Tissue-engineered endothelial and epithelial implants differentially and synergistically regulate airway repair.
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ABSTRACT:
SUBMITTER: Zani BG
PROVIDER: S-EPMC2383974 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAirway remodeling might explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling might regulate HLF ECM expression.ObjectivesWe sought to determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents.MethodsPrimary AECs were obtained from well-characterized atopic asthmatic (n = 10) and healthy (n = 10) children intubated during anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface for 3 weeks and then cocultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1), collagen III (COL3A1), hyaluronan synthase (HAS) 2, and fibronectin expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs were assessed by using RT-PCR. TGF-β1 and TGF-β2 concentrations in media were measured by using ELISA.ResultsCOL1A1 and COL3A1 expression by HLFs cocultured with AECs from asthmatic patients was greater than that by HLFs cocultured with AECs from healthy subjects (2.2-fold, P < .02; 10.8-fold, P < .02). HAS2 expression by HLFs cocultured with AECs from asthmatic patients was 2.5-fold higher than that by HLFs cocultured with AECs from healthy subjects (P < .002). Fibronectin expression by HLFs cocultured with AECs from asthmatic patients was significantly greater than that by HLFs alone. TGF-β2 activity was increased in cocultures of HLFs with AECs from asthmatic patients (P < .05), whereas PGES2 was downregulated in AEC-HLF cocultures (2.2-fold, P < .006).ConclusionsHLFs cocultured with AECs from asthmatic patients showed differential expression of the ECM constituents COL1A1 and COL3A1 and HAS2 compared with HLFs cocultured with AECs from healthy subjects. These findings support a role for altered ECM production in asthmatic airway remodeling, possibly regulated by unbalanced AEC signaling.
| S-EPMC4149938 | biostudies-literature
Project description:Fibrin deposition mediated through activation of tissue factor (TF) in the airspace is central to the pathogenesis of acute lung injury. Defining the mechanisms of TF regulation in the lung is critical to understanding pulmonary fibrin formation. Tumor necrosis factor-? (TNF-?) up-regulates TF in the injured lung, and there is emerging evidence that another cytokine, interferon-? (IFN-?), also modulates expression. The effects of TNF-? and IFN-? on regulation of TF were studied in alveolar epithelial A549 cells. In addition, potential mechanisms of modulation of TF expression by the 2 cytokines were analyzed with the hypothesis that IFN-? acts synergistically with TNF-? to up-regulate alveolar epithelial TF through modulation of TNF receptor (TNFR) expression. TNF-? but not IFN-? treatment increased TF mRNA, protein, and cell surface TF activity. The combination of IFN-? and TNF-? treatment augmented the effects of TNF-? on TF up-regulation and also increased release of procoagulant microparticles (MPs) from A549 cells. IFN-? modulated expression of both TNF-? receptors. Studies utilizing neutralizing antibodies against the two TNF receptors showed that the TF effects were mediated primarily through augmentation of TNFR1-dependent cellular responses. These findings have important implications for regulation of fibrin formation in the lung in the setting of acute inflammation.
| S-EPMC3652424 | biostudies-literature
Project description:Vascular endothelial growth factor A (VEGF) is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration, and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2). We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO) synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell homeostasis in normoxia and hypoxia.
| S-EPMC5030275 | biostudies-literature
Project description:Despite a common understanding that Gli TFs are utilized to convey a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. Using the mouse model (Mus musculus), we demonstrated that rather than being driven by a Hh threshold, robust Gli3 transcriptional activity during skeletal and glossal development required interaction with the basic helix-loop-helix TF Hand2. Not only did genetic and expression data support a co-factorial relationship, but genomic analysis revealed that Gli3 and Hand2 were enriched at regulatory elements for genes essential for mandibular patterning and development. Interestingly, motif analysis at sites co-occupied by Gli3 and Hand2 uncovered mandibular-specific, low-affinity, 'divergent' Gli-binding motifs (dGBMs). Functional validation revealed these dGBMs conveyed synergistic activation of Gli targets essential for mandibular patterning and development. In summary, this work elucidates a novel, sequence-dependent mechanism for Gli transcriptional activity within the craniofacial complex that is independent of a graded Hh signal.
| S-EPMC7556880 | biostudies-literature
Project description:Hantaviruses cause two human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantaviruses infect human endothelial cells but cause little or no damage to the infected endothelium. We analyzed with Affymetrix DNA Arrays (Santa Clara, CA) the endothelial cell transcriptional responses directed by hantaviruses associated with HPS [New York-1 virus (NY-1V)], HFRS [Hantaan virus (HTNV)], or by a hantavirus not associated with human disease [Prospect Hill virus (PHV)]. Hantavirus infections induced 117 cellular genes and repressed 25 genes by >3-fold, 4 days postinfection (p.i.). Although >80% of cells were infected by each virus 1 day p.i., PHV induced or repressed 67 genes at this early time compared with three genes altered by HTNV or NY-1V. The early high-level induction of 24 IFN-stimulated genes by PHV (4- to 229-fold) represents a fundamental difference in the temporal regulation of cellular responses by pathogenic and nonpathogenic hantaviruses. Because all hantaviruses induced >23 IFN-stimulated genes at late times p.i., pathogenic hantaviruses appear to suppress early cellular IFN responses that are activated by nonpathogenic hantaviruses. At late times p.i., 13 genes were commonly induced by HTNV and NY-1V that were not induced by PHV. In contrast to NY-1V, HTNV uniquely induced a variety of chemokines and cell adhesion molecules (i.e., IL-8, IL-6, GRO-beta, ICAM), as well as two complement cascade-associated factors that may contribute to immune components of HFRS disease. NY-1V failed to induce most cellular chemokines directed by HTNV (3/14) or genes primarily activated by NF-kappaB. However, NY-1V uniquely induced beta3 integrin-linked potassium channels, which could play a role in HPS-associated vascular permeability. These studies provide a basic understanding of hantavirus-directed cellular responses that are likely to differentiate pathogenic and nonpathogenic hantaviruses, contribute to HFRS and HPS pathogenesis, and provide insight into disease mechanisms and potential therapeutic interventions.
| S-EPMC129784 | biostudies-literature
Project description:RhoGTPases are known regulators of intracellular actin dynamics that are important for maintaining endothelial barrier function. RhoA is most extensively studied as a key regulator of endothelial barrier function, however the function of the 2 highly homologous family-members (> 88%) RhoB and RhoC in endothelial barrier function is still poorly understood. This study aimed to determine whether RhoA, RhoB and RhoC have overlapping or distinct roles in barrier function and permeability in resting and activated endothelium. By using primary endothelial cells in combination with siRNA transfection to establish individual, double or triple knockdown of the RhoA/B/C RhoGTPases, we found that RhoB, but not RhoA or RhoC, is in resting endothelium a negative regulator of permeability. Loss of RhoB accounted for an accumulation of VE-cadherin at cell-cell contacts. Thrombin-induced loss of endothelial integrity is mediated primarily by RhoA and RhoB. Combined loss of RhoA/B showed decreased phosphorylation of Myosin Light Chain and increased expression of VE-cadherin at cell-cell contacts after thrombin stimulation. RhoC contributes to the Rac1-dependent restoration of endothelial barrier function. In summary, this study shows that these highly homologous RhoGTPases differentially control the dynamics of endothelial barrier function.
| S-EPMC6748378 | biostudies-literature
Project description:BackgroundThe airway epithelium can express factors that drive subepithelial airway remodeling. TGF-?2, vascular epithelial growth factor (VEGF), a disintegrin and metalloprotease 33 (ADAM33), and periostin are hypothesized to be involved in subepithelial remodeling and are overexpressed in adult asthmatic airways. Epidemiologic data suggest that lung function deficits in asthmatic patients are acquired in childhood.ObjectivesWe sought to determine whether airway epithelial cells (AECs) from asthmatic children differentially express TGF-?2, VEGF, ADAM33, or periostin compared with cells from atopic nonasthmatic and healthy children intrinsically or in response to IL-4/IL-13 stimulation.MethodsBronchial and nasal epithelial cells were obtained from brushings from well-characterized asthmatic (n = 16), atopic nonasthmatic (n = 9), and healthy (n = 15) children after achievement of anesthesia for elective procedures. After differentiation at an air-liquid interface (ALI) for 3 weeks, conditioned media were sampled and RNA was extracted from unstimulated and IL-4/IL-13-stimulated cultures. TGF-?2 and VEGF levels were measured with ELISA. ADAM33 and periostin expression was assessed by using real-time PCR.ResultsTGF-?2 and VEGF production was significantly greater in bronchial and nasal ALI cultures from asthmatic children than in cultures from atopic nonasthmatic and healthy children. TGF-?2 levels increased significantly in asthmatic cultures after IL-4/IL-13 stimulation. Within-subject correlation between nasal and bronchial ALI production of TGF-?2 (r = 0.64, P = .001) and VEGF (r = 0.73, P < .001) was good. Periostin expression was 3.7-fold higher in bronchial cells (P < .001) and 3.9-fold higher in nasal cells (P < .004) from asthmatic children than in cells from atopic nonasthmatic or healthy children. ADAM33 was not differentially expressed by AECs from asthmatic patients compared with that from cells from atopic nonasthmatic or healthy children.ConclusionAECs from asthmatic children differentially express TGF-?2, VEGF, and periostin compared with cells from atopic nonasthmatic and healthy children. Nasal epithelial cells might be a suitable surrogate for bronchial cells that could facilitate investigation of the airway epithelium in future longitudinal pediatric studies.
| S-EPMC3604976 | biostudies-literature
Project description:Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.
| S-EPMC3582145 | biostudies-literature
Project description:Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair. 8 samples in total. Parthenogenetic stem cells (PSC): - PSC_1 - PSC_2 - PSC_3 Embryoid body (EB) assays of parthenogenetic stem cells: - EB_PSC_1 - EB_PSC_2 Embryonic stem cells (ESC): - ESC_1 - ESC_2 - ESC_3
2013-02-23 | E-GEOD-30868 | biostudies-arrayexpress
Project description:Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-D-mannoside β1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.
| S-EPMC3207438 | biostudies-literature