Thioredoxin-1 and its mimetic peptide improve systolic cardiac function and remodeling after myocardial infarction.

Myocardial infarction (MI) is characterized by a significant loss of cardiomyocytes (CMs), and it is suggested that reactive oxygen species (ROS) are involved in cell cycle arrest, leading to impaired CM renewal. Thioredoxin-1 (Trx-1) scavenges ROS and may play a role in restoring CM renewal. However, the truncated form of Trx-1, Trx-80, can compromise its efficacy by exerting antagonistic effects. Therefore, a Trx-1 mimetic peptide called CB3 was tested as an alternative way to restore CMs. This study aimed to investigate the effects of Trx-1, Trx-80, and CB3 on mice with experimental MI and study the underlying mechanism of CB3 on CMs. Mouse cardiac parameters were quantified by echocardiography, and infarction size and fibrosis determined using Trichrome and Picro-Sirius Red staining. The study found that Trx-1 and CB3 improved mouse cardiac function, reduced the size of cardiac infarct and fibrosis, and decreased the expression of cardiac inflammatory markers. Furthermore, CB3 polarized macrophages into M2 phenotype, reduced apoptosis and oxidative stress after MI, and increased CM proliferation in cell culture and in vivo. CB3 effectively protected against myocardial infarction and could represent a new class of compounds for treating MI.

Anti-inflammatory and anticoagulant effects of polyphenol-rich extracts from Thymus atlanticus: An in vitro and in vivo study.

ETHNOPHARMACOLOGICAL EVIDENCE: Thymus atlanticus (TA) is used in traditional medicine in Morocco to treat chronic inflammatory diseases, after local and oral treatment. AIM OF STUDY: This study aimed to investigate the in vitro and in vivo anti-inflammatory and anticoagulant activities of an aqueous extract (AE) and polyphenol fraction (PF) derived from TA. MATERIALS AND METHODS: The effect of AE and PF on monocyte chemoattractant protein-1 (MCP-1) production by naïve and LPS-stimulated peritoneal macrophages isolated from C57Bl/6 mice was assessed by ELISA assay. The effect of chronic administration of the extracts at three different doses by oral rout for 2 weeks on blood coagulation and inflammation induced by carrageenan in Wistar rats was evaluated. In addition, the in vitro anticoagulant effect was tested on blood plasma collected from healthy rats using the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) tests. The acute toxicity of AE was investigated. Phytochemical analysis was carried out by HPLC. RESULTS: Analysis by HPLC indicated rosmarinic acid as the main phenolic acid in TA extracts. Compared to control macrophages, MCP-1 level was lower in medium supplemented with AE at 50 and 500 µg/mL and PF at 500 µg/mL, but higher in medium with PF at 50 µg/mL. Rosmarinic and chicoric acids, served as controls, significantly decreased MCP-1 production. Chronic oral administration of TA extracts prevented inflammation induced by carrageenan and induced a significant prolongation of blood coagulation time, in a dose dependant manner, in Wistar rats. The results of the in vitro assay showed that the coagulation time was significantly prolonged in plasma incubated with extracts in APTT, PT and TT tests. Lethal dose 50 of AE in mice was 27.90 ± 1.19 g/kg. CONCLUSION: This study indicated TA as an herb with anti-inflammatory and anticoagulant proprieties and supports the traditional use of this plant for the treatment of inflammatory diseases.

A thioredoxin-mimetic peptide exerts potent anti-inflammatory, antioxidant, and atheroprotective effects in ApoE2.Ki mice fed high fat diet.

Aims: Oxidative stress and inflammation play a pathogenic role in atherosclerosis. Thioredoxin-1 (Trx-1) is an anti-oxidative, anti-inflammatory protein with atheroprotective effects. However, in vivo cleavage of Trx-1 generates a truncated pro-inflammatory protein, Trx-80, which compromises the therapeutic use of Trx-1. Here we analysed whether the thioredoxin-mimetic peptide (TxMP), CB3 might exert anti-oxidative, anti-inflammatory, and atheroprotective effects in ApoE2.Ki mice. Methods and results: We synthesized a small TxMP, Ac-Cys-Pro-Cys-amide, CB3 and characterized its antioxidant and anti-inflammatory effects on cultured peritoneal murine macrophages. CB3 significantly and dose-dependently reduced the level of reactive oxygen species in lipopolysaccharides (LPS)-activated macrophages. In addition, it efficiently lowered LPS-induced inflammatory process through NF-κB inhibition, as evidenced by the reduced secretion of monocyte chemoattractant protein-1, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α by macrophages. Nevertheless, CB3 did not affect cholesterol accumulation in macrophages. A daily-administered dose of 10 µg/g body weight CB3 to ApoE2.Ki mice on high fat diet did not affect plasma of total cholesterol and triglycerides levels but significantly reduced the plasma levels of pro-inflammatory cytokines (IL-33 and TNF-α) and oxidative markers. In contrast, it significantly induced the plasma levels of anti-inflammatory proteins (adiponectin, IL-10). In addition, CB3 reduced the number of pro-inflammatory M1 macrophages in spleen and decreased the ratio of M1/M2 macrophages in atherosclerotic lesion areas. Finally, CB3 significantly reduced the surface area of aortic lesions. Conclusions: Our results clearly showed that similar to the full length Trx-1, CB3 exerts protective effects, by reducing inflammation and oxidative stress in macrophages and in ApoE2.Ki mice. The atheroprotective effect of CB3 opens promising therapeutic approaches for treatment of atherosclerosis.

The Oxygen Paradox, the French Paradox, and age-related diseases.

A paradox is a seemingly absurd or impossible concept, proposition, or theory that is often difficult to understand or explain, sometimes apparently self-contradictory, and yet ultimately correct or true. How is it possible, for example, that oxygen "a toxic environmental poison" could be also indispensable for life (Beckman and Ames Physiol Rev 78(2):547-81, 1998; Stadtman and Berlett Chem Res Toxicol 10(5):485-94, 1997)?: the so-called Oxygen Paradox (Davies and Ursini 1995; Davies Biochem Soc Symp 61:1-31, 1995). How can French people apparently disregard the rule that high dietary intakes of cholesterol and saturated fats (e.g., cheese and paté) will result in an early death from cardiovascular diseases (Renaud and de Lorgeril Lancet 339(8808):1523-6, 1992; Catalgol et al. Front Pharmacol 3:141, 2012; Eisenberg et al. Nat Med 22(12):1428-1438, 2016)?: the so-called, French Paradox. Doubtless, the truth is not a duality and epistemological bias probably generates apparently self-contradictory conclusions. Perhaps nowhere in biology are there so many apparently contradictory views, and even experimental results, affecting human physiology and pathology as in the fields of free radicals and oxidative stress, antioxidants, foods and drinks, and dietary recommendations; this is particularly true when issues such as disease-susceptibility or avoidance, "healthspan," "lifespan," and ageing are involved. Consider, for example, the apparently paradoxical observation that treatment with low doses of a substance that is toxic at high concentrations may actually induce transient adaptations that protect against a subsequent exposure to the same (or similar) toxin. This particular paradox is now mechanistically explained as "Adaptive Homeostasis" (Davies Mol Asp Med 49:1-7, 2016; Pomatto et al. 2017a; Lomeli et al. Clin Sci (Lond) 131(21):2573-2599, 2017; Pomatto and Davies 2017); the non-damaging process by which an apparent toxicant can activate biological signal transduction pathways to increase expression of protective genes, by mechanisms that are completely different from those by which the same agent induces toxicity at high concentrations. In this review, we explore the influences and effects of paradoxes such as the Oxygen Paradox and the French Paradox on the etiology, progression, and outcomes of many of the major human age-related diseases, as well as the basic biological phenomenon of ageing itself.

Human Plasma Thioredoxin-80 Increases With Age and in ApoE-/- Mice Induces Inflammation, Angiogenesis, and Atherosclerosis.

BACKGROUND: Thioredoxin (TRX)-1, a ubiquitous 12-kDa protein, exerts antioxidant and anti-inflammatory effects. In contrast, the truncated form, called TRX80, produced by macrophages induces upregulation of proinflammatory cytokines. TRX80 also promotes the differentiation of mouse peritoneal and human macrophages toward a proinflammatory M1 phenotype. METHODS: TRX1 and TRX80 plasma levels were determined with a specific ELISA. A disintegrin and metalloproteinase domain-containing protein (ADAM)-10, ADAM-17, and ADAM-10 activities were measured with SensoLyte 520 ADAM10 Activity Assay Kit, Fluorimetric, and InnoZyme TACE Activity Kit, respectively. Western immunoblots were performed with specific antibodies to ADAM-10 or ADAM-17. Angiogenesis study was evaluated in vitro with human microvascular endothelial cells-1 and in vivo with the Matrigel plug angiogenesis assay in mice. The expression of macrophage phenotype markers was investigated with real-time polymerase chain reaction. Phosphorylation of Akt, mechanistic target of rapamycin, and 70S6K was determined with specific antibodies. The effect of TRX80 on NLRP3 inflammasome activity was evaluated by measuring the level of interleukin-1ß and -18 in the supernatants of activated macrophages with ELISA. Hearts were used for lesion surface evaluation and immunohistochemical studies, and whole descending aorta were stained with Oil Red O. For transgenic mice generation, the human scavenger receptor (SR-A) promoter/enhancer was used to drive macrophage-specific expression of human TRX80 in mice. RESULTS: In this study, we observed a significant increase of plasma levels of TRX80 in old subjects compared with healthy young subjects. In parallel, an increase in expression and activity of ADAM-10 and ADAM-17 in old peripheral blood mononuclear cells compared with those of young subjects was observed. Furthermore, TRX80 was found to colocalize with tumor necrosis factor-α, a macrophage M1 marker, in human atherosclerotic plaque. In addition, TRX80 induced the expression of murine M1 macrophage markers through Akt2/mechanistic target of rapamycin-C1/70S6K pathway and activated the inflammasome NLRP3, leading to the release of interleukin-1ß and -18, potent atherogenic cytokines. Moreover, TRX80 exerts a powerful angiogenic effect in both in vitro and in vivo mouse studies. Finally, transgenic mice that overexpress human TRX80 specifically in macrophages of apoE-/- mice have a significant increase of aortic atherosclerotic lesions. CONCLUSIONS: TRX80 showed an age-dependent increase in human plasma. In mouse models, TRX80 was associated with a proinflammatory status and increased atherosclerosis.

Inhibition of the Inflammasome NLRP3 by Arglabin Attenuates Inflammation, Protects Pancreatic ß-Cells from Apoptosis, and Prevents Type 2 Diabetes Mellitus Development in ApoE2Ki Mice on a Chronic High-Fat Diet.

Intraperitoneal injection of arglabin (2.5 ng/g of body weight, twice daily, 13 weeks) into female human apolipoprotein E2 gene knock-in (ApoE2Ki) mice fed a high-fat Western-type diet (HFD) reduced plasma levels of glucose and insulin by ∼20.0% ± 3.5% and by 50.0% ± 2.0%, respectively, in comparison with vehicle-treated mice. Immunohistochemical analysis revealed the absence of active caspase-3 in islet sections from ApoE2Ki mice fed a HFD and treated with arglabin. In addition, arglabin reduced interleukin-1ß (IL-1ß) production in a concentration-dependent manner in Langerhans islets isolated from ApoE2Ki mice treated with lipopolysaccharide (LPS) and with cholesterol crystals. This inhibitory effect is specific for the inflammasome NOD-like receptor family, pyrin domain-containing 3 (NLRP3) because IL-1ß production was abolished in Langerhans islets isolated from Nlrp3(-/-) mice. In the insulin-secreting INS-1 cells, arglabin inhibited, in a concentration-dependent manner, the maturation of pro-IL-1ß into biologically active IL-1ß probably through the inhibition of the maturation of procaspase-1 into active capsase-1. Moreover, arglabin reduced the susceptibility of INS-1 cells to apoptosis by increasing Bcl-2 levels. Similarly, autophagy activation by rapamycin decreased apoptosis susceptibility while autophagy inhibition by 3-methyladenin treatment promoted apoptosis. Arglabin further increased the expression of the autophagic markers Bcl2-interacting protein (Beclin-1) and microtubule-associated protein 1 light chain 3 II (LC3-II) in a concentration-dependent manner. Thus, arglabin reduces NLRP3-dependent inflammation as well as apoptosis in pancreatic ß-cells in vivo and in the INS-1 cell line in vitro, whereas it increases autophagy in cultured INS-1 cells, indicating survival-promoting properties of the compound in these cells. Hence, arglabin may represent a new promising compound to treat inflammation and type 2 diabetes mellitus development.

AMPK Signaling Involvement for the Repression of the IL-1ß-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1ß-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleotide (AICAR) treatment strongly repressed IL-1ß-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1ß-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1ß induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

Anti-inflammatory and antiatherogenic effects of the NLRP3 inflammasome inhibitor arglabin in ApoE2.Ki mice fed a high-fat diet.

BACKGROUND: This study was designed to evaluate the effect of arglabin on the NLRP3 inflammasome inhibition and atherosclerotic lesion in ApoE2Ki mice fed a high-fat Western-type diet. METHODS AND RESULTS: Arglabin was purified, and its chemical identity was confirmed by mass spectrometry. It inhibited, in a concentration-dependent manner, interleukin (IL)-1ß and IL-18, but not IL-6 and IL-12, production in lipopolysaccharide and cholesterol crystal-activated cultured mouse peritoneal macrophages, with a maximum effect at ≈50 nmol/L and EC50 values for both cytokines of ≈ 10 nmol/L. Lipopolysaccharide and cholesterol crystals did not induce IL-1ß and IL-18 production in Nlrp3(-/-) macrophages. In addition, arglabin activated autophagy as evidenced by the increase in LC3-II protein. Intraperitoneal injection of arglabin (2.5 ng/g body weight twice daily for 13 weeks) into female ApoE2.Ki mice fed a high-fat diet resulted in a decreased IL-1ß plasma level compared with vehicle-treated mice (5.2±1.0 versus 11.7±1.1 pg/mL). Surprisingly, arglabin also reduced plasma levels of total cholesterol and triglycerides to 41% and 42%, respectively. Moreover, arglabin oriented the proinflammatory M1 macrophages into the anti-inflammatory M2 phenotype in spleen and arterial lesions. Finally, arglabin treatment markedly reduced the median lesion areas in the sinus and whole aorta to 54% (P=0.02) and 41% (P=0.02), respectively. CONCLUSIONS: Arglabin reduces inflammation and plasma lipids, increases autophagy, and orients tissue macrophages into an anti-inflammatory phenotype in ApoE2.Ki mice fed a high-fat diet. Consequently, a marked reduction in atherosclerotic lesions was observed. Thus, arglabin may represent a promising new drug to treat inflammation and atherosclerosis.

NLRP3 inflammasome: from a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases.

IL-1ß production is critically regulated by cytosolic molecular complexes, termed inflammasomes. Different inflammasome complexes have been described to date. While all inflammasomes recognize certain pathogens, it is the distinctive feature of NLRP3 inflammasome to be activated by many and diverse stimuli making NLRP3 the most versatile, and importantly also the most clinically implicated inflammasome. However, NLRP3 activation has remained the most enigmatic. It is not plausible that the intracellular NLRP3 receptor is able to detect all of its many and diverse triggers through direct interactions; instead, it is discussed that NLRP3 is responding to certain generic cellular stress-signals induced by the multitude of molecules that trigger its activation. An ever increasing number of studies link the sensing of cellular stress signals to a direct pathophysiological role of NLRP3 activation in a wide range of autoinflammatory and autoimmune disorders, and thus provide a novel mechanistic rational, on how molecules trigger and support sterile inflammatory diseases. A vast interest has created to unravel how NLRP3 becomes activated, since mechanistic insight is the prerequisite for a knowledge-based development of therapeutic intervention strategies that specifically target the NLRP3 triggered IL-1ß production. In this review, we have updated knowledge on NLRP3 inflammasome assembly and activation and on the pyrin domain in NLRP3 that could represent a drug target to treat sterile inflammatory diseases. We have reported mutations in NLRP3 that were found to be associated with certain diseases. In addition, we have reviewed the functional link between NLRP3 inflammasome, the regulator of cellular redox status Trx/TXNIP complex, endoplasmic reticulum stress and the pathogenesis of diseases such as type 2 diabetes. Finally, we have provided data on NLRP3 inflammasome, as a critical regulator involved in the pathogenesis of obesity and cardiovascular diseases.

PCSK9 polymorphism in a Tunisian cohort: identification of a new allele, L8, and association of allele L10 with reduced coronary heart disease risk.

The c.61_63dupCTG (L10) allele of rs72555377 polymorphism in PCSK9 has been reported to be associated with low-density lipoprotein-cholesterol (LDL-C) levels and with a decreased risk of coronary artery disease (CAD). We investigated the effect of two known alleles for rs72555377, L10 and L11, on the risk of CAD in a Tunisian cohort (218 patients diagnosed by angiography and 125 control subjects). Two subgroups of patients were defined by their level of stenosis: ≥50% for CAD and <50% for no-CAD. The genotypes were obtained by the size measurement of fluorescent-labeled PCR products. We identified a novel allele for the rs72555377 polymorphism: an in-frame deletion, c.61_63delCTG (L8). The frequency of the L10 allele was significantly higher in the no-CAD subgroup than in the CAD subgroup (0.210 vs 0.114, p = 0.045), and than in the subgroup of CAD patients presenting a stenosis ≥50% in two or three major coronary arteries (0.210 vs 0.125, p = 0.028). Multiple regression analysis showed that the L10 allele was significantly associated with a reduced risk of CAD (p = 0.049, OR = 0.51[0.26-1.00]), and with its reduced severity (p = 0.045, OR = 0.44[0.20-0.98]). The L10 allele is associated with a reduced risk and severity of CAD, seemingly independently of its LDL-lowering effect, suggesting a direct effect of PCSK9 on atherogenesis.

Effect of the PPARγ C161T gene variant on serum lipids in ischemic stroke patients with and without type 2 diabetes mellitus.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor involved in the regulation of lipid metabolism, diabetes, obesity, atherogenesis and inflammation. PPARγ genetic variation has been associated with metabolic and cardiovascular diseases. The aim of this study was to explore, for the first time, the relationship between PPARγ C161T polymorphism and the risk of ischemic stroke (IS) among patients with type 2 diabetes mellitus (T2DM). A total of 196 patients with IS (117 diabetics and 79 nondiabetics) and 192 controls were recruited to enroll in this study. PPARγ C161T genotyping was performed by PCR-RFLP technique. The 161T allele as compared with C allele was found to be higher in controls than in IS patients (with or without T2DM). After adjusting for multiple risk factors, the T allele carriers had significantly reduced IS risk (OR=0.575, 95% CI 0.348-0.951, p=0.030) compared to the CC homozygotes which increased significantly the risk in IS patients with T2DM (OR=1.85, 95% CI 1.23-2.62). Moreover, the triglycerides (TG) and ApoB levels in CC homozygote carriers were significantly higher than those in T allele carriers. These results indicate that the C161T of PPARγ may reduce the risk of IS by modulation of adipose metabolism especially TG and ApoB in IS patients with T2DM.

Effect of genetic polymorphism +294T/C in peroxisome proliferator-activated receptor delta on the risk of ischemic stroke in a Tunisian population.

PPARδ +294T/C polymorphism was investigated in diabetics, in normolipidemic healthy controls, in dyslipidemic and nondyslipidemic coronary artery disease patients but never in ischemic stroke patients. The aim of this study was to explore, for the first time, the relationship between the genetic polymorphism of PPARδ and the risk of ischemic stroke among patients with diabetes. The study group consisted of 196 patients with ischemic stroke and 192 controls. Plasma concentrations of total cholesterol, triglycerides, low-, and high-density lipoprotein did not differ significantly between subjects carrying the TT genotype and those carrying the CC/TC genotype in both ischemic stroke patients (with or without diabetes) and control groups. The +294C allele (CC + CT genotypes) as compared with TT genotypes was found to be higher in total ischemic stroke patients than in controls. On the other hand, no interaction between diabetes and PPAR +294T/C polymorphism on the risk of ischemic stroke was found (p = 0.089). The PPARδ +294T/C polymorphism was associated with the risk of ischemic stroke in Tunisian subjects. This polymorphism has no influence on plasma lipoprotein concentrations and body mass index either in healthy subjects or in ischemic stroke patients with or without diabetes both in males and females.

Truncated thioredoxin (Trx-80) promotes pro-inflammatory macrophages of the M1 phenotype and enhances atherosclerosis.

Vascular cells are particularly susceptible to oxidative stress that is believed to play a key role in the pathogenesis of cardiovascular disorders. Thioredoxin-1 (Trx-1) is an oxidative stress-limiting protein with anti-inflammatory and anti-apoptotic properties. In contrast, its truncated form (Trx-80) exerts pro-inflammatory effects. Here we analyzed whether Trx-80 might exert atherogenic effects by promoting macrophage differentiation into the M1 pro-inflammatory phenotype. Trx-80 at 1 µg/ml significantly attenuated the polarization of anti-inflammatory M2 macrophages induced by exposure to either IL-4 at 15 ng/ml or IL-4/IL-13 (10 ng/ml each) in vitro, as evidenced by the expression of the characteristic markers, CD206 and IL-10. By contrast, in LPS-challenged macrophages, Trx-80 significantly potentiated the differentiation into inflammatory M1 macrophages as indicated by the expression of the M1 cytokines, TNF-α and MCP-1. When Trx-80 was administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/g body weight (b.w.) challenged either with LPS at 30 µg/30 g (b.w.) or IL-4 at 500 ng/30 g (b.w.), it significantly induced the M1 phenotype but inhibited differentiation of M2 macrophages in thymus and liver. When ApoE2.Ki mice were challenged once weekly with LPS for 5 weeks, they showed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. Such effect was potentiated when mice received daily, in addition to LPS, the Trx-80. Moreover, the Trx-80 treatment led to a significantly increased aortic lesion area. The ability of Trx-80 to promote differentiation of macrophages into the classical proinflammatory phenotype may explain its atherogenic effects in cardiovascular diseases.

The thioredoxin system as a therapeutic target in human health and disease.

The thioredoxin (Trx) system comprises Trx, truncated Trx (Trx-80), Trx reductase, and NADPH, besides a natural Trx inhibitor, the thioredoxin-interacting protein (TXNIP). This system is essential for maintaining the balance of the cellular redox status, and it is involved in the regulation of redox signaling. It is also pivotal for growth promotion, neuroprotection, inflammatory modulation, antiapoptosis, immune function, and atherosclerosis. As an ubiquitous and multifunctional protein, Trx is expressed in all forms of life, executing its function through its antioxidative, protein-reducing, and signal-transducing activities. In this review, the biological properties of the Trx system are highlighted, and its implications in several human diseases are discussed, including cardiovascular diseases, heart failure, stroke, inflammation, metabolic syndrome, neurodegenerative diseases, arthritis, and cancer. The last chapter addresses the emerging therapeutic approaches targeting the Trx system in human diseases.

Thioredoxin-1 promotes anti-inflammatory macrophages of the M2 phenotype and antagonizes atherosclerosis.

OBJECTIVE: Oxidative stress is believed to play a key role in cardiovascular disorders. Thioredoxin (Trx) is an oxidative stress-limiting protein with anti-inflammatory and antiapoptotic properties. Here, we analyzed whether Trx-1 might exert atheroprotective effects by promoting macrophage differentiation into the M2 anti-inflammatory phenotype. METHODS AND RESULTS: Trx-1 at 1 µg/mL induced downregulation of p16(INK4a) and significantly promoted the polarization of anti-inflammatory M2 macrophages in macrophages exposed to interleukin (IL)-4 at 15 ng/mL or IL-4/IL-13 (10 ng/mL each) in vitro, as evidenced by the expression of the CD206 and IL-10 markers. In addition, Trx-1 induced downregulation of nuclear translocation of activator protein-1 and Ref-1, and significantly reduced the lipopolysaccharide-induced differentiation of inflammatory M1 macrophages, as indicated by the decreased expression of the M1 cytokines, tumor necrosis factor-α and monocyte chemoattractant protein-1. Consistently, Trx-1 administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/30 g body weight challenged either with lipopolysaccharide at 30 µg/30 g body weight or with IL-4 at 500 ng/30 g body weight significantly induced the M2 phenotype while inhibiting differentiation of macrophages into the M1 phenotype in liver and thymus. ApoE2.Ki mice challenged once weekly with lipopolysaccharide for 5 weeks developed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. In contrast, however, daily injections of Trx-1 shifted the phenotype pattern of lesional macrophages in these animals to predominantly M2 over M1, and the aortic lesion area was significantly reduced (from 100%±18% to 62.8%±9.8%; n=8; P<0.01). Consistently, Trx-1 colocalized with M2 but not with M1 macrophage markers in human atherosclerotic vessel specimens. CONCLUSIONS: The ability of Trx-1 to promote differentiation of macrophages into an alternative, anti-inflammatory phenotype may explain its protective effects in cardiovascular diseases. These data provide novel insight into the link between oxidative stress and cardiovascular diseases.

Effect of mutations in LDLR and PCSK9 genes on phenotypic variability in Tunisian familial hypercholesterolemia patients.

BACKGROUND: Autosomal dominant hypercholesterolemia (ADH) is commonly caused by mutations in the low-density lipoprotein (LDL) receptor gene (LDLR), in the apolipoprotein B-100 gene (APOB), or in the proprotein convertase subtilisin kexine 9 gene (PCSK9). ADH subjects carrying a mutation in LDLR present highly variable plasma LDL-cholesterol (LDL-C). This variability might be due to environmental factors or the effect of some modifying genes such as PCSK9 and APOE. AIMS: We investigated the molecular basis of thirteen Tunisian ADH families and attempted to determine the impact of PCSK9 and APOE gene variations on LDL-cholesterol levels and on the variable phenotypic expression of the disease. METHODS AND RESULTS: Fifty six subjects were screened for mutations in the LDLR gene through direct sequencing. The causative mutation was found to segregate with the disease in each family and a new frameshift mutation, p.Met767CysfsX21, was identified in one family. The distribution of total- and LDL-cholesterol levels, adjusted for age and gender, among homozygous and heterozygous ADH patients varied widely. Within seven families, nine subjects presented low LDL-cholesterol levels despite carrying a mutation in the LDLR gene. To identify the molecular actors underlying this phenotypic variability, the PCSK9 gene was screened using direct sequencing and/or enzymatic restriction analysis, and the apo E genotypes were determined. A new missense variation (p.Pro174Ser) in the PCSK9 gene was identified and characterized as a new putative loss-of-function mutation. CONCLUSION: Genetic variations in PCSK9 and APOE genes could explain only part of the variability observed in the phenotypic expression in Tunisian ADH patients carrying mutations in the LDLR gene. Other genetic variants and environmental factors very probably act to fully explain this phenotypic variability.

Amino-functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages.

Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH(2)) of ∼100 nm in diameter, but not carboxyl- or nonfunctionalized particles, trigger NLRP3 inflammasome activation and subsequent release of proinflammatory interleukin 1ß (IL-1ß) by human macrophages. PS-NH(2) induced time-dependent proton accumulation in lysosomes associated with lysosomal destabilization, release of cathepsin B, and damage of the mitochondrial membrane. Accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. Upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (TXNIP). Liberated TXNIP, in turn, interacted with the NLRP3 protein, resulting in a conformational change of the pyrin domain of the NLRP3 protein, as was predicted by molecular modeling. Consequently, this prompted assembly of the NLRP3 inflammasome complex with recruitment and activation of caspase-1, inducing IL-1ß release by cleavage of pro-IL-1ß. The central role of the NLRP3 inflammasome for cytokine production was confirmed by in vitro knockdown of NLRP3 and of the adaptor protein ASC, confirming that other inflammasomes were not activated by PS-NH(2). The PS-NH(2)-mediated proinflammatory macrophage activation could be antagonized by the radical scavenger N-acetyl-L-cysteine, which prevented mitochondrial damage, caspase-1 activation, and the subsequent release of IL-1ß. Our study reveals the molecular mechanism of NLRP3 inflammasome activation by amino-functionalized nanoparticles and suggests a strategy as to how such adverse effects could be antagonized.

Peroxisome proliferator-activated receptor gamma induces apoptosis and inhibits autophagy of human monocyte-derived macrophages via induction of cathepsin L: potential role in atherosclerosis.

Macrophages play a pivotal role in the pathophysiology of atherosclerosis. These cells express cathepsin L (CatL), a cysteine protease that has been implicated in atherogenesis and the associated arterial remodeling. In addition, macrophages highly express peroxisome proliferator-activated receptor (PPAR) γ, a transcription factor that regulates numerous genes important for lipid and lipoprotein metabolism, for glucose homeostasis, and inflammation. Hence, PPARγ might affect macrophage function in the context of chronic inflammation such as atherogenesis. In the present study, we examined the effect of PPARγ activation on the expression of CatL in human monocyte-derived macrophages (HMDM). Activation of PPARγ by the specific agonist GW929 concentration-dependently increased the levels of CatL mRNA and protein in HMDM. By promoter analysis, we identified a functional PPAR response element-like sequence that positively regulates CatL expression. In addition, we found that PPARγ-induced CatL promotes the degradation of Bcl2 without affecting Bax protein levels. Consistently, degradation of Bcl2 could be prevented by a specific CatL inhibitor, confirming the causative role of CatL. PPARγ-induced CatL was found to decrease autophagy through reduction of beclin 1 and LC3 protein levels. The reduction of these proteins involved in autophagic cell death was antagonized either by the CatL inhibitor or by CatL knockdown. In conclusion, our data show that PPARγ can specifically induce CatL, a proatherogenic protease, in HMDM. In turn, CatL inhibits autophagy and induces apoptosis. Thus, the proatherogenic effect of CatL could be neutralized by apoptosis, a beneficial phenomenon, at least in the early stages of atherosclerosis.

Matrix metalloproteinase-1 and matrix metalloproteinase-12 gene polymorphisms and the outcome of coronary artery disease.

OBJECTIVES: In this study, we investigated the association between matrix metalloproteinase-1 (MMP-1) G-1607GG, MMP-12 A-82G and MMP-12 A1082G genotypes and haplotypes and the prognosis of coronary artery disease (CAD). METHODS: A total of 129 Tunisian patients with CAD were followed prospectively for a median of 2.5 years. Genotypes were determined by a PCR-based restriction fragment length polymorphism. Two endpoints were considered: restenosis and incidence of clinical vascular events (restenosis, myocardial infarction, stroke, cardiac death). RESULTS: Genotypes of MMP-1 G-1607GG, MMP-12 A-82G and MMP-12 A1082G were not associated with the incidence of restenosis or clinical events. Analysis of haplotypes consisting of alleles of MMP-1 G-1607GG and MMP-12 A1082G showed that the rate of clinical events was significantly higher in patients carrying the GG-A haplotype than those with other haplotypes (0.637 vs. 0.424, respectively, odds ratio=1.45; 95% confidence interval=1.04-2.04; P<0.05; P adjusted for multiple risk factors). However, after Bonferroni correction for multiple comparisons, this difference did not reach statistical significance (P=0.093), showing that there was a tendency for the association between the GG-A haplotype and future clinical events in patients with CAD. CONCLUSION: These findings showed a trend of the GG-A haplotype of MMP-1 G-1607GG/MMP-12 A1082G towards the prediction of future clinical events in patients with CAD and suggested a possible importance of these loci in the prediction of the prognosis of CAD. Studies with large sample size are warranted to better investigate this association, as MMP genotyping could aid in identifying patients who are likely to have unfavourable prognosis.