## 4 Discussion
The superimposition of photoacoustic wave and the applied ultrasound wave can produce a stronger mechanical effect to activate endothelial cells. However, strong cavitation produced by PUT can damage endothelial cells, as demonstrated in the previous study [3], resulting in the dysfunction of endothelial cells and thus reduced levels of NO and PGI\({}_{2}\) released. This can explain our observation in Figure 11 showing the apoptosis of cells. [PERSON] et al. showed that on inhibiting the production of endothelial NO, a substantial fall in resting flow and an elevation in peripheral resistance was observed in the human forearm [83]. Reduced production of PGI\({}_{2}\) was shown to result in the accumulation of plaques in blood vessels, which might result in the shutting down of the blood vessels [84; 85]. As PUT was already shown to be capable of shutting the blood vessels, this study focuses on understanding the effects on the release of NO and PGI\({}_{2}\) as they are vasoactive agents [4]. The changes in these levels were only expected to be associated with endothelial dysfunction. PUT achieves therapeutic effect through cavitation produced in the blood vessel, so monitoring cavitation can provide more detailed information into the mechanism. However, the most frequently used passive cavitation detection technique is more sensitive in detecting inertial cavitation, which is more likely to damage endothelial cells. While we did observe inertial cavitation at a high energy level, we expect that the apoptosis of endothelial cells is more likely caused by non-inertial cavitation. It is also worth noting that PUT can produce either non-inertial or inertial cavitation based on the applied energy levels [68]. Previous studies reported that the maximum depth of the PUT treatment was approximately 1 cm [86]. A high laser fluence at the surface or targeted laser delivery using optical fiber is required to achieve greater treatment depths. Further, the cavitation nuclei will also influence the result. The limitations of the study are the use of the complete medium containing phenol red in place of blood, and the fluid flow was in a straight channel, while the blood vessels are not perfectly straight. The use of phenol red can also offer optical absorption similar to real blood, a material property that is fundamental to PUT [3]. Though the phenol red compensates for the absorption of the blood, it cannot account for the scattering properties of blood. The size of the channel is 450 \(\upmu\)m in diameter, which would represent a large vessel because using a vessel diameter smaller than that would result in a smaller number of cells in the channel. The smaller number of cells could affect the sensitivity of the assay to measure the amount of NO and PGI\({}_{2}\) released from the cells. Moreover, the state of cells post-treatment is not thoroughly evaluated. In the future, the effect of ultrasound and PUT on other biochemical productions related to endothelial cells can be studied using this vessel model. The other limitation of our study is that the vessel model was produced under a static condition. While the advantage of the static culture is that it does not cause any mechanotransduction associated with shear stress on the endothelial cells during cell culture, the real in vivo situation always involved shear stress [87]. Further study will be needed to investigate the effect of different cell culture conditions. Moreover, the current study focuses on understanding the effects of PUT on the release of NO and PGI\({}_{2}\). Werecognize that there are many other biomarkers related to the endothelial cell damage and dysfunction [88; 89]. The characterization of these markers may provide a full picture of endothelial dysfunction, which is, however, out of the scope of the current study.