Abstract
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.