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1.  A novel molecular pathway for Snail-dependent, SPARC-mediated invasion in non-small cell lung cancer pathogenesis 
Definition of the molecular pathogenesis of lung cancer allows investigators an enhanced understanding of the natural history of the disease, thus fostering development of new prevention strategies. In addition to regulating epithelial-to-mesenchymal transition (EMT), the transcription factor Snail exerts global effects on gene expression. Our recent studies reveal that Snail is upregulated in non-small cell lung cancer (NSCLC), is associated with poor prognosis, and promotes tumor progression in vivo. Herein, we demonstrate that overexpression of Snail leads to upregulation of Secreted Protein, Acidic and Rich in Cysteine (SPARC) in models of premalignancy and established disease, as well as in lung carcinoma tissues in situ. Snail overexpression leads to increased SPARC-dependent invasion in vitro, indicating that SPARC may play a role in lung cancer progression. Bioinformatic analysis implicates TGF-β, ERK1/2, and miR-29b as potential intermediaries in Snail-mediated upregulation of SPARC. Both the TGF-β1 ligand and TGF-βR2 are upregulated following Snail overexpression. Treatment of human bronchial epithelial cell (HBEC) lines with TGF-β1 and inhibition of TGF-β1 mRNA expression modulated SPARC expression. Inhibition of MEK phosphorylation downregulated SPARC. MiR-29b is downregulated in Snail overexpressing cell lines, while overexpression of miR-29b inhibited SPARC expression. In addition, miR-29b was upregulated following ERK inhibition, suggesting a Snail-dependent pathway by which Snail activation of TGF-β and ERK signaling results in downregulation of miR-29b and subsequent upregulation of SPARC. Our discovery of pathways responsible for Snail-induced SPARC expression contributes to the definition of NSCLC pathogenesis.
doi:10.1158/1940-6207.CAPR-13-0263
PMCID: PMC3919444  PMID: 24253315
NSCLC; Snail; SPARC; invasion; parallel progression
2.  CXCR3 Ligands Are Associated with the Continuum of Diffuse Alveolar Damage to Chronic Lung Allograft Dysfunction 
Rationale: After lung transplantation, insults to the allograft generally result in one of four histopathologic patterns of injury: (1) acute rejection, (2) lymphocytic bronchiolitis, (3) organizing pneumonia, and (4) diffuse alveolar damage (DAD). We hypothesized that DAD, the most severe form of acute lung injury, would lead to the highest risk of chronic lung allograft dysfunction (CLAD) and that a type I immune response would mediate this process.
Objectives: Determine whether DAD is associated with CLAD and explore the potential role of CXCR3/ligand biology.
Methods: Transbronchial biopsies from all lung transplant recipients were reviewed. The association between the four injury patterns and subsequent outcomes were evaluated using proportional hazards models with time-dependent covariates. Bronchoalveolar lavage (BAL) concentrations of the CXCR3 ligands (CXCL9/MIG, CXCL10/IP10, and CXCL11/ITAC) were compared between allograft injury patterns and “healthy” biopsies using linear mixed-effects models. The effect of these chemokine alterations on CLAD risk was assessed using Cox models with serial BAL measurements as time-dependent covariates.
Measurements and Main Results: There were 1,585 biopsies from 441 recipients with 62 episodes of DAD. An episode of DAD was associated with increased risk of CLAD (hazard ratio, 3.0; 95% confidence interval, 1.9–4.7) and death (hazard ratio, 2.3; 95% confidence interval, 1.7–3.0). There were marked elevations in BAL CXCR3 ligand concentrations during DAD. Furthermore, prolonged elevation of these chemokines in serial BAL fluid measurements predicted the development of CLAD.
Conclusions: DAD is associated with marked increases in the risk of CLAD and death after lung transplantation. This association may be mediated in part by an aberrant type I immune response involving CXCR3/ligands.
doi:10.1164/rccm.201305-0861OC
PMCID: PMC3863740  PMID: 24063316
lung transplantation; chronic lung allograft dysfunction; bronchiolitis obliterans syndrome; diffuse alveolar damage; CXC chemokines
3.  Aspergillus Colonization of the Lung Allograft is a Risk Factor for Bronchiolitis Obliterans Syndrome 
Multiple infections have been linked with the development of bronchiolitis obliterans syndrome (BOS) post-lung transplantation. Lung allograft airway colonization by Aspergillus species is common among lung transplant recipients. We hypothesized that Aspergillus colonization may promote the development of BOS and may decrease survival post-lung transplantation. We reviewed all lung transplant recipients transplanted in our center between 1/2000 and 6/2006. Bronchoscopy was performed according to a surveillance protocol and when clinically indicated. Aspergillus colonization was defined as a positive culture from bronchoalveolar lavage or two sputum cultures positive for the same Aspergillus species, in the absence of invasive pulmonary Aspergillosis. We found that Aspergillus colonization was strongly associated with BOS and BOS related mortality in Cox regression analyses. Aspergillus colonization typically preceded the development of BOS by a median of 261 days (95% CI 87 to 520). Furthermore, in a multivariate Cox regression model, Aspergillus colonization was a distinct risk factor for BOS, independent of acute rejection. These data suggest a potential causative role for Aspergillus colonization in the development of BOS post-lung transplantation and raise the possibility that strategies aimed to prevent Aspergillus colonization may help delay or reduce the incidence of BOS.
doi:10.1111/j.1600-6143.2009.02635.x
PMCID: PMC4214373  PMID: 19459819
4.  Molecular Imaging of the Kinetics of Vascular Endothelial Growth Factor Gene Expression in Ischemic Myocardium 
Circulation  2004;110(6):685-691.
Background
Angiogenic gene therapy is a promising treatment paradigm for patients with ischemic heart disease. In this study, we used micro–positron emission tomography (microPET) to monitor the transgene expression, function, and effects in a whole-body system.
Methods and Results
Adenovirus with cytomegalovirus promoter driving an angiogenic gene (vascular endothelial growth factor [VEGF]) linked to a PET reporter gene (herpes simplex virus type 1 mutant thymidine kinase; Ad-CMV-VEGF121-CMV-HSV1-sr39tk) was used to transfect rat embryonic cardiomyoblasts in vitro. Expression of both genes correlated strongly (r=0.98; P<0.001). Afterward, rats underwent ligation of the left anterior descending artery followed by injection of 1×1010 pfu of Ad-CMV-VEGF121-CMV-HSV1-sr39tk (study; n=35) or Ad-null (control; n=15) at the peri-infarct region. Noninvasive microPET imaging was used to assess the uptake of 9-(4-[18F]-fluoro-hydroxymethylbutyl)guanine ([18F]-FHBG) PET reporter probe by cells expressing the HSV1-sr39tk PET reporter gene. Cardiac transgene expression peaked at day 1 and declined over the next 2 weeks. Repeat adenoviral injections at day 60 yielded no detectable signal. The in vivo reporter gene expression (% injected dose/g of [18F]-FHBG) correlated well with ex vivo gamma counting (r=0.92), myocardial tissue HSV1-sr39TK enzyme activity (r=0.95), and myocardial tissue VEGF level (r=0.94; P<0.001 for all). The VEGF121 isoform induced significant increases in capillaries and small blood vessels. However, the level of neovasculature did not translate into significant improvements in functional parameters such as myocardial contractility by echocardiography, perfusion by nitrogen-13 ammonia imaging, and metabolism by [18F]-fluorodeoxyglucose imaging.
Conclusions
Taken together, these findings establish the feasibility of molecular imaging for monitoring angiogenic gene expression with a PET reporter gene and probe noninvasively, quantitatively, and repetitively. The principles demonstrated here can be used to evaluate other therapeutic genes of interest in animal models before future clinical trials are initiated.
doi:10.1161/01.CIR.000013815302213.22
PMCID: PMC4154794  PMID: 15302807
gene therapy; angiogenesis; imaging; myocardium; nuclear medicine
5.  Molecular Imaging of Cardiac Cell Transplantation in Living Animals Using Optical Bioluminescence and Positron Emission Tomography 
Circulation  2003;108(11):1302-1305.
Background
The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques.
Methods and Results
For optical bioluminescence charged-coupled device imaging, rats (n = 20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3 × 106 to 5 × 105) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3 × 106 cells: day 1 (627 000±15%), day 2 (346 100±21%), day 4 (112 800±20%), day 8 (78 860±24%), day 12 (67 780±12%), and day 16 (62 200±5% p · s−1 · cm2−1 · sr−1). For micro–positron emission tomography imaging, rats (n = 20) received cardiomyoblasts (3 × 106) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48±0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01±0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays.
Conclusions
The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.
doi:10.1161/01.CIR.0000091252.20010.6E
PMCID: PMC4154815  PMID: 12963637
transplantation; heart failure; genes; imaging; nuclear medicine
6.  A Fluorescence Lifetime Imaging Classification Method to Investigate the Collagen to Lipid Ratio in Fibrous Caps of Atherosclerotic Plaque 
Lasers in surgery and medicine  2012;44(7):564-571.
Background and Objective
This study describes a novel fluorescence lifetime imaging (FLIM) classification method to determine the ratio of collagen to lipid content in the fibrous cap of atherosclerotic plaques. Additionally, an analytical process to assess risk of plaque rupture based on this ratio is proposed. Collagen to lipid ratio has been shown to be an important parameter to evaluate structural integrity of the fibrous cap. FLIM and other time-resolved fluorescence techniques have recently been applied to the study of atherosclerosis based on the ability to assess biochemical composition.
Study Design/Materials and Methods
Autofluorescence of specimens retrieved during carotid endarterectomy procedures was measured through three optical filters, F377: 377/50 nm, F460: 460/66 nm, and F510: 510/ 84 nm (center wavelength/bandwidth). A Laguerre deconvolution technique was used for the evaluation of fluorescence decay dynamics. The resulting decay parameters (average fluorescence lifetime and 4 Laguerre coefficients at each of the recorded bandwidths) were used for sample characterization. Linear discriminant analysis (LDA) was used to classify each image into collagen or lipid-rich regions based on these parameters. Ultimately, a risk-level was assigned based on the ratio of collagen to lipid on the surface of the fibrous cap.
Results
FLIM images were acquired in 18 carotid plaque specimens at 43 locations. Classification of collagen and lipid-rich regions within the fibrous cap was performed with sensitivity and specificity of 80% and 82%, respectively.
Conclusions
Results from this study show that an LDA method of classifying regions of FLIM images of carotid plaque into collagen and lipid-rich regions is capable of being automated and used to rate the risk of plaque rupture based on autofluorescence decay dynamics and without the need for fluorescence intensity or contrast agents. Lasers Surg. Med. 44:564–571, 2012.
doi:10.1002/lsm.22059
PMCID: PMC4083183  PMID: 22886522
carotid plaque; fluorescence lifetime imaging microscopy (FLIM); vulnerable plaque
7.  Low-Level Vagus Nerve Stimulation Upregulates Small Conductance Calcium Activated Potassium Channels in the Stellate Ganglion 
Background
Small conductance calcium activated potassium (SK) channels are responsible for afterhyperpolarization that suppresses nerve discharges.
Objectives
To test the hypotheses that low-level vagus nerve stimulation (LL-VNS) leads to the upregulation of SK2 proteins in the LSG.
Methods
Six dogs (Group 1) underwent 1-wk LL-VNS of the left cervical vagus nerve. Five normal dogs (Group 2) were used as control. SK2 protein levels were examined by western blotting. The ratio between SK2 and glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) levels was used as an arbitrary unit (AU).
Results
We found higher SK2 expression in Group 1 (0.124 ± 0.049 AU) than Group 2 (0.085 ± 0.031 AU, P < 0.05). Immunostaining showed that the density of nerve structures stained with SK2 antibody was also higher in Group 1 (11,546 ± 7,271 μm2/mm2) than in Group 2 (5,321 ± 3,164 μm2/mm2, P < 0.05). There were significantly more ganglion cells without immunoreactivity to TH in Group 1 (11.4 ± 2.3%) than Group 2 (4.9 ± 0.7%; P < 0.05). The TH-negative ganglion cells mostly stained positive for choline acetyltransferase (ChAT) (95.9 ± 2.8% in Group 1 and 86.1 ± 4.4% in Group 2, P = 0.10). Immunofluorescence confocal microscopy revealed a significant decrease in the SK2 staining in the cytosol but an increase in the SK2 staining on the membrane of the ganglion cells in Group 1 compared to Group 2.
Conclusion
Left LL-VNS results in the upregulation of SK2 proteins, increased SK2 protein expression in the cell membrane and the increased TH-negative (mostly ChAT-positive) ganglion cells in the LSG. These changes may underlie the antiarrhythmic efficacy of LL-VNS in ambulatory dogs.
doi:10.1016/j.hrthm.2013.01.029
PMCID: PMC3671581  PMID: 23357541
Autonomic nervous system; Vagus nerve stimulation; Stellate ganglion; Small conductance calcium activated potassium channel; Western blot
8.  Electroanatomic Remodeling of the Left Stellate Ganglion After Myocardial Infarction 
Objectives
The purpose of this study was to evaluate the changes of left stellate ganglionic nerve activity (SGNA) and left thoracic vagal nerve activity (VNA) after acute myocardial infarction (MI).
Background
Whether MI results in remodeling of extracardiac nerve activity remains unclear.
Methods
We implanted radiotransmitters to record the SGNA, VNA, and electrocardiogram in 9 ambulatory dogs. After baseline monitoring, MI was created by 1-h balloon occlusion of the coronary arteries. The dogs were then continuously monitored for 2 months. Both stellate ganglia were stained for growth-associated protein 43 and synaptophysin. The stellate ganglia from 5 normal dogs were used as control.
Results
MI increased 24-h integrated SGNA from 7.44 ± 7.19 Ln(Vs)/day at baseline to 8.09 ± 7.75 Ln(Vs)/day after the MI (p < 0.05). The 24-h integrated VNA before and after the MI was 5.29 ± 5.04 Ln(Vs)/day and 5.58 ± 5.15 Ln(Vs)/day, respectively (p < 0.05). A significant 24-h circadian variation was noted for the SGNA (p < 0.05) but not the VNA. The SGNA/VNA ratio also showed significant circadian variation. The nerve densities from the left SG were 63,218 ± 34,719 μm2/mm2 and 20,623 ± 4,926 μm2/mm2 for growth-associated protein 43 (p < 0.05) and were 32,116 ± 8,190 μm2/mm2 and 16,326 ± 4,679 μm2/mm2 for synaptophysin (p < 0.05) in MI and control groups, respectively. The right SG also showed increased nerve density after MI (p < 0.05).
Conclusions
MI results in persistent increase in the synaptic density of bilateral stellate ganglia and is associated with increased SGNA and VNA. There is a circadian variation of the SGNA/VNA ratio. These data indicate significant remodeling of the extracardiac autonomic nerve activity and structures after MI.
doi:10.1016/j.jacc.2011.11.030
PMCID: PMC3975658  PMID: 22381432
acute myocardial infarction; autonomic nervous system; nerve recordings
9.  Sympathetic nerve fibers and ganglia in canine cervical vagus nerves: Localization and quantitation 
Background
Cervical vagal nerve (CVN) stimulation may improve left ventricular ejection fraction in patients with heart failure.
Objectives
To test the hypothesis that sympathetic structures are present in the CVN and to describe the location and quantitate these sympathetic components of the CVN.
Methods
We performed immunohistochemical studies of the CVN from 11 normal dogs and simultaneously recorded stellate ganglion nerve activity, left thoracic vagal nerve activity, and subcutaneous electrocardiogram in 2 additional dogs.
Results
A total of 28 individual nerve bundles were present in the CVNs of the first 11 dogs, with an average of 1.87 ± 1.06 per dog. All CVNs contain tyrosine hydroxylase-positive (sympathetic) nerves, with a total cross-sectional area of 0.97 ± 0.38 mm2. The sympathetic nerves were nonmyelinated, typically located at the periphery of the nerve bundles and occupied 0.03%–2.80% of the CVN cross-sectional area. Cholineacetyltransferase-positive nerve fibers occupied 12.90%–42.86% of the CVN cross-sectional areas. Ten of 11 CVNs showed tyrosine hydroxylase and cholineacetyltransferase colocalization. In 2 dogs with nerve recordings, we documented heart rate acceleration during spontaneous vagal nerve activity in the absence of stellate ganglion nerve activity.
Conclusions
Sympathetic nerve fibers are invariably present in the CVNs of normal dogs and occupy in average up to 2.8% of the cross-sectional area. Because sympathetic nerve fibers are present in the periphery of the CVNs, they may be susceptible to activation by electrical stimulation. Spontaneous activation of the sympathetic component of the vagal nerve may accelerate the heart rate.
doi:10.1016/j.hrthm.2012.12.015
PMCID: PMC3758134  PMID: 23246597
Cervical vagus nerves; Sympathetic nerves; Ganglion cells; Heart failure; Vagal nerve stimulation
10.  Interaction between Pseudomonas and CXC Chemokines Increases Risk of Bronchiolitis Obliterans Syndrome and Death in Lung Transplantation 
Rationale: Pseudomonas aeruginosa is the most commonly isolated gram-negative bacterium after lung transplantation and has been shown to up-regulate glutamic acid–leucine–arginine–positive (ELR+) CXC chemokines associated with bronchiolitis obliterans syndrome (BOS), but the effect of pseudomonas on BOS and death has not been well defined.
Objectives: To determine if the influence of pseudomonas isolation and ELR+ CXC chemokines on the subsequent development of BOS and the occurrence of death is time dependent.
Methods: A three-state model was developed to assess the likelihood of transitioning from lung transplant (state 1) to BOS (state 2), from transplant (state 1) to death (state 3), and from BOS (state 2) to death (state 3). This Cox semi-Markovian approach determines state survival rates and cause-specific hazards for movement from one state to another.
Measurements and Main Results: The likelihood of transition from transplant to BOS was increased by acute rejection, CXCL5, and the interaction between pseudomonas and CXCL1. The pseudomonas effect in this transition was due to infection rather than colonization. Movement from transplant to death was facilitated by pseudomonas infection and single lung transplant. Transition from BOS to death was affected by the length of time in state 1 and by the interactions between any pseudomonas isolation and CXCL5 and aspergillus, either independently or in combination.
Conclusions: Our model demonstrates that common post-transplantation events drive movement from one post-transplantation state to another and influence outcomes differently depending upon when after transplantation they occur. Pseudomonas and the ELR+ CXC chemokines may interact to negatively influence lung transplant outcomes.
doi:10.1164/rccm.201207-1228OC
PMCID: PMC3733405  PMID: 23328531
transplantation; lung; BOS; pseudomonas; chemokine
11.  LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin 
Cancer cell  2013;23(2):143-158.
SUMMARY
The LKB1 (also called STK11) tumor suppressor is mutationally inactivated in ~20% of non-small cell lung cancers (NSCLC). LKB1 is the major upstream kinase activating the energy-sensing kinase AMPK, making LKB1-deficient cells unable to appropriately sense metabolic stress. We tested the therapeutic potential of metabolic drugs in NSCLC and identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1-deficient NSCLC cells. Therapeutic trials in Kras-dependent mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations showed selective response to phenformin as a single agent, resulting in prolonged survival. This study suggests phenformin as a cancer metabolism-based therapeutic to selectively target LKB1-deficient tumors.
doi:10.1016/j.ccr.2012.12.008
PMCID: PMC3579627  PMID: 23352126
12.  Blockade of P-selectin is Sufficient to Reduce MHC I Antibody-Elicited Monocyte Recruitment In Vitro and In Vivo 
Donor specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody-mediated rejection. Intracapillary macrophage infiltration is a hallmark of antibody-mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc-independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody-mediated rejection, in which B6.RAG1-/- recipients of BALB/c cardiac allografts were passively transferred with donor specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P-selectin, which supports monocyte binding. In the experimental model, donor specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL-1-Ig abolished antibody-induced monocyte infiltration in the allograft, but had little effect on antibody-induced endothelial injury. Our data suggest that antagonism of P-selectin may ameliorate accumulation of macrophages in the allograft during antibody-mediated rejection.
doi:10.1111/ajt.12016
PMCID: PMC3563267  PMID: 23279566
Endothelial cells; HLA antibody; signal transduction; monocytes; P-selectin
13.  Anti-MHC Class I Antibody Activation of Proliferation and Survival Signaling in Murine Cardiac Allografts1 
Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2Kb/Db) received a fully MHC-incompatible BALB/c (H-2Kd/Dd) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2Kd Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments.
PMCID: PMC3883756  PMID: 18250428
14.  Canonical Wnt/β-catenin signaling in epicardial fibrosis of failed pediatric heart allografts with diastolic dysfunction 
Background
Failed pediatric heart allografts with diastolic dysfunction exhibit severe epicardial fibrosis. The molecular mechanism underlying this process is poorly understood. Canonical Wnt/β-catenin signaling plays an important role in epithelial-mesenchymal transition and is implicated in fibrosing diseases. In this study, we tested the hypothesis that canonical Wnt/β-catenin signaling is activated in epicardial fibrosis of end-stage dysfunctional pediatric allografts.
Methods
Fourteen explanted heart grafts of 12 patients who had undergone 14 heart transplantations were used for immunohistochemical staining of β-catenin and its nuclear binding partners, T-cell factor/lymphoid enhancer factor (TCF/LEF) family transcriptional factors. Fourteen age-matched native hearts from patients who undergone first heart transplantation without evidence of epicardial fibrosis were used as controls.
Results and Conclusions
Epicardial fibroblasts from explanted allografts demonstrated nuclear accumulation of β-catenin. These cells also showed nuclear positivity for TCF-4. No TCF-3 expression was present in the epicardium. TCF-1 and LEF-1 were observed in lymphocytes, but not in other cell types of the epicardium. These findings suggest an association between canonical Wnt/beta-catenin signaling and epicardial fibrosis of failed pediatric heart allografts. Should activation of this pathway be shown to be causal to epicardial fibrosis in this setting, then inhibition of this pathway may help to prevent this devastating process.
doi:10.1016/j.carpath.2012.03.004
PMCID: PMC3427707  PMID: 22475572
Heart transplant; Pediatric; Epicardium; Fibrosis; Allograft; Heart failure; Wnt signaling; LEF/TCF; β-Catenin
15.  CXCR3 Chemokine Ligands during Respiratory Viral Infections Predict Lung Allograft Dysfunction 
Community-acquired respiratory viruses (CARV) can accelerate the development of lung allograft dysfunction, but the immunologic mechanisms are poorly understood. The chemokine receptor CXCR3 and its chemokine ligands, CXCL9, CXCL10, and CXCL11 have roles in the immune response to viruses and in the pathogenesis of bronchiolitis obliterans syndrome, the predominant manifestation of chronic lung allograft rejection. We explored the impact of CARV infection on CXCR3/ligand biology and explored the utility of CXCR3 chemokines as biomarkers for subsequent lung allograft dysfunction. Seventeen lung transplant recipients with CARV infection had bronchoalveolar lavage fluid (BALF) available for analysis. For comparison, we included 34 BALF specimens (2 for each CARV case) that were negative for infection and collected at a duration post-transplant similar to a CARV case. The concentration of each CXCR3 chemokine was increased during CARV infection. Among CARV infected patients, a high BALF concentration of either CXCL10 or CXCL11 was predictive of a greater decline in FEV1 6 months later. CXCR3 chemokine concentrations provide prognostic information and this may have important implications for the development of novel treatment strategies to modify outcomes following CARV infection.
doi:10.1111/j.1600-6143.2011.03859.x
PMCID: PMC3833088  PMID: 22152000
Lung transplantation; community-acquired respiratory viruses; respiratory viral infection; chemokines; rejection
16.  Asymptomatic Antibody-mediated Rejection After Heart Transplantation Predicts Poor Outcomes 
Background
Antibody-mediated rejection (AMR) has been associated with poor outcome after heart transplantation. The diagnosis of AMR usually includes endomyocardial biopsy findings of endothelial cell swelling, intravascular macrophages, C4d+ staining, and associated left ventricular dysfunction. The significance of AMR findings in biopsy specimens of asymptomatic heart transplant patients (normal cardiac function and no symptoms of heart failure) is unclear.
Methods
Between July 1997 and September 2001, AMR was found in the biopsy specimens of 43 patients. Patients were divided into 2 groups: asymptomatic AMR (AsAMR, n = 21) and treated AMR (TxAMR with associated left ventricular dysfunction, n = 22). For comparison, a control group of 86 contemporaneous patients, without AMR, was matched for age, gender, and time from transplant. Outcomes included 5-year actuarial survival and development of cardiac allograft vasculopathy (CAV). Patients were considered to have AMR if they had ≥ 1 endomyocardial biopsy specimen positive for AMR.
Results
The 5-year actuarial survival for the AsAMR (86%), TxAMR (68%), and control groups (79%) was not significantly different (p = 0.41). Five-year freedom from CAV (≥ 30% stenosis in any vessel) was AsAMR, 52%; TxAMR, 68%; and control, 79%. Individually, freedom from CAV was significantly lower in the AsAMR group compared with the control group (p = 0.02). There was no significant difference between AsAMR vs TxAMR and TxAMR vs control for CAV.
Conclusions
Despite comparable 5-year survival with controls after heart transplantation, AsAMR rejection is associated with a greater risk of CAV. Trials to treat AsAMR to alter outcome are warranted.
doi:10.1016/j.healun.2009.01.015
PMCID: PMC3829690  PMID: 19416767
17.  Extra-Cardiac Neural Remodeling in Humans with Cardiomyopathy 
Background
Intra-myocardial nerve sprouting after myocardial infarction is associated with ventricular arrhythmias (VAs). Whether human stellate ganglia remodel in association with cardiac pathology is unknown. The purpose of this study was to determine whether cardiac pathology is associated with remodeling of the stellate ganglia in humans.
Methods and Results
Left stellate ganglia (LSG) were collected from patients undergoing sympathetic denervation for intractable ventricular arrhythmias, and from cadavers, along with intact hearts. Clinical data on patients and cadaveric subjects were reviewed. We classified ganglia from normal; scarred; and non-ischemic cardiomyopathic hearts without scar as NL (n=3); SCAR (n=24); and NICM (n=7), respectively. Within LSG, neuronal size, density, fibrosis, synaptic density and nerve sprouting were determined. Nerve density and sprouting were also quantified in cadaveric hearts. Mean neuronal size in NL, SCAR, and NICM groups were; 320±4μm2, 372±10μm2,and 435±10μm2 (p=0.002). No significant differences in neuronal density and fibrosis were present between the groups. Synaptic density in SCAR and NICMganglia were 57.8±11.2um2/ mm2 (p=0.039) and 44.5±7.9um2/ mm2 (p=0.084) respectively, compared to the NL, 17.8±7um2/ mm2 (overall p=0.162). There were no significant differences in LSG nerve sprouting or myocardial nerve density between the groups.
Conclusions
Neuronal hypertrophy withinLSGis associated with chronic cardiomyopathy in humans. Ganglionic and myocardial nerve sprouting and nerve density were not significantly different. These changes may be related to increased cardiac sympathetic signaling and VAs. Further studies are needed to determine the electrophysiologic consequences of extra-cardiac neuronal remodeling in humans.
doi:10.1161/CIRCEP.112.972836
PMCID: PMC3529182  PMID: 22923270
cardiomyopathy; nervous system; autonomic; nervous system; sympathetic; ventricular arrhythmia
18.  Correction: The CD44high Tumorigenic Subsets in Lung Cancer Biospecimens Are Enriched for Low miR-34a Expression 
PLoS ONE  2013;8(9):10.1371/annotation/9ae8db84-5d44-4a9c-bb80-47c9d68820ea.
doi:10.1371/annotation/9ae8db84-5d44-4a9c-bb80-47c9d68820ea
PMCID: PMC3790883  PMID: 24116237
19.  DISTINGUISHING EPICARDIAL FAT FROM SCAR: ANALYSIS OF ELECTROGRAMS USING HIGH DENSITY ELECTROANATOMIC MAPPING IN A NOVEL PORCINE INFARCT MODEL 
Background
The presence of epicardial fat can confound the quantification of scar during transpericardial electroanatomic mapping. The electrogram (EGM) characteristics of epicardial fat have not been systematically compared with infarct scar using gross and histopathologic analysis as a gold standard.
Methods
A closed-chest infarction was created in 40–50 kg pigs by occlusion of the circumflex artery for 150 minutes using an angioplasty balloon. This artery was chosen to minimize any potential overlap of epicardial fat with infarct and to spare any septal involvement. After 4–12 weeks of infarct healing, epicardial mapping was performed. EGMs in low voltage regions (<1.5mV) were analyzed and bipolar amplitude, duration, number of deflections, and the presence of late potentials were recorded. Statistical analysis was performed using unpaired t-test and chi square analysis. Gross and histopathologic examination was used to confirm areas of fat and infarct scar.
Results
Seven porcine hearts were analyzed after high-density epicardial mapping (364±92 points) was performed 48±19 days after infarction. The mean bipolar EGM amplitude was similar in fat and scar (0.77±0.34 vs 0.75±0.38mV; P=NS). The mean EGM duration was longer in scar than fat (68.8±18.9 vs 50.1±11.6 ms; P<0.0001) and exhibited more fractionation (8.5±3.1 vs 4.7±1.8 deflections; P<0.0001). The presence of late potentials was 99% specific for scar. Further, areas of fat >4 mm in thickness registered low voltage bipolar EGMs.
Conclusion
Scar from healed myocardial infarction exhibits more fractionation and longer EGM duration when compared to fat. Late potentials are highly specific for locating infarct scars.
doi:10.1016/j.hrthm.2009.11.023
PMCID: PMC3766735  PMID: 20185114
Epicardial; fat; infarction; electrogram; late potential
20.  MODE AND MECHANISMS OF DEATH FOLLOWING ORTHOTOPIC HEART TRANSPLANTATION 
Background
Ventricular fibrillation (VF) is the primary mechanism of cardiac arrest in the vast majority of sudden death patients. Whether similar modes and mechanisms of death can be generalized to denervated hearts in orthotopic heart transplantation (OHT) patients is unknown.
Objective
The purpose of this study was to determine the mode and mechanisms of death in patients who have undergone cardiac transplantation.
Methods
We analyzed the outcomes of 628 patients who underwent OHT between January 1994 and December 2004. The mode of death was classified as either sudden death (SD) or non-sudden death (NSD). The first documented rhythm taken at the time of arrest was also reviewed to determine the mechanism of cardiac arrest.
Results
During a mean follow up of 76 months, 194 patients expired. Of these, the mode of death could be determined in 116 patients (60%). Forty-one patients (35%) died from SD and 75 patients (65%) died of NSD. The first documented rhythm of death was available in 91 patients (26 SD and 65 NSD). The terminal rhythms in patients who died suddenly were: asystole (34%), pulseless electrical activity (PEA) (20%), and VF (10%). In NSD patients, the terminal rhythms were asystole (73%), followed by VF (7%), and PEA (7%), p<0.001 compared to SD patients.
Conclusion
SD represented the mode of death in 35% of OHT patients. The main mechanisms underlying SD in this population were asystole and PEA, suggesting that denervation of the donor heart, among other post-transplant changes, may alter susceptibility to VF.
doi:10.1016/j.hrthm.2009.01.005
PMCID: PMC3766739  PMID: 19324311
heart transplantation; sudden death; ventricular fibrillation
21.  The CD44high Tumorigenic Subsets in Lung Cancer Biospecimens Are Enriched for Low miR-34a Expression 
PLoS ONE  2013;8(9):e73195.
Cellular heterogeneity is an integral part of cancer development and progression. Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including “de-differentiation” to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials). We observed that many biomarkers that are used to identify Cancer Stem Cells (CSC) can label cell subsets in an advanced clinical stage of lung cancer (malignant pleural effusions, or MPE). Thus, CSC-biomarkers may be useful for live sorting functionally distinct cell subsets from individual tumors, which may enable investigators to hone in on the molecular basis for functional heterogeneity. We demonstrate that the CD44hi (CD44-high) cancer cell subsets display higher clonal, colony forming potential than CD44lo cells (n = 3) and are also tumorigenic (n = 2/2) when transplanted in mouse xenograft model. The CD44hi subsets express different levels of embryonal (de-differentiation) markers or chromatin regulators. In archived lung cancer tissues, ALDH markers co-localize more with CD44 in squamous cell carcinoma (n = 5/7) than Adeno Carcinoma (n = 1/12). MPE cancer cells and a lung cancer cell line (NCI-H-2122) exhibit chromosomal abnormalities and 1p36 deletion (n = 3/3). Since miR-34a maps to the 1p36 deletion site, low miR-34a expression levels were detected in these cells. The colony forming efficiency of CD44hi cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples. In addition the highly tumorigenic CD44hi cells are enriched for cells in the G2 phase of cell cycle.
doi:10.1371/journal.pone.0073195
PMCID: PMC3760902  PMID: 24019906
22.  Elevated neutrophil gelatinase-associated lipocalin contributes to erlotinib resistance in non-small cell lung cancer 
Purpose: The EGFR tyrosine kinase inhibitors (TKIs) demonstrate efficacy in NSCLC patients whose tumors harbor activating EGFR mutations. However, patients who initially respond to EGFR TKI treatment invariably develop resistance to the drugs. Known mechanisms account for approximately 70% of native and acquired EGFR TKI resistance. In the current study we investigated a novel mechanism of NSCLC resistance to erlotinib. Experimental Design: The mechanisms of acquired erlotinib resistance were evaluated by microarray analysis in thirteen NSCLC cell lines and in vivo in mice. Correlations between plasma neutrophil gelatinase associated lipocalin (NGAL) levels, erlotinib response and the EGFR mutational status were assessed in advanced stage NSCLC patients treated with erlotinib. Results: In 5 of 13 NSCLC cell lines NGAL was significantly upregulated. NGAL knockdown in erlotinib-resistant cells increased erlotinib sensitivity in vitro and in vivo. NGAL overexpression in erlotinib-sensitive cells augmented apoptosis resistance. This was mediated by NGAL-dependent modulation of the pro-apoptotic protein Bim levels. Evaluation of the plasma NGAL levels in NSCLC patients that received erlotinib revealed that patients with lower baseline NGAL demonstrated a better erlotinib response. Compared to patients with wild type EGFR, patients with activating EGFR mutations had lower plasma NGAL at baseline and weeks 4 and 8. Conclusions: Our studies uncover a novel mechanism of NGAL-mediated modulation of Bim levels in NSCLC that might contribute to TKI resistance in lung cancer patients. These findings provide the rationale for the further investigations of the utility of NGAL as a potential therapeutic target or diagnostic biomarker.
PMCID: PMC3745436  PMID: 23977408
Lung cancer; effectors of apoptosis; survival factors; EGFR; erlotinib resistance
23.  Neural Control of Ventricular Rate in Ambulatory Dogs with Pacing Induced Sustained Atrial Fibrillation 
Background
We hypothesize that inferior vena cava-inferior atrial ganglionated plexus nerve activity (IVC-IAGPNA) is responsible for the ventricular rate (VR) control during atrial fibrillation (AF) in ambulatory dogs.
Methods and Results
We recorded bilateral cervical vagal nerve activity (VNA) and IVC-IAGPNA during baseline sinus rhythm and during pacing-induced sustained AF in 6 ambulatory dogs. Integrated nerve activities and average VR were measured every 10-s over 24-hour periods. LVNA was associated with VR reduction during AF in 5 dogs (from 211 bpm, 95% confidence interval [CI], 186 to 233 to 178 bpm [95% CI, 145 to 210], p<0.001) and RVNA in 1 dog (208 bpm [95% CI, 197 to 223] to 181 bpm [95% CI, 163 to 200], p<0.01). There were good correlations between IVC-IAGPNA and LVNA in the former 5 dogs, and between IVC-IAGPNA and RVNA in the latter dog. IVC-IAGPNA was associated with VR reduction in all dogs studied. RVNA was associated with baseline sinus rate reduction from 105 bpm (95% CI, 95 to 116) to 77 bpm (95% CI, 64 to 91, p<0.01) in 4 dogs while LVNA was associated with sinus rate reduction from 111 bpm (95% CI, 90 to 1250) to 81 bpm (95% CI, 67 to 103, p<0.01) in 2 dogs.
Conclusions
IVC-IAGPNA is invariably associated with VR reduction during AF. In comparison, right or left VNA was associated with VR reduction only when it co-activates with the IVC-IAGPNA. The vagus nerve that controls VR during AF may be different than that controls sinus rhythm.
doi:10.1161/CIRCEP.111.967737
PMCID: PMC3405983  PMID: 22586260
atrial fibrillation; atrioventricular node; ECG; nervous system, autonomic; ventricular rate
24.  Acute Myocardial Infarction Induces Bilateral Stellate Ganglia Neural Remodeling in Rabbits 
Cardiovascular Pathology  2011;21(3):143-148.
Introduction
Myocardial infarction (MI) results in cardiac nerve sprouting in the myocardium. Whether or not similar neural remodeling occurs in the stellate ganglia (SG) is unknown. We aimed to test the hypothesis that MI induces bilateral SG nerve sprouting.
Methods
Acute MI was created by coronary artery ligation in rabbits (n=12). Serum nerve growth factor (NGF) level was measured by enzyme-linked immunosorbent assay (ELISA). The hearts and bilateral SGs were harvested for immunohistochemistry after 1 week in 6 rabbits, and after 1 month in 6 rabbits. Immunostaining for tyrosine hydroxylase (TH), growth-associated protein 43 (GAP43), cholineacetyltransferase (ChAT) and synaptophysin (SYN) was performed to determine the magnitude of nerve sprouting. Tissues from 6 normal rabbits were used as controls. Nerve density was determined by computerized morphometry.
Results
MI results in increased serum NGF levels at 1 week (1519.8±632.2 ng/ml) that persists to 1 month (1361.2±176.3 ng/ml) as compared to controls (89.6±34.9 ng/ml), (p=.0002, and , p=.0001, respectively). Immunostaining demonstrated nerve sprouting and hyperinnervation in both SGs after MI. The nerve densities (µm2/ganglion cell) in SG 1 week after MI, 1 month after MI and in control groups, respectively, were: GAP43, 278±96, 225±39 and 149±57 (p=.01); SYN, 244±152, 268±115 and 102±60 (p=.02); TH, 233±71, 180±50 and 135±68 (p=.047); ChAT, 244±100, 208±46 and 130±41 µm2/cell (p=.01).
Conclusions
MI increases serum NGF levels and induces nerve sprouting and hyperinnervation in bilateral SGs for at least 1 month after MI. The hyperinnervation includes both postganglionic adrenergic axons and preganglionic cholinergic axons in the SG.
doi:10.1016/j.carpath.2011.08.001
PMCID: PMC3267867  PMID: 22001051
Myocardial Infarction; Ventricular Arrhythmia; Autonomic Nervous System; Stellate Ganglion; Nerve Sprouting; Sudden Cardiac Death
25.  IL-1β is Crucial for Induction of Coronary Artery Inflammation in a Mouse Model of Kawasaki Disease 
Circulation  2012;125(12):1542-1550.
Background
Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease in US children. Untreated, children may develop coronary artery aneurysms, myocardial infarction and sudden death as a result of the illness. Up to a third of KD patients fail to respond to intravenous gammaglobulin (IVIG), the standard therapy, and alternative treatments are being investigated. Genetic studies have indicated a possible role for IL-1β in KD. We therefore explored the role of IL-1β in a murine model of KD.
Methods and Results
Using an established mouse model of KD that involves injection of Lactobacillus casei cell wall extract (LCWE), we investigated the role of IL- 1β and caspase-1 (activated by the inflammasome and required for IL-1β maturation) in coronary arteritis, and evaluated the efficacy of IL-1 receptor antagonist (IL-1Ra) as a potential treatment. LCWE-induced IL-1β maturation and secretion was dependent on the NLRP3 inflammasome in macrophages. Both caspase1-deficient and IL-1R-deficient mice were protected from LCWE-induced coronary lesions. Injection of recombinant IL-1β to caspase-1-deficient mice restored the ability of LCWE to cause coronary lesions in response to LCWE. Furthermore, daily injections of the IL-1Ra prevented LCWE-mediated coronary lesions, up to three days after LCWE injection.
Conclusions
Our results strongly suggest that caspase-1 and IL-1β play critical roles in the development of coronary lesions in this KD mouse model, blocked by IL-1Ra. Therefore, anti-IL-1β treatment strategies may constitute an effective, more targeted treatment of KD to prevent coronary lesions.
doi:10.1161/CIRCULATIONAHA.111.072769
PMCID: PMC3337219  PMID: 22361326
Kawasaki disease

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