DZ2002 alleviates corneal angiogenesis and inflammation in rodent models of dry eye disease via regulating STAT3-PI3K-Akt-NF-κB pathway.

Dry eye disease (DED) is a prevalent ocular disorder with a multifactorial etiology. The pre-angiogenic and pre-inflammatory milieu of the ocular surface plays a critical role in its pathogenesis. DZ2002 is a reversible type III S-adenosyl-L-homocysteine hydrolase (SAHH) inhibitor, which has shown excellent anti-inflammatory and immunosuppressive activities in vivo and in vitro. In this study, we evaluated the therapeutic potential of DZ2002 in rodent models of DED. SCOP-induced dry eye models were established in female rats and mice, while BAC-induced dry eye model was established in female rats. DZ2002 was administered as eye drops (0.25%, 1%) four times daily (20 µL per eye) for 7 or 14 consecutive days. We showed that topical application of DZ2002 concentration-dependently reduced corneal neovascularization and corneal opacity, as well as alleviated conjunctival irritation in both DED models. Furthermore, we observed that DZ2002 treatment decreased the expression of genes associated with angiogenesis and the levels of inflammation in the cornea and conjunctiva. Moreover, DZ2002 treatment in the BAC-induced DED model abolished the activation of the STAT3-PI3K-Akt-NF-κB pathways in corneal tissues. We also found that DZ2002 significantly inhibited the proliferation, migration, and tube formation of human umbilical endothelial cells (HUVECs) while downregulating the activation of the STAT3-PI3K-Akt-NF-κB pathway. These results suggest that DZ2002 exerts a therapeutic effect on corneal angiogenesis in DED, potentially by preventing the upregulation of the STAT3-PI3K-Akt-NF-κB pathways. Collectively, DZ2002 is a promising candidate for ophthalmic therapy, particularly in treating DED.

Promotion of corneal angiogenesis by sensory neuron-derived calcitonin gene-related peptide.

The normal cornea has no blood vessels but has abundant innervation. There is emerging evidence that sensory nerves, originated from the trigeminal ganglion (TG) neurons, play a key role in corneal angiogenesis. In the current study, we examined the role of TG sensory neuron-derived calcitonin gene-related peptide (CGRP) in promoting corneal neovascularization (CNV). We found that CGRP was expressed in the TG and cultured TG neurons. In the cornea, minimal CGRP mRNA was detected and CGRP immunohistochemical staining was exclusively co-localized with corneal nerves, suggesting corneal nerves are likely the source of CGRP in the cornea. In response to intrastromal suture placement and neovascularization in the cornea, CGRP expression was increased in the TG. In addition, we showed that CGRP was potently pro-angiogenic, leading to vascular endothelial cell (VEC) proliferation, migration, and tube formation in vitro and corneal hemangiogenesis and lymphangiogenesis in vivo. In a co-culture system of TG neurons and VEC, blocking CGRP signaling in the conditioned media of TG neurons led to decreased VEC migration and tube formation. More importantly, subconjunctival injection of a CGRP antagonist CGRP8-37 reduced suture-induced corneal hemangiogenesis and lymphangiogenesis in vivo. Taken together, our data suggest that TG sensory neuron and corneal nerve-derived CGRP promotes corneal angiogenesis.

Knockdown of lncRNA TUG1 suppresses corneal angiogenesis through regulating miR-505-3p/VEGFA.

OBJECTIVES: Vascular endothelial growth factor A (VEGFA) is one of the major factors initiating and regulating angiogenesis. LncRNA taurine up-regulated gene 1 (TUG1) has been implicated in the pathological neovascularization. The aim of this study is to explore the function of TUG1 in regulating VEGFA-mediated angiogenesis in endothelial cells. METHODS: A total of 12 corneal neovascularization (CRNV) samples were collected form patient undergoing corneal transplantation at Tongji Hospital, Wuhan, China. qRT-PCR and Western blotting were performed to examine gene expression and protein levels. Human umbilical vein endothelial cells (HUVECs) were used as an in vitro angiogenesis model. CCK-8 proliferation assay was used to determine cell proliferation capacity and wound healing was performed to analyze cell migration ability. Dual luciferase reporter assay was used for functional interaction validation between miR-505-3p and its targets. The in vitro angiogenic potential was evaluated by tube formation assay. RESULTS: TUG1 and VEGFA were upregulated in CRNV tissues and VEGFA-treated HUVECs. TUG1 knockdown inhibited proliferation, migration and tube formation capacity of HUVECs. TUG1 regulated the angiogenesis of HUVECs by modulating VEGFA expression through targeting miR-505-3p. CONCLUSIONS: Our results suggest that lncRNA TUG1 promotes the angiogenesis of HUVECs through modulating miR-505-3p/VEGFA axis.

Consensus guidelines for the use and interpretation of angiogenesis assays.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Animal models of ocular angiogenesis: from development to pathologies.

Pathological angiogenesis in the eye is an important feature in the pathophysiology of many vision-threatening diseases, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration, as well as corneal diseases with abnormal angiogenesis. Development of reproducible and reliable animal models of ocular angiogenesis has advanced our understanding of both the normal development and the pathobiology of ocular neovascularization. These models have also proven to be valuable experimental tools with which to easily evaluate potential antiangiogenic therapies beyond eye research. This review summarizes the current available animal models of ocular angiogenesis. Models of retinal and choroidal angiogenesis, including oxygen-induced retinopathy, laser-induced choroidal neovascularization, and transgenic mouse models with deficient or spontaneous retinal/choroidal neovascularization, as well as models with induced corneal angiogenesis, are widely used to investigate the molecular and cellular basis of angiogenic mechanisms. Theoretical concepts and experimental protocols of these models are outlined, as well as their advantages and potential limitations, which may help researchers choose the most suitable models for their investigative work.-Liu, C.-H., Wang, Z., Sun, Y., Chen, J. Animal models of ocular angiogenesis: from development to pathologies.

Effects of ranibizumab (Lucentis®) and bevacizumab (Avastin®) on human corneal endothelial cells.

BACKGROUND: Ingrowth of newly formed blood and lymph vessels (angiogenesis) from the limbus region into the cornea can be treated successfully by subconjunctival application of antiangiogenic agents. Currently, there are several angiogenesis inhibitors from various manufacturers available, such as vascular endothelial growth factor (VEGF) antibodies. The aim of the study was to investigate potential cytotoxic effects of two anti-VEGF agents, ranibizumab (Lucentis®) and bevacizumab (Avastin®) on the human corneal endothelium. METHODS: Human donor corneas, not suitable for corneal transplantation, were organ-cultured in the presence of either ranibizumab (Lucentis®) or bevacizumab (Avastin®) at different concentrations (group 1: 250 µg / ml, group 2: 25 µg / ml, group 3: 2.5 µg / ml) for a period of up to 4 weeks. Microscopic imaging for endothelial cell counting, detection of morphologic alterations of the endothelium, and molecular biology testing (Enzyme-linked Immunosorbent Assay [ELISA]) for metabolic changes was performed. RESULTS: Background-corrected results showed neither a significant lactate dehydrogenase (LDH) change with increasing culturing time nor a significant difference between ranibizumab (Lucentis®) and bevacizumab (Avastin®) treatment. The endothelial cell density revealed also no statistically significant difference between the two treatment groups with ranibizumab (Lucentis®) and bevacizumab (Avastin®) at all concentrations tested in this study. CONCLUSIONS: In this study, the anti-angiogenic agents ranibizumab (Lucentis®) and bevacizumab (Avastin®) demonstrated no cytotoxic effects on the corneal endothelium of human organ-cultured donor corneas over the limited study time period of 4 weeks. However, based on the study design (in-vitro) and the limited follow-up period, no conclusions on potential long-term effects can be drawn.

Efficacy and safety of combined UV-light corneal crosslinking and fine-needle diathermy to regress pathological murine corneal (lymph)angiogenesis in vivo.

PURPOSE: To compare safety and efficacy of isolated and combined UV-light corneal crosslinking (CXL) and fine-needle diathermy (FND) to regress pathological corneal vessels in vivo. METHODS: Mice with inflamed and pathologically vascularized corneas received CXL or FND as monotherapy or a combination of both treatments. Corneal pathological blood and lymphatic vessels, immune cells and the morphology of anterior segment structures were evaluated. RESULTS: All three approaches were able to regress blood and lymphatic vessels in mice. A comparative analysis of the three methods revealed that the FND monotherapy and the CXL + FND combination were significantly more effective than the CXL monotherapy, one and 2 weeks after therapy and especially in regressing lymphatic vessels. Furthermore, the combination therapy induced significantly less immune cell recruitment compared to the monotherapies. All three methods were safe to use in regards of corneal integrity. CONCLUSIONS: A combination of FND and CXL led to regression of pathological corneal lymphatic and blood vessels and reduced the infiltration of immune cells into inflamed murine corneas. This approach offers a new effective, safe and clinically usable strategy to treat eyes with mature pathological blood vessels and even more so for lymphatic vessels, for example prior to high-risk corneal transplantation.

Fine-needle diathermy for corneal vascularisation.

Background: Corneal angiogenesis occurs as a sequel to an insult and it brings with it cells that mediate immunity as well as repair and aids in flushing toxins out. These vessels are formed in haste and leak lipid and cells, ultimately resulting in loss of transparency, lipid keratopathy and immunogenicity. So, they may need treatment prior to an optical keratoplasty. Purpose: To demonstrate the procedure of Fine Needle Diathermy (FND) to treat corneal neovascularization, its indications and contraindications. Synopsis: FND uses coagulating current from a monopolar cautery unit to occlude the afferent and efferent blood vessels. FND works best at the stage of mature vessel formation. The needle is placed across a tuft of vessels or parallel to a single large vessel, being mindful of the depth and direction. FND is avoided in necrotic tissue where the blood vessel is needed for healing process. Occlusion of the vessel in these situations may result in tissue melt. Highlights: Corneal neovascularization follows the stages of latent phase, active neovascularization, mature vessel formation and then regression. The treatment modality depends on the stage of angiogenesis. FND works best for neovascularization due to infectious keratitis. Keratoplasty is best performed 3 to 4 months later when regression of corneal vascularization occurs. Video Link: https://youtu.be/2RK6d_a2Gdc.

Paxillin promotes the migration and angiogenesis of HUVECs and affects angiogenesis in the mouse cornea.

Neonatal vascular ophthalmopathy is a refractory ophthalmologic disease, and is a major cause of blindness. Occurrence of neonatal vascular ophthalmopathy may be associated with Paxillin, a cellular adhesion molecule which promotes the migration of endothelial cells and angiogenesis. To explore the role of PXN in corneal angiogenesis, human umbilical vein endothelial cells were divided into five groups: i) Control group; ii) Empty vector-transfected control group; iii) PXN knockdown group (shPXN group); iv) PXN-negative control (NC) group; and v) PXN over-expressed group (overExp group). PXN protein levels, migration and tube formation were assessed in the different experimental groups. Mice were divided into four groups: i) Control; ii) Model; iii) shPXN; and iv) overExp groups. Tube formation was significantly increased in the overExp group compared with the empty vector-transfected control group (P<0.01). Tube formation was significantly decreased in the shPXN group compared with the PXN-NC group (P<0.01). In mice, blood corpuscles were significantly decreased in the shPXN group. PXN promoted the migration of endothelial cells and corneal angiogenesis. The results of the present study suggest a role for PXN in corneal angiogenesis and provide a theoretical basis and potential target for the treatment of corneal angiogenesis.

MSCs inhibits the angiogenesis of HUVECs through the miR-211/Prox1 pathway.

The aim of this study was to investigate the effect of mesenchymal stem cells (MSCs) on the angiogenesis of human umbilical vein endothelial cells (HUVECs). MSCs were subconjunctival injected into rat corneal alkali burn models. Their impacts on the degree of corneal neovascularization (CNV) and corneal opacity were evaluated at 3, 6, 9 and 12 days after injection. An in vitro experiment of MSCs affecting HUVECs angiogenesis was performed and evaluated using the tube formation assay. The results showed that both CNV and corneal opacity were decreased in rats after MSCs injection. In HUVECs, angiogenesis of cells was inhibited by miR-211 overexpression. miR-211 negatively regulated Prox1 expression. Knockdown of miR-211 blocked the decrease of Prox1 expression induced by MSCs and the inhibitory effect of MSCs on the angiogenesis of HUVECs. The critical role of miR-211 in MSCs inhibition of corneal angiogenesis was confirmed in rat experiments. We concluded that MSCs inhibited the angiogenesis of HUVEC through miR-211 mediating the down-regulation of Prox1.

Short-term uvb-irradiation leads to putative limbal stem cell damage and niche cell-mediated upregulation of macrophage recruiting cytokines.

Ultraviolet light B (UVB)-irradiation is linked to various ocular pathologies such as limbal stem cell defects in pterygium. Despite the large circumstantial evidence linking UVB irradiation and limbal epithelial stem cell damage, the precise molecular responses of limbal stem cells to UVB irradiation are unclear. Here the effect of UVB irradiation on the putative stem cell phenotype, limbal niche cells and the subsequent effects on corneal (lymph)angiogenic privilege were investigated. Primary human limbal epithelial stem cells and fibroblasts were irradiated with 0.02 J/cm(2) of UVB, a low dose corresponding to 3 min of solar irradiation. UVB irradiation caused significant reduction of limbal epithelial and limbal fibroblast proliferation for 24 h, but apoptosis of limbal epithelial stem cells only. Moreover, UVB induced stem-like character loss of limbal epithelial cells, as their colony forming efficiency and putative stem cell marker expression significantly decreased. Interestingly, limbal epithelial cells co-cultured with UVB-irradiated limbal fibroblasts also exhibited loss of stem cell character and decrease of colony forming efficiency. Conditioned media from limbal epithelial cells inhibited lymphatic endothelial cell proliferation and tube network complexity; however this effect diminished following UVB irradiation. In contrast, pro-inflammatory and macrophage-recruiting cytokines such as TNFα, IFNγ and MCP1 were significantly upregulated following cell irradiation of limbal fibroblasts. These data demonstrate the key role of the limbal stem cell niche in response to UVB and subsequent (lymph)angiogenic and inflammatory events. These data suggest that the known pro(lymph)angiogenic effect of UVB irradiation in pterygium is not linked to a direct up-regulation of pro-angiogenic cytokines, but rather to indirect macrophage-recruiting cytokines being upregulated after UVB irradiation.

S100A proteins as molecular targets in the ocular surface inflammatory diseases.

The S100 proteins are calcium-binding proteins that are exclusively expressed in vertebrates, where they interact with enzymes, cytoskeletal proteins, receptors, transcription factors, and nucleic acids to regulate proliferation, differentiation, apoptosis, inflammation, cell migration, energy metabolism, and Ca(2+) homeostasis. In this review, we focus on the S100A8 and S100A9 members of the family that are involved in the regulation of neutrophil chemotaxis and inflammation related to ocular surface diseases such as dry eye, meibomian gland dysfunction, pterygium, and corneal neovascularization. In our previous studies, we have found that the levels of S100A8 and S100A9 were elevated in these inflammatory ocular diseases. For instance, S100A8 and A9 were found to be upregulated in pterygium tissues at both transcript and protein levels. These findings are consistent with the role of S100A8 and S100A9 proteins in activating the innate immune system in the eye via Toll-like receptors (TLRs) and altering the immune tolerance of the eye-associated lymphoid system. Recently, use of S100A8-targeting antibody has shown promising results in targeting corneal neovascularization. Injection of S100A8 has been shown to inhibit eosinophilic infiltration and thus may have potential therapeutic implications in allergic diseases.

Gamma knife radiosurgery inhibits angiogenesis of meningiomas: in vivo rat corneal assay.

OBJECTIVE: The aim of this study is to reveal inhibitory effect of gamma knife irradiation on angiogenesis of meningiomas using rat corneal angiogenesis assay. METHODS: A total of 72 rats were divided into three preliminary groups. Each group, consisting of 24 rats, was implanted to World Health Organization (WHO) grade I (typical), grade II (atypical), and grade III (malignant) meningioma. Each of these three preliminary groups of 24 rats, were then divided into four subgroups, each consisting of 6 rats and subsequently irradiated by gamma knife with dose prescriptions of 0, 14, 18, and 22 Gy. The numbers of vessels that developed around the micropockets of the corneas were counted and photographed on days 5, 10, 15, and 20. RESULTS: For WHO grade I meningiomas, 18 and 22 Gy doses (P < 0.001), and for grade II meningiomas, the 22-Gy (P = 0.021) dose were found to inhibit tumor-induced angiogenesis compared with the radiation-free control group. For grade III meningiomas, there was no statistical difference with the control group in any of the doses applied. Our findings demonstrate that gamma knife irradiation may suppress the angiogenic activity of WHO grades I and II meningiomas but not of the grade III meningiomas. CONCLUSIONS: For the first time, this study provides an experimental data to show the antiangiogenic effect of gamma knife irradiation on meningiomas.

Bevacizumab for ocular neovascular diseases: a systematic review / Bevacizumabe para doenças oculares neovasculares: revisão sistemática

CONTEXT AND OBJECTIVE: Many eye diseases involve increased local levels of vascular endothelial growth factor (VEGF), and there are several therapeutic strategies for them. Thus, the aim of this study was to evaluate the effectiveness and safety of bevacizumab for treating eye diseases involving increased local levels of VEGF, as the assumed pathophysiological mechanism. DATA SOURCES: The following databases were systematically searched for evidence: PubMed, CENTRAL (Cochrane Library), Literatura Latino-Americana e do Caribe em Ciências da Saúde (Lilacs) and reference lists, without language restrictions. Only randomized controlled trials were included. The primary outcome of interest was visual acuity, irrespective of the evaluation method. DATA SYNTHESIS: A total of 667 eyes in nine randomized trials were included. Meta-analysis showed that the proportion of patients with age-related macular degeneration who presented improvements from baseline regarding best-corrected visual acuity was higher among those treated with bevacizumab than among those in the photodynamic therapy group (risk ratio, RR, 0.49; 95 percent confidence interval, CI, 0.31 to 0.78; P = 0.01). CONCLUSIONS: The evidence available demonstrates that bevacizumab alone or combined with other treatments is more effective than other options, including photodynamic therapy, focal photocoagulation and triamcinolone. The use of bevacizumab instead of photodynamic therapy could reduce treatment costs by more than 99 percent and could significantly increase access to treatment. However, long-term studies are still needed in order to reduce uncertainty concerning the safety of this medication for all ocular neovascular diseases in which bevacizumab has the potential to improve visual acuity.

CONTEXTO E OBJETIVOS: Muitas doenças oculares envolvem o aumento dos níveis locais de fator de crescimento do endotélio vascular (FCEV), uma diversidade de estratégias terapêuticas para tais condições. Assim, o objetivo do presente estudo é avaliar a efetividade e a segurança de bevacizumabe para o tratamento de pacientes com doença ocular que envolva o aumento dos níveis locais de FCEV, como mecanismo patofisiológico assumido. FONTE DAS INFORMAÇÕES: Foi realizada busca sistemática pelas evidências disponíveis nas seguintes bases de dados da eletrônicas: PubMed, CENTRAL (The Cochrane Library), Literatura Latino-Americana e do Caribe em Ciências da Saúde (Lilacs), além de referências bibliográficas de estudos relevantes, sem restrições de língua. Foram incluídos apenas ensaios controlados e aleatórios. Acuidade visual, independentemente do método de avaliação, foi considerada o desfecho primário de interesse. SÍNTESE DOS DADOS: Foi incluído um total de 667 olhos testados em nove ensaios clínicos aleatórios. A metanálise demonstrou que a proporção de pacientes com degeneração macular relacionada à idade que melhoraram a acuidade visual foi maior entre os tratados com bevacizumabe do que entre os pacientes em terapia fotodinâmica (risco relativo [RR] 0.49, 95 por cento intervalo de confiança [IC] 0,31 a 0,78, P = 0,01). CONCLUSÕES: A evidência disponível demonstra que bevacizumabe isolado ou combinado com outras terapias é mais eficaz que terapia fotodinâmica, fotocoagulação focal e triancinolona. O uso de bevacizumabe em vez da terapia fotodinâmica poderia reduzir os custos do tratamento em mais de 99 por cento e aumentar significativamente o acesso ao tratamento. Entretanto, o aspecto de segurança do fármaco ainda necessita ser avaliado por estudos em longo prazo com todas as doenças neovasculares em que bevacizumabe tenha o potencial de melhorar acuidade visual.