Effects And Mechanisms Of Cytochrome P450 Epoxygenase 2J2 Overexpression On Abdominal Aortic Aneurysm In Apoe-deficient Mice Induced By Angitension â…¡ Chronic Infusion

Background and purposeArachidonic acid (AA) is the most abundant substances in vivo and it is the precursor of various important biological active substances. AA exist maninly in the form of combined esterified fatty acids in the membrane on the inside. When cells are subject to various physiological or pathological stimulus, actived phospholidase A2 then release arachidonic acid from cell membrane, which make it accessible for metabolism by three signaling pathways:lipoxygenases, cyclooxygenases and cytochromes P450. After years of research on AA, the third channel AA metabolism (cytochrome P450 pathway), has been a more and more important metabolic pathway. Cyclooxygenases convert free arachidonic acid to be prostaglandins, prostacylin, and thromboxanes, which paly important roles in numerous physiological or pathophysiological processes. Lipoxygenases induce the free arachidonic acid to be converted to be leukotrienes, which are important mediators of inflammatory and allergic reactions. One form of cytochrome P450 enzymes (CYP2J and CYP2C) generates four different epoxyeicosatrienoic acids (EETs), respectively,5,6-EET, 8,9-EET,11,12-EET and 14,15-EET. Hydrolysis of EETs mainly by the soluble form oxide hydrolase (Soluble epoxide hydrolase, sEH) metabolites generated the corresponding Dihydroxyeicosatrienoic acids (DHET) which have been considered to have minor biological effects. Many studies confirm that the inhibition of sEH can significantly enhance the biological effects of EETs. What should be noted here is that DHETs which be considered to be no role ever been shown to also play an important role in lipid metabolism in the recent studies.The cytochrome P450 enzymes comprise a large superfamily of proteins. Cytochrome P450 epoxygenases widely distributed in the body, especially liver. Only 2J2 among 2J family is found in human tissue. And CYP2J2 has the abundant expression in heart, kidney and vascular endothelial cells. In the past few decades, mang reports have show that cytochrome P450 enzymes-EETs system plays an important biological role in the cardiovascular system and renal system. These effects include:①EETs are with a strong expansion effects of vascular function to regulate blood pressure and the mechanism is through activation of calcium-sensitive potassium channels, causing hyperpolarization of vascular smooth muscle cells and thus causes vasodilation. More, at least in part, vasodilation effects of EETs are through activation and (or) increased eNOS expression which increas NO synthesis and release;②EETs promote endothelial cell proliferation and angiogenesis, the mechanism including the activation of MAPK and PI3K/AKT signaling pathways, and eNOS/NO pathway is also played an important role.③cytochrome P450 enzymes overexpression in order to increase EETs biosynthesis, significantly reduced the tumor necrosis factor (TNF-a) induced endothelial cell apoptosis. The mechanism includes at least inhibition of the ERK1/2 dephosphorylation, and activation of PI3K/AKT signaling pathway.④EETs inhibit inflammation and protect the biological function of vascular endothelial cells. EETs inhibit inflammatory cell adhesion to endothelial cells, reducing the infiltration of inflammatory cells, inhibiting TNF-a induced endothelial cell expression of inflammatory adhesion molecules. The anti-inflammation effects of EETs at least in part are through inhibition of nuclear factor NFkB nuclear translocation. EETs were confirmed to be PPARy ligands, which activate PPARy and thus play important anti-inflammatory effects. And the PPARy antagonist (GW9662) significantly attenuates the anti-inflammatory effects of EETs.⑤A large number of studies confirm that EETs can inhibit transforming growth factorβ(TGFP)-induced aortic smooth muscle cell migration.⑥Recent findings in our laboratory showed that EETs significantly inhibited the progression of diabetic nephropathy, inhibited the apoptosis of tongue squamous cell carcinoma cell line (TCA) induced by arsenic trioxide (arsenic trioxide, ATO). The mechanisms in these effects include at least inhibit the cell generation of reactive oxygen species, inhibit the increase in NADPH subunit expression, inhibit the downregulation of catalase (CAT) and superoxide dismutase (SOD) expression.Many studies confirm that application of specific inhibitors of sEH (AUDA etc.) which notably inhibit the biological activity of sEH and significantly increase the concentrations of EETs in tissue and serum, has significant protective effects in the cardiovascular system and renal system:dilation of blood vessels to lower blood pressure; inhibition of kidney damage caused by hypertension; inhibition of endothelial cell injury and atherosclerotic plaque formation; inhibition of elevation in levels of low density lipoprotein (LDL); inhibits ischemia-induced brain damage and vascular injury; inhibition of the load and(or) angiotensin-induced cardiac hypertrophy; attenuates myocardial ischemia reperfusion injury and so on. Using adeno-associated viral vectors to obtain the cytochrome P450 enzymes overexpression which elevated the concentration of EETs in serum and organs also obtained similar biological effects to sEH inhibition.More and more studies confirm that inflammation and oxidative stress play important roles in the occurrence and development of abdominal aortic aneurysms. Recently, reserchers conducted a series of extensive research in the model of abdominal aortic aneurysm in APOE gene knockout mice induced by angiotensinⅡ. The findings suggest that inflammation chemokine expression and mononuclear macrophage infiltration was significantly increased in angiotensinⅡ-treated mice. ROS (reactive oxygen species, ROS) has been shown to increase the level of vascular disease in a variety of diseases. AngⅡcould induce ROS generation. ROS promote smooth muscle cell proliferation and migration, recruitment of inflammatory cells, vascular endothelial cell apoptosis and increased expression of inflammatory adhesion molecules. ROS can activate the matrix metalloproteinase (matrix metalloproteinases, MMPs) in blood vessel which lead to AAA formation. Inhibition of ROS production and activity of MMPs in the AAA model can block the formation and development of aneurysm.Vascular smooth muscle cells are major effector cells in the vascular media. The loss and disordered arrangement of smooth muscle cell is particularly prominent in the local tissue of AAA. AngⅡcan induce oxidative stress in vascular smooth muscle cells, leading to cell aging and apoptosis. In addition, vascular smooth muscle cells are the main source of extracellular matrix cells. The loss of vascular smooth muscle cells decreased the extracellular matrix, which causes vascular wall thinner. the inflammatory chemokines induce vascular smooth muscle cells to produce a large number of ROS and inflammatory cells in the local tissue also release ROS. ROS could activated vascular smooth muscle cells to produce and release cyclophilin A (CypA) which is a major oxidative stress inducing factor (SOXY). CypA in turn stimulate smooth muscle cell proliferation and migration, endothelial cell adhesion molecule expression. Also CypA is chemotaxis of inflammatory cells which could aggravate oxidative stress, upregulated the expression of inflammatory chemokines and increase inflammatory cell infiltration. Cyclophilin A can significantly increase the activity of MMPs, which will ultimately contribute to the formation of AAA. Inhibition of CypA (gene level or drug) expression has been shown to significantly reduce angiotensin II-induced AAA.Then can we take some measures, which can inhibit or block the inflammatory response and oxidative stress and improve the function of smooth muscle cells, to prevent the occure of abdominal aortic aneurysms? Cytochrome P450 epoxygenase overexpression can significantly inhibit TNF-a induced apoptosis in vascular endothelial cells;James Liao et al reported that cytochrome P450 epoxygenase overexpression inhibit inflammation. Recent studies have found that inhibition of sEH activity can significantly inhibit the angiotensinⅡ-induced ApoE knockout mouse atherosclerosis and abdominal aortic aneurysm formation. Cytochrome P450 epoxygenase overexpression also protect brain and heart from ischemia-reperfusion injury, which is through inhibition of oxidative stress at least in part. So, can EETs be used for the prevention of abdominal aortic aneurysms?In this experiment, we suppose that the increases in the level of endogenous EETs could activate endogenous protective mechanisms of the vascular wall through the inhibition of inflammation and oxidative stress in the initiation and progress. And the effects could prevent the formation of abdominal aortic aneurysms. Thus, we conducted experiments to confirm the effects of CYP2J2 overexpression on the formation of abdominal aortic aneurysm induced by angiotensin II in ApoE knockout mice, and the possible mechanisms which exist in the protective effects of CYP 2J2 overexpression.Experimental methods and resultsExperimental methods:1. CYP2J2 gene was cloned into the recombinant adeno-associated virus-mediated vector (rAAV), the pXX2, pXX6 and pXXUFl-2J2 plasmid were extracted by alkaline lysis method and purified by silica purification. In 293 cells, three plasmids (pXX2, pXX6 and pXXUFl-2J2) were packaged into rAAV-2J2 virus through cotransfection, and using the same method to package the green fluorescent protein GFP(rAAV-GFP virus). And the real-time PCR method was used to detect the virus titer.2.32 Male APOE deficient mice (ApoE-/-),8 weeks old, weight 22-28 grams, were randomly divided into four groups after adaptive feeding a week:sline group (sline, n=8), AngiotensinⅡgroup (AngiotensinⅡ, n=8), AngiotensinⅡ+GFP group (GFP, n=8), AngiotensinⅡ+CYP2J2 group (2J2, n=8). The micro infusion pumps containing AngⅡ(1.44mg/kg/d) were implanted subcutaneously in mice by posterior approach of neck, while the saline group preparation of experimental animals (Subcutaneous output micro-injection pump saline). Then 4 ApoE-/- mouse model of abdominal aortic aneurysm was ok after 4 weeks. Specific experimental method is as follows:4 weeks before surgery, according to body weight of mice, the mice were given the virus solution carrying the gene GFP or 2J2 (1×1011p.fu) treatment by intravenous injection respectly. The mice in saline group and AngiotensinⅡgroup were given an equal volume of saline injection, GFP group received rAAV-GFP virus solution,2J2 group received rAAV-2J2 virus solution. The night before surgery, according weight of mice, micro pumps were filled with Ang II solution or saline, and then immersed in saline at 37 degrees constant temperature cell incubator for 12 hours. These operations require console in sterile complete. Surgical procedure:Mouse was anesthetized with 2% isoflurane anesthesia, and then the mice were fixed in the sterile surgical operation table, belly down. The vertical neck incision was done on the skin of neck, the length of lcm. Then the pumps were implanted into mouse skin, and suture. Mice were then placed in waiting incubators after surgery until they were awake. The mice should be observed every day to guard against accidents in mice.4 weeks after, the experimental animals were sacrificed, isolated from mouse aorta and photographs, to retain the appropriate blood, urine and tissue samples for use. Among them, serum specimens from blood after centrifugation, frozen -80 degrees to prepare for testing. After centrifugation, the urine was frozen -80 degrees to prepare for testing. Aortic specimens were frozen as part of -80 degrees, part of the OTC fixed stationary to prepare for cutting frozen sections of frozen, there are still some organizations placed in 4% formaldehyde solution fixation, dehydration, dipping wax, embedded in paraffin at room temperature. All animal procedures are in line with NIH guide management and use of laboratory animals, and animal management practices of the Chinese Academy of Sciences to develop standards.3. Proteins were extracted from frozen aorta tissue for western blot to detect protein expression levels of CYP2J2 protein. And ELISA methods to test DHET content in blood and urine.4. Blood samples were centrifuged to measure lipid levels.5. The paraffin section of aorta of mice were prepared to carry out HE stain, Sirius Red (Sirius Red) staining, Masson trichrome (Masson, s Trichrome) staining, Prussian blue staining and Van Giessen staining in order to observe changes in aortic structure, and distribution of collagen deposition and iron deposition.6. Immunohistochemical staining was carried out to detect matrix metalloproteinase (MMP) 2 and MMP-9, and semi-quantitative analysis of expression levels. Simultaneously, we extracted proteins to perform western blot (MMP-2 and MMP-9).7. Detection of serum and aortic tissue inflammation-related indicators and CYP2J2 overexpression on inflammation:Elisa method to detect the serum levels of inflammatory factors (IL-1β, IL-4, IL-6, IL-10, sVCAM-1, sE-selection, etc.); immunohistochemical staining of inflammatory cells in aortic tissue to detect chemokine MCP-1 and macrophage marker protein ED-1 expression; Western blot to detect MCP-1, ED-1, VCAM-1, PPARγ, and IkBa levels. And the expression of CypA was also detected.8. Detection of oxidative stress in mouse blood, urine and aortic:Detection kit was eued to detect the level of lipid peroxidation malondialdehyde (MDA) level and total superoxide dismutase activity (SOD) in mice of each group; Western blot was done to detect PI3K/AKT/eNOS, CAT and the expression of NADPH subunits (gp91, P47, P67, Mn-SOD, CuZn-SOD, CAT, etc..)Results:(1) Western Blot and ELISA confirmed that rAAV-2J2 gene delivery through tail vein injection in mice get lasting and stable expression 8weeks after gene delivery.(2) Lipid data:Plasma total cholesterol levels:plasma total cholesterol levels of mice in angiotensinⅡgroup (17.1±2.3 mmol/l) is higher than saline group (15.9±1.6 mmol/l) mice, and GFP group (16.9±2.5 mmol/l) has no different to angiotensinⅡgroup. But total cholesterol of mice in 2J2 group (9.7±1.12 mmol/l) was significantly lower than GFP group and AngiotensinⅡgroup, the difference was statistically significant (n=8, p<0.05).Plasma triglyceride levels:plasma triglyceride levels of mice in angiotensinⅡgroup(1.88±0.33 mmol/l) is higher than saline group(1.74±0.16 mmol/l) mice, and GFP group (1.86±0.31mmol/l) has no different to angiotensinⅡgroup. But triglyceride of mice in 2J2 group (1.70±0.21 mmol/l) was significantly lower than GFP group and AngiotensinⅡgroup, the difference was statistically significant (n=8, p<0.05).Low-density lipoprotein:plasma Low-density lipoprotein levels of mice in angiotensinⅡgroup (17.72±1.83 mmol/l) is higher than saline group (15.42±1.12 mmol/l) mice, and GFP group(17.4±1.81 mmol/l) has no different to angiotensinⅡgroup. But Low-density lipoprotein levelsof mice in 2J2 group (16.36±1.45 mmol/l) was significantly lower than GFP group and Angiotensin II group, the difference was statistically significant (n=8, p<0.05).High density lipoprotein:plasma high density lipoprotein levels of mice in angiotensinⅡgroup (0.33±0.07 mmol/l) is lower than saline group (0.44±0.03 mmol/l) mice, and GFP group (0.33±0.07 mmol/l) has no different to angiotensinⅡgroup. But total high density lipoprotein of mice in 2J2 group (0.37±0.04 mmol/l) was significantly higher than GFP group and AngiotensinⅡgroup, the difference was statistically significant (n=8, p<0.05).(3) The effects of CYP2J2 overexpression on the incidence of aneurysm and the diameter of the abdominal aorta:The incidence of aneurysm:the incidence of aneurysms (75%,6/8) in mice in AngiotensinⅡgroup is higher than that in saline group (0%,0/8), and GFP group (75%, 6/8) is similar to AngiotensinⅡgroup, but the incidence of aneurysm (25%,2/8) in 2J2-treated mice was significantly lower than the GFP group and Angiotensin group, the difference was statistically significant (n=8, p<0.05).Abdominal aortic diameter in angiotensinⅡand GFP mice (2.3±0.35 mm VS 2.27± 0.34 mm) was significantly higher than saline mice (0.96±0.09 mm), but abdominal aortic diameter in 2J2-treated mice (1.33±0.21 mm) was significantly lower than GFP group and AngiotensinⅡgroup, the difference was statistically significant (n=8,p<0.05).(4) Morphology changes:Angiotensin 11 and GFP mice were significantly thicker lining the abdominal aorta:common plaque, and irregular thickening of the membrane, significantly hypertrophy extravascular membrane, under the more common endometrial bleeding and endometrial acute intimal surface often interrupted by the uplift of the plaque. Prussian blue staining of the intima and adventitia in the wall of iron deposition can be seen clearly, location, and CD68-positive inflammatory cells overlap. Outer membrane has a certain degree of thickening, mainly composed of fibroblasts and inflammatory cells. Saline group were seen lining the blood vessels and plaque to acute bleeding and did not see the iron deposition, were significantly higher than the wall of angiotensin II and GFP group to be thin. Wall can also be found CYP2J2 mice less collagen deposition, but the collagen bundles are mostly complete and orderly arranged, the outer membrane significantly higher than that of angiotensinⅡand GFP group and rare thin iron deposition and inflammatory cell infiltration. That CYP2J2 overexpression significantly inhibited the chronic infusion of AngiotensinⅡinduced vascular structural changes and iron deposition and infiltration of inflammatory cells(5) Detection of MMP-2 and MMP-9 expression:vessel walls of mice in AngiotensinⅡgroup and GFP mice have a large number of MMP-1 and MMP-9 expression, whereas overexpression of CYP2J2 significantly reduced the expression of MMPs. Western blot results also confirmed this result.(6) Results of inflammation-related test:ELISA:detection of inflammatory factors in serum shows that 2J2 overexpression significantly inhibited the serum levels of proinflammatory factors observed in GFP and AngiotensinⅡgroup.The serum levels of IL-1βin 2J2-treated mice (41±4.5 pg/ml) was significantly lower than GFP (64±7 pg/ml) and AngiotensinⅡgroup (62±8 pg/ml), but higher than the saline group (22±2 pg/ml);The serum levels of IL-4 in 2J2-treated mice (24.3±2.85 pg/ml) was significantly lower than GFP (19.6±3.27 pg/ml) and AngiotensinⅡgroup (19.2±3.52 pg/ml), but higher than the saline group (28±1.7 pg/ml);The serum levels of IL-6 in 2J2-treated mice (35.2±4 pg/ml) was significantly lower than GFP (52.5±7 pg/ml) and AngiotensinⅡgroup (53.5±6 pg/ml), but higher than the saline group (26.6±2pg/ml);The serum levels of IL-10 in 2J2-treated mice (92±11 pg/ml) was significantly higher than that of GFP (62±16.5 pg/ml) and AngiotensinⅡgroup (63.2±15pg/ml), but higher than the saline group (138±8.4 pg/ml);The serum levels of sVCAM-lin 2J2-treated mice (3.72±0.384 ng/ml) was significantly lower than GFP (4.84±0.67 ng/ml) and AngiotensinⅡgroup (4.89±0.58 ng/ml), but higher than the saline group (2.45±0.22 ng/ml);The serum levels of sE-selection in 2J2-treated mice (39.66±4.39 ng/ml) was significantly lower than GFP (51.71±8.27 ng/ml) and AngiotensinⅡgroup (51.91±8.72 ng/ml), but higher than the saline group (23.25±3.14 ng/ml).Immunohistochemical detection of monocyte-macrophage chemotactic factor (MCP-1) and macrophage marker protein (ED-1):compared with the GFP group and AngiotensinⅡgroup,2J2 overexpression significantly reduced the tissue MCP-1 expression and the proportion of ED-1 positive cells to total cells.Western blot showed that aortic VCAM-1, ICAM-1 and CypA expression in GFP group and AngiotensinⅡgroup significantly increased, but the expression of PPARγand IkBa was significantly reduced.2J2 overexpression significantly inhibited the above changes.(7) The relevant indicators of oxidative stress in serum, urine and kidney.Detection of serum levels of MDA:compared with the serum MDA levels in saline group (0.46±0.05 umol/l), serum MDA levels in GFP (0.73±0.098 umol/l) and AngiotensinⅡgroup (0.76±0.116 umol/l) was significantly higher.2J2 group serum MDA levels (0.63±0.069 umol/l) was significantly lower than GFP group and AngiotensinⅡgroup.Detection of SOD activity in serum:compared with the serum SOD activity in saline group (63±3.5 U/ml), serum levels of SOD activity in the GFP (31±6 U/ml) and AngiotensinⅡgroup (29±7 U/ml) decreased significantly.2J2 serum SOD activity (43±5 U/ml) was significantly higher than GFP group and AngiotensinⅡgroup.Detection of the levels of MDA in homogenates of aortic tissue:compared with the MDA levels in saline group (3.77±0.87 nmol/mg protein), MDA levels in GFP (13.51±1.63 nmol/mg protein) and AngiotensinⅡgroup (13.46±1.74 nmol/mg protein) was significantly higher. MDA levels in 2J2-treated mice (9.17±1.40 nmol/mg protein) were significantly lower than GFP group and AngiotensinⅡgroup.Detection of SOD activity in in homogenates of aortic tissue:compared with the SOD activity in saline group (97±4.59 U/mg protein), SOD activity in the GFP (62.2±9.89 U/mg protein) and Angiotensin II group (59.2±9.96 U/mg protein) decreased significantly. SOD activity in 2J2-treated mice (79.2±7.16 U/mg protein) was significantly higher than GFP group and AngiotensinⅡgroup.Urinary NOx excretion:compared to saline group (0.59±0.032 umol/24h/100mg BW), urinary NOx excretion in GFP (0.373±0.069 umol/24h/100mg BW) and AngiotensinⅡgroup (0.376±0.067 umol/24h/100mg BW) decreased significantly. Urinary NOX excretion in 2J2-treated mice (0.468±0.048 umol/24h/100mg BW) was significantly higher than the total GFP group and urine (NOx) excretion.The concentration of NO in serum:compared to saline group (45±2.03 umol/1), the concentration of NO in GFP (27.7±4.74 umol/l)) and AngiotensinⅡgroup (28.3±4.63 umol/l)) decreased significantly. the concentration of NO in 2J2-treated mice (37.17±3.10 umol/l)) was significantly higher than the total GFP group and urine (NOx) excretion. Prussian blue staining to detect the deposition of iron revealed that 2J2 overexpression notably attenuated the deposition of iron, which was increased in aortic by AngiotensinⅡ. Immunohistochemistry to detect the P47 expression in aortic, which was upregulated by AngiotensinⅡWestern blot to detect the expression of oxidative stress related proteins (Mn-SOD, CuZn-SOD, CAT, P67 phox, P47 phox, gp91 phox, PI3K, AKT, and eNOS) in aortic tissue of mice:Compared with saline group, the proteins (P67 phox, P47 phox and gp91 phox)expression of aortic in GFP group and AngiotensinⅡwas significantly increased, while the inhibitors of oxidative stress(Mn-SOD, CuZn-SOD, CAT, PI3K, AKT, and eNOS) was significantly reduced,2J2 overexpression significantly inhibited the changes of related protein expression mentioned obove.(8) The expression of CypA was notably upregulated in aortic by AngiotensinⅡchronic infusion. But CYP2J2 over-exoression significantly attenuated the rise of the expression of CypA.Statistical AnalysisData are presented as mean±SEM. Comparisons were carried out with One-way ANOVA and Newman-Keuls tests for post hoc analyses. Significance was accepted at a value of P<0.05.Conclusion:1. Recombinant adeno-associated virus mediated CYP2J2 gene delivery in mice received a long-term and stable expression, and significantly increased the content of EETs in blood and urine.2. CYP2J2 overexpression significantly inhibited the AngiotensinⅡ-induced incidence of abdominal aorta and dilatation of the aorta.3. CYP2J2 overexpression inhibits intimal thickening and fracture, reducing the degree of medial hypertrophy, attenuated the structural damage of elastin, inhibited the hypertrophy of outer membrane. CYP2J2 overexpression inhibited the up-regulation of matrix metalloproteinase (MMP2 and MMP9).4. CYP2J2 overexpression significantly decreased the serum levels of proinflammation factor in the APOE deficient mice, and significantly reduced expression of inflammatory chemokines and infiltration of monocyte macrophages in aortic.5. CYP2J2 overexpression significantly inhibited oxidative stress-associated indicators in blood, urine and aortic tissue of the APOE deficient mice, including the expression of NADPH subunits, MDA levels in blood and aortic tissue, decline of SOD levels in blood and aortic and total urinary excretion of nitric oxide.6. CYP2J2 overexpression significantly inhibited the upregulation of angiotensin-induced CypA expression.7. The results obtained in the current experiment offer a new way of strategy for the abdominal aortic aneurysm (AAA) treatment in the future and we expected it to be a new targets for drug development and gene therapy about AAA.