Effect of Pharmacological Activation of Vascular Endothelial Growth Factor (VEGF) on Regenerative Physiology in Larval Axolotls (<i>Ambystoma mexicanum</i>)

Vascular Endothelial Growth Factor (VEGF) is a potent angiogenic factor essential for vascular endothelial cell growth. In biological systems, these cells comprise blood vessels that play a fundamental role in bone formation, wound healing, and development. Along with aiding normal growth and development, we see VEGF stimulation after injury. Angiogenesis drives epimorphic tissue regeneration in vertebrate animals that can regrow anatomically identical portions of lost tissue. One unique example of this regenerative pattern is evident in the model organism known as the axolotl salamander ( Ambystoma mexicanum ). This species has extensive regenerative capabilities, including regrowth of entire appendages and scarless wound healing. Although axolotls have been the subject of widespread biomedical study, little information is known about their angiogenic and regenerative response given increased VEGF stimulation. Our work seeks to assess the extent to which blood vessel formation can be pharmacologically enhanced in this species and what impact, if any, this has on the regeneration of other tissue types. We hypothesized that artificially increasing VEGF stimulation during the regenerative period would enhance angiogenesis and quicken regenerative rates. To test our hypothesis, we used the drug GS4012. This compound is a potent inducer of VEGF-driven vessel formation. We administered this drug after distal tail amputation and tracked regenerative rates over a 6-week period. We used 10 larval white strain axolotls (snout-vent length 3-5cm; RRID AGSC_101L) obtained from the Ambystoma Genetic Stock Center. Each axolotl was anesthetized prior to amputation. Once the axolotl was unresponsive, we used an ethanol cleaned razor blade to remove 15% of the distal tail. After amputation, the axolotls were split into two groups of five which became our control and treatment groups. A 10 millimolar (mM) stock solution of GS4012 (Sigma Aldrich, St. Louis, MO) in dimethyl sulfoxide (DMSO) was diluted in 40% Holtfreter’s solution to make a 1 micromolar (µM) treatment solution. The control axolotls were housed in an equivalent volume of DMSO in 40% Holtfreter’s solution. When analyzing our threshold images to quantify vascularization and early angiogenesis, we found that throughout the entire regeneration process, the treatment group had more visually prominent blood vessels (38% of the regenerating tail area contained vessels in GS4012 treated larvae versus 30% in the control group). After six weeks of drug treatment, we found that complete tail regeneration (i.e., regrowth of 100% of amputated length) was not achieved by any axolotl in either group. In fact, we only had an average of 16.8% regrowth in our treatment group and 12.6% regrowth in our control group. Based on this preliminary data, we conclude that at the dosage used in these experiments, GS4012 modestly enhanced vascularization and early angiogenesis but that contrary to our initial hypothesis, this increased vascularity did not have a significant effect on regenerative rates and tissue outgrowth. Investigations are ongoing to determine the influence of artificially induced microvessels at different stages of the regenerative process, such as the role of precocious vascularity during the normally avascular stage of the blastema versus the critical need for at least some microcirculation during subsequent tail outgrowth. Comparative studies of the role of VEGF within the highly regenerative environment of axolotl tail regrowth versus the less regenerative environments of other species may shed light on the physiology of wound healing and ultimately contribute to a greater understanding of the role of angiogenic therapies in human biomedicine. We gratefully acknowledge the Ambystoma Genetic Stock Center, which provided the larvae and is supported by NIH P40-OD019794. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.