Background Statins reduce aneurysm growth in mouse models of Marfan syndrome, although the mechanism is unknown. In addition to reducing cholesterol, statins block farnesylation and geranylgeranylation, which participate in membrane-bound G-protein signaling, including Ras. We dissected the prenylation pathway to define the effect of statins on aneurysm reduction. Methods and Results Fbn1^C1039G/+ mice were treated with (1) pravastatin (HMG-CoA [3-hydroxy-3-methylglutaryl coenzyme A] reductase inhibitor), (2) manumycin A ( MA ; FPT inhibitor), (3) perillyl alcohol ( GGPT 1 and -2 inhibitor), or (4) vehicle control from age 4 to 8 weeks and euthanized at 12 weeks. Histological characterization was performed. Protein analysis was completed on aortic specimens to measure ERK (extracellular signal-regulated kinase) signaling. In vitro Fbn1^C1039G/+ aortic smooth muscle cells were utilized to measure Ras-dependent ERK signaling and MMP (matrix metalloproteinase) activity. Pravastatin and MA significantly reduced aneurysm growth compared with vehicle control (n=8 per group). In contrast, PA did not significantly decrease aneurysm size. Histology illustrated reduced elastin breakdown in MA -treated mice compared with vehicle control (n=5 per group). Although elevated in control Marfan mice, both phosphorylated c-Raf and phosphorylated ERK 1/2 were significantly reduced in MA -treated mice (4-5 per group). In vitro smooth muscle cell studies confirmed phosphorylated cR af and phosphorylated ERK 1/2 signaling was elevated in Fbn1^C1039G/+ smooth muscle cells (n=5 per group). Fbn1^C1039G/+ smooth muscle cell Ras-dependent ERK signaling and MMP activity were reduced following MA treatment (n=5 per group). Corroborating in vitro findings, MMP activity was also decreased in pravastatin-treated mice. Conclusions Aneurysm reduction in Fbn1^C1039G/+ mice following pravastatin and MA treatment was associated with a decrease in Ras-dependent ERK signaling. MMP activity can be reduced by diminishing Ras signaling.

Aortic root aneurysm formation and subsequent dissection and/or rupture remain the leading cause of death in patients with Marfan syndrome. Our laboratory has reported that miR-29b participates in aortic root/ascending aorta extracellular matrix remodeling during early aneurysm formation in Fbn1C1039G/+ Marfan mice. Herein, we sought to determine whether miR-29b suppression can reduce aneurysm formation long-term. Fbn1C1039G/+ Marfan mice were treated with retro-orbital LNA-anti-miR-29b inhibitor or scrambled-control-miR before aneurysms develop either (1) a single dose prenatally (pregnant Fbn1C1039G/+ mice at 14.5 days post-coitum) (n = 8-10, each group) or (2) postnatally every other week, from 2 to 22 weeks of age, and sacrificed at 24 weeks (n = 8-10, each group). To determine if miR-29b blockade was beneficial even after aneurysms develop, a third group of animals were treated every other week, starting at 8 weeks of age, until sacrificed (n = 4-6, each group). miR-29b inhibition resulted in aneurysm reduction, increased elastogenesis, decreased matrix metalloproteinase activity and decreased elastin breakdown. Prenatal LNA-anti-miR-29b inhibitor treatment decreased aneurysm formation up to age 32 weeks, whereas postnatal treatment was effective up to 16 weeks. miR-29b blockade did not slow aortic growth once aneurysms already developed. Systemic miR-29b inhibition significantly reduces aneurysm development long-term in a Marfan mouse model. Drug administration during aortic wall embryologic development appears fundamental. miR-29b suppression could be a potential therapeutic target for reducing aneurysm formation in Marfan syndrome patients.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1^C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin-enhanced chemiluminescence (LGCL), Verhoeff's elastin-Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1^C1039G/+ AS- or DES-derived smooth muscle cells (SMC) were treated with anti-TGF-β antibody, angiotensin II (AngII), anti-TGF-β antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1^C1039G/+ AS aorta, but absent in normal-sized DES aorta. Fbn1^C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1^C1039G/+ -derived AS SMC had increased NADPH activity compared to DES-derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF-β dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF-β dependent.