Etiology-independent activation of the LTß-LTßR-RELB axis drives aggressiveness and predicts poor prognosis in HCC.

BACKGROUND AND AIMS: HCC is the most common primary liver tumor, with an increasing incidence worldwide. HCC is a heterogeneous malignancy and usually develops in a chronically injured liver. The NF-κB signaling network consists of a canonical and a noncanonical branch. Activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of noncanonical NF-κB and its downstream effectors is not established. APPROACH AND RESULTS: Four human HCC cohorts (total n = 1462) and 4 mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro , NF-κB signaling, proliferation, and cell death were measured, proving a pro-proliferative role of v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) activated by means of NF-κB-inducing kinase. In vivo , lymphotoxin beta was identified as the predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico , RELB activity and RELB-directed transcriptomics were validated on the The Cancer Genome Atlas HCC cohort using inferred protein activity and Gene Set Enrichment Analysis. In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to v-rel avian reticuloendotheliosis viral oncogene homolog A, nuclear enrichment of noncanonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence. CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent, and cross-species consistent activation of a lymphotoxin beta/LTßR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation.

Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling.

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.

Subclinical hepatitis E virus infection in laboratory ferrets in the UK.

Ferrets are widely used for experimental modelling of viral infections. However, background disease in ferrets could potentially confound intended experimental interpretation. Here we report the detection of a subclinical infection of ferret hepatitis E virus (FRHEV) within a colony sub-group of female laboratory ferrets that had been enrolled on an experimental viral infection study (non-hepatitis). Lymphoplasmacytic cuffing of periportal spaces was identified on histopathology but was negative for the RNA and antigens of the administered virus. Follow-up viral metagenomic analysis conducted on liver specimens revealed sequences attributed to FRHEV and these were confirmed by reverse-transcriptase polymerase chain reaction. Further genomic analysis revealed contiguous sequences spanning 79-95 % of the FRHEV genome and that the sequences were closely related to those reported previously in Europe. Using in situ hybridization by RNAScope, we confirmed the presence of HEV-specific RNA in hepatocytes. The HEV open reading frame 2 (ORF2) protein was also detected by immunohistochemistry in the hepatocytes and the biliary canaliculi. In conclusion, the results of our study provide evidence of background infection with FRHEV in laboratory ferrets. As this infection can be subclinical, we recommend routine monitoring of ferret populations using virological and liver function tests to avoid incorrect causal attribution of any liver disease detected in in vivo studies.

Promotion of cholangiocarcinoma growth by diverse cancer-associated fibroblast subpopulations.

Cancer-associated fibroblasts (CAF) are a poorly characterized cell population in the context of liver cancer. Our study investigates CAF functions in intrahepatic cholangiocarcinoma (ICC), a highly desmoplastic liver tumor. Genetic tracing, single-cell RNA sequencing, and ligand-receptor analyses uncovered hepatic stellate cells (HSC) as the main source of CAF and HSC-derived CAF as the dominant population interacting with tumor cells. In mice, CAF promotes ICC progression, as revealed by HSC-selective CAF depletion. In patients, a high panCAF signature is associated with decreased survival and increased recurrence. Single-cell RNA sequencing segregates CAF into inflammatory and growth factor-enriched (iCAF) and myofibroblastic (myCAF) subpopulations, displaying distinct ligand-receptor interactions. myCAF-expressed hyaluronan synthase 2, but not type I collagen, promotes ICC. iCAF-expressed hepatocyte growth factor enhances ICC growth via tumor-expressed MET, thus directly linking CAF to tumor cells. In summary, our data demonstrate promotion of desmoplastic ICC growth by therapeutically targetable CAF subtype-specific mediators, but not by type I collagen.

Enhanced prognostic stratification of neoadjuvant treated lung squamous cell carcinoma by computationally-guided tumor regression scoring.

INTRODUCTION: The amount of residual tumor burden after neoadjuvant chemotherapy is an important prognosticator, but for non-small cell lung carcinoma (NSCLC), no official regression scoring system is yet established. Computationally derived histological regression scores could provide unbiased and quantitative readouts to complement the clinical assessment of treatment response. METHODS: Histopathologic tumor regression was microscopically assessed on whole cases in a neoadjuvant chemotherapy-treated cohort (NAC, n = 55 patients) of lung squamous cell carcinomas (LSCC). For each patient, the slide showing the least pathologic regression was selected for subsequent computational analysis and histological features were quantified: percentage of vital tumor cells (cTu.Percentage), total surface covered by vital tumor cells (cTu.Area), area of the largest vital tumor fragment (cTu.Size.max), and total number of vital tumor fragments (cTu.Fragments). A chemo-naïve LSCC cohort (CN, n = 104) was used for reference. For 23 of the 55 patients [18F]-Fluorodeoxyglucose (FDG) PET/CT measurements of maximum standard uptake value (SUVmax), background subtracted lesion activity (BSL) and background subtracted volume (BSV) were correlated with pathologic regression. Survival analysis was carried out using Cox regression and receiver operating characteristic (ROC) curve analysis using a 3-years cutoff. RESULTS: All computational regression parameters significantly correlated with relative changes of BSV FDG PET/CT values after neoadjuvant chemotherapy. ROC curve analysis of histological parameters of NAC patients showed that cTu.Percentage was the most accurate prognosticator of overall survival (ROC curve AUC = 0.77, p-value = 0.001, Cox regression HR = 3.6, p = 0.001, variable cutoff < = 30 %). CONCLUSIONS: This study demonstrates the prognostic relevance of computer-derived histopathologic scores. Additionally, the analysis carried out on slides displaying the least pathologic regression correlated with overall pathologic response and PET/CT values. This might improve the objective histopathologic assessment of tumor response in neoadjuvant setting.

Prognostic Immune Cell Profiling of Malignant Pleural Effusion Patients by Computerized Immunohistochemical and Transcriptional Analysis.

Malignant pleural effusion (MPE) is a severe condition of advanced tumors without effective therapy. We used digitalized immunohistochemical and transcriptional approaches to investigate the prognostic influence of immune cells and expression variance of associated immunomodulatory molecules in MPE. Cytology tissue microarrays were constructed from MPE cell blocks of 155 patients with five tumor entities. Immune cells lineage markers were quantified by computational cytopathology on immunohistochemistry. mRNA expression analysis of nine lineage markers and 17 immunomodulators was performed by NanoString. Immunohistochemically quantified high B cells to leukocytes ratio (hazard ratio (HR) = 0.70, p-value = 0.043) and low neutrophils to leukocytes ratio (HR = 1.78, p-value = 0.003) were favorable prognosticators for overall survival independent of tumor entity. Correspondingly, patients with high B cells but low neutrophils gene expression signature showed longer median overall survival of 500 days (HR = 2.29, p-value = 0.009). Regarding targetable molecule expressions, lung adenocarcinomas were characterized by high PD-L1, but mesothelioma by high LAG-3. Ovarian carcinoma was least immunogenic. Independent of tumor entity, the condition of the immune system in MPE liquids is able to provide additional prognostic cytologic information. Combined analysis of lineage specific markers and related immunomodulators may direct immune-based therapeutic decisions.

Microfluidics-assisted multiplexed biomarker detection for in situ mapping of immune cells in tumor sections.

Because of the close interaction between tumors and the immune system, immunotherapies are nowadays considered as the most promising treatment against cancer. In order to define the diagnosis and the subsequent therapy, crucial information about the immune cells at the tumor site is needed. Indeed, different types or activation status of cells may be indicative for specific and personalized treatments. Here, we present a quantitative method to identify ten different immuno-markers in the same tumor cut section, thereby saving precious samples and enabling correlative analysis on several cell families and their activation status in a tumor microenvironment context. We designed and fabricated a microfluidic chip with optimal thermomechanical and optical properties for fast delivery of reagents on tissue slides and for fully automatic imaging by integration with an optical microscope. The multiplexing capability of the system is enabled by an optimized cyclic immunofluorescence protocol, with which we demonstrated quantitative sequential immunostaining of up to ten biomarkers on the same tissue section. Furthermore, we developed high-quality image-processing algorithms to map each cell in the entire tissue. As proof-of-concept analyses, we identified coexpression and colocalization patterns of biomarkers to classify the immune cells and their activation status. Thanks to the quantitativeness and the automation of both the experimental and analytical methods, we believe that this multiplexing approach will meet the increasing clinical need of personalized diagnostics and therapy in cancer pathology.

Microfluidic-Based Immunohistochemistry Combined With Next-Generation Sequencing on Diagnostic Tissue Sections for Detection of Tumoral BRAF V600E Mutation.

OBJECTIVES: Tailored diagnostics requires immunohistochemistry (IHC) and next generation sequencing (NGS). Here we combined on a single paraffin-embedded slide microfluidic-based IHC (micro-IHC) and NGS for BRAF V600E mutation detection in BRAFomas. METHODS: For micro-IHC, we performed the primary antibody incubation step of conventional chromogenic IHC in a LabSat device (Lunaphore Technologies SA). Tumor areas immunoreactive for pan-cytokeratin, pan-melanoma, and BRAF V600E mutation-specific antibody were H-scored, microdissected, and analyzed by NGS. RESULTS: After 2 minutes, pan-cytokeratin and BRAF micro-IHC increased exponentially (half-time values: 1.7 and 3.2 minutes). Pan-melanoma displayed a higher half-time value of 15 minutes. There was no significant difference in H-score and staining quality, respectively, between conventional and micro-IHC. BRAF V600E mutation was detected in all pan-cytokeratin and pan-melanoma stained samples without amplification but in only 40% of BRAF V600E stained samples with amplification. CONCLUSIONS: Micro-IHC enables short antibody incubation times and subsequent NGS. Preprocessing is critical for preservation of DNA quality.

Microfluidics-based immunofluorescence for fast staining of ALK in lung adenocarcinoma.

BACKGROUND: Anaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed. The microfluidic tissue processor (MTP) device is based on a chip-confined low-volume technology allowing for rapid immunohistochemistry/immunofluorescence (IHC/IF) stainings of formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples. METHODS: A novel ALK IF protocol was developed for the MTP device using the primary mouse anti-human ALK antibody clone 5A4. FFPE tumor whole sections from 14 resected lung adenocarcinoma patients documented to be ALK positive (ALK+) by automated chromogenic IHC and/or FISH were used. MTP-derived IF immunoreactivity was measured by computerized analysis of digitalized images on individual frames of tumor epithelia and surrounding stroma, using an ImageJ plug-in. RESULTS: The 5A4 antibody yielded saturated immunoreactivity at an incubation time of 4 min on a titration curve ranging from 2 to 32 min. Total staining time on the MTP device was 18 min including secondary IgG Alexa Fluor 647. MTP-based ALK IF confirmed all 12 cases; with epithelial signal above stromal staining based on computerized pixel-based measurement. MTP-IF (mean intensity levels 458 to 1301) and chromogenic IHC (H-score 120 to 300) showed an equal range of variation of 2.8 and 2.5 folds, respectively, and a trend for direct correlation (p-value 0.051). CONCLUSION: The newly developed protocol for immunofluorescent detection of ALK protein with the MTP device confirms chromogenic IHC results on FFPE lung adenocarcinoma specimens. MTP-based IF is fast and reliable. We foresee this study to be a first step opening the road for further realization of microfluidic-based assays for rapid simultaneous detection of targetable oncogenic and immune-system related markers in their topographical context to investigate tumour heterogeneity and micro-environmental interactions.

Selective M2 Macrophage Depletion Leads to Prolonged Inflammation in Surgical Wounds.

BACKGROUND: A prolonged inflammatory phase is seen in aberrant wound healing and in chronic wounds. Macrophages are central to wound healing. Distinct macrophage subtypes have differing roles both in initial inflammation and in later tissue repair. Broadly, these cells can be divided into M1 and M2 macrophages. M2 macrophage proliferation and differentiation is regulated by colony-stimulating factor 1 (CSF-1) signalling and can be blocked by GW2580, a competitive cFMS kinase inhibitor, thereby allowing for analysis of the effect of M2 blockade on progression of surgical wounds. MATERIALS AND METHODS: Macrophage Fas-induced apoptosis (MaFIA) transgenic mice with a macrophage-specific promoter used to express green fluorescent protein (GFP) were used to allow for cell tracking. The animals were treated by oral gavage with GW2580. Surgical wounds were created and harvested after 2 weeks for analysis. RESULTS: GW2580-treated mice had significantly more GFP+ cells in the surgical scar than vehicle-treated animals (GW2580, 68.0 ± 3.1%; vehicle, 42.8 ± 1.7%; p < 0.001), and GW2580 treatment depleted CD206+ M2 macrophages in the scar (GW2580, 1.4%; vehicle, 19.3%; p < 0.001). Treated animals showed significantly higher numbers of neutrophils (vehicle, 18.0%; GW2580, 51.3%; p < 0.01) and M1 macrophages (vehicle, 3.8%; GW2580, 12.8%; p < 0.01) in the scar compared to vehicle-treated animals. The total collagen content in the area of the scar was decreased in animals treated with GW2580 as compared to those treated with vehicle alone (GW2580, 67.1%; vehicle, 79.9%; p < 0.005). CONCLUSIONS: Depletion of M2 macrophages in surgical wounds via CSF-1 signalling blockade leads to persistent inflammation, with an increase in neutrophils and M1 macrophages and attenuated collagen deposition.

A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.

Systemic and Cardiac Depletion of M2 Macrophage through CSF-1R Signaling Inhibition Alters Cardiac Function Post Myocardial Infarction.

The heart hosts tissue resident macrophages which are capable of modulating cardiac inflammation and function by multiple mechanisms. At present, the consequences of phenotypic diversity in macrophages in the heart are incompletely understood. The contribution of cardiac M2-polarized macrophages to the resolution of inflammation and repair response following myocardial infarction remains to be fully defined. In this study, the role of M2 macrophages was investigated utilising a specific CSF-1 receptor signalling inhibition strategy to achieve their depletion. In mice, oral administration of GW2580, a CSF-1R kinase inhibitor, induced significant decreases in Gr1lo and F4/80hi monocyte populations in the circulation and the spleen. GW2580 administration also induced a significant depletion of M2 macrophages in the heart after 1 week treatment as well as a reduction of cardiac arginase1 and CD206 gene expression indicative of M2 macrophage activity. In a murine myocardial infarction model, reduced M2 macrophage content was associated with increased M1-related gene expression (IL-6 and IL-1ß), and decreased M2-related gene expression (Arginase1 and CD206) in the heart of GW2580-treated animals versus vehicle-treated controls. M2 depletion was also associated with a loss in left ventricular contractile function, infarct enlargement, decreased collagen staining and increased inflammatory cell infiltration into the infarct zone, specifically neutrophils and M1 macrophages. Taken together, these data indicate that CSF-1R signalling is critical for maintaining cardiac tissue resident M2-polarized macrophage population, which is required for the resolution of inflammation post myocardial infarction and, in turn, for preservation of ventricular function.

Synthetic chemically modified mrna-based delivery of cytoprotective factor promotes early cardiomyocyte survival post-acute myocardial infarction.

To extend the temporal window for cytoprotection in cardiomyocytes undergoing apoptosis after hypoxia and myocardial infarction (MI), a synthetic chemically modified mRNA (modRNA) was used to drive delivery of insulin-like growth factor-1 (IGF1) within the area at risk in an in vivo murine model of MI. Delivery of IGF1 modRNA, with a polyethylenimine-based nanoparticle, augmented secreted and cell-associated IGF1, promoting cardiomyocyte survival and abrogating cell apoptosis under hypoxia-induced apoptosis conditions. Translation of modRNA-IGF1 was sufficient to induce downstream increases in the levels of Akt and Erk phosphorylation. Downregulation of IGF1 specific miRNA-1 and -133 but not miR-145 expression was also confirmed. As a proof of concept, intramyocardial delivery of modRNA-IGF1 but not control modRNA-GFP significantly decreased the level of TUNEL positive cells, augmented Akt phosphorylation, and decreased caspase-9 activity within the infarct border zone 24 h post-MI. These findings demonstrate the potential for an extended cytoprotective effect of transient IGF1 driven by synthetic modRNA delivery.

Potent endothelial progenitor cell-conditioned media-related anti-apoptotic, cardiotrophic, and pro-angiogenic effects post-myocardial infarction are mediated by insulin-like growth factor-1.

AIMS: We have previously reported the cardioprotective effects of endothelial progenitor cell (EPC)-conditioned media (CM) therapy post-myocardial infarction (MI). In the present study, we have determined the insulin-like growth factor-1 (IGF-1) contribution to EPC CM effects on cardiomyocyte survival, contractility, and angiogenesis in vivo. METHODS AND RESULTS: Conditioned media from porcine EPC were administered intracoronary in the presence and absence of specific neutralizing antibodies to IGF-1 or control IgG in a porcine model of MI. X-vivo (non-conditioned) medium was used as a control. Functional, histological, and biochemical parameters were evaluated at 24 h and 8-week post-therapy. Conditioned media therapy significantly abrogated infarct zone (IZ) apoptosis, hypocontractility, and impaired left ventricular (LV) relaxation observed in control infarcts acutely (24 h post-MI). At 8 weeks following treatment, CM therapy augmented LV contractility and relaxation, IZ angiogenesis and inhibited infarct size expansion, wall expansion, and wall thinning. All of these acute and chronic beneficial effects of CM therapy were vitiated by neutralizing antibodies to IGF-1 but not by control IgG. Moreover, the addition of neutralizing IGF-1 antibody to control medium had no effect on these structural or functional changes in the heart post-treatment. CONCLUSION: Insulin-like growth factor-1 within the EPC CM mediates potent acute myocardial repair and chronic remodelling effects post-MI. These findings may provide a rationale for comparative trials of specific growth factors vs. current progenitor cell strategies.

Potent long-term cardioprotective effects of single low-dose insulin-like growth factor-1 treatment postmyocardial infarction.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) is recognized as an important regulator of cardiac structure and cardiomyocyte homeostasis. The prosurvival and antiapoptotic effects of IGF-1 have been investigated in vitro and in rodent models of myocardial infarction (MI). However, the clinical application of IGF-1 has been hampered by dose-dependent side effects both acutely and during chronic administration. We hypothesized that single, low-dose IGF-1 (LD-IGF-1) administered locally and early in the reperfusion phase after acute MI in a large animal model would avoid significant side effects but would have prosurvival effects that would manifest in long-term structural and functional improvement after MI treatment. METHODS AND RESULTS: Forty-four female Landrace pigs underwent intracoronary administration of LD-IGF-1 or saline 2 hours into the reperfusion phase of acute left anterior descending artery occlusion MI. In the area of infarction, IGF-1 receptor and signaling responses were activated at 30 minutes and cardiomyocyte cell death attenuated at 24 hours after LD-IGF-1 but not saline treatment. Hemodynamic and structural studies using pressure-volume loop, CT, and triphenyltetrazolium chloride analysis 2 months post-MI confirmed a marked reduction in infarct size, attenuation of wall thinning, and augmentation of wall motion in the LD-IGF-1-treated but not in the saline-treated animals. These regional structural benefits were associated with global reductions in left ventricular volumes and significant improvement in left ventricular systolic and diastolic function. CONCLUSIONS: One-time LD-IGF-1 effects potent acute myocardial salvage in a preclinical model of left anterior descending artery occlusive MI, extending to long-term benefits in MI size, wall structure, and function and underscoring its potential as an adjunctive therapeutic agent.

Developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered stem cells in a murine model of airway injury.

Interest has increased in the use of exogenous stem cells to optimize lung repair and serve as carriers of a therapeutic gene for genetic airway diseases such as cystic fibrosis. We investigated the survival and engraftment of exogenous stem cells after intratracheal injection, in a murine model of acute epithelial airway injury already used in gene therapy experiments on cystic fibrosis. Embryonic stem cells and mesenchymal stem cells were intratracheally injected 24 hr after 2% polidocanol administration, when epithelial airway injury was maximal. Stem cells were transfected with reporter genes immediately before administration. Reporter gene expression was analyzed in trachea-lungs and bronchoalveolar lavage, using nonfluorescence, quantitative, and sensitive methods. Enzyme-linked immunosorbent assay quantitative results showed that 0.4 to 5.5% of stem cells survived in the injured airway. Importantly, no stem cells survived in healthy airway or in the epithelial lining fluid. Using 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining, transduced mesenchymal stem cells were detected in injured trachea and bronchi lumen. When the epithelium was spontaneously regenerated, the in vivo amount of engrafted mesenchymal stem cells from cell lines decreased dramatically. No stem cells from primary culture were located within the lungs at 7 days. This study demonstrated the feasibility of intratracheal cell delivery for airway diseases with acute epithelial injury.

Cardiac cell therapy: overexpression of connexin43 in skeletal myoblasts and prevention of ventricular arrhythmias.

Cell-based therapies have great potential for the treatment of cardiovascular diseases. Recently, using a transgenic mouse model Roell et al. reported that cardiac engraftment of connexin43 (Cx43)-overexpressing myoblasts in vivo prevents post-infarct arrhythmia, a common cause of death in patients following heart attack. We carried out a similar study but in a clinically relevant context via transplantation of autologous connexin43-overexpressing myoblasts in infarcted rats. Seven days after coronary ligation, rats were randomized into three groups: a control group injected with myoblasts, a null group injected with myoblasts transduced with an empty lentivirus vector (null) and a Cx43 group injected with myoblasts transduced with a lentivirus vector encoding connexin43. In contrast to Roell's report, arrhythmia occurrence was not statistically different between groups (58%, 64% and 48% for the control (n= 12), null (n= 14) and Cx43 (n= 23) groups, respectively, P= 0.92). Using ex vivo intramural monophasic action potential recordings synchronous electrical activity was observed between connexin43-overexpressing myoblasts and host cardiomyocytes, whereas such synchrony did not occur in the null-transduced group. This suggests that ex vivo connexin43 gene transfer and expression in myoblasts improved intercellular electrical coupling between myoblasts and cardiomyocytes. However, in our model such electrical coupling was not sufficient to decrease arrhythmia induction. Therefore, we would suggest a note of caution on the use of combined Cx43 gene and cell therapy to prevent post-infarct arrhythmias in heart failure patients.