Keratin 6a reorganization for ubiquitin-proteasomal processing is a direct antimicrobial response.

Skin and mucosal epithelia deploy antimicrobial peptides (AMPs) to eliminate harmful microbes. We reported that the intermediate filament keratin 6a (K6a) is constitutively processed into antimicrobial fragments in corneal epithelial cells. In this study, we show that K6a network remodeling is a host defense response that directly up-regulates production of keratin-derived AMPs (KAMPs) by the ubiquitin-proteasome system (UPS). Bacterial ligands trigger K6a phosphorylation at S19, S22, S37, and S60, leading to network disassembly. Mutagenic analysis of K6a confirmed that the site-specific phosphorylation augmented its solubility. K6a in the cytosol is ubiquitinated by cullin-RING E3 ligases for subsequent proteasomal processing. Without an appreciable increase in K6a gene expression and proteasome activity, a higher level of cytosolic K6a results in enhanced KAMP production. Although proteasome-mediated proteolysis is known to produce antigenic peptides in adaptive immunity, our findings demonstrate its new role in producing AMPs for innate immune defense. Manipulating K6a phosphorylation or UPS activity may provide opportunities to harness the innate immunity of epithelia against infection.

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.

MicroRNA-184 Regulates Corneal Lymphangiogenesis.

PURPOSE: MicroRNAs are a class of small noncoding RNAs that negatively regulate gene expression by binding to complimentary sequences of target messenger RNA. Their roles in corneal lymphangiogenesis are largely unknown. This study was to investigate the specific role of microRNA-184 (mir-184) in corneal lymphangiogenesis (LG) in vivo and lymphatic endothelial cells (LECs) in vitro. METHODS: Standard murine suture placement model was used to study the expressional change of mir-184 in corneal inflammatory LG and the effect of synthetic mir-184 mimic on this process. Additionally, a human LEC culture system was used to assess the effect of mir-184 overexpression on cell functions in vitro. RESULTS: Expression of mir-184 was significantly downregulated in corneal LG and, accordingly, its synthetic mimic suppressed corneal lymphatic growth in vivo. Furthermore, mir-184 overexpression in LECs inhibited their functions of adhesion, migration, and tube formation in vitro. CONCLUSIONS: These novel findings indicate that mir-184 is involved critically in LG and potentially could be used as an inhibitor of the process. Further investigation holds the promise for divulging new therapies for LG disorders, which occur inside and outside the eye.

Integrin Alpha-9 Mediates Lymphatic Valve Formation in Corneal Lymphangiogenesis.

PURPOSE: We recently reported that corneal lymphatic vessels develop integrin alpha-9 (Itga-9)-positive valves during inflammatory lymphangiogenesis. The purpose of this study was to further investigate the role of Itga-9 in corneal lymphatic valve formation in vivo and lymphatic endothelial cell (LEC) functions in vitro. METHODS: Standard murine suture placement model was used to study the effect of Itga-9 blockade on lymphatic valve formation in vivo using Itga-9 neutralizing antibody. Whole-mount corneas were harvested for immunofluorescent microscopic analysis. Additionally, human LEC culture system was used to examine the effect of Itga-9 gene knockdown on cell functions using small interfering RNAs (siRNAs). RESULTS: Itga-9 blockade in vivo significantly reduced the number of lymphatic valves formed in the inflamed cornea. Moreover, Itga-9 gene knockdown in human LECs suppresses cell functions of proliferation, adhesion, migration, and tube formation. CONCLUSIONS: Itga-9 is critically involved in corneal lymphatic valve formation. Further investigation of the Itga-9 pathway may provide novel strategies to treat lymphatic-related diseases occurring both inside and outside the eye.

Mutation of threonine 34 in mouse podoplanin-Fc reduces CLEC-2 binding and toxicity in vivo while retaining antilymphangiogenic activity.

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared with Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.

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.

Corneal lymphatic valve formation in relation to lymphangiogenesis.

PURPOSE: We have recently provided evidence showing that luminal lymphatic valves are formed right after the onset of corneal inflammatory lymphangiogenesis (LG). The purpose of this study was to further characterize the long-term time course, spatial distribution, directional orientation, and functional implications of the valve formation in relation to corneal LG. METHODS: Corneal LG was induced in normal adult BALB/c mice by a modified suture placement model with equal distribution in the nasal and temporal side. Whole-mount corneas were harvested every 2 weeks for up to 8 weeks post suturing for immunofluorescent microscopic assays. Quantitative analysis on both lymphatic vessels and valves was performed by using National Institutes of Health ImageJ software. Corneal lymphatic live imaging was performed to show functional drainage of the valves. RESULTS: Lymphatic vessel invasion areas at 4, 6, and 8 weeks were significantly less than the peak at 2 weeks post corneal suturing. In contrast, the ratio of lymphatic valves to vessel invasion area was at its lowest at 2 weeks with a peak approximately at 6 weeks post suturing. Lymphatic valves were more localized in the nasal quadrant at all time points studied, and most of the well-formed valves were directionally oriented toward the limbus. The lymphatic valves function to guide lymphatic drainage outside the cornea. CONCLUSIONS: This study presents new insights into corneal lymphatic valve formation and function in inflammatory LG. Further investigation on lymphatic valves may provide novel strategies to interfere with lymphatic maturation and function and to treat lymphatic-related disorders.

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.

Conjunctival lymphatic response to corneal inflammation in mice.

Due to its unique characteristics, the cornea has been widely used for vascular research. However, it has never been studied whether lymphatic vessels in the conjunctiva, its neighboring tissue, are affected by corneal lymphangiogenesis (LG). The purpose of this study was to investigate whether the distribution pattern of conjunctival lymphatic vessels changes during LG using a standardized two-suture placement model. Our data from immunofluorescent microscopic studies demonstrate, for the first time, that conjunctival lymphatic vessels were more distributed in the nasal side under both normal and inflamed conditions. Additionally, under the inflamed condition, conjunctival lymphatic vessels showed a higher density and more branching points, indicating that LG occurs in the conjunctiva in response to corneal inflammation. This study not only provides novel insights into lymphatic events in the ocular surface but also offers new guidelines for developing therapeutic strategies to treat lymphatic diseases at related sites.

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.

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.

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.

Very late antigen-1 mediates corneal lymphangiogenesis.

PURPOSE: To investigate the specific role of very late antigen-1 (VLA-1; also known as integrin α1ß1) 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.

A study on the functional interaction between the GH/PRL family of polypeptides with their receptors in zebrafish: Evidence against GHR1 being the receptor for somatolactin.

The growth hormone (GH)/prolactin (PRL) family of polypeptide hormones plays important roles in many aspects of vertebrate physiology. In fish, there is an additional member in this family called somatolactin (SL). Specifically, zebrafish contains five ligands (GH, SLα, SLß, PRL1 and PRL2) and four cognate receptors including two GH receptors (GHR1 and GHR2) and two PRL receptors (PRLR1 and PRLR2). There is much controversy regarding whether one of the two GHRs in teleosts is in fact the receptor of SL. A multitude of different assay methods were employed to study the functional interaction among these ligands and their receptors in zebrafish. These include assessment of the binding between the ligands and the extracellular domains of the receptors using His-tag pulldown assays, activation of receptor-evoked promoter activities by treatment of the receptor-transfected cells with the recombinant hormones, and phosphorylation of post-receptor signaling factors by treatment of receptor-transfected cells with the recombinant hormones. The results showed that the zebrafish GH can only interact with the GHRs and the zebrafish PRLs can only interact with the PRLRs. The zebrafish SLs, found to be biologically active in another assay, were found to be ineffective in interacting with the zebrafish GHRs and PRLRs. Our data argue against the hypothesis that GHR1 is the SL receptor.

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.

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.

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 alpha5 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.

Discovery of a novel prolactin in non-mammalian vertebrates: evolutionary perspectives and its involvement in teleost retina development.

BACKGROUND: The three pituitary hormones, viz. prolactin (PRL), growth hormone (GH) and somatolactin (SL), together with the mammalian placental lactogen (PL), constitute a gene family of hormones with similar gene structure and encoded protein sequences. These hormones are believed to have evolved from a common ancestral gene through several rounds of gene duplication and subsequent divergence. PRINCIPAL FINDINGS: In this study, we have identified a new PRL-like gene in non-mammalian vertebrates through bioinformatics and molecular cloning means. Phylogenetic analyses showed that this novel protein is homologous to the previously identified PRL. A receptor transactivation assay further showed that this novel protein could bind to PRL receptor to trigger the downstream post-receptor event, indicating that it is biologically active. In view of its close phylogenetic relationship with PRL and also its ability to activate PRL receptor, we name it as PRL2 and the previously identified PRL as PRL1. All the newly discovered PRL2 sequences possess three conserved disulfide linkages with the exception of the shark PRL2 which has only two. In sharp contrast to the classical PRL1 which is predominantly expressed in the pituitary, PRL2 was found to be mainly expressed in the eye and brain of the zebrafish but not in the pituitary. A largely reduced inner nuclear layer of the retina was observed after morpholino knockdown of zebrafish PRL2, indicating its role on retina development in teleost. SIGNIFICANCE: The discovery of this novel PRL has revitalized our understanding on the evolution of the GH/PRL/SL/PL gene family. Its unique expression and functions in the zebrafish eye also provide a new avenue of research on the neuroendocrine control of retina development in vertebrates.