Multiple lines of evidence suggest that intraplaque (IP) neovascularization promotes atherosclerotic plaque growth, destabilization, and rupture. However, pharmacological inhibition of IP... Show moreMultiple 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. Show less
Venous grafts are often used to bypass occlusive atherosclerotic lesions; however, poor patency leads to vein graft disease. Deficiency of TLR4, an inflammatory regulator, reduces vein graft... Show moreVenous grafts are often used to bypass occlusive atherosclerotic lesions; however, poor patency leads to vein graft disease. Deficiency of TLR4, an inflammatory regulator, reduces vein graft disease. Here, we investigate the effects of the accessory molecule and TLR4 analogue RadioProtective 105 (RP105) on vein graft disease. RP105 deficiency resulted in a 90% increase in vein graft lesion area compared to controls. In a hypercholesterolemic setting (LDLr(-/-)/RP105(-/-) versus LDLr(-/-) mice), which is of importance as vein graft disease is usually characterized by excessive atherosclerosis, total lesion area was not affected. However we did observe an increased number of unstable lesions and intraplaque hemorrhage upon RP105 deficiency. In both setups, lesional macrophage content, and lesional CCL2 was increased. In vitro, RP105(-/-) smooth muscle cells and mast cells secreted higher levels of CCL2. In conclusion, aggravated vein graft disease caused by RP105 deficiency results from an increased local inflammatory response. Show less