Several Cre recombinase transgenic mouse models have been generated for cardiac fibroblast (CF) tracking and heart regulation. However, there is still no consensus on the ideal mouse model to optimally identify and/or regulate these cells. Here, a comparative evaluation of the efficiency and specificity of the indirect reporter Cre-loxP system was carried out in three of the most commonly used fibroblast reporter transgenic mice (Pdgfra-CreERT2, Col1a1-CreERT2 and PostnMCM) under healthy and ischemic conditions, to determine their suitability in in vivo studies of cardiac fibrosis. We demonstrate optimal Cre recombinase activity in CF (but also, although moderate, in endothelial cells (ECs)) derived from healthy and infarcted hearts in the PDGFRa-creERT2 mouse strain. In contrast, no positive reporter signal was found in CF derived from the Col1a1-CreERT2 mice. Finally, in the PostnMCM line, fluorescent reporter expression was specifically detected in activated CF but not in EC, which leads us to conclude that it may be the most reliable model for future studies on cardiovascular disease. Importantly, no lethality or cardiac fibrosis were induced after tamoxifen administration at the established doses, either in healthy or infarcted mice of the three fibroblast reporter lineages. This study lays the groundwork for future efficient in vivo CF tracking and functional analyses.

The use of allogeneic adipose-derived mesenchymal stromal cells (alloADSCs) represents an attractive approach for treating myocardial infarction (MI). Furthermore, adding a natural support improves alloADSCs engraftment and survival in heart tissues, leading to a greater therapeutic effect. We aimed to examine the safety and immunological reaction induced by epicardial implantation of a clinical-grade collagen scaffold (CS) seeded with alloADSCs for its future application in humans. Thus, cellularized scaffolds were myocardially or subcutaneously implanted in immunosuppressed rodent models. The toxicological parameters were not significantly altered, and tumor formation was not found over the short or long term. Furthermore, biodistribution analyses in the infarcted immunocompetent rats displayed cell engraftment in the myocardium but no migration to other organs. The immunogenicity of alloADSC-CS was also evaluated in a preclinical porcine model of chronic MI; no significant humoral or cellular alloreactive responses were found. Moreover, CS cellularized with human ADSCs cocultured with human allogeneic immune cells produced no alloreactive response. Interestingly, alloADSC-CS significantly inhibited lymphocyte responses, confirming its immunomodulatory action. Thus, alloADSC-CS is likely safe and does not elicit any alloreactive immunological response in the host. Moreover, it exerts an immunomodulatory action, which supports its translation to a clinical setting.

Adeno-associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac-specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation-factor-1-alpha or the cardiac-specific troponin-T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. The transduction efficacy and specificity, the time-course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV-TnT, which was significantly higher after intramyocardial injection regardless of ischemia-induction. On the contrary, high hepatic transgene expression levels were detected with the elongation-factor-1-alpha-promoter, independently of the administration route and heart damage. Moreover, tissue-specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.

Single nucleotide polymorphisms (SNPs) may modulate individual susceptibility to carcinogens. We designed a genome-wide association study to characterize individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced non-small cell lung cancer (NSCLC), and we validated our results. We hypothesized that this strategy would enrich the frequencies of the alleles that contribute to the observed traits. We genotyped 2.37 million SNPs in 95 extreme phenotype individuals, that is: heavy smokers that either developed NSCLC at an early age (extreme cases); or did not present NSCLC at an advanced age (extreme controls), selected from a discovery set (n=3631). We validated significant SNPs in 133 additional subjects with extreme phenotypes selected from databases including >39,000 individuals. Two SNPs were validated: rs12660420 (p(combined)=5.66x10(-5); ORcombined=2.80), mapping to a noncoding transcript exon of PDE10A; and rs6835978 (p(combined)=1.02x10(-4); ORcombined=2.57), an intronic variant in ATP10D. We assessed the relevance of both proteins in early-stage NSCLC. PDE10A and ATP10D mRNA expressions correlated with survival in 821 stage I-II NSCLC patients (p=0.01 and p<0.0001). PDE10A protein expression correlated with survival in 149 patients with stage I-II NSCLC (p=0.002). In conclusion, we validated two variants associated with extreme phenotypes of high and low risk of developing tobacco-induced NSCLC. Our findings may allow to identify individuals presenting high and low risk to develop tobacco-induced NSCLC and to characterize molecular mechanisms of carcinogenesis and resistance to develop NSCLC.

PET/CT imaging of 18F-FDG-labeled CSC allows quantifying biodistribution and acute retention of implanted cells in a clinically relevant pig model of chronic myocardial infarction. Similar levels of acute retention are achieved when cells are IM or IC administered. However, acute cell retention does not correlate with cell engraftment, which is improved by IM injection.

Background & Aims: Cardiotrophin-1 (CT-1) is a hepatoprotective cytokine that modulates fat and glucose metabolism in muscle and adipose tissue. Here we analyzed the changes in hepatic fat stores induced by recombinant CT-1 (rCT-1) and its therapeutic potential in non-alcoholic fatty liver disease (NAFLD). Methods: rCT-1 was administered to two murine NAFLD models: ob/ob and high fat diet-fed mice. Livers were analyzed for lipid composition and expression of genes involved in fat metabolism. We studied the effects of rCT-1 on lipogenesis and fatty acid (FA) oxidation in liver cells and the ability of dominant negative inhibitor of AMP-activated protein kinase (AMPK) to block these effects. Results: CT-1 was found to be upregulated in human and murine steatotic livers. In two NAFLD mouse models, treatment with rCT-1 for 10 days induced a marked decrease in liver triglyceride content with augmented proportion of poly-unsaturated FA and reduction of monounsaturated species. These changes were accompanied by attenuation of inflammation and improved insulin signaling. Chronic administration of rCT-1 caused downregulation of lipogenic genes and genes involved in FA import to hepatocytes together with amelioration of ER stress, elevation of NAD(+)/ NADH ratio, phosphorylation of LKB1 and AMPK, increased expression and activity of sirtuin1 (SIRT1) and upregulation of genes mediating FA oxidation. rCT-1 potently inhibited de novo lipogenesis and stimulated FA oxidation in liver cells both in vitro and in vivo. In vitro studies showed that these effects are mediated by activated AMPK. Conclusions: rCT-1 resolves hepatic steatosis in obese mice by mechanisms involving AMPK activation. rCT-1 deserves consideration as a potential therapy for NAFLD. (c) 2013 European Association for the Study of the Liver.

Cardiotrophin-1 (CT-1) is a cytokine with antiobesity properties and with a role in lipid metabolism regulation and adipose tissue function. The aim of this study was to analyze the molecular mechanisms involved in the lipolytic actions of CT-1 in adipocytes. Recombinant CT-1 (rCT-1) effects on the main proteins and signaling pathways involved in the regulation of lipolysis were evaluated in 3T3-L1 adipocytes and in mice. rCT-1 treatment stimulated basal glycerol release in a concentration- and time-dependent manner in 3T3-L1 adipocytes. rCT-1 (20 ng/ml for 24 h) raised cAMP levels, and in parallel increased protein kinase (PK)A-mediated phosphorylation of perilipin and hormone sensitive lipase (HSL) at Ser660. siRNA knock-down of HSL or PKA, as well as pretreatment with the PKA inhibitor H89, blunted the CT-1-induced lipolysis, suggesting that the lipolytic action of CT-1 in adipocytes is mainly mediated by activation of HSL through the PKA pathway. In ob/ob mice, acute rCT-1 treatment also promoted PKA-mediated phosphorylation of perilipin and HSL at Ser660 and Ser563, and increased adipose triglyceride lipase (desnutrin) content in adipose tissue. These results showed that the ability of CT-1 to regulate the activity of the main lipases underlies the lipolytic action of this cytokine in vitro and in vivo, and could contribute to CT-1 antiobesity effects.

A pre-emulsified mixture of linseed and algae oils (15/10) and stabilized with 686 ppm of a lyophilized water extract of Melissa officinalis, was successfully applied in dry fermented sausages to increase the ¿-3 PUFA content. The objective of this work was to evaluate the stability of this modified formulation during the storage and to compare it to that of a traditional formulation. Traditional and modified products were stored during 90 days at 4 ºC in aerobic conditions. Fatty acid profiles, TBARS and volatile compounds derived from oxidation were analyzed at 0, 30 and 90 days of storage. The fatty acid profiles did not significantly change along the storage period. The stabilizing effect of the natural antioxidants of M. officinalis could contribute to detect no losses of ¿-3 PUFA in Modified (30 days: 2.13 g/100 g of product, 90 days: 2.33 g/100 g of product), whereas in Control products a slightly significant reduction was detected (30 days: 0.34 g/100 g of product, 90 days: 0.29 g/100 g of product). After 90 days, the increases of TBARS and hexanal content were much higher in Control than in Modified (Control: 1.41 mg MDA/kg & 17,915 ng dodecane/kg of dry matter; Modified: 0.48 mg MDA/kg & 2,496 ng dodecane/kg of dry matter). In conclusion, the lyophilized water extract of M. officinalis protected high ¿-3 PUFA of dry fermented sausages from oxidation along the storage time, guaranteeing the nutritional improvements achieved with the modified formulation.

Cardiotrophin-1 (CT-1/CTF1) is a member of the interleukin-6 (IL-6) family of cytokines that stimulates STAT-3 phosphorylation in cells bearing the cognate receptor. We report that Ctf1(-/-) mice (hereby referred to as CT-1(-/-) mice) are resistant to the hepatic engraftment of MC38 colon carcinoma cells, while these cells engraft normally in the mouse subcutaneous tissue. Tumor intake in the liver could be enhanced by the systemic delivery of a recombinant adenovirus encoding CT-1, which also partly rescued the resistance of CT-1(-/-) mice to the hepatic engraftment of MC38 cells. Moreover, systemic treatment of wild-type (WT) mice with a novel antibody-neutralizing mouse CT-1 also reduced engraftment of this model. Conversely, experiments with Panc02 pancreatic cancer and B16-OVA melanoma cells in CT-1(-/-) mice revealed rates of hepatic engraftment similar to those observed in WT mice. The mechanism whereby CT-1 renders the liver permissive for MC38 metastasis involves T lymphocytes and natural killer (NK) cells, as shown by selective depletion experiments and in genetically deficient mice. However, no obvious changes in the number or cell killing capacity of liver lymphocytes in CT-1(-/-) animals could be substantiated. These findings demonstrate that the seed and soil concept to understand metastasis can be locally influenced by cytokines as well as by the cellular immune system