LCA inhibits IFNγ gene expression in Jurkat T cells

<p><b>(A)</b> Cell viability of Jurkat T cells measured by flowcytometry analysis and expressed as percentage of control in response to incubation with unconjugated bile acids and <b>(B)</b> taurine-conjugated bile acids for 24 hours. <b>(C)</b> IFNγ mRNA expression of PMA/Ionomycin (P/I)-activated Jurkat T cells treated for 24 hours with 10 μM of different bile acid species. LCA, lithocholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid; DCA, deoxycholic acid; TLCA, taurolithocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TCA, taurocholic acid; AU, Arbitrary units. Results represent the mean ± SEM. *Statistically significant, P<0.05. Experiments were performed in triplicates and repeated at least twice.</p

Characterizing the LCA sensor in CD4⁺ Th cells.

<p><b>(A)</b> Table showing mRNA expression of Membrane and Nuclear bile acid receptors in Jurkat T cells and primary human CD4⁺ Th cells. “+” indicates that the gene is expressed at low levels, i.e. Real-time RT-PCR Ct cycles of 30–35; “++” indicates that the gene is expressed at Ct cycles of 25–30, and “+++” indicates high expression of the gene at Ct cycles of <25. <b>(B)</b> mRNA expression of <i>TGR5</i>, <i>FPR3</i>, <i>S1PR2 PXR</i> and <i>VDR</i> in resting “C” and in Dynabeads T-activator stimulated primary human CD4⁺ Th cells “A”. <b>(C)</b> mRNA expression of VDR in Jurkat T cells transfected with silencing RNA against the VDR (siVDR; light grey bar) or scrambled siRNA (siSCR; dark grey bar). <b>(D)</b> <i>IFN</i>γ and <b>(E)</b> <i>T-BET</i> mRNA expression in Jurkat T cells transfected with siRNA against the VDR (siVDR; light grey bars) or scrambled siRNA (siScr; dark grey bars) in response to P/I stimulation and in combination with 10 μM LCA or vehicle treatment. LCA, lithocholic acid; P/I, PMA/ionomycin; AU, Arbitrary units. Results represent the mean ± SEM. NS, Not significant. *Statistically significant, P<0.05. Experiments were performed in triplicates and repeated at least twice.</p

LCA inhibits CD4⁺ Th cell activation.

<p><b>(A)</b><i>IFN</i>γ mRNA expression and <b>(B)</b> <i>TNF</i>α mRNA expression in Jurkat T cells in response to 10 μM LCA treatment (light grey lines) or vehicle (dark grey lines) with P/I activation (triangles) or in resting Jurkat T cells (squares). <b>(C)</b> <i>IFN</i>γ and <b>(D)</b> <i>TNF</i>α mRNA expression in P/I-activated Jurkat T cells in response to increasing concentrations of LCA. <b>(E)</b> Secreted TNFα protein levels of Jurkat T cells in the supernatant in response to 10 μM LCA treatment (light grey lines) or vehicle (dark grey lines) with P/I activation (triangles) or in resting Jurkat T cells (squares). <b>(F)</b> mRNA expression of <i>IFN</i>γ, <i>IL-6</i>, <i>CD40L</i>, <i>TNF</i>α in primary mouse CD4⁺ Th cells activated with P/I and treated with 10 μμM LCA (light grey bars) or vehicle (dark grey bars). <b>(G)</b> mRNA expression of <i>IFN</i>γ, <i>IL-6</i>, <i>CD40L</i>, <i>TNF</i>α in primary human CD4⁺ Th cells stimulated with Dynabeads T-activator (T-act) and treated with 10 μM LCA (light grey bars) or vehicle (dark grey bars). P/I, PMA/ionomycin; LCA, lithocholic acid; AU, Arbitrary units. Results represent the mean ± SEM. *Statistically significant, P<0.05. Experiments were performed in triplicates and repeated at least twice.</p

Stents Eluting 6-Mercaptopurine Reduce Neointima Formation and Inflammation while Enhancing Strut Coverage in Rabbits

<div><p>Background</p><p>The introduction of drug-eluting stents (DES) has dramatically reduced restenosis rates compared with bare metal stents, but in-stent thrombosis remains a safety concern, necessitating prolonged dual anti-platelet therapy. The drug 6-Mercaptopurine (6-MP) has been shown to have beneficial effects in a cell-specific fashion on smooth muscle cells (SMC), endothelial cells and macrophages. We generated and analyzed a novel bioresorbable polymer coated DES, releasing 6-MP into the vessel wall, to reduce restenosis by inhibiting SMC proliferation and decreasing inflammation, without negatively affecting endothelialization of the stent surface.</p><p>Methods</p><p>Stents spray-coated with a bioresorbable polymer containing 0, 30 or 300 μg 6-MP were implanted in the iliac arteries of 17 male New Zealand White rabbits. Animals were euthanized for stent harvest 1 week after implantation for evaluation of cellular stent coverage and after 4 weeks for morphometric analyses of the lesions.</p><p>Results</p><p>Four weeks after implantation, the high dose of 6-MP attenuated restenosis with 16% compared to controls. Reduced neointima formation could at least partly be explained by an almost 2-fold induction of the cell cycle inhibiting kinase p27^Kip1. Additionally, inflammation score, the quantification of RAM11-positive cells in the vessel wall, was significantly reduced in the high dose group with 23% compared to the control group. Evaluation with scanning electron microscopy showed 6-MP did not inhibit strut coverage 1 week after implantation.</p><p>Conclusion</p><p>We demonstrate that novel stents coated with a bioresorbable polymer coating eluting 6-MP inhibit restenosis and attenuate inflammation, while stimulating endothelial coverage. The 6-MP-eluting stents demonstrate that inhibition of restenosis without leaving uncovered metal is feasible, bringing stents without risk of late thrombosis one step closer to the patient.</p></div

Aortic SMAD2 signaling.

<p>A) Phosphorylation of Smad2 (pSmad2) and localization in the nucleus of vascular cells in the aortic wall (positive area/total aortic wall area) is expressed in arbitrary units (AU). pSmad2 was significantly reduced by losartan treatment, as compared to placebo-treated Marfan mice. The other anti-inflammatory drugs did not affect the number of pSmad2-positive nuclei. B) An example of pSmad2 staining in placebo-treated Marfan mice and reduced pSmad2 in losartan-treated Marfan mice. A = adventitia, L = lumen, line indicates media.</p

Proposed mechanism.

<p>Losartan is currently the only drug that effectively inhibits aortic root dilatation in mice and men, and specifically targets the angiotensin-II receptor type 1. Losartan clearly decreases TGF-β/pSmad2 signaling, decreases total leukocyte and macrophage influx into the vessel wall, and diminishes aortic root dilatation. TGF-β is known to polarize macrophages into a repair phenotype and at the same time induces collagen synthesis and matrix metalloproteinase activity to degrade extracellular matrix proteins (ECM). Methylprednisolone and abatacept decreased macrophage influx significantly, which resulted in increased GAG accumulation in the aortic vessel wall, thus disturbing ECM homeostasis, which may be potentially harmful.</p

Aortic wall thickness, elastin breaks and GAG accumulation.

<p>A) The area of the aortic media of placebo-treated Marfan mice was significantly thickened compared to wall thickness in wildtype mice. Methylprednisolone showed a trend towards enhanced thickening of the aortic media in Marfan mice (* p = 0.066). B) There were significantly more elastic lamina breaks in the aortic wall of Marfan mice compared to wildtype mice. Methylprednisolone revealed a trend towards enhanced elastic lamina breaks in the aortic media in Marfan mice (* p = 0.076). C) There was enhanced alcian blue positive area in the aortic media of methylprednisolone-treated mice, as compared to Marfan placebo mice, as a marker for medial necrosis. Abatacept showed a trend towards increased GAG accumulation as visualized by alcian blue (* p = 0.066). D) Alcian blue staining (blue) is present in the media (black line) in placebo-treated Marfan mice, yet it is more pronounced in the Methylprednisolone-treated aortic root. Pink stain = cytoplasm, red dots = nuclei, A = adventitia, L = lumen.</p

No Beneficial Effect of General and Specific Anti-Inflammatory Therapies on Aortic Dilatation in Marfan Mice

<div><p>Aims</p><p>Patients with Marfan syndrome have an increased risk of life-threatening aortic complications, mostly preceded by aortic dilatation. In the <i>FBN1</i>^C1039G/+ Marfan mouse model, losartan decreases aortic root dilatation. We recently confirmed this beneficial effect of losartan in adult patients with Marfan syndrome. The straightforward translation of this mouse model to man is reassuring to test novel treatment strategies. A number of studies have shown signs of inflammation in aortic tissue of Marfan patients. This study examined the efficacy of anti-inflammatory therapies in attenuating aortic root dilation in Marfan syndrome and compared effects to the main preventative agent, losartan.</p><p>Methods and Results</p><p>To inhibit inflammation in <i>FBN1</i>^C1039G/+ Marfan mice, we treated the mice with losartan (angiotensin II receptor type 1 inhibitor), methylprednisolone (corticosteroid) or abatacept (T-cell-specific inhibitor). Treatment was initiated in adult Marfan mice with already existing aortic root dilatation, and applied for eight weeks. Methylprednisolone- or abatacept-treated mice did not reveal a reduction in aortic root dilatation. In this short time frame, losartan was the only treatment that significantly reduced aorta inflammation, transforming growth factor-beta (TGF-β) signaling and aortic root dilatation rate in these adult Marfan mice. Moreover, the methylprednisolone-treated mice had significantly more aortic alcian blue staining as a marker for aortic damage.</p><p>Conclusion</p><p>Anti-inflammatory agents do not reduce the aortic dilatation rate in Marfan mice, but possibly increase aortic damage. Currently, the most promising therapeutic drug in Marfan syndrome is losartan, by blocking the angiotensin II receptor type 1 and thereby inhibiting pSmad2 signaling.</p></div

Scanning electron microscopy of stent surface 1 week after implantation.

<p>For qualitative <i>en face</i> assessment of stent coverage, high dose 6-MP eluting stents as well as Cypher rapamycin-eluting stents were implanted (N = 2). Stents excised after one week were cut longitudinally and sputtered with gold particles. 6-MP eluting stents (A,B) show good coverage, as opposed to rapamycin-eluting stents (C, D), which are completely exposed.</p

Inflammatory cells in the aortic vessel wall.

<p>Immunohistochemical staining (positive area/total aortic wall area) for leukocytes (A; CD45) and macrophages (B; Mac3) revealed that placebo-treated Marfan mice contained significantly more leukocytes and macrophages in the aortic wall as compared to wildtype mice. Losartan significantly reduced both leukocyte and macrophage influx. While methylprednisolone revealed a trend in decreased leukocytes (* p = 0.050), abatacept did not. Yet, all drugs significantly decreased the macrophage influx. Each group of mice comprises 11 mice, except Marfan placebo with n = 12, with equal male/female distribution.</p