Objectives: Marfan syndrome (MFS) is characterized by increased expression of matrix metalloproteinases (MMP) associated with medial elastolysis and aortic aneurysm formation predominantly in the ascending aorta. Long-term expression of MMP antagonist tissue inhibitor of metalloproteinases 1 (TIMP-1) via adeno-associated viruses (AAV-TIMP-1) comprises a potential treatment option for MFS. We aim to provide a causal therapy for the aortopathy of MFS using locally applied gene therapy in a translational murine model.
Methods: Through a mini thoracotomy AAV-TIMP-1, AAV-enhanced green fluorescent protein (AAV-EGFP) vectors and control carrier were circumferentially applied around the adventitia of the ascending aorta in adult male mgR/mgR Marfan mice. Thirty days post-operatively, the ascending aorta was harvested and prepared for cryostat sections. Subsequently, Van Giesson differential staining was carried out to assess the elastic fibers in the aortic media. Additionally, aortic cryosections were used for endothelial marker CD31, tight junction proteins ZO-1 and Occludin IF-stainings to measure endothelial dysfunction. For evaluation of MMP activity, in situ zymography was performed with aortic sections. For clinical outcome measures, survival subgroups were monitored until the time of death.
Results: AAV-TIMP-1-treated mice exhibited significantly reduced elastic fiber degradation compared to the AAV-EGFP and control group (Islands of Damage = 4.13±2.42 vs 19.22±10.72 vs 25.0±7.5; p<0.01 and Elastin fiber breaks = 29.13±13.63 vs 118.2±35.69 vs 118.4±47.24; p<0.01). Evaluation of tight junction integrity through ZO-1 and Occludin staining showed a severe reduction of these proteins in Marfan mice treated with AAV-EGFP (Mean grey value ZO-1 = 11.29±3.96; Occludin = 13.57±3.46) and control (ZO-1 = 10.77±2.72; Occludin = 12.24±2.37) compared to the AAV-TIMP-1-treated mice (Mean grey value ZO-1 = 20.14±3.96; Occludin = 24.98±7.15) not only in the endothelial layer, but also in the aortic media (ZO-1 p<0.01; Occludin p<0.01). Furthermore, in situ zymography showed a significant reduction of MMP acitivity in AAV-TIMP-1-treated mice (Mean grey value = 18.46±4.81) compared to AAV-EGFP-treated mice (Mean grey value = 26.40±5.61) and control (Mean grey value = 25.48±3.80) (p<0.01). Median survival was 193 (115, 244) days in AAV-TIMP-1-treted mice, 81 (64, 158 days in AAV-EGFP-treated mice and 129 (72, 161) days in the control group (Gehan-Breslow-Wilcoxon test p=0.03).
Conclusion: Local administration of AAV-TIMP-1 effectively reduces elastic fiber degradation as well as MMP activitiy and prevents endothelial dysfunction in the ascending aorta of fibrillin-1 deficient Marfan mice. AAV-TIMP-1-treated Marfan mice showed more prolonged survival compared to AAV-EGFP and control groups. Thus, the local overexpression of MMP inhibitors via gene therapy represents a valuable treatment option for the life-threatening aortopathy of Marfan syndrome.
