Due to the tissue specificity of the reference genes[24–27], the results for each examined target gene are not identical relative to the individual reference genes (especially in the heart). However, for both housekeeping genes, the relative levels of target gene expression presented almost the same pattern of changes.
Our results suggest that, in the case of chicken breast muscle tissue, oral AgNano administration influences FGF2 and VEGFA expression on the mRNA level, without leading to changes on the protein level. It could be suspected, at least in the case of FGF2, that such a difference might be caused by an unspecific isoform of this protein. Currently, only low molecular weight recombinant FGF2 protein is available on the market. However, the FGF2 antibody is not differentially specific against the two isoforms. Assuming this, our results could mean that all the histological changes (like multipotent mesenchymal cell differentiation, satellite cell proliferation and angiogenesis) that have been noted in previous in vitro and in vivo studies[28–32] should not be expected in our case, or at least may not be caused directly by changes of FGF2 and VEGFA protein expression. This effect could be considered beneficial in terms of angiogenesis, which is a fundamental process affecting tumour growth and metastasis[19, 33–37]. Nevertheless, this remains to be clarified by applying histological and immunohistochemical methods of examination of the tissue.
The opposite effect of AgNano administration was observed in the case of the cardiac muscle, where, without any impact on relative heart weight (Table 2), the expression of both genes was affected.
FGF2 expression on the mRNA level was down-regulated in the in ovo injected group which obtained 20 ppm of AgNano in the drinking water, compared to the control group. Similar results were noted regarding protein expression, where the FGF2 level was lower in the AgNano-treated group compared to the control group. The results obtained for FGF2 in our case are not in agreement with the findings of Bougioukas et al., which suggested possible neovascularisation in the heart caused by this protein. However, according to another study, the expression of this gene remains unchanged during angiogenesis caused by bradycardia, suggesting that FGF2 does not play a direct role in this process. On the other hand, some evidence exists that FGF2 (the high molecular weight isoform) can prevent endothelial cell migration and angiogenesis and promote cardiac myocyte hypertrophy[40–42]. From this point of view, our findings may support possible targeted therapies with AgNano application.
It has been shown that VEGFA mRNA and protein are increased in the muscle during angiogenesis caused by repeated exercise[43–45]. Also, the mechanisms associated with bradycardia provide a signal for the enhancement of VEGFA expression, which is responsible for the myocardial angiogenesis. We have demonstrated that AgNano at a concentration of 20 ppm administered to broiler chickens in the drinking water caused up-regulation of VEGFA expression in the heart on the mRNA and protein level. VEGFA modulates angiogenesis in dystrophic muscle[17, 18] and in the heart[21, 46]. To reveal whether AgNano might influence the heart angiogenesis in our experiment, resulting in the neovascularisation of the cardiac muscle, histological examination of the tissue should be performed.
Recent data indicate that regulation of VEGFA and FGF2 expression occurs on the transcriptional as well as translational levels, depending on the tissue and cell type[8, 33–35, 47, 48]. Our results support these findings, showing that VEGFA and FGF2 expression can be regulated pre- and post-transcriptionally. Transcriptional and translational regulation of gene expression comprises the cascade of interactions between different biological molecules, including DNA, proteins, mRNAs and miRNAs[49–51]. Our results demonstrate that, in the case of FGF2 expression in the muscle, regulatory mechanisms prevent the increase of the level of the protein product of the gene.
Experimental data show that in ovo treatment can provide a basis for muscle growth and stimulate coronary development[20, 21]. However, some of our unpublished data suggest that AgNano injected in ovo at early stages of the embryonic development seem not to evoke long-lasting influence on gene expression, which is prolonged after the hatching period. Therefore, our aim was rather to observe whether in ovo injection could change the effect of AgNano distributed with drinking water. Answering this question, we assumed that indeed, in ovo injection up-regulated both the heart expression of FGF2 as well as the muscle expression of VEGFA in broilers treated with drinking solution of 20 ppm of AgNano. However, the AgNano drinking solution itself (both concentrations) evoked up-regulation of FGF2 expression in the muscle, without enhancing the effect of in ovo injection.
Changes in the expression of investigated genes on the protein level may lead to harmful or beneficial histological changes. When examining silver residues in organs after AgNano administration, our unpublished results have shown minor Ag deposition in the breast muscle tissue and very slight deposition in the hearts of birds treated with a 20-ppm solution of colloidal silver, compared to the control group. However, these results could not inform us regarding the form of Ag deposited in the tissues. Supporting data from histological and TEM examinations of these tissues are needed to elucidate whether AgNano or rather silver ions exert a direct action.