BACKGROUND It is hypothesized that activation of extracellular signal-related kinase (ERK) is critical in activating matrix metalloproteinases (MMPs) during abdominal aortic aneurysm (AAA) formation. STUDY DESIGN C57BL/6 male mice underwent either elastase or heat-inactivated elastase aortic perfusion (n = 9 per group). Mouse aortic smooth muscle cells were transfected with ERK-1 and 2 siRNA along with or without elastase treatment. Mouse and human aortic tissue were analyzed by Western blots, zymograms, and immunohistochemistry, and statistical analysis was done using Graphpad and Image J softwares. RESULTS Western blot and immunohistochemistry documented increased phospho-mitogen-activated protein kinase kinase-1/2 (pMEK-1/2; 153%, p = 0.270 by Western) and pERK (171%, p = 0.004 by Western blot) in the elastase perfused aortas. Male ERK-1−/− mice underwent elastase perfusion, and aortic diameter was determined at day 14. ERK-1−/− mice failed to develop AAA, and histologic analysis depicted intact collagen and elastin fibers in the aortas. Zymography of aortas of elastase-treated ERK-1−/− mice showed lower levels of proMMP2 (p < 0.005) and active MMP2 (p < 0.0001), as well as proMMP9 (p = 0.037) compared with C57BL/6 mice. siRNA transfection of ERK-1 and -2 significantly reduced formation of pro- and active MMP2 (p < 0.01 for both isoforms) in aortic smooth muscle cells treated with elastase in vitro. Human AAA tissue had significantly elevated levels of pMEK-1/2 (150%, p = 0.014) and pERK (159%, p = 0.013) compared with control tissues. CONCLUSIONS The MAPK (mitogen-activated protein kinase)/ERK pathway is an important modulator of MMPs during AAA formation. Targeting the ERK pathway by reagents that inhibit either the expression or phosphorylation of ERK isoforms could be a potential therapy to prevent AAA formation.

INTRODUCTION Estrogen receptor alpha (ERα) has been identified in the vessel wall, offering vasoprotective effects when upregulated. Estrogens are known to mediate the inflammatory mileu, and inflammation has long been associated with abdominal aortic aneurysm (AAA) formation. Therefore it is theorized that increased estrogen receptor in females contributes to their relative resistance to AAAs. The study’s objective was determining gender differences in ERα levels during experimental AAA formation. METHODS Infrarenal aortas of male and female C57 mice (n=18 and n=16, respectively) were infused with 0.4% elastase. Diameters were measured at day 0 and day 14. Aortic mRNA expression of ERα was determined on day 3 by RTPCR, while ERα protein levels were measured via Western blot. Immunohistochemistry using rabbit antibody for ERα was performed on day 14 samples and quantified. Zymography was done for MMP2 and 9 activity levels. Samples of human AAAs were collected and Western blot performed. Data were compared for significance using a student t-test. RESULTS Infrarenal aortic diameter increased in elastase-perfused males (ME) by 80% at 14 days post perfusion, while females (FE) increased by only 35% (p=0.0012). FE had 10x greater ERα mRNA expression compared with ME at day three (p=0.003). Similarly, ERα protein levels were 100% higher in FE compared to ME on day 14 (p=0.035). ERα protein levels were 80% higher in female human patients with AAA than in their male counterparts (p=0.029). ERα visualized via immunohistochemistry was 1.5 fold higher in FE than ME (p=0.029). MMP2 and 9 activity levels were decreased in female as compared with male aortas. CONCLUSION(S) This study demonstrates an increase in aortic wall ERα in females compared with males that correlates inversely with MMP activity and AAA formation. These findings, coupled with observations that exogenous estrogen inhibits AAA formation in males, further suggest that estrogen supplementation may be important to prevent AAA formation and growth.

It is hypothesized that differential AKT phosphorylation between sexes is important in abdominal aortic aneurysm (AAA) formation. Male C57BL/6 mice undergoing elastase treatment showed a typical AAA phenotype (80% over baseline, P < 0.001) and significantly increased phosphorylated AKT-308 (p308) and total-AKT (T-AKT) at day 14 compared with female mice. Elastase-treated Raw cells produced increased p308 and significant amounts of matrix metalloproteinase 9 (MMP-9), and these effects were suppressed by LY294002 treatment, a known AKT inhibitor. Male and female rat aortic smooth muscle cells treated with elastase for 1, 6, or 24 hours demonstrated that the p308/T-AKT and AKT-Ser-473/T-AKT ratios peaked at 6 hours and were significantly higher in the elastase-treated cells compared with controls. Similarly, male cells had higher phosphorylated AKT/T-AKT levels than female cells. LY294002 also inhibited elastase-induced p308 formation more in female smooth muscle cells than in males, and the corresponding cell media had less pro-MMP-9. AKT siRNA significantly decreased secretion of pro-MMP-9, as well as pro-MMP-2 and active MMP-2 from elastase-treated male rat aortic smooth muscle cells. IHC of male mice AAA aortas showed increased p308, AKT-Ser-473, and T-AKT compared with female mice. Aortas from male AAA patients had a significantly higher p308/T-AKT ratio than female AAA tissues. These data suggest that AKT phosphorylation is important in the upstream regulation of MMP activity, and that differential phosphorylation may be important in sex differences in AAA.

Background In humans, there is a 4:1 male:female ratio in the incidence of abdominal aortic aneurysms (AAAs). c-Jun-N-terminal kinase (JNK) is an important upstream regulator of several enzymes involved in AAA formation, including the matrix metalloproteinases (MMPs). The purpose of this study was to determine if there is a gender difference between males and females in JNK during AAA formation. Materials and Methods Male and female C57/B6 mice underwent aortic perfusion with elastase or heat inactivated elastase with aortas harvested at day 3 and 14 for phenotype determination, RT-PCR, Western blot, and zymography. Additionally, in vitro experiments using siRNA were conducted to define JNK regulation of matrix metalloproteinases (MMPs). A t-test was used to compare between groups. Results Males formed larger AAAs at day 14 compared to females (p<0.001), with significantly higher levels of JNK1 protein, proMMP9, proMMP2, and active MMP2. At day 3, males had more JNK1 mRNA, protein, and MMP activity. Knockdown of JNK 1 or 2 in vitro decreased MMP activity, while knockdown of JNK 1 and 2 together blocked all MMP activity. Conclusion Alterations in JNK between genders is partially responsible for the differential rates of experimental AAA formation, likely through differential regulation of MMPs.

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.