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C-reactive protein (CRP) is a risk factor for cardiovascular events and functions to amplify vascular inflammation through promoting endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1) is the primary endothelial receptor for oxLDL, and both its expression and function are associated with vascular inflammation. As a scavenger receptor, LOX-1 is capable of binding to a variety of structurally unrelated ligands. Evidence is provided that demonstrates that CRP can act as a novel ligand for LOX-1. The direct interaction between these two proteins was demonstrated with purified protein in both ELISA and AlphaScreen assays. This interaction could be disrupted with known LOX-1 ligands, such as oxLDL and carrageenan. Moreover, the CRP interaction with cell surface-expressed LOX-1 was confirmed in cell-based immunofluorescent-binding studies. Mutagenesis studies demonstrated that the arginine residues forming the basic spine structure on the LOX-1 ligand-binding interface were dispensable for CRP binding, suggesting a novel ligand-binding mechanism for LOX-1, distinct from that used for oxLDL binding. The treatment of human endothelial cells with CRP led to the activation of proinflammatory genes including IL-8, ICAM-I, and VCAM-1. The inductions of these genes by CRP were LOX-1 dependent, as demonstrated by their attenuation in cells transfected with LOX-1 small-interfering RNA. Our study identifies and characterizes the direct interaction between LOX-1 and CRP and suggests that this interaction may mediate CRP-induced endothelial dysfunction. endothelial cells; scavenger receptor; oxidized low-density lipoprotein receptor- 1; C-reactive protein

The nuclear hormone receptor farnesoid X receptor (FXR) plays a critical role in the regulation of bile acid, triglyceride (TG), and cholesterol homeostasis. WAY-362450 (FXR-450/XL335) is a potent synthetic FXR agonist as characterized in luciferase reporter assays and in mediating FXR target gene regulation in primary human and immortalized mouse hepatocytes. In vivo, WAY-362450 dose dependently decreased serum TG levels after 7 days of oral dosing in western diet-fed low-density lipoprotein receptor-/- mice and in the diabetic mouse strains KK-Ay and db/db comparable to that achieved with the peroxisome proliferator activated receptor-[alpha] agonist, fenofibrate. WAY-362450 treatment also reduced serum cholesterol levels via reductions in LDLc, VLDLc, and HDLc lipoprotein fractions that were not accompanied by hepatic cholesterol accumulation. This cholesterol lowering was dependent on FXR as demonstrated in a hypothyroid-induced hypercholesterolemia setting in FXR-/- mice. In fructose-fed models, WAY-362450 also decreased TG and VLDLc levels in rats and hamsters but significantly increased HDLc levels in rats while reducing HDLc levels in hamsters. The differential effect of WAY-362450 on HDLc is likely due to a murine-specific induction of endothelial lipase and scavenger receptor-BI that does not occur in rats. These studies demonstrate a consistent ability of WAY-362450 to lower both serum TG and cholesterol levels and suggest that synthetic FXR agonists may have clinical utility in the treatment of mixed dyslipidemia. triglyceride; very low-density lipoprotein; high-density lipoprotein; low-density lipoprotein

Bile acids are synthesized in the liver, stored in gallbladder, and secreted into the intestine to aid in the absorption of lipid-soluble nutrients. In addition, bile acids also actively participate in regulation of gene expression through their ability to act as ligands for the nuclear receptor farnesoid X receptor or by activating kinase signaling pathways. Under cholestatic conditions, elevated levels of bile acids in the liver induce hepatic inflammation, and because bile acid levels are also elevated in the circulation, they might also induce vascular inflammation. To test this hypothesis, primary human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells were treated with bile acids, and the expression of ICAM-1, VCAM-1, and E-selectin were monitored. The three major bile acids found in the circulation, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, all strongly induced both the mRNA and protein expression of ICAM-1 and VCAM-1. To delineate the mechanism, the experiments were conducted in the presence of various kinase inhibitors. The results demonstrate that the bile acid-mediated induction of adhesion molecule expression occurs by stimulation of NF-KB and p38 MAPK signaling pathways through the elevation in reactive oxygen species. The bile acid-induced cell surface expression of ICAM-1 and VCAM-1 was sufficient to result in the increased adhesion of THP-1 monocytes to the HUVEC, suggesting that elevated levels of bile acids in the circulation may cause endothelium dysfunction and contribute to the initiation of early events associated with vascular lesion formation. nuclear factor-[kappa]B; human umbilical vein endothelial cells: progressive familial intrahepatic cholestasis doi:10.1152/ajpheart.01184.2005

Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3-5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-[alpha], VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7[alpha]-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time- dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-I promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-[alpha] cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events. inflammation

Inflammation is now recognized as a key component in a number of diseases such as atherosclerosis, rheumatoid arthritis, and inflammatory bowel disease. The transcription factor NF-[kappa]B has been shown to be involved in both the early and late stages of the inflammatory-proliferative process. In this report, we describe the identification of the pathway-selective estrogen receptor (ER) ligand, WAY-169916, that inhibits NF-[kappa]B transcriptional activity but is devoid of conventional estrogenic activity. This pathway-selective ligand does not promote the classic actions of estrogens such as stimulation of uterine proliferation or ER-mediated gene expression, but is a potent antiinflammatory agent, as demonstrated in the HLA-B27 transgenic rat model of inflammatory bowel disease. Our results indicate the potential utility of pathway-selective ER ligands such as WAY-169916 in the treatment of chronic inflammatory diseases. inflammation | therapy

A well-established model of bowel inflammation is the HLA-B27 transgenic rat that exhibits a spontaneous disease phenotype resulting in chronic diarrhea caused by immune cell activation. Estrogens have previously been shown to modulate the immune system, and both estrogen receptors (ER[alpha] and ER[beta]) are present in the intestine and cells of the immune system. Therefore, the ability of estrogen to ameliorate disease progression in the HLA-B27 transgenic rat was determined. HLA-B27 transgenic rats with chronic diarrhea were treated with 17[alpha]-ethynyl-17[beta]-estradiol (EE) for 5 days. EE treatment dramatically improved stool scores after only 3 days. Histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon were also improved by EE treatment. Because neutropbil infiltration into the colon is involved in the development and propagation of disease, myeloperoxidase (MPO) activity was measured. MPO levels were reduced by 80% by EE treatment. Cotreatment with the pure ER antagonist ICI-182780 (ICI) blocked the effects of EE on stool character, MPO activity, and histology scores, strongly suggesting that the activity of EE is mediated through ER. Mast cell proteases can promote neutrophil infiltration, and gene expression analysis demonstrated that mast cell protease 1, 3, and 4 mRNA were all decreased in colons from estrogen-treated rats. In addition, a direct effect of estrogen on bone marrow-derived mast cell activity was demonstrated, suggesting that ER-mediated inactivation of mast cells may contribute to the improvement in the clinical sign and histological scores in this model. mast cells: estrogen receptor

Retinoic acid (RA) plays an important role in normal embryogenesis; however, excessive doses are teratogenic. At present, the molecular mechanisms responsible for these effects of RA are not well understood. The action of retinoids are believed to be mediated by two classes of proteins, nuclear receptors (retinoic acid receptors (RARs) and retinoid X receptors (RXRs)) and small cellular retinol-binding and retinoic acid-binding proteins (CRBP-I, CRBP-II, CRABP-I and CRABP-II). Teratogenic doses of RA increase the level of RAR-beta(sub 2) mRNA, RAR-alpha(sub 2) mRNA, CRBP-I mRNA and CRABP-II mRNA in mouse conceptuses and embryos. The elevation in the level of only RAR-beta(sub 2) mRNA correlates with the target tissues, as well as developmental stages that are sensitive to the teratogenic effects of RA. In addition, we have screened a few other natural and synthetic retinoids with similar results. These results are consistent with the possibility that RAR-beta(sub 2) may mediate at least some of the effects of retinoids during abnormal development.

Bile acids are synthesized in the liver, stored in gallbladder, and secreted into the intestine to aid in the absorption of lipid-soluble nutrients. In addition, bile acids also actively participate in regulation of gene expression through their ability to act as ligands for the nuclear receptor farnesoid X receptor or by activating kinase signaling pathways. Under cholestatic conditions, elevated levels of bile acids in the liver induce hepatic inflammation, and because bile acid levels are also elevated in the circulation, they might also induce vascular inflammation. To test this hypothesis, primary human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells were treated with bile acids, and the expression of ICAM-1, VCAM-1, and E-selectin were monitored. The three major bile acids found in the circulation, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, all strongly induced both the mRNA and protein expression of ICAM-1 and VCAM-1. To delineate the mechanism, the experiments were conducted in the presence of various kinase inhibitors. The results demonstrate that the bile acid-mediated induction of adhesion molecule expression occurs by stimulation of NF-κB and p38 MAPK signaling pathways through the elevation in reactive oxygen species. The bile acid-induced cell surface expression of ICAM-I and VCAM-I was sufficient to result in the increased adhesion of THP-I monocytes to the HUVEC, suggesting that elevated levels of bile acids in the circulation may cause endothelium dysfunction and contribute to the initiation of early events associated with vascular lesion formation. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3–5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-α, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependant repression of cholesterol 7α-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time-dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-α cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events. [ABSTRACT FROM AUTHOR]

Reports study findings that the estrogen receptor alpha regulates expression of the orphan receptor small heterodimer partner (SHP). Importance of the mutation of the human SHP promoter to the basal activity; Indications that direct regulation of SHP expression may provide a basis for some of the numerous biological effects of estrogen.

Pathway selective ligands of the estrogen receptor inhibit transcriptional activation of proinflammatory genes mediated by NF-kappaB. Substituted 2-cyanopropanoic acid derivatives were developed leading to the discovery of WAY-204688, an orally active, pathway selective, estrogen receptor dependent anti-inflammatory agent. This propanamide was shown to be orally active in preclinical models of inflammatory diseases, such as rheumatoid arthritis, without the proliferative effect associated with traditional estrogens.

Inflammation is recognized as a key component in a number of diseases, including rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. Although well known for their classic effects on the reproductive tract and action by means of estrogen response elements in gene promoters, estrogens are also known to possess anti-inflammatory activity. This was originally highlighted with the observation that pregnancy ameliorates symptoms of rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Furthermore, the antagonistic cross talk between nuclear factor kappaB and estrogen receptor signaling pathways has been well documented. Recently, novel estrogen receptor ligands, pathway-selective ligands and estrogen receptor beta-selective ligands have been identified which demonstrate potent anti-inflammatory activity; these ligands are being analyzed for their therapeutic potential in pathogenic inflammation.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that produces synovial proliferation and joint erosions. The pathologic lesions of RA are driven through the production of inflammatory mediators in the synovium mediated, in part, by the transcription factor NF-kappaB. We have identified a non-steroidal estrogen receptor ligand, WAY-169916, that selectively inhibits NF-kappaB transcriptional activity but is devoid of conventional estrogenic activity. The activity of WAY-169916 was monitored in two models of arthritis, the HLA-B27 transgenic rat and the Lewis rat adjuvant-induced model, after daily oral administration. In both models, a near complete reversal in hindpaw scores was observed as well as marked improvements in the histological scores. In the Lewis rat adjuvant model, WAY-169916 markedly suppresses the adjuvant induction of three serum acute phase proteins: haptoglobin, alpha1-acid glycoprotein (alpha1-AGP), and C-reactive protein (CRP). Gene expression experiments also demonstrate a global suppression of adjuvant-induced gene expression in the spleen, liver, and popliteal lymph nodes. Finally, WAY-169916 was effective in suppressing tumor necrosis factor-alpha-mediated inflammatory gene expression in fibroblast-like synoviocytes isolated from patients with RA. Together, these data suggest the utility of WAY-169916, and other compounds in its class, in treating RA through global suppression of inflammation via selective blockade of NF-kappaB transcriptional activity.

Hormonal status can influence diverse metabolic pathways. Small heterodimer partner (SHP) is an orphan nuclear receptor that can modulate the activity of several transcription factors. Estrogens are here shown to directly induce expression of the SHP in the mouse and rat liver and in human HepG2 cells. SHP is rapidly induced within 2 h following treatment of mice with ethynylestradiol (EE) or the estrogen receptor alpha (ERalpha)-selective compound propyl pyrazole triol (PPT). SHP induction by these estrogens is completely absent in ERalphaKO mice. Mutation of the human SHP promoter defined HNF-3, HNF-4, GATA, and AP-1 sites as important for basal activity, whereas EE induction required two distinct elements located between -309 and -267. One of these elements contains an estrogen response element half-site that bound purified ERalpha, and ERalpha with a mutated DNA binding domain was unable to stimulate SHP promoter activity. This ERalpha binding site overlaps the known farnesoid X receptor (FXR) binding site in the SHP promoter, and the combination of EE plus FXR agonists did not produce an additive induction of SHP expression in mice. Surprisingly, induction of SHP by EE did not inhibit expression of the known SHP target genes cholesterol 7alpha-hydroxylase (CYP7A1) or sterol 12alpha-hydroxylase (CYP8B1). However, the direct regulation of SHP expression may provide a basis for some of the numerous biological effects of estrogens.