Combined blockade of VEGFR-2 and VEGFR-3 inhibits inflammatory lymphangiogenesis in early and middle stages.

Lymphatic research has experienced exponential growth in recent years largely because of the advancement of technologies and the discoveries of several lymphatic endothelial specific molecules, including lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1), Prox-1, and VEGFR-3. The lymphatic network penetrates most tissues in the body, and its dysfunction has been found in a broad spectrum of disorders, such as cancer metastasis, inflammation, transplant rejection, obesity, and hypertension.1–6 To date, there is still little effective treatment for lymphatic diseases; it is, therefore, a field with an urgent demand for new therapeutic protocols. Given its accessible location and transparent nature, the cornea provides an optimal site for lymphatic research. Although the normal cornea does not have lymphatic vessels, lymphangiogenesis (LG; the development of new lymphatic vessels) can be induced in this tissue after inflammatory, traumatic, chemical, or infectious damage.7–9 LG also constitutes the afferent arm of the immune reflex arc of corneal transplantation immunity,7,9,10 and, most recently, it has been demonstrated that LG is a primary mediator of corneal transplant rejection.11 The VEGF family is the backbone of a complex network controlling blood and lymphatic vessel processes. A number of previous studies have shown that VEGFR-3 mediates LG in the cornea and other tissues, and its inhibition suppresses transplant rejection, tumor growth, and metastasis.11–17 The specific role of VEGFR-2 in LG, however, has been much less studied. It is known that this factor is critically involved in hemangiogenesis (HG; the development of new blood vessels) and that its inhibition suppresses HG and tumor growth.18–21 Most recently, it has been indicated that VEGFR-2 also plays a role in corneal LG,12,22,23 though the underlying mechanisms still remain largely unknown. Although results from previous corneal studies on LG are promising, there is still a large knowledge gap when translating these results to clinic settings. For example, almost all the previous studies focused on how to prevent LG by testing an intervention started right before, or on the same day as, a pathologic stimulation, which happens only infrequently in patients. Indeed, many patients who need LG treatment miss their first chance to receive emergent care, possibly because of difficult living conditions, transportation problems, or insufficient knowledge about the disease process itself. Indeed, LG is a progressive event that can occur insidiously at its early phases. In this study, we first demonstrate that VEGFR-2 is involved in suture-induced inflammatory LG but to a lesser extent than VEGFR-3. More important, we reveal that there is a discrete time window for therapeutic intervention of corneal inflammatory LG. We show that a combined blockade of VEGFR-2 and VEGFR-3 suppresses both early- and middle-stage LG while demonstrating no effect on late-stage LG. Close correlation between the timing and outcomes of the treatment is also defined. It is hoped that our results will provide a useful guide for understanding of the LG processes and the development of novel therapeutic protocols for LG-related diseases at various stages.