Reduced Plaque Size Research Articles

Abstract 267: Histomorphometric Evaluation of Estrogen Effects on Coronary Artery Atherosclerosis: Inhibition of Plaque Formation, Reduction in Plaque Size and Promotion of Positive Remodeling Independent of Plaque Complications

Introduction: Whether and to what extent postmenopausal estrogen therapy has beneficial effects on progression of coronary artery atherosclerosis is controversial. In previous studies with monkey models we have shown that the stage of plaque progression modulates estrogen benefits and have led to the “timing hypothesis”. In this study, we used a histomorphometric approach to understand better the effects of estrogen therapy on critical components of atherosclerosis pathogenesis. Our hypothesis was that estrogen therapy would inhibit the initial development of atherosclerotic plaques, result in smaller but not less complicated plaques and among subjects that developed plaques, it would promote positive arterial remodeling. Methods: Forty-eight surgically postmenopausal monkeys (Macaca fascicularis) were fed a moderately atherogenic diet and randomly assigned to receive no treatment (CTL) or a women’s equivalent dose of conjugated equine estrogen (CEE) (0.45 mg/day). The experimental period lasted for 20 months (equivalent to 5 years in humans). At the end of the experiment, prevalence of atherosclerotic plaques was determined. From those monkeys with affected arteries, plaque extent, severity and prevalence of stenosis were determined. Results: Prevalence: The CEE treated group had significantly fewer monkeys with coronary artery atherosclerosis compared to the CTL group (CTL: 57 % vs. CEE: 33 % p=0.005). Extent: Among affected monkeys, the mean plaque size was significantly smaller in the CEE treated group compared to the CTL group (CTL: 0.30 ± 0.048 mm2 vs. CEE: 0.153 ± 0.028 mm2 p=0.015). Remodeling: The proportion of stenotic plaques was lower in the CEE group compared to the CTL group (CTL: 11.14 % vs. CEE: 1.34 % p= 0.008). Severity: CEE treatment did not have a beneficial effect on coronary artery severity (% of complicated [AHA grade≥ III] plaques- CTL: 52.34 % vs. 49.61 %, p=0.397). Conclusions: These histomorphometric findings support previous evidence that beginning of estrogen therapy in the perimenopausal transition prevents plaque initiation. Among affected cases, estrogen treated monkeys had smaller plaques but were equally complicated. Notably, estrogen also promoted positive arterial remodeling among affected arteries.

Abstract 676: 14q32 MicroRNA Inhibition Reduces Atherosclerotic Lesion Formation and Increases Plaque Stability

Introduction: Atherosclerosis is a multifactorial disease involving inflammatory, metabolic and lipid-related processes. As microRNAs (miRs) regulate expression of up to hundreds of target genes, we aimed to identify miRs that target genes involved in all processes of atherosclerosis. Using www.targetscan.org, we performed a Reverse Target Prediction on a set of 150 atherosclerosis-related genes. We found enrichment of binding sites for several miRs from a single gene cluster on human chromosome 14. Methods: ApoE-/- mice fed a Western type diet received semi-constrictive collars around both carotid arteries to induce atherosclerotic lesion formation. Gene Silencing Oligonucleotides (GSOs) were used to inhibit 3 14q32 miRs: miR-329, miR-494 and miR-495. Mice were injected with 1 mg GSO at day 4 after collar placement and again with 0.5 mg GSO at day 18. Mice were sacrificed at day 7 and day 28 for analysis of carotid arteries, liver, spleen, bone marrow and blood samples. Results: Atherosclerotic plaque analysis revealed a striking decrease of lesion size in mice treated with GSO-494 and GSO-495 (GSO-Control: 47 ± 11*103 μm2; GSO-494: 16 ± 3*103 μm2; GSO-495: 22 ± 5*103 μm2), whereas a trend towards reduced plaque size was observed in mice treated with GSO-329 (27 ± 6*103 μm2). Moreover, plaque stability was increased in mice treated with GSO-494 and GSO-495, determined by both a decrease in necrotic core size (GSO-494: 80% decrease; GSO-495: 60% decrease compared to GSO-Control) and an increase in plaque collagen content (GSO-Control: 6.6 ± 1.6%; GSO-494: 12.7 ± 2.1%; GSO-495: 15.8 ± 3.1%). Furthermore, inhibition of miR-494 and miR-495 resulted in decreased plasma cholesterol levels (GSO-Control: 30.4 ± 1.1 mM; GSO-494: 26.4 ± 0.7 mM; GSO-495: 26.4 ± 1.6) and decreased VLDL fractions. Conclusions: Inhibition of miR-494, miR-495 and, to a lesser extent, miR-329, leads to reduced plaque size, increased plaque stability and reduced plasma cholesterol levels. This makes 14q32 miRs promising novel therapeutic targets in atherosclerosis.

Vaccinia virus F5 is required for normal plaque morphology in multiple cell lines but not replication in culture or virulence in mice

Vaccinia virus (VACV) gene F5L was recently identified as a determinant of plaque morphology that is truncated in Modified Vaccinia virus Ankara (MVA). Here we show that F5L also affects plaque morphology of the virulent VACV strain Western Reserve (WR) in some, but not all cell lines, and not via previously described mechanisms. Further, despite a reduction in plaque size for VACV WR lacking F5L there was no evidence of reduced virus replication or spread in vitro or in vivo. In vivo we examined two mouse models, each with more than one dose and measured signs of disease and virus burden. These data provide an initial characterization of VACV F5L in a virulent strain of VACV. Further they show the necessity of testing plaque phenotypes in more than one cell type and provide an example of a VACV gene required for normal plaque morphology but not replication and spread.

Initial characterization of Vaccinia Virus B4 suggests a role in virus spread

Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus.

Targeted inhibition of ATP‐binding cassette transporter A1 protein degradation promotes reverse cholesterol transport and reduces atherosclerosis in mice (832.15)

Objective ATP‐Bing Cassette Transporter A1 (ABCA1) is a key protein in the rate‐limiting step of reverse cholesterol transport, which maintains plasma HDL levels. Transcriptional regulating ABCA1 promotes HDL generation and RCT is well established, however, little is known about the posttranspcriptional inhibition of ABCA1 degradation on macrophage RCT and atherosclerosis. We therefore investigate whether suppressing ABCA1 degradation would promote RCT and reduce atherosclerosis.Methods and results Pulse‐chase analysis revealed that a small molecular (IMM‐H007) significantly inhibits ABCA1 degradation and facilitates its cell‐surface localization in THP‐1 macrophages. In vitro tests described that IMM‐H007 promote cholesterol efflux via ABCA1‐mediated pathway. Animals fed a western diet containing 1.25% cholesterol with IMM‐H007 treatment showed an increase in circulating HDL levels and enhanced RCT to the plasma, liver, and feces. Additional, elevated hepatic ABCA1 expression and functional HDL were also observed in mice after IMM‐H007 administration. Furthermore, suppressing ABCA1 degradation gave rise to the reduction of plaque size, lipid content and increase markers of plaque stability in apoE‐/‐ mice.Conclusion Our findings reveal that raising HDL levels by inhibiting ABCA1 protein degradation enhances reverse cholesterol transport and decreases plaque formation, suggesting that it is a promising HDL therapeutic strategy to protect against atherosclerotic. IMM‐H007 could be a leading compound for development of drugs for raising HDL quantity and function.

Cholesterol Lowering Modulates T Cell Function In Vivo and In Vitro

BackgroundThe lipid milleu exacerbates the inflammatory response in atherosclerosis but its effect on T cell mediated immune response has not been fully elucidated. We hypothesized that lipid lowering would modulate T cell mediated immune function.Methods and ResultsT cells isolated from human PBMC or splenic T cells from apoE-/- mouse had higher proliferative response to T cell receptor (TCR) ligation in medium supplemented with 10% fetal bovine serum (FBS) compared to medium with 10% delipidated FBS. The differences in proliferation were associated with changes in lipid rafts, cellular cholesterol content, IL-10 secretion and subsequent activation of signaling molecule activated by TCR ligation. Immune biomarkers were also assessed in vivo using male apoE-/- mice fed atherogenic diet (AD) starting at 7 weeks of age. At 25 weeks of age, a sub-group was switched to normal diet (ND) whereas the rest remained on AD until euthanasia at 29 weeks of age. Dietary change resulted in a lower circulating level of cholesterol, reduced plaque size and inflammatory phenotype of plaques. These changes were associated with reduced intracellular IL-10 and IL-12 expression in CD4+ and CD8+ T cells.ConclusionOur results show that lipid lowering reduces T cell proliferation and function, supporting the notion that lipid lowering modulates T cell function in vivo and in vitro.

Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus

To determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus.

Suppressive Effects of Irbesartan on Inflammation and Apoptosis in Atherosclerotic Plaques of apoE−/− Mice: Molecular Imaging with 14C-FDG and 99mTc-Annexin A5

ObjectivesTo investigate the effects of irbesartan on inflammation and apoptosis in atherosclerotic plaques by histochemical examination and molecular imaging using 14C-FDG and 99mTc-annexin A5.BackgroundIrbesartan has a peroxisome proliferator-activated receptor gamma (PPARγ) activation property in addition to its ability to block the AT1 receptor. Accordingly, irbesartan may exert further anti-inflammatory and anti-apoptotic effects in atherosclerotic plaques. However, such effects of irbesartan have not been fully investigated. Molecular imaging using 18F-FDG and 99mTc-annexin A5 is useful for evaluating inflammation and apoptosis in atherosclerotic plaques.MethodsFemale apoE−/− mice were treated with irbesartan-mixed (50 mg/kg/day) or irbesartan-free (control) diet for 12 weeks (n = 11/group). One week after the treatment, the mice were co-injected with 14C-FDG and 99mTc-annexin A5, and cryostat sections of the aortic root were prepared. Histochemical examination with Movat's pentachrome (plaque size), Oil Red O (lipid deposition), Mac-2 (macrophage infiltration), and TUNEL (apoptosis) stainings were performed. Dual-tracer autoradiography was carried out to evaluate the levels of 14C-FDG and 99mTc-annexin A5 in plaques (%ID×kg). In vitro experiments were performed to investigate the mechanism underlying the effects.ResultsHistological examination indicated that irbesartan treatment significantly reduced plaque size (to 56.4%±11.1% of control), intra-plaque lipid deposition (53.6%±20.2%) and macrophage infiltration (61.9%±20.8%) levels, and the number of apoptotic cells (14.5%±16.6%). 14C-FDG (43.0%±18.6%) and 99mTc-annexin A5 levels (45.9%±16.8%) were also significantly reduced by irbesartan treatment. Irbesartan significantly suppressed MCP-1 mRNA expression in TNF-α stimulated THP-1 monocytes (64.8%±8.4% of un-treated cells). PPARγ activation was observed in cells treated with irbesartan (134%±36% at 3 µM to 3329%±218% at 81 µM) by a PPARγ reporter assay system.ConclusionsRemissions of inflammation and apoptosis as potential therapeutic effects of irbesartan on atherosclerosis were observed. The usefulness of molecular imaging using 18F-FDG and 99mTc-annexin A5 for evaluating the therapeutic effects of irbesartan on atherosclerosis was also suggested.

EC4, a truncation of soluble N-cadherin, reduces vascular smooth muscle cell apoptosis and markers of atherosclerotic plaque instability

Atherosclerotic plaque instability is precipitated by vascular smooth muscle cell apoptosis in the fibrous cap, weakening it and leading to plaque rupture. We previously showed that reducing smooth muscle cell apoptosis with soluble N-cadherin (SNC) increased features of plaque stability. We have now identified the active site of SNC and examined whether a truncated form containing this site retains the antiapoptotic effect. SNC was mutated to prevent interaction with N-cadherin or fibroblast growth factor receptor (FGFR). Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect. Therefore, we made a truncated form consisting of the EC4 domain. EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis in vitro by ~70%, similar to SNC. Elevation of plasma levels of EC4 in male apolipoprotein E–deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%. EC4 reduced plaque size and the incidence of buried fibrous layers and the macrophage:smooth muscle cell ratio (surrogate markers of plaque instability). Interaction of EC4 with FGFR induced potent antiapoptotic signaling in vitro and in vivo. EC4 modulates atherosclerosis in mice demonstrating its therapeutic potential for retarding plaque size and instability.

Traitements médicaux et chirurgicaux des courbures de verge congénitales et acquises : revue de la littérature

The aim of the current study was to provide an overview about the surgical and medical management of acquired and congenital penile's curvature. [corrected] A systematic review of the literature was done from the PubMed database by searching the following keywords alone or in combination: Congenital penile curvature; Congenital penile deviation; Acquired penile curvature; Acquired penile deviation; Peyronie's disease. The treatment of congenital curvature is only surgical. The most common technique is the Nesbit's technique which consists in making elliptical excisions of the tunica albuginea. There are also incison or plication procedures which are efficient as well. Acquired curvature is most of the time represented by the Peyronie's disease or is post-traumatic. Among oral treatments available, the Potaba is the only drug which has proved a significant reduction in penile plaque size. Injections of interferon and nicardipine have also shown their efficacy. Ionotophoresis and extracorporeal shock-wave therapy may be beneficial for penile pain. Other therapies (vacuum, traction devices, topical Verapamil) can be interesting but other studies are necessary to recommend them. Surgical treatment is recommended during the fibrotic phase. The most common technique is also the Nesbit's technique. In case of severe curvature (curve superior to 60°), small penis, major deformations, graft techniques can be used. Moreover, if there is a sexual dysfunction, penile prosthesis is recommended. Other studies are necessary to prove the efficacy of most of the drugs already available in the treatment of the penile curvature. It seems to be interesting to combine the different treatments to improve the results of those therapies.

Extracorporeal Shock Wave Therapy in Peyronie's Disease: Results of a Placebo-Controlled, Prospective, Randomized, Single-Blind Study

Extracorporeal shock wave therapy (ESWT) for treatment of Peyronie's disease (PD) is controversial. To study the efficacy of ESWT by a placebo-controlled, randomized trial. Patients with PD (n=102) were randomly assigned (n=51) to each group (ESWT or placebo). All patients were given 6 weekly treatments. Patients in the ESWT-group received 2,000 shock waves per session, using the Piezoson 100 lithotripter (Richard Wolf, Knittlingen, Germany). Patients in the placebo-group were treated with interposition of a plastic membrane, which prevented any transmission of shock waves. Primary end point was decrease of pain between baseline and after 4 weeks follow-up. Secondary end points were changes in deviation, plaque size, and sexual function. Pain was assessed by a visual analog scale. Deviation was measured by a goniometer after artificial erection using Alprostadil (Viridal®, Schwarz Pharma, Monheim, Germany). Plaque size was measured with a ruler and sexual function assessed by a scale regarding the ability to perform sexual intercourse. Overall, only 45 patients experienced pain at baseline. In the subgroup analysis of these patients, pain decreased in 17/20 (85.0%) patients in the ESWT group and 12/25 (48.0%) patients in the placebo group (P=0.013, relative risk [RR]=0.29, 95% confidence interval: 0.09-0.87). Penile deviation was not reduced by ESWT (P=0.66) but worsened in 20/50 (40%) and 12/49 (24.5%) patients of the ESWT and placebo-group, respectively (P=0.133). Plaque size reduction was not different between the two groups (P=0.33). Additional, plaque size increased in five patients (10.9%) of the ESWT group only. An improvement in sexual function could not be verified (P=0.126, RR=0.46). Despite some potential benefit of ESWT in regard to pain reduction, it should be emphasized that pain usually resolves spontaneously with time. Given this and the fact that deviation may worsen with ESWT, this treatment cannot be recommended.

Reprogramming macrophages to an anti‐inflammatory phenotype by helminth antigens reduces murine atherosclerosis

Atherosclerosis is a lipid-driven inflammatory disease of the vessel wall, characterized by the chronic activation of macrophages. We investigated whether the helminth-derived antigens [soluble egg antigens (SEAs)] could modulate macrophage inflammatory responses and protect against atherosclerosis in mice. In bone marrow-derived macrophages, SEAs induce anti-inflammatory macrophages, typified by high levels of IL-10 and reduced secretion of proinflammatory mediators. In hyperlipidemic LDLR(-/-) mice, SEA treatment reduced plaque size by 44%, and plaques were less advanced compared with PBS-injected littermate controls. The atheroprotective effect of SEAs was found to be mainly independent of cholesterol lowering and T-lymphocyte responses but instead could be attributed to diminished myeloid cell activation. SEAs reduced circulating neutrophils and inflammatory Ly6C(high) monocytes, and macrophages showed high IL-10 production. In line with the observed systemic effects, atherosclerotic lesions of SEA-treated mice showed reduced intraplaque inflammation as inflammatory markers [TNF-α, monocyte chemotactic protein 1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD68], neutrophil content, and newly recruited macrophages were decreased. We show that SEA treatment protects against atherosclerosis development by dampening inflammatory responses. In the future, helminth-derived components may provide novel opportunities to treat chronic inflammatory diseases, as they diminish systemic inflammation and reduce the activation of immune cells.

Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus

Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection.

Reduced susceptibility to all neuraminidase inhibitors of influenza H1N1 viruses with haemagglutinin mutations and mutations in non-conserved residues of the neuraminidase

We characterized human H1N1 influenza isolate A/Hokkaido/15/02, which has haemagglutinin and neuraminidase mutations that reduce drug susceptibility to oseltamivir, zanamivir and peramivir. One wild-type and three mutant viruses were isolated by plaque purification. Viruses were tested in MUNANA-based enzyme assays, cell culture and receptor binding assays. Two viruses had a neuraminidase Y155H mutation that reduced susceptibility in the enzyme inhibition assay to all inhibitors by 30-fold to >100-fold. The Y155H mutation reduced plaque size and affected the stability, Km and pH activity profile of the enzyme. In contrast to previous mutants, this neuraminidase demonstrated a slower rate of inhibitor binding in the IC50 kinetics assay. One virus had both the Y155H mutation and a haemagglutinin D225G mutation that rescued the small-plaque phenotype of the Y155H virus and affected receptor binding and drug susceptibility in cell culture and binding assays. We also isolated a third mutant virus, with both neuraminidase V114I and haemagglutinin D225N mutations, which affected susceptibility in the enzyme inhibition assay and receptor binding, respectively, but to lesser extents than the Y155H and D225G mutations. Neither Y155 nor V114 is conserved across neuraminidase subtypes. Furthermore, Y155 is not conserved even among avian and swine N1 viruses. Structurally, both residues reside far from the neuraminidase active site. D225 forms part of the receptor binding site of the haemagglutinin. We believe this is the first demonstration of a specific haemagglutinin mutation correlating with reduced drug susceptibility in plaque assays in both Madin Darby Canine Kidney and SIAT cells.

Identification and Functional Analysis of Membrane Proteins gD, gE, gI, and pUS9 of Infectious Laryngotracheitis Virus

Herpesvirus envelope proteins are of particular interest for development of attenuated live, marker, and subunit vaccines, as well as development of diagnostic tools. The unique short genome region of the chicken pathogen infectious laryngotracheitis virus (ILTV, Gallid herpesvirus 1) contains a cluster of six conserved alphaherpesvirus genes encoding membrane proteins, of which up to now only glycoproteins gG and gJ have been analyzed in detail. We have now prepared monospecific rabbit antisera against ILTV gD, gE, and gI, and the ILTV type II membrane protein pUS9, each of which showed specific immunofluorescence reactions, and detected proteins of approximately 65 and 70 kDa (gD), 62 kDa (gI), 75 kDa (gE), or 37 kDa (pUS9) in western blot analyses of infected chicken cells. The proteins gD, gI, and gE, but not pUS9, were identified as abundant virion proteins, and gE and gI were shown to be N-glycosylated. We also isolated gE-, gI-, and pUS9-deleted ILTV recombinants, whereas it was not possible to purify gD-negative ILTV to homogeneity, indicating that gD, like in other alphaherpesviruses, is essential for receptor binding and virus entry. The pUS9-deleted ILTV exhibited almost wild-type-like replication properties in cell culture. The gE- and gI-negative viruses showed significantly reduced plaque sizes, whereas virus titers were barely affected. Since homologous gene-deletion mutants of other alphaherpesviruses are in use as live vaccines, the generated ILTV recombinants might be also suitable for this application.

Therapeutic Silencing of MicroRNA-33 Inhibits the Progression of Atherosclerosis in Ldlr −/− Mice—Brief Report

To study the efficacy of anti-miRNA-33 therapy on the progression of atherosclerosis. Ldlr(-/-) mice were injected subcutaneously with PBS, control, or anti-miR-33 oligonucleotides weekly and fed a Western diet for 12 weeks. At the end of treatment, the expression of miR-33 target genes was increased in the liver and aorta, demonstrating effective inhibition of miR-33 function. Interestingly, plasma high-density lipoprotein (HDL)-cholesterol was significantly increased in anti-miR-33-treated mice but only when they were fed a chow diet. However, HDL isolated from anti-miR-33-treated mice showed an increase cholesterol efflux capacity compared with HDL isolated from nontargeting oligonucleotide-treated mice. Analysis of atherosclerosis revealed a significant reduction of plaque size and macrophage content in mice receiving anti-miR-33. In contrast, no differences in collagen content and necrotic areas were observed among the 3 groups. Long-term anti-miR-33 therapy significantly reduces the progression of atherosclerosis and improves HDL functionality. The antiatherogenic effect is independent of plasma HDL-cholesterol levels.

Abstract 476: Shear Stress-induced Atherosclerotic Plaque Regression is Reversed by Regulation of Macrophage Mobility via Matrix Metalloproteinase Inhibition

Atherosclerotic plaques form in regions of low blood flow, whereas vessels exposed to high shear stress remain lesion-free. We hypothesized that exposing established atherosclerotic plaques to elevated shear stress leads to lesion regression by facilitating inflammatory cell movement within the plaque. We developed a model of arteriovenous fistula (AVF) in mice, where the right carotid artery is anastomosed into the jugular vein. LDLR-/- mice were placed on a high-fat diet. Control mice were sacrificed at week 12, which coincided with sham and AVF surgery. Sham and AVF mice were kept on a high-fat diet for a further 4 weeks. This procedure increases the shear stress in the brachiocephalic artery (BCA) and leads to a 51% plaque regression in AVF. All groups had comparable lipid levels. However, BCA plaque macrophage, smooth muscle cell and collagen content was halved in AVF. We observed greater gelatinase activity in plaques of AVF mice, suggesting a role for matrix metalloproteinases (MMPs) in plaque regression. MMP-9 and MMP-3 expression was increased in AVF plaques whereas MMP-2 and MMP-14 expression was decreased (p<0.05). A separate group of mice was therefore treated post-surgery with an MMP inhibitor, doxycycline, or with a TIMP-1 over-expressing plasmid. Both prevented the reduction in plaque size in the AVF group. To better define the mechanism of plaque regression in the AVF, we devised an endothelial cell (EC)-macrophage co-culture system where the ECs were exposed to high, low or no shear stress, and macrophages exposed to the EC effluent. There was a 2.5 fold increase in the migration of macrophages exposed to high shear effluent vs. low shear (p<0.05). This coincided with a 3-fold increase in the number of macrophages expressing activated β1 integrin in the high shear conditions. Uptake of apoptotic cells by macrophages was also 25% higher in the high shear vs. static (p<0.05). When repeated using the MMP inhibitor, GM6001, the high shear increase in migration was blocked in the presence of MMP inhibition; however, it had no effect on cell phagocytosis. Our findings suggest that shear stress acting on ECs may influence the cells within the plaque by increasing MMP activity allowing for better macrophage motility, an important feature of regressing plaques.

Abstract 461: Inhibition of Four- and- a- Half LIM Domain Protein-2 Decreases Atherosclerosis in Apoe -/- Mice model: Role of Immune T- Cells

OBJECTIVE Four-and-a-half LIM domain protein-2 (FHL2), a member of the FHL family of proteins, is expressed in endothelial and vascular smooth muscle cells. FHL2 negatively regulates endothelial cell survival and migration, but its role in atherogenesis is unknown. METHODS AND RESULTS To investigate the role of FHL2 in atherosclerosis, we crossed FHL2 knockout (FHL2-/-) with apolipoprotein (Apo) E-deficient (ApoE -/-) mice, and fed them a high fat diet for 7 weeks. Despite a total plasma cholesterol levels comparable to the ApoE-/- mice, FHL2-/-ApoE-/- mice showed a strong increase in plasma HDL-cholesterol as compared to ApoE -/- mice (P<0.01; n=6) and had significantly smaller atherosclerotic plaques in the aortic sinus (0.14±0.02 vs. 0.29±0.04 mm 2 ) and aorta (6.9±0.9 vs 10.3±1%) (P<0.05; n=6), assessed by oil red O staining, compared with ApoE-/- mice. The relative monocyte/ macrophage content within atherosclerotic plaques, determined by MOMA-2 immunostaining, was equivalent in both animal groups. However, we observed an enhanced relative content of collagen (16±2 vs 8.6±3 %) and smooth muscle cells (4.5±0.8 vs 1.8±0.5%) within the plaques in the aortic sinus of FHL2-/-ApoE-/- mice compared with ApoE-/- mice, determined by Sirius red and alpha-actin staining respectively. These results suggest that absence of FHL2 promotes smaller and more stable plaques. The reduced plaque size in FHL2-/-ApoE-/- mice could be explained, at least in part, by the 40% reduction in ICAM-1 mRNA expression observed in aortas (p<0.05 vs ApoE-/-). Moreover, although the relative monocyte/ macrophage content in spleens was equivalent in both animal groups, FACS analysis of T cells in spleens showed a significant increase in CD4+CD25+Foxp3+ regulatory T cell numbers, in FHL2-/-ApoE-/- compared with ApoE-/- mice (19.5±1.6 vs. 16.2±2% of CD3+CD4+ cells, respectively) (P<0.005). CONCLUSIONS These results suggest that FHL2 may play an important role in atherosclerosis by promoting plaque formation, through upregulation of adhesion molecule expression and suppression of regulatory T cells.

Abstract 446: Therapeutic Silencing of MicroRna-33 In Mice Inhibits the Progression of Atherosclerosis in Ldlr-/- Mice

Objective- To study the efficacy of anti-miRNA-33 therapy on the progression of atherosclerosis. Methods and Results- LDLr -/- mice were injected subcutaneously with PBS, control or miR-33 antisense oligonucleotides (ASO) weekly and fed a Western diet for 12 weeks. As expected, hepatic levels of miR-33 were significantly decreased in anti-miR-33 treated mice. Accordingly, the expression of miR-33 target genes was increased in the liver and aorta. Interestingly, plasma HDL cholesterol (HDL-C) was significantly increased in anti-miR-33 treated mice but only when they were fed a chow diet. Analysis of atherosclerosis revealed a significant reduction of plaque size and macrophage content in mice receiving miR-33 ASO. In contrast, no differences in collagen content and necrotic areas were observed between the three groups. Conclusions- Long-term anti-miR-33 therapy significantly reduces the progression of atherosclerosis. The miR-33 ASO anti-atherogenic effect is independent of plasma HDL-C.

Four-dimensional microglia response to anti-Aβ treatment in APP/PS1xCX3CR1/GFP mice.

Senile plaques, mainly composed of amyloid-β (Aβ), are a major hallmark of Alzheimer disease (AD), and immunotherapy is a leading therapeutic approach for Aβ clearance. Although the ultimate mechanisms for Aβ clearance are not well known, characteristic microglia clusters are observed in the surround of senile plaques, and are implicated both in the elimination of Aβ as well as the deleterious inflammatory effects observed in AD patients after active immunization. Therefore, analyzing the direct effect of immunotherapy on microglia, using longitudinal in vivo multiphoton microscopy can provide important information regarding the role of microglia in immunotherapy. While microglia were observed to surround senile plaques, topical anti-Aβ antibody administration, which led to a reduction in plaque size, directed microglia toward senile plaques, and the overall size of microglia and number of processes were increased. In some cases, we observed clusters of microglia in areas of the brain that did not have detectable amyloid aggregates, but this did not predict the deposition of new plaques in the area within a week of imaging, indicating that microglia react to but do not precipitate amyloid aggregation. The long-term presence of large microglial clusters in the surrounding area of senile plaques suggests that microglia cannot effectively remove Aβ unless anti-Aβ antibody is administered. All together, these data suggest that although there is a role for microglia in Aβ clearance, it requires an intervention like immunotherapy to be effective.