CPP altered the morphology of HDFs.
In order to test whether the hypochlorous acid (HOCl) probe CPP could potentially promote the differentiation of HDFs into endothelial cells, we first investigated whether CPP affects the cell viability of HDFs. Using the sulforhodamine B (SRB) assay, we observed that CPP did not significantly affect the viability of HDFs (Fig. 1A-1B). Secondly, we investigated whether treatment with CPP affects the morphology of HDFs. We observed that HDFs treated with CPP for 6 days and 10 days were elongated, curved and formed circular patterns (Fig. 1C), which resembles the function of endothelial cells.
From the above results we draw a conclusion that CPP, as a hypochlorous acid probe, can affect the morphology of HDFs. In order to identify whether other hypochlorous acid probes can also promote HDFs to form circular patterns, we treated HDFs with other HOCl probes for 10days. Interestingly, these HOCl probes failed HDFs to elongated, curved and formed circular patterns (Fig. 1D). Taken together, these data indicate that we may have found a new inducer that can induce HDFs to change their morphology and possess endothelial cell functions.
CPP promotes the expression of endothelial cell marker CD133.
In order to prove that HDFs indeed differentiated into VECs after treatment with CPP, we treated HDFs with different doses of CPP for 10 days. Cells treated with CPP had significantly decreased protein levels of the endothelial cell marker CD133 (Fig. 2A, 2B). Next, we further text the expression of CD133 in CPP-treated HDFs by qPCR. Consistent with western blot results, CPP can significantly increase the mRNA level of CD133 (Fig. 2C).
Furthermore, we used immunofluorescence staining to detect the level of CD133 in HDFs and found that CPP can promote the increase of CD133 levels. Collectively, CD133, as an important regulator for the maintenance of endothelial progenitor cell stemness, plays an important role in the process of endothelial cell differentiation[20]. From the above data, we found that CPP can significantly promote the expression of CD133.
CPP promotes the expression of endothelial cell marker CD31.
CD31, as the main marker of VECs, is the main factor to identify whether there is vascular endothelial cell production. In order to prove that CPP induce HDFs to differentiate into VECs. We treated HDFs with CPP at 1, 10 and 20 µM for 10 days, the protein level of CD31 was measured by western blot. As expected, the protein level of CD31 was significantly increased (Fig. 3A, 3B). Next, we tested the mRNA level of CD31 by qPCR, we also proved that the mRNA level of CD31 was increased (Fig. 3C). These results were verified by immunofluorescence staining (Fig. 3D). Together, these data showed that CPP promoted the expression of CD31.
Moreover, we quantified the percentage of CD31-positive cells using immunofluorescence staining, and found that nearly 80% of cells after 10 days of treatment with 10 or 20 µM CPP expressed CD31, and less than 10% of CD31-positive cells was observed in the control group (Fig. 4A). Taken together, these data demonstrate that CPP efficiently induced the differentiation of HDFs into VECs in vitro.
CPP reduced the level of HDFs marker Vimentin.
In the process of transforming one type of cell into another, the level of the marker protein of the source cell will decrease, and the level of the marker protein of the target cell will increase. Therefore, in order to further prove that CPP induces the differentiation of HDFs into VECs, we detected Vimentin, a marker protein of HDFs. After CPP treated HDFs at 1, 10 and 20 µM for 10 days, western blot was used to detect the protein level of Vimentin. We found that CPP significantly reduced the protein level of Vimentin at 10µM (Fig. 5A, 5B). Interestingly, these results were verified by qRT-PCR analysis and by immunofluorescence staining (Fig. 5C, 5D).
CPP promotes the angiogenesis of VECs derived from HDFs in vitro.
Next, we tested the angiogenic ability of HDFs treated with CPP using an in vitro Matrigel tube formation assay. HDFs treated with CPP at concentrations of 10 or 20 µM for 12 days, we observed the formation of HDFs into tubules in vitro on 4 days, 8 days, 10 days, and 12 days. We found that CPP-treated HDFs began to form tubules on day 10, and the number of tubes has increased significantly on day 12. In contrast, tube-like structures didn’t appear in the control group without treatment of CPP (Fig. 6).
CPP induces HDFs to differentiate into VECs by promoting the expression of pro-angiogenic factors.
As a secretory cell, VECs can secrete a variety of cytokines. Studies have shown that Vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2) and Platelet derived growth factor (PDGF-BB) are closely related to the maturation of VECs. In order to clarify the mechanism of CPP-induced differentiation of HDFs into VECs, we analyzed whether CPP(0,1,10,20 µM) treatment enhanced the expression levels of vascular endothelial function related factors in HDFs, and qRT-PCR analysis revealed that mRNA levels of VEGF, FGF-2 and PDGF-BB were strongly increased in cells treated with CPP for 10 days (Fig. 7A-C). Next, in order to further prove that CPP promoted the secretion of VEGF, FGF-2 and PDGF-BB, western blot was further verified. As expected, CPP significantly promoted the increase of VEGF, FGF-2 and PDGF-BB protein levels (Fig. 7D-I). Through the above results, we preliminarily proved that VECs derived from CPP-induced HDFs have their typical secretory function.