https://doi.org/10.1007/s00392-024-02526-y
1Universitäts-Herzzentrum Freiburg - Bad Krozingen Klinik für Kardiologie und Angiologie Freiburg im Breisgau, Deutschland; 2Universitätsklinikum Freiburg Institut für Pharmakologie Freiburg im Breisgau, Deutschland; 3Universitäts-Herzzentrum Freiburg / Bad Krozingen Klinik für Kardiologie und Angiologie Bad Krozingen, Deutschland; 4Universitätsspital Basel Klinik für Innere Medizin Basel, Schweiz
Peripheral Artery Disease (PAD) affects around 20% of Europe's population older than 55 years of age, making it one of the most widespread cardiovascular diseases. Up until today, treatment options - especially non-interventional - remain unsatisfactory in both target and outcome. Bone Morphogenetic Proteins (BMPs) are members of the Transforming Growth Factor beta (TGF-beta) superfamily. Increasing evidence suggests their crucial role in vascular remodelling. Upon activation of the BMP ligand-receptor complex, the intracellular signalling pathway is activated through Smad proteins. While Smads 1/5/7 interact with Smad 4 to induce BMP-signalling, Smad 6 generally inhibits the pathway, resulting in an autoregulated system. Previous data shows that overexpression of Smad 6 inhibited Vascular Endothelial Growth Factor (VEGF)-stimulated neovascularization in endothelial cells. Therefore, the current project aims to target Smad 6 specifically using RNA - interference (RNAi) to promote endogenous neovascularization at sites of ischemia.
Methods and Results:
Six- to eight-week-old C57/BL6J wildtype mice were used for the study. The mice were treated with a unilateral femoral artery ligation combined with a sham procedure on the respective other hind limb. Sufficient ischemia was ensured by Laser Doppler Perfusion Imaging (LDPI). Silencing Smad 6 RNA (siSmad 6) or control (siNeg) embedded in hydrogel was subcutaneously injected around the surgical area. Post-interventional revascularization was evaluated on days 3, 7, 10, 14 and 21 using LDPI. In comparison to the control group, the mice injected with siSmad 6 demonstrated accelerated revascularization following day 7 after induction of ischemia, with an increase of perfusion by 40% on day 10. In immunofluorescent evaluation, a two-fold-increase in total capillary-quantity as well as a 1.6-fold-increase in inflammatory cells, i.e., leukocytes, could be demonstrated in ischemic regions. Molecular methods confirmed increased inflammatory responses as well as a significant increase in vascular smooth muscle gene transcription, thereby indicating increased arterialization of preformed capillaries. Western blots for downstream BMP-pathway regulators indicated a total increase in pathway activity following injection of siSmad 6. Immunofluorescent demonstration of significantly increased quantities of pERK-positive cells in the experimental group suggests the increase in pathway-activity to be linked to MAPK/ERK-pathway signalling.
Conclusion:
Our results demonstrate that local inhibition of Smad 6 promotes revascularization in a murine hindlimb ischemia model. This might open up new options in future PAD treatment.
Funding:
This project was funded by grants from the German Research Foundation (DFG) to J.S.E. (ES 582/3-1) and Q.Z. (ZH 231/5-1) as well as a dissertation scholarship from the German Cardiac Society (DGK) to T.F.