On April 20, a research paper co-authored by Prof. Zhou Bin's lab, School of Life Science, Hangzhou Institute for Advanced Study (HIAS), UCAS, was published online in theinternational academic journalCirculation Research under the title "Genetic Lineage Tracing of Pericardial Cavity Macrophages in the Injured Heart". In this study, the newly established dual homologous recombinase-mediated genetic lineage tracing technology was used to efficiently and specifically label pericardial macrophages in mice. It was found that after cardiac injury, pericardial macrophages would immediately gather on the damaged surface and participate in the post-epicardial injury response and inflammatory process, but would not penetrate into the myocardium for myocardial repair. With new genetic tools, this study reassesses the function of pericardial macrophages and provides another powerful genetic tool for a deeper and clearer understanding of their function.
Cardiovascular diseases remain the leading cause of death worldwide. In today's society, its high incidence and high mortality rate are the main reasons that threaten human health and life. Most organs have a degree of self-repair at the cellular level, but the self-repair ability of the heart is very limited due to the low self-proliferation of adult cardiomyocytes and the absence of endogenous cardiac stem cells (eCSCs). The massive death of cardiomyocytes resulting from myocardial ischemia will eventually lead to severe heart damage, heart failure, and even death. Despite significant advances in understanding and treating acute myocardial infarction (MI), mortality remains high. Therefore, it is particularly urgent and important to find new therapeutic targets and create new therapies. In recent years, a large number of studies have shown that macrophages have become the key to myocardial repair and regeneration, including monocyte-differentiated macrophages and cardiac resident macrophages.
Recently, Paul Kubes's group found that Gata6+ macrophages located in the pericardial cavity play an important role in structural cardiac remodeling and MI repair in mice. In their study, they modified a mouse model of coronary artery ligation to maintain the integrity of the pericardial cavity. In this case, Gata6+ pericardial macrophages were found to enter the myocardium through the epicardium, lose GATA6 expression, but continue to exert anti-fibrotic function. Conditional knockout of Gata6 with Lyz2-Cre will cause the loss of this group of macrophages, resulting in increased cardiac fibrosis and decreased cardiac function after MI. Their study demonstrated that Gata6+ macrophages in the pericardial cavity can promote cardiac repair and serve as a potential target for cardiac immunotherapy. However, in these studies, researchers mainly used Gata6H2B-Venus and Lyz2-Cre mice, combined with the bone marrow transplant experiment,to label and trace pericardial macrophages, which could not achieve in situ specific labeling of Gata6+ macrophages in the pericardial cavity. Previous findings lack convincing genetic evidence due to the limitations and nonspecificity of single-gene tracer systems. As a result, there is great controversy about whether pericardial macrophages have the ability to promote cardiac repair, and new specific genetic tracers are needed to elucidate.
Through flow cytometry and immunofluorescence assay, researchers of Zhou Bin's group first proved the newly established dual homologous recombinase-mediated genetic lineage tracing technology can effectively and specifically label pericardial macrophages in CD45-Dre, Gata6-iCreER, and R26-tdTomato tool mice. Based on that, they administered tamoxifen-induced labeled Gata6+ macrophages to those mice and left them for a week for pericardium intact MI model, and samples were collected for testing 7 days later. According to immunofluorescence staining and flow cytometry, tdTomato-positive cells clustered on the surface of the injured area with thickened epicardial mesothelium in response to the inflammatory response there. But few tdTomato-positive cells were found in the myocardium. In vivo genetic evidence suggests that tdTomato-positive pericardial macrophages in pericardium intact MI model, while able to respond to an inflammatory response and cluster in the injured epicardial layer, rarely enter the heart.
To further clarify the function of pericardial macrophages, researchers constructed diphtheria toxin-mediated clearance of pericardial macrophages in CD45-Dre, Gata6-iCreER and R26-tdTomato/iDTR mice and conditional knockout of Gata6 in CD45-Dre, Gata6-iCreER/flox and R26-tdTomato mice. Injection of diphtheria toxin after tamoxifen induction in CD45-Dre, Gata6-iCreER, and R26-tdTomato/iDTR mice can specifically clear GATA6+ pericardial macrophages. GATA6 is essential for the survival, proliferation and function of pericardial macrophages. Knockdown of Gata6 by tamoxifen treatment in CD45-Dre, Gata6-iCreER/flox and R26-tdTomato mice can result in a large reduction of pericardial macrophages. According to the experimental results, whether the clearance of most GATA6+ macrophages or conditional knockdown of GATA6, the cardiac function (EF, FS and PV-loop) of the mice 28 days after MI was not significantly different from that of the control group. Similarly, there was no significant difference in the area of fibrosis. This indicates that pericardial macrophages have no significant effect on myocardial tissue repair and regeneration after cardiac injury.
In addition, to rule out differences in experimental models, researchers constructed mouse models of bone marrow transplantation to further clarify the function of pericardial macrophages in cardiac injury. Bone marrow cells from CD45-Dre, Gata6-iCreER and R26-tdTomato mice were transplanted into irradiated recipient C57BL/6J mice to construct corresponding chimeric mice. Flow cytometry and immunofluorescence staining experiments showed that the recombination efficiency of pericardial macrophages in recipient mice induced by tamoxifen was as high as 50% after 8 weeks of transplantation, while cardiac resident macrophages were not labeled. Subsequently, the chimeric mice were administered the same pericardium intact MI. After 7 days, immunofluorescence staining and flow cytometry results once again demonstrated that the labeled pericardial macrophages did not migrate into cardiomyocytes, but instead concentrated in large numbers in the thickened epicardial mesothelial layer in response to the inflammatory response.
In conclusion, researchers can use the established dual homologous recombinase-mediated genetic lineage tracing technology to specifically label a subset of cardiac macrophages-pericardial macrophages. Out of the traditional thinking and technical limitations, this system does not rely on in vitro transplantation experiments, and can genetically label pericardial macrophages in mice to study their cell migration and function. Different from the previously discovered GATA6+ macrophages, this study revealed that in a pericardium intact cardiac injury model, pericardial macrophages will gather at the injury site for epicardial inflammatory response, but will not migrate into the myocardium for cardiac repair and regeneration. Unlike previous technical means and methods, the newly constructed genetic lineage tracing technology can mark pericardial macrophages in depth and obtain more accurate experimental results in mice. This study provides an important genetic tool for the research field of pericardial macrophages, a more reliable technical means to explore the role of pericardial macrophages in cardiac inflammation and repair, and new therapeutic targets for patients with heart disease.
Macrophages freed in the pericardial cavity will respond to the inflammatory response in the case of cardiac injury and gather in a large number in the thickened epicardial mesothelium, but cannot penetrate into the myocardium for cardiac repair.
Jin Hengwei, a postdoctoral fellow of Zhou Bin's research group at CAS Center for Excellence in Molecular Cell Science (CEMCS), and Liu Kuo, a postdoctoral fellow at HIAS, UCAS, are the co-first authors of the paper, and Zhou Bin, a research fellow at CEMCS/HIAS, UCAS, Prof. He Ben at Shanghai Chest Hospital, and Prof. Qiao Zengyong at Shanghai Fengxian District Central Hospital are the co-corresponding authors of the paper. The research was completed with strong support from the animal platform and cell platform of CEMCS, CAS, the National Center for Protein Science • Shanghai (NCPSS), and financial support from the Chinese Academy of Sciences, the National Natural Science Foundation of China, the Ministry of Science and Technology of the People's Republic of China, and Science and Technology Commission of Shanghai Municipality.
Article link: https://www.ahajournals.org/doi/abs/10.1161/CIRCRESAHA.122.320567
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Source | School of Life Science
Typesetter: Yu Xuan
Executive Editor | Wang Xia
Published in Zhejiang
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