Journal: Cardiovascular Research

Article Title: Exogenous CXCL4 infusion inhibits macrophage phagocytosis by limiting CD36 signalling to enhance post-myocardial infarction cardiac dilation and mortality

doi: 10.1093/cvr/cvy211

Figure Lengend Snippet: Macrophages were the major source of CXCL4 in Day 5 LV infarct, and exogenous CXCL4 infusion decreased 7 day post-MI survival. (A) Infarct CXCL4 mRNA expression significantly increased at Day 3 (n = 19), peaked at Days 5 (n = 17) and 7 (n = 24), returning to baseline levels by Day 28 post-MI (n = 7), compared with Day 0 (n = 32). One-way ANOVA was used. (B) Infarct CXCL4 protein increased by approximately 12-fold at Day 5 post-MI. Arrows indicate positive staining (brown dots). Scale bar, 50 µm. n = 6–9/group. *P < 0.05 vs. Day 0. Unpaired t-test was used. (C) Representative images of multiplex staining of CXCL4 (green), macrophages (orange), and neutrophils (red). Nuclei were stained with DAPI (blue). Scale bar, 50 µm. The quantitative analysis of multiplex staining of CXCL4, macrophages, and neutrophils revealed that 72 ± 5% of infarct CXCL4 co-localized to macrophages, 4 ± 1% to neutrophils, and 24 ± 4% to other cell types. n = 6/group. (D) Within infarct macrophages, 55 ± 6% of macrophages were CXCL4+, while 45 ± 6% were CXCL4−. n = 6/group. (E) Compared with Day 0 macrophages, Day 5 infarct macrophages produced 70-fold more CXCL4 protein. n = 5/group. *P < 0.05 vs. Day 0. Unpaired t-test was used. (F) M1 peritoneal macrophages demonstrated lower CXCL4 mRNA levels than M0, evaluated by RNA-seq. n = 4/group. *FDR adjusted P = 0.001 vs. M0. One-way ANOVA was used. (G) CXCL4 infusion decreased 7 day post-MI survival to 10%, compared with 47% for the saline group. n = 15–30/group. *P < 0.05 vs. saline. The survival rate was analysed by Kaplan–Meier survival analysis and compared by the log-rank test. (H) Lung oedema index, the ratio of wet lung weight to tibia length, was significantly higher in CXCL4 treated non-surviving mice than saline controls. n = 8–27/group. *P < 0.05 vs. saline. Unpaired t-test was used.

Article Snippet: 21 , 23 , 32 The following antibodies were used: rat anti-mouse neutrophil antibody (1:100, CL8993AP, Cedarlane) and macrophage antibody Mac3 (1:100, CL8493AP, Cedarlane).

Techniques: Expressing, Staining, Multiplex Assay, Produced, RNA Sequencing, Saline

Journal: Cardiovascular Research

Article Title: Exogenous CXCL4 infusion inhibits macrophage phagocytosis by limiting CD36 signalling to enhance post-myocardial infarction cardiac dilation and mortality

doi: 10.1093/cvr/cvy211

Figure Lengend Snippet: The impact of exogenous CXCL4 infusion on the inflammatory response. (A) The volcano plot showed that out of 84 inflammatory mediators examined by gene array, CXCL4 treated mice displayed lower Ccl3 and Ltb and higher Ccr4 in Day 5 LV infarct. n = 9/group. Unpaired t-test were used. FC, fold change. (B) Ccr4 gene levels positively correlated with end systolic dimension (ESD). n = 17. Pearson correlation analysis was performed. (C and D) Infarct neutrophil and macrophage numbers were similar between saline and CXCL4 groups. Arrows indicate positive staining. n = 9/group. Unpaired t-test was used. (E) In vitro, CXCL4 treatment up-regulated Ccl3 expression in peritoneal macrophages. n = 6–9/group. *P < 0.05 vs. negative control. Paired t-test was used.

Article Snippet: 21 , 23 , 32 The following antibodies were used: rat anti-mouse neutrophil antibody (1:100, CL8993AP, Cedarlane) and macrophage antibody Mac3 (1:100, CL8493AP, Cedarlane).

Techniques: Saline, Staining, In Vitro, Expressing, Negative Control

Journal: Cardiovascular Research

Article Title: Exogenous CXCL4 infusion inhibits macrophage phagocytosis by limiting CD36 signalling to enhance post-myocardial infarction cardiac dilation and mortality

doi: 10.1093/cvr/cvy211

Figure Lengend Snippet: Exogenous CXCL4 infusion decreased macrophage phagocytosis by inhibiting CD36 expression. (A) Isolated macrophages (CD11b+Ly6G−) from Day 5 infarct were all Mac-3+, a specific marker for macrophages, indicating high purity. (B–D) Macrophage (DAPI+) phagocytosis of myocytes (green) and neutrophils (red) were decreased after CXCL4 treatment. Yellow arrows indicate phagocytic macrophages. n = 7–9/group. Scale bar, 50 µm. (E) Out of six phagocytosis markers measured, Cd36 expression was lower in macrophages isolated from the Day 5 infarct of the CXCL4 group. n = 6/group. (F) CXCL4 infusion significantly reduced CD36 protein levels in Day 5 LV infarct. n = 9/group. (G) CXCL4 infusion significantly reduced CD36 protein levels in Day 5 infarct macrophages. n = 5–6/group. *P < 0.05 vs. saline. Unpaired t-test was used.

Article Snippet: 21 , 23 , 32 The following antibodies were used: rat anti-mouse neutrophil antibody (1:100, CL8993AP, Cedarlane) and macrophage antibody Mac3 (1:100, CL8493AP, Cedarlane).

Techniques: Expressing, Isolation, Marker, Saline

Journal: Cardiovascular Research

Article Title: Exogenous CXCL4 infusion inhibits macrophage phagocytosis by limiting CD36 signalling to enhance post-myocardial infarction cardiac dilation and mortality

doi: 10.1093/cvr/cvy211

Figure Lengend Snippet: A diagram illustrating the mechanisms of action of CXCL4 in regulating LV remodelling following MI. MI triggers macrophage infiltration into the infarct area, which releases abundant CXCL4. CXCL4 decreases CD36 expression in MMP-9 independent (direct) and dependent (cleavage of CD36) modes to inhibit macrophage phagocytosis of dead myocytes and neutrophils, which results in impaired removal of debris and adverse remodelling post-MI. In addition, CXCL4 up-regulates Ccr4 and Itgb4 and down-regulates Adamts8, which may result in LV dilation. The cell images are from Servier Medical Art (www.servier.com).

Article Snippet: 21 , 23 , 32 The following antibodies were used: rat anti-mouse neutrophil antibody (1:100, CL8993AP, Cedarlane) and macrophage antibody Mac3 (1:100, CL8493AP, Cedarlane).

Techniques: Expressing

Journal:

Article Title: Inhibition of Mist1 Homodimer Formation Induces Pancreatic Acinar-to-Ductal Metaplasia

doi: 10.1128/MCB.24.7.2673-2681.2004

Figure Lengend Snippet: Mist1MB-expressing cells do not express the gap junction protein Cx32. Pancreas sections from Mist1MB mice were processed for Cx32 immunofluorescence (green) and Mist1MB-Myc immunofluorescence (red). Mist1MB-expressing cells (identified by the red nuclei and arrows) do not contain detectable levels of the Cx32 gap junction protein (green spots), whereas Mist1MB-negative cells (cells to the right of the broken line) continue to generate normal gap junction plaques. Nuclei in panels A and E were also stained with the DNA fluorochrome DAPI.

Article Snippet: Primary antibodies included rabbit Mist1 (diluted 1:100), rabbit c-Myc (diluted 1:200; Santa Cruz), mouse Myc 9E10 (diluted 1:100; Developmental Studies Hybridoma Bank), mouse connexin32 (diluted 1:50; Developmental Studies Hybridoma Bank) (diluted 1:100; Chemicon), rabbit connexin32 (diluted 1:200; Zymed Laboratories), goat clusterin (diluted 1:100; Santa Cruz), mouse CK20 (diluted 1:100; DAKO), and mouse CK19 (TROMA 3; diluted 1:500; gift of Rolf Kemler).

Techniques: Expressing, Immunofluorescence, Staining