Prostaglandin Endoperoxide H Synthase-2 (PGHS-2) Variants and Risk of Obesity and Microvascular Dysfunction Among Tunisians: Relevance of rs5277 (306G/C) and rs5275 (8473T/C) Genetic Markers.

The purpose of this study was to determine the impact of six PGHS-2 genetic variants on obesity development and microvascular dysfunction. The study included 305 Tunisian subjects (186 normal weights, 35 overweights and 84 obeses). PCR analyses were used for allelic discrimination between polymorphisms. Prostaglandin (PGE2, PGI2), leptin, and matrix metalloproteinase (MMP1, 2, 3, 9) levels were evaluated by ELISA. Fatty acid composition was performed by gas chromatography-mass spectrometry. Our results revealed that subjects carrying the PGHS-2 306CC (rs5277) and 8473CC (rs5275) genotypes present higher anthropometric values compared to wild-type genotypes (306GG, BMI (Kg/m2): 27.11 ± 0.58; WC (cm): 93.09 ± 1.58; 306CC, BMI: 33.83 ± 2.46; WC: 109.93 ± 5.41; 8473TT, BMI: 27.75 ± 0.68; WC: 93.96 ± 1.75; 8473CC, BMI: 33.72 ± 2.2; WC: 117.89 ± 2.94). A reduced microvascular reactivity and a higher PGE2 level were also found in individuals with the 306CC and 8473CC genotypes in comparison to 306GG and 8473TT carriers (306GG, Peak Ach-CVC (PU/mmHg): 0.46 ± 0.03; PGE2 (pg/ml): 7933.1 ± 702; 306CC, Peak Ach-CVC: 0.24 ± 0.01; PGE2: 13,380.3 ± 966.2; 8473TT, Peak Ach-CVC: 0.48 ± 0.05; PGE2: 7086.41 ± 700.31; 8473CC, Peak Ach-CVC: 0.23 ± 0.01; PGE2: 13,175.7 ± 1165.8). Fatty acid analysis showed a significant increase of palmitic acid (PA) (34.2 ± 2.09 vs. 16.82% ± 1.76, P < 0.001), stearic acid (SA) (25.76 ± 3.29 vs. 9.05% ± 2.53, P < 0.001), and linoleic acid (LA) (5.25 ± 1.18 vs. 0.5% ± 0.09, P < 0.001) levels in individuals carrying the PGHS-2 306CC genotype when compared to GG genotype individuals. Subjects with the 8473CC genotype showed also a significant increase of PA, SA ,and LA levels when compared to TT genotype carriers (PA: 38.02 ± 1.51 vs. 12.65% ± 1.54, P < 0.001; SA: 32.96 ± 1.87 vs. 1.38% ± 0.56, P < 0.001; LA: 26.84 ± 2.09 vs. 3.7% ± 1.54, P < 0.001). Logistic regression analysis revealed that PGHS-2 306CC and 8473CC variants are significantly associated with obesity status (OR 6.25, CI (1.8-21.6), P = 0.004; OR 3.01, CI (1.13-8.52), P = 0.03, respectively). Haplotypes containing the C306:T8473 (OR 2.91; P = 0.01) and G306:C8473 (OR 5.25; P = 0.002) combinations were associated with an enhanced risk for obesity development in the studied population. In conclusion, our results highlight that PGHS-2 306G/C and 8473T/C variants could be useful indicators of obesity development, inflammation, and microvascular dysfunction among Tunisians.

Concomitant elevations of MMP-9, NGAL, proMMP-9/NGAL and neutrophil elastase in serum of smokers with chronic obstructive pulmonary disease.

A growing body of evidence points towards smoking-related phenotypic differences in chronic obstructive pulmonary disease (COPD). As COPD is associated with systemic inflammation, we determined whether smoking status is related to serum levels of matrix metalloproteinase-9 (pro- and active MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) and the proMMP-9/NGAL complex in patients with COPD. Serum samples were collected in 100 stable-phase COPD patients (82 smokers, 18 never-smokers) and 28 healthy adults (21 smokers, 7 never-smokers). Serum levels of studied factors were measured in ELISA. Our data provide the first evidence of simultaneously elevated serum levels of MMP-9, NGAL and proMMP-9/NGAL in COPD smokers. While the triad discriminated between smokers and non-smokers in the COPD group, MMP-9 and proMMP-9/NGAL (but not NGAL) discriminated between smokers with and without COPD. Adjustment for age and smoking pack-years did not alter the findings. Serum MMP-9, NGAL and proMMP-9/NGAL levels were not correlated with the GOLD stage or FEV1 decline. Furthermore, serum levels of neutrophil elastase (NE) and MMP-3 (but not of IL-6 and MMP-12) were also higher in COPD smokers than in healthy smokers before and after adjustment for age and pack-years. Among COPD smokers, levels of MMP-9, NGAL and proMMP-9/NGAL were positively correlated with NE (P < 0.0001) but not with the remaining factors. Gelatin zymography detected proMMP-9 in serum samples of healthy and COPD smoking groups. Our results suggest that associated serum levels of proMMP-9, NGAL, proMMP-9/NGAL and NE may reflect the state of systemic inflammation in COPD related to cigarette smoking.

MMP-2 (-1306 C/T) Polymorphism Affects Serum Matrix Metalloproteinase (MMP)-2 Levels and Correlates with Chronic Obstructive Pulmonary Disease Severity: A Case-Control Study of MMP-1 and -2 in a Tunisian Population.

OBJECTIVE: The aim of this study was to determine the role of MMP-1 (-1607 1G/2G; -519 A/G) and MMP-2 (-1306 C/T; -735 C/T) polymorphisms in the development and severity of chronic obstructive pulmonary disease (COPD) in Tunisian patients. We also evaluated the impact of these genetic variants on serum levels of the corresponding proteins. METHODS: The study included 138 patients with COPD and 216 healthy controls. Pulmonary function was evaluated using body plethysmography, and COPD severity was determined based on forced expiratory volume in 1 s (FEV1%). MMP-1 and MMP-2 variants were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while serum matrix metalloproteinase (MMP)-1 and -2 levels were determined by enzyme-linked immunosorbent assay (ELISA) and activity of MMP-2 was determined by gelatin zymography. RESULTS: No significant associations were found between genetic variations in MMP-1 and MMP-2 variants and the risk of development of COPD. Additionally, no significant impact of the MMP-1 (-1607 1G/2G; -519 A/G) and MMP-2 (-735 C/T) polymorphisms was observed on the respective protein levels and clinical parameters of the disease. Interestingly, a significant correlation was identified between the MMP-2 (-1306) C/T and disease severity [p = 0.01; Bonferroni corrected p value (p c) = 0.04]. Increased levels of MMP-2 were also identified in patients with the MMP-2 (-1306) CC genotype compared with those with CT and TT genotypes (105 [84.69-121.5] vs. 86.29 [80.99-92.62] ng/ml; p = 0.01, p c = 0.04). Additionally, MMP-2 activity was enhanced in patients carrying the CC genotype compared with those carrying the T variant (p = 0.01, p c = 0.02). CONCLUSION: Our data suggest that, although MMP-1 (-1607 1G/2G; -519 A/G) and MMP-2 (-735 C/T) may not affect COPD risk and clinical parameters, the MMP-2 (-1306C/T) variant was correlated to COPD severity. These findings could be related to alterations in the level and activity of MMP-2 in serum from patients carrying the (-1306) CC genotype.

Functional G894T (rs1799983) polymorphism and intron-4 VNTR variant of nitric oxide synthase (NOS3) gene are susceptibility biomarkers of obesity among Tunisians.

OBJECTIVE: The endothelial nitric oxide synthase (NOS3) has been shown to play a role in the modulation of lipolysis. The goal of this study was to examine the impact of the G894T (rs1799983) and a 27 bp variable number of tandem repeats (VNTR 4a/b) of NOS3 gene on obesity in a sample of the Tunisian population. RESEARCH METHODS AND PROCEDURES: The study included 211 normal weight subjects and 183 obese patients. NOS3 G894T and 4a/b variants were determined by PCR analysis and examined for association with obesity-related traits. The effect of obesity on forearm skin blood flow (FSBF) response to acetylcholine, an endothelium-dependent vasodilator was determined by laser Doppler iontophoresis. RESULTS: In case-control studies, both G894T and 4a/b variants were associated with obesity. A significantly increased risk of obesity was found with the NOS3(G894T) TT genotype (OR:2.62, P=0.04). This association remains significant after adjustments for age and gender (OR: 2.93, P=0.03). A higher risk was also observed for carriers of the G894T allele (OR: 1.72, P=0.001). Stratified analysis by gender revealed that obese men (but not women) had significantly higher frequency of TT genotypes compared to controls (9.9% vs. 2.9%, P=0.01). Carriers of the 4b allele presented a significantly higher risk of obesity than non-carriers even after adjustments for age and gender (OR (95%CI): 1.72 (1.16-2.56), P=0.004). Correlations with anthropometric parameters revealed that carriers of TT and bb genotypes had significantly higher body mass index compared to those homozygous for the G and a alleles (P=0.0004). CONCLUSION: This study provides the first evidence for the association of G894T and 4a/b variants with body mass index and the risk of obesity in Tunisians. These polymorphisms did not exhibit, however any significant association with both metabolic traits and vascular function.

Matrix Metalloproteinase-9 (279R/Q) Polymorphism is Associated with Clinical Severity and Airflow Limitation in Tunisian Patients with Chronic Obstructive Pulmonary Disease.

BACKGROUND: The aim of this study was to investigate the role of matrix metalloproteinase-9 (MMP-9) C-1562T and 279R/Q (836G>A) polymorphisms in the development of chronic obstructive pulmonary disease (COPD) in Tunisians and to determine their impact on disease progression and airflow obstruction. METHODS: Pulmonary functional tests were evaluated by body plethysmography. MMP-9 genotypes were determined in patients with COPD (n = 138) and healthy controls (n = 216) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Serum MMP-9 and interleukin-6 (IL-6) levels were determined by enzyme-linked immunosorbent assays (ELISA) and activity of MMP-9 was evaluated by gelatin zymography. RESULTS: No significant association was found between genetic variations in MMP-9 C-1562T and 279R/Q polymorphisms and the risk of development of COPD. However, a significant correlation was retrieved between the 279 R/Q polymorphism and disease severity (P = 0.02). In addition, homozygous Q (A) genotype was associated with a poorer lung function with a fall in forced expiratory volume in 1 s (FEV1) (%) and forced vital capacity (FVC%) among COPD patients compared with both AG and GG individuals (52.06 ± 19.6 vs. 59.08 ± 17.19, P = 0.03 and 72.41 ± 21.42 vs. 82.98 ± 16.48, P = 0.002, respectively). Using ELISA, a higher level of MMP-9 was found in patients with the CT genotype (P = 0.03), while no significant impact of the 279R/Q polymorphism was observed (P = 0.48). In contrast, by using zymography gel analysis, MMP-9 activity was enhanced in individuals carrying the R(G) allele in comparison with those homozygous for the Q(A) variant (P = 0.02). CONCLUSION: Our results support a role for the 279R/Q polymorphism in physiological alterations that may affect progression and severity of COPD. These findings could be related to the decreased activity of MMP-9 among COPD patients carrying the 279Q variant.

Assessment of the clinical significance of antigenic and functional levels of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinomas.

OBJECTIVES: To determine the clinical significance of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinoma patients. DESIGN AND METHODS: Serum levels of α1-Pi, tryptic specific inhibitory capacity and α1-Pi circulating immune complexes were determined using radial immunodiffusion, BAPNA assays and ELISA, respectively. 2-DE-MS and immunohistochemistry were performed to examine α1-Pi protein expression. RESULTS: A decreased serum level of α1-Pi was found among breast cancer patients in comparison to controls. In addition, we found a significantly decreased mean level of α1-Pi in the node metastatic group when compared to node negative patients. However, the functional activity of the inhibitor did not decrease proportionately. Through 2-DE analyses, a differential expression of α1-Pi isoforms according to tumor stage and node metastatic development was found. CONCLUSIONS: Both α1-Pi levels and specific activity could be a source of complementary clinical information and may provide useful information for a better understanding of the mechanisms of metastasis.

An elongation factor-like protein (EF-Tu) elicits a humoral response in infiltrating ductal breast carcinomas: an immunoproteomics investigation.

OBJECTIVES: In the current study, we have used an immunoproteomics approach to identify proteins that commonly elicit a humoral response in patients with infiltrating ductal carcinomas of the breast. DESIGN AND METHODS: Sera obtained at the time of diagnosis from 40 patients with invasive breast cancer and 42 healthy controls were screened for the presence of IgG antibodies to MCF-7 cell line proteins using a serological proteomics-based approach. RESULTS: An immunoreactive protein detected in sera from 21 of 40 patients was isolated and subsequently identified as elongation factor-Tu. CONCLUSIONS: The immunoproteomic approach implemented here offers a powerful tool for determining novel tumor antigens that induce a humoral immune response in cancer patients. From our findings, the immunoreactive EF-Tu protein and/or the related circulating antibodies may display clinical usefulness as potential diagnostic markers and provide a means for a better understanding of the molecular mechanisms underlying breast cancer development.

Functional vascular endothelial growth factor -2578 C/A polymorphism in relation to nasopharyngeal carcinoma risk and tumor progression.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and a potent regulator of angiogenesis and inflammatory processes in nasopharyngeal carcinomas. In the current report, we designed a case-controlled study to evaluate whether a genetically predetermined variation in the VEGF expression may affect susceptibility and prognosis. METHODS: A PCR and restriction fragment length polymorphism analysis was used to determine the variation of the -2578 C/A promoter region in a Tunisian population of patients with nasopharyngeal carcinomas (NPC) and in healthy control subjects. RESULTS: A significantly risk of NPC was observed for carriers of VEGF -2578 C allele (OR=1.4; P=0.03). Regarding prognostic indicators, a significant association was found between -2578 C allele carriers and the aggressive forms of NPC as defined by large tumor size (OR=2.29; P=0.0002) and advanced tumor stages (OR=1.97; P=0.02). Moreover, an association was ascertained between the VEGF polymorphism and gender. CONCLUSIONS: This is the first report on the studies of functional VEGF polymorphisms in NPC and our preliminary data suggest that this genetic variant may play a role in mediating susceptibility to NPC, as well as, in neoplastic progression, supporting our hypothesis for VEGF involvement in NPC etiology.

Matrix metalloproteinase-1 (-1607) 1G/2G and -9 (-1562) C/T promoter polymorphisms: susceptibility and prognostic implications in nasopharyngeal carcinomas.

BACKGROUND: Matrix metalloproteinases (MMPs) are proteolytic enzymes that play important roles in tumor invasion and metastasis by degrading extracellular matrix components. Genetic variations in promoter regions of MMP genes, affecting their expression, have been associated with susceptibility to cancers. The aim of this study was to investigate the susceptibility and prognostic implications of the MMP-1 (-1607) 1G/2G and MMP-9 (-1562) C/T polymorphisms in nasopharyngeal carcinomas. METHODS: The variation of the MMP-1 and MMP-9 promoter regions in 174 patients with NPC and 171 healthy control subjects was investigated. Association of the clinico-pathologic parameters and the genetic markers with the rates of the nasopharyngeal carcinoma-specific overall survival and the disease-free survival were assessed using univariate and multivariate analyses. RESULTS: No association was found between genetic variation in MMP-9 and the risk of NPC occurrence. In contrast, a significantly increased risk of NPC was associated with the homozygous MMP-1 (-1607) 2G2G genotype (OR=2.27; p=0.02). A significant association was also found between the 2G2G genotype and the aggressive forms of NPC as defined by large tumor size (T3-T4), lymph node metastasis and advanced stages (III-IV) at the time of diagnosis. Moreover, an association was ascertained between the MMP-1 polymorphism and gender (OR=2.90; p=0.02). In univariate analysis, the MMP-1 (-1607) 2G allele showed a significant association with reduced disease-free survival for NPC patients (p=0.03). CONCLUSIONS: The genetic variation in MMP-1 may represent a marker for the increased risk of nasopharyngeal carcinoma.