Optical imaging innovations for atherosclerosis research: multiphoton microscopy and optical nanoscopy.

Cardiovascular disease is the leading cause of death and morbidity worldwide. Improving vascular prevention and therapy based on a refined mechanistic pervasion of atherosclerosis as the underlying pathology could limit the effect of vascular disease in aging societies. During the past decades, microscopy has contributed greatly to a better understanding of vascular physiology and pathology by allowing imaging of living specimen with subcellular resolution and high specificity. An important advance has been accomplished through the application of multiphoton microscopy in the vascular domain, a technological development that enabled multidimensional and dynamic imaging deep into the cellular architecture of intact tissue under physiological conditions. To identify and validate new targets for treating atherosclerosis, novel imaging strategies with nanoscale resolution will be essential to visualize molecular processes in intracellular and extracellular compartments. This review will discuss the current use of 2-photon microscopy and will provide an overview and outlook on options for introducing nanoscopic optical imaging modalities in atherosclerosis research.

Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice.

RATIONALE: Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbß3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity. OBJECTIVE: This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. METHODS AND RESULTS: JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbß3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbß3 signaling in vitro. CONCLUSIONS: Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease.

Platelets and their chemokines in atherosclerosis-clinical applications.

The concept of platelets as important players in the process of atherogenesis has become increasingly accepted due to accumulating experimental and clinical evidence. Despite the progress in understanding the molecular details of atherosclerosis, particularly by using animal models, the inflammatory and thrombotic roles of activated platelet s especially in the human system remain difficult to dissect, as often only the complications of atherosclerosis, i.e., stroke and myocardial infarction are definable but not the plaque burden. Platelet indices including platelet count and mean platelet volume (MPV) and soluble mediators released by activated platelets are associated with atherosclerosis. The chemokine CXCL4 has multiple atherogenic activities, e.g., altering the differentiation of T cells and macrophages by inhibiting neutrophil and monocyte apoptosis and by increasing the uptake of oxLDL and synergizing with CCL5. CCL5 is released and deposited on endothelium by activated platelets thereby triggering atherogenic monocyte recruitment, which can be attenuated by blocking the corresponding chemokine receptor CCR5. Atheroprotective and plaque stabilizing properties are attributed to CXCL12, which plays an important role in regenerative processes by attracting progenitor cells. Its release from luminal attached platelets accelerates endothelial healing after injury. Platelet surface molecules GPIIb/IIIa, GP1bα, P-selectin, JAM-A and the CD40/CD40L dyade are crucially involved in the interaction with endothelial cells, leukocytes and matrix molecules affecting atherogenesis. Beyond the effects on the arterial inflammatory infiltrate, platelets affect cholesterol metabolism by binding, modifying and endocytosing LDL particles via their scavenger receptors and contribute to the formation of lipid laden macrophages. Current medical therapies for the prevention of atherosclerotic therapies enable the elucidation of mechanisms linking platelets to inflammation and atherosclerosis.

Atherogenic mononuclear cell recruitment is facilitated by oxidized lipoprotein-induced endothelial junctional adhesion molecule-A redistribution.

BACKGROUND: Junctional adhesion molecule (JAM-) A is a transmembrane protein expressed in many cell types and maintains junctional integrity in endothelial cells. Upon inflammatory stimulation, JAM-A relocates to the apical surface and might thereby facilitate the recruitment of leukocytes. OBJECTIVE: Although inflammatory JAM-A redistribution is an established process, further effort is required to understand its exact role in the transmigration of mononuclear cells, particularly under atherogenic conditions. METHODS: By the use of RNA interference and genetic deletion, the role of JAM-A in the transmigration of T cells and monocytes through aortic endothelial cells was investigated. JAM-A-localization and subsequent mononuclear cell rolling, adhesion and transmigration were explored during endothelial inflammation, induced by oxidized LDL or cytokines. RESULTS: RNA interference or genetic deletion of JAM-A in aortic endothelial cells resulted in a decreased transmigration of mononuclear cells. Treatment of the endothelial cells with oxLDL resulted in an increase of both permeability and apical JAM-A presentation, as shown by bead adhesion and confocal microscopy experiments. Redistribution of JAM-A resulted in an increased leukocyte adhesion and transmigration, which could be inhibited with antibodies against JAM-A or by lovastatin-treatment, but not with the peroxisome proliferator activated receptor gamma-agonist pioglitazone. CONCLUSIONS: This study demonstrates that redistribution of JAM-A in endothelial cells after stimulation with pro-atherogenic oxidized lipoproteins results in increased transmigration of mononuclear cells. This inflammatory dispersal of JAM-A could be counteracted with statins, revealing a novel aspect of their mechanism of action.

Endothelial junctional adhesion molecule-a guides monocytes into flow-dependent predilection sites of atherosclerosis.

BACKGROUND: Junctional adhesion molecule (JAM)-A expressed in endothelial, epithelial, and blood cells can regulate permeability and leukocyte extravasation. Atherosclerosis develops at sites of disturbed flow in large arteries, but the mechanisms guiding inflammatory cells into these predilection sites remain unknown. METHODS AND RESULTS: To characterize cell-specific functions of JAM-A in atherosclerosis, we used apolipoprotein E-deficient mice with a somatic or endothelium-specific deficiency in JAM-A and bone marrow chimeras with JAM-A-deficient leukocytes. We show that impaired JAM-A expression in endothelial cells reduced mononuclear cell recruitment into the arterial wall and limited atherosclerotic lesion formation in hyperlipidemic mice. In contrast, JAM-A deficiency in bone marrow cells impeded monocyte de-adhesion, thereby increasing vascular permeability and lesion formation, whereas somatic JAM-A deletion revealed no significant effects. Regions with disturbed flow displayed a focal enrichment and luminal redistribution of endothelial JAM-A and were preferentially protected by its deficiency. The functional expression and redistribution of endothelial JAM-A was increased by oxidized low-density lipoprotein, but confined by atheroprotective laminar flow through an upregulation of microRNA (miR)-145, which repressed JAM-A. CONCLUSIONS: Our data identify endothelial JAM-A as an important effector molecule integrating atherogenic conditions to direct inflammatory cell entry at predilection sites of atherosclerosis.

Clearance of fetuin-A--containing calciprotein particles is mediated by scavenger receptor-A.

RATIONALE: Fetuin-A is a liver-derived plasma protein involved in the regulation of calcified matrix metabolism. Biochemical studies showed that fetuin-A is essential for the formation of protein-mineral complexes, called calciprotein particles (CPPs). CPPs must be cleared from circulation to prevent local deposition and pathological calcification. OBJECTIVE: We studied CPP clearance in mice and in cell culture to identify the tissues, cells, and receptors involved in the clearance. METHODS AND RESULTS: In mice, fetuin-A-containing CPPs were rapidly cleared by the reticuloendothelial system, namely Kupffer cells of the liver and marginal zone macrophages of the spleen. Macrophages from scavenger receptor-AI/II (SR-A)-deficient mice cleared CPPs less efficiently than macrophages from wild-type mice, suggesting that SR-AI/II is involved in CPP binding and endocytosis. Accordingly, we found reduced clearance of CPPs in SR-A/MARCO-deficient mice. CONCLUSIONS: We could demonstrate that fetuin-A-containing CPPs facilitate the clearance of mineral debris by macrophages via SR-A. Since the same receptor also contributes to the uptake of modified low-density lipoprotein particles in atherosclerosis, defective endocytosis of both types of particle may impinge on lipid as well as mineral debris clearance in calcifying atherosclerosis.