Fig 1: (A) TUNEL assay was conducted on aortic root sections from male and female mice treated with PPE or vehicle to determine the number of dead cells in the atherosclerotic plaque. Bar, 50 µm (B) In-situ efferocytosis assay was conducted by immunostaining macrophages with anti-F4/80 antibody (red) and dead cells using TUNEL reagent (green). The number of TUNEL + nuclei that were either associated with an F4/80 + macrophage (white arrow) or were lying free (white arrowheads) was quantified. The associated: free ratio was used as a measure of lesional macrophage efferocytosis efficiency. Bar, 10 µm (C) Immunostaining of aortic root sections with anti-F4/80 and anti-Mertk antibody. The mean fluorescence intensity of the staining was quantified in ImageJ by drawing an ROI around the F4/80 + intimal region of the plaque. Bar, 50 µm. Non-parametric Mann-Whitney U test was conducted to determine statistical significance. *, p < 0.05; **, p < 0.01.
Fig 2: Tumor vasculature and components of stroma in lung tumors are targeted by anti-angiogenesis. Formalin fixed paraffin embedded lung sections (5 µm) stained with anti-CD31 (endothelial cells), anti-desmin (smooth muscle cells), anti-F4-80 (macrophages), anti-VEGFR1 and anti-VEGFR2 as described in the materials and methods. The slides were scanned on Nanozoomer instrument using 20× magnification. Irrespective of treatment, AIs treated lungs showed lower CD31 and desmin density. F4-80 staining in vehicle treated mice showed greater infiltration of macrophages. However, AIs particularly axitinib and sunitinib significantly reduced macrophage infiltration in the tumors. AIs had differential effects on VEGFR1 and VEGFR2 expression since all treatments reduced VEGFR2 expression in the tumor vasculature, VEGFR1 expression was mainly inhibited by PF-210 and sunitinib. Pictures are the representative images of each staining from each treatment.
Fig 3: Aortic root sections from male and female mice treated with PPE or vehicle were immunostained with (A) anti-F4/80 antibody (B) anti-CD3 antibody, or (C) anti-smooth muscle actin antibody followed by fluorescence microscopy for imaging. The number of F4/80+ (macrophages), sm-actin + (smooth muscle cells), and CD3+ (T-cells) cells in control or PPE-treated groups were quantified using ImageJ. The dotted lines demarcate the intimal region. Bar, 50 µm n = 5 mice per group per sex. Analysis of statistical significance was conducted by Mann-Whitney test. *, p < 0.05; **, p < 0.01.
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