Alveolar Morphogenesis Research Articles

Dynamic Hippo pathway activity underlies mesenchymal differentiation during lung alveolar morphogenesis.

Alveologenesis, the final stage in lung development, substantially remodels the distal lung, expanding the alveolar surface area for efficient gas exchange. Secondary crest myofibroblasts (SCMF) exist transiently in the neonatal distal lung and are crucial for alveologenesis. However, the pathways that regulate SCMF function, proliferation and temporal identity remain poorly understood. To address this, we purified SCMFs from reporter mice, performed bulk RNA-seq and found dynamic changes in Hippo-signaling components during alveologenesis. We deleted the Hippo effectors Yap/Taz from Acta2-expressing cells at the onset of alveologenesis, causing a significant arrest in alveolar development. Using single cell RNA-seq, we identified a distinct cluster of cells in mutant lungs with altered expression of marker genes associated with proximal mesenchymal cell types, airway smooth muscle and alveolar duct myofibroblasts. In vitro studies confirmed that Yap/Taz regulates myofibroblast-associated gene signature and contractility. Together, our findings show that Yap/Taz is essential for maintaining functional myofibroblast identity during postnatal alveologenesis.

Endothelial cells regulate alveolar morphogenesis by constructing basement membranes acting as a scaffold for myofibroblasts

Alveologenesis is a spatially coordinated morphogenetic event, during which alveolar myofibroblasts surround the terminal sacs constructed by epithelial cells and endothelial cells (ECs), then contract to form secondary septa to generate alveoli in the lungs. Recent studies have demonstrated the important role of alveolar ECs in this morphogenetic event. However, the mechanisms underlying EC-mediated alveologenesis remain unknown. Herein, we show that ECs regulate alveologenesis by constructing basement membranes (BMs) acting as a scaffold for myofibroblasts to induce septa formation through activating mechanical signaling. Rap1, a small GTPase of the Ras superfamily, is known to stimulate integrin-mediated cell adhesions. EC-specific Rap1-deficient (Rap1iECKO) mice exhibit impaired septa formation and hypo-alveolarization due to the decreased mechanical signaling in myofibroblasts. In Rap1iECKO mice, ECs fail to stimulate integrin β1 to recruit Collagen type IV (Col-4) into BMs required for myofibroblast-mediated septa formation. Consistently, EC-specific integrin β1-deficient mice show hypo-alveolarization, defective mechanical signaling in myofibroblasts, and disorganized BMs. These data demonstrate that alveolar ECs promote integrin β1-mediated Col-4 recruitment in a Rap1-dependent manner, thereby constructing BMs acting as a scaffold for myofibroblasts to induce mechanical signal-mediated alveologenesis. Thus, this study unveils a mechanism of organ morphogenesis mediated by ECs through intrinsic functions.

FOXF1 Regulates Alveolar Epithelial Morphogenesis through Transcriptional Activation of Mesenchymal WNT5A.

Mutations in the FOXF1 (forkhead box F1) gene, encoding the mesenchymal FOX (forkhead box) transcription factor, are linked to alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with the loss of alveolar capillaries and lung hypoplasia. Although proangiogenic functions of FOXF1 have been extensively studied, the role of FOXF1 in mesenchymal-epithelial signaling during lung development remains uncharacterized. Herein, we used murine lung organoids to demonstrate that the S52F FOXF1 mutation (found in patients with ACDMPV) stimulates canonical WNT/β-catenin signaling in type 2 alveolar epithelial cells (AEC2s), leading to increased proliferation of AEC2s and decreased differentiation of AEC2s into type 1 alveolar epithelial cells (AEC1s). Alveolar organoids containing Foxf1WT/S52F lung fibroblasts and wild-type epithelial cells grew faster on Matrigel and exhibited AEC2 hyperplasia. AEC2 hyperplasia and loss of AEC1s were found in the lungs of Foxf1WT/S52F embryos, a mouse model of ACDMPV. Activation of canonical WNT/β-catenin signaling in AEC2s of lung organoids and Foxf1WT/S52F mice was associated with decreased expression of noncanonical WNT5A (Wnt family member 5A) ligand in lung fibroblasts. Mechanistically, FOXF1 directly activates the Wnt5a gene transcription through an evolutionarily conserved +6320/+6326 region located in the first intron of the Wnt5a gene. Site-directed mutagenesis of the +6320/+6326 region prevented the transcriptional activation of the Wnt5a enhancer by FOXF1. Treatment with exogenous WNT5A ligand inhibited the effects of the S52F FOXF1 mutation on canonical WNT/β-catenin signaling in alveolar organoids, preventing aberrant AEC2 expansion and restoring differentiation of AEC1s. Activation of either FOXF1 or WNT5A may provide an attractive strategy to improve lung function in patients with ACDMPV.

Mechanical Tension‐induced Vascular and Alveolar Morphogenesis during Lung Regeneration

Mechanical forces altered after unilateral pneumonectomy (PNX) control post‐PNX vascular and alveolar morphogenesis. This study aims to understand the mechanosensitive mechanism of lung vascular and alveolar regeneration in the post‐PNX mouse lungs. Vascular formation is stimulated at the subpleural region in the remaining mouse lung after unilateral PNX, where mechanical stretch is enhanced, while inhibition of mechanical tension by insertion of prosthesis attenuates these effects. The focal adhesion protein, paxillin senses mechanical stretch after PNX and the levels of paxillin are upregulated in SPB‐positive alveolar epithelial type II (ATII) cells and endothelial cells (ECs), which stimulates the expression of angiogenic factor angiopoietin‐1 (Ang1) in ATII cells and Ang2 in ECs to induce EC directional motility to the subpleural region. Cellular stretching increases the expression of paxillin, which induces expression of Ang2 in ECs and Ang1 in ATII cells in vitro. Post‐PNX lung growth and vascular formation toward the subpleural region are suppressed in Pxnfl/fl‐Sftpc‐CreERT2 mouse lungs, in which paxillin expression is attenuated in ATII cells. Structure of lamellar bodies that control surfactant homeostasis is also disrupted in post‐PNX Pxnfl/fl‐Sftpc‐CreERT2 mouse lung ATII cells. These results suggest that cooperative action of mechanosensitive paxillin signaling in ECs and ATII cells mediates vascular and alveolar epithelial formation during post‐PNX lung growth.

Obesity in Angiogenesis and Lung Regeneration

Obesity is associated with impairment of wound healing and tissue regeneration. Angiogenesis, the formation of new blood capillaries, plays a key role in organ regeneration and repair. We and other groups have reported that inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and that obesity is accompanied by endothelial cell dysfunction. The aim of this project is to investigate the effects of obesity on post‐PNX lung vascular and alveolar morphogenesis. We have found that post‐PNX regenerative lung growth is inhibited in Lepob/ob obese mice compared to Lepob/+ mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the lung tissue collected from post‐PNX mice compared to those from sham‐operated control mice, while these effects are attenuated in post‐PNX Lepob/ob mice. The levels of adiponectin, one of the adipokines that exhibits pro‐angiogenic and vascular protective properties, increase in the remaining mouse lungs after unilateral PNX, while these effects are attenuated in Lepob/ob obese mice. Regenerative lung growth, vascular and alveolar morphogenesis, and VEGF levels in the post‐PNX mouse lungs are inhibited in adiponectin knockout mice. These results suggest that obesity inhibits post‐PNX regenerative lung growth through adiponectin‐VEGF signaling. Modulation of adiponectin‐VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese people.

Obesity in Angiogenesis and Regenerative Lung Growth after Pneumonectomy

Obese population is rapidly growing worldwide. Obesity is associated with impairment of wound healing and tissue regeneration. Angiogenesis, the formation of new blood capillaries, plays a key role in organ regeneration and repair. We and other groups have reported that inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and that obesity is accompanied by endothelial cell dysfunction. However, the effects of obesity on lung regeneration remain unclear. Compensatory lung growth and vascular and alveolar morphogenesis after PNX are suppressed in leptin-deficient ob/ob mice treated with high-fat diet compared to that in control lean mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the lung tissue collected from post-PNX mice compared to those from sham-operated control mice, while these effects are attenuated in post-PNX leptin-deficient ob/ob mice with high-fat diet. Among various VEGF isoforms, VEGF164, but not VEGF188 and VEGF120, increases in the post-PNX mouse lungs, while these effects are inhibited in obese mice. These results suggestthat obesity inhibits post-PNX regenerative lung growth through VEGF164 signaling. Modulation of VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese people.

Obesity in Vascular and Alveolar Morphogenesis after Pneumonectomy

Obese population is rapidly growing worldwide. Obesity is associated with chronic lung diseases including chronic obstructive pulmonary disease (COPD). Impairment of lung regeneration and repair contributes to the pathogenesis of COPD. Angiogenesis – the formation of new blood capillaries ‐ plays a key role in organ regeneration. Inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and obesity is accompanied by endothelial cell (EC) dysfunction. However, the effects of obesity on lung vascular and alveolar regeneration remain unclear. Compensatory lung growth and vascular and alveolar morphogenesis after PNX are suppressed in leptin‐deficient ob/ob mice treated with high‐fat diet compared to that in control lean mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the brown adipose tissue collected from post‐PNX mice compared to those from sham‐operated control mice, while these effects are attenuated in post‐PNX leptin‐deficient ob/ob mice with high‐fat diet. The serum collected from post‐PNX control mice stimulates EC sprouting, while EC sprouting is inhibited when treated with serum collected from leptin‐deficient ob/ob mice after PNX. These results suggest that obesity may inhibit post‐PNX regenerative lung growth through VEGF signaling. Modulation of VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese population.Support or Funding InformationNIH R01HL142578

LRP5 in age-related changes in vascular and alveolar morphogenesis in the lung

Aging is associated with impaired angiogenesis and lung alveolar regeneration, which contributes to the increased susceptibility to chronic lung diseases (CLD). We have reported that the Wnt ligand co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), stimulates angiogenesis and lung alveolar regeneration. However, the role of LRP5 in age-related decline in vascular and alveolar morphogenesis remains unclear. In this report, we have demonstrated that vascular and alveolar structures are disrupted in the 24-month (24M) old mouse lungs. The expression of LRP5 and the major angiogenic factors, VEGFR2 and Tie2, is lower in endothelial cells (ECs) isolated from 24M old mouse lungs compared to those from 2M old mouse lungs. Vascular and alveolar formation is attenuated in the hydrogel implanted on the 24M old mouse lungs, while overexpression of LRP5, which restores angiogenic factor expression, reverses vascular and alveolar morphogenesis in the gel. Compensatory lung growth after unilateral pneumonectomy is inhibited in 24M old mice, which is reversed by overexpression of LRP5. These results suggest that LRP5 mediates age-related inhibition of angiogenesis and alveolar morphogenesis. Modulation of LRP5 may be a novel intervention to rejuvenate regenerative ability in aged lung and will lead to the development of efficient strategies for aging-associated CLD.

Effects of vascular formation during alveolar bone process morphogenesis in mice.

The alveolar bone process is the thickened ridge of bone that bears the teeth and is known to have dynamic functional interactions with surrounding tissues. However, the detailed morphological changes that occur during alveolar bone process development and the underlying molecular mechanisms behind this morphogenesis have not been elucidated. In this study, we examined the detailed morphological changes of the alveolar bone process during mouse development using HE and MTC staining. In addition, we evaluated the precise localization pattern of various signaling molecules involved in blood vessel formation including CD31, α-SMA, VEGF, periostin, and TGF-β. Innervation of the alveolar bone process was examined following injection of the nerve terminal dye AM1-43. The morphological and immunohistochemical data suggested that there is an intimate relationship between alveolar bone process development and blood vessel formation. To more closely examine the role of blood vessels in alveolar bone process formation, we microinjected mice with a clinically available anti-VEGF antibody, bevacizumab, at PN5 and analyzed the effects 5days later. Compared to the control animals, anti-VEGF treated animals showed a disruption of the integration of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these data, we conclude that specific morphogenesis of the alveolar bone process is closely associated with blood vessel formation.

Effects of vascular formation during alveolar bone process morphogenesis in mice

The alveolar bone process is the thickened ridge of bone that bears the teeth and is known to have dynamic functional interactions with surrounding tissues. However, the detailed morphological changes that occur during alveolar bone process development and the underlying molecular mechanisms behind this morphogenesis have not been elucidated. In this study, we examined the detailed morphological changes of the alveolar bone process during mouse development using HE and MTC staining. In addition, we evaluated the precise localization pattern of various signaling molecules involved in blood vessel formation including CD31, α-SMA, VEGF, periostin, and TGF-β. Innervation of the alveolar bone process was examined following injection of the nerve terminal dye AM1-43. The morphological and immunohistochemical data suggested that there is an intimate relationship between alveolar bone process development and blood vessel formation. To more closely examine the role of blood vessels in alveolar bone process formation, we microinjected mice with a clinically available anti-VEGF antibody, bevacizumab, at PN5 and analyzed the effects 5 days later. Compared to the control animals, anti-VEGF treated animals showed a disruption of the integration of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these data, we conclude that specific morphogenesis of the alveolar bone process is closely associated with blood vessel formation.

The innate immune response in fetal lung mesenchymal cells targets VEGFR2 expression and activity.

In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). With the use of Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Vegfr2 Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express Vegfr2 and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. With the use of an experimental coculture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of three-dimensional structures. Both LPS and tyrosine kinase inhibition reduced three-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme.

Type IV collagen drives alveolar epithelial-endothelial association and the morphogenetic movements of septation.

BackgroundType IV collagen is the main component of the basement membrane that gives strength to the blood–gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown.ResultsWe have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal–epithelial septal-like structures in co-culture.ConclusionsOur study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0281-2) contains supplementary material, which is available to authorized users.

Epithelial Notch signaling regulates lung alveolar morphogenesis and airway epithelial integrity

Abnormal enlargement of the alveolar spaces is a hallmark of conditions such as chronic obstructive pulmonary disease and bronchopulmonary dysplasia. Notch signaling is crucial for differentiation and regeneration and repair of the airway epithelium. However, how Notch influences the alveolar compartment and integrates this process with airway development remains little understood. Here we report a prominent role of Notch signaling in the epithelial-mesenchymal interactions that lead to alveolar formation in the developing lung. We found that alveolar type II cells are major sites of Notch2 activation and show by Notch2-specific epithelial deletion (Notch2(cNull)) a unique contribution of this receptor to alveologenesis. Epithelial Notch2 was required for type II cell induction of the PDGF-A ligand and subsequent paracrine activation of PDGF receptor-α signaling in alveolar myofibroblast progenitors. Moreover, Notch2 was crucial in maintaining the integrity of the epithelial and smooth muscle layers of the distal conducting airways. Our data suggest that epithelial Notch signaling regulates multiple aspects of postnatal development in the distal lung and may represent a potential target for intervention in pulmonary diseases.

VEGF and endothelium-derived retinoic acid regulate lung vascular and alveolar development.

Prevention or treatment of lung diseases caused by the failure to form, or destruction of, existing alveoli, as observed in infants with bronchopulmonary dysplasia and adults with emphysema, requires understanding of the molecular mechanisms of alveolar development. In addition to its critical role in gas exchange, the pulmonary circulation also contributes to alveolar morphogenesis and maintenance by the production of paracrine factors, termed "angiocrines," that impact the development of surrounding tissue. To identify lung angiocrines that contribute to alveolar formation, we disrupted pulmonary vascular development by conditional inactivation of the Vegf-A gene during alveologenesis. This resulted in decreased pulmonary capillary and alveolar development and altered lung elastin and retinoic acid (RA) expression. We determined that RA is produced by pulmonary endothelial cells and regulates pulmonary angiogenesis and elastin synthesis by induction of VEGF-A and fibroblast growth factor (FGF)-18, respectively. Inhibition of RA synthesis in newborn mice decreased FGF-18 and elastin expression and impaired alveolarization. Treatment with RA and vitamin A partially reversed the impaired vascular and alveolar development induced by VEGF inhibition. Thus we identified RA as a lung angiocrine that regulates alveolarization through autocrine regulation of endothelial development and paracrine regulation of elastin synthesis via induction of FGF-18 in mesenchymal cells.

Bronchioalveolar morphogenesis of human bronchial epithelial cells depending upon hepatocyte growth factor.

Lung alveolar regeneration occurs in adult human lungs as a result of proliferation, differentiation and alveolar morphogenesis of stem cells. It is increasingly being believed that bronchial epithelial cells (BECs) have a potential as stem cells, because they are potent to differentiate into multiple central and peripheral lung cell types in three-dimensional (3D) cultures, and they develop multiple foci with well-differentiated histogenesis after transformed into neoplastic cells. In this study, we investigated morphogenic abilities of HBE135 human BECs immortalized by E6/E7 oncogene in 3D cultures. When HBE135 cells were cultured alone or co-cultured with endothelial cells, the cells formed spherical colonies without branching. However, in co-culture with lung fibroblast MRC-9 cells, HBE135 cells formed colonies with bronchioalveolar-like complex branching, suggesting that MRC-9-derived soluble factor(s) are responsible for the branching formation. MRC-9 cells, not endothelial cells, were found to highly express hepatocyte growth factor (HGF), a soluble molecule involved in liver and kidney regeneration. An anti-HGF neutralizing antibody severely suppressed the complex branching formation, but addition of HGF could not sufficiently compensate the morphogenic effects of MRC-9 cells, suggesting that MCR-9-derived HGF was necessary but insufficient for the bronchioalveolar structure formation. Immunohistochemistry revealed that Met, a cognate receptor for HGF, was highly expressed and phosphorylated in neoplastic BECs from lung adenocarcinomas with well-differentiated, not poorly differentiated, histogenesis. These results are consistent with the notion that BECs have an aspect of stem cells. This aspect appears to become manifest through HGF-Met signalling pathway activation.

Epithelial-mesenchymal co-culture model for studying alveolar morphogenesis

Division of large, immature alveolar structures into smaller, more numerous alveoli increases the surface area available for gas exchange. Alveolar division requires precise epithelial-mesenchymal interactions. However, few experimental models exist for studying how these cell-cell interactions produce changes in 3-dimensional structure. Here we report an epithelial-mesenchymal cell co-culture model where 3-dimensional peaks form with similar cellular orientation as alveolar structures in vivo. Co-culturing fetal mouse lung mesenchyme with A549 epithelial cells produced tall peaks of cells covered by epithelia with cores of mesenchymal cells. These structures did not form when using adult lung fibroblasts. Peak formation did not require localized areas of cell proliferation or apoptosis. Mesenchymal cells co-cultured with epithelia adopted an elongated cell morphology closely resembling myofibroblasts within alveolar septa in vivo. Because inflammation inhibits alveolar formation, we tested the effects of E. coli lipopolysaccharide on 3-dimensional peak formation. Confocal and time-lapse imaging demonstrated that lipopolysaccharide reduced mesenchymal cell migration, resulting in fewer, shorter peaks with mesenchymal cells present predominantly at the base. This epithelial-mesenchymal co-culture model may therefore prove useful in future studies of mechanisms regulating alveolar morphogenesis.

Extracellular Matrix Structure and Tissue Stiffness Control Postnatal Lung Development through the Lipoprotein Receptor–Related Protein 5/Tie2 Signaling System

Physical properties of the tissues and remodeling of extracellular matrix (ECM) play an important role in organ development. Recently, we have reported that low-density lipoprotein receptor-related protein (LRP) 5/Tie2 signaling controls postnatal lung development by modulating angiogenesis. Here we show that tissue stiffness modulated by the ECM cross-linking enzyme, lysyl oxidase (LOX), regulates postnatal lung development through LRP5-Tie2 signaling. The expression of LRP5 and Tie2 is up-regulated twofold in lung microvascular endothelial cells when cultured on stiff matrix compared to those cultured on soft matrix in vitro. LOX inhibitor, β-aminopropionitrile, disrupts lung ECM (collagen I, III, and VI, and elastin) structures, softens neonatal mouse lung tissue by 20%, and down-regulates the expression of LRP5 and Tie2 by 20 and 60%, respectively, which leads to the inhibition of postnatal lung development (30% increase in mean linear intercept, 1.5-fold increase in air space area). Importantly, hyperoxia treatment (Postnatal Days 1-10) disrupts ECM structure and stiffens mouse lung tissue by up-regulating LOX activity, thereby increasing LRP5 and Tie2 expression and deregulating alveolar morphogenesis in neonatal mice, which is attenuated by inhibiting LOX activity. These findings suggest that appropriate physical properties of lung tissue are necessary for physiological postnatal lung development, and deregulation of this mechanism contributes to postnatal lung developmental disorders, such as bronchopulmonary dysplasia.

Single primary fetal lung cells generate alveolar structures in vitro

Organ morphogenesis, including lung morphogenesis, involves a series of cellular behaviors that are difficult to observe and document in vivo due to current limitations in imaging techniques. Therefore, in vitro models are necessary to study these cellular behaviors as well as basic developmental processes relevant to in vivo morphogenesis. Here, we describe a novel in vitro three-dimensional (3D) culture system for assessing mouse lung alveolar morphogenesis using primary fetal mouse lung cells cultured in Matrigel supplemented with fibroblast growth factor 10 and hepatocyte growth factor. In our in vitro 3D culture system, single primary mouse fetal lung cells successfully grew, developed lumen, and formed multivesicular epithelial structures, resulting in a morphology that was highly similar to that of lung alveoli. This culture system is a useful tool for investigating the cellular and molecular mechanisms involved in lung alveolar morphogenesis.

Receptor for advanced glycation end products contributes to postnatal pulmonary development and adult lung maintenance program in mice.

The role of the receptor for advanced glycation end products (RAGE) in promoting the inflammatory response through activation of NF-κB pathway is well established. Recent findings indicate that RAGE may also have a regulative function in apoptosis, as well as in cellular proliferation, differentiation, and adhesion. Unlike other organs, lung tissue in adulthood and during organ development shows relatively high levels of RAGE expression. Thus a role for the receptor in lung organogenesis and homeostasis may be proposed. To evaluate the role of RAGE in lung development and adult lung homeostasis, we generated hemizygous and homozygous transgenic mice overexpressing human RAGE, and analyzed their lungs from the fourth postnatal day to adulthood. Moderate RAGE hyperexpression during lung development influenced secondary septation, resulting in an impairment of alveolar morphogenesis and leading to significant changes in morphometric parameters such as airspace number and the size of alveolar ducts. An increase in alveolar cell apoptosis and a decrease in cell proliferation were demonstrated by the terminal deoxy-nucleotidyltransferase-mediated dUTP nick end labeling reaction, active caspase-3, and Ki-67 immunohistochemistry. Alterations in elastin organization and deposition and in TGF-β expression were observed. In homozygous mice, the hyperexpression of RAGE resulted in histological changes resembling those changes characterizing human bronchopulmonary dysplasia (BPD). RAGE hyperexpression in the adult lung is associated with an increase of the alveolar destructive index and persistent inflammatory status leading to "destructive" emphysema. These results suggest an important role for RAGE in both alveolar development and lung homeostasis, and open new doors to working hypotheses on the pathogenesis of BPD and chronic obstructive pulmonary disease.

Ozone Exposure During thE Early Postnatal Period Alters the Timing and Pattern of Alveolar Growth and Development in Nonhuman Primates

Exposure to oxidant air pollutants in early childhood, with ozone as the key oxidant, has been linked to significant decrements in pulmonary function in young adults and exacerbation of airway remodeling in asthma. Development of lung parenchyma in rhesus monkeys is rapid during the first 2 years of life (comparable to the first 6 years in humans). Our hypothesis is that ozone inhalation during infancy alters alveolar morphogenesis. We exposed infant rhesus monkeys biweekly to 5, 8 hr/day, cycles of 0.5 ppm ozone with or without house dust mite allergen from 1 to 3 or 1 to 6 months of age. Monkeys were necropsied at 3 and 6 months of age. A morphometric approach was used to quantify changes in alveolar volume and number, the distribution of alveolar size, and capillary surface density per alveolar septa. Quantitative real time PCR was used to measure the relative difference in gene expression over time. Monkeys exposed to ozone alone or ozone combined with allergen had statistically larger alveoli that were less in number at 3 months of age. Alveolar capillary surface density was also decreased in the ozone exposed groups at 3 months of age. At 6 months of age, the alveolar number was similar between treatment groups and was associated with a significant rise in alveolar number from 3 to 6 months of age in the ozone exposed groups. This increase in alveolar number was not associated with any significant increase in microvascular growth as measured by morphometry or changes in angiogenic gene expression. Inhalation of ozone during infancy alters the appearance and timing of alveolar growth and maturation. Understanding the mechanism involved with this altered alveolar growth may provide insight into the parenchymal injury and repair process that is involved with chronic lung diseases such as severe asthma and COPD.