Models of atherosclerosis, hyperlipidemia, lipoprotein oxidation and blood vessel inflammation and methods for making and using them
The invention provides genetically altered fish of the family Cyprinidae, or genus Danio , including zebrafish ( Danio rerio ) and host cells from these animals, where the fish have been genetically altered to lack or have a modified gene related to lipid metabolism, for example, an ApoE, ApoAI and/or LDL-R gene. In another aspect, the invention is directed to drug design or discovery using the animal or cell models of the invention and/or wild type zebrafish, and by administering an altered diet and/or environment to the animal of invention. The invention also provides methods for screening for a compound capable of ameliorating or preventing or reversing: atherosclerosis; hyperlipidemia; lipoprotein oxidation; the accumulation of lipid in a blood vessel wall; vascular inflammation associated with lipid accumulation in a blood vessel wall; acute atherosclerosis-associated events; heart attack; stroke.
1. A method for screening for a compound capable of ameliorating or reversing:
an atherosclerosis or
a vascular inflammation associated with lipid accumulation in a blood vessel wall,
the method comprising:
(a) providing a test compound;
(b) providing a genetically altered fish of the family Cyprinidae of the genus Danio having an induced vascular inflammation and atherosclerosis,
wherein the vascular inflammation and atherosclerosis is induced by
growing or subjecting the genetically altered Danio fish to an enriched cholesterol diet for a time sufficient to lead to cholesterol or lipid accumulation in an aorta and vascular inflammation,
wherein the genetically altered Danio fish is a transgenic having an enhanced green fluorescence protein (EGFP) constitutively expressed in endothelial cells such that the Danio fish is optically transparent with a fluorescent vasculature (fli:EGFP), and the Danio fish has genetic alterations comprising knockdown of an ApoE, ApoAI and/or LDL-R gene locus;
(c) administering the test compound to the genetically altered Danio fish of (b); and
(d) determining if the test compound ameliorates or reverses the induced vascular inflammation or the atherosclerosis in the genetically altered Danio fish,
wherein identifying a compound that can ameliorate or reverse the induced vascular inflammation or the atherosclerosis in the genetically altered Danio fish, identified the compound as a compound capable of ameliorating or reversing:
an atherosclerosis or
a vascular inflammation associated with lipid accumulation in a blood vessel wall.
2. The method of claim 1 , wherein the enriched cholesterol diet further comprises a diet selected from the group consisting of:
a high fat diet,
an enriched fat diet,
and
a combination thereof.
3. The method of claim 1 , wherein the test compound is selected from the group consisting of a small molecule, a polypeptide, a peptide, a nucleic acid, an siRNA, a polysaccharide, a lipid and a combination thereof.
4. The method of claim 1 , wherein the test compound is designed to target and/or increase or decrease the activity of: a matrix metalloproteinase, a lipoxygenase, a cyclooxygenase, a phospholipase, a toll-like receptor, a NADPH oxidase, a nuclear receptor, a transcription factor NF-κB associated gene expression, or a combination thereof.
5. The method of claim 1 , wherein the test compound is designed to target and/or increase or decrease: macrophage lipid uptake, endothelial adhesion molecules and/or monocyte recruitment, smooth muscle cell growth and migration, apoptosis of vascular cells, phagocytosis of apoptotic cells, activation of T- and B-1 cells in the lesions, or a combination thereof.
6. The method of claim 1 , wherein the genetically altered Danio fish further comprises:
(a) an expressed exogenous gene or an exogenous set of lipid metabolism genes;
(b) a completely or partially deleted, or “knocked out” endogenous lipid metabolism gene or set of lipid metabolism genes; or
(c) a combination of (a) and (b).
7. The method of claim 1 , wherein the genetically altered Danio fish is a zebrafish ( Danio rerio ).
8. The method of claim 1 , wherein in the genetically altered Danio fish the LDL-R gene or gene locus is completely knocked out.
9. The method of claim 1 , wherein the ApoE, ApoAI and/or LDL-R gene locus has at least one nucleic acid residue altered or removed.
10. The method of claim 1 , wherein the ApoE, ApoAI and/or LDL-R gene locus has an inserted heterologous sequence.
11. The method of claim 1 , wherein the ApoE, ApoAI and/or LDL-R gene locus has a rearranged endogenous sequence.
12. The method of claim 1 , wherein the ApoE, ApoAI and/or LDL-R gene locus is modified such that no amount or a less amount of ApoE, ApoAI and/or LDL-R message and/or polypeptide is expressed.
13. The method of claim 1 , wherein in the ApoE, ApoAI and/or LDL-R gene locus is modified such that an ApoE, ApoAI and/or LDL-R polypeptide has no or less than wild type activity.
14. The method of claim 1 , wherein the ApoE gene or gene locus is completely knocked out.
15. The method of claim 1 , wherein in the ApoAI gene or gene locus is completely knocked out.