Additional file 1: Tables S1-S6. of Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and tolerance

Table S1. Top 25 up- and downregulated genes in Nur77-KO vs WT BMM. Table S2. Top 25 canonical pathways associated with differentially expressed genes in Nur77-KO vs WT BMM. Table S3. Top 20 gene sets identified with GSEA that are upregulated in Nur77-KO vs WT BMM. Table S4. Upstream Regulators in Nur77-KO vs WT BMM. Table S5. Top 25 up- and downregulated genes in LPS-stimulated Nur77-KO vs WT BMM. Table S6. Top 25 canonical pathways associated with differentially expressed genes in LPS-stimulated Nur77-KO vs WT BMM. (PDF 140 kb

FHL2 deficiency enhances SMC proliferation via activation of ERK1/2.

<p><b>A</b>, Serum-starved SMCs were stimulated with or without FCS and treated with or without PD98059 (ERK1/2 inhibitor, 25µM). Cells were pulse-labeled with BrdU to measure DNA synthesis. <b>B</b>, SMCs from WT and FHL2-KO were seeded at equal density. 1 day after seeding, cells were treated with or without PD98059 and cells were counted manually. <b>C–D</b>, Western blot analysis (C) and quantification (D) for pERK1/2 in serum-starved SMCs after overexpression with or with out FHL2 followed by FCS stimulation for the indicated time periods, showing enhanced and prolonged activation of ERK1/2 in FHL2-defeicient SMCs and reduced activation of ERK1/2 in FHL2-KO SMCs after overexpression of FHL2. Data represent means±SD. *<i>P</i><0.05 for FHL2-KO versus WT.</p

FHL2-KO SMCs migrate faster.

<p><b>A</b>, A scratch was made in a confluent layer of serum-starved SMCs that were stimulated with PDGF (20 ng/ml). Images were captured every 10 min using a live cell microscope and representative images at 0, 16 and 32 h are shown. Movies of the movement are in the online supplement. <b>B</b>, Quantitative analysis of SMC migration in the scratch wound assay showing that FHL2-KO SMCs migrated 1.8 fold faster than WT SMCs. <b>C</b>, SMCs were treated with or without PD98059 and cell migration was evaluated using a trans-well assay. Cells were labeled with a fluorescent dye and seeded in the upper chamber. Cell migration was measured as fluorescence after 3 h. <b>D</b>, SMC migration was evaluated using a trans-well assay after knock-down of FHL2 using lentiviral particles encoding shCtrl, shFHL2#1 and shFHL2#2 in WT SMCs. Cell migration was measured as fluorescence after 3 h. Data represent means±SD. *<i>P</i><0.05 for shCtrl versus shFHL2. <b>E</b>, Schematic representation of FHL2 function in the modulation of SMC-rich lesion formation. FHL2 modulates SMC-rich lesion formation by inhibiting proliferation and migration of SMCs via the ERK1/2-CyclinD1signaling pathway.</p

FHL2 deficiency enhances cell proliferation <i>in vivo</i>.

<p><b>A</b>, To assess the extent of proliferation in the vascular lesions, representative sections of injured carotid arteries from WT and FHL2-KO mice ligated for 1, 2 and 4 weeks were immunostained for Ki67. n = 7 for 1 and 2 weeks and n = 14 for 4 weeks. <b>B–C</b>, qRT-PCR was performed to assess mRNA expression of Ki67 (B) and PCNA (C) in the ligated vessels from WT and FHL2-KO mice for the indicated time periods. Data are means±SD. *<i>P</i><0.05 for FHL2-KO versus WT mice.</p

Deficiency of FHL2 accelerates neointima formation after carotid artery ligation.

<p><b>A, C</b> and <b>E</b>; Representative cross sections of hematoxylin/eosin-stained carotid arteries from WT and FHL2-KO mice ligated for 1 (A), 2 (C) and 4 weeks (E). <b>B, D</b> and <b>F</b>; Quantitative analysis of neointima/media ratio and neointimal area in histological sections from WT and FHL2-KO mice ligated for 1 (B), 2 (D) and 4 weeks (F), revealed increased lesion formation in FHL2-KO mice. n = 7 for 1 and 2 weeks and n = 14 for 4 weeks. Three consecutive sections per mouse at each location were employed in the analysis. Lesions were characterized at 1.7, 2.0 and 2.3 mm from the reference point at 1, 2 and 4 weeks, respectively. Values are mean±SEM. *<i>P</i><0.05 for FHL2-KO versus WT mice.</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

FHL2 regulates cell proliferation via modulation of CycinD1 expression.

<p><b>A</b>, SMCs were transduced with lentiviral particles encoding FHL2 and assayed for CyclinD1 mRNA expression, showing that FHL2 inhibits its expression. <b>B</b>, Serum-starved WT SMCs were transduced with lentiviral particles encoding shCtrl, shCyclinD1 #1 and shCyclinD1 #2 and were pulse-labeled with BrdU to measure DNA synthesis. <b>C</b>, The CyclinD1 promoter-reporter plasmid showed higher induction in FHL2-KO SMCs stimulated with FCS than in WT SMCs. The ERK1/2 inhibitor PD98059 partly reduces this induction. Data represent means±SD. *<i>P</i><0.05 for FHL2-KO versus WT.</p

Characteristics of 6-MP eluting stents: 6-MP release, coating quality, and biological activity of eluted 6-MP.

<p>The cumulative release of 6-MP was assayed in vitro for the 30 μg DES (diamonds, n = 3) and the 300 μg 6-MP eluting stents (squares, n = 3) up to 38 days, and expressed as absolute release (A) as well as percentage release (B). A polymer-only stent (C) and a stent loaded with 300 μg 6-MP before (D,E) and after expansion (F) show smooth coating surfaces by SEM. The stability of 6-MP after storage was measured after elution from the stents and quantified by its ability to activate Nur77 in a luciferase reporter assay (G). 6-MP was retrieved from stents after 4 (4m, n = 6 measurements) and 16 months (16m, n = 6) and the activity was compared with a negative control (white bar, n = 9) and freshly dissolved 6-MP at 50 μM (black bar, n = 9). * depicts value that is statistically different from other groups analyzed by ANOVA. Error bars represent standard error.</p

Adventitial capillaries.

<p>Higher 6-MP release resulted in higher numbers of capillaries (Jonkheere’s independent samples trend test, P = 0.02). Stents loaded with 300 μg 6-MP (n = 9) induced 30% more capillaries than polymer-only stents (N = 10, Mann-Whitney U test, P = 0.02). Low 6-MP dose (N = 10) failed to increase the number.</p

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