Abstract：
Sustained hemodynamic pressure overload (PO) produced by murine transverse aortic constriction (TAC) causes myocardial fibrosis; removal of TAC (unTAC) returns left ventricle (LV) hemodynamic load to normal and results in significant, but incomplete regression of myocardial fibrosis. However, the cellular mechanisms that result in these outcomes have not been defined. The objective was to determine temporal changes in myocardial macrophage phenotype in TAC and unTAC and determine whether macrophage depletion alters collagen degradation after unTAC. Myocardial macrophage abundance and phenotype were assessed by immunohistochemistry, flow cytometry, and gene expression by RT-PCR in control (non-TAC), 2 wk, 4 wk TAC, and 2 wk, 4 wk, and 6 wk unTAC. Myocardial cytokine profiles and collagen-degrading enzymes were determined by immunoassay and immunoblots. Initial collagen degradation was detected with collagen-hybridizing peptide (CHP). At unTAC, macrophages were depleted with clodronate liposomes, and endpoints were measured at 2 wk unTAC. Macrophage number had a defined temporal pattern: increased in 2 wk and 4 wk TAC, followed by increases at 2 wk unTAC (over 4 wk TAC) that then decreased at 4 wk and 6 wk unTAC. At 2 wk unTAC, macrophage area was significantly increased and was regionally associated with CHP reactivity. Cytokine profiles in unTAC reflected a proinflammatory milieu versus the TAC-induced profibrotic milieu. Single-cell sequencing analysis of 2 wk TAC versus 2 and 6 wk unTAC revealed distinct macrophage gene expression profiles at each time point demonstrating unique macrophage populations in unTAC versus TAC myocardium. Clodronate liposome depletion at unTAC reduced CHP reactivity and decreased cathepsin K and proMMP2. We conclude that temporal changes in number and phenotype of macrophages play a critical role in both TAC-induced development and unTAC-mediated partial, but incomplete, regression of myocardial fibrosis.NEW & NOTEWORTHY Our novel findings highlight the dynamic changes in myocardial macrophage populations that occur in response to PO and after alleviation of PO. Our data demonstrated, for the first time, a potential benefit of macrophages in contributing to collagen degradation and the partial regression of interstitial fibrosis following normalization of hemodynamic load.
摘要：
小鼠横主动脉缩窄(TAC)产生的持续血液动力学压力超负荷(PO)导致心肌纤维化;去除TAC(unTAC)使左心室(LV)血液动力学负荷恢复正常,但心肌纤维化不完全消退。然而,导致这些结局的细胞机制尚未确定.目的是确定TAC和unTAC中心肌巨噬细胞表型的时间变化，并确定巨噬细胞耗竭是否会改变unTAC后的胶原降解。通过免疫组织化学评估心肌巨噬细胞丰度和表型，流式细胞术,在对照（非TAC）中通过RT-PCR和基因表达，2周，4周TAC，2周,4周,和6周unTAC。通过免疫测定和免疫印迹测定心肌细胞因子谱和胶原降解酶。用胶原杂交肽(CHP)检测初始胶原降解。在UNTAC，巨噬细胞被氯膦酸盐脂质体耗尽，和终点在2周测量unTAC。巨噬细胞数量具有确定的时间模式：在2周和4周TAC中增加，随后在2周unTAC（超过4周TAC）增加，然后在4周和6周unTAC减少。在2周unTAC，巨噬细胞面积显着增加，并与CHP反应性区域相关。与TAC诱导的促纤维化环境相比，unTAC中的细胞因子谱反映了促炎环境。2wkTAC与2和6wkunTAC的单细胞测序分析显示，在每个时间点不同的巨噬细胞基因表达谱，表明unTAC与TAC心肌中独特的巨噬细胞群。在unTAC处的氯膦酸盐脂质体消耗降低了CHP反应性并降低了组织蛋白酶K和proMMP2。我们得出结论，巨噬细胞数量和表型的时间变化在TAC诱导的发育和unTAC介导的部分，但不完整,心肌纤维化的消退。新的和注意我们的新发现强调了对PO的反应和PO缓解后发生的心肌巨噬细胞群的动态变化。我们的数据表明，第一次,在血流动力学负荷正常化后，巨噬细胞在促进胶原降解和间质纤维化部分消退方面的潜在益处。
