ANALYSIS OF THE CORRELATIONS BETWEEN OXIDATIVE STRESS, GELATINASES AND THEIR TISSUE INHIBITORS IN THE HUMAN SUBJECTS WITH OBSTRUCTIVE SLEEP APNEA SYNDROME

Obstructive sleep apnea syndrome (OSAS) is a sleep disorder characterized by repeated partial or complete obstructions of upper airways during sleep with consequent apnea or hypopnea, intermittent arterial oxygen desaturation and sleep disruption (1). OSAS affects especially middle-aged and elderly subjects and its prevalence is increasing worldwide (2).

OSAS is significantly and independently associated with an increased risk of cardiovascular diseases, cerebrovascular events and all-cause mortality and some studies have demonstrated that the incidence of cardiovascular events is related to its severity (3-5). Atherosclerosis is common in OSAS (6), and the elevated mortality is associated with the severity of the atherosclerosis (7). The mechanisms leading to the development and the progression of atherosclerotic plaques involve multiple factors, including oxidative stress, endothelial dysfunction, and inflammatory factors. The continued hypoxia-reoxygenation episodes have a key role in the pathogenesis of the endothelial dysfunction: the intermittent hypoxia may induce the production of reactive oxygen species (ROS) that contribute to the generation of adhesion molecules, leukocyte activation, and to an enhanced systemic inflammation (8).

In particular, evaluating the oxidative/antioxidant status of subjects with OSAS, several authors have observed an increase in lipid (9-11) and protein oxidation (9, 12) and a decrease in nitric oxide (NO) metabolites (13), and in antioxidant defenses (9, 10, 14), even if other authors did not find any difference in plasma lipid peroxidation, total antioxidant capacity and protein carbonyl levels between OSAS subjects and controls (15). In addition, an altered expression of some matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs) has been described in subjects with OSAS (16-22). MMPs, and in particular gelatinases (MMP-2 and MMP-9), are involved in the atherosclerotic lesion development and progression (23, 24). MMPs and oxidative stress seem to be strongly correlated in subjects with high cardiovascular risk (25, 26, 28-31). The link between oxidative stress and MMPs has been demonstrated in several experimental models (32-34): peroxynitrite, in the presence of gluthation, activates some MMPs via the S-glutathiolation of the cystein in the propeptide domain (26, 34) but, at higher concentrations, can lead to the inactivation of MMP-2 (34). Also hydrogen peroxide (H2O2) activates MMP-2 and promotes the expression of MMP-2 and MMP-9 in human venous endothelial cells (35). The activation of MMPs via S-nitrosylation is still unclear, even if a role of NO has been suggested by some authors (27). In addiction, ROS can influence MMP transcription influencing the activity of the mitogen-activated protein kinase (MAPK), of the MAPK phosphatase or of the histone deacetylase (36).

Previously we have evaluated the behavior of lipid peroxidation and protein oxidation (37), the nitric oxide metabolites and the erythrocyte deformability (38) and also the gelatinases and their inhibitors in OSAS subjects (in press); the aim of this research was to examine some parameters of the oxidative status and their possible relationships with gelatinases and TIMPs in the same group of subjects with OSAS.

MATERIALS AND METHODS

Patients

We consecutively recruited 48 subjects (36 men and 12 women; mean age 50.3 ± 14.68 years) with obstructive sleep apnea syndrome from those with suspected OSAS referred to our center. OSAS was diagnosed after a 1-night cardiorespiratory sleep study: apneas were defined as the cessation of airflow for ≥10 seconds and hypopneas were defined as a transient reduction of breathing ≥ 50% with an oxygen desaturation of ≥3% or as a reduction of breathing ≥ 30% with an oxygen desaturation of ≥4% for ≥10 seconds. Obstructive apneas and hypopneas were distinguished from central events by the detection of respiratory efforts during the event. AHI was defined as the number of obstructive apneas and hypopneas per hour of sleep. Patients with an AHI ≥5 were considered as affected by OSAS and then they were subdivided according to the AHI value in two subgroups: Low (L = 21 subjects with AHI < 30) and High (H = 27 subjects with AHI > 30). Therefore the Low subgroup included subjects with mild to moderate OSAS, while the H subgroup included the subjects with severe OSAS. Means and S.D. of age, BMI, waist and neck circumference, AHI, oxygen desaturation index (ODI), and mean nocturnal SO2 (mSO2) are reported in Table 1. Twenty-three of the OSAS subjects had arterial hypertension, 10 had diabetes mellitus and 6 had cardiovascular disease (history of myocardial infarction or stroke). Each subject gave the informed consent and the study was approved by the Ethical Committee.

Table 1. Means ± S.D. of age, anthropometric characteristic and OSAS parameters in the two subgroups of OSAS patients.

***P < 0.001 versus L-OSAS (Student’s ‘t’ test for unpaired data). BMI, body mass index; mSO2, mean oxygen saturation; AHI, apnea/hypopnea index; ODI, oxygen desaturation index.

On fasting venous blood, collected by puncture from the antecubital vein of each subject after the night of cardiorespiratory sleep study and immediately transferred to glass tube anticoagulated with EDTA-K3, we evaluated lipid peroxidation, protein carbonyl (PC) groups, total antioxidant status (TAS), nitric oxide metabolites (NOx), gelatinases (MMP-2 and -9) and their tissue inhibitors (TIMP-1 and -2).

Lipid peroxidation

Lipid peroxidation was evaluated in plasma by detection of thiobarbituric acid-reactive substances (TBARS), generated by peroxidative processes, which include lipid peroxides and malonildialdehyde. The evaluation of TBARS was made by fluorimetry, using 1,1,3,3-tetramethoxypropane as standard.

Protein carbonyl (PC) groups

The PC groups were measured by an enzyme-linked immunosorbent assay (ELISA) kit (BioCell PC test kit, Enzo Life Sciences AG, Switzerland).

Total antioxidant status (TAS)

TAS was obtained using an Assay kit (Calbiochem, La Jolla, USA) which relies on the ability of plasma antioxidant substances to inhibit the oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline sulfonic acid) (ABTS) to the radical cation ABTS.+ by a peroxidase (39). The radical concentration was measured by spectrophotometry.

Nitric oxide metabolites (NOx)

Considering that in vivo NO has a very short life (less than 0.1 s) and it is converted into nitrite (NO2–), which has a half-life of few minutes, and into the more stable nitrate (NO3–), NOx represents almost only the nitrate concentration. In the laboratory method adopted by us at first nitrate was converted into nitrite by a nitrate reductase, and then nitrite was assessed by spectrophotometry after addition of Griess reagent.

Gelatinases and their inhibitors

Plasma concentrations of gelatinases (MMP-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) were evaluated using respectively the Human MMP-2 ELISA and Human MMP-9 ELISA kit (Boster Biological Technology, LTD) and the Human TIMP-1 ELISA and Human TIMP-2 ELISA kit (Boster Biological Technology, LTD).

Statistical analysis

Data were expressed as means ± S.D. The statistical difference between the L subgroup and the H subgroup of OSAS subjects was estimated using the Student’s “t” test for unpaired data; the correlations were performed employing the linear regression test. The null hypothesis was rejected for P values < 0.05.

RESULTS

First of all, the L and H subgroup of OSAS subjects are significantly different regarding the neck circumference, the mean oxygen saturation and the oxygen desaturation index (Table 1).

In the H subgroup of OSAS subjects we found a significant increase in lipid peroxidation and protein oxidation and a significant decrease in total antioxidant status and in NO metabolites in comparison with the L subgroup (Table 2). Similarly, in the H subgroup of OSAS subjects we observed a significant increase in the plasma concentration of MMP-9 and TIMP-1 in comparison with the L subgroup, while regarding the plasma concentration of MMP-2 and TIMP-2 no statistical difference was observed between the two subgroups (Table 2).

Table 2. Means ± S.D. of oxidative parameters, nitric oxide metabolites, gelatinases and their inhibitors in the two subgroups of OSAS patients.

**P < 0.01 ***P < 0.001 versus L-OSAS (Student’s ‘t’ test for unpaired data).

Considering the aim of this research, we examined all the correlations among the parameters of oxidative status and the parameters of the metalloproteinases profile. From this statistical evaluation was evident that in the L subgroups MMP-9 was positively correlated with TAS and NOx (Table 3, Figs. 3 and 4) while in the H subgroup we found a positive correlation between MMP-9 and TBARS (Table 3, Fig. 1), a positive correlation between MMP-2 and TAS and a negative correlation between TIMP-1 and TBARS (Table 3). In the whole group of OSAS subjects only MMP-9 was positively correlated with TBARS and carbonyl groups while it was negatively correlated with NOx (Table 3, Figs. 1, 2 and 4).

Fig. 1. Correlations between MMP-9 and TBARS in the two subgroups and in the whole group of OSAS patients.

Fig. 2. Correlations between MMP-9 and PC in the two subgroups and in the whole group of OSAS patients.

Table 3. Values of r for linear correlations between oxidative parameters, nitric oxide metabolites, gelatinases and their inhibitors in the two subgroups and in the whole group of OSAS patients.

#P = 0.05, *P < 0.0,5 **P < 0.01, ***P < 0.001 (linear regression).

Fig. 3. Correlations between MMP-9 and TAS in the two subgroups and in the whole group of OSAS patients.

Fig. 4. Correlations between MMP-9 and NOx in the two subgroups and in the whole group of OSAS patients.

In addition we evaluated the correlations among the indicators of oxidative stress, the MMPs profile, and the parameters of OSAS severity in the entire group of OSAS subjects. We found a positive correlation between TBARS and AHI and between TBARS and ODI and a negative correlation between TBARS and mSO2 (Table 4). PC were positively correlated with AHI and ODI and negatively correlated with mSO2 (Table 4), while TAS was negatively correlated with AHI and ODI and positively correlated with mSO2 (Table 4). Regarding the NOx, we noted a negative correlation with AHI and ODI and a positive correlation with mSO2 (Table 4). We also observed a positive correlation between MMP-9 and AHI and between MMP-9 and ODI and a negative correlation between MMP-9 and mSO2 (Table 4); no significant correlation among MMP-2, TIMP-1, TIMP-2 and polysomnographic parameters was found.

Table 4. Values of r for linear correlations between the OSAS parameters and oxidative parameters, nitric oxide metabolites, gelatinases and their inhibitors in the whole group of OSAS patients.

#P = 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 (linear regression).

DISCUSSION

The data of this study confirm the results previously described by us and in fact lipid peroxidation, protein oxidation, total antioxidant status and NO metabolites are significantly influenced by the degree of severity of this syndrome (37, 38). The behavior of oxidative status is dependent in particular on the hypoxia-reoxygenation episodes that characterize OSAS (8, 40). An increased mitochondrial ROS synthesis in endothelial cells exposed to hypoxia has been proved (8). As it is known, in vitro hypoxia induces leukocyte activation (41) and ROS production and some authors (8) have also described an increased ROS synthesis by monocytes and granulocytes from OSAS subjects. ROS indirectly influence several nuclear transcription factors such as NF-κB that leads to an increased production of cytokines and adhesion molecules, and the hypoxia-inducible factor-1α (HIF-1α), that increases the sympathetic activity (8, 40). All these considerations seem to find an equilibrium point when we observe the close positive correlation between TBARS and carbonyl groups (data not shown) as well as the strong negative correlation between TBARS and TAS (data not shown) and between carbonyl groups and TAS (data not shown), especially in the entire group of OSAS subjects.

The increase in NF-κB is associated with the endothelial dysfunction, confirmed by decreased levels of activated endothelial NO synthases (eNOS) (42). This last datum contributes to explain the behavior of NO metabolites in OSAS subjects and in particular why its trend is dependent on its severity degree. As the oxygen is a substrate of NOS, the frequent episodes of desaturation decrease NOS activity; in addition, hypoxia is also responsible for alterations in gene regulation, so it could suppress the transcription of the eNOS gene (43).

On cultured human umbilical vein endothelial cells, an intermittent hypoxia causes significant lower levels of NO, NOS activity and NOS mRNA expression (44), while in animal models it has been proved that the intermittent hypoxia down-regulates the eNOS expression inducing NF-κB activity and the consequent overproduction of TNF-α, which inhibits eNOS expression (45). In OSAS an increased NF-κB may also reduce the levels of activated eNOS and all these premises seem to be confirmed by the negative correlation between TBARS and NOx (data not shown) and between carbonyl groups and NOx (data not shown) in the entire group of OSAS subjects.

As well as for the parameters of the oxidative status, also MMP-9 and TIMP-1 are influenced by the degree of severity of this syndrome; this finding agrees with the data obtained by some authors (16, 18, 19, 22) in adults with OSAS, although it differs from what found by other authors in children with OSAS (17).

The activity of MMPs is regulated by the four TIMPs: TIMP-1 inhibits in particular MMP-9 while TIMP-2 inhibits especially MMP-2 (46) and this prerequisite explains easily the positive correlation between MMP-9 and TIMP-1 and between MMP-2 and TIMP-2 observed in the entire group of OSAS subjects (data not shown). The trend of the gelatinases and their tissue inhibitors may be imputable to their cosecretion or to a compensatory effect (47) and it influences the extracellular matrix remodeling (48, 49).

However, the principal aim of this study has regarded the possible interrelationships between the parameters reflecting the oxidative stress and the gelatinases in OSAS subjects. The intermittent hypoxia that induces the ROS overproduction may contribute to the generation of mediators of inflammation and at the same time may activate, together with other proteases, the MMPs (25, 26, 50). We believe that in OSAS the behavior of the gelatinases is dependent especially on their overproduction stimulated by the hypoxia-reoxygenation events and by some cytokines, such as IL-6 and TNF-α (21, 51-53) and this physiopathological consideration substantiates the significant positive correlation found among TBARS, carbonyl groups and MMP-9 in the whole group of OSAS subjects.

Bearing in mind that OSAS is a clinical condition accompanied by different complications, such as arterial hypertension, coronary disease and cerebrovascular events (3-5, 54), it should be considered if and how the oxidative stress and the MMPs might play a role in the development of these complications. At the same time the literature data underline how the use of cPAP may reduce lipid peroxidation and protein oxidation (55-60) and may increase TAS (61) and NO (58, 62-67) as well as the same treatment may reduce the plasma levels or the production of MMP-9 (18, 19).

Considering the prognosis of these subjects, especially of those with severe OSAS, another aspect that deserves to be underlined is if oxidative stress and gelatinases may be contemplated as pharmacological target in this clinical condition.

In conclusion, we found an alteration of the parameters of the oxidative status and of the MMP profile in OSAS subjects that seems to be more evident in the subgroup of subjects with a severe degree of the disease evaluated according to the AHI. The data of this study moreover show interesting statistical correlations among lipid peroxidation, protein oxidation and MMP-9.

Conflict of interests: None declared.

REFERENCES

1. Epstein LJ, Kristo D, Strollo PJ, Jr, et al. Adult obstructive sleep apnea task force of the American Academy of Sleep Medicine. Clinical guideline for the evaluation, management and long-term care of obstructive sleep apnea in adults. J Clin Sleep Med 2009; 5: 263-276.
2. Qaseem A, Holty JE, Owens DK, et al. Management of obstructive sleep apnea in adults: a clinical practice guideline from the American College of Physicians. Ann Intern Med 2013; 159: 471-483.
3. Wang X, Ouyang Y, Wang Z, Zhao G, Liu L, Bi Y. Obstructive sleep apnea and risk of cardiovascular disease and all-cause mortality: a meta-analysis of prospective cohort studies. Int J Cardiol 2013; 169: 207-214.
4. Marin JM, Carrizo SJ, Vicente E, Agusti AG. Long-term cardiovascular outcomes in men with obstructive sleep apnoea-hypopnoea with or without treatment with continuous positive airway pressure: an observational study. Lancet 2005; 365: 1046-1053.
5. Yaggi HK, Concato J, Kernan WN, Lichtman JH, Brass LM, Mohsenin V. Obstructive sleep apnea as a risk factor for stroke and death. N Engl J Med 2005; 353: 2034-2041.
6. Quercioli A, Mach F, Montecucco F. Inflammation accelerates atherosclerotic processes in obstructive sleep apnea syndrome (OSAS). Sleep Breath 2010; 14: 261-269.
7. Toraldo DM, Peverini F, De Benedetto M, De Nuccio F. Obstructive sleep apnea syndrome: blood viscosity, blood coagulation abnormalities, and early atherosclerosis. Lung 2013; 191: 1-7.
8. Lavie L, Lavie P. Molecular mechanisms of cardiovascular disease in OSAHS: the oxidative stress link. Eur Respir J 2009; 33: 1467-1484.
9. Celec P, Hodosy J, Behuliak M, et al. Oxidative and carbonyl stress in patients with obstructive sleep apnea treated with continuous positive airway pressure. Sleep Breath 2012; 16: 393-398.
10. Murri M, Alcazar-Ramirez J, Garrido-Sanchez L, et al. Oxidative stress and metabolic changes after continuous positive airway pressure treatment according to previous metabolic disorders in sleep apnea-hypopnea syndrome patients. Transl Res 2009; 154: 111-121.
11. Cofta S, Wysocka E, Piorunek T, Rzymkowska M, Batura-Gabryel H, Torlinski L. Oxidative stress markers in the blood of persons with different stages of obstructive sleep apnea syndrome. J Physiol Pharmacol 2008; 59 (Suppl. 6): 183-190.
12. Kimoff RJ, Hamid Q, Divangahi M, et al. Increased upper airway cytokines and oxidative stress in severe obstructive sleep apnoea. Eur Resp J 2010; 38: 89-97.
13. Suzuki YJ, Jain V, Park AM, Day RM. Oxidative stress and oxidant signaling in obstructive sleep apnea and associated cardiovascular diseases. Free Radic Biol Med 2006; 40: 1683-1692.
14. Katsoulis K, Kontakiotis T, Spanogiannis D, et al. Total antioxidant status in patients with obstructive sleep apnea without comorbidities: the role of the severity of the disease. Sleep Breath 2011; 15: 861-866.
15. Ntalapascha M, Makris D, Kyparos A, et al. Oxidative stress in patients with obstructive sllep apnea syndrome. Sleep Breath 2013; 17: 549-555.
16. Chuang LP, Chen NH, Lin SW, Chang YL, Chao IJ, Pang JH. Increased matrix metalloproteinases-9 after sleep in plasma and in monocytes of obstructive sleep apnea patients. Life Sci 2013; l93: 220-225.
17. Kaditis AG, Alexopoulos EI, Karathanasi A, et al. Adiposity and low-grade systemic inflammation modulate matrix metalloproteinase-9 levels in Greek children with sleep apnea. Pediatr Pulmonol 2010; 45: 693-699.
18. Tamaki S, Yamauchi M, Fukuoka A, et al. Production of inflammatory mediators by monocytes in patients with obstructive sleep apnea syndrome. Intern Med 2009; 48: 1255-1262.
19. Tazaki T, Minoguchi K, Yokoe T, et al. Increased levels and activity of matrix metalloproteinase-9 in obstructive sleep apnea syndrome. Am J Respir Crit Care Med 2004; 170: 1354-1359.
20. Volna J, Kemlink D, Kalousova M, et al. Biochemical oxidative stress-related markers in patients with obstructive sleep apnea. Med Sci Monit 2011; 17: CR491-CR497.
21. Vuralkan E, Mutlu M, Firat IH, et al. Changes in serum levels of MDA and MMP-9 after UPF in patients with OSAS. Eur Arch Otorhinolaryngol 2014; 271: 1329-1334.
22. Ye J, Liu H, Li Y, Liu X, Zhu JM. Increased serum levels of C-reactive protein and matrix metalloproteinase-9 in obstructive sleep apnea syndrome. Chin Med J (Engl) 2007; 120: 1482-1486.
23. Liu P, Sun M, Sader S. Matrix metalloproteinases in cardiovascular disease. Can J Cardiol 2006; 22: 25B-30B.
24. Hansson J, Vasan RS, Arnlov J, et al. Biomarkers of extracellular matrix metabolism (MMP-9 and TIMP-1) and risk of stroke, myocardial infarction, and cause-specific mortality: cohort study. PLoS One 2011; 6: e16185. doi: 10.1371/journal.pone.0016185.
25. Kameda K, Matsunaga T, Abe N, et al. Correlation of oxidative stress with activity of matrix metalloproteinase in patients with coronary artery disease. Possible role for left ventricular remodelling. Eur Heart J 2003; 24: 2180-2185.
26. Kandasamy AD, Chow AK, Ali MA, Schulz R. Matrix metalloproteinase-2 and myocardial oxidative stress injury: beyond the matrix. Cardiovasc Res 2010; 85: 413-423.
27. Jacob-Ferreira AL, Schulz R. Activation of intracellular matrix metalloproteinase-2 by reactive oxygen-nitrogen species: consequences and therapeutic strategies in the heart. Arch Biochem Biophys 2013; 540: 82-93.
28. Hayden MR, Sowers JR, Tyagi SC. The central role of vascular extracellular matrix and basement membrane remodeling in metabolic syndrome and type 2 diabetes: the matrix preloaded. Cardiovasc Diabetol 2005; 4: 9.
29. Bittner A, Alcaino H, Castro PF, et al. Matrix metalloproteinase-9 activity is associated to oxidative stress in patients with acute coronary syndrome. Int J Cardiol 2010; 143: 98-100.
30. Kelly PJ, Morrow JD, Ning M, et al. Oxidative stress and matrix metalloproteinase-9 in acute ischemic stroke: the biomarker evaluation for antioxidant therapies in stroke (BEAT-Stroke) study. Stroke 2008; 39: 100-114.
31. Pawlak K, Tankiewicz J, Mysliwiec M, Pawlak D. Systemic levels of MMP2/TIMP2 and cardiovascular risk in CAPD patients. Nephron Clin Pract 2010; 115: c251-c258.
32. Brown DJ, Lin B, Chwa M, Atilano SR, Kim DW, Kenney MC. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis 2004; 10: 281-288.
33. Donnini S, Monti M, Roncone R, et al. Peroxynitrite inactivates human-tissue inhibitor of metalloproteinase-4. FEBS Lett 2008; 582: 1135-1140.
34. Viappiani S, Nicolescu AC, Holt A, et al. Activation and modulation of 72kDa matrix metalloproteinase-2 by peroxynitrite and glutathione. Biochem Pharmacol 2009; 77: 826-834.
35. Koken T, Gursoy F, Kahraman A. Long-term alcohol consumption increases pro-matrix metalloproteinase-9 levels via oxidative stress. J Med Toxicol 2010; 6: 126-130.
36. Kar S, Subbaram S, Carrico PM, Melendez JA. A critical link between free radicals, matrix remodeling and degenerative disease. Respir Physiol Neurobiol 2010; 174: 299-306.
37. Hopps E, Canino B, Calandrino V, Montana M, Lo Presti R, Caimi G. Lipid peroxidation and protein oxidation are related to the severity of OSAS. Eur Rev Med Pharmacol Sci 2014; 18: 3773-3778.
38. Canino B, Hopps E, Calandrino V, Montana M, Lo Presti R, Caimi G. Nitric oxide metabolites and erythrocyte deformability in a group of subjects with obstructive sleep apnea syndrome. Clin Hemorheol Microcirc 2015; 59: 45-52.
39. Lowry OH, Rosebrough NJ, Farr AL. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
40. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci 1993; 89: 407-412.
41. Zirlik S, Hildner KM, Targosz A, et al. Melatonin and omentin: influence factors in the obstructive sleep apnoea syndrome? J Physiol Pharmacol 2013; 64: 353-360.
42. Kent BD, Ryan S, McNicholas WT. Obstructive sleep apnea and inflammation: relationship to cardiovascular co-morbidity. Respir Physiol Neurobiol 2011; 178: 475-481.
43. Jelic S, Padeletti M, Kawut SM, et al. Inflammation, oxidative stress, and repair capacity of the vascular endothelium in obstructive sleep apnea. Circulation 2008; 117: 2270-2278.
44. Ciftci TU, Kokturk O, Demirtas S, Gulbahar O, Bukan N. Consequences of hypoxia-reoxygenation phenomena in patients with obstructive sleep apnea syndrome. Ann Saudi Med 2011; 31: 14-18.
45. Zhao HY, Chen BY, Cao J, Feng J, Guo MN. Effects of obstructive sleep apnea style intermittent hypoxia on endothelin-1, nitric oxide, and nitric oxide synthase in endothelium: experiment with human umbilical vein endothelial cells. Zhonghua Yi Xue Za Zhi 2007; 87: 2189-2192.
46. Wang, Yan B, Son D, Ye X, Liu SF. Chronic intermittent hypoxia down-regulates endothelial nitric oxide synthase expression by an NF-kB-dependent mechanism. Sleep Med 2013; 14: 165-171.
47. Nagareddy P, Rajput P, Vasudevan H, et al. Inhibition of matrix metalloproteinase-2 improves endothelial function and prevents hypertension in insulin resistant rats. Br J Pharmacol 2012; 165: 705-715.
48. Wiera G, Szczot M, Wojtowicz T, Lebida K, Koza P, Mozrzymas JW. Impact of matrix metalloproteinase-9 overexpression on synaptic excitatory transmission and its plasticity in rat CA3-CA1 hippocampal pathway. J Physiol Pharmacol 2015; 66: 309-315.
49. Kiczak L, Tomaszek A, Bania J, et al. Matrix metalloproteinase 9/neutrophil gelatinase associated lipocalin/tissue inhibitor of metalloproteinases type 1 complexes are localized within cardiomyocytes and serve as a reservoir of active metalloproteinase in porcine female myocardium. J Physiol Pharmacol 2014; 65: 365-375.
50. Deardorff R, Spinale FG. Cytokines and matrix metalloproteinases as potential biomarkers in chronic heart failure. Biomark Med 2009; 3: 513-523.
51. Amalinei C, Caruntu ID, Balan RA. Biology of metalloproteinases. Rom J Morphol Embryol 2007; 48: 323-334.
52. Kossakowska AE, Edwards DR, Prusinkiewicz C, et al. Interleukin-6 regulation of matrix metalloproteinase (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) expression in malignant non-Hodgkin’s lymphomas. Blood 1999; 94: 2080-2089.
53. Kondo S, Kubota S, Shimo T, et al. Connective tissue growth factor increased by hypoxia may initiate angiogenesis in collaboration with matrix metalloproteinases. Carcinogenesis 2002; 23: 769-776.
54. Saren P, Welgus HG, Kovanen PT. TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages. J Immunol 1996; 157: 4159-4165.
55. Dong JY, Zhang YH, Qin LQ. Obstructive sleep apnea and cardiovascular risk: meta-analysis of prospective cohort studies. Atherosclerosis 2013; 229: 489-495.
56. Barcelo A, Miralles C, Barbe F, Vila M, Pons S, Agusti AG. Abnormal lipid peroxidation in patients with sleep apnoea. Eur Resp J 2000; 16: 644-647.
57. Lavie L, Vishnevsky A, Lavie P. Evidence for lipid peroxidation in obstructive sleep apnea. Sleep 2004; 27: 123-128.
58. Murri M, Garcia-Delgado R, Alcazar-Ramirez J, et al. Assessment of cellular and plasma oxidative stress in SAHS patients before and after continuous positive airway pressure treatment. Clin Lab 2010; 56: 397-406.
59. Oyama J, Yamamoto H, Maeda T, Ito A, Node K, Makino N. Continuous positive airway pressure therapy improves vascular dysfunction and decreases oxidative stress in patients with the metabolic syndrome and obstructive sleep apnea syndrome. Clin Cardiol 2011; 35: 231-236.
60. Del Ben M, Fabiani M, Loffredo A, et al. Oxidative stress mediated arterial dysfunction in patients with obstructive sleep apnoea and the effect of continuous positive airway pressure treatment. BMC Pulm Med 2012; 12: 36. doi: 10.1186/1471-2466-12-36.
61. Karamanli H, Ozol D, Uzur KS, et al. Influence of CPAP treatment on airway and systemic inflammation in OSAS patients. Sleep Breath 2014; 18: 251-256.
62. Mancuso M, Bonanni E, Lo Gerfo A, et al. Oxidative stress biomarkers in patients with untreated obstructive sleep apnea syndrome. Sleep Med 2012; 13: 632-636.
63. Ip MS, Lam B, Chan LY, et al. Circulating nitric oxide is suppressed in obstructive sleep apnea and is reversed by nasal continuous positive airway pressure. Am J Resp Crit Care Med 2000; 162: 2166-2171.
64. Schulz R, Schmidt D, Lopes-Ribeiro X, et al. Decrease plasma levels of nitric oxide derivatives in obstructive sleep apnoea: response to cPAP therapy. Thorax 2000; 55: 1046-1051.
65. Lavie L, Hefetz A, Luboshitzky R, Lavie O. Plasma levels of nitric oxide and L-arginine in sleep apnea patients. J Mol Neurosci 2003; 21: 57-63.
66. Ohike Y, Kozaki K, Iijima K, et al. Amelioration of vascular endothelial dysfunction in ovstructive sleep apnea syndrome by nasal continuous positive airway pressure. Circ J 2005; 69: 221-226.
67. Alonso-Fernandez A, Garcia-Rio F, Arias MA, et al. Effects of cPAP upon oxidative stress and nitrate deficiency in sleep apnoea. A randomized trial. Thorax 2009; 64: 581-586.