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.

Mesenchymal stem cells (MSC) are not universal and may be subject to dynamic changes upon local milieus in vivo and after isolation and cultivation in vitro. Here, we demonstrate that MSC derived from murine pericardial adipose tissue (pMSC) constitute two cohorts of population distinguished by the level of CD73 expression (termed as CD73^high and CD73^low pMSC). Transplantation of two types of cells into mouse hearts after myocardial infarction (MI) revealed that the CD73^high pMSC preferentially brought about structural and functional repair in comparison to the PBS control and CD73^low pMSC. Furthermore, the CD73^high pMSC displayed a pronounced anti-inflammatory activity by attenuating CCR2⁺ macrophage infiltration and upregulating several anti-inflammatory genes 5 days after in vivo transplantation and ex vivo cocultivation with peritoneal macrophages. The immunomodulatory effect was not seen in cocultivation experiments with pMSC derived from CD73 knockout mice (CD73^-/-) but was partially blocked by pretreatment of the A2b receptor antagonist, PSB603. The results highlight a heterogeneity of the CD73 expression that may be related to its catalytic products on the modulation of the local immune response and thus provide a possible explanation to the inconsistency of the regenerative results when different sources of donor cells were used in stem cell-based therapy.

Introduction: Adipose tissue-derived stromal cells (ADSCs) are abundant and easy to obtain, but the diversity of differentiation potential from different locations may vary with the developmental origin of their mesenchymal compartment. We therefore aim to compare the myogenic differentiation and reparative activity of ADSCs derived from the pericardial tissue to ADSCs of subcutaneous origin. Methods: Pericardial and inguinal adipose tissues from Wistar rats were surgically obtained, and the stromal fraction was isolated after enzymatic digestion. The phenotypic epitopes of the resultant two types of ADSCs were analyzed with flow cytometry, and the expression of transcriptional factors was analyzed with immunostaining. Furthermore, their potential toward adipogenic, osteogenic, and myogenic differentiation also was compared. Finally, the reparative activity and the resultant functional benefits were examined by allograft transplantation into an infarcted model in rats. Results: ADSCs from two adipose sources showed identical morphology and growth curve at the initial stage, but inguinal ADSCs (ingADSCs) sustained significantly vigorous growth after 25 days of cultivation. Although both ADSCs shared similar immunophenotypes, the pericardial ADSCs (periADSC) intrinsically exhibited partial expression of transcription factors for cardiogenesis (such as GATA-4, Isl-1, Nkx 2.5, and MEF-2c) and more-efficient myogenic differentiation, but less competent for adipogenic and osteogenic differentiation. After in vivo transplantation, periADSCs exhibited significantly vigorous reparative activity evidenced by thickening of ventricular wall and pronounced vasculogenesis and myogenesis, although the majority of prelabeled cells disappeared 28 days after transplantation. The structural repair also translated into functional benefits of hearts after infarction. Conclusions: Although two sources of ADSCs are phenotypically identical, pericADSCs constituted intrinsic properties toward myogenesis and vasculogenesis, and thus provided more potent reparative effects after transplantation; therefore, they represent an attractive candidate cell donor for cardiac therapy.

Background: Cellular dedifferentiation is a regenerative prerequisite that warrants cell cycle reentry and appropriate mitotic division during de novo formation of cardiomyocytes. In the light of our previous finding that expression of injury-responsive element, Wilms Tumor factor 1 (WT1), in pericardial adipose stromal cells (ADSC) conferred a compelling reparative activity with concomitant IL-6 upregulation, we then aim to unravel the mechanistic network that governs the process of regenerative dedifferentiation after ADSC-based therapy. Methods and results: WT1-expressing ADSC (eGFP:WT1) were irreversibly labeled in transgenic mice (WT1-iCre/Gt(ROSA)26Sor-eGFP) primed with myocardial infarction. EGFP:WT1 cells were enzymatically isolated from the pericardial adipose tissue and cytometrically purified (ADSC^gfp+). Bulk RNA-seq revealed upregulation of cardiac-related genes and trophic factors in ADSC^gfp+ subset, of which IL-6 was most abundant as compared to non-WT1 ADSC (ADSC^gfp−). Injection of ADSC^gfp+ subset into the infarcted hearts yielded striking structural repair and functional improvement in comparison to ADSC^gfp− subset. Notably, ADSC^gfp+ injection triggered significant quantity of dedifferentiated cardiomyocytes recognized as round-sharp, marginalization of sarcomeric proteins, expression of molecular signature of non-myogenic genes (Vimentin, RunX1), and proliferative markers (Ki-67, Aurora B and pH3). In the cultured neonatal cardiomyocytes, spontaneous dedifferentiation was accelerated by adding tissue extracts from the ADSC-treated hearts, which was neutralized by IL-6 antibody. Genetical lack of IL-6 in ADSC dampened cardiac dedifferentiation and reparative activity. Conclusions: Taken collectively, our results revealed a previous unappreciated effect of IL-6 on cardiac dedifferentiation and regeneration. The finding, therefore, fulfills the promise of stem cell therapy and may represent an innovative strategy in the treatment of ischemic heart disease.

Injury may induce a sequential activation of intrinsic reparative activity that supports the maintenance of tissue homeostasis. In the present experiments, we investigated whether myocardial infarction (MI) was able to reinstate the expression of Wilms’ tumor factor 1 (WT1) as a key hallmark of fetal reprograming in the pericardial adipose-derived stem cells (pADSC). We characterized the immunophenotypical markers, cardiac potential, and reparative activity of WT1-expressing pADSC (WT1^pos) isolated MI Wistar rats with an intact pericardial sac in which cardiac transudate was accumulated, sampled, and analyzed. The WT1^pos cells formed colony-like aggregates in culture that subsequently generated phase-bright cells that homogenously constituted WT1 expression (> 98%). The WT1^pos cells shared identical surface markers with canonical pADSC, but enhanced transcripts for cardiogenesis (isl-1, gata-4, Sox2 and Tbx18) as well as cardiac commitment (endothelial: 28%; cardiomyogenic: 12.3%) in defined conditions. Remarkably, cardiac transplantation of WT1^pos cells promoted regional angiogenesis and myogenesis which led to significant functional amelioration of the infarcted hearts. Furthermore, we demonstrated that WT1^pos cells uniquely secreted hepatocyte growth factor (HGF) as a key antiapoptotic factor that promotes cardiac repair. Injury-associated fetal reprogramming in pADSC facilitates cardiac differentiation and promotes the reparative activity by enhancing HGF production. As such, injury-“conditioned” pADSC may represent a useful autologous cell donor from infarcted patients for cell-based therapy.