Steffes Lea C, Kumar Maya E
Department of Pediatrics, Division of Pulmonary Medicine, Stanford University School of Medicine, Palo Alto, CA, USA.
Bio Protoc. 2022 Jan 5;12(1):e4273. doi: 10.21769/BioProtoc.4273.
Pulmonary hypertension (PH) is a heterogenous and incurable disease marked by varying degrees of pulmonary vascular remodeling. This vascular remodeling, which includes thickening of the smooth muscle layer (an early finding) and formation of occlusive neointimal lesions (a late finding) in the pulmonary arteries, is a major driver of morbidity and mortality in PH. Available PH therapies consist of vasodilators that do not specifically target lesion formation or expansion and neither prevent progression nor reverse disease. This paucity of curative treatments highlights the need for new drug discovery targeting crucial steps of artery remodeling in PH. The cell dynamics and molecular signals driving neointimal lesion formation have been difficult to elucidate as classic mouse models of PH do not develop neointima. Here, we detail the methods to generate a robust and non-genetic mouse model of PH with medial thickening and neointimal lesion formation in the pulmonary arteries, through chronic exposure to an inflammatory stimulus-house dust mite (HDM). This model rapidly generates human-like pulmonary arterial lesions following a reproducible time course, allowing scrutiny of the cellular and molecular mechanisms controlling each stage of artery remodeling. Further, we outline optimal tissue handling, sectioning, and staining methodologies for detailed quantitative analysis of artery medial thickening and neointimal lesion formation and expansion. Finally, we present a method for staged pharmacologic intervention to identify molecules and pathways required at each step of the pulmonary arterial remodeling process. The advantages of this mouse model of PH over currently available animal models are five-fold. (i) It allows the use of the full range of genetic and single cell tools available in mice to manipulate and study the process of vascular remodeling seen in human disease, including the formation of neointimal lesions in a controlled and cell specific manner. (ii) The vascular lesions develop in a stereotyped manner with predictable timing, allowing for pharmacologic manipulation at discrete stages of vessel remodeling. (iii) It is rapid, with development of PH and vascular remodeling in a timeframe of two to eight weeks. (iv) It uses simple techniques and requires neither surgery, unusual equipment, or extensive personnel training. (v) The staining and quantitation methodologies we present are a significant improvement over those currently in use in the field. We hope that dissemination of this model and the associated detailed methods will speed up the development of novel and more effective PH therapeutics. Graphic abstract: Chronic perivascular inflammation induces medial thickening and neointima formation in pulmonary arteries, following a stereotyped time course, and allowing staged pharmacologic intervention during specific remodeling events, as well as quantitative assessment of vascular changes.
肺动脉高压（PH）是一种异质性且无法治愈的疾病，其特征为不同程度的肺血管重塑。这种血管重塑包括肺动脉平滑肌层增厚（早期表现）和闭塞性新生内膜病变形成（晚期表现），是PH发病和死亡的主要驱动因素。现有的PH治疗方法包括血管扩张剂，这些药物并非特异性针对病变形成或扩展，既不能阻止疾病进展，也无法逆转病情。治疗方法的匮乏凸显了针对PH动脉重塑关键步骤研发新药的必要性。由于经典的PH小鼠模型不会形成新生内膜，因此驱动新生内膜病变形成的细胞动力学和分子信号一直难以阐明。在此，我们详细介绍了通过长期暴露于炎症刺激物——屋尘螨（HDM），生成一种强大的、非基因的PH小鼠模型的方法，该模型会出现肺动脉中层增厚和新生内膜病变形成。该模型在可重复的时间进程后迅速产生类似人类的肺动脉病变，从而能够仔细研究控制动脉重塑各个阶段的细胞和分子机制。此外，我们概述了用于动脉中层增厚以及新生内膜病变形成和扩展的详细定量分析的最佳组织处理、切片和染色方法。最后，我们介绍了一种分阶段药物干预方法，以确定肺动脉重塑过程中每个步骤所需的分子和途径。这种PH小鼠模型相较于目前可用的动物模型具有五个优势。（i）它允许使用小鼠中所有可用的基因和单细胞工具来操纵和研究人类疾病中所见的血管重塑过程，包括以可控且细胞特异性的方式形成新生内膜病变。（ii）血管病变以可预测的时间顺序呈模式化发展，从而能够在血管重塑的离散阶段进行药物操纵。（iii）它速度快，在两到八周的时间内即可发生PH和血管重塑。（iv）它使用简单的技术，既不需要手术、特殊设备，也不需要大量人员培训。（v）我们介绍的染色和定量方法比该领域目前使用的方法有显著改进。我们希望传播这个模型及相关详细方法将加速新型、更有效PH治疗方法的开发。图形摘要：慢性血管周围炎症会导致肺动脉中层增厚和新生内膜形成，遵循模式化的时间进程，并允许在特定重塑事件期间进行分阶段药物干预以及对血管变化进行定量评估。
