AAV-mediated TIMP-1 overexpression in aortic tissue reduces the severity of allograft vasculopathy in mice
Section snippets
AAV production
The cotransfection of adenoviral helper plasmid pDG∆VP, endothelial-specific AAV9 capsid variant p5E18VD2/9-SLRSPPS, and either genome plasmid pDS-H1-hTIMP-1-CMV or pDS-H1-enhanced green fluorescence protein (EGFP)-CMV generates the vascular-targeted AAV9SLR vectors used in this work.15 Iodixanol gradient ultracentrifugation was used to purify the AAV9SLR vectors. Genomic titers were determined by quantitative real-time polymerase chain reaction.15,17, 18, 19, 20
SMC isolation and culture
Primary SMCs were isolated
In vitro TIMP-1 overexpression reduces the activity of secreted MMP9 and decreases SMCs migration
To prove the overexpression of TIMP-1 in the supernatant of transduced SMCs, the media was subjected to western blot analysis 3 days after the application of AAV. As illustratively shown in Figure 1A, our results demonstrate a marked expression of the investigated human protein in AAV-TIMP-1 overexpressing cells, which was absent in the control AAV-EGFP treated SMCs.
MMP activity was shown to enhance SMCs migration, a main process leading to vascular remodeling in neointima formation.25 Hence,
Discussion
As heart transplantation remains the gold standard therapy for terminal heart failure, AV—as the main factor for long-term organ deterioration—needs to be further investigated. The period during allograft harvesting and implantation represents an optimal timing for specific solid organ therapy, there by reducing systemic effects in the recipient. Recently, we demonstrated a novel therapeutic approach by AAV-mediated long-term inhibition of AP-1 through decoys in murine aortic allografts that
Disclosure statement
The manuscript has not been published previously and is not under consideration for publication elsewhere. All authors read the manuscript, declare that they have no conflict of interest, and are all in agreement with the content of the manuscript.
The authors are indebted to Antje Weber and Franziska Mohr for expert technical assistance
This work was supported by the Dietmar Hopp Foundation, St. Leon-Rot, Germany. (Project No.: 23011198)
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