Hypoxia-Inducible Factor Signaling in Macrophages Promotes Lymphangiogenesis in Leishmania major Infection.

Vascular remodeling is a phenomenon seen in the cutaneous lesions formed during infection with Leishmania parasites. Within the lesion, Leishmania major infection leads to the infiltration of inflammatory cells, including macrophages, and is associated with hypoxic conditions and lymphangiogenesis in the local site. This low-oxygen environment is concomitant with the expression of hypoxic inducible factors (HIFs), which initiate the expression of vascular endothelial growth factor-A (VEGF-A) in macrophages during the infection. Here, we found that macrophage hypoxia is elevated in the skin, and the HIF target Vegfa is preferentially expressed at the site of infection. Further, transcripts indicative of both HIF-1α and HIF-2α activation were increased at the site of infection. Given that HIF mediates VEGF-A and that VEGF-A/VEGFR-2 signaling induces lymphangiogenesis, we wanted to investigate the link between myeloid HIF activation and lymphangiogenesis during L. major infection. We show that myeloid aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF/VEGF-A signaling promotes lymphangiogenesis (the generation of newly formed vessels within the local lymphatic network), which helps resolve the lesion by draining away inflammatory cells and fluid. Concomitant with impaired lymphangiogenesis, we find the deletion of myeloid ARNT/HIF signaling leads to an exacerbated inflammatory response associated with a heightened CD4+ Th1 immune response following L. major infection. Altogether, our data suggest that VEGF-A-mediated lymphangiogenesis occurs through myeloid ARNT/HIF activation following Leishmania major infection and this process is critical in limiting immunopathology.

In Vivo Ear Sponge Lymphangiogenesis Assay.

Lymphangiogenesis, the formation of lymphatic vessels from preexisting ones, is an important process in wound-healing physiology. Deregulation of lymphangiogenesis and lymphatic vascular remodeling have been implicated in a range of inflammatory conditions, such as lymphedema, lymphadenopathy, tumor growth, and cancer metastasis. Any attempt in understanding various parameters of the lymphangiogenic process and developing desirable therapeutic targets requires recapitulating these conditions in in vivo models. One pitfall with some experimental models is the absence of immune response, an important regulatory factor for lymphangiogenesis. We overcome this issue by using immune competent mice. In this chapter, by using Angiopoietin-2 (Ang2), a protein that belongs to the Ang/Tie signaling pathway, we describe the ear sponge assay with important adaptations, highlighting a reproducible and quantitative tool for assessment of in vivo lymphangiogenesis.

Enhanced Meningeal Lymphatic Drainage Ameliorates Neuroinflammation and Hepatic Encephalopathy in Cirrhotic Rats.

BACKGROUND & AIMS: Hepatic encephalopathy (HE) is a serious neurologic complication in patients with liver cirrhosis. Very little is known about the role of the meningeal lymphatic system in HE. We tested our hypothesis that enhancement of meningeal lymphatic drainage could decrease neuroinflammation and ameliorate HE. METHODS: A 4-week bile duct ligation model was used to develop cirrhosis with HE in rats. Brain inflammation in patients with HE was evaluated by using archived GSE41919. The motor function of rats was assessed by the rotarod test. Adeno-associated virus 8-vascular endothelial growth factor C (AAV8-VEGF-C) was injected into the cisterna magna of HE rats 1 day after surgery to induce meningeal lymphangiogenesis. RESULTS: Cirrhotic rats with HE showed significantly increased microglia activation in the middle region of the cortex (P < .001) as well as increased neuroinflammation, as indicated by significant increases in interleukin 1ß, interferon γ, tumor necrosis factor α, and ionized calcium binding adaptor molecule 1 (Iba1) expression levels in at least 1 of the 3 regions of the cortex. Motor function was also impaired in rats with HE (P < .05). Human brains of patients with cirrhosis with HE also exhibited up-regulation of proinflammatory genes (NFKB1, IbA1, TNF-α, and IL1ß) (n = 6). AAV8-VEGF-C injection significantly increased meningeal lymphangiogenesis (P = .035) and tracer dye uptake in the anterior and middle regions of the cortex (P = .006 and .003, respectively), their corresponding meninges (P = .086 and .006, respectively), and the draining lymph nodes (P = .02). Furthermore, AAV8-VEGF-C decreased microglia activation (P < .001) and neuroinflammation and ameliorated motor dysfunction (P = .024). CONCLUSIONS: Promoting meningeal lymphatic drainage and enhancing waste clearance improves HE. Manipulation of meningeal lymphangiogenesis could be a new therapeutic strategy for the treatment of HE.

Adipose-Derived Stem Cells Promote Intussusceptive Lymphangiogenesis by Restricting Dermal Fibrosis in Irradiated Tissue of Mice.

Currently, there is no definitive treatment for lymphatic disorders. Adipose-derived stem cells (ADSCs) have been reported to promote lymphatic regeneration in lymphedema models, but the mechanisms underlying the therapeutic effects remain unclear. Here, we tested the therapeutic effects of ADSC transplantation on lymphedema using a secondary lymphedema mouse model. The model was established in C57BL/6J mice by x-irradiation and surgical removal of the lymphatic system in situ. The number of lymphatic vessels with anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) immunoreactivity increased significantly in mice subjected to transplantation of 7.5 × 105 ADSCs. X-irradiation suppressed lymphatic vessel dilation, which ADSC transplantation could mitigate. Proliferative cell nuclear antigen staining showed increased lymphatic endothelial cell (LEC) and extracellular matrix proliferation. Picrosirius red staining revealed normal collagen fiber orientation in the dermal tissue after ADSC transplantation. These therapeutic effects were not related to vascular endothelial growth factor (VEGF)-C expression. Scanning electron microscopy revealed structures similar to the intraluminal pillar during intussusceptive angiogenesis on the inside of dilated lymphatic vessels. We predicted that intussusceptive lymphangiogenesis occurred in lymphedema. Our findings indicate that ADSC transplantation contributes to lymphedema reduction by promoting LEC proliferation, improving fibrosis and dilation capacity of lymphatic vessels, and increasing the number of lymphatic vessels via intussusceptive lymphangiogenesis.

Mast Cells, Angiogenesis and Lymphangiogenesis in Human Gastric Cancer.

Gastric cancer is diagnosed in nearly one million new patients each year and it remains the second leading cause of cancer-related deaths worldwide. Although gastric cancer represents a heterogeneous group of diseases, chronic inflammation has been shown to play a role in tumorigenesis. Cancer development is a multistep process characterized by genetic and epigenetic alterations during tumour initiation and progression. The stromal microenvironment is important in maintaining normal tissue homeostasis or promoting tumour development. A plethora of immune cells (i.e., lymphocytes, macrophages, mast cells, monocytes, myeloid-derived suppressor cells, Treg cells, dendritic cells, neutrophils, eosinophils, natural killer (NK) and natural killer T (NKT) cells) are components of gastric cancer microenvironment. Mast cell density is increased in gastric cancer and there is a correlation with angiogenesis, the number of metastatic lymph nodes and the survival of these patients. Mast cells exert a protumorigenic role in gastric cancer through the release of angiogenic (VEGF-A, CXCL8, MMP-9) and lymphangiogenic factors (VEGF-C and VEGF-F). Gastric mast cells express the programmed death ligands (PD-L1 and PD-L2) which are relevant as immune checkpoints in cancer. Several clinical undergoing trials targeting immune checkpoints could be an innovative therapeutic strategy in gastric cancer. Elucidation of the role of subsets of mast cells in different human gastric cancers will demand studies of increasing complexity beyond those assessing merely mast cell density and microlocalization.

Tumor-Associated Lymphatic Vessel Features and Immunomodulatory Functions.

The lymphatic system comprises a network of lymphoid tissues and vessels that drains the extracellular compartment of most tissues. During tumor development, lymphatic endothelial cells (LECs) substantially expand in response to VEGFR-3 engagement by VEGF-C produced in the tumor microenvironment, a process known as tumor-associated lymphangiogenesis. Lymphatic drainage from the tumor to the draining lymph nodes consequently increases, powering interstitial flow in the tumor stroma. The ability of a tumor to induce and activate lymphatic growth has been positively correlated with metastasis. Much effort has been made to identify genes responsible for tumor-associated lymphangiogenesis. Inhibition of lymphangiogenesis with soluble VEGFR-3 or with specific monoclonal antibodies decreases tumor spread to LNs in rodent models. Importantly, tumor-associated lymphatics do not only operate as tumor cell transporters but also play critical roles in anti-tumor immunity. Therefore, metastatic as well as primary tumor progression can be affected by manipulating tumor-associated lymphatic remodeling or function. Here, we review and discuss our current knowledge on the contribution of LECs immersed in the tumor microenvironment as immunoregulators, as well as a possible functional remodeling of LECs subsets depending on the organ microenvironment.

[VEGF-C and lymphatic vessels: a double-edged sword in tumor development and metastasis]. / VEGF-C et vaisseaux lymphatiques - Une épée à double tranchant dans le développement tumoral et la dissémination métastatique.

The lymphatic system is made up of vessels that drain interstitial fluids throughout the body. The circulation of the lymph (liquid in the lymphatic system) in the lymphatic vessels is unidirectional: tissues to the lymph nodes and then to the veins. Ganglia are mechanical filters but also immune barriers that can block the progression of certain pathogens as well as cancer cells. However, most studies on the lymphatic system and cancer highlight the role of the lymphatic network in metastatic dissemination as tumor cells use this network to reach other organs. However, recent studies describe a beneficial role of the lymphatic system and of the vascular endothelial growth factor C (VEGF-C) which is one of the main factors responsible for the development of lymphatic vessels in cancer. In this review, we will illustrate this ambivalent and emerging role of VEGF-C and the lymphatic system in cancer aggressiveness.

Modulation of Immunity by Lymphatic Dysfunction in Lymphedema.

The debilitating condition known as secondary lymphedema frequently occurs after lymphadenectomy and/or radiotherapy for the treatment of cancer. These therapies can damage lymphatic vessels leading to edema, fibrosis, inflammation and dysregulated adipogenesis, which result in profound swelling of an affected limb. Importantly, lymphedema patients often exhibit impaired immune function which predisposes them to a variety of infections. It is known that lymphadenectomy can compromise the acquisition of adaptive immune responses and antibody production; however the cellular mechanisms involved are poorly understood. Here we discuss recent progress in revealing the cellular and molecular mechanisms underlying poor immune function in secondary lymphedema, which has indicated a key role for regulatory T cells in immunosuppression in this disease. Furthermore, the interaction of CD4+ T cells and macrophages has been shown to play a role in driving proliferation of lymphatic endothelial cells and aberrant lymphangiogenesis, which contribute to interstitial fluid accumulation in lymphedema. These new insights into the interplay between lymphatic vessels and the immune system in lymphedema will likely provide opportunities for novel therapeutic approaches designed to improve clinical outcomes in this problematic disease.

S1P promotes inflammation-induced tube formation by HLECs via the S1PR1/NF-κB pathway.

Inflammation-induced lymphangiogenesis is a widely accepted concept. However, most of the inflammatory factors and their related mechanisms have not been clarified. It has been reported that sphingosine-1-phosphate (S1P) is not only closely related to the chronic inflammatory process but also affects angiogenesis. Therefore, we investigated the inflammatory effects of S1P on human lymphatic endothelial cells (HLECs). Our results showed that S1P promotes tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) secretion in HLECs. We also confirmed that S1P-stimulated TNF-α and IL-1ß secretion is mediated through S1P receptor 1 (S1PR1). Using TNF-α siRNA and IL-1ß siRNA, we found that TNF-α and IL-1ß play essential roles in S1P-induced HLEC proliferation, migration, and tube formation. S1P induces phosphorylation of NF-κB p65 and activation of NF-κB nuclear translocation. A S1PR1 antagonist (W146) and NF-κB inhibitor (BAY11-7082) inhibited S1P-induced TNF-α and IL-1ß secretion and prevented NF-κB nuclear translocation. Taken together, the results demonstrated for the first time that S1P promotes the secretion of TNF-α and IL-1ß in HLECs via S1PR1-mediated NF-κB signaling pathways, thus affecting lymphangiogenesis. The study provides a new strategy for finding treatments for lymphangiogenesis-related diseases.

Antibody-mediated delivery of VEGF-C potently reduces chronic skin inflammation.

VEGF-C is an important mediator of lymphangiogenesis and has been shown to alleviate chronic inflammation in a variety of disease models. In this study, we investigated whether targeted delivery of VEGF-C to sites of inflammation and site-specific activation of lymphatic vessels would represent a clinically feasible strategy for treating chronic skin inflammation. To this end, we generated a fusion protein consisting of human VEGF-C fused to the F8 antibody (F8-VEGF-C), which is specific for the alternatively spliced, angiogenesis-marking extradomain A (EDA) of fibronectin. In two mouse models of psoriasis-like skin inflammation, mediated by transgenic VEGF-A overexpression or repeated application of imiquimod, intravenous treatment with F8-VEGF-C but not with untargeted VEGF-C significantly reduced ear skin edema and was as effective as the clinically used TNF-α receptor-Fc fusion protein (TNFR-Fc). Treatment with F8-VEGF-C led to a marked expansion of lymphatic vessels in the inflamed skin and significantly improved lymphatic drainage function. At the same time, treatment with F8-VEGF-C significantly reduced leukocyte numbers, including CD4+ and γδ T cells. In sum, our results reveal that targeted delivery of VEGF-C and site-specific induction of lymphatic vessels represent a potentially new and promising approach for the treatment of chronic inflammatory diseases.

Lymphatic Vessels: A Potential Approach to the Treatment of Atherosclerosis?

Many basic and clinical studies have demonstrated that atherosclerosis is a chronic inflammatory disease. Although there are many factors affecting atherosclerosis, the role of lymphatic vessels in this disease has been neglected. Traditionally, lymphatic vessels have been considered to be passages for transporting interstitial fluid to the blood circulation. However, as early as the last century, researchers found that there are numerous lymphatic vessels surrounding sites of atherosclerosis; however, the relationship between lymphatic vessels and atherosclerosis is not clear. With further research, lymphatic vessels were determined to be involved in the induction and resolution of arterial inflammation and also to play a positive role in plaque cholesterol transport. There are abundant immune cells around atherosclerosis, and these immune cells not only have a significant impact on plaque formation but also affect local lymphangiogenesis (IAL). This promotion of IAL seems to relieve the progression of atherosclerosis. Therefore, research into the relationship between lymphatic vessels and atherosclerosis is of great importance for improving atherosclerosis treatment. This review highlights what is known about the relationship between lymphatic vessels and atherosclerosis, including the effect of immune cells on IAL, and reverse cholesterol transport. In addition, we present some of our views on the improvement of atherosclerosis treatment, which have significant clinical value in research.

Immunohistochemical characterization of lymphangiogenesis-related biomarkers in primary and recurrent gliomas: A STROBE compliant article.

Glial tumors constitute the majority of primary intracranial brain tumors. The expression of specific markers of lymphangiogenesis in gliomas still remains unclear.A total of 40 surgical specimens from 20 patients with recurrent gliomas were included in the study. The expression of D2-40, vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor-3 (VEGR-3) was detected by immunohistochemistry (IHC). The clinicopathologic data (p53 and Ki67) were also collected and analyzed.At relapse malignant transformation rate was 65% (13/20 cases). D2-40, VEGF-C, VEGF-D, and VEGFR-3 were expressed in 20%, 30%, 60%, and 20% of primary and 45%, 30%, 75%, and 35% of recurrent glioma tumors (P < .01, P = 1.00, P = .03, P = .03). In 13 cases with increased malignancy grade, the expression of Ki67 and p53 were higher at relapse compared with the primary tumors (P = .001, P = .045). Multivariate survival analysis showed VEGF-D was an independent prognostic factor for malignant transformation (HR = 0.376, P = .045).Glioma is easy to relapse with tumor progression. VEGF-D was an independent prognostic factor for malignant transformation.

IL-4 dysregulates microRNAs involved in inflammation, angiogenesis and apoptosis in epidermal keratinocytes.

IL-4 plays an important role in the pathogenesis of atopic dermatitis (AD) by dysregulating many key factors at the transcriptional level. In this study, a microRNA array technique and IL-4 transgenic mice were used to demonstrate that IL-4 dysregulates microRNAs involved in inflammation, angiogenesis, lymphangiogenesis and apoptosis. Of the 372 common microRNAs examined, 26 and one microRNAs were found to be up- and down-regulated, respectively. MicroRNA-101-5p, -122-5p, -142-3p, -204-5p, -335-3p, -376a-3p, -378a-5p, -639 and -9-5p are among the most significantly up-regulated microRNAs. MicroRNA-147a, the only one that was down- regulated in the present study, attenuates TLR-induced inflammatory responses. These dysregulated microRNAs may provide post-transcriptional regulation of key genes in AD.

Dendritic cells in inflammatory angiogenesis and lymphangiogenesis.

Lymph node (LN) expansion during inflammation is essential to establish immune responses and relies on the development of blood and lymph vessels. Human dendritic cells (DCs), subdivided into two main subsets, namely conventional DCs (cDCs) and plasmacytoid DCs (pDCs), are professional antigen presenting cells endowed with the capability to produce soluble mediators regulating inflammation and tissue repair. cDCs support angiogenesis in secondary LNs both directly and indirectly through the secretion of vascular endothelial growth factor-A (VEGF)-A and VEGF-C and the production of several other mediators endowed with angiogenic properties. Finally, cDCs can affect neovascular formation via a transdifferentiation process. At variance with cDCs, the angiogenic properties of pDCs still remain poorly explored.

CD4+ T cells are activated in regional lymph nodes and migrate to skin to initiate lymphedema.

T cell-mediated responses have been implicated in the development of fibrosis, impaired lymphangiogenesis, and lymphatic dysfunction in secondary lymphedema. Here we show that CD4+ T cells are necessary for lymphedema pathogenesis by utilizing adoptive transfer techniques in CD4 knockout mice that have undergone tail skin and lymphatic excision or popliteal lymph node dissection. We also demonstrate that T cell activation following lymphatic injury occurs in regional skin-draining lymph nodes after interaction with antigen-presenting cells such as dendritic cells. CD4+ T cell activation is associated with differentiation into a mixed T helper type 1 and 2 phenotype, as well as upregulation of adhesion molecules and chemokines that promote migration to the skin. Most importantly, we find that blocking T cell release from lymph nodes using a sphingosine-1-phosphate receptor modulator prevents lymphedema, suggesting that this approach may have clinical utility.

CXCL5 Facilitates Melanoma Cell-Neutrophil Interaction and Lymph Node Metastasis.

Chemokines influence tumor metastasis by targeting tumor, stromal, and hematopoietic cells. Characterizing the chemokine mRNA expression profile of human primary melanoma samples, we found CXCL5 significantly up-regulated in stage T4 primary melanomas when compared to thin melanomas (T1 stage). To characterize the role of CXCL5 in melanoma progression, we established a metastasizing murine xenograft model using CXCL5-overexpressing human melanoma cells. CXCL5 had no effect on melanoma proliferation in vitro and on primary tumor growth in vivo, but CXCL5-overexpressing tumors recruited high amounts of neutrophils and exhibited significantly increased lymphangiogenesis in our severe combined immune-deficient mouse model. Recruited neutrophils were found in close proximity to or within lymphatic vessels, often in direct contact with melanoma cells. Clinically, CXCL5-overexpressing melanomas had significantly increased lymph node metastases. We were able to translate these findings to human patient samples and found a positive correlation between CXCL5 expression, numbers of neutrophils in stage T4 primary melanoma, and the occurrence of subsequent locoregional metastasis.

Tumor Regulation of Lymph Node Lymphatic Sinus Growth and Lymph Flow in Mice and in Humans.

The lymphatic vasculature collects and drains fluid and cells from the periphery through lymph nodes (LNs) for immune monitoring, and then returns lymph to the bloodstream. During immune responses LNs enlarge and remodel, featuring extensive growth of lymphatic sinuses (lymphangiogenesis). This LN lymphangiogenesis also arises in cancer, and is associated with altered lymph drainage through LNs. Studies of mouse solid tumor models identified lymphatic sinus growth throughout tumor-draining LNs (TDLNs), and increased lymph flow through the expanded sinuses. Mice developing B cell lymphomas also feature LN lymphangiogenesis and increased lymph flow, indicating that these changes occur in lymphoma as well as in solid tumors. These LN alterations may be key to promote tumor growth and metastasis to draining LNs and distant organs. Lymphatic sinus growth within the TDLN may suppress anti-tumor-immune responses, and/or the increased lymph drainage could promote metastasis to draining LNs and distant organs. Investigations of human cancers and lymphomas are now identifying TDLN lymphatic sinus growth and increased lymph flow, that correlate with metastasis and poor prognosis. Pathology assessment of TDLN lymphangiogenesis or noninvasive imaging of tumor lymph drainage thus could potentially be useful to assist with diagnosis and treatment decisions. Moreover, the expanded lymphatic sinuses and increased lymph flow could facilitate vaccine or drug delivery, to manipulate TDLN immune functioning or to treat metastases. The insights obtained thus far should encourage further investigation of the mechanisms and consequences of TDLN lymphatic sinus growth and lymph flow alterations in mouse cancer models, and in human cancer patients.

Comprehensive Modeling of Corneal Alkali Injury in the Rat Eye.

PURPOSE: To characterize the molecular, clinical, and histopathological profiles in the rat cornea after alkali injury over a 21-day period. METHODS: Alkali injury was induced in one eye of male Lewis rats. Corneal opacity and corneal neovascularization were assessed daily. Real-time qRT-PCR analysis and immunohistochemical staining were conducted to examine inflammation, neovascularization, and fibrosis. RESULTS: We found that within 2 hours of chemical exposure, corneal opacification rapidly developed with an acute increase in various cytokine expressions, while several cytokines demonstrated a secondary peak by day 7. Early neutrophil infiltration peaked at day 1 post-injury while macrophage infiltration peaked at day 7. Throughout the time course of the study, corneal opacity persisted and neovascularization, lymphangiogenesis, and fibrosis progressed. CONCLUSIONS: This study highlights the molecular, clinical, and histopathological changes throughout the progression of alkali injury in the rat cornea. These profiles will assist in the development of new strategies and therapies for ocular alkali injury.

[Novel Adjuvant Therapy for Ocular Melanoma]. / Neue adjuvante Therapien beim okulären Melanom.

Background Malignant melanoma is the most common cancer of the eye in adults that originates either in the intra-ocular uveal tract or extra-ocular conjunctiva. Although the primary tumor can be treated successfully, no effective therapy for both metastatic conjunctival and uveal melanoma currently exits. Tumor-associated lymphangiogenesis and immune cell infiltration play a pivotal role in the development and therapeutic targeting of metastases. Project description Here, we provide an overview of current translational research on lymphangiogenesis and its therapeutic inhibition as well as modulation of immune cell infiltration by passive and active immunotherapy in melanoma of the eye. Specifically, our previous and ongoing work on lymphangiogenesis and immune cells in ocular melanoma within the clinical research unit FOR 2240 "(Lymph)Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" is summarized. Conclusions Translational research on the modulation of tumor-associated lymphangiogenesis and immune cell infiltration could provide novel targets for adjuvant therapy in melanoma of the eye.

[Anti(lymph)angiogenic Strategies to Improve Corneal Graft Survival]. / Antihämangiogene/antilymphangiogene Strategien zur Verbesserung des Transplantatüberlebens nach Keratoplastik.

Corneal transplantation (keratoplasty) is the most frequently performed form of transplantation worldwide. A rejection reaction against the transplant is the main complication occurring after transplantation in an already vascularized, so-called "high-risk" recipient eye. Our group has shown that clinically invisible lymphatic vessels play a crucial role in the induction of a rejection reaction against the corneal graft, and that anti-(lymph)angiogenic therapies in the mouse model of keratoplasty can significantly improve transplant survival. The underlying mechanisms, which improve transplant survival through anti-lymphangiogenic therapies have not been well understood. We assume that the blockade of lymph vessel sprouting leads to a tolerance (and not to a simple ignorance) of the transplant, in which the antigen-presenting cells are held longer in the cornea and, thus, an immunomodulation of these cells occurs. Therefore, an important goal of our project is to find out whether and when transplant tolerance comes from a corneal anti-lymphangiogenic therapy. We assume that the antigen-presenting cells will have a different maturity level and that more tolerogenic effector cells (regulatory T cells, Tregs) develop in the absence of lymphatic vessels. Current anti(lymph)angiogenic therapies have the disadvantage that they are primarily effective on actively growing vessels. Most patients who receive high-risk keratoplasty often present in the clinic with already established, mature corneal blood and lymphatic vessels. At present, there are no lymph vessel regressing strategies, and the mechanisms regulating the maturation of the lymphatics are largely unknown. Therefore, our second goal is to develop new strategies for the regression of existing, pathological lymphatic vessels in the cornea. We are testing both destructive strategies, such as photodynamic therapy and diathermy as well as strategies for the molecular destabilization of the lymph vessel endothelium. Thus, our project identifies the precise mechanisms by which anti-lymphangiogenic therapies improve transplant survival, and we are developing new strategies to push back mature lymphatics in the high-risk setting.