Free Cholesterol Deposition in the Cornea of Human Apolipoprotein A-II Transgenic Mice With Functional Lecithin:Cholesterol Acyltransferase Deficiency Josep Julve-Gil, Elena Ruiz-P6rez, Ricardo P. Casaroli-Marano, Africa MarzaI-Casacuberta, Joan Caries Escol/~-Gil, Francesc Gonzalez-Sastre, and Francisco Bianco-Vaca We have developed several lines of transgenic animals that overexpress different levels of human apolipoprotein A-il (apoA-ll). The 11.1 transgenic line has human apoA-II in plasma at threefold the level in normolipidemic humans and a functional lecithin:cholesterol acyltransferase (LCAT) deficiency. The latter is a biochemical phenotype similar to that of fish-eye disease (FED), which is characterized by free cholesterol (FC) and phospholipid accumulation in the cornea, leading to opacity and impaired vision. To assess whether the metabolic alterations in these mice also lead to lipid accumulation in the cornea, we fed them on a long-term regular chow or high-fat/high-cholesterol (HF/HC) diet. The 11.1 transgenic mice showed a moderate accumulation of FC in the cornea, but only when fed the regular chow diet. This FC accumulation was less severe than the accumulation described in FED, which may explain the lack of corneal opacity in these mice. Electron microscopy and immunoblotting analysis of the cornea of 11.1 transgenic mice in comparison to control mice showed (1) a mild but nevertheless more intense intracytoplasmatic lipid particle deposition in the epithelial cells and (2) a decrease of immunoreactive apoA-I in the area of Bowman's layer and at the superficial stroma. The serum capacity to cause cholesterol efflux from rat fibroblasts was decreased in 11.1 transgenic mice, but only in those fed a regular chow diet. We conclude that 11.1 human apoA-II transgenic mice may be a useful model for studies of early lipid deposition in the cornea and its possible prevention. Copyright © 1999 by W,B. Saunders Company T HERE ARE TWO MAIN FORMS of lipid accumulation in the cornea, both associated with alterations in lipoprotein metabolism. One is arcus lipoides, which occurs in the periph- ery of the cornea, does not interfere with vision, and is associated with an elevation of plasma apolipoprotein B (apoB)- containing lipoproteins. 1 The second form, known as corneal opacity, is characterized by a diffuse cloudiness of the corneal stroma and interferes with vision.1 Corneal opacities are usually associated with inherited high-density lipoprotein (HDL) defi- ciency. One such disorder is due to a total or partial deficiency of the enzyme lecithin:cholesterol acyltransferase (LCAT)] -4 which is responsible for catalyzing the esterification of plasma cholesterol using a fatty acid from phosphatidylcholine as a source. 2-4 Impaired LCAT activity results in a failure to esterify cholesterol in plasma, HDL deficiency, and an accumulation of free cholesterol (FC) and phospholipids in the tissues. 2-4 The magnitude and site of lipid deposition differs depending on the form of inherited LCAT deficiency. One form is a complete familial LCAT deficiency (FLD) and the other is a partial LCAT deficiency, also known as fish-eye disease (FED). Both FLD and FED have a similar molecular basis and are characterized by lipid deposition in the cornea, but FLD has clinically important depositions in the red blood cells and kidney. 2-4 The lack of animal models for LCAT deficiency has pre- cluded investigation into the deposition of lipids in the cornea and other tissues. We have reported previously that transgenic mice overexpressing human apoA-II show functional LCAT deficiency related to a decreased plasma apoA-I concentration 5 and, as such, represent a phenotype similar to that of FED patients. 2-4 The present study was undertaken to determine whether these mice develop corneal opacities and/or FC accumu- lation over a protracted time course, and to gain insight into the molecular mechanisms that may be implicated in the process. MATERIALS AND METHODS Animals The C57BL/6 human apoA-II transgenic mice used in these studies have been described previously.S,6 Transgenic and control mice used for the corneal studies were fed ad libitum with regular chow diet or a high-fat/high-cholesterol (HF/HC) diet. The animals were 3 months old when introduced into the study, and then spent either 16 months on a regular chow diet or 9 months on a HF/HC diet. Lipid Measurements in Plasma and Isolated Lipop;vteins and LCAT Activity Lipoproteins from pooled plasma were isolated by sequential ultracen- trifugation as previously described. 5 Total cholesterol, FC, and triglycer- ide (corrected for free glycerol) levels were measured colorimetrically using commercial kits (Boehringer, Mannheim, Germany). Plasma human apoA-II concentrations were determined using a commercial immunoassay (hnmuno, Vienna, Austria). Mouse apoA-I was deter- mined by radial immunodiffusion. 7 Endogenous plasma LCAT activity and LCAT activity for an exogenous substrate were measured as previously described. 5 Lipid Extraction and Determination of Cholesterol in the Cornea The mice were killed and the eyes excised. A comeal-scleral button was cut from the eye, and the corneal tissue was removed from any remaining sclera and stored at -80°C for lipid analysis. Pooled samples From the Servei de Bioqufmica and Institut de Recerca, Hospital de la Santa Creu i Sant Pau, Barcelona; Departament de Biologia CeLlular, Universitat de Barcelona, Barcelona; and Departament de Bioqufmica i Biologia Molecular, Universitat Autbnoma de Barcelona, Barcelona, Spain. Submitted March 25, 1997; accepted October 23, 1998. Supported by grants from the Fondo de Investigaci6n Sanitaria (94/1304) and Fundacid d'Investigaci6 Cardiovascular-Murat6 de TV3 (to F.B.-V.) and the Promoci6n General deI Conocimiento (PB93/1269. to EG.-S.). During this study, A.M.-C. was supported by the Comis- sionat per a Universitats i Recerca and J.C.E.-G. was a predoctoral fellow of the Ministerio de Educaci6n y Cultura. Address reprint requests to Francisco Blanco-Vaca, MD, PhD, Servei de Bioqufmica, Hospital de la Santa Creu i Sant Pau, C~ Antoni M Claret 167, 08025 Barcelona, Spain. Copyright © 1999 by W.B. Saunders Company 0026-0495/99/4804-0002510.00/0 Metabolism, Vol 48, No 4 (April), 1999:pp 415-421 415