# Effects and Mechanism of Plasma-Activated Medium on Angiogenesis of Vascular Endothelial Cells
## 1 Introduction  
After the cell culture medium is treated with low temperature plasma (LTP) jet (atmospheric pressure plasma jet, APPJ) [1] for a period of time, the ions, electrons, neutral particles (atoms, gas molecules, etc. ), ultraviolet rays, RONS and other substances react with the culture medium [2] and produce plasma-activated medium (PAM). PAM can increase the level of oxidative stress in cells and thus promote or inhibit the proliferation of cells. Related studies have shown that short-term PAM treatment can promote the proliferation of fibroblasts [3], induce osteoblast differentiation [4], inhibit over 20 kinds of cancer cells cultured in vitro [5], including melanoma cells [6], prostate cancer cells [7], glioma cells [8], lymphoma cells [9], and liver cancer cells [10]. The main mechanisms of action include inducing apoptosis and necrosis of tumor cells, inhibiting proliferation signaling pathway, damaging DNA structure, and affecting migration and adhesion of tumor cells [11]. Studies have also shown that under the same treatment mode and time, when LTP simultaneously treats normal cells and tumor cells, it inhibits the activity of tumor cells, but significantly reduces the invasion, metastasis and adhesion of tumor cells, and has no obvious damage to normal cells [12, 13]. What's more, PAM can be kept stable for several days under low temperature (\(-80\)\({}^{\circ}\)C), dark and sealed storage, and the concentration of active ingredients remains unchanged [14]. Angiogenesis [15] refers to the process of growing new capillaries from existing blood vessels, which is an indispensable link in physiological phenomena such as growth anddevelopment, inflammatory response, wound healing, and also an important process of tumor development and progression of angioproliferative diseases. Angiogenesis is a complex and highly regulated process mediated by the expressions of a series of closely regulated pro-angiogenic and anti-angiogenic factors and their receptors on various vascular components (such as endothelial cells, pericellular cells) and stromal components [16]. Studies have shown that in the early stage of tumor growth, when the tumor diameter is less than 1 mm, there is no angiogenesis, and nutrients are mainly absorbed by diffuse oxygen supply. However, when the tumor volume increases to 2-3 mm\({}^{3}\), tumor cells are induced by hypoxia or oncogenes, angiogenesis factors are activated [17], and the angiogenesis process is initiated. Tumor angiogenesis process is chaotic and endothelial structure is incomplete. New blood vessels supply oxygen and nutrients necessary for tumor proliferation, which provide necessary conditions for tumor proliferation, growth, metastasis and diffusion [18]. Therefore, angiogenesis is considered one of the indicators of tumor invasion, metastasis and poor clinical prognosis [19; 20]. Hypoxia conditions, specific and non-specific acting factors, and some cytokines, chemokines, active oxygen and active nitrogen substances can affect the formation of neovascularization [16]. As a product of cellular aerobic metabolism [21], RONS are an indispensable and important factor in the regulation of angiogenesis. As an intracellular signaling molecule, low-concentration reactive oxygen species (ROS) that does not exceed the level of cellular oxidative stress, can participate in the regulation of cell proliferation, promote cell migration and differentiation, stimulate the expression of VEGF in endothelial cells [22], effectively promote tissue regeneration, and increase the number of generated blood vessels [23]. In contrast, a large number of RONS-mediated chain reactions have a strong damaging effect, damaging intracellular lipids and protein macromolecules, causing endothelial cell apoptosis, and inhibiting angiogenesis. LTP, as a diverse supplier of RONS, has a significant impact on angiogenesis. Domestic and foreign scholars have selected different plasma discharge devices to study the direct effects of different cellular angiogenesis.