Exposure of blood to tissue factor (TF) rapidly initiates the coagulation serine protease cascades. TF is expressed by macrophages and other types of cell within atherosclerotic lesions and plays an important role in thrombus formation after plaque rupture. Macrophage TF expression is induced by pro‐inflammatory stimuli including lipopolysaccharide (LPS), interleukin‐1β and tumor necrosis factor‐α. Here we demonstrate that activation of liver X receptors (LXRs) LXRα and LXRβ suppresses TF expression. Treatment of mouse peritoneal macrophages with synthetic LXR agonist T0901317 or GW3965 reduced TF expression induced by pro‐inflammatory stimuli. LXR agonists also suppressed TF expression and its activity in human monocytes. Human and mouse TF promoters contain binding sites for the transcription factors AP‐1, NFκB, Egr‐1 and Sp1, but no LXR‐binding sites could be found. Cotransfection assays with LXR and TF promoter constructs in RAW 264.7 cells revealed that LXR agonists suppressed LPS‐induced TF promoter activity. Analysis of TF promoter also showed that inhibition of TF promoter activity by LXR was at least in part through inhibition of the NFκB signaling pathway. In addition, in vivo, LXR agonists reduced TF expression within aortic lesions in an atherosclerosis mouse model as well as in kidney and lung in mice stimulated with LPS. These findings indicate that activation of LXR results in reduction of TF expression, which may influence atherothrombosis in patients with vascular disease.

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Objective: Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (ca. 2.1-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO × hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO × hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [ 3 H]-cholesterol and confirmed significant increases of [ 3 H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a–treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a–immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. Conclusions: This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. This is also the first report describing that a small-molecule direct LCAT activator achieved HDL cholesterol elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.

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