Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

CXCL4 mediates organ fibrosis after ischemic injury via a macrophage-fibroblast axis

G. Schäfer1

1Medizinische Klinik 2, Uniklinik RWTH Aachen, Aachen;

Fibrosis is a key feature of ischemic organ injury and is characterized by expansion of myofibroblasts and disproportionate interstitial accumulation of extracellular matrix (ECM). While an adequate myofibroblast response is necessary for post-ischemic tissue integrity and repair, excessive myofibroblast activation leads to excessive scar formation replacing functional parenchymal tissue, subsequently aggravating organ failure. Although myofibroblasts are considered the main source of ECM in fibrotic disease, newer studies have shown immune cells to be master regulators in myofibroblast activation, orchestrating inflammatory processes with the potential to either ameliorate or aggravate fibrosis. To elucidate macrophage-fibroblast crosstalk after myocardial infarction (MI), we analyzed immune cells from a publicly available murine left ventricular MI single cell RNA sequencing (scRNAseq) dataset (Forte et al., 2020). Scoring of immune cells based on their expression of ECM regulator genes revealed that macrophages have the highest ECM regulator expression. Subsequent correlation analysis identified C-X-C motif chemokine 4 (Cxcl4) as one of the top genes, that correlates with ECM regulator gene expression in immune cells. To test whether Cxcl4 affects development of fibrosis we subjected Cxcl4-/- and wildtype (WT) mice to myocardial infarction or sham surgery. Loss of Cxcl4 protected mice from development of post-ischemic fibrosis in heart, decreased infarction size and preserved cardiac function with higher left ventricular ejection fraction. To test, whether the observed phenotype is conserved across organs, we performed kidney ischemia reperfusion injury (IRI) in Cxcl4-/- and WT mice. Indeed, Cxcl4-/- mice developed significantly less fibrosis after renal ischemia. To better understand the effect of Cxcl4 on macrophage gene expression, we sub-clustered macrophages from the aforementioned MI scRNAseq dataset into Cxcl4-high and Cxcl4-low macrophages. Surprisingly, differential expression analysis revealed Cxcl4-high macrophages to upregulate characteristic genes for a M2-like polarization and identified a platelet-specific gene signature in Cxcl4-high macrophages, pointing towards a possible platelet-macrophage interaction. To validate these findings, we cocultured murine WT and Cxcl4-/- platelets with WT PBMCs and performed fluorescence activated cell sorting of monocytes-platelet aggregates. RT-qPCR analysis confirmed that Cxcl4 proficient platelets induce an M2-like polarization of macrophages consistent with our findings in scRNAseq data. Interestingly, we found that loss of Cxcl4 in platelets leads to decreased platelet-macrophage interactions. Imaging of cocultures of Cxcl4-/-/WT-platelets with WT-PBMCs using confocal microscopy verified decreased uptake of Cxcl4-/- platelets by WT PBMCs. To examine the influence of platelets on post-ischemic fibrosis, we induced a hematopoietic Cxcl4-KO (hCxcl4-/-) by bone marrow transplantation of Cxcl4-/-/WT hematopoietic stem cells into lethally irradiated WT mice and subjected them to IRI surgery. As hypothesized, hCxcl4-/- mice revealed less M2-like phenotypic macrophage polarization and were protected from fibrosis. To decipher the mechanism of macrophage-myofibroblast crosstalk, we performed single nuclear RNA sequencing in IRI/Sham kidneys of Cxcl4-/-/WT mice. Preliminary data of ligand-receptor analysis confirmed increased profibrotic macrophage-myofibroblast interactions in WT kidneys.

https://dgk.org/kongress_programme/jt2022/aP778.html