Xiao-Hui Bai

Pre‐Implantation Multiple Cytokine mRNA Expression Analysis of Donor Lung Grafts Predicts Survival After Lung Transplantation in Humans

While current donor selection with clinical findings is generally effective, the imprecise nature of the assessment forces clinicians to remain on the conservative side. A reliable biological marker would assist donor selection and would improve donor organ utilization. We collected biopsies from 169 donor lungs before implantation. Expression levels of IL‐6, IL‐8, IL‐10, TNF‐α, IFN‐γ and IL‐1β were measured by quantitative real‐time RT‐PCR (qRT‐PCR). Seventeen cases died within 30 days after transplantation. No donor factor was significantly associated with 30‐day mortality. Univariate analysis of the 84 cases for development of the prediction model showed that IL‐6, IL‐8, TNF‐α and IL‐1β were risk factors for mortality and IL‐10 and IFN‐γ were protective factors. We analyzed the cytokine expression ratios of risk to protective cytokines. A stepwise logistic regression for 30‐day mortality demonstrated that a model containing the ratio of IL‐6/IL‐10 was the most predictive (p = 0.0013). When applied to the remaining 85 cases for validation, the test of model fit was significant (p = 0.039). Using the cytokine ratio, we were able to define three risk groups with striking differences in survival (p = 0.0003). Multi‐cytokine analysis of the donor lung graft with qRT‐PCR shows significant promise as a strategy to biologically evaluate the donor lung prior to implantation.

Prostaglandin E1 protects lung transplants from ischemia-reperfusion injury: a shift from pro- to anti-inflammatory cytokines.

INTRODUCTION Prostaglandin E1 (PGE1) has been demonstrated to reduce ischemia-reperfusion (IR) injury following lung transplantation. However, the cytoprotective mechanisms remain largely unknown. The purpose of this study was to determine whether the mechanism through which PGE1 improves IR injury is related to the level of apoptosis or the release of inflammatory cytokines. METHODS In a rat single-lung-transplant model, animals were randomly allocated into four groups of five animals each. Group 1 received normal saline (NS) in the preservation solution and during the 2-hr reperfusion period. Group 2 received NS in the preservation solution and PGE1 during the reperfusion period. Group 3 received PGE1 in the preservation solution and NS during the reperfusion period. Group 4 received PGE1 in the preservation solution and during the reperfusion period. RESULTS The two groups that received PGE1 during the reperfusion period had a significantly higher partial pressure of oxygen (PaO2), lower wet-dry weight ratio, and lower peak airway pressure at the end of the reperfusion period than did the two groups that received NS. In the two groups that received PGE1 during the reperfusion period, we observed significantly higher levels of interleukin (IL)-10 in the transplanted lung tissue and plasma and significantly lower levels of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and IL-12 in lung tissue. The levels of IL-4 and macrophage inflammatory protein-2 (MIP-2) were not significantly different between groups. The number of apoptotic cells and the expression of Bcl-2 were not significantly different between groups. CONCLUSIONS PGE1 does not decrease the amount of apoptosis after reperfusion and does not significantly upregulate Bcl-2. We have demonstrated that PGE1 administered during the reperfusion period reduces IR injury and improves lung function through a mechanism that is likely mediated by a shift between pro- and anti-inflammatory cytokine release.

XB130, a Novel Adaptor Protein for Signal Transduction

Adaptor proteins are important mediators in signal transduction. In the present study, we report the cloning and characterization of a novel adaptor protein, XB130. This gene is located on human chromosome 10q25.3 and encodes a protein of 818 amino acids. It contains several Src homology (SH)2- and SH3-binding motifs, two pleckstrin homology domains, a coiled-coil region, and a number of potential tyrosine or serine/threonine phosphorylation sites. Endogenous XB130 interacts with c-Src tyrosine kinase. Their co-expression in COS-7 cells resulted in activation of c-Src and elevated tyrosine phosphorylation of multiple proteins, including XB130 itself. XB130 expression in HEK293 cells enhanced serum response element- and AP-1-dependent transcriptional activation mediated by c-Src. XB130ΔN, an N-terminal deletion mutant lacking a putative SH3-binding motif and several putative SH2-binding sites, reduced its ability to mediate Src signal transduction. Down-regulation of endogenous XB130 with siRNA reduced c-Src activity, IL-8 production, EGF-induced phosphorylation of Akt and GSK3β, and altered cell cycles in human lung epithelial cells. These data suggest that XB130 as an adaptor may play an important role in the regulation of signal transduction and cellular functions.

Activating Transcription Factor 3 Confers Protection against Ventilator-induced Lung Injury

RATIONALE Ventilator-induced lung injury (VILI) significantly contributes to mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury. Understanding the molecular basis for response to cyclic stretch (CS) and its derangement during high-volume ventilation is of high priority. OBJECTIVES To identify specific molecular regulators involved in the development of VILI. METHODS We undertook a comparative examination of cis-regulatory sequences involved in the coordinated expression of CS-responsive genes using microarray analysis. Analysis of stretched versus nonstretched cells identified significant enrichment for genes containing putative binding sites for the transcription factor activating transcription factor 3 (ATF3). To determine the role of ATF3 in vivo, we compared the response of ATF3 gene-deficient mice to wild-type mice in an in vivo model of VILI. MEASUREMENTS AND MAIN RESULTS ATF3 protein expression and nuclear translocation is increased in the lung after mechanical ventilation in wild-type mice. ATF3-deficient mice have greater sensitivity to mechanical ventilation alone or in conjunction with inhaled endotoxin, as demonstrated by increased cell infiltration and proinflammatory cytokines in the lung and bronchoalveolar lavage, and increased pulmonary edema and indices of tissue injury. The expression of stretch-responsive genes containing putative ATF3 cis-regulatory regions was significantly altered in ATF3-deficient mice. CONCLUSIONS ATF3 deficiency confers increased sensitivity to mechanical ventilation alone or in combination with inhaled endotoxin. We propose ATF3 acts to counterbalance CS and high volume-induced inflammation, dampening its ability to cause injury and consequently protecting animals from injurious CS.

XB130, a tissue-specific adaptor protein that couples the RET/PTC oncogenic kinase to PI 3-kinase pathway

XB130 is a recently cloned 130 kDa-adaptor protein and Src kinase substrate, structurally similar to actin-filament-associated protein. Here we show that XB130 is predominantly expressed in the thyroid. Given that XB130 is a thyroid-specific tyrosine kinase substrate, we asked whether it is targeted by RET/PTC, a genetically rearranged, constitutively active, thyroid-specific tyrosine kinase that plays a pathogenic role in papillary thyroid cancer. RET/PTC induced robust tyrosine phosphorylation of XB130, which promoted its subsequent association with the p85α subunit of phosphatidylinositol 3-kinase (PI 3-kinase). We identified tyrosine 54 of XB130 as the major target of RET/PTC-mediated phosphorylation and a critical binding site for the SH2 domains of p85α. Importantly, downregulation of XB130 in TPC1 papillary thyroid cancer cells, harboring the RET/PTC1 kinase, strongly reduced Akt activity without altering ERK1/2 phosphorylation, and concomitantly inhibited cell-cycle progression and survival in suspension. In conclusion, XB130 is a novel substrate of the RET/PTC kinase that links RET/PTC signaling to PI 3-kinase activation, and thereby plays an important role in sustaining proliferation and survival of thyroid tumor cells.

Long pentraxin PTX3 deficiency worsens LPS-induced acute lung injury

ObjectiveLong pentraxin PTX3 is an inflammatory mediator and a component of the humoral arm of innate immunity. PTX3 expression is increased in animals with acute lung injury (ALI) and in patients with sepsis or acute respiratory distress syndrome and is considered to be a potential biomarker for these diseases. However, the role of PTX3 in the pathogenesis of ALI is not fully understood. We hypothesized that PTX3, as an important immune modulator, may determine the severity of ALI.MethodsLipopolysaccharide (LPS) was intra-tracheally administrated to PTX3 knock-out (PTX3-KO) and wild-type (WT) mice. Lung injury, neutrophil infiltration, cell death, fibrin deposition, and tissue factor expression in the lung were determined. Local and systemic inflammatory responses were assessed by measuring cytokines in the lung and plasma.ResultsLPS instillation induced ALI in both PTX3-KO and WT mice. Interestingly, PTX3 deficiency significantly increased the magnitude/extent of lung injury compared to that in WT mice. The severe lung injury was accompanied by elevated neutrophil infiltration, cell death, and fibrin deposition in the lung. PTX3 deficiency also enhanced LPS-induced tissue factor expression/activation in the lung and increased tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in the plasma.ConclusionOur data suggest that the endogenously expressed PTX3 plays a protective role in the pathogenesis of ALI and that a lack of PTX3 may enhance neutrophil recruitment, cell death, activation of coagulation cascades, and inflammatory responses in the lung.

The Protein Kinase C Cascade Regulates Recruitment of Matrix Metalloprotease 9 to Podosomes and Its Release and Activation

ABSTRACT Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.

Claudin 1 Mediates TNFα-Induced Gene Expression and Cell Migration in Human Lung Carcinoma Cells

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFβ1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFβ1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies.

Lipopolysaccharide accelerates caspase-independent but cathepsin B-dependent death of human lung epithelial cells.

Caspase‐independent cell death has drawn increasing attention. In the present study, we found that lipopolysaccharide (LPS) accelerated spontaneous death of human lung epithelial A549 cells in a serum‐ and cell density‐dependent manner: while serum starvation has been demonstrated to induce apoptosis in the same cell line, LPS‐induced cell death was only observed in the presence of serum; in addition, the cell death was not observed when the cells were seeded at 10‐ or 100‐fold lower density. The apoptotic features were demonstrated by TUNEL assay, DNA laddering and Annexin V staining. However, treatment of cells with two commonly used pan‐caspase inhibitors, zVAD.fmk or BOC‐D.fmk, failed to block cell death. In contrast, two cathepsin B inhibitors, Ca074‐Me or N‐1845, reduced cell death significantly. A time‐dependent activation of cathepsin B, but not caspase 3, was observed in both control and LPS‐treated cells. Although LPS did not further activate cathepsin B or its release, it increased expression and translocation of apoptosis inducing factor from mitochondria to the nucleus, and increased release of cytochrome c from mitochondria. LPS‐induced cell death was significantly attenuated by either N‐acetyl‐L‐cysteine or pyrrolidine‐dithiocarbamate, both free radical scavengers. Disruption of lipid raft formation with filipin or methyl‐β‐cyclodextrin also reduced apoptosis significantly, suggesting that lipid raft‐dependent signaling is essential. These data imply that confluent cells undergo spontaneous cell death mediated by cathepsin B; LPS may accelerate this caspase‐independent cell death through release of mitochondrial contents and reactive oxygen species. J. Cell. Physiol. 209: 457–467, 2006.

α1-Antitrypsin inhibits ischemia reperfusion-induced lung injury by reducing inflammatory response and cell death

BACKGROUND Pulmonary ischemia-reperfusion (IR)-induced lung injury is a severe complication that increases the likelihood of primary graft dysfunction and early death after lung transplantation. Inflammatory cytokine release and cell death play a critical role in the development of IR-induced lung injury. α1-Antitrypsin (A1AT) is a protease inhibitor clinically used for the treatment of A1AT-deficiency emphysema. On the basis of a literature review, we hypothesize that A1AT may have the potential to reduce IR-induced lung injury through its anti-inflammatory and anti-apoptotic effects. METHODS A human pulmonary cell culture model was used to simulate IR processes in lung transplantation. Effects of A1AT on cell death and cytokine production were examined. A rat pulmonary IR model, in which the left pulmonary hilum was clamped for 90 minutes, followed by reperfusion for 2 hours, was used to determine the effects of A1AT on acute lung injury, function, cell death, and inflammatory response. RESULTS A1AT significantly inhibited cell death and inflammatory cytokine release dose-dependently in vitro and significantly improved lung oxygenation and lung mechanics and reduced pulmonary edema in vivo. Moreover, A1AT inhibited neutrophil infiltration in the lung and reduced cell death and significantly reduced IR-induced inflammatory mediators in plasma, including interleukin (IL)-1α, IL-4, IL-12p70, monocyte chemotactic protein 1, and tumor necrosis factor-α. CONCLUSIONS Considering its current clinical use, our findings indicate that administration of A1AT may be an effective and safe therapy for the treatment of IR injury in human lung transplantation.