Different flavonoids present in the micronized purified flavonoid fraction (Daflon 500 mg) contribute to its anti-hyperpermeability effect in the hamster cheek pouch microcirculation.

AIM: This study evaluated microcirculatory effects of the flavonoid substances that constitute the micronized purified flavonoid fraction (MPFF) (Daflon 500 mg) in comparison to diosmin. METHODS AND RESULTS: In groups of 3 male hamsters, oral treatment with MPFF or diosmin (15 min before anesthesia) did not alter blood pressure. At 10 or 30 mg/kg, both MPFF and diosmin significantly decreased the leaky sites caused by ischemia/reperfusion (I/R) (30 min) in the hamster cheek pouch; the effect was significantly higher with MPFF (39+/-1% and 52+/-1%, respectively) than diosmin (18+/-1% and 37+/-3%, respectively). Eight groups of 3 hamsters each were treated with the components of MPFF. Diosmetin only decreased the number leaky sites at 30 mg/kg (decrease: 15+/-2%). The decrement at 10 and 30 mg/kg averaged at: 17+/-3% and 44+/-1%, respectively, for hesperidin; 19+/-1% and 46+/-2%, respectively, for linarin; and 30+/-1% and 44+/-1%, respectively, for isorhoifolin. Hesperidin, linarin, and isorhoifolin each displayed an anti-leakage effect comparable to or greater than diosmin. MPFF decreases permeability more than any of its single constituents, suggesting that the flavonoids present in its formulation have a synergistic action. CONCLUSION: These results illustrate that MPFF is more potent than single diosmin in this model of hyperpermeability and that each of the flavonoid substances present in MPFF contribute to its action.

Imbalance in the synthesis of collagen type I and collagen type III in smooth muscle cells derived from human varicose veins.

Varicose veins have a thickening wall. Their smooth muscle cells are disorganized as regards proliferation and production of extracellular matrix protein. An imbalance between the synthesis of collagen type I protein (collagen I) and collagen type III protein (collagen III) could explain the lack of elasticity of varicose veins. Therefore, collagen synthesis was compared in the media and in cultured smooth muscle cells derived from human control and varicose saphenous veins. An increase in total collagen synthesis was observed in the media and in smooth muscle cells derived from varicose veins. This augmentation was due to an overproduction of collagen I in cultured cells from varicose veins consistent with an increase in the release of collagen I metabolites in the media. A concomitant decrease in collagen III was observed in cultures of smooth muscle cells from varicose veins. The increase in the synthesis of collagen I in cells from varicose veins was correlated with an overexpression of the gene since mRNAs for collagen I were augmented without change in mRNA-half-life. This augmentation in the synthesis of collagen I was reduced by the addition of exogenous collagen III in cultures from varicose veins. These findings suggest a dysregulation of the synthesis of collagen I and III in smooth muscle cells derived from varicose veins.

S17834, a new inhibitor of cell adhesion and atherosclerosis that targets nadph oxidase.

microdant stress is involved in the events that accompany endothelial cell expression of adhesion molecules and leukocyte adherence in many disease states, including atherosclerosis. A recently discovered benzo(b)pyran-4-one derivative, S17834 (10 to 50 micromol/L), reduced tumor necrosis factor-stimulated vascular cell adhesion molecule-1 (VCAM) mRNA accumulation and protein expression in human umbilical vein endothelial cells. Intercellular cell adhesion molecule-1 and E-selectin were also inhibited by S17834, but platelet endothelial cell adhesion molecule-1 was not. Adherence of U937 monocytic cells to the endothelial cells as well as to plastic plates coated with soluble VCAM, intercellular cell adhesion molecule-1, P-selectin, and E-selectin was also decreased. Consistent with an antioxidant mechanism of action, S17834 (10 to 50 micromol/L) inhibited tumor necrosis factor-stimulated release of superoxide from endothelial cells measured by cytochrome c reduction. S17834 had no effect on superoxide produced by xanthine oxidase, indicating that rather than by acting as a scavenger of superoxide anion, the drug acts by inhibiting the production of free radicals. Indeed, S17834 inhibited NADPH oxidase activity of endothelial cell membranes. The ability to inhibit superoxide anion production appears to be key in the effect of S17834 on superoxide anion production and VCAM expression, because these actions were mimicked by adenovirus-mediated overexpression of superoxide dismutase. Furthermore, these actions may be relevant in vivo, because S17834 reduced aortic superoxide anion levels by 40% and aortic atherosclerotic lesions by 60% in apolipoprotein E-deficient mice. These results indicate that S17834 inhibits adhesion molecule expression and adherence of leukocytes to endothelial cells as well as aortic atherogenesis and that perhaps these effects can be explained by its ability to inhibit endogenous superoxide anion production.

Histochemical evidence for inducible nitric oxide synthase in advanced but non-ruptured human atherosclerotic carotid arteries.

In response to cytokine stimulation, the inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide with potential consequences in the pathophysiology of atherosclerosis. Previous investigations have demonstrated the presence of iNOS in human atherosclerotic lesions. The goal of this study was to evaluate the occurrence of the expression of iNOS in ruptured versus non-ruptured human carotid atherosclerotic plaques. Using plastic-embedded sections, we performed in situ hybridization and immunohistochemistry on very advanced atherosclerotic lesions type V (non-ruptured) and type VI (ruptured) from 12 atheromatous carotid arteries from endarterectomy and six non-atherosclerotic internal mammary arteries from aorto-coronary bypass. Only one internal mammary artery expressed iNOS in the endothelium. In contrast, iNOS mRNA and protein were repeatedly expressed in advanced lesions type V in 5/7 cases, particularly in inflammatory regions. Specific cell markers identified iNOS-positive cells as macrophages and T-lymphocytes but also as smooth muscle cells and endothelial cells adjacent to these inflammatory regions. Nitration of protein tyrosines was not always associated to iNOS expression but more likely to the presence of inflammatory cells. In complicated lesions type VI, the occurrence of iNOS mRNA and protein expression diminished drastically (1/5 cases). Combined expression of iNOS mRNA and protein is frequently found in advanced but non-ruptured human atherosclerotic carotid lesions while it becomes rare after the plaque has ruptured. These findings suggest that iNOS could be an active participant in the plaque rupture event.

TGF-(beta)1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation.

Somatic stem cells are largely quiescent in spite of their considerable proliferative potential. Transforming growth factor-(beta)1 (TGF-(beta)1) appears to be a good candidate for controlling this quiescence. Indeed, various mutations in the TGF-beta signalling pathway are responsible for neoplasic proliferation of primitive stem/progenitor cells in human tissues of various origins. In hemopoietic single cell culture assays, blocking autocrine and endogeneous TGF-(beta)1 triggers the cell cycling of high proliferative potential undifferenciated stem/progenitor cells. However, it has never been demonstrated whether TGF-(beta)1 has an apoptotic effect or a differentiating effect on these primitive cells, as already described for more mature cells. Using single cell experiments both in liquid or semi-solid culture assays and dye tracking experiments by flow cytometry, we demonstrate that low, physiological concentrations of TGF-(beta)1, which specifically maintain primitive human hemopoietic stem/progenitor cells in quiescence, have a reversible effect and do not induce apoptosis. We moreover demonstrate that these low concentrations prevent the rapid loss of the mucin-like protein CD34, a most common marker of immature hematopoietic stem/progenitor cells, which is progressively lost during differentiation. TGF-(beta)1 not only up-modulated the CD34 antigen before S phase entry but also maintained a high level of CD34 expression on cells which had escaped cell cycle inhibition, suggesting that proliferation inhibition and differentiation control by TGF-(beta)1 may be independent. These data provide additional evidence that TGF-(beta)1 acts as a key physiological factor ensuring the maintenance of a stem cell reserve.

Abnormal deposition of extracellular matrix proteins by cultured smooth muscle cells from human varicose veins.

The aim of the present study was to verify whether the modifications of the extracellular matrix, described in varicose veins, are also present in cultures of smooth muscle cells from human varicose veins. The accumulation of collagen type III and fibronectin was determined by immunofluorescence in cultures of smooth muscle cells at passage 2-3 during the proliferation phase. After 5 days of culture, the immunostaining of both collagen type III and fibronectin was weaker in cells from varicose than in those of control veins while the expression of collagen type III and fibronectin messenger ribonucleic acids was not significantly different. Collagen type I and III synthesis were quantified by tritiated proline incorporation in control and varicose cell layers at postconfluence. Collagen type I deposition was similar in both types of cell layers while collagen type III was decreased in cell layers from varicose veins. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) were also quantified by enzyme immunoassays in supernatants from smooth muscle cell cultures at postconfluence. No significant difference was observed in the synthesis of any of the MMPs (-1, -2 and -9) or their inhibitors (-1 and -2) tested. These data illustrate that smooth muscle cells cultured from varicose veins deposit less collagen type III and fibronectin than control cells despite comparable levels of mRNAs for these proteins suggesting dysregulation of posttranslational steps in the synthesis of both proteins by smooth muscle cells from varicose veins.