Increased PAI-1 in females compared with males is protective for abdominal aortic aneurysm formation in a rodent model

Abstract

The serine proteases, along with their inhibitor plasmin activator inhibitor-1 (PAI-1), have been shown to play a role in abdominal aortic aneurysm (AAA) formation. The aim of this study is to determine if PAI-1 may be a protective factor for AAA formation and partially responsible for the gender difference observed in AAAs. Male and female wild-type (WT) C57BL/6 and PAI-1(-/-) mice 8-12 wk of age underwent aortic perfusion with porcine pancreatic elastase. Animals were harvested 14 days following perfusion and analyzed for phenotype, PAI-1 protein levels, and matrix metalloproteinase (MMP)-9 and -2 activity. WT males had an average increase in aortic diameter of 80%, whereas females only increased 32% (P < 0.001). PAI-1(-/-) males increased 204% and females 161%, significantly more than their WT counterparts (P < 0.001). Western blot revealed 61% higher PAI-1 protein levels in the WT females compared with the WT males (P = 0.01). Zymography revealed higher levels of pro-MMP-2 and active MMP-2 in the PAI-1(-/-) males and females compared with their WT counterparts. PAI-1(-/-) females had significantly higher serum plasmin levels compared with WT females (P = 0.003). In conclusion, WT female mice are protected from aneurysm formation and have higher levels of PAI-1 compared with males during experimental aneurysm formation. Additionally, both male and female PAI-1(-/-) animals develop significantly larger aneurysms than WT animals, correlating with higher pro- and active MMP-2 levels. These findings suggest that PAI-1 is protective for aneurysm formation in the elastase model of AAA and plays a role in the gender differences seen in AAA formation.

Fig. 1.

A: changes in aortic diameter for the four groups studied, represented as percentage increase from baseline. WT, wild type; PAI-1, plasminogen activator inhibitor-1. B: PAI-1 mRNA expression in WT male and female mice at day 1 following elastase perfusion represented as relative expression compared with β-actin. C: PAI-1 protein levels in WT male and female mice 14 days following elastase perfusion. Samples have been normalized to β-actin and are represented by the average density of the bands on Western blot. D: representative images of immunostaining for PAI-1 in WT male and female mice document increased PAI-1 in females (arrows) with little PAI-1 staining in males.

Fig. 2.

Urokinase-type plasminogen activator (uPA) protein levels in aortic tissue at baseline and 14 days following elastase perfusion in WT and PAI-1^−/− mice, represented as the relative intensity of the bands on Western blot.

Fig. 3.

Changes in matrix metalloproteinase (MMP) levels. Representative lanes from zymograms for pro-MMP-9, pro-MMP-2, and active MMP-2 for each of the four groups studied are shown. Graphs represent the average relative intensity of the various MMP bands for each group.

Fig. 4.

Representative hematoxylin- and eosin-stained sections for each of the four groups studied at low (×4) and high (×20) magnification. Arrows demonstrate white blood cell infiltration in the aortic wall.

Fig. 5.

Representative images of MAC-2 antibody staining for macrophages in each of the four groups studied at low (×4) and high (×20) magnification. Red staining demonstrates the macrophages in each sample (arrows).

Fig. 6.

Serum plasmin levels at baseline and day 14 following elastase perfusion as measured by enzyme-linked immunosorbent assay.