Marcelo H. Petri

The role of the FPR2/ALX receptor in atherosclerosis development and plaque stability

Aims The formyl peptide receptor (FPR) subtype FPR2/ALX transduces pro-inflammatory responses and participates in the resolution of inflammation depending on activation. The aim of the present study was to unravel the role of FPR2/ALX signalling in atherosclerosis. Methods and results Expression of FPR2/ALX was analysed in 127 human carotid atherosclerotic lesions and revealed that this receptor was expressed on macrophages, smooth muscle cells (SMCs), and endothelial cells. Furthermore, FPR2/ALX mRNA levels were significantly up-regulated in atherosclerotic lesions compared with healthy vessels. In multiple regression, age, creatinine, and clinical signs of increased cerebral ischaemia were independent predictors of FPR2/ALX expression. To provide mechanistic insights into these observations, we generated Ldlr−/−xFpr2−/− mice, which exhibited delayed atherosclerosis development and less macrophage infiltration compared with Ldlr−/−xFpr2+/+ mice. These findings were reproduced by transplantation of Fpr2−/− bone marrow into Ldlr−/− mice and further extended by in vitro experiments, demonstrating a lower inflammatory state in Fpr2−/− macrophages. FPR2/ALX expression correlated with chemo- and cytokines in human atherosclerotic lesions and leucocytes. Finally, atherosclerotic lesions in Ldlr−/−xFpr2−/− mice exhibited decreased collagen content, and Fpr2−/− SMCs exhibited a profile of increased collagenase and decreased collagen production pathways. Conclusion FPR2/ALX is proatherogenic due to effects on bone marrow-derived cells, but promoted a more stable plaque phenotype through effects on SMCs. Taken together, these results suggest a dual role of FPR2/ALX signalling in atherosclerosis by way of promoting disease progression and but increasing plaque stability.

Intermittent hypoxia-activated cyclooxygenase pathway: role in atherosclerosis

Intermittent hypoxia, the main stimulus of obstructive sleep apnoea (OSA), induces inflammation, leading to early atherosclerosis. Whether the cyclooxygenase (COX) pathway contributes to intermittent hypoxia-induced atherosclerosis remains to be determined. We studied the effects of 8-weeks of intermittent hypoxia exposure on COX-pathway gene expression and atherosclerosis, and the influence of COX-1 inhibition by SC-560 on atherosclerosis progression in aortas of apolipoprotein E-/- mice. Urinary 11-dehydrothromboxane B2 (11-dTXB2) was assessed in 50 OSA subjects free of cardiovascular risk factor matched for age and body mass index with 25 controls, and 56 OSA with cardiovascular risk factor. Intermittent hypoxia significantly increased atherosclerotic lesion sizes, mRNA levels of COX-1 and thromboxane synthase (TXBS). Lesion sizes correlated to COX-1 (r = 0.654, p = 0.0003) and TXBS (r = 0.693, p<0.0001) mRNA levels. COX-1 inhibition reduced lesion progression in intermittent hypoxia mice only (p = 0.04). Urinary 11-dTXB2 was similar in OSA subjects free of cardiovascular risk factor and controls, but was increased by 13% (p = 0.007) in OSA subjects with cardiovascular risk factor compared with those without. Although OSA itself was not associated with increased urinary 11-dTXB2 concentration, the COX-1 pathway was activated in intermittent hypoxia-exposed mice and in OSA subjects presenting with cardiovascular risk factor, and may contribute to intermittent hypoxia-induced atherogenesis. COX-1 inhibition could be of clinical interest in the prevention of cardiovascular morbidity in OSA.

Leukotriene B4 pathway activation and atherosclerosis in obstructive sleep apnea

Leukotriene B4 (LTB4) production increases in obstructive sleep apnea syndrome (OSA) and is linked to early vascular remodeling, the mechanism of which is unknown. The objective of this study was to to determine the molecular mechanisms of LTB4 pathway activation in polymorphonuclear cells (PMNs) and early vascular remodeling in OSA and the specific contribution of intermittent hypoxia (IH). PMNs were isolated from 120 OSA patients and 33 healthy subjects and used for measurements of LTB4 production, determination of mRNA and protein expression levels, or exposed for four cycles of in vitro IH. PMNs derived from OSA patients exhibited increased LTB4 production, for which apnea-hypopnea index was an independent predictor (P=0.042). 5-Lipoxygenase-activating protein (FLAP) mRNA and protein increased significantly in PMNs from OSA patients versus controls and were associated with carotid luminal diameter and intima-media thickness. LTB4 (10 ng/ml) increased IL-6 (P=0.006) and MCP-1 (P=0.002) production in OSA patient monocytes. In vitro exposure of PMNs from controls to IH enhanced FLAP mRNA levels (P= 0.027) and induced a 2.7-fold increase (P=0.028) in LTB4 secretion compared with PMNs exposed to normoxia. In conclusion, upregulation of FLAP in PMNs in response to IH may participate in early vascular remodeling in OSA patients, suggesting FLAP as a potential therapeutic target for the cardiovascular morbidity associated with OSA.

Implications in the difference of anti-Mi-2 and -p155/140 autoantibody prevalence in two dermatomyositis cohorts from Mexico City and Guadalajara

IntroductionAutoantibodies and clinical manifestations in polymyositis/dermatomyositis (PM/DM) are affected by both genetic and environmental factors. The high prevalence of DM and anti-Mi-2 in Central America is thought to be associated with the high UV index of the area. The prevalences of autoantibodies and the clinical manifestations of PM/DM were evaluated comparing two cohorts in Mexico.MethodsNinety-five Mexican patients with PM/DM (66 DM, 29 PM; 67 Mexico City, 28 Guadalajara) were studied. Autoantibodies were characterized by immunoprecipitation using 35S-methionine labeled K562 cell extract. Clinical information was obtained from medical records.ResultsDM represented 69% of PM/DM and anti-Mi-2 was the most common autoantibody (35%), followed by anti-p155/140 (11%); however, anti-Jo-1 was only 4%. The autoantibody profile in adult-onset DM in Mexico City versus Guadalajara showed striking differences: anti-Mi-2 was 59% versus 12% (P = 0.0012) whereas anti-p155/140 was 9% versus 35% (P = 0.02), respectively. A strong association of anti-Mi-2 with DM was confirmed and when clinical features of anti-Mi-2 (+) DM (n = 30) versus anti-Mi-2 (-) DM (n = 36) were compared, the shawl sign (86% versus 64%, P < 0.05) was more common in the anti-Mi-2 (+) group (P = 0.0001). Levels of creatine phosphokinase (CPK) were higher in those who were anti-Mi-2 (+) but they responded well to therapy.ConclusionsAnti-Mi-2 has a high prevalence in Mexican DM and is associated with the shawl sign and high CPK. The prevalence of anti-Mi-2 and anti-p155/140 was significantly different in Mexico City versus Guadalajara, which have a similar UV index. This suggests roles of factors other than UV in anti-Mi-2 antibody production.

IL-17 Increased in the Third Trimester in Healthy Women with Term Labor

Citation Martínez‐García EA, Chávez‐Robles B, Sánchez‐Hernández PE, Nuñez‐Atahualpa L, Martín‐Máquez BT, Muñoz‐Gómez A, González‐López L, Gámez‐Nava JI, Salazar‐Páramo M, Dávalos‐Rodríguez I, Petri MH, Zuñiga‐Tamayo D, Vargas‐Ramírez R, Vázquez‐Del Mercado M. IL‐17 Increased in the third trimester in healthy women with term labor. Am J Reprod Immunol 2011; 65: 99–103

Inhibitors of MAPK Pathway ERK1/2 or p38 Prevent the IL-1β-induced Up-regulation of SRP72 Autoantigen in Jurkat Cells

Phosphorylation is the most important post-translational event at a cellular level that is regulated by protein kinases. MAPK is a key player in the important cellular signaling pathway. It has been hypothesized that phosphorylation might have a role in the induction of break tolerance against some autoantigens such as SRP72. The aim of this study was to explore the pathways of phosphorylation and overexpression of the SRP72 polypeptide, using an in vitro model of Jurkat cells stimulated by recombinant human (rh)IL-1β in the presence of MAPK inhibitors. We used Jurkat cells as a substrate stimulated with rhIL-1β in the presence of MAPK inhibitors at different concentrations in a time course in vitro experiment by immunoprecipitation, immunoprecipitation-Western blotting, and real time PCR. Our results showed that rhIL-1β causes up-regulation of protein expression and phosphorylation of SRP72 in Jurkat cells. Inhibitors of the MAPK pathway ERK1/2 or p38α/β down-regulate the expression of SRP72 autoantigen in Jurkat cells stimulated by rhIL-1β. Our results highlight the importance of studying the pathways of activation and overexpression of autoantigens. It will be necessary to perform careful research on various kinases pathways, including MAPK in dermatomyositis and other rheumatic diseases, to help to explain the routes of activation and inhibition of autoantigens. The understanding of this process may help to develop new therapies to prevent and control the loss of tolerance toward own normal proteins.

Autoantibodies to transcription intermediary factor (TIF)1β associated with dermatomyositis

IntroductionMyositis specific autoantibodies are associated with unique clinical subsets and are useful biomarkers in polymyositis/dermatomyositis (PM/DM). A 120 kD protein recognized by certain patients with DM was identified and clinical features of patients with this specificity were characterized.MethodsThe 120 kD protein recognized by a prototype serum was purified and identified by mass spectrometry and immunological methods. Autoantibody to this 120 kD protein was screened in sera from 2,356 patients with various diagnoses from four countries, including 254 PM/DM, by immunoprecipitation of 35S-methionine labeled K562 cell extracts. Clinical information of patients with this specificity was collected.ResultsThe 120 kD protein, which exactly comigrated with PL-12, was identified as transcription intermediary factor TIF1β (TRIM28) by mass spectrometry and validated by immunoassays. By immunofluorescence, anti-TIF1β positivity showed a fine-speckled nuclear staining pattern. Four cases of anti-TIF1β were identified; all are women, one each in a Japanese, African American, Caucasian, and Mexican individual. Three had a diagnosis of DM and one case was classified as having an undifferentiated connective tissue disease with an elevated CPK but without significant muscle symptoms. This individual also had a history of colon cancer, cervical squamous metaplasia and fibroid tumors of the uterus. Myopathy was mild in all cases and resolved without treatment in one case. The anti-TIF1β specificity was not found in other conditions.ConclusionsAnti-TIF1β is a new DM autoantibody associated with a mild form of myopathy. Whether it has an association with malignancy, as in the case of anti-TIF1γ, or other unique features will need to be evaluated in future studies.

Genotype Ser413/Ser of PAI-2 polymorphism Ser413/Cys is associated with anti-phospholipid syndrome and systemic lupus erythematosus in a familial case: comparison with healthy controls.

Background: We describe a family with a 7‐year‐old proband case diagnosed with systemic lupus erythematosus (SLE) plus secondary anti‐phospholipid syndrome (APS) as well as two affected paternal aunts. We compared the frequency of these polymorphisms with healthy controls. Objectives: To evaluate the mode of inheritance in this familial case of APS and SLE and the possible association of plasminogen activator inhibitor‐1 (PAI‐1) −675 4G/5G and PAI‐2 Ser413/Cys polymorphisms. To compare the genotype frequency of these polymorphisms with the results found in a Mexican Mestizo population. Methods:PAI‐1 −675 4G/5G and PAI‐2 Ser413/Cys were determined by the polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) technique using Bsl I and Mwo I on four generations of the family studied. PAI‐2 Ser413/Cys polymorphism was also determined in 50 healthy individuals of Mexican Mestizo origin. Results: The family pedigree demonstrated that this family did not follow a Mendelian inheritance pattern. When the PAI‐2 Ser413/Cys polymorphism was examined, we found that 60% (3/5) of the relatives homozygous to Ser413/Ser were affected with SLE and/or APS (p = 0.027). The proband case was 4G/5G genotype for the PAI‐1 −675 4G/5G polymorphism. No differences between healthy controls of the Mexican Mestizo population and the family studied for the PAI‐2 Ser413/Cys polymorphism or PAI‐1 −675 4G/5G polymorphisms were found. Conclusions: Our data indicate that this family did not follow the Mendelian inheritance pattern. The Ser413/Ser genotype demonstrated in 60% of the affected members (3/5) of this family might increase the risk for autoimmune syndromes such as APS or SLE.

Aspirin‐triggered lipoxin A4 inhibits atherosclerosis progression in apolipoprotein E−/− mice

Atherosclerosis is characterized by a chronic non‐resolving inflammation in the arterial wall. Aspirin‐triggered lipoxin A4 (ATL) is a potent anti‐inflammatory mediator, involved in the resolution of inflammation. However, the therapeutic potential of immune targeting by means of ATL in atherosclerosis has not previously been explored. The aim of the present study was to determine the effects of ATL and its receptor Fpr2 on atherosclerosis development and progression in apolipoprotein E deficient (ApoE−/−) mice.

Docosahexaenoic acid supplementation modifies fatty acid incorporation in tissues and prevents hypoxia induced-atherosclerosis progression in apolipoprotein-E deficient mice.

The n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), displays anti-inflammatory properties that may prevent atherosclerosis progression. Exposure of apolipoprotein-E deficient (ApoE(-/-)) mice to chronic intermittent hypoxia (CIH) accelerates atherosclerosis progression. Our aim was to assess DHA-supplementation influence on fatty acid incorporation in different tissues/organs and on atherosclerosis progression in ApoE(-/-) mice exposed to CIH. ApoE(-/-) mice were exposed to CIH or normoxia (N) and randomized to four groups (N control, CIH control, N+DHA, and CIH+DHA). DHA-supplementation enhanced DHA and reduced arachidonic acid (AA) contents in tissues/organs. CIH control mice exhibited increased atherosclerosis lesion sizes compared to N control mice. DHA prevented CIH induced atherosclerosis but did not improve atherosclerosis burden in N mice. Aortic matrix metalloproteinase-2 (MMP-2) expression was decreased in CIH+DHA mice (p=0.007). DHA-supplementation prevented CIH-induced atherosclerosis acceleration. This was associated with a decrease of AA incorporation and of aortic MMP-2 gene expression.