Trimetazidine demonstrated cardioprotective effects through mitochondrial pathway in a model of acute coronary ischemia.

Myocardial ischemia affects mitochondrial function leading to ionic imbalance and susceptibility to ventricular fibrillation. Trimetazidine (TMZ), a metabolic agent, is clinically used as an anti-anginal therapy. This study was conducted to compare the effect of TMZ 20 mg immediate release (IR) and TMZ 35 mg modified release (MR), two bioequivalent marketed formulations of TMZ, on cardioprotection during acute ischemia in pigs. A 4-day oral treatment with TMZ 20 mg IR (800 mg, tid) or TMZ 35 mg MR (1,400 mg, bid) had no effect on ventricular fibrillation threshold (VFT) prior to ischemia but significantly prevented the decrease in VFT observed in placebo-treated groups after a 1-min left anterior descending coronary artery occlusion. This effect occurred without modifying cardiac hemodynamic and conduction parameters. In both TMZ-treated groups, a significant reduction of the ischemic area as well as a protection of cardiomyocytes were observed. Cardiac enzymatic activity (phosphorylase, succinate dehydrogenase, ATPase) was increased in TMZ-treated groups. Both formulations preserved mitochondrial structure and improved mitochondrial function as demonstrated by a twofold increase of oxidative phosphorylation, by a reduction of reactive oxygen species (ROS) production (>30 %) and by a trend to increase the mitochondrial calcium retention capacity. In this model of ischemia, both TMZ formulations, leading to equivalent TMZ plasma exposures, demonstrated similar cardioprotective effects. This protection could be attributed to a preservation of mitochondrial structure and function, which plays a central role in ATP and ROS production and consequently could be considered as a target of cardioprotection.

Venous hypertension, inflammation and valve remodeling.

OBJECTIVES: To identify possible mechanisms for destruction of valves in chronic venous hypertension and the results of treatment with an anti-inflammatory micronized purified flavonoid fraction. MATERIAL AND METHODS: The saphenous vein valves in a rat model of venous hypertension caused by a femoral arterial-venous fistula were studied. Studies included femoral venous pressure, valve morphology, femoral venous reflux and selected molecular inflammatory markers as examined by immunohistochemistry. The effects of treatment with the anti-inflammatory micronized purified flavonoid fraction (S 5628, Servier, 50 and 100 mg/kg/day) were investigated. RESULTS: The femoral venous pressure was elevated close to arterial values for a period of 3 weeks. We then examined the morphology of the veins and selected molecular inflammatory markers were assessed. The results show that in this model venous reflux develops in response to venous hypertension. This can be inhibited by the administration of the anti-inflammatory micronized purified flavonoid fraction (S 5628, Servier, 50 and 100 mg/kg/day). The valve becomes incompetent by a combination of venous dilation and shortening of the valve leaflets. This is not inhibited by treatment with S 5628. The valve leaflets are infiltrated with granulocytes, monocytes and T-lymphocytes, and the endothelial cells express enhanced levels of P-selectin and ICAM-1. Cells in the valves are subject to extensive apoptosis although no enhancement of MMP 2,9 expression could be detected at the three-week time point examined in this study. CONCLUSIONS: These results indicate that in this model chronic elevation of venous pressure is associated with an inflammatory reaction in venous valves, a process that may lead to their dysfunction, reflux, and upstream elevation of venous pressure. These effects are mitigated by the anti-inflammatory micronized purified flavonoid fraction in a dose dependent manner.

The inflammatory reaction during venous hypertension in the rat.

OBJECTIVE: Numerous studies have examined arterial occlusion followed by reperfusion but few studies have reported about venular occlusion which, in contrast to arterial occlusion, is associated with elevation of the capillary blood pressure. Here we examine leukocytes infiltration and tissue injury in rat mesentery during local venular occlusion and venous hypertension followed by reperfusion, and determine the level of protection offered by pretreatment with micronized, purified flavonoid fraction (MPFF). METHODS: Leukocyte rolling, adhesion, migration, and parenchymal cell death as detected by propidium iodide labeling were determined during venular occlusion using a micropipette followed by reperfusion in the rat mesenteric microcirculation pretreated with 0, 50, or 100 mg/kg MPFF for 7 days. Spontaneous leukocyte activation by nitroblue tetrazolium reduction and expression of CD18 and CD62L on naive donor neutrophils incubated with plasma from each treatment group were determined. RESULTS: Venous occlusion led to elevated levels of leukocyte rolling, adhesion, and migration as well as parenchymal cell death. These injurious processes were significantly inhibited by MPFF in a those-dependent fashion. MPFF reduced spontaneous leukocyte NBT reduction and the neutropil expression of CD62L, even though CD18 was not affected. CONCLUSION: These results suggested that microvascular occlusion in venules with elevation of the micropressure followed by reperfusion is a highly cytotoxic process in the rat mesentery which can be attenuated by MPFF pretreatment.

Inflammation in chronic venous insufficiency. Is the problem insurmountable?

One of the hallmarks of venous insufficiency is an elevated venous pressure. While a number of mechanisms have been proposed for vascular and parenchymal cell damage following venous pressure elevation, such as white cell infiltration, a key question remains as to what degree venous occlusion and flow interruption per se may constitute a risk factor in venous disease. To gain an insight into this mechanism, we examined the effect of venous occlusion followed by reperfusion. A draining venule (circa 50 micrometer) in the rat mesentery was occluded with a micropipette (1 h) followed by reperfusion (1 h). The procedure serves to raise the microvascular pressure to about 31 mm Hg during the occlusion while the flow is completely stopped in the local venous and capillary network. Parenchymal cell death in the mesentery was monitored by propidium iodide (PI) labeling. The number of PI-positive cells significantly increased predominantly during reperfusion. A 1-week treatment with a micronized purified flavonoid fraction (100 mg/kg/day) served to significantly reduce parenchymal cell death as well as leukocyte rolling, adhesion to postcapillary venule, and migration into the tissue both during occlusion and reperfusion. The results indicate, that even in an initially symptomless tissue, flow reduction combined with microvascular pressure elevation during venous occlusion results in tissue damage not only during reperfusion (as in arterial occlusion) but also during occlusion.

Leukocyte adhesion after oxidant challenge in the hamster cheek pouch microcirculation.

Oral administration of S-5682 (Daflon 500 mg, 90% diosmin, 10% hesperidin) inhibits oxidant-induced increase in macromolecular permeability in the postcapillary venules of the hamster cheek pouch microcirculation. In this study, the effect of S-5682 on leukocyte-endothelium interaction was evaluated using the same experimental model. Hamsters kept on a standard diet were divided into 5 groups (n = 6) and treated orally, twice a day, with placebo (10% lactose solution), S-5682, 5, 20 or 80 mg/kg/day (suspended in 10% lactose solution) or alpha-tocopherol, 1 mg/kg/day, for 10 days prior to the oxidant challenge with tert-butylhydroperoxide (TBOOH). Topical application of TBOOH (10(-4) M for 5 min) to hamsters given acridine orange prior to TBOOH resulted in increases in the number of rolling and sticking (no movement for at least 30 s) leukocytes in postcapillary venules. No changes in the number of rolling leukocytes could be observed in the treated groups compared with the placebo group (p > 0.05). On the contrary, leukocyte adhesion was inhibited in groups treated with S-5682 (5, 20 and 80 mg/kg/day) or alpha-tocopherol: placebo 105 +/- 3/6 mm(2) (mean +/- SEM); S-5682, 5 mg/kg/day 68 +/- 3/6 mm(2) (p < 0.01), 20 mg/kg/day 55 +/- 3/6 mm(2) (p < 0.001) and 80 mg/ kg/day 39 +/- 2/6 mm(2) (p < 0.001) and alpha-tocopherol 36 +/- 1/6 mm(2) (p < 0.001). The inhibition of oxidant-induced leukocyte adhesion by S-5682 was similar to that seen for ischemia-reperfusion and the higher dose of S-5682 was as effective as alpha-tocopherol in inhibiting it.

Effect of a micronized purified flavonoid fraction on in vivo platelet functions in the rat.

The aim of this study was to investigate the effects of a micronized purified flavonoid fraction (MPFF) on in vivo rat platelet functions. Platelet aggregation and disaggregation were evaluated by a noninvasive, automated isotope monitoring system (AimsPlus). Indium-labeled platelets were injected into anesthetized rats and stimulated by adenosine diphosphate (ADP) (10 microg/kg, i.v.) or collagen (50 microg/kg, i.v.). Fibrinogen binding to ex vivo ADP-activated platelets was determined by flow cytometry. MPFF (100 mg/kg, p.o.) significantly reduced ADP-induced platelet aggregation (p<0.05) and increased platelet disaggregation (p<0.05) compared with controls. Moreover, MPFF inhibited collagen-induced platelet aggregation (p<0.001) and increased platelet disaggregation (p<0.01). In addition, fibrinogen binding to 2.5 or 5 microM ADP-stimulated platelets also was reduced significantly (p<0.05 and 0.01, respectively). These results show that MPFF inhibits in vivo rat platelet functions.

Microvascular reactivity after ischemia/reperfusion in the hamster cheek pouch: beneficial effects of different oral doses of S-5682 (Daflon 500 mg).

Daflon 500 mg (S-5682) is a purified, micronized flavonoid fraction containing 90% diosmin and 10% hesperidin that is currently used to treat chronic venous insufficiency and hemorrhoidal disease. Thus, it seemed of interest to evaluate the effects of S-5682 on ischemia/reperfusion, ie, the changes in mean internal diameter and blood flow of arterioles and venules and the functional capillary density (FCD) during reperfusion after ninety minutes of total ischemia in the hamster cheek pouch microvasculature. Different doses of S-5682 (5, 20, 80, and 160 mg/kg body weight/day), suspended in 10% lactose solution or vehicle (10% lactose) were administered orally to male hamsters for ten days twice a day. The cheek pouch preparation was placed under an intravital microscope coupled to a closed-circuit TV system. A ninety-minute local ischemia was obtained by a cuff mounted around the neck of the everted pouch where it left the mouth of the hamster. Mean arteriolar and venular internal diameters were determined by means of an image-shearing device, IPM model 907; red blood cell (RBC) velocity was measured by the dual-slit photometric technique; microvessel volume flow was calculated from diameters and RBC velocities; and FCD was defined as the number of red-cell-perfused capillaries per observation field. During reperfusion, placebo-treated animals showed significant vasodilatation concomitant with a decrease in blood flow and FCD compared with preischemic values and an impairment of the myogenic response. In S-5682-treated animals, there was a significant dose-dependent improvement in all these parameters including the myogenic tonus. These results clearly demonstrated that oral administration of different doses of S-5682 for ten days improved the microvascular reactivity and FCD after ischemia/reperfusion in a dose-dependent fashion in the hamster cheek pouch microvasculature.

Oxidant-induced increase in vascular permeability is inhibited by oral administration of S-5682 (Daflon 500 mg) and alpha-tocopherol.

The aim was to study the effect of oral administration of three different doses of S-5682 and alpha-tocopherol on an oxidant-induced injury by tert-butylhydroperoxide (TBOOH) resulting in increased plasma leakage from postcapillary venules in the hamster cheek pouch microcirculation. Hamsters were on a standard laboratory animal diet with normal vitamin E and C content. Five groups of hamsters (n = 6) were treated orally with placebo (10% lactose solution), S-5682 (5, 20 or 80 mg/kg/day) suspended in 10% lactose solution, or alpha-tocopherol (1 mg/kg/day) for 10 days prior to oxidant challenge with TBOOH. Topical application of 10(-4) M TBOOH for 5 min to hamsters given FITC-dextran 30 min prior to TBOOH resulted in reversible increases in the number (mean +/- SE) of leaks in postcapillary venules: placebo, 117+/-7 leaks/cm2; S-5682, 5 mg/kg/day, 68+/-3 leaks/cm2 (p < 0.01); S-5682, 20 mg/kg/day, 41+/-3 leaks/cm2 (p < 0.01); S-5682, 80 mg/kg/day, 25+/-2 leaks/cm2 (p < 0.001), and alpha-tocopherol, 1 mg/kg/day, 18+/-1 leaks/cm2 (p < 0.001). The efficacy of inhibition of oxidant-induced leakage by S-5682 was similar to that seen with the same dose (20 mg/kg/day) of histamine, bradykinin, leukotriene B4 or ischemia/reperfusion-induced leakage, suggesting a common pathway for the induction of plasma leakage by these mediators. The maximal dose of S-5682 (80 mg/kg/day) was as effective as alpha-tocopherol (1 mg/kg/day) in inhibiting plasma leakage.

Effects of oral administration of different doses of purified micronized flavonoid fraction on microvascular reactivity after ischaemia/reperfusion in the hamster cheek pouch.

1. The effects of a purified micronized flavonoid fraction (S5682) on mean internal diameter and blood flow of arterioles and venules, as well as the functional capillary density (FCD) were evaluated in the hamster cheek pouch microvasculature before and after 90 min of total ischaemia. 2. Male hamsters were treated for ten days, twice a day, with oral doses of S5682 (5, 20, 80 and 160 mg kg-1 day-1) or placebo (10% lactose solution). The cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. Local ischaemia was obtained by a cuff mounted around the neck of the everted pouch where it leaves the mouth of the hamster. 3. Measurements were performed before ischaemia, at the onset of reperfusion and 10, 20, 30, 45 and 60 min thereafter. Diameters were measured by means of an image shearing device. Red blood cell (RBC) velocity was analysed by use of the dual-slit photometric technique. Blood flow was calculated from diameters and RBC velocities. FCD, defined as the number of capillaries with flowing blood per field of observation, was also assessed. 4. During reperfusion, placebo-treated animals showed a significant vasodilatation, a decrease in blood flow and FCD and S5682-treated animals showed a clear trend, dose-dependent, towards maintaining these parameters closer to the value found before ischaemia. 5. In conclusion, our results indicate that S5682 improves the microvascular reactivity and FCD after ischaemia/reperfusion. These data suggest that S5682 could function as an antioxidant, which may explain its beneficial therapeutic effect in chronic venous insufficiency where oxidative stress is involved in the pathological mechanism.