Catherine Martel

A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation

Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show this effect is mediated through inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show they accumulate in atherosclerotic lesions where they directly affect plaque macrophages. Finally we demonstrate that a three-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a one-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.

Requirements for Membrane Attack Complex Formation and Anaphylatoxins Binding to Collagen-Activated Platelets

Background The activation of complement during platelet activation is incompletely understood. Objectives: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets. Methods C5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation. Results C5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg2+-EGTA and significantly reduced by the thrombin inhibitor hirudin (−37%, p<0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg2+-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (−71% and −44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface. Conclusions C5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis.

Pexelizumab fails to inhibit assembly of the terminal complement complex in patients with ST-elevation myocardial infarction undergoing primary percutaneous coronary intervention. Insight from a substudy of the Assessment of Pexelizumab in Acute Myocardial Infarction (APEX-AMI) trial.

BACKGROUNDnReasons for pexelizumab lack of benefit in ST-elevation myocardial infarction patients undergoing primary percutaneous coronary intervention remain unclear. In a substudy of the APEX-AMI trial, we explored the hypothesis that early complement activation preceding drug administration explained the failure.nnnMETHODSnA panel of terminal complement complex proteins and fragments and biomarkers of inflammation, apoptosis, and high-risk features were assessed in serum obtained before and 24 hours after administration of placebo or pexelizumab and primary percutaneous coronary intervention (n = 356) and in human umbilical vein endothelial cell cultures coincubated with serum (n = 45).nnnRESULTSnIn the placebo group, C5a and sC5b-9 levels increased by 37% (7.9-14.2 ηg/mL, P = .007) and 96% (442-845 ηg/mL, P < .0001), respectively, during the first 24 hours. Pexelizumab prevented the increase in C5a (P = .01 vs placebo), but not that of sC5b-9 (502-1,157 ηg/mL, not significant vs placebo). Levels of C-reactive protein, interleukin (IL) 6, IL-1ß, Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) or Chemokine C-C motif ligand 5 (CCL5), and N-terminal probrain natriuretic peptide increased significantly in both groups; those of IL-10, IL-12, IL-1ra, and Interferon gamma-induced protein 10 (IP-10) or C-X-C motif chemokine 10 (CXCL10) decreased. Pexelizumab halved the increase in IL-6 (+92% vs 156%, P = .01) without effects on other markers, including C-reactive protein and N-terminal probrain natriuretic peptide. In cell culture, pexelizumab inhibited C5a, sC5b-9, and membrane-bound C5b-9 by 92%, 75%, and 78%, respectively (all P < .0001), without influencing cytokine levels and cell apoptosis.nnnCONCLUSIONSnThe blockage of both C5a and terminal complement in cell culture, but of C5a only in vivo with minimal effects on inflammation and risk biomarkers, supports the hypothesis that late administration of pexelizumab after the ischemia/reperfusion insult precluded adequate myocardial protection, resulting in a negative trial.

Effect of Pericardial Blood Processing on Postoperative Inflammation and the Complement Pathways

BACKGROUNDnThe objective of the present study was to determine the effect of processing of pericardial blood with a cell-saving device (CS) and vacuum-assisted cardiopulmonary bypass (VACPB) on reduction of postoperative inflammation.nnnMETHODSnOne hundred patients who underwent on-pump coronary artery bypass grafting surgery were included in a prospective randomized study. Patients were randomly assigned into four groups of 25 patients, each in a two-by-two factorial design: group A had no CS and no VACPB, group B had VACPB alone, group C had CS alone, and group D had CS and VACPB. The complement factors C4a, C3a, and C5a, and the terminal complex sC5b-9, MBL (mannose-binding lectin), and Bb were measured in plasma preoperatively and at 30 and 240 minutes after termination of CPB.nnnRESULTSnMean age, CPB, and aortic cross-clamping times were similar in all groups. At 30 and 240 minutes after CPB, C3a, sC5b-9, and Bb were increased and C5a and MBL levels were decreased compared with preoperative levels in all groups. At 240 minutes, Bb levels were lower in patients with CS (p = 0.0002).nnnCONCLUSIONSnThe present study shows that contemporary CPB remains associated with a striking activation of all complement pathways and its terminal component. The use of CS decreases the activation of the complement alternative pathway.

Complement activity and pharmacological inhibition in cardiovascular disease

While complement is the most important component of humoral autoimmunity, and inflammation plays a key role in atherosclerosis, relatively few studies have looked at complement implications in atherosclerosis and its complications. C-reactive protein is a marker of inflammation and is also involved in atherosclerosis; it activates complement and colocalizes with activated complement proteins within the infarcting myocardium and the active atherosclerotic plaques. As new agents capable of modulating complement activity are being developed, new targets for the management of atherosclerosis are emerging that are related to autoimmunity and inflammation. The present paper reviews the putative roles of the various complement activation pathways in the development of atherosclerosis, in ST segment elevation and non-ST segment elevation acute coronary syndromes, and in coronary artery bypass graft surgery. It also provides a perspective on new therapeutic interventions being developed to modulate complement activity. These interventions include the C1 esterase inhibitor, which may be consumed in some inflammatory states resulting in the loss of one of the mechanisms inhibiting activation of the classical and lectin pathways; TP10, a recombinant protein of the soluble complement receptor type 1 (sCR1) which inhibits the C3 and C5 convertases of the common pathway by binding C3b and C4b; a truncated version of the soluble complement receptor type 1 CRI lacking the C4b binding site which selectively inhibits the alternative pathway; and pexelizumab, a monoclonal antibody selectively blocking C5 to prevent the activation of the terminal pathway that is involved in excessive inflammation and autoimmune responses.

Angiopoietin-1 but not angiopoietin-2 promotes neutrophil viability: Role of interleukin-8 and platelet-activating factor.

We previously reported the expression of angiopoietin receptor Tie2 on human neutrophils. Both angiopoietins (Ang1 and Ang2) induce platelet activating factor (PAF) synthesis from endothelial cells (ECs) and neutrophils. Both angiopoietins can also modulate EC viability and since PAF can promote pro-survival activity on neutrophils, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Neutrophils were isolated from venous blood of healthy volunteers and neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers (annexin-V and propidium iodide (P.I.), respectively). Basal neutrophil viability from 0 to 24 h post-isolation decreased from 98% to ≈45%. Treatment with anti-apoptotic mediators such as interleukin-8 (IL-8; 25 nM) and PAF (100 nM) increased neutrophil basal viability by 34 and 26% (raising it from 43 to 58 and 55%) respectively. Treatment with Ang1 (0.001-50 nM) increased neutrophil viability by up to 41%, while Ang2 had no significant effect. Combination of IL-8 (25 nM) or PAF (100 nM) with Ang1 (10 nM) further increased neutrophil viability by 56 and 60% respectively. We also observed that Ang1, but not Ang2 can promote IL-8 release and that a pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 81% respectively. Pretreatment with a selective PAF receptor antagonist (BN 52021), did abrogate PAF pro-survival activity, without affecting Ang1-induced neutrophil viability. Our data are the first ones to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.

Cholesterol and Lipoprotein Metabolism Early Career Committee Contribution

Great strides have been made toward understanding the mechanisms that maintain and disrupt cholesterol homeostasis. Over a century’s worth of research has led to the development of amazing drugs such as statins that potently reduce hypercholesterolemia and consequently decrease atherosclerosis development and CVD risk. In spite of the significant advances, it is clear from the research highlighted in this article that there is still much to learn about the impact of cholesterol on CVD. We believe that current and future research on cholesterol homeostasis will steer us towards new targets and treatments that will further diminish CVD morbidity and mortality in the United States and around the world.

Cholesterol and Lipoprotein Metabolism

Great strides have been made toward understanding the mechanisms that maintain and disrupt cholesterol homeostasis. Over a century’s worth of research has led to the development of amazing drugs such as statins that potently reduce hypercholesterolemia and consequently decrease atherosclerosis development and CVD risk. In spite of the significant advances, it is clear from the research highlighted in this article that there is still much to learn about the impact of cholesterol on CVD. We believe that current and future research on cholesterol homeostasis will steer us towards new targets and treatments that will further diminish CVD morbidity and mortality in the United States and around the world.

Thrombospondin-1-derived peptide RFYVVMWK improves the adhesive phenotype of CD34sup+/sup cells from atherosclerotic patients with type 2 diabetes.

CD34+ progenitor cells are growing in use for vascular repair. However, in diabetic individuals with cardiovascular diseases, these cells have dysfunctional engraftment capabilities, which compromise their use for autologous cell therapy. The thrombospondin-1-derived peptide RFYVVMWK has previously been reported to stimulate cell adhesiveness through CD47 and integrin activation pathways. Our aim was to test whether RFYVVMWK preconditioning could modulate CD34+ cell phenotype and enhance its proadhesive properties in diabetic patients. Peripheral blood mononuclear CD34+ cells isolated from 40 atherosclerotic patients with type 2 diabetes (T2D; n = 20) or without (non-T2D; n = 20) were preconditioned with 30 μM RFYVVMWK or truncated peptide RFYVVM. CD34+ cell adhesion was assessed on a vitronectin–collagen matrix and on TNF-α or IL-1β-stimulated HUVEC monolayers. Adhesion receptors, platelet/CD34+ cell conjugates, and cell viability were analyzed by flow cytometry and confocal microscopy. RFYVVMWK increased the adhesion of T2D CD34+ cells by eightfold to the vitronectin–collagen matrix (p < 0.001) corresponding to a threefold increase compared to unstimulated non-T2D CD34+ cells. The peptide induced the formation of platelet/CD34+ conjugates and increased the expression of TSP-1, CD29, CD51/CD61, and CD62P in both T2D and non-T2D cells. However, RFYVVMWK treatment did not affect the viability/apoptosis of CD34+ progenitor cells. In conclusion, priming CD34+ cells with RFYVVMWK may enhance their vascular engraftment during autologous proangiogenic cell therapy.

Translational and Therapeutic Approaches to the Understanding and Treatment of Dyslipidemia

Circulating lipoproteins containing cholesterol and triglycerides are major risk factors for many cardiovascular diseases (CVD). Many therapeutic strategies targeting lipid metabolism have been successfully used to treat dyslipidemia and subsequently reduce cardiovascular risk. Most notably, statins, which effectively lower low-density lipoprotein-cholesterol (LDL-C), have been widely used and have been shown to substantially reduce cardiovascular events. More recently, a new class of LDL-lowering drugs, the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, has been developed with highly promising results. Many other therapeutic approaches aiming to modulate lipids are also being considered. These include antisense oligonucleotides (ASOs) designed to inhibit well-characterized gene targets involved in dyslipidemia, modulators of high-density lipoprotein (HDL) function and reverse cholesterol transport (RCT) pathways, and triglyceride-lowering drugs. We herein highlight recent basic, translational, and clinical studies published in ATVB that further elucidates the mechanisms and therapeutic potential of these approaches.nnLDL-receptor (LDLR) is a cell surface receptor that was found to be mutated in patients with familial hypercholesterolemia (FH).1 FH is a genetic disorder typically characterized by hypercholesterolemia, ectopic cholesterol deposition, and premature CVD. Hepatic LDLR is essential for the uptake of circulating LDL-C, which cannot be effectively cleared in FH patients. Conversely, increasing LDLR levels, particularly in the liver, enhances LDL-C clearance. Recent evidence identified PCSK9, a protein secreted by the liver, as a mediator of LDLR degradation by interacting with its extracellular domain and promoting its trafficking to the lysosome, rather than allowing normal recycling to the cell surface.2,3 PCSK9 gain-of-function mutations in humans were associated with decreased LDLR levels, and thus increased circulating LDL-C, causing autosomal dominant hypercholesterolemia.4,5 In contrast, loss-of-function mutations in PCSK9 were associated with lower LDL-C levels and reduced CVD events.6 Therefore, PCSK9 became a …