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Polyphenols have shown ability to modulate DNA damage, inflammation, and the gut microbiota. Cardoon stalks (Cynara cardunculus var. L. altilis DC) and sweet Italian green peppers fruits (Capsicum annuum L.) are vegetables with different polyphenolic profile (chlorogenic acids vs flavonoids respectively). Thus, the main aim of this project was to evaluate the potential genoprotective and/or anti-inflammatory and prebiotic activity of cardoon stalks and sweet Italian green pepper fruits in the gastrointestinal (GI) tract, by simulating an intake situation. A total of 30 polyphenols were identified and quantified by HPLC-MS/MS in white and red cardoon stalks. Red cardoon almost doubled the polyphenol content of white cardoon, and was therefore selected for the assessment of its biological activity. Besides, the impact of 4 heat treatments (i.e., blanching, different frying conditions, traditional boiling and sous-vide cooking) on the antioxidant capacity and/or the polyphenol profile and content of cardoon stalks, was evaluated. Then, sous-vide cooking was selected for red cardoon, and griddling for green pepper, as they favour a high polyphenol bioaccessibility after GI digestion. Bioaccessible polyphenols of GI-digested green pepper did not show genoprotection against oxidatively generated damage in HT-29 cells, regardless of the application of a heat treatment (griddling). Besides, high polyphenol concentrations caused a slight pro-oxidant effect on DNA. GI-digested raw red cardoon and raw green pepper had

Myocardial infarction (MI) leads to an irreversible loss of cardiac myocytes, which compromises cardiac function. The cellular and molecular mechanisms involved in the ischemic event remain under study, being the cardiac exosomes transfer of non-coding RNAs a key process in this pathology. In this context, a comparative analysis of the exosomal compartment of human cardiac progenitor cells (CPC) was performed in comparison with human bone marrow-mesenchymal stem cells (MSC) and human dermal fibroblasts (HDF). A total of 481 differentially expressed microRNAs (miR) were found, being miR-935 the most differentially expressed in CPC. Analysis of miR-935 in vitro overexpression promoted a positive trend to increment cardiomyocyte survival, in response to oxidative stress. Furthermore, when miR-935 regulation was analyzed in a mouse model of MI disease, miR downregulation was shown five days post-infarct in different cardiac subpopulations. In order to study the role of miR-935 in MI and to achieve an optimal miR-935 overexpression in our mouse model, we developed a robust viral-based method for cardiac-specific miR overexpression. Cardiotropic Serotype 9-Adeno-Associated Virus (AAV9) were used for this purpose and their biodistribution was first determined in mice. Ubiquitous EF1α or the cardiac-specific TnT promoters were combined with Luciferase (Luc) or GFP reporters and administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. 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 EF1α-promoter, independently of the administration route and heart damage. Luc expression increased with both promoters in a time-dependent manner, reaching a peak by day 3-7 that was stable for at least 60 days. Moreover, tissue-specific GFP expression was found in cardiomyocytes with the TnT-vector, while minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that MI greatly increased the transcriptional activity of AAV genomes. Thus, we found AAV9-TnT as a robust and stable vector for cardiac-specific delivery after intramyocardial injection. Finally, the therapeutic potential of this vector in combination with miR-935 was evaluated in a mouse model of myocardial ischemia. Intramyocardial injection of AAV9-TnT-miR935 vector did not significantly improve heart function 60 days post-infarct. However, a slight positive trend in the ejection fraction and a decreased adverse cardiac remodeling are observed, suggesting miR-935 putative cardioprotective role.

Stem cells allow to investigate about the basic mechanisms that regulate embryonic development, cellular plasticity, and organ maintenance and regeneration. Induced pluripotent stem cells (iPSCs) are a powerful source of cells for diverse applications such as developmental and disease modeling, drug discovery and regenerative medicine. Cardiac development is coordinated by complex interactions between cardiac progenitor cell populations, different molecular signaling pathways, and spatially and temporally regulated gene expression. Cardiovascular progenitors (CVPs), with similar potential to these present in early stages of embryonic development, can be obtained from iPSCs by mimicking signaling during cardiogenesis, creating an ideal cell source to treat the damaged heart. However, the mechanisms and conditions for long-term self-renewal and maintenance of CVPs remain elusive. The generation of new iPSC models for tracing CVP lineages permits to delve into the biology of CVPs and discover novel potential regulators of their fate. We have established three different Cre/LoxP mouse models for lineage tracing of CVPs and their cell progeny by the expression of ZsGreen (ZsG) protein: Ai6-Mesp1-Cre (Mesp1 tracer), Ai6-Isl1-Cre (Isl1 tracer) and Ai6-Mef2c-AHF-Cre (AHF tracer) mice. Multiple iPSC clones have been derived from Ai6-Isl1-Cre and Ai6-Mef2c-AHF-Cre reporter mice. Several generated iPSC lines have been fully characterized, demonstrating embryonic stem-like features. iPSCs encoded the expected genomic insertions, showed normal karyotypes, transgenes were silenced, and expressed endogenous pluripotency-associated markers. Moreover, iPSCs were capable to differentiate into the three germ layers both in vitro and in vivo. We have verified the utility of established AHFiPSCs to track CVPs and their differentiated progeny. Upon differentiation, ZsG+ cells derived from AHFiPSCs appeared from embryoid body (EB) day 6 onwards, expressed cardiovascular-related markers, and were able to differentiate into cardiomyocytes, endothelial and smooth muscle cells. Comparative gene expression analysis using four different AHFiPSC lines revealed distinct molecular signatures in three particular stages of differentiation: undifferentiated iPSCs (AHFiPS-D0), sorted ZsG+ cells at day 6 (AHFiPS-D6.ZsG+) and sorted ZsG+ cells at day 13 of differentiation (AHFiPS-D13.ZsG+), that expressed pluripotency-, CVP- and cardiac/vascular lineages-associated markers, respectively. Gapdh and Polr2a have been determined the most stable housekeeping genes along the differentiation of AHFiPSCs, being optimal for accurate normalization of gene expression in our samples. We have identified novel regulators of transcription specifically upregulated in CVPs: Lin28a (and its paralog Lin28b), Lhx1 and Nr6a1. The expression of these genes was also found increased in the corresponding CVP-enriched samples from two different public analyses using mouse and human pluripotent stem cells. Moreover, using bioinformatic analysis of biological pathways we have found p53 as an interconnecting molecule of all these selected regulators of transcription. In order to explore novel insights into the biological role of the selected regulators in CVP fate, we have generated an inducible vector (pTRE-CDS-IRES-Puro-REX1-Blast) to carry out gain-of-function (GOF) analyses. This Tet-On system worked properly in AHFiPSC clones, but unfortunately it failed to work in EBs of certain size along differentiation. In contrast, this GOF system correctly functioned in human iPSCs differentiated in monolayer cultures. We have established four CBiPS1sv-4F-5 cell lines carrying Tet-On systems for the inducible expression of LIN28A, LIN28B, NR6A1 and LHX1, and preliminary results indicated that these regulators of transcription might have a role in CVP fate determination.