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www.nature.com/scientificreports/ with green light showed enhanced levels of NRG1-b1, but also serpin F1. The expression of CXCL16 was increased after red light stimulation. Experiments were performed with pooled supernatants from three donors. Values represent means ± standard deviation from two technical replicates. effects observed 72 hours after stimulation. This is in line with recent data from our lab which showed that treat- ment by extracorporeal shock waves also had effects on EC and ASC proliferation several days after one-time treatment7, 27. These data may indicate that long-term cellular effects can be initiated by light therapy. LLLT can either act directly on cells but also could initiate the release of specific mediators. As a first step to evaluate the secretom we analysed light-induced ROS formation. Low levels of ROS are important for signaling but higher doses can induce detrimental effects. We showed that, in contrast to red and green light, blue light led to a significant formation of ROS, which might correlate with the negative effects of blue light on proliferation and migration. Vasculogenesis involves complex steps of proliferation and migration of endothelial cells and the maturation of primitive vascular structures. We therefore performed 2D as well as 3D migration experiments with human endothelial cells. The reduction of artificially created 2D scratch wounds was trendwise reduced by red LED light and reached significance with green LED light treatment. The positive effect of red light is in line with the report of Teuschl et al. showing in scratch wound models that LLLT with red LED light increased myoblast, fibroblast and keratinocyte migration in vitro while blue LED light was ineffective28. Similar effects were seen when quan- tifying the migration of HUVEC in a 3D assay with enhanced migration in the green and red light stimulated groups while blue light was not effective. Fushimi et al. indicated similar effects in human keratinocytes and showed that red and green LED light stimulation enhances wound healing29. As endothelial cells are important for angiogenesis we aimed to investigate light-induced network forma- tion in two separate in vitro models which mimick early events in angiogenic and vasculogenic processes. In a Matrigel assay no effects of either wavelength were detected. However, this assay only reflects early vasculo- genic events within approx. 24 hrs. Amongst other disadvantages, Matrigel contains an undefined mixture of pro-angiogenic cytokines derived from a mouse brain tumor. Thus, we also investigated the effects of LLLT in an in vitro 3D co-culture model developed in our lab. Using this model we have recently shown that mechanical stimulation with shockwaves enhances HUVEC vascular tube formation7. In order to evaluate if light of different wavelengths may have similar stimulating effects, fibrin clots containing HUVEC and ASC at a ratio of 1:0.01 were stimulated with LLLT and the precursors of vascularization was quantified over 7 days. Since in previous studies it has been shown that a 1:1 seeding ratio usually leads to mature capillary network within one week even without any stimulation, a seeding ratio of 1:0.01 was chosen in order to observe the stimulating effects of LLLT on vasculogenic processes. At 1:0.01 ratio there was no network formation within 7 days of culture. However, it was possible to detect and quantify single-cell specific changes in morphology. Light treatment at all used wavelengths led to significant stretching and elongation of HUVEC on day 4. This data is supported by Ricci et al. who investigated the effects of laser light on endothelial cell morphology and suggested that LLLT influ- ences the organization of endothelial cytoskeletons11. In this model also proliferation was significantly enhanced by red and green light determined by analysis of the cell-occupied area which is in accordance with the data obtained from the 2D proliferation assay. The positive trend of increased proliferation continued until day 7. Stretching and elongation of cells was still significantly enhanced by green and red light on day 7. In the present study positive effects on regenerative processes were demonstrated for green and red light while blue light was not effective. The discrepancy between the positive effects of blue LED light in vivo5 and inefficiency in vitro as shown in this study might indicate different underlying mechanisms. Dungel et al. showed that blue light is most efficient to release the important mediator nitric oxide (NO) bound to mitochondrial complexes13. In in vivo situations of ischemic wound healing NO is reduced from nitrite via oxygen-independent nitrite reductases. Thus, NO-mediated effects induced by blue light may play an important role in in vivo settings. The formation of NO was analysed in cell culture supernatants immediately after illumination as well as on day 4. Although not significant, both red and green light induced higher NO levels, which might be associated with the positive effects of these wavelengths on proliferation and migration. The finding that NO and ROS levels are indirectly correlated might once more point to different pathways activated by different wavelengths. Red light has also been reported to act via photobiomodulation of complexes in the respiratory chain of mitochondria, mainly cytochrome c oxi- dase. The role of green light in these processes has to be further elucidated. By observing expressed protein levels after LLLT, we found that pro-angiogenic factors such as HGF, NRG1-b1, PIGF, CXCL16 and DPPIV as well as the anti-angiogenic factor serpin F1 were upregulated after LLLT. Ieda et al. showed that HGF improves the recovery of hind limb ischemia by promoting angiogenesis30. Further, endothelial-derived NRG has an important function in ischemia-induced angiogenesis and arteriogenesis31. DPPIV serves as a mediator of angiogenesis and tissue remodelling and serves as a necessary protein involved in the proliferation of smooth muscle cells32. Interestingly, the expression of the analysed proteins did not correlate very well with the observed effects on migration and wound healing. The anti-angiogenic factor Serpin F1 was upregulated only in the green light group, while blue light that was ineffective upregulated only pro-angiogenic factors. However, the co-culture model does not nec- essarily reflect in vivo angiogenesis but rather vasculogenic events. The formation of vascular structures includes remodeling processes and a balance of pro- and anti-angiogenic factors. Our in vitro findings suggest that an interplay of pro-angiogenic, remodelling, but also anti-angiogenic factors such as serpin F1 could be the driving factors of LLLT-mediated wound healing and angiogenesis in vivo. In summary, LLLT by red and green LED light increased migration and proliferation of endothelial cells consistently in several independent models. Especially the positive effects of green light are novel and have to be tested in further studies. The difference in the effects of blue light in vivo compared to in vitro raises interest- ing issues on the mechanisms of light therapy. Certainly in vivo situations are much more complex with many SCientifiC REpORTS | 7: 10700 | DOI:10.1038/s41598-017-11061-y 7PDF Image | impact of wavelengths of LED light-therapy on endothelial cells
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