Richard W. James

Antagonism of RANTES Receptors Reduces Atherosclerotic Plaque Formation in Mice

Abstract— Increasing evidence supports the involvement of inflammation in the early phases of atherogenesis. Recruitment of leukocytes within the vascular wall, controlled by chemokines, is an essential process in the development of this common disease. In this study, we report that blocking a chemokine pathway in vivo with the CC chemokine antagonist Met-RANTES reduces the progression of atherosclerosis in a hypercholesterolemic mouse model. The reduction of lesions was correlated with a diminution of expression of several major chemokines and chemokine receptors, a decrease in leukocyte infiltration, and an increase of collagen-rich atheroma, features associated with stable atheroma. Treatment was well tolerated and serum lipid profiles were not affected. Whereas genetically engineered mice with deletion of either a CC chemokine or its receptor have demonstrated resistance to disease, to our knowledge, this is the first demonstration that treatment with a chemokine receptor antagonist limits the progression of atherosclerosis in vivo. Thus, our findings indicate that blockade of chemokine receptor/ligand interactions might become a novel therapeutic strategy to reduce the evolution of this common disease.

Reduced Connexin43 Expression Inhibits Atherosclerotic Lesion Formation in Low-Density Lipoprotein Receptor–Deficient Mice

Background—Gap junctions allow the direct exchange of ions and small molecules between cells in contact, thus coordinating physiological processes such as cell growth and differentiation. We have recently demonstrated increased expression of the gap junction protein connexin43 (Cx43) in specific subsets of cells in atherosclerotic lesions. Because the development of atherosclerosis depends critically on paracrine cell-to-cell interactions, we hypothesized that direct intercellular communication via gap junctions may be another factor controlling atherogenesis. Methods and Results—The role of Cx43 in atherogenesis was examined by use of both a genetic and a pharmacological approach. First, atherosclerosis-susceptible LDL receptor–deficient (LDLR−/−) mice with normal (Cx43+/+) or reduced (Cx43+/−) levels of Cx43 were fed a cholesterol-rich diet for 14 weeks. The progression of atherosclerosis was reduced by 50% (P <0.01) in the thoracoabdominal aorta and in the aortic roots of Cx43+/−LDLR−/− mice compared with Cx43+/+LDLR−/− controls. Atheroma in Cx43+/−LDLR−/− mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells. Next, we observed that HMG-CoA reductase inhibitors, or “statins,” lipid-lowering drugs well known for their pleiotropic antiatherogenic effects, reduced Cx43 expression in primary human vascular cells in vitro. Atheroma of LDLR−/− mice treated orally with pravastatin contained fewer inflammatory cells and exhibited thicker fibrous caps than controls. This was associated with reduced Cx43 expression in lesions of statin-treated mice. Conclusions—These data indicate a critical role for Cx43-mediated gap junctional communication in atherosclerotic plaque formation.

Native and reconstituted HDL activate Stat3 in ventricular cardiomyocytes via ERK1/2: Role of sphingosine-1-phosphate

AIMS High-density lipoprotein (HDL) has been reported to have cardioprotective properties independent from its cholesterol transport activity. The influence of native HDL and reconstituted HDL (rHDL) on Stat3, the transcription factor playing an important role in myocardium adaptation to stress, was analysed in neonatal rat ventricular cardiomyocytes. We have investigated modulating the composition of rHDL as a means of expanding its function and potential cardioprotective effects. METHODS AND RESULTS Stat3 phosphorylation and activation were determined by western blotting and electrophoretic mobility shift assay (EMSA). In ventricular cardiomyocytes, HDL and the HDL constituent sphingosine-1-phosphate (S1P) induce a concentration- and time-dependent increase in Stat3 activation. They also enhance extracellular signal-regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. U0126, a specific inhibitor of MEK1/2, the upstream activator of ERK1/2, abolishes HDL- and S1P-induced Stat3 activation, whereas the p38 MAPK blocker SB203580 has no significant effect. Inhibition of the tyrosine kinase family Src (Src) caused a significant reduction of Stat3 activation, whereas inhibition of phosphatidylinositol 3-kinase (PI3K) had no effect. S1P and rHDL containing S1P have a similar strong stimulatory action on Stat3, ERK1/2, and p38 MAPK comparable to native HDL. S1P-free rHDL has a much weaker effect. Experiments with agonists and antagonists of the S1P receptor subtypes indicate that HDL and S1P activate Stat3 mainly through the S1P2 receptor. CONCLUSION In ventricular cardiomyocytes, addition of S1P to rHDL enhances its therapeutic potential by improving its capacity to activate Stat3. Activation of Stat3 occurs mainly via the S1P constituent and the lipid receptor S1P2 requiring stimulation of ERK1/2 and Src but not p38 MAPK or PI3K. The study underlines the therapeutic potential of tailoring rHDL to confront particular clinical situations.

Monoclonal antibodies directed against the neurotransmitter binding site of nicotinic acetylcholine receptor

The pathogenesis of the disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) involves an autoimmune reaction to nicotinic acetylcholine receptor (nAcChR) [l--5], where anti-nAcChR antibodies effectively impair transmission at the neuromuscular junction [ 1,6]. Hence, the immunogenic properties of nAcChR are of major consideration. The ability to differentiate the antigenic determinants of nAcChR would be of great use by permitting a itetailed analysis of the importance of different determinants in MG, as well as providing a new dimension for structural and functional studies of nAcChR. The lymphocyte hybridization procedure provides an opportunity to develop such antigenic site-specific monoclonal antibodies (mAb) [7]. We have applied the procedure to nAcChR and report the isolation, in particular, of mAbs directed: (i) at the neurotransmitter-binding site; (ii) close to the neurotransmitter-binding site. These mAbs should help delineate the contribution of this region of nAcChR to the pathogenesis of MG.

Native and reconstituted HDL protect cardiomyocytes from doxorubicin-induced apoptosis

AIMS We analysed the impact of native and reconstituted HDL on doxorubicin-induced cardiomyocyte apoptosis. While it is an effective anti-cancer agent, doxorubicin has serious cardiotoxic side effects. HDL has been shown to protect cardiomyocytes, notably against oxidative stress. METHODS AND RESULTS Cultured neonatal rat ventricular cardiomyocytes were subjected to doxorubicin-induced stress, monitored as caspase3 activation, apoptotic DNA fragmentation and cell viability. The protective effects of HDL and sphingosine-1-phosphate (S1P) were investigated using native HDL, reconstituted HDL of varied composition and agonists and antagonists of S1P receptors. Anti-apoptotic signalling pathways were identified with specific inhibitors. Native and reconstituted HDL significantly decreased doxorubicin-induced cardiomyocyte apoptosis, essentially due to the S1P component of HDL. The latter was mediated by the S1P2 receptor, but not the S1P1 or S1P3 receptors. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) signalling pathway was required for the anti-apoptotic effects of HDL and S1P. The transcription factor Stat3 also played an important role, as inhibition of its activity compromised the protective effects of HDL and S1P on doxorubicin-induced apoptosis. CONCLUSION HDL and its sphingosine-1-phosphate component can protect cardiomyocytes against doxorubicin toxicity and may offer one means of reducing cardiotoxic side effects during doxorubicin therapy. The study identified anti-apoptotic pathways that could be exploited to improve cardiomyocyte survival.

HDL-associated paraoxonase-1 can redistribute to cell membranes and influence sensitivity to oxidative stress.

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant capacity of the lipoprotein. In previous studies, we proposed that HDL promoted PON1 secretion by transfer of the enzyme from its plasma membrane location to HDL transiently anchored to the hepatocyte. This study examined whether PON1 can be transferred into cell membranes and retain its enzymatic activities and functions. Using Chinese hamster ovary and human endothelial cells, we found that recombinant PON1 as well as PON1 associated with purified human HDL was freely exchanged between the external medium and the cell membranes. Transferred PON1 was located in the external face of the plasma membrane of the cells in an enzymatically active form. The transfer of PON1 led to a gain of function by the target cells, as revealed by significantly reduced susceptibility to oxidative stress and significantly increased ability to neutralize the bacterial virulence agent 3-oxo-C(12)-homoserine lactone. The data demonstrate that PON1 is not a fixed component of HDL and suggest that the enzyme could also exert its protective functions outside the lipoprotein environment. The observations may be of relevance to tissues exposed to oxidative stress and/or bacterial infection.

When are pro-inflammatory cytokines SAFE in heart failure?

The cytokine hypothesis presently suggests that an excessive production of pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF) and interleukin 6 (IL6), contributes to the pathogenesis of heart failure. The concept, successfully proved in genetically modified animal models, failed to translate to humans. Recently, accumulation of apparently paradoxical experimental data demonstrates that, under certain conditions, production of pro-inflammatory cytokines can initiate the activation of a pro-survival cardioprotective signalling pathway. This novel path that involves the activation of a transcription factor, signal transducer and activator of transcription 3 (STAT3), has been termed the survival activating factor enhancement (SAFE) pathway. In this review, we will discuss whether targeting the SAFE pathway may be considered as a preventive and/or therapeutic measure for the treatment of heart failure.

Anti-apolipoprotein A-1 IgG as an independent cardiovascular prognostic marker affecting basal heart rate in myocardial infarction

AIMS To assess the prognostic value of anti-apolipoprotein A-1 (anti-apoA-1) IgG after myocardial infarction (MI) and its association with major cardiovascular events (MACEs) at 12 months and to determine their association with resting heart rate (RHR), a well-established prognostic feature after MI. Anti-apoA-1 IgG have been reported in MI without autoimmune disease, but their clinical significance remains undetermined. METHODS AND RESULTS A total of 221 consecutive patients with MI were prospectively included, and all completed a 12-month follow-up. Major cardiovascular events consisted in death, MI, stroke, or hospitalization either for an acute coronary syndrome or heart failure. Resting heart rate was obtained on Holter the day before discharge under the same medical treatment. Neonate rat ventricular cardiomyocytes (NRVC) were used in vitro to assess the direct anti-apoA-1 IgG effect on RHR. During follow-up, 13% of patients presented a MACE. Anti-apoA-1 IgG positivity was 9% and was associated with a higher RHR (P = 0.0005) and higher MACE rate (adjusted OR, 4.3; 95% CI, 1.46-12.6; P = 0.007). Survival models confirmed the significant nature of this association. Patients with MACE had higher median anti-apoA-1 IgG values at admission than patients without (P = 0.007). On NRVC, plasma from MI patients and monoclonal anti-apoA-1 IgG induced an aldosterone and dose-dependent positive chronotropic effect, abrogated by apoA-1 and therapeutic immunoglobulin (IVIG) pre-incubation. CONCLUSIONS In MI patients, anti-apoA-1 IgG is independently associated with MACE at 1-year, interfering with a currently unknown aldosterone-dependent RHR determinant. Knowing whether anti-apoA-1 IgG assessment could be of interest to identify an MI patient subset susceptible to benefit from apoA-1/IVIG therapy remains to be demonstrated.

Relationship between paraoxonase-1 activity, its Q192R genetic variant and clopidogrel responsiveness in the ADRIE study.

*Division of Angiology and Hemostasis, Division of Endocrinology, Diabetology and Nutrition, Department of Medical Specialties, GenevaUniversity Hospitals and Faculty of Medicine, Geneva, Switzerland; Hematology Laboratory, §Division of Cardiology, Be´ziers Hospital, Be´ziers;–Hematology Laboratory, Montpellier University Hospital, Montpellier, France; **Division of Laboratory Medicine, Department of Genetics andLaboratory Medicine, Geneva University Hospitals, Geneva; and Division of General Internal Medicine, Geneva University Hospital and Facultyof Medicine, Geneva, SwitzerlandTo cite this article: Fontana P, James R, Barazer I, Berdague´ P, Schved JF, Rebsamen M, Vuilleumier N, Reng JL. Relationship betweenparaoxonase-1 activity, its Q192R genetic variant and clopidogrel responsiveness in the ADRIE study. J Thromb Haemost 2011; 9: 1664–6.

Paraoxonase 1: genetics and activities during aging.

The increasing longevity of the population, one of the most important issues throughout the planet, is a very complex phenomenon (trait), likely resulting from a variety of environmental determinants interacting with and modulated by genetic mechanisms, mostly devoted to maintenance and repair. In fact, the genes involved in longevity impact upon basic processes such as inflammation, glucose and energy utilization, and oxidative stress. Based on the free radical theory of aging, in the past few years we have focused our attention on an enzyme that protects lipids from peroxidative damage-paraoxonase 1 (PON1). PON1 has been widely investigated, especially for its involvement in atherosclerosis and age-related diseases. In this review, we summarize data on the role played by PON1 on aging and its possible involvement in human longevity, focusing on the relationship between genetic polymorphisms and enzyme activity and its capability to counteract oxidative stress.