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

Morphology and morphometric analyses of sectioned arteries stented for 4 weeks.

<p>Representative HE-stained sections of arteries with stents eluting 0 (A), 30 (B) or 300 μg 6-MP (C). HE staining at low (D) and high (E) magnification with adventitia (a), media (m) and intima (i); strut holes are indicated by (s). Lawson-Van Gieson staining clearly reveals the lamina and thus the different layers of the vessel wall (F). The outer diameter of the stented arteries (G) was measured and found to be highly similar within and between the groups, indicating high reproducibility of stent implantation and expansion. The injury score was low in all stents and similar between groups (H). Lumen stenosis (I) was significantly different across the groups (Jonkheere’s independent samples trend test), with 16% reduction of stenosis in the 300 μg 6-MP eluting stents compared with the polymer-only group (Mann-Whitney U test). N = 10 for 0 and 30 μg, N = 9 for 300 μg. * depicts value that is statistically different from other groups. Error bars represent standard error.</p