Don Yuen

Cutting Edge: Lymphatic Vessels, Not Blood Vessels, Primarily Mediate Immune Rejections After Transplantation

The purpose of this study was to determine the relative importance of blood vessels (hemangiogenesis) versus lymphatic vessels (lymphangiogenesis) in mediating immunological responses after transplantation. Using the murine model of corneal transplantation, graft survival was compared in differentially prevascularized and avascular recipient beds. Donor corneas (C57BL/6) were transplanted into uninflamed or inflamed avascular, prehemvascularized only or prehemvascularized and prelymphvascularized recipient murine eyes (BALB/C). Selective inhibition of lymphangiogenesis was achieved using antivascular endothelial growth factor receptor 3 Abs and anti-integrin α5 small molecules. Grafts placed into only prehemvascularized recipient beds had a similarly good graft survival compared with grafts placed into completely avascular, normal recipients, whereas the pre-existence of lymphatic vessels significantly deteriorated corneal graft survival (p < 0.05). Lymphatic vessels seem to contribute significantly to graft rejection after (corneal) transplantation. That may allow for selective, temporary, perioperative antilymphangiogenic treatment to promote graft survival without affecting blood vessels, even after solid organ transplantation.

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds (LOPAC)1280 collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.

Differential distribution of blood and lymphatic vessels in the murine cornea.

PURPOSE Because of its unique characteristics, the cornea has been widely used for blood and lymphatic vessel research. However, whether limbal or corneal vessels are evenly distributed under normal or inflamed conditions has never been studied. The purpose of this study was to investigate this question and to examine whether and how the distribution patterns change during corneal inflammatory lymphangiogenesis (LG) and hemangiogenesis (HG). METHODS Corneal inflammatory LG and HG were induced in two most commonly used mouse strains, BALB/c and C57BL/6 (6-8 weeks of age), by a standardized two-suture placement model. Oriented flat-mount corneas together with the limbal tissues were used for immunofluorescence microscope studies. Blood and lymphatic vessels under normal and inflamed conditions were analyzed and quantified to compare their distributions. RESULTS The data demonstrate, for the first time, greater distribution of both blood and lymphatic vessels in the nasal side in normal murine limbal areas. This nasal-dominant pattern was maintained during corneal inflammatory LG, whereas it was lost for HG. CONCLUSIONS Blood and lymphatic vessels are not evenly distributed in normal limbal areas. Furthermore, corneal LG and HG respond differently to inflammatory stimuli. These new findings will shed some light on corneal physiology and pathogenesis and on the development of experimental models and therapeutic strategies for corneal diseases.

Combined blockade of VEGFR-3 and VLA-1 markedly promotes high-risk corneal transplant survival.

PURPOSE. High-risk corneal transplantation refers to grafting performed on inflamed and highly vascularized host beds. It represents a clinical dilemma because the rejection rate can be as high as 90%, irrespective of current treatment modalities. This study was conducted to investigate whether combined blockade of VEGFR-3 (vascular endothelial growth factor receptor-3) and VLA-1 (very late antigen-1) promotes high-risk transplant survival and how it correlates with corneal lymphangiogenesis and hemangiogenesis before and after transplantation. METHODS. High-risk corneal transplantation was performed between normal C57BL/6 (donor) and inflamed BALB/c (recipient) mice. The recipients were randomized to receive intraperitoneal injections of VEGFR-3 and VLA-1-neutralizing antibodies or their controls twice a week for up to 8 weeks after transplantation. Corneal grafts were evaluated by ophthalmic slit-lamp biomicroscopy and analyzed by Kaplan-Meier survival curve. Additionally, whole-mount corneas before and after transplantation were examined by immunofluorescent microscopic assays, and the correlation between lymphatic or blood vessel distribution and transplant outcome was analyzed. RESULTS. The combined blockade markedly promotes 90% survival of high-risk transplants. This strategy specifically modified host beds by selective inhibition of lymphangiogenesis but not hemangiogenesis. A strong correlation was also identified between high-risk transplant rejection and severe lymphatic invasion reaching the donor-graft border. CONCLUSIONS. These novel findings not only provide a new and potentially powerful strategy to promote high-risk transplant survival, they also confirm a critical role of high-degree lymphangiogenesis in mediating high-risk transplant rejection. Results from this study may also shed new light on our understanding and management of other lymphatic- and immune-related diseases in general.

Combined Blockade of VEGFR-2 and VEGFR-3 Inhibits Inflammatory Lymphangiogenesis in Early and Middle Stages

PURPOSE Lymphangiogenesis (LG) accompanies many corneal diseases after inflammatory, infectious, or chemical insults and is a primary mediator of transplant rejection. The purpose of this study was to investigate whether there is a time window for therapeutic intervention of corneal LG and whether a combined blockade of VEGFR-2 and VEGFR-3 effectively suppresses early-, middle-, or late-stage LG. METHODS Corneal inflammatory neovascularization was induced by a standard suture placement model in mice. Neutralizing antibodies against VEGFR-3 and/or VEGFR-2 were administrated systemically with the treatment started at postoperative day 0, day 7, or day 14. Whole mount corneas were sampled for immunofluorescence microscopic studies using LYVE-1 (a lymphatic marker) antibodies. Digital images were analyzed by software. RESULTS Both VEGFR-3 and VEGFR-2 were involved in corneal suture-induced inflammatory LG. Their combined blockade led to a significant inhibition of both early- and middle-stage LG while demonstrating no effect on late-stage LG. CONCLUSIONS Corneal inflammatory LG has a discrete time window for intervention therapy. Although it is important to start the treatment as soon as possible, interventions initiated in the middle of the LG process are still effective. These novel findings will shed some light on our understanding of inflammatory LG and the development of new therapeutic protocols for LG-related diseases at different stages.

Very Late Antigen-1 Mediates Corneal Lymphangiogenesis

PURPOSE To investigate the specific role of very late antigen-1 (VLA-1; also known as integrin α1β1) in corneal inflammatory lymphangiogenesis in vivo and lymphatic endothelial cell functions in vitro. METHODS A standard suture-induced corneal inflammatory lymphangiogenesis model was used in normal adult BALB/c mice to test the effect of systemic administration of VLA-1-neutralizing antibody on lymphatic formation and macrophage infiltration in vivo. Additionally, a human lymphatic endothelial cell culture system was used to examine the effect of VLA-1 gene depletion on lymphatic endothelial cell functions in vitro using small interfering RNAs. RESULTS These data demonstrated, for the first time, that VLA-1 blockade significantly suppressed corneal lymphangiogenesis and macrophage infiltration during inflammation. Moreover, VLA-1 gene depletion led to a marked inhibition of lymphatic endothelial cell processes of adhesion, proliferation, and capillary tube formation. CONCLUSIONS These novel findings together indicate that VLA-1 is critically involved in the processes of lymphangiogenesis. Further investigation on this factor may provide novel therapies for corneal inflammation, transplant rejection, and other lymphatic-related disorders in the body.

Novel characterization of lymphatic valve formation during corneal inflammation.

Lymphatic research has progressed rapidly in recent years. Though lymphatic dysfunction has been found in a wide array of disorders from transplant rejection to cancer metastasis, to date, there is still little effective treatment for lymphatic diseases. The cornea offers an optimal site for lymphatic research due to its accessible location, transparent nature, and lymphatic-free but inducible features. However, it still remains unknown whether lymphatic valves exist in newly formed lymphatic vessels in the cornea, and how this relates to an inflammatory response. In this study, we provide the first evidence showing that lymphatic valves were formed in mouse cornea during suture-induced inflammation with the up-regulation of integrin alpha 9. The number of corneal valves increased with the progression of inflammatory lymphangiogenesis. Moreover, we have detected lymphatic valves at various developmental stages, from incomplete to more developed ones. In addition to defining the average diameter of lymphatic vessels equipped with lymphatic valves, we also report that lymphatic valves were more often located near the branching points. Taken together, these novel findings not only provide new insights into corneal lymphatic formation and maturation, but also identify a new model for future investigation on lymphatic valve formation and possibly therapeutic intervention.

Role of Angiopoietin-2 in Corneal Lymphangiogenesis

PURPOSE Lymphatic research has progressed rapidly in recent years. Lymphatic dysfunction has been found in myriad disorders from cancer metastasis to transplant rejection; however, effective treatment for lymphatic disorders is still limited. This study investigates the role of angiopoietin-2 (Ang-2) in corneal inflammatory lymphangiogenesis (LG) in vivo and in lymphatic endothelial cell (LEC) functions in vitro. METHODS Standard suture placement model was used to study Ang-2 expression in inflamed cornea, and corneal LG and hemangiogenesis (HG) responses in Ang-2 knockout mice. Moreover, human LEC culture system was used to examine the effect of Ang-2 gene knockdown on LEC functions using small interfering RNAs (siRNAs). The effect of siRNA treatment on corneal LG was also assessed in vivo. RESULTS Angiopoietin-2 was expressed on lymphatic vessels and macrophages in inflamed cornea. While corneal LG response was abolished in Ang-2 knockout mice, the HG response was also significantly suppressed with disorganized patterning. Moreover, anti-Ang-2 treatment inhibited LEC proliferation and capillary tube formation in vitro and corneal LG in vivo. CONCLUSIONS Angiopoietin-2 is critically involved in lymphatic processes in vivo and in vitro. Further investigation of the Ang-2 pathway may provide novel insights and therapeutic strategies for lymphatic-related disorders, which occur both inside and outside the eye.

Intravital Imaging Reveals Dynamics of Lymphangiogenesis and Valvulogenesis

Lymphatic research signifies a field of rapid progression in recent years. Though lymphatic dysfunction has been found in a myriad of disorders, to date, few effective treatments are available for lymphatic diseases. It is therefore urgent to develop new experimental approaches and therapeutic protocols. The cornea offers an ideal site for lymphatic research due to its transparent nature, accessible location, and lymphatic-free but –inducible features. Moreover, we have recently discovered that corneal lymphatic vessels develop luminal valves as lymphangiogenesis proceeds. This tissue thus provides an optimal tool to study both lymphangiogenesis and valvulogenesis upon a pathological insult. In this paper, we show that the modified Prox-1-GFP mice carrying wildtype C57BL/6 background provide a valuable tool for intravital imaging of corneal lymphatic vessels and valves and can be used to study pathological lymphangiogenesis induced by various insults. Further, we demonstrate the multifaceted dynamics of lymphangiogenesis and valvulogenesis associated with transplantation, from the initiation to regression phases, and report several novel and critical phenomena and mechanisms that cannot be detected by conventional ex vivo approaches. Further investigation holds the great potential for divulging new mechanisms and therapeutic strategies for lymphangiogenesis and lymphangiogenesis-related diseases at various stages and inside or outside the eye.

Increased lymphangiogenesis and hemangiogenesis in infant cornea.

BACKGROUND Corneal lymphangiogenesis (LG) and hemangiogenesis (HG) accompany many diseases after inflammatory, infectious, traumatic or chemical insults. They also contribute to transplant rejection. It is known that corneal transplants in infants or children have a higher rejection rate than in adults. However, it has never been studied whether infant corneas differ from adult corneas in inflammatory LG, HG, or both, which is the focus of this study. METHODS AND RESULTS Corneal inflammatory LG and HG were induced by a standard suture placement model in C57BL/6 mice of 3 weeks and 8 weeks of age, respectively. Corneal LG, HG, and macrophage infiltration were assessed by immunofluorescent microscopic studies using specific antibodies against CD31 (a panendothelial cell marker), LYVE-1 (a lymphatic marker), and F4/80 (a macrophage marker). Blood vessels were also examined by ophthalmic slit-lamp microscopic assays in vivo. Digital images were analyzed by NIH Image J software. It was found, for the first time, that infant corneas exhibited a higher level of LG, HG, and macrophage infiltration during inflammation. Infant lymphatic and blood vessels demonstrated greater density and invasion area but similar branching points. Additionally, infant lymphatic vessels were also of larger diameter. CONCLUSIONS Infant and adult corneas differ greatly in their inflammatory responses of LG, HG, and macrophage infiltration. These novel findings will shed some light on our understanding of the LG and HG processes, as well as the development of new therapeutic protocols for corneal diseases, particularly, in infants or children, where an early restoration of sight is critically important in preventing amblyopia or permanent vision loss.