Monocyte Chemoattractant Protein-1 (MCP-1) Regulates Macrophage Cytotoxicity in Abdominal Aortic Aneurysm

<div><p>Aims</p><p>In abdominal aortic aneurysm (AAA), macrophages are detected in the proximity of aortic smooth muscle cells (SMCs). We have previously demonstrated in a murine model of AAA that apoptotic SMCs attract monocytes and other leukocytes by producing MCP-1. Here we tested whether infiltrating macrophages also directly contribute to SMC apoptosis.</p><p>Methods and Results</p><p>Using a SMC/RAW264.7 macrophage co-culture system, we demonstrated that MCP-1-primed RAWs caused a significantly higher level of apoptosis in SMCs as compared to control macrophages. Next, we detected an enhanced Fas ligand (FasL) mRNA level and membrane FasL protein expression in MCP-1-primed RAWs. Neutralizing FasL blocked SMC apoptosis in the co-culture. <i>In situ</i> proximity ligation assay showed that SMCs exposed to primed macrophages contained higher levels of receptor interacting protein-1 (RIP1)/Caspase 8 containing cell death complexes. Silencing RIP1 conferred apoptosis resistance to SMCs. In the mouse elastase injury model of aneurysm, aneurysm induction increased the level of RIP1/Caspase 8 containing complexes in medial SMCs. Moreover, TUNEL-positive SMCs in aneurysmal tissues were frequently surrounded by CD68⁺/FasL⁺ macrophages. Conversely, elastase-treated arteries from MCP-1 knockout mice display a reduction of both macrophage infiltration and FasL expression, which was accompanied by diminished apoptosis of SMCs.</p><p>Conclusion</p><p>Our data suggest that MCP-1-primed macrophages are more cytotoxic. MCP-1 appears to modulate macrophage cytotoxicity by increasing the level of membrane bound FasL. Thus, we showed that MCP-1-primed macrophages kill SMCs through a FasL/Fas-Caspase8-RIP1 mediated mechanism.</p></div

MCP-1 is necessary for FasL expression in infiltrated macrophage following aneurysm induction.

<p>(<b>A</b>) WT or MCP-1^−/− mice were subjected to aneurysm induction by elastase. Inactive elastase-treated arteries were used as control. Cross-sections harvested 3 days after surgery were stained for FasL (red) and macrophages (CD68, green). Fourth column represents increased magnification of highlighted region in the third column. Arrows indicate cells positive for both CD68 and FasL. L indicates lumen. Scale bar, 50 μm. (<b>B</b>) Cross-sections harvested 7 days after surgery were stained for TUNEL (red) and SMCs (SM-αA, green). Scale bar, 100 μm. (<b>C</b>) A semi-quantitative analysis of TUNEL-positive cells in the media of WT or MCP-1^−/− arteries on day 7 after elastase or heat-inactivated elastase infusion. Data are presented as mean ± SEM. n = 3, *<i>p</i><0.05, **<i>p</i><0.01, One-way ANOVA.</p

MCP-1-primed macrophages show higher cytotoxicity.

<p>(<b>A</b>) RAW macrophages (after 24 hours starvation) and SMCs were exposed to various culture conditions with or without addition of MCP-1 (100 ng/ml) for 24 hours. SMCs were also co-cultured with MCP-1-primed RAWs (100 ng/ml, 24 h), or naïve RAWs (PBS treated), or cell-free conditioned medium harvested from MCP-1-primed RAWs for 24 hours. Early apoptotic cells were identified as PE Annexin-V⁺/7-AAD⁻ by flow cytometric analysis. H₂O₂ (700 μM, 4 h) treatment was used as a positive control. (<b>B</b>) Co-culture was stained with the monocyte/macrophage marker CD11b prior to PE Annexin-V/7-AAD staining. (<b>C</b>) Representative bright field images of SMCs, RAWs, SMCs/RAWs co-culture (7 days), and SMCs/MCP-1 primed RAWs co-culture (7 days). Scale bar, 50 μm. Magnification, 20X. Data are mean±SEM. n = 3∼6, *<i>p</i><0.05, **<i>p</i><0.01 vs. untreated SMCs, #<i>p</i><0.05, N.S.  =  not significant, One-way ANOVA.</p

Activation of Caspase 8/Caspase 3-mediated cell death pathway in the co-culture.

<p>(<b>A</b>) Representative immunoblots of cleaved caspase-8, -9, -3, and –PARP in SMCs, RAWs, and co-cultures as indicated. RAWs were primed by treatment of MCP-1 (100 ng/ml) for 24 h prior to co-culture. Cells were harvested after 3 days of co-culture or individual culture. (<b>B</b>) Quantifications of Western blots for cleaved caspase-8, -9, -3, and -PARP from SMCs co-cultured with naïve or MCP-1 (100 ng/ml, 24 h) primed RAWs. Data are mean±SEM. n = 3∼6, #<i>p</i><0.05, N.S.  =  not significant, Two-tailed Student's <i>t</i>-test.</p

Medial accumulation of macrophages coincides with SMC apoptosis in elastase-induced mouse AAA.

<p>Mice were subjected to aneurysm induction by elastase. Inactive elastase-treated arteries were used as control. Aortas were harvested 3 days after surgery and double stained for (<b>A</b>) SMCs (SM-αA, red) and macrophages (CD68, green), or for (<b>B</b>) apoptotic cells (TUNEL, red) and SMCs (SM-αA, green), or for (<b>C</b>) apoptotic cells (TUNEL, red) and macrophages (CD68, green). Sections were counterstained with DAPI (blue). L indicates Lumen. Scale bar, 50 μm. Magnification, 40X. (<b>D</b>) Quantification of TUNEL-positive cells in the media of elastase or heat-inactivated elastase injured arteries on day 3 after surgery. Data are mean ± SEM. n = 3, *<i>p</i><0.05, Two-tailed Student's <i>t</i>-test.</p

Increased RIP1/Caspase 8 containing complexes in SMCs exposing to primed RAWs and aneurysmal tissues.

<p>(<b>A</b>) SMCs were exposed to MCP-1 primed RAWs or naïve RAWs for 3 days. Left: RIP1/Caspase 8 containing complex formation was examined by in situ proximity ligation assay (PLA). Middle: co-immunostaining for SMCs (SM-αA, green) and RIP1/Caspase 8 containing complex (red spots). Nuclei (DAPI, blue). Right: magnified view of the boxed areas in the middle panel. Scale bar, 100 μm. (<b>B</b>) Increased RIP1/Caspase 8 containing complex in elastase-treated aortas. Arteries were harvested 3 days after aneurysm induction by elastase infusion. Heat-inactivated elastase served as a control. Nuclei (DAPI, blue), SMCs (SM-αA, green), RIP1/Caspase 8 containing complex (red spots). Higher magnified views of highlighted regions were shown on the right. L indicates lumen. Scale bar, 50 μm.</p

Receptor interacting protein-1 (RIP-1) underlies SMC apoptosis in the co-culture.

<p>(<b>A</b>) Inhibition of RIP1 with necrostatin-1 (nec-1, 40 μM) profoundly diminished apoptosis in the co-culture. (<b>B</b>) Efficiency of siRNA-mediated knockdown of RIP1 was examined by real-time PCR analysis at 24 hours and 72 hours after siRNA transfection to SMC. (<b>C</b>) SiRNA-mediated knockdown of RIP1 in SMCs prior to exposing SMCs to macrophages significantly attenuated apoptosis in the co-culture. Apoptosis was evaluated by Western blot analysis of cleaved caspase-8 and -3. Data are mean±SEM. n = 3∼4, #<i>p</i><0.05, ***<i>p</i><0.001, Two-tailed Student's <i>t</i>-test (A and C), One-way ANOVA (B).</p

Chiral Surfactant-Type Catalyst for Asymmetric Reduction of Aliphatic Ketones in Water

A novel chiral surfactant-type catalyst is developed. Micelles formed in water by association of the catalysts themselves, and this was confirmed by TEM analyses. Asymmetric transfer hydrogenation of aliphatic ketones catalyzed by the chiral metallomicellar catalyst gave good to excellent conversions and remarkable stereoselectivities (up to 95% ee). Synergistic effects between the metal-catalyzed center and the hydrophobic microenvironment of the core in the metallomicelle led to high enantioselectivities

Table1_A novel small molecule RK-019 inhibits FGFR2-amplification gastric cancer cell proliferation and induces apoptosis in vitro and in vivo.DOCX

Gastric cancer (GC) is one of the most malignant cancers and is estimated to be fifth in incidence ratio and the third leading cause of cancer death worldwide. Despite advances in GC treatment, poor prognosis and low survival rate necessitate the development of novel treatment options. Fibroblast growth factor receptors (FGFRs) have been suggested to be potential targets for GC treatment. In this study, we report a novel selective FGFR inhibitor, RK-019, with a pyrido [1, 2-a] pyrimidinone skeleton. In vitro, RK-019 showed excellent FGFR1-4 inhibitory activities and strong anti-proliferative effects against FGFR2-amplification (FGFR2-amp) GC cells, including SNU-16 and KATO III cells. Treatment with RK-019 suppressed phosphorylation of FGFR and its downstream pathway proteins, such as FRS2, PLCγ, AKT, and Erk, resulting in cell cycle arrest and induction of apoptosis. Furthermore, daily oral administration of RK-019 could attenuate tumor xenograft growth with no adverse effects. Here, we reported a novel specific FGFR inhibitor, RK-019, with potent anti-FGFR2-amp GC activity both in vitro and in vivo.</p

Image2_A novel small molecule RK-019 inhibits FGFR2-amplification gastric cancer cell proliferation and induces apoptosis in vitro and in vivo.TIF

Gastric cancer (GC) is one of the most malignant cancers and is estimated to be fifth in incidence ratio and the third leading cause of cancer death worldwide. Despite advances in GC treatment, poor prognosis and low survival rate necessitate the development of novel treatment options. Fibroblast growth factor receptors (FGFRs) have been suggested to be potential targets for GC treatment. In this study, we report a novel selective FGFR inhibitor, RK-019, with a pyrido [1, 2-a] pyrimidinone skeleton. In vitro, RK-019 showed excellent FGFR1-4 inhibitory activities and strong anti-proliferative effects against FGFR2-amplification (FGFR2-amp) GC cells, including SNU-16 and KATO III cells. Treatment with RK-019 suppressed phosphorylation of FGFR and its downstream pathway proteins, such as FRS2, PLCγ, AKT, and Erk, resulting in cell cycle arrest and induction of apoptosis. Furthermore, daily oral administration of RK-019 could attenuate tumor xenograft growth with no adverse effects. Here, we reported a novel specific FGFR inhibitor, RK-019, with potent anti-FGFR2-amp GC activity both in vitro and in vivo.</p