| BackgroundCardio-cerebrovascular disease caused by atherosclerosis (As) is a severe, life-threatening illness, and has become one of the major killers in Western countries. Unravelling the molecular pathogenesis of As is therefore very important. However, its pathogenesis is complex, and to date, is not completely understood. Several recent studies have demonstrated that oxidative stress is closely involved in the pathogenesis of As, based on which an oxidative stress theory of As has been proposed.Recently, many efforts have focused on studying the involvement of macrophage oxidative stress in cardiovascular progression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the major source of reactive oxygen species (ROS) produced by phagocytic cells including neutrophil and monocyte/macrophage. It is composed of five subunits, i.e, p47phox, p67phox, the small G proteins racl (present in monocytes) and rac2 (present in neutrophils), p22phox, and gp91phox. The latter two are membrane-associated and together constitute the cytochrome b558, whereas the other subunits are located in the cytoplasm of resting cells. The p47phox contains two tandem Src homology 3 (SH3) domains, the interaction between which in resting cells can inhibit the binding of p47phox to p22phox. Upon activation, p47phox is phosphorylated, and meanwhile, the cytosolic components translocate to the plasma membrane, where they bind to cytochrome b558, thus forming the active NADPH oxidase and initiating the respiratory electron transport chain.Upon stimulation by chemokines released from endothelial cells, macrophages are recruited into the intima of the wall of the artery, resulting in the production of ROS through activation of NADPH oxidase and subsequent atherosclerotic lesions. Studies using animal models have demonstrated that the monocyte/macrophage NADPH oxidase is likely the main source of O-2 at atherosclerosic lesions The concentration of O2- is increased in atherosclerosic plaques, which is coupled with an elevated expression of phagocytic gp91phox and p22phox as well as of non-phagocytic NOX4. Furthermore, gp91phox is found to be extensively expressed in the scapular region in patients with coronary atherosclerosis, consistent with the distribution pattern of macrophages. These findings suggest that macrophage oxidative stress plays a key role in the pathogenesis of As.It is well known that low-density lipoprotein (LDL) can be oxidized by ROS to oxidized LDL (Ox-LDL). Lipid intake by macrophage is mediated by specific Ox-LDL receptors. LOX-1 is a newly identified Ox-LDL receptor expressed on bovine vascular endothelial cells, which is of the SR type. Unlike other SR members, LOX-1 is a membrane-bound glycoprotein, belonging to the C-type lectin subfamily. Further studies revealed that LOX-1 is also present on the surface of macrophage and vascular smooth muscle cells (VSMCs), and similarly, has the capacity to recognize Ox-LDL and contributes to the foam cell formation in As. Cominacini et al first reported that the binding of LOX-1 by Ox-LDL can induce the ROS production in endothelial cells, as well as promote the release of O2- and H2O2. The increased ROS not only functions as a second messenger, but also can directly inhibit the production of endothelial NO and inactivate the NF-κB signaling, consequently causing the peroxidation of plasma lipid. Based on these findings, we hypothethized that LOX-1 likely played a crucial role in As caused by macrophage oxidative stress; i.e., the binding of LOX-1 by Ox-LDL could induce the production of ROS through activation of macrophage NADPH oxidase, and excessive ROS could in turn activate intracellular signaling pathways contributing to the development and progression of As.PurposeWe aimed to extensively investigate the important role of LOX-1 in As by macrophage oxidative stress. The effects of LOX-1 on the ROS production of macrophage as well as on the expression of the subunit genes of NADPH oxidase were studied. Using flow cytometry, real-time PCR and Western blot techniques, we sought to determine whether Ox-LDL could induce the activation of macrophage NADPH through LOX-1 receptor, consequently causing the generation of ROS and oxidative stress, and to test whether the MAPK signaling was involved in these processes. To further unravel the role of LOX-1 in vivo, we knocked down its expression through adenoviral delivery of its specific shRNAs in an ApoE knockout mice model, and investigated the effects of LOX-1 inhibition on the oxidative stress-related As. This project would unravel the roles of LOX-1 in oxidative stress and its underlying molecular mechanisms, and also likely provide a potential therapeutic target for the treatment of As based on the knowledge of the LOX-1 signaling.Methods1. Generation of LOX-1-siRNA-expressing plasmidsThe LOX-1 siRNA sequence was designed by using the RNA interference (RNAi) design algorithm. A negative control sequence that is not predicted to target any known gene was included. The synthesized oligonucleotides were annealed, and inserted into the linearized pGenesil-1 expression vector. The recombinant plasmids were identified by enzymatic digestion and direct DNA sequencing.2. Transfection of LOX-1-siRNA-expressing plasmidsMouse macrophages were cultured in serum-free medium, and at approximately 60% subconfluence cells were randomly divided into 2 groups (1×107 cells for each group):the control and the transfection groups. The used plasmid/lipofectamine ratios included 1:1,1:2 and 1:3. At 48 h posttransfection, the transfected cells were observed under fluorescent microscopy for detection of green fluorescence. The transfection rate was measured using flow cytometry, and cell viability was determined by MTT assay. Based on the above experiments, we attempted to establish an optimized condition for transfection of macrophages.Macrophages were transfected under the optimized condition, and quantitative real-time-PCR and Western blotting were conducted to analyze the LOX-1 mRNA and protein expression levels, respectively. The most efficient LOX-1-siRNA was applied in the following experiments.3. The influence of LOX-1 on Ox-LDL-induced macrophage oxidative stress(1) Measurement of MDA and SOD. Five groups were used:normal cell control group, Ox-LDL (50 mg/L) group, LOX-1-siRNA and Ox-LDL (50 mg/L) group, and (p)LOX-1-siRNA group (LOX-1-siRNA treatment for 24 h followed by the addition of Ox-LDL (50 mg/L) for another 24 h), and pCon group (empty vector). After the indicated treatments, cells were harvested and subjected to the measurement of MDA and SOD.(2) ROS analysis. Seven groups were used:the negative control group without fluorescent probes; normal cell group, normal cell with fluorescent probes group; positive control group (treatment with H2O2), Ox-LDL group (treatment with 50mg/L Ox-LDL for 16 h), LOX-1-siRNA+Ox-LDL group (LOX-1-siRNA treatment for 24 h followed by the addition of Ox-LDL (50 mg/L) for another 16 h) and pCon group (empty vector). After the treatments, intracellular ROS levels were determined by fluorescent microscopy and flow cytometry.(3) Analysis of the expression of the components of NADPH oxidase. The studied genes included NOX1, NOX2, Racl, p47phox, and p22phox. GAPDH was used as an internal control. Their mRNA and protein levels were quantitated by real-time PCR and Western blot analysis, respectively.4. The effects of LOX-1 on the MAPK signaling in macrophage oxidative stress(1) The effects of different durations of Ox-LDL treatment on the MAPK signaling. Cells were untreated or treated with 50 mg/L Ox-LDL for different times (15,30,60,90, and 120 min). After the treatments, cells were harvested and subjected to Western blot analysis for detection of the protein levels of P-p38MAPK, p38 MAPK, P-ERK, ERK, P-JNK, and JNK.(2) The influence of LOX-1-siRNA on the MAPK signaling. Four groups were used:the control group, Ox-LDL (50mg/L) group, LOX-1-siRNA+Ox-LDL (50 mg/L) group and empty vector group. After the treatments, cells were harvested and subjected to Western blot analysis for detection of the protein levels of P-p38MAPK, p38 MAPK, P-ERK, ERK, P-JNK, and JNK.5. The role of LOX-1 in oxidative stress-caused As (1) Eight-week-old ApoE-/- mice were fed with high lipid diet for 5 weeks. After the treatments, animals were randomly divided into 3 groups (n=60 for each group) to receive an injection through the tail vein with 200μl of PBS (the control group), Ad-siRNA-Neg (5×1012 pful/mL; the empty vector group), or Ad-siRNA-LOX-1 (5×1012 pful/mL; the RNAi group). The injection was repeated after 10 (for both the control and empty vector groups) or 14 (for the RNAi group) days. Animals received an additional 4-week high lipid feeding.(2) Analysis of the Ad-siRNA-LOX-1 distribution in vivo. At 7 and 14 days after an initial injection,5 mice were randomly selected, whose liver, kidney and artery tissues were collected for preparation of frozen sections following anaesthesia with injection with pentobarbital sodium (0.4 ml/100 g; i.p.). The distribution of Ad5-EGFP-LOX-1-sIRNA in vivo was analyzed under fluorescent microscopy.(3) Measurement of LOX-1 mRNA and protein expression in artery tissues. At 4 weeks after the delivery of Ad-siRNA-LOX-1, LOX-1 mRNA and protein levels were determined using Real-time PCR and Western blotting, respectively.(4) Analysis of the serum lipid levels. The contents of total cholesterol (TC), triglycerides, as well as low-and high-density lipoprotein (LDL and HDL, respectively) were measured using the enzymatic colorimetric methods.(5) Movat pentachrome staining. The changes of plaque morphology were analyzed using Image Pro-Plus 5.1. Additionally, the average plaque size as well as the ratio between the plaque area and lumen area was determined.(6) The diameter of the left carotid artery and the intima-media thickness were measured using Vevo 770 High-Resolution Imaging System (VisualSonics Inc., Toronto, Ontario, Canada) for 3 times, and the average values were obtained.(7) Immunostaining for CD68. CD68 immunostaining were performed to detect the percentage of macrophage in plaques.(8) Gene expression analysis. The mRNA and protein levels of LOX-1, NOX1, NOX2, rac1, p47phox, and p22phox were determined by real-time PCR and Western blot analysis, respectively.Results1. Generation of LOX-1-siRNA expressing plasmidThe plasmids targeting LOX-1, LOX-1-siRNA1 and LOX-1-siRNA2, were created, which were confirmed by enzymatic digestion and direct DNA sequencing.2. Transfection of LOX-1 siRNA-expressing plasmids(1) RAW264.7 cells were grown well in DMEM containing 10% FBS. They were mainly circular or polygonal shapes, with pseudopods and 1-2 nuclei. Cells had a fast growth rate and usually reached confluence in 5-6 days.(1) Under the optimized condition (1.0μg plasmids and 2μl transfection agent), cells were transfected using Lipofectamine 2000 with LOX-1-siRNA expressing plasmids. After transfection, the endogenous LOX-1 mRNA level was significantly reduced. LOX-1-siRNA2 showed more robust inhibitory capacity, and therefore was selected for further experiments.3. The influence of LOX-1 on Ox-LDL-induced macrophage oxidative stress(1)The influence of LOX-1 on the MDA of macrophage and the SOD activity:The MDA concentration in the Ox-LDL group was significantly higher than that in the control group, whereas, conversely, the SOD activity in the former group is considerably lower than that in the latter, suggesting an imbalance the oxidation and anti-oxidation states in macrophage, a hallmark of oxidative stress. Compared to the Ox-LDL group, the Ox-LDL plus LOX-1-siRNA group showed a decreased MDA but increased SOD activity.(2)Analysis of the effects of LOX-1 on the intracellular ROS levels by fluorescent microscopy: The results showed that upon the treatment of Ox-LDL the intracellular ROS level was significantly increased as compared to the control group. By contrast, the presence of LOX-1-siRNA significantly resulted in a reduction in the ROS level.(3)Analysis of the intracellular ROS levels by flow cytometry:The ROS levels in the positive control and mock groups were110.0±2.1 and 33.8±2.8, respectively. After the treatment with Ox-LDL for 16 h, the ROS amount (70.9±3.1) was significantly increased (P<0.05 compare to the mock group). However, the pretreatment with LOX-1-siRNA for 24 h caused a significant reduction in the ROS level (38.7±2.3; P<0.05 compare to the mock group). There was no statistical significance between the empty vector and mock groups (P>0.05).(4)The results of real-time PCR analysis revealed that after treatment with 50 mg/L Ox-LDL for 16h, the mRNA levels of NOX1, NOX2, Rac1, p47phox, and p22phox were significantly increased in comparison with the control group (P<0.05). The pretreatment with LOX-1-siRNA for 48 h could significantly downregulate these gene expression levels compared to the Ox-LDL group (P<0.05). Similar results with regards to the influence of LOX-1-siRNA on the NOX1, NOX2, Rac1, p47phox, and p22phox expression were found using Western blot analysis.4. The effects of LOX-1 on the MAPK signaling in macrophage oxidative stressCompared to the control, the treatment with Ox-LDL for 15 min could induce significant phosphorylations of p38MAPK and JNK (P<0.05), and considerable phosphorylations of ERK1/2 were detected after the Ox-LDL treatment for 60 min (P<0.05). The pretreatment with LOX-1-siRNA for 48 h significantly abrogated the inductive effects of Ox-LDL (P<0.05 compared to the Ox-LDL group). There was no statistically significant difference between the control and empty vector groups (P>0.05). 5. The role of LOX-1 in oxidative stress-caused As(1) The body weight of all mice was slightly increased when the experiments finished, but no statistically significant difference was found among the studied groups (P>0.05). Animals in the Ad-siRNA-LOX-1 group showed decreased levels of TC, LDL and HDL, but the decrease was not statistically significantly (P>0.05).(2) The delivery of LOX-1-siRNA through the tail vein caused a wide distribution of the recombinant adenovirus in vivo, including the liver, kidney and artery tissues.(3) The results of real-time PCR and Western blot analyses consistently showed that the administration of LOX-1-siRNA into ApoE KO mice resulted in a significant reduction of endogenous LOX-1 mRNA in artery tissues, as compared to the ApoE KO and empty vector controls (P<0.05).(4) The analysis using Movat staining revealed that the plaque area, the external elastic membrane area, and the plaque area/lumen area were slightly reduced, but not statistically significant in the RNAi group compared to the control and empty vector groups (P>0.05). However, the delivery of LOX-1-siRNA resulted in a significantly increase in the fiber cap thickness (5.41±0.46 vs.4.78±0.25 (control) and 4.81±0.34 (empty vector); P<0.05 for both comparisons).(5) Ultrasound examination of the left common carotid artery revealed that there was no statistically significant difference in the average artery size among the studied groups. However, as compared to the control and empty vector groups, the RNAi group showed significantly decreased intima-media thickness of the left carotid artery (0.29±0.06 vs 0.28±0.02 and 0.2±0.01, respectively; P<0.05 for both comparisons).(6) As compared to the control and empty vector groups, the proportion of the CD68+ macrophages was considerably reduced in the RNAi group (P<0.05).(7) The results of real-time PCR and Western blot analyses consistently showed that the mRNA and protein levels of NOX1, NOX2, p22phox, p47phox, and racl were significantly reduced in the RNAi group as compared to the control and empty vector groups (P<0.05).Conclusions(1) LOX-1-siRNA expressing plasmids have been successfully generated, which show high inhibitory activity with regard to knockdown mouse endogenous LOX-1.(2) Ox-LDL is able to promote the LOX-1 expression in macrophages and activate the NADPH oxidase, which in turn upregulates the expression of NOX1, NOX2, Rac1, p47phox, and p22phox and triggers the generation of ROS, consequently causing oxidative stress.(3) Ox-LDL can bind to LOX-1 and induce the release of ROS in vivo, which subsequently activates the MAPK signaling pathways. (4) The studies using the ApoE KO mouse model show that the involvement of LOX-1 in the pathogenesis of As is likely through regulation of the plaque stability, and the activation of NADPH oxidase and the production of ROS may be associated with this function of LOX-1.
|
PDF Full Text Request