Tan Truong

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.

Ocular surface health during 30-day continuous wear: rigid gas-permeable versus silicone hydrogel hyper-O2 transmitted contact lenses.

PURPOSE To determine the effects on corneal epithelial permeability and ocular response of 30 nights of continuous wear (CW) of gas permeable (GP) and silicone hydrogel (SiH) contact lenses. METHODS Ninety-one subjects successfully completed 30 days of CW of either GP (n = 42) or SiH (n = 49) contact lenses. Epithelial permeability (P(dc)) was measured by scanning fluorometer at an afternoon (PM) baseline session and again the next morning (AM). One randomly selected eye of each subject was patched overnight and the patch removed immediately before the AM visit. P(dc) measurements and ocular examinations were conducted at baseline and after 30 days of CW. RESULTS Epithelial permeability increased significantly after 30 days of CW in the patched eyes of the GP group (P = 0.022) and in the unpatched eyes of the SiH group (P = 0.004). The increase was driven primarily by the Asian subjects in each group (GP, P = 0.015; SiH, P = 0.001). There was no significant increase in either lens group in the non-Asian subjects. Multivariate models suggest that the change in AM P(dc) from baseline to 30 days of CW was also related to lens type (P = 0.035), time awake before measurement (P = 0.001), palpebral aperture size (P = 0.003), lens deposits (P = 0.020), and horizontal lens bearing (P = 0.003). CONCLUSIONS Subclinical increases in epithelial permeability can be caused by contact lens CW, despite the elimination of hypoxia. GP lenses permit recovery of the epithelium more quickly than do SiH lenses. Asians appear to be more susceptible to contact lens-induced epithelial changes than do non-Asians.

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.

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.

Integrin Alpha 9 Blockade Suppresses Lymphatic Valve Formation and Promotes Transplant Survival

Purpose The lymphatic pathway mediates transplant rejection. We recently reported that lymphatic vessels develop luminal valves in the cornea during lymphangiogenesis, and these valves express integrin alpha 9 (Itga-9) and play a critical role in directing lymph flow. In this study, we used an allogeneic corneal transplantation model to investigate whether Itga-9 blockade could suppress valvulogenesis after transplantation, and how this effect would influence the outcomes of the transplants. Methods Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice. The recipients were randomized to receive subconjunctival injections of either Itga-9 blocking antibody or isotype control twice a week for 8 weeks. Corneal grafts were assessed in vivo by ophthalmic slit-lamp biomicroscopy and analyzed using Kaplan-Meier survival curves. Additionally, whole-mount full-thickness corneas were evaluated ex vivo by immunofluorescent microscopy on both lymphatic vessels and valves. Results Anti–Itga-9 treatment suppressed lymphatic valvulogenesis after transplantation. Our treatment did not affect lymphatic vessel formation or their nasal polarized distribution in the cornea. More importantly, Itga-9 blockade led to a significant promotion of graft survival. Conclusions Lymphatic valvulogenesis is critically involved in transplant rejection. Itga-9 targeting may offer a new and effective strategy to interfere with the immune responses and promote graft survival.

Sox10+ Cells Contribute to Vascular Development in Multiple Organs—Brief Report

Objective— Previous genetic lineage tracing studies showed that Sox10+ cells differentiate into vascular mural cells, limited to neural crest–derived blood vessels in craniofacial tissues, aortic arch, pulmonary arch arteries, brachiocephalic, carotid arteries, and thymus. The purpose of this study was to investigate the contribution of Sox10+ cells to the vascular development in other tissues and organs and their relationship with neural crest. Approach and Results— Using genetic lineage tracing technique based on Cre/LoxP system, we examined blood vessels in the adult organs of the mice expressing Sox10-Cre/Rosa-LoxP-red fluorescent protein or Wnt1-Cre/Rosa-LoxP-red fluorescent protein by immunohistological analysis. In addition to previously reported tissues and organs derived from neural crest, we showed that Sox10+ cells also contributed to vascular mural cells in the lung, spleen, and kidney, which are derived from non-neural crest origin as evidenced by red fluorescent protein-negative blood vessels in these 3 organs of Wnt1-Cre/Rosa-LoxP-red fluorescent protein mice. Conclusions— This study demonstrates that Sox10+ cells contribute to pericytes and smooth muscle cells in most parts of the body, including those from neural crest and non-neural crest, which has significant implications in vascular remodeling under physiological and pathological conditions.