Department of Plastic Surgery, State Key Laboratory of Trauma and Chemical Poisoning, Southwest Hospital, Army Medical University (Third Military Medical University), Chongqing, 400038, China.
Mol Med. 2024 Sep 27;30(1):162. doi: 10.1186/s10020-024-00925-5.
During wound healing, fibroblast to myofibroblast transition is required for wound contraction and remodeling. While hypoxia is an important biophysical factor in wound microenvironment, the exact regulatory mechanism underlying hypoxia and fibroblast-to-myofibroblast transition remains unclear. We previously found that tetraspanin CD9 plays an important role in oxygen sensing and wound healing. Herein, we investigated the effects of physiological hypoxia on fibroblast-to-myofibroblast transition and the biological function and mechanism of CD9 in it.
Human skin fibroblasts (HSF) and mouse dermis wounds model were established under physiological hypoxia (2% O). The cell viability and contractility of HSF under hypoxia were evaluated by CCK8 and collagen gel retraction, respectively. The expression and distribution of fibroblast-to-myofibroblast transition markers and CD9 in HSF were detected by Western blotting and immunofluorescence. CD9 slicing and overexpressing HSFs were constructed to determine the role of CD9 by small interfering RNA and recombinant adenovirus vector. The association of TβR2 and TβR1 was measured by immunoprecipitation to explore the regulatory mechanism. Additionally, further validation was conducted on mouse dermis wounds model through histological analysis.
Enhanced fibroblast-to-myofibroblast transition and upregulated CD9 expression was observed under hypoxia in vitro and in vivo. Besides, reversal of fibroblast-to-myofibroblast transition under hypoxia was observed when silencing CD9, suggesting that CD9 played a key role in this hypoxia-induced transition. Moreover, hypoxia increased fibroblast-to-myofibroblast transition by activating TGF-β1/Smad2/3 signaling, especially increased interaction of TβR2 and TβR1. Ultimately, CD9 was determined to directly affect TβR1-TβR2 association in hypoxic fibroblast.
Collectively, these findings suggest that CD9 promotes TβR2-TβR1 association, thus driving the transition of human dermal fibroblasts to myofibroblast under hypoxia.
在伤口愈合过程中，成纤维细胞向肌成纤维细胞的转化对于伤口收缩和重塑是必需的。虽然缺氧是伤口微环境中的一个重要生物物理因素，但缺氧和成纤维细胞向肌成纤维细胞转化的确切调节机制仍不清楚。我们之前发现四跨膜蛋白 CD9 在氧感应和伤口愈合中起着重要作用。在此，我们研究了生理缺氧对成纤维细胞向肌成纤维细胞转化的影响以及 CD9 在其中的生物学功能和机制。
在生理缺氧（2% O ）下建立人皮肤成纤维细胞（HSF）和小鼠真皮伤口模型。通过 CCK8 和胶原凝胶回缩分别评估 HSF 在缺氧下的细胞活力和收缩性。通过 Western blot 和免疫荧光检测 HSF 中成纤维细胞向肌成纤维细胞转化标志物和 CD9 的表达和分布。构建 CD9 切割和过表达 HSF，通过小干扰 RNA 和重组腺病毒载体确定 CD9 的作用。通过免疫沉淀测量 TβR2 和 TβR1 的关联，以探索调节机制。此外，通过组织学分析在小鼠真皮伤口模型上进行了进一步验证。
体外和体内观察到缺氧增强了成纤维细胞向肌成纤维细胞的转化，上调了 CD9 的表达。此外，沉默 CD9 时观察到缺氧下成纤维细胞向肌成纤维细胞的逆转，表明 CD9 在这种缺氧诱导的转化中起着关键作用。此外，缺氧通过激活 TGF-β1/Smad2/3 信号增加了成纤维细胞向肌成纤维细胞的转化，特别是增加了 TβR2 和 TβR1 的相互作用。最终，确定 CD9 直接影响缺氧成纤维细胞中 TβR1-TβR2 的关联。
综上所述，这些发现表明 CD9 促进了 TβR2-TβR1 的关联，从而在缺氧下促进人真皮成纤维细胞向肌成纤维细胞的转化。
