Impact of cathepsin D activity and C224T polymorphism (rs17571) on chronic obstructive pulmonary disease: correlations with oxidative and inflammatory markers.

BACKGROUND: Cathepsin D (CTSD) is an aspartyl proteinase that plays an important role in protein degradation, antigen processing and apoptosis. It has been associated with several pathologies such as cancer, Alzheimer's disease and inflammatory disorders. Its function in lung diseases remains, however, controversial. In the current study, we determined CTSD activity in serum of patients with chronic obstructive pulmonary disease (COPD) and evaluated the correlations between this proteinase and inflammatory and oxidative parameters. We also investigated the impact of a CTSD C224T polymorphism on enzyme activity and clinicopathological parameters. METHODS: Our population included 211 healthy controls and 138 patients with COPD. CTSD activity, MMPs (-1/-7/-12), cytokines (IL-6, TNF-α), malondialdehyde (MDA), nitric oxide and peroxynitrite levels were measured in patients and controls using standard methods. Genotyping of CTSD C224T polymorphism was determined using PCR-RFLP. RESULTS: Our results showed an increased CTSD activity in COPD patients compared to healthy controls (4.87 [3.99-6.07] vs. 3.94 [2.91-5.84], respectively, p < 0.001). COPD smokers presented also a higher CTSD activity when compared to nonsmokers (4.91[3.98-6.18] vs. 4.65[4.16-5.82], respectively, p = 0.01), while no differences were found when subjects were compared according to their GOLD stages. The activity of this proteinase was not dependent on the C224T polymorphism because we did not found any influence of this SNP on proteinase activity among patients and controls. Furthermore, our data provide the first evidence of the interrelationships between CTSD activity and both MMPs and TNF-α levels (MMP-1[r = - 0.4; p = 0.02], MMP-7[r = 0.37; p = 0.04], MMP-12[r = 0.43; p = 0.02], TNF-α [r = 0.89, p = 0.001]) in COPD smokers. There were no correlations, however, between CTSD activity and oxidative stress parameters in controls and patients. CONCLUSION: Our findings suggest that CTSD could be a relevant marker for COPD disease. Alteration of CTSD activity may be related to increased MMPs and TNF-α levels, particularly in COPD smokers.

Alterations in acetylcholinesterase and butyrylcholinesterase activities in chronic obstructive pulmonary disease: relationships with oxidative and inflammatory markers.

This work focused on finding a relationship between acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities and the development and severity of COPD. The possible link of these enzymes to oxidative and inflammatory processes was also investigated. The study included 229 healthy controls and 153 COPD patients. Erythrocyte AChE and plasma BChE activities were determined using spectrophotometric methods. Markers related to the oxidative status including thiobarbituric acid-reactive substances (TBARS), total protein carbonyls (PCs), advanced oxidation protein products (AOPP), reduced glutathione, nitric oxide, and peroxynitrite were measured. We also evaluated the activity of glutathione peroxidase, catalase, and superoxide dismutase in the plasma and erythrocytes. Serum levels of IL-6 and TNF-α were measured by the enzyme-linked immunosorbent assay. COPD patients showed increased AChE and BChE activities in comparison to healthy controls. Interestingly, AChE activity was higher in COPD smokers than in nonsmokers, while no difference was revealed for BChE. In addition, our results showed an inverse correlation between AChE activity and the levels of IL-6 in COPD smokers. Positive correlations were found, in COPD smokers, between plasma BChE activity and the levels of several biomarkers of protein oxidative damage including AOPP and PC. Our findings suggest that the alterations in AChE and BChE activities may be related to the oxidative and inflammatory processes in COPD patients rendering these enzymes as markers of COPD disease.

MMP-3 (-1171 5A/6A; Lys45Glu) variants affect serum levels of matrix metalloproteinase (MMP)-3 and correlate with severity of COPD: A study of MMP-3, MMP-7 and MMP-12 in a Tunisian population.

BACKGROUND: The present study aimed to examine the role of matrix metalloproteinase (MMP)-3 [(-1171) 5A/6A; Lys45Glu (A/G)], MMP-7 [(-181) A/G] and MMP-12 [(-82) A/G; Asn357Ser (A/G)] variants in the development and severity of chronic obstructive pulmonary disease (COPD) in Tunisians. METHODS: Plethysmography was performed in all participants to measure forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and FEV1/FVC parameters. Genotyping of MMP-3, MMP-7 and MMP-12 polymorphisms was carried out in 138 patients with COPD and 216 healthy controls using a polymerase chain reaction-restriction fragment length polymorphism. Serum levels of MMPs and cytokines (interleukin-6, tumor necrosis factor-α) were determined by an enzyme-linked immunosorbent assay. RESULTS: No significant correlations were observed between genetic variations in MMP-3, MMP-7 and MMP-12 and the risk of development of COPD. Additionally, no impact of MMP-7 (-181) A/G and MMP-12 [(-82) A/G; Asn357Ser (A/G)] polymorphisms was observed on the respective protein levels and clinical parameters of the disease. Interestingly, both MMP-3 (-1171) 5A/6A and Lys45Glu (A/G) variants were associated with respiratory function, as well as with serum levels of MMP-3 in COPD patients. A relationship was found between the (-1171) 6A and 45Glu (G) alleles of the MMP-3 gene and enhanced airflow limitation among COPD patients. Additionally, carriers of the 6A6A and 45 GG genotypes present higher MMP-3 levels than noncarriers. CONCLUSIONS: MMP-3 (-1171) 5A/6A and Lys45Glu (A/G) polymorphisms were associated with the decline of lung function among COPD patients. These results could be linked to the upregulation of MMP-3 in serum from COPD patients carrying the (-1171) 6A and 45 G homozygous genotypes.

The -786 T/C polymorphism of NOS3 gene is a susceptibility marker of COPD among Tunisians that correlates with nitric oxide levels and airflow obstruction.

OBJECTIVE: The goal of this study was to examine the role of G894T (rs1799983), -786T/C (rs3918161) and a 27 bp variable number of tandem repeats (VNTR) 4B/4A of NOS3 gene on the risk and severity of COPD. METHODS: The study included 194 controls and 138 COPD patients. NOS3 G894T, -786T/C and 4B/4A variants were determined by PCR analysis based on the banding pattern on gel electrophoresis. Pulmonary function was evaluated using body plethysmography. The levels of nitric oxide, peroxynitrite and lipid peroxides (T-BARS) were determined using spectrophotometric methods. Levels of serum IL-6, TNF-α and TGFß were determined by ELISA. RESULTS: In case-control studies, both G894T and -786T/C variants were associated with COPD risk. A significantly increased risk of COPD was found with the NOS3894T and -786C alleles (OR:1.93, P=0.001; OR:2.05, P=0.001, respectively). No significant impact of the G894T and 4B/4A SNPs was found on COPD severity, while a significant correlation was retrieved between the NOS3 -786T/C variation and advanced stages (OR: 1.89, P=0.009). In addition, COPD patients with the -786CC genotype exhibited lower FEV1% values in comparison to -786TT carriers (48±3.28 vs. 58.06±2.3, P=0.01, respectively). Patients having the -786CC genotype presented lower plasma levels of nitric oxide and higher T-BARS in comparison to -786TT individuals (173.22±13.4 vs. 228.93±16.8, P=0.01; 1.8±0.15 vs. 1.22±0.15, P=0.01, respectively). CONCLUSION: This study provides the first evidence for the association of G894T, -786T/C variants with COPD risk among Tunisians. The -786T/C variation correlates with enhanced airflow limitation. This finding could be related to altered levels of nitric oxide and enhanced lipid peroxides among patients carrying the -786CC genotype.

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.

Alteration in systemic markers of oxidative and antioxidative status in Tunisian patients with asthma: relationships with clinical severity and airflow limitation.

OBJECTIVE: This study aims to determine the systemic oxidant-antioxidant status in Tunisian patients with asthma. METHODS: We evaluated the levels of malondialdehyde (MDA) as thiobarbituric acid complexes, total protein carbonyls (PCs) and advanced oxidation protein products (AOPP). The levels of total thiols, protein sulfhydryls, glutathione (GSH), together with hydrogen peroxide, ascorbic acid, iron and total antioxidant status (TAS) were colorimetrically estimated. Glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities were assessed in plasma and erythrocytes by spectrophotometry. We also determined the levels of nitric oxide (NO) and peroxynitrite in plasma from asthmatic patients and healthy controls. The volume of fractionated exhaled NO (FeNO) was evaluated by the Medisoft HypAir method. Estimation of DNA damage was determined using the comet assay. RESULTS: Asthmatic patients showed increased levels of MDA in comparison to healthy controls (p < 0.001), while no significant difference was found in protein carbonyls (p = 0.79) and AOPP (p = 0.98). Patients with asthma also had significantly lower levels of total thiols (355.9 ± 15.72 versus 667.9 ± 22.65, p < 0.001), protein sulfhydryls (333.99 ± 16.41 versus 591.95 ± 24.28, p < 0.001) and glutathione (p < 0.001). They also showed decreased GSH-Px activity (p < 0.001), whereas no significant differences in measurements of catalase and SOD enzyme activities were observed between the two groups (respectively, p = 0.06 and p = 0.55). In addition, ascorbic acid and nitric oxide levels were decreased in asthmatics in comparison to controls (p < 0.01). CONCLUSIONS: Our findings highlight that oxidative stress and defective anti-oxidative status are major alterations in Tunisian patients with asthma.

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.

Increased oxidative stress and altered levels of nitric oxide and peroxynitrite in Tunisian patients with chronic obstructive pulmonary disease: correlation with disease severity and airflow obstruction.

This study was aimed to evaluate the oxidant-antioxidant imbalance in the pathogenesis of chronic obstructive pulmonary disease (COPD) in Tunisians. We assessed 16 parameters related to the oxidative status that include malondialdehyde (MDA), total protein carbonyls (PCs), and advanced oxidation protein products (AOPP). We also examined the activity of glutathione peroxydase (GSH-Px), catalase, and superoxide dismutase (SOD) in the plasma and erythrocytes. Levels of total thiols, reduced glutathione (GSH), total antioxidant status (TAS), hydrogen peroxide, ascorbic acid, iron, and protein sulfhydryls were determined using spectrophotometry. We also evaluated the level of nitric oxide (NO) and peroxynitrite in plasma from COPD patients and healthy controls. Estimation of DNA damage was determined using the comet assay. Pulmonary functional tests were performed by body plethysmography. Levels of MDA, PC, DNA damage, and AOPP were significantly increased while total thiols, GSH, and TAS were decreased in COPD patients. GSH-Px activity was higher in COPD patients while no difference was found for catalase and SOD. We also observed a lower level of NO and peroxynitrite in COPD patients. Decreased levels of peroxynitrite were found to correlate with disease progression, as well as with forced expiratory volume in 1 s/forced vital capacity among COPD patients. Multivariate analysis revealed that NO is associated with pathological pathways that help to predict patient outcome independently of the degree of airflow obstruction. These results indicate the presence of a systemic oxidative stress and highlight the importance of NO and peroxynitrite as major effectors in COPD development and airflow obstruction.