Dual Genetic Lineage Tracing Reveals Capillary to Artery Formation in the Adult Heart

HomeCirculationVol. 145, No. 15Dual Genetic Lineage Tracing Reveals Capillary to Artery Formation in the Adult Heart Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBDual Genetic Lineage Tracing Reveals Capillary to Artery Formation in the Adult Heart Maoying Han, BS, Zixin Liu, BS, Lingjuan He, PhD, Xufeng Li, BS, Lei Liu, BS, Xiuzhen Huang, BS, Mingjun Zhang, BS, Yan Yan, MD, PhD, Kathy O. Lui, PhD and Bin Zhou, MD, PhD Maoying HanMaoying Han The State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (M.H., Z.L., X.H., M.Z., B.Z.), University of Chinese Academy of Sciences, Shanghai, China. Science and Technology, ShanghaiTech University, China (M.H., L.L., B.Z.). , Zixin LiuZixin Liu The State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (M.H., Z.L., X.H., M.Z., B.Z.), University of Chinese Academy of Sciences, Shanghai, China. , Lingjuan HeLingjuan He School of Life Science, Westlake University, Hangzhou, China (L.H.). , Xufeng LiXufeng Li School of Life Science, Hangzhou Institute for Advanced Study (X.L., B.Z.), University of Chinese Academy of Sciences, Shanghai, China. , Lei LiuLei Liu Science and Technology, ShanghaiTech University, China (M.H., L.L., B.Z.). , Xiuzhen HuangXiuzhen Huang The State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (M.H., Z.L., X.H., M.Z., B.Z.), University of Chinese Academy of Sciences, Shanghai, China. , Mingjun ZhangMingjun Zhang The State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (M.H., Z.L., X.H., M.Z., B.Z.), University of Chinese Academy of Sciences, Shanghai, China. , Yan YanYan Yan Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China (Y.Y.). , Kathy O. LuiKathy O. Lui https://orcid.org/0000-0002-1616-3643 Department of Chemical Pathology and Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, China (K.O.L.). and Bin ZhouBin Zhou Correspondence to: Bin Zhou, MD, PhD, Yueyang Road A2112, Shanghai, 200031, China. Email E-mail Address: [email protected] https://orcid.org/0000-0001-5278-5522 The State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (M.H., Z.L., X.H., M.Z., B.Z.), University of Chinese Academy of Sciences, Shanghai, China. School of Life Science, Hangzhou Institute for Advanced Study (X.L., B.Z.), University of Chinese Academy of Sciences, Shanghai, China. Science and Technology, ShanghaiTech University, China (M.H., L.L., B.Z.). Originally published11 Apr 2022https://doi.org/10.1161/CIRCULATIONAHA.121.056249Circulation. 2022;145:1179–1181In patients with ischemic vascular disease, substantial generation of collateral arteries is often associated with fewer ischemic complications and reduced angina pectoris.1 Collateral arteries are mainly derived from preexisting arteries by rapid lumen size growth and vascular wall thickening.1 A recent study on neonatal heart regeneration revealed that endothelial cells (ECs) migrated away from arteries after myocardial infarction (MI) and formed new collateral arteries through an artery reassembly model, which is nearly absent in adults.2 Although capillary ECs can undergo angiogenesis, migrate, and coalesce to form coronary arteries (capillary to artery formation) in the developing heart,3 they form few coronary arteries in adults after MI.4 We designed a dual genetic system that can robustly trace the contribution of capillary ECs to coronary arteries in the adult mouse heart. Mice studies were done in accordance with the Institutional Animal Care and Use Committee. All the supporting data, materials, experimental procedures, and protocols are available on request.To detect the arteries derived from capillaries, we attempted to design a system that enables visualization of these few arteries in the whole-mount heart. Apln (apelin) is active in sprouting capillaries but not in arteries of the adult heart after MI.5 A major technical difficulty has been to label Apln+ capillaries efficiently in the adult heart, as Apln expression is significantly reduced in the adult compared with developing capillaries.3 A second challenge is illuminating the newly formed capillary-derived arteries without labeling all the background capillaries. We created a design that could demonstrate coronary artery formation from Apln+ capillaries with high efficiency and resolution (Figure [A]). In this design, we generated 3 new mouse lines: Cdh5-DreER, Apln-CrexER, and Cx40-LSL-GFP (Figure [A]). After tamoxifen induction, Cdh5-DreER would remove the ER cassette in coronary ECs through rox recognition, yielding the Apln-Cre genotype. If Apln expression is activated in adult capillaries after injury, Apln-Cre would remove the stop cassette of Cx40-LSL-GFP through loxP recognition, leading to the Cx40-GFP genotype. Therefore, if these Apln+ ECs contribute to arterial ECs expressing Cx40, GFP (green fluorescent protein) could be detected to mark these arteries (Figure [A]).Download figureDownload PowerPointFigure. Apln+ capillary endothelial cells contribute to coronary arteries after myocardial infarction. A, Schematic showing genetic tracing strategy. B, Schematic showing the experimental strategy used to trace new arteries derived from Apln+ (apelin) capillaries after myocardial infarction (MI) induction is shown in the left panel. Whole-mount fluorescence images of MI heart compared with sham control are presented in the middle panel. Immunostaining for αSMA (α-smooth muscle actin) and GFP (green fluorescent protein) on MI heart section and their quantification are shown in the right panel. Arrowheads indicate αSMA+ arteries. Data are mean±SEM; n=5; Student t test used for comparison; *P<0.05. C, Schematic showing the experimental strategy in adults is shown in the left panel. Whole-mount fluorescence images of hearts after transverse aortic constriction (TAC), MI/reperfusion (MI/R), or MI are shown in the right panel. D, Immunostaining for GFP and smooth muscle cell markers on injured heart sections. Arrowheads indicate GFP+ vessels. The right panel shows quantification of the percentage of GFP+ arteries in different models. Data are mean±SEM; n = 5; 1-way analysis of variance was performed followed by the Tukey multiple comparisons test; *P<0.05. E, Immunostaining for FABP4 (fatty acid-binding protein 4), tdTomato, and GFP on heart sections. F, Cartoon showing new arteries derived from capillaries in the MI model, but minimal in the TAC or MI/R model. Scale bars: yellow, 1 mm; white, 100 µm. NS indicates nonsignificant.We first induced tamoxifen at postnatal day 0, performed MI at postnatal day 2, and then collected hearts at postnatal day 6 for analysis (Figure [B]). Whole-mount fluorescence of neonatal hearts showed that GFP+ arteries formed after MI that were barely noticeable in the sham control (Figure [B]). Immunostaining for vascular smooth muscle cell marker αSMA (α-smooth muscle actin) and GFP on tissue sections confirmed increased GFP+ arteries in the MI hearts compared with sham (Figure [B]). No GFP was detected across tissues in the absence of tamoxifen injection. These data demonstrated that at least a subset of coronary arteries derived from Apln+ capillaries could be sensitively detected using this tracing system. Our results suggested that capillary ECs contribute to collateral arteries during neonatal heart repair.Having established a sensitive genetic approach in neonates, we next used it to determine capillary to artery formation in adults. We injected tamoxifen in 8-week-old tracing mice, and after a 2-week washout period, they were subjected to 1 of 3 heart surgeries: transverse aortic constriction (TAC), MI/reperfusion (MI/R), or MI (Figure [C]). Whole-mount fluorescence of hearts collected at 2 weeks after injuries showed few but readily detectable GFP+ vascular structures in the MI-treated hearts that were not apparent in the TAC or MI/R hearts (Figure [C]). Immunostaining for GFP on TAC- or MI/R-treated heart sections further showed that very few GFP+ vascular ECs were observed in the injured myocardium (Figure [D]). To ascertain that the visualization was not limited by inefficient labeling of Apln+ capillaries in TAC or MI/R hearts, we crossed the triple knock-in mice with R26-LSL-tdTomato to label Apln+ capillaries by the tdTomato reporter (Figure [E]). Immunostaining for the capillary EC marker FABP4 and tdTomato on TAC- or MI/R-treated heart sections showed efficient tdTomato labeling of FABP4+ (fatty acid-binding protein 4) capillaries by the tracing system (Figure [E]). No hematopoietic cells were labeled in the Apln or Cdh5 (cadherin 5) tracing models, excluding contribution of hematopoietic cells to coronary vessels in our model. To confirm that the observed GFP+ vessels were indeed arteries, we stained the MI-treated heart sections with αSMA and GFP, and found that a subset of coronary arteries contained some GFP+ ECs (Figure [D]). We also confirmed that a subset of the GFP+ vessels expressed the more mature vascular smooth muscle cell markers SM-MHC (smooth muscle myosin heavy chain) and SM22 (Figure [D]).We generated a new genetic tracing system enabling more efficient and sensitive detection of the contribution of capillary ECs to arterial ECs in the mouse heart. Our study suggests that capillary ECs contribute to collateral arteries in the neonatal heart. Our results also showed that at least a subset of coronary arteries in MI hearts constitute some ECs derived from capillaries. The contribution of capillary ECs to arterial ECs in the adult heart was only observed after severe MI (Figure [F]), indicating that specific signaling pathways or environmental cues could be involved in the induction process.Article InformationAcknowledgmentsThe authors thank Shanghai Biomodel Organism Co, Ltd, for Cdh5-DreER, Apln-CrexER, and Cx40-LSL-GFP mice generation.Sources of FundingThis work was supported by the National Key Research & Development Program of China (2019YFA0110403, 2019YFA0802000, and 2018YFA0107900), National Science Foundation of China (31730112, 82088101, 31922032, and 81872241), and Shanghai Municipal Science and Technology Commission (19JC1415700 and 20QC1401000).Nonstandard Abbreviations and AcronymsαSMAα-smooth muscle actinAplnapelinCdh5cadherin 5ECendothelial cellFABP4+fatty acid-binding protein 4MImyocardial infarctionMI/Rmyocardial infarction/reperfusionGFPgreen fluorescent proteinSM-MHCsmooth muscle myosin heavy chainTACtransverse aortic constrictionDisclosures None.FootnotesCirculation is available at www.ahajournals.org/journal/circ*M. Han and Z. Liu contributed equally.For Sources of Funding and Disclosures, see page 1181.Correspondence to: Bin Zhou, MD, PhD, Yueyang Road A2112, Shanghai, 200031, China. Email [email protected]ac.cnReferences1. Helisch A, Schaper W. Arteriogenesis: the development and growth of collateral arteries.Microcirculation. 2003; 10:83–97. doi: 10.1038/sj.mn.7800173CrossrefMedlineGoogle Scholar2. Das S, Goldstone AB, Wang H, Farry J, D’Amato G, Paulsen MJ, Eskandari A, Hironaka CE, Phansalkar R, Sharma B, et al.. A unique collateral artery development program promotes neonatal heart regeneration.Cell. 2019; 176:1128–1142.e18. doi: 10.1016/j.cell.2018.12.023CrossrefMedlineGoogle Scholar3. Tian X, Hu T, Zhang H, He L, Huang X, Liu Q, Yu W, He L, Yang Z, Zhang Z, et al.. Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries.Cell Res. 2013; 23:1075–1090. doi: 10.1038/cr.2013.83CrossrefMedlineGoogle Scholar4. He L, Liu Q, Hu T, Huang X, Zhang H, Tian X, Yan Y, Wang L, Huang Y, Miquerol L, et al.. Genetic lineage tracing discloses arteriogenesis as the main mechanism for collateral growth in the mouse heart.Cardiovasc Res. 2016; 109:419–430. doi: 10.1093/cvr/cvw005CrossrefMedlineGoogle Scholar5. Liu Q, Hu T, He L, Huang X, Tian X, Zhang H, He L, Pu W, Zhang L, Sun H, et al.. Genetic targeting of sprouting angiogenesis using Apln-CreER.Nat Commun. 2015; 6:6020. doi: 10.1038/ncomms7020CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails April 12, 2022Vol 145, Issue 15 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.121.056249PMID: 35404681 Originally publishedApril 11, 2022 Keywordscoronary arteriescapillariesmyocardial infarctionPDF download Advertisement SubjectsBasic Science ResearchCoronary Circulation