Abstract
Decorin, a small multifunctional proteoglycan, is expressed by sprouting endothelial cells (ECs) during inflammation-induced angiogenesis in vivo and by human ECs co-cultured with fibroblasts in a collagen lattice. To investigate how decorin is induced, human EA.hy 926 ECs and/or human umbilical vein ECs were treated with interleukin (IL)-10 and IL-6. Both treatments induced decorin mRNA in human ECs. IL-6 and IL-10 led to a dose-dependent mRNA increase with a maximum at 10 and 50 ng/ml, respectively. The combination of both interleukins together had a stronger effect than one alone. Immunostaining demonstrated that both interleukins caused decorin synthesis in ECs and the formation of capillary-like structures in a collagen lattice. However, immunoprecipitations of interleukin-treated ECs cultured on plastic were negative. Only interleukin-stimulated ECs grown on a collagen type I matrix or growth factor-reduced Matrigel were able to synthesize the proteoglycan. Acid-soluble collagen type I did not support decorin protein synthesis. The addition of antibodies to alpha(1) or alpha(2) integrins or the alpha(2) integrin inhibitor rhodocetin led to an inhibition of synthesis. These data show that IL-10 and IL-6 induce decorin mRNA transcription, but additional signals from the extracellular matrix are necessary for its translation.
Highlights
Decorin is the most well known member of the small leucinerich repeat proteoglycans (SLRPs) family
Induction of Decorin mRNA in Endothelial Cells by IL-10 and IL-6 —In previous experiments we excluded a large number of growth factors and cytokines associated with angiogenesis as inducers of decorin synthesis in endothelial cells [2]
Semiquantitative reverse transcription-PCR showed that IL-10 can dose dependently increase decorin mRNA production in endothelial cells in a concentration range between 12.5 and 50 ng/ml
Summary
Materials—Human IL-10 and IL-6 were from TEBU (Frankfurt, Germany). Chondroitin ABC lyase was from Seikagaku Kogyo (Tokyo, Japan). Collagen type I was the major protein in the preparation, but the presence of collagen type III could not be ruled out (result not shown) This collagen was used to coat cell culture dishes. For inhibition experiments EA.hy 926 cells (2.5 ϫ 106) were preincubated with the respective antibodies (5 ng/ml) and seeded on collagen type I matrices in Waymouth medium (see above) with 1% heat-inactivated fetal calf serum. Immunohistochemistry—Immunostaining of collagen gels was performed as described previously [2], except that in addition to chondroitin ABC lyase (2 h, 37 °C) in 5% bovine serum albumin, 10% goat serum (Sigma) in phosphate-buffered saline, proteinase XXIV (0.005%, 10 min, 37 °C, Sigma) was used to unmask epitopes
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