Lymphangiogenesis Guidance Mechanisms and Therapeutic Implications in Pathological States of the Cornea.

Corneal lymphangiogenesis is one component of the neovascularization observed in several inflammatory pathologies of the cornea including dry eye disease and corneal graft rejection. Following injury, corneal (lymph)angiogenic privilege is impaired, allowing ingrowth of blood and lymphatic vessels into the previously avascular cornea. While the mechanisms underlying pathological corneal hemangiogenesis have been well described, knowledge of the lymphangiogenesis guidance mechanisms in the cornea is relatively scarce. Various signaling pathways are involved in lymphangiogenesis guidance in general, each influencing one or multiple stages of lymphatic vessel development. Most endogenous factors that guide corneal lymphatic vessel growth or regression act via the vascular endothelial growth factor C signaling pathway, a central regulator of lymphangiogenesis. Several exogenous factors have recently been repurposed and shown to regulate corneal lymphangiogenesis, uncovering unique signaling pathways not previously known to influence lymphatic vessel guidance. A strong understanding of the relevant lymphangiogenesis guidance mechanisms can facilitate the development of targeted anti-lymphangiogenic therapeutics for corneal pathologies. In this review, we examine the current knowledge of lymphatic guidance cues, their regulation of inflammatory states in the cornea, and recently discovered anti-lymphangiogenic therapeutic modalities.

Therapeutic Strategies for Restoring Perturbed Corneal Epithelial Homeostasis in Limbal Stem Cell Deficiency: Current Trends and Future Directions.

Limbal stem cells constitute an important cell population required for regeneration of the corneal epithelium. If insults to limbal stem cells or their niche are sufficiently severe, a disease known as limbal stem cell deficiency occurs. In the absence of functioning limbal stem cells, vision-compromising conjunctivalization of the corneal epithelium occurs, leading to opacification, inflammation, neovascularization, and chronic scarring. Limbal stem cell transplantation is the standard treatment for unilateral cases of limbal stem cell deficiency, but bilateral cases require allogeneic transplantation. Herein we review the current therapeutic utilization of limbal stem cells. We also describe several limbal stem cell markers that impact their phenotype and function and discuss the possibility of modulating limbal stem cells and other sources of stem cells to facilitate the development of novel therapeutic interventions. We finally consider several hurdles for widespread adoption of these proposed methodologies and discuss how they can be overcome to realize vision-restoring interventions.

Lymphatic Vessel Regression and Its Therapeutic Applications: Learning From Principles of Blood Vessel Regression.

Aberrant lymphatic system function has been increasingly implicated in pathologies such as lymphedema, organ transplant rejection, cardiovascular disease, obesity, and neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. While some pathologies are exacerbated by lymphatic vessel regression and dysfunction, induced lymphatic regression could be therapeutically beneficial in others. Despite its importance, our understanding of lymphatic vessel regression is far behind that of blood vessel regression. Herein, we review the current understanding of blood vessel regression to identify several hallmarks of this phenomenon that can be extended to further our understanding of lymphatic vessel regression. We also summarize current research on lymphatic vessel regression and an array of research tools and models that can be utilized to advance this field. Additionally, we discuss the roles of lymphatic vessel regression and dysfunction in select pathologies, highlighting how an improved understanding of lymphatic vessel regression may yield therapeutic insights for these disease states.

Corneal lymphangiogenesis as a potential target in dry eye disease - a systematic review.

Dry eye disease (DED) is a common ocular surface condition causing symptoms of significant discomfort, visual disturbance, and pain. With recent advancements, DED has become recognized as a chronic self-perpetuating inflammatory condition triggered by various internal and environmental factors. DED has been shown to arise from the activation of both the innate and adaptive immune systems, leading to corneal epithelium and lacrimal gland dysfunction. While the cornea is normally avascular and thus imbued with angiogenic and lymphangiogenic privilege, various DED models have revealed activated corneal antigen-presenting cells in regional lymph nodes, suggesting the formation of new corneal lymphatic vessels in DED. The recent availability of reliable lymphatic cell surface markers such as LYVE-1 has made it possible to study lymphangiogenesis. Accordingly, numerous studies have been published within the last decade discussing the role of lymphangiogenesis in DED pathology. We systematically review the literature to identify and evaluate studies presenting data on corneal lymphangiogenesis in DED. There is considerable evidence supporting corneal lymphangiogenesis as a central mediator of DED pathogenesis. These findings suggest that anti-lymphangiogenic therapeutic strategies may be a viable option for the treatment of DED, a conclusion supported by the limited number of reported clinical trials examining anti-lymphangiogenic modalities in DED.

Simultaneous fluorescence imaging of distinct nerve and blood vessel patterns in dual Thy1-YFP and Flt1-DsRed transgenic mice.

Blood vessels and nerve tissues are critical to the development and functionality of many vital organs. However, little is currently known about their interdependency during development and after injury. In this study, dual fluorescence transgenic reporter mice were utilized to observe blood vessels and nervous tissues in organs postnatally. Thy1-YFP and Flt1-DsRed (TYFD) mice were interbred to achieve dual fluorescence in the offspring, with Thy1-YFP yellow fluorescence expressed primarily in nerves, and Flt1-DsRed fluorescence expressed selectively in blood vessels. Using this dual fluorescent mouse strain, we were able to visualize the networks of nervous and vascular tissue simultaneously in various organ systems both in the physiological state and after injury. Using ex vivo high-resolution imaging in this dual fluorescent strain, we characterized the organizational patterns of both nervous and vascular systems in a diverse set of organs and tissues. In the cornea, we also observed the dynamic patterns of nerve and blood vessel networks following epithelial debridement injury. These findings highlight the versatility of this dual fluorescent strain for characterizing the relationship between nerve and blood vessel growth and organization.

Proteomics-Based Characterization of the Effects of MMP14 on the Protein Content of Exosomes from Corneal Fibroblasts.

BACKGROUND: Exosomes secreted by corneal fibroblasts contain matrix metalloproteinase (MMP) 14, which is known to influence pro-MMP2 accumulation on exosomes. Accordingly, we hypothesized that the enzymatic activity of MMP14 may alter the protein content of corneal fibroblast- secreted exosomes. OBJECTIVE: The aim of this study was to investigate the effects of MMP14 on the composition and biological activity of corneal fibroblast-derived exosomes. METHODS: Knock out of the catalytic domain (ΔExon4) of MMP14 in corneal fibroblasts was used to determine the effect of MMP14 expression on the characteristics of fibroblast-secreted exosomes. The amount of secreted proteins and their size distribution were measured using Nano Tracking Analysis. Proteins within exosomes from wild-type (WT) and ΔExon4-deficient fibroblasts were identified by liquid chromatography-tandem mass spectrometry (MS/MS) proteomics analysis. The proteolytic effects of MMP14 were evaluated in vitro via MS identification of eliminated proteins. The biological functions of MMP14-carrying exosomes were investigated via fusion to endothelial cells and flow cytometric assays. RESULTS: Exosomes isolated from WT and ΔExon4-deficient fibroblasts exhibited similar size distributions and morphologies, although WT fibroblasts secreted a greater amount of exosomes. The protein content, however, was higher in ΔExon4-deficient fibroblast-derived exosomes than in WT fibroblast-derived exosomes. Proteomics analysis revealed that WT-derived exosomes included proteins that regulated cell migration, and ΔExon4 fibroblast-derived exosomes contained additional proteins that were cleaved by MMP14. CONCLUSION: Our findings suggest that MMP14 expression influences the protein composition of exosomes secreted by corneal fibroblasts, and through those biological components, MMP14 in corneal fibroblasts derived-exosomes may regulate corneal angiogenesis.

Transgenic models for investigating the nervous system: Currently available neurofluorescent reporters and potential neuronal markers.

Recombinant DNA technologies have enabled the development of transgenic animal models for use in studying a myriad of diseases and biological states. By placing fluorescent reporters under the direct regulation of the promoter region of specific marker proteins, these models can localize and characterize very specific cell types. One important application of transgenic species is the study of the cytoarchitecture of the nervous system. Neurofluorescent reporters can be used to study the structural patterns of nerves in the central or peripheral nervous system in vivo, as well as phenomena involving embryologic or adult neurogenesis, injury, degeneration, and recovery. Furthermore, crucial molecular factors can also be screened via the transgenic approach, which may eventually play a major role in the development of therapeutic strategies against diseases like Alzheimer's or Parkinson's. This review describes currently available reporters and their uses in the literature as well as potential neural markers that can be leveraged to create additional, robust transgenic models for future studies.

Quantification of Angiogenesis and Lymphangiogenesis in the Dual ex vivo Aortic and Thoracic Duct Assay.

BACKGROUND: Lymphatic vessel formation (lymphangiogenesis) plays important roles in cancer metastasis, organ rejection, and lymphedema, but the underlying molecular events remain unclear. Furthermore, despite significant overlap in the molecular families involved in angiogenesis and lymphangiogenesis, little is known about the crosstalk between these processes. The ex vivo aortic ring assay and lymphatic ring assay have enabled detailed studies of vessel sprouting, but harvesting and imaging clear thoracic duct samples remain challenging. Here we present a modified ex vivo dual aortic ring and thoracic duct assay using tissues from dual fluorescence reporter Prox1- GFP/Flt1-DsRed (PGFD) mice, which permit simultaneous visualization of blood and lymphatic endothelial cells. OBJECTIVE: To characterize the concurrent sprouting of intrinsically fluorescent blood and lymphatic vessels from harvested aorta and thoracic duct samples. METHODS: Dual aorta and thoracic duct specimens were harvested from PGFD mice, grown in six types of endothelial cell growth media (one control, five that each lack a specific growth factor), and visualized by confocal fluorescence microscopy. Linear mixed models were used to compare the extent of vessel growth and sprouting over a 28-day period. RESULTS: Angiogenesis occurred prior to lymphangiogenesis in our assay. The control medium generally induced superior growth of both vessel types compared with the different modified media formulations. The greatest decrease in lymphangiogenesis was observed in vascular endothelial growth factor-C (VEGF-C)-devoid medium, suggesting the importance of VEGF-C in lymphangiogenesis. CONCLUSION: The modified ex vivo dual aortic ring and thoracic duct assay represents a powerful tool for studying angiogenesis and lymphangiogenesis in concert.

Application of corneal injury models in dual fluorescent reporter transgenic mice to understand the roles of the cornea and limbus in angiogenic and lymphangiogenic privilege.

The role of the corneal epithelium and limbus in corneal avascularity and pathological neovascularization (NV) is not well understood. To investigate the contributions of the corneal and limbal epithelia in angiogenic and lymphangiogenic privilege, we designed five injury models involving debridement of different portions of the cornea and limbus and applied them to the dual-fluorescence reporter Prox1-GFP/Flt1-DsRed mouse, which permits in vivo imaging of blood and lymphatic vessels via fluorescence microscopy. Debridement of the whole cornea resulted in significant hemangiogenesis (HA) and lymphangiogenesis (LA), while that of the whole limbus yielded minimal corneal HA or LA. Following hemilimbal plus whole corneal debridement, corneal NV occurred only through the non-injured aspect of the limbus. Overall, these results suggest that the integrity of the corneal epithelium is important for (lymph)angiogenic privilege, whereas the limbus does not act as a physical or physiologic barrier to invading vessels. In CDh5-CreERT2VEGFR2lox/PGFD mice, conditional deletion of vascular endothelial growth factor receptor 2 in vascular endothelial cells abolished injury-induced HA and LA, demonstrating the utility of this transgenic mouse line for identifying important factors in the process of neovascularization.

MMP14-Containing Exosomes Cleave VEGFR1 and Promote VEGFA-Induced Migration and Proliferation of Vascular Endothelial Cells.

Purpose: Investigate the impact matrix metalloproteinase 14 (MMP14) delivered via exosomes produced by corneal fibroblasts on vascular endothelial growth factor receptor 1 (VEGFR1) cleavage on endothelial cells, and other key processes of angiogenesis. Methods: Proteolysis of VEGFR1 and R2 by the catalytic domain of MMP14 was investigated via immunocytochemistry with anti-VEGFR1, anti-VEGFR2, and anti-MMP14 antibodies. Exosomes were isolated via precipitation and serial ultracentrifugation from wild-type (WT) and MMP14 exon4-deficient corneal fibroblasts. Transmission electron microscopy and nanotracking analysis were used to characterize the isolated exosomes. The presence of MMP14 in exosomes from WT fibroblasts was confirmed by Western blotting. VEGFR1 cleavage upon treatment with WT-derived exosomes, Δexon4-derived exosomes, or the pan-MMP inhibitor GM60001 was examined via in vitro proteolysis analysis using recombinant mouse (rm) VEGFR1/R2. Endothelial cell migration and proliferation were investigated using a Boyden chamber assay and BrdU incorporation, respectively. Results: WT-derived exosomes specifically cleaved rmVEGFR1 in vitro, whereas Δexon4-derived exosomes did not. Treatment with the pan-MMP inhibitor GM6001 effectively inhibited VEGFR1 cleavage by WT-derived exosomes, confirming the role of MMP14 in this cleavage. WT-derived exosomes induced greater endothelial cell migration (P < 0.01) and proliferation (P < 0.5) compared to Δexon4-derived exosomes. Conclusions: MMP14-containing exosomes may be involved in the regulation of corneal neovascularization through degradation of VEGFR1 and VEGFA-induced endothelial cell proliferation and migration.

Fluorescent reporter transgenic mice for in vivo live imaging of angiogenesis and lymphangiogenesis.

The study of lymphangiogenesis is an emerging science that has revealed the lymphatic system as a central player in many pathological conditions including cancer metastasis, lymphedema, and organ graft rejection. A thorough understanding of the mechanisms of lymphatic growth will play a key role in the development of therapeutic strategies against these conditions. Despite the known potential of this field, the study of lymphatics has historically lagged behind that of hemangiogenesis. Until recently, significant strides in lymphatic studies were impeded by a lack of lymphatic-specific markers and suitable experimental models compared to those of the more immediately visible blood vasculature. Lymphangiogenesis has also been shown to be a key phenomenon in developmental biological processes, such as cell proliferation, guided migration, differentiation, and cell-to-cell communication, making lymphatic-specific visualization techniques highly desirable and desperately needed. Imaging modalities including immunohistochemistry and in situ hybridization are limited by the need to sacrifice animal models for tissue harvesting at every experimental time point. Moreover, the processes of mounting and staining harvested tissues may introduce artifacts that can confound results. These traditional methods for investigating lymphatic and blood vasculature are associated with several problems including animal variability (e.g., between mice) when replicating lymphatic growth environments and the cost concerns of prolonged, labor-intensive studies, all of which complicate the study of dynamic lymphatic processes. With the discovery of lymphatic-specific markers, researchers have been able to develop several lymphatic and blood vessel-specific, promoter-driven, fluorescent-reporter transgenic mice for visualization of lymphatics in vivo and in vitro. For instance, GFP, mOrange, tdTomato, and other fluorescent proteins can be expressed under control of a lymphatic-specific marker like Prospero-related homeobox 1 (Prox1), which is a highly conserved transcription factor for determining embryonic organogenesis in vertebrates that is implicated in lymphangiogenesis as well as several human cancers. Importantly, Prox1-null mouse embryos develop without lymphatic vessels. In human adults, Prox1 maintains lymphatic endothelial cells and upregulates proteins associated with lymphangiogenesis (e.g., VEGFR-3) and downregulates angiogenesis-associated gene expression (e.g., STAT6). To visualize lymphatic development in the context of angiogenesis, dual fluorescent-transgenic reporters, like Prox1-GFP/Flt1-DsRed mice, have been bred to characterize lymphatic and blood vessels simultaneously in vivo. In this review, we discuss the trends in lymphatic visualization and the potential usage of transgenic breeds in hemangiogenesis and lymphangiogenesis research to understand spatial and temporal correlations between vascular development and pathological progression.

Potential lymphangiogenesis therapies: Learning from current antiangiogenesis therapies-A review.

In recent years, lymphangiogenesis, the process of lymphatic vessel formation from existing lymph vessels, has been demonstrated to have a significant role in diverse pathologies, including cancer metastasis, organ graft rejection, and lymphedema. Our understanding of the mechanisms of lymphangiogenesis has advanced on the heels of studies demonstrating vascular endothelial growth factor C as a central pro-lymphangiogenic regulator and others identifying multiple lymphatic endothelial biomarkers. Despite these breakthroughs and a growing appreciation of the signaling events that govern the lymphangiogenic process, there are no FDA-approved drugs that target lymphangiogenesis. In this review, we reflect on the lessons available from the development of antiangiogenic therapies (26 FDA-approved drugs to date), review current lymphangiogenesis research including nanotechnology in therapeutic drug delivery and imaging, and discuss molecules in the lymphangiogenic pathway that are promising therapeutic targets.

Angiogenesis and lymphangiogenesis in corneal transplantation-A review.

Corneal transplantation has been proven effective for returning the gift of sight to those affected by corneal disorders such as opacity, injury, and infections that are a leading cause of blindness. Immune privilege plays an important role in the success of corneal transplantation procedures; however, immune rejection reactions do occur, and they, in conjunction with a shortage of corneal donor tissue, continue to pose major challenges. Corneal immune privilege is important to the success of corneal transplantation and closely related to the avascular nature of the cornea. Corneal avascularity may be disrupted by the processes of angiogenesis and lymphangiogenesis, and for this reason, these phenomena have been a focus of research in recent years. Through this research, therapies addressing certain rejection reactions related to angiogenesis have been developed and implemented. Corneal donor tissue shortages also have been addressed by the development of new materials to replace the human donor cornea. These advancements, along with other improvements in the corneal transplantation procedure, have contributed to an improved success rate for corneal transplantation. We summarize recent developments and improvements in corneal transplantation, including the current understanding of angiogenesis mechanisms, the anti-angiogenic and anti-lymphangiogenic factors identified to date, and the new materials being used. Additionally, we discuss future directions for research in corneal transplantation.

Prox1-GFP/Flt1-DsRed transgenic mice: an animal model for simultaneous live imaging of angiogenesis and lymphangiogenesis.

The roles of angiogenesis in development, health, and disease have been studied extensively; however, the studies related to lymphatic system are limited due to the difficulty in observing colorless lymphatic vessels. But recently, with the improved technique, the relative importance of the lymphatic system is just being revealed. We bred transgenic mice in which lymphatic endothelial cells express GFP (Prox1-GFP) with mice in which vascular endothelial cells express DsRed (Flt1-DsRed) to generate Prox1-GFP/Flt1-DsRed (PGFD) mice. The inherent fluorescence of blood and lymphatic vessels allows for direct visualization of blood and lymphatic vessels in various organs via confocal and two-photon microscopy and the formation, branching, and regression of both vessel types in the same live mouse cornea throughout an experimental time course. PGFD mice were bred with CDh5CreERT2 and VEGFR2lox knockout mice to examine specific knockouts. These studies showed a novel role for vascular endothelial cell VEGFR2 in regulating VEGFC-induced corneal lymphangiogenesis. Conditional deletion of vascular endothelial VEGFR2 abolished VEGFA- and VEGFC-induced corneal lymphangiogenesis. These results demonstrate the potential use of the PGFD mouse as a powerful animal model for studying angiogenesis and lymphangiogenesis.

Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization.

Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.

MMP14 Regulates VEGFR3 Expression on Corneal Epithelial Cells.

Vascular endothelial growth factor receptor 3 (VEGFR3) regulates the growth and differentiation of blood and lymphatic vessels. To determine whether matrix metalloproteinase 14 (MMP14) modulates VEGFR3 expression in the corneal epithelium to influence the avascularity of the cornea, VEGFR3 expression was compared between wild-type and MMP14-deficient (MMP14 Δexon4) corneal epithelial cells. Western blot analysis showed that VEGFR3 protein expression was higher on MMP14 Δexon4 corneal epithelial cells than on wild-type cells, and quantitative RT-PCR analysis showed that VEGFR3 gene expression was highly induced in MMP14 Δexon4 corneal epithelial cells but not in wild-type corneal epithelial cells or wild-type and MMP14 Δexon4 corneal keratocytes. Unlike in epithelial cells, MMP14 Δexon4 keratocytes did not express relatively higher levels of VEGFR3 than wild-type keratocytes. Interestingly, in vitro proteolysis experiments showed that MMP14 does not cleave VEGFR3 in vitro as it does VEGFR1, indicating that other genes may be involved in the modulation of VEGFR3 expression by MMP14. Using proteomic analysis to identify candidate factors, we found that 39 nuclear proteins were differentially expressed between wildtype and MMP14 Δexon4 corneal epithelial cells. These findings suggest that MMP14 may regulate VEGFR3 expression at the transcriptional level on corneal epithelial cells but not on corneal keratocytes.

Proangiogenic Interactions of Vascular Endothelial MMP14 With VEGF Receptor 1 in VEGFA-Mediated Corneal Angiogenesis.

PURPOSE: Matrix metalloproteinase 14 (MMP14) has been shown to be required for corneal angiogenesis. We hypothesized that the proangiogenic activity of MMP14 may be based on its selective binding to, and cleaving of, vascular endothelial growth factor receptor 1 (VEGFR1), but not VEGFR2 or VEGFR3. METHODS: Recombinant human (rh)VEGFR1, R2, and R3 were incubated with human MMP14, and the reaction mixtures were analyzed by SDS-PAGE and Coomassie blue staining. Surface plasmon resonance was used to determine the equilibrium constants (KD) for binding between MMP14 and VEGFA versus rhVEGFR1, R2, and R3. Extracellular signal-regulated kinase (ERK) phosphorylation was assayed in vascular endothelial cells after incubation with VEGF and various concentrations of MMP14. Ex vivo aortic ring tube formation assays and VEGFA micropocket corneal neovascularization assays were performed using Flk1Cre/Flk1mCherry/MMP14lox and Flk1mCherry/MMP14lox control mice. RESULTS: Maxtrix metalloproteinase 14 increased VEGFA-induced ERK phosphorylation in a time- and concentration-dependent manner in vascular endothelial cells. Aortic ring assays showed diminished vessel sprouting in vitro in response to VEGFA, but not to basic fibroblast growth factor, in mice with conditional deletion of vascular MMP14 (Flk1creMMP14lox) compared with that in MMP14lox control mice. In addition, diminished VEGFA-induced corneal angiogenesis was seen in flk1creMMP14lox mice compared with MMP14lox mice in vivo. CONCLUSIONS: Our findings indicate that VEGFR1 interaction with MMP14 and the enzymatic activity of MMP14 are necessary for VEGFA-induced angiogenesis. Additionally, selective cleavage of VEGFR1 by MMP14 may play an important role in VEGFA-induced corneal angiogenesis.

Limited versus total epithelial debridement ocular surface injury: Live fluorescence imaging of hemangiogenesis and lymphangiogenesis in Prox1-GFP/Flk1::Myr-mCherry mice.

BACKGROUND: Immunohistochemical staining experiments have shown that both hemangiogenesis and lymphangiogenesis occur following severe corneal and conjunctival injury and that the neovascularization of the cornea often has severe visual consequences. To better understand how hemangiogenesis and lymphangiogenesis are induced by different degrees of ocular injury, we investigated patterns of injury-induced corneal neovascularization in live Prox1-GFP/Flk1::myr-mCherry mice, in which blood and lymphatic vessels can be imaged simultaneously in vivo. METHODS: The eyes of Prox1-GFP/Flk1::myr-mCherry mice were injured according to four models based on epithelial debridement of the: A) central cornea (a 1.5-mm-diameter circle of tissue over the corneal apex), B) total cornea, C) bulbar conjunctiva, and D) cornea+bulbar conjunctiva. Corneal blood and lymphatic vessels were imaged on days 0, 3, 7, and 10 post-injury, and the percentages of the cornea containing blood and lymphatic vessels were calculated. RESULTS: Neither central corneal nor bulbar conjunctival debridement resulted in significant vessel growth in the mouse cornea, whereas total corneal and corneal+bulbar conjunctival debridement did. On day 10 in the central cornea, total cornea, bulbar conjunctiva, and corneal+bulbar conjunctival epithelial debridement models, the percentage of the corneal surface that was occupied by blood vessels (hemangiogenesis) was 1.9±0.8%, 7.14±2.4%, 2.29±1%, and 15.05±2.14%, respectively, and the percentage of the corneal surface that was occupied by lymphatic vessels (lymphangiogenesis) was 2.45±1.51%, 4.85±0.95%, 2.95±1.27%, and 4.15±3.85%, respectively. CONCLUSIONS: Substantial corneal debridement was required to induce corneal neovascularization in the mouse cornea, and the corneal epithelium may therefore be partially responsible for maintaining corneal avascularity. GENERAL SIGNIFICANCE: Our study demonstrates that GFP/Flk1::myr-mCherry mice are a useful model for studying coordinated hemangiogenic and lymphangiogenic responses.

Scanning Electron Microscopic Analysis of Biofilm Formation in Explanted Human Boston Type I Keratoprostheses.

PURPOSE: To describe the morphological distribution of host tissue and microbial biofilms on the intraocular surfaces of Boston type I keratoprostheses (KPros) explanted because of corneal melt. METHODS: Retrospective study of scanning electron microscopy (SEM) images from 4 explanted Boston type I KPros composed of polymethylmethacrylate and titanium. SEM images of KPro-associated ocular surfaces were reviewed for the presence of inflammatory cells, microbes, and/or biofilm formation. One sterile type I KPro was also imaged to serve as a (device only) control. RESULTS: All 4 KPros were explanted because of culture-negative, clinically "sterile" donor corneal melt with impending KPro extrusion. In all cases, the rough, irregular surfaces of the device harbored more adherent corneal epithelium and stromacytes, inflammatory cells, and bacteria than the smooth, polished surface of the KPro optic. Two KPros showed not only evidence of prior bacterial colonization but marked biofilm formation. CONCLUSIONS: SEM images of explanted KPros explanted for "sterile" corneal melt demonstrated evidence of biofilm formation despite negative donor corneal cultures and the absence of clinical suspicion for infection. These results suggest that "sterile" corneal melt may be due to inflammatory host responses to low microbial burdens as seen in biofilms and/or released antigens after antibiotic-induced lysis. There was increased adherence of host tissue cells and microbial biofilms on the nonpolished surfaces of the KPro. Polishing the intraocular polymethylmethacrylate and titanium KPro surfaces may decrease microbial adhesion and biofilm formation in human subjects with KPros, but what impact this will have on rates of postoperative endophthalmitis is unknown.

Understanding lymphangiogenesis in knockout models, the cornea, and ocular diseases for the development of therapeutic interventions.

A major focus of cancer research for several decades has been understand the ability of tumors to induce new blood vessel formation, a process known as angiogenesis. Unfortunately, only limited success has been achieved in the clinical application of angiogenesis inhibitors. We now know that lymphangiogenesis, the growth of lymphatic vessels, likely also plays a major role in tumor progression. Thus, therapeutic strategies targeting lymphangiogenesis or both lymphangiogenesis and angiogenesis may represent promising approaches for treating cancer and other diseases. Importantly, research progress toward understanding lymphangiogenesis is significantly behind that related to angiogenesis. A PubMed search of "angiogenesis" returns nearly 80,000 articles, whereas a search of "lymphangiogenesis" returns 2,635 articles. This stark contrast can be explained by the lack of molecular markers for identifying the invisible lymphatic vasculature that persisted until less than 2 decades ago, combined with the intensity of research interest in angiogenesis during the past half century. Still, significant strides have been made in developing strategies to modulate lymphangiogenesis, largely using ocular disease models. Here we review the current knowledge of lymphangiogenesis in the context of knockout models, ocular diseases, the biology of activators and inhibitors, and the potential for therapeutic interventions targeting this process.