Figure 3

Saquinavir inhibits cathepsin L activity and intimal hyperplasia. (A-C) Administration of SQV ameliorates intimal hyperplasia in wild-type mice. SQV (saquinavir 5mg/kg and ritonavir 1.25mg/kg) or DSMO control was administered intraperitoneally once daily 3 d before surgery and 28 d after surgery. The ratios of intima and media area and vessel medial size were quantified by planimetry at 28 d. Representative hematoxylin and eosin stained sections of carotid arteries from control mice (n = 7) and SQV-treated mice (n = 8) are shown. The white arrows indicate internal elastic lamina. The black arrows indicate luminal side of the artery. (D) Arterial enzyme activity for cathepsin L was measured by a fluorescence-based assay and normalized to tissue extract protein concentration at indicated time points following wire injury. Cathepsin L activity of the injured and the uninjured carotid arteries from control and SQV-treated mice are displayed (n = 4 at each time point). *P <0.05 versus sham group. #P <0.05 versus the wild-type injured group. (E,F) Tissue lysates from aortic arteries (30 µg) were prepared from DMSO control and SQV-treated mice at 7 d after injury and subjected to western blot assay for cathepsin L (n = 4). *P <0.05 versus sham group. #P< 0.05 versus the wild-type injured group. (G) Quantification of THP-1 cell adhesion to HUVEC. THP-1 with treatment as indicated was labeled with the calcein AM and added to confluent monolayers of HUVEC. The plate was read at 485/538 nm in a fluorescent plate reader. Quantitative analysis of binding of THP-1 cells to HUVEC is presented with bar graphs of fluorescence values. Data represent the means ± SEM. *P <0.05 versus control. #P <0.05 versus LPS group. Scale bar represents 100 µm.