Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding.

Culture conditions can modify the composition of the extracellular matrix of cultured calf aortas smooth muscle cells. In the absence of ascorbate the major components of the matrix are microfibrillar proteins; deposition of collagen occurs upon ascorbate supplementation and, with increased time of exposure of cells to ascorbate, collagen becomes the dominant protein of the extracellular matrix (greater than 80%). Collagen accumulation follows a sigmoidal time-course, suggesting that it is a cooperative phenomenon. Covalent crosslinks are not required for collagen accumulation in the matrix. Microfibrillar proteins and increased amounts of proteoglycans and fibronectin accumulate concurrently with collagen but elastin deposition was not observed either with or without ascorbate feeding. Addition of ascorbate leads to a general stimulation of incorporation of [14C]proline into cellular protein and to changes in cell growth parameters and morphology: cell-doubling time decreases from 62 to 47 h and plating efficiency increases approximately fourfold. We conclude that the composition of the extracellular matrix assembled by cultured cells is subject to experimental manipulation and that changes in endogenously deposited matrix may have significant effects on cellular functions.

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Ascorbic Acid Increases the Thrombogenicity of Cellular Matrices

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646447 ◽

1991 ◽

Vol 66 (04) ◽

pp. 505-509 ◽

Cited By ~ 4

Author(s):

Georg A Hindriks ◽

Jan J Sixma ◽

Philip G de Groot

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Strong Increase ◽

Aggregate Formation ◽

Lower Shear ◽

The Matrix ◽

Proline Incorporation

SummaryWe have studied the influence of ascorbate on extracellular matrix formation in cultured human endothelial cells, smooth muscle cells and fibroblasts and measured the influence of the changed composition of their isolated extracellular matrices on their affinity for platelets. When endothelial cells were grown for a week in the presence of ascorbate, no influence on proline incorporation in their extracellular matrix was found. In accordance, no influence on platelet adhesion or aggregate formation on these matrices was detected. When smooth muscle cells were cultured in the presence of ascorbate, a strong increase in the amount of collagen types I and III in the extracellular matrix was found. When these matrices were perfused with whole blood, a significant enhanced increase in aggregate formation was observed. No influence was seen on the total coverage of the matrix with platelets. When fibroblasts were grown in the presence of ascorbate, no significant increase in proline incorporation in their matrix was measured. However, an increased adhesion of platelets was seen to the matrices at lower shear rates. We conclude that ascorbate feeding has a significant effect on endogenous deposited matrices of smooth muscle cells and fibroblasts, and that the changed composition had profound effects on platelet interaction with these matrices.

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Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells

Journal of Cell Science ◽

10.1242/jcs.111.22.3379 ◽

1998 ◽

Vol 111 (22) ◽

pp. 3379-3387 ◽

Cited By ~ 4

Author(s):

A.J. Putnam ◽

J.J. Cunningham ◽

R.G. Dennis ◽

J.J. Linderman ◽

D.J. Mooney

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Cultured Cells ◽

Muscle Cells ◽

Cellular Protein ◽

Bovine Brain ◽

Applied Strain ◽

Microtubule Assembly ◽

Mechanical Forces ◽

Mechanical Stimuli

Mechanical forces clearly regulate the development and phenotype of a variety of tissues and cultured cells. However, it is not clear how mechanical information is transduced intracellularly to alter cellular function. Thermodynamic modeling predicts that mechanical forces influence microtubule assembly, and hence suggest microtubules as one potential cytoskeletal target for mechanical signals. In this study, the assembly of microtubules was analyzed in rat aortic smooth muscle cells cultured on silicon rubber substrates exposed to step increases in applied strain. Cytoskeletal and total cellular protein fractions were extracted from the cells following application of the external strain, and tubulin levels were quantified biochemically via a competitive ELISA and western blotting using bovine brain tubulin as a standard. In the first set of experiments, smooth muscle cells were subjected to a step-increase in strain and the distribution of tubulin between monomeric, polymeric, and total cellular pools was followed with time. Microtubule mass increased rapidly following application of the strain, with a statistically significant increase (P<0.05) in microtubule mass from 373+/−32 pg/cell (t=0) to 514+/−30 pg/cell (t=15 minutes). In parallel, the amount of soluble tubulin decreased approximately fivefold. The microtubule mass decreased after 1 hour to a value of 437+/−24 pg/cell. In the second set of experiments, smooth muscle cells were subjected to increasing doses of externally applied strain using a custom-built strain device. Monomeric, polymeric, and total tubulin fractions were extracted after 15 minutes of applied strain and quantified as for the earlier experiments. Microtubule mass increased with increasing strain while total cellular tubulin levels remained essentially constant at all strain levels. These findings are consistent with a thermodynamic model which predicts that microtubule assembly is promoted as a cell is stretched and compressional loads on the microtubules are presumably relieved. Furthermore, these data suggest microtubules are a potential target for translating changes in externally applied mechanical stimuli to alterations in cellular phenotype.

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Analysis of the glycopeptides derived from the extracellular matrix secreted by cultured bovine aortic smooth muscle cells

Biochemistry and Cell Biology ◽

10.1139/o87-079 ◽

1987 ◽

Vol 65 (7) ◽

pp. 595-601

Author(s):

James R. A. Leushner

Keyword(s):

Extracellular Matrix ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Minor Components ◽

Proliferating Cells ◽

Cell Behaviour ◽

Aortic Smooth Muscle Cells ◽

Aortic Smooth Muscle ◽

The Matrix

Modulation of smooth muscle cell behaviour in culture has been associated with changes in the extracellular matrix. In the present study cultures of bovine aortic smooth muscle cells were compared in the rapidly proliferating and confluent phases of growth. The extracellular matrix was similar in both phases of growth and consisted of glycoproteins ranging from molecular weight 20 000 to over 200 000. The glycopeptides derived from these components displayed several differences. N-linked heteropolysaccharides of the biantennary and complex (more than two branches) types were predominant in the matrix of the confluent phase. Larger amounts of high mannose glycopeptides were present in the preparations from proliferating cells. O-Glycosidic glycopeptides were minor components in both preparations, but a slight increase was noted in the confluent phase of growth. Some of the changes in glycopeptides were interpreted in terms of the levels of the major components of the matrix such as the interstitial procollagens and fibronectin. The results indicate that processing of oligosaccharides associated with secreted glycoproteins of the extracellular matrix correlates with the state of growth of smooth muscle cells in culture.

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