| Background and Objectives: Atherosclerosis is a kind of inflammation-relateddiseases. TNF-α and other cytokines play important roles in the occurrence and development procedure of Atherosclerosis and other cardiovascular damages. TNF-α can be induced by disturbance of lipid metabolism, infection and abnormity of haemodynamics. TNF-α causes the injury and functional changes of vessel endothelial cells, including induction of the expression of adhesion molecules, release of cytokines and increase in vessel permeability. All these facilitate occurrence and development of atherosclerosis. Furthermore, TNF-α is associated with pathology progress of congestive heart failure, viral myocarditis, dilated cardiomyopathy and rejection of transplanted heart. Cytochrome P450 (CYP) arachidonic acid epoxygenase-derived epoxyeicosatrienoic acids participate in the homostasis of caidiovascular systems, including modulating blood pressure, promoting angiogenesis and inhibiting migration of smooth muscle cells. Previous studies have shown EETs have anti-inflammatory properties. Expogenous EETs can inhibit the expression of vascular cell adhesion molecule-1 protein and the adhesion of peripheral blood mononuclear cells to endothelium. It remains elucidated. However, whether endogenous EETs induced by overexpression of epoxygenase gene inhibit the inflammation and prevent farther cardiovascular damages induced by TNF-α and what is the associated mechanisms. Therefor, this work was carried out to address the issues.Methods:1. In Vitro Study: Human umbilical vein endothelial cells (HUVECs) were infected with recombinant adeno-associated viruses (rAAV) containing CYP2C11, CYP2J2 or CYP F87V to increase endogenous levels of EETs and incubated with or without the inhibitors of various cell signal transduction pathways. TNF-α was added subsequently and 6 hours later, the expression of VCAM-1 was investigated by western blot and the adhesion assays of PBMC were performed.2. In Vivo Study: Male Sprague-Dawley rats were divided into five groups randomly and injected intravenously with a construct containing with CYP2C11, CYP2J2 or CYPF87V cDNA and empty plasmid and saline was used as control, respectively. Two weeks later, TNF-α was injected intravenously (10μg/Kg) . Six to eight hours after injection of TNFα, animal haemodynamic index were recorded using a cardiac catheter via right , the aortic ring response to vasoactive drugs was tested using 4-channel instrument; the plasma levels of sVCAM-1, sICAM-1, sE-selectin, IL-1β, IL-6 and IL-10 were measured, respectively, using ELISA kits, and the expression of VCAM-1 in aortic endothelium was probed using Western Blots. The apoptosis in heart and aortic vessel was observed using TUNEL kits and and the expression of Bcl-2 and p-ERK in aortic endothelium and myocardium was probed using Western Blots.Results: 1. In vitro, TNF-α Induced significant increase in the expression ofVCAM-1 and PKCε protein in HUVEC and the adhesion of PBMC endothelial cells buttransfection with rAAV-CYP2C11 and rAAV-CYP2J2 markedly attenuated the effects. 2. In vivo, hemodynomic records showed that TNF-α infusion induced a significantincrease in left ventricular end diastolic pressure (LVEDP) in rats compared with control animals (1.31±0.22 vs 3.66±0.26 mmHg, p<0.05). CYP epoxygenase Gene delivery with CYP2C11, CYP2J2 and CYPF87V decreased the TNF-α induced increase in LVEDP (2.61±0.28, 2.55±0.88 and 2.73±0.49 mmHg, respectively, P<0.05 vs control, respectively ). As well TNF-α infusion led to decrease in the maximum left ventricular pressure ascending speed (+dp/dt max) compared with control rats (5606.5±277.7 mmHg·s-1 in control vs 1919.5±391.4 mmHg·s-1 in TNFα). Delivery of CYP2C11, CYP2J2 and CYPF87V attenuated the depression of +dp/dt max induced by TNF-α (2721.4±305.7, 2921.5±79.9, 3774.1±495.5 mmHg·s-1, respectively, p<0.05 vs control, respectively) .3. The relaxation response of aortic ring to acetylcholine was decreased by TNF-α. butforced overexpression of CYP2C11, CYP2J2 and CYPF87V attenuated the toxic effects in part.4. TNF-α infusion significantly elevated the plasma levels of sVCAM-1, sE-selectin,IL-1β and IL-6 , in contrast decreased the plasma levels of IL-10. Overexpression of CYP2C11, CYP2J2 and CYPF87V, however, reversed partially the both the elevation in the plasma levels of sVCAM-1, sE-selectin, IL-1β and IL-6 and the decrease in plasma IL-10 level.5. Western Blots showed that the expression of VCAM-1 and PKCε protein in aorticendothelium was upregulated by TNF-α However, overexpression of CYP2C11, CYP2J2 and CYPF87V inhibited the effects.6. There appears no significant apoptosis in the myocardium of TNF-α group.Western Blots showed that CYP2C11, CYP2J2 and CYPF87V delivery can upregulate the expression of Bcl-2 and p-ERK protein in aortic endothelium and myocardium.Conclusion: (1) These findings suggest that overexpression of CYP2C11 and CYP2J2 genes showed anti-inflammatory properties, which is related with inhibition of protein kinase C. (2) The delivery of CYP2C11, CYP2J2 and CYPF87V Genes can ameliorate hemodynamics, improved the relaxation reaction of vessels to acetylcholine, and the mechanisms are involved in inhibiting the inflammation and the expression of angiotensin II type 1 receptor in tissues. These results provide a new insight into understanding endogenous protection via CYP epoxygenase and EETs and development of new strategies for the treatment of atherosclerosis.
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