Demetrio Marino

Pro-apoptotic effect of fluvastatin on human smooth muscle cells.

The antiatherosclerotic effect of statins has been attributed to their hypocholesterolemic action. We therefore evaluated the effect, in vitro, of the addition of the serum of patients taking fluvastatin on human smooth muscle cells in order to ascertain the effect of the drug on cell proliferation and apoptosis. We found that the addition of serum from patients treated with fluvastatin for 6 days caused a significant reduction in cell proliferation, increased cell apoptosis and reduced the B cell leukemia-2 (bcl-2) concentration. It is concluded that the induction of apoptosis by statins could be a supplementary mechanism in the prevention of atherosclerotic lesions in humans.

Effects of homocysteine on proliferation, necrosis, and apoptosis of vascular smooth muscle cells in culture and influence of folic acid.

BACKGROUND It is known that hyperhomocysteinemia is associated with an increased risk of vascular disease, yet little is known about the pathogenic mechanisms underlying the action of homocysteine (Hcy) itself. METHODS We evaluated the effects of Hcy on cell proliferation, apoptosis, and necrosis in smooth muscle cells (SMCs) cultured for 24 h with different amounts of Hcy. The percentage of apoptotic and necrotic cells from the culture was evaluated using two different techniques: annexin V-FITC and propidium iodide (PI) fluorescence and apoptosis TUNEL assay. RESULTS The addition of 10 microM/l of Hcy to the medium was followed by a significant increase in cell proliferation and death, through apoptosis and necrosis, respectively. Notwithstanding this apparent balance, a significant increase was found in the total number of cells present in Hcy-treated culture, thus demonstrating a positive dose-dependent correlation with Hcy concentrations in the culture medium. The addition of folic acid to the culture medium significantly reduced both Hcy concentrations in media and the effects of Hcy on the proliferation/apoptosis/necrosis balance of cells in culture. The percentages for apoptotic cells and for cells with a necrotic morphology continued to increase as Hcy concentrations increased, although the absolute values were lower in the culture treated than in that not treated with folic acid. CONCLUSIONS In the presence of folic acid, at increasing concentrations of Hcy, the total number of cells in culture showed increases far less relevant with respect to the control. Also the percentage of apoptotic cells to that of cells with a necrotic morphology, although conserving the tendency to increase to growth of the concentrations of Hcy, have shown absolute values that were lower in the folic acid-treated cultures.

Cell Proliferation/Cell Death Balance in Renal Cell Cultures after Exposure to a Static Magnetic Field

The effect of a static magnetic field (MF) of 0.5 mT of intensity on the cell proliferation/cell death balance was investigated in renal cells (VERO) and cortical astrocyte cultures from rats. Magnetic stimulation was delivered by magnetic disks at known intensities. The percentage of apoptotic and necrotic cells was evaluated using flow cytometry and morphological analysis following Hoechst chromatin staining. An index of cell proliferation was determined using sulfonated tetrazolium (WST-1). Control cultures were prepared without exposure to MFs. After 2, 4 and 6 days of exposure to a MF, we observed a gradual decrease in apoptosis and proliferation and a gradual increase in cells with a necrotic morphology with respect to the control group. In astrocyte cultures, over a 6-day exposure period. A gradual increase was observed in apoptotic, proliferating, and necrotic cells. Our findings suggest that the effect of exposure to MFs varies, depending on the cell type; MFs may also have a nephropathogenic effect.

Effect of interleukin 8 and ICAM-1 on calcium-dependent outflow of K+ in erythrocytes from subjects with essential hypertension.

SUMMARY Introduction: The pathogenic mechanisms underlying the increase in peripheral resistance and the contraction of smooth muscular fibre cells in essential hypertension are not yet clearly understood. However, it is now known that immune system activation plays a role in the pathogenesis of some forms of arterial hypertension, and recent data show that the Ca2+ influx in some cells (i.e. red blood cells, leukocytes, platelets, smooth muscular fibre cells) is increased in subjects with essential hypertension, thus revealing a possible alteration in cellular membrane. The end-points of this study were therefore to ascertain whether red blood cells used as a cellular membrane model have a greater Ca2+ dependent K+ flow (Gardos effect) in hypertensive patients than in normotensive controls, to point out a different regulation of ionic channels, and whether IL-8 and the adhesion molecule ICAM-1 influence the membranous outflow. Material and methods: The study was conducted on 87 Caucasian subjects. Of these, 50 (25 men, 25 women; mean age 43 ± 3 years, mean body mass index (BMI) 27 ± 0.5 and 22.3 ± 0.3 kg m2, respectively) had mild-to-moderate hypertension (mean arterial blood pressure 120±8mmHg).The other 37 (18 men, 19 women; mean age 39 ± 3 years; BMI 23.8 ± 0.5 kg m2 and 22.8 ± 0.5 kg m2, respectively were normotensive healthy volunteers (mean arterial blood pressure 89 ± 2 mm Hg).All the patients and subjects were untreated for at least 4 weeks before blood sampling. Results: Ca2+-dependent K+ outflow was found to be greater in samples from patients with essential hypertension than in those from normotensive controls. lL-8 and ICAM-1 significantly enhanced the Ca2+-dependent K+ outflow in red blood cells from hypertensive subjects but had an inhibitory effect on cells from controls. In the experimental model, the presence of TMB-8, a membrane calcium antagonist, significantly reduced the Ca2+-dependent K+ efflux. Conclusion: Vasoconstriction in subjects with essential hypertension may therefore depend on a different regulation of ionic flow that probably supports an increased Ca2+ inflow in smooth muscle fibre cells. Under certain pathological conditions, some immune system components (i.e. interleukins, adhesion molecules) may directly enhance membrane permeability to Ca2+, thus inducing vasoconstriction in the smooth muscle cells.

Does Captopril Have a Direct Pro-Apoptotic Effect?

The beneficial effect of ACE inhibitors on cardiovascular remodelling from hypertension and ischemic disease may be due to the effect of these drugs on the proliferation/cell death balance. We therefore investigated the effect of the addition of captopril in vitro, on the onset of apoptosis in human vascular myocytes, by using a propidium iodide fluorescence analysis and a morphological analysis using the acridine orange technique. Captopril (0.23 mM) caused an increase in apoptotic phenomena that was more than 3.5-fold than in controls both at the 24th (7.7 vs. 2%) and the 48th h (10.1 vs. 3.8%). The addition of propranolol strengthened the effect on apoptosis. The induction of apoptotic phenomena may be a mechanism by which ACE inhibitors affect cardiovascular remodelling and it might also explain the favorable effect these drugs have on diseases such as IgA nephropathy and diabetic nephropathy.

Alterations in Induced Potassium Calcium Efflux in the Erythrocytes of Patients with Autosomal Dominant Polycystic Kidney Disease and Hypertension

Prof. Michele Buemi, Via Salita Villa Contino 30, I-98100 Messina (Italy) Dear Sir, Autosomal dominant polycystic kidney disease (ADPKD) can be frequently complicated by chronic renal failure and hypertension [1]· In the literature there is little consensus on the pathogenesis of hypertension in patients with ADPKD. In fact, although it would seem obvious that the incidence of hypertension would increase as renal insufficiency becomes more severe, it has also been demonstrated that there is no correlation between the severity of hypertension and the severity of renal involvement, and that 15-20% of subjects with ADPKD without renal function impairment have hypertension and that blood pressure alterations can be found in ADPKD patients without renal function impairment before puberty [2]. Importantly, the genetic trait appears to be dominant. The main genie locus responsible for ADPKD is PKD-1, localized on the short arm of chromosome 16, which presents a 100% penetration [3]. However, another locus is involved in the disease. Called PKD-2, it is linked to chromosome 4, which is altered in 10% of patients, and modifications in it could give rise to an altered cellular membrane structure, and therefore to anomalous functioning of the ionic transport systems [4, 5]. Recently Vareesangthip et al. [6] identified altered Na/Li countertransport in ADPKD patients, and Rutherford et al. [7] found that patients with essential hypertension had an altered ionic transport system. However, this alteration is present in the erythrocytes of all ADPKD patients, and so it does not explain why hypertension develops in only 15-20% of such patients without renal function impairment. We therefore evaluated induced Ca2+/K+ flow in the erythrocytes of ADPKD subjects in order to ascertain whether ADPKD patients with hypertension but without renal function impairment also had alterations in other ionic transport systems. Our study was conducted on 40 subjects divided into four groups that were matched for age and gender as follows; 10 healthy controls (5 M, 5 F; mean age 52 ± 7 years); 10 with essential