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.
Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 expression and how its ...production is regulated are largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. We found that IL-6, measured in various cell types in post-MI hearts at the protein level and by quantitative PCR and RNAscope, was preferentially formed by cardiac fibroblasts (CFs). Single-cell RNA-Seq (scRNA-Seq) in infarcted mouse and human hearts confirmed this finding. We found that adenosine stimulated fibroblast IL-6 formation via the adenosine receptor A2bR in a Gq-dependent manner. CFs highly expressed Adora2b and rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans, scRNA-Seq revealed that Adora2B was also mainly expressed by fibroblasts. We assessed global IL-6 production in isolated hearts from mice lacking CD73 on T cells (CD4-CD73-/-), a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4-CD73-/- mice, suggesting adenosine-mediated modulation. Together, these findings demonstrate that post-MI IL-6 was mainly derived from activated CFs and was controlled by T cell-derived adenosine. We show that purinergic metabolic cooperation between CFs and T cells is a mechanism that modulates IL-6 formation by the heart and has therapeutic potential.
To investigate the tolerability and safety of two sublingual tree pollen extracts approved in 2018, a non-interventional study (NIS) was performed. This NIS was an 8-month observational study ...conducted at 84 sites throughout Germany. Study participants received either a sublingual liquid allergen extract of birch pollen (SBPE) or a liquid allergen extract consisting of a mixture of birch, hazel, and alder tree pollen (STPE). Data from 432 patients were analyzed for the occurrence of adverse events and patient compliance. At least one local reaction occurred in 69 (22.2%) patients, whereas systemic reactions were only observed in 27 (6.3%) patients. STPE-treated patients developed systemic reactions more frequently than SBPE-treated patients (SBPE: 9 (4.3%) vs. STPE: 18 (8.0%)). Only one patient developed a systemic grade III reaction. Severe systemic grade IV reactions were not observed. A total of 348 (98.6%) of the patients who completed all visits were satisfied or very satisfied with the sublingual immunotherapy (SLIT), and 322 (71%) patients completed all visits. Both investigated products were well tolerated by the patients and demonstrated a good safety profile. AEs were observed less frequently than in the preceding clinical phase III trial, and no new safety concerns were identified.
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 (A2BR). Since tissue hypoxia is well known to be a potent stimulus for the generation of extracellular adenosine, the present study explored the crosstalk of A2BR 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 A2BR 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 A2BR signaling was only minor, hypoxia and A2BR activation cumulatively increased VEGFA expression. Normoxic A2BR 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 A2BR signaling, which may result in a sustained HIF‐1α activity. The A2BR‐mediated normoxic HIF‐1α induction was also observed in cardiac fibroblasts from healthy mouse hearts, suggesting that this mechanism is also functional in other A2BR‐expressing cell types. Altogether, we identified A2BR‐mediated HIF‐1α induction as novel aspect in the HIF‐1α‐adenosine crosstalk, which modulates EpiSC activity and can amplify HIF‐1α‐mediated cardioprotection.
Cardiac fibroblasts (CF) migrate and differentiate into myofibroblasts (activated cardiac fibroblasts ‐ aCF) in response to myocardial infarction (MI), thereby promoting structural and functional ...repair. However, the CF population is believed to be heterogeneous and difficult to capture by conventionally used markers. An additional problem concerns the potential contamination in CF preparations by endothelial and immune cells. Furthermore, contamination with epicardial mesenchymal progenitor cells (epicardium‐derived cells ‐ EPDC), formed on the epicardial surface after MI, is another critically factor, which so far has not been evaluated.
We have established a novel CF isolation protocol using enzymatic digestion of the murine heart via the coronaries (Langendorff) and the simultaneous enzymatic removal of the EPDC layer from the outside in a single procedure (within 12 min). Subsequently, the cell suspension derived from the EPDC‐depleted heart is separated from cardiomyocytes by centrifugation and endothelial cells (CD31+) as well as immune cells (CD45+) are removed by magnetic bead purification. Analysis of cellular composition of the non‐cardiomyocyte fraction in the control heart by flow cytometry revealed that endothelial cells (CD31+) are the most abundant cell type (45.3 ± 1.4%) within the non‐cardiomyocyte fraction, followed by CF (CD31−/CD45−) and immune cells (CD45+) accounting for 40.4 ± 1.9% and 12.1 ± 0.3%, respectively. Purified CF (CD31−/CD45−) were further analyzed for common CF markers: Pdgfrα and the MEFSK4 epitope was expressed on the majority of isolated CF (72.2 ± 2.9% and 52.1 ± 4.6%, respectively), while CD90+, Sca1+ and Vimentin+ cells amounted to 23.3 ± 2%, 12.4 ± 0.7% and 35.5 ± 2.1%, respectively, suggesting a heterogeneous CF population. Expression of non‐fibroblast markers revealed that the fraction of pericytes (Ng‐2: 1.1 ± 0.3 %) and smooth muscle expressing cells (α‐SMA: 12.3 ± 0.5%) was rather small. In addition we studied the expression of 23 genes encoding for activation markers (a‐Sma, Tnc, Postn, Col1a1, Col3a1, Fn), cytokines and growth factors (Il‐6, Il‐1b, Il‐10, Vegfa, Vegfc, Tgfb1, Fgf1) as well as cardiac genes and transcription factors (Wt‐1, Tbx‐18, Mef2c, Nkx2.5, Gata‐4, Tnnt2, Hand2, Tbx‐5, Tbx‐20, Tcf‐21) between CF of the unstressed heart, aCF and EPDC isolated 5 days after MI (n=4). Analysis revealed that expression of WT‐1, an established epicardial marker, was 25‐fold/86‐fold higher in EPDC compared to aCF/CF, respectively. Expression of activation markers in aCF and EPDC were in a similar range and higher as compared to quiescent CF. Cardiac genes and transcription factors were expressed in CF, aCF and EPDC, however, with large individual differences: troponin T was 21‐fold higher expressed in CF/aCF when compared to EPDC, while Tbx‐5 was 4‐fold higher in EPDC when compared to CF/aCF. These profound differences between EPDC and aCF/CF highlight that the removal of EPDC is an essential prerequisite for studies of the CF phenotype after MI.
In summary, we report a rapid isolation technique for cardiac fibroblasts pre and post MI, which are largely free of contaminating immune cell, endothelial cells, pericytes and EPDC. This protocol will permit the separate analysis of infarct‐induced phenotype changes in freshly isolated as well as cultured CF and EPDC.
Support or Funding Information
Funded by DFG (IRTG1902)
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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
receptor (A
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 A
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 A
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 A
R signaling was only minor, hypoxia and A
R activation cumulatively increased VEGFA expression. Normoxic A
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 A
R signaling, which may result in a sustained HIF-1α activity. The A
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 A
R-expressing cell types. Altogether, we identified A
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.
Abstract
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.
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+/BrdU+ cardiomyocytes that was paralleled by progressively restored ejection fraction. USSC induced significant T-cell enrichment in ischemic tissue with enhanced expression of T-cell related cytokines. Inhibition of T-cell activation by anti-CD28 monoclonal antibody, fully abolished the regenerative response which was restored by adoptive T-cell transfer. Secretome analysis of USSC and lineage tracing studies suggest that USSC secrete paracrine factors over an extended period of time which boosts a T-cell driven endogenous regenerative response mainly from adult cardiomyocytes.
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•Transplantation of USSC into infarcted hearts resulted in unprecedented thickening of the left ventricular wallwith de novo formation of cardiomyocytes.•USSC induced T-cell enrichment with expression of T-cell related cytokines, which foresters the formation of cardiac progenitor-like cells.•Secretome analysis of USSC and lineage tracing studies suggest that USSC secrete paracrine factors which boosts the regenerative response.
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 <0.8 mm could be visualized unequivocally in the murine inferior vena cava as hot spots in vivo by simultaneous acquisition of anatomic matching 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.
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