Substrate stiffness modulates cardiac fibroblast activation, senescence, and proinflammatory secretory phenotype.
Substrate stiffness modulates cardiac fibroblast activation, senescence, and proinflammatory secretory phenotype.
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基质硬度调节心脏成纤维细胞活化、衰老和促炎分泌表型。
DOI：
10.1152/ajpheart.00483.2023
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
McKinsey,TimothyA
中科院分区：
文献类型：
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作者：
Felisbino,MarinaB;Rubino,Marcello;Travers,JoshuaG;Schuetze,KatherineB;Lemieux,MadeleineE;Anseth,KristiS;Aguado,BrianA;McKinsey,TimothyA
原代心脏成纤维细胞（CF），心脏的主要细胞外基质（ECM）产生细胞的体外培养物，用于确定心脏纤维化的分子机制。然而，组织培养聚苯乙烯（TCPS）的超生理刚度触发CFs转化为活化的肌纤维母细胞样状态，细胞的连续传代导致复制性衰老的诱导。这些表型开关混淆了用培养的CF获得的实验数据的解释。为了避免TCPS诱导的CF活化和衰老，我们使用聚乙二醇（PEG）水凝胶作为细胞培养平台，分别采用低刚度和高刚度配方模拟健康和纤维化心脏。低水凝胶硬度将活化的CF转化为静止状态，含有α-平滑肌肌动蛋白（α-SMA）的应力纤维的丰度降低。出乎意料的是，较低的基质硬度伴随着CF衰老，其特征在于衰老相关的β-半乳糖苷酶（SA-β-Gal）活性升高和p16和p21表达增加，这是衰老的抗增殖标志物。使用动态硬化水凝胶与光可调的交联能力，我们证明，过早的，基板诱导的CF衰老是部分可逆的。RNA测序分析显示，在低硬度水凝胶上培养的CF的广泛转录重编程，编码ECM蛋白的促纤维化基因的表达减少，伴随着NF-κ B反应性炎症基因表达的增加，其代表衰老相关分泌表型（SASP）。我们的研究结果表明，基质硬度的改变深刻地影响CF细胞的状态转换，并建议CFs在体内改变表型的机制取决于心肌微环境的硬度，他们residence.NEW & NOTEWORTHYOUR的研究结果突出了与不同硬度的水凝胶培养心脏成纤维细胞相关的优点和陷阱。这些发现还定义了心脏成纤维细胞中的刚度依赖性信号传导和转录网络。
In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These phenotypic switches confound the interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we used poly(ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic hearts, respectively. Low hydrogel stiffness converted activated CFs into a quiescent state with a reduced abundance of α-smooth muscle actin (α-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated β-galactosidase (SA-β-Gal) activity and increased expression of p16 and p21, which are antiproliferative markers of senescence. Using dynamically stiffening hydrogels with phototunable cross-linking capabilities, we demonstrate that premature, substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low-stiffness hydrogels, with a reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-κB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment in which they reside.NEW & NOTEWORTHYOur findings highlight the advantages and pitfalls associated with culturing cardiac fibroblasts on hydrogels of varying stiffness. The findings also define stiffness-dependent signaling and transcriptional networks in cardiac fibroblasts.