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Regenerating the injured heart remains one of the most vexing challenges in cardiovascular medicine. Cell therapy has shown potential for treatment of myocardial infarction, but low cell retention so far has limited its success. Here we show that intramyocardial injection of highly apoptosis-resistant unrestricted somatic stem cells (USSC) into infarcted rat hearts resulted in an unprecedented thickening of the left ventricular wall with cTnT

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

Myocardial infarction (MI) activates the epicardium to form epicardial stromal cells (EpiSC) that reside in the epicardial hypoxic microenvironment. Paracrine factors secreted by EpiSC were shown to modulate the injury response of the post-MI heart and improve cardiac function. We have previously reported that the expression of the angiogenic cytokines vascular endothelial growth factor A (VEGFA) and IL-6 is strongly upregulated in EpiSC by adenosine acting via the A

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of WT1+ cells the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, that can be classified in three groups each containing a cluster of proliferating cells. Two groups expressed cardiac specification makers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of HIF-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

Myocardial infarction (MI) activates the epicardium to form epicardial stromal cells (EpiSC) that reside in the epicardial hypoxic microenvironment. Paracrine factors secreted by EpiSC were shown to modulate the injury response of the post-MI heart and improve cardiac function. We have previously reported that the expression of the angiogenic cytokines vascular endothelial growth factor A (VEGFA) and IL-6 is strongly upregulated in EpiSC by adenosine acting via the A2B receptor (A2B R). Since tissue hypoxia is well known to be a potent stimulus for the generation of extracellular adenosine, the present study explored the crosstalk of A2B R activation and hypoxia-hypoxia-inducible factor 1 alpha (HIF-1α) signaling in cultured EpiSC, isolated from rat hearts 5 days after MI. We found substantial nuclear accumulation of HIF-1α after A2B R activation even in the absence of hypoxia. This normoxic HIF-1α induction was PKC-dependent and involved upregulation of HIF-1α mRNA expression. While the influence of hypoxia on adenosine generation and A2B R signaling was only minor, hypoxia and A2B R activation cumulatively increased VEGFA expression. Normoxic A2B R activation triggered an HIF-1α-associated cell-protective metabolic switch and reduced oxygen consumption. HIF-1α targets and negative regulators PHD2 and PHD3 were only weakly induced by A2B R signaling, which may result in a sustained HIF-1α activity. The A2B R-mediated normoxic HIF-1α induction was also observed in cardiac fibroblasts from healthy mouse hearts, suggesting that this mechanism is also functional in other A2B R-expressing cell types. Altogether, we identified A2B R-mediated HIF-1α induction as novel aspect in the HIF-1α-adenosine crosstalk, which modulates EpiSC activity and can amplify HIF-1α-mediated cardioprotection.

Aims Myocardial infarction (MI) leads to activation of cardiac fibroblasts (aCFs) and at the same time induces the formation of epicardium-derived cells at the heart surface. To discriminate between the two cell populations, we elaborated a fast and efficient protocol for the simultaneous isolation and characterization of aCFs and epicardial stromal cells (EpiSCs) from the infarcted mouse heart. Methods and results For the isolation of aCFs and EpiSCs, infarcted hearts (50 min ischaemia/reperfusion) were digested by perfusion with a collagenase-containing medium for only 8 min, while EpiSCs were enzymatically removed from the outside by applying mild shear forces via a motor driven device. Cardiac fibroblasts (CFs) isolated from unstressed hearts served as control. Viability of isolated cells was >90%. Purity of EpiSCs was confirmed by immunofluorescence staining and qPCR of various mesenchymal markers including Wilms-tumor-protein-1. Microarray analysis of CFs, aCFs, and EpiSCs on day 5 post-MI revealed a unique gene expression pattern in the EpiSC fraction, which was enriched for epithelial markers and epithelial to mesenchymal transition-related genes. Compared to aCFs, 336 significantly altered gene entities were identified in the EpiSC fraction. qPCR analysis showed high expression of Serpinb2, Cxcl13, Adora2b, and Il10 in EpiSCs relative to CFs and aCFs. Furthermore, microarray data identified Ddah1 and Cemip to be highly up-regulated in aCFs compared to CFs. Immunostaining of the infarcted heart revealed a unique distribution of Dermokine, Aquaporin-1, Cytokeratin, Lipocalin2, and Periostin within the epicardial cell layer. Conclusions We describe the simultaneous isolation of viable, purified fractions of aCFs and EpiSCs from the infarcted mouse heart. In this study, several differentially expressed markers for aCFs and EpiSCs were identified, underlining the importance of cell separation to study heterogeneity of stromal cells in the healing process after MI.

Background— Noninvasive detection of deep venous thrombi and subsequent pulmonary thromboembolism is a serious medical challenge, since both incidences are difficult to identify by conventional ultrasound techniques. Methods and Results— Here, we report a novel technique for the sensitive and specific identification of developing thrombi using background-free 19 F magnetic resonance imaging, together with α2-antiplasmin peptide (α2 AP )–targeted perfluorocarbon nanoemulsions (PFCs) as contrast agent, which is cross-linked to fibrin by active factor XIII. Ligand functionality was ensured by mild coupling conditions using the sterol-based postinsertion technique. Developing thrombi with a diameter 1 H and 19 F magnetic resonance images at 9.4 T with both excellent signal-to-noise and contrast-to-noise ratios (71±22 and 17±5, respectively). Furthermore, α2 AP -PFCs could be successfully applied for the diagnosis of experimentally induced pulmonary thromboembolism. In line with the reported half-life of factor XIIIa, application of α2 AP -PFCs >60 minutes after thrombus induction no longer resulted in detectable 19 F magnetic resonance imaging signals. Corresponding results were obtained in ex vivo generated human clots. Thus, α2 AP -PFCs can visualize freshly developed thrombi that might still be susceptible to pharmacological intervention. Conclusions— Our results demonstrate that 1 H/ 19 F magnetic resonance imaging, together with α2 AP -PFCs, is a sensitive, noninvasive technique for the diagnosis of acute deep venous thrombi and pulmonary thromboemboli. Furthermore, ligand coupling by the sterol-based postinsertion technique represents a unique platform for the specific targeting of PFCs for in vivo 19 F magnetic resonance imaging.