A cyclic peptide significantly improves thyroid function, thyrotropin-receptor antibodies and orbital mucine /collagen content in a long-term Graves' disease mouse model.

BACKGROUND: BALB/c mice which received long-term immunizations of adenovirus (Ad) expressing thyrotropin receptor A-subunits (TSHR) developed stable Graves' disease (GD). TSHR-derived cyclic peptide 19 (P19) was identified as effective therapy in this model. METHODS: In Ad-TSHR mice, we investigated shorter disease intervals up to 4 months for histological alterations of the orbits, fine tuning of anti-TSHR antibodies (Ab) and free thyroxine (fT4) hormone levels by using novel detection methods in an independent laboratory. Therapy (0.3 mg/kg P19 or vehicle) was given intravenously after the fourth Ad-TSHR immunization (week 11) and continued until week 19. RESULTS: Thyrotropin binding inhibitory immunoglobulins (TBII, bridge immunoassay), blocking (TBAb) and stimulating (TSAb) TSHR-Ab (both cell-based bioassays) and serum levels of fT4 were significantly elevated at week 11 in Ad-TSHR-immunized mice versus none in control mice. For the first time, TSAb, TBAb, and thyroperoxidase-Ab were detected in 17 of 19, 12/19 and 6/19 Ad-TSHR immunized mice, respectively at week 21. Also, for the first time, this study showed that P19 treatment markedly reduced serum TBII (p < 0.0001), serum fT4 (p = 0.02), and acidic mucins and collagen content in the orbital tissue of Ad-TSHR-immunized mice. CONCLUSION: P19 significantly improved thyroid function, confirming previous results in an independent second laboratory. A relevant shift of anti-TSHR antibody subpopulations in response to P19 therapy may help explain its immunological effects. Moreover, P19 exerted a beneficial effect on mucine and collagen content of orbital tissue. Hence, P19 offers a potential novel therapeutic approach for GD and associated orbitopathy.

Prostaglandin F2α and EP2 agonists, and a ROCK inhibitor modulate the formation of 3D organoids of Grave's orbitopathy related human orbital fibroblasts.

3D organoid cultures were used to elucidate the periocular effects of several anti-glaucoma drugs including a prostaglandin F2α analogue (bimatoprost acid; BIM-A), EP2 agonist (omidenepag; OMD) or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (ripasudil; Rip) on Grave's orbitopathy (GO) related orbital fatty tissue. 3D organoids were prepared from GO related human orbital fibroblasts (GHOFs) obtained from patients with GO. The effects of either 100 nM BIM-A, 100 nM OMD or 10 µM Rip on the 3D GHOFs organoids were examined with respect to organoid size, physical properties by a micro-squeezer, and the mRNA expression of extracellular matrix (ECM) proteins including collagen (COL) 1, COL 4, COL 6, and fibronectin (FN), ECM regulatory genes including lysyl oxidase (LOX), Connective Tissue Growth Factor (CTGF) and inflammatory cytokines including interleukin-1ß (IL1ß) and interleukin-6 (IL6). The size of the 3D GHOFs organoids decreased substantially in the presence of BIM-A, but also increased substantially in the presence of the others (OMD or Rip). The physical stiffness of the 3D GHOFs organoids was significantly decreased by Rip. BIM-A caused significantly the down-regulation of three ECM genes, Col 1, Col 6 and Fn, and two ECM regulatory genes and the up-regulation of IL6. In the presence of OMD, two ECM genes, Col 1 and Fn, and LOX were significantly down-regulated but IL1ß and IL6 were significantly up-regulated. In the case of Rip, Col 1, FN and CTGF were significant down-regulated. Our present findings indicate that anti-glaucoma drugs modulate the structures and physical properties 3D GHOFs organoids in different manners by modifying the gene expressions of ECM, ECM regulatory factors and inflammatory cytokines. The results indicate that the benefits and demerits of anti-glaucoma medications need to be scrutinized carefully, in cases of patients with GO.

Effect of Prunella vulgaris polysaccharides on cultured orbit fibroblasts in vitro from patients with thyroid-associated ophthalmopathy.

To observe the effect of Prunella vulgaris polysaccharides (PVP) on cultured orbit fibroblasts in vitro from patients with thyroid-associated ophthalmopathy (TAO). PVP at different concentrations were used to treat different groups of fibroblasts from TAO patients and normal persons. Dexamethasone (Dex) was used as a positive control drug, and interferon-γ (IFN-γ) was used as a positive stimulant. The effects of PVP on the proliferation of orbital fibroblasts, the secretion of hyaluronic acid (HA), the expression of intercellular adhesion molecule-1 (ICAM-1/CD54) and apoptosis in orbital fibroblasts were determined. The experimental results showed when the concentration of PVP was greater than 400 µg/mL, it could significantly inhibit the proliferation of orbital fibroblasts from patients with TAO (P < 0.05). However, no definite inhibitory effect was observed in the orbital fibroblasts from the normal people. Dex could significantly inhibit the proliferation of orbital fibroblasts from patients with TAO and the normal people (P < 0.05). In contrast, every concentration of IFN-γ could promote the orbital fibroblasts from patients with TAO and the normal people proliferation. No groups had statistically significant stimulatory effect on HA secretion by orbital fibroblasts from normal people (P > 0.05). But the Dex group, IFN-γ+PVP-1600 group and IFN-γ+Dex group could significantly inhibit the secretion of HA from orbital fibroblasts of TAO patients. And there were no groups had statistically significant stimulatory effect on the expression of ICAM-1/CD54 in orbital fibroblasts from TAO patients (P > 0.05). PVP and Dex at all concentrations could significantly promote orbital fibroblast co-cultured with IFN-γ apoptosis (P < 0.05). But without IFN-γ, PVP and Dex at all concentrations could only significantly promote orbital fibroblast from TAO patients apoptosis (P < 0.05). These results suggest that PVP exerts its therapeutic effect by inhibiting the proliferation of orbital fibroblasts and promoting the apoptosis of orbital fibroblasts in TAO patients. In addition, in this process, HA secretion is suppressed. But the participation of IFN-γ is required. This effect is similar to that of Dex. And in the MTT experiment, the efficacy of PVP showed selectivity for TAO patients. This is different from Dex. This may be a feature of PVP that deserves attention.

Chitosan inhibits inflammation and adipogenesis of orbital fibroblasts in Graves ophthalmopathy.

Purpose: The aim of this study was to investigate the roles of chitosan in inflammation and adipogenesis of primary cultured orbital fibroblasts in Graves ophthalmopathy (GO). Methods: Cell viability, apoptosis, and cell cycle were determined with the Cell Counting Kit-8 (CCK-8), the Annexin V-FITC/PI kit, and flow cytometry, respectively. Inflammation of orbital fibroblasts was stimulated by interleukin-1 beta (IL-1ß). The levels of IL-6 and prostaglandin E-2 (PGE-2) were measured using an enzyme-linked immunosorbent assay (ELISA). The expression of cyclooxygenase-2 (COX-2) was measured with real-time PCR and western blot assay. Phosphorylation of c-Jun N-terminal kinase (JNK) was evaluated with western blot assay. An inhibitor of JNK was used to investigate the signal transduction pathway of cytokine production. Orbital fibroblasts differentiated to adipose cells in differentiation medium. Adipose cells were dyed with Oil Red O. FABP4, adiponectin, C/EBPα, PPAR-γ, and phosphorylation of AKT were evaluated with western blot assay. Results: The results showed that IL-1ß statistically significantly increased the expression of IL-6, PGE-2, and COX-2 in orbital fibroblasts. Phosphorylation of JNK was promoted by IL-1ß. IL-6 and PGE-2 were modulated by the JNK signaling pathway as determined with the inhibition experiments. Chitosan downregulated expression of IL-1ß-stimulated IL-6, COX-2, and PGE-2 and downregulated phosphorylation of JNK. Chitosan inhibited the production of adipose cells dyed by Oil Red O. Chitosan statistically significantly decreased the protein levels of FABP4, adiponectin, C/EBPα, and PPAR-γ with downregulation of AKT phosphorylation during adipocyte differentiation. Conclusions: Chitosan statistically significantly inhibits inflammation and adipogenesis, as well as related signaling pathways, of orbital fibroblasts in GO. This indicates a possible therapeutic effect of chitosan on Graves ophthalmopathy.

In Vivo Effects of Retrobulbar Bimatoprost Injection on Orbital Fat.

PURPOSE: Recent publications have reported the adverse effects of prostaglandin analogues on the periocular tissues. These medications may cause periorbital lipodystrophy, enophthalmos, and deepening of the superior sulcus deformity. While these effects may have adverse consequences for some patients, the atrophy of the periorbital fat may have a useful role in diseases that lead to orbital and periorbital fat hypertrophy such as thyroid eye disease. In this pilot study, the authors investigated the effects of retrobulbar bimatoprost injection on the intraocular pressure and orbital fat in a rat animal model. METHODS: Three rats were sedated and intraocular pressure was measured. A 0.1 ml aliquot of bimatoprost was injected into the right orbit of all rats. In the left orbit, 0.1 ml of phosphate-buffered saline was injected as a control. Three weeks later, all rats were sedated and intraocular pressure was measured before euthanizing. Routine histologic staining was performed and thin sections through the intraconal orbital fat were obtained. Density of intraconal adipocytes was measured and adipocyte heterogeneity was determined using a computer image analysis algorithm. RESULTS: The specimens injected with bimatoprost demonstrated atrophy of orbital fat with significantly increased adipocyte density (p = 0.009) and heterogeneity (p = 0.008) when compared with control. Intraocular pressure was not significantly decreased at 3 weeks after injection of retrobulbar bimatoprost. CONCLUSIONS: In this pilot study, orbital injection of bimatoprost demonstrated atrophy of intraconal adipocytes when compared with control orbits injected with saline. The orbits injected with bimatoprost were noted to have smaller, more heterogeneous adipocytes that were densely packed in the intraconal space. The study limitations include the small sample size, which limited the ability for us to make conclusions about the effect on intraocular pressure. Nevertheless, the findings presented suggest that retrobulbar bimatoprost may present a nonsurgical alternative to induce atrophy of the orbital fat without inducing inflammation or hypotony.

The effects of vascular endothelial growth factor (VEGF) on human orbital preadipocyte.

PURPOSE: To investigate the presence of the Vascular Endothelial Growth Factor (VEGF) and its receptor (VEGFR) in human orbital preadipocytes, and to evaluate the effect of VEGF on human orbital preadipocyte differentiation and adipogenesis in vitro. RESULTS: Four isoforms of VEGF (VEGF121, 155, 189, and 206), VEGFR-1, VEGF-2, and neuropilin-1 were expressed in human orbital preadipocytes. Treatment with 100 ng/ml VEGF induced higher expressions of C/EBPα and LPL than the non-treated control (p = 0.03 and p = 0.01) or treatment with 50ng/ml (p = 0.04 for both). At both concentrations VEGF enhanced the accumulation of intra-cytoplasmic lipid versus the control, and treatment with 100 ng/ml VEGF induced more lipid accumulation than treatment with 50 ng/ml VEGF (p = 0.03). CONCLUSIONS: VEGF and VEGFR were observed in human orbital preadipocytes, and exogenous VEGF enhanced adipogenesis in these cells. These results suggest that VEGF plays a role as an autocrine or paracrine growth factor during human orbital preadipocyte differentiation.

Hyaluronic Acid Gel Re-Injection for Enophthalmos Correction in Silent Sinus Syndrome.

A 43-year-old female with residual enophthalmos following functional endoscopic surgery (FESS) due to silent sinus syndrome (SSS) was initially successfully treated with a 2-ml intraorbital injection of hyaluronic acid gel (HAG). The enophthalmos partially recurred 22 months after the injection. HAG was re-injected with good functional and cosmetic results. Functional (kinetic) computed tomography was performed to visualize HAG distribution in the orbit.

Orbital MRI appearance after remote retrobulbar alcohol injection.

An 88-year-old woman with a blind painful OS underwent a retrobulbar alcohol injection. Nine months following the procedure, she was evaluated in the clinic for discomfort surrounding her left brow and forehead and was found to have dysesthesia in the left V1 nerve distribution. There was no evidence of orbital inflammation on history or ocular examination. An MRI of the orbits showed intraconal abnormal signal with enhancement and enophthalmos. The chronic effect of retrobulbar alcohol injection on the orbital contents may mimic orbital inflammatory disease on the MRI, but the absence of clinical correlates should allow for appropriate diagnosis.

Eighteen-point abobotulinum toxin a upper face rejuvenation: an eye plastic perspective on 845 subjects.

PURPOSE: To report the method and results of 18-point Abobotulinum toxin A (ABO-BTA, Dysport) upper face rejuvenation on 845 subjects. METHODS: In a retrospective chart review, all subjects (the first cycle of injection) with ABO-BTA upper face rejuvenation from 2003 to 2009 were included. Excluded were subjects with facial spastic disorders, injection after upper face lifting, and aberrant regeneration of facial nerves. Upper face rejuvenation included 18 points of injection at forehead creases (4), frown lines (5), bunny line (1), crow's feet (4 on both sides), and lower eyelid crease (4 on both sides). They were revisited in 10 to 14 days for assessment of the effects and possible touch-up injection. Demographics, year of injection, topical anesthetic usage, touch-up injection, and adverse effects (AE) were recorded. RESULTS: There were 845 subjects (85.8% women) whose age was below 40 in 49.3%. All but 68 (8%) were happy with the touch-up visit, 10 to 14 days after injection. Touch-up injection was performed in 8% of subjects mainly for the eyebrow asymmetry. AE (22/845, 2.6%) were bruise (15/845, 1.8%), blepharoptosis (3/845, 0.3%), remained eyebrow asymmetry after touch-up injection (2/845, 0.2%), and headache (2/845, 0.2%). They were significantly more in subjects with touch-up injection, younger than 40 years, and in the first and second year of experience (especially for the bruise). CONCLUSIONS: Eighteen-point ABO-BTA upper face rejuvenation had a low rate of AE in this series in which majority was bruise at the lateral canthal area. They were significantly more in the first years of experience, subjects younger than 40, and who had touch-up injections.

Calcium hydroxyl-apatite (Radiesse) for the correction of periorbital hollows, dark circles, and lower eyelid bags.

PURPOSE: To describe the authors experience with calcium hydroxyl-apatite (CaHa) injections for the aesthetic correction of tear trough, infraorbital hollows, deep upper sulcus, dark circles and lower eyelid bags. METHODS: The records of 63 patients (127 eyelids) injected with CaHa for aesthetic rejuvenation of the periocular region between March 2012 and March 2013 were retrospectively evaluated. All injections were carried out using a 25-gauge cannula after adding 0.5 ml of 2% lidocaine to 1.5 ml vials of the original product. Postoperative visits were scheduled at 1 week and 1 month. Any previous treatment was recorded, and necessity of retreatments and side effects was evaluated. Patient satisfaction was recorded at 1 month with self-evaluation of the treatment result as "worsened," "unchanged," or "improved." Standard pre- and postinjection photographs were taken and compared to analyze the success of the procedure. Pictures were retrospectively graded by the authors on a similar improvement scale of 1 (worse), 2 (no change), and 3 (improvement). RESULTS: Fifty-eight/sixty-three patients were women (92%), with an average age of 42 years (range; 18-57 years). Chief complaints were "hollows" in 94% of patients, "dark circles" in 33%, lower eyelid "bags" in 17%, and deep upper sulcus in 4.7%. Twenty-three patients (36.5%) required an additional correction 1 month after the primary treatment. Satisfaction was as high as 98% among patients treated primarily for hollowness, and the overall satisfaction rate was 92%. Associated dark circles were satisfactorily treated in 68% of the patients. Temporary side effects involved mild erythema and swelling for 2 to 3 days and pseudoxanthalesma effect in 22 eyelids (17.4%) lasting <6 weeks. In 2 patients, erythema lasted longer than 4 weeks. The 2 worse complications in this series were migration of the product above the medial canthal tendon in 1 patient and overcorrection in another patient. These complications were all managed conservatively and resolved spontaneously within 6 to 8 weeks. No case of irregular contour, palpable lumpiness, or unevenness were encountered. In the end, only 1 patient thought she was worsened after the treatment. CONCLUSIONS: Treatment of the periocular region with CaHa injections is a safe and effective treatment with high patient satisfaction and low complication rate. Advanced technical skills may have to be acquired for the specific treatment of this area using this particulate material.

Cyclosporin A inhibits PDGF-BB induced hyaluronan synthesis in orbital fibroblasts.

Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 µM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production.

Anatomical position of hyaluronic Acid gel following injection to the infraorbital hollows.

PURPOSE: To examine with histology the anatomical location of hyaluronic acid gel injected to the infraorbital hollows of cadaver specimens. METHODS: The authors dissected 5 fresh hemifacial cadaver specimens following preperiosteal injection of hyaluronic acid gel to the infraorbital hollows. Following tissue fixation, full-thickness soft tissue sections were obtained along the medial, central, and lateral lower eyelid/midface of each specimen. Histologic examination of the anatomical location of hyaluronic acid gel was performed using hematoxylin and eosin and Hale colloidal iron stains. RESULTS: Histologic examination of the central and lateral lower eyelid/midface sections revealed a significant portion of hyaluronic acid gel in either a postorbicularis or a subcutaneous plane in 8 of 10 sections. Only 2 sections displayed hyaluronic acid gel solely within a preperiosteal plane. The medial sections revealed hyaluronic acid gel resting in either a preperiosteal or an intraorbicularis plane. Soft tissue structures such as deep fat compartment septa and the orbicularis oculi muscle appeared to play a significant role in influencing the resting position of hyaluronic acid gel. CONCLUSIONS: In most specimens, the location of a significant portion of hyaluronic acid gel following injection to the infraorbital hollows differed from the intended injection plane. Soft tissue structures including fat compartment septa and the orbicularis oculi muscle appear to influence the resting position of hyaluronic acid gel. Careful attention should be used to avoid overfilling the thin soft tissue layers of the medial infraorbital hollows or tear trough.

Double-blind, bilateral pain comparison with simultaneous injection of 2% lidocaine versus buffered 2% lidocaine for periocular anesthesia.

PURPOSE: Determine if raising the pH of 2% lidocaine with epinephrine 1:100 000 to a physiologic level decreases pain perception during periocular, subcutaneous anesthesia. DESIGN: Double-blind, prospective, randomized study. Simultaneous unilateral injections of buffered and unbuffered lidocaine solutions were given before surgery to patients having bilateral, periocular surgery. PARTICIPANTS: Fifty-four consecutive patients (27 male and 27 female; mean age, 68 years; standard deviation, 11 years). INTERVENTION: Patients were given simultaneous injections of buffered and unbuffered 2% lidocaine with epinephrine 1:100 000. The needles were inserted simultaneously and the anesthesia was injected for a 20-second count for a total volume of 1.0 ml per injected side. MAIN OUTCOME MEASURES: After the simultaneous injections, the patients were asked to rate the pain on each side on a Likert-type visual analog scale of 0 to 10. RESULTS: Sixty-five percent of patients preferred the buffered lidocaine with a scaled pain reduction of 0.9 (P = 0.0005). Additionally, for the patients who believed that the buffered solution was less painful, the mean decrease in scaled pain rating was 2, for a 51% reduction in pain level (P = 0.001). No gender differences were noted. CONCLUSIONS: Buffering 2% lidocaine with epinephrine 1:100 000 with sodium bicarbonate 8.4% offers a clinically and statistically significant reduction in pain experienced by two-thirds of patients receiving periocular subcutaneous anesthesia.

Anterior filler displacement following injection of calcium hydroxylapatite gel (Radiesse) for anophthalmic orbital volume augmentation.

OBJECTIVE: To describe the complication of anterior filler displacement following injection of calcium hydroxylapatite gel (Radiesse) for anophthalmic enophthalmos correction. METHODS: Retrospective case series of patients who experienced anterior filler displacement following orbital injection of calcium hydroxylapatite. Data includes patient demographics, indication for injection, route and volume of injection, description of postinjection complications, and final outcome. RESULTS: Four cases of anterior filler displacement and expansion following injection of calcium hydroxylapatite were identified. The patients' ages ranged from 33 to 64 years old. All 4 patients underwent multiple prior orbital surgeries and suffered from anophthalmic enophthalmos. Injectable calcium hydroxylapatite was delivered transcutaneously, to the deep extraconal orbital space, via 27-gauge, 1.25-inch retrobulbar needles. Each patient received an initial 1.3 ml of filler, with 1 patient receiving an additional 0.8 ml. Within 1 week, all patients experienced prominent, edematous lower eyelids. A CT scan of 1 patient radiographically documented anterior migration of the filler material. Two patients required transconjunctival excision of the filler and infiltrated orbital fat. Histopathologic examination of 1 specimen revealed chronic foreign body granulomatous inflammation. Two patients were treated medically, with resolution of clinical findings over 6 to 9 months. CONCLUSIONS: Anterior filler displacement is a potential complication of orbital volume augmentation with injectable calcium hydroxylapatite. Patients should be counseled regarding this possibility when considering options for the treatment of anophthalmic enophthalmos. A history of multiple prior orbital surgeries, with associated tissue disruption and scarring, may be a risk factor for filler displacement.

The effect of cancer therapies on pediatric anophthalmic sockets.

PURPOSE: To determine the impact of chemotherapy or external beam radiotherapy (EBRT) on pediatric anophthalmic sockets. DESIGN: A retrospective, nonrandomized, interventional cohort study. PARTICIPANTS: A total of 135 sockets of 133 children undergoing enucleation from late 1999 to early 2009 at the St. Jude Children's Research Hospital were included. METHODS: A retrospective chart review of outcomes after enucleation in patients treated with systemic chemotherapy or orbital EBRT either before or after removal of the eye compared with patients who received no other treatment. MAIN OUTCOME MEASURES: Incidence of implant exposure, migration, extrusion, socket contracture, and pyogenic granuloma formation. RESULTS: Retinoblastoma was the primary diagnosis in 128 eyes (95%). Median follow-up was 3.6 years (range, 0.1-9.3 years). Event-free course was observed in 94 sockets (69.6%). Complications included implant exposure (n = 28, 20.7%), socket contracture (n = 16, 11.9%), pyogenic granuloma (n = 9, 6.7%), implant extrusion (n = 3, 2.2%), and migration (n = 2, 1.5%). Exposure resolved in 21 sockets (77.8%) and improved in 2 sockets (11.1%); 1 patient with exposure died. Use of prior, adjuvant, or subsequent chemotherapy increased the long-term risk of exposure (odds ratio [OR] = 3.7; 95% confidence interval [CI], 1.4-9.4), and contracture (OR could not be calculated, P<0.0001). External beam radiotherapy greatly increased the risk of contracture (OR 24.0; 95% CI, 6.9-82.8) and exposure (OR 2.89; 95% CI, 1.1-7.9). CONCLUSIONS: In this unique pediatric population with cancer, chemotherapy and EBRT had an additive effect, significantly increasing the incidence of exposure and socket contracture. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Nattokinase Attenuates Retinal Neovascularization Via Modulation of Nrf2/HO-1 and Glial Activation.

Purpose: Nattokinase (NK), an active ingredient extracted from traditional food Natto, has been studied for prevention and treatment of cardiovascular diseases due to various vasoprotective effects, including fibrinolytic, antihypertensive, anti-atherosclerotic, antiplatelet, and anti-inflammatory activities. Here, we reported an antineovascular effect of NK against experimental retinal neovascularization. Methods: The inhibitory effect of NK against retinal neovascularization was evaluated using an oxygen-induced retinopathy murine model. Expressions of Nrf2/HO-1 signaling and glial activation in the NK-treated retinae were measured. We also investigated cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) after NK administration. Results: NK treatment significantly attenuated retinal neovascularization in the OIR retinae. Consistently, NK suppressed VEGF-induced cell proliferation and migration in a concentration-dependent manner in cultured vascular endothelial cells. NK ameliorated ischemic retinopathy partially via activating Nrf2/HO-1. In addition, NK orchestrated reactive gliosis and promoted microglial activation toward a reparative phenotype in ischemic retina. Treatment of NK exhibited no cell toxicity or anti-angiogenic effects in the normal retina. Conclusions: Our results revealed the anti-angiogenic effect of NK against retinal neovascularization via modulating Nrf2/HO-1, glial activation and neuroinflammation, suggesting a promising alternative treatment strategy for retinal neovascularization.

LncRNA LPAL2/miR-1287-5p/EGFR Axis Modulates TED-Derived Orbital Fibroblast Activation Through Cell Adhesion Factors.

CONTEXT: The activation of orbital fibroblasts, the prime targets in thyroid eye disease (TED), is central to its underlying pathogenesis. OBJECTIVE: We aimed to investigate the mechanism of TED orbital fibroblast activation from the perspective of noncoding RNA regulation. METHODS: Immunofluorescence (IF) staining was applied to evaluate the fibrotic changes in target cells. Cell proliferation was evaluated by 5-ethoxy 2-deoxyuridine and colony-formation assays. Collagen I concentration was determined by enzyme-linked immunosorbent assay. Human microarray analysis was performed on 3 TED and 3 healthy control orbital tissue samples. RESULTS: Bioinformatics analysis showed that cell adhesion signaling factors were differentially expressed in TED tissues, including intercellular adhesion molecule (ICAM)-1, ICAM-4, vascular cell adhesion molecule, and CD44, which were all upregulated in diseased orbital tissues. Long noncoding RNA LPAL2 level was also upregulated in orbital tissues and positively correlated with ICAM-1 and ICAM-4 expression. Stimulation of the TED orbital fibroblasts by transforming growth factor-ß1 (TGF-ß1) significantly increased the expression of ICAM-1, ICAM-4, and LPAL2. Knockdown of LPAL2 in orbital fibroblasts inhibited TGF-ß1-induced increases in cell adhesion factor levels and orbital fibroblast activation. Microarray profiling was performed on TED and normal orbital tissues to identify differentially expressed microRNAs, and miR-1287-5p was remarkably reduced within diseased orbital samples. miR-1287-5p was directly bound to the epidermal growth factor receptor (EGFR) 3' untranslated region and LPAL2, and LPAL2 modulated EGFR/protein kinase B (AKT) signaling through targeting miR-1287-5p. CONCLUSION: The LPAL2/miR-1287-5p axis modulated TGF-ß1-induced increases in cell adhesion factor levels and TED orbital fibroblast activation through EGFR/AKT signaling.

Octreotide inhibits secretion of IGF-1 from orbital fibroblasts in patients with thyroid-associated ophthalmopathy via inhibition of the NF-κB pathway.

PURPOSE: We investigated the effect of octreotide, a long-acting somatostatin (SST) analogue, on IGF-1 secretion and its possible mechanism of action in orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO). MATERIALS AND METHODS: OFs were isolated from the orbital fat of patients with TAO or healthy individuals. The expression level of insulin-like growth factor (IGF)-1, at the protein and mRNA level, was determined with ELISA and quantitative RT-PCR, respectively. The expression pattern of somatostatin receptor (SSTR) 2, which has the highest affinity for octreotide, was examined by flow cytometry. The activity of NF-κB pathway was determined by examining the levels of phosphorylation of IKKα/ß and p65, and degradation of IκB via western blot analysis, and by measuring the activity of NF-kB-dependent luciferase via transfection with plasmids containing luciferase and NF-κB binding site. RESULTS: OFs from patients with TAO showed significantly higher levels of IGF-1 secretion and NF-κB activity even in the absence of stimulation, compared to those from controls. Treatment with octreotide reduced the level of IGF-1 secretion in OFs from patients with TAO, but not in OFs from controls. OFs from patients with TAO expressed higher levels of SSTR2 on the cell surface, compared to controls. In addition, the expression of IGF-1 at the protein and mRNA level was dependent on the activity of NF-κB pathway in OFs from patients with TAO. Furthermore, treatment with octreotide reduced on the activity of NF-κB pathway in OFs from patients with TAO. CONCLUSION: OFs from patients with TAO showed significantly higher levels of IGF-1 secretion via up-regulation of NF-κB activity. Treatment with octreotide inhibited the secretion of IGF-1 by reducing the NF-κB pathway in OFs, which expressed higher levels of SSRT2 on the cell surface, from patients with TAO.

Diffuse anterior retinoblastoma without retinal involvement.

PURPOSE: To present a unique case of an 8.5-year-old child with unilateral, anterior, pseudouveitis. He was found to have unilateral, invasive, small blue cell tumor of the anterior segment that was diagnosed as diffuse infiltrating retinoblastoma despite lack of retinal involvement on fundus examination or histopathologic analysis. DESIGN: Interventional case report. PARTICIPANTS: One patient. INTERVENTION: The patient was treated with topical prednisolone acetate 1% and oral prednisone with no improvement in anterior chamber reaction. The patient underwent fine-needle aspiration biopsy (FNAB) of anterior chamber fluid, the results of which were consistent with a primitive neuroectodermal neoplasm, either retinoblastoma or medulloepithelioma. Retinoblastoma was favored strongly, and the patient underwent enucleation followed by chemotherapy with vincristine, carboplatin, and etoposide, and radiation to the eye socket of 4140 cGy total was performed. MAIN OUTCOME MEASURES: The patient is alive and tumor free with follow-up of 5 years. RESULTS: Microscopic examination demonstrated cells similar to those seen on the FNAB infiltrating the iris stroma, trabecular meshwork, Schlemm's canal, and the inner portion of sclera in the region of the angle. No calcifications were identified. Serial sections of the entire globe were performed to determine the origin of the tumor. No retinal involvement was identified, and tumor was not seen to arise from the ciliary epithelium. Immunohistochemistry demonstrated positive staining with synaptophysin and negative staining with leukocyte common antigen and CD34. CONCLUSIONS: This patient represents a case of diffuse anterior retinoblastoma with lack of obvious retinal involvement. Morphologic features typical of medulloepithelioma were not found on pathologic analysis. Although the patient lacked a retinal focus, he is alive at 5 years without evidence of recurrence of tumor.