Hypoxia Induced The Differentiation Of Tbx18-positive Epicardial Cells To CoSMCs

Coronary vascular smooth muscle cells(Co SMCs) provide structural support for coronary vessels and play important roles in the pathophysiological process of cardiovascular disease. Understanding the origin and differentiation mechanism of Co SMCs is very important to cardiovascular biology.Research in mammalian embryos indicate that embryonic epicardial progenitor cells(EPCs) is the main source of Co SMCs. Epithelial-mesenchymal transition(EMT) is the critical mechanism, by which the EPCs migrate into the mesenchyme and differatiate to the specified epicardium-derived cells(EPDCs). Various signalling pathways and cell types were shown to regulate the EMT process of EPCs, which induces the differentiation to Co SMCs. The embryonic EPCs contains a pool of progenitor cells expressing a variety of progenitor cell markers, including Tbx18, WT1, and Tcf21. The T-box family is shown to play a crucial role in the process of embryonic heart development. Tbx18-positive EPCs is a subset of progenitor cells which expressed the T-box transcription factor Tbx18. The lineage tracing studies using Cre-Lox P technology confirmed that Tbx18-positive EPCs are pluripotent progenitor cells, which could differentiate into Co SMCs, fibroblasts, and sinoatrial node cells. The early cardiovascular system is formed in a hypoxic microenvironment. The cellular hypoxic response was shown to be regulated by hypoxia inducible factor-1α(HIF-1α). HIF-1α is expressed in the early embryonic heart and is associated with coronary vascular development. Hypoxia could regulate the EMT process of tumor cells by HIF-1α. The adaptation of the cardiovascular system to hypoxia promotes the expression of specific genes, which support the formation and development of the coronary circulatory system. Previous work has indicated that the knockout of HIF-1α caused coronary vascular malformations, interstitial cell death, cardiac abnormalities and the death of E11.0 embryos. Thus, it has important clinical significance to make clear the differentiation mechanism of Tbx18+ EPCs to Co SMCs.Genetic lineage tracing technique,such as Cre-Lox P system,has been widely used in the field of development. In our research, to visualize Tbx18-positive EPCs and their differentiation, we identified the Tbx18 fate tracing models by genotype analysis and further cultured Tbx18-positive EPCs in vitro. We further exposed Tbx18-positive epicardial cells to hypoxia in vitro and in vivo and studied the role of hypoxia in the EMT process of Tbx18-positive EPCs and their differentiation to Co SMCs. Part ⅠThe establishment of in vitro fate-tracing Tbx18+ EPCsObjective: Using Tbx18:Cre/R26REYFP/Lac Z fate-tracing mice to culture highly purified Tbx18-positive EPCs in vitro, which lays the foundation for the research on the mechanism of differentiation of Tbx18+ EPCs in vitro.Methods: Tbx18:Cre knock-in mice and the Cre-lineage reporter R26 REYFP, R26 RLacz mice were maintained on a C57BL/6 background in our laboratory. We crossed the female Tbx18:Cre knock-in mice with the male R26 REYFP mice or R26 RLac Z mice separately, and then screened out Tbx18:Cre/R26REYFP/Lac Z double heterozygous mice by the PCR method. We used Tbx18:Cre/R26REYFP/Lac Z fate-tracing hearts of E11.5 to culture highly purified Tbx18-positive EPCs in vitro. Fluorescence microscopy and Lacz staining methods were used to detect the YFP autofluorescence of Tbx18:Cre/R26 REYFP fate-tracing EPCs and the Lacz staining of Tbx18 :Cre/R26 RLacz fate-tracing EPCs seperately. The expression of Tbx18 gene was detected by immunofluorescence and q RT-PCR techniques.Results: We bred Tbx18:Cre knock-in mice and the Cre-lineage reporter R26 REYFP, R26 RLacz mice on a C57BL/6 background. We established highly pure cultures of Tbx18-positive EPCs using Tbx18 genetic lineage tracing mice. The Tbx18-positive EPCs obtained from Tbx18:Cre/R26REYFP/Lacz hearts showed YFP autofluorescence or stained positive for Lacz separately.Conclusion: Fate-tracing Tbx18-positive EPCs were successfully cultured in vitro by using Tbx18 genetic lineage tracing mice. They were similar in their shape and arrangement to that on the heart surface, which was conducive to studying the mechanism underlying EMT of the Tbx18-positive EPCs. Part ⅡEffect of hypoxia on the differentiation of Tbx18-positive EPCs to Co SMCs in vitroObjective: To investigate the effect of hypoxia on the in vitro differentiation of Tbx18-positive EPCs to Co SMCs and the the related mechanism.Methods: We used Co Cl2 to induce the hypoxic response in Tbx18+ EPCs. The immunofluorescence and immunohistochemistry were used to analyze the expression of the SMCs markers α-SMA and Myh11 in Tbx18+ EPCs cultured in Co Cl2-induced hypoxia. We analyzed the percent of differentiation in Tbx18+ EPCs when seperately cultured in hypoxia group, hypoxia combined with 2ME2 group, normoxia group,and control group. We used immunofluorescence and q RT-PCR methods to observe the expression of the EMT major regulators in Tbx18-positive EPCs cultured in all groups. A transwell assay was further used in our research to clarify the role of hypoxia and the requirement for HIF-1α in epicardial cell migration.Resurts: Co Cl2-induced hypoxia successfully induced the differentiation of Tbx18-poxitive epicardial cells to Co SMCs in vitro. As the time of hypoxia intervention was prolonged, the differentiation rates increased gradually. After blocking HIF-1α with 2ME2, the differentiation rate was significantly decreased. Hypoxia caused the lost of ZO-1 expression in the cell-cell junctions and the increased expression of Snail in Tbx18+ EPCs. The effect of hypoxia on the expression of ZO-1 and Snail was inhibited after blocking HIF-1α. Hypoxia enhanced the migration of Tbx18+ EPCs.Conclusion: Hypoxia successfully induced the differentiation of Tbx18+ EPCs to Co SMCs in vitro. The effect of hypoxia on the Tbx18+ EPCs differentiation was mainly achieved through the HIF-1α regulation of the EMT process. Meanwhile, snail was likely to be a downstream target of HIF-1α during the hypoxia-induced EMT progress. PART Ⅲ Construction of the hypoxia model of E14.5 embryonic epicardium and premature differentiation of Tbx18+ EPCs to Co SMCs in vivoObjective: To explore the differentiation potential of Tbx18+ EPCs to Co SMCs in vivo. To investigate the effect of in vivo hypoxia on the differentiation of Tbx18+ EPCs and the related mechanism.Methods: We isolated Tbx18:Cre/R26REYFP/Lac Z tracing embryos and observed the YFP or Lac Z expression in the coronary vessels in vivo. we constructed a hypoxia model of the embryonic epicardium at E14.5, by the persistent inhalation of 15% O2 of the pregnant female mice. We observed the hypoxia of E14.5 fetal epicardium using Hypoxyprobe-1. Immunofluorescence confocal technique was used to detect the expression of SMCs markers and EMT regulatory factor in the Tbx18+ EPCs of hypoxia model in vivo. We investigated the effect of hypoxia on the migration of Tbx18+ EPCs in vivo with Lacz staining of E14.5 Tbx18:Cre/R26 RLac Z hearts.Results: Immunofluorescence showed that the Co SMC-specific markers α-SMA and Myh11 merged with YFP fluorescence in sections of E16.5 and neonatal Tbx18:Cre/ R26 REYFP mice. Analysis of Lacz staining in E18.5 Tbx18:Cre/R26 RLac Z embryos also showed that a subset of Co SMCs was positive for Lacz staining. Hypoxia probe staining was markedly increased and merged with YFP fluorescence in the E14.5 epicardium after 3 h of maternal hypoxia. With the prolonged inhalation time, the epicardium of E14.5 fetuses continued to be hypoxic. SMCs markers and snail were prematurely expressed and merged with YFP fluorescence in the epicardium of E14.5 Tbx18:Cre/R26 REYFP hearts. However, the in vivo migration of these cells into the myocardium was not significantly altered after 24 h of hypoxia intervention in the E14.5 heart.Conclusion: Lineage tracing models confirmed a part of Co SMCs originated from Tbx18+ EPCs. Hypoxia led to the premature differentiation of Tbx18+ EPCs to Co SMCs in vivo.