Abstract-The oxidized low density lipoprotein (LDL) hypothesis of atherosclerosis proposes that LDL undergoes oxidation in the interstitial fluid of the arterial wall. We have shown that aggregated (vortexed) nonoxidized LDL was taken up by J774 mouse macrophages and human monocyte-derived macrophages and oxidized intracellularly, as assessed by the microscopic detection of ceroid, an advanced lipid oxidation product. Confocal microscopy showed that the ceroid was located in the lysosomes. To confirm these findings, J774 macrophages were incubated with acetylated LDL, which is internalized rapidly to lysosomes, and then incubated (chase incubation) in the absence of any LDL. Key Words: atherosclerosis Ⅲ ceroid Ⅲ lysosome Ⅲ iron Ⅲ oxidized low density lipoprotein T he local oxidation of low density lipoprotein (LDL) within atherosclerotic lesions is widely believed to be of importance in the pathogenesis of atherosclerosis. 1 LDL is thought to be oxidized within the extracellular space of atherosclerotic lesions and then to be bound by scavenger receptors and taken up by macrophages, which become cholesterol-laden foam cells, a major feature of atherosclerotic lesions. 2 Among many other effects, oxidized LDL increases the expression of cellular adhesion molecules and chemokines, 3,4 increases the production of metalloproteinases, 5 which probably destabilize the fibrous caps over advanced lesions, and induces apoptosis in cells. 6 The mechanisms by which LDL is oxidized in atherosclerotic lesions remain uncertain, despite a great deal of work. 7 The oxidation hypothesis of atherosclerosis needs to address the high antioxidant capacity of extracellular fluids. Even a few percent of serum or interstitial fluid can inhibit greatly the oxidation of LDL by cells. 8,9 We postulated that LDL oxidation might occur not within the interstitial fluid of atherosclerotic lesions but within lysosomes in macrophages in atherosclerotic lesions. Materials and Methods LDL Isolation and ModificationBlood was taken from healthy volunteers with EDTA as the anticoagulant (final concentration 3 mmol/L). LDL (1.019 to 1.063 g/mL) was isolated from the plasma by sequential density ultracentrifugation at 4°C, as described previously. 10 LDL was stored in the dark under argon at 4°C and used within 1 month. Aggregation of LDL was achieved by vortexing 11 or acetylation. 12 Acetylation of LDL was confirmed by agarose gel electrophoresis (Paragon gels; Beckman), as seen by an increase of about 4.5 in electrophoretic mobility relative to native LDL. Cell CultureCell culture media (DMEM, RPMI 1640, and Ham's F-10) and phosphate buffered saline (PBS) (without calcium or magnesium) were obtained from Gibco Life Technologies. The media used in this study were supplemented with 20% (v/v) fetal calf serum, Glutamax (2 mmol/L), penicillin (50 IU/mL), streptomycin (50 g/mL), and amphotericin B (0.95 g/mL), unless otherwise stated. Humidified 95% air/5% carbon dioxide at 37°C was used for cell culture. J774 cells were regularly cultured in supplement...

Key Points• Oxidized LDL stimulates rapid change in platelet shape through ligation of CD36.• Ligation of CD36 by oxidized LDL simultaneously activates tyrosine and Rho kinase-dependent signaling pathways.Oxidized low-density lipoproteins (oxLDL) generated in the hyperlipidemic state may contribute to unregulated platelet activation during thrombosis. Although the ability of oxLDL to activate platelets is established, the underlying signaling mechanisms remain obscure. We show that oxLDL stimulate platelet activation through phosphorylation of the regulatory light chains of the contractile protein myosin IIa (MLC). oxLDL, but not native LDL, induced shape change, spreading, and phosphorylation of MLC (serine 19) through a pathway that was ablated under conditions that blocked CD36 ligation or inhibited Src kinases, suggesting a tyrosine kinase-dependent mechanism. Consistent with this, oxLDL induced tyrosine phosphorylation of a number of proteins including Syk and phospholipase C g2. Inhibition of Syk, Ca 21 mobilization, and MLC kinase (MLCK) only partially inhibited MLC phosphorylation, suggesting the presence of a second pathway. oxLDL activated RhoA and RhoA kinase (ROCK) to induce inhibitory phosphorylation of MLC phosphatase (MLCP). Moreover, inhibition of Src kinases prevented the activation of RhoA and ROCK, indicating that oxLDL regulates contractile signaling through a tyrosine kinase-dependent pathway that induces MLC phosphorylation through the dual activation of MLCK and inhibition of MLCP. These data reveal new signaling events downstream of CD36 that are critical in promoting platelet aggregation by oxLDL. (Blood. 2013;122(4):580-589)

Neural restoration has proven to be difficult after brain stroke, especially in its chronic stage. This is mainly due to the generation of an unpropitious niche in the injured area, including loss of vascular support but production of numerous inhibitors against neuronal regeneration. Reconstruction of a proper niche for promoting local angiogenesis, therefore, should be a key approach for neural restoration after stroke. In the present study, a new biomaterial composite that could be implanted in the injured area of the brain was created for experimental therapy of brain ischemia in the mouse. This composite was made using a hyaluronic acid (HA)-based biodegradable hydrogel scaffold, mixed with poly(lactic-co-glycolic acid) (PLGA) microspheres containing vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1), two factors that stimulate angiogenesis. In addition, the antibody of Nogo receptor (NgR-Ab), which can bind to multiple inhibitory myelin proteins and promote neural regeneration, was covalently attached to the hydrogel, making the hydrogel more bioactive and suitable for neural survival. This composite (HA-PLGA) was implanted into the mouse model with middle cerebral artery occlusion (MCAO) to explore a new approach for restoration of brain function after ischemia. A good survival and proliferation of human umbilical artery endothelial cells (HUAECs) and neural stem cells (NSCs) were seen on the HA hydrogel with PLGA microspheres in vitro. This new material was shown to have good compatibility with the brain tissue and inhibition to gliosis and inflammation after its implantation in the normal or ischemic brain of mice. Particularly, good angiogenesis was found around the implanted HA-PLGA hydrogel, and the mouse models clearly showed a behavioral improvement. The results in this present study indicate, therefore, that the HA-PLGA hydrogel is a promising material, which is able to induce angiogenesis in the ischemic region by releasing VEGF and Ang1, thus creating a suitable niche for neural restoration in later stages of stroke. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

This study examined sustained co-delivery of vascular endothelial growth factor (VEGF), angiopoietin-1 and basic fibroblast growth factor (bFGF) encapsulated in angiogenic microspheres. These spheres were delivered to sites of spinal cord contusion injury in rats, and their ability to induce vessel formation, neural regeneration and improve hindlimb motor function was assessed. At 2–8 weeks after spinal cord injury, ELISA-determined levels of VEGF, angiopoietin-1, and bFGF were significantly higher in spinal cord tissues in rats that received angiogenic microspheres than in those that received empty microspheres. Sites of injury in animals that received angiogenic microspheres also contained greater numbers of isolectin B4-binding vessels and cells positive for nestin or β III-tubulin (P < 0.01), significantly more NF-positive and serotonergic fibers, and more MBP-positive mature oligodendrocytes. Animals receiving angiogenic microspheres also suffered significantly less loss of white matter volume. At 10 weeks after injury, open field tests showed that animals that received angiogenic microspheres scored significantly higher on the Basso-Beattie-Bresnahan scale than control animals (P < 0.01). Our results suggest that biodegradable, biocompatible PLGA microspheres can release angiogenic factors in a sustained fashion into sites of spinal cord injury and markedly stimulate angiogenesis and neurogenesis, accelerating recovery of neurologic function.