Airway contractility in the precision-cut lung slice after cryopreservation.

An emerging tool in airway biology is the precision-cut lung slice (PCLS). Adoption of the PCLS as a model for assessing airway reactivity has been hampered by the limited time window within which tissues remain viable. Here we demonstrate that the PCLS can be frozen, stored long-term, and then thawed for later experimental use. Compared with the never-frozen murine PCLS, the frozen-thawed PCLS shows metabolic activity that is decreased to an extent comparable to that observed in other cryopreserved tissues but shows no differences in cell viability or in airway caliber responses to the contractile agonist methacholine or the relaxing agonist chloroquine. These results indicate that freezing and long-term storage is a feasible solution to the problem of limited viability of the PCLS in culture.

Airway smooth muscle: a potential target for asthma therapy.

PURPOSE OF REVIEW: Asthma is a major public health problem that afflicts nearly one in 20 people worldwide. Despite available treatments, asthma symptoms remain poorly controlled in a significant minority of asthma patients, especially those with severe disease. Accordingly, much ongoing effort has been directed at developing new therapeutic strategies; these efforts are described in detail below. RECENT FINDINGS: Although mucus hypersecretion is an important component of asthma pathobiology, the primary mechanism of morbidity and mortality in asthma is excessive narrowing of the airway. The key end- effector of excessive airway narrowing is airway smooth muscle (ASM) contraction; overcoming ASM contraction is therefore a prominent therapeutic strategy. Here, we review exciting new advances aimed at ASM relaxation. SUMMARY: Exciting advances in ASM biology have identified new therapeutic targets for the prevention or reversal of bronchoconstriction in asthma.

Dilatation of the constricted human airway by tidal expansion of lung parenchyma.

RATIONALE: In the normal lung, breathing and deep inspirations potently antagonize bronchoconstriction, but in the asthmatic lung this salutary effect is substantially attenuated or even reversed. To explain these findings, the prevailing hypothesis focuses on contracting airway smooth muscle and posits a nonlinear dynamic interaction between actomyosin binding and the tethering forces imposed by tidally expanding lung parenchyma. OBJECTIVE: This hypothesis has never been tested directly in bronchial smooth muscle embedded within intraparenchymal airways. Our objective here is to fill that gap. METHODS: We designed a novel system to image contracting intraparenchymal human airways situated within near-normal lung architecture and subjected to dynamic parenchymal expansion that simulates breathing. MEASUREMENTS AND MAIN RESULTS: Reversal of bronchoconstriction depended on the degree to which breathing actually stretched the airway, which in turn depended negatively on severity of constriction and positively on the depth of breathing. Such behavior implies positive feedbacks that engender airway instability. OVERALL CONCLUSIONS: These findings help to explain heterogeneity of airflow obstruction as well as why, in people with asthma, deep inspirations are less effective in reversing bronchoconstriction.

Temporal gene expression analysis of human coronary artery endothelial cells treated with Simvastatin.

Increasing evidence indicates that the beneficial "pleiotropic" effects of statins on clinical events involve nonlipid mechanisms including the modification of blood vessel endothelial cell function. However, the involved molecular events and pathways are not completely understood. In the present study, Affymetrix microarrays were used to monitor the temporal gene expression of human coronary artery endothelial cells (HCAEC) treated with simvastatin (Sim) to gain insight into statins' direct effects on the endothelial function. We isolated and labeled mRNA from HCAEC treated with Sim for 0, 3, 6, 12, 24, and 48 h and hybridized these samples to Affymetrix GeneChip HG-U95Av2 to analyze the temporal gene expression profile. Out of 12,625 genes present on the HG-U95Av2 GeneChip, expression of 5,432 genes was detected. There were 1,475 of 5,432 genes that displayed the differential expression compared to baseline (0 h). Fifty-four genes were upregulated (< or = twofold) while 61 genes were downregulated ( > or = twofold) at 24-48 h after the Sim treatment. Many new target genes and pathways modulated by Sim were uncovered. This study indicates that many aspects of the pleiotropic effect of Sim on the endothelial cell function can be mediated by transcriptional control. Physiological function of 22% of 115 differentially expressed genes in Sim-treated HCAEC are currently unknown. These newly identified genes could be useful for new mechanistic study and new therapeutic modalities. Expressions of 13 out of 18 genes (> 70%) in the cell cycle/proliferation control process were significantly inhibited by the Sim treatment. CDC25B and ITGB4 gene expressions were validated by RT-PCR and Western blotting. Sim's inhibitory effect of on HCAEC growth was confirmed by the measurement of [3H]thymidine incorporation into the DNA synthesis. Further in-depth analysis of this effect may shed light on molecular mechanisms of Sim's beneficial inhibition of neointima formation in the atherosclerotic artery stenosis.

Disrupting actin-myosin-actin connectivity in airway smooth muscle as a treatment for asthma?

Breathing is known to functionally antagonize bronchoconstriction caused by airway muscle contraction. During breathing, tidal lung inflation generates force fluctuations that are transmitted to the contracted airway muscle. In vitro, experimental application of force fluctuations to contracted airway smooth muscle strips causes them to relengthen. Such force fluctuation-induced relengthening (FFIR) likely represents the mechanism by which breathing antagonizes bronchoconstriction. Thus, understanding the mechanisms that regulate FFIR of contracted airway muscle could suggest novel therapeutic interventions to increase FFIR, and so to enhance the beneficial effects of breathing in suppressing bronchoconstriction. Here we propose that the connectivity between actin filaments in contracting airway myocytes is a key determinant of FFIR, and suggest that disrupting actin-myosin-actin connectivity by interfering with actin polymerization or with myosin polymerization merits further evaluation as a potential novel approach for preventing prolonged bronchoconstriction in asthma.

Pluripotent allospecific CD8+ effector T cells traffic to lung in murine obliterative airway disease.

Long-term success in lung transplantation is limited by obliterative bronchiolitis, whereas T cell effector mechanisms in this process remain incompletely understood. Using the mouse heterotopic allogeneic airway transplant model, we studied T cell effector responses during obliterative airways disease (OAD). Allospecific CD8+ IFN-gamma+ T cells were detected in airway allografts, with significant coexpression of TNF-alpha and granzyme B. Therefore, using IFN-gamma as a surrogate marker, we assessed the distribution and kinetics of extragraft allo-specific T cells during OAD. Robust allospecific IFN-gamma was produced by draining the lymph nodes, spleen, and lung mononuclear cells from allograft, but not isograft recipients by Day 14, and significantly decreased by Day 28. Although the majority of allospecific T cells were CD8+, allospecific CD4+ T cells were also detected in these compartments, with each employing distinct allorecognition pathways. An influx of pluripotent CD8+ effector cells with a memory phenotype were detected in the lung during OAD similar to those seen in the allografts and secondary lymphoid tissue. Antibody depletion of CD8+ T cells markedly reduced airway lumen obliteration and fibrosis at Day 28. Together, these data demonstrate that allospecific CD8+ effector T cells play an important role in OAD and traffic to the lung after heterotopic airway transplant, suggesting that the lung is an important immunologic site, and perhaps a reservoir, for effector cells during the rejection process.

Pre-B-cell-colony-enhancing factor is critically involved in thrombin-induced lung endothelial cell barrier dysregulation.

Prior genomic and genetic studies identified pre-B-cell colony-enhancing factor (PBEF) as a novel candidate gene and biomarker in acute lung injury (ALI). As increased vascular permeability is a cardinal feature of ALI, we assessed the role of PBEF in in vitro vascular barrier regulation using confluent human pulmonary artery endothelial cell (HPAEC) monolayers. Reductions in PBEF protein expression (>70%) by siRNA significantly attenuated EC barrier dysfunction induced by the potent edemagenic agent, thrombin, reflected by reductions in transendothelial electric resistance (TER, approximately 60% reduction). Furthermore, PBEF siRNA blunted thrombin-mediated increases in Ca(2+) entry, polymerized actin formation, and myosin light chain phosphorylation, events critical to the thrombin-mediated permeability response. Finally, PBEF siRNA also significantly inhibited thrombin-stimulated increase of IL-8 secretion in HPAEC, a chemokine known to induce actin fiber formation and intercellular gap formation of endothelial cells. Taken together, these studies demonstrate that PBEF may be required for complete expression of the thrombin-induced inflammatory response and reveal potentially novel role for PBEF in the regulation of EC Ca(2+)-dependent cytoskeletal rearrangement and endothelial barrier dysfunction. Ongoing studies will continue to address the molecular mechanisms by which PBEF contributes to ALI susceptibility.