Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Doctoral School of Health Sciences, University of Debrecen, Debrecen, Hungary.
Invest Ophthalmol Vis Sci. 2024 Nov 4;65(13):13. doi: 10.1167/iovs.65.13.13.
To study the impact of myofibroblast differentiation (MD) on hyaluronan (HA) turnover in orbital fibroblasts (OFs) focusing on the expression of its key enzymes and their potential implications in the pathogenesis of thyroid eye disease (TED).
Primary cultures of OFs were established from tissue samples (TED OFs, n = 4; non-TED OFs, n = 5). MD was induced by TGF-β1 (5 ng/mL). Measurements were performed after 24- and 72-hour treatments. The proliferation rate was determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. HA level and size were measured using an aggrecan-based ELISA-like method and agarose gel electrophoresis, respectively. mRNA expressions of myofibroblast markers and enzymes with a role in HA metabolism were determined using real-time PCR.
Upregulation of type I collagen alpha1 chain, alpha-smooth muscle actin, and fibronectin indicated that OFs underwent MD after stimulation by TGF-β. After 72 hours, proliferation of untreated cultures declined, but it remained higher in myofibroblasts. Pericellular HA content, but not HA in the supernatant of myofibroblasts, increased compared to untreated cells. TGF-β was a potent stimulator of hyaluronan synthase 1 (HAS1) expression. The expression of hyaluronidase-1 and cell migration-inducing protein (CEMIP) diminished following MD, whereas the expression of transmembrane protein 2, the regulator of HA catabolism through CEMIP, was elevated. The size distribution of HA shifted toward a high-molecular-weight form following treatment with TGF-β.
OFs undergoing MD are characterized by decreased HA turnover as a consequence of the inhibition of hyaluronidases and HAS1 induction. Our results suggest that hyaluronidases could be potential targets in the treatment of TED.
研究肌成纤维细胞分化（MD）对眼眶成纤维细胞（OFs）中透明质酸（HA）代谢的影响，重点关注其关键酶的表达及其在甲状腺眼病（TED）发病机制中的潜在意义。
从组织样本中建立 OFs 的原代培养物（TED-OFs，n=4；非-TED-OFs，n=5）。用 TGF-β1（5ng/ml）诱导 MD。在 24 小时和 72 小时处理后进行测量。通过 5-溴-2'-脱氧尿苷（BrdU）掺入测定增殖率。使用基于 aggrecan 的 ELISA 样方法和琼脂糖凝胶电泳分别测量 HA 水平和大小。使用实时 PCR 测定与 HA 代谢相关的肌成纤维细胞标志物和酶的 mRNA 表达。
I 型胶原 alpha1 链、alpha-平滑肌肌动蛋白和纤维连接蛋白的上调表明 OFs 在 TGF-β刺激后发生 MD。72 小时后，未经处理的培养物的增殖下降，但在肌成纤维细胞中仍较高。与未经处理的细胞相比，细胞周围的 HA 含量增加，而肌成纤维细胞上清液中的 HA 含量增加。TGF-β是透明质酸合酶 1（HAS1）表达的有力刺激物。MD 后，透明质酸酶-1 和细胞迁移诱导蛋白（CEMIP）的表达减少，而透明质酸代谢调节剂跨膜蛋白 2 的表达增加。经 TGF-β处理后，HA 的大小分布向高分子量形式转移。
发生 MD 的 OFs 的特征是由于透明质酸酶的抑制和 HAS1 的诱导，HA 周转率降低。我们的结果表明，透明质酸酶可能是治疗 TED 的潜在靶点。
