Lymphatic Regeneration: New Insights From VEGFR-3 Blockade

The lymphatic network is uniquely organized for the continuous removal of interstitial fluid and proteins, and for adsorption of dietary fat from the small intestine. Malfunction of the lymphatic system leads to lymphoedema ( 1 ) . Lymphatic vessels are a conduit for lymphocytes and antigen-presenting cells, and importantly, for tumor cells that metastasize to distant sites through the lymphatic system. Recent progress in the field has come from the identification of molecules that characterize and regulate lymphatic vessel development and function ( 1 , 2 ) . A critical advance came from the pioneering work by Alitalo and colleagues, who identified vascular endothelial growth factor (VEGF)-C, a member of VEGF growth factor family, as a specific lymphangiogenic growth factor that promotes lymphatic sprouting ( 1 ) . VEGF-C is produced by a variety of human tumors, and deregulated expression of VEGF-C induces tumor lymphangiogenesis and promotes lymphatic metastasis ( 3 ) . Lymphangiogenesis induced by VEGF-C is driven mainly by the activation of the tyrosine kinase-linked receptor VEGFR-3 ( 1 ) . VEGFR-3 is expressed almost exclusively by the lymphatic endothelium in normal adults. However, during mouse development, VEGFR-3 is widely expressed in blood endothelial cells, before the lymphatic vessels emerge ( 2 ) . Consistent with VEGFR-3 expression in blood endothelial cells early during development, VEGFR-3 knockout mice are embryonically lethal and display defects in remodeling the primary vascular plexus. In addition to VEGF-C, VEGF-D also activates VEGFR-3, and VEGF-D overexpression leads to accelerated lymphangiogenesis and spread of tumor cells to lymph nodes ( 4 ) . In turn, inactivation of VEGF-C and VEGF-D by the VEGFR3-Ig fusion protein inhibited tumor lymphangiogenesis and tumor metastasis to lymph nodes in a xenograft model of human lung cancer ( 3 ) . In contrast to blood capillaries, lymphatic capillaries are lined by a single, non-fenestrated endothelial cell layer that lacks pericytes, smooth muscle cells, and a basal membrane ( 1 ) . Newly described lymphatic endothelial cell markers include lymphatic vessel endothelial hyaluronan receptor 1, Prox-1, and VEGFR-3 ( 2 ) . Recently, VEGFR-3 proved to also be expressed in blood capillaries of normal breast tissue, neuroendocrine organs, and chronic wounds ( 5 , 6 ) . In addition, monocytes, macrophages, and some dendritic cells express this receptor ( 1 ) . Furthermore, VEGFR-2, which binds VEGF-A, VEGF-C, and VEGF-D, and is the principal mitogenic receptor in blood endothelial cells, is also expressed in lymphatic capillaries and mediates lymphangiogenesis ( 1 ) . In this issue of the Journal, Pytowski et al. ( 7 ) report on the generation of a new antibody to murine VEGFR-3 that blocks the binding of VEGF-C to VEGFR-3. Using this antibody, mF4–31C1, in a murine model of adult lymphatic regeneration ( 8 ) , the authors confirm the important role of VEGF-C and VEGFR-3 in mediating lymphatic sprouting. Even in the presence of VEGF-C-overexpressing breast carcinoma cells, lymphangiogenesis was blocked by mF4–31C1. Importantly, the pre-existing lymphatic vessels seemed unaffected morphologically and functionally by a prolonged VEGFR-3 blockade. In addition, blood capillaries, including those resulting from neovascularization of regenerating skin, were only slightly reduced in number. This work adds support for the development of VEGFR-3 inhibitors as anti-cancer agents. Systemic administration of the anti-VEGFR-3 antibody may reduce tumor-associated lymphangiogenesis and cancer metastasis, without affecting physiologic lymphatic flow. In Kaposi's sarcoma, the most common neoplasm in AIDS, both VEGF-C and VEGFR-3 are highly expressed in the tumor lesions, which are believed to consist of lymphatic endothelial cells ( 9 ) . Suppression of VEGFR-3 signaling may contribute to reduce Kaposi's sarcoma progression. Other potential targets of VEGFR-3 inhibition are immune disorders, including inflammatory bowel disease. In ulcerative colitis and Crohn's disease, there is a substantial increase in the number of lymphatic capillaries in the lamina propria and submucosa of the small and large intestine, which are believed to contribute to disease morbidity ( 10 ) . VEGFR-3 signaling is also involved in corneal transplant rejection by corneal dendritic cells ( 11 ) and perhaps other forms of graft rejection. Activated macrophages and corneal dendritic cells express VEGFR-3, and blockade of VEGFR-3 signaling in mice inhibited donor-specific delayed-type hypersensitivity, leading to a statistically significantly improved corneal transplant survival in mice ( 11 ) . In addition to the potential therapeutic applications of VEGFR-3 antagonists, the study by Pytowski et al. represents an exciting advance for scientists in the field. Yet the study raises a number of questions for future investigation, particularly about the function of VEGFR-3 in pre-existing lymphatic vessels and angiogenesis. Besides regulating fluid homeostasis, the lymphatic system is important for immune surveillance and fat absorption. Pytowski et al. analyzed lymphatic flow patterns but not immune cell trafficking or nutritional effects after extended VEGFR-3 blockade. If the VEGFR-3 blocking antibody does not alter these functions, what is the role of VEGFR-3 on mature lymphatic vessels? In vitro studies have revealed a role for VEGF-C/VEGFR-3 in lymphatic cell survival ( 12 ) . Under what circumstances does VEGF-C exert pro-survival effects on lymphatic endothelial cells? Second, despite VEGFR-3 expression on sprouting blood capillaries in skin wounds and in tumor vessels ( 13 ), why did prolonged VEGFR-3 inhibition only minimally affect blood capillary regeneration? One might have expected a substantial reduction in blood capillary formation, particularly in the presence of VEGF-C overexpression. Is this minimal regeneration attributable to compensatory VEGF-C stimulation of VEGFR-2 in blood endothelial cells ( 14 ) ? Or is it perhaps attributable to modulation of VEGFR-2 signaling by VEGFR-3 in blood endothelial cells as noted previously in lymphatic endothelial cells ( 15 ) ? What is the function VEGF-D, which binds to and activates VEGFR-3 and VEGFR-2, in the context of VEGFR-3 blockade ( 16 ) ? Third, does VEGFR-3 inhibition affect the survival and function of circulating endothelial progenitors? A subset of circulating CD34 + cells coexpress VEGFR-3 and CD133, and these cells are a functionally unique population of progenitor cells that can differentiate into lymphatic or vascular endothelial cells ( 17 ) . Future work pursuing these important questions may yield additional insight in lymphatic endothelial cell function and therapeutic uses of VEGFR-3 inhibition.