Evaluation of cellular responses in 3T3 and EAhy926 cell lines to PDGF (Platelet-Derived Growth Factor) with chitosan uptake

ACEBEDO, SOFÍA LORENA; DI SANTO, MARIANA CAROLINA; SPAGNUOLO, CARLA CECILIA CECILIA; ALAIMO, AGUSTINA; PÉREZ, OSCAR EDGARDO

Buenos Aires

Jornada; IX Jornadas de Jóvenes Investigadores; 2019

Facultad de Ciencias Veterinarias. UBA

PDGFs (Platelet-Derived Growth Factors) are released from blood platelets when they are activated as a consequence of tissue damage. Then, cellular proliferation and migration is stimulated in target cells -including fibroblasts and endotelials type cells - in order to repair vascular and connective tissue. Chitosan is a promising polysaccharide studied for nano-technology applications because of their biocompatibility, biodegradability and controlled drug- release. Little is known about chitosan effect when it is combined in culture media with potential therapeutical proteins, like growth factors. The aim of this work was to analyze changes in some cellular responses induced by recombinant PDGF-AA and PDGF-BB when free chitosan is added to culture media. Standardin vitro culture of 3T3 and EAhy926 cell lines was performed and known concentrations of PDGF and chitosan were mixed as a media additive. Here, we analyze possible cytotoxic effect of chitosan by means of crystal violet assay, the cellular uptake rate of chitosan at 1, 4 and 24 hs post-treatment with chitosan fluorescent-dyied and their possible co-localization with lysosomes by confocal microscopy. Changes in mitochondrial dynamics were tested in cells exposed for 48 hs to PDGF alone or in combination with chitosan. Results showed that chitosan not only has cytotoxic effect in 3T3 and EAhy926 but also cell lines improved the cellular maintenance in an in vitro model. The cells incorporated chitosan in intracellular bodies at 1h and 4 h post-treatment with a maximum at 24 hs in both cell lines, been particularly notable in EAhy926 type. On the other hand, we found that chitosan co-localized with lysosomes at 24 hs after-treatment. PDGF-AA and PDGF-BB induced a marked mitochondrial fragmentation after 24 hs post-treatment (a 40 % increase related to control) and chitosan was no able to enhance this effect. In EAhy926 line, PDGF induced a particular mitochondrial morphology with these fragmented organelles organized around the nucleus that accounted by 50% of fragmented mitochondrial phenotype. In contrast, in 3T3 line, PDGF induced an organelle fragmentation more distributed throughout cytoplasm and a distinctive mitochondrial phenotype with an intermediate tubular state. We conclude that chitosan incorporated in culture media at optimum serum condition and physiological pH had no effect in PDGF-induced mitochondrial fission frequency and it can easily incorporated into the cell and then targeted to lysosomes for degradation.