Co-culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors.

Abstract

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors.