Source: UNIVERSITY OF NEW HAMPSHIRE submitted to
ROLE OF OXIDIZED LDL RECEPTORS (LOX-1) IN ATHEROGENESIS IN NORMAL AND DIABETIC ANIMALS
 
PROJECT DIRECTOR: Foxall, T. L.
 
PERFORMING ORGANIZATION
ANIMAL & NUTRITIONAL SCIENCE
UNIVERSITY OF NEW HAMPSHIRE
DURHAM,NH 03824
 
NON TECHNICAL SUMMARY: A. Cardiovascular disease is the number one cause of mortality in American men and women. B. Diabetes accelerates the development of atherosclerosis. C. The number of Americans with diabetes is on the increase. A. This project examines the role of diabetes in the development of atherosclerosis. B. The purpose of this study is to discover some of the cellular and molecular mechanisms by which high glucose levels can accelerate the development of arterial lesions.
 
OBJECTIVES: 1) To characterize the expression of LOX-1 receptor in each major lesion stage of aortic atherosclerosis. 2) To correlate the expression of LOX-1 with markers of oxidative stress and inflammation, plasma and tissue lipids, glycosylation of vascular proteins, and both glucose and insulin levels. 3)To determine if diabetes affects the role of LOX-1 in early atherosclerosis.
 
APPROACH: Hamsters will be placed on either a low fat-low cholesterol rodent chow diet or a high fat-high cholesterol diet known to induce aortic atherosclerosis. Plasma lipid profiles and circulating malondialdehyde (MDA) will be measured every 2 weeks until the end of the study. Half of the animals in each diet treatment will be made diabetic by administering streptozotocin. Blood glucose levels will be measured to establish that the hamsters are hyperglycemic. At the time they are killed, blood insulin levels will also be determined. Animals will be treated for 8, 24 and 48 weeks. At each time point, the animals will be anethestized, blood samples drawn, and the arterial tree will be flushed with saline and fixed with formalin. Five aortic arches from each group will be stained for neutral lipids using oil red O and mounted as en face preparations to quantify the area of lipid lesion. Five additional arches from each group will be processed into paraffin for histopathology and immunohistochemistry. Using commercially available antibodies, sections of each aorta will be reacted for the presence of LOX-1 receptor, hydroxynonenal (a product of LDL oxidation), cyclooxygenase-1 and -2, carboxymethyl lysine (a product of protein glycosylation), NF kappa B (the transcription factor for LOX-1 mediated effects) and the transcription factor PPAR gamma (a nuclear receptor acitivated by oxidatively modified lipids). Primary antibodies will be detected using a multispecies Level-2 Ultra Streptavidin Detection System alkaline phosphatase kit. Reaction will be visualized using AEC which is a red chromagen. Positive and negative controls will be run for each IHC assay. Postive controls will consist of tissues known to contain the specific antigen while negative controls will consist of the assay protocol minus the primary antibody (PBS and 1 percent BSA as a substitute), as well as peptide neutralization of the primary antibody with specific peptides prior to their use in IHC. In order to obtain more specific information about the comparative degree of LOX-1 receptor expression, the aortic arch protein from individuals in each treatment group will be characterized by Western blot analysis. Aortic arches will be homogenized in lysis buffer, and the protein determined in each sample. Following protein separation on a polyacrylamide gel, proteins will be transferred to a nitrocellulose membrane. The membrane will be incubated with blocking buffer and then the primary antibody to LOX-1 receptor and incubated with secondary antibody conjugated to horseradish peroxidase, washed, and incubated with luminol reagent and visulized on film. Histolopathology results will be photodocumented and compared for extent and character of aortic atherosclerotic lesions. IHC results will be scored and compared between groups. The plasma lipid and malondialdehyde values will be averaged and compared. Correlations will be made to determine whether there are relationships between oxidation products, extent of lesions, transcription factors, products of protein glycosylation, glucose levels, and nature of lesions between diabetic and nondiabetic animals.
 
CRIS NUMBER: 0169009 SUBFILE: CRIS
PROJECT NUMBER: NH00378 SPONSOR AGENCY: CSREES
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2001 TERMINATION DATE: Sep 30, 2004

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
701384010105.1100%

CLASSIFICATION HEADINGS
KA701 - Nutrient Composition of Food
S3840 - Laboratory animals
F1010 - Nutrition and metabolism
G5.1 - Ensure Access to Nutritious Food


RESEARCH EFFORT CATEGORIES
BASIC 60%
APPLIED 20%
DEVELOPMENTAL 20%

KEYWORDS: atherosclerosis; arteries; diabetes; oxidation; glycosylation; transcription; hamsters; animal models; receptors; atherogenesis; low density lipoproteins; laboratory animals; human nutrition; nutrient composition; lesions; oxidative stress; human health; inflammation; plasma levels; lipids; tissue analysis; glucose; insulin; diet; dietary fats; malondialdehyde

PROGRESS: Oct 1, 2001 TO Sep 30, 2004
This investigation of the effects of hyperglycemia on atherosclerosis involved four hamster treatment groups: Control(C)fed a low fat diet of standard chow, hyperlipidemic(L)fed a high fat diet of supplemented chow, hyperglycemic (G) as induced by streptozotocin (Stz), and hyperlipidemic/hyperglycemic(H) fed a high fat diet and given Stz. Hyperglycemia is the primary clinical manifestation of diabetes. The hamsters injected with Stz developed hyperglycemia as indicated by significantly higher fasting glucose levels. Glucose values between the G and H groups were not significantly different. Human diabetic dyslipidemia is characterized by hypertriglyceridemia, decreased plasma high-density lipoprotein (HDL) and increased small, dense low-density lipoprotein (LDL) particles. A higher triglyceride/HDL ratio is indicative of increased levels of small dense LDL. Small dense LDL particles more easily infiltrate the subendothelial space where they aggregate and oxidize. Increased non-HDL (VLDL and LDL) and decreased HDL are proatherogenic. A higher total cholesterol (TC)/HDL ratio is indicative of this proatherogenic lipid profile. Stz injection induced a diabetic dyslipidemia profile in this model by significantly decreasing HDL and increasing triglycerides and the triglyceride/HDL ratio but none of the hamsters in the G group developed atherosclerotic lesions by 20 weeks. The L group developed small foam cell lesions. This lesion development may be attributed to higher non-HDL-C but not to higher triglycerides, as there was no significant difference in the triglycerides between the L and G groups. Hyperlipidemia/hyperglycemia significantly accelerated lesion development beyond all other groups. The H group showed large fibro-fatty lesions by 20 weeks and this lesion effect was attributed to a hyperglycemia mediated increase in non-HDL-C, triglycerides and the increased triglyceride/HDL ratio. Thus, hyperglycemia increased the proatherogenic LDL and altered it to a more readily oxidizable form in the hyperlipidemic/hyperglycemic hamster. This is substantiated by the significantly elevated plasma lipid hydroperoxides in the H group. Immunohistochemistry (IHC) for the LDL receptor (LDL-R), malondialdehyde (MDA), and the lectin-like oxidized LDL receptor (LOX-1) was performed. LDL-R, a cell surface receptor that mediates the uptake of LDL, is down regulated when the cell contains enough cholesterol. The order of LDL-R IHC expression from most to least is C>G>L>H. MDA is a major product of endogenous lipid peroxidation. The order of MDA IHC expression is H>L>G>C. LOX-1 is an unregulated receptor for oxidized LDL (ox-LDL). The order of LOX-1 IHC expression is also H>L>G>C. The hyperglycemic condition promoted the expression of LOX-1 that led to the formation of more advanced lesions.

IMPACT: 2001-10-01 TO 2004-09-30 The elevated glucose (hyperglycemia) of diabetes type II will have significantly adverse health effects on an increasingly obese human population. Understanding how this state promotes cardiovascular disease may lead to therapies to lessen the impact of diabetes. The receptor that was the focus of this study may be the target for pharmaceutical intervention to reduce the major cause of illness and death in diabetics. We have established that the hyperlipemic/hyperglycemic hamster is a valid model for future studies in this area.

PUBLICATION INFORMATION: 2001-10-01 TO 2004-09-30
No publications reported this period

PROJECT CONTACT INFORMATION
NAME: Foxall, T. L.
PHONE: 603-862-2354
FAX: 603-862-3758