Journal: International Journal of Molecular Sciences

Article Title: Enhanced Integrin Activation of PLD2-Deficient Platelets Accelerates Inflammation after Myocardial Infarction

doi: 10.3390/ijms21093210

Figure Lengend Snippet: Increased inflammatory response in PLD2-deficient mice 24 h after I/R. ( A ) Increased PLD1 and PLD2 protein expression in cardiac sections of the left ventricle 24 h after I/R. Left: Determination of either PLD1- or PLD2-expressing cells. Positive cells werecounted. Right: Representative images of paraffin-embedded heart sections before and 24 h after I/R stained for either PLD1 or PLD2, n = 4-6, scale bar 50 µm. ( B ) Cardiac sections were stained with hematoxylin and eosin 24 h after I/R to analyze the migration of inflammatory cells into the infarct border zone. Left: Quantitative analysis of the migration of inflammatory cells. Right: Representative images are shown:, n = 4, scale bar = 50 μm. ( C ) Quantitative analysis of pro-inflammatory cytokine IL-1β in the left ventricle using real-time PCR, n = 3 and ( D ) in the plasma 24 h post I/R, n=9-11. ( E ) Quantitative analysis of IL-6 in the plasma of Pld2 +/+ and Pld2 −/− mice 24 h after I/R, n = 15. ( F ) Quantitative analysis of TGF-β plasma levels 72 h after I/R, n = 5. ( G ) Flow cytometric analysis of platelet–leukocyte and ( H ) platelet–neutrophil aggregate formation in the plasma of healthy mice and 24 h and 72 h post I/R, n = 6. ( I ) Flow cytometric quantification of MAC-1 exposure on macrophages 24 h after I/R, n = 6. Bar graphs depict mean values ± SEM. Statistical analysis was performed using two-way ANOVA with Sidak’s post-hoc test ( A ) and a two-tailed Student’s t -test ( B – I ). * p < 0.05, ** p < 0.01 and *** p < 0.001. AMI = acute myocardial infarction.

Article Snippet: For the analysis of either PLD1- or PLD2-expressing cells in the left ventricle, streptavidin-biotin-immunoperoxidase staining of cardiac sections of paraffin-embedded hearts before and 24 h after ischemia/reperfusion was performed using rabbit anti mouse PLD1 antibody (Cell Signaling, Danvers, Massachusetts, USA) and rabbit anti mouse PLD2 antibody (Acris, Rockville, MD, USA), using a horseradish peroxidase (HRP)-labeled second antibody kit (LSAB2 System-HRP; DAKO, Santa Clara, California, USA) and Diaminobenzidine (DAB) reagent (DAKO) as chromogen.

Techniques: Expressing, Staining, Migration, Real-time Polymerase Chain Reaction, Clinical Proteomics, Two Tailed Test