Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis.

BACKGROUND: Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed. METHODS: We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements. RESULTS: Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls. CONCLUSIONS: These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis.

Atherosclerosis is driven by the expansion of cholesterol-loaded 'foamy' macrophages in the arterial intima. Factors regulating foamy macrophage differentiation and survival in plaque remain poorly understood. Here we show, using trajectory analysis of integrated single-cell RNA sequencing data and a genome-wide CRISPR screen, that triggering receptor expressed on myeloid cells 2 (Trem2) is associated with foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up oxidized low-density lipoprotein (oxLDL). Myeloid-specific deletion of Trem2 showed an attenuation of plaque progression, even when targeted in established atherosclerotic lesions, and was independent of changes in circulating cytokines, monocyte recruitment or cholesterol levels. Mechanistically, we link Trem2-deficient macrophages with a failure to upregulate cholesterol efflux molecules, resulting in impaired proliferation and survival. Overall, we identify Trem2 as a regulator of foamy macrophage differentiation and atherosclerotic plaque growth and as a putative therapeutic target for atherosclerosis.