Background: Activated fibroblasts (myofibroblasts) play a critical role in cardiac fibrosis; however, their origin in the diseased heart remains unclear, warranting further investigation. Recent studies suggest the contribution of bone marrow fibroblast progenitor cells (BM-FPCs) in pressure overload–induced cardiac fibrosis. We have previously shown that interleukin-10 (IL10) suppresses pressure overload–induced cardiac fibrosis; however, the role of IL10 in inhibition of BM-FPC–mediated cardiac fibrosis is not known. We hypothesized that IL10 inhibits pressure overload–induced homing of BM-FPCs to the heart and their transdifferentiation to myofibroblasts and thus attenuates cardiac fibrosis.

Methods: Pressure overload was induced in wild-type (WT) and IL10 knockout (IL10KO) mice by transverse aortic constriction. To determine the bone marrow origin, chimeric mice were created with enhanced green fluorescent protein WT mice marrow to the IL10KO mice. For mechanistic studies, FPCs were isolated from mouse bone marrow.

Results: Pressure overload enhanced BM-FPC mobilization and homing in IL10KO mice compared with WT mice. Furthermore, WT bone marrow (from enhanced green fluorescent protein mice) transplantation in bone marrow–depleted IL10KO mice (IL10KO chimeric mice) reduced transverse aortic constriction–induced BM-FPC mobilization compared with IL10KO mice. Green fluorescent protein costaining with α-smooth muscle actin or collagen 1α in left ventricular tissue sections of IL10KO chimeric mice suggests that myofibroblasts were derived from bone marrow after transverse aortic constriction. Finally, WT bone marrow transplantation in IL10KO mice inhibited transverse aortic constriction–induced cardiac fibrosis and improved heart function. At the molecular level, IL10 treatment significantly inhibited transforming growth factor-β–induced transdifferentiation and fibrotic signaling in WT BM-FPCs in vitro. Furthermore, fibrosis-associated microRNA (miRNA) expression was highly upregulated in IL10KO-FPCs compared with WT-FPCs. Polymerase chain reaction–based selective miRNA analysis revealed that transforming growth factor-β–induced enhanced expression of fibrosis-associated miRNAs (miRNA-21, -145, and -208) was significantly inhibited by IL10. Restoration of miRNA-21 levels suppressed the IL10 effects on transforming growth factor-β–induced fibrotic signaling in BM-FPCs.

Conclusions: Our findings suggest that IL10 inhibits BM-FPC homing and transdifferentiation to myofibroblasts in pressure-overloaded myocardium. Mechanistically, we show for the first time that IL10 suppresses Smad–miRNA-21–mediated activation of BM-FPCs and thus modulates cardiac fibrosis.

背景：活化的成纤维细胞（肌成纤维细胞）在心脏纤维化中起关键作用。然而，它们在患病心脏中的起源尚不清楚，有待进一步研究。最近的研究表明，骨髓成纤维细胞祖细胞（BM-FPC）在压力超负荷引起的心脏纤维化中的作用。先前我们已经证明白介素10（IL10）可以抑制压力超负荷引起的心脏纤维化。但是，IL10在抑制BM-FPC介导的心脏纤维化中的作用尚不清楚。我们假设IL10抑制了BM-FPC向心脏的压力超负荷诱导的归巢以及它们向成纤维细胞的转分化，从而减弱了心脏纤维化。

方法：野生型（WT）和IL10基因敲除（IL10KO）小鼠通过主动脉横向收缩诱发压力超负荷。为了确定骨髓起源，用增强的绿色荧光蛋白WT小鼠骨髓生成IL10KO小鼠的嵌合小鼠。为了进行机理研究，从小鼠骨髓中分离了FPC。

结果：与野生型小鼠相比，IL10KO小鼠的压力超负荷增强了BM-FPC的动员和归巢。此外，与IL10KO小鼠相比，将WT骨髓（来自增强型绿色荧光蛋白小鼠）移植到骨髓耗竭的IL10KO小鼠（IL10KO嵌合小鼠）中，减少了横向主动脉缩窄引起的BM-FPC动员。IL10KO嵌合小鼠左心室组织切片中与α平滑肌肌动蛋白或1α胶原蛋白共绿的绿色荧光蛋白提示，成肌纤维细胞源于横断主动脉缩窄后的骨髓。最后，在IL10KO小鼠中进行WT骨髓移植可抑制横向主动脉缩窄引起的心脏纤维化并改善心脏功能。在分子水平上 IL10处理可显着抑制WT BM-FPCs中转化生长因子-β诱导的转分化和纤维化信号传导。此外，与WT-FPC相比，IL10KO-FPC中与纤维化相关的microRNA（miRNA）的表达高度上调。基于聚合酶链反应的选择性miRNA分析显示，IL10显着抑制了转化生长因子-β诱导的与纤维化相关的miRNA（miRNA-21，-145和-208）的表达增强。miRNA-21水平的恢复抑制了IL10对BM-FPCs中转化生长因子-β诱导的纤维化信号转导的影响。基于聚合酶链反应的选择性miRNA分析显示，IL10显着抑制了转化生长因子-β诱导的与纤维化相关的miRNA（miRNA-21，-145和-208）的表达增强。miRNA-21水平的恢复抑制了IL10对BM-FPCs中转化生长因子-β诱导的纤维化信号转导的影响。基于聚合酶链反应的选择性miRNA分析显示，IL10显着抑制了转化生长因子-β诱导的与纤维化相关的miRNA（miRNA-21，-145和-208）的表达增强。miRNA-21水平的恢复抑制了IL10对BM-FPCs中转化生长因子-β诱导的纤维化信号转导的影响。

结论：我们的研究结果表明，IL10抑制压力超负荷心肌中BM-FPC归巢和转分化为成肌纤维细胞。从机制上讲，我们首次证明IL10抑制Smad–miRNA-21介导的BM-FPC活化，从而调节心脏纤维化。