In order to study the pathophysiological alterations of the ciliary body (CB) during persistent hypotony, it is necessary to develop an animal model without CB injury. In this study, we successfully established a modified model of persistent hypotony without CB injury in New Zealand rabbits. A 23-gauge pars plana vitrectomy (PPV) was performed and a trocar-formed fistula was allowed to remain in situ, to produce a continuous outflow of intraocular fluid. Both eyes underwent PPV with normal intraocular pressure (IOP); eyes with no surgical intervention were used as controls. The IOP was monitored and used to evaluate the reliability of the model. Secondary changes of hypotony were evaluated by slit-lamp biomicroscopy and B scans while morphological changes of the CB were observed by haematoxylin and eosin staining. The mean IOP in the hypotony groups were consistently lower than 6 mmHg. Furthermore, there were no significant differences in IOP between the PPV control group and normal eyes. Collectively, our data indicate that this model successfully simulates the secondary changes of hypotony, including a reduction in corneal size, corneal oedema, anterior chamber inflammation, morphological alterations of the CB, cataract, retinal detachment, and choroidal detachment. The morphological structure of the CB tissue changed dramatically after persistent hypotony, indicating that normal IOP may be required in order to maintain normal function in the CB. This model of persistent hypotony potentially represents a valuable tool for future studies aiming to investigate the pathophysiological mechanisms underlying CB dysfunction and other secondary changes that occur during hypotony.

BACKGROUND:Angiogenesis is tightly linked to inflammation. Cytokines of interleukin 1 (IL-1) family are key mediators in modulating inflammatory responses.METHODS:In this study, we examined the role of IL-38, a member of the IL-1 family, in mediating inflammation-induced angiogenesis.RESULTS:The results showed that the angiogenesis was attenuated by topical administration of IL-38 to the injured corneas in a mouse model of alkali-induced corneal neovascularization (CNV). Further study showed that the expression of inflammatory cytokines TNF-α, IL-6, IL-8 and IL-1β was decreased in the IL-38-treated corneas. Moreover, the angiogenic activities including the proliferation, migration and tube formation of human retinal endothelial cells were reduced by IL-38 treatment in vitro.CONCLUSION:The data indicate that IL-38 modulates inflammation-induced angiogenesis.

Toll-like receptors (TLRs) play an important role in inflammatory and immunological responses, which are intimately related to neovascularization. However, the precise mode of action of TLR3 in neovascularization still remains ambiguous. In this study, we sought to investigate the role of TLR3 in pathological corneal neovascularization (CNV) using a mouse model of alkali-induced CNV. CNV was attenuated in TLR3-deficient mice, and the absence of TLR3 led to decreased production of stromal cell-derived factor 1 (SDF-1), a well-characterized cytokine that regulates the recruitment of endothelial progenitor cells (EPCs) to the sites of neo-angiogenic niches in the injured tissues. Topical administration of polyinosinic-polycytidylic acid [poly (I:C)], a synthetic ligand for TLR3, to the injured cornea promoted CNV in wild type (WT) mice but not in TLR3-deficient mice. In addition, the effect of poly (I:C) on WT mice was abolished by addition of SDF-1 receptor antagonist AMD 3100. Furthermore, poly (I:C) treatment in vitro enhanced the migration of EPCs, whereas the enhanced migration was abolished by AMD 3100. These results indicate an essential role of TLR3 signalling in CNV that involves up-regulating SDF-1 production and recruiting EPCs to the sites of injury for neovascularization. Thus, targeting the TLR3 signalling cascade may constitute a novel therapeutic approach for treating neovascularization-related diseases.

Pulmonary fibrosis (PF) is a fatal respiratory disease with no effective medical treatments available. TGF-β/Smads signaling has been implicated to play an essential in the pathogenesis of PF, in which Smad3 act as the integrator of pro-fibrosis signals. In this study, we determined the effect of SIS3, a specific inhibitor of Smad3, in an experimental mouse model of lung fibrosis. We observed that SIS3 treatment significantly reduced bleomycin (BLM)-induced pathological changes and collagen deposition in the lung as indicated by Masson staining, real-time PCR and hydroxyproline content assay. As expected, the levels of Smad3 phosphorylation were decreased in the lung of mice treated with SIS3. Furthermore, SIS3 treatment also suppressed BLM-induced infiltration of inflammatory cells in the lung. Taken together, our results suggest that SIS3 ameliorated BLM-induced PF in mouse lungs. Thus, targeting Smad3 with SIS3 may be an effective approach for treatment of fibrotic disorders.

In proliferative diabetic retinopathy (PDR), activated Müller glial cells (MGCs) exhibit increased motility and a fibroblast-like proliferation phenotype that contribute to the formation of fibrovascular membrane. In this study, we investigated the capacity of high glucose (HG) to regulate the expression of cell surface receptors that may participate in the proinflammatory responses of MGCs. We found that MGCs express a G-protein coupled chemoattractant receptor formyl peptide receptor 2 (Fpr2) and fibroblast growth factor receptor 1 (FGFR1), which mediated MGC migration and proliferation in response to corresponding ligands. HG upregulated Fpr2 through an NF-κB pathway in MGCs, increased the activation of MAPKs coupled to Fpr2 and FGFR1, which also further enhanced the production of vascular endothelial growth factor by MGCs in the presence of HG. In vivo, Fpr2 was more highly expressed by retina MGCs of diabetic mice and the human counterpart FPR2 was detected in the retina MGCs in fibrovascular membrane of PDR patients. To support the potential pathological relevance of Fpr2, an endogenous Fpr2 agonist cathelin-related antimicrobial peptide was detected in mouse MGCs and the retina, which was upregulated by HG. These results suggest that Fpr2, together with FGFR1, may actively participate in the pathogenesis of PDR thus may be considered as one of the potential therapeutic targets.

OBJECTIVE To explore the expression profile of angiogenic factors associated with proliferative diabetic retinopathy (PDR). METHODS Undiluted vitreous humor samples were obtained from 10 diabetic patients with PDR (10 eyes) and 9 non-diabtic patients (9 eyes). The concentrations of 60 angiogenic factors in the vitreous humor samples were measured by RayBio Angiogenic Cytokine Antibody Array. Some differentially expressed factors were further confirmed in vitreous humor by enzyme-linked immunosorbent assay (ELISA). RESULTS Compared with the non-diabetic controls, 20 differentially expressed factors with more than 1.50 fold changes were detected in patients with PDR. The median concentration of vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), angiopoietin (ANG)-1, ANG-2, urokinase-type plasminogen activator receptor (uPAR), Follistatin and matrix metalloproteinases 9 (MMP-9) was significantly increased in vitreous samples from PDR compared to controls (P < 0.05). However, (MCP)-1, Angiogenin and Leptin was significantly lower in PDR eyes compared to controls (P < 0.05). In the verification assay using ELISA, ANG-1, ANG-2, IL-6, VEGF, MMP-9, hepatocyte growth factor (HGF) and placenta growth factor (PIGF) concentration were increased in patients with PDR compared to controls (all P-values < 0.05). CONCLUSION This is the first report of a comprehensive multiplex analysis to identify angiogenic factors associated with PDR. These angiogenic factors may contribute to the pathogenesis of PDR and may be targets for therapeutic strategies of PDR.

Interleukin 38 (IL-38) is a novel identified cytokine of IL-1 family in which some members are important in inflammation and angiogenesis. However, the role of IL-38 in regulating angiogenesis is unknown. The aim of the present study is to explore the effect of IL-38 on angiogenesis. Oxygen-induced retinopathy (OIR) of C57BL/6 J mice was induced by exposure of hyperoxia (75% oxygen) from postnatal day 7 (P7) to P12 and then returned to room air. The mice were injected with IL-38. At P17, neovascular region (tufts) and avascular area of the retinas were analyzed. The data showed that administration of IL-38 in vivo inhibited retinal angiogenesis significantly. Furthermore, the addition of IL-38 to the cell cultures attenuated the proliferation, scratch wound healing and tube formation of vascular endothelial cells induced by VEGF significantly. Our findings suggest that IL-38 is an antiangiogenic cytokine in pathophysiological settings and may have therapeutic potential for angiogenesis related diseases.

IL-37 is a novel pro-angiogenic cytokine that potently promotes endothelial cell activation and pathological angiogenesis in our previous study, but the mechanisms behind the pro-angiogenic effect of IL-37 are less well understood. Extending our observations, we found that TGF-β interacts with IL-37, and potently enhances the binding affinity of IL-37 to the ALK1 receptor complex, thus allowing IL-37 to signal through ALK1 to activate pro-angiogenic responses. We further show that TGF-β and ALK1 are required in IL-37 induced pro-angiogenic response in ECs and in the mouse model of Matrigel plug and oxygen-induced retinopathy. The result suggests that IL-37 induces pro-angiogenic responses through TGF-β, which may act as the bridging molecule that mediates IL-37 binding to the TGF-β receptor complex.

Listeria monocytogenes is a Gram-positive intracellular bacterium that was transmitted through contaminated food and causes sepsis and even death. IL-37 has been described as an important anti-inflammatory factor, but little is known about the function of IL-37 in host defense against Liseria monocytogenes (Lm) infection. In mice model of systemic infection, we found that mice treated with IL-37 were more sensitive to Lm infection compared with PBS-treated mice. This reduced resistance to Lm in IL-37-treated mice is accompanied with increased bacterial burden and liver damage. Serum levels of colony-stimulating factors were decreased in IL-37-treated mice. IL-37 treatment reduced bactericidal ability of bone marrow derived macrophages (BMDMs) in vitro, which contribute to the inability of IL-37-treated mice to combat Lm infection. Furthermore, increased apoptosis was observed in Lm-infected macrophages treated with IL-37. Increased macrophage apoptosis reduced percentage in liver macrophages was observed in IL-37-treated mice following Lm infection. These results indicate the negative regulatory effect of IL-37 on host resistance during immune defense against Lm.

Immune responses play a key role in the pathogenesis and progression of polypoidal choroidal vasculopath (PCV) and age-related macular degeneration (AMD). In this study, we determined the Th cell-associated immune responses by measuring the cytokine expression of peripheral blood mononuclear cells (PBMC) in both PCV and neovascular AMD (nAMD) patients.

PURPOSE:Allergic conjunctivitis (AC) is an immunoglobulin E (IgE)-mediated and helper T cell 2 (Th2)--cell-mediated disease characterized by conjunctival eosinophilic infiltration. Previous study shows that IL-28A had anti-allergic activity in airway disease. In this study, we examined the effect of IL-28A on a mouse model of ovalbumin (OVA)-induced experimental allergic conjunctivitis (EAC).METHODS:Mouse EAC was induced by topical application of OVA after intraperitoneal (IP) sensitization with OVA in aluminum hydroxide (ALUM). Interleukin-28A was administered 1 hour before OVA challenge. Allergic conjunctivitis symptoms, eosinophil infiltration in the conjunctiva, antigen-specific IgE in the serum, and Th2 cytokine production by lymph node cells and splenocytes were subsequently analyzed.RESULTS:Topical application of IL-28A to OVA-induced EAC reduced clinical symptoms, serum OVA-specific IgE, and the infiltration of eosinophils in the conjunctiva. In addition, topical administration of IL-28A suppressed the expression of IL-4, IL-5, and IL-13 (Th2-type cytokine) but promoted the expression of IFN-γ (Th1-type cytokine) by splenocytes and cervical lymph node cells in EAC mice. Immunofluorescence staining showed decrease expression of IL-4 and IL-5 in IL-28A-treated EAC conjunctiva.CONCLUSIONS:Interleukin-28A shows therapeutic potential for allergic conjunctival inflammation.

Purpose. To investigate the effect of 2-Methoxyestradiol (2ME2) on experimental autoimmune uveitis (EAU) and the mechanism. Method. C57BL/6 male mice were used to establish the EAU model. 2ME2 was abdominal administrated in D0–D13, D0–D6, and D7–D13 and control group was given vehicle from D0–D13. At D14, pathological severity was scored. Lymphocyte reaction was measured using MTT assay. T cell differentiation in draining lymph nodes and eye-infiltrating cells was tested by flow cytometry. Proinflammatory cytokines production from lymphocytes was determined by ELISA. Result. The disease scores from 2ME2 D0–D13, 2ME2 D0–D6, 2ME2 D7–D13, and vehicle groups were 0.20±0.12, 1.42±0.24, 2.25±0.32, and 2.42±0.24. Cells from all 2ME2 treated groups responded weaker than control (p<0.05). The inhibitory effect of 2ME2 on lymphocyte proliferation attenuated from 2ME2 D0–D13 to 2ME2 D0–D6 and to 2ME2 D7–D13 groups (p<0.05). 2ME2 treated mice developed fewer Th1 and Th17 cells both in draining lymph nodes and in eyes than control (p<0.05). Lymphocytes from 2ME2 group secreted less IFN-γ and IL-17A than those from control (p<0.05). Conclusion. 2ME2 ameliorated EAU progression and presented a better effect at priming phase. The possible mechanism could be the inhibitory impact on IRBP specific lymphocyte proliferation and Th1 and Th17 cell differentiation.

PURPOSE:Interleukin-37 is suggested as a novel proangiogenic factor in our previous study. In this study, the role of IL-37 was investigated in proliferative diabetic retinopathy (PDR).METHODS:Vitreous fluids from 10 patients with PDR and 8 controls were collected. The levels of IL-37 were determined by ELISA and the relationship between IL-37 and VEGF-A/Ang-2 was analyzed. The effects of IL-37 on chorioretinal endothelial cell (RF/6A) proliferation, migration, and tube formation were determined by BrdU incorporation assay, Boyden chamber assay, scratch-wound assay and tube formation assay.RESULTS:The concentration of IL-37 in the PDR group was 95.09 ± 5.22 pg/mL and 34.91 ± 5.61 pg/mL in control group (P = 0.001). The level of IL-37 was highly related to the level of Ang-2 (P = 0.009, r = 0.772) and VEGF-A (P = 0.003, r = 0.827) in the PDR group, and VEGF expression in RF/6A cell was upregulated by IL-37 at low concentration. Interleukin-37 remarkably promoted RF/6A cell proliferation and migration. Interleukin-37 (1 ng/mL) remarkably stimulated tube formation with an increase of 85.3% for total tubule length and 74.1% for branching points compared with PBS control.CONCLUSIONS:The level of IL-37 is elevated in vitreous fluids of patients with PDR and correlates with the level of VEGF-A and Ang-2. Interleukin-37 stimulates proangiogenic response of retinal endothelial cells in vitro, suggesting the involvement of IL-37 in the pathogenesis of PDR.

Formyl-peptide receptors are a family of 7 transmembrane domain, Gi-protein-coupled receptors that possess multiple functions in many pathophysiologic processes because of their expression in a variety of cell types and their capacity to interact with a variety of structurally diverse, chemotactic ligands. Accumulating evidence demonstrates that formyl-peptide receptors are critical mediators of myeloid cell trafficking in the sequential chemotaxis signal relays in microbial infection, inflammation, and immune responses. Formyl-peptide receptors are also involved in the development and progression of cancer. In addition, one of the formyl-peptide receptor family members, Fpr2, is expressed by normal mouse-colon epithelial cells, mediates cell responses to microbial chemotactic agonists, participates in mucosal development and repair, and protects against inflammation-associated tumorigenesis. These novel discoveries greatly expanded the current understanding of the role of formyl-peptide receptors in host defense and as potential molecular targets for the development of therapeutics.

Dendritic cells (DCs) control immune responses and are central to the development of immune memory and tolerance. DCs initiate and orchestrate immune responses in a manner that depends on signals they receive from microbes and cellular environment. Although DCs consist mainly of bone marrow-derived and resident populations, a third tissue-derived population resides the spleen and lymph nodes (LNs), different subsets of tissue-derived DCs have been identified in the blood, spleen, lymph nodes, skin, lung, liver, gut and kidney to maintain the tolerance and control immune responses. Tissue-resident DCs express different receptors for microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs), which were activated to promote the production of pro- or anti-inflammatory cytokines. Malfunction of DCs contributes to diseases such as autoimmunity, allergy, and cancer. It is therefore important to update the knowledge about resident DC subsets and diseases associated with DC malfunction.

Background Hyperglycemia-induced inflammation causes the dysfunction of blood vessels, and Toll-like receptor 4 (TLR4) plays a key role in inflammation-induced angiogenesis. However, the impact of TLR4 on the pathogenesis of diabetic retinopathy (DR) is poorly understood. In this study, we examined the expression of TLR4 in retinal vascular endothelial cells of patients with DR and diabetic mice, and explored the role of TLR4 in mediating inflammatory responses by human microvascular endothelial cells (HMEC-1) under high-glucose condition. Methods The expression of TLR4 in retinal vascular endothelial cells of patients with proliferative diabetic retinopathy and diabetic mice induced by streptozotocin was examined using immunofluorescence. HMEC-1 cells were cultured and the expression of TLR4, MyD88 and Interleukin-1β (IL-1β) was examined under high-glucose condition. Endothelial cells with TLR4 silencing and antagonist of TLR4 as well as endothelial cells from TLR4 deficient mice were used to study the effect of activated TLR4 on inflammation induced by high-glucose treatment. Results We observed that TLR4 was detected in CD31-labled human retinal vascular endothelia and its expression was markedly increased in fibrovascular membranes from DR patients and in retinal vascular endothelial cells of diabetic mice. The expression of TLR4, MyD88 and IL-1β was enhanced by high glucose in cultured HMEC-1 and the expression of TLR4 and IL-1β was inhibited by TLR4 siRNA knock-down and TLR4 antagonist. The expression of IL-1β by endothelial cells from TLR4 deficient mice under high glucose condition was decreased. Conclusions Our results revealed that hyperglycemia induced overexpression and activation of TLR4 in endothelial cells. This effect may lead to inflammatory responses contribute to the pathogenesis of diabetic retinopathy.

Endothelial progenitor cells (EPCs) promote both physiological and pathological neovascularization. Recently we found high-mobility group box-1 (HMGB1)–Toll-like receptor 4 (TLR4) signaling pathway promotes corneal neovascularization (CNV) induced by alkali in a mouse model. However, it is still unclear whether HMGB1–TLR4 promotes the mobility of EPCs. In this study, we explored the role of HMGB1–TLR4 signaling in EPC recruitment by modulating the activity of HMGB1–TLR4 signaling in the corneas of alkali-induced CNV mouse model. The level of EPC recruitment in injured corneas, as detected by flow cytometry, is increased and reaches the peak level 4days after injury. Activating TLR4 with exogenous HMGB1 or LPS enhances the EPC recruitment, whereas inhibiting the activity of HMGB1 and TLR4 with A-box (selective HMGB1 antagonist) or LPS-RS (selective TLR4 antagonist), respectively, reverses this phenotype. Moreover, the TLR4 mediated EPC recruitment is associated with up-regulation of stromal cell-derived factor-1 (SDF-1), a pivotal cytokine in EPC mobilization. Activation of TLR4 or HMGB1 leads to increased SDF-1 expression, while blocking TLR4 or HMGB1 inhibits the expression of SDF-1. Topical administration of AMD-3100, an antagonist of SDF-1 receptor, suppresses the TLR4-mediated EPC recruitment and ameliorates CNV. Our results indicated that activation of HMGB1–TLR4 signaling pathway promotes EPC recruitment in CNV, at least in part through up-regulation of SDF-1.