## 2 Materials and Methods
### Ultrasound and Laser System Setup for Assessing NO and PGI\({}_{2}\) Release  
The schematic of the experimental set up with ultrasound/laser and flow system is shown in Figure 1. A 532 nm laser system (Surelite SLI-30, Continuum, Santa Clara, CA, USA), with a pulse repetition rate of 30 Hz and a pulse duration of 5 ns, was used to produce photoacoustic signals and align the focal region of a 500 kHz high-intensity focused ultrasound (HIFU) transducer (H107, Sonic Concepts, Bothell, WA, USA) on the channel before each treatment. A pulse delay generator (Model DG355, Stanford Research Systems, Sunnyvale, CA, USA) was used to trigger the laser system, ultrasound system, and oscilloscope with a repetition rate of 30 Hz. During the alignment process (switch 1 position), the laser beam was used to illuminate the channel, and the produced photoacoustic signal was detected by using the HIFU transducer. The signal was acquired by a digital oscilloscope (DPO 3034, Tektronix Inc., Beaverton, OR, USA). The HIFU transducer was scanned across the channel until the maximum photoacoustic signal was detected, indicating the focal region of the HIFU transducer was on the channel and overlaid the laser beam. During treatment (switch 2 position), a function generator (33250A, Agilent Technologies, Santa Clara, CA, USA) supplied ultrasound signals. Its output was first amplified by 50 dB using a power amplifier (350L RF Power Amplifier, ENI Technology Inc., Rochester, NY, USA). It was then connected to the HIFU transducer through an impedance matching circuit (Impedance Matching Network H107, Sonic Concepts, Bothell, WA, USA). The focal spot of the HIFU transducer, whose focal length and focal width are 21.02 and 3.02 mm, respectively, was focused at the center of the designated channel. The radius of curvature of the HIFU transducer is 63.2 mm. Ultrasound bursts with 10% duty cycle were used to minimize the generation of heat. Each burst contained 1650 cycles and was repeated at a 30 Hz pulse repetition rate. Figure 1: Schematic of the experimental setup for treatment in vitro. PA: power amplifier. DG: delay generator. PR: pulse-receiver. MN: matching network for HIFU transducer. FG: function generator. HIFU: high-intensity focused ultrasound. Position 1 is for detecting photoacoustic signals during the alignment process and Position 2 is for treatment. For PUT treatment, the laser intensities were carefully controlled to the desired laser fluence on the sample surface, and the same ultrasound parameters used in ultrasound-only treatments were used. In PUT, laser treatment and ultrasound were applied synchronously [6]. The laser spot diameter used for the treatment was 6 mm. An optical power meter was used to check the laser power before treatment for each sample. For NO measurement, the treatment pressures were 0.1, 0.5, 1, 1.5, and 2 MPa in ultrasound-only treatment group, and laser fluences were 5, 10, 15, and 20 mJ/cm\({}^{2}\) in laser-only treatment group with six samples (\(n=6\)) in each group. In the first PUT group (\(n=9\)), the 0.1, 0.5, and 1 MPa ultrasound pressures and laser intensity of 10 mJ/cm\({}^{2}\) were applied synchronously. In the second PUT group (\(n=6\)), the 5, 10, 15, and 20 mJ/cm\({}^{2}\) laser fluences and ultrasound intensity of 0.5 MPa were applied synchronously. For PGL\({}_{2}\) measurement, the treatment pressures were 0.5, 1, 1.5, and 2 MPa in ultrasound-only treatment group, and laser fluences were 5, 10, 15, and 20 mJ/cm\({}^{2}\) in laser-only treatment group. The same laser fluence range and ultrasound pressure of 0.5 MPa were applied synchronously in PUT group with six samples (\(n=6\)) in each group. As the change in NO began to decrease from 1.5 MPa for ultrasound only group, 0.1 MPa for PGL\({}_{2}\) measurements was not considered. Moreover, when changing ultrasound for constant laser, there was no significant change in any group except 0.1 MPa. Hence, the variation in ultrasound pressure for constant laser fluence was not carried out for PGL\({}_{2}\) measurements.