A decline in mucosal vascularity is a histological hallmark of oral submucous fibrosis (OSF), a premalignant disease that is largely induced by betel quid chewing. However, the lack of available models has challenged studies of angiogenesis in OSF. Here, we found that the expression of thrombospondin 1 (THBS1), an endogenous angiostatic protein, was elevated in the stroma of tissues with OSF. Using a fibroblast-attached organoid (FAO) model, the overexpression of THBS1 in OSF was stably recapitulated in vitro. In the FAO model, treatment with arecoline, a major pathogenic component in areca nuts, enhanced the secretion of transforming growth factor (TGF)-β1 by epithelial cells, which then promoted the expression of THBS1 in fibroblasts. Furthermore, human umbilical vein endothelial cells (HUVECs) were incorporated into the FAO to mimic the vascularized component. Overexpression of THBS1 in fibroblasts drastically suppressed the sprouting ability of endothelial cells in vascularized FAOs (vFAOs). Consistently, treatment with arecoline reduced the expression of CD31 in vFAOs, and this effect was attenuated when the endothelial cells were preincubated with neutralizing antibody of CD36, a receptor of THBS1. Finally, in an arecoline-induced rat OSF model, THBS1 inhibition alleviated collagen deposition and the decline in vascularity in vivo. Overall, we exploited an assembled organoid model to study OSF pathogenesis and provide a rationale for targeting THBS1.

粘膜血管减少是口腔粘膜下纤维化（OSF）的组织学标志，口腔粘膜下纤维化是一种主要由咀嚼槟榔引起的癌前疾病。然而，缺乏可用的模型对 OSF 血管生成的研究提出了挑战。在这里，我们发现血小板反应蛋白 1 (THBS1)（一种内源性血管抑制蛋白）的表达在 OSF 组织的基质中升高。使用成纤维细胞附着类器官 (FAO) 模型，在体外稳定地再现了 OSF 中 THBS1 的过表达。在FAO模型中，槟榔中的主要致病成分槟榔碱的处理增强了上皮细胞转化生长因子（TGF）-β1的分泌，从而促进了成纤维细胞中THBS1的表达。此外，人脐静脉内皮细胞（HUVEC）被纳入FAO以模拟血管化成分。成纤维细胞中 THBS1 的过度表达极大地抑制了血管化FAO（vFAO）中内皮细胞的萌芽能力。一致地，槟榔碱治疗降低了 vFAO 中 CD31 的表达，并且当内皮细胞与 THBS1 受体 CD36 的中和抗体预孵育时，这种效应减弱。最后，在槟榔碱诱导的大鼠 OSF 模型中，THBS1 抑制减轻了体内胶原沉积和血管分布的下降。总的来说，我们利用组装的类器官模型来研究 OSF 发病机制，并为靶向 THBS1 提供理论依据。