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Anne Chetty, Heber C Nielsen Tufts Medical Center, Tufts University, Boston, MA, USACorrespondence: Heber C NielsenDepartment of Pediatrics, Tufts Medical Center, Boston, MA, USAEmail heber.nielsen@tufts.eduAbstract: Airway smooth muscle (ASM) cell dysfunction is an important component of several obstructive pulmonary diseases, particularly asthma. External stimuli such as allergens, dust, air pollutants, and change in environmental temperatures provoke ASM cell hypertrophy, proliferation, and migration without adequate mechanistic controls. ASM cells can switch between quiescent, migratory, and proliferative phenotypes in response to extracellular matrix proteins, growth factors, and other soluble mediators. While some aspects of airway hypertrophy and remodeling could have beneficial effects, in many cases these contribute to a clinical phenotype of difficult to control asthma. In this review, we discuss the factors responsible for ASM hypertrophy and proliferation in asthma, focusing on cytokines, growth factors, and ion transporters, and discuss existing and potential approaches that specifically target ASM hypertrophy to reduce the ASM mass and improve asthma symptoms. The goal of this review is to highlight strategies that appear ready for translational investigations to improve asthma therapy.Keywords: airway smooth muscle cells, hypertrophy, proliferation, airway remodeling

Anne Chetty,1 Azeem Sharda,1 Rod Warburton,2 Ellen O Weinberg,3 Jinghui Dong,4 Min Fang,5 G Gary Sahagian,5 Tiangmeng Chen,3 Chang Xue,3 John J Castellot,3,6 Philip G Haydon,4 Heber C Nielsen1,6 1Department of Pediatrics, Tufts Medical Center, Boston, MA, USA; 2Department of Medicine, Tufts Medical Center, Boston, MA, USA; 3Department of Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, MA, USA; 4Department of Neuroscience, Tufts University School of Medicine, Boston, MA, USA; 5Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, USA; 6Graduate Program in Cell, Molecular and Developmental Biology, Tufts University School of Medicine, Boston, MA, USABackground: Purinergic receptors control cell proliferation, apoptosis, migration, inflammation, and cytokine secretion. Increased expression of specific purinergic receptors is reported in asthma. The role of purinergic P2Y6 receptors (P2Y6R) in asthma is controversial. Hypothesis: P2Y6R activation in asthma improves pulmonary function and reduces inflammation and smooth muscle amount. Methods: Female mice (C57/BL6, age 30 days) were randomly assigned to receive intranasal house dust mite (HDM) antigen (40 or 80 µg) or saline, 5 days/week, for 6 weeks. Randomly selected subgroups received intraperitoneal P2Y6R agonist prodrug (GC021109; 10 or 100 μg/kg weight/dose) simultaneously with HDM. After 6 weeks, lung function was measured. Lung lavage fluid (LLF) was used to measure total cell count, total protein, and cytokines. Immunohistochemistry for alpha smooth muscle actin (α-SMA) was done. Airway wall thickness was measured on micro-computed tomography (micro-CT) images. Results: Pulmonary function testing revealed a HDM dose-dependent airway hyperresponsiveness. Airway resistance was increased 2-fold while compliance was decreased by 50% at the higher HDM dose (P

Objective: Osteoarthritis (OA) has recently been suggested to be associated with diabetes. However, this association often disappears when accounting for body mass index (BMI), suggesting that mechanical stress may be a confounding factor. We investigated the combined influence of glucose level and loading stress on OA progression using a novel whole joint-in-motion (JM) culture system., Design: Whole mouse knee joints were placed in an enclosed chamber with culture media and actuated to recapitulate leg movement, with a dynamic stress regimen of 0.5 Hz, 8 h/day for 7 days. These joints were treated with varying levels of glucose and controlled for osmolarity and diffusion. Joint movement and joint space were examined by X-ray fluoroscopy and microCT. Cartilage matrix levels were quantified by blinded Mankin scoring and immunohistochemistry., Results: Culturing in the JM device facilitated proper leg extension and flexion movements, and adequate mass transport for analyzing the effect of glucose on cartilage. Treatment with higher levels of glucose either via media supplementation or intra-articular injection caused a significant decrease in levels of glycosaminoglycan (GAG) and an increase in aggrecan neoepitope in articular cartilage, but only under dynamic stress. Additionally, collagen II level was slightly reduced by high glucose levels., Conclusions: High levels of glucose and dynamic stress have permissive effects on articular cartilage GAG loss and aggrecan degradation, implicating that mechanical stress confounds the association of diabetes with OA. The JM device supports novel investigation of mechanical stress on the integrity of an intact living mouse joint to provide insights into OA pathogenesis., (Copyright © 2022 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Long bone growth requires the precise control of chondrocyte maturation from proliferation to hypertrophy during endochondral ossification, but the bioenergetic program that ensures normal cartilage development is still largely elusive. We show that chondrocytes have unique glucose metabolism signatures in these stages, and they undergo bioenergetic reprogramming from glycolysis to oxidative phosphorylation during maturation, accompanied by an upregulation of the pentose phosphate pathway. Inhibition of either oxidative phosphorylation or the pentose phosphate pathway in murine chondrocytes and bone organ cultures impaired hypertrophic differentiation, suggesting that the appropriate balance of these pathways is required for cartilage development. Insulin-like growth factor 2 (IGF2) deficiency resulted in a profound increase in oxidative phosphorylation in hypertrophic chondrocytes, suggesting that IGF2 is required to prevent overactive glucose metabolism and maintain a proper balance of metabolic pathways. Our results thus provide critical evidence of preference for a bioenergetic pathway in different stages of chondrocytes and highlight its importance as a fundamental mechanism in skeletal development., (© 2022. The Author(s).)

Background: Alternative splicing (AS) creates different protein isoforms, an important mechanism regulating cell-specific function. Little is known about AS in lung development, particularly in alveolar type II (ATII) cells. ErbB4 receptor isoforms Jma and Jmb have significant and opposing functions in the brain, heart, and lung development and/or disease. However, the regulators of ErbB4 AS are unknown. ErbB4 AS regulators in fetal mouse ATII cells control its function in ATII cell maturation., Methods: Candidate ErbB4 AS regulators were found using in silico analysis. Their developmental expression was studied in fetal mouse ATII cells. The effects of splice factor downregulation and upregulation on ATII cell maturation were analyzed., Results: ErbB4-Jma increased significantly in ATII cells after gestation E16.5. In silico analysis found four candidate splice factors: FOX2, CUG/CELF1, TIAR, and HUB. Fetal ATII cells expressed these factors in distinct developmental profiles. HUB downregulation in E17.5 ATII cells increased Jma isoform levels and Sftpb gene expression and decreased Jmb. HUB overexpression decreased Jma and Sftpb., Conclusions: ErbB4 AS is developmentally controlled by HUB in fetal ATII cells, promoting ATII differentiation. Regulated AS expression during ATII cell differentiation suggests novel therapeutic strategies to approach human disease., Impact: Alternative splicing (AS) of the ErbB4 receptor, involving mutually exclusive exon inclusion, creates Jma and Jmb isoforms with distinct differences in receptor processing and function. The Jma isoform of ErbB4 promotes differentiation of fetal lung alveolar type II cells. The AS is mediated in part by the RNA-binding protein HUB. The molecular mechanism of AS for ErbB4 has not been previously described. The regulation of ErbB4 AS has important implications in the development of organs, such as the lung, brain, and heart, and for disease, including cancer., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Background: Expressed breast milk (EBM) protein content is highly variable between mothers and often below published values that are still used for EBM protein fortification strategies. This approach may result in significant protein deficit and suboptimal protein energy (P/E) ratio. The study aim was to determine whether individualized EBM protein analysis and fortification will reduce preterm infant protein deficits and improve growth and neurodevelopmental outcome. Study Methods: In a single-center randomized, blinded study of infants born at 24 0/7-29 6/7 weeks, mother-specific protein values measured by a milk analyzer were used to individualize infant-specific protein intake (interventional group, IG), and compared this to a standardized protein fortification scheme based on published values of EBM protein content of 1.4 g/dL (control group, CG). For IG, milk analyzer protein values of mother's EBM were used to adjust protein content of the EBM. The CG EBM protein content was adjusted using the standard published value of 1.4 g/dL and not based on milk analyzer values. EBM protein content, protein intake, protein/energy (P/E) ratio, weight (WT), head circumference (HC), length (L), growth velocity (GV) from 2 to 6 weeks of age, WT, HC and L Z-Scores at 32- and 35-weeks PMA, and lean body mass (35 weeks PMA skin fold thickness) were measured. Neurodevelopment was assessed by Bayley III at average 24 months corrected gestational age (CGA). Results: EBM protein content before fortification was significantly below published values of 1.4 g/dL at all time points in both CG and IG. CG protein deficit was significantly decreased and progressively worsened throughout the study. Individualized protein fortification in IG avoided protein deficit and optimized P/E ratio. Although no significant change in short-term GV (at 6 weeks of age) was seen between groups, IG infants born at <27 weeks had significant improvements in WT and L z-scores, and leaner body mass at 32 and 35 weeks PMA. IG exhibited significantly improved cognitive scores at 24 months CGA. Conclusions: Infant-specific protein supplementation of mother's EBM optimized P/E ratio by eliminating protein deficit and improved growth z scores at 32- and 35-weeks PMA and neurocognitive testing at 24 months., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Khaira, Pert, Farrell, Sibley, Harvey-Wilkes, Nielsen and Volpe.)

We investigate the launch of negative upward streamers from sprite glows. This phenomenon is readily observed in high-speed observations of sprites and underlies the classification of sprites into carrot or column types. First, we describe how an attachment instability leads to a sharply defined region in the upper part of the streamer channel. This region has an enhanced electric field, low conductivity and strongly emits in the first positive system of molecular nitrogen. We identify it as the sprite glow. We then show how, in the most common configuration of a carrot sprite, several upward streamers emerge close to the lower boundary of the glow, where negative charge gets trapped and the lateral electric field is high enough. These streamers cut off the current flowing toward the glow and lead to the optical deactivation of the glow above. Finally, we discuss how our results naturally explain angel sprites., (©2019. The Authors.)

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are common and significant morbidities of prematurely born infants. These diseases have in common altered and pathologic vascular formation in the face of incomplete organ development. Therefore, it is reasonable to question whether factors affecting angiogenesis could have a joint pathogenic role for both diseases. Inhibition or induced expression of a single angiogenic factor is unlikely to be 100% causative or protective of either of BPD or ROP. It is more likely that interactions of multiple factors leading to disordered angiogenesis are present, increasing the likelihood of common pathways in both diseases. This review explores this possibility by assessing the evidence showing involvement of specific angiogenic factors in the vascular development and maldevelopment in each disease. Theoretical interactions of specific factors mutually contributing to BPD and ROP are proposed and, where possible, a timeline of the proposed relationships between BPD and ROP is developed. It is hoped that future research will be inspired by the theories put forth in this review to enhance the understanding of the pathogenesis in both diseases.

Osteoarthritis (OA) is a leading cause of chronic disability whose mechanism of pathogenesis is largely elusive. Local inflammation is thought to play a key role in OA progression, especially in injury-associated OA. While multiple inflammatory cytokines are detected, the timing and extent of overall inflammatory activities in early OA and the manner by which joint inflammation correlates with cartilage structural damage are still unclear. We induced OA via destabilization of the medial meniscus (DMM) in NFκB luciferase reporter mice, whose bioluminescent signal reflects the activity of NFκB, a central mediator of inflammation. Bioluminescence imaging data showed that DMM and sham control joints had a similar surge of inflammation at 1-week post-surgery, but the DMM joint exhibited a delay in resolution of inflammation in subsequent weeks. A similar trend was observed with synovitis, which we found to be mainly driven by synovial cell density and inflammatory infiltration rather than synovial lining thickness. Interestingly, an association between synovitis and collagen structural damage was observed in early OA. Using Second Harmonic Generation (SHG) imaging, we analyzed collagen fiber organization in articular cartilage. Zonal differences in collagen fiber thickness and organization were observed as soon as OA initiated after DMM surgery, and persisted over time. Even at 1-week post-surgery, the DMM joint showed a decrease in collagen fiber thickness in the deep zone and an increase in collagen fiber disorganization in the superficial zone. Since we were able detect and quantify collagen structural changes very early in OA development by SHG imaging, we concluded that SHG imaging is a highly sensitive tool to evaluate pathological changes in OA. In summary, this study uncovered a dynamic profile of inflammation and joint cartilage damage during OA initiation and development, providing novel insights into OA pathology.

Lung immaturity is the major cause of morbidity and mortality in premature infants, especially those born <28 weeks of gestation. These infants are at high risk of developing respiratory distress syndrome (RDS), a lung disease caused by insufficient surfactant production and immaturity of saccular/alveolar type II epithelial cells in the lung. RDS treatment includes oxygen and respiratory support that improve survival but also increase the risk for bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by arrested alveolarization, airway hyperreactivity, and pulmonary hypertension. The mechanisms regulating normal alveolar development and how injury disrupts normal development to cause BPD are not well understood. We examined the role of the matricellular protein CCN5 (Cysteine-rich protein 61/Connective tissue growth factor/Nephroblastoma-overexpressed protein) in the development of BPD. Cultured non-proliferating alveolar type II cells expressed low levels of CCN5 protein, and displayed higher levels during proliferation. siRNA targeting of CCN5 reduced alveolar type II cell proliferation and migration in cell culture. In a mouse model of hyperoxia-induced BPD, CCN5 protein was increased only in proliferating alveolar type I cells. Alveolar epithelial cells co-expressing markers of type II cells and type I cells also appeared. The results suggest that hyperoxic injury in immature lungs induces proliferation of type I cells and trans-differentiation of type II cells into type I cells. We propose that the mechanism of the injury response in BPD includes CCN5 expression. Study of CCN5 in neonatal alveolar injury will further our understanding of BPD pathophysiology while providing a mechanistic foundation for therapeutic approaches.

One of the first "tools" used for systematically evaluating successful newborn transitional physiology at birth was the Apgar Score, devised by Virginia Apgar in 1953. This objective assessment tool allowed clinicians to immediately gauge the relative success of a newborn infant making the transition from the in utero liquid immersive environment to the ex utero gas environment in the delivery room during the first minutes after birth. The scoring system, although eponymous, is generally summarized as an acronym based on Appearance, Pulse, Grimace, Activity, and Respiration, criteria evaluated and scored at 1 and 5 min after birth. This common clinical appraisal is a guide for determining the elements of integrated physiology involved as the infant makes the transition from a "sea water" environment of 3% oxygen to a "land" environment in 21% oxygen. Appearance determines the perfusion of the skin with oxygenated blood-turning it pink; Pulse is the rate of heart beat, reflecting successful oxygen delivery to organs; Grimace , or irritability, is a functional marker for nervous system integration; Activity represents locomotor capacity; and, of course, Respiration represents pulmonary function as well as the successful neuro-feedback-mediated drive to breathe, supplying oxygen by inspiring atmospheric gas. Respiration, locomotion, and metabolism are fundamental processes adapted for vertebrate evolution from a water-based to an atmosphere-based life and are reflected by the Apgar Score. These physiologic processes last underwent major phylogenetic changes during the water-land transition some 300-400 million years ago, during which specific gene duplications occurred that facilitated terrestrial adaptation, in particular the parathyroid hormone-related protein receptor, the β-adrenergic receptor, and the glucocorticoid receptor. All these genetic traits and the gene regulatory networks they comprise represent the foundational substructure of the Apgar Score. As such, these molecular elements can be examined using a Molecular Apgar evaluation of keystone evolutionary events that predict successful evolutionary adaptation of physiologic functions necessary for neonatal transition and survival.

Background: Inflammation is a major cause of cartilage destruction and leads to the imbalance of metabolic activities in the arthritic joint. Pigment epithelium-derived factor (PEDF) has been reported to have both pro- and anti-inflammatory activities in various cell types and to be upregulated in the arthritic joint, but its role in joint destruction is unclear. Our aim was to investigate the role of PEDF in cartilage degeneration under inflammatory conditions., Methods: PEDF was ectopically expressed in primary human articular chondrocytes, and catabolic gene expression and protein secretion in response to the pro-inflammatory cytokine interleukin 1 beta (IL-1β) were evaluated. Metatarsal bones from PEDF-deficient and wild type mice were cultured in the presence or absence of IL-1β. Cartilage matrix integrity and matrix metalloproteinases MMP-1, MMP-3, and MMP-13 were evaluated. PEDF-deficient and wild type mice were evaluated in the monosodium iodoacetate (MIA) inflammatory joint destruction animal model to determine the role of PEDF in inflammatory arthritis in vivo. Student's t-tests and Mann-Whitney tests were employed where appropriate, for parametric and non-parametric data, respectively., Results: We showed that PEDF protein levels were higher in human osteoarthritis samples compared to normal samples. We demonstrated that ectopic PEDF expression in primary human articular chondrocytes exacerbated catabolic gene expression in the presence of IL-1β. In whole bone organ cultures, IL-1β induced MMP-1, MMP-3 and MMP-13 protein production, and caused significant cartilage matrix loss. Interestingly, Toluidine Blue staining showed that PEDF-deficient bones from 29 week old animals, but not 10 week old animals, had reduced matrix loss in response to IL-1β compared to their wild type counterparts. In addition, PEDF-deficiency in 29 week old animals preserved matrix integrity and protected against cell loss in the MIA joint destruction model in vivo., Conclusion: We conclude that PEDF exacerbates cartilage degeneration in an age-dependent manner under an inflammatory setting. This is the first study identifying a specific role for PEDF in joint inflammation and highlights the multi-faceted activities of PEDF.

Glucocorticoid induction of pulmonary surfactant involves a mesenchyme-derived protein first characterized in 1978 by Smith and termed fibroblast-pneumocyte factor (FPF). Despite a number of agents having been postulated as being FPF, its identity has remained obscure. In the past decade, three strong candidates for FPF have arisen. This review examines the evidence that keratinocyte growth factor (KGF), leptin or neuregulin-1β (NRG-1β) act as FPF or components of it. As with FPF production, glucocorticoids enhance the concentration of each of these agents in fibroblast-conditioned media. Moreover, each stimulates the synthesis of surfactant-associated phospholipids and proteins in type II pneumocytes. Further, some have unique activities, for example, KGF also minimizes lung injury through enhanced epithelial cell proliferation and NRG-1β enhances surfactant phospholipid secretion and β-adrenergic receptor activity in type II cells. However, even though these agents have attributes in common with FPF, it is inappropriate to specify any one of these agents as FPF. Rather, it appears that each contributes to separate mesenchymal-epithelial signaling mechanisms involved in different aspects of lung development. Given that the production of pulmonary surfactant is essential for postnatal survival, it is reasonable to suggest that several mechanisms independently regulate surfactant synthesis., Competing Interests: Statement: The authors have nothing to disclose regarding financial ties or conflicts of interest.

The temporal origins of childhood asthma are incompletely understood. We hypothesize that allergen sensitization which begins in early infancy causes IgE-mediated airway and vascular remodeling, and airway hyper-responsiveness. Mice were sensitized with ovalbumin (OVA) without or with anti-IgE antibody from postnatal day (P) 10 through P42. We studied airway resistance in response to Methacholine (MCh) challenge, bronchoalveolar lavage fluid (BAL) inflammatory cell content, immunohistochemistry for inflammation, alpha-smooth muscle actin (alpha-SMA) and platelet/endothelial cell adhesion molecule (PECAM) proteins, and Western blotting for vascular endothelial growth factor (VEGF) protein. Compared to controls, mice treated with OVA had increased airway resistance (baseline: 192% of control; MCH 12 mg/mL 170% of control; P less than 0.0.5). OVA treatment also increased lung alpha-SMA, VEGF and PECAM compared to controls. Inflammatory cells in the BAL and perivascular and peribronchiolar inflammatory cell infiltrates increased over controls with OVA exposure. These changes were counteracted by anti-IgE treatment. We conclude that mice sensitized in early infancy develop an IgE-mediated hyper-reactive airway disease with airway and vascular remodeling. Preventive approaches in early infancy of at-risk individuals may reduce childhood asthma.

ErbB4 receptor and thyroid transcription factor (TTF)-1 are important modulators of fetal alveolar type II (ATII) cell development and injury. ErbB4 is an upstream regulator of TTF-1, promoting its expression in MLE-12 cells, an ATII cell line. Both proteins are known to promote surfactant protein-B gene (SftpB) and protein (SP-B) expression, but their feedback interactions on each other are not known. We hypothesized that TTF-1 expression has a feedback effect on ErbB4 expression in an in-vitro model of isolated mouse ATII cells. We tested this hypothesis by analyzing the effects of overexpressing HER4 and Nkx2.1, the genes of ErbB4 and TTF-1 on TTF-1 and ErbB4 protein expression, respectively, as well as SP-B protein expression in primary fetal mouse lung ATII cells. Transient ErbB4 protein overexpression upregulated TTF-1 protein expression in primary fetal ATII cells, similarly to results previously shown in MLE-12 cells. Transient TTF-1 protein overexpression down regulated ErbB4 protein expression in both cell types. TTF-1 protein was upregulated in primary transgenic ErbB4-depleted adult ATII cells, however SP-B protein expression in these adult transgenic ATII cells was not affected by the absence of ErbB4. The observation that TTF-1 is upregulated in fetal ATII cells by ErbB4 overexpression and also in ErbB4-deleted adult ATII cells suggests additional factors interact with ErbB4 to regulate TTF-1 levels. We conclude that the interdependency of TTF-1 and ErbB4 is important for surfactant protein levels. The interactive regulation of ErbB4 and TTF-1 needs further elucidation.

Sprites are large, luminous electrical discharges in the upper atmosphere caused by intense cloud-to-ground lightning flashes, manifesting an impulsive coupling mechanism between lower and upper atmospheric regions. Their dynamics are governed by filamentary streamer discharges whose propagation properties have been well studied by past work. However, how they are initiated is still under active debate. It has recently been concluded that ionospheric/mesospheric inhomogeneities are required for their initiation, but it is an open question as to what the sources of those inhomogeneities are. Here we present numerical simulation results to demonstrate that naturally-existing, small-scale mesospheric structures such as those created by gravity waves via instability and breaking are viable sources. The proposed theory is supported by a recent, unique high-speed observation from aircraft flying at 14-km altitude. The theory naturally explains many aspects of observed sprite streamer initiation and has important implications for future observational work.

Bronchopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. Studies show the importance of proangiogenic factors for alveolarization, but the importance of antiangiogenic factors is unknown. We proposed that hyperoxia increases the potent angiostatin, pigment epithelium-derived factor (PEDF), in neonatal lungs, inhibiting alveolarization and microvascularization. Wild-type (WT) and PEDF(-/-) mice were exposed to room air (RA) or 0.9 fraction of inspired oxygen from Postnatal Day 5 to 13. PEDF protein was increased in hyperoxic lungs compared with RA-exposed lungs (P < 0.05). In situ hybridization and immunofluorescence identified PEDF production primarily in alveolar epithelium. Hyperoxia reduced alveolarization in WT mice (P < 0.05) but not in PEDF(-/-) mice. WT hyperoxic mice had fewer platelet endothelial cell adhesion molecule (PECAM)-positive cells per alveolus (1.4 ± 0.4) than RA-exposed mice (4.3 ± 0.3; P < 0.05); this reduction was absent in hyperoxic PEDF(-/-) mice. The interactive regulation of lung microvascularization by vascular endothelial growth factor and PEDF was studied in vitro using MFLM-91U cells, a fetal mouse lung endothelial cell line. Vascular endothelial growth factor stimulation of proliferation, migration, and capillary tube formation was inhibited by PEDF. MFLM-91U cells exposed to conditioned medium (CM) from E17 fetal mouse lung type II (T2) cells cultured in 0.9 fraction of inspired oxygen formed fewer capillary tubes than CM from T2 cells cultured in RA (hyperoxia CM, 51 ± 10% of RA CM, P < 0.05), an effect abolished by PEDF antibody. We conclude that PEDF mediates reduced vasculogenesis and alveolarization in neonatal hyperoxia. Bronchopulmonary dysplasia likely results from an altered balance between pro- and antiangiogenic factors.

Hoxb5 and Hoxa5 transcription factor proteins uniquely impact lung morphogenesis at the developmental time point when extremely preterm infants are born. The effect of O2 exposure (0.4 FiO2) used in preterm infant care on these Hox proteins is unknown. We used ex vivo fetal mouse lung organ cultures to explore the effects of 0.4 FiO2 on lung airway and vascular formation in the context of Hoxb5 and Hoxa5 expression and regulation. Compared to room air, 48 h (h) 0.4 FiO2 adversely attenuated airway and microvasculature formation while reducing lung growth and epithelial cell volume, and increasing mesenchymal volume. 0.4 FiO2 decreased pro-angiogenic Hoxb5 and VEGFR2 while not altering protein levels of angiostatic Hoxa5. Lungs returned to RA after 24 h 0.4FiO2 had partial structural recovery but remained smaller and less developed. Mesenchymal cell apoptosis increased and proliferation decreased with time in O2 while epithelial cell proliferation significantly increased. Hoxb5 overexpression led to prominent peri-airway VEGFR2 expression and promoted lung vascular and airway patterning. Hoxa5 overexpression had the opposite effects. We conclude that 0.4 FiO2 exposure causes a profound loss of airway and lung microvascular development that occurs partially via reduction in pro-angiogenic Hoxb5 while angiostatic Hoxa5 expression is maintained.

Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast-Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts.

Lung immaturity is the major cause of morbidity and mortality in premature infants, especially those born <28 weeks gestation. Proper lung development from 23-28 weeks requires coordinated cell proliferation and differentiation. Infants born at this age are at high risk for respiratory distress syndrome (RDS), a lung disease characterized by insufficient surfactant production due to immaturity of the alveoli and its constituent cells in the lung. The ErbB4 receptor and its stimulation by neuregulin (NRG) plays a critical role in surfactant synthesis by alveolar type II epithelial cells. In this review, we first provide an introduction to normal human alveolar development, followed by a discussion of the neuregulin and ErbB4-mediated mechanisms regulating alveolar development and surfactant production.

Sprites are spectacular optical emissions in the mesosphere induced by transient lightning electric fields above thunderstorms. Although the streamer nature of sprites has been generally accepted, how these filamentary plasmas are initiated remains a subject of active research. Here we present observational and modelling results showing solid evidence of pre-existing plasma irregularities in association with streamer initiation in the D-region ionosphere. The video observations show that before streamer initiation, kilometre-scale spatial structures descend rapidly with the overall diffuse emissions of the sprite halo, but slow down and stop to form the stationary glow in the vicinity of the streamer onset, from where streamers suddenly emerge. The modelling results reproduce the sub-millisecond halo dynamics and demonstrate that the descending halo structures are optical manifestations of the pre-existing plasma irregularities, which might have been produced by thunderstorm or meteor effects on the D-region ionosphere.

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Many signaling pathways are mediated by Shc adapter proteins that, in turn, are expressed as three isoforms with distinct functions. The p66(Shc) isoform antagonizes proliferation, regulates oxidative stress, and mediates apoptosis. It is highly expressed in the canalicular but not the later stages of mouse lung development, and its expression persists in bronchopulmonary dysplasia, a chronic disease associated with premature birth. These observations suggest that p66(Shc) has a developmental function. However, constitutive p66(Shc) deletion yields no morphological phenotype, and the structure of the Shc gene precludes its inducible deletion. To elucidate its function in lung development, we transfected p66(Shc) or nonsilencing small-interfering RNA (siRNA) into the epithelia of embryonic day 11 mouse lungs that were then cultured for 3 days and analyzed morphometrically. To assess cellular proliferation and epithelial differentiation, lung explants were immunostained and immunoblotted for p66(Shc), proliferating cell nuclear antigen (PCNA), the proximal airway differentiation antigens Clara cell 10-kDa protein (CC10) and thyroid transcription factor (TTF)-1, and the alveolar surfactant proteins (SP)-A, -B, and -C. Explants transfected with nonsilencing siRNA demonstrated specific epithelial uptake and normal morphological development relative to uninjected controls. In contrast, transfection with p66(Shc) siRNA significantly increased lumenal cross-sectional areas, decreased branching, and increased epithelial proliferation (P < 0.05 for all). Relative to controls, the expression of SP-B, SP-C, CC10, and TTF-1 was decreased by p66(Shc) knockdown. SP-A was not expressed in either control or treated lungs. These data suggest that p66(Shc) attenuates epithelial proliferation while promoting both distal and proximal epithelial maturation.

The Eunice Kennedy Shriver National Institute of Child Health and Human Development convened an Asthma Group in response to the Best Pharmaceuticals for Children Act. The overall goal of the Best Pharmaceuticals for Children Act Program is to improve pediatric therapeutics through preclinical and clinical drug trials that lead to drug-labeling changes. Although significant advances have been made in the understanding and management of asthma in adults with appropriately labeled medications, less information is available on the management of asthma in children. Indeed, many medications are inadequately labeled for use in children. In general, the younger the child, the less information there is available to guide clinicians. Because asthma often begins in early childhood, it is incumbent on us to continue to address the primary questions raised in this review and carefully evaluate the medications used to manage asthma in children. Meanwhile, continued efforts should be made in defining effective strategies that reduce the risk of exacerbations. If the areas of defined need are addressed in the coming years, namely prevention of exacerbations and progression of disease, as well as primary intervention, we will see continuing reduction in asthma mortality and morbidity along with improved quality of life for children with asthma., (Published by Mosby, Inc.)

Background: MicroRNAs play important roles in regulating biological processes, including organ morphogenesis and maturation. However, little is known about specific pathways regulated by miRNA during lung development. Between the canalicular and saccular stages of the developing lung several important cellular events occur, including the onset of surfactant synthesis, microvascular remodeling and structural preparation for subsequent alveolarization. The miRNAs that are actively regulated, and the identity of their targets during this important developmental interval in the lung remain elusive., Results: Using TLDA low density real-time PCR arrays, the expression of 376 miRNAs in male and female fetal mouse lungs of gestational days E15 - E18 were profiled. Statistical analyses identified 25 and 37 miRNAs that changed significantly between sexes and with gestation, respectively. In silico analysis using Ingenuity Pathway Analysis (IPA) identified specific pathways and networks known to be targets of these miRNAs which are important to lung development. Pathways that are targeted by sex regulated miRNAs include retinoin, IGFR1, Tp53 and Akt. Pathways targeted by gestation-regulated miRNAs include VEGFA and mediators of glucose metabolism., Conclusion: MiRNAs are differentially regulated across time and between sexes during the canalicular and saccular stages of lung development. Sex-associated differential miRNA expression may regulate the differences in structural and functional male and female lung development, as shown by networks generated using in silico analysis. These data provide a valuable resource to further enhance the understanding of miRNA control of lung development and maturation.

Androgens enhance airway branching but delay alveolar maturation contributing to increased respiratory morbidity in prematurely born male infants. Hoxb5 protein positively regulates airway branching in developing lung. In other organs, androgen regulation intersects with Hox proteins and TGF β -SMAD signaling, but these interactions have not been studied in the lung. We hypothesized that androgen alteration of airway branching early in lung development requires Hoxb5 expression and that these androgen-Hoxb5 interactions occur partially through regional changes in TGF β signaling. To evaluate acute effects of androgen and TGF β on Hoxb5, E11 whole fetal mouse lungs were cultured with dihydrotestosterone (DHT) with/without Hoxb5 siRNA or TGF β inhibitory antibody. Chronic in utero DHT exposure was accomplished by exposing pregnant mice to DHT (subcutaneous pellet) from E11 to E18. DHT's ability to enhance airway branching and alter phosphorylated SMAD2 cellular localization was partially dependent on Hoxb5. Hoxb5 inhibition also changed the cellular distribution of SMAD7 protein. Chronic in utero DHT increased Hoxb5 and altered SMAD7 mesenchymal localization. TGF β inhibition enhanced airway branching, and Hoxb5 protein cellular localization was more diffuse. We conclude that DHT controls lung airway development partially through modulation of Hoxb5 protein expression and that this level of regulation involves interactions with TGF β signaling.

Epithelial-mesenchymal interactions play a crucial role in branching morphogenesis, but very little is known about how endothelial cells contribute to this process. Here, we examined how anti-angiogenic miR-221 and pro-angiogenic miR-130a affect airway and vascular development in the fetal lungs. Lung-specific effects of miR-130a and miR-221 were studied in mouse E14 whole lungs cultured for 48 hours with anti-miRs or mimics to miR-130a and miR-221. Anti-miR 221 treated lungs had more distal branch generations with increased Hoxb5 and VEGFR2 around airways. Conversely, mimic 221 treated lungs had reduced airway branching, dilated airway tips and decreased Hoxb5 and VEGFR2 in mesenchyme. Anti-miR 130a treatment led to reduced airway branching with increased Hoxa5 and decreased VEGFR2 in the mesenchyme. Conversely, mimic 130a treated lungs had numerous finely arborized branches extending into central lung regions with diffusely localized Hoxa5 and increased VEGFR2 in the mesenchyme. Vascular morphology was analyzed by GSL-B4 (endothelial cell-specific lectin) immunofluorescence. Observed changes in airway morphology following miR-221 inhibition and miR-130a enhancement were mirrored by changes in vascular plexus formation around the terminal airways. Mouse fetal lung endothelial cells (MFLM-91U) were used to study microvascular cell behavior. Mimic 221 treatment resulted in reduced tube formation and cell migration, where as the reverse was observed with mimic 130a treatment. From these data, we conclude that miR-221 and miR-130a have opposing effects on airway and vascular morphogenesis of the developing lung.

Maturation of pulmonary fetal type II cells to initiate adequate surfactant production is crucial for postnatal respiratory function. Little is known about specific mechanisms of signal transduction controlling type II cell maturation. The ErbB4 receptor and its ligand neuregulin (NRG) are critical for lung development. ErbB4 is cleaved at the cell membrane by the γ-secretase enzyme complex whose active component is either presenilin-1 (PSEN-1) or presenilin-2. ErbB4 cleavage releases the 80kDa intracellular domain (4ICD), which associates with chaperone proteins such as YAP (Yes-associated protein) and translocates to the nucleus to regulate gene expression. We hypothesized that PSEN-1 and YAP have a development-specific expression in fetal type II cells and are important for ErbB4 signaling in surfactant production. In primary fetal mouse E16, E17, and E18 type II cells, PSEN-1 and YAP expression increased at E17 and E18 over E16. Subcellular fractionation showed a strong cytosolic and a weaker membrane location of both PSEN-1 and YAP. This was enhanced by NRG stimulation. Co-immunoprecipitations showed ErbB4 associated separately with PSEN-1 and with YAP. Their association, phosphorylation, and co-localization were induced by NRG. Confocal immunofluorescence and nuclear fractionation confirmed these associations in a time-dependent manner after NRG stimulation. Primary ErbB4-deleted E17 type II cells were transfected with a mutant ErbB4 lacking the γ-secretase binding site. When compared to transfection with wild-type ErbB4, the stimulatory effect of NRG on surfactant protein mRNA expression was lost. We conclude that PSEN-1 and YAP have crucial roles in ErbB4 signal transduction during type II cell maturation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The following are the proceedings of a symposium held at the Second International Congress for Respiratory Science in Bad Honnef, Germany. The goals of the symposium were to delineate the blood-gas barrier phenotype across vertebrate species; to delineate the interrelationship between the evolution of the blood-gas barrier, locomotion and metabolism; to introduce the selection pressures for the evolution of the surfactant system as a key to understanding the physiology of the blood-gas barrier; to introduce the lung lipofibroblast and its product, leptin, which coordinately regulates pulmonary surfactant, type IV collagen in the basement membrane and host defense, as the cell-molecular site of selection pressure for the blood-gas barrier; to drill down to the gene regulatory network(s) involved in leptin signaling and the blood-gas barrier phenotype; to extend the relationship between leptin and the blood-gas barrier to diving mammals., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The ErbB4 receptor has an important function in fetal lung maturation. Deletion of ErbB4 leads to alveolar hypoplasia and hyperreactive airways similar to the changes in bronchopulmonary dysplasia (BPD). BPD is a chronic pulmonary disorder affecting premature infants as a consequence of lung immaturity, lung damage, and abnormal repair. We hypothesized that proper ErbB4 function is needed for the timely progression of fetal lung development. An ErbB4 transgenic cardiac rescue mouse model was used to study the effect of ErbB4 deletion on fetal lung structure, surfactant protein (SP) expression, and synthesis, and inflammation. Morphometric analyses revealed a delayed structural development with a significant decrease in saccular size at E18 and more pronounced changes at E17, keeping these lungs in the canalicular stage. SP-B mRNA expression was significantly down regulated at E17 with a subsequent decrease in SP-B protein expression at E18. SP-D protein expression was significantly decreased at E18. Surfactant phospholipid synthesis was significantly decreased on both days, and secretion was down regulated at E18. We conclude that pulmonary ErbB4 deletion results in a structural and functional delay in fetal lung development, indicating a crucial regulatory role of ErbB4 in the timely progression of fetal lung development., (2010. Published by Elsevier B.V.)

In many organs, integrins and cadherins are partly regulated by Hox genes, but their interactions in airway morphogenesis and congenital lung diseases are unknown. We previously showed that the Hox protein HoxB5 is abnormally increased in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), congenital lung lesions with abnormal airway branching. We now report on alpha(2)-, alpha(3)-, and beta(1)-integrin and E-cadherin expression in normal human lung and in BPS and CCAM tissue previously shown to have abnormal HoxB5 expression and on the relationship of cell adhesion molecule expression to Hoxb5 regulation. alpha(2)-, alpha(3)-, and beta(1)-integrins and E-cadherin expression in normal human lung and BPS and CCAM were evaluated using Western blot and immunohistochemistry. Fetal mouse lung fibroblasts with Hoxb5-specific siRNA downregulation were evaluated for alpha(2)-integrin protein levels by Western blot. Compared with normal human lung, a previously undetected alpha(2)-integrin isoform potentially lacking essential cytoplasmic sequences was significantly increased in BPS and CCAM, and alpha(2)-integrin spatial and cellular expression was more intense. E-cadherin protein levels were also significantly increased, whereas alpha(3) increased in CCAM compared with canalicular, but not with alveolar, stage lung. beta(1)-integrin levels were unchanged. We conclude that in BPS and CCAM, altered alpha(2)-integrin cytoplasmic signaling contributes to abnormal cellular behavior in these lung lesions. Aberrant cell adhesion molecule and Hox protein regulation are likely part of the mechanism involved in the development of BPS and CCAM.

ErbB4 is a predominant heterodimer for other ErbB receptors in late fetal lung development where it participates in regulating type II cell surfactant synthesis. To further elucidate the role of ErbB4 in pulmonary alveolar epithelial cell function, the authors hypothesized that ErbB4 participates in maintaining adult lung type II cell homeostasis. The authors used small interfering RNA (siRNA) to down-regulate endogenous, ErbB4 receptors in the adult rat lung epithelial L2 cell line and measured neuregulin 1beta (NRG1beta)-, and fibroblast conditioned medium (FCM)-induced effects on L2 cell surfactant phospholipid synthesis and proliferation. Under control conditions, total and phosphorylated ErbB4 were significantly increased after both NRG1beta and FCM treatment, as were surfactant phospholipids synthesis and cell proliferation. Down-regulation of ErbB4 with siRNA reduced stimulation of NRG1beta- and FCM-induced ErbB4 phosphorylation, decreased endogenous surfactant phospholipid synthesis, and blocked NRG1beta- and FCM-stimulated surfactant phospholipid synthesis. NRG1beta- and FCM-induced cell proliferation was not affected. The authors conclude that ErbB4 participates in maintaining adult lung alveolar epithelial cell surfactant synthesis and proliferation with development-specific functions.

Matrix metalloprotease-9 (MMP-9) is increased in lung injury following hyperoxia exposure in neonatal mice, in association with impaired alveolar development. We studied the role of MMP-9 in the mechanism of hyperoxia-induced functional and histological changes in neonatal mouse lung. Reduced alveolarization with remodeling of ECM is a major morbidity component of oxidant injury in developing lung. MMP-9 mediates oxidant injury in developing lung causing altered lung remodeling. Five-day-old neonatal wild-type (WT) and MMP-9 (-/-) mice were exposed to hyperoxia for 8 days. The lungs were inflation fixed, and sections were examined for morphometry. The mean linear intercept and alveolar counts were evaluated. Immunohistochemistry for MMP-9 and elastin was performed. MMP-2, MMP-9, type I collagen, and tropoelastin were measured by Western blot analysis. Lung quasistatic compliance was studied in anaesthetized mice. MMP-2 and MMP-9 were significantly increased in lungs of WT mice exposed to hyperoxia compared with controls. Immunohistochemistry showed an increase in MMP-9 in mesenchyme and alveolar epithelium of hyperoxic lungs. The lungs of hyperoxia-exposed WT mice had less gas exchange surface area and were less compliant compared with room air-exposed WT and hyperoxia-exposed MMP-9 (-/-) mice. Type I collagen and tropoelastin were increased in hyperoxia-exposed WT with aberrant elastin staining. These changes were ameliorated in hyperoxia-exposed MMP-9 (-/-) mice. MMP-9 plays an important role in the structural changes consequent to oxygen-induced lung injury. Blocking MMP-9 activity may lead to novel therapeutic approaches in preventing bronchopulmonary dysplasia.

Background: Hox transcription factors modulate signaling pathways controlling organ morphogenesis and maintain cell fate and differentiation in adults. Retinoid signaling, key in regulating Hox expression, is altered in pulmonary hypoplasia. Information on pattern-specific expression of Hox proteins in normal lung development and in pulmonary hypoplasia is minimal. Our objective was to determine how pulmonary hypoplasia alters temporal, spatial, and cellular expression of Hoxa5, Hoxb4, and Hoxb6 proteins compared to normal lung development., Methods: Temporal, spatial, and cellular Hoxa5, Hoxb4, and Hoxb6 expression was studied in normal (untreated) and nitrofen-induced hypoplastic (NT-PH) lungs from gestational day 13.5, 16, and 19 fetuses and neonates using Western blot and immunohistochemistry., Results: Modification of protein levels and spatial and cellular Hox expression patterns in NT-PH lungs was consistent with delayed lung development. Distinct protein isoforms were detected for each Hox protein. Expression levels of the Hoxa5 and Hoxb6 protein isoforms changed with development and were altered further in NT-PH lungs. Compared to normal lungs, GD19 and neonatal NT-PH lungs had decreased Hoxb6 and increased Hoxa5 and Hoxb4. Hoxa5 cellular localization changed from mesenchyme to epithelia earlier in normal lungs. Hoxb4 was expressed in mesenchyme and epithelial cells throughout development. Hoxb6 remained mainly in mesenchymal cells around distal airways., Conclusions: Unique spatial and cellular expression of Hoxa5, Hoxb4, and Hoxb6 participates in branching morphogenesis and terminal sac formation. Altered Hox protein temporal and cellular balance of expression either contributes to pulmonary hypoplasia or functions as a compensatory mechanism attempting to correct abnormal lung development and maturation in this condition., (Copyright 2008 Wiley-Liss, Inc.)

We examined the cytoprotective effect of interleukin-6 (IL-6) and interleukin-11 (IL-11) during oxidant injury in neonatal lung and the regulators of cell death in vitro and in vivo after oxidant exposure. Type II cells from day 21 fetal neonatal rat lungs were treated with varying concentrations of either IL-6 or IL-11 for 24 hr prior to exposure to H(2)O(2). Three-day-old transgenic lung-specific IL-11 and IL-6 overexpressing and wild type (WT) mouse pups were exposed to hyperoxia or room air for 3 days. Type II cells exposed to either IL-6 or IL-11 prior to oxidant injury exhibited improved survival compared to controls, 67% +/- 2.6 survivals in IL-6 pretreated cells compared to 48% +/- 1.6 in control; 63% +/- 3 survivals in IL-11 pretreated cells compared to 49% +/- 2.6 in control. The number of TUNEL positive cells in hyperoxia-exposed lungs was increased compared to room air animals (27 +/- 0.9 vs. 4 +/- 0.4; mean +/- SEM; P < 0.05). In contrast, the number of TUNEL positive cells was reduced in hyperoxia-exposed lungs from IL-11 (+) mice (15.2 +/- 2.2; mean +/- SEM; P < 0.05). There was an enhanced accumulation of Bcl-2 and reduction of Bax protein in hyperoxia-exposed IL-11 (+) compared to room air-exposed mice. This was not seen in hyperoxia-exposed IL-6 (+) pups. An increase in caspase-3 was seen in hyperoxia-exposed lungs of WT pups compared to IL-11 (+) pups. IL-11 and IL-6 provide protective effects against oxidant-mediated injury in fetal type II cells and IL-11 provides protection in vivo by down-regulation of caspase-mediated cell death., ((c) 2008 Wiley-Liss, Inc.)

Insufficient fetal surfactant production leads to respiratory distress syndrome among preterm infants. Neuregulin signals the onset of fetal surfactant phospholipid synthesis through formation of erbB receptor dimers. We hypothesized that erbB4 downregulation in fetal type II epithelial cells will downregulate not only fetal surfactant phospholipid synthesis, but also affect proliferation and erbB receptor localization. We tested these hypotheses using small interfering RNA (siRNA) directed against the erbB4 gene to silence erbB4 receptor function in cultures of primary day 19 fetal rat lung type II cells. ErbB4 siRNA treatment inhibited erbB4 receptor protein expression, fibroblast-conditioned medium induced erbB4 phosphorylation, and fetal surfactant phospholipid synthesis. Cell proliferation, measured as thymidine incorporation, was also inhibited by erbB4 siRNA treatment. Downregulation of erbB4 receptor protein changed erbB1 localization at baseline and after stimulation, as determined by confocal microscopy and subcellular fractionation. We conclude that erbB4 is an important receptor in the control of fetal lung type II cell maturation.