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Peripheral arterial disease (PAD) is characterized by stenosis and occlusion of the arteries leading to poor blood supply to the limb. Patients with PAD suffer pain at rest, intermittent claudication, skin ulcers, and gangrene. The end-stage of the disease could lead to limb amputation despite optimal medical and surgical management. The delivery of angiogenic factors to restore tissue perfusion is an attractive strategy, both as a primary and adjunctive treatment for PAD. We synthesized multigenic plasmid constructs expressing combinations of VEGF, FGF2, and DsRed genes:pVax1-VEGF-FGF2-DsRed, pVax1-VEGF-DsRed, pVax1-FGF2-DsRed, and pVax1-DsRed. In the constructed vectors, gene sequences are linked through the furin-containing 2A-peptide sequence of picornaviruses. Plasmid vector pVax1 is approved by the FDA for use in clinical trials. Previously, we confirmed the functionality of the developed non-viral constructs and the synthesis of VEGF, FGF2, and DsRed proteins by transfected cells. At this stage, we injected plasmid constructs into rat muscles after hind limb ischemia. Quantitative analysis of serum cytokines and chemokines of experimental and control groups on 3, 14, and 21 days after plasmids injection showed no significant differences in the secretion of the 18 cytokines studied. We observed a gradual increase in volumetric blood flow in the experimental groups, despite decreased expression of VEGF and FGF2 on 14 and 21 days. On day 21, the maximum increase in volumetric blood flow was in the pVax1-VEGF-FGF2-DsRed group. In turn, the maximum number of capillaries at 21 days was in the pVax1-VEGF-DsRed group. Capillary density was increased in pVax1-VEGF-FGF2-DsRed and pVax1-FGF2-DsRed groups compared to control groups. We also observed low expression levels of caspase-3 and caspase-9 in the muscles of the experimental groups. Thus, co-expression of VEGF and FGF2 stimulates angiogenic and regenerative processes in a rat model of hind limb ischemia. In addition, the results of this study are consistent with our previous work and confirm the effectiveness of using systems based on 2A-peptide sequences for transgene co-expression. The study of the serum cytokine profile showed the absence of adverse immune effects, indicating the safety of the non-viral constructs used. These results suggest the possibility of using these non-viral constructs to enhance therapeutic angiogenesis in the treatment of ischemic diseases. This paper has been supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030). Full article

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. Full article