Apoptosis of human macrophages by Flt-4 signaling: implications for atherosclerotic plaque pathology.

BACKGROUND Neointimal inflammation and angiogenesis are important contributors of progression and destabilization of the atherosclerotic plaque. While the role of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1) in this process has clearly been defined, expression of the VEGF-R3 (Flt-4) has only been documented on lymphatic and tumor endothelium. This study examined Flt-4 expression in human atherosclerotic plaque and explored its implications for atherosclerotic disease. METHODS AND RESULTS Carotid artery thrombendartherectomy specimens from 10 patients with unstable plaque were stained for Flt-4 and its specific growth factors VEGF-C and VEGF-D. Microvascular endothelial cells (MVEC) stained positive for VEGF-C and -D, but not for Flt-4. Interestingly, macrophages within inflammatory perivascular regions coexpressed Flt-4, VEGF-C and VEGF-D. In vitro studies confirmed the expression of Flt-4, VEGF-C and VEGF-D in human monocytes and cultured macrophages. Treatment of macrophages with VEGF-D induced apoptosis as determined by annexin V staining, by immunoblotting of activated caspase 3, and by the ratio of Bcl-2/Bax as well as by DNA fragmentation. Immunohistochemical studies of advanced human carotid atherosclerotic plaque confirmed the coexpression of Flt-4 with activated caspase 3 and TUNEL staining in macrophages, indicating an ongoing apoptotic process. CONCLUSION Human monocytes/macrophages express VEGF-C and -D and their receptor Flt-4 in vitro and in vivo within advanced atherosclerotic lesions. Flt-4, in turn, mediates monocyte/macrophage apoptosis and may this way alter plaque stability.