Atherosclerosis is
associated with a compromised endothelial barrier,
facilitating the accumulation of immune cells and macromolecules in
atherosclerotic lesions. In this study, we investigate endothelial
barrier integrity and the enhanced permeability and retention (EPR)
effect during atherosclerosis progression and therapy in Apoe–/– mice using hyaluronan
nanoparticles (HA-NPs). Utilizing ultrastructural and en face plaque imaging, we uncover a significantly decreased junction continuity
in the atherosclerotic plaque-covering endothelium compared to the
normal vessel wall, indicative of disrupted endothelial barrier. Intriguingly,
the plaque advancement had a positive effect on junction stabilization,
which correlated with a 3-fold lower accumulation of in vivo administrated HA-NPs in advanced plaques compared to early counterparts.
Furthermore, by using super-resolution and correlative light and electron
microscopy, we trace nanoparticles in the plaque microenvironment.
We find nanoparticle-enriched endothelial junctions, containing 75%
of detected HA-NPs, and a high HA-NP accumulation in the endothelium-underlying
extracellular matrix, which suggest an endothelial junctional traffic
of HA-NPs to the plague. Finally, we probe the EPR effect by HA-NPs
in the context of metabolic therapy with a glycolysis inhibitor, 3PO,
proposed as a vascular normalizing strategy. The observed trend of
attenuated HA-NP uptake in aortas of 3PO-treated mice coincides with
the endothelial silencing activity of 3PO, demonstrated in
vitro. Interestingly, the therapy also reduced the plaque
inflammatory burden, while activating macrophage metabolism. Our findings
shed light on natural limitations of nanoparticle accumulation in
atherosclerotic plaques and provide mechanistic insight into nanoparticle
trafficking across the atherosclerotic endothelium. Furthermore, our
data contribute to the rising field of endothelial barrier modulation
in atherosclerosis.
Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell–cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue.
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