Bone-Targeted Nanoparticle Drug Delivery System-Mediated Macrophage Modulation for Enhanced Fracture Healing.

Despite decades of progress, developing minimally invasive bone-specific drug delivery systems (DDS) to improve fracture healing remains a significant clinical challenge. To address this critical therapeutic need, nanoparticle (NP) DDS comprised of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) functionalized with a peptide that targets tartrate-resistant acid phosphatase (TRAP) and achieves preferential fracture accumulation has been developed. The delivery of AR28, a glycogen synthase kinase-3 beta (GSK3ß) inhibitor, via the TRAP binding peptide-NP (TBP-NP) expedites fracture healing. Interestingly, however, NPs are predominantly taken up by fracture-associated macrophages rather than cells typically associated with fracture healing. Therefore, the underlying mechanism of healing via TBP-NP is comprehensively investigated herein. TBP-NPAR28 promotes M2 macrophage polarization and enhances osteogenesis in preosteoblast-macrophage co-cultures in vitro. Longitudinal analysis of TBP-NPAR28 -mediated fracture healing reveals distinct spatial distributions of M2 macrophages, an increased M2/M1 ratio, and upregulation of anti-inflammatory and downregulated pro-inflammatory genes compared to controls. This work demonstrates the underlying therapeutic mechanism of bone-targeted NP DDS, which leverages macrophages as druggable targets and modulates M2 macrophage polarization to enhance fracture healing, highlighting the therapeutic benefit of this approach for fractures and bone-associated diseases.

Rare Pulmonary Connective Tissue Type Mast Cells Regulate Lung Endothelial Cell Angiogenesis.

Within the human lung, mast cells typically reside adjacent to the conducting airway and assume a mucosal phenotype (MCT). In rare pathologic conditions, connective tissue phenotype mast cells (MCTCs) can be found in the lung parenchyma. MCTCs accumulate in the lungs of infants with severe bronchopulmonary dysplasia, a chronic lung disease associated with preterm birth, which is characterized by pulmonary vascular dysmorphia. The human mast cell line (LUVA) was used to model MCTCs or MCTs. The ability of MCTCs to affect vascular organization during fetal lung development was tested in mouse lung explant cultures. The effect of MCTCs on in vitro tube formation and barrier function was studied using primary fetal human pulmonary microvascular endothelial cells. The mechanistic role of MCTC proteases was tested using inhibitors. MCTCLUVA but not MCTLUVA was associated with vascular dysmorphia in lung explants. In vitro, the addition of MCTCLUVA potentiated fetal human pulmonary microvascular endothelial cell interactions, inhibited tube stability, and disrupted endothelial cell junctions. Protease inhibitors ameliorated the ability of MCTCLUVA to alter endothelial cell angiogenic activities in vitro and ex vivo. These data indicate that MCTCs may directly contribute to disrupted angiogenesis in bronchopulmonary dysplasia. A better understanding of factors that regulate mast cell subtype and their different effector functions is essential.

Collagen VI Deficiency Results in Structural Abnormalities in the Mouse Lung.

Collagen VI (COL6) is known for its role in a spectrum of congenital muscular dystrophies, which are often accompanied by respiratory dysfunction. However, little is known regarding the function of COL6 in the lung. We confirmed the presence of COL6 throughout the basement membrane region of mouse lung tissue. Lung structure and organization were studied in a previously described Col6a1-/- mouse, which does not produce detectable COL6 in the lung. The Col6a1-/- mouse displayed histopathologic alveolar and airway abnormalities. The airspaces of Col6a1-/- lungs appeared simplified, with larger (29%; P < 0.01) and fewer (31%; P < 0.001) alveoli. These airspace abnormalities included reduced isolectin B4+ alveolar capillaries and surfactant protein C-positive alveolar epithelial type-II cells. Alterations in lung function consistent with these histopathologic changes were evident. Col6a1-/- mice also displayed multiple airway changes, including increased branching (59%; P < 0.001), increased mucosal thickness (34%; P < 0.001), and increased epithelial cell density (13%; P < 0.001). Comprehensive transcriptome analysis revealed that the loss of COL6 is associated with reductions in integrin-paxillin-phosphatidylinositol 3-kinase signaling in vivo. In vitro, COL6 promoted steady-state phosphorylated paxillin levels and reduced cell density (16% to 28%; P < 0.05) at confluence. Inhibition of phosphatidylinositol 3-kinase, or its downstream effectors, resulted in increased cell density to a level similar to that seen on matrices lacking COL6.

CX3CR1 as a respiratory syncytial virus receptor in pediatric human lung.

BACKGROUND: Data on the host factors that contribute to infection of young children by respiratory syncytial virus (RSV) are limited. The human chemokine receptor, CX3CR1, has recently been implicated as an RSV receptor. Here we evaluate a role for CX3CR1 in pediatric lung RSV infections. METHODS: CX3CR1 transcript levels in the upper and lower pediatric airways were assessed. Tissue localization and cell-specific expression was confirmed using in situ hybridization and immunohistochemistry. The role of CX3CR1 in RSV infection was also investigated using a novel physiological model of pediatric epithelial cells. RESULTS: Low levels of CX3CR1 transcript were often, but not always, expressed in both upper (62%) and lower airways (36%) of pediatric subjects. CX3CR1 transcript and protein expression was detected in epithelial cells of normal human pediatric lung tissues. CX3CR1 expression was readily detected on primary cultures of differentiated pediatric/infant human lung epithelial cells. RSV demonstrated preferential infection of CX3CR1-positive cells, and blocking CX3CR1/RSV interaction significantly decreased viral load. CONCLUSION: CX3CR1 is present in the airways of pediatric subjects where it may serve as a receptor for RSV infection. Furthermore, CX3CR1 appears to play a mechanistic role in mediating viral infection of pediatric airway epithelial cells in vitro.

A novel in vitro model of primary human pediatric lung epithelial cells.

BACKGROUND: Current in vitro human lung epithelial cell models derived from adult tissues may not accurately represent all attributes that define homeostatic and disease mechanisms relevant to the pediatric lung. METHODS: We report methods for growing and differentiating primary Pediatric Human Lung Epithelial (PHLE) cells from organ donor infant lung tissues. We use immunohistochemistry, flow cytometry, quantitative RT-PCR, and single cell RNA sequencing (scRNAseq) analysis to characterize the cellular and transcriptional heterogeneity of PHLE cells. RESULTS: PHLE cells can be expanded in culture up to passage 6, with a doubling time of ~4 days, and retain attributes of highly enriched epithelial cells. PHLE cells can form resistant monolayers, and undergo differentiation when placed at air-liquid interface. When grown at Air-Liquid Interface (ALI), PHLE cells expressed markers of airway epithelial cell lineages. scRNAseq suggests the cultures contained 4 main sub-phenotypes defined by expression of FOXJ1, KRT5, MUC5B, and SFTPB. These cells are available to the research community through the Developing Lung Molecular Atlas Program Human Tissue Core. CONCLUSION: Our data demonstrate that PHLE cells provide a novel in vitro human cell model that represents the pediatric airway epithelium, which can be used to study perinatal developmental and pediatric disease mechanisms.

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues.

Human lung morphogenesis begins by embryonic life and continues after birth into early childhood to form a complex organ with numerous morphologically and functionally distinct cell types. Pulmonary organogenesis involves dynamic changes in cell proliferation, differentiation, and migration of specialized cells derived from diverse embryonic lineages. Studying the molecular and cellular processes underlying formation of the fully functional lung requires isolating distinct pulmonary cell populations during development. We now report novel methods to isolate four major pulmonary cell populations from pediatric human lung simultaneously. Cells were dissociated by protease digestion of neonatal and pediatric lung and isolated on the basis of unique cell membrane protein expression patterns. Epithelial, endothelial, nonendothelial mesenchymal, and immune cells were enriched by fluorescence-activated cell sorting. Dead cells and erythrocytes were excluded by 7-aminoactinomycin D uptake and glycophorin-A (CD235a) expression, respectively. Leukocytes were identified by membrane CD45 (protein tyrosine phosphatase, receptor type C), endothelial cells by platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cadherin (CD144), and both were isolated. Thereafter, epithelial cell adhesion molecule (CD326)-expressing cells were isolated from the endothelial- and immune cell-depleted population to enrich epithelial cells. Cells lacking these membrane markers were collected as "nonendothelial mesenchymal" cells. Quantitative RT-PCR and RNA sequencing analyses of population specific transcriptomes demonstrate the purity of the subpopulations of isolated cells. The method efficiently isolates major human lung cell populations that we announce are now available through the National Heart, Lung, and Blood Institute Lung Molecular Atlas Program (LungMAP) for their further study.

Exome sequencing identifies gene variants and networks associated with extreme respiratory outcomes following preterm birth.

BACKGROUND: Previous studies have identified genetic variants associated with bronchopulmonary dysplasia (BPD) in extremely preterm infants. However, findings with genome-wide significance have been rare, and not replicated. We hypothesized that whole exome sequencing (WES) of premature subjects with extremely divergent phenotypic outcomes could facilitate the identification of genetic variants or gene networks contributing disease risk. RESULTS: The Prematurity and Respiratory Outcomes Program (PROP) recruited a cohort of > 765 extremely preterm infants for the identification of markers of respiratory morbidity. We completed WES on 146 PROP subjects (85 affected, 61 unaffected) representing extreme phenotypes of early respiratory morbidity. We tested for association between disease status and individual common variants, screened for rare variants exclusive to either affected or unaffected subjects, and tested the combined association of variants across gene loci. Pathway analysis was performed and disease-related expression patterns were assessed. Marginal association with BPD was observed for numerous common and rare variants. We identified 345 genes with variants unique to BPD-affected preterm subjects, and 292 genes with variants unique to our unaffected preterm subjects. Of these unique variants, 28 (19 in the affected cohort and 9 in unaffected cohort) replicate a prior WES study of BPD-associated variants. Pathway analysis of sets of variants, informed by disease-related gene expression, implicated protein kinase A, MAPK and Neuregulin/epidermal growth factor receptor signaling. CONCLUSIONS: We identified novel genes and associated pathways that may play an important role in susceptibility/resilience for the development of lung disease in preterm infants.

Engineering models of head and neck and oral cancers on-a-chip.

Head and neck cancers (HNCs) rank as the sixth most common cancer globally and result in over 450 000 deaths annually. Despite considerable advancements in diagnostics and treatment, the 5-year survival rate for most types of HNCs remains below 50%. Poor prognoses are often attributed to tumor heterogeneity, drug resistance, and immunosuppression. These characteristics are difficult to replicate using in vitro or in vivo models, culminating in few effective approaches for early detection and therapeutic drug development. Organs-on-a-chip offer a promising avenue for studying HNCs, serving as microphysiological models that closely recapitulate the complexities of biological tissues within highly controllable microfluidic platforms. Such systems have gained interest as advanced experimental tools to investigate human pathophysiology and assess therapeutic efficacy, providing a deeper understanding of cancer pathophysiology. This review outlines current challenges and opportunities in replicating HNCs within microphysiological systems, focusing on mimicking the soft, glandular, and hard tissues of the head and neck. We further delve into the major applications of organ-on-a-chip models for HNCs, including fundamental research, drug discovery, translational approaches, and personalized medicine. This review emphasizes the integration of organs-on-a-chip into the repertoire of biological model systems available to researchers. This integration enables the exploration of unique aspects of HNCs, thereby accelerating discoveries with the potential to improve outcomes for HNC patients.

Identifying novel radioprotective drugs via salivary gland tissue chip screening.

During head and neck cancer treatment, off-target ionizing radiation damage to the salivary glands commonly causes a permanent loss of secretory function. Due to the resulting decrease in saliva production, patients have trouble eating, speaking and are predisposed to oral infections and tooth decay. While the radioprotective antioxidant drug Amifostine is approved to prevent radiation-induced hyposalivation, it has intolerable side effects that limit its use, motivating the discovery of alternative therapeutics. To address this issue, we previously developed a salivary gland mimetic (SGm) tissue chip platform. Here, we leverage this SGm tissue chip for high-content drug discovery. First, we developed in-chip assays to quantify glutathione and cellular senescence (ß-galactosidase), which are biomarkers of radiation damage, and we validated radioprotection using WR-1065, the active form of Amifostine. Following validation, we tested other reported radioprotective drugs, including, Edaravone, Tempol, N-acetylcysteine (NAC), Rapamycin, Ex-Rad, and Palifermin, confirming that all drugs but NAC and Ex-Rad exhibited robust radioprotection. Next, a Selleck Chemicals library of 438 FDA-approved drugs was screened for radioprotection. We discovered 25 hits, with most of the drugs identified with mechanisms of action other than antioxidant activity. Hits were down-selected using EC 50 values and pharmacokinetics and pharmacodynamics data from the PubChem database leading to testing of Phenylbutazone (anti-inflammatory), Enoxacin (antibiotic), and Doripenem (antibiotic) for in vivo radioprotection in mice using retroductal injections. Results confirm that Phenylbutazone and Enoxacin exhibited equivalent radioprotection to Amifostine. This body of work demonstrates the development and validation of assays using a SGm tissue chip platform for high-content drug screening and the successful in vitro discovery and in vivo validation of novel radioprotective drugs with nonantioxidant primary indications pointing to possible, yet unknown novel mechanisms of radioprotection.

Identifying novel radioprotective drugs via salivary gland tissue chip screening.

During head and neck cancer treatment, off-target ionizing radiation damage to the salivary glands commonly causes a permanent loss of secretory function. Due to the resulting decrease in saliva production, patients have trouble eating, speaking and are predisposed to oral infections and tooth decay. While the radioprotective antioxidant drug Amifostine is FDA approved to prevent radiation-induced hyposalivation, it has intolerable side effects that limit its use, motivating the discovery of alternative therapeutics. To address this issue, we previously developed a salivary gland mimetic (SGm) tissue chip platform. Here, we leverage this SGm tissue chip for high-content drug discovery. First, we developed in-chip assays to quantify glutathione and cellular senescence (ß-galactosidase), which are biomarkers of radiation damage, and we validated radioprotection using WR-1065, the active form of Amifostine. Other reported radioprotective drugs including Edaravone, Tempol, N-acetylcysteine (NAC), Rapamycin, Ex-Rad, and Palifermin were also tested to validate the ability of the assays to detect cell damage and radioprotection. All of the drugs except NAC and Ex-Rad exhibited robust radioprotection. Next, a Selleck Chemicals library of 438 FDA-approved drugs was screened for radioprotection. We discovered 25 hits, with most of the drugs identified exhibiting mechanisms of action other than antioxidant activity. Hits were down-selected using EC50 values and pharmacokinetic and pharmacodynamic data from the PubChem database. This led us to test Phenylbutazone (anti-inflammatory), Enoxacin (antibiotic), and Doripenem (antibiotic) for in vivo radioprotection in mice using retroductal injections. Results confirm that Phenylbutazone and Enoxacin exhibited radioprotection equivalent to Amifostine. This body of work demonstrates the development and validation of assays using a SGm tissue chip platform for high-content drug screening and the successful in vitro discovery and in vivo validation of novel radioprotective drugs with non-antioxidant primary indications pointing to possible, yet unknown novel mechanisms of radioprotection.

Slow hydrogel matrix degradation enhances salivary gland mimetic phenotype.

We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland cells encapsulated in matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) hydrogels within arrays of ∼320 µm diameter spherical cavities molded in PDMS. The SGm provides a functional and physiologically relevant platform well-suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would benefit from improved retention of acinar cell phenotype and function. We hypothesized that tuning biochemical cues presented within the PEG hydrogel matrix would improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed >2-fold increase in sphere number formed at 48 h, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of adhesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm over 14 days of culture. Furthermore, combining this matrix with optimized media conditions synergistically prolonged the retention of the acinar cell phenotype in SGm. STATEMENT OF SIGNIFICANCE: Salivary gland (SG) dysfunction occurs due to off-target radiation due to head and neck cancer treatments. Progress in understanding gland dysfunction and developing therapeutic strategies for the SG are hampered by the lack of in vitro models, as salivary gland cells rapidly lose critical secretory function within 24 hours in vitro. Herein, we identify properties of poly(ethylene glycol) hydrogel matrices that enhance the secretory phenotype of SG tissue mimetics within the previously-described SG-microbubble tissue chip environment. Combining slow-degrading hydrogels with media conditions optimized for secretory marker expression further enhanced functional secretory response and secretory marker expression.

Tissue Engineered Neurovascularization Strategies for Craniofacial Tissue Regeneration.

Craniofacial tissue injuries, diseases, and defects, including those within bone, dental, and periodontal tissues and salivary glands, impact an estimated 1 billion patients globally. Craniofacial tissue dysfunction significantly reduces quality of life, and successful repair of damaged tissues remains a significant challenge. Blood vessels and nerves are colocalized within craniofacial tissues and act synergistically during tissue regeneration. Therefore, the success of craniofacial regenerative approaches is predicated on successful recruitment, regeneration, or integration of both vascularization and innervation. Tissue engineering strategies have been widely used to encourage vascularization and, more recently, to improve innervation through host tissue recruitment or prevascularization/innervation of engineered tissues. However, current scaffold designs and cell or growth factor delivery approaches often fail to synergistically coordinate both vascularization and innervation to orchestrate successful tissue regeneration. Additionally, tissue engineering approaches are typically investigated separately for vascularization and innervation. Since both tissues act in concert to improve craniofacial tissue regeneration outcomes, a revised approach for development of engineered materials is required. This review aims to provide an overview of neurovascularization in craniofacial tissues and strategies to target either process thus far. Finally, key design principles are described for engineering approaches that will support both vascularization and innervation for successful craniofacial tissue regeneration.

Encapsulation of Primary Salivary Gland Acinar Cell Clusters and Intercalated Ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-Degradable Hydrogels to Maintain Tissue Structure and Function.

Progress in the development of salivary gland regenerative strategies is limited by poor maintenance of the secretory function of salivary gland cells (SGCs) in vitro. To reduce the precipitous loss of secretory function, a modified approach to isolate intact acinar cell clusters and intercalated ducts (AIDUCs), rather than commonly used single cell suspension, is investigated. This isolation approach yields AIDUCs that maintain many of the cell-cell and cell-matrix interactions of intact glands. Encapsulation of AIDUCs in matrix metalloproteinase (MMP)-degradable PEG hydrogels promotes self-assembly into salivary gland mimetics (SGm) with acinar-like structure. Expression of Mist1, a transcription factor associated with secretory function, is detectable throughout the in vitro culture period up to 14 days. Immunohistochemistry also confirms expression of acinar cell markers (NKCC1, PIP and AQP5), duct cell markers (K7 and K5), and myoepithelial cell markers (SMA). Robust carbachol and ATP-stimulated calcium flux is observed within the SGm for up to 14 days after encapsulation, indicating that secretory function is maintained. Though some acinar-to-ductal metaplasia is observed within SGm, it is reduced compared to previous reports. In conclusion, cell-cell interactions maintained within AIDUCs together with the hydrogel microenvironment may be a promising platform for salivary gland regenerative strategies.

The critical role of collagen VI in lung development and chronic lung disease.

Type VI collagen (collagen VI) is an obligate extracellular matrix component found mainly in the basement membrane region of many mammalian tissues and organs, including skeletal muscle and throughout the respiratory system. Collagen VI is probably most recognized in medicine as the genetic cause of a spectrum of muscular dystrophies, including Ullrich Congenital Myopathy and Bethlem Myopathy. Collagen VI is thought to contribute to myopathy, at least in part, by mediating muscle fiber integrity by anchoring myoblasts to the muscle basement membrane. Interestingly, collagen VI myopathies present with restrictive respiratory insufficiency, thought to be due primarily to thoracic muscular weakening. Although it was recently recognized as one of the (if not the) most abundant collagens in the mammalian lung, there is a substantive knowledge gap concerning its role in respiratory system development and function. A few studies have suggested that collagen VI insufficiency is associated with airway epithelial cell survival and altered lung function. Our recent work suggested collagen VI may be a genomic risk factor for chronic lung disease in premature infants. Using this as motivation, we thoroughly assessed the role of collagen VI in lung development and in lung epithelial cell biology. Here, we describe the state-of-the-art for collagen VI cell and developmental biology within the respiratory system, and reveal its essential roles in normal developmental processes and airway epithelial cell phenotype and intracellular signaling.

Development of a functional salivary gland tissue chip with potential for high-content drug screening.

Radiation therapy for head and neck cancers causes salivary gland dysfunction leading to permanent xerostomia. Limited progress in the discovery of new therapeutic strategies is attributed to the lack of in vitro models that mimic salivary gland function and allow high-throughput drug screening. We address this limitation by combining engineered extracellular matrices with microbubble (MB) array technology to develop functional tissue mimetics for mouse and human salivary glands. We demonstrate that mouse and human salivary tissues encapsulated within matrix metalloproteinase-degradable poly(ethylene glycol) hydrogels formed in MB arrays are viable, express key salivary gland markers, and exhibit polarized localization of functional proteins. The salivary gland mimetics (SGm) respond to calcium signaling agonists and secrete salivary proteins. SGm were then used to evaluate radiosensitivity and mitigation of radiation damage using a radioprotective compound. Altogether, SGm exhibit phenotypic and functional parameters of salivary glands, and provide an enabling technology for high-content/throughput drug testing.

3D iPSC modeling of the retinal pigment epithelium-choriocapillaris complex identifies factors involved in the pathology of macular degeneration.

The retinal pigment epithelium (RPE)-choriocapillaris (CC) complex in the eye is compromised in age-related macular degeneration (AMD) and related macular dystrophies (MDs), yet in vitro models of RPE-CC complex that enable investigation of AMD/MD pathophysiology are lacking. By incorporating iPSC-derived cells into a hydrogel-based extracellular matrix, we developed a 3D RPE-CC model that recapitulates key features of both healthy and AMD/MD eyes and provides modular control over RPE and CC layers. Using this 3D RPE-CC model, we demonstrated that both RPE- and mesenchyme-secreted factors are necessary for the formation of fenestrated CC-like vasculature. Our data show that choroidal neovascularization (CNV) and CC atrophy occur in the absence of endothelial cell dysfunction and are not necessarily secondary to drusen deposits underneath RPE cells, and CC atrophy and/or CNV can be initiated systemically by patient serum or locally by mutant RPE-secreted factors. Finally, we identify FGF2 and matrix metalloproteinases as potential therapeutic targets for AMD/MDs.

Lymphocyte-Specific Biomarkers Associated With Preterm Birth and Bronchopulmonary Dysplasia.

Many premature babies who are born with neonatal respiratory distress syndrome (RDS) go on to develop Bronchopulmonary Dysplasia (BPD) and later Post-Prematurity Respiratory Disease (PRD) at one year corrected age, characterized by persistent or recurrent lower respiratory tract symptoms frequently related to inflammation and viral infection. Transcriptomic profiles were generated from sorted peripheral blood CD8+ T cells of preterm and full-term infants enrolled with consent in the NHLBI Prematurity and Respiratory Outcomes Program (PROP) at the University of Rochester and the University at Buffalo. We identified outcome-related gene expression patterns following standard methods to identify markers for oxygen utilization and BPD as outcomes in extremely premature infants. We further identified predictor gene sets for BPD based on transcriptomic data adjusted for gestational age at birth (GAB). RNA-Seq analysis was completed for CD8+ T cells from 145 subjects. Among the subjects with highest risk for BPD (born at <29 weeks gestational age (GA); n=72), 501 genes were associated with oxygen utilization. In the same set of subjects, 571 genes were differentially expressed in subjects with a diagnosis of BPD and 105 genes were different in BPD subjects as defined by physiologic challenge. A set of 92 genes could predict BPD with a moderately high degree of accuracy. We consistently observed dysregulation of TGFB, NRF2, HIPPO, and CD40-associated pathways in BPD. Using gene expression data from both premature and full-term subjects (n=116), we identified a 28 gene set that predicted the PRD status with a moderately high level of accuracy, which also were involved in TGFB signaling. Transcriptomic data from sort-purified peripheral blood CD8+ T cells from 145 preterm and full-term infants identified sets of molecular markers of inflammation associated with independent development of BPD in extremely premature infants at high risk for the disease and of PRD among the preterm and full-term subjects.

Type VI collagen promotes lung epithelial cell spreading and wound-closure.

Basement membrane (BM) is an essential part of the extracellular matrix (ECM) that plays a crucial role in mechanical support and signaling to epithelial cells during lung development, homeostasis and repair. Abnormal composition and remodeling of the lung ECM have been associated with developmental abnormalities observed in multiple pediatric and adult respiratory diseases. Collagen VI (COL6) is a well-studied muscle BM component, but its role in the lung and its effect on pulmonary epithelium is largely undetermined. We report the presence of COLVI immediately subjacent to human airway and alveolar epithelium in the pediatric lung, in a location where it is likely to interact with epithelial cells. In vitro, both primary human lung epithelial cells and human lung epithelial cell lines displayed an increased rate of "wound healing" in response to a scratch injury when plated on COL6 as compared to other matrices. For the 16HBE cell line, wounds remained >5-fold larger for cells on COL1 (p<0.001) and >6-fold larger on matrigel (p<0.001), a prototypical basement membrane, when compared to COL6 (>96% closure at 10 hr). The effect of COL6 upon lung epithelial cell phenotype was associated with an increase in cell spreading. Three hours after initial plating, 16HBE cells showed >7-fold less spreading on matrigel (p<0.01), and >4-fold less spreading on COL1 (p<0.01) when compared to COL6. Importantly, the addition of COL6 to other matrices also enhanced cell spreading. Similar responses were observed for primary cells. Inhibitor studies indicated both integrin ß1 activity and activation of multiple signaling pathways was required for enhanced spreading on all matrices, with the PI3K/AKT pathway (PI3K, CDC42, RAC1) showing both significant and specific effects for spreading on COL6. Genetic gain-of-function experiments demonstrated enhanced PI3K/AKT pathway activity was sufficient to confer equivalent cell spreading on other matrices as compared to COL6. We conclude that COL6 has significant and specific effects upon human lung epithelial cell-autonomous functions.

MicroRNA expression profiling defines the impact of electronic cigarettes on human airway epithelial cells.

While all forms of tobacco exposure have negative health effects, the significance of exposure to electronic cigarettes (eCig) is not fully understood. Here, we studied the global effects of eCig on the micro RNA (miRNA) transcriptome in human lung epithelial cells. Primary human bronchial epithelial (NHBE) cells differentiated at air-liquid interface were exposed to eCig liquid. Exposure of NHBE to any eCig liquid resulted in the induction of oxidative stress-response genes including GCLM, GCLC, GPX2, NQO1 and HO-1. Vaporization of, and/or the presence of nicotine in, eCig liquid was associated with a greater response. We identified 578 miRNAs dysregulated by eCig exposure in NHBE, and 125 miRNA affected by vaporization of eCig liquid. Nicotine containing eCig vapor displayed the most profound effects upon miRNA expression. We selected 8 miRNAs (29A, 140, 126, 374A, 26A-2, 147B, 941 and 589) for further study. We validated increased expression of multiple miRNAs, including miR126, following eCig exposure. We also found significant reduction in the expression of two miR126 target genes, MYC and MRGPRX3, following exposure. These data demonstrated that eCig exposure has profound effects upon gene expression in human lung epithelial cells, some of which are epigenetically programmed at the level of miRNA regulation.