Accelerated atherosclerosis diminishes the long term patency of vascular interventions, such as percutaneous coronary intervention and implantation of saphenous vein grafts. However, the cause of this accelerated atherosclerosis is unclear. In this study, we tested the hypothesis that intimal hyperplasia formed following vascular intervention promotes retention of atherogenic lipoproteins. Intimal hyperplasia was surgically induced in the mouse common carotid artery. The surgery was combined with different mouse models of hypercholesterolemia to obtain different cholesterol levels and to control the onsets of hypercholesterolemia. Three weeks after surgery, samples were immunostained for apoB lipoproteins, smooth muscle cells and leukocytes. Already at mild hypercholesterolemia (193 mg/dL), pronounced apoB lipoprotein retention was found in the extracellular matrix in both intimal hyperplasia and the injured underlying media. In contrast, minimal retention was detected in the uninjured proximal region of the same vessel, or in vessels from mice with normal cholesterol levels (81 mg/dL). Induction of aggravated hypercholesterolemia 3 weeks after surgery, when a mature intimal hyperplasia had been formed, caused a very rapid development of atherosclerotic lesions. Mechanistically, we show that lipoprotein retention was almost exclusively dependent on electrostatic interactions to proteoglycan glycosaminoglycans, and the lipoprotein retention to intimal hyperplasia could be inhibited in vivo using glycosaminoglycan‐binding antibodies. Thus, formation of intimal hyperplasia following vascular intervention makes the vessel wall highly susceptible for lipoprotein retention and accelerated atherosclerosis. The increased lipoprotein retention in intimal hyperplasia can be targeted by blocking the interaction between apoB lipoproteins and glycosaminoglycans in the extracellular matrix.

Immunofluorescence microscopy is a unique method to reveal the spatial location of proteins in tissues and cells. By combining antibodies that are labeled with different fluorochromes, the location of several proteins can simultaneously be visualized in one sample. However, because of the risk of bleed-through signals between fluorochromes, standard multicolor microscopy is restricted to a maximum of four fluorescence channels, including one for nuclei staining. This is not always enough to address common scientific questions. In particular, the use of a rapidly increasing number of marker proteins to classify functionally distinct cell populations and diseased tissues emphasizes the need for more complex multistainings. Hence, multicolor microscopy should ideally offer more channels to meet the current needs in biomedical science. Here we present an enhanced multi-fluorescence setup, which we call Filter-Dense Multicolor Microscopy (FDMM). FDMM is based on condensed filter sets that are more specific for each fluorochrome and allow a more economic use of the light spectrum. FDMM allows at least six independent fluorescence channels and can be applied to any standard fluorescence microscope without changing any operative procedures for the user. In the present study, we demonstrate an FDMM setup of six channels that includes the most commonly used fluorochromes for histology. We show that the FDMM setup is specific and robust, and we apply the technique on typical biological questions that require more than four fluorescence microscope channels.

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