High-throughput screening for modulators of cellular contractile force.

When cellular contractile forces are central to pathophysiology, these forces comprise a logical target of therapy. Nevertheless, existing high-throughput screens are limited to upstream signalling intermediates with poorly defined relationships to such a physiological endpoint. Using cellular force as the target, here we report a new screening technology and demonstrate its applications using human airway smooth muscle cells in the context of asthma and Schlemm's canal endothelial cells in the context of glaucoma. This approach identified several drug candidates for both asthma and glaucoma. We attained rates of 1000 compounds per screening day, thus establishing a force-based cellular platform for high-throughput drug discovery.

Stiffness-activated GEF-H1 expression exacerbates LPS-induced lung inflammation.

Acute lung injury (ALI) is accompanied by decreased lung compliance. However, a role of tissue mechanics in modulation of inflammation remains unclear. We hypothesized that bacterial lipopolysacharide (LPS) stimulates extracellular matrix (ECM) production and vascular stiffening leading to stiffness-dependent exacerbation of endothelial cell (EC) inflammatory activation and lung barrier dysfunction. Expression of GEF-H1, ICAM-1, VCAM-1, ECM proteins fibronectin and collagen, lysyl oxidase (LOX) activity, interleukin-8 and activation of Rho signaling were analyzed in lung samples and pulmonary EC grown on soft (1.5 or 2.8 kPa) and stiff (40 kPa) substrates. LPS induced EC inflammatory activation accompanied by expression of ECM proteins, increase in LOX activity, and activation of Rho signaling. These effects were augmented in EC grown on stiff substrate. Stiffness-dependent enhancement of inflammation was associated with increased expression of Rho activator, GEF-H1. Inhibition of ECM crosslinking and stiffening by LOX suppression reduced EC inflammatory activation and GEF-H1 expression in response to LPS. In vivo, LOX inhibition attenuated LPS-induced expression of GEF-H1 and lung dysfunction. These findings present a novel mechanism of stiffness-dependent exacerbation of vascular inflammation and escalation of ALI via stimulation of GEF-H1-Rho pathway. This pathway represents a fundamental mechanism of positive feedback regulation of inflammation.

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.

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.

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.

Microarray analysis of regional cellular responses to local mechanical stress in acute lung injury.

Human acute lung injury is characterized by heterogeneous tissue involvement, leading to the potential for extremes of mechanical stress and tissue injury when mechanical ventilation, required to support critically ill patients, is employed. Our goal was to establish whether regional cellular responses to these disparate local mechanical conditions could be determined as a novel approach toward understanding the mechanism of development of ventilator-associated lung injury. We utilized cross-species genomic microarrays in a unilateral model of ventilator-associated lung injury in anesthetized dogs to assess regional cellular responses to local mechanical conditions that potentially contribute pathogenic mechanisms of injury. Highly significant regional differences in gene expression were observed between lung apex/base regions as well as between gravitationally dependent/nondependent regions of the base, with 367 and 1,544 genes differentially regulated between these regions, respectively. Major functional groupings of differentially regulated genes included inflammation and immune responses, cell proliferation, adhesion, signaling, and apoptosis. Expression of genes encoding both acute lung injury-associated inflammatory cytokines and protective acute response genes were markedly different in the nondependent compared with the dependent regions of the lung base. We conclude that there are significant differences in the local responses to stress within the lung, and consequently, insights into the cellular responses that contribute to ventilator-associated lung injury development must be sought in the context of the mechanical heterogeneity that characterizes this syndrome.

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.

Molecular cloning and characterization of canine pre-B-cell colony-enhancing factor.

During our previous attempt to search for the candidate genes to acute lung injury (ALI), we unexpectedly identified PBEF as the most highly upregulated gene in a canine model of ALI by crosshybridizing canine lung cRNA to the Affymetrix human gene chip HG-U133A. The result suggested that PBEF may be a potential biomarker in ALI. To extend and translate that finding, we have performed the molecular cloning and characterization of canine PBEF cDNA in this study. Deduced amino acid sequence alignment revealed that the PBEF gene is evolutionarily highly conserved, with the canine PBEF protein sequence 96% identical to human PBEF and 94% identical to both murine and rat PBEF counterparts. Canine PBEF protein was successfully expressed both by in vitro transcription coupled with translation in a cell-free system and by transfection of canine PBEF cDNA into the human lung type II alveolar adenocarcinoma cell line A549. The expressed canine PBEF protein was visualized by either an anti-V5 tag peptide polyclonal antibody or an anti-canine PBEF peptide polyclonal antibody. RT-PCR assay indicates that canine PBEF is expressed in canine lung, brain, heart, liver, spleen, kidney, pancreas, and muscle, with liver showing the highest expression,followed by muscle. Isolation of the canine PBEF cDNA and expression of its recombinant protein may provide molecular tools to study the molecular mechanism of ALI in the canine model and to elucidate the potential role of PBEF as an ALI biomarker.

Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1.

Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding angiogenic growth factors, which are secreted by hypoxic cells and stimulate endothelial cells, leading to angiogenesis. To determine whether HIF-1 also mediates cell-autonomous responses to hypoxia, we have compared gene expression profiles in arterial endothelial cells cultured under nonhypoxic versus hypoxic conditions and in nonhypoxic cells infected with adenovirus encoding beta-galactosidase versus a constitutively active form of HIF-1alpha (AdCA5). There were 245 gene probes that showed at least 1.5-fold increase in expression in response to hypoxia and in response to AdCA5; 325 gene probes showed at least 1.5-fold decrease in expression in response to hypoxia and in response to AdCA5. The largest category of genes down-regulated by both hypoxia and AdCA5 encoded proteins involved in cell growth/proliferation. Many genes up-regulated by both hypoxia and AdCA5 encoded cytokines/growth factors, receptors, and other signaling proteins. Transcription factors accounted for the largest group of HIF-1-regulated genes, indicating that HIF-1 controls a network of transcriptional responses to hypoxia in endothelial cells. Infection of endothelial cells with AdCA5 under nonhypoxic conditions was sufficient to induce increased basement membrane invasion and tube formation similar to the responses induced by hypoxia, indicating that HIF-1 mediates cell-autonomous activation of endothelial cells.

Deficiency of different nitric oxide synthase isoforms activates divergent transcriptional programs in cardiac hypertrophy.

Decreased nitric oxide synthase (NOS) activity induces left ventricular hypertrophy (LVH), but the transcriptional pathways mediating this effect are unknown. We hypothesized that specific NOS isoform deletion (NOS3 or NOS1) would activate different transcriptional programs in LVH. We analyzed cardiac expression profiles (Affymetrix MG-U74A) from NOS-/- mice using robust multi-array average (RMA). Of 12,422 genes analyzed, 47 genes were differentially expressed in NOS3-/- and 67 in NOS1(-/-) hearts compared with wild type (WT). Only 16 showed similar changes in both NOS-/- strains, most notably decreased heat-shock proteins (HSP10, 40, 70, 86, 105). Hypertrophied NOS1-/- hearts had unique features, including decreased myocyte-enriched calcineurin interacting protein and paradoxical downregulation of fetal isoforms (alpha-skeletal actin and brain natriuretic peptide). Cluster analyses demonstrated that NOS1 deletion caused more pronounced changes in the myocardial transcriptome than did NOS3 deletion, despite similar cardiac phenotypes. These findings suggest that the transcriptional basis for LVH varies depending on the inciting biochemical stimulus. In addition, NOS isoforms appear to play distinct roles in modulating cardiac structure.

An improved RSP method to detect HpaI polymorphism in the apolipoprotein C-1 gene promoter.

BACKGROUND: An apolipoprotein C1 gene promoter polymorphism (CGTT insertion at position -317) is associated with familial dysbetalipoprotemia, cardiovascular diseases, and Alzheimer's disease. Restriction site polymorphism (RSP) assays were previously established to detect this polymorphism. In this study, we introduce an improved RSP assay to detect this polymorphism. METHODS: This method included newly designed primers and only one round of PCR amplification which yields one short and specific APOC1 fragment followed by HpaI digestion. Briefly, It consists of three steps: 1) one round of PCR amplification of DNA sample, 2) HpaI enzyme digestion of PCR products, and 3) electrophoresis on an agarose gel to visualize the genotypes. This improved RSP method was applied to genotype 92 human samples collected from The Johns Hopkins Hospital. RESULTS: The observed allele frequencies for H1 and H2 from 92 genotyped human subjects were 0.707 and 0.293 respectively. The H2 allele frequency in the black subjects (0.350) was significantly (p = 0.024) higher than that in the white subjects (0.177). This method was more economical and convenient than the methods previously reported to detect this mutation in the APOC1 gene. CONCLUSIONS: This assay will be readily applied to screen large sample sizes for population studies in a simple and cost effective way.

Microarray, SAGE and their applications to cardiovascular diseases.

The wealth of DNA data generated by the human genome project coupling with recently invented high-throughput gene expression profiling techniques has dramatically sped up the process for biomedical researchers on elucidating the role of genes in human diseases. One powerful method to reveal insight into gene functions is the systematic analysis of gene expression. Two popular high-throughput gene expression technologies, microarray and Serial Analysis of Gene Expression (SAGE) are capable of producing large amounts of gene expression data with the potential of providing novel insights into fundamental disease processes, especially complex syndromes such as cardiovascular disease, whose etiologies are due to multiple genetic factors and their interplay with the environment. Microarray and SAGE have already been used to examine gene expression patterns of cell-culture, animal and human tissues models of cardiovascular diseases. In this review, we will first give a brief introduction of microarray and SAGE technologies and point out their limitations. We will then discuss the major discoveries and the new biological insights that have emerged from their applications to cardiovascular diseases. Finally we will touch upon potential challenges and future developments in this area.