AAV-mediated TIMP-1 overexpression in aortic tissue reduces the severity of allograft vasculopathy in mice

https://doi.org/10.1016/j.healun.2020.01.1338Get rights and content

BACKGROUND

Allograft vasculopathy (AV) is the primary limiting factor for long-term graft survival. An increased activity of matrix metalloproteinases (MMPs) contributes to neointima formation in AV and represents a potential therapeutic target. Adeno-associated virus (AAV)-mediated gene therapy comprises a potentially benign vector model for the long-term expression of MMP antagonists.

METHODS

Aortic allografts from DBA/2 mice were incubated with control buffer, AAV-enhanced green fluorescence protein (EGFP), or tissue inhibitor of metalloproteinases 1 (TIMP-1)-loaded AAV (AAV-TIMP-1) and transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg body weight) was administered daily. Explantation as well as histomorphometric and immunohistochemical evaluation was performed after 30 days. Matrix metalloproteinase (MMP) activity was visualized by gelatin in situ zymography.

RESULTS

Intima-to-media area ratio and neointima formation were significantly reduced in the AAV-TIMP-1 treatment group compared with those in the control group (by 40%; p < 0.001) and the AAV-EGFP group (by 38.2%; p < 0.001). TIMP-1 overexpression positively affected several pathomechanisms for the development of AV both in vitro and in vivo as compared to that in the control groups: endothelium integrity was preserved as shown by zona occludens 1 and occludin staining; MMP9 expression and activity were significantly reduced (p = 0.01); and smooth muscle cell migration was significantly reduced as smooth muscle actin positive cells predominantly remained in the aortic media in the treatment group (p = 0.001). Moreover, macrophage infiltration was markedly reduced by 49% in the AAV-TIMP-1 group (p < 0.001).

CONCLUSION

Immediate post-harvesting allograft incubation with AAV-TIMP-1 reduces neointima formation and macrophage infiltration, constituting a possible adjunct therapeutic strategy to preserve graft function after transplantation.

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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