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      Abnormal Deposition of Extracellular Matrix Proteins by Cultured Smooth Muscle Cells from Human Varicose Veins

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          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.

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          Analysis of the connective tissue matrix and proteolytic activity of primary varicose veins.

          Valvular incompetence and venous wall abnormalities have been suggested as primary etiologic factors responsible for the development of varicose veins. This study was conducted to evaluate the connective tissue constituents of greater saphenous varicosities. Proteolytic activity, a factor that can lead to matrix degradation and cause weakening and dilation of the venous wall, was also assessed. The collagen and elastin contents of 16 nonthrombophlebitic greater saphenous varicose veins (VV) and seven normal greater saphenous veins (NV) were quantified. In addition, four duplex scanning-confirmed competent segments of greater saphenous veins (i.e., potential varicose veins [PV]) affected by varicosis at alternate sites were analyzed. Proteolytic activity was determined by zymography and radiolabeled substrate assay. The content of collagen was significantly increased in the VV and PV compared with NV (VV = 189 +/- 7 mg/gm, PV = 189 +/- 9 mg/gm vs NV = 144 +/- 10 mg/gm, p < 0.05). Conversely, the elastin content in the VV and PV was significantly reduced (VV = 53 +/- 3 mg/gm, PV = 50 +/- 4 mg/gm vs NV = 74 +/- 4 mg/gm, p < 0.05). The collagen to elastin ratio demonstrated an alteration in VV and PV compared with NV (VV = 3.7 +/- 0.3, PV = 3.9 +/- 0.4 vs NV = 2.0 +/- 0.2, p < 0.05). Casein and gelatin zymography did not demonstrate significant qualitative differences in the enzymatic activities among the three groups. Quantitative analysis of the elastase activity in the venous tissues was similarly not appreciably altered (VV = 5.1 +/- 0.2 U/gm, PV = 5.3 +/- 0.2 U/gm vs NV = 5.7 +/- 0.3 U/gm). A significant increase in the collagen content and a significant reduction in the elastin content of VV were demonstrated. The net increase in the collagen/elastin ratio is indicative of an imbalance in the connective tissue matrix. The biochemical profile of PV was similar to VV and significantly different from NV. These preliminary data support the presence of connective tissue abnormalities before valvular insufficiency. In addition, the absence of an increase in the proteolytic activity excludes enzymatic matrix degradation as an essential component in the formation of venous varicosities.
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            A colorimetric microtiter assay for the quantitation of cytokine activity on adherent cells in tissue culture.

             Yona Keisari (1992)
            A colorimetric microtiter assay was developed for the quantitation of adherent cells in culture, which is based on the staining of cells with a commercially available staining kit. Adherent L929, A375 cell lines and human monocytes were stained with Hemacolor reagents and the color eluted with SDS 0.5% was determined spectrophotometrically with an ELISA plate reader at 630 nm. The method enabled the detection of cells and was linear up to 3 x 10(4) L929 cells/well. The Hemacolor staining assay was compared to the crystal violet staining assay and the MTT reduction assay, and was found to be sensitive, accurate and reproducible, and has the advantage of enabling microscopic inspection of the stained cells prior to color elution. The assay was found to be suitable for the determination of cytotoxic cytokines, and the enumeration of adherent monocytes. This method might be also used for the quantitation of cytotoxic drugs, and the cytophatic activity of viruses.
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              In vitro evaluation of endothelial and smooth muscle function of primary varicose veins.

              Experiments were designed to study functional changes in superficial leg veins of patients with primary varicosity. Excess segments were taken from 19 patients undergoing elective vein resection. Excess segments of greater saphenous veins taken from 13 patients undergoing coronary or lower extremity arterial bypass were used as controls. These rings, some with endothelium deliberately removed, were suspended for the measurement of isometric force in organ chambers. Segments of veins were also evaluated with respect to their total protein content, endothelin content, and histologic structure. In varicose veins, maximal contractions in response to potassium chloride (n = 9), norepinephrine (n = 9), and endothelin (n = 4) were reduced 71.1%, 78.2%, and 75.6%, respectively, compared with contractions in control veins (p < 0.01). In addition, no differences were detected in the maximal tension or tissue sensitivity (EC50) between segments of nonvaricose greater saphenous vein and adjacent varicose tributaries from the same patient. Rings with and without endothelium contracted similarly. In varicose veins, endothelium-dependent relaxations produced by the calcium ionophore A23187 were attenuated 89.7% compared with relaxations in controls (n = 6, p < 0.01). In veins from patients with primary varicosity, endothelium-independent relaxations produced by nitric oxide (n = 6) and forskolin (n = 3) were diminished 86.8% (p < 0.01) and 65.6% (p < 0.05), respectively, compared with relaxations in control veins. In primary varicose veins, protein content was decreased (n = 17, p < 0.01) and endothelin content increased (n = 17, p = 0.1) compared with those values in control veins (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                April 1998
                16 April 1998
                : 35
                : 2
                : 115-123
                a Division of Angiology, Institut de Recherches Servier, Suresnes, and b Département de Chirurgie Cardiaque, Hôpital Broussais, Paris, France
                25573 J Vasc Res 1998;35:115–123
                © 1998 S. Karger AG, Basel

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                Page count
                Figures: 7, References: 39, Pages: 9
                Research Paper


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