Inflammation, Oxidative Stress, Senescence in Atherosclerosis: Thioredoxine-1 as an Emerging Therapeutic Target.

Atherosclerosis is a leading cause of cardiovascular diseases (CVD) worldwide and intimately linked to aging. This pathology is characterized by chronic inflammation, oxidative stress, gradual accumulation of low-density lipoproteins (LDL) particles and fibrous elements in focal areas of large and medium arteries. These fibrofatty lesions in the artery wall become progressively unstable and thrombogenic leading to heart attack, stroke or other severe heart ischemic syndromes. Elevated blood levels of LDL are major triggering events for atherosclerosis. A cascade of molecular and cellular events results in the atherosclerotic plaque formation, evolution, and rupture. Moreover, the senescence of multiple cell types present in the vasculature were reported to contribute to atherosclerotic plaque progression and destabilization. Classical therapeutic interventions consist of lipid-lowering drugs, anti-inflammatory and life style dispositions. Moreover, targeting oxidative stress by developing innovative antioxidant agents or boosting antioxidant systems is also a well-established strategy. Accumulation of senescent cells (SC) is also another important feature of atherosclerosis and was detected in various models. Hence, targeting SCs appears as an emerging therapeutic option, since senolytic agents favorably disturb atherosclerotic plaques. In this review, we propose a survey of the impact of inflammation, oxidative stress, and senescence in atherosclerosis; and the emerging therapeutic options, including thioredoxin-based approaches such as anti-oxidant, anti-inflammatory, and anti-atherogenic strategy with promising potential of senomodulation.

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.

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.

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.

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.

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.

25-hydroxycholesterol provokes oligodendrocyte cell line apoptosis and stimulates the secreted phospholipase A2 type IIA via LXR beta and PXR.

In several neurodegenerative diseases of the CNS, oligodendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investigated oxysterol effects on these myelinating glial cells. Natural oxysterol 25-hydroxycholesterol (25-OH; 1 and 10 microM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22(S)-OH (1 and 10 microM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol-induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2-IIA) over-expression partially rescued 158N cells from oxysterol-induced apoptosis. In fact, 25-OH, 24(S)-OH, and T0901317 stimulated sPLA2-IIA promoter and sPLA2 activity in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2-IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 microM) and by LXR beta at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggesting that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR pathway which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.

Inhibition of interleukin-1beta-induced group IIA secretory phospholipase A2 expression by peroxisome proliferator-activated receptors (PPARs) in rat vascular smooth muscle cells: cooperation between PPARbeta and the proto-oncogene BCL-6.

The inflammation that occurs during atherosclerosis is characterized by the release of large amounts of group IIA secretory phospholipase A2 (sPLA2-IIA). This study was designed to define the function of the three peroxisome proliferator-activated receptors (PPARs) on sPLA2 expression in vascular smooth muscle cells (VSMCs). We found that PPAR ligands decreased sPLA2-IIA activity and inhibited mRNA accumulation under inflammatory conditions. Furthermore, interleukin-1beta-induced sPLA2-IIA promoter activity was inhibited by the three PPAR ligands and in a similar way when cells were cotransfected with PPARalpha, PPARbeta, or PPARgamma, plus retinoid X receptor alpha (RXRalpha). Our study revealed that the regulation of sPLA2-IIA gene transcription by PPARalpha/RXR and PPARgamma/RXR heterodimers requires an interaction with a PPAR response element (PPRE) of the sPLA2-IIA promoter. In contrast, PPARbeta operates through a PPRE-independent mechanism. In addition, we demonstrated that VSMCs expressed the transcriptional repressor BCL-6. Overexpression of BCL-6 markedly reduced sPLA2-IIA promoter activity in VSMCs, while a dominant negative form of BCL-6 abrogated sPLA2 repression by PPARbeta. The PPARbeta agonist induced a BCL-6 binding to the sPLA2 promoter in VSMCs under inflammatory conditions. The knockdown of BCL-6 by short interfering RNA abolished the inhibitory effect of the PPARbeta ligand on sPLA2 activity and prostaglandin E2 release. Thus, the inhibition of sPLA2-IIA activity by PPARbeta agonists may provide a promising approach to impacting the initiation and progression of atherosclerosis.

Modifications of arterial phenotype in response to amine oxidase inhibition by semicarbazide.

Semicarbazide-sensitive amine oxidase (SSAO)-deficient mice present no alteration in elastin cross-linking processes and carotid mechanical properties. In contrast, previous studies have shown that SSAO inhibitors induced marked anomalies in arterial structure and function. The aim of the present study was to examine the effect of semicarbazide (SCZ), an efficient SSAO inhibitor, on the arterial phenotype of the carotid artery in relation to modulation of SSAO and lysyl oxidase activities in growing rats. We first show that after 6 weeks of SCZ treatment (100 mg/kg per day), SSAO activity was reduced by 90%, whereas lysyl oxidase activity was only partially inhibited (<60%) in carotid artery, compared with controls. There was significant growth inhibition and no difference in mean arterial pressure but an increase in pulse pressure with a smaller arterial diameter in SCZ-treated rats. SCZ decreased aortic insoluble elastin without a change in total collagen. In addition, extracellular proteins other than insoluble elastin and collagen were increased in SCZ-treated rats. All of the elastic lamellae presented globular masses along their periphery, and focal disorganization was observed in the ascending aorta. Carotid artery mechanical strength was lower in SCZ-treated rats, and the elastic modulus-wall stress curve was shifted leftward compared with controls, indicating increased stiffness. Thus, SCZ modifies arterial geometry and mechanical properties, alters elastic fiber structure, and reduces the content of cross-linked elastin. Because these abnormalities are essentially absent in SSAO-deficient mice, our results suggest that lysyl oxidase inhibition is responsible for the major part of the vascular phenotype of SCZ-treated rats.

Carotid arterial stiffness, elastic fibre network and vasoreactivity in semicarbazide-sensitive amine-oxidase null mouse.

OBJECTIVE: We examined the arterial phenotype of semicarbazide-sensitive amine-oxidase null mouse (SSAO -/-) using various techniques including high resolution echotracking. METHODS AND RESULTS: SSAO -/- mice showed no change in arterial pressure under anesthesia. The in vivo arterial diameter, only measured in the carotid artery (CA), was higher in SSAO -/- than in SSAO +/+ animals. Elastic modulus-wall stress curves and CA rupture pressure were similar between SSAO -/- and +/+ mice, indicating no change in arterial wall stiffness or mechanical strength. There was no significant difference in insoluble elastin, total collagen content and elastic lamellar morphology between the two genotypes. No alteration in vascular reactivity was observed in aortic rings and mesenteric arteries from SSAO -/- mice. Aortic lysyl oxidase (LO) activity remained unaltered, indicating that SSAO invalidation is not accompanied by a compensatory increase in LO activity. CONCLUSION: This is the first functional study of arteries lacking SSAO. Our results indicate that SSAO -/- mice present an increased arterial diameter associated with normal arterial mechanical properties, suggesting that SSAO deficiency might contribute to arterial wall remodeling. However, these results argue against the hypothesis that SSAO intervenes in elastic fibre organization, elastin cross-linking processes and vasoreactivity.

Semicarbazide-sensitive amine oxidase in annulo-aortic ectasia disease: relation to elastic lamellae-associated proteins.

Lysyl oxidases (Lox), which are members of the amine oxidase family, are involved in the maturation of elastic lamellae and collagen fibers. Modifications of amine oxidases in idiopathic annulo-aortic ectasia disease (IAAED) have never been investigated. Our aim was to examine the expression of several proteins that might interfere with elastic fiber organization in control (n=10) and IAAED (n=18) aortic tissues obtained at surgery. Expression of amine oxidases and semicarbazide-sensitive amine oxidase (SSAO), and cellular phenotypic markers were examined by immunohistopathology and confocal microscopy. The expression of these proteins was assessed in relation to clinical and histomorphological features of the arterial wall. In control aorta, SSAO staining was expressed along elastic lamellae, whereas in aneurysmal areas of IAAED, SSAO was markedly decreased, in association with severe disorganization of elastic lamellae. Smooth muscle myosin heavy chain was also decreased in IAAED compared with controls, indicating smooth muscle cell dedifferentiation. Multiple regression analysis showed that elastic lamellar thickness (ELT) was correlated positively with the SSAO:elastin ratio and negatively with the Lox:elastin ratio, and that the clinical features of IAAED (aneurysm, thoracic aorta diameter, and aortic insufficiency) were positively correlated with ELT but not with SSAO. The relationship between SSAO expression and ELT suggests that this amine oxidase may be involved in elastic fiber organization. However, in advanced IAAED, the deficit in SSAO expression could be secondary to the decrease and fragmentation of elastic fibers and/or to vascular smooth muscle cell dedifferentiation.

In vitro suppression of the lipogenic pathway by the nonnucleoside reverse transcriptase inhibitor efavirenz in 3T3 and human preadipocytes or adipocytes.

A serious metabolic syndrome combining insulin-resistance, dyslipidemia, central adiposity, and peripheral lipoatrophy has arisen in HIV-infected patients receiving highly active antiretroviral therapy. The aim of this work was to examine the effects of the nonnucleoside reverse transcriptase inhibitor (NNRTI) efavirenz on adipocyte differentiation and metabolism. When induced to differentiate in the presence of efavirenz (5-50 microm), 3T3-F442A preadipocytes failed to accumulate cytoplasmic triacylglycerol droplets. This phenomenon was rapidly reversible and was also readily detectable in the 3T3-L1 preadipose cell line and in primary cultures of human preadipocytes. When applied to mature 3T3-F442A adipocytes, efavirenz induced a delayed and moderate reduction in cell triglyceride content. Measurement of [(3)H]deoxyglucose uptake, basal and agonist-stimulated lipolysis, and cell viability indicated that these pathways are not involved in efavirenz effects on triacylglycerol accumulation. By contrast, we found that the NNRTI induced a dramatic dose- and time-dependent decrease in gene and protein expression of the lipogenic transcription factor sterol regulatory element-binding protein-1c (SREBP-1c). Adipose conversion was only altered at the highest efavirenz concentrations, as suggested by the mild reduction in peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer-binding protein-alpha. CCAAT/enhancer-binding protein-beta remained unchanged. The inhibition of SREBP-1c expression was accompanied by a sharp reduction in the expression of SREBP-1c target genes and in the adipocyte lipogenic activity in efavirenz-treated cells. Finally, the inhibitory effect of efavirenz on cell triglyceride accumulation was prevented by directly providing free fatty acids to the cells and was reversed by overexpression of a dominant positive form of SREBP-1c, reinforcing the implication of this transcription factor in the antilipogenic effect of the drug. When considered together, these results demonstrate for the first time that the NNRTI efavirenz induces a strong inhibition of the SREBP-1c-dependent lipogenic pathway that might contribute to adipose tissue atrophy.

Regulation of semicarbazide-sensitive amine oxidase expression by tumor necrosis factor-alpha in adipocytes: functional consequences on glucose transport.

Membrane-associated semicarbazide-sensitive amine oxidase (SSAO) is mainly present in the media of aorta and in adipose tissue. Recent works have reported that SSAO activation can stimulate glucose transport of fat cells and promote adipose conversion. In this study, the murine 3T3-L1 preadipose cell line was used to investigate SSAO regulation by tumor necrosis factor-alpha (TNF-alpha), a cytokine that is synthesized in fat cells and known to be involved in obesity-linked insulin resistance. SSAO mRNA and protein levels, and enzyme activity were decreased by TNF-alpha in a dose- and time-dependent manner, without any change of SSAO affinity for substrates or inhibitors. SSAO inhibition caused by TNF-alpha was spontaneously reversed along the time after TNF-alpha removal. The decrease in SSAO expression also occurred in white adipose tissue of C57BL/6 mice treated with mTNF-alpha. Overall, we demonstrated that reduction in SSAO expression induced by the cytokine had marked repercussions on amine-stimulated glucose transport, in a dose- and time-dependent manner. This effect was more pronounced than the inhibiting effect of TNF-alpha on insulin-stimulated glucose transport. Moreover, the peroxisome proliferator-activated receptor gamma agonists thiazolidinediones did not reverse either TNF-alpha effect on amine-sensitive glucose transport or the inhibition of SSAO activity, whereas they antagonized TNF-alpha effects on insulin-sensitive glucose transport. These results demonstrate that TNF-alpha can strongly down-regulate SSAO expression and activity, and through this mechanism can dramatically reduce amine-stimulated glucose transport. This suggests a potential role of this regulatory process in the pathogenesis of glucose homeostasis dysregulations observed during diseases accompanied by TNF-alpha overproduction, such as cachexia or obesity.

Semicarbazide-sensitive amine oxidase in vascular smooth muscle cells: differentiation-dependent expression and role in glucose uptake.

Cultured vascular smooth muscle cells (VSMCs) derived from rat aortic media were used to examine semicarbazide-sensitive amine oxidase (SSAO) expression during their differentiation process. In a defined serum-free medium permissive for in vitro VSMC differentiation, there was a large increase in SSAO mRNA and protein levels and in the related enzyme activity during the course of cell culture. This pattern of expression was concomitant with that of some smooth muscle-specific mRNA markers of differentiation. mRNAs in differentiated cultured VSMCs were comparable to those detected in total aorta and media. Pharmacological properties of SSAO present in VSMCs were similar to enzyme activities previously described in the aortic wall. In this model, we also demonstrated that methylamine, a physiological substrate of SSAO, activated 2-deoxyglucose transport in a time- and dose-dependent manner. This methylamine effect was reproduced by other SSAO substrates and was prevented by the SSAO inhibitor semicarbazide. It was antagonized in the presence of catalase, suggesting that SSAO-activated glucose transport was mediated through H(2)O(2) production. In addition, methylamine promoted glucose transporter 1 accumulation at the cell surface. Thus, we demonstrate for the first time the differentiation-dependent expression of SSAO in VSMCs and its role in the regulation of VSMC glucose uptake.