Genomic regions with replicated linkage to asthma-related phenotypes likely harbor multiple susceptibility loci with relatively minor effects on disease susceptibility. The 11q13 chromosomal region has repeatedly been linked to asthma with five genes residing in this region with reported replicated associations. Cortactin, an actin-binding protein encoded by the CTTN gene in 11q13, constitutes a key regulator of cytoskeletal dynamics and contractile cell machinery, events facilitated by interaction with myosin light chain kinase; encoded by MYLK, a gene we recently reported as associated with severe asthma in African Americans. To evaluate potential association of CTTN gene variation with asthma susceptibility, CTTN exons and flanking regions were re-sequenced in 48 non-asthmatic multiethnic samples, leading to selection of nine tagging polymorphisms for case-control association studies in individuals of European and African descent. After ancestry adjustments, an intronic variant (rs3802780) was significantly associated with severe asthma (odds ratio [OR]: 1.71; 95% confidence interval [CI]: 1.20-2.43; p = 0.003) in a joint analysis. Further analyses evidenced independent and additive effects of CTTN and MYLK risk variants for severe asthma susceptibility in African Americans (accumulated OR: 2.93, 95% CI: 1.40-6.13, p = 0.004). These data suggest that CTTN gene variation may contribute to severe asthma and that the combined effects of CTTN and MYLK risk polymorphisms may further increase susceptibility to severe asthma in African Americans harboring both genetic variants.
CTTN; MLCK; cytoskeleton; SNP; asthma
Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane rafts formation (via methyl-β-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (13M, 5 min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting >3-fold change. S1P-induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85α phosphatidylinositol 3-kinase (p85αPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.
endothelial cells; cell membrane rafts; 2-DE; Mass spectrometry; sphingosine 1-phosphate (S1P)
Acute lung injury represents the result of multiple pathways initiated by local or systemic insults and is characterized by profound vascular permeability, pulmonary edema, and life-threatening respiratory failure. Permeability-reducing therapies are of potential clinical utility but are currently unavailable. We hypothesized that polyethylene glycol (PEG) compounds, inert and non-toxic polymers that serve as a surrogate mucin lining in intestinal epithelium, may attenuate agonist-mediated lung endothelial cell (EC) barrier dysfunction. High molecular weight PEG (PEG15-20) produced rapid, dose-dependent increases in transendothelial electrical resistance (TER) in human lung endothelium cultured on gold microelectrodes, reflecting increased paracellular integrity. The maximal effective concentration of 8% PEG induced a sustained 125% increase in TER (40 hrs), results similar to barrier-enhancing agonists such as sphingosine 1-phosphate (40% increase in TER). Maximal PEG barrier enhancement was achieved at 45–60 min and PEG effectively reversed both thrombin- and LPS-induced EC barrier dysfunction. Consistent with the increase in TER, immunofluorescent studies demonstrated that PEG produced significant cytoskeletal rearrangement with formation of well-defined cortical actin rings and lamellipodia containing the actin-binding proteins, cortactin and MLCK, known participants in cell-matrix and cell-cell junctional adhesion. Finally, PEG challenge induced rapid alterations in levels of MAP kinase and MLC phosphorylation. In summary, PEG joins a number of EC barrier-regulatory agents which rapidly activate barrier-enhancing signal transduction pathways which target the cytoskeleton and provides a potential therapeutic strategy in inflammatory lung injury.
PEG; LPS; thrombin; endothelium; barrier function
Acute lung injury (ALI) results from loss of alveolar-capillary barrier integrity and the evolution of high-permeability pulmonary edema resulting in alveolar flooding and significant morbidity and mortality. HMGB1 is a late mediator of sepsis which uniquely participates in the evolution of sepsis and sepsis-induced ALI. The molecular events by which HMGB1 contributes to ALI remain poorly characterized. We characterized the role of HMGB1 in endothelial cell (EC) cytoskeletal rearrangement and vascular permeability, events essential to paracellular gap formation and barrier dysfunction characteristic of ALI. Initial experiments demonstrated HMGB1-mediated dose-dependent (5–20 μg/ml) decreases in transendothelial cell electrical resistance (TER) in human pulmonary artery EC, a reflection of loss of barrier integrity. Furthermore, HMGB1 produced dose-dependent increases in paracellular gap formation in concert with loss of peripheral organized actin fibers, dissociation of cell-cell junctional cadherins, and development of central stress fibers, a phenotypic change associated with increased contractile activity and increased EC permeability. Using siRNA strategies directed against known HMGB1 receptors (RAGE, TLR2, TLR4), we systematically determined that the receptor for advanced glycation end products (RAGE) is the primary receptor signaling HMGB1-induced TER decreases and paracellular gap formation via p38 MAP kinase activation and phosphorylation of the actin-binding protein, Hsp27. These studies add to understanding of HMGB1-induced inflammatory events and vascular barrier disruption and offer the potential for clinical intervention in sepsis-induced ALI.
HMGB1; RAGE; acute lung injury; endothelium; MAP kinase; Hsp27
The integration of molecular, genomic, and clinical medicine in the post-genome era provides the promise of novel information on genetic variation and pathophysiologic cascades. The current challenge is to translate these discoveries rapidly into viable biomarkers that identify susceptible populations and into the development of precisely targeted therapies. In this article, we describe the application of comparative genomics, microarray platforms, genetic epidemiology, statistical genetics, and bioinformatic approaches within examples of complex pulmonary pathobiology. Our search for candidate genes, which are gene variations that drive susceptibility to and severity of enigmatic acute and chronic lung disorders, provides a logical framework to understand better the evolution of genomic medicine. The dissection of the genetic basis of complex diseases and the development of highly individualized therapies remain lofty but achievable goals.
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are complex illnesses involving the interplay of both environmental (such as mechanical ventilation) and genetic factors. To understand better the underlying mechanisms of pathogenesis associated with ALI, we recently identified several candidate genes by global expression profiling in preclinical models of ALI and relevant single-nucleotide polymorphisms. We summarize here several strategies successfully used to identify novel ALI candidate genes and detail the validation of variants in these genes as contributing factors to ALI pathobiology, conclusions based on functional analyses, and specific genetic association studies conducted in ALI cohorts. Continued insights into ALI pathogenesis and identification of genetic variants, which confer ALI risk and severity, promise to reveal novel molecular therapeutic targets that can be translated into personalized treatments to reduce the very high, unacceptable mortality of this disorder.
acute lung injury; SNP; microarray; PBEF; inflammation
Acute lung injury (ALI) is a common and frequently devastating illness characterized by acute hypoxemic respiratory failure, profound inflammation, and flooding of the alveoli. Despite recent advances in ALI care, the morbidity and mortality of ALI continues to be unacceptably high. ALI-inciting events (e.g., sepsis, trauma, aspiration, pneumonia) are quite common, yet only a fraction of patients develop the syndrome. This heterogeneity of patients presenting with ALI has sparked interest in identifying the role of genetic factors that contribute to ALI susceptibility and prognosis. Recent advances in high-throughput sequencing and expression technologies now provide the tools to perform large-scale genomic analyses in complex disorders such as ALI; gene expression profiling and pathway analysis provide further insight into previously described molecular pathways involved in the syndrome. In this article, we describe the use of genomewide association studies, ortholog in silico techniques, utility of consomic rat methods, and candidate gene approaches using expression profiling and pathway analyses. These methods have confirmed suspected ALI candidate genes (e.g., IL-6 and MIF), but more impressively have identified novel genes (e.g., GADD45α and PBEF) not previously suspected in ALI. The analysis of the molecular pathways (e.g., the cytoskeleton in vascular barrier regulation) has identified additional genes contributing to the development and severity of ALI (e.g., MLCK), thereby providing therapeutic targets in this devastating illness.
acute lung injury; genetics; ventilator-induced lung injury
The multifunctional non-muscle isoform of myosin light chain kinase (nmMLCK) is critical to the rapid dynamic coordination of the cytoskeleton involved in cancer cell proliferation and migration. We identified 45 nmMLCK-influenced genes by bioinformatic filtering of genome–wide expression in wild type and nmMLCK knockout (KO) mice exposed to preclinical models of murine acute inflammatory lung injury, pathologies that are well established to include nmMLCK as an essential participant. To determine whether these nmMLCK-influenced genes were relevant to human cancers, the 45 mouse genes were matched to 38 distinct human orthologs (M38 signature) (GeneCards definition) and underwent Kaplan-Meier survival analysis in training and validation cohorts. These studies revealed that in training cohorts, the M38 signature successfully identified cancer patients with poor overall survival in breast cancer (P<0.001), colon cancer (P<0.001), glioma (P<0.001), and lung cancer (P<0.001). In validation cohorts, the M38 signature demonstrated significantly reduced overall survival for high-score patients of breast cancer (P = 0.002), colon cancer (P = 0.035), glioma (P = 0.023), and lung cancer (P = 0.023). The association between M38 risk score and overall survival was confirmed by univariate Cox proportional hazard analysis of overall survival in the both training and validation cohorts. This study, providing a novel prognostic cancer gene signature derived from a murine model of nmMLCK-associated lung inflammation, strongly supports nmMLCK-involved pathways in tumor growth and progression in human cancers and nmMLCK as an attractive candidate molecular target in both inflammatory and neoplastic processes.
Environmental nontuberculous mycobacteria (NTM) are ubiquitous organisms with which humans commonly interact. The epidemiologic characteristics of NTM diseases including mortality rate and its associated factors remain largely unknown. In this study, we explored the geographical area of exposure and mortality and comorbid conditions of affected persons to determine environment, host, and host-pathogen interactive factors.
We analyzed mortality related to nontuberculous mycobacterial infections from 1999 through 2010 by examining multiple-cause-of-death data from the National Center for Health Statistics. Among those who died with these diseases, we analyzed age-adjusted mortality rates, trends, associations with demographic variables, and comorbid conditions and correlated this information with similar data for tuberculosis-related mortality during the same time.
Measurements and Mean Results
From 1999 through 2010, nontuberculous mycobacterial disease was reported as an immediate cause of death in 2,990 people in the United States with a combined overall mean age-adjusted mortality rate of 0.1 per 100,000 person-years. A significant increase in the number of NTM related deaths was seen from 1999 through 2010 (R2 = 0.72, p<0.0001), but it was not significant after adjustment for age. Persons aged 55 years and older, women, those living in Hawaii and Louisiana, and those of non-Hispanic, white ethnicity had higher mortality rates. Compared to tuberculosis-related mortality, chronic obstructive pulmonary disease, bronchiectasis, HIV, interstitial lung diseases, and tobacco use were significantly more common in persons with nontuberculous mycobacteria-related deaths.
Nontuberculous mycobacteria-related death numbers are rising and are unevenly distributed. The strong association of nontuberculous mycobacterial disease with age suggests that its prevalence will increase as the United States population ages.
Lung transplantation remains the only viable treatment option for the majority of patients with advanced lung diseases. However, 5-year post-transplant survival rates remain low primarily secondary to chronic rejection. Novel insights from global gene expression profiles may provide molecular phenotypes and therapeutic targets to improve outcomes after lung transplantation.
Whole-genome gene expression profiling was performed in a cohort of patients that underwent lung transplantation as well as healthy controls using the Affymetrix Human Exon 1.0ST Array. To explore the potential roles of microRNAs (miRNAs) in regulating lung transplantation-associated gene dysregulation, miRNA expression levels were also profiled in the same samples using the Exiqon miRCURY™ LNA Array.
In a cohort of 18 lung transplant patients, 364 dysregulated genes were identified in Caucasian lung transplant patients relative to normal individuals. Pathway enrichment analysis of the dysregulated genes pointed to Gene Ontology biological processes such as “defense response”, “immune response” and “response to wounding”. We then compared the expression profiles of potential regulating miRNAs, suggesting that dysregulation of a number of lung transplantation-associated genes (e.g., ATR, FUT8, LRRC8B, NFKBIA) may be attributed to the dysregulation of their respective regulating miRNAs.
Following human lung transplantation, a substantial proportion of genes, particularly those genes involved in certain biological processes like immune response, were dysregulated in patients relative to their healthy counterparts. This exploratory analysis of the relationships between miRNAs and their gene targets in the context of lung transplantation warrants further investigation and may serve as novel therapeutic targets in lung transplant complications.
lung transplant; gene expression; microRNA; pathway; gene ontology
Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity.
Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry.
Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible.
We observed that Cn is involved in the recovery from thrombin-induced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation.
endothelium; barrier function; isoforms of catalytic subunit of calcineurin; stress fibers; immunofluorescence
Exposure to particulate air pollution is associated with increased cardiopulmonary morbidity and mortality, although the pathogenic mechanisms are poorly understood. We previously demonstrated that particulate matter (PM) exposure triggers massive oxidative stress in vascular endothelial cells (ECs), resulting in the loss of EC integrity and lung vascular hyperpermeability. We investigated the protective role of hydrogen sulfide (H2S), an endogenous gaseous molecule present in the circulation, on PM-induced human lung EC barrier disruption and pulmonary inflammation. Alterations in EC monolayer permeability, as reflected by transendothelial electrical resistance (TER), the generation of reactive oxygen species (ROS), and murine pulmonary inflammatory responses, were studied after exposures to PM and NaSH, an H2S donor. Similar to N-acetyl cysteine (5 mM), NaSH (10 μM) significantly scavenged PM-induced EC ROS and inhibited the oxidative activation of p38 mitogen-activated protein kinase. Concurrent with these events, NaSH (10 μM) activated Akt, which helps maintain endothelial integrity. Both of these pathways contribute to the protective effect of H2S against PM-induced endothelial barrier dysfunction. Furthermore, NaSH (20 mg/kg) reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in a murine model of PM-induced lung inflammation. These data suggest a potentially protective role for H2S in PM-induced inflammatory lung injury and vascular hyperpermeability.
particulate matter; hydrogen sulfide; endothelial permeability; Akt
Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAQ™) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. Our results demonstrated that S1P mediates rapid and specific recruitment (1 µm, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S1P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S1P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.
Strategies to improve pulmonary endothelial barrier function are needed to reverse the devastating effects of vascular leak in acute respiratory distress syndrome (ARDS). FTY720 is a pharmaceutical analogue of the potent barrier-enhancing phospholipid, sphingosine 1-phosphate (S1P). FTY720 decreases vascular permeability through an incompletely characterized mechanism that differs from S1P. Here we describe its barrier-promoting effects on intracellular signaling and junctional assembly formation in human pulmonary endothelium.
Permeability of cultured human pulmonary endothelial cells was assessed by transendothelial electrical resistance (TER) and dextran transwell assays. Junctional complex formation was assessed by membrane fractionation and immunofluorescence. Pharmacologic inhibitors and siRNA were utilized to determine the effects of individual components on permeability.
Unlike S1P, FTY720 failed to induce membrane translocation of adherens junction or tight junction proteins. β-catenin, occludin, claudin-5, or ZO-1/ZO-2 siRNAs did not alter FTY720-induced barrier enhancement. FTY720 induced FAK phosphorylation and focal adhesion formation with FAK siRNA partially attenuating the prolonged phase of barrier enhancement. Inhibition of Src, PKA, PKG, PKC, or PP2A failed to alter FTY720-induced barrier enhancement. FTY720 increased c-Abl tyrosine kinase activity, and c-Abl siRNA attenuated peak barrier enhancement after FTY720.
FTY720 enhances endothelial barrier function by a novel pathway involving c-Abl signaling.
c-Abl tyrosine kinase; vascular endothelium; FTY720; junctional complexes; vascular permeability
Pulmonary hypertension (PH) is a disorder characterized by vascular remodeling and proliferation, a phenotype dependent upon unimpeded growth factor and kinase pathway activation with strong similarities to malignant tumors. This chapter details our novel application of the multikinase inhibitor, sorafenib, in rodent models of PH to improved hemodynamic parameters and attenuates PH structural changes1. Sorafenib is a Raf kinase inhibitor and our biochemical and genomic evidence supported the potential involvement of the MAPK cascade system and TGFB3 in PH development and the response to therapy. Integration of expression genomic analyses coupled with intense bioinformatics identified gene expression and ontology signatures in the development of PH and implicated the role of cytoskeletal protein such as caldesmon or nmMLCK as potentially key participants in PH-induced vascular remodeling and proliferation. Our studies suggest the PKI sorafenib as a potentially novel treatment for severe PH with the MAPK cascade a potential canonical target profoundly effecting vascular cytoskeletal rearrangements and remodeling1.
Vascular remodeling; sorafenib; endothelium; cytoskeleton; caldesmon
Previous studies in our lab have identified Pre-B-cell colony enhancing factor as a novel biomarker in acute lung injury. This study continues to elucidate the underlying molecular mechanism of Pre-B-cell colony enhancing factor (PBEF) in the pathogenesis of acute lung injury in pulmonary cell culture models. Our results revealed that IL-1β induced PBEF expression in pulmonary vascular endothelial cells at the transcriptional level and a -1535 T-variant in the human PBEF gene promoter significantly attenuated its binding to an IL-1β induced unknown transcription factor. This may underlie the reduced expression of PBEF and thus the less susceptibility to acute lung injury in those -1535T carriers. Furthermore, overexpression of PBEF significantly augmented IL-8 secretion and mRNA expression by more than 6 fold and 2 fold in A549 cells and HPAEC, respectively. It also significantly augmented IL-1β mediated cell permeability by 44% in A549 cells and 65% in endothelial cells. The knockdown of PBEF expression significantly inhibited IL-1β-stimulated IL-8 secretion and mRNA level by 60% and 70%, respectively; and the knockdown of PBEF expression also significantly attenuated IL-1β-induced cell permeability by 29% in epithelial cells and 24% in endothelial cells. PBEF expression also affected the expression of two other inflammatory cytokines (IL-16 and CCR3 genes). These results suggest that PBEF is critically involved in pulmonary vascular and epithelial inflammation and permeability, which are hallmark features in the pathogenesis of acute lung injury. This study lend further support that PBEF is a potential new target in acute lung injury.
Pulmonary inflammation; permeability; PBEF; Acute lung injury; IL-1β; IL-8; Epithelial cells; Endothelial cells; Inflammatory cytokines
Growth arrest and DNA damage protein 45b (Gadd45b) functions as an intrinsic neuroprotective molecule protecting retinal ganglion cells (RGCs) from injury. This study was performed to elucidate further the induction pathway of Gadd45b expression in RGCs.
The induction of Gadd45b expression in response to TGFβNFκB signaling was investigated in RGC5 cultures in vitro and murine retina in vivo. Gadd45b mRNA and protein expression were detected by quantitative real-time RT-PCR, immunoblot assay, immunohistochemistry, and immunocytochemistry. Activation of NFκB and TGFβ/Gadd45b signaling were assessed by measuring phosphorylation of NFκB and using specific inhibitors. Gadd45b siRNA was transfected into RGC5 to silence Gadd45b mRNA expression.
Expression of TGFβ receptors I and II was detected in RGC5 in vitro and RGCs in vivo. TGFβ induced abundant Gadd45b mRNA and protein expression, exhibiting a dose-dependent response in vitro. Exogenous TGFβ1 induced upregulation of Gadd45b expression in RGCs in murine retina in vivo. TGFβ stimulated phosphorylation of NFκB, and inhibition of NFκB phosphorylation blocked induction of Gadd45b by TGFβ in RGC5 cells. Induction of Gadd45b by TGFβ increased the resistance of RGC5 cells against TNFα cytotoxicity and paraquat oxidative stress.
TGFβ signaling induced Gadd45b expression in RGCs. Modulation of the TGFβ/NFκB/Gadd45b signaling pathway may provide a means to enhance the neuroprotective effect of Gadd45b in RGCs.
Gadd45b is an intrinsic neuroprotective molecule protecting RGCs from injuries. The present study identified TGFβ/NFκB as the induction pathway of Gadd45b in RGCs. Study on TGFβ/NFκB/Gadd45b signaling pathway may provide means to enhance the neuroprotective effect of Gadd45b in RGCs.
AHA Scientific Statements; genetics
Multiple events are involved in the development of acute inflammation and injury in the lungs. A progressive rise of oxidative stress due to altered reduction-oxidation (redox) homeostasis appears to be one of the hallmarks of lung pathologies such as injury, inflammation and ischemia/reperfusion. However, despite the growing evidence that alteration of the redox balance in the lungs, antioxidant therapy may attenuate acute lung injury and inflammation. We studied the effect of thiol antioxidant compound, amifostine, on acute lung dysfunction and pulmonary endothelial barrier compromise induced by gram-negative bacterial wall lypopolysacharide (LPS). In vitro, LPS as well as other producers of reactive oxygen spices (ROS), interleukin-6 (IL-6) and hydrogen peroxide (H2O2), induced significant reorganization of actin cytoskeleton accompanied by formation of stress fibers and paracellular gaps and associated with decreased transendothelial electrical resistance, a hallmark of endothelial barrier dysfunction. These disruptive effects were inhibited by pretreatment of endothelial monolayer with amifostine. Moreover, amifostine inhibited LPS-mediated ROS production and significantly suppressed LPS-, IL-6-, and H2O2-induced activation of redox sensitive signaling mechanisms including p38 and Erk1/2 MAP kinases, and NFκB pathway. In the murine model of LPS-induced acute lung injury, intraperitoneal administration of amifostine reduced LPS-induced oxidative stress and neutrophil recruitment to the lungs. These studies demonstrate for the first time that amifostine dramatically reduces endothelial cell barrier dysfunction and acute lung injury caused by bacterial products via inhibition of oxidative stress and redox-sensitive inflammatory pathways, and may therefore be considered for therapeutic treatment of lung inflammation.
permeability; endothelium; lung; LPS; ROS; MAPK
Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of diphospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.
ERM; Endothelial cells; Barrier function; Cytoskeleton; S1P; Rac1
Sickle cell disease (SCD) is a hemoglobinopathy that affects one in 500 African Americans. Although it is well established that patients with SCD have left ventricular (LV) diastolic dysfunction, it is not clear whether they have subtle LV systolic dysfunction despite preserved ejection fraction (EF). We used three-dimensional speckle tracking echocardiography (3D STE) to assess changes in both systolic and diastolic LV function in SCD.
Transthoracic real-time 3D images were obtained (Philips iE33) in 56 subjects, including 28 stable outpatients with SCD (age 33±7 years) and 28 normal controls (age 35±9 years). 3D-STE was performed using prototype software (4DLV Analysis, TomTec) to obtain LV volume and deformation time-curves, from which indices of systolic and diastolic LV function were calculated.
In SCD patients, 3D-STE derived LV filling parameters were significantly different from normal controls, reflecting an increase in both rapid and atrial filling volumes and prolonged active relaxation, depicted by a decrease in filling volume fractions at fixed times and an increase in rapid filling duration. Global LV systolic function was not only preserved but increased compared to controls, as reflected by significantly increased global longitudinal strain. Importantly, twist angle and torsion as well as radial and circumferential components of 3D strain were similar in both groups.
3D-STE was able to confirm diastolic dysfunction, as expected in some patients with SCD. However, 3D-STE strain analysis did not reveal any changes in LV systolic function. These findings provide novel insight into the pathophysiology of the cardiovascular complications of SCD.
three-dimensional echocardiography; left ventricle; diastole; ventricular function
Pulmonary hypertension (PH) and cancer pathology share growth factor- and MAPK stress-mediated signaling pathways resulting in endothelial and smooth muscle cell dysfunction and angioproliferative vasculopathy. In this study, we assessed sorafenib, an antineoplastic agent and inhibitor of multiple kinases important in angiogenesis [VEGF receptor (VEGFR)-1–3, PDGF receptor (PDGFR)-β, Raf-1 kinase] as a potential PH therapy. Two PH rat models were used: a conventional hypoxia-induced PH model and an augmented PH model combining dual VEGFR-1 and -2 inhibition (SU-5416, single 20 mg/kg injection) with hypoxia. In addition to normoxia-exposed control animals, four groups were maintained at 10% inspired O2 fraction for 3.5 wk (hypoxia/vehicle, hypoxia/SU-5416, hypoxia/sorafenib, and hypoxia/SU-5416/sorafenib). Compared with normoxic control animals, rats exposed to hypoxia/SU-5416 developed hemodynamic and histological evidence of severe PH while rats exposed to hypoxia alone displayed only mild elevations in hemodynamic values (pulmonary vascular and right ventricular pressures). Sorafenib treatment (daily gavage, 2.5 mg/kg) prevented hemodynamic changes and demonstrated dramatic attenuation of PH-associated vascular remodeling. Compared with normoxic control rats, expression profiling (Affymetrix platform) of lung RNA obtained from hypoxia [false discovery rate (FDR) 6.5%]- and hypoxia/SU-5416 (FDR 1.6%)-challenged rats yielded 1,019 and 465 differentially regulated genes (fold change >1.4), respectively. A novel molecular signature consisting of 38 differentially expressed genes between hypoxia/SU-5416 and hypoxia/SU-5416/sorafenib (FDR 6.7%) was validated by either real-time RT-PCR or immunoblotting. Finally, immunoblotting studies confirmed the upregulation of the MAPK cascade in both PH models, which was abolished by sorafenib. In summary, sorafenib represents a novel potential treatment for severe PH with the MAPK cascade a potential canonical target.
microarrays; SU-5416; bioinformatics
Increasing evidence supports the contribution of genetic influences on susceptibility/severity in acute lung injury (ALI), a devastating syndrome requiring mechanical ventilation with subsequent risk for ventilator-associated lung injury (VALI). To identify VALI candidate genes, we determined that Brown Norway (BN) and Dahl salt-sensitive (SS) rat strains were differentially sensitive to VALI (tidal volume of 20 ml/kg, 85 breaths/min, 2 h) defined by bronchoalveolar lavage (BAL) protein and leukocytes. We next exploited differential sensitivities and phenotyped both the VALI-sensitive BN and the VALI-resistant SS rat strains by expression profiling coupled to a bioinformatic-intense candidate gene approach (Significance Analysis of Microarrays, i.e., SAM). We identified 106 differentially expressed VALI genes representing gene ontologies such as “transcription” and “chemotaxis/cell motility.” We mapped the chromosomal location of the differentially expressed probe sets and selected consomic SS rats with single BN introgressions of chromosomes 2, 13, and 16 (based on the highest density of probe sets) while also choosing chromosome 20 (low probe sets density). VALI exposure of consomic rats with introgressions of BN chromosomes 13 and 16 resulted in significant increases in both BAL cells and protein (compared to parental SS strain), whereas introgression of BN chromosome 2 displayed a large increase only in BAL protein. Introgression of BN chromosome 20 had a minimal effect. These results suggest that genes residing on BN chromosomes 2, 13, and 16 confer increased sensitivity to high tidal volume ventilation. We speculate that the consomic-microarray-SAM approach is a time- and resource-efficient tool for the genetic dissection of complex diseases including VALI.
rodent mechanical ventilation; consomics; bioinformatics; microarrays; candidate gene approach
Low tidal volume ventilation, although promoting atelectasis, is a protective strategy against ventilator-induced lung injury. Deep inflation (DI) recruitment maneuvers restore lung volumes, but potentially compromise lung parenchymal and vascular function via repetitive overdistention. Our objective was to examine cardiopulmonary physiological and transcriptional consequences of recruitment maneuvers. C57/BL6 mice challenged with either PBS or LPS via aspiration were placed on mechanical ventilation (5 h) using low tidal volume inflation (TI; 8 μl/g) alone or in combination with intermittent DIs (0.75 ml twice/min). Lung mechanics during TI ventilation significantly deteriorated, as assessed by forced oscillation technique and pressure–volume curves. DI mitigated the TI-induced alterations in lung mechanics, but induced a significant rise in right ventricle systolic pressures and pulmonary vascular resistances, especially in LPS-challenged animals. In addition, DI exacerbated the LPS-induced genome-wide lung inflammatory transcriptome, with prominent dysregulation of a gene cluster involving vascular processes, as well as increases in cytokine concentrations in bronchoalveolar lavage fluid and plasma. Gene ontology analyses of right ventricular tissue expression profiles also identified inflammatory signatures, as well as apoptosis and membrane organization ontologies, as potential elements in the response to acute pressure overload. Our results, although confirming the improvement in lung mechanics offered by DI, highlight a detrimental impact in sustaining inflammatory response and exacerbating lung vascular dysfunction, events contributing to increases in right ventricle afterload. These novel insights should be integrated into the clinical assessment of the risk/benefit of recruitment maneuver strategies.
mechanical ventilation; microarray; pulmonary hypertension; right ventricle; acute lung injury
Staphylococcus aureus pneumonia causes significant morbidity and mortality. Alpha-hemolysin (Hla), a pore-forming cytotoxin of S. aureus, has been identified through animal models of pneumonia as a critical virulence factor that induces lung injury. In spite of considerable molecular knowledge of how this cytotoxin injures the host, the precise host response to Hla in the context of infection remains poorly understood. We employed whole-genome expression profiling of infected lungs to define the host response to wild-type S. aureus compared with the response to an Hla-deficient isogenic mutant in experimental pneumonia. These data provide a complete expression profile at 4 and at 24 h postinfection, revealing a unique response to the toxin-expressing strain. Gene ontogeny analysis revealed significant differences in the extracellular matrix and cardiomyopathy pathways, both of which govern cellular interactions in the tissue microenvironment. Evaluation of individual transcript responses to Hla-secreting staphylococci was notable for upregulation of host cytokine and chemokine genes, including the p19 subunit of interleukin-23. Consistent with this observation, the cellular immune response to infection was characterized by a prominent Th17 response to the wild-type pathogen. These findings define specific host mRNA responses to Hla-producing S. aureus, coupling the pulmonary Th17 response to the secretion of this cytotoxin. Expression profiling to define the host response to a single virulence factor proved to be a valuable tool in identifying pathways for further investigation in S. aureus pneumonia. This approach may be broadly applicable to the study of bacterial toxins, defining host pathways that can be targeted to mitigate toxin-induced disease.