Proangiogenic Effect of 2A-Peptide Based Multicistronic Recombinant Constructs Encoding VEGF and FGF2 Growth Factors

Dilara Z Gatina,Valeriya V Solovyeva,Margarita N Zhuravleva,Andrey P Kiyasov,Albert A Rizvanov,Ilnur I Salafutdinov,Adelya F Mullakhmetova,Catrin S Rutland,Ekaterina E Garanina,Gulnaz E Synbulatova

doi:10.3390/ijms22115922

Abstract

Coronary artery disease remains one of the primary healthcare problems due to the high cost of treatment, increased number of patients, poor clinical outcomes, and lack of effective therapy. Though pharmacological and surgical treatments positively affect symptoms and arrest the disease progression, they generally exhibit a limited effect on the disease outcome. The development of alternative therapeutic approaches towards ischemic disease treatment, especially of decompensated forms, is therefore relevant. Therapeutic angiogenesis, stimulated by various cytokines, chemokines, and growth factors, provides the possibility of restoring functional blood flow in ischemic tissues, thereby ensuring the regeneration of the damaged area. In the current study, based on the clinically approved plasmid vector pVax1, multigenic constructs were developed encoding vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF2), and the DsRed fluorescent protein, integrated via picornaviruses’ furin-2A peptide sequences. In vitro experiments demonstrated that genetically modified cells with engineered plasmid constructs expressed the target proteins. Overexpression of VEGF and FGF2 resulted in increased levels of the recombinant proteins. Concomitantly, these did not lead to a significant shift in the general secretory profile of modified HEK293T cells. Simultaneously, the secretome of genetically modified cells showed significant stimulating effects on the formation of capillary-like structures by HUVEC (endothelial cells) in vitro. Our results revealed that when the multicistronic multigene vectors encoding 2A peptide sequences are created, transient transgene co-expression is ensured. The results obtained indicated the mutual synergistic effects of the growth factors VEGF and FGF2 on the proliferation of endothelial cells in vitro. Thus, recombinant multicistronic multigenic constructs might serve as a promising approach for establishing safe and effective systems to treat ischemic diseases.

Highlights

In bi-cistronic and tri-cistronic vectors the furin cleavage site (Fu)-2A-peptide sequence was incorporated between the target genes
We found that the expression plasmids increase the expression of mRNA vascular endothelial growth factor (VEGF)- and FGF2-modified cells compared with the empty vector and non-transfected cells
Our data demonstrated that the expression of mRNA and FGF2 and VEGF proteins in vitro was higher than that observed in the control groups

Summary

Introduction

Ischemic diseases remain one of the leading causes of death in the world’s developed countries [1] This disease group is characterized by a lack of oxygen and nutrient supply due to impaired micro- and macro-blood supply to a tissue, organ, or limb [2]. The lack of an adequate blood supply stimulates the activation of angiogenesis processes due to the release of proangiogenic factors [3]. In this regard, a strategy of supportive angiogenic therapy was proposed, which underlies the introduction of exogenous growth factors that promote vasculogenesis and blood circulation restoration [4]

Methods

Results

Discussion

Conclusion