# Effects and Mechanism of Plasma-Activated Medium on Angiogenesis of Vascular Endothelial Cells
## 4 Discussion  
The results of MTT and cell cycle experiments in this study showed that PAM produced by LTP treatment for 15-30 s could promote the proliferation and cell viability of HUVECs after 24 h culture, and the proportion of cells in S phase increased. With the extension of LTP treatment, the activity of HUVECs was inhibited by PAM culture for 24 h, and the proportion of cells in S phase decreased significantly. This verified that the change of LTP treatment time could change the proliferation state of cells [3], which was also consistent with the previous research results that \"short-term LTP treatment promoted normal cell proliferation, while long-term treatment inhibited proliferation\" [29]. [PERSON] et al. [25] showed that short-term DBD-LTP treatment could enhance endothelial cell proliferation, suggesting that LTP may be a new method of time-dependent factor in endothelium-mediated angiogenesis. Angiogenesis is a multifactorial, continuous and complex process in which the key steps are the proliferation, migration and differentiation of endothelial cells and the formation, maturation and remodeling of blood vessels [30]. Therefore, cell migration, adhesion and angiogenesis experiments are important to quantify the angiogenic ability of cells. The results of this study showed that PAM produced by short-term LTP treatment could promote the angiogenesis, migration, infiltration and adhesion of HUVECs. However, PAM produced by long-term LTP treatment inhibited the angiogenesis, migration, infiltration and adhesion of HUVECs. This, on the one hand, is related to the direct inhibition of cell viability by PAM, and on the other hand, the RONS produced by PAM directly inhibits the tube formation and migration ability of cells. For further explanation, we speculate that PAM treatment could gradually decrease connexin 43 (CX43) gap junction expression of HUVECs, which in turn lowers cell migration speed and decreases cell angiogenesis ability. [PERSON] et al. [31, 32] found that CX43 is crucial for endothelial cell proliferation, migration and monocyte-endothelial cell adhesion. However, as the generation of LTP is often affected by environmental conditions, the temperature, humidity and gas flow rate (the remaining gas amount in the cylinder affects the stability of gas flow rate) will affect the effects of PAM. Moreover, the differences in cell state, passage times and experimental operation errors make the stability of the effects of 15-30 s PAM on promoting cell migration poor, and there is no significant difference between the results of penetration experiment in 30 s and 0 s groups. Therefore, in future studies, we will consider shortening the treatment interval and optimizing the experimental device, in order to find more accurate discharge conditions that promote the proliferation and angiogenesis of HUVECs. VEGF is a strong and specific positive promoter that promotes angiogenesis [33]. bFGF, as one of the most important non-specific angiogenesis factors [34], can directly interact with HUVECs, promote the proliferation and migration of endothelial cells, and play a positive regulatory role in angiogenesis [35]. VEGF family members bind to receptors on the cell surface and are activated through dimerization and auto-phosphorylation to trigger the activation of MEK and ERK signal transducers. Activated MEK further phosphorylates ERK1 and ERK2 [36], which is closely related to cell growth, proliferation, differentiation, and migration. Overexpression of VEGFR directly leads to pathological angiogenesis [37], and VEGFR mediates different functions in different tissues [38]. VEGF-VEGF-2 and MEK-ERK signaling pathways are the most important angiogenic signaling pathways. The experimental results in Figures 6 and 7 showed that PAM produced by LTP could promote the secretion of VEGF and bFGF, and enhance the expression of VEGFR-2 and the MEK-ERK signaling pathway when the treatment time was short.