Z33 - Low-Intensity Ultrasound Increases Pro-angiogenic Gene Expression in 3D Macrophage and Endothelial Cell Co-Culture

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

JB

Josephine Bochicchio

Undergraduate Research Assistant
Bucknell University
Lewisburg, United States

Co-Author(s)

Evelyn Hergenhan

Undergraduate Research Assistant
Bucknell University, United States

Primary Investigator(s)

Olivia Boerman

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

Introduction: : The goal of this work was to investigate the effects of ultrasound on pro-angiogenic gene expression using reverse transcription polymerase chain reaction (RT-PCR). Low-frequency, low-intensity therapeutic ultrasound has been clinically shown to advance healing in chronic wounds. [1] Over 6.5 million patients are affected every year by chronic wounds, and current treatments, such as pressure bandages and debridement, focus on maintaining the wound rather than promoting healing. [2] One possible way that ultrasound aids in wound healing is by promoting tissue repair through angiogenesis, or blood vessel formation. [3] An increase or decrease of pro-healing cytokines, measurable through gene expression, affects the process of wound healing. [4] Vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) are two pro-angiogenic genes that are expressed by endothelial cells, or the cells that line the inside of blood vessels, which are promising in promoting tissue repair. [5] The purpose of this study was to analyze the effects of low-frequency, low-intensity ultrasound on the expression of both VEGFA and FGF2 using RT-PCR. Human umbilical vein endothelial cells (HUVECs) and macrophages, a type of immune cell, were cultured together to simulate the dynamics between tissue repair and inflammation within chronic wounds.

Materials and

Methods: : 1x106 macrophage cells and 6x105 HUVECs were seeded together on 3D porcine collagen scaffolds. Seeded scaffolds were exposed to ultrasound (30kHz, 100 mW/cm2, and 25 Hz pulse repetition frequency) for 15 minutes. Cells were collected and lysed 18 hrs (n=6) and 24 hrs (n=3) after ultrasound exposure.

RNA was extracted following a Qiagen RNeasy Micro Kit instruction manual. RNA concentrations were quantified using a NanoDrop. RNA was converted using a ThermoFisher’s High-Capacity RNA-to-cDNA kit.

A 20 µL solution was prepared using 10 µL of TaqMan Fast Advanced Master Mix, the TaqMan assay, nuclease-free water, and 2 µL of cDNA. VEGFA and FGF2 assays were used as the genes of interest, and GAPDH was used as the control. The gene samples were amplified using the QuantStudio3 PCR machine on the Comparative CT-TaqMan setting adjusted to a 45-cycle run. The data was exported and analyzed using the delta-delta CT method. The data was graphed to represent the ultrasound intensity compared to fold change for each group of samples. A student’s t-test was conducted for statistical analysis and a p value of less than 0.5 was considered to be significant.

Results, Conclusions, and Discussions:: We saw an increase in VEGFA gene expression in ultrasound groups both 18 and 24 hours following exposure (Figure 1A-1B). Moreover, we saw an increase in gene expression of FGF2 (Figure 1C-1D). Our results are consistent with a previous study by Hanawa et al., who found that both VEGF and FGF expression increased after exposure to low-intensity pulsed ultrasound. [6] Although we saw an increase in both VEGFA and FGF2 gene expressions, it is important to acknowledge that this data was not significant, which may be a result of high standard deviations due to relatively low sample number. Even so, these data suggest an increase in pro-angiogenic gene expressions following ultrasound exposure.

These results suggest that VEGFA and FGF2 gene expression increase following exposure to low-frequency, low-intensity ultrasound 18 and 24 hours after exposure. These genes suggest that ultrasound may stimulate angiogenesis in chronic wounds. Although further experiments are necessary to more rigorously test this hypothesis, we can appreciate the increase in VEGFA and FGF2 gene expression following ultrasound exposure, indicating that ultrasound stimulates angiogenesis in a 3D endothelial-macrophage co-culture and may be a possible mechanism of ultrasound-mediated chronic wound healing. These data give us insight into the biomechanical pathway by which ultrasound promotes angiogenesis, supporting its potential as an efficient treatment for not only chronic wounds but also other medical conditions.

Acknowledgements and/or References (Optional): : [1] Samuels, J. A.; Weingarten, M. S.; Margolis, D. J.; Zubkov, L.; Sunny, Y.; Bawiec, C. R.; Conover, D.; Lewin, P. A. Low-Frequency ( < 100 KHz), Low-Intensity ( < 100 MW/Cm2) Ultrasound to Treat Venous Ulcers: A Human Study and in Vitro Experiments. The Journal of the Acoustical Society of America 2013, 134 (2), 1541–1547.
https://doi.org/10.1121/1.4812875.

[2] Sen, C. K.; Gordillo, G. M.; Roy, S.; Kirsner, R.; Lambert, L.; Hunt, T. K.; Gottrup, F.; Gurtner, G. C.; Longaker, M. T. Human Skin Wounds: A Major and Snowballing Threat to Public Health and the Economy. Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society 2009, 17 (6), 763–771.
https://doi.org/10.1111/j.1524-475X.2009.00543.x.

[3] Aleksandrowicz, H.; Owczarczyk-Saczonek, A.; Placek, W. Venous Leg Ulcers: Advanced Therapies and New Technologies. Biomedicines 2021, 9 (11), 1569. https://doi.org/10.3390/biomedicines9111569.

[4] Koh, T. J.; DiPietro, L. A. Inflammation and Wound Healing: The Role of the Macrophage. Expert Reviews in Molecular Medicine 2011, 13 (23). https://doi.org/10.1017/s1462399411001943.

[5] Laddha, A. P., & Kulkarni, Y. A. (2019). VEGF and FGF-2: Promising targets for the treatment of respiratory disorders. Respiratory medicine, 156, 33–46. https://doi.org/10.1016/j.rmed.2019.08.003

[6] Hanawa, K., Ito, K., Aizawa, K., Shindo, T., Nishimiya, K., Hasebe, Y., Tuburaya, R., Hasegawa, H., Yasuda, S., Kanai, H., & Shimokawa, H. (2014). Low-intensity pulsed ultrasound induces angiogenesis and ameliorates left ventricular dysfunction in a porcine model of chronic myocardial ischemia. PloS one, 9(8), e104863.
https://doi.org/10.1371/journal.pone.0104863