Department of Surgery, Division of Otolaryngology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Laryngoscope. 2024 Jan;134(1):287-296. doi: 10.1002/lary.30873. Epub 2023 Jul 17.
Subglottic stenosis (SGS) may result from prolonged intubation where fibrotic scar tissue narrows the airway. The scar forms by differentiated myofibroblasts secreting excessive extracellular matrix (ECM). TGF-β1 is widely accepted as a regulator of fibrosis; however, it is unclear how biomechanical pathways co-regulate fibrosis. Therefore, we phenotyped fibroblasts from pediatric patients with SGS to explore how key signaling pathways, TGF-β and Hippo, impact scarring and assess the impact of inhibiting these pathways with potential therapeutic small molecules SB525334 and DRD1 agonist dihydrexidine hydrochloride (DHX).
Laryngeal fibroblasts isolated from subglottic as well as distal control biopsies of patients with evolving and maturing subglottic stenosis were assessed by α-smooth muscle actin immunostaining and gene expression for α-SMA, FN, HGF, and CTGF markers. TGF-β and Hippo signaling pathways were modulated during TGF-β1-induced fibrosis using the inhibitor SB525334 or DHX and analyzed by RT-qPCR for differential gene expression and atomic force microscopy for ECM stiffness.
SGS fibroblasts exhibited higher α-SMA staining and greater inflammatory cytokine and fibrotic marker expression upon TGF-β1 stimulation (p < 0.05). SB525334 restored levels to baseline by reducing SMAD2/3 nuclear translocation (p < 0.0001) and pro-fibrotic gene expression (p < 0.05). ECM stiffness of stenotic fibroblasts was greater than healthy fibroblasts and was restored to baseline by Hippo pathway modulation using SB525334 and DHX (p < 0.01).
We demonstrate that distinct fibroblast phenotypes from diseased and healthy regions of pediatric SGS patients respond differently to TGF-β1 stimulation, and SB525334 has the superior potential for subglottic stenosis treatment by simultaneously modulating TGF-β and Hippo signaling pathways.
NA Laryngoscope, 134:287-296, 2024.
声门下狭窄（SGS）可能是由于长时间插管导致纤维组织疤痕导致气道变窄所致。疤痕是由分化的肌成纤维细胞分泌过多细胞外基质（ECM）形成的。TGF-β1 被广泛认为是纤维化的调节剂；然而，尚不清楚生物力学途径如何共同调节纤维化。因此，我们对患有 SGS 的儿科患者的成纤维细胞进行表型分析，以探讨关键信号通路 TGF-β 和 Hippo 如何影响瘢痕形成，并评估用潜在的治疗性小分子 SB525334 和 DRD1 激动剂二氢去氧皮质酮盐酸盐（DHX）抑制这些途径的影响。
通过α-平滑肌肌动蛋白免疫染色和α-SMA、FN、HGF 和 CTGF 标志物的基因表达，评估来自声门下和患有进行性和成熟性声门下狭窄患者的远端对照活检的喉成纤维细胞。在 TGF-β1 诱导的纤维化过程中，使用抑制剂 SB525334 或 DHX 调节 TGF-β 和 Hippo 信号通路，并通过 RT-qPCR 分析差异基因表达，通过原子力显微镜分析 ECM 硬度。
SGS 成纤维细胞在 TGF-β1 刺激下表现出更高的α-SMA 染色和更高的炎症细胞因子和纤维化标志物表达（p<0.05）。SB525334 通过减少 SMAD2/3 核易位（p<0.0001）和促纤维化基因表达（p<0.05）将水平恢复到基线。使用 SB525334 和 DHX 调节 Hippo 通路可使狭窄成纤维细胞的 ECM 硬度恢复到基线（p<0.01）。
我们证明了来自小儿 SGS 患者患病和健康区域的不同成纤维细胞表型对 TGF-β1 刺激的反应不同，SB525334 通过同时调节 TGF-β 和 Hippo 信号通路，具有更好的治疗声门下狭窄的潜力。
无。喉镜，134:287-296,2024。
