Abstract 476: TLR4 Activation in Microvascular Endothelial Cells Triggers a Robust Inflammatory Response and Crosstalk with Mononuclear Cells

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Although coronary artery disease is a macrovascular disease, microvessels are developed in atherosclerotic plaques from the vasa vasorum and contribute to inflammation and intraplaque hemorrhage. As the major types of cells in vascular system, both macrovascular endothelial cells (MAC ECs), which cover the luminal surface of atherosclerotic plaques, and microvascular endothelial cells (MIC ECs), which line small size vessels and capillaries inside lesions, are important players in atherosclerosis. In recent years, a large number of studies have provided evidence that toll-like receptor (TLR)4, an innate immune receptor, plays an essential role in atherosclerosis. However, the inflammatory responses by MIC and MAC ECs to TLR4 activation have not been fully characterized and compared. In this study, ELISA, real-time PCR and gene expression arrays showed that TLR4 activation by lipopolysaccharide (LPS) stimulated a much more robust expression of pro-inflammatory genes including cytokines, chemokines, growth factors and adhesion molecules in human dermal MIC ECs than human aortic MAC ECs. For example, TLR4 activation increased interleukin (IL)-6 secretion from MIC and MAC ECs by 185- and 17-fold of that from control cells (150,916 vs 814 pg/ml for MIC ECs and 501 vs 29 pg/ml for MAC ECs), respectively. Also, TLR4 activation increased chemokine CXCL10 secretion from MIC and MAC ECs by 5,405- and 12-folds of that from control cells, respectively. Furthermore, TLR4 activation in MIC ECs triggered a crosstalk with U937 mononuclear cells through MIC EC-released IL-6 to upregulate matrix metalloproteinase (MMP)-1 expression in U937 cells. In contrast, TLR4 activation in MAC ECs did not trigger the crosstalk with mononuclear cells. To explore the mechanisms underlying the different degrees of responsiveness to TLR4 activation by MIC and MAC ECs, we found that TLR4 and CD14 expression in MIC ECs was significantly higher than that in MAC ECs. Finally, we found that the nuclear factor kappa B (NFκB) activity in response to TLR4 activation increased by10- and 3-fold, respectively, in MIC and MAC ECs. Taken together, this study showed that TLR4 activation in MIC ECs triggered a more robust inflammatory response than that in MAC ECs and facilitated MIC ECs to crosstalk with mononuclear cells. Given the major contribution of cytokines and MMPs to atherosclerotic plaque destabilization, our study indicates that MIC ECs may play a major role in plaque vulnerability through a TLR4-dependent mechanism.