Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, PC32
Sphingosine 1-phosphate-induced release of TIMP-2 from vascular smooth muscle cells inhibits angiogenesis in coronary artery endothelial cells
K. S. Mascall1, G. R. Small1, G. F. Nixon1
1. School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
Following myocardial infarction resulting from thrombus formation, angiogenesis occurs and permits reperfusion of damaged myocardium. Sphingosine 1-phosphate (S1P) is a naturally occurring lipid mediator released from platelets and is found at sites of thrombosis. S1P could be involved in regulating angiogenesis following myocardial infarction and may therefore influence reperfusion. The aims of this study was to determine the effects of S1P in human coronary artery endothelial cell (EC) angiogenesis and delineate the subsequent mechanisms. An in vitro model of angiogenesis was developed using a co-cultures of human coronary artery ECs, human coronary smooth muscle cells (SMCs) and human fibroblasts. After 14 days, endothelial tubule formation was visualized by immunofluorescence labelling with anti-CD146 antibody. Fluorescent images were analysed using ImageJ software. S1P (1 μM) significantly inhibited tubule formation in these co-cultures by 80 ± 7% (mean ± s.e.m., n=7, p<0.05 Student’s t-test). When SMCs were omitted from the co-culture (ECs and fibroblasts only) EC tubules were still observed as previously, but S1P had no significant effect on tubule formation. In order to determine if this inhibitory effect of S1P was due to a soluble factor released from SMCs, conditioned medium from SMCs incubated with S1P was used on co-cultures of ECs and fibroblasts. S1P-treated conditioned medium from SMCs also inhibited endothelial tubule formation compared to untreated conditioned medium (64 ± 7%, n=4, p<0.05). As tissue inhibitor of metalloproteinases (TIMPs) are known inhibitors of angiogenesis and released by several different cell types, we examined TIMP-2 activity in conditioned medium from SMCs. In conditioned medium from SMCs treated with 1 μM S1P there was a significant increase in TIMP-2 activity as determined by reverse zymography compared to untreated conditioned medium (1.35 ± 0.07 fold increase, n=4, p<0.05). In co-cultures of ECs, SMCs and fibroblasts containing anti-TIMP-2 blocking antibody, S1P had no effect on tubule formation (n=4). Confocal microscopy of co-cultured treated with S1P revealed that vascular endothelial (VE)-cadherin localization to endothelial-endothelial cell junctions was disrupted thereby inhibiting tubule formation. This S1P-inhibitory effect was reversed by inclusion of TIMP-2 blocking antibody in the medium. In conclusion, S1P-induced inhibition of angiogenesis in human artery endothelial cells is mediated by a release of TIMP-2 from SMCs. This reduces the integrity of intercellular junctions between nascent endothelial cells by inhibiting VE-cadherin localization. S1P may therefore inhibit the angiogenic response following myocardial infarction.
Where applicable, experiments conform with Society ethical requirements