No abstract

The endothelial cell (EC) dysfunction is a common characteristic of various pathologies that include atherosclerosis, hypertension, and Fabry's disease. Aware of the role of eNO and ACE in EC dysfunction, we questioned whether polymorphism of eNOS and/or ACE gene may be a common denominator in these pathologies. Patients with CHD (108), HT (109), Fabry's disease (37) and healthy subjects (control, 141) were genotyped for the eNOSG894T by RFLP‐PCR technique and for eNOS4b/a, and ACEI/D polymorphisms by PCR amplification. The results of these studies were statistically evaluated. Compared to controls, the frequency of the eNOSG894T (T allele) was higher in CHD (P=0.03) and Fabry (P=0.01), while the eNOS4b/a (a allele) in CHD (P=0.01) and HT patients (P=0.01). The proportion of the ACEI/D was similar in all subjects. In CHD patients at “low risk” of atherogenic factors, the frequency of the T and a alleles of eNOS gene was high (P=0.03 and 0.02, respectively). Carriers of the T allele of eNOSG894T were over‐represented (P=0.04) in Fabry subgroup with renal failure. Compared to women, the eNOS894T alleles were more frequent (P=0.03) in men with CHD and HT, whereas ACE I/D in men (P=0.03) with HT. These findings suggest: (i) the frequency of eNOSG894T and/or eNOS4b/a is significantly associated with coronary dysfunction; (ii) eNOS4b/a confers a relatively high risk of hypertension in subjects with atherogenic risk factors; (iii) the frequency of eNOSG894T is high in Fabry hemizygotes with renal complications. Therefore, eNOS gene polymorphism represent a frequent risk factor for vascular abnormalities in CHD, HT and Fabry's disease, afflictions which have in common, the endothelial dysfunction.

The objective of this study was to evaluate whether administration of L-arginine, the substrate for nitric oxide synthesis, was able to ameliorate the endothelial dysfunction and the morphological changes induced by the combined insult of hyperlipemia and hyperglycemia. To this purpose, golden Syrian hamsters were rendered simultaneously hyperlipemic and diabetic (HD group) for 24 weeks, and then orally treated with 622.14 mg/kg per day L-arginine, for 12 weeks (HD + L-arg group). The following assays were carried out: (1) spectrophotometric: concentrations of circulating glucose, cholesterol, and creatinine, the activity of angiotensin-converting enzyme (ACE), and the osmotic fragility of erythrocyte plasmalemma; (2) myographic: the endothelium-dependent and -independent relaxation of the resistance arteries (i.d. 210-250 microm) to 10(-8) to 10(-4) M acetylcholine (ACh) or sodium nitroprusside (SNP); and (3) electron-microscopic: the ultrastructure of the resistance arteries, myocardium, and kidney glomeruli, which are main targets of hypertensive complications. The results showed that oral supplementation with L-arginine in simultaneous hyperlipemia-hyperglycemia induced in hamsters had favorable effects on: (1) homeostasis, i.e., diminished the concentration of circulating glucose (by ~63%) and cholesterol (by approximately 10%), reduced the ACE activity (by approximately 45%), and lowered the osmotic fragility of erythrocyte plasmalemma (as marker for the oxidative stress in plasma); (2) mesenteric resistance arteries, which showed (in 10(-4) M ACh) an improved endothelium-dependent relaxation (72.40+/-4.6% in the HD + L-arg group vs 61.90+/-1.45% in the HD group) and a reduced thickness (approximately 1.32-fold) of the smooth muscle cells' extracellular matrix; and (3) the heart, which displayed approximately 16% diminishing of the thickness of the left ventricular wall, and an apparently normal structure of the myocardium; the restoration of the thickness of the pericapillary extracellular matrix to almost normal dimensions was also observed. Administration of L-arginine did not modify the high level of plasma creatinine determined for the HD group (approximately 48% increased vs control group) and had no effect on the thickened, nodular basal lamina of the kidney capillaries. The results indicate that endothelial dysfunction established in combined hyperlipemia-diabetes is distinctive for each vascular bed (mesenteric arterioles, heart capillaries, kidney glomerular capillaries), and there is a reversible stage of the dysfunction in which L-arginine oral supplementation induced beneficial effects.

The gene encoding endothelial nitric oxide synthase (eNOS) is involved in abnormalities in nitric oxide (NO) synthesis that mediates functional damage of vascular cells, especially of endothelial cells (ECs), a common characteristic in cardiovascular diseases. In Fabry's disease, the characteristic mutation in the alpha-galactosidase A (alpha-gal A) gene induces large deposits of glycosphingolipids, particularly concentrated in ECs, a process associated with endothelial dysfunction. To determine whether in addition to alpha-gal A gene mutations, eNOS genetic variations are implicated in this process, we examined the genotypes of the missense Glu298Asp (G894T) variant in exon 7 and 27-bp tandem repeats in intron 4 (4b/a) in 19 patients with Fabry's disease, and 39 normal volunteers. The results showed that both varials have a significant association with Fabry's disease. The frequencies of mutant Glu/Asp + Asp/Asp genotypes and Asp allele are significantly higher in Fabry's disease (68.4%, p = 0.044, and 47.4%, p = 0.022, respectively) than in controls (46.7% and 25%, respectively). The frequencies of eNOS 4b/a polymorphisms are also significantly different in Fabry's disease when compared to controls. The mutant 4b/a + 4a/a genotype frequencies are 55.5% (p = 0.032) and 4a allele 27.8% (p = 0.05) compared with controls (23.1% and 12.8%, respectively). These results indicate that more than half of the patients with Fabry's disease carry the Glu298Asp variant ( approximately 68%) and/or the 4b/a polymorphism ( approximately 55%). To the best of our knowledge, this is the first report showing an influence of eNOS gene polymorphisms in patients with Fabry's disease.

Clotrimazole (CLT) is a drug known to interfere with cellular calcium homeostasis, which in turn is reported to intervene in cell proliferation and in the reactivity of small blood vessels. Experiments were designed to test the influence of CLT on the proliferative and vasorelaxant effect of bradykinin (BK) and on calcium homeostasis in smooth muscle cells (SMC). To this purpose two model systems were employed: (i) cultured human smooth muscle cells (HSMC), and (ii) isolated resistance arteries maintained in an organ bath. The effect of various concentrations of CLT (2-15 microM) on BK-induced proliferation of HSMC was quantitated by spectrometry following [3H]-thymidine incorporation, and intracellular calcium [Ca+]i was determined by spectrofluorimetry using Fura 2-AM assay. In other experiments the roles of BK receptor (AB2) and of thapsigargin were assessed. The reactivity of the resistance arteries was measured by the myograph technique, and the effects of BK, CLT, and NO synthase blocker, L-NAME were evaluated. The results showed that 10 microM CLT: (i) inhibits the BK-induced proliferation of HSMC by 45-50%: (ii) prevents the rise of [Ca2+]i induced by BK (120.8 +/- 12.4 nM vs. 235.8 +/- 34.1 nM), an cffect similar to that of "classic" L-type calcium channels blockers: (iii) reduces the release of Ca2+ entry induced by thapsigargin suggesting a possible inhibition of the capacitative Ca2+ entry. Organ bath assays showed that CLT enhanced the BK-induced relaxation of the resistance arteries by an endothelium NO-independent pathway. Together, these data suggest that the mechanism of action of CLT on SMC implies mainly a modification of intracellular calcium homeostasis, with a minor contribution of BK B2 receptors. These new distinctive features of CLT effects suggest the potential use of this drug in the therapy of cardiovascular diseases associated with SMC increased proliferation and impeded relaxation in small arteries, such as atherosclerosis and restenosis.