Effect of PPAR-ß/δ agonist GW0742 treatment in the acute phase response and blood-brain barrier permeability following brain injury.

The systemic response to ischemic stroke is associated with the hepatic acute phase response (APR) that modulates leukocytes recruitment to the injured brain. The inappropriate recruitment of leukocytes to the brain parenchyma can result in blood-brain barrier (BBB) breakdown. Emerging data suggest that peroxisome proliferator-activated receptor beta/delta (PPAR-ß/δ) activation has a potential neuroprotective role in ischemic stroke. However, mechanisms of PPAR-ß/δ mediated protection in ischemic insults remain unclear. In the present study, we determined for the first time, the effects of GW0742, a PPAR-ß/δ agonist on the APR following brain injury and assessed the effects on BBB permeability and tight junction integrity via claudin-5, occludin, and zona occludens-1 expression. C57/BL6 mice were exposed to 1 hour of ischemia and received 10 minutes before reperfusion either a vehicle solution or GW0742. Hepatic expression of chemokines (C-X-C motif ligand: CXCL1, CXCL2, and CXCL10), serum amyloid A-1, tumor necrosis factor alpha, interleukin-1ß, and interleukin-6 was measured, and the extent of brain and hepatic neutrophil infiltration was determined. The results showed that GW0742 treatment decreased infarct volume and edema, reactant production and neutrophil recruitment to the brain and liver, which is a hallmark of the APR. GW0742 significantly reduced BBB leakage and metalloproteinase 9 expression and upregulated the expression of tight junction proteins. These findings may help to guide the experimental and clinical therapeutic use of PPAR-ß/δ agonists against brain injury.

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.

Gene Variant and Level of IL-1ß in Ischemic Stroke Patients With and Without Type 2 Diabetes Mellitus.

Evidence is emerging that inflammation plays a key role in the pathophysiology of ischemic stroke (IS). The aim of this study was to explore, for the first time, the relationship between IL-1ß -31 T/C polymorphism and the risk of ischemic stroke (IS) among patients with type 2 diabetes mellitus (T2DM). One hundred ninety-six patients with IS (117 diabetics and 79 nondiabetics) and 192 controls were recruited to enroll in this study. IL-1ß genotyping was performed by PCR-RFLP technique. After adjusting for sex, age, smoking, obesity, dyslipidemia, and hypertension, there was no significant difference in the distribution of IL-1ß -31 T/C genotypes and allele frequencies between IS patients with or without type 2 diabetes mellitus and control group (p > 0.05). Moreover, a significant positive correlation between serum IL-1ß level and glucose (p1 = 0.044) was showed. In addition, serum levels of IL-1ß were found to be higher among TT genotype carriers than TC and CC genotype carriers in ischemic stroke patients with or without T2DM but these differences were not significant. These results indicate that IL-1ß gene polymorphism might not be a risk factor in the development of ischemic stroke in Tunisian population.

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.

Genomic characterization of two deletions in the LDLR gene in Tunisian patients with familial hypercholesterolemia.

Autosomal Dominant Hypercholesterolemia (ADH) is due to defects in the LDL receptor gene (LDLR), the apolipoprotein B-100 gene (APOB) or the proprotein convertase subtilisin/kexin type 9 gene (PCSK9). The aim of this study was to identify and to characterize the ADH-causative mutations in two Tunisian families. Analysis of the LDLR gene was performed by direct sequencing, multiplex ligation-dependent probe amplification (MLPA) and by long range PCR and sequencing. The PCSK9 gene was analysed by direct sequencing and the APOB gene was screened for the most common mutation: p.Arg3527Gln. In the LDLR gene, we found two large deletions and characterized their exact extent and breakpoint sequences. The first one is a deletion of 12,684 bp linking intron 1 to intron 5: g.11205052_11217736del12684. The second deletion spans 2364 bp from intron 4 to 6: g.11216885_11219249del2364. Sequence analysis of each deletion breakpoint indicates that intrachromatid non-allelic homologous recombination (NAHR) between Alu elements is involved. These two large rearrangements in the LDLR gene are the first to be described in the Tunisian population, increasing the spectrum of ADH-causative mutations.

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.

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.

Moderate phenotypic expression of familial hypercholesterolemia in Tunisia.

BACKGROUND: Autosomal Dominant Hypercholesterolemia (ADH) is an autosomal dominant disease caused by mutations in the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Xanthomas and coronary heart diseases (CHD) at an early age are the major clinical manifestations of the disease. METHODS: 16 families with familial hypercholesterolemia from different regions in Tunisia participated in the study. Mutations within the LDLR gene were screened through DNA sequencing. Lipids values were measured by standard enzymatic methods. RESULTS: We present here thirty five homozygotes and fifty six heterozygotes. Homozygotes presented extensive xanthomatosis, variable clinical manifestations of CHD, and total cholesterol levels in males and females of 17.26+/-4.18 and 17.64+/-2.59 mmol/L respectively. HDL-cholesterol levels were 0.62+/-0.24 and 1.00+/-0.61 mmol/L for males and females, respectively. None of the heterozygotes had tendon xanthomas (except for one female aged 62), eight had corneal arcus, and nine developed CHD mean between 46 and 88 years old. Total cholesterol levels in males and females ranged from 4.60 to 8.90 and from 4.30 to 10.50 mmol/L, respectively. CONCLUSION: Tunisian FH heterozygotes are characterized by a moderate clinical and biological expression of the disease.

[Metalloproteinases: therapeutic target in atherosclerosis]. / Les métalloprotéases: cibles thérapeutiques dans l'athérosclérose.

Matrix metalloproteinases are a family of enzymes which collectively can cleave all components of the extracellular matrix. In physiological situations, the expression of matrix metalloproteinases is very low. The increase of their expression leads to several diseases as atherosclerosis, restenosis, rheumatoid arthritis and cancers. In atherosclerosis, metalloproteinases are implicated in the rupture of the atheromatous plaque and contribute to acute vascular accident. Consequently, several studies hypothesized that the inhibition of matrix metalloproteinases activity could reduce the volume of the atheromatous plaque and prevent its destabilisation and therefore could be useful in the treatment of atherosclerosis. However, clinical results have so far been inconclusive because matrix metalloproteinases inhibitors are not very specific. The development of selective inhibitors and gene transfer approaches may better suit the treatment of atherosclerosis.

Apolipoprotein E knockout mice over-expressing human tissue inhibitor of metalloproteinase 1 are protected against aneurysm formation but not against atherosclerotic plaque development.

AIMS: We investigated the effect of plasma levels of human tissue inhibitor of metalloproteinase (hTIMP)-1 on arterial lesion development and aneurysm formation in apolipoprotein-E-deficient mice (ApoE(-/-)). METHODS: Control and transgenic mice were fed either a chow diet or a high-fat diet for 90 and 180 days. RESULTS: hTIMP-1 has a tendency to decrease atherosclerotic lesions, but did not attain significance (approximately 6% reduction in hTIMP-1(+/+), p = 0.075, and approximately 4% in hTIMP-1(+/0), p = 0.088 vs. control). Immunohistological and histological analyses revealed a reduction in macrophage accumulation (23% of control in hTIMP(+/0), p = 0.065, and 49% of control in hTIMP(+/+), p < 0.05) but not in collagen degradation within the lesion in transgenic mice. Moreover, elastin degradation in sites of pseudo-microaneurysms was reduced in transgenic mice (37% of control in hTIMP-1(+/0), p < 0.05, and 50% of control in hTIMP-1(+/+), p < 0.05). DNA array analysis of matrix metalloproteinase (MMP) expression followed by real-time PCR quantification revealed a significant up-regulation of MMP-3, MMP-12 and MMP-13 in arterial lesions of ApoE(-/-) mice fed a high-fat diet in comparison with the same mice fed a chow diet. CONCLUSION: These data show that hTIMP-1 reduces aneurysm formation in ApoE(-/-) mice but does not protect them against the development of arterial lesions.