Figure 1.

TNF-α–induced VCAM-1 expression within heart-associated ECs in vivo is dependent on FAK activity. PBS or TNF-α (0.02 mg/kg) was tail vein injected into mice, and, after 6 h, tissues were analyzed by immunoblotting or staining. Where indicated, FAK-I (100 mg/kg, PND-1186) was administered 3 h before starting experiments. (A) Immunoblotting of heart lysates shows increased VCAM-1 expression and FAK Y397 or FAK Y576 phosphorylation upon TNF-α stimulation in vivo. FAK-I addition prevents VCAM-1 production and FAK tyrosine phosphorylation but no change in FAK expression. Internal loading controls for each gel are shown by anti-actin immunoblotting. (B) Heart-associated VCAM-1 or FAK activation (pY397) was determined by immunoblotting (see Fig. S1) and expressed as a ratio to actin or total FAK, as determined by densitometry, respectively. Values are means (±SD) from six mice, representing two independent experiments (***, P < 0.001). (C) In vivo signaling assays were performed as in A, and heart sections were analyzed by combined staining for activated FAK (pY576) and ECs (CD31). Bar, 20 µm. (D) Mean correlation of pixel intensities from anti–pY576 FAK and anti-CD31 staining of heart sections, as shown in C. (E) Visualization of EC-associated VCAM-1 expression. Heart sections were analyzed by combined staining for VCAM-1 and ECs (CD31). A merged image is shown. Bar, 20 µm. (F) Mean correlation of pixel intensities from anti–VCAM-1 and anti-CD31 staining of heart sections, as shown in C. (D and F) 10 full-frame images were analyzed per experimental group for calculations of VCAM-1 and pY576 FAK associated with CD31 staining (±SEM; ***, P < 0.001; ****, P < 0.0001).