Lymphatic vessels drain fluid, antigens, and immune cells from the interstitial space to lymph nodes (LNs) and eventually back into the systemic blood circulation [
1]. Initial lymphatic capillaries consist of a monolayer of lymphatic endothelial cells (LECs) attached to a thin basement membrane, and unlike larger lymphatic vessels, they are deprived of smooth muscle cells (SMCs). They collect interstitial fluid, forming lymph, and drain to the pre-collecting and then collecting lymphatic vessels that are surrounded by SMCs and segmented with bicuspid valves. This physiological process regulates the tissue fluid homeostasis, leukocyte recirculation, and transport of antigen-presenting cells to the lymph nodes, crucial for adaptive immunity [
2,
3]. Post-developmental lymphangiogenesis occurs primarily during inflammation [
4,
5], both in the inflamed tissue as well as its draining lymph node, and in lymph nodes following infection and vaccination [
6,
7] or after tissue transplantation [
8]. Although its precise immunological functions remain unclear [
4,
9], inflammatory lymphangiogenesis has been associated with both immune tolerance as well as immunogenicity. In melanoma, tumor-driven lymphangiogenesis promoted tolerance induction by suppression and deletion of tumor-reactive T cells [
10], and lymphangiogenesis has been shown to help resolve inflammation after lung injury [
11]. However, corneal lymphangiogenesis is a major risk factor for corneal transplant rejection [
12‐
15], and blocking lymphangiogenesis after experimental islet transplantation helped prevent rejection [
16]. VEGF-C or VEGF-D-driven lymphangiogenesis can be prevented by function-blocking antibodies against the lymphatic receptor VEGFR-3 [
17‐
19]. However, anti-lymphangiogenic treatment has no influence on the function of pre-existing lymphatic vessels. Thus, there is great potential, for both fundamental lymphatic research as well as immunotherapeutic strategies, for methods that can locally destroy lymphatic vessels, and particularly lymphatic collectors.
Photodynamic therapy (PDT) is a clinical treatment for ablating unwanted or pathological blood vessels based on the administration of a non-toxic photosensitizer followed by sub-thermal light exposure that induces photosensitizer toxicity via generation of reactive oxygen species (ROS), resulting in thrombosis and vascular occlusion [
20‐
23]. This strategy can be used to treat cancer, as well as non-oncological conditions, such as age-related macular degeneration (AMD) [
24] or polypoidal choroidal vasculopathy [
25]. Visudyne
®, a liposomal formulation of verteporfin (the benzoporphyrin derivative monoacid ring A), is a clinically used photosensitizer administrated intravenously in patients for photodynamic treatment of pathologic cornea neovascularization [
26,
27] AMD [
28] or polypoidal choroidal vasculopathy [
25]. Light-activated verteporfin induces a cascade of reactions leading to the formation of highly reactive and toxic ROS, mainly singlet oxygen [
29,
30]. Although the effects of PDT on the blood vessels have been extensively studied during the past several decades [
20,
23] the use of PDT for lymphatic ablation has only begun to be explored, namely for targeting peritumoral lymphatics and the in-transit tumor cells they contained to treat metastatic disease [
31].
Our goal was to determine whether lymphatic vessels could be specifically targeted in skin, and to identify the optimal conditions to selectively close lymphatic collecting vessels without injuring the blood vasculature. More specifically, we focused on (1) the timing and the mechanism of PDT-induced lymphatic-specific closure using optimal light fluencies and photosensitizer doses, and (2) the kinetics of ensuing lymphatic regeneration. Such information, although presumably specific to mouse dermal lymphatics, is necessary to further develop lymphatic-specific PDT, for example to control metastatic spread or perform basic lymphatic research.