Marisa J. Linn

Neovascularization during venous thrombosis organization: A preliminary study

PURPOSE Thrombus organization after venous thromboembolism leading to recanalization occurs at a variable rate. The angiogenic chemokine interleukin-8 (IL-8) has been found in thrombus months after thrombus initiation. We hypothesize that thrombus organization involves neovascularization and leukocyte influx and that IL-8 administered at thrombus induction will promote thrombus organization. METHODS A group of rats underwent inferior vena caval occlusive thrombosis. At thrombus induction and every 24 hours, the rats were administered IL-8 (1 microgram) or serum albumin. The rats were killed at either day 4, day 8, or day 12, and, at death, colloidal carbon was perfused via the heart. The inferior vena cava was isolated, measured, weighed, and formalin fixed. The sections were stained with anti-polymorphonuclear leukocyte antibody, the endothelial marker factor VIII-related antigen, and with hematoxylin and eosin. Thrombus neovascularization (colloidal carbon) with morphometric analysis was normalized to the total thrombus area. In addition, the rats underwent perfusion with fluorescein isothiocyanate dextran (molecular weight, 150,000) at death to correlate with colloidal carbon perfusion, and thrombus fluorescence was determined. RESULTS Thrombus cellularity initially involved neutrophils, followed by monocytes. Significantly more neutrophils, monocytes, and cells that were defined as spindle shaped (fibroblasts and endothelial cells) were noted in the animals treated with IL-8. Neovascularization was significantly increased at day 4 in the animals treated with IL-8 versus the animals treated with serum albumin and was corroborated with a significant increase in thrombus fluorescein isothiocyanate dextran fluorescence at day 4 in the rats treated with IL-8. Colloidal carbon perfusion was noted within vascular channels without extravasation and colocalized with factor VIII-related antigen. CONCLUSION This study shows that thrombus organization involves neovascularization and that IL-8 augments thrombus organization.

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness.

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.

Rhinovirus Infection of Allergen-Sensitized and -Challenged Mice Induces Eotaxin Release from Functionally Polarized Macrophages

Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus (RV)-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated OVA-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared with sham-infected, OVA-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13, and IFN-γ. Administration of anti–eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemical analysis showed eotaxin-1 in the lung macrophages of virus-infected, OVA-treated mice, and confocal fluorescence microscopy revealed colocalization of rhinovirus, eotaxin-1, and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from OVA-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from OVA-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2, and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from OVA-sensitized and -challenged mice reduced eosinophilic inflammation and airways responsiveness following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.

Elastase- and LPS-exposed mice display altered responses to rhinovirus infection

Viral infection is associated with approximately one-half of acute exacerbations of chronic obstructive pulmonary disease (COPD), which in turn, accelerate disease progression. In this study, we infected mice exposed to a combination of elastase and LPS, a constituent of cigarette smoke and a risk factor for development of COPD, with rhinovirus serotype 1B, and examined animals for viral persistence, airway resistance, lung volume, and cytokine responses. Mice exposed to elastase and LPS once a week for 4 wk showed features of COPD such as airway inflammation and obstruction, goblet cell metaplasia, reduced lung elastance, increased total lung volume, and increased alveolar chord length. In general, mice exposed to elastase or LPS alone showed intermediate effects. Compared with rhinovirus (RV)-infected PBS-exposed mice, RV-infected elastase/LPS-exposed mice showed persistence of viral RNA, airway hyperresponsiveness, increased lung volume, and sustained increases in expression of TNFalpha, IL-5, IL-13, and muc5AC (up to 14 days postinfection). Furthermore, virus-induced IFNs, interferon response factor-7, and IL-10 were deficient in elastase/LPS-treated mice. Mice exposed to LPS or elastase alone cleared virus similar to PBS-treated control mice. We conclude that limited exposure of mice to elastase/LPS produces a COPD-like condition including increased persistence of RV, likely due to skewing of the immune response towards a Th2 phenotype. Similar mechanisms may be operative in COPD.

Airway smooth muscle hyperplasia and hypertrophy correlate with glycogen synthase kinase-3β phosphorylation in a mouse model of asthma

Increased airway smooth muscle (ASM) mass, a characteristic finding in asthma, may be caused by hyperplasia or hypertrophy. Cell growth requires increased translation of contractile apparatus mRNA, which is controlled, in part, by glycogen synthase kinase (GSK)-3beta, a constitutively active kinase that inhibits eukaryotic initiation factor-2 activity and binding of methionyl tRNA to the ribosome. Phosphorylation of GSK-3beta inactivates it, enhancing translation. We sought to quantify the contributions of hyperplasia and hypertrophy to increased ASM mass in ovalbumin (OVA)-sensitized and -challenged BALB/c mice and the role of GSK-3beta in this process. Immunofluorescent probes, confocal microscopy, and stereological methods were used to analyze the number and volume of cells expressing alpha-smooth muscle actin and phospho-Ser(9) GSK-3beta (pGSK). OVA treatment caused a 3-fold increase in ASM fractional unit volume or volume density (Vv) (PBS, 0.006 +/- 0.0003; OVA, 0.014 +/- 0.001), a 1.5-fold increase in ASM number per unit volume (Nv), and a 59% increase in volume per cell (Vv/Nv) (PBS, 824 +/- 76 microm(3); OVA, 1,310 +/- 183 mum(3)). In OVA-treated mice, there was a 12-fold increase in the Vv of pGSK (+) ASM, a 5-fold increase in the Nv of pGSK (+) ASM, and a 1.6-fold increase in Vv/Nv. Lung homogenates from OVA-treated mice showed increased GSK-3beta phosphorylation and lower GSK-3beta activity. Both hyperplasia and hypertrophy are responsible for increased ASM mass in OVA-treated mice. Phosphorylation and inactivation of GSK-3beta are associated with ASM hypertrophy, suggesting that this kinase may play a role in asthmatic airway remodeling.

Longitudinal measures of lung function in infants with bronchopulmonary dysplasia

We previously demonstrated that infants with a history of bronchopulmonary dysplasia (BPD) exhibit airflow obstruction and air trapping. The purpose of this study was to assess longitudinal changes in pulmonary function in infants with a history of BPD over the first 3 years of life, and the relationship to somatic growth. Spirometry was measured using the raised volume rapid thoracoabdominal compression technique, and lung volumes measured by plethysmography. Eighteen infants (mean gestational age ± SD 27.3 ± 2.2 weeks, birthweight 971 ± 259 g) underwent two lung function studies. Average age at first test was 58.8 weeks. Spirometry demonstrated significant reductions in forced expiratory volume in 0.5 sec (FEV0.5, 76.0 ± 15.9% predicted, Z‐score −2.13 ± 1.69), forced expiratory flow at 75% of expired forced vital capacity (FEF75, 54.8 ± 31.1%, −3.58 ± 2.73), and FEF25–75 (67.8 ± 33.3%, −1.79 ± 1.76). Group mean total lung capacity (TLC) was in the low normal range (82.9 ± 13.5% predicted) and residual volume (RV)/TLC was mildly elevated (122.4 ± 38.2% predicted). Repeat testing was performed an average of 32.7 weeks after initial testing. At re‐evaluation, group mean lung volumes and flows tracked at or near their previous values; thus, in general, there was a lack of catch‐up growth. However, compared to infants with below average or average somatic growth (as represented by g/day), infants with above average growth showed significantly greater improvements in percent predicted FVC, FEV0.5, TLC, and RV/TLC (all P < 0.05, ANOVA). We conclude that longitudinal measures of pulmonary function in infants and young children with BPD demonstrate significant airflow obstruction and modest restriction, which tends to persist with time. On the other hand, infants with above average somatic growth showed greater lung growth than their peers. Additional studies examining the effects of various nutritional regimens on lung function are warranted. Pediatr Pulmonol. 2011; 46:369–375.

Glycogen synthase kinase-3β/β-catenin signaling regulates neonatal lung mesenchymal stromal cell myofibroblastic differentiation

In bronchopulmonary dysplasia (BPD), alveolar septa are thickened with collagen and α-smooth muscle actin-, transforming growth factor (TGF)-β-positive myofibroblasts. We examined the biochemical mechanisms underlying myofibroblastic differentiation, focusing on the role of glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. In the cytoplasm, β-catenin is phosphorylated on the NH(2) terminus by constitutively active GSK-3β, favoring its degradation. Upon TGF-β stimulation, GSK-3β is phosphorylated and inactivated, allowing β-catenin to translocate to the nucleus, where it activates transcription of genes involved in myofibroblastic differentiation. We examined the role of β-catenin in TGF-β1-induced myofibroblastic differentiation of neonatal lung mesenchymal stromal cells (MSCs) isolated from tracheal aspirates of premature infants with respiratory distress. TGF-β1 increased β-catenin expression and nuclear translocation. Transduction of cells with GSK-3β S9A, a nonphosphorylatable, constitutively active mutant that favors β-catenin degradation, blocked TGF-β1-induced myofibroblastic differentiation. Furthermore, transduction of MSCs with ΔN-catenin, a truncation mutant that cannot be phosphorylated on the NH(2) terminus by GSK-3β and is not degraded, was sufficient for myofibroblastic differentiation. In vivo, hyperoxic exposure of neonatal mice increases expression of β-catenin in α-smooth muscle actin-positive myofibroblasts. Similar changes were found in lungs of infants with BPD. Finally, low-passage unstimulated MSCs from infants developing BPD showed higher phospho-GSK-3β, β-catenin, and α-actin content compared with MSCs from infants not developing this disease, and phospho-GSK-3β and β-catenin each correlated with α-actin content. We conclude that phospho-GSK-3β/β-catenin signaling regulates α-smooth muscle actin expression, a marker of myofibroblast differentiation, in vitro and in vivo. This pathway appears to be activated in lung mesenchymal cells from patients with BPD.

Reduced platelet-derived growth factor receptor expression is a primary feature of human bronchopulmonary dysplasia

Animal studies have shown that platelet-derived growth factor (PDGF) signaling is required for normal alveolarization. Changes in PDGF receptor (PDGFR) expression in infants with bronchopulmonary dysplasia (BPD), a disease of hypoalveolarization, have not been examined. We hypothesized that PDGFR expression is reduced in neonatal lung mesenchymal stromal cells (MSCs) from infants who develop BPD. MSCs from tracheal aspirates of premature infants requiring mechanical ventilation in the first week of life were studied. MSC migration was assessed in a Boyden chamber. Human lung tissue was obtained from the University of Rochester Neonatal Lung Biorepository. Neonatal mice were exposed to air or 75% oxygen for 14 days. PDGFR expression was quantified by qPCR, immunoblotting, and stereology. MSCs were isolated from 25 neonates (mean gestational age 27.7 wk); 13 developed BPD and 12 did not. MSCs from infants who develop BPD showed lower PDGFR-α and PDGFR-β mRNA and protein expression and decreased migration to PDGF isoforms. Lungs from infants dying with BPD show thickened alveolar walls and paucity of PDGFR-α-positive cells in the dysmorphic alveolar septa. Similarly, lungs from hyperoxia-exposed neonatal mice showed lower expression of PDGFR-α and PDGFR-β, with significant reductions in the volume of PDGFR-α-positive alveolar tips. In conclusion, MSCs from infants who develop BPD hold stable alterations in PDGFR gene expression that favor hypoalveolarization. These data demonstrate that defective PDGFR signaling is a primary feature of human BPD.

Neonatal Rhinovirus Infection Induces Mucous Metaplasia and Airways Hyperresponsiveness

Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13–driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 d postinfection. Within this time frame, IFN-α, -β, and -γ peaked 1 d postinfection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 d after initial infection. Compared with sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335+, TCR-β+ cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B, and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production, or airways responsiveness 28 d postinfection. Intraperitoneal administration of anti–IL-13 neutralizing Ab attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia, and hyperresponsiveness, which are mediated, at least in part, by IL-13.

Mesenchymal Stromal Cells from Neonatal Tracheal Aspirates Demonstrate a Pattern of Lung-Specific Gene Expression

We have previously isolated mesenchymal stromal cells (MSCs) from the tracheal aspirates of premature neonates with respiratory distress. Although isolation of MSCs correlates with the development of bronchopulmonary dysplasia, the physiologic role of these cells remains unclear. To address this, we further characterized the cells, focusing on the issues of gene expression, origin, and cytokine expression. Microarray comparison of early passage neonatal lung MSC gene expression to cord blood MSCs and human fetal and neonatal lung fibroblast lines demonstrated that the neonatal lung MSCs differentially expressed 971 gene probes compared with cord blood MSCs, including the transcription factors Tbx2, Tbx3, Wnt5a, FoxF1, and Gli2, each of which has been associated with lung development. Compared with lung fibroblasts, 710 gene probe transcripts were differentially expressed by the lung MSCs, including IL-6 and IL-8/CXCL8. Differential chemokine expression was confirmed by protein analysis. Further, neonatal lung MSCs exhibited a pattern of Hox gene expression distinct from cord blood MSCs but similar to human fetal lung fibroblasts, consistent with a lung origin. On the other hand, limiting dilution analysis showed that fetal lung fibroblasts form colonies at a significantly lower rate than MSCs, and fibroblasts failed to undergo differentiation along adipogenic, osteogenic, and chondrogenic lineages. In conclusion, MSCs isolated from neonatal tracheal aspirates demonstrate a pattern of lung-specific gene expression, are distinct from lung fibroblasts, and secrete pro-inflammatory cytokines.