Catherine Pavoine

M2 Kupffer cells promote M1 Kupffer cell apoptosis: A protective mechanism against alcoholic and nonalcoholic fatty liver disease

Alcoholic and nonalcoholic fatty liver disease (ALD and NAFLD) are the predominant causes of liver‐related mortality in Western countries. We have shown that limiting classical (M1) Kupffer cell (KC) polarization reduces alcohol‐induced liver injury. Herein, we investigated whether favoring alternatively activated M2 KCs may protect against ALD and NAFLD. Ongoing alcohol drinkers and morbidly obese patients, with minimal hepatic injury and steatosis, displayed higher hepatic expression of M2 genes, as compared to patients with more severe liver lesions; individuals with limited liver lesions showed negligible hepatocyte apoptosis but significant macrophage apoptosis. Experiments in mouse models of ALD or NAFLD further showed that BALB/c or resveratrol‐treated mice fed alcohol or a high‐fat diet displayed preponderant M2 KC polarization, M1 KC apoptosis, and resistance to hepatocyte steatosis and apoptosis, as compared to control C57BL6/J mice. In vitro experiments in isolated KC, peritoneal, and Raw264.7 macrophages demonstrated that M1 macrophage apoptosis was promoted by conditioned medium from macrophages polarized into an M2 phenotype by either interleukin (IL)4, resveratrol, or adiponectin. Mechanistically, IL10 released from M2 cells promoted M1 death, and anti‐IL10 antibodies blunted the proapoptic effects of M2‐conditioned media. IL10 secreted by M2 KCs promoted selective M1 death by a mechanism involving activation of arginase in high inducible nitric oxide synthase‐expressing M1 KCs. In alcohol‐exposed mice, neutralization of IL10 impaired M1 apoptosis. Conclusion: These data uncover a novel mechanism regulating the M1/M2 balance that relies on apoptotic effects of M2 KCs towards their M1 counterparts. They suggest that promoting M2‐induced M1 KC apoptosis might prove a relevant strategy to limit alcohol‐ and high fat‐induced inflammation and hepatocyte injury. (Hepatology 2014;58:130–142)

Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice

Activation of Kupffer cells plays a central role in the pathogenesis of alcoholic liver disease. Because cannabinoid CB2 receptors (CB2) display potent anti‐inflammatory properties, we investigated their role in the pathogenesis of alcoholic liver disease, focusing on the impact of CB2 on Kupffer cell polarization and the consequences on liver steatosis. Wild‐type (WT) mice fed an alcohol diet showed an induction of hepatic classical (M1) and alternative (M2) markers. Cotreatment of alcohol‐fed mice with the CB2 agonist, JWH‐133, decreased hepatic M1 gene expression without affecting the M2 profile. In keeping with this, genetic ablation of CB2 enhanced hepatic induction of M1 gene signature and blunted the induction of M2 markers. CB2 also modulated alcohol‐induced fatty liver, as shown by the reduction of hepatocyte steatosis in JWH‐133‐treated mice and its enhancement in CB2−/− animals. Studies in isolated Kupffer cells and cultured macrophages further demonstrated that CB2 inhibits M1 polarization and favors the transition to an M2 phenotype. In addition, conditioned‐medium experiments showed that preventing M1 polarization in CB2‐activated macrophages protects from lipid accumulation in hepatocytes. Heme oxygenase‐1 (HO‐1) mediated the anti‐inflammatory effects of CB2 receptors. Indeed, alcohol‐fed mice treated with JWH‐133 showed increased hepatic expression of macrophage HO‐1, as compared to vehicle‐treated counterparts. In keeping with this, JWH‐133 induced HO‐1 expression in cultured macrophages, and the HO‐1 inhibitor, zinc protoporphyrin, blunted the inhibitory effect of JWH‐133 on lipopolysaccharide‐induced nuclear factor‐kappa B activation and M1 polarization. Altogether, these findings demonstrate that CB2 receptors display beneficial effects on alcohol‐induced inflammation by regulating M1/M2 balance in Kupffer cells, thereby reducing hepatocyte steatosis via paracrine interactions between Kupffer cells and hepatocytes. These data identify CB2 agonists as potential therapeutic agents for the management of alcoholic liver disease. (HEPATOLOGY 2011;)

Sphingomyelinases: their regulation and roles in cardiovascular pathophysiology

Sphingomyelinases (SMases) hydrolyse sphingomyelin, releasing ceramide and creating a cascade of bioactive lipids. These lipids include sphingosine and sphingosine-1-phosphate, all of which have a specific signalling capacity. Sphingomyelinase activation occurs in different cardiovascular system cell types, namely cardiac myocytes, endothelial and vascular smooth muscle cells, mediating cell proliferation, cell death, and contraction of cardiac and vascular myocytes. Three main types of SMases contribute to cardiovascular physiology: the lysosomal and secreted acidic SMases (L- and S-ASMases, respectively) and the membrane neutral SMase (NSMase). These three enzymes have common activators, including ischaemia/reperfusion stress and proinflammatory cytokines, but they differ in their enzymatic properties and subcellular locations that determine the final effect of enzyme activation. This review focuses on the recent advances in the understanding of ASMase and NSMase pathways and their specific contribution to cardiovascular pathophysiology. Current knowledge indicates that the inhibitors of the different SMase types are potential tools for the treatment of cardiovascular diseases. Acid SMase inhibitors could be tools against post-ischaemia reperfusion injury and in the treatment of atherosclerosis. Neutral SMase inhibitors could be tools for the treatment of atherosclerosis, heart failure, and age-related decline in vasomotion. However, the design of bioavailable and more specific SMase-type inhibitors remains a challenge.

The cannabinoid receptor type 2 promotes cardiac myocyte and fibroblast survival and protects against ischemia/reperfusion-induced cardiomyopathy

Post‐myocardial infarction (MI) heart failure is a major public health problem in Western countries and results from ischemia/reperfusion (IR)‐induced cell death, remodeling, and contractile dysfunction. Ex vivo studies have demonstrated the cardioprotective anti‐inflammatory effect of the cannabinoid type 2 (CB2) receptor agonists within hours after IR. Herein, we evaluated the in vivo effect of CB2 receptors on IR‐induced cell death, fibrosis, and cardiac dysfunction and investigated the target role of cardiac myocytes and fibroblasts. The infarct size was increased 24 h after IR in CB2 /_ vs. wild‐type (WT) hearts and decreased when WT hearts were injected with the CB2 agonist JWH133 (3 mg/kg) at reperfusion. Compared with WT hearts, CB2–/– hearts showed widespread injury 3 d after IR, with enhanced apoptosis and remodeling affecting the remote myocardium. Finally, CB2 –/– hearts exhibited exacerbated fibrosis, associated with left ventricular dysfunction 4 wk after IR, whereas their WT counterparts recovered normal function. Cardiac myocytes and fibroblasts isolated from CB2 –/– hearts displayed a higher H2O2‐ induced death than WT cells, whereas 1 μM JWH133 triggered survival effects. Furthermore, H2O2‐induced myofibroblast activation was increased in CB2 –/– fibroblasts but decreased in 1 μM JWH133‐treated WT fibroblasts, compared with that in WT cells. Therefore, CB2 receptor activation may protect against post‐IR heart failure through direct inhibition of cardiac myocyte and fibroblast death and prevention of myofibroblast activation.—Defer, N.,Wan, J., Souktani, R., Escoubet, B., Perier, M., Caramelle, P., Manin, S., Deveaux, V., Bourin, M.‐C., Zimmer, A., Lotersztajn, S., Pecker, F., Pavoine, C. The cannabinoid receptor type 2 promotes cardiac myocyte and fibroblast survival and protects against ischemia/reperfusion‐induced cardiomyopathy. FASEB J. 23, 2120–2130 (2009)

TNFR1 and TNFR2 Signaling Interplay in Cardiac Myocytes

Tumor necrosis factor α (TNFα) plays a major role in chronic heart failure, signaling through two different receptor subtypes, TNFR1 and TNFR2. Our aim was to further delineate the functional role and signaling pathways related to TNFR1 and TNFR2 in cardiac myocytes. In cardiac myocytes isolated from control rats, TNFα induced ROS production, exerted a dual positive and negative action on [Ca2+] transient and cell fractional shortening, and altered cell survival. Neutralizing anti-TNFR2 antibodies exacerbated TNFα responses on ROS production and cell death, arguing for a major protective role of the TNFR2 pathway. Treatment with either neutralizing anti-TNFR1 antibodies or the glutathione precursor, N-acetylcysteine (NAC), favored the emergence of TNFR2 signaling that mediated a positive effect of TNFα on [Ca2+] transient and cell fractional shortening. The positive effect of TNFα relied on TNFR2-dependent activation of the cPLA2 activity, independently of serine 505 phosphorylation of the enzyme. Together with cPLA2 redistribution and AA release, TNFα induced a time-dependent phosphorylation of ERK, MSK1, PKCζ, CaMKII, and phospholamban on the threonine 17 residue. Taken together, our results characterized a TNFR2-dependent signaling and illustrated the close interplay between TNFR1 and TNFR2 pathways in cardiac myocytes. Although apparently predominant, TNFR1-dependent responses were under the yoke of TNFR2, acting as a critical limiting factor. In vivo NAC treatment proved to be a unique tool to selectively neutralize TNFR1-mediated effects of TNFα while releasing TNFR2 pathways.

Glutathione Deficiency in Cardiac Patients Is Related to the Functional Status and Structural Cardiac Abnormalities

Background The tripeptide glutathione (L-gamma-glutamyl-cysteinyl-glycine) is essential to cell survival, and deficiency in cardiac and systemic glutathione relates to heart failure progression and cardiac remodelling in animal models. Accordingly, we investigated cardiac and blood glutathione levels in patients of different functional classes and with different structural heart diseases. Methods Glutathione was measured using standard enzymatic recycling method in venous blood samples obtained from 91 individuals, including 15 healthy volunteers and 76 patients of New York Heart Association (NYHA) functional class I to IV, undergoing cardiac surgery for coronary artery disease, aortic stenosis or terminal cardiomyopathy. Glutathione was also quantified in right atrial appendages obtained at the time of surgery. Results In atrial tissue, glutathione was severely depleted (−58%) in NYHA class IV patients compared to NYHA class I patients (P = 0.002). In patients with coronary artery disease, this depletion was related to the severity of left ventricular dysfunction (P = 0.006). Compared to healthy controls, blood glutathione was decreased by 21% in NYHA class I patients with structural cardiac disease (P<0.01), and by 40% in symptomatic patients of NYHA class II to IV (P<0.0001). According to the functional NYHA class, significant depletion in blood glutathione occurred before detectable elevation in blood sTNFR1, a marker of symptomatic heart failure severity, as shown by the exponential relationship between these two parameters in the whole cohort of patients (r = 0.88). Conclusions This study provides evidence that cardiac and systemic glutathione deficiency is related to the functional status and structural cardiac abnormalities of patients with cardiac diseases. These data also suggest that blood glutathione test may be an interesting new biomarker to detect asymptomatic patients with structural cardiac abnormalities.

Structural Localization and Expression of CXCL12 and CXCR4 in Rat Heart and Isolated Cardiac Myocytes

CXCL12 (SDF-1), which binds CXCR4, is involved in several physiological and pathophysiological processes. In heart, this axis seems to play a key role in cardiogenesis and is involved in the neovascularization of ischemic tissues. Rats have three known CXCL12 mRNA isoforms, of which only α and γ are present in the normal heart. However, little is known about CXCL12 protein expression and localization. We investigated the pattern of protein expression and the localization of both CXCR4 and CXCL12 in the heart, using isolated cardiomyocytes and a rat myocardial infarction model. Western blots showed that cardiomyocytes contained a specific 67-kDa CXCR4 isoform and a 12-kDa CXCL12 isoform. Confocal and electron microscopy clearly showed that CXCR4 was present at the plasmalemma and CXCL12 in continuity of the Z-line, in the proximal part of T-tubules. In conclusion, we provide the first description of the expression and fine localization of CXCR4 and CXCL12 proteins in normal rat heart and cardiomyocytes. These results suggest that the CXCL12/CXCR4 axis may be involved in cardiomyocyte calcium homeostasis regulation. Our work and the well-known chemoattraction properties of the CXCL12/CXCR4 axis highlight the importance of deciphering the function of this axis in both normal and pathological hearts.

The Cytosolic Phospholipase A2 Pathway, a Safeguard of β2-Adrenergic Cardiac Effects in Rat

We have recently demonstrated that in human heart, β2-adrenergic receptors (β2-ARs) are biochemically coupled not only to the classical adenylyl cyclase (AC) pathway but also to the cytosolic phospholipase A2 (cPLA2) pathway (Pavoine, C., Behforouz, N., Gauthier, C., Le Gouvello, S., Roudot-Thoraval, F., Martin, C. R., Pawlak, A., Feral, C., Defer, N., Houel, R., Magne, S., Amadou, A., Loisance, D., Duvaldestin, P., and Pecker, F. (2003) Mol. Pharmacol. 64, 1117–1125). In this study, using Fura-2-loaded cardiomyocytes isolated from adult rats, we showed that stimulation of β2-ARs triggered an increase in the amplitude of electrically stimulated [Ca2+]i transients and contractions. This effect was abolished with the PKA inhibitor, H89, but greatly enhanced upon addition of the selective cPLA2 inhibitor, AACOCF3. The β2-AR/cPLA2 inhibitory pathway involved Gi and MSK1. Potentiation of β2-AR/AC/PKA-induced Ca2+ responses by AACOCF3 did not rely on the enhancement of AC activity but was associated with eNOS phosphorylation (Ser1177) and l-NAME-sensitive NO production. This was correlated with PKA-dependent phosphorylation of PLB (Ser16). The constraint exerted by the β2-AR/cPLA2 pathway on the β2-AR/AC/PKA-induced Ca2+responses required integrity of caveolar structures and was impaired by Filipin III treatment. Immunoblot analyses demonstrated zinterol-induced translocation of cPLA and its cosedimentation with MSK1, eNOS, PLB, and sarcoplasmic reticulum Ca2+ pump (SERCA) 2a in a low density caveolin-3-enriched membrane fraction. This inferred the gathering of β2-AR signaling effectors around caveolae/sarcoplasmic reticulum (SR) functional platforms. Taken together, these data highlight cPLA as a cardiac β2-AR signaling pathway that limits β2-AR/AC/PKA-induced Ca2+ responses in adult rat cardiomyocytes through the impairment of eNOS activation and PLB phosphorylation.

Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling

Background— Stromal interaction molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiomyocytes. STIM1 is thought to act as an initiator of cardiac hypertrophic response at the level of the sarcolemma, but the pathways underpinning this effect have not been examined. Methods and Results— To determine the mechanistic role of STIM1 in cardiac hypertrophy and during the transition to heart failure, we manipulated STIM1 expression in mice cardiomyocytes by using in vivo gene delivery of specific short hairpin RNAs. In 3 different models, we found that Stim1 silencing prevents the development of pressure overload–induced hypertrophy but also reverses preestablished cardiac hypertrophy. Reduction in STIM1 expression promoted a rapid transition to heart failure. We further showed that Stim1 silencing resulted in enhanced activity of the antihypertrophic and proapoptotic GSK-3&bgr; molecule. Pharmacological inhibition of glycogen synthase kinase-3 was sufficient to reverse the cardiac phenotype observed after Stim1 silencing. At the level of ventricular myocytes, Stim1 silencing or inhibition abrogated the capacity for phosphorylation of AktS473, a hydrophobic motif of Akt that is directly phosphorylated by mTOR complex 2. We found that Stim1 silencing directly impaired mTOR complex 2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTOR complex 2. Conclusions— These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiomyocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2, which further results in repression of GSK-3&bgr; activity.

Emergence of Orai3 activity during cardiac hypertrophy

AIMS Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca(2+)) influx pathway that emerges during the hypertrophic remodelling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca(2+) influx in cardiomyocytes. METHODS AND RESULTS We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The level of STIM1 and Orai1 were significantly greater in the macromolecular complex precipitated by the Orai3 antibody in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent, i.e. store-independent and store-operated Ca(2+) entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca(2+) entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current. CONCLUSION Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca(2+) entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy.