The Maintenance of Lymphatic Vessels in the Cornea Is Dependent on the Presence of Macrophages

Purpose.: It has been shown previously that the presence in the cornea of antigen-presenting cells (APC), such as macrophages (MPS) and lymphangiogenesis, is a risk for corneal transplantation. We sought to determine whether the existence of lymphatic vessels in the non-inflamed cornea is associated with the presence of MPS. Methods.: Flat mounts were prepared from corneas of untreated C57BL/6, CD11b−/−, F4/80−/−, and BALB/c mice, and after suture placement or corneal transplantation, observed by immunofluorescence for the presence of lymphatic vessels using LYVE-1 as a marker of lymphatic endothelium. Innate immune cells were detected in normal mouse corneas using CD11b, F4/80, CD40, as well as MHC-class II. Digital images of the flat mounts were taken using a spot image analysis system, and the area covered by lymphatic vessels was measured using NIH Image software. Results.: The number of spontaneous lymphatic vessels in C57BL/6 corneas was significantly greater than in BALB/c corneas (P = 0.03). There were more CD11b+ (P < 0.01) and CD40+, MHC-class II (+) cells in the C57BL/6 corneas than in BALB/c mouse corneas. MPS depletion via clodronate liposome in C57BL/6 mice led to fewer spontaneous lymphatic vessels and reduced inflammation-induced lymphangiogenesis relative to control mice. Mice deficient in CD11b or F4/80 had fewer spontaneous lymphatic vessels and less lymphangiogenesis than control C57BL/6 mice. Conclusions.: C57BL/6 mouse corneas have more endogenous CD11b+ cells and lymphatic vessels. The endogenous lymphatic vessels, along with pro-inflammatory MPS, account for the high risk of corneal graft rejection in C57BL/6 mice. CD11b+ and F4/80+ MPS appear to have an important role in of the formation of new lymphatic vessels

Rare Histological Type of Adenoma of the Nonpigmented Ciliary Epithelium

We report the rare case of an adenoma of the nonpigmented ciliary epithelium (NPCE). A 67-year-old healthy man presented with a regularly shaped and nonpigmented mass at the iris root of his right eye. His best-corrected visual acuity was 1.5 with normal intraocular pressure. During observation, the size of the tumor remained stable for 1.5 years but then rapidly grew, extending through the iris, and gradually enlarged to the point of compressing the iris. Ultimately, an iridocyclectomy with scleral resection under a lamellar scleral flap was performed. The histopathologic features of the resected tissue were consistent with adenoma of the NPCE. Histopathological analysis showed that the tumor consisted of both tubular and solid components. There were solid lesions inside of the ciliary epithelium and tubular lesions outside. We observed positive immunoreactivity to vimentin and cytokeratin CK (AE1/AE3) and negative reactivity to S-100 and CD68, both rarely associated with adenoma of NPCE. During 1 year of follow-up after the iridocyclectomy, no signs of tumor recurrence were observed

Intracellular Thiol Redox Status Regulates Lymphangiogenesis and Dictates Corneal Limbal Graft Survival

Purpose.: Compounds regulating intracellular thiol redox status, such as N,N-diacetyl-L-cystine dimethylester (NM2), were shown to prolong corneal graft survival in a penetrating keratoplasty (PKP) model. However, the effect of NM2 on hemangiogenesis and lymphangiogenesis has not been investigated. The effect of manipulating ambient thiol redox status on riskier (higher rejection rate) transplantation models, such as limbal graft survival and hemangiogenesis and lymphangiogenesis in a corneal suture model, were investigated. Methods.: C57BL/10 mice that received BALB/c corneas were treated by subconjunctival injection of NM2, and limbal graft survival was assessed. Sutured C57BL/6 received daily intraperitoneal injections of NM2, glutathione diethylester (GSH-OEt), or PBS. Lymphatic endothelial cell (LEC) and peritoneal mps were treated with NM2 or GSHOEt, and then VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression were analyzed. Supernatants were collected for analysis of TNF-α and VEGF-A levels by ELISA. Results.: Significantly less cellular infiltration was detected in mice with corneal limbal transplant–treated NM2-treated mice. Hemangiogenesis and lymphangiogenesis were suppressed in the NM2-treated mice. NM2 treatment of mps led to reduced levels of VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression compared with PBS- or GSHOEt- treated mps, lower levels of TNF-α, and increased secretion of VEGF. Moreover, NM2-treated LECs had reduced levels of LYVE-1 and Prox-1. Conclusions.: Reduction of ambient redox status reduced inflammatory cell infiltrates. Consequently, reduced inflammatory response might have contributed to both the observed prolonged corneal limbal graft survival and the attenuated hemangiogenesis and lymphangiogenesis in cornea

Thrombospondin 1 inhibits inflammatory lymphangiogenesis by CD36 ligation on monocytes

Lymphangiogenesis plays an important role in tumor metastasis and transplant outcome. Here, we show that thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein and naturally occurring inhibitor of angiogenesis inhibits lymphangiogenesis in mice. Compared with wild-type mice, 6-mo-old TSP-1–deficient mice develop increased spontaneous corneal lymphangiogenesis. Similarly, in a model of inflammation-induced corneal neovascularization, young TSP-1–deficient mice develop exacerbated lymphangiogenesis, which can be reversed by topical application of recombinant human TSP-1. Such increased corneal lymphangiogenesis is also detected in mice lacking CD36, a receptor for TSP-1. In these mice, repopulation of corneal macrophages with predominantly WT mice via bone marrow reconstitution ameliorates their prolymphangiogenic phenotype. In vitro, exposure of WT macrophages to TSP-1 suppresses expression of lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and VEGF-D, but not of a primarily hemangiogenic factor VEGF-A. Inhibition of VEGF-C is not detected in the absence or blockade of CD36. These findings suggest that TSP-1, by ligating CD36 on monocytic cells, acts as an endogenous inhibitor of lymphangiogenesis

Mitochondrial DNA as a biomarker for acute central serous chorioretinopathy: A case-control study

The literature suggests that stress may play a pivotal role in the precipitation of acute central serous chorioretinopathy (CSC) because chorioretinal integrity can be affected by the psychosocial state of the patient, indicating the need for a biomarker. Not only physical stress but also psychological stress causes many types of physical disorders. However, little is known about the pathophysiology of stress-induced disease. The objective of this study was to investigate whether serum factors might be involved in the development of stress-induced ocular diseases. Methods: This observational case series included 33 eyes of 33 consecutive patients with treatment-naïve acute CSC. Fifty eyes of 50 age-matched healthy volunteers were included in this study as non-CSC controls. Serum samples were collected from all participants, and the levels of mitochondrial DNA (mtDNA) were measured by quantitative real-time (RT)-PCR. Serum levels of high-mobility group box (HMGB) 1 and 8-hydroxy-2′-deoxyguanosine (8-OHdG), biological markers of acute/chronic inflammation and oxidative stress, were also measured. The relationships between serum mtDNA, 8-OHdG, and HMGB1 concentrations were investigated by multivariate regression analysis, alongside an assessment of clinical data. Results: In the treatment-naïve acute CSC group, the serum mtDNA levels (36.5 ± 32.4 ng/mL) were significantly higher than the levels in the control group (7.4 ± 5.9 ng/mL; p < 0.001). Serum levels of 8-OHdG and HMGB1 in treatment-naïve acute CSC patients measured 0.12 ± 0.08 ng/mL and 18.1 ± 35.0 ng/mL, respectively, indicating that HMGB1 levels were elevated in CSC compared with the control group. Multivariable regression analysis demonstrated that increased serum mtDNA levels were significantly associated with the height of serous retinal detachment. Conclusion: We showed serum mtDNA and HMGB1 level elevation and its relation to the clinical activities of CSC, indicating that serum mtDNA and HMGB1 could serve as biomarkers for the acute phase of the disease. The use of these biomarkers makes it possible to predict disease onset and determine disease severity

PAX6-positive microglia evolve locally in hiPSC-derived ocular organoids

Microglia are the resident immune cells of the central nervous system (CNS). They govern the immunogenicity of the retina, which is considered to be part of the CNS; however, it is not known how microglia develop in the eye. Here, we studied human-induced pluripotent stem cells (hiPSCs) that had been expanded into a self-formed ectodermal autonomous multi-zone (SEAM) of cells that partially mimics human eye development. Our results indicated that microglia-like cells, which have characteristics of yolk-sac-like linage cells, naturally develop in 2D eye-like SEAM organoids, which lack any vascular components. These cells are unique in that they are paired box protein 6 (PAX6)-positive, yet they possess some characteristics of mesoderm. Collectively, the data support the notion of the existence of an isolated, locally developing immune system in the eye, which is independent of the body’s vasculature and general immune system

Inhibition of Hemangiogenesis and LymphangiogenesisafterNormal-Risk Corneal Transplantation by Neutralizing VEGF Promotes Graft Survival

purpose. To evaluate the occurrence and time course of hem- and lymphangiogenesis after normal-risk corneal transplantation in the mouse model and to test whether pharmacologic strategies inhibiting both processes improve long-term graft survival. methods. Normal-risk allogeneic (C57BL/6 to BALB/c) and syngeneic (BALB/c to BALB/c) corneal transplantations were performed and occurrence and time course of hem- and lymphangiogenesis after keratoplasty was observed, by using double immunofluorescence of corneal flatmounts (with CD31 as a panendothelial and LYVE-1 as a lymphatic vascular endothelium–specific marker). A molecular trap designed to eliminate VEGF-A (VEGF TrapR1R2; 12.5 mg/kg) was tested for its ability to inhibit both processes after keratoplasty and to promote long-term graft survival (intraperitoneal injections on the day of surgery and 3, 7, and 14 days later). results. No blood or lymph vessels were detectable immediately after normal-risk transplantation in either donor or host cornea, but hem- and lymphangiogenesis were clearly visible at day 3 after transplantation. Both vessel types reached donor tissue at 1 week after allografting and similarly after syngeneic grafting. Early postoperative trapping of VEGF-A significantly reduced both hem- and lymphangiogenesis and significantly improved long-term graft survival (78% vs. 40%; P < 0.05). conclusions. There is concurrent, VEGF-A-dependent hem- and lymphangiogenesis after normal-risk keratoplasty within the preoperatively avascular recipient bed. Inhibition of hem- and lymphangiogenesis (afferent and efferent arm of an immune response) after normal-risk corneal transplantation improves long-term graft survival, establishing early postoperative hem- and lymphangiogenesis as novel risk factors for graft rejection even in low-risk eyes

Increased prostaglandin e2 has a positive correlation with plasma calcium during goldfish reproduction

We recently demonstrated that prostaglandin E2 PG¿ increases osteoclastic activity and induces bone resorption in both in vitro and in vivo experiments using goldfish. In the fish reproductive period, the plasma calcium (Ca) level in female teleosts increases remarkably to make vitellogenin, which is a major component of egg protein and a Ca-binding protein. In this period, however, there is no reported relationship between PGE2 and Ca metabolism in fish. To clarify the Ca metabolism in fish reproduction, we examined plasma PGE2 and Ca levels and measured tartrate-resistant acid phosphatase (TRAP) activities as an indicator of osteoclastic activity in goldfish. Plasma PGE2 levels in the reproductive stage significantly increased as compared with those in non-reproductive stages. Also, both plasma Ca and TRAP increased in the reproductive stage. Significant positive correlations were recognized between plasma Ca and the gonad somatic index (r=0.81, p<0.001), plasma Ca and plasma PGE2 levels (r=0.635, p<0.05), and plasma Ca and plasma TRAP activities (r=0.584, p<0.05) from the analysis using samples of both reproductive and nonreproductive stages. Taking these data into consideration, we suggested that PGE, acts on osteoclasts and increases plasma Ca as a result of osteoclastic bone resorption, and we concluded that PGE, is an important hormone in Ca metabolism during fish reproduction

Diagnosis of choroidal disease with deep learning-based image enhancement and volumetric quantification of optical coherence tomography

Purpose: The purpose of this study was to quantify choroidal vessels (CVs) in pathological eyes in three dimensions (3D) using optical coherence tomography (OCT) and a deep-learning analysis. Methods: A single-center retrospective study including 34 eyes of 34 patients (7 women and 27 men) with treatment-naïve central serous chorioretinopathy (CSC) and 33 eyes of 17 patients (7 women and 10 men) with Vogt-Koyanagi-Harada disease (VKH) or sympathetic ophthalmitis (SO) were imaged consecutively between October 2012 and May 2019 with a swept source OCT. Seventy-seven eyes of 39 age-matched volunteers (26 women and 13 men) with no sign of ocular pathology were imaged for comparison. Deep-learning-based image enhancement pipeline enabled CV segmentation and visualization in 3D, after which quantitative vessel volume maps were acquired to compare normal and diseased eyes and to track the clinical course of eyes in the disease group. Region-based vessel volumes and vessel indices were utilized for disease diagnosis. Results: OCT-based CV volume maps disclose regional CV changes in patients with CSC, VKH, or SO. Three metrics, (i) choroidal volume, (ii) CV volume, and (iii) CV index, exhibit high sensitivity and specificity in discriminating pathological choroids from healthy ones. Conclusions: The deep-learning analysis of OCT images described here provides a 3D visualization of the choroid, and allows quantification of features in the datasets to identify choroidal disease and distinguish between different diseases. Translational Relevance: This novel analysis can be applied retrospectively to existing OCT datasets, and it represents a significant advance toward the automated diagnosis of choroidal pathologies based on observations and quantifications of the vasculature

Thrombospondin 1 inhibits inflammatory lymphangiogenesis by CD36 ligation on monocytes

By engaging CD36 on murine macrophages, thrombospondin-1 prevents excessive macrophage VEGF-C production and corneal neovascularization