Lymphedema is a debilitating disorder that occurs commonly after lymph node dissection for cancer treatment. Despite its common occurrence and profound impact on quality of life, there is no effective surgical cure. Although clinical reports have shown that autogenous tissue transfer (i.e. transfer of healthy tissues) can bypass damaged lymphatics and improve lymphedema, the precise mechanisms governing lymphatic regeneration in these circumstances remain unknown. The findings of the current study suggest that lymphatic regeneration after tissue transfer occurs as a result of spontaneous reconnection of existing lymphatics as well as ingrowth of new lymphatic vessels from the recipient. In addition, our studies support the hypothesis that these processes occur in the setting of VEGF-C expression and recipient-derived macrophage infiltration beginning at the periphery of the graft. Similar findings have been reported in blood vessel regeneration in skin grafts.
lymphangiogenesis has been studied in a variety of human diseases from chronic renal transplant rejection to tumor metastasis. The role of recipient and donor tissues in the regulation of lymphatic regeneration has only been evaluated in a few studies, however. Kerjaschki et al.
demonstrated that the majority (>95%) of lymphatics in the transplanted kidney are donor-derived while a relatively small number of lymphatic vessels (4.5%) in these tissues were of recipient origin.
These results are in contrast to our finding that by 6 weeks following skin grafting, the majority of lymphatic vessels present in the transplanted tissues were of recipient origin (),
indicating that inosculation with ingrowth of new lymphatic channels is a critical mechanism regulating lymphatic regeneration after skin/subcutaneous tissue transfer but not in solid organ transplantation. Although spontaneous reconnection of existing lymphatics occurs in transplanted skin/subcutaneous tissues, this mechanism is the primary mechanism regulating lymphatic regeneration after solid organ transplantation. It is possible that ingrowth of lymphatic vessels in kidney transplants from surrounding tissues is actively inhibited by the presence of the kidney capsule. Indeed, fibrous capsule and fascial tissues have long been considered to be important barriers to lymphatic tumor metastasis and are used routinely as surgical landmarks during oncological procedures. Alternatively, differences in lymphatic regeneration in solid organs as compared to skin and subcutaneous tissues may reflect volumetric differences in tissues transferred since skin grafts are relatively low volume (and therefore easily infiltrated by surrounding tissues) whereas solid organ transplantation involves a much larger volume of tissues that allows ingrowth of lymphatics only at the periphery of the tissues. Finally, in our model the skin graft was transferred as an avascular tissue with infiltration of local blood vessels and eventual incorporation into the recipient tissues. Kidney transplantation is performed by reconnecting the vascular supply of the tissues thereby minimizing the need for vessel ingrowth.
Previous studies have demonstrated that lymphangiogenesis in the mouse tail model is dependent on gradients of interstitial flow and expression of VEGF-C.
These studies have shown that gradients of VEGF-C expression coordinate and regulate lymphatic endothelial cell migration and differentiation resulting in tubule formation and lymphatic regeneration in a distal to proximal direction. In the current study, we also found that 2 weeks following surgery, VEGF-C expression was localized to the peripheral edges of the skin graft especially in the distal margin. These expression patterns correlated with the ingrowth of new lymphatic vessels from the recipient into the donor tissues. Contrary to previous studies with lymphatic regeneration in collagen gel scaffolds, however, we also found lymphatic ingrowth from the proximal aspect of the wound albeit at a slightly slower rate, indicating that lymphatic regeneration across a tissue graft occurs as a result of coordinated lymphatic ingrowth from both margins of the wound. This finding may be due to a higher potential for lymphatic transport via interstitial fluid flow in skin grafts as compared to acellular scaffolds such as type I collagen. Alternatively, it is likely that the skin graft becomes incorporated into recipient tissues more quickly and efficiently than lymphatic/tissue regeneration that occurs in collagen gel scaffolds resulting in improved interstitial flow and expression of VEGF-C even in the proximal portion of the graft.
In the current study we found that by 6 weeks after surgery, VEGF-C expression was highest within the center of the transplanted tissues. This response correlated with the patterns of lymphatic vessel regeneration after tissue transfer, beginning from distal and proximal margins and invading into the center of the donor tissues, implying that lymphatic regeneration occurs as a consequence of VEGF-C expression within the graft. This finding is intriguing and is supported by previous studies demonstrating improved lymphangiogenesis in surgical skin flaps after adenoviral VEGF-C transfer.
However, in contrast to these previous studies, we found that endogenous expression of VEGF-C is adequate for promoting rapid and efficient lymphatic regeneration after tissue transfer suggesting that gene therapeutic interventions to augment lymphangiogenic cytokine expression are not necessary for lymphatic regeneration in uncomplicated tissue transfer or surgical interventions.
Macrophages are critical regulators of lymphangiogenesis.
Macrophage expression of VEGF-C is a major regulator of lymphangiogenesis in tumors and during inflammation after kidney transplantation or corneal injury.
In addition, macrophages are thought to directly contribute to lymphatic vessel formation by trans-differentiating into lymphatic endothelial cells.
Our study showed that macrophages served a dual role in lymphatic regeneration after tissue transfer. Immunohistochemical localization of VEGF-C and F4/80 demonstrated that the patterns of VEGF-C expression correlated with accumulation of F4/80+
), supporting the hypothesis that macrophages play a critical role in VEGF-C expression during incorporation and lymphatic regeneration of autogenous tissues. In addition, we found that recipient-derived macrophages may directly contribute to lymphatic formation at the peripheral edges of the graft in the early time period by demonstrating F4/80+
tubular structures at the 2 week time point. Similar to previous studies on inflammatory lymphangiogenesis, these newly formed vessels likely underwent remodeling with loss of F4/80 expression at the later time point since no double positive lymphatic vessels could be identified in sections obtained from animals sacrificed 6 weeks after surgery.
Perhaps the most significant finding of our study was the fact that autogenous tissue transplantation could be used to rapidly bypass damaged lymphatic vessels with resultant restoration of lymphatic flow. Skin-grafted animals had marked reductions in tail swelling and significantly improved lymphatic function, with significant decreases in dermal thickness and tissue fibrosis. The finding that tissue transfer can bypass damaged lymphatic vessels is supported by anecdotal surgical reports demonstrating that tissue transfer can aid in treatment of some patients with lymphedema and provides a mechanism for this observation. Our findings suggest that improvements in extremity swelling occur as a result of both spontaneous lymphatic regeneration and reconnection of local and transferred lymphatic vessels thereby effectively bypassing lymphatic channels damaged during lymphadenectomy. These findings provide a rationale for the development of tissue engineered lymphatic constructs designed specifically for this purpose and may represent a novel means of treating patients with lymphedema.