目的:声门下狭窄(SGS)可能是由于长时间插管导致纤维性瘢痕组织使气道变窄。通过分化的肌成纤维细胞分泌过量的细胞外基质(ECM)形成瘢痕。TGF-β1被广泛接受为纤维化的调节因子;然而,目前尚不清楚生物力学通路如何共同调节纤维化。因此,我们用SGS对儿科患者的成纤维细胞进行表型分析,以探索关键信号通路,TGF-β和河马,影响瘢痕形成,并评估用潜在的治疗性小分子SB525334和DRD1激动剂盐酸二氢吡嗪(DHX)抑制这些途径的影响。
方法:通过α-平滑肌肌动蛋白免疫染色和α-SMA的基因表达评估了从声门下和远端对照活检中分离的喉成纤维细胞,FN,HGF,和CTGF标记。使用抑制剂SB525334或DHX在TGF-β1诱导的纤维化期间调节TGF-β和Hippo信号通路,并通过RT-qPCR分析差异基因表达和原子力显微镜分析ECM硬度。
结果:SGS成纤维细胞在TGF-β1刺激后表现出较高的α-SMA染色和较高的炎性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)将水平恢复至基线。狭窄成纤维细胞的ECM硬度大于健康成纤维细胞,并且通过使用SB525334和DHX的Hippo途径调节恢复到基线(p<0.01)。
结论:我们证明了来自儿童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信号通路具有声门下狭窄治疗的优越潜力。
方法:N/A喉镜,2023年。
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.