Functional characteristics and molecular regulation of lymphangiogenesis during gecko tail regeneration: evidence for the roles of VEGF-C, VEGF-D and the receptor VEGFR-3.

Abstract

The Australian marbled gecko, Christinus marmoratus has the ability to voluntarily shed its tail (autotomy) and subsequently regenerate the lost tail. The lymphatic vessels of the gecko tail are severed during autotomy and yet the regenerated tail is not lymphoedematous, indicating that the mechanisms for interstitial fluid drainage are maintained, presumably by the growth of new lymphatic vessels(lymphangiogenesis). In contrast, disruption to the lymphatic system in humans can readily result in lymphoedema due to inadequate lymphatic regenerative capacity. Hence, the regenerating gecko tail offers an excellent model to study the process of and fundamental molecular mechanisms behind lymphatic regeneration. Here, I examine lymphangiogenesis within regenerating gecko tails. I hypothesise that physiological function of lymphatic vasculature is recovered by tail regeneration. Further, I hypothesise that lymphatic regeneration is, in part, regulated by vascular endothelial growth factor C (VEGF-C) and VEGF-D via binding to their receptor, VEGFR-3, a key lymphangiogenic pathway in mammals. Lymphatic uptake and transport, of different sized radiolabelled tracers, were examined using lymphoscintigraphy. Basic lymphatic function is apparent at 6 weeks of regeneration, however lymph clearance and velocity are not restored to near original levels until 12 weeks of regeneration. Differential clearance and lymph velocity between tracers are likely influenced by changes in the cellular matrix and lymphatic vessel permeability. Molecular control of lymphangiogenesis within regenerating gecko tails was studied by identifying and characterising VEGF-C, VEGF-D and VEGFR-3 in gecko tail tissue extracts. This is the first study to demonstrate the presence of these genes within any reptile. Sequence alignments and molecular modelling highlight conservation of many lymphangiogenic functional residues within the gecko proteins at both a sequence and structural level. Real time PCR established differential expression profiles of VEGF-C, VEGF-D and VEGFR-3 mRNA throughout tail regeneration, with up-regulation during the early, late and mid-phases of regeneration, respectively. These data are consistent with mammalian studies in wound healing and suggest differing roles during gecko tail regeneration and potentially the lymphangiogenic process following autotomy. Sites of expression of VEGF-C and VEGF-D in regenerating gecko tails, demonstrated by immunohistochemistry, include keratinocytes and fibroblasts. Positive staining lining blood and lymphatic-like vessels is demonstrated for VEGF-D and VEGF-C, respectively indicating possible associations of the proteins with VEGFRs on endothelial cell surfaces and hence angiogenic and lymphangiogenic capabilities. Strong positive staining of VEGF-C and VEGFR-3 is also observed in adipose tissue in both regenerated and original tail tissue suggesting potential roles in adipogenesis and lymphangiogenesis during fat store expansion. Positive immunostaining using the LYVE-1 lymphatic endothelium marker demonstrates that lymphangiogenesis does occur during tail regeneration. Technical limitations, possibly related to antibody cross-reactivity prevented detection of VEGFR-3 staining on lymphatic (or blood) endothelial cells in all regenerated and original tails. A suspected lack of mammalian-derived antibody reactivity to the reptilian proteins was also encountered with ELISA and western blotting analyses, with both yielding inconclusive results. In conclusion, this study demonstrates that adequate lymphatic vasculature and function are restored during gecko tail regeneration. Furthermore, this study provides several lines of evidence, through sequence conservation and mRNA and tissue expression profiles, that VEGF-C, VEGF-D and VEGFR-3 play a role in lymphatic regeneration in a reptile.