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

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.

Reactive astrocytes transduce inflammation in a blood-brain barrier model through a TNF-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin

“…Key endocytosis molecules were downregulated by TNF in both culture conditions, for instance, PACSIN3 (important regulator of internalization of plasma membrane proteins 42 ) and RAB5C (a small GTPases key regulator of trafficking of integrin complex in endothelial cells 43 ). Exposure to TNF also caused the downregulation of the proangiogenic genes such as AGGF1, FGF2, and ANG [44][45][46] . Further, while many overlapping DEGs could be identified, we observed key differences in gene ontology.…”

Reactive astrocytes transduce blood-brain barrier dysfunction through a TNFα-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin

“…Key endocytosis molecules were downregulated by TNFα in both culture conditions, for instance, PACSIN3 (important regulator of internalization of plasma membrane proteins 42 ) and RAB5C (a small GTPases key regulator of trafficking of integrin complex in endothelial cells 43 ). Exposure to TNFα also caused the downregulation of the proangiogenic genes such as AGGF1, FGF2, and ANG [44][45][46] . Further, while many overlapping DEGs could be identified, we observed key differences in gene ontology.…”

Angiogenic Modification of Microfibrous Polycaprolactone by pCMV-VEGF165 Plasmid Promotes Local Vascular Growth after Implantation in Rats

“…For example, the VEGF165 plasmid with cytomegalovirus (CMV) promoter as a gene therapeutic drug (pCMV-VEGF165) is used to stimulate therapeutic angiogenesis in lower-limb ischemia [ 24 , 25 ]. Techniques for functionalization of material surfaces with plasmid-based gene therapy were used in bone implants [ 26 , 27 , 28 ].…”