SummaryThe physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular 125 I-LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE À/À mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic in vivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the agedependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease.Key words: autophagy; lipids; atherosclerosis; mouse.Cardiovascular disease, driven in part by the accumulation of modified lipids within the vessel wall, represents the leading cause of death in developed nations (Go et al., 2013). Considerable attention and study has been directed at the molecular consequences following the subendothelial deposition of lipids. These consequences include the recruitment of inflammatory cells and the subsequent engulfment of this deposited subendothelial lipid by resident macrophages (Chinetti-Gbaguidi et al., 2014;Randolph, 2014). In contrast, relatively little is known regarding the steps proximal to lipid deposition and what regulatory role, if any, the endothelium might play in this process. While evidence is limited for the case of the endothelium, in other cell types it appears that autophagy may play a critical role in maintaining overall lipid homeostasis. For instance, mice bearing ATG5-deficient macrophages appear to exhibit increased plaque formation when bred with pro-atherogenic mouse strains (Liao et al., 2012;Razani et al., 2012). While the basis for this increased plaque burden is undoubtedly complex, evidence suggests that macrophage-specific ATG5 deletion results in impaired lipophagy (Sergin & Razani, 2014). Lipophagy refers to the specific degradation of lipids by the autophagic machinery. This concept was perhaps first described in the liver where genetic disruption of macroautophagy led to the accumulation of lipid droplets (Singh...

Patches of atrophy of the retinal pigment epithelium (RPE) have not been described in rodent models of retinal degeneration, as they have the clinical setting using fundus autofluorescence. We hypothesize that prelabeling the RPE would increase contrast and allow for improved visualization of RPE loss in vivo. Here, we demonstrate a new technique termed “delayed near-infrared analysis (DNIRA)” that permits ready detection of rat RPE, using optical imaging in the near-infrared (IR) spectrum with aid of indocyanine green (ICG) dye. Using DNIRA, we demonstrate a fluorescent RPE signal that is detected using confocal scanning laser ophthalmoscopy up to 28 days following ICG injection. This signal is apparent only after ICG injection, is dose dependent, requires the presence of the ICG filters (795/810 nm excitation/emission), does not appear in the IR reflectance channel, and is eliminated in the presence of sodium iodate, a toxin that causes RPE loss. Rat RPE explants confirm internalization of ICG dye. Together with normal retinal electrophysiology, these findings demonstrate that DNIRA is a new and safe noninvasive optical imaging technique for in vivo visualization of the RPE in models of retinal disease.