Single Functional Group Platform for Multistimuli Responsivities: Tertiary Amine for CO2/pH/ROS-Triggered Cargo Release in Nanocarriers.

The design of multistimuli-responsive soft nanoparticles (NPs) often presents synthetic complexities and limited breadth in exploiting changes surrounding physiological environments. Nanocarriers that could collectively take advantage of several endogenous stimuli can offer a powerful tool in nanomedicine. Herein, we have capitalized on the chemical versatility of a single tertiary amine to construct miktoarm polymer-based nanocarriers that respond to dissolved CO2, varied pH, reactive oxygen species (ROS), and ROS + CO2. Curcumin (Cur), an anti-inflammatory phytopharmaceutic, was loaded into micelles, and we validated the sensitivity of the tertiary amine in tuning Cur release. An in vitro evaluation indicated that Cur encapsulation strongly suppressed its toxicity at high concentrations, significantly inhibited nigericin-induced secretion of interleukin-1ß by THP-1 macrophages, and the proportion of M2/M1 (anti-inflammatory/pro-inflammatory macrophages) was higher for Cur-loaded NPs than for free Cur. Our approach highlights the potential of a simple-by-design strategy in expanding the scope of polymeric NPs in drug delivery.

Adenylate cyclase type 9 antagonizes cAMP accumulation and regulates endothelial signalling involved in atheroprotection.

AIMS: The adenylate cyclase type 9 (ADCY9) gene appears to determine atherosclerotic outcomes in patients treated with dalcetrapib. In mice, we recently demonstrated that Adcy9 inactivation potentiates endothelial function and inhibits atherogenesis. The objective of this study was to characterize the contribution of ADCY9 to the regulation of endothelial signalling pathways involved in atherosclerosis. METHODS AND RESULTS: We show that ADCY9 is expressed in the endothelium of mouse aorta and femoral arteries. We demonstrate that ADCY9 inactivation in cultured endothelial cells paradoxically increases cAMP accumulation in response to the adenylate cyclase activators forskolin and vasoactive intestinal peptide (VIP). Reciprocally, ADCY9 overexpression decreases cAMP production. Using mouse femoral artery arteriography, we show that Adcy9 inactivation potentiates VIP-induced endothelial-dependent vasodilation. Moreover, Adcy9 inactivation reduces mouse atheroma endothelial permeability in different vascular beds. ADCY9 overexpression reduces forskolin-induced phosphorylation of Ser157-vasodilator-stimulated phosphoprotein (VASP) and worsens thrombin-induced fall of RAP1 activity, both leading to increased endothelial permeability. ADCY9 inactivation in thrombin-stimulated human coronary artery endothelial cells results in cAMP accumulation, increases p-Ser157-VASP, and inhibits endothelial permeability. MLC2 phosphorylation and actin stress fibre increases in response to thrombin were reduced by ADCY9 inactivation, suggesting actin cytoskeleton regulation. Finally, using the Miles assay, we demonstrate that Adcy9 regulates thrombin-induced endothelial permeability in vivo in normal and atherosclerotic animals. CONCLUSION: Adcy9 is expressed in endothelial cells and regulates local cAMP and endothelial functions including permeability relevant to atherogenesis.

Inhibition of the 3CL Protease and SARS-CoV-2 Replication by Dalcetrapib.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) 3CL protease is a promising target for inhibition of viral replication by interaction with a cysteine residue (Cys145) at its catalytic site. Dalcetrapib exerts its lipid-modulating effect by binding covalently to cysteine 13 of a cholesteryl ester transfer protein. Because 12 free cysteine residues are present in the 3CL protease, we investigated the potential of dalcetrapib to inhibit 3CL protease activity and SARS-CoV-2 replication. Molecular docking investigations suggested that dalcetrapib-thiol binds to the catalytic site of the 3CL protease with a delta G value of -8.5 kcal/mol. Dalcetrapib inhibited both 3CL protease activity in vitro and viral replication in Vero E6 cells with IC50 values of 14.4 ± 3.3 µM and an EC50 of 17.5 ± 3.5 µM (mean ± SD). Near-complete inhibition of protease activity persisted despite 1000-fold dilution after ultrafiltration with a nominal dalcetrapib-thiol concentration of approximately 100 times below the IC50 of 14.4 µM, suggesting stable protease-drug interaction. The inhibitory effect of dalcetrapib on the SARS-CoV-2 3CL protease and viral replication warrants its clinical evaluation for the treatment of COVID-19.

Variants at the APOE /C1/C2/C4 Locus Modulate Cholesterol Efflux Capacity Independently of High-Density Lipoprotein Cholesterol.

Background Macrophage cholesterol efflux to high-density lipoproteins ( HDLs ) is the first step of reverse cholesterol transport. The cholesterol efflux capacity ( CEC ) of HDL particles is a protective risk factor for coronary artery disease independent of HDL cholesterol levels. Using a genome-wide association study approach, we aimed to identify pathways that regulate CEC in humans. Methods and Results We measured CEC in 5293 French Canadians. We tested the genetic association between 4 CEC measures and genotypes at >9 million common autosomal DNA sequence variants. These analyses yielded 10 genome-wide significant signals ( P<6.25×10-9) representing 7 loci. Five of these loci harbor genes with important roles in lipid biology ( CETP , LIPC , LPL , APOA 1/C3/A4/A5, and APOE /C1/C2/C4). Except for the APOE /C1/C2/C4 variant ( rs141622900, P nonadjusted=1.0×10-11; P adjusted=8.8×10-9), the association signals disappear when correcting for HDL cholesterol and triglyceride levels. The additional 2 significant signals were near the PPP 1 CB / PLB 1 and RBFOX 3/ ENPP 7 genes. In secondary analyses, we considered candidate functional variants for 58 genes implicated in HDL biology, as well as 239 variants associated with blood lipid levels and/or coronary artery disease risk by genome-wide association study . These analyses identified 27 significant CEC associations, implicating 5 additional loci ( GCKR , LIPG , PLTP , PPARA , and TRIB 1). Conclusions Our genome-wide association study identified common genetic variation at the APOE /C1/C2/C4 locus as a major determinant of CEC that acts largely independently of HDL cholesterol. We predict that HDL -based therapies aiming at increasing CEC will be modulated by changes in the expression of apolipoproteins in this gene cluster.

ADCY9 (Adenylate Cyclase Type 9) Inactivation Protects From Atherosclerosis Only in the Absence of CETP (Cholesteryl Ester Transfer Protein).

BACKGROUND: Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined. METHODS: Adcy9-inactivated ( Adcy9Gt/Gt) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtg Adcy9Gt/Gt and CETPtg Adcy9WT), were submitted to an atherogenic protocol (injection of an AAV8 [adeno-associated virus serotype 8] expressing a PCSK9 [proprotein convertase subtilisin/kexin type 9] gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated. RESULTS: Adcy9Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9Gt/Gt mice compared to WT animals ( P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9Gt/Gt mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9Gt/Gt ( P<0.01 for all). Aortic endothelium from Adcy9Gt/Gt mice allowed significantly less adhesion of splenocytes compared to WT ( P<0.05). Adcy9Gt/Gt mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume ( P<0.01 for both). Feed efficiency was increased in Adcy9Gt/Gt compared to WT mice ( P<0.01), which was accompanied by prolonged cardiac RR interval ( P<0.05) and improved nocturnal heart rate variability ( P=0.0572). Adcy9 inactivation-induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtg Adcy9Gt/Gt mice ( P>0.05 versus CETPtg Adcy9WT). CONCLUSIONS: Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.

Expression of the Frizzled receptors and their co-receptors in calcified human aortic valves.

The cellular mechanisms that induce calcific aortic stenosis are yet to be unraveled. Wnt signaling is increasingly being considered as a major player in the disease process. However, the presence of Wnt Frizzled (Fzd) receptors and co-receptors LRP5 and 6 in normal and diseased human aortic valves remains to be elucidated. Immunohistochemistry and quantitative polymerase chain reaction were used to determine Fzd receptor expression in normal and calcified human aortic valve tissue, as well as human aortic valve interstitial cells (HAVICs) isolated from calcified and normal human aortic valves. There was significantly higher mRNA expression of 4 out of the 10 Fzd receptors in calcified aortic valve tissues and 8 out of the 10 in HAVICs, and both LRP5/6 co-receptors in calcified aortic valves (P < 0.05). These results were confirmed by immunohistochemistry, which revealed abundant increase in immunoreactivity for Fzd3, 7, and 8, mainly in areas of lipid core and calcified nodules of diseased aortic valves. The findings of abundant expression of Fzd and LRP5/6 receptors in diseased aortic valves suggests a potential role for both canonical and noncanonical Wnt signaling in the pathogenesis of human aortic valve calcification. Future investigations aimed at targeting these molecules may provide potential therapies for aortic valve stenosis.

Dalcetrapib and anacetrapib differently impact HDL structure and function in rabbits and monkeys.

Inhibition of cholesteryl ester transfer protein (CETP) increases HDL cholesterol (HDL-C) levels. However, the circulating CETP level varies and the impact of its inhibition in species with high CETP levels on HDL structure and function remains poorly characterized. This study investigated the effects of dalcetrapib and anacetrapib, the two CETP inhibitors (CETPis) currently being tested in large clinical outcome trials, on HDL particle subclass distribution and cholesterol efflux capacity of serum in rabbits and monkeys. New Zealand White rabbits and vervet monkeys received dalcetrapib and anacetrapib. In rabbits, CETPis increased HDL-C, raised small and large α-migrating HDL, and increased ABCA1-induced cholesterol efflux. In vervet monkeys, although anacetrapib produced similar results, dalcetrapib caused opposite effects because the LDL-C level was increased by 42% and HDL-C decreased by 48% (P < 0.01). The levels of α- and preß-HDL were reduced by 16% (P < 0.001) and 69% (P < 0.01), resulting in a decrease of the serum cholesterol efflux capacity. CETPis modulate the plasma levels of mature and small HDL in vivo and consequently the cholesterol efflux capacity. The opposite effects of dalcetrapib in different species indicate that its impact on HDL metabolism could vary greatly according to the metabolic environment.

Development of a new bioactivatable fluorescent probe for quantification of apolipoprotein A-I proteolytic degradation in vitro and in vivo.

BACKGROUND AND AIMS: The potential benefits of high-density lipoproteins (HDL) against atherosclerosis are attributed to its major protein component, apolipoprotein A-I (apoA-I). Most of the apoA-I in the vascular wall appears to be in its lipid-poor form. The latter, however, is subjected to degradation by proteases localized in atherosclerotic plaques, which, in turn, has been shown to negatively impact its atheroprotective functions. Here, we report the development and in vivo use of a bioactivatable near-infrared full-length apoA-I-Cy5.5 fluorescent probe for the assessment of apoA-I-degrading proteolytic activities. METHODS: Fluorescence quenching was obtained by saturation of Cy5.5 fluorophore molecules on apoA-I protein. ApoA-I cleavage led to near-infrared fluorescence enhancement. In vitro proteolysis of the apoA-I probe by a variety of proteases including serine, cysteine, and metalloproteases resulted in an up to 11-fold increase in fluorescence (n = 5, p ≤ 0.05). RESULTS: We detected activation of the probe in atherosclerotic mice aorta sections using in situ zymography and showed that broad-spectrum protease inhibitors protected the probe from degradation, resulting in decreased fluorescence (-54%, n = 6 per group, p ≤ 0.0001). In vivo, the injected probe showed stronger fluorescence emission in the aorta of human apoB transgenic Ldlr-/- atherosclerotic mice (ATX) as compared to wild-type mice. In vivo observations were confirmed by ex vivo aorta imaging quantification where a 10-fold increase in fluorescent signal in ATX mice (p ≤ 0.05 vs. control mice) was observed. CONCLUSIONS: The use of this probe in different applications may help to assess new molecular mechanisms of atherosclerosis and may improve current HDL-based therapies by enhancing apoA-I functionality.

Thrombospondin-1-Derived Peptide RFYVVMWK Improves the Adhesive Phenotype of CD34+ 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-1ß-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.

Role of Noncanonical Wnt Signaling Pathway in Human Aortic Valve Calcification.

OBJECTIVE: The mechanisms underlying the pathogenesis of aortic valve calcification remain unclear. With accumulating evidence demonstrating that valve calcification recapitulates bone development, the crucial roles of noncanonical Wnt ligands WNT5a, WNT5b, and WNT11 in osteogenesis make them critical targets in the study of aortic valve calcification. APPROACH AND RESULTS: Using immunohistochemistry, real-time qPCR, Western blotting, and tissue culture, we examined the tissue distribution of WNT5a, WNT5b, and WNT11 in noncalcified and calcified aortic valves and their effects on human aortic valve interstitial cells (HAVICs). Only focal strong immunostaining for WNT5a was seen in and around areas of calcification. Abundant immunostaining for WNT5b and WNT11 was seen in inflammatory cells, fibrosis, and activated myofibroblasts in areas of calcified foci. There was significant correlation between WNT5b and WNT11 overall staining and presence of calcification, lipid score, fibrosis, and microvessels (P<0.05). Real-time qPCR and Western blotting revealed abundant expression of both Wnts in stenotic aortic valves, particularly in bicuspid valves. Incubation of HAVICs from noncalcified valves with the 3 noncanonical Wnts significantly increased cell apoptosis and calcification (P<0.05). Treatment of HAVICs with the mitogen-activated protein kinase-38ß and GSK3ß inhibitors significantly reduced their mineralization (P<0.01). Raman spectroscopy identified the inorganic phosphate deposits as hydroxyapatite and showed a significant increase in hydroxyapatite deposition in HAVICs in response to WNT5a and WNT11 (P<0.05). Similar crystallinity was seen in the deposits found in HAVICs treated with Wnts and in calcified human aortic valves. CONCLUSIONS: These findings suggest a potential role for noncanonical Wnt signaling in the pathogenesis of aortic valve calcification.

Genotype-Dependent Effects of Dalcetrapib on Cholesterol Efflux and Inflammation: Concordance With Clinical Outcomes.

BACKGROUND: Dalcetrapib effects on cardiovascular outcomes are determined by adenylate cyclase 9 gene polymorphisms. Our aim was to determine whether these clinical end point results are also associated with changes in reverse cholesterol transport and inflammation. METHODS AND RESULTS: Participants of the dal-OUTCOMES and dal-PLAQUE-2 trials were randomly assigned to receive dalcetrapib or placebo in addition to standard care. High-sensitivity C-reactive protein was measured at baseline and at end of study in 5243 patients from dal-OUTCOMES also genotyped for the rs1967309 polymorphism in adenylate cyclase 9. Cholesterol efflux capacity of high-density lipoproteins from J774 macrophages after cAMP stimulation was determined at baseline and 12 months in 171 genotyped patients from dal-PLAQUE-2. Treatment with dalcetrapib resulted in placebo-adjusted geometric mean percent increases in high-sensitivity C-reactive protein from baseline to end of trial of 18.1% (P=0.0009) and 18.7% (P=0.00001) in participants with the GG and AG genotypes, respectively, but the change was -1.0% (P=0.89) in those with the protective AA genotype. There was an interaction between the treatment arm and the genotype groups (P=0.02). Although the mean change in cholesterol efflux was similar among study arms in patients with GG genotype (mean: 7.8% and 7.4%), increases were 22.3% and 3.5% with dalcetrapib and placebo for those with AA genotype (P=0.005). There was a significant genetic effect for change in efflux for dalcetrapib (P=0.02), but not with placebo. CONCLUSIONS: Genotype-dependent effects on C-reactive protein and cholesterol efflux are supportive of dalcetrapib benefits on atherosclerotic cardiovascular outcomes in patients with the AA genotype at polymorphism rs1967309. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov; Unique Identifiers: NCT00658515 and NCT01059682.

HDL mimetic peptide CER-522 treatment regresses left ventricular diastolic dysfunction in cholesterol-fed rabbits.

OBJECTIVES: High-density lipoprotein (HDL) infusions induce rapid improvement of experimental atherosclerosis in rabbits but their effect on ventricular function remains unknown. We aimed to evaluate the effects of the HDL mimetic peptide CER-522 on left ventricular diastolic dysfunction (LVDD). METHODS: Rabbits were fed with a cholesterol- and vitamin D2-enriched diet until mild aortic valve stenosis and hypercholesterolemia-induced LV hypertrophy and LVDD developed. Animals then received saline or 10 or 30mg/kg CER-522 infusions 6 times over 2weeks. We performed serial echocardiograms and LV histology to evaluate the effects of CER-522 therapy on LVDD. RESULTS: LVDD was reduced by CER-522 as shown by multiple parameters including early filling mitral deceleration time, deceleration rate, Em/Am ratio, E/Em ratio, pulmonary venous velocities, and LVDD score. These findings were associated with reduced macrophages (RAM-11 positive cells) in the pericoronary area and LV, and decreased levels of apoptotic cardiomyocytes in CER-522-treated rabbits. CER-522 treatment also resulted in decreased atheromatous plaques and internal elastic lamina area in coronary arteries. CONCLUSIONS: CER-522 improves LVDD in rabbits, with reductions of LV macrophage accumulation, cardiomyocyte apoptosis, coronary atherosclerosis and remodelling.

Fabricating Water Dispersible Superparamagnetic Iron Oxide Nanoparticles for Biomedical Applications through Ligand Exchange and Direct Conjugation.

Stable superparamagnetic iron oxide nanoparticles (SPIONs), which can be easily dispersed in an aqueous medium and exhibit high magnetic relaxivities, are ideal candidates for biomedical applications including contrast agents for magnetic resonance imaging. We describe a versatile methodology to render water dispersibility to SPIONs using tetraethylene glycol (TEG)-based phosphonate ligands, which are easily introduced onto SPIONs by either a ligand exchange process of surface-anchored oleic-acid (OA) molecules or via direct conjugation. Both protocols confer good colloidal stability to SPIONs at different NaCl concentrations. A detailed characterization of functionalized SPIONs suggests that the ligand exchange method leads to nanoparticles with better magnetic properties but higher toxicity and cell death, than the direct conjugation methodology.

Magnetic resonance imaging/fluorescence dual modality protocol using designed phosphonate ligands coupled to superparamagnetic iron oxide nanoparticles.

A simple and versatile methodology to tailor the surface of superparamagnetic iron oxide nanoparticles (SPIONs), and render additional fluorescence capability to these contrast agents, is reported. The dual modality imaging protocol was developed by designing multi-functional scaffolds with a combination of orthogonal moieties for aqueous dispersion and stealth, to covalently link them to SPIONs, and carry out post-functionalization of nanoparticles. SPIONs stabilized with ligands incorporating surface-anchoring phosphonate groups, ethylene glycol backbone for aqueous dispersion, and free surface exposed OH moieties were coupled to near-infrared dye Cy5.5A. Our results demonstrate that design of multi-tasking ligands with desired combination and spatial distribution of functions provides an ideal platform to construct highly efficient dual imaging probes with balanced magnetic, optical and cell viability properties.

Lipoprotein(a) levels, genotype, and incident aortic valve stenosis: a prospective Mendelian randomization study and replication in a case-control cohort.

BACKGROUND: Although a previous study has suggested that a genetic variant in the LPA region was associated with the presence of aortic valve stenosis (AVS), no prospective study has suggested a role for lipoprotein(a) levels in the pathophysiology of AVS. Our objective was to determine whether lipoprotein(a) levels and a common genetic variant that is strongly associated with lipoprotein(a) levels are associated with an increased risk of developing AVS. METHODS AND RESULTS: Serum lipoprotein(a) levels were measured in 17 553 participants of the European Prospective Investigation into Cancer (EPIC)-Norfolk study. Among these study participants, 118 developed AVS during a mean follow-up of 11.7 years. The rs10455872 genetic variant in LPA was genotyped in 14 735 study participants, who simultaneously had lipoprotein(a) level measurements, and in a replication study of 379 patients with echocardiography-confirmed AVS and 404 controls. In EPIC-Norfolk, compared with participants in the bottom lipoprotein(a) tertile, those in the top lipoprotein(a) tertile had a higher risk of AVS (hazard ratio, 1.57; 95% confidence interval, 1.02-2.42) after adjusting for age, sex, and smoking. Compared with rs10455872 AA homozygotes, carriers of 1 or 2 G alleles were at increased risk of AVS (hazard ratio, 1.78; 95% confidence interval, 1.11-2.87, versus hazard ratio, 4.83; 95% confidence interval, 1.77-13.20, respectively). In the replication study, the genetic variant rs10455872 also showed a positive association with AVS (odds ratio, 1.57; 95% confidence interval, 1.10-2.26). CONCLUSIONS: Patients with high lipoprotein(a) levels are at increased risk for AVS. The rs10455872 variant, which is associated with higher lipoprotein(a) levels, is also associated with increased risk of AVS, suggesting that this association may be causal.

Evaluation of links between high-density lipoprotein genetics, functionality, and aortic valve stenosis risk in humans.

OBJECTIVE: Studies have shown that high-density lipoprotein (HDL)-raising compounds induce regression of aortic valve stenosis (AVS) in animal models. However, whether patients with AVS have an impaired HDL metabolism is unknown. APPROACH AND RESULTS: A total of 1435 single nucleotide polymorphisms in genes associated with HDL cholesterol levels (in or around GALNT2, LPL, ABCA1, APOA5, SCARB1, LIPC, CETP, LCAT, LIPG, APOC4, and PLTP) were genotyped in 382 patients with echocardiography-confirmed AVS (aortic jet velocity ≥2.5 m/s) and 401 controls. After control for multiple testing, none of the genetic variants showed a positive association with case/control status (adjusted P≥0.05 for all single nucleotide polymorphisms tested). In a subsample of this cohort, HDL cholesterol levels, apolipoprotein AI levels, lecithin-cholesterol acyltransferase activity, pre-ß-HDL, HDL size, and 4 parameters of cholesterol efflux capacity were measured in apolipoprotein B-depleted serum samples from 86 patients with and 86 patients without AVS. Cholesterol efflux capacity was measured using J774 macrophages with and without stimulation of ATP-binding cassette A-1 expression by cAMP, and HepG2 hepatocytes for scavenger receptor class B type 1-mediated efflux. None of these parameters were different between cases and controls. However, compared with patients without coronary artery disease, sera from patients with coronary artery disease had lower HDL cholesterol levels, scavenger receptor class B type 1-mediated efflux, and HDL size (P≤0.003), independently of the presence or absence of AVS. CONCLUSIONS: Results of the present study suggest that, based on HDL genetics and HDL functionality, HDL metabolism does not seem to predict the risk of AVS. Because of our limited sample size, additional studies are needed to confirm these findings.

What is the role of erythropoietin in acute myocardial infarct? Bridging the gap between experimental models and clinical trials.

Erythropoietin (EPO) is the main hormone that regulates erythropoiesis. Beyond its well-known hematopoietic action, EPO has diverse cellular effects in non-hematopoietic tissues. It has been shown to inhibit apoptosis by activating pro-survival pathways in the myocardium, to mobilize endothelial progenitor cells and to inhibit migration of inflammatory cells. EPO has also been shown to have potent pro-angiogenic properties. Numerous experimental data support the cardioprotective effects of EPO in animal models of acute myocardial infarct (AMI). However, these findings are not supported by recent clinical trials designed to investigate the safety and efficacy of EPO in patients with AMI. In this article, we begin by providing a comprehensive review of the cardioprotective effects of EPO in experimental animal models and the molecular mechanisms underlying these effects. We then discuss the EPO data obtained through clinical trials. We compare similarities and differences between the animal and human studies as well as between the different clinical studies in terms of sample size and study design including the dose, the route and the timing of administration as well as confounding factors such as comorbidities and concomitant treatments. Finally, we question the gap between the experimental and the translational clinical data and propose further developments to address these discrepancies and clearly evaluate the role of EPO in the clinical setting of MI.

Pharmacological control of platelet-leukocyte interactions by the human anti-P-selectin antibody inclacumab--preclinical and clinical studies.

BACKGROUND AND OBJECTIVE: Elevated levels of platelet-leukocyte aggregates (PLAs) have been reported in several cardiovascular diseases and suggested to contribute to disease pathology. Our aim was to characterize the effects of inclacumab, a novel human anti-P-selectin antibody, on the interactions between leukocytes and platelets in preclinical and clinical studies. EXPERIMENTAL APPROACHES: Dual-label flow cytometry was used to detect the effect of inclacumab on agonist-induced platelet-leukocyte/platelet-monocyte aggregates in cynomolgus monkeys and humans, following ex vivo and in vivo administration. Platelet-dependent leukocyte activation and leukocyte adhesion to a platelet monolayer were also investigated after ex vivo administration of inclacumab to human blood. RESULTS: Treatment of cynomolgus monkeys with inclacumab profoundly inhibited thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP)-induced PLAs with an IC50 (<2 µg/mL) similar to the in vitro spiking experiments. Maximal inhibition of PLAs persisted for ≥28 days following single dose of inclacumab. In human blood, inclacumab was about 2-fold more potent in inhibiting TRAP-induced PLAs (IC50: 0.7 µg/mL) compared to monkeys. PLA formation was suppressed independently of the inducing platelet agonist. Inclacumab also inhibited the activation of the leukocyte integrin Mac-1 and leukocyte adhesion to a platelet monolayer under flow conditions. In clinical studies, inclacumab inhibited TRAP-induced PLA formation in a dose-dependent manner following single and multiple dose administration to healthy volunteers. It also reduced elevated circulating PLA levels in patients with peripheral arterial disease. CONCLUSION: By inhibiting platelet-leukocyte interactions, demonstrated in multiple preclinical and clinical studies, inclacumab may provide an effective treatment for cardiovascular diseases.

Differential distribution of 4-hydroxynonenal adducts to sulfur and nitrogen residues in blood proteins as revealed using Raney nickel and gas chromatography-mass spectrometry.

Quantification of 4-hydroxy-2-nonenal (HNE) bound to circulating proteins may prove to be useful in evaluating the role of this bioactive lipoperoxidation by-product in the pathogenesis of various diseases. Recently, we developed a quantitative gas chromatography-mass spectrometry (GCMS) assay of total protein-bound HNE (HNE-P) in blood after reduction with NaB(2)H(4) and cleavage with Raney nickel. Whereas it has been assumed that Raney nickel cleaves only Michael adducts of HNE to cysteine via a thioether bond (HNE-SP), results from this study demonstrate that our GCMS method also detects with precision picomoles of HNE adducts via nitrogen residues (HNE-NP). Specifically, evidence was obtained using various study models, including polyamino acids consisting of cysteine, lysine, and histidine and a biologically relevant molecule, albumin. Furthermore, we show that dinitrophenylhydrazine treatment before Raney nickel treatment can be used to discriminate and quantify the various HNE-P molecular species in plasma and blood samples from normal rats, which range between 0.15 and 3 pmol/mg protein or 10 to 600 nM. However, whereas HNE-SP predominated in whole blood, we detected HNE-NP only in plasma. We also identified another significant MS signal, which we attribute to protein-bound 1,4-dihydroxynonane (DHN-P) presumably formed from the enzymatic reduction of HNE-P. The distribution profile of all these species in plasma differed from that observed when physiologically relevant concentrations of albumin and HNE were incubated in vitro. Furthermore, interestingly, hypercholesterolemic rabbits showed higher plasma levels of HNE-NP, but not of DHN-P. Beyond documenting the presence of various types of HNE-P in circulating proteins, our results emphasize the importance of enzymatic mechanisms in situ as a factor determining their distribution in the various blood compartments under various conditions.