The A445C Variant in Apelin Receptor and Diabetic Retinopathy in Tunisian Patients.

BACKGROUND: Apelin and apelin receptor (APLN/APLNR) are involved in the retinal neovascularization of diabetic retinopathy (DR). METHODS: This study investigated the impact of the APLNR A445C variant on the risk of DR in a sample of the Tunisian population (100 patients with DR and 105 healthy controls) using PCR-RFLP. RESULTS: The genotype frequencies of the APLNR A445C variant were not significantly different between the patient and control groups. The genotype was not associated with DR (OR = 1.49; 95% CI [0.49 - 4.48], p = 0.47 for the AC heterozygous genotype and OR = 1.57; 95% CI [0.43 - 5.71], p = 0.49 for the CC homozygous genotype). Furthermore, the clinical and biochemical parameters according to the APLNR A445C genotypes revealed that only total cholesterol (TC) was significantly higher in the DR group with the CC genotype compared to the AA genotype (p < 0.02). CONCLUSIONS: The APLNR A445C polymorphism was not associated with DR in a sample of the Tunisian population, but the CC genotype carrier patients with DR had a high TC concentration.

Alpha Adducin G460T Variant is a Risk Factor for Hypertension in Tunisian Population.

BACKGROUND: Adducin is a membrane cytoskeletal protein, consisting of three subunits: α, ß, and γ subunits encoded by three different genes (ADD1, ADD2, ADD3). A specific mutation G460T of the α-adducin gene (ADD1) is associated with high renal tubular sodium reabsorption. This mutation is associated with salt sensitivity and may influence the risk of hypertension. In this study, we investigated the relationship between the G460T polymorphism of the ADD1 and essential hypertension (EH) in the Tunisian population. METHODS: The case-controlled study included 280 patients with hypertension and 257 healthy controls. The G460T polymorphism of ADD1 was determined by polymerase chain reaction-restriction fragment length polymorphism analysis method. RESULTS: In the whole population, the genotypic frequencies of the a-adducin G460T polymorphism in hypertensive and control groups (GG, GT, TT) were 78.6%, 17.5%, 3.9% and 87.5%, 11.29%, 1.16%, respectively (χ2 = 9.13, p < 0.01). The genotype was associated with hypertension, OR = 1.79, 95% CI (1.04 - 3.41), p < 0.03 for GT heterozygous and OR = 1.93, 95% CI (1.39 - 2.22), p < 0.03 for TT homozygous. Moreover, when we stratified the population according to gender, the genotypic frequencies were significantly associated with G460T polymorphism in men (p < 0.01) and in women (p < 0.05). Furthermore, no relationship was found between clinical characteristics and ADD1 G460T genotypes. CONCLUSIONS: The present study shows that the a-adducin G460T polymorphism is associated with EH. Our results suggest that this variant can be considered a genetic risk factor for hypertension in the Tunisian population.

G protein beta3 subunit gene C825T and angiotensin converting enzyme gene insertion/deletion polymorphisms in hypertensive Tunisian population.

BACKGROUND: We investigated the interaction between the G protein beta3 subunit (GNB3) C825T variant and angiotensin converting enzyme (ACE) Insertion/Deletion (I/D) polymorphism in hypertensive Tunisian population. METHODS: Analyses of ACE and GNB3 genotypes were performed in 388 hypertensive patients and 425 healthy controls by polymerase chain reaction-restriction fragment length polymorphism. The plasma ACE activity was determined by a spectrophotometric method. RESULTS: The ACE genotype distribution and allele frequencies were not significantly different between the hypertensive and normotensive subjects (p > 0.05). This polymorphism was not associated with hypertension (HTA) (OR = 0.93, 95% CI = 0.75 - 1.15; p = 0.50). In cases, subjects carrying the DD genotype exhibited higher plasma ACE activity than those with ID and II genotypes (p = 0.001). In this group, a linear regression analysis revealed that the ACE I/D polymorphism is independently associated with plasma ACE activity (p = 0.017). The genotypic distribution and allelic frequencies of the GNB3 C825T polymorphism were not significantly different between the two groups. This polymorphism was found to have no effect on the risk of HTA (OR = 1.14, 95% CI = 0.93 - 1.39; p = 0.21). We did not observe a significant interaction between the GNB3 gene and the ACE gene with HTA. CONCLUSIONS: In this study, the I/D polymorphism is a significant independent predictor for variability of plasma ACE activity but the ACE I/D and GNB3 C825T polymorphisms are not significant factors for HTA in the Tunisian population. Moreover, we found no interaction between ACE D allele and GNB3 825T allele solely or combined with respect to HTA in the Tunisian population.

Contribution of the ACE (rs1799752) and CYP11B2 (rs1799998) Gene Polymorphisms to Atrial Fibrillation in the Tunisian Population.

BACKGROUND: This study investigated the association of angiotensin-converting enzyme (ACE I/D) and aldosterone synthase (CYP11B2-344C/T) gene polymorphisms in the renin-angiotensin-aldosterone system (RAAS) with atrial fibrillation (AF) in the Tunisian population. MATERIALS AND METHODS: The study population included 120 patients with AF and 123 age-matched controls. Genotyping of the I/D polymorphism in the ACE gene and the -344C/T polymorphism in the CYP11B2 gene was performed by polymerase chain reaction (PCR) and PCR-RFLP methods, respectively. RESULTS: The genotype distribution of the ACE I/D and CYP11B2-344C/T polymorphisms was significantly different between AF patients and control participants (p < 0.01 and p < 0.006 respectively). In addition, ACE I/D increased the risk of AF significantly by 3.41-fold for the DD genotype (OR = 3.41; 95% CI [1.39-8.34]; p < 0.007), and after adjusting for confounding factors (age, diabetes, hypertension, and dyslipidemia), the risk was higher (OR = 5.71; 95% CI [1.48-21.98]; p < 0.01). Likewise, the CYP11B2-344C/T polymorphism increased the incidence of AF for the TT genotype (OR = 3.66; 95% CI [1.62-8.27]; p < 0.002) and the CT genotype (OR = 2.68; 95% CI [1.22-5.86]; p < 0.01). After adjusting for confounding factors (age, diabetes, hypertension and dyslipidemia), the risk remained higher for the TT genotype (OR = 3.58; 95% CI [1.08-11.77]; p < 0.03). Furthermore, the haplotype-based association of the ACE I/D and CYP11B2-344C/T polymorphisms showed that the D-T haplotype increased the risk for AF. CONCLUSION: Our study suggests a significant association of the ACE (I/D) and CYP11B2-344C/T polymorphisms with AF in the Tunisian population.

Association of BglII Polymorphism in ITGA2 and (894G/T and -786T/C) Polymorphisms in eNOS Gene With Stroke Susceptibility in Tunisian Patients α2 Gene Polymorphism in α2ß1 Integrin and eNOS Gene Variants and Stroke.

BACKGROUND: This study investigated the association of BglII polymorphism in α2ß1 integrin gene (ITGA2) and eNOS (894G/T and -786T/C) polymorphisms with ischemic stroke (IS) in Tunisian patients. METHODS: The study comprised 210 patients with IS and 208 controls. The genotypes of the BglII polymorphism in ITGA2 and eNOS (894G/T and -786T/C) polymorphisms were determined using the PCR-RFLP. The χ2 test was used and the genotype data comparison included heterozygous groups. Haplotype estimation and multiple logistic regression analysis were performed to analyze the significance of polymorphisms. RESULTS: The genotype distribution of the BglII polymorphism was significantly different between cases and controls (p < 0.004). This polymorphism was associated with the risk of IS (OR = 3.38, p < 0.001) for the BglII(+/+) genotype. Likewise, the genotype distributions of eNOS (894G/T and -786T/C) polymorphisms were significantly different between the two groups (p < 0.005 and p < 0.01, respectively). The 894G/T polymorphism increased the risk of IS for the TT genotype (OR = 2.23, p < 0.008) and the GT genotype (OR = 1.74, p < 0.009). In addition, the -786T/C variant in the eNOS gene was a risk factor for IS for CC homozygous (OR = 2.52, p < 0.005). T-C Haplotype (OR = 3.06) from combination of the eNOS (894G/T and -786T/C) and T-C-BglII(+) haplotype (OR = 2.76) from combination of eNOS and ITGA2 polymorphisms represented high risks for IS. CONCLUSIONS: This study suggests that the BglII variant in ITGA2 is associated with IS susceptibility. Furthermore, the 894G/T and -786T/C polymorphisms in the eNOS gene may be considered as genetic risk factors for IS in the Tunisian population.

Impaired nitric oxide production in patients with primary open-angle glaucoma.

BACKGROUND: Glaucoma is an optic neuropathy induced by many factors. Vascular dysfunction is involved in the mechanism underlying glaucoma. AIM: To determine the involvement of nitric oxide (NO), which is implicated in the regulation of ocular blood flow, in primary open angle glaucoma (POAG). Furthermore, lactate and uric acid (UA) levels were investigated. METHODS: Concentrations of NO, UA and lactate in plasma and aqueous humor (AH) were measured in 214 Tunisian patients (100 patients with POAG and 114 subjects with cataract as control group). NO metabolites, nitrate and nitrite (NOx) production were determined using the Griess reaction. UA and lactate concentrations were measured using enzymatic- colorimetric methods. RESULTS: NOx concentrations in patients with POAG were significantly lower compared to cataract group in plasma (5.23±1.55 µmol/L vs 18.35±6.87 µmol/L, p=0.01) and AH (20.54±7.41 µmol/L vs 45.25±10.92 µmol/L, p=0.02). Plasma and AH levels of lactate and UA were significantly higher in glaucoma patients than in control subjects. CONCLUSIONS: In the present study, decreased NO and increased UA and lactate levels were found in the AH and plasma of POAG patients compared to control subjects. These data suggest a possible involvement of these factors in glaucoma.

Atrial natriuretic peptide and brain natriuretic peptide release in human essential hypertension.

Hypertension is associated with an increase in vasoactive peptides, but conflicting results are reported concerning their causes of elevation. In this study, cardiac vasodilator hormones atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and vasoconstrictor hormones (renin, aldosterone, cortisol, metanephrins) were determined in 36 hypertensive subjects (HT) without left ventricular hypertrophy (LVH), 19 healthy subjects without family hypertension (NTFN) and 35 healthy subjects with family hypertension (NTFH). Plasma levels of ANP and BNP were significantly higher (p<0.04) in HT subjects (28.1 +/- 6.1 and 22.7 +/- 6.8 pg/ml) compared to NTFN (13.4 +/- 3.3 and 6.1 +/- 1.5 pg/ml) and NTFH (12.5 +/- 1.4 and 7.2 +/- 1.3 pg/ml) subjects, respectively. No significant differences were observed in ANP and BNP concentrations between NTFN and NTFH. Measurement of vasoconstrictor hormones showed no significant differences between the three groups. Plasma ANP and BNP concentrations were significantly correlated in both HT (r=0.73; P<0.001), NTFN (r=0.71; P<0.002) and NTFH (r=0.53; P<0.003) subjects. ANP values were significantly related to systolic blood pressure (r=0.34; P<0.05) in the HT group while BNP values were not. The echocardiographic findings were not correlated with ANP or BNP in the HT patients. This suggests that natriuretic peptides increase is related to the blood pressure elevation rather than LVH to reduce detrimental high BP effects.

The Role of Genetic Variants (rs869109213 and rs2070744) Of the eNOS Gene and BglII in the α2 Subunit of the α2ß1 Integrin Gene in Diabetic Retinopathy in a Tunisian Population.

Purpose: In this study, we investigated the association of two polymorphisms (rs869109213 and rs2070744) in the eNOS gene and one polymorphism BglII in the α2ß1 integrin gene (ITGA2) with the risk of diabetic retinopathy (DR) in a Tunisian population. Methods: The study investigated of 110 type 2 diabetes mellitus (T2DM) and 127 DR patients. The genotypes of the eNOS 4b/4a (rs869109213) and -786T/C (rs2070744) polymorphisms and of the BglII polymorphism of ITGA2 were studied using the PCR or PCR-RFLP method. Results: The genotype distributions of the two polymorphisms in eNOS 4b4a and eNOS (-786T/C) were significantly different between T2DM and DR patients (p < .004 and p = .033, respectively). These polymorphisms were associated with the risk of DR (OR = 2.65, 95%CI [1.45-4.84], p = .002) for the eNOS 4b4a genotype and (OR = 2.43, 95%CI [1.06 - 5.56], p = .036) for the CC genotype of the eNOS gene (-786T/C). Similarly, the genotype distribution of the BglII polymorphism was significantly different between the two groups studied (p = .037). This polymorphism was associated with an increased risk of DR (OR = 4.03, 95% CI [1.17 - 7.85], p = .022) for BglII(+/+). Conclusion: The present study suggests that the polymorphisms 4b4a and -786T/C in the eNOS gene might be associated with DR. In addition, the BglII polymorphism in the ITGA2 gene was a risk factor for DR.

Effect and mechanisms underlying scorpion toxin action from Androctonus australis garzonii on atrial natriuretic peptide in rat atria: an in vitro study.

Scorpion envenomation is considered public health problem in Northern African countries. The mechanisms of cardiac dysfunction following scorpion envenomation are not fully understood. This study examined the effect and mechanisms underlying scorpion toxin action from Androctonus australis garzonii on atrial natriuretic peptide (ANP) release from rat atrium using in vitro organ perifusion. Male Sprague Dawley rats were used in this study. Three experiments were conducted. In experiment 1, atrial tissues were exposed either to Krebs-bicarbonate buffer medium (control) or to scorpion toxin (10(-8) M to 10(-6) M). In experiment 2, animals were chemically sympathectomized with a single intraperitoneal injection of 6-hydroxydopamine (6-OHDOPA) at a dose of 40 microg/g 24 h before sacrifice. Vehicle-treated rats served as control. Atrial tissues were collected and perifused in the presence of 10(-6) M scorpion toxin. In experiment 3, atrial tissues were exposed to 10(-6) M scorpion toxin either in the absence or presence of 10(-6) M propranolol (a beta-adrenoceptor blocker), or 10(-6) M tetrodotoxin (a sodium channel blocker). ANP levels released in the perifusion medium were determined by radioimmunoassay. The scorpion toxin at 10(-6) M induced a significant (p<0.01) increase (106%) in ANP levels. This effect was decreased (20%) by 6-OHDOPA. Propranolol and tetrodotoxin significantly (p<0.01) inhibited 55% and 60%, respectively, the toxin-induced ANP release. The data show that the North African scorpion toxin from Androctonus australis garzonii increases the ANP release in rat atrium through stimulation of sympathetic cardiac nerves and sodium channels activation.

Aging and photoperiod affect the daily rhythm pattern of atrial natriuretic peptide in the rat atrium.

This study investigates the release characteristics of atrial natriuretic peptide (ANP) from young (10 weeks) and old (22 months) rat atrium. Levels of ANP release from samples of atrium were studied by organ perifusion. Rats were exposed to light:dark (LD) cycles of 12:12 or 18:6 and sacrificed at different zeitgeber time (ZT) points: ZT0, ZT6, ZT8, ZT12, ZT16, and ZT19 for LD 12:12 or ZT0, ZT9, ZT16, ZT18, ZT20, and ZT 21.5 for LD 18:6. The heart was collected, and the right atrium was removed, weighed, and perifused with Krebs-bicarbonate buffer for 100 min, including a period of 50 min for stabilization of secretion rate. ANP concentrations released by atrium did not differ between the two age groups either under LD 12:12 or under LD 18:6, except at the light:dark transition under LD 12:12 conditions where ANP levels were significantly (P<0.05) lower in young compared to old rats. ANP exhibited daily variations in concentrations under LD 12:12, with a peak during the beginning of photophase (ZT0) in young rats and a peak at the beginning of scotophase (ZT12) in old animals. These variations were strongly modified under LD 18:6, where the pattern of the release exhibited a peak during the light phase at ZT16 in both young and old rats. This strongly suggests that the atrial ANP rhythm is dependent on the environmental light:dark cycle. Moreover, the total ANP levels released by atria in old rats were significantly increased under LD 18:6 compared to standard LD 12:12. This observation strongly suggests that old animals are more sensitive to a photoperiodic change. In conclusion, our results show that ANP concentrations in the rat atrium exhibit daily variations which are significantly affected by the daylength (photoperiod) change in aged rats.

Effect of benzalkonium chloride on trabecular meshwork cells in a new in vitro 3D trabecular meshwork model for glaucoma.

PURPOSE: To validate a new culture model of primary human trabecular meshwork cells (p-hTMCs) using Matrigel®, in order to mimic in vitro 3D-TM organization, and to investigate the proinflammatory effect of benzalkonium chloride (BAK) in 3D p-hTMC cultures. METHODS: p-hTMCs, seeded onto Matrigel®-coated inserts were stimulated with BAK (10-4%), dexamethasone (DEX) (10-6M) or transforming growth factor-beta 2 (TGF-ß2) (5ng/ml) for 48h and observed with confocal microscopy. The BAK effect at 10-4% or 5.10-3% on the gene expressions of interleukin-6 (IL-6), interleukin-8 (IL-8) and matrix metalloproteinase (MMP-9) was investigated using qRT-PCR in 2D and 3D p-hTMC cultures. RESULTS: p-hTMCs seeded in Matrigel® were able to organize themselves in a 3D-spatial conformation in the different conditions tested with cross-linked actin network (CLAN) formation in presence of DEX or TGF-ß2 and intercellular space contraction with TGF-ß2. IL-6 and IL-8 gene expressions increased in presence of BAK in 2D and in 3D p-hTMC cultures. BAK 10-4% only showed a tendency to stimulate MMP-9 expression in p-hTMCs after 24h-recovery. CONCLUSIONS: We investigated this new 3D-TM in vitro model in Matrigel® matrix for pathophysiological and toxicological purposes. It appears as a new promising tool for a better understanding of TM behavior in physiological and stress conditions, as well as toxicological evaluations of antiglaucoma eyedrops and preservatives.

The mechanism of the angiotensin-converting enzyme inhibitor quinapril is not related to bradykinin level in heart tissue.

In order to examine the effect of the angiotensin-converting enzyme inhibitor (ACEi) quinapril, we performed a sensitive and specific radioimmunoassay (RIA) to quantify bradykinin, BK-(1-9), in heart and kidney tissues. The BK-(1-9) level was unaffected in the heart of sham and water-deprived rats treated for 2h with quinapril (10mg/kg), but was significantly higher in the kidneys in the two groups. In these conditions, circulating and tissue angiotensin II (Ang II) levels were significantly decreased by quinapril. Moreover, our results indicated that acute treatment with this dose of quinapril induced kinin-mediated effects which were not related to its action on bradykinin degradation in rat hearts.

Scorpion toxin of Androctonus australis garzonii induces neuropeptide Y release via bradykinin stimulation in rat atria and kidneys.

The ability of scorpion toxins to produce hemodynamic alterations is well documented but all mediators implied in cardiovascular disturbances are not known. In the present investigation we studied the effect of North African Androctonus australis garzonii scorpion toxin on neuropeptide Y (NPY) release from rat atria and kidneys by a perifusion system in vitro. To further understand the mechanisms of the scorpion toxin action on NPY release, the effects of icatibant (HOE 140, a selective bradykinin-B2 receptor antagonist), tetrodotoxin (TTX, a sodium channel antagonist) and diltiazem (a calcium channel antagonist), and the effect of the scorpion toxin on bradykinin (BK, a potent vasorelaxant peptide of the kinin group) release were studied in both tissues. We showed that the scorpion toxin (10(-6)M) increased the NPY release from both atria (35%) and kidneys (40%). This increase was significantly (p<0.001) inhibited by HOE 140 (10(-5)M). The scorpion toxin (10(-6)M) enhanced BK secretion in both atria (52%) and kidneys (55%). Diltiazem (10(-5)M) and TTX (10(-5)M) decreased by 45-75% NPY levels induced by scorpion toxin in both organs. The results show that A. australis garzonii scorpion toxin stimulates NPY release from both rat atria and kidneys, and suggest that the toxin induces NPY release via BK stimulation through B2 receptors. This effect appears to involve calcium and sodium channel activation.