The Alvira Lab
Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that nuclear factor kappa-B (NFkB) promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NFkB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate pro-angiogenic NFkB signaling in the developing pulmonary vasculature. Proteomic analysis of the lung secretome was performed using twodimensional difference gel electrophoresis (2D-DIGE). More NFkB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PEC) and TGFBI-regulated genes identified using RNA-sequencing. Alveolarization and pulmonary angiogenesis was assessed in WT 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 PEC. Proteomic analysis identified transforming growth factor beta-induced protein (TGFBI) as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NFkB via avb3 integrins. RNA-sequencing identified Csf3 as a TGFBIregulated gene that enhances nitric oxide production in PEC. 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 NFkB-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. More 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.
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 (NFkB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFkB in the pulmonary endothelium, and its downstream targets are not known. More In this study, we sought to delineate the specific roles for the NFkB activating kinases, IKKa and IKKb, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKa or IKKb demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKa induced mild impairments in angiogenic function, silencing IKKb induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKb regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKa and IKKb regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKb as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKb activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.
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. More The NF-kB pathway promotes vascular growth in the developing mouse lung, and we hypothesized that IUGR disrupts NF-kB-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-kB signaling and significant alterations in extracellular matrix (ECM) pathways in IUGR PAEC, including decreases in collagen 41 and laminin 4, components of the basement membrane and putative NF-B targets. In comparison with controls, immunostaining of active NF-B complexes, NF-kB-DNA binding, baseline expression of NF-B subunits p65 and p50, and LPS-mediated inducible activation of NF-kB signaling were decreased in IUGR PAEC. Although pharmacological NF-B 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-kB signaling as central to the disrupted angiogenesis observed in IUGR, likely by impairing both intrinsic PAEC angiogenic function and NF-kB-mediated regulation of ECM components necessary for vascular development. These data further suggest that strategies that preserve endothelial NF-B activation may be useful in lung diseases marked by disrupted angiogenesis such as IUGR.
ABSTRACT: 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. More 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 Ca2+ entry and contractility were increased in mSMC from pregnant compared to nonpregnant rats. Cell membrane TRPV4 expression was specifically increased, whereas the expression of b-arrestin-1 and b-arrestin-2, molecules that can sequester TRPV4 in the cytoplasm, decreased. Physical interaction of b-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 b-arrestin-1 or b-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.
ABSTRACT: 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 essentialfor alveolarization. More However, the transcriptional regulators mediating pulmonary angiogenesis remain unknown. We previously demonstratedthat NFkB, a transcription factor traditionally associated withinflammation, plays a unique protective role in the neonatal lung.Therefore, we hypothesized that constitutive NFkB activity is essential for postnatal lung development. Blocking NFkB activity in6-day-old neonatal mice induced the alveolar simplification similar to that observed in BPD and significantly reduced pulmonary capillarydensity. Studies to determine the mechanism responsible for this effect identified greater constitutive NFkB in neonatal lung and in primary pulmonary endothelial cells (PEC) compared with adult. Moreover, inhibiting constitutive NFkB 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, NFkB was found to be a direct regulator of the angiogenic mediator, VEGFreceptor-2, in the neonatal pulmonary vasculature. Taken together, our data identify an entirely novel role for NFkB in promoting physiological angiogenesis and alveolarization in the developing lung. Our data suggest that disruption of NFkB signaling may contribute to the pathogenesis of BPD and that enhancement of NFkB may representa viable therapeutic strategy to promote lung growth and regeneration in pulmonary diseases marked by impaired angiogenesis.
ABSTRACT: A significant portion of lung de- velopment is completed postnatally during alveolarization, rendering the immature lung vulnerable to inflammatory stimuli that can disrupt lung structure and function. Although the NFkB pathway has well- recognized pro-inflammatory functions, novel anti-inflammatory and developmental roles for NFkB have recently been described. More Thus, to determine how NFkB modulates alveolarization during inflamma- tion, we exposed postnatal day 6 mice to vehicle (PBS), systemic lipopolysaccharide (LPS), or the combination of LPS and the global NFkB pathway inhibitor BAY 11-7082 (LPS BAY). LPS impaired alveolarization, decreased lung cell proliferation, and reduced epithe- lial growth factor expression. BAY exaggerated these detrimental effects of LPS, further suppressing proliferation and disrupting pul- monary 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 inflam- matory protein-2 (MIP-2). Experiments using primary neonatal pul- monary 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 NFkB pathway in the early alveolar lung that functions to mitigate the detrimental effects of inflammation on pulmonary angiogenesis and alveolariza- tion. Furthermore, these data suggest that neutralization of MIP-2 may represent a novel therapeutic target that could be beneficial in pre- serving lung growth in premature infants exposed to inflammatory stress.
ABSTRACT: In contrast to other organs, the lung completes a significant portion of its development after term birth. During this stage of alveolarization, division ofthe 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. More 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 k-B (NFkB) family of transcriptionfactors are ubiquitously expressed, and function to regulate diverse cellular processes including proliferation, survival, and immunity. Extensive evidence suggests that activation of NFkB is important in the regulation of inflammation
and in the control of angiogenesis. Therefore, NFkB-mediated downstreameffects likely influence the lung response to injury and may also mediatenormal alveolar development. This review summarizes the main biologic functions of NFkB, 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 NFkB in the lung, and of NFkB-mediated angiogenic effects. Finally, this review summarizes the clinical and experimental data that support a role for NFkB in mediating postnatal angiogenesis and alveolarization, and discusses the challenges that remain in developing therapies that can selectively block the detrimental
functions of NFkB yet preserve the beneficial effects.