# Cold Atmospheric Pressure Plasma in Wound Healing and Cancer Treatment
## 4 Properties of CAP Stimulate Tissue Regeneration but May also Induce Cell Death
A further study also observed a CAP stimulated expression of key genes (IL-6, IL-8, MCP-1, TGF-81, TGF-82) involved in wound healing cascades, as well as elevated production of collagen type I and alpha-SMA [60]. The same group found CAP induced expression of IL-8, TGF-81, and TGF-82, as well as upregulation of antimicrobial peptides of the \(\upbeta\)-defensin family [61]. At the same time, the 2 min CAP applied in this study induced gene expression of key regulators important for inflammation and wound healing, while not causing proliferation, migration, or cell death in keratinocytes. Using in vitro models that mimic various steps of angiogenesis, it has been demonstrated that CAP triggered the generation of nitric oxide [62]. Furthermore, CAP enhanced cell migration and the assembly of endothelial cells into vessel-like structures--hallmarks of the proliferation phase of wound healing [62]. In the same study, CAP treatment of a mouse model with third-degree burn wounds was associated with enhanced angiogenesis, accelerated wound healing, and increased cellular proliferation. The enzyme that catalyzes nitric oxide synthesis, endothelial NO synthase (eNOS), as well as expression of pro-angiogenic markers, PDGF8 and CD31, were significantly increased in mouse wounds after CAP. In vitro CAP stimulated pro-angiogenic VEGFA/VEGFR2 signaling [62]. Hence, this study demonstrates the ability of CAP to modulate endothelial nitric oxide synthase signaling and enhances neovascularization in burn wounds. The ability of CAP to induce wound angiogenesis by activating angiogenesis-related molecules has also been shown in keratinocytes, fibroblasts, and endothelial cells [63]. In keratinocytes, significantly increased expression of Artemin, EGF, EG-VEGF, Endothelin-1, FGF-2, IL-8 (CXCL8), and urokinase-type plasminogen activator (uPA) was observed. In fibroblasts, Angiogenin, Endostatin (Col18A1), MCP-1 (CCL2), MMP-9, TIMP-1, uPA, and VEGF had been increased after CAP treatment, and in endothelial cells, the expression of Angiopoietin-2, Angiostatin, Amphiregulin, Endostatin, FGF-2, FGFR1, and VEGFR had been activated. By transferring the conditioned medium from CAP-treated fibroblasts and keratinocytes to endothelial cells, the authors demonstrated that CAP modulates pro-angiogenic factors via autocrine and paracrine mechanisms. CAP induced wound angiogenesis was confirmed in vivo by an immunohistochemistry assay assessing the platelet and endothelial cell adhesion molecule CD31 in CAP-treated and placebo-treated tissues. Increased mRNA levels of CD31 and FGF-2 have also been observed in the CAP treated tissue [63]. Positive effects of CAP on angiogenesis by the release of FGF-2 or by the generation of nitric oxide have also been observed by others [64; 65]. The effects of CAP on paracrine cross-talk between dermal keratinocytes and fibroblasts have also been investigated in a recent study using an in vitro co-culture model. This study identified YAP and its targets CTGF and Cyr61 as drivers for paracrine signaling and CAP mediated wound healing [66]. The authors conclude that the secretion of CTGF and Cyr61 from fibroblasts influences the activation of keratinocytes through paracrine signaling and hence improves cell migration upon wound onset. Intriguingly,plasma treatment promotes the antioxidant phase II response elements in keratinocytes, which varies to a certain degree upon changes in the feed gas composition and subsequent RONS chemistry [67; 68]. Interestingly, the frequent exposure of the same human keratinocyte culture in vitro to plasma pointed to several adaptation processes in these cells, e.g., in antioxidant defense [69]. Enhanced migration of fibroblasts and keratinocytes after CAP treatment has also been observed in vitro in scratch assays [70]. This was correlated with the downregulation of the gap junctional protein connexin 43, as well as the downregulation of E-cadherin and several integrins.