Perivascular Collar Placement Research Articles

Study on the Mechanism of Improving Mice with Atherosclerosis Using Dendrocrepine

Aim: To discuss Den on apoptosis and Nrf2/ARE in atherosclerotic vulnerable plaque of apolipoprotein in E (ApoE)-/- mice. Methods: Randomly dividing ApoE-/- mice as 5 groups including Normal, Model, Den-L (10 mg/kg), Den-M (20 mg/kg) and Den-H (40 mg/kg) groups. The atherosclerotic vulnerable plaque model was established by high-fat feeding and right common carotid artery catheterization (perivascular carotid collar placement, PCR), and Den was given by difference concentration Den, the pathological changes of right common carotid arery, apoptosis of vascular smooth muscle, Bax, Bcl-2 and Caspase-3 proteins expression using IHC and WB assay, Nrf2, ARE and MDA, 8-OHdG and TAC levels were detected. Results: Model group showed typical pathological changes of vulnerable plaque, the apoptosis cell number, Bax, Caspase-3 and MDA, 8-OHdG significantly increased, the Bcl-2, Nrf2, ARE and TAC levels significantly decreased (P < 0.001, respectively); Compared with model group, the plaque of Den groups were reduced and tended to be stable, the apoptosis cell number, Bax, Caspase3 and MDA, 8-OHdG levels significantly decreased, Bcl-2, Nrf2, ARE and TAC levels significantly increased (P<0.05, respectively). Conclusion: Den improves atherosclerotic vulnerable plaque of ApoE-/- mice, which is related to the inhibition of apoptosis and Nrf2/ARE pathway.

Comparative Pharmacokinetics of Seven Major Compounds in Normal and Atherosclerosis Mice after Oral Administration of Simiao Yong'an Decoction.

Simiao Yong'an decoction (SMYAD), a classic traditional Chinese medicine formula, has been used to treat atherosclerosis (AS) in clinical in China, but its therapeutic mechanism and pharmacodynamic material basis are not clear. In this study, the AS model was caused by a high-fat diet and perivascular carotid collar placement (PCCP), and SMYAD was orally administered to the model and normal mice. A rapid, sensitive, selective, and reliable method using ultrahigh-performance liquid chromatography (UHPLC) system combined with a Q Exactive HF-X mass spectrometer (UHPLC-Q Exactive HF-X MS) was established and validated for the simultaneous determination of seven compounds, including harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin in the plasma of normal and AS mice. The specificity, linearity, precision, accuracy, recovery, and stability of the method were all within the acceptable criteria. The results showed that some pharmacokinetic behaviors of harpagide, chlorogenic acid, and isoliquiritigenin were significantly different among the two groups of mice. The specific parameter changes were harpagide (AUC0–t and AUC0–∞ were 11075.09 ± 2132.38 and 16221.95 ± 5622.42 ng·mL−1·h, respectively; CLz/F was 2.45 ± 0.87 L/h/mg), chlorogenic acid (t1/2 was 21.59 ± 9.16 h; AUC0–∞ was 2637.51 ± 322.54 ng·mL−1·h; CLz/F was 13.49 ± 1.81 L/h/mg) and isoliquiritigenin (AUC0–t and AUC0–∞ were 502.25 ± 165.65 and 653.68 ± 251.34 ng·mL−1·h, respectively; CLz/F was 62.16 ± 23.35 L/h/mg) were altered under the pathological status of AS. These differences might be partly ascribed to the changes in gastrointestinal microbiota, nonspecific drug transporters, and cytochrome P450 activity under the AS state, providing research ideas and experimental basis for pharmacological effects and pharmacodynamic material basis.

A host lipase prevents lipopolysaccharide-induced foam cell formation

SummaryAlthough microbe-associated molecular pattern (MAMP) molecules can promote cholesterol accumulation in macrophages, the existence of a host-derived MAMP inactivation mechanism that prevents foam cell formation has not been described. Here, we tested the ability of acyloxyacyl hydrolase (AOAH), the host lipase that inactivates gram-negative bacterial lipopolysaccharides (LPSs), to prevent foam cell formation in mice. Following exposure to small intraperitoneal dose(s) of LPSs, Aoah−/− macrophages produced more low-density lipoprotein receptor and less apolipoprotein E and accumulated more cholesterol than did Aoah+/+ macrophages. The Aoah−/− macrophages also maintained several pro-inflammatory features. Using a perivascular collar placement model, we found that Aoah−/− mice developed more carotid artery foam cells than did Aoah+/+ mice after they had been fed a high fat, high cholesterol diet, and received small doses of LPSs. This is the first demonstration that an enzyme that inactivates a stimulatory MAMP in vivo can reduce cholesterol accumulation and inflammation in arterial macrophages.

Urate-lowering therapy alleviates atherosclerosis inflammatory response factors and neointimal lesions in a mouse model of induced carotid atherosclerosis.

Hyperuricemia (HU) is a cause of gout. Clinical studies show a link between HU and cardiovascular disease. However, the role of soluble serum urate (SU) on atherosclerosis development remains elusive. We aimed to use a new HU mouse model [Uricase/Uox knockout (KO)] to further investigate the relationship between HU and atherosclerosis. A mouse model by perivascular collar placement of induced carotid atherosclerosis was established in male Uox‐ KO mice. The Uox‐ KO mice had elevated SU levels and enhanced levels of atherosclerosis inflammatory response proteins. In contrast, Uox‐ KO mice with carotid atherosclerosis showed severe neointimal changes in histology staining consistent with increases in intimal area and increases in proliferating cell nuclear antigen (PCNA)‐ and F4/80‐positive cells. Allopurinol reduced neointimal areas induced by the perivascular collar in hyperuricemic mice, accompanied by decreased expression of PCNA‐ and F4/80‐positive cells. Urate‐lowering treatment alleviated atherosclerosis inflammatory response factors and reactive oxygen species (ROS) intensities in both collar placement Uox‐ KO mice and urate‐stimulated human umbilical vein endothelial cells (HUVECs). In vitro results using HUVECs showed ROS was induced by urate and ROS induction was abrogated using antioxidants. These data demonstrate that urate per se does not trigger atherosclerosis intima lesions in male mice. Urate worsens carotid neointimal lesions induced by the perivascular collar and urate‐lowering therapy partially abrogates the effects. The current study warrants clinical studies on the possible benefits of urate‐lowering therapy in atherosclerosis patients with HU.

Silencing of Non-POU-domain-containing octamer-binding protein stabilizes atherosclerotic plaque in apolipoprotein E-knockout mice via NF-κB signaling pathway

BackgroundIt remains unknown whether Non-POU-domain-containing octamer-binding protein (NonO) plays a causative role in plaque destabilization. We hypothesized that NonO gene silencing may stabilize atherosclerotic plaque by increasing P4Hα1 expression and inhibiting the inflammation. Methods and resultsVulnerable atherosclerotic plaques were induced in ApoE−/− mice by high fat diet, perivascular collar placement and mental stress.Compared with normal carotid arteries, those contained vulnerable plaques had high NonO expression. In another in vivo experiment, mice contained vulnerable plaques were randomly divided into 5 groups to receive physiological saline, si-N.C-lentivirus (LV), si-NonO-LV, pGC-GFP-LV and NonO-LV, respectively. NonO overexpression increased while NonO silencing decreased the incidence of carotid plaque disruption. NonO overexpression enhanced macrophage infiltration and lipid deposition but reduced the content of vascular smooth muscle cells and collagen in plaques, leading to an increased plaque vulnerability index, whereas NonO silencing exhibited the opposite effect. In addition, NonO overexpression increased the expression of proinflammatory cytokines and matrix metalloproteinases and decreased the expression of P4Hα1 both in vivo and in vitro, whereas NonO silencing showed the contrary effect. NonO co-immunoprecipitated with NF-κB p65, and promoted its nuclear translocation and phosphorylation, and these effects were reversed by NonO silencing. ConclusionNonO may promote plaque destabilization and increase the incidence of plaque disruption in ApoE−/− mice by inducing the expression of inflammatory cytokines and matrix metalloproteinases and suppressing that of P4Hα1. The mechanism may involve the interaction of NonO with NF-κB leading to enhanced NF-κB nuclear translocation and phosphorylation.

NLRP3 Inflammasome Inhibition by MCC950 Reduces Atherosclerotic Lesion Development in Apolipoprotein E-Deficient Mice-Brief Report.

Inflammasomes are multiprotein complexes, and their activation has been associated with cardiovascular disease. Inflammasome activation leads to secretion of caspase-1 by innate immune cells, resulting in the activation of interleukin-1β. Recently, a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, was described. In this study, we investigated the effect of MCC950 on atherosclerotic lesion development in apoE-/- mice. First, we determined the efficacy of MCC950 in vitro. Bone marrow-derived macrophages and dendritic cells were stimulated with lipopolysaccharide and cholesterol crystals resulting in high levels of interleukin-1β release, which was inhibited by MCC950. In vivo MCC950 treatment reduced lipopolysaccharide-induced interleukin-1β secretion, without affecting the tumor necrosis factor-α response. Subsequently, atherosclerotic plaques were induced in Western-type diet fed apoE-/- mice by semiconstrictive perivascular collar placement at the carotid arteries, after which the mice received MCC950 (10 mg/kg) or vehicle control 3× per week intraperitoneally for 4 weeks. After euthanize, atherosclerotic plaque size and volume were quantified in hematoxylin-eosin-stained 10-µm cryosections throughout the artery. MCC950 treatment significantly reduced the development of atherosclerotic lesions as determined by maximal stenosis, average plaque size, and plaque volume. Although the amount of collagen and the necrotic core size were not affected, the number of macrophages in the plaque was significantly reduced on treatment. In addition, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA expression was significantly reduced in the carotids of MCC950-treated mice. These findings show that specific inhibition of the NLRP3 inflammasome using MCC950 can be a promising therapeutic approach to inhibit atherosclerotic lesion development.

AG1296 enhances plaque stability via inhibiting inflammatory responses and decreasing MMP-2 and MMP-9 expression in ApoE−/− mice

BackgroundAtherosclerosis is a chronic process that progresses to unstable plaques. Plaque rupture leads to deleterious consequences such as acute coronary syndrome, thrombosis and stroke. AG1296 is a potent tyrosine kinase inhibitor which is able to block PDGF-PDGFR signaling pathway. This study aims to assess the effect of AG1296 on plaque stability and explore the potential mechanisms. MethodsAtherosclerotic plaques were induced in carotid arteries in ApoE−/− mice by perivascular collar placement. All mice were randomly divided into PBS and AG1296 groups. 3 weeks after the surgery, the carotid arteries were harvested for histological analysis. ResultsIn AG1296 group, plaque area decreased by 41.5% (p = 0.0041) and the contents of macrophages and lipids decreased by 43.5% (p = 0.0003) and 35.6% (p = 0.0032) respectively. The contents of smooth muscle cells increased by 22.3% (p = 0.0214) in AG1296 group. Vulnerable index decreased by 48.3% (p = 0.0002). The inflammation factors IL-6 and TNF- alpha decreased by 49.0% (p = 0.0008) and 51.8% (p < 0.0001) and matrix metalloproteinases MMP-2 and MMP-9 decreased by 54.1% (p = 0.0004) and 37.1% (p < 0.0001) in AG1296 group. M1 macrophage markers (MCP-1) were downregulated by 30.3% (p = 0.0007) and M2 macrophage markers (ARG-1) were increased by 55.2% (p = 0.0009) in AG1296 group. ConclusionAG1296 inhibited the atherosclerotic plaque progression and enhanced plaque stability by inhibiting inflammatory responses, reducing the expression of matrix metalloproteinases and promoting macrophages from proinflammatory phenotype to anti-inflammatory phenotype.

The absence of PCSK9 determines a lower neointimal formation in response to perivascular carotid collar placement

The absence of PCSK9 determines a lower neointimal formation in response to perivascular carotid collar placement

PCSK9 knock-out mice are protected from neointimal formation in response to perivascular carotid collar placement

Background and aimsProprotein convertase subtilisin kexin type 9 (PCSK9) induces degradation of the low-density lipoprotein-receptor (LDLR). Smooth muscle cells (SMCs) in human atherosclerotic plaques and cultured SMCs express PCSK9. The present study aimed at defining the role of PCSK9 on vascular response to injury. MethodsCarotid neointimal lesions were induced by positioning a non-occlusive collar in PCSK9 knock-out (PCSK9−/−) and wild type littermate (PCSK9+/+) mice. ResultsIn PCSK9−/− mice, we observed a significantly less intimal thickening (p < 0.05), a lower intimal media ratio (p < 0.02), and a tendency to higher lumen area, compared to PCSK9+/+ mice. When compared with PCSK9−/−, lesions of PCSK9+/+ mice had a higher content of SMCs (p < 0.05) and collagen (p < 0.05), while no difference was observed in the accumulation of macrophages. PCSK9 was detectable in both left and right carotids artery in regions occupied by medial and neointimal SMCs. SMCs freshly isolated from PCSK9−/−, when compared to PCSK9+/+ cells, showed higher levels of α-smooth muscle actin (α-SMA; 2.24 ± 0.36 fold; p < 0.01) and myosin heavy chain II (MHC-II; 8.65 ± 1.55 fold; p < 0.01), and lower levels of caldesmon mRNA(−54 ± 14%; p < 0.01).PCSK9−/− cells also showed a slower proliferation rate, and an impaired migratory capacity and G1/S progression of the cell cycle. The reconstitution of PCSK9 expression, by retroviral infection of PCSK9−/− SMCs, led to a downregulation of α-SMA (−56 ± 2%; p < 0.01), MHC-II (−45% ± 25.5 fold: p = 0.06) and calponin (−25% ± 0.8 fold: p < 0.05) and induction of caldesmon mRNA (1.46 ± 0.3 fold; p < 0.05). Proliferation rate of SMCs PCSK9−/− was significantly lower compared to PCSK9 reconstituted cells. ConclusionsTaken together, the present results suggest that PCSK9, by sustaining SMC synthetic phenotype, proliferation, and migration, may play a pro-atherogenic role in the arterial wall.

Effects of vitamin C treatment on collar-induced intimal thickening.

Vitamin C has efficient antioxidant properties and is involved in important physiological processes such as collagen synthesis. As such, vitamin C deficiency leads to serious complications, including vascular diseases. The aim of this study was to investigate the effects of vitamin C treatment on collar-induced intimal thickening. Rabbits were fed a normocholesterolemic diet and a non-occlusive silicon collar was placed around the left carotid artery for 3, 7, and 14 days. The rabbits were treated with or without vitamin C (150 mg/kg/day). Collar-induced intimal thickening became apparent at day 7. The effect of the collar on intimal thickening was more prominent at day 14. Vitamin C treatment significantly inhibited collar-induced intimal thickening at day 14. The placement of the collar around the carotid artery decreased maximum contractile responses against contractile agents (KCl, phenylephrine, 5-hydroxytryptamine). The effect of the collar on contractile responses was enhanced as days elapsed. Decreased contractile responses of collared carotid arteries normalized at day 14 in the vitamin C treatment group. Vitamin C treatment also restored sensitivity to phenylephrine. The collar also significantly decreased acetylcholine-induced relaxations at day 3 and day 7. Acetylcholine-induced relaxations normalized in collared-arteries in the placebo group at day 14. Vitamin C treatment significantly increased acetylcholine-induced relaxations of both normal and collared carotid arteries at day 14. MMP-9 expression increased in collared arteries at day 3 and day 7 but did not change at day 14. MMP-2 expression increased in collared arteries at day 14. However, vitamin C treatment reduced collar-stimulated expression of MMP-2 at day 14. These findings indicate that vitamin C may have potentially beneficial effects on the early stages of atherosclerosis. Furthermore these results, for the first time, may indicate that vitamin C can also normalize decreased contractile response through perivascular collar placement.

Low dose tunicamycin enhances atherosclerotic plaque stability by inducing autophagy

After decades of indolent progression, atherosclerosis may cause unheralded events, such as myocardial infarction, acute coronary syndrome and stroke due to sudden rupture of atherosclerotic plaques, and pharmacologically modulating plaque stability would reduce the risk of cardiovascular diseases. Endoplasmic reticulum stress (ERS) is responsible for the vulnerability of plaques. However, the underlying mechanism has not been fully elucidated. In this work, ApoE−/− mice underwent perivascular carotid collar placement surgeries or sham operations were given higher (3.0mg/kg) and lower (0.3mg/kg) doses of tunicamycin (TM), and plaque stability was evaluated. It was shown that lower TM-treated animals exhibited reduced plaque areas and necrotic cores as well as fibrous cap thickness accompanied by a lower percentage of infiltrates and foam cells than the sham-operated and higher TM treated animals. Lower TM had a profound inhibitory effect on plasma inflammatory response and lipid profile in atherosclerotic ApoE−/− mice. In addition, we found that the ApoE−/− mice presented higher autophagy activity in response to lower TM administration while apoptosis was reduced. An in vitro study in murine macrophages revealed that lower TM could markedly reduce lipid uptake and accumulation and cell apoptosis while significantly upregulated the expression of Atg7. However, higher TM had adverse effects. Finally, mild induction of ERS by lower TM inhibits AKT-TSC-mTOR cascades to increase cellular autophagy. However, high TM failed to enhance autophagy and equilibrate elevated CHOP-mediated cell death in spite of the inhibition of AKT-TSC-mTOR signaling. In conclusion, lower TM stabilized plaques by activating autophagy through AKT-TSC-mTOR signaling.

CD137 signaling regulates the expression of nuclear factor of activated T cells c1 through miR-145a-5p in ApoE(-)/(-) mice

To investigate if miR-145a-5p participates the modulation process of CD137 signaling on the expression of nuclear factor of activated T cells c1 (NFATc1) in ApoE(-)/(-) mice. Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE(-)/(-) mice. After surgery, the mice were randomly divided into the following groups: CD137 activated group (CD137 group, n = 6), CD137 inhibited group (anti-CD137 group, n = 6) and control group (n = 6). The mRNA expression of miR-145a-5p in plaque and cells was measured by real-time quantitative PCR (RT-PCR). Immunofluorescence was used to observe the distribution of NFATc1 in plaque and the expression of NFATc1 at mRNA and protein levels were detected by qRT-PCR, Western blot, respectively. The mouse vascular smooth muscle cells (VSMCs) were isolated and transfected with miR-145a-5p mimics or inhibitors by Lipofectamine. The eukaryotic expression vector and luciferase vector including p3xFLAG-NFATc1, p3xFLAG-NFATc1-3'UTR, psicheck2-NFATc1, psicheck2-NFATc1-Mut were constructed through molecular cloning and homologous recombination techniques, 293T cells were transfected with the miR-145a-5p mimics or inhibitors and the protein level and fluorescence intensity were then measured, respectively. In vivo or in vitro, the level of miR-145a-5p was significantly decreased (0.21 ± 0.06 vs. 1.00 ± 0.00, P < 0.05, 0.22 ± 0.07 vs. 0.50 ± 0.12, P < 0.05) while the opposite effects were observed in anti-CD137 group. NFATc1 expression was decreased or increased in VSMCs transfected with miR-145a-5p mimics or inhibitors, respectively (all P < 0.05). miR-145a-5p mimics decreased the expression of p3xFLAG-NFATc1-3'UTR and the fluorescence intensity (0.56 ± 0.08 vs. 1.00 ± 0.00, P < 0.05). CD137 signaling participates the regulation process on the expression of NFATc1 through miR-145a-5p in ApoE(-)/(-) mice.

Prolyl-4-hydroxylase- 1 improves the stability of advanced plaques but accelerates the atherosclerotic lesion formation of early plaques

To investigate the changes in Prolyl 4-hydroxylase (P4Hα1) expression during the pathological course of atherosclerotic plaque development and to test the effect of P4Hα1 overexpression on vulnerability of early and advanced plaque in apolipoprotein E-deficient (ApoE−/−) mice. In vivo , the changes in P4Hα1 level were assessed during the pathological course of spontaneously induced atherosclerotic plaque in the aortic root of ApoE−/− mice at 10, 12, 14, and 16 weeks of age and fed a high-fat diet. A lentivirus-mediated P4Hα1 construct was transfected in carotid plaque induced in mice by perivascular collar placement for 2 and 10 weeks. After 2 weeks of lenti-P4Hα1 treatment, both early and advanced plaque exhibited stable characteristics, with increased collagen and smooth muscle cell content, reduced macrophage content, and no change in lipid content. Lenti-P4Hα1 treatment of early and advanced plaque substantially increased interstitial collagen content in the fibrous cap of plaque. It increased the carotid plaque size and the relative en face lesion area of the aorta in early but not advanced plaque, with reduced phosphatase and tensin homolog expression. Furthermore, the treatment lowered the expression of inflammatory cytokines and the production and activity of matrix metalloproteinase-9 and -2 in both early and advanced plaque. P4Hα1 overexpression had a differential effect at various stages of plaque pathological progression. P4Hα1 overexpression might be a valuable therapeutic modality in stabilizing advanced but not early plaque because of the adverse event of accelerated lesion formation.

Perivascular adipose tissue-derived adiponectin inhibits collar-induced carotid atherosclerosis by promoting macrophage autophagy.

ObjectivesAdiponectin (APN) secreted from perivascular adipose tissue (PVAT) is one of the important anti-inflammatory adipokines to inhibit the development of atherosclerosis, but the underlying mechanism has not been clarified. In this study, we aimed to elucidate how APN regulates plaque formation in atherosclerosis.Methods and ResultsTo assess the role of APN secreted by PVAT in atherosclerosis progression, we performed PVAT transplantation experiments on carotid artery atherosclerosis model: ApoE knockout (ApoE−/−) mice with a perivascular collar placement around the left carotid artery in combination with a high-fat diet feeding. Our results show that the ApoE−/− mice with PVAT derived from APN knockout (APN−/−) mice exhibited accelerated plaque volume formation compared to ApoE−/− mice transplanted with wild-type littermate tissue. Conversely, autophagy in macrophages was significantly attenuated in ApoE−/− mice transplanted with APN-/- mouse-derived PVAT compared to controls. Furthermore, in vitro studies indicate that APN treatment increased autophagy in primary macrophages, as evidenced by increased LC3-I processing and Beclin1 expression, which was accompanied by down-regulation of p62. Moreover, our results demonstrate that APN promotes macrophage autophagy via suppressing the Akt/FOXO3a signaling pathway.ConclusionsOur results indicate that PVAT-secreted APN suppresses plaque formation by inducing macrophage autophagy.

Effects of OX40–OX40L Interaction on the Nuclear Factor of Activated T Cells c1 in ApoE-Deficient Mice

We previously reported the emerging role of OX40-OX40L interaction in inflammation and atherosclerosis. However, the mechanism by which OX40-OX40L interaction contributes to pathogenesis is poorly understood. This study investigated the effects of OX40-OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE(-/-) mice. Atherosclerotic plaque was induced via rapid perivascular carotid collar placement in ApoE(-/-) mice. The expression levels of OX40, OX40L, and NFATc1 in the lymphocytes were measured via real-time polymerase chain reaction and flow cytometry. The presence of NFATc1 in the atherosclerotic plaque was detected via immunohistochemistry, and the level of IL-4 was measured via enzyme-linked immunosorbent assay. The expression level of NFATc1 significantly increased in atherosclerotic lesion and in the leukocytes from the ApoE(-/-) mice. After stimulating OX40-OX40L interaction, the mRNA and protein expression levels of NFATc1 in the lymphocytes significantly increased. Meanwhile, anti-OX40LmAb significantly suppressed the expression of NFATc1 in the leukocytes and substantially elevated the level of IL-4. NFATc1 inhibitor markedly suppressed IL-4 production. This study suggests that OX40-OX40L interaction regulates the expression of NFATc1, which may play a critical role in atherosclerotic plaque formation, and may therefore have implications with pathophysiology of atherosclerosis.

Passive Immunization with Hypochlorite-oxLDL Specific Antibodies Reduces Plaque Volume in LDL Receptor-Deficient Mice

AimsNew strategies to overcome complications of cardiovascular diseases are needed. Since it has been demonstrated that atherosclerosis is an inflammatory disease, modulation of the immune system may be a promising approach. Previously, it was suggested that antibodies may confer protective effects on the development of atherosclerosis. In this study, we hypothesised that passive immunization with anti-oxLDL IgM antibodies specific for hypochlorite (HOCl) may be athero-protective in mice.Methods and ResultsMonoclonal mouse IgM antibodies were produced and the antibody with specificity for hypochlorite-oxLDL (HOCl-oxLDL) (Moab A7S8) was selected. VH sequence determination revealed that Moab A7S8 is a natural IgM antibody. Atherosclerosis in LDLr−/− mice was induced by a perivascular collar placement around the right carotid artery in combination with feeding a high-fat diet. Subsequently, the mice were treated every six days with 500 µg Moab A7S8, non-relevant IgM or with PBS and the carotid arteries and aortic roots were studied for atherosclerosis. Passive immunization with this Moab A7S8 resulted in a significant reduced plaque volume formation in LDLr−/− mice when compared with PBS treatment (P = 0.002 and P = 0.035). Cholesterol levels decreased by 20% when mice were treated with Moab A7S8 compared to PBS. Furthermore, anti-oxLDL specific IgM and IgG antibody production increased significantly in the Moab A7S8 treated mice in comparison with PBS treated mice.ConclusionOur data show that passive immunization with a natural IgM antibody, directed to HOCl-oxLDL, can reduce atherosclerotic plaque development. We postulate that specific antibody therapy may be developed for use in human cardiovascular diseases.

Lysophosphatidic acid triggers mast cell-driven atherosclerotic plaque destabilization by increasing vascular inflammation

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability.

An animal model of atherosclerotic plaque disruption and thrombosis in rabbit using pharmacological triggering to plaques induced by perivascular collar placement

Limited availability of suitable animal model of plaque disruption and thrombosis has hampered the study of mechanism and preclinical evaluation of plaque-stabilizing therapies. This study aims to develop an animal model of atherosclerotic plaque disruption and thrombosis in rabbit femoral artery. Silastic collars were placed around the bilateral femoral arteries of rabbits, which had been fed with atherogenic diet for 7 days. After 28 days on the same diet, the rabbits received pharmacological triggering by intraperitoneal injection of Russell's viper venom (RVV, 0.15 mg/kg) followed by intravenous injection of histamine (0.02 mg/kg), and the animals were then processed for imageological and histological examinations. Perivascular collar placement of the femoral artery in high-cholesterol-fed rabbits for 28 days induced marked intimal hyperplasia, which was a lipid- and collagen-rich lesion that contained substantial amount of macrophages and smooth muscle cells. Subsequent histological analysis showed that the pharmacological triggering evoked plaque disruption and platelet- and fibrin-rich thrombi in the collared femoral arteries. We demonstrated, for the first time, a rabbit model of plaque disruption and thrombosis induced by the combination of perivascular collar placement, RVV, and histamine injections. This model can be rapidly formed, easily operated, and site controlled.

Nitric Oxide-Donating Atorvastatin Attenuates Neutrophil Recruitment During Vascular Inflammation Independent of Changes in Plasma Cholesterol

Polymorphonuclear neutrophils, the first leukocytes to infiltrate the inflamed tissue, can make important contributions to vascular inflammatory processes driving the development of atherosclerosis. We herein investigated the effects of atorvastatin and NCX 6560 (a nitric oxide (NO)-donating atorvastatin derivative that has completed a successful phase 1b study) on neutrophilic inflammation in carotid arteries of normocholesterolemic rabbits subjected to perivascular collar placement. Atorvastatin or NCX 6560 were administered orally (5 mg/kg/day or equimolar dose) to New Zealand White rabbits for 6 days, followed by collar implantation 1 h after the last dose. Twenty-four hours later carotids were harvested for neutrophil quantification by immunostaining. Treatment with NCX 6560 was associated with a lower neutrophil infiltration (-39.5 %), while atorvastatin did not affect neutrophil content. The result was independent of effects on plasma cholesterol or differences in atorvastatin bioavailability, which suggests an important role of NO-related mechanisms in mediating this effect. Consistent with these in vivo findings, in vitro studies showed that NCX 6560, as compared to atorvastatin, had greater inhibitory activity on processes involved in neutrophil recruitment, such as migration in response to IL-8 and IL-8 release by endothelial cells and by neutrophils themselves. Pretreatment with NCX 6560, but not with atorvastatin, reduced the ability of neutrophil supernatants to promote monocyte chemotaxis, a well-known pro-inflammatory activity of neutrophils. Experimental data suggest a potential role of NO-releasing statins in the control of the vascular inflammatory process mediated by polymorphonuclear neutrophils.

OX40-OX40L INTERACTION TARGETS NFATC1 IN APOE−/− MICE DURING ATHEROGENESIS

ObjectivesWe investigated the effect of OX40-OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE−/− mice.MethodsAtherosclerotic plaque was induced by rapid perivascular carotid collar placement in...