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.

Differential expression of matrix metalloproteinases after stent implantation and balloon angioplasty in the hypercholesterolemic rabbit.

BACKGROUND: Intimal hyperplasia is the principal mechanism of in-stent restenosis. Matrix metalloproteinases (MMPs) play a key role in intimal growth after balloon angioplasty (BA). Little is known, however, about MMP expression after stent implantation (ST). We investigated whether MMP9 and MMP2 are differentially expressed after ST and BA. METHODS AND RESULTS: Hypercholesterolemic rabbits underwent ST and BA in the right and left iliac arteries, respectively. The expression of MMPs and their inhibitors (TIMPs) was studied at various time points in the injured arteries by use of zymography, reverse transcription-polymerase chain reaction, and immunohistochemistry. MMP2, but not MMP9, was constitutively expressed in uninjured arteries. MMP9 expression was rapidly induced after injury, whereas the increase in MMP2 expression was delayed. At all time points, pro-MMP9 activity and MMP9 mRNA levels were >/=2-fold (ANOVA, P=0.002) and >/=3-fold (P<0.0001) higher after ST than after BA, respectively. Active MMP9 was detected only after ST. Although the increases in MMP2 mRNA levels were of similar magnitudes after ST and BA, pro-MMP2 activity was slightly higher 7 and 30 days after ST, and MMP2 activity was >/=2-fold higher 7 to 60 days after ST (P=0.002). No difference in TIMP expression was observed between stented and balloon-injured arteries. Cellular distributions of MMPs and TIMP1 were similar after ST and BA. Early inflammatory cell recruitment and 30-day intimal growth were more severe after ST. CONCLUSIONS: Stent implantation results in more intense and sustained expression of MMP9 and activation of MMP2 than balloon angioplasty.

Extracellular matrix remodeling in the vascular wall.

The extracellular matrix provides a structural framework essential for the functional properties of vessel walls. The three dimensional organization of the extracellular matrix molecules--elastin, collagens, proteoglycans and structural glycoproteins--synthesized during fetal development--is optimal for these functions. Early in life, the vessel wall is subjected to injury: lipid deposition, hypoxia, enzyme secretion and reactive oxygen species production during inflammatory processes, and the extracellular matrix molecules are hydrolyzed by proteases--matrix metalloproteinases, leukocyte elastase, etc. In uninjured arteries and veins, some proteases are constitutively expressed, but through the control of their activation and/or their inhibition by inhibitors, these proteases have a very low activity. During the occurrence of vascular pathologies--atherosclerosis, hypertension, varicosis, restenosis, etc.--the balance between proteases and their inhibitors is temporally destroyed through the induction of matrix metalloproteinase gene expression or the secretion of enzymes by inflammatory cells. Smooth muscle cells, the most numerous cells in vascular walls, have a high ability to respond to injury through their ability to synthesize extracellular matrix molecules and protease inhibitors. However, the three dimensional organization of the newly synthesized extracellular matrix is never functionally optimal. In some other pathologies--aneurysm--the injury overcomes the responsive capacity of smooth muscle cells and the quantity of extracellular matrix decreases. In conclusion, care should be taken to maintain the vascular extracellular matrix reserve and any therapeutic manipulation of the protease/inhibitor balance must be perfectly controlled, because an accumulation of abnormal extracellular matrix may have unforeseen adverse effects.

Smooth muscle cell modulation and cytokine overproduction in varicose veins. An in situ study.

The exact aetiology and physiopathology of varicose disorders remain unclear. The aim of the present work was to study, in situ, the morphology and composition of cellular and matrix components in varicose veins compared with control veins and to identify factors that could contribute to varicose remodelling. A combined histological, immunohistochemical, and biochemical approach was used. Longitudinal sections of varicose (n=12) and control veins (n=9) were studied to assess the organization, structure, and phenotype of smooth muscle cells; the localization of microvascular endothelial cells; the distribution of connective tissue proteins; and the localization of cytokines. These cytokines were further quantified by ELISA. Considerable heterogeneity of the varicose vein wall was observed, with a succession of hypertrophic and atrophic segments, presenting severe disorganization of the medial layer and numerous areas of intimal thickening. In hypertrophic portions, medial smooth muscle cells showed marked alterations suggesting modulation from a contractile to a proliferative and synthetic phenotype; furthermore, the number of vasa vasorum was increased. In contrast, in atrophic portions, both cellular and matrix components were decreased. TGFbeta1 (p< or =0.005) and bFGF (p< or =0.001) were increased and VEGF was not significantly modified in varicose veins when the results were expressed per mg of DNA. These results show that phenotypic modulation of smooth muscle cells, altered extracellular matrix metabolism, and angiogenesis are the main mechanisms contributing to the morphological and functional modifications of varicose remodelling. The increased expression of bFGF and TGFbeta1 by varicose vein cells may play a pivotal role in the hypertrophy of the venous wall, but the exact mechanism leading to aneurysmal dilatations remains to be elucidated.

Increased TIMP/MMP ratio in varicose veins: a possible explanation for extracellular matrix accumulation.

Primary varicose veins are functionally characterized by venous back-flow and blood stagnation in the upright position. Dilatation and tortuosity provide evidence for progressive venous wall remodelling, with disturbance of smooth muscle cell/extracellular matrix organization. Affected areas are not uniformly distributed, some areas being hypertrophic, whereas others are atrophic or unaffected. In 12 varicose veins and ten control veins, the proteolytic enzyme/inhibitor balance which may participate in the remodelling of the venous wall was investigated. For this purpose, the presence and enzymatic activity of matrix metalloproteinases (MMP-2, MMP-9), tissue inhibitors of MMPs (TIMP-1, TIMP-2), urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PAs), and plasminogen activator inhibitor-1 (PAI-1) were quantified by western blot and gelatin or plasminogen-casein zymography. In addition, MMP-2, TIMP-1, TIMP-2, and PAI-1 levels were measured by ELISA. A high TIMP-1 level and a low MMP-2 level/activity were found in varicose veins (p<0.005), resulting in a three-fold increase in the TIMP-1/MMP-2 ratio in varicose versus control veins. Levels of PAs (uPA and tPA) as well as PAI-1 were both lower in varicose veins (p<0.005), with minimal change in the PAI/PA ratio. These results demonstrate that varicose veins are characterized by a higher than normal TIMP/MMP ratio, which may facilitate extracellular matrix accumulation in the diseased venous wall.