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Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis. Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1 [ABSTRACT FROM AUTHOR]

Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell-associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α-induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular [Ca.sup.2+] mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD., Introduction Platelet-leukocyte-endothelial cell interactions can contribute to vaso-occlusion in thromboinflammatory disease (1, 2). During venular inflammation, neutrophils roll over the activated endothelium through interaction between selectins and their ligands (3). [...]

Integrins have a critical role in thrombosis and haemostasis (1). Antagonists of the platelet integrin [α.sub.IIb][β.sub.3] are potent anti-thrombotic drugs, but also have the life-threatening adverse effect of causing bleeding (2,3). It is therefore desirable to develop new antagonists that do not cause bleeding. Integrins transmit signals bidirectionally (4,5). Inside-out signalling activates integrins through a talin-dependent Mechanism (6,7). Integrin ligation mediates thrombus formation and outside-in signaling (8,9), which requires G[α.sub.13] and greatly expands thrombi. Here we show that G[α.sub.13] and talin bind to mutually exclusive but distinct sites within the integrin [β.sub.3] cytoplasmic domain in opposing waves. The first talin-binding wave mediates inside-out signalling and also ligand-induced integrin activation, but is not required for outside-in signalling. Integrin ligation induces transient talin dissociation and G[α.sub.13] binding to an EXE motif (in which X denotes any residue), which selectively mediates outside-in signalling and platelet spreading. The second talin-binding wave is associated with clot retraction. An EXE-motif-based inhibitor of G[α.sub.13]-integrin interaction selectively abolishes outside-in signalling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without affecting bleeding time. Thus, we have discovered a new mechanism for the directional switch of integrin signalling and, on the basis of this mechanism, designed a potent new antithrombotic drug that does not cause bleeding., Integrin signalling involves the binding of several molecules to the cytoplasmic domain of integrin β-subunits including talin (6,7), kindlins (10,11), SRC (12,13) and G[α.sub.13] (8) (Fig. 1a). Co-immunoprecipitation of G[α.sub.13] [...]