RESUMEN
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.
Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Neovascularización Patológica/genética , Factor A de Crecimiento Endotelial Vascular/genética , Inductores de la Angiogénesis/farmacología , Proliferación Celular/genética , Enfermedad de la Arteria Coronaria/terapia , Células Endoteliales/metabolismo , Células Endoteliales/patología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Furina/genética , Regulación de la Expresión Génica/genética , Genes/genética , Vectores Genéticos , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Patológica/terapia , Neovascularización Fisiológica/genética , Péptidos/genética , Péptidos/farmacología , Plásmidos/genética , Plásmidos/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Liquid chromatography (LC) - mass spectrometry quantitative analysis of substances in biological samples is usually performed in the multiple reaction monitoring (MRM) variant. In complex biological matrices, strong interferences can be observed when using the LC-MRM method. Interference levels can be significantly reduced by using LC - multiple reaction monitoring cubed (MRM3). 6-sulfatoxymelatonin (6-SM) is a metabolite of melatonin, an important regulator of many biological processes. The quantitative analysis of 6-SM in urine allows monitoring of the melatonin level in the blood. The aim of the present work was to evaluate the LC-MRM3 method for the quantitative determination of 6-SM in urine. We found that for 6-SM in aqueous solutions, under some parameters of the MRM3 experiment, the effect of degradation of the MRM3 signal is observed. When analyzing 6-SM in urine, this signal degradation effect was significantly reduced. We have shown that optimization of such parameters of the MRM3 method as the linear ion trap fill time, the number of scans to sum, and the range of triple-stage scan allows obtaining the LC-MRM3 method, which is comparable to the LC-MRM in sensitivity and significantly exceeds it in selectivity.