Mastrolonardo Eric V, Sussman Sarah, Yun Bo, Jegede Victor, Amin Dev R, Rosenbloom Joel, South Andrew P, Bar-Ad Voichita, Wermuth Peter J, Luginbuhl Adam J
Department of Otolaryngology-Head and Neck Surgery, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania, USA.
Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
Wound Repair Regen. 2025 Jul-Aug;33(4):e70075. doi: 10.1111/wrr.70075.
We tested if hyperoxic conditions can reduce the proportion of active myofibroblasts, which are assumed to be a major driver of head and neck radiation-induced fibrosis, as measured by expression levels of pro-fibrotic genes. Radiated, non-cancerous soft tissue from the head and neck and skin/soft tissue from non-radiated flap donor site were collected from each patient. Myofibroblast density was quantified using immunofluorescence staining with α-SMA and DAPI and visualisation under confocal microscopy and compared between baseline non-radiated and radiated tissue from the same patient. From each tissue specimen, fibroblast cell lines were cultured and exposed to either normoxic, hypoxic, or hyperoxic conditions for 10 days. Total RNA was extracted and reverse-transcribed, and gene expression levels were quantified using RT-PCR. Relative gene expression levels of pro-fibrotic genes COL1A1, COL3A1, FN-EDA, α-SMA, HIF-1α, VEGFα, and VEGFR were compared between normoxic, hypoxic, and hyperoxic treatment groups. Three patients with six total tissue samples were acquired. Radiated tissue contained a higher density of myofibroblasts (calculated as cells/mm) and demonstrated higher expression of pro-fibrotic genes than non-radiated donor site tissue. Hyperoxia decreases expression levels of pro-fibrotic genes in radiated and non-radiated tissue, while hypoxia increases pro-fibrotic gene expression levels in radiated and non-radiated tissue. Study findings indicate that hypoxia is a driver of myofibroblast activation and that subjects with radiation-induced fibrosis of the head and neck have increased expression of myofibroblastic phenotype. Hyperoxygenation can reduce the proportion of active myofibroblasts, revealing a potential therapeutic method to halt chronic fibrotic pathways.
我们测试了高氧条件是否能降低活性肌成纤维细胞的比例，活性肌成纤维细胞被认为是头颈部辐射诱导纤维化的主要驱动因素，通过促纤维化基因的表达水平来衡量。从每位患者身上收集头颈部的放射性非癌性软组织以及非放射性皮瓣供区的皮肤/软组织。使用α-SMA和DAPI进行免疫荧光染色并在共聚焦显微镜下观察，对肌成纤维细胞密度进行定量，并比较同一患者基线非辐射组织和辐射组织之间的差异。从每个组织标本中培养成纤维细胞系，并将其暴露于常氧、低氧或高氧条件下10天。提取总RNA并进行逆转录，使用RT-PCR对基因表达水平进行定量。比较常氧、低氧和高氧治疗组之间促纤维化基因COL1A1、COL3A1、FN-EDA、α-SMA、HIF-1α、VEGFα和VEGFR的相对基因表达水平。共纳入3例患者，共6个组织样本。辐射组织中肌成纤维细胞密度更高（以细胞/mm计算），并且与非辐射供区组织相比，促纤维化基因的表达更高。高氧降低了辐射组织和非辐射组织中促纤维化基因的表达水平，而低氧则增加了辐射组织和非辐射组织中促纤维化基因的表达水平。研究结果表明，低氧是肌成纤维细胞活化的驱动因素，并且头颈部辐射诱导纤维化的患者肌成纤维细胞表型的表达增加。高氧可以降低活性肌成纤维细胞的比例，揭示了一种阻止慢性纤维化途径的潜在治疗方法。
