The Study Of Meprin Regulation Of The Development And Progression Of Atheroscleosis In Mice

During atherosclerosis, the arterial wall gradually thickens to form an atherosclerotic plaque, resulting in the narrowing of the lumen of the artery. Consequently, the amount of blood supplied to the organ is reduced, most commonly affecting the heart and the brain. Plaques can abruptly rupture, causing a blood clot and often myocardial infarction (heart attack) or stroke. Intensive study of the cellular and molecular mechanisms that underlie atherogenesis (that is, the formation of atherosclerotic plaques) and plaque rupture has led to a consensus view of these processes. Initiation and progression of the lesion are highly complex processes, and many aspects of atherogenesis remain incompletely understood. Furthermore, in most cases, mechanistic insights have yet to be translated into therapeutic approaches. The insights of mechanism of atherosclerosis may lead to the development of successful therapeutic interventions for atherosclerotic cardiovascular disease.Meprin is a member of the astacin family of zinc metalloendopeptidases. It is highly expressed in the kidney, intestinal brush border membranes, and leukocytes and in some cancer cells. It consists of two subunits,αandβ, which form disulfide-bridged homodimers and heterodimers that differ in oligomerization potentials and substrate specificity. Meprin-αforms heterogeneous multimers and is secreted. Meprin-βrestricts the oligomerization potential of meprin to tetramers and attaches meprin oligomers to the plasma membrane. Its substrates include bioactive peptides and extracellular matrix proteins. Meprin proteins have been implicated in cancer and intestinal inflammation. Like neutral endopeptidase 24.11 (NEP), meprin could hydrolyze and inactive several endogenous growth factors, vasoactive peptides, cytokines, and extracellular matrix proteins circulating in peripheral blood or produced at vascular walls. Endothelial meprin could therefore metabolize vasoactive peptides and reduce their local concentrations at the arterial walls, resulting in attenuation of the effects of the peptides on vascular functions. Meprin inhibition may potentiate vascular responsiveness to these endogenous vasoactive peptides.The natriuretic peptide (NP) family has an important role in the regulation of blood pressure homeostasis and salt and water balance. Studies have demonstrated that NPs and other factors in the vascular wall are the substrates of meprin, particularly atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) (meprin has no effect on C-type natriuretic peptide (CNP)). NPs can also stimulate the endothelial release of vasodilators such as prostaglandins, endothelium-derived relaxing factor(s), and nitric oxide, which can inhibit the proliferation and apoptosis of vascular smooth muscle cells (VSMC), and modulation of the level of reactive oxygen species (ROS) in different cell types. These activities have been strongly associated with experimental hypertension, cardiac hypertrophy, thrombosis, restenosis, and atherosclerosis.Considerable evidence suggests that NP signaling may have a direct role in the development of atherosclerosis: most data support the anti-proliferative action of NP on smooth muscle cells (SMCs), suggesting an anti-atherogenic action of NP. Mice lacking the NPs receptor type-A (NPRA) and ApoE (Npr1–/– ApoE–/–) have increased atherosclerosis compared with ApoE-deficient mice that are of the wild-type for Npr1 (Npr1+/+ ApoE–/–). One could therefore expect that a meprin inhibitor could potentiate the vascular actions of NPs or other kinins by inhibition of their degradation at the vascular wall.This study initially aimed to assess the influence of chronic meprin inhibition by daily administration of the meprin inhibitor Actinonin(AC) on the development of atherosclerotic changes in ApoE–/–, and then aimed to clarify elevation of the level of NPs contributed to the anti-atherogenic effects of meprin inhibition.Methods:1. Male ApoE–/– mice (C57/Bl6 genetic background; purchased from Beijing University, Beijing, China) were used for this study. Twenty-eight ApoE–/– mice were maintained in a room set at 22°C with a 12-hour light/dark cycle. They received drinking water ad libitum. We assessed the influence of chronic meprin inhibition by daily administration of actinonin (5 mg/kg body weight per day; i.p.) on development of atherosclerotic changes in ApoE–/– mice. Mice were fed a high-fat (21% fat), cholesterol-rich (1% cholesterol) Western-type diet for 16 weeks starting at 10 weeks of age. At 20 weeks of age, randomly selected ApoE–/– mice were treated with Western-type-diet chow pellets supplemented with commercially available actinonin (meprin-I group) for 6 weeks; the diet of control ApoE–/– mice was supplemented with saline (placebo group). 2. Natriuretic peptides were measured in snap-frozen carotid arteries and plasma from experimental animals. Levels of natriuretic peptides were measured by RIA commercial kits specific for ANP and mouse BNP (Phoenix Biotech, Beijing, China) according to the manufacturer's instructions. Plasma was obtained through centrifugation of the blood for 10 minutes at 5500g at 4°C and stored at -80°C. Total plasma cholesterol and triglyceride concentrations were measured using enzymatic assayon a Cobas Mira Plus automated analyzer.3. For morphometric studies, at 26 weeks of age, rats were anesthetized and killed. Left carotid arteries were serially sectioned (5-μm) and, beginning from a random start site within the first 75μm, a section was stained every 75μm with hematoxylin and eosin (H&E). Images were captured with a Leica microscope and lesion area quantified using an image analysis system.4. Carotid artery superoxide levels were measured with Dihydroethidium (DHE) on serial frozen sections (10μm). NADPH oxidase activity in vascular was assessed by lucigenin-enhanced chemiluminescence as described previously with some modifications. An in-situ cell death detection POD kit was used with slight modification. After development using diaminobenzidine, sections were counterstained with methyl green. Four serial sections from each mouse were stained.5. Cells were cultured in 25-mm plastic tissue culture flasks and grown in DMEM supplemented with 10% fetal calf serum (FCS), 100μg/ml streptomycin and 100 U/ml penicillin in a humidified atmosphere of 5% CO2 and 95% air at 37 ?C. Cells, harvested once a week with 0.25% trypsin-0.02% EDTA and refed every other day, were used as confluent monolayers after 6–8 days at passage level 4–9 and cell number was plated at a density of 5×105 cells/cm2. Intracellular ROS were detected by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H2-DCF-DA). Apoptosis was measured by TUNEL assay according to the manufacturer's instructions. Proliferation was measured by [3H]-thymidine incorporation assay as previously described.Results:1. We first aimed to confirm that ApoE–/– administrated with actinonin resulted in increased levels of NPs in plasma and vascular tissues. BNP levels of meprin inhibition (meprin-I) in ApoE–/– mice were significantly increased by 2.5-fold in plasma and 3.3-fold in the vascular wall compared with those observed in ApoE–/– control mice. ANP levels were also significantly increased by 1.9-fold in plasma and 1.8-fold in the vascular wall. Total cholesterol, triglyceride levels, body weights and blood pressure were similar in meprin-I ApoE–/– mice and ApoE–/– mice.2. Treatment with actinonin changed the size of atherosclerotic lesions in carotid arteries compared with placebo group (meprin-I group), which suggested actinonin exerted apparent anti-atherogenic actions. These inhibitory effects of actinonin on atherosclerosis were significantly displayed among the three groups when entire right carotid arteries were stained by Oil-red-O.3. To evaluate lesion composition in the three groups, we immunostained lesions for macrophages, SMCs, as well as collagen and calculated the percentage of lesional area occupied. Treatment of ApoE–/– mice with actinonin resulted in dramatic reduction in lesion size, monocyte/macrophage content, and augmentation of collagen deposition compared with placebo group. These results suggest that treatment with actinonin halted genesis/progression of plaques, and altered the phenotype of lesions in ApoE–/– mice.4. In mice treated with actinonin, MMP-9 activity was significantly reduced compared with placebo. This result was consistent with actinonin-induced augmentation of collagen deposition. Actinonin markedly suppressed superoxide levels in the carotid arteries of ApoE-deficient mice. Consistent with decreased in-situ production of ROS, NADPH oxidase activity was also significantly reduced in tissues from actinonin-treated ApoE–/– mice. Actinonin treatment significantly reduced the number of TUNEL-positive cells in the carotid arteries of ApoE–/– mice fed a high-fat diet.5. Exogenous ANP failed to affect proliferation rates of THP-1 cells. BNP significantly inhibited proliferation of THP-1 cells. Exogenous ANP and BNP significantly affected serum-induced VSMC proliferation compared with untreated cells. Neutral endopeptidase inhibition (NEP-I) increased ANP- and BNP-inhibited VSMC proliferation rate, but actinonin increased only BNP-inhibited VSMC proliferation rate. The rate of lipopolysaccharide-induced apoptosis, as detected by TUNEL assay in THP-1 cells and VSMC, was reduced by treatment with ANP and BNP compared with untreated cells. NEP-I increased ANP- and BNP-inhibited cell apoptosis, and actinonin increased the effects of BNP on cell apoptosis only. These effects of NEP-I and actinonin work in parallel with ROS production in VSMC and THP-1 cells. Conclusions:1. The present study demonstrated that meprin inhibition by actinonin suppresses formation of atherosclerotic plaques, and preserves vascular wall function in ApoE–/– mice fed a high-cholesterol diet.2. In the current study, meprin inhibition for 6 weeks reduced ROS production and NAD(P)H oxidase activity in the vascular wall by 25%. Reduced oxidant stress may contribute to the anti-atherogenic effects of meprin inhibition.3. Apoptosis of VSMCs, endothelial cells and macrophages may promote plaque growth, pro-coagulation and may induce rupture, the major consequence of atherosclerosis in humans. The fact that increased levels of ROS induce cell apoptosis has been demonstrated in these cells. In the present study, meprin inhibition also reduced apoptosis measured by an in-situ cell death assay.4. Treatment of ApoE–/– mice with actinonin resulted in dramatic reduction in lesion size, monocyte/macrophage content, and augmentation of collagen deposition compared with placebo group. Treatment with actinonin may halt genesis/progression of plaques, and altered the phenotype of lesions in ApoE–/– mice.