Poster K14 - Low-frequency (20 kHz), Low-intensity (100 mW/cm²) Ultrasound Upregulates ROCK RNA Expression in Endothelial Cells

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Andie McCullough

Undergraduate Researcher
Bucknell University, United States

Co-Author(s)

Kim Hergenhan

Undergraduate Researcher
Bucknell University, United States

Primary Investigator(s)

Olivia Boerman, PhD (she/her/hers)

Assistant Professor of Biomedical Engineering
Bucknell University
Lewisburg, Pennsylvania, United States

Introduction: This study aims to determine the biological pathway through which ultrasound stimulates angiogenesis using reverse transcription polymerase chain reaction (RT-PCR). Studies have found that 5% to 8% of the world suffers from venous disease, a condition that leads to the formation of venous ulcers, a type of chronic wound that can become life-threatening.¹ Current treatment of chronic wounds has an annual cost of $25 billion for the American health system.³ Low-intensity, low-frequency ultrasound has shown promise as a potential alternative treatment. The application of low-intensity, low-frequency ultrasound has been shown to decrease the healing time of chronic wounds.³ Angiogenesis, the creation of blood vessels, is a critical step in the healing process, as blood vessels form up to 60% of tissue mass found in healing wounds and are critical in promoting tissue regeneration.² GTP binding protein Rho and its downstream effector Rho-kinase (ROCK) are two key genes involved in angiogenesis. ROCKs have been found to mediate important cell functions including cell migration and proliferation.⁵ The Rho/ROCK pathway is a known mechanotransduction pathway, meaning this pathway is stimulated by external mechanical forces.⁶ Ultrasound is an external mechanical force presented as peak positive and negative pressure. Rho/ROCK is a promising pathway through which ultrasound may modulate endothelial cell function and ultimately assist wound healing. The purpose of this study was to determine the effect of ultrasound on the RNA expression of GTP binding protein Rho and its downstream effector Rho-kinase (ROCK) using RT-PCR.

Materials and

Methods: 600,000 human umbilical vein endothelial cells (HUVECs) were seeded in collagen matrices and exposed to 50 mW/cm2, 100 mW/cm2, and 150 mW/cm2 intensity ultrasound for 15 minutes. Cells were collected and lysed 12, 18, and 24 hours after exposure.

RNA was extracted using a Qiagen RNeasy Micro Kit following manufacturer’s instructions and RNA was quantified using a SpectraDrop to determine RNA concentrations. RNA was synthesized using ThermoFisher’s High-Capacity RNA-to-cDNA kit.

Two microliters of cDNA were added to an 18 µL solution, consisting of 10 µLs of TaqMan Fast Advanced Master Mix, the TaqMan assay, and nuclease-free water. ROCK1 and RHO TaqMan gene expression assays were used as genes of interest and GAPDH was used as an exogenous control. Samples were amplified using the QuantStudio3 PCR machine on the Comparative CT-TaqMan setting adjusted to run 45 cycles. The data was exported and analyzed using the delta-delta CT method. The data was plotted to show the ultrasound intensity compared to fold change for each group of samples.

Results, Conclusions, and Discussions: Although ROCK showed no significant changes 6 hours or 12 hours after ultrasound exposure (Figure 1A and 1B), ROCK significantly increased (p < 0.05) by 2.5 fold in the 100 mW/cm² group as compared to the control group 18 hours after ultrasound exposure (Figure 1C). Fold changes of ultrasound groups at 24 hours were below the control which suggests a decrease in RNA expression of ROCK 24 hours after ultrasound exposure (Figure 1D).
These results suggest that ROCK may be a possible mechanotransduction pathway through which ultrasound effectuates healing. These findings agree with previous studies in which ROCK was shown to respond to ultrasound stimulus.⁴

These results demonstrate that ROCK gene expression increases following low-intensity, low-frequency ultrasound 18 hours after ultrasound application. ROCK’s involvement in angiogenesis supports that ultrasound may induce healing through the use of the Rho/ROCK pathway. Further investigation is necessary to verify the mechanism behind chronic wound healing using ultrasound, but these findings suggest that gene expression of Rho-kinase is involved.

Acknowledgements (Optional):