Depletion of B2 but not B1a B cells in BAFF receptor-deficient ApoE mice attenuates atherosclerosis by potently ameliorating arterial inflammation

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE(-/-) mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE(-/-) mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R(-/-) ApoE(-/-) (BaffR.ApoE DKO) and BAFF-R(+/+)ApoE(-/-) (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE(-/-) mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation.     

A critical function of Th17 proinflammatory cells in the development of atherosclerotic plaque in mice

Considerable evidence supports that the CD4(+) T cell-mediated immune response contributes to the development of atherosclerotic plaque. However, the effects of Th17 cells on atherosclerosis are not thoroughly understood. In this study, we evaluated the production and function of Th17 and Th1 cells in atherosclerotic-susceptible ApoE(-/-) mice. We observed that the proportion of Th17 cells, as well as Th1, increased in atherosclerotic ApoE(-/-) mice compared with nonatherosclerotic wild-type littermates. In ApoE(-/-) mice with atherosclerosis, the expression of IL-17 and retinoic acid-related orphan receptor γt was substantially higher in the arterial wall with plaque than in the arterial wall without plaque. Increased Th17 cells were associated with the magnitude of atherosclerotic plaque in ApoE(-/-) mice. Importantly, treatment of ApoE(-/-) mice with neutralizing anti-IL-17 Ab dramatically inhibited the development of atherosclerotic plaque, whereas rIL-17 application significantly promoted the formation of atherosclerotic plaque. These data demonstrate that Th17 cells play a critical role in atherosclerotic plaque formation in mice, which may have implications in patients with atherosclerosis.     

Divergent coronary flow responses to uridine adenosine tetraphosphate in atherosclerotic ApoE knockout mice

Uridine adenosine tetraphosphate (Up₄A) exerts potent relaxation in porcine coronary arteries that is reduced following myocardial infarction, suggesting a crucial role for Up₄A in the regulation of coronary flow (CF) in cardiovascular disorders. We evaluated the vasoactive effects of Up₄A on CF in atherosclerosis using ApoE knockout (KO) mice ex vivo and in vivo. Functional studies were conducted in isolated mouse hearts using the Langendorff technique. Immunofluorescence was performed to assess purinergic P2X₁ receptor (P2X₁R) expression in isolated mouse coronary arteries. In vivo effects of Up₄A on coronary blood flow (CBF) were assessed using ultrasound. Infusion of Up₄A (10^?9–10^?5 M) into isolated mouse hearts resulted in a concentration-dependent reduction in CF in WT and ApoE KO mice to a similar extent; this effect was exacerbated in ApoE KO mice fed a high-fat diet (HFD). The P2X₁R antagonist MRS2159 restored Up₄A-mediated decreases in CF more so in ApoE KO + HFD than ApoE KO mice. The smooth muscle to endothelial cell ratio of coronary P2X₁R expression was greater in ApoE KO + HFD than ApoE KO or WT mice, suggesting a net vasoconstrictor potential of P2X₁R in ApoE KO + HFD mice. In contrast, Up₄A (1.6 mg/kg) increased CBF to a similar extent among the three groups. In conclusion, Up₄A decreases CF more in ApoE KO + HFD mice, likely through a net upregulation of vasoconstrictor P2X₁R. In contrast, Up₄A increases CBF in vivo regardless of the atherosclerotic model.     

Alterations in the mouse and human proteome caused by Huntington's disease

Huntington's disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In our effort to identify proteins involved in processes upstream or downstream of the disease-causing huntingtin, we studied the proteome of a well established mouse model by large gel two-dimensional electrophoresis. We could demonstrate for the first time at the protein level that alpha1-antitrypsin and alphaB-crystalline both decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver and testes in mice. Reduced expression of the serine protease inhibitors alpha1-antitrypsin and contraspin was found in liver, heart, and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. In three brain regions obtained post-mortem from Huntington's disease patients, alpha1-antitrypsin expression was also altered. Reduced expression of the major urinary proteins not found in the brain was seen in the liver of affected mice, demonstrating that the disease exerts its influence outside the brain of transgenic mice at the protein level. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington's disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.     

Atorvastatin Upregulates the Expression of miR-126 in Apolipoprotein E-knockout Mice with Carotid Atherosclerotic Plaque

Carotid atherosclerosis (AS) is a chronic inflammatory disease of the carotid arterial wall, which is very important in terms of the occurrence of cerebral vascular accidents. Studies have demonstrated that microRNAs (miRNAs) and their target genes are involved in the formation of atherosclerosis and that atorvastatin might reduce atherosclerotic plaques by regulating the expression of miRNAs. However, the related mechanism is not yet known. In this study, we first investigated the effects of atorvastatin on miR-126 and its target gene, i.e., vascular cell adhesion molecule-1 (VCAM-1) in apolipoprotein E-knockout (ApoE?/?) mice with carotid atherosclerotic plaque in vivo. We compared the expressions of miR-126 and VCAM-1 between the control, atherosclerotic model and atorvastatin treatment groups of ApoE?/? mice using RT-PCR and Western blot. We found the miR-126 expression was significantly down-regulated, and the VCAM-1 expression was significantly up-regulated in the atherosclerotic model group, which accelerated the progression of atherosclerosis in the ApoE?/? mice. These results following atorvastatin treatment indicated that miR-126 expression was significantly up-regulated, VCAM-1 expression was significantly down-regulated and atherosclerotic lesions were reduced. The present results might explain the mechanism by which miR-126 is involved in the formation of atherosclerosis in vivo. Our study first indicated that atorvastatin might exert its anti-inflammatory effects in atherosclerosis by regulating the expressions of miR-126 and VCAM-1 in vivo.     

Lack of acyl-CoA:diacylglycerol acyltransferase 1 reduces intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E knockout mice

Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in several tissues. The final step in TG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Lack of whole body DGAT1 is associated with reduced lipid-induced inflammation. Since one major component of atherosclerosis is chronic inflammation we hypothesized that DGAT1 deficiency might ameliorate atherosclerotic lesion development. We therefore crossbred Apolipoprotein E-deficient (ApoE(-/-)) mice with Dgat1(-/-) mice. ApoE(-/-) and ApoE(-/-)Dgat1(-/-) mice were fed Western-type diet (WTD) for 9weeks and thereafter examined for plaque formation. The mean atherosclerotic lesion area was substantially reduced in ApoE(-/-)Dgat1(-/-) compared with ApoE(-/-) mice in en face and aortic valve section analyses. The reduced lesion size was associated with decreased cholesterol uptake and absorption by the intestine, reduced plasma TG and cholesterol concentrations and increased cholesterol efflux from macrophages. The expression of adhesion molecules was reduced in aortas of ApoE(-/-)Dgat1(-/-) mice, which might be the reason for less migration capacities of monocytes and macrophages and the observed decreased amount of macrophages within the plaques. From our results we conclude that the lack of DGAT1 is atheroprotective, implicating an additional application of DGAT1 inhibitors with regard to maintaining cholesterol homeostasis and attenuating atherosclerosis.     

Deletion of L-selectin increases atherosclerosis development in ApoE-/- mice

Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel(-/-)) animals were crossed with mice lacking Apolipoprotein E (ApoE(-/-)). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE(-/-)L-sel(-/-)) mice and the corresponding ApoE(-/-) controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE(-/-)L-sel(-/-) mice as compared to ApoE(-/-) controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE(-/-)L-sel(-/-) animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE(-/-)L-sel(-/-) and ApoE(-/-) mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE(-/-)L-sel(-/-) as compared to ApoE(-/-) mice. Leukocyte rolling on lesions in the aorta was similar in ApoE(-/-)L-sel(-/-) and ApoE(-/-) animals. ApoE(-/-)L-sel(-/-) mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE(-/-) controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis.     

Low circulating insulin-like growth factor I increases atherosclerosis in ApoE-deficient mice

Some clinical studies have suggested that lower IGF-I levels may be associated with an increased risk of ischemic heart disease. We generated atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice with 6T alleles (6T/ApoE(-/-) mice) with a 20% decline in circulating IGF-I and fed these mice and control ApoE(-/-) mice with normal chow or a Western diet for 12 wk to evaluate the effect of low serum IGF-I on atherosclerosis progression. We found that the 6T/ApoE(-/-) phenotype was characterized by an increased atherosclerotic burden, elevated plaque macrophages, and increased proinflammatory cytokine TNF-α levels compared with ApoE(-/-) controls. 6T/ApoE(-/-) mice had similar body weight, blood pressure, serum total cholesterol levels, total plaque and smooth muscle cell apoptosis rates, and circulating levels of endothelial progenitor cells as ApoE(-/-) mice. 6T/ApoE(-/-) mice fed with normal chow had reduced vascular endothelial nitric oxide synthase mRNA levels and a trend to increased aortic expression of chemokine (C-C motif) receptor (CCR)1, CCR2, and monocyte chemoattractant protein-1/chemokine (C-C motif) ligand 2. Western diet-fed 6T/ApoE(-/-) mice had a trend to increased expression of macrophage scavenger receptor-1/scavenger receptor-A, osteopontin, ATP-binding cassette (subfamily A, member 1), and angiotensin-converting enzyme and elevated circulating levels of the neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 (KC). Our data establish a link between lower circulating IGF-I and increased atherosclerosis that has important clinical implications.     

Npp1 promotes atherosclerosis in ApoE knockout mice

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PP(i)), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in human beings. NPP1 and PP(i) have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification. Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high-fat/high-cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) mice demonstrated decreased Opn expression. The 28-week-old ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) had significantly smaller atherosclerotic lesions compared with wild-type congenic ApoE(-/-) mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE(-/-) mice without Npp1 deficiency. We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.     

Long-term high-fat diet links the regulation of the insulin-sensitizing fibroblast growth factor-21 and visfatin

High-fat diet (HFD) is associated with insulin resistance, hyperinsulinemia, elevated plasma free fatty acid (FFA), and increased risk for atherosclerotic vascular disease. However, the mechanisms underlying the HFD-induced insulin resistance have not been fully clarified. The aim of present study is to evaluate the effects of long-term HFD on the regulation of the insulin-sensitizing fibroblast growth factor-21 (FGF-21) and visfatin in ApoE(-/-) mice. A total of twenty male ApoE(-/-) mice were randomly divided into normal chow diet (NC) or HFD (HF) group for 16 weeks. Euglycemic-hyperinsulinemic clamp was performed to evaluate insulin sensitivity in this animal model. Both mRNA and protein contents of FGF-21 and visfatin were assayed by Quantitative real-time PCR and Western blot. Long-term HFD resulted in the marked abnormality of glucose and lipid metabolism as well as a large decrease in whole-body insulin sensitivity. Accompanied by abnormal glucose-lipid metabolism and aggravated insulin resistance, FGF-21, β-klotho, FGFR1, FGFR3 and FGFR4 mRNA expressions were markedly up-regulated, whereas visfatin mRNA expression was markedly down-regulated in liver and/or adipose tissue of HFD-fed mice. In addition, Western blotting also revealed both up-regulation of the FGF-21 protein and down-regulation of visfatin protein in liver, adipose tissue and plasma of HFD-fed mice. Both FGF-21 and visfatin expression and secretion are regulated by a potent regulator, long-term HFD. And these adipokines are associated with glucose-lipid metabolism and insulin resistance.     

Proteomic analysis of urinary biomarker candidates for nonmuscle invasive bladder cancer

Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen β chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen β chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.     

Atherosclerosis induced by endogenous and exogenous toll-like receptor (TLR)1 or TLR6 agonists

Atherosclerosis is a chronic inflammatory vascular disease. Toll-like receptors (TLRs) are major initiators of inflammation. TLR2 promotes atherosclerosis in LDL receptor (LDLr)-deficient mice fed a high-fat diet (HFD). TLR2 forms heterodimers with TLR1 or TLR6 to enable inflammatory responses in the presence of distinct ligands. Here we asked whether TLR1 and/or TLR6 are required. We studied atherosclerotic disease using either TLR1- or TLR6-deficient mice. Deficiency of TLR1 or TLR6 did not diminish HFD-driven disease. When HFD-fed LDLr-deficient mice were challenged with Pam3 or MALP2, specific exogenous ligands of TLR2/1 or TLR2/6, respectively, atherosclerotic lesions developed with remarkable intensity in the abdominal segment of the descending aorta. In contrast to atherosclerosis induced by the endogenous agonists, these lesions were diminished by deficiency of either TLR1 or TLR6. The endogenous ligand(s) that arise from consumption of a HFD and promote disease via TLR2 are unknown. Either TLR1 or TLR6 are redundant for this endogenous ligand detection, or they are both irrelevant to endogenous ligand detection. However, the exogenous ligands Pam3 and MALP2 promote severe abdominal atherosclerosis in the descending aorta that is dependent on TLR1 and TLR6, respectively.     

Serum amyloid A directly accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice

Although serum amyloid A (SAA) is an excellent marker for coronary artery disease, its direct effect on atherogenesis in vivo is obscure. In this study we investigated the direct effect of SAA on promoting the formation of atherosclerosis in apolipoprotein E-deficient (ApoE?/?) mice. Murine SAA lentivirus was constructed and injected into ApoE?/? mice intravenously. Then, experimental mice were fed a chow diet (5% fat and no added cholesterol) for 14 wks. The aortic atherosclerotic lesion area was larger with than without SAA treatment. With increased SAA levels, the plasma levels of interleukin-6 and tumor necrosis factor-α were significantly increased. Macrophage infiltration in atherosclerotic regions was enhanced with SAA treatment. A migration assay revealed prominent dose-dependent chemotaxis of SAA to macrophages. Furthermore, the expression of monocyte chemotactic protein-1 and vascular cell adhesion molecule-1 (VCAM-1) was upregulated significantly with SAA treatment. SAA-induced VCAM-1 production was detected in human aortic endothelial cells in vitro. Thus, an increase in plasma SAA directly accelerates the progression of atherosclerosis in ApoE?/? mice. SAA is not only a risk marker for atherosclerosis but also an active participant in atherogenesis.     

Heterozygous mutation of ataxia-telangiectasia mutated gene aggravates hypercholesterolemia in apoE-deficient mice

Individuals with a heterozygous mutation at the ataxia-telangiectasia mutated gene (ATM) have been reported to be predisposed to ischemic heart disease. This report examined for the first time the effect of a heterozygous ATM mutation (ATM(+)(/-)) on plasma lipid levels and atherosclerosis intensity using ATM(+/-), ATM(+)(/+) (wild type), ATM(+)(/+)/LDLR(-)(/-) (low density lipoprotein receptor knockout), ATM(+)(/-)/LDLR(-)(/-), ATM(+)(/+)/ApoE(-)(/-) (apolipoprotein E knockout), and ATM(+)(/-)/ApoE(-)(/-) mice. Our data demonstrated that the plasma cholesterol and triglyceride levels in ATM(+)(/-) and ATM(+)(/-)/LDLR(-)(/-) mice were approximately the same as those in ATM(+)(/+) and ATM(+)(/+)/LDLR(-)(/-) control mice, respectively. In contrast, the plasma cholesterol level was significantly higher in ATM(+)(/-)/ApoE(-)(/-) mice than in ATM(+)(/+)/ApoE(-)(/-) control mice. In addition, the ATM(+)(/-)/ApoE(-)(/-) mice showed higher plasma apoB-48 levels, slower clearance for plasma apoB-48-carrying lipoproteins, and more advanced atherosclerotic lesions in the aorta compared with the ATM(+)(/+)/ApoE(-)(/-) mice. These novel results suggest that the product of ATM is involved in an apoE-independent pathway for catabolism of apoB-48-carrying remnants; therefore, superimposition of a heterozygous ATM mutation onto an ApoE deficiency background reduces the clearance of apoB-48-carrying lipoproteins from the blood circulation and promotes the formation of atherosclerosis.     

Extracellular matrix components in atherosclerotic arteries of Apo E/LDL receptor deficient mice: an immunohistochemical study

During accelerated vascular remodeling such as in atherosclerosis, the composition of the extracellular matrix becomes altered. The matrix components of the diseased artery influence cellular processes such as adhesion, migration and proliferation. Furthermore, in atherosclerosis, the inability of the cells within the lesion to produce a mechanically stable matrix may lead to plaque rupture. In this immunohistochemical study of atherosclerotic mice aorta, we have reviewed the presence of ECM components with roles in maintaining tissue structure and function. These components include osteopontin and COMP as well as the leucine rich repeats proteins decorin, PRELP, and fibromodulin. Immunohistochemistry demonstrated presence of osteopontin, COMP, decorin, PRELP and fibromodulin in lesion areas of ApoE/LDLr deficient mice. Some advanced lesions exhibited areas of cartilage-like morphology and were shown to represent cartilage by their content of the cartilage specific proteins collagen II and aggrecan. The results suggest that cartilage-associated cell/collagen binding ECM proteins may be involved in the pathogenesis of atherosclerosis.     

Lack of the antioxidant glutathione peroxidase-1 does not increase atherosclerosis in C57BL/J6 mice fed a high-fat diet

Oxidative stress is thought to contribute to the initiation and progression of atherosclerosis. As glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that detoxifies lipid hydroperoxides, we tested the impact of Gpx1 deficiency on atherosclerotic processes and antioxidant enzyme expression in mice fed a high-fat diet (HFD). After 12 weeks of HFD, atherosclerotic lesions at the aortic sinus were of similar size in control and Gpx1-deficient mice. However, after 20 weeks of HFD, lesion size increased further in control but not in Gpx1-deficient mice, even though plasma and aortic wall markers of oxidative damage did not differ between groups. In control mice, the expression of Gpx1 increased and that of Gpx3 decreased at the aortic sinus after 20 weeks of HFD, with no change in the expression of Gpx2, Gpx4, catalase, peroxiredoxin-6, glutaredoxin-1 and -2, or thioredoxin-1 and -2. By comparison, in Gpx1-deficient mice, the expression of antioxidant genes was unaltered except for a decrease in glutaredoxin-1 and an increase in glutaredoxin-2. These changes were associated with increased expression of the proinflammatory marker monocyte chemoattractant protein-1 in control mice but not in Gpx1-deficient mice. In summary, a specific deficiency in Gpx1 was not accompanied by an increase in markers of oxidative damage or increased atherosclerosis in a murine model of HFD-induced atherogenesis.     

Tanshinone II-A attenuates and stabilizes atherosclerotic plaques in apolipoprotein-E knockout mice fed a high cholesterol diet

Tanshinone II-A (Tan), a bioactive diterpene isolated from Salvia miltiorrhiza Bunge (Danshen), possesses anti-oxidant and anti-inflammatory activities. The present study investigated whether Tan can decrease and stabilize atherosclerotic plaques in Apolipoprotein-E knockout (ApoE(-/-)) mice maintained on a high cholesterol diet (HCD). Six week-old mice challenged with a HCD were randomly assigned to 4 groups: (a) C57BL/6J; (b) ApoE(-/-); (c) ApoE(-/-)+Tan-30 (30 mg/kg/d); (d) ApoE(-/-)+Tan-10 (10mg/kg/d). After 16 weeks of intervention, Tan treated mice showed decreased atherosclerotic lesion size in the aortic sinus and en face aorta. Furthermore, immunohistochemical analysis revealed that Tan rendered the lesion composition a more stable phenotype as evidenced by reduced necrotic cores, decreased macrophage infiltration, and increased smooth muscle cell and collagen contents. Tan also significantly reduced in situ superoxide anion production, aortic expression of NF-κB and matrix metalloproteinase-9 (MMP-9). In vitro treatment of RAW264.7 macrophages with Tan significantly suppressed oxidized LDL-induced reactive oxygen species production, pro-inflammatory cytokine (IL-6, TNF-α, MCP-1) expression, and MMP-9 activity. Tan attenuates the development of atherosclerotic lesions and promotes plaque stability in ApoE(-/-) mice by reducing vascular oxidative stress and inflammatory response. Our findings highlight Tan as a potential therapeutic agent to prevent atherosclerotic cardiovascular diseases.     

Extracellular proteolysis in the development and progression of atherosclerosis

Clinical complications of atherosclerosis are often triggered by the rupture of unstable plaques, while thinning of the atherosclerotic vessel wall owing to elastin and collagen degradation and media necrosis may result in aneurysm formation and bleeding. Proteolysis, mediated via the plasminogen/plasmin and/or matrix metalloproteinase (MMP) systems may contribute to neovascularization and rupture of plaques, or to ulceration and rupture of aneurysms. In an in vivo model of atherosclerosis, using mice that had a combined deficiency of apolipoprotein E (ApoE) and urokinase-type plasminogen activator (u-PA) and that were maintained on a cholesterol-rich diet, it was observed that u-PA deficiency protects against aneurysm formation. This was explained by the findings that plasmin, generated from plasminogen by u-PA, activates several macrophage-secreted proMMPs (e.g. proMMP-3, -9, -12 and -13), which in turn cause extracellular matrix degradation. A potential role for MMP-3 (stromelysin-1) was confirmed in a subsequent study using mice with a combined deficiency of ApoE and MMP-3, that were kept on a cholesterol-rich diet. The results suggest that MMP-3 contributes to plaque destabilization, possibly by degrading extracellular matrix components, but also promotes aneurysm formation by degrading the elastic lamina. These effects may be mediated by MMP-3 directly or by activation of other proMMPs or other (proteolytic) systems. A functional role of MMPs is further supported by the finding that deficiency in TIMP-1 (tissue inhibitor of MMPs type 1) reduces atherosclerotic plaque size but enhances aneurysm formation. Taken together, these results suggest that u-PA has an important role in the structural integrity of the atherosclerotic vessel wall, which is likely to involve triggering the activation of MMPs and, furthermore, they suggest that increased u-PA levels are a risk factor for aneurysm formation.     

Suppression of atherogenesis by overexpression of glutathione peroxidase-4 in apolipoprotein E-deficient mice

Accumulation of oxidized lipids in the arterial wall contributes to atherosclerosis. Glutathione peroxidase-4 (GPx4) is a hydroperoxide scavenger that removes oxidative modifications from lipids such as free fatty acids, cholesterols, and phospholipids. Here, we set out to assess the effects of GPx4 overexpression on atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. The results revealed that atherosclerotic lesions in the aortic tree and aortic sinus of ApoE(-/-) mice overexpressing GPx4 (hGPx4Tg/ApoE(-/-)) were significantly smaller than those of ApoE(-/-) control mice. GPx4 overexpression also diminished signs of advanced lesions in the aortic sinus, as seen by a decreased occurrence of fibrous caps and acellular areas among hGPx4Tg/ApoE(-/-) animals. This delay of atherosclerosis in hGPx4Tg/ApoE(-/-) mice correlated with reduced aortic F(2)-isoprostane levels (R(2)=0.75, p<0.01). In addition, overexpression of GPx4 lessened atherogenic events induced by the oxidized lipids lysophosphatidylcholine and 7-ketocholesterol, including upregulated expression of adhesion molecules in endothelial cells and adhesion of monocytes to endothelial cells, as well as endothelial necrosis and apoptosis. These results suggest that overexpression of GPx4 inhibits the development of atherosclerosis by decreasing lipid peroxidation and inhibiting the sensitivity of vascular cells to oxidized lipids.