P596Therapeutic neovascularization by AAV2/9-based gene transfer of Thymosin B4: results of a preclinical pig study

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Abstract

In patients suffering from ischemic cardiomyopathy, lacking interventional or surgical treatment options, induction of neovascularisation via cell therapy and vascular growth factor application is a novel therapeutic approach. Tβ4, a small 4.9-kDa peptide, has been identified as a major G-actin-sequestering factor to control cytoskeletal reorganisation and possesses a number of other biological functions, including its proangiogenic activity. Here, we investigated the role of long term overexpression of Thymosin β4 (Tβ4) in pigs under normo- and hypercholesterinemic diet in a model of chronic ischemia.

Methods

To induce chronic ischemia, pigs underwent a catheter-based implantation of reduction stent graft into the circumflex artery, leading to complete occlusion on day 28. Pressure-regulated retroinfusion of saline or AAV2/9-Tβ4 (5x10E12 viral particles) was performed on day 28 and compared with ubiquitous Tβ4 transgenic pigs. On day 28 and day 56, global myocardial function (LVEDP) was measured. Subendocardial segment shortening (SES) for determination of regional myocardial function and post mortem angiography for collateral growth quantification were performed on day 56. Tissue samples from the ischemic and non-ischemic regions were harvested for analysis of capillary density using alkaline phosphatase staining.

Results

(Mean ± SD vs. control, p < 0.05) Four weeks after treatment with rAAV2/9-Tβ4, the normocholesterinemic pigs exhibited in ischemic region a significantly enhanced number of capillaries (335 ± 13 vs. 66 ± 7 capillaries/field) and collaterals (9 ± 1 vs. 3 ± 1), which were associated with improved global (LVEDP: 12 ± 1 vs. 19 ± 1 mmHg) and regional myocardial function (SES at 150 beats/min: 73 ± 5 vs. 9 ± 6 % LAD region). Similar results were obtained in transgenic pigs with ubiquitous Tβ4 overexpression. In the model of hypercholesterinemia, the significant improvement of neovascularization and myocardial function could also be achieved with the regional application of rAAV2/9-Tβ4, however, at a lower level (collaterals: 6 ± 1 vs. 3 ± 1; LVEDP: 14 ± 1 vs. 18 ± 1 mmHg).

Conclusion

rAAV2/9-Tβ4 transduction enables capillary growth in vivo. This process provides enhanced collateralization and perfusion, thereby improving myocardial function. A comparable effect is seen in the dyslipidemic hearts, indicating a therapeutic potential of the rAAV2/9-Tβ4 for real world no-option patients with ischemic cardiomyopathy.

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