Role of the CD40 receptor/CD154 ligand dyad in the control of smooth muscle cells phenotype

Abstract

In the pathogenesis of atherosclerosis, circulating leukocytes adhere to endothelial cells, migrate through them and enter the vessel wall. CD40, a member of the tumor necrosis factor receptor family, that is expressed by both leukocytes and endothelial cells plays an important role in this process. Upon their emigration into the vessel wall leukocytes come into contact with the vascular smooth muscle cells (SMC). These cells also play a major role in the development and progression of atherosclerosis and express CD40 under pro-inflammatory conditions. The aim of the present study was to answer the question which role CD40 expression in SMC might play in the context of atherosclerosis. To address this complex question, four different experimental approaches were taken: (i) analysis of CD40 expression itself in human cultured SMC, (ii) analysis of CD154-induced gene expression in these cells, as well as (iii) the signal transduction pathways involved therein, and (iv) investigating possible functional consequences of these changes in gene expression. A first result achieved was that under pro-inflammatory conditions, CD40 expression is markedly up-regulated in human cultured SMC. Moreover, activation of CD40 in these cells resulted in the differential expression of 36 genes, as judged by DNA microarray and confirmed by RT-PCR analysis. Most of these gene products were up-regulated and comprised pro-inflammatory molecules, namely chemokines and their receptors, cytokines and their receptors, and adhesion molecules. The expression of these gene products in human SMC could be demonstrated for the first time. To elucidate the signalling pathways linking CD40-CD154 interaction to gene expression in the SMC, CD40-induced matrix metalloproteinase-3 (MMP-3) expression was chosen as a readout. By using different pharmacological inhibitors, it was demonstrated that the tyrosine kinase c-Src and the mitogen-activated protein kinase p38 are involved in CD40 signalling to the nucleus in these cells. Moreover, MMP-3 expression was verified to be up-regulated on the level of transcription, although the transcription factor(s) responsible therefor could not be identified as yet. By using two different experimental approaches, a SMC-monocyte interaction assay and analysing changes in gene expression in these cells upon exposure to the conditioned medium of CD154-stimulated SMC, it was further shown that monocyte activation is mediated by a humoral factor rather than a cell-to-cell contact. Further investigations, employing neutralizing antibodies, revealed that granulocyte-macrophage colony-stimulating factor is a likely candidate for the monocyte-activating humoral factor. Collectively, these findings suggest that vascular SMC like endothelial cells contribute to the maintenance of an inflammatory response in the vessel wall as, e.g., in atherosclerosis when stimulated via the CD40/CD154 receptor/ligand dyad. Elucidating the transcriptional mechanism by which CD40 activation in these cells is translated into the release of pro-inflammatory mediators represents a valuable therapeutic goal in this context.