Timo Rademakers

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.

Abrogated transforming growth factor beta receptor II (TGFβRII) signalling in dendritic cells promotes immune reactivity of T cells resulting in enhanced atherosclerosis

AIMS The importance of transforming growth factor beta (TGFβ) as an immune regulatory cytokine in atherosclerosis has been established. However, the role of TGFβ signalling in dendritic cells (DCs) and in DC-mediated T cell proliferation and differentiation in atherosclerosis is unknown. METHODS AND RESULTS Here, we investigated the effect of disrupted TGFβ signalling in DCs on atherosclerosis by using mice carrying a transgene resulting in functional inactivation of TGFβ receptor II (TGFβRII) signalling in CD11c(+) cells (Apoe(-/-)CD11cDNR). Apoe(-/-)CD11cDNR mice exhibited an over two-fold increase in the plaque area compared with Apoe(-/-) mice. Plaques of Apoe(-/-)CD11cDNR mice showed an increase in CD45(+) leucocyte content, and specifically in CD3(+), CD4(+) and CD8(+) cells, whereas macrophage content was not affected. In lymphoid organs, Apoe(-/-)CD11cDNR mice had equal amounts of CD11c(+) cells, and CD11c(+)CD8(+) and CD11c(+)CD8(-) subsets, but showed a subtle shift in the CD11c(+)CD8(-) population towards the more inflammatory CD11c(+)CD8(-)CD4(-) DC subset. In addition, the number of plasmacytoid-DCs decreased. Maturation markers such as MHCII, CD86 and CD40 on CD11c(hi) cells did not change, but the CD11cDNR DCs produced more TNFα and IL-12. CD11c(+) cells from CD11cDNR mice strongly induced T-cell proliferation and activation, resulting in increased amounts of effector T cells producing high amounts of Th1 (IFN-γ), Th2 (IL-4, IL-10), Th17 (IL-17), and Treg (IL-10) cytokines. CONCLUSION Here, we show that loss of TGFβRII signalling in CD11c(+) cells induces subtle changes in DC subsets, which provoke uncontrolled T cell activation and maturation. This results in increased atherosclerosis and an inflammatory plaque phenotype during hypercholesterolaemia.

Endothelial JAM-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.

Plaque-Associated Vasa Vasorum in Aged Apolipoprotein E–Deficient Mice Exhibit Proatherogenic Functional Features In Vivo

Objective—Neovascularization of human atherosclerotic plaques is implicated in plaque progression and destabilization, although its functional implications are yet unresolved. Here, we aimed to elucidate functional and morphological properties of plaque microvessels in mice in vivo. Methods and Results—Atherosclerotic carotid arteries from aged (>40 weeks) apolipoprotein E–deficient mice were imaged in vivo using multiphoton laser scanning microscopy. Two distinct groups of vasa vasorum microvessels were observed at sites of atherosclerosis development (median diameters of 18.5 and 5.9 &mgr;m, respectively), whereas microvessels within the plaque could only rarely be found. In vivo imaging showed ongoing angiogenic activity and injection of fluorescein isothiocyanate-dextran confirmed active perfusion. Plaque vasa vasorum showed increased microvascular leakage, combined with a loss of endothelial glycocalyx. Mean blood flow velocity in plaque-associated vasa vasorum was reduced by ±50% compared with diameter-matched control capillaries, whereas mean blood flow was reduced 8-fold. Leukocyte adhesion and extravasation were increased 6-fold in vasa vasorum versus control capillaries. Conclusion—Using a novel in vivo functional imaging strategy, we showed that plaque-associated vasa vasorum were angiogenically active and, albeit poorly, perfused. Moreover, plaque-associated vasa vasorum showed increased permeability, reduced blood flow, and increased leukocyte adhesion and extravasation (ie, characteristics that could contribute to plaque progression and destabilization).

Multi-photon microscopy in cardiovascular research

Multiphoton laser scanning microscopy has proven profound value for ex vivo 3D histology and in vivo imaging of motionless tissue. The development of triggering systems and fast imaging methods, combined with advanced preparation procedures solved the challenging task of intravital imaging of the fast pulsating heart and major arteries in animals and further increased the popularity of intravital multiphoton imaging in cardiovascular research. This review article will highlight the potential of multiphoton microscopy for the visualization and characterization of dynamical and structural processes involved in cardiac and vascular diseases, both in an ex vivo and an intravital animal setting. Examples will be given how multiphoton microscopy can be applied to imaging of atherosclerotic plaque development and progression at subcellular level as well as to intravital imaging of inflammatory processes in the heart. In addition to highlighting the potential of multiphoton microscopy in preclinical cardiovascular research, we will discuss how this tool and its applications may be clinically translated to support disease diagnosis and therapy in patients.

Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis

During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE−/− mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3+ T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3+ T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.

Plaque lymphangiogenesis: To drain or not to drain

Objectives: To investigate the actions of nutraceuticals on key macrophage processes associated with atherosclerosis. Background: Atherosclerosis is an inflammatory disorder of the vasculature orchestrated by the action of cytokines. Macrophages play a pivotal role in atherosclerosis and represent promising therapeutic targets. Current therapies against atherosclerosis are associated with substantial residual risk together with other issues such as adverse side effects. In addition, there have been numerous disappointments on many pharmaceutical agents identified from drug discovery programs. This has initiated interest in nutraceuticals as preventative or therapeutic agents in atherosclerosis but requires an in-depth understanding of their actions. The purpose of this study was to delineate the effects of nutraceuticals on key macrophage processes associated with atherosclerosis together with the molecular mechanisms underlying their actions. Methods: The studies used a combination of macrophage cell lines and primary cultures. Gene expression was monitored by real time quantitative PCR and western blot analysis. The production of reactive oxygen species was determined using a kit from Abcam. Foam cell formation was monitored by uptake of fluorescently labeled modified LDL, intracellular lipid profile and cholesterol efflux. Inflammasome activation was evaluated by following the release of interleukin (IL)-1beta. Cell viability was assessed by release of lactate dehydrogenase. Results: The studies focused on key components in olive oil and omega-6 polyunsaturated fatty acids. These attenuated the expression of key markers of inflammation induced by several pro-atherogenic cytokines, the uptake of modified LDL, macropinocytosis and foam cell formation in macrophages. In addition, they stimulated macrophage cholesterol efflux. A differential effect was observed for other parameters such as production of reactive oxygen species and production of IL-1beta via inflammasome activation. The mechanisms underlying such actions will be presented. Conclusions: The studies provide novel insights into the actions of nutraceuticals on key macrophage pprocesses associated with atherosclerosisroces