Faculty Bibliography

Apolipoprotein B (apoB) is the essential protein required for the assembly and secretion of chylomicrons from the small intestine and VLDLs from the liver. These lipoproteins, as well as their remnants and LDL, play key roles in the transport of dietary and endogenously synthesized lipids throughout the body. However, they can be involved in the initiation of atherosclerotic lesions in the vessel wall. Therefore, it is not surprising that the assembly of apoB-containing lipoproteins in the small intestine and liver is a highly regulated process. In particular, cotranslational and posttranslational targeting of apoB for degradation, regulated largely by the availability of the core lipids carried in the lipoprotein, by the types of dietary fatty acids consumed, and by the hormonal milieu, determines the number of chylomicrons or VLDL that are secreted. In this review, we summarize both older and more recent findings on the pathways of apoB degradation, focusing on events in the liver.

Atherosclerosis is the leading cause of death worldwide. To date, the use of statins to lower LDL levels has been the major intervention used to delay or halt disease progression. These drugs have an incomplete impact on plaque burden and risk, however, as evidenced by the substantial rates of myocardial infarctions that occur in large-scale clinical trials of statins. Thus, it is hoped that by understanding the factors that lead to plaque regression, better approaches to treating atherosclerosis may be developed. A transplantation-based mouse model of atherosclerosis regression has been developed by allowing plaques to form in a model of human atherosclerosis, the apoE-deficient mouse, and then placing these plaques into recipient mice with a normolipidemic plasma environment. Under these conditions, the depletion of foam cells occurs. Interestingly, the disappearance of foam cells was primarily due to migration in a CCR7-dependent manner to regional and systemic lymph nodes after 3 days in the normolipidemic (regression) environment. Further studies using this transplant model demonstrated that liver X receptor and HDL are other factors likely to be involved in plaque regression. In conclusion, through the use of this transplant model, the process of uncovering the pathways regulating atherosclerosis regression has begun, which will ultimately lead to the identification of new therapeutic targets

OBJECTIVE: Macrophage apoptosis plays important roles in atherosclerosis. Bcl-2 is a key cell survival molecule, but its role in macrophage apoptosis in atherosclerosis is not known. The goal herein was to determine the effect of macrophage-targeted deletion of Bcl-2 on macrophage apoptosis in atherosclerotic lesions of Apoe(-/-) mice. METHODS AND RESULTS: Bcl2(flox)-LysMCre mice were created as a model of macrophage Bcl-2 deficiency. Macrophages from these mice were more susceptible to apoptosis than those from control Bcl2(WT)-LysMCre mice. The mice were bred onto the Apoe(-/-) background and fed a Western-type diet for 4 or 10 weeks. Apoptotic cells were equally very rare in the lesions of both groups of the 4-week-diet mice, and there was no difference in lesion area. However, Bcl2(flox)-LysMCre;Apoe(-/-) plaques from the 10-week-diet protocol had a 40% to 45% increase in apoptotic cells and, in female mice, a approximately 25% increase in plaque necrosis (P<0.05) compared with Bcl2(WT)-LysMCre lesions. CONCLUSIONS: Macrophage Bcl-2 plays a protective role against macrophage apoptosis specifically in advanced atherosclerotic lesions of Apoe(-/-) mice

Purpose: Unlike other DMARDs methotrexate diminishes the risk of Atherosclerotic Cardiovascular Disease (ASCVD) in patients with Rheumatoid Arthritis and adenosine, acting at adenosine A2A receptors, has been shown to mediate the anti-inflammatory effects of methotrexate. Adenosine inhibits the first step in formation of atherosclerotic plaque, foam cell formation in macrophages and this effect appears to be mediated by enhanced expression of cholesterol 27-hydroxylase, an enzyme involved in reverse cholesterol transport. We therefore asked whether the effect of adenosine A2A receptors on foam cell formation in vitro are mediated by apoE or 27-hydroxylase (27OH'ase), proteins involved in reverse cholesterol transport. Method: THP-1 cells, a human monocytoid cell line, were infected with lentiviral vectors expressing siRNA for either apoE or 27OH'ase or scrambled RNA and infected cell lines were selected by incubation with puromycin. Foam cell formation was induced in THP-1 cells by incubation with interferon- (500U/ml) and % foam cells enumerated in 5 high power fields. 3H-Cholesterol efflux was measured after loading with label. Results: Specific lentiviral siRNA infection markedly reduces apoE (p< 0.0001, apoE siRNA vs. control, n=3) or 27OH'ase mRNA (p< 0.0001, 27-hydroxylase siRNA vs. control, n=3) and protein (p< 0.0107, 27-hydroxylase siRNA vs. control n= 3) in THP-1 cells. Despite diminished apoE expression CGS-21680 (1muM), an adenosine A2A receptor agonist, inhibits IFN-induced foam cell formation (p< 0.0002, IFN CGS vs. IFN alone, n= 4) but has no effect on foam cell formation in 27OH'ase KD cells. CGS21680 increases cholesterol efflux in wild type and apoE1 KD cells (from 9.5% to 17.5+2.5% and from 10.0+2% to 17.5 +2%, respectively) but not 27OH'ase KD cells. Conclusion: Adenosine A2A receptor-mediated increases in reverse cholesterol transport leading to diminished foam cell formation explains the anti-atherosclerotic effects of methotrexate

Introduction: A critical event in the development of cardiac fibrosis is the transformation of fibroblasts into myofibroblasts. Fibroblasts isolated from healthy hearts and grown under standard tissue culture conditions express alpha-SMA and have been referred to as myofibroblasts. However, recent data suggest the in vitro transformation does not fully replicate the in vivo activation process. The purpose of this study was to investigate the potential of activated fibroblasts to contribute to an arrhythmogenic substrate through paracrine and direct coupling effects. Methods: Confluent neonatal rat myocyte monolayers were treated with media (CM) conditioned by cardiac fibroblasts isolated from ventricles of healthy (Fb) and infarcted (MI-Fb) hearts and optically mapped 16-20 hours later. To study the combined paracrine and direct coupling effect, Fb and MI-Fb were plated on top of myocyte monolayers. Results: Treatment with both Fb CM (16.6+/-0.4 cm/s) and MIFb CM (15.8+/-0.4 cm/s) significantly decreased conduction velocity (CV) compared to homocellular myocyte monolayers (Myo; 19.7+/-0.7 cm/s). Action potential duration (APD70) was significantly reduced by MI-Fb CM (143.6+/-1.7 ms) treatment compared to Myo (159.4+/-4.0 ms) and Fb CM (153.4+/-2.7 ms). In heterocellular cultures, Fb significantly decreased (17.0+/-0.5 cm/s) and MI-Fb increased (22.0+/-0.6 cm/s) average CV compared to Myo. In addition, CV was significantly faster with MI-Fb compared to Fb (p=1.95E-8). Fb (145.0+/-3.9 ms) and MIFb (131.1+/-3.7 ms) significantly reduced APD70 compared to Myo (159.4+/-4.0 ms), and APD70 was significantly shorter with MI-Fb compared to Fb (p=0.01). Analysis of Cx43 levels showed a significant upregulation of Cx43 in MI-Fb compared to Fb. Conclusions: These data demonstrate Fb exert predominantly paracrine effects while MI-Fb affect myocyte electrophysiology through a combination of paracrine and direct coupling mechanisms. Moreover, APD shortening and increased Cx43 levels in MI-Fb could contribute to the greater incidence of arrhythmias observed in fibrotic hearts. These findings may lead to the development of new anti-arrhythmic therapeutic approaches targeting the fibroblast activation process

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose towards arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization, but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiologic effects of GJR. We paced wildtype (normal Cx43 abundance) and heterozygous Cx43 knockout mice (Cx43(+/-), 66% mean reduction in Cx43) for six hours at 10-15% above their average sinus rate. We investigated the electrophysiologic effects of pacing on the whole animal using programmed electrical stimulation, and in isolated ventricular myocytes with patch clamp studies. Cx43(+/-) myocytes had significantly shorter action potential durations (APD) and increased steady state and inward rectifier potassium currents (Iss and IK1, respectively) compared to wildtype littermate cells. In Cx43(+/-) hearts, pacing resulted in significant prolongation of ventricular effective refractory period and action potential duration, and significant diminution of Iss compared to unpaced Cx43(+/-) hearts. However, these changes were not seen in paced wildtype mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiologic changes which may contribute to the worsened prognosis often associated with pacing in the failing heart. Key words: Connexin43, ventricular myocytes, mouse, gap junction

Magnetic resonance (MR) imaging is becoming a pivotal diagnostic method to identify and characterize vulnerable atherosclerotic plaques. We previously reported a reconstituted high-density lipoprotein (rHDL) nanoparticle platform enriched with Gd-based amphiphiles as a plaque-specific MR imaging contrast agent. Further modification can be accomplished by inserting targeting moieties into this platform to potentially allow for improved intraplaque macrophage uptake. Since studies have indicated that intraplaque macrophage density is directly correlated to plaque vulnerability, modification of the rHDL platform may allow for better detection of vulnerable plaques. In the current study we incorporated a carboxyfluoresceine-labeled apolipoprotein E-derived lipopeptide, P2fA2, into rHDL. The in vitro macrophage uptake and in vivo MR efficacy were demonstrated using murine J774A.1 macrophages and the apolipoprotein E knock-out (apoE(-/-)) mouse model of atherosclerosis. The in vitro studies indicated enhanced association of murine macrophages to P2fA2 enriched rHDL (rHDL-P2A2) nanoparticles, relative to rHDL, using optical techniques and MR imaging. The in vivo studies showed a more pronounced and significantly higher signal enhancement of the atherosclerotic wall 24 h after the 50 micromol Gd/kg injection of rHDL-P2A2 relative to administration of rHDL. The normalized enhancement ratio for atherosclerotic wall of rHDL-P2A2 contrast agent injection was 90%, while that of rHDL was 53% 24 h post-injection. Confocal laser scanning microscopy revealed that rHDL-P2A2 nanoparticles co-localized primarily with intraplaque macrophages. The results of the current study confirm the hypothesis that intraplaque macrophage uptake of rHDL may be enhanced by the incorporation of the P2fA2 peptide into the modified HDL particle

High density lipoprotein (HDL) is an important natural nanoparticle that may be modified for biomedical imaging purposes. Here we developed a novel technique to create unique multimodality HDL mimicking nanoparticles by incorporation of gold, iron oxide, or quantum dot nanocrystals for computed tomography, magnetic resonance, and fluorescence imaging, respectively. By including additional labels in the corona of the particles, they were made multifunctional. The characteristics of these nanoparticles, as well as their in vitro and in vivo behavior, revealed that they closely mimic native HDL.