| Background and Objective:Inflammation, oxidative stress and extracellular matrix （ECM） remodeling areaccelerators of atherosclerosis （AS）; moreover, excessive matrix degradation by matrixmetalloproteinases （MMPs） could convert silent atherosclerotic plaque into a vulnerableplaque. Histological markers, such as a thin and collagen-poor fibrous cap, large lipid core,abundant macrophages, and few smooth muscle cells （SMC）, are signs of a vulnerable lesion.It is now becoming increasingly apparent that AS could be a relatively benign disease ifvulnerable plaque rupture and thrombosis would be prevented. Thus, stabilizingatherosclerotic plaque, along with retarding AS development, is important in AS therapy.Extracellular matrix metalloproteinase inducer （EMMPRIN）, initially found on thesurface of tumor cells, is a member of the immunoglobulin （Ig） superfamily, consisting of ashort cytoplasmic domain, a transmembrane domain, and two Ig-like extracellular domains.EMMPRIN is expressed on numerous cell types and serves as a cell surface receptor formultiple ligands including cyclophilin, monocarboxylate transporter, integrins, andEMMPRIN itself. Binding to these various partners enables EMMPRIN to induce MMPs andto mediate various functions. Three decades of research on EMMPRIN have discovered thatthis transmembrane glycoprotein is a pleiotropic molecule, critical to the physicaldevelopment of retinal, sperm, immune, and nervous systems. Notably, EMMPRIN is alsoinvolved in multiple pathological processes, including carcinoma invasion and migration,cyclophilin A （CyPA）-mediated inflammation and oxidative stress.MMPs contribute both to the formation and the destabilization of atherosclerotic plaque.Although there are reports with contradictory results regarding the role of MMP activity,MMPs have been considered putative therapeutic targets for the prevention ofatherothrombosis and ischemic events. Because of the limitations of direct MMP inhibitors,preventing MMP up-regulation may be more beneficial for plaque stability. EMMPRIN, an upstream regulator of MMPs, had rekindled the discussion of anti-MMP therapy for AS.EMMPRIN was first related to AS by Major who found EMMPRIN was present in humanatheromas. Now, it has been reported that EMMPRIN is implicated in the formation of foamcells, the induction of inflammatory factors and MMPs,atheroma inflammation and theactivation of platelet. In our previous work, we demonstrated that angiotensin II, a knownatherogenic factor, could upregulate EMMPRIN expression in atherosclerotic plaque andmacrophages. On these grounds, we suggest that EMMPRIN may affect AS. Consistent withour idea, data from recent studies show that EMMPRIN can mediate monocyte recruitment tothe vascular wall and abdominal aortic aneurysm formation. Furthermore, nitric oxideexhibited a cardioprotective function in repressing EMMPRIN.Research on theEMMPRIN-related protein CyPA also demonstrated that genetic ablation of CyPA in anapolipoprotein E knockout (ApoE-/-) background inhibited the progression of AS. In view ofthis above, it seems plausible that EMMPRIN may be an atherogenic agent, affectingatherosclerotic plaque development or stability.Methods1. Animals and proceduresAnimal procedures were approved by the Experimental Animal Ethics Committee ofThird Military Medical University prior to performing the study, and was conformed by theGuidance for the Care and Use of Laboratory Animals, published by the U.S. NationalInstitute of Health.Male ApoE-/- mice were maintained at22°C,12/12-h light/dark cycle, specificpathogen-free conditions and with access to water ad libitum.For the first part of the study,12mice （male,6-8weeks old,18-22g） were randomizedto a normal diet group （n=3） or a high-fat diet （21%fat,0.15%cholesterol） group （n=9）. Atthe end of4,8, and12weeks feeding of the high-fat diet group （3mice at each time point）,and at12weeks in the normal diet group, mice were anesthetized with overdose use ofsodium pentobarbitone （i.p.60mg/kg）, loss of sensibility was detected by pricking theanimal’s feet and legs with forceps, and then death confirmed by exsanguination. Theseprotocols complied fully with the recommendations of the American Veterinary MedicalAssociation Panel on Euthanasia. EMMPRIN protein expression in the aorta was assessed. For antibody treatment regimens,40mice were randomized to the ND control group（normal diet, n=10）, HFD group （high-fat diet:21%fat,0.15%cholesterol, n=10）, E-ABgroup （high-fat diet+EMMPRIN antibody, n=10）, or the I-AB group （high-fat diet+isotypeantibody, n=10）, and fed for12weeks. Starting at12weeks, an anti-mouse EMMPRIN（clone RL73.2） functional-grade purified function-blocking antibody （eBioscience, USA） orrat IgG2a K isotype control （eBioscience） was injected intraperitoneally （100μg per mouse,twice per week,4weeks） into the mice （the time, dose and toxicity of treatment wereaccording to reference and determined in preliminary optimization experiments）. At the end ofthe treatment, the40mice were anesthetized with overdose use of sodium pentobarbitone （i.p.60mg/kg）, loss of sensibility was detected by pricking the animal’s feet and legs with forceps,and then death was confirmed by exsanguination. The plasma, aorta, and innominate arterywere harvested.2. Plasma lipoprotein profile and inflammatory cytokines.Plasma was obtained from orbital blood. Total cholesterol （TC）, triglycerides （TG）, HDL,and LDL in the plasma （200ul） were measured using an enzymatic assay （Roche, China）.Circulating levels of tumor necrosis factor-alpha （TNF-α）, interleukin-6（IL-6）, and monocytechemotactic protein （MCP-1） were quantified using an ELISA assay kit.3. Histology and morphometry3.1Aortic lesion analysisAtherosclerotic lesion burden was assessed along the luminal surface of the entire aortaby oil red O staining as described previously. Six aortas in each group were measured. Theresults are reported as a percentage of the total aorta area containing lesions.3.2Histological analysisFor morphological analysis, abdominal aortas or innominate arteries （50%of mice fromeach group） were fixed and embedded in paraffin. Cross sections （6μm） were stained withHE staining （hematoxylin and eosin）, Masson’s trichrome, or immunostaining. Cryosectionsobtained from parts of the innominate arteries （50%of mice from each group） were stainedwith oil red O. Images were captured using a microscope.3.3ImmunohistochemistryImmunohistochemistry （IHC） was performed to detect EMMPRIN and CyPA in macrophages, and SMC in the plaque as described previously. Primary anti-mouse antibodies（CD1471:150; CyPA1:200; α-smooth muscle actin1:200; Santa Cruz; MAC3, a macrophagemarker1:150; Abcam） were used. Images were captured using a microscope （Leica） and thepercentage of positive area was calculated; six sections were selected from each group.3.4ROS production in atherosclerotic lesionsDihydroethidium （DHE） staining was used to assess vascular lesion superoxideproduction in situ, as described by Sukhanov. Cryosections from innominate arteries wereincubated with DHE and imaged with a fluorescence microscope. The same series of sectionswere pre-incubated with the superoxide scavenger N-acetylcysteine （NAC,10mM） for acontrol. The intensity of red fluorescence （normalized to plaque area）, representing in situsuperoxide production in the atherosclerotic plaque, was measured using the Image-Pro Plus6.0software; six sections were selected from five arteries.4. Western blotting analysisWestern blotting was used to measure protein expression in the aorta. Proteins fromaortic tissue were seperated using10%or12%sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and transferred to nitrocellulose membranes. Primary antibodies againstmouse EMMPRIN （1:200）, CyPA （1:400）, β-actin （1:1000）, and fluorescent labeled secondaryantibody （1:5000） were used. Binding was detected using the Odyssey Infrared ImagingSystem and quantified using the Quantity One Software; three or four aortas in each groupwere measured.5. Gelatinolytic activity assayTo investigate the in situ gelatinolytic activity of the atherosclerotic lesion, cryosectionsfrom innominate arteries were assessed using an in situ zymography fluorescence staining kit.Control sections were incubated in the presence of the MMP inhibitor, EDTA （20mM）. Sixsections were selected from five arteries. Analysis of the intensity of green fluorescence（normalized to plaque area） was conducted using the Image-Pro Plus6.0software.To assess gelatinolytic activity of total aortas, aorta protein supernatants wereelectrophoresed in10%polyacrylamide gels containing0.1%（w/v） gelatin. After incubationat37°C for42h in a developing buffer, the gel was stained with0.25%Coomassie brilliantblue. Proteolysis was detected as a white zone in a dark-blue field. 6. Migration analysisMacrophage migration was evaluated using the scratch assay, as described previously.Briefly,1.0×106RAW264.7cells in three test groups （control, EMMPRIN antibody10μg/mL, isotype control10μg/mL） were seeded onto dishes and incubated for24h. Themonolayer was scratched using a pipette tip and the cells were allowed to migrate for theindicated time. Distances between the edges of the scratch were quantitatively measured toevaluate cell migration.7. Statistical analysisAll values are expressed as means±SD. Raw data was analyzed with the SPSS software（ver.17.0）. Statistical analysis for multiple group comparisons was performed using one-wayANOVA, followed by Tukey’s multiple comparisons procedure. P values <0.05wereconsidered to indicate statistical significance.Result1. Expression of EMMPRIN in atherosclerotic lesionsIn preparation for our studies investigating AS, ApoE-/- mice were fed a21%fat and0.15%cholesterol diet for AS formation. After the model was established, we first assessedEMMPRIN expression in atherosclerotic lesions in ApoE-/- mice. EMMPRIN levels detectedby immunoblotting were increased substantially in the aorta of mice after12weeks of thehigh-fat diet. We found that EMMPRIN was mostly expressed on the intimal cells in the×40magnification fields. Measurements of the EMMPRIN-positive area displayed similarcharacteristics to the EMMPRIN expression detected by Western blotting.2. Effects of EMMPRIN antibody administration on body weight, plasma lipoproteinprofiles, and inflammatory cytokinesTo evaluate the effects of antibody administration on mice, body weight and plasmalipids were measured. Significant increases in TC, TG, and LDL and a decrease in HLD wereobserved in mice with the high-fat diet; however, there was no significant change in lipids inthe antibody treatment groups.As proinflammatory cytokines have been shown to play a role in the progression of AS,we measured levels of circulating TNF-α, IL-6, and MCP-1. Serum IL-6and MCP-1decreased55.9%and22.9%in the mice of the E-AB group, respectively, compared with the HFD group. No significant change was detected in serum levels of TNF-α, and mice in theI-AB group showed no decrease in serum cytokines. The results indicated that the circulatingIL-6and MCP-1levels were reduced by EMMPRIN functional blockage.3. Effect of EMMPRIN function blockage on atherosclerotic lesions in ApoE-/- mice3.1. To study the functional role of EMMPRIN in AS, a function-blocking antibody forEMMPRIN or an isotype control was administered to mice eight times beginning at12weeksof high-fat diet feeding, and the effect of treatment was monitored. The administration ofantibodies in the last4weeks of feeding did not affect the body weight, compared with thecontrol group.3.2To assess whether the EMMPRIN antibody affected AS progression, we comparedatherosclerotic lesions in mice from the different groups. The en face of the aortic tree wasstained with oil red O. Few lesions were found in the aortas in the ND group （2.47%±1.17%of the total）; the mean atherosclerotic lesion volume in HFD group mice increasedsignificantly, accompanied by hyperlipidemia （55.43%±11.22%）. Although EMMPRINblocking antibody treatment did not change the lipid profiles, we found oil red O staining inthe E-AB group （30.70%±9.48%） was notably reduced, compared with that of the HFD group,and reduced atherosclerotic lesions were not observed in mice treated with the isotypeantibody （53.79%±13.79%）. To further assess the lipid deposition in plaque, we quantified theoil red O-positive area in cross-sections of innominate arterial lesions. Consistent with the enface staining results, lipid burden decreased in the plaque of EMMPRIN antibody-treatedmice. These data demonstrated that EMMPRIN antibody administration limits atheroscleroticlesion progression, and these changes occurred independently of circulating lipids levels.3.3To survey the effects of the EMMPRIN antibody on the composition ofatherosclerotic plaque in detail, we stained for collagen, macrophages, and SMC oninnominate arterial atherosclerotic lesions, and calculated the occupied percentages of each.We found increased α-smooth muscle actin （α-SMC） accumulation （9.15±2.72%vs.5.08±1.66%） and collagen content （62.25±10.69%vs.28.50±6.40%） in the plaques of E-ABmice compared with the HFD group. Treatment with the EMMPRIN antibody dramaticallyreduced the macrophage content （MAC3-positive area decreased to21.56±8.10%） comparedwith the HFD group （MAC3-positive area41.69±6.31%）. These results suggested that treatment with EMMPRIN functional blocking antibody altered the phenotype of the plaquein ApoE-/- mice.3.4Because inflammation is a key marker of advanced atherosclerotic lesions, weapplied DHE staining to assess ROS production in plaques. Representative images show theinnominate arteries stained with DHE in the presence or absence of the antioxidant NAC.EMMPRIN antibody administration markedly suppressed superoxide levels in atheroscleroticlesions.Together, these results confirmed our experimental findings that EMMPRIN functionblockage ameliorated AS in ApoE-/- mice.4. Mechanism of action of EMMPRIN function blockage on atherosclerotic lesions inApoE-/- micewe used the in vitro scratch test to assess whether the different circulating monocytenumber was due to a preventative effect on monocyte migration induced by the EMMPRINantibody. With12-and24-h tests, we found less cell migration in the EMMPRIN antibodytreatment group. It is our belief that reduced migration resulted in a lower macrophagecontent in plaque.Western blotting results showed no change in protein expression of EMMPRIN in theaorta in mice with EMMPRIN antibody administration. However, gelatin zymographydemonstrated that with EMMPRIN antibody treatment, MMP-2and-9activities decreasedconsiderably, compared with the HFD control, but no decrease was observed in the I-ABgroup. In situ zymography on innominate arterial cryosections was performed for HFD, E-AB,and I-AB mice, but not for ND mice because plaque formation rarely occurred in these micewith a normal diet for16weeks. Gelatinolytic activity, after normalizing to plaque area, wasmuch lower in atheromas from E-AB group mice versus HFD and I-AB mice. Addition of theMMP inhibitor, EDTA, abolished the proteolytic activity, indicating the presence ofgelatinolytic MMPs.The above results illustrate that EMMPRIN antibody could decrease the capacity of cellmigration in vitro and capacity for gelatin degradation in vivo, primarily throughdown-regulation of MMP-2and MMP-9, and, as a consequence, preventing monocytemigration into the intima and ECM degradation in atherosclerotic plaques. Conclusion1.The present study demonstrated an increase in EMMPRIN levels in atheroscleroticplaque.2. EMMPRIN antibody intervention ameliorated atherosclerosis in ApoE-/- mice.3. The atheroprotective effect of anti-EMMPRIN intervention appeared to involve theregulation of MMP expression and atherogenic cytokine induction.y the down-regulation ofmetalloproteinase activity. |
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