Reversible and irreversible differentiation of cardiac fibroblasts

Aim: Differentiation of cardiac fibroblasts (Fb) into myofibroblasts (MyoFb) is responsible for connective tissue buildup in myocardial remodeling. We examined reversibility of MyoFb differentiation. Methods and Results: Adult rat cardiac Fb were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different Fb phenotypes. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fiber formation decorated with alpha-smooth muscle actin (a-SMA). Transforming growth factor-ß1 (TGF-ß1) promoted terminal differentiation into a-SMA positive MyoFb showing near absence of proliferation i.e. non-p-MyoFb (2-fold increase in cell number after 12 days vs 11-fold for p-MyoFb). SD-208, a TGF-ß-receptor-I kinase blocker, inhibited p-MyoFb differentiation as shown by stress fiber absence, low levels of a-SMA protein expression, and high levels of proliferation (32-fold increase after 12 days). Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and 4-fold contraction. Fb produced low levels of collagen and secreted high levels of IL-10. Non-p-MyoFb showed high collagen production and high MCP-1 and TIMP-1 secretion. Transcriptome analysis indicated differential gene expression between all phenotypes. Dedifferentiation of p-MyoFb, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress, shown by stress fiber de-polymerization in 30% of p-MyoFb vs in 8% of non-p-MyoFb. Stress fiber de-polymerization could be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2 day culture in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D culture. Conclusions: Both reduction in mechanical strain and TGF-ß-receptor-I kinase inhibition can reverse p-MyoFb differentiation but not in non-p-MyoFb.研究目的：心脏成纤维细胞（cardiac fibroblasts, Fb）向肌成纤维细胞（myofibroblasts, MyoFb）分化是心肌重构过程中结缔组织增生的核心机制，本研究旨在探讨MyoFb分化的可逆性。方法与结果：将成年大鼠心脏Fb接种于可提供机械应力的塑料基质上培养，通过调整培养条件获取不同表型的Fb。Fb可自发分化为增殖型肌成纤维细胞（proliferating MyoFb, p-MyoFb），形成由α-平滑肌肌动蛋白（alpha-smooth muscle actin, α-SMA）标记的应力纤维。转化生长因子-β1（transforming growth factor-β1, TGF-β1）可促进Fb终末分化为α-SMA阳性的MyoFb，这类细胞几乎无增殖能力，即非增殖型肌成纤维细胞（non-p-MyoFb）：12天后细胞数仅增加2倍，而p-MyoFb的细胞数则增加11倍。TGF-β受体I激酶抑制剂SD-208可抑制p-MyoFb的分化，具体表现为无应力纤维形成、α-SMA蛋白表达水平低下，且增殖能力显著升高（12天后细胞数增加32倍）。接种于胶原基质中的原始Fb无基质收缩现象，而p-MyoFb和non-p-MyoFb分别可引发2.5倍和4倍的基质收缩。原始Fb的胶原合成水平较低，且分泌高水平的IL-10；non-p-MyoFb则表现出高胶原合成能力，并分泌高水平的MCP-1与TIMP-1。转录组分析显示，所有细胞表型间均存在显著的差异基因表达。尽管维持机械应力环境，SD-208可诱导p-MyoFb发生去分化，但无法诱导non-p-MyoFb去分化：具体表现为30%的p-MyoFb出现应力纤维解聚，而non-p-MyoFb的解聚比例仅为8%。将细胞接种于无约束的三维胶原基质中培养2天，可通过解除机械应力诱导p-MyoFb与non-p-MyoFb发生应力纤维解聚。仅p-MyoFb在长期三维培养后可实现真正的去分化。结论：降低机械应力与抑制TGF-β受体I激酶均可逆转p-MyoFb的分化，但无法逆转non-p-MyoFb的分化。