Decreased Nitric Oxide Synthesis in Human Endothelial Cells Cultured on Type I Collagen

A specific antiserum to purified rabbit tissue inhibitor of metalloproteinases (TIMP) was raised in sheep, characterized and used to investigate the role of TIMP in a model system. Chondrocytes and endothelial cells cultured on 14C-labelled type I collagen films and stimulated to produce collagenase were unable to degrade the films unless the anti-TIMP antibody was added. The degradation induced was inhibited by a specific anti-rabbit collagenase antibody. It was concluded that TIMP is a major regulatory factor in cell-mediated collagen degradation.

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Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum

Biochemical Journal ◽

10.1042/bj2680225 ◽

1990 ◽

Vol 268 (1) ◽

pp. 225-230 ◽

Cited By ~ 29

Author(s):

A E Canfield ◽

R P Boot-Handford ◽

A M Schor

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Endothelial Cells ◽

Cell Shape ◽

Type I Collagen ◽

Three Dimensional ◽

Type I ◽

Mrna Levels ◽

Collagen Gels ◽

Cells Cultured

Endothelial cells plated on the surface of a two-dimensional substratum (gelatin-coated dishes, dishes coated with native type I collagen or collagen gels) form a cobblestone monolayer at confluence, whereas cells plated within a three-dimensional gel matrix elongate into a sprouting morphology and self-associate into tube-like structures. In this study, we have compared the synthesis of thrombospondin by quiescent endothelial cells displaying (a) the same morphological phenotype (cobblestone) on different substrata (gelatin and collagen) and (b) different morphological phenotypes (cobblestone and sprouting) on the same substratum (collagen). We demonstrate that thrombospondin is a major biosynthetic product of confluent, quiescent cells cultured on dishes coated with either gelatin or collagen, and that the synthesis of this protein is markedly decreased when cells are plated on or in three-dimensional collagen gels. Moreover, we demonstrate that cells plated in gel (sprouting) secrete less thrombospondin than do cells plated on the gel surface (cobblestone). The regulation of thrombospondin synthesis is reversible and occurs at the level of transcription, as steady-state mRNA levels for thrombospondin decrease in a manner comparable with the levels of protein secreted by these cells. We also show that mRNA levels for laminin B2 chains are increased when cells are cultured on and in collagen gels compared with on gelatin-coated dishes, suggesting that the syntheses of thrombospondin and laminin are regulated by different mechanisms. When cells are cultured on gelatin- or collagen-coated dishes, thrombospondin gene expression is directly proportional to the proliferative state of the cultures. By contrast, the synthesis of thrombospondin by cells cultured on collagen gels remains at equally low levels whether they are labelled when they are sparse and rapidly proliferating or when they are confluent and quiescent. Fibronectin synthesis was found to increase with increasing confluency of the cells plated on all three substrata. These results demonstrate that thrombospondin gene expression is modulated by cell shape, cell proliferation and the nature of the substratum used for cell culture.

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Mitochondrial Arginase II Modulates Nitric-Oxide Synthesis through Nonfreely Exchangeable l-Arginine Pools in Human Endothelial Cells

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.106.103747 ◽

2006 ◽

Vol 318 (3) ◽

pp. 1368-1374 ◽

Cited By ~ 57

Author(s):

Gökce Topal ◽

Annie Brunet ◽

Laurence Walch ◽

Jean-Luc Boucher ◽

Monique David-Dufilho

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Nitric Oxide Synthesis ◽

Human Endothelial Cells ◽

Arginase Ii

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Thiamine pyrophosphate diminishes nitric oxide synthesis in endothelial cells

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000650 ◽

2020 ◽

pp. 1-9

Author(s):

Susana Alcázar-Leyva ◽

Estrella Zapata ◽

Demetrio Bernal-Alcántara ◽

Patricia Gorocica ◽

Noé Alvarado-Vásquez

Keyword(s):

Nitric Oxide ◽

Cell Proliferation ◽

Endothelial Cells ◽

High Glucose ◽

Thiamine Pyrophosphate ◽

Diabetic Patients ◽

Protective Agent ◽

Nitric Oxide Synthesis ◽

Umbilical Cord Vein ◽

Cells Cultured

Abstract. Although thiamine pyrophosphate (TPP) is considered a protective agent for endothelial cells, it is still unknown if this is associated with nitric oxide (NO) synthesis. Our aim was to evaluate the synthesis of NO in endothelial cells incubated with TPP and high glucose concentrations. Endothelial cells from the umbilical cord vein from newborns (n = 20), were incubated with 5, 15 or 30 mmol/L glucose, in absence or presence of 0.625 mg/ml of TPP. Our results showed a significant increase in cell proliferation (> 40%; P < 0.05), and cell viability (> 90%; P < 0.001) after 48 h in endothelial cells cultured with glucose plus TPP. Likewise, in the presence of glucose and TPP an important rise in the consumption of glucose by the endothelial cells was observed after 24 h (> 7%; P < 0.001) and 48 h (> 10%; P < 0.05). Additionally, the levels of lactate after incubation with glucose and TPP showed only slight variations after 48 h (P < 0.05). However, these changes were clearly different from those observed in the absence of TPP. Interestingly, we found that the changes mentioned were linked with reduced levels of nitrites both at 24 h (< 171 pmol/μg protein; P < 0.001), and 48 h (< 250 pmol/μg protein; P < 0.05), which was associated with a reduced expression of mRNA of eNOS in endothelial cells incubated with TPP and high glucose. In conclusion, the presence of TPP regulates the consumption of glucose and the synthesis of NO, which would explain its protective effect in the endothelium of diabetic patients.

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