Foam Cell Specific LXRα Ligand

Objective The liver X receptor α (LXRα) is a ligand-dependent nuclear receptor and the major regulator of reverse cholesterol transport in macrophages. This makes it an interesting target for mechanistic study and treatment of atherosclerosis. Methods and Results We optimized a promising stilbenoid structure (STX4) in order to reach nanomolar effective concentrations in LXRα reporter-gene assays. STX4 displayed the unique property to activate LXRα effectively but not its subtype LXRβ. The potential of STX4 to increase transcriptional activity as an LXRα ligand was tested with gene expression analyses in THP1-derived human macrophages and oxLDL-loaded human foam cells. Only in foam cells but not in macrophage cells STX4 treatment showed athero- protective effects with similar potency as the synthetic LXR ligand T0901317 (T09). Surprisingly, combinatorial treatment with STX4 and T09 resulted in an additive effect on reporter-gene activation and target gene expression. In physiological tests the cellular content of total and esterified cholesterol was significantly reduced by STX4 without the undesirable increase in triglyceride levels as observed for T09. Conclusions STX4 is a new LXRα-ligand to study transcriptional regulation of anti-atherogenic processes in cell or ex vivo models, and provides a promising lead structure for pharmaceutical development

Hormetic shifting of redox environment by pro-oxidative resveratrol protects cells against stress

AbstractResveratrol has gained tremendous interest owing to multiple reported health-beneficial effects. However, the underlying key mechanism of action of this natural product remained largely controversial. Here, we demonstrate that under physiologically relevant conditions major biological effects of resveratrol can be attributed to its generation of oxidation products such as reactive oxygen species (ROS). At low nontoxic concentrations (in general <50µM), treatment with resveratrol increased viability in a set of representative cell models, whereas application of quenchers of ROS completely truncated these beneficial effects. Notably, resveratrol treatment led to mild, Nrf2-specific gene expression reprogramming. For example, in primary epidermal keratinocytes derived from human skin this coordinated process resulted in a 1.3-fold increase of endogenously generated glutathione (GSH) and subsequently in a quantitative reduction of the cellular redox environment by 2.61mVmmol GSH per g protein. After induction of oxidative stress by using 0.78% (v/v) ethanol, endogenous generation of ROS was consequently reduced by 24% in resveratrol pre-treated cells. In contrast to the common perception that resveratrol acts mainly as a chemical antioxidant or as a target protein-specific ligand, we propose that the cellular response to resveratrol treatment is essentially based on oxidative triggering. In physiological microenvironments this molecular training can lead to hormetic shifting of cellular defense towards a more reductive state to improve physiological resilience to oxidative stress

STX4 is a novel selective LXRα agonist.

<p><b>A</b>, Chemical structure of STX4. <b>B</b>, Transcriptional activation of LXRα by T0901317 (T09), 22-R-hydroxycholesterol or STX4 in a reporter gene assay (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057311#pone-0057311-t001" target="_blank">Table 1</a>). Data are expressed as mean±SD (n = 3). <b>C</b>, Validation of LXRα specificity. Transcriptional activation of LXRβ by T09 or STX4 in a reporter gene assay. STX4 does not activate the LXRβ subtype (mean±SD, n = 3). <b>D</b>, Cytotoxicity of STX4 in macrophages (mean±SD, n = 3). STX4 does not reduce cellular viability up to 25 µM.</p

Data of oxygen- and pH-dependent oxidation of resveratrol

AbstractWe show here if under physiologically relevant conditions resveratrol (RSV) remains stable or not. We further show under which circumstances various oxidation products of RSV such as ROS can be produced. For example, in addition to the widely known effect of bicarbonate ions, high pH values promote the decay of RSV. Moreover, we analyse the impact of reduction of the oxygen partial pressure on the pH-dependent oxidation of RSV. For further interpretation and discussion of these focused data in a broader context we refer to the article “Hormetic shifting of redox environment by pro-oxidative resveratrol protects cells against stress” (Plauth et al., in press) [1]

Physiological analyses of STX4 treatment confirm unique STX4 properties in foam cells.

<p><b>A</b>, Cholesterol content in foam cells after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). <b>B</b>, Cholesterol content in macrophages after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). <b>C</b>, Triglyceride content in foam cells after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). <b>D</b>, Triglyceride content in macrophages after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). ***P<0.001 vs. DMSO; n.s., not significant.</p

STX4 specifically regulates gene expression in foam cells.

<p><b>A</b>, Genome-wide gene expression and subsequent gene set enrichment analysis (GSEA) of macrophages and foam cells after treatment with T09 (10 µM) or STX4 (10 µM). Regulation of lipid-derived Reactome and KEGG pathways is shown. <b>B</b>, Validation of gene expression microarray analysis by quantitative PCR. For all tested samples, array observations significantly correlated with qPCR results. <b>C</b>, Gene distance matrix of genome-wide gene expression analysis of macrophages and foam cells after treatment with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Pairwise distances were calculated for comparison of two treatments and cell types. Colored squares show the distance in Euclidean space, ranging from exactly the same profile (black) to completely different (red). In macrophages STX4 expression is very different to that of T09 (P<0.0001), whereas in foam cells STX4-induced gene expression is similar to that of T09 (P<0.0001).</p

STX4 specifically targets diseased, oxysterol-loaden foam cells.

<p><b>A</b>, Gene expression in foam cells after siRNA-mediated, single and combined LXRα/β knockdown (mean±SEM, n = 4, fold-change vs. DMSO, logarithmised). Left, efficiency of LXRα, LXRβ and LXRα/β knockdown (kd). Middle, LXRα, LXRβ and LXRα/β knockdown (kd). Middle, single and combined knockdown influence on gene expression of LXRα, LXRβ and ABCA1 upon T09 treatment. Right, single and combined knockdown influence on gene expression of LXRα, LXRβ and ABCA1 upon STX4 treatment. *P<0.05, **P<0.01, ***P<0.001 vs. negative siRNA. <b>B</b>, Gene expression in THP-1 macrophages and foam cells after treatment with T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 4). <b>C</b>, Western blot analysis of LXRα and APOE content in foam cells and STX4 treated foam cells. Bar plot displays the results of densitometry analysis (mean±SEM, n = 3). *P<0.05, **P<0.01 vs. foam cell.<b>D</b>, Transcriptional activation of LXRα by STX4 in the presence or absence of 200 nM T09 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057311#pone-0057311-t001" target="_blank">table <b>1</b></a>). Data are expressed as mean±SD (n = 3). <b>E</b>, Gene expression in THP-1 macrophages after treatment with different concentrations of T09 or STX4 in the presence or absence of 1 µM T09. Data are expressed as mean±SEM (n = 2–3). *P<0.05, **P<0.01, ***P<0.001 vs. DMSO; n.s., not significant.</p

Results from competitive reporter-gene assays.

<p>Results from competitive reporter-gene assays.</p