Inducing Vascular Endothelial Growth Factor to Investigate Tissue Regeneration and Stimulation of Early Angiogenesis in Axolotl Salamanders

Vascular Endothelial Growth Factor (VEGF) is described as a potent angiogenic factor essential for vascular endothelial cell growth.VEGF is produced by a myriad of cell types such as tumor cells, macrophages, and platelets.After production, VEGF works to regulate vital bodily processes by forming new blood vessels.These new blood vessels in combination with the VEGF signaling pathway ultimately play a fundamental role in bone formation, hematopoiesis, wound healing, and development [1].In biological systems, the formation of new blood vessels, also referred to as angiogenesis, is very important for development and growth.Studies show that even tumor growth is limited by blood vessel supply.Without proper angiogenesis, avascular solid tumors remain small and asymptomatic [2].Interestingly, in some cases, angiogenesis appears to be less critical, perhaps even detrimental during early tissue regeneration.Although this seems to be the case for early regeneration, as time goes on, angiogenesis is required for complete appendage replacement in regenerative species [3][4][5].Mescher (1996) hypothesized that a transient avascular period is essential for epimorphic tissue regeneration [6].We sought to test this hypothesis by artificially inducing VEGF to possibly stimulate early angiogenesis.The drug GS4012 is a potent inducer of VEGF and VEGF-driven vessel formation.It has been widely used as a pro-angiogenic compound in zebrafish, but little information is known of its role in animals that can regenerate more extensively [7].Axolotl salamanders, also known as Ambystoma mexicanum, have extensive regenerative capacities, including regrowth of entire appendages.Our goal was to expose axolotls to GS4012 and assess how early angiogenesis and regeneration may be affected.Light microscopy on anesthetized live animals allowed us to trace the regrowth of individual animals over the regenerative period.Larval white strain axolotls (snout vent length 3-5cm; RRID AGSC_101L) were purchased from the Ambystoma Genetic Stock Center.All animal procedures were conducted in accordance with an approved IACUC protocol.Upon receiving axolotls (n=10), we individually housed them in tanks containing 40% Holtfreter's solution.After the axolotls had time to acclimate to their environment, we amputated 15% of each of their tails.After amputation, the axolotls were split into two groups of five which became our control and treatment groups.A 10mM stock solution of GS4012 in dimethyl sulfoxide (DMSO) was diluted in 40% Holtfreter's solution to make a 1M treatment solution.The five treatment axolotls were then removed from their original tanks and added to new tanks containing the 1M drug mixture.The control axolotls were also removed from their original tanks and added to new tanks containing an equivalent volume of DMSO in 40% Holtfreter's solution.Concentrations were based on effective dose in zebrafish [7].It is important to note that after fourteen days of treatment, concentrations for both the control and treatment groups were increased from 1M to 1.5M.We believed that increasing the concentrations may help with the angiogenic process.Following amputation and drug administration, live anesthetized axolotls were imaged using a Bausch & Lomb stereomicroscope once every week.Images were captured using a Celestron ocular camera attachment and Micam computer software.Images focused on the regenerating portion of the tail.Lower magnification images were taken to track regeneration, while higher magnification images were taken to identify vasculature.After the images were recorded, ImageJ [8] software was used for regeneration measurements, thresholding, image calibration, and the addition of scale bars.No further image manipulation was performed.For thresholding, we chose random areas within images of the regenerating tail on each axolotl starting the third week of images and averaged the total amount of vessels we saw.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.For the first fourteen days, the treatment group seemed to be regenerating, on average, slightly more rapidly than the control group, though this did not rise to the level of statistical significance.When we reached the third week of treatment, we noticed that every axolotl had plateaued, and growth was significantly slowed (Fig. 1).For the next three weeks, none of the axolotls grew any significant portion of their tail, and most actually decreased in length (Fig. 2).None of the axolotls decreased so much to pass the point of amputation.Without clear differences between the regeneration patterns in both groups, it is hard to conclude that GS4012 had any significant effect on tissue regeneration despite some enhancement of blood vessel formation.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.In the control group we saw, on average, 29.9% of the regenerating tail contained vessels.In the treatment group we saw, on average, 38.5% of the regenerating tail contained vessels.With this preliminary data, we conclude that GS4012 enhanced vascularization and early angiogenesis (Fig. 3).Microscopic examination of the tissue structure of live animals over the course of regeneration was valuable in that it minimizes the number of animals required in research.The rapidly evolving field of regenerative biology provides the framework to develop new therapies and medical procedures for tissue healing and biomimetics.Studying axolotl salamanders helps us to further understand epimorphic tissue regeneration and its biomedical implications in hopes of someday harnessing this technology for humans.We plan to continue studying axolotl salamanders, especially angiogenesis and its role in tissue regeneration [9].