Department of Bioengineering, University of California San Diego, La Jolla, CA 92093-0412, USA.
Cells. 2021 Apr 23;10(5):1000. doi: 10.3390/cells10051000.
Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during the progression of pulmonary arterial hypertension (PAH), a disease that currently has no effective anti-fibrotic treatments. We conducted in-vitro experiments in PAAFs cultured on hydrogels attached to custom-made equibiaxial stretchers at 10% stretch and substrate stiffnesses representing the mechanical conditions of mild and severe stages of PAH. The expression of collagens α(1)I and α(1)III and elastin messenger RNAs (, , ) were upregulated by increased stretch and substrate stiffness, while lysyl oxidase-like 1 and α-smooth muscle actin messenger RNAs (, ) were only significantly upregulated when the cells were grown on matrices with an elevated stiffness representative of mild PAH but not on a stiffness representative of severe PAH. Fibronectin messenger RNA () levels were significantly induced by increased substrate stiffness and transiently upregulated by stretch at 4 h, but was not significantly altered by stretch at 24 h. We modified our published computational network model of the signaling pathways that regulate profibrotic gene expression in PAAFs to allow for differential regulation of mechanically-sensitive nodes by stretch and stiffness. When the model was modified so that stiffness activated integrin β, the Macrophage Stimulating 1 or 2 (MST1\2) kinases, angiotensin II (Ang II), transforming growth factor-β (TGF-β), and syndecan-4, and stretch-regulated integrin β, MST1\2, Ang II, and the transient receptor potential (TRP) channel, the model correctly predicted the upregulation of all six genes by increased stiffness and the observed responses to stretch in five out of six genes, although it could not replicate the non-monotonic effects of stiffness on and expression. Blocking Ang II Receptor Type 1 (ATR) with losartan in-vitro uncovered an interaction between the effects of stretch and stiffness and angiotensin-independent activation of expression by stretch in PAAFs grown on 3-kPa matrices. This novel combination of in-vitro and in-silico models of PAAF profibrotic cell signaling in response to altered mechanical conditions may help identify regulators of vascular adventitial remodeling due to changes in stretch and matrix stiffness that occur during the progression of PAH in-vivo.
肺小动脉壁成纤维细胞（PAAFs）在肺动脉高压（PAH）进展过程中是重要的纤维化血管重构调节剂，而目前针对这种疾病还没有有效的抗纤维化治疗方法。我们在培养于水凝胶上的 PAAFs 中进行了体外实验，这些水凝胶附着在定制的等双轴拉伸器上，拉伸率为 10%，基质硬度代表了 PAH 的轻度和重度阶段的力学条件。胶原 α(1)I 和 α(1)III 以及弹力蛋白信使 RNA（，）的表达随拉伸和基质硬度的增加而上调，而赖氨酰氧化酶样 1 和α-平滑肌肌动蛋白信使 RNA（，）仅在细胞生长于代表轻度 PAH 的基质硬度升高的基质上时才显著上调，但在代表重度 PAH 的基质硬度上则没有显著上调。纤维连接蛋白信使 RNA（）水平随基质硬度的增加而显著诱导，并在 4 小时时短暂上调，但在 24 小时时不受拉伸的显著影响。我们修改了我们之前发表的 PAAFs 中调节促纤维化基因表达的信号通路的计算网络模型，以便通过拉伸和硬度对机械敏感节点进行差异调节。当模型被修改为使硬度激活整合素β时，模型可以激活巨噬细胞刺激素 1 或 2（MST1\2）激酶、血管紧张素 II（Ang II）、转化生长因子-β（TGF-β）和 syndecan-4，以及拉伸调节的整合素β、MST1\2、Ang II 和瞬时受体电位（TRP）通道，该模型正确预测了所有六个基因随基质硬度增加而上调，以及五个基因中六个基因对拉伸的观察到的反应，尽管它无法复制基质硬度对和表达的非单调效应。在体外用氯沙坦阻断 Ang II 受体 1（ATR）发现，在生长于 3-kPa 基质的 PAAFs 中，拉伸和刚度的作用以及 Ang II 独立激活拉伸引起的表达之间存在相互作用。这种新型的 PAAF 促纤维化细胞信号对改变的机械条件的体外和计算机模型的组合，可能有助于确定由于体内 PAH 进展过程中拉伸和基质硬度的变化导致的血管外膜重塑的调节剂。
