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Cardiovascular Engineering

Effects of Low Intensity Pulsed Ultrasound and Recombinant TGF-B1 on VEGF Secretion from Endothelial Cells

(G-262) Effects of Low Intensity Pulsed Ultrasound and Recombinant TGF-B1 on VEGF Secretion from Endothelial Cells

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row G - Poster # 262

Presenting Author(s)

Connor Kozick

Undergraduate Biomedical Engineering Student
Bucknell University
Danville, Pennsylvania, United States

Introduction:: Chronic wounds last on average 12-13 months and affect roughly 1-2% of the worldwide adult population. Low intensity pulsed ultrasound (LIPUS) has been demonstrated to clinically advance the healing of chronic wounds by significantly advancing chronic wound healing. However, the biological mechanisms of this ultrasound-assisted healing is not yet understood. Angiogenesis, the creation of blood vessels, is an essential process to progress the healing of chronic wounds. It is hypothesized that the LIPUS may induce angiogenesis. Vascular Endothelial Growth Factor (VEGF) is an established protein that has been shown to be increased in secretion by endothelial cells during angiogenesis, making VEGF a reliable indicator of angiogenesis. Transforming growth factor beta 1 (TGF-B1) is another protein associated with angiogenesis of endothelial cells. The combination of TGF-B1 and LIPUS may be a promising dual-prong therapy. Therefore, the purpose of this study was to investigate the effect of TGF-B1 alone, LIPUS alone, and TGF-B1 and LIPUS on endothelial cell secretion of VEGF.

Materials and Methods:: 80,000 Human Umbilical Vein Endothelial Cells (HUVECs) were seeded per well in a 12-well plate. Each well consisted of 2 mL total of vascular growth media. 24 hours after being seeded, 10 ng of recombinant TGF-B1 was added to the TGF-B1 alone group (n=3) and ultrasound plus TGF-B1 group (n=3). Additionally, 150 mW/cm2 was applied for 15 minutes to the ultrasound alone group (n=3) and ultrasound plus TGF-B1 group (n=3). 48 hours after applying the treatment, a Human VEGF ELISA development kit from Peprotech was used to quantify VEGF secretion by the HUVECs. A standard dilution from 4000 pg/mL to 0 pg/mL allowed the absorbance of the samples to be calculated by applying a line of best fit of the standard curve. A single factor analysis of variance (ANOVA) was then run to determine the statistical significance between the means of the four variables by seeking a p-value less than 0.05.

Results, Conclusions, and Discussions:: There were no statistically significant differences between the ultrasound alone, TGFB-1 alone, and ultrasound and TGFB-1 groups (Fig. 1). Although not significant, endothelial cells treated with 150 mW/cm2 of ultrasound secreted more VEGF than the control group. We can also appreciate that only 10 ng of recombinant TGF-B1 yielded VEGF concentrations greater than those of the control, and even higher than the LIPUS samples. Interestingly, the LIPUS and TGF-B1 combined group yielded an average concentration of VEGF very similar to the average of the control. This result may indicate a more complicated mechanism of action when 10 ng of TGF-B1 is applied in addition to 150 mW/cm2 of pulsed ultrasound. The signaling pathways of TGF-B1 are altered in the presence of LIPUS, limiting the secretion of VEGF by endothelial cells, thus not increasing angiogenesis.Trends from the experiment show that the application of 150 mW/cm2 of LIPUS and 10 ng of TGF-B1 on HUVECs separately increases the secretion of VEGF, suggesting an increase in angiogenesis. However, when LIPUS and TGF-B1 are used together at the same intensity and concentration respectively, the secretion of VEGF by HUVECs decreases.

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