A Notch3-Marked Subpopulation of Vascular Smooth Muscle Cells Is the Cell of Origin for Occlusive Pulmonary Vascular Lesions

Abstract

Background: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. Methods: We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. Results: Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. Conclusions: This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

Progenitor/Stem Cells in Vascular Remodeling during Pulmonary Arterial Hypertension

“…Indeed, lineage tracing approaches in the context of PH are lacking to prove the involvement of the different stem and progenitor cells. In particular, lineage tracing investigations in mouse PH models with more severe vascular remodeling, such as House Dust Mite mouse model [138], left pneumonectomy/monocrotaline pyrrole mouse model [58], or mice with a EC specific-loss of prolyl-4 hydroxylase 2 (PHD2) [197], could help us identify the fate of progenitor cells and dissect the hierarchy between the different cell populations while getting closer to human pathology pattern. In addition, genome editing advances using CRISPR/Cas9 promote new opportunities to generate Cre reporter rats for lineage tracing studies in MCT and Sugen/hypoxia rat models.…”

“…The Notch pathway is activated within weeks 1-2 of CH, leading to pulmonary vascular remodeling [142] and increased Hes/Hey family target genes expression [137]. Interestingly, in the study of Steffes et al [138], Notch 3 expression was restricted to some specific SMC in pulmonary arterioles which participate in neointima formation. This is reminiscent of the scarce acti-vated SMC observed by Sheikh et al in the pulmonary arteriolar wall during CH-induced remodeling that participates in neomuscularization [44].…”

“…The role of the Notch pathway in regulating PH-associated vascular pulmonary remodeling was demonstrated using γ-secretase inhibitors, which prevent Notch cleavage necessary for intracellular signaling [136][137][138]. However, the impact of the different Notch receptors is somewhat debated.…”

Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

“…Furthermore, Notch3 and its target gene HES5 are highly expressed in lung SMCs of PAH patients, and their expression levels are related to disease severity (Li et al , 2009). Also, the Notch3-positive SMCs population has been identified as the origin of occlusive neointimal lesions of PAH (Steffes et al , 2020). Of note, pharmacological and genetical inhibition of Notch3 signaling diminished medial thickening and ameliorated PH in mice (Li et al , 2009; Steffes et al , 2020).…”

“…Also, the Notch3-positive SMCs population has been identified as the origin of occlusive neointimal lesions of PAH (Steffes et al , 2020). Of note, pharmacological and genetical inhibition of Notch3 signaling diminished medial thickening and ameliorated PH in mice (Li et al , 2009; Steffes et al , 2020). Therefore, Notch3 expressed in SMCs might be a major receptor for Notch ligands expressed in senescent ECs.…”