) and SM22α-ET-1(+/+) (control) littermates. After exposure to hypoxia (10% O2, 21-24 days), RVSP was and vascular remodeling were less in SM22α-ET-1(-/-) mice compared with control littermates (P < 0.01). Loss of ET-1 decreased PASMC proliferation and migration and increased apoptosis under normoxic and hypoxic conditions. Exposure to selective ET-1 receptor antagonists had no effect on either the hypoxia-induced hPASMC proliferative or migratory response. SMC-specific ET-1 deletion attenuates hypoxia-induced increases in pulmonary vascular tone and structural remodeling. The observation that loss of ET-1 inhibited SMC proliferation, survival, and migration represents evidence that ET-1 derived from SMC plays a previously undescribed role in modulating the response of the pulmonary circulation to hypoxia. Thus PASMC ET-1 may modulate vascular tone independently of ET-1 produced by EC., (Copyright © 2015 the American Physiological Society.)

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.)

Purpose of Review: Advances in medical therapy have increased survival of extremely premature infants and changed the pathology of bronchopulmonary dysplasia (BPD) from one of acute lung injury to a disease of disrupted lung development. With this evolution, new questions emerge regarding the molecular mechanisms that control postnatal lung development, the effect of early disruptions of postnatal lung development on long-term lung function, and the existence of endogenous mechanisms that permit lung regeneration after injury., Recent Findings: Recent data demonstrate that a significant component of alveolarization, the final stage of lung development, occurs postnatally. Further, clinical and experimental studies demonstrate that premature birth disrupts alveolarization, decreasing the gas exchange surface area of the lung and causing BPD. BPD is associated with significant short-term morbidity, and new longitudinal, clinical data demonstrate that survivors of BPD have long-standing deficits in lung function and may be at risk for the development of additional lung disease as adults. Unfortunately, current care is mainly supportive with few effective therapies that prevent or treat established BPD. These studies underscore the need to further elucidate the mechanisms that direct postnatal lung growth and develop innovative strategies to stimulate lung regeneration., Summary: Despite significant improvements in the care and survival of extremely premature infants, BPD remains a major clinical problem. Although efforts should remain focused on the prevention of preterm labor and BPD, novel research aimed at promoting postnatal alveolarization offers a unique opportunity to develop effective strategies to treat established BPD.

In contrast to other organs, the lung completes a significant portion of its development after term birth. During this stage of alveolarization, division of the alveolar ducts into alveolar sacs by secondary septation, and expansion of the pulmonary vasculature by means of angiogenesis markedly increase the gas exchange surface area of the lung. However, postnatal completion of growth renders the lung highly susceptible to environmental insults such as inflammation that disrupt this developmental program. This is particularly evident in the setting of preterm birth, where impairment of alveolarization causes bronchopulmonary dysplasia, a chronic lung disease associated with significant morbidity. The nuclear factor κ-B (NFκB) family of transcription factors are ubiquitously expressed, and function to regulate diverse cellular processes including proliferation, survival, and immunity. Extensive evidence suggests that activation of NFκB is important in the regulation of inflammation and in the control of angiogenesis. Therefore, NFκB-mediated downstream effects likely influence the lung response to injury and may also mediate normal alveolar development. This review summarizes the main biologic functions of NFκB, and highlights the regulatory mechanisms that allow for diversity and specificity in downstream gene activation. This is followed by a description of the pro and anti-inflammatory functions of NFκB in the lung, and of NFκB-mediated angiogenic effects. Finally, this review summarizes the clinical and experimental data that support a role for NFκB in mediating postnatal angiogenesis and alveolarization, and discusses the challenges that remain in developing therapies that can selectively block the detrimental functions of NFκB yet preserve the beneficial effects., (Copyright © 2014 The Authors Birth Defects Research Part A: Clinical and Molecular Teratology Published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.)

Neonatal chronic lung disease, also known as bronchopulmonary dysplasia (BPD), is the most common complication of premature birth, affecting up to 30% of very low birth weight infants. Improved medical care has allowed for the survival of the most premature infants and has significantly changed the pathology of BPD from a disease marked by severe lung injury to the "new" form characterized by alveolar hypoplasia and impaired vascular development. However, increased patient survival has led to a paucity of pathologic specimens available from infants with BPD. This, combined with the lack of a system to model alveolarization in vitro, has resulted in a great need for animal models that mimic key features of the disease. To this end, a number of animal models have been created by exposing the immature lung to injuries induced by hyperoxia, mechanical stretch, and inflammation and most recently by the genetic modification of mice. These animal studies have 1) allowed insight into the mechanisms that determine alveolar growth, 2) delineated factors central to the pathogenesis of neonatal chronic lung disease, and 3) informed the development of new therapies. In this review, we summarize the key findings and limitations of the most common animal models of BPD and discuss how knowledge obtained from these studies has informed clinical care. Future studies should aim to provide a more complete understanding of the pathways that preserve and repair alveolar growth during injury, which might be translated into novel strategies to treat lung diseases in infants and adults.

Rationale: Hypoxia-inducible factor-1α (HIF-1α), an oxygen (O2)-sensitive transcription factor, mediates transcriptional responses to low-O2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1α in modulating pulmonary vascular tone., Objective: To investigate the role of smooth muscle cell (SMC)-specific HIF-1α in regulating pulmonary vascular tone., Methods and Results: Mice with an SMC-specific deletion of HIF-1α (SM22α-HIF-1α(-/-)) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1α modulates pulmonary vascular tone and the response to hypoxia. SM22α-HIF-1α(-/-) mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22α-HIF-1α(-/-) mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1α decreased myosin light chain phosphorylation in HIF-1α-null SMCs., Conclusions: In both normoxia and hypoxia, PASMC HIF-1α maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1α expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.

Bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy, is characterized by arrested alveolar development. Pulmonary angiogenesis, mediated by the vascular endothelial growth factor (VEGF) pathway, is essential for alveolarization. However, the transcriptional regulators mediating pulmonary angiogenesis remain unknown. We previously demonstrated that NF-κB, a transcription factor traditionally associated with inflammation, plays a unique protective role in the neonatal lung. Therefore, we hypothesized that constitutive NF-κB activity is essential for postnatal lung development. Blocking NF-κB activity in 6-day-old neonatal mice induced the alveolar simplification similar to that observed in BPD and significantly reduced pulmonary capillary density. Studies to determine the mechanism responsible for this effect identified greater constitutive NF-κB in neonatal lung and in primary pulmonary endothelial cells (PEC) compared with adult. Moreover, inhibiting constitutive NF-κB activity in the neonatal PEC with either pharmacological inhibitors or RNA interference blocked PEC survival, decreased proliferation, and impaired in vitro angiogenesis. Finally, by chromatin immunoprecipitation, NF-κB was found to be a direct regulator of the angiogenic mediator, VEGF-receptor-2, in the neonatal pulmonary vasculature. Taken together, our data identify an entirely novel role for NF-κB in promoting physiological angiogenesis and alveolarization in the developing lung. Our data suggest that disruption of NF-κB signaling may contribute to the pathogenesis of BPD and that enhancement of NF-κB may represent a viable therapeutic strategy to promote lung growth and regeneration in pulmonary diseases marked by impaired angiogenesis.

Previously, we observed that hypoxia increases the expression of the β1-subunit (KCNMB1) of the calcium-sensitive potassium channel (BK(Ca)). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC). In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1-α (HIF-1α) and KCNMB1 are increased. Knockdown of HIF-1α using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1α binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between -3,540 bp and -3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1α transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the β1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1α increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at -3,540 to -3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone.

Rationale: Marfan syndrome (MFS) is a systemic connective tissue disorder notable for the development of aortic root aneurysms and the subsequent life-threatening complications of aortic dissection and rupture. Underlying fibrillin-1 gene mutations cause increased transforming growth factor-β (TGF-β) signaling. Although TGF-β blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF-β causes aneurysms remain ill-defined., Objective: We investigated the role of microRNA-29b (miR-29b) in aneurysm formation in MFS., Methods and Results: Using quantitative polymerase chain reaction, we discovered that miR-29b, a microRNA regulating apoptosis and extracellular matrix synthesis/deposition genes, is increased in the ascending aorta of Marfan (Fbn1(C1039G/+)) mice. Increased apoptosis, assessed by increased cleaved caspase-3 and caspase-9, enhanced caspase-3 activity, and decreased levels of the antiapoptotic proteins, Mcl-1 and Bcl-2, were found in the Fbn1(C1039G/+) aorta. Histological evidence of decreased and fragmented elastin was observed exclusively in the Fbn1(C1039G/+) ascending aorta in association with repressed elastin mRNA and increased matrix metalloproteinase-2 expression and activity, both targets of miR-29b. Evidence of decreased activation of nuclear factor κB, a repressor of miR-29b, and a factor suppressed by TGF-β, was also observed in Fbn1(C1039G/+) aorta. Furthermore, administration of a nuclear factor κB inhibitor increased miR-29b levels, whereas TGF-β blockade or losartan effectively decreased miR-29b levels in Fbn1(C1039G/+) mice. Finally, miR-29b blockade by locked nucleic acid antisense oligonucleotides prevented early aneurysm development, aortic wall apoptosis, and extracellular matrix deficiencies., Conclusions: We identify increased miR-29b expression as key to the pathogenesis of early aneurysm development in MFS by regulating aortic wall apoptosis and extracellular matrix abnormalities.

Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) β increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-β would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-β signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-β by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-β suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-β, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

At birth, pulmonary vasodilation occurs concomitant with the onset of air-breathing life. Whether and how Rho kinase (ROCK) modulates the perinatal pulmonary vascular tone remains incompletely understood. To more fully characterize the separate and interactive effects of ROCK signaling, we hypothesized that ROCK has discrete effects on both pulmonary artery (PA): 1) endothelial cell (PAEC) nitric oxide (NO) production and contractile state; and 2) smooth muscle cell tone independent of endothelial NO synthase (eNOS) activity. To test these hypotheses, NO production and endothelial barrier function were determined in fetal PAEC under baseline hypoxia and following exposure to normoxia with and without treatment with Y-27632, a specific pharmacological inhibitor of ROCK. In acutely instrumented, late-gestation ovine fetuses, eNOS was inhibited by nitro-l-arginine infusion into the left PA (LPA). Subsequently, fetal lambs were mechanically ventilated (MV) with 100% oxygen in the absence (control period) and presence of Y-27632. In PAEC, treatment with Y-27632 had no effect on cytosolic calcium but did increase normoxia-induced NO production. Moreover, acute normoxia increased PAEC barrier function, an effect that was potentiated by Y-27632. In fetal lambs, MV during the control period had no effect on LPA flow. In contrast, MV after Y-27632 increased LPA flow and fetal arterial P(O)₂ (Pa(O₂)) and decreased PA pressure. In conclusion, ROCK activity modulates vascular tone in the perinatal pulmonary circulation via combined effects on PAEC NO production, barrier function, and smooth muscle tone. ROCK inhibition may represent a novel treatment strategy for neonatal pulmonary vascular disease.

Defective lung septation and angiogenesis, quintessential features of neonatal chronic lung disease (CLD), typically result from lengthy exposure of developing lungs to mechanical ventilation (MV) and hyperoxia. Previous studies showed fewer alveoli and microvessels, with reduced VEGF and increased transforming growth factor-beta (TGFbeta) signaling, and excess, scattered elastin in lungs of premature infants and lambs with CLD vs. normal controls. MV of newborn mice with 40% O(2) for 24 h yielded similar lung structural abnormalities linked to impaired VEGF signaling, dysregulated elastin production, and increased apoptosis. These studies could not determine the relative importance of cyclic stretch vs. hyperoxia in causing these lung growth abnormalities. We therefore studied the impact of MV for 24 h with air on alveolar septation (quantitative lung histology), angiogenesis [CD31 quantitative-immunohistochemistry (IHC), immunoblots], apoptosis [TdT-mediated dUTP nick end labeling (TUNEL), active caspase-3 assays], VEGF signaling [VEGF-A, VEGF receptor 1 (VEGF-R1), VEGF-R2 immunoblots], TGFbeta activation [phosphorylated Smad2 (pSmad2) quantitative-IHC], and elastin production (tropoelastin immunoblots, quantitative image analysis of Hart's stained sections) in lungs of 6-day-old mice. Compared with unventilated controls, MV caused a 3-fold increase in alveolar area, approximately 50% reduction in alveolar number and endothelial surface area, >5-fold increase in apoptosis, >50% decrease in lung VEGF-R2 protein, 4-fold increase of pSmad2 protein, and >50% increase in lung elastin, which was distributed throughout alveolar walls rather than at septal tips. This study is the first to show that prolonged MV of developing lungs, without associated hyperoxia, can inhibit alveolar septation and angiogenesis and increase apoptosis and lung elastin, findings that could reflect stretch-induced changes in VEGF and TGFbeta signaling, as reported in CLD.

Peroxisome proliferator-activated receptor (PPAR)-gamma is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPARgamma in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPARgamma in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPARgamma(flox/flox) mice to induce EC loss of PPARgamma (Tie2 PPARgamma(-/-)), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPARgamma(-/-) mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPARgamma(-/-) mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPARgamma(-/-) vs. WT mice in RA had increased platelet-derived growth factor receptor-beta (PDGF-Rbeta) expression and signaling, despite an elevation in the PPARgamma target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-Rbeta signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPARgamma(-/-) mice. Thus the disruption of PPARgamma signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-Rbeta signaling is sufficient to reverse PAH in this genetic model.

Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3' UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin 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.

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 PPARgamma 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 PPARgamma that is independent of Smad1/5/8 phosphorylation. Both BMP-2 and a PPARgamma 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 PPARgamma antagonist, and PASMCs isolated from PPARgamma- and apoE-deficient mice, we demonstrated that the antiproliferative effect of BMP-2 was BMP-RII, PPARgamma, and apoE dependent. Furthermore, we created mice with targeted deletion of PPARgamma 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, PPARgamma-mediated events could protect against PAH, and PPARgamma agonists may reverse PAH in patients with or without BMP-RII dysfunction.