Three-Dimensional Imaging of Intraplaque Neovascularization in a Mouse Model of Advanced Atherosclerosis

Abstract

Multiple lines of evidence suggest that intraplaque (IP) neovascularization promotes atherosclerotic plaque growth, destabilization, and rupture. However, pharmacological inhibition of IP neovascularization remains largely unexplored due to the limited number of animal models that develop IP neovessels and the lack of reliable methods for visualizing IP angiogenesis. Here, we applied 3D confocal microscopy with an optimized tissue-clearing process, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, to visualize IP neovessels in apolipoprotein E-deficient (ApoE −/−) mice carrying a heterozygous mutation (C1039+/−) in the fibrillin-1 gene. Unlike regular ApoE −/− mice, this mouse model is characterized by the presence of advanced plaques with evident IP neovascularization. Plaques were stained with antibodies against endothelial marker CD31 for 3 days, followed by incubation with fluorescently labeled secondary antibodies. Subsequent tissue clearing with dichloromethane (DCM)/methanol, DCM, and dibenzyl ether allowed easy visualization and 3D reconstruction of the IP vascular network while plaque morphology remained intact.

A Switch from Cell-Associated to Soluble PDGF-B Protects against Atherosclerosis, despite Driving Extramedullary Hematopoiesis

“…In the current study, no intra-plaque vessels were found in hypercholesterolemic mice, as expected. Our results coincide with numerous studies that did not observe plaque angiogenesis in the widely used Ldlr -/- or ApoE -/- mouse models of atherosclerosis [ 31 , 32 , 33 , 34 ]. This may be related to scarcity of plaque neovascularization in mouse models or difficulties with the conventional detection of intraplaque microvessels through CD31 endothelial cell imaging [ 34 ].…”

Assessment of Microvessel Permeability in Murine Atherosclerotic Vein Grafts Using Two-Photon Intravital Microscopy

“…We here confirm in vivo the abundant presence of intimal microvessels in advance atherosclerotic lesions in the ApoE3*Leiden VG model that was previously shown with histology [8,9]. This is a rare feature seldomly seen in other atherosclerotic mouse models [19,20]. These microvessel networks are characterized by typical vessel features of ongoing angiogenesis, as observed by the irregular microvessel architecture with accentuated turns and branching.…”

The tandem stenosis mouse model: Towards understanding, imaging, and preventing atherosclerotic plaque instability and rupture

“…Importantly, this model recreates several clinical consequences of atherosclerosis, including MI, stroke, and a high incidence of spontaneous death (70% by 35 weeks). Similarly to the TS model, this approach has now been used to interrogate biological mechanisms, as well as test diagnostic and therapeutic approaches (De Dominicis et al, 2020; De Wilde et al, 2015; Kurdi et al, 2019; Luyckx et al, 2018; Perrotta, Pintelon, et al, 2020; Perrotta, Van der Veken, et al, 2020; Roth et al, 2015, 2019; Roth, Rombouts, et al, 2016; Roth, Schrijvers, et al, 2016; Van der Donckt, Roth, et al, 2015; Van der Veken et al, 2018).…”