Marfan syndrome is a rare genetic disorder characterized by mutations in the fibrillin-1 gene. The disease is associated with cardiovascular complications, mainly formation of aortic aneurysms with a high risk of rupture or dissection. This often leads to death at a young age for affected patients. It was shown that increased matrix-metalloproteinase expression (MMPs) and elevated levels of proinflammatory cytokines, in particular interleukin-6 (IL-6), play a central role in the pathogenesis. Regnase-1 is a newly identified endoribonuclease which cleaves the mRNA of proinflammatory targets and therefore decreases their stability and expression.

We therefore aimed to determine the role of regnase-1 in progression of thoracic aneurysms and to examine the effects of its overexpression on inflammatory parameters and elastin degradation. 

Aortic smooth muscle cells (SMC) from mgR/mgR (Marfan) mice were utilized as an in vitro system. Regnase-1 overexpression was achieved by transduction with vascular-targeted AAV vectors, while cells treated with AAV encoding enhanced green fluorescent protein (EGFP) were used as control. Interferon γ (IFNγ) was added to cell culture medium to induce expression of pro-inflammatory cytokines. As a readout, we measured the degree of elastin and gelatin degradation and monitored the expression of several inflammatory parameters using real-time qPCR, Western Blot, macrophage migration assay and enzyme-linked immunosorbent assay (ELISA). Furthermore, 8 week-old mgR/mgR mice were intravenously injected with vascular-targeted AAV vectors expressing either regnase-1 or EGFP. Aortic diameter was measured using echocardiography 4 weeks after injection. Additionally, thoracic aortae were analysed in regards to elastin architecture and expression of MMPs and levels of circulating proinflammatory cytokines were measured in serum samples.

AAV9-mediated regnase-1 overexpression in murine Marfan SMCs decreased elastin degradation and reduced IL-6 and MMP9 expression as well as NFkB activation in vitro. Moreover, we could prove a marked decline in IL-6 level and macrophage migration towards the supernatant of transduced SMCs. Treated Marfan mice presented with efficient transduction of both aortic endothelial cells and SMC following injection of vascular-targeted AAV vectors. Importantly, echocardiographic measurements showed that regnase-1 overexpression significantly reduced aortic diameter at distinct locations. In addition, elastin structure was markedly improved in the regnase-1 treatment group compared to controls. Moreover, our approach led to decreased protein expression of MMPs as shown by immunohistochemistry. Finally, we could demonstrate that plasma levels of proinflammatory cytokines such as IL-6, macrophage chemoattractant protein 1 and transforming growth factor beta were decreased in mice with regnase-1 overexpression in the aorta. 

Here we show that AAV-mediated regnase-1 overexpression in the aorta of Marfan mice ameliorates pathological changes in the aortic wall and potentially prevents progression of aortic aneurysms.