Identification And Validation Of LOX-1 As A Potential Anti-atherosclerotic Drug Target

Atherosclerosis(AS) is a chronic, complex vascular inflammatory disease characterized by lipid deposition and even lipid plaque formation in the vascular intima. The rupture of atherosclerotic plaques was the main cause of death in atherosclerotic patients.Both the etiology and the pathology of the disease remain to a large extent obscure. Several mechanisms had been proposed such as the lipid infiltrative theory, the thrombosis hypothesis, the platelet aggregation theory, and the clone hypothesis. Now, most researchers believed that the pathogenesis of AS is the complicated results of the interaction among the vascular cells, extracellular matrix, blood constituents(especially monocytes, platelets and low density lipoprotein(LDL)), local hemodynamic environment and heredity. Therefore, there is no single etiological factor for AS. In recent years, the"response-to-injury"theory was widely accepted. The endothelial dysfunction and injury was the initial step of AS in"response-to-injury"theory. Recent studies revealed that the ox-LDL derived from the oxidative modification of LDL was the main risk factor which played an important role in the activation, injury and dysfunction of endothelial cells(ECs).Lectin-like oxidized low density lipoprotein receptor-1(LOX-1) was a new kind of ox-LDL receptor discovered by scientists in 1997. Ox-LDL could not be recognized by LDL receptor but could be recognized by macrophage scavenger receptors(SRs), CD36 etc. However, these receptors are absent in ECs. Accumulated studies showed that LOX-1 was expressed in ECs and was the main receptor for ox-LDL. LOX-1 might play an important role in several kinds of cardiovascular diseases including hypertension, myocardial ischemia-reperfusion injury, diabetes mellitus, glomerulosclerosis and especially in AS.Drug targets are the biomacromolecules or special biomolecules which could bind specific drugs and produce specific pharmacological effects. The discovery of new drug target is the basis of new drug development and new drug mechanism exploration. Though the advent of pharmacogenomics era opens the door for new drug target research, the identification and validation of new drug target still faces many difficulties. The traditional drug targets such as enzymes and receptors are still the main targets for drug research. As far as AS is concerned, no specific drug target has been discovered which partly accounts for the difficulty in its prediction and treatment. Identification and validation of new drug target is very important in anti-atherosclerotic drug discovery. Several potential drug targets had been proposed such as COXs, HDL, NADPH oxidase and CD40 etc, which point out new directions for anti-atherosclerotic drug research.Combining the recent advances in LOX-1 research, the present study was designed to explore the possibility of LOX-1 as a potential anti-atherosclerotic drug target.Effects of Atherosclerotic Risk Factors on LOX-1 ExpressionAs the main receptor for ox-LDL in ECs, the expression of LOX-1 could be regulated by many factors. Endothelial cells, vascular smooth muscle cells(VSMCs) and monocytes were the main three kinds of cells involved in the development of AS. Ox-LDL and advanced glycation end products(AGEs) were risk factors of AS. Using primary cultured rat aortic VSMCs and U937 cells, the effects of ox-LDL and AGEs on LOX-1 expression were studied.The results showed that ox-LDL upregulated LOX-1 expression in rat VSMCs in a dosedependent and time-dependent manner. Ox-LDL increased intracellular reactive oxygen species(ROS) production in VSMCs. Apocynin, a NADPH oxidase inhibitor, aspirin, a cycloxygenase inhibitor, and nordihydroguaiaretic acid(NDGA), a lipoxidase inhibitor could significantly decrease ox-LDL induced intracellular ROS production and LOX-1 expression while rotenone, an inhibitor of mitochondrial respiratory chain, and allopurinol, an inhibitor of xanthine oxidase(XO), showed no effect. These results revealed that there were multiple causes of ROS formation in ox-LDL treated VSMCs which might play an important role in ox-LDL induced LOX-1 expression.Both AGEs and ox-LDL induced LOX-1 expression in U937 cells in a dose-dependent and time-dependent manner. Furthermore, U937 cells showed enhanced phagocytotic ability for ox-LDL and increased adhesion to EAhy926 cells after AGEs and ox-LDL treatment. These results suggested that ox-LDL and AGEs played an important role in the formation of foam cells and LOX-1 might take part in this process.Role of LOX-1 in Ox-LDL Induced Endothelial Cell ApoptosisApoptosis plays an important role in the pathogenesis of AS. Previous studies suggested that ox-LDL induced several kinds of cell apoptosis. As the main receptor for ox-LDL in ECs, we supposed that LOX-1 might play an important role in ox-LDL induced apoptosis in ECs. Thus the role of LOX-1 in ox-LDL induced human umbilican vein endothelial cells(HUVECs) apoptosis was studied.Our results showed that ox-LDL increased intracellular ROS and superoxide anion(O₂^-) formation in a dose-dependent and time-dependent manner in HUVECs. Ox-LDL dosedependently induced HUVECs apoptosis. Inhibitors of ROS production and anti-LOX-1 mAb could inhibit ox-LDL induced apoptosis in different degrees. These results suggested that LOX-1 took part in ox-LDL induced apoptosis in ECs and the formation of intracellular ROS might play an import role.High-throughput Screening for Identifying LOX-1 LigandsIn order to identify LOX-1 mimic ligands, a competitive fluorescence polarization-based high throughput screening method was established. A total of 12700 chemicals were screened and 3 chemicals(6291, 6302 and 6306) with high affinity for human LOX-1 protein were discovered. The stability and toxicity of LOX-1 ligands were studied.The results revealed that the screened LOX-1 ligands(6291, 6306) were unstable and had little cytotoxicity. However, they showed toxicity to both rats and mice.Biological Identification of LOX-1 LigandPrevious studies revealed that the binding of ox-LDL to LOX-1 induced intracellular ROS production. Ox-LDL could decrease intracellular nitric oxide(NO) concentration due to the increase of intracellular O₂^-. Ox-LDL also activated NF-kB. Using CHO-K₁ cells trans-fected with human LOX-1 gene(LOX-1-CHO-K₁ cells), the effects of the screened LOX-1 ligand 6306 on ROS and O₂^-production and NF-κB expression were studied.Results showed that 6306 had similar effects on the ROS and O₂^-production to ox-LDL in LOX-1-CHO-K₁ cells. 6306 could significantly reduce the NO₂^-level in the supernatant of cultured cell. The expression of NF-κB was also increased after 6306 treatment. These results suggested that 6306 might activate LOX-1 and result in effects similar to those of ox-LDL.Effects of LOX-1 Ligand on Experimental Rat Atherosclerotic ModelTo explore the role of LOX-1 in the pathogenesis of AS and to test if LOX-1 ligand could be new potential anti-atherosclerotic lead compound, a rat AS model induced by high-fat diet was established and the effects of LOX-1 ligand 6302 on this model were studied.The results revealed that LOX-1 ligand 6302 could attenuate high-fat diet induced rat aortic intima injury and inhibit atherosclerotic plaque formation. The serum levels of total cholesterol(TC), triglyceride(TG), and low density lipoprotein-cholesterol(LDL-C) were decreased while the high density lipoprotein-cholesterol(HDL-C) level was increased in 6302 treated atherosclerotic model.The serum malondialdehyde(MDA) level was also reduced. However, results from RT-PCR showed that 6302 did not affect the expression of LOX-1 in rat aortic tissue. These results suggested that LOX-1 inhibition might have some beneficial effects on AS.In summary, the present study revealed that AS risk factors ox-LDL and AGEs could significantly up-regulate LOX-1 expression; LOX-1 took part in ox-LDL induced apoptosis in ECs; LOX-1 ligand 6302 could attenuate high-fat diet induced AS. LOX-1 is one of the new potential drug targets for anti-atherosclerotic therapy.