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Periostin and matrix stiffness combine to regulate myofibroblast differentiation and fibronectin synthesis during palatal healing.
Matrix Biology ( IF 6.9 ) Pub Date : 2020-08-07 , DOI: 10.1016/j.matbio.2020.07.002
Georgia Nikoloudaki 1 , Paige Snider 2 , Olga Simmons 2 , Simon J Conway 2 , Douglas W Hamilton 3
Affiliation  

Although the matricellular protein periostin is prominently upregulated in skin and gingival healing, it plays contrasting roles in myofibroblast differentiation and matrix synthesis respectively. Palatal healing is associated with scarring that can alter or restrict maxilla growth, but the expression pattern and contribution of periostin in palatal healing is unknown. Using periostin-knockout (Postn−/−) and wild-type (WT) mice, the contribution of periostin to palatal healing was investigated through 1.5 mm full-thickness excisional wounds in the hard palate. In WT mice, periostin was upregulated 6 days post-wounding, with mRNA levels peaking at day 12. Genetic deletion of periostin significantly reduced wound closure rates compared to WT mice. Absence of periostin reduced mRNA levels of pivotal genes in wound repair, including α-SMA/acta2, fibronectin and βigh3. Recruitment of fibroblasts and inflammatory cells, as visualized by immunofluorescent staining for fibroblast specific factor-1, vimentin, and macrophages markers Arginase-1 and iNOS was also impaired in Postn−/−, but not WT mice. Palatal fibroblasts isolated from the hard palate of mice were cultured on collagen gels and prefabricated silicon substrates with varying stiffness. Postn−/− fibroblasts showed a significantly reduced ability to contract a collagen gel, which was rescued by the exogenous addition of recombinant periostin. As the stiffness increased, Postn−/− fibroblasts increasingly differentiated into myofibroblasts, but not to the same degree as the WT. Pharmacological inhibition of Rac rescued the deficient myofibroblastic phenotype of Postn−/− cells. Low stiffness substrates (0.2 kPa) resulted in upregulation of fibronectin in WT cells, an effect which was significantly reduced in Postn−/− cells. Quantification of immunostaining for vinculin and integrinβ1 adhesions revealed that Periostin is required for the formation of focal and fibrillar adhesions in mPFBs. Our results suggest that periostin modulates myofibroblast differentiation and contraction via integrinβ1/RhoA pathway, and fibronectin synthesis in an ECM stiffness dependent manner in palatal healing.



中文翻译:

骨膜素和基质刚度相结合,可在腭愈合过程中调节肌成纤维细胞分化和纤连蛋白合成。

尽管基质细胞蛋白骨膜素在皮肤和牙龈愈合中显着上调,但它分别在肌成纤维细胞分化和基质合成中起不同作用。腭愈合与可以改变或限制上颌骨生长的瘢痕形成有关,但骨膜素在腭愈合中的表达模式和贡献尚不清楚。使用骨膜素敲除(Postn -/-) 和野生型 (WT) 小鼠,通过硬腭中 1.5 mm 全层切除伤口研究骨膜素对腭愈合的贡献。在 WT 小鼠中,periostin 在受伤后 6 天上调,mRNA 水平在第 12 天达到峰值。与 WT 小鼠相比,periostin 的基因缺失显着降低了伤口闭合率。骨膜素的缺乏降低了伤口修复中关键基因的 mRNA 水平,包括 α-SMA/ acta2、纤连蛋白βigh3。Postn 中成纤维细胞和炎症细胞的募集,如通过成纤维细胞特异性因子-1、波形蛋白和巨噬细胞标志物 Arginase-1 和 iNOS 的免疫荧光染色可见,在Postn -/-,但不是 WT 小鼠。从小鼠硬腭分离的腭成纤维细胞在胶原凝胶和具有不同硬度的预制硅基板上培养。Postn -/-成纤维细胞显示出收缩胶原凝胶的能力显着降低,这通过外源性添加重组骨膜素得以挽救。随着硬度的增加,Postn -/-成纤维细胞逐渐分化为肌成纤维细胞,但与 WT 的分化程度不同。Rac 的药理学抑制挽救了Postn -/-细胞的肌成纤维细胞表型缺陷。低刚度底物 (0.2 kPa) 导致 WT 细胞中纤连蛋白的上调,这种效应在Postn中显着降低-/-细胞。对 vinculin 和 integrinβ1 粘连的免疫染色定量显示,Periostin 是 mPFB 中局灶性和纤维状粘连形成所必需的。我们的研究结果表明,骨膜素通过整合素β1/RhoA 途径调节肌成纤维细胞的分化和收缩,并在腭愈合过程中以 ECM 刚度依赖性方式调节纤连蛋白的合成。

更新日期:2020-08-07
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