Growth Of Atherosclerotic Lesions Research Articles

Abstract 3066: Pro-Atherogenic Macrophages Induce The Upregulation Of MLKL And Cell Death In Vascular Smooth Muscle Cells During Atherogenic Stimulation

Background: A hallmark of advanced, rupture-prone atherosclerotic plaques is the presence of a necrotic core - a dense mass of pro-inflammatory and cellular debris. Traditionally, it has been thought that most of the foam cells within this core are derived from macrophages. However, recent advances in single cell technologies have revealed that vascular smooth muscle cells have the capacity to transdifferentiate into "macrophage-like foam cells", accounting for almost half of the cell content in advanced plaques. Our lab and others have discovered that a primary cause of the necrotic core is necroptotic cell death, where the mixed lineage kinase domain like pseudokinase MLKL is a key executioner of this process. While the contribution of necroptotic macrophage-derived foam cells to atherosclerotic lesion growth and instability has been fairly well-studied, the role of necroptotic vascular smooth muscle cell-derived foam cells in this process remains unclear. Hypothesis: MLKL regulates cell death differently in macrophages and vascular smooth muscle cells when challenged with pro-atherogenic stimuli. Methods and Results: Bone marrow-derived macrophages (BMDMs) and aortic vascular smooth muscle cells (VSMCs) were isolated from wildtype mice and treated with pro-atherogenic stimuli in the presence or absence of apoptotic and necroptotic inhibitors, zVAD and Necrostatin-1 (Nec1). BMDMs underwent cell death in response to treatment with oxLDL or LPS plus zVAD within 24 hours (as measured by LDH release and SYTOX assays) while VSMCs survive up to 72 hours. However, when VSMCs are treated with BMDM conditioned media in addition to pro-atherogenic stimuli, VSMCs begin to show higher relative rates of cell death compared to controls. Confocal microscopy and Western blots revealed that VSMCs treated with cell death ligands in conjunction with BMDM conditioned media showed an increase in relative expression of total and pMLKL- a hallmark of necroptosis, compared to VSMCs without conditioned media. Conclusions: Overall, this work suggests VSMCs are inherently resistant to necroptosis unless treated with conditioned media from BMDMs, which may suggest that macrophages have the capacity to prime necroptosis in VSMCs, likely via secreted factors.

Clonal Hematopoiesis: Connecting Aging and Inflammation in Atherosclerosis.

Clonal hematopoiesis (CH) is a prevalent condition that results from the acquisition of somatic mutations in hematopoietic stem cells. When these mutations occur in "driver" genes, they can potentially confer fitness advantages to the cell, leading to a clonal expansion. While most clonal expansions of mutant cells are generally considered to be asymptomatic since they do not impact overall blood cell numbers, CH carriers display long-term risks of all-cause mortality and age-associated diseases including cardiovascular disease (CVD). This review summarizes recent findings in CH related to aging, atherosclerotic CVD, and inflammation, emphasizing epidemiological and mechanistic studies, and potential therapeutic options to treat CVDs that are promoted by CH. Epidemiological studies have revealed associations between CH and CVDs. Experimental studies with CH models employing the Tet2- and Jak2-mutant mouse lines display inflammasome activation and a chronic inflammatory state that leads to accelerated atherosclerotic lesion growth. A body of evidence suggests that CH represents a new causal risk factor for CVD. Studies also indicate that understanding an individual's CH status could provide guidance for personalized approaches to treat atherosclerosis and other CVDs with anti-inflammatory drugs.

Continuous Hypobaric Hypoxia may Promote Atherosclerosis Progression in Apolipoprotein E-deficient Mice.

Background: Intermittent normobaric hypoxia can promote the progression of atherosclerotic plaques. However, the effect of continuous hypobaric hypoxia (CHH), which is a major feature of high-altitude environment, on atherosclerosis has not been investigated thoroughly. Materials and Methods: After eight weeks of high-cholesterol diet, 30 male ApoE-/- mice were randomly divided into control and CHH groups. Mice in the CHH group lived in a hypobaric chamber with an oxygen content of 10% and air pressure of 364 mmhg (equal to 5,800 m altitude above sea level) for 4 weeks, while mice in the control group lived in normoxia condition. Then all mice were euthanized and the atherosclerotic lesion size and plaque stability in the aortic root were assessed. Intraplaque angiogenesis was characterized by immunostaining of CD31 and endomucin, which are identified as specific markers of vascular endothelial cells. Immunohistochemistry and qRT-PCR were performed to measure inflammatory cytokines. Results: Four weeks of CHH exposure promoted the growth of atherosclerotic lesions (p=0.0017) and decreased the stability of atherosclerotic plaques. In CHH group, plaque smooth muscle cells and collagen contents decreased, while plaque macrophages and lipids contents increased significantly (p<0.001). The contents of CD31 (p=0.0379) and endomucin (p=0.0196) in the plaque was higher in the CHH group and correlated with angiogenesis progression. Further, the content of monocyte chemotactic protein-1 (p=0.0376) and matrix metalloproteinase-2 was significantly higher (p=0.0212) in the CHH group. Conclusions: CHH may accelerate atherosclerosis progression in ApoE-/- mice by promoting angiogenesis and inflammation.

Evaluation of Plaque Characteristics and Inflammation Using Magnetic Resonance Imaging.

Atherosclerosis is an inflammatory disease of large and medium-sized arteries, characterized by the growth of atherosclerotic lesions (plaques). These plaques often develop at inner curvatures of arteries, branchpoints, and bifurcations, where the endothelial wall shear stress is low and oscillatory. In conjunction with other processes such as lipid deposition, biomechanical factors lead to local vascular inflammation and plaque growth. There is also evidence that low and oscillatory shear stress contribute to arterial remodeling, entailing a loss in arterial elasticity and, therefore, an increased pulse-wave velocity. Although altered shear stress profiles, elasticity and inflammation are closely intertwined and critical for plaque growth, preclinical and clinical investigations for atherosclerosis mostly focus on the investigation of one of these parameters only due to the experimental limitations. However, cardiovascular magnetic resonance imaging (MRI) has been demonstrated to be a potent tool which can be used to provide insights into a large range of biological parameters in one experimental session. It enables the evaluation of the dynamic process of atherosclerotic lesion formation without the need for harmful radiation. Flow-sensitive MRI provides the assessment of hemodynamic parameters such as wall shear stress and pulse wave velocity which may replace invasive and radiation-based techniques for imaging of the vascular function and the characterization of early plaque development. In combination with inflammation imaging, the analyses and correlations of these parameters could not only significantly advance basic preclinical investigations of atherosclerotic lesion formation and progression, but also the diagnostic clinical evaluation for early identification of high-risk plaques, which are prone to rupture. In this review, we summarize the key applications of magnetic resonance imaging for the evaluation of plaque characteristics through flow sensitive and morphological measurements. The simultaneous measurements of functional and structural parameters will further preclinical research on atherosclerosis and has the potential to fundamentally improve the detection of inflammation and vulnerable plaques in patients.

Effect of transport parameters on atherosclerotic lesion growth: A parameter sensitivity analysis

BackgroundAtherosclerosis is a degenerative disease of the arterial wall. It results in the formation of progressively growing plaque lesions that can harden and narrow their host arteries. Current computational models of the inflammatory process that govern atherosclerosis growth are reliant on a number of parameters that can freely vary and whose precise values are not well known. MethodsTo identify the significance of variation in such parameters, a parametric sensitivity study had been conducted on the blood density, blood viscosity, plasma viscosity and bulk flow low density lipoprotein (LDL) concentration. Using computational modeling, the significance of variation in these parameters was assessed on the transport of LDL. The simulation was performed via the 2k factorial experimental design, which was conducted to identify the significance of the select parameters on the intima LDL concentration and endothelial LDL coverage area. ResultsResults identified the blood viscosity and bulk flow LDL concentration are the dominant parameters for the atherosclerotic lesion growth. The coverage of LDL on the arterial wall surface was strongly dependent on the blood viscosity. The significance of these findings was discussed. ConclusionThis statistical study identifies two dominating blood factors, LDL concentration and blood viscosity, and how they influence atherosclerosis which will serves as a guideline for further investigation on the atherosclerosis topic.

Coronary inflammation: why searching, how to identify and treat it.

Inflammation plays an important role in the development of atherosclerotic lesions. A variety of stimuli promote atherosclerosis, including increased LDL cholesterol in blood, exposure to tobacco, diabetes mellitus, hypertension, or rheological stress. Inflammatory cells have an established role in the growth of atherosclerotic lesions. Macrophages recognize and internalise ox-LDL to eventually become lipid-laden foam cells, the hallmark cellular component of atheroma. Infiltrating CD4-T cells have a role too, by interacting with ox-LDL and other antigens. Cytokines secreted by inflammatory cells stimulate smooth muscle cells migration whilst macrophages produce metalloprotease that lead to fibrous cap rupture. The necrotic debris of died macrophages and smooth muscle cells help to continue the inflammatory process. The inflammatory response can also directly activate platelets and promote thrombus formation at the surface of complicated coronary plaques. The CANTOS trial can be waived as an innovative study promoting a novel approach of personalized medicine. In patients with previous myocardial infarction, high-sensitivity C-reactive protein level of 2 mg and normal LDL level (<70 mg/dL), canakinumab a therapeutic monoclonal antibody targeting interleukin-1β, at a dose of 150 mg every 3 months, led to a significant reduction of the primary efficacy end point: nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death at 48 months. Based on the CANTOS results, patients on statins and residual inflammatory risk as assessed by means of a high-sensitivity CRP >2 mg/l at baseline have a high risk of future cardiac events, comparable to that of statin-treated patients with suboptimal cholesterol LDL level. The inhibition of interleukin-1β by means of canakinumab, which is only one of many potential anti-inflammatory pathways, open new perspectives, showing that a selective inhibition of the inflammatory pathway may be beneficial in reducing cardiovascular risk. In a process of personalized medicine, there is need to accurately identify patients at high risk of events, to be treated with potent statins or anti-inflammatory drugs. Perhaps in the near future a more specific assessment of coronary inflammations, possibly obtained with imaging modalities (either invasive or non-invasive), will better select patients at risk of events. In this scenario, in the setting of secondary prevention, OCT may serve the scope of identifying vulnerable plaques with local aggregates of inflammatory cells. Future studies are needed to understand the clinical effectiveness of strategies based on invasive coronary assessment.

418 Ultrasound molecular imaging of the role of von willebrand factor-mediated platelet adhesion in atherogenesis

Abstract Funding Acknowledgements JSPS Overseas Research Fellowship Background Platelets are known to be both pro-inflammatory and pro-mitogenic. However, the role of platelet-endothelial interactions in the initiation and growth of atherosclerotic lesions is not well understood. Purpose We used contrast-enhanced ultrasound (CEU) molecular imaging of the arterial endothelium to test the hypothesis that platelet attachment to endothelial Von Willebrand Factor (VWF) promotes atherogenesis. Methods We studied wild-type mice (WT), low-density lipoprotein deficient mice fed western diet to produce atherosclerosis (LDLR-/-), and LDLR-/- mice also deficient for ADAMTS-13 (LDLR-/-ADAMTS13-/-) which is the enzyme responsible for proteolytic cleavage of endothelial-associated VWF. Mice were studied at 20 weeks and 30 weeks of age. A subset of LDLR-/- mice were treated with recombinant ADAMTS13 1 hr prior to study. Proximal aortic CEU molecular imaging of P-selectin, vascular cell adhesion molecule (VCAM)-1, von Willebrand factor (VWF), and platelet GPIbα was performed. Aortic distensibility was assessed using high-frequency (30 MHz) transthoracic echocardiography and tail cuff blood pressure systems. NF-κB of aorta was assessed by ELISA kit. Plaque size and composition were assessed by histology. Platelets and macrophage immunohistochemistry were also performed on confocal microscopy. Results Aortic molecular imaging signal for P-selectin, VCAM-1, VWF, and platelet adhesion was significantly higher in LDLR-/- than WT mice, and increased by 2-fold between 20 and 30 wks of age. Signal for VWF and platelet adhesion was abolished 1 h after administration of ADAMTS13, confirming that platelet adhesion was VWF-mediated. At 20 and 30 wks of age, molecular imaging signal for all targets was 2-fold higher (p &amp;lt; 0.01) in LDLR-/-ADAMTS13-/- versus LDLR-/- mice. The LDLR-/-ADAMTS13-/- mice also had lower aortic distensibility (p &amp;lt; 0.05), had a 2-fold higher NF-κB signal (p &amp;lt; 0.05), and had a 2-fold greater total plaque area (p &amp;lt; 0.01). Fluorescent immunohistochemistry confirmed that the LDLR-/-ADAMTS13-/- mice also had greater platelets (p &amp;lt; 0.05) and increased macrophage content (p &amp;lt; 0.05) than LDLR-/- mice in aortic plaque. Conclusion In early to mid-stage atherosclerosis, abnormal regulation of endothelial-associated VWF results in platelet adhesion and secondary up-regulation of endothelial inflammatory adhesion molecules, thereby promoting atherosclerotic plaque progression. These results indicate an important role of platelet-endothelial interactions in early atherogenesis. Abstract 418 Figure

4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Endoplasmic reticulum stress plays an important role in cardiovascular disease (CVD) and atherosclerosis. We aimed to assess the ability of 4-phenylbutyrate (4-PBA), a small chemical chaperone administered via drinking water, to reduce atherosclerotic lesion size in chow-fed apolipoprotein (Apo) e-/- mice and to identify mechanisms that contribute to its antiatherogenic effect. Chow-fed 17-wk-old female Apoe-/- mice treated with 4-PBA-supplemented drinking water for 5 wk exhibited smaller lesions as well as increased plasma levels of heat shock protein (HSP) 25, the mouse homolog of human HSP27, compared with controls. In addition, 4-PBA inhibited cell death and increased HSP27 expression as measured by real-time PCR and immunoblotting, as well as induced nuclear localization of its transcription factor, heat shock factor 1, in human monocyte/macrophage (THP-1) cells. Furthermore, HSP27 small interfering RNA diminished the protective effect of 4-PBA on THP-1 macrophage attachment and differentiation. In summary, drinking water containing 4-PBA attenuated early lesion growth in Apoe-/- mice fed a chow diet and increased expression of HSP25 and HSP27 in macrophages and HSP25 in the circulation of Apoe-/- mice. Given that increased expression of HSP27 is inversely correlated with CVD risk, our findings suggest that 4-PBA protects against the early stages of atherogenesis in part by enhancing HSP27 levels, leading to inhibition of both macrophage cell death and monocyte-macrophage differentiation.-Lynn, E. G., Lhoták, Š., Lebeau, P., Byun, J. H., Chen, J., Platko, K., Shi, C., O'Brien, E. R., Austin, R. C. 4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

The Contribution of Homocysteine Metabolism Disruption to Endothelial Dysfunction: State-of-the-Art.

Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid formed during the metabolism of the essential amino acid methionine. Hcy is considered a risk factor for atherosclerosis and cardiovascular disease (CVD), but the molecular basis of these associations remains elusive. The impairment of endothelial function, a key initial event in the setting of atherosclerosis and CVD, is recurrently observed in hyperhomocysteinemia (HHcy). Various observations may explain the vascular toxicity associated with HHcy. For instance, Hcy interferes with the production of nitric oxide (NO), a gaseous master regulator of endothelial homeostasis. Moreover, Hcy deregulates the signaling pathways associated with another essential endothelial gasotransmitter: hydrogen sulfide. Hcy also mediates the loss of critical endothelial antioxidant systems and increases the intracellular concentration of reactive oxygen species (ROS) yielding oxidative stress. ROS disturb lipoprotein metabolism, contributing to the growth of atherosclerotic vascular lesions. Moreover, excess Hcy maybe be indirectly incorporated into proteins, a process referred to as protein N-homocysteinylation, inducing vascular damage. Lastly, cellular hypomethylation caused by build-up of S-adenosylhomocysteine (AdoHcy) also contributes to the molecular basis of Hcy-induced vascular toxicity, a mechanism that has merited our attention in particular. AdoHcy is the metabolic precursor of Hcy, which accumulates in the setting of HHcy and is a negative regulator of most cell methyltransferases. In this review, we examine the biosynthesis and catabolism of Hcy and critically revise recent findings linking disruption of this metabolism and endothelial dysfunction, emphasizing the impact of HHcy on endothelial cell methylation status.

ERV1/ChemR23 Signaling Protects Against Atherosclerosis by Modifying Oxidized Low-Density Lipoprotein Uptake and Phagocytosis in Macrophages.

In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein-coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. Lipidomic plasma analysis were performed after EPA supplementation in Apoe-/- mice. Erv1/Chemr23-/- xApoe-/- mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe-/- mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1-mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.

HDL is essential for atherosclerotic lesion regression in Apoe knockout mice by bone marrow Apoe reconstitution

Background and aimsAlthough studies in mice have suggested that lesion regression is feasible, the underlying mechanisms remain largely unknown. Here we determined the impact of high-density lipoprotein (HDL) on atherosclerosis regression outcome. MethodsAtherosclerotic lesion dynamics were studied upon bone marrow transplantation-mediated re-introduction of apolipoprotein E (Apoe) in Apoe knockout mice. Probucol was used to pharmacologically deplete HDL. ResultsRestoration of Apoe function was associated with an initial growth of atherosclerotic lesions and parallel decrease in lesional macrophage foam cell content (47 ± 4% at 4 weeks versus 72 ± 2% at baseline: p < 0.001), despite the fact that cholesterol levels were markedly reduced. Notably, significant lesion regression was detected from 4 weeks onwards, when plasma cholesterol levels had returned to the normolipidemic range. As a result, lesions were 41% smaller (p < 0.05) at 8 weeks than at 4 weeks after bone marrow transplantation. Regressed lesions contained an even lower level of macrophage foam cells (33 ± 5%: p < 0.001) and were rich in collagen. Probucol co-treatment was associated with a 3.2-fold lower (p < 0.05) plasma HDL-cholesterol level and a more pro-inflammatory (CCR2+) monocyte phenotype. Importantly, probucol-treated mice exhibited atherosclerotic lesions that were larger than those of regular chow diet-fed bone marrow transplanted mice at 8 weeks (186 ± 15*103 μm2 for probucol-treated versus 120 ± 19*103 μm2 for controls: p < 0.05). ConclusionsWe have shown that probucol-induced HDL deficiency impairs the ability of established lesions to regress in response to reversal of the genetic hypercholesterolemia in Apoe knockout mice. Our studies thus highlight a crucial role for HDL in the process of atherosclerosis regression.

Blockade of vascular endothelial growth factor receptor 2 inhibits intraplaque haemorrhage by normalization of plaque neovessels.

BackgroundPlaque angiogenesis is associated with atherosclerotic lesion growth, plaque instability and negative clinical outcome. Plaque angiogenesis is a natural occurring process to fulfil the increasing demand of oxygen and nourishment of the vessel wall. However, inadequate formed, immature plaque neovessels are leaky and cause intraplaque haemorrhage.ObjectiveBlockade of VEGFR2 normalizes the unbridled process of plaque neovessel formation and induces maturation of nascent vessels resulting in prevention of intraplaque haemorrhage and influx of inflammatory cells into the plaque and subsequently increases plaque stability.Methods and ResultsIn human carotid and vein graft atherosclerotic lesions, leaky plaque neovessels and intraplaque haemorrhage co‐localize with VEGF/VEGFR2 and angiopoietins. Using hypercholesterolaemic ApoE3*Leiden mice that received a donor caval vein interposition in the carotid artery, we demonstrate that atherosclerotic vein graft lesions at t28 are associated with hypoxia, Hif1α and Sdf1 up‐regulation. Local VEGF administration results in increased plaque angiogenesis. VEGFR2 blockade in this model results in a significant 44% decrease in intraplaque haemorrhage and 80% less extravasated erythrocytes compared to controls. VEGFR2 blockade in vivo results in a 32% of reduction in vein graft size and more stable lesions with significantly reduced macrophage content (30%), and increased collagen (54%) and smooth muscle cell content (123%). Significant decreased VEGF, angiopoietin‐2 and increased Connexin 40 expression levels demonstrate increased plaque neovessel maturation in the vein grafts. VEGFR2 blockade in an aortic ring assay showed increased pericyte coverage of the capillary sprouts.ConclusionInhibition of intraplaque haemorrhage by controlling neovessels maturation holds promise to improve plaque stability.

Abstract 186: Deletion of Ppard in Cd11c+ Cells Attenuates Atherosclerosis in Apoe Knockout Mice

Atherosclerosis is a chronic sterile inflammation of the vascular wall triggered by hyperlipidemia. The role of dendritic cells (DCs) in the development of atherosclerosis has not been recognised until the last decade. DCs can engulf lipids to adopt a foam cell-like appearance that may constitute the earliest stages of plaque formation. DCs may also recruit T cells to the inflamed vessel wall via secretion of chemokines and stimulate T cell responses via cytokines. PPARD is a nuclear receptor, which acts as a sensor of native and oxidized fatty acids. We examined whether PPARD regulates DC function in response to hyperlipidemia and affect atherosclerotic lesion growth. We used Ppard fl/fl mice and Itgax-cre (CD11c-Cre) mice crossed with Apoe -/- mice to generate Ppard f/f ;CD11c Cre/+ ;Apoe -/- as DC-specific knockout of PPARD on Apoe-KO background (Ppard DC-KO ) and Ppard f/f ;Apoe -/- (Ppard DC-WT ) as controls. Ppard DC-KO and Ppard DC-WT were fed with high cholesterol diet for 4 months. Tissues were dissected and digested to obtain single cell suspension for flow cytometric analysis. Bone marrow derived DCs were isolated and cultured in RPMI with serum and GM-CSF and maturated with LPS.Atherosclerotic lesion size and collagen deposition decreased in Ppard DC-KO mice. Less CD4 and CD8 T lymphocytes were found in the atherosclerotic lesion of Ppard DC-KO mice comparing to Ppard DC-WT mice. Ppard deletion in DCs reduced DC infiltration especially CD11b + CD103 - DCs in the atherosclerotic lesion. Production of IFNγ from CD4 + T cells decreased in Ppard DC-KO mice. In BMDCs from Ppard DC-KO mice, LPS and palmitic acid induced expression of co-stimulatory molecules CD80 and CD86, as well as TNF were decreased. LDL uptake was attenuated in BMDCs from PpardDC-KO mice. Our results suggested that PPARD may be involved in DC mediated T cell activation in response to hyperlipidemia. Deletion of PPARD in DCs attenuated plaque formation in atherosclerosis. Whether lipid sensing and uptake by PPARD is required for the enhanced inflammatory response in atherosclerotic mice requires further study. (This study is supported by Hong Kong Health Bureau HMRF 05162906 and 01150057, Hong Kong RGC T402/13-N and CRF C4024-16W, and CUHK Direct grants)

Clonal hematopoiesis of indeterminate potential (CHIP): A potential contributor to atherlosclerotic cardio/cerebro-vascular diseases?

At least 10% of the elderly population above the age of 70 carry a condition termed clonal hematopoiesis indeterminate potential (CHIP) due to oligoclonal expansion of mutated hematopoietic stem cells. Although CHIP is known to predispose patients to a higher risk of malignant blood disorders, the recent revelation of its association with higher morbidity and mortality of atherosclerotic cardiovascular disease and ischemic stroke is rather surprising. Two independent research groups published studies indicating that Tet2 mutated monocytes from mice modeling CHIP had a causal role in accelerating the growth of atherosclerotic lesions due to their pro-inflammation activities. This important discovery points to CHIP as a risk factor and raises the prospect of novel treatment to minimize the adverse cardio/cerebro-vascular events.

Apo A-I (Apolipoprotein A-I) Vascular Gene Therapy Provides Durable Protection Against Atherosclerosis in Hyperlipidemic Rabbits.

Gene therapy that expresses apo A-I (apolipoprotein A-I) from vascular wall cells has promise for preventing and reversing atherosclerosis. Previously, we reported that transduction of carotid artery endothelial cells with a helper-dependent adenoviral (HDAd) vector expressing apo A-I reduced early (4 weeks) fatty streak development in fat-fed rabbits. Here, we tested whether the same HDAd could provide long-term protection against development of more complex lesions. Fat-fed rabbits (n=25) underwent bilateral carotid artery gene transfer, with their left and right common carotids randomized to receive either a control vector (HDAdNull) or an apo A-I-expressing vector (HDAdApoAI). Twenty-four additional weeks of high-fat diet yielded complex intimal lesions containing lipid-rich macrophages as well as smooth muscle cells, often in a lesion cap. Twenty-four weeks after gene transfer, high levels of apo A-I mRNA (median ≥250-fold above background) were present in all HDAdApoAI-treated arteries. Compared with paired control HDAdNull-treated arteries in the same rabbit, HDAdApoAI-treated arteries had 30% less median intimal lesion volume (P=0.03), with concomitant reductions (23%-32%) in intimal lipid, macrophage, and smooth muscle cell content (P≤0.05 for all). HDAdApoAI-treated arteries also had decreased intimal inflammatory markers. VCAM-1 (vascular cell adhesion molecule-1)-stained area was reduced by 36% (P=0.03), with trends toward lower expression of ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein 1), and TNF-α (tumor necrosis factor-α; 13%-39% less; P=0.06-0.1). In rabbits with severe hyperlipidemia, transduction of vascular endothelial cells with an apo A-I-expressing HDAd yields at least 24 weeks of local apo A-I expression that durably reduces atherosclerotic lesion growth and intimal inflammation.

Abstract 28: Apolipoprotein A-I Vascular Gene Therapy Provides Durable Protection from Atherosclerosis in Hyperlipidemic Rabbits

Background: Gene therapy, delivered directly to the vascular wall, could potentially protect against atherosclerotic lesion growth or regress existing lesions. Previously, we demonstrated that infusion of HDAdApoAI [a helper-dependent adenoviral vector (HDAd) expressing apolipoprotein (apo) A-I] in carotid arteries of fat-fed rabbits reduced early carotid atherosclerosis development (4 wk after vector infusion). Here we tested whether the same HDAd could protect long-term against more severe atherosclerosis. Methods: 25 fat-fed rabbits underwent bilateral carotid gene transfer [HDAdApoAI on one side; control vector (HDAdNull) on the other, with sides randomized]. Postoperatively, diets were adjusted to maintain plasma cholesterols of 200-800mg/dl. This protocol yields large lipid- and macrophage-rich lesions. 6 mo later, carotids were harvested for analyses of DNA, RNA, protein, cholesterol, and histology (H&amp;E, oil red O), or immunostaining for macrophages, muscle actin, T-cells, ICAM-1 and VCAM-1. Results: Vector genomes persisted equally in HDAdApoAI and HDAdNull-infused arteries; however, apoA-I mRNA was detected only in HDAdApoAI-infused arteries. HDAdApoAI-infused arteries had ~60% less median intimal lesion volume than HDAdNull-infused arteries, with concomitant reductions (40-75%) in intimal lipid, macrophage, smooth muscle cell, VCAM-1 and ICAM-1. We used within-rabbit paired analyses to control for high correlations of lesion size and composition between left and right carotids of the same rabbit. 19 of 25 rabbits had smaller lesions in HDAdApoAI-infused vs HDAdNull-infused arteries; median decreased intimal area between paired carotids in the same rabbit was 30% ( P &lt;0.03). Within individual rabbits, intimal lipid, macrophage, smooth muscle cell, VCAM-1 and ICAM-1 were all less in HDAdApoAI arteries (median 23-36% less; P &lt;0.05 for all except ICAM-1). Conclusions: Vascular gene therapy with HDAdApoAI reduced lesion development and decreased intimal lipid, macrophage, and adhesion molecule expression 6 mo after treatment. Vector genomes and apo A-I mRNA remained high at 6 mo. A single application of vascular gene therapy durably retards development of atherosclerosis, even in a setting of severe hyperlipidemia.

Macrophages and Their Contribution to the Development of Atherosclerosis.

Atherosclerosis can be regarded as chronic inflammatory disease driven by lipid accumulation in the arterial wall. Macrophages play a key role in the development of local inflammatory response and atherosclerotic lesion growth. Atherosclerotic plaque is a complex microenvironment, in which different subsets of macrophages coexist executing distinct, although in some cases overlapping functions. According to the classical simplified nomenclature, lesion macrophages can belong to pro-inflammatory or anti-inflammatory or alternatively activated types. While the former promote the inflammatory response and participate in lipid accumulation, the latter are responsible for the inflammation resolution and plaque stabilisation. Atherosclerotic lesion dynamics depends therefore on the balance between these macrophages populations. The diverse functions of macrophages make them an attractive therapeutic target for the development of novel anti-atherosclerotic treatments. In this chapter, we discuss different types of macrophages and their roles in atherosclerotic lesion dynamics and describe the results of several experiments studying macrophage polarisation in atherosclerosis.

Local Vascular Gene Therapy With Apolipoprotein A-I to Promote Regression of Atherosclerosis.

Gene therapy, delivered directly to the blood vessel wall, could potentially prevent atherosclerotic lesion growth and promote atherosclerosis regression. Previously, we reported that a helper-dependent adenoviral (HDAd) vector expressing apolipoprotein A-I (apoA-I) in carotid endothelium of fat-fed rabbits reduced early (4 weeks) atherosclerotic lesion growth. Here, we tested whether the same HDAd-delivered to the existing carotid atherosclerotic lesions-could promote regression. Rabbits (n=26) were fed a high-fat diet for 7 months, then treated with bilateral carotid gene transfer. One carotid was infused with an HDAd expressing apoA-I (HDAdApoAI) and the other with a control nonexpressing HDAd (HDAdNull). The side with HDAdApoAI was randomized. Rabbits were then switched to regular chow, lowering their plasma cholesterols by over 70%. ApoA-I mRNA and protein were detected in HDAdApoAI-transduced arteries. After 7 weeks of gene therapy, compared with HDAdNull-treated arteries in the same rabbits, HDAdApoAI-treated arteries had significantly less vascular cell adhesion molecule-1 expression (28%; P=0.04) along with modest but statistically insignificant trends toward decreased intimal lesion volume, lipid and macrophage content, and intercellular adhesion molecule-1 expression (9%-21%; P=0.1-0.4). Post hoc subgroup analysis of rabbits with small-to-moderate-sized lesions (n=20) showed that HDAdApoAI caused large reductions in lesion volume, lipid content, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression (30%-50%; P≤0.04 for all). Macrophage content was reduced by 30% (P=0.06). There was a significant interaction (P=0.02) between lesion size and treatment efficacy. Even when administered on a background of aggressive lowering of plasma cholesterol, local HDAdApoAI vascular gene therapy may promote rapid regression of small-to-moderate-sized atherosclerotic lesions.

Abstract 14013: E-selectin-targeted Novel Nano Polymers Stabilize Atherosclerotic Plaque and Improve Cardiac Remodeling

Introduction: Inflammation is implicated in the initiation and progression of cardiovascular disease. Endothelial activation with up-regulation of E-selectin adhesion molecules stimulates leukocyte infiltration leading to atherosclerotic plaque growth, plaque vulnerability and acute myocardial infarction. Hypothesis: Novel E-selectin-targeted polymers, with or without dexamethasone, can prevent plaque progression and adverse cardiac remodeling in atherosclerosis prone ApoE deficient ApoE (-/-) mice. Methods and Results: To target and modulate cardiovascular inflammation we used novel N-(2-hydroxypropyl)-meth-acrylamide polymers conjugated with peptides that bind E-selectin with high affinity, with and without dexamethasone 1mg/kg (P-ESBP-Dex, P-ESBP). Five-month-old ApoE (-/-) mice were fed a high-fat diet (HFD) for 8 weeks. Plaque growth was assessed by vascular ultrasound (US) 4 and 8 weeks after onset of HFD. We used a novel software application designed to measure in-vivo vessel wall anatomy and motion in small-animal models. Following the baseline vascular US, mice were randomized to 4 treatment groups: P-ESBP-Dex, P-ESBP, free dexamethasone or saline, delivered by 4 weekly intraperitoneal injections. Left ventricular remodeling and function were assessed by echocardiography in parallel to the vascular US. We found that both P-ESBP and P-ESBP-Dex selectively targeted atherosclerotic lesions, and reduced wall thickness of the ascending aorta. Furthermore, compared with saline controls, P-ESBP significantly decreased the area of necrotic core in the aortic plaques (74.2±5.9 mm2 vs. 34.7±15.5 mm2, p&lt;0.05). Notably, the addition of dexamethasone to P-ESBP did not improve their therapeutic effect. Finally, P-ESBP attenuated left ventricular diastolic and systolic dilatation, compared with controls. Conclusion: E-selectin binding polymers reduce the growth of atherosclerotic lesions, contribute to plaque stability and prevent adverse cardiac remodeling in ApoE (-/-) mice. We suggest a novel nanomedicine-based therapy for atherosclerosis, unstable plaque, and to prevent adverse cardiac remodeling.

Abstract 13062: Pathogenic IgG Antibodies in Hyperlipidemic Mice Promote Atherosclerosis

Background: B2 cells promote atherosclerosis, but the exact mechanisms by which they promote atherosclerosis are not known. Antibodies can be pathogenic in a number of chronic inflammatory diseases. Here, we examined the role of antibodies in the development of atherosclerosis in hyperlipidemic mice. Methods and Results: As an interaction between B cells and CD4 T cells is important for plasma cell development and antibody production, we generated mixed chimeric LDLR -/- mice with B cells selectively deficient in MHC II or CD40 molecules. B cells deficient in either molecule decreased atherosclerosis by ~30-40%. This was associated with reduced spleen germinal centre B cells and plasma cells, markedly reduced plasma total immunoglobulins, reduced spleen CD4 T cell activation and decreased IFN-γ+ CD4 T cells. To confirm that the observed effects on atherosclerosis were largely due to impaired plasma cell generation and antibody production, we also deleted B cell-specific Blimp-1, a transcription factor critically important for B cell differentiation into antibody-producing plasma cells. Chimeric LDLR -/- mice transplanted with CD23 CRE -Blimp-1 fl/fl bone marrow were completely devoid of spleen plasma cells and plasma total immunoglobulins but CD4+ T cell activation was unaffected; atherosclerosis in these mice was reduced by nearly 50%. To exclude the possibility that the reduction in atherosclerosis was due to deletion of plasma cells, we then purified IgG from hyperlipidemic mice with atherosclerosis and transferred the IgG into immunoglobulin/plasma cell-deficient CD23 CRE -Blimp-1 fl/fl bone marrow transplanted LDLR -/- mice. Atherosclerotic lesions doubled in size in mice that received IgG from atherosclerotic mice, compared to transfer of IgG purified from non-atherosclerotic mice. Conclusions: Our study indicates that IgG antibodies generated in hyperlipidemic mice during atherosclerosis development accelerates growth of atherosclerotic lesions. Targeting pathogenic IgG antibodies in atherosclerosis has a potential to reduce lesion progression and the incidence of heart attacks and strokes.