Endogenous VEGF Is Required for Visual Function: Evidence for a Survival Role on Müller Cells and Photoreceptors

Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical.Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective.These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution

TGF-β Is Required for Vascular Barrier Function, Endothelial Survival and Homeostasis of the Adult Microvasculature

Pericyte-endothelial cell (EC) interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-β signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-β signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-β signaling was inhibited by systemic expression of soluble endoglin (sEng) and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-β signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG). Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-β signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-β signaling. These results implicate constitutive TGF-β signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-β signaling in vasoproliferative and vascular degenerative retinal diseases

Phase 3, Randomized, 20-Month Study of the Efficacy and Safety of Bimatoprost Implant in Patients with Open-Angle Glaucoma and Ocular Hypertension (ARTEMIS 2)

Objective- To evaluate the intraocular pressure (IOP)-lowering efficacy and safety of 10 and 15 µg bimatoprost implant in patients with open-angle glaucoma (OAG) or ocular hypertension (OHT). Methods- This randomized, 20-month, multicenter, masked, parallel-group, phase 3 trial enrolled 528 patients with OAG or OHT and an open iridocorneal angle inferiorly in the study eye. Study eyes were administered 10 or 15 µg bimatoprost implant on day 1, week 16, and week 32, or twice-daily topical timolol maleate 0.5%. Primary endpoints were IOP and IOP change from baseline through week 12. Safety measures included treatment-emergent adverse events (TEAEs) and corneal endothelial cell density (CECD). Results- Both 10 and 15 µg bimatoprost implant met the primary endpoint of noninferiority to timolol in IOP lowering through 12 weeks. Mean IOP reductions from baseline ranged from 6.2–7.4, 6.5–7.8, and 6.1–6.7 mmHg through week 12 in the 10 µg implant, 15 µg implant, and timolol groups, respectively. IOP lowering was similar after the second and third implant administrations. Probabilities of requiring no IOP-lowering treatment for 1 year after the third administration were 77.5% (10 µg implant) and 79.0% (15 µg implant). The most common TEAE was conjunctival hyperemia, typically temporally associated with the administration procedure. Corneal TEAEs of interest (primarily corneal endothelial cell loss, corneal edema, and corneal touch) were more frequent with the 15 than the 10 µg implant and generally were reported after repeated administrations. Loss in mean CECD from baseline to month 20 was ~ 5% in 10 µg implant-treated eyes and ~ 1% in topical timolol-treated eyes. Visual field progression (change in the mean deviation from baseline) was reduced in the 10 µg implant group compared with the timolol group. Conclusions- The results corroborated the previous phase 3 study of the bimatoprost implant. The bimatoprost implant met the primary endpoint and effectively lowered IOP. The majority of patients required no additional treatment for 12 months after the third administration. The benefit-risk assessment favored the 10 over the 15 µg implant. Studies evaluating other administration regimens with reduced risk of corneal events are ongoing. The bimatoprost implant has the potential to improve adherence and reduce treatment burden in glaucoma

GLAUCOMA DRAINAGE DEVICE COMPLICATED BY FUNGAL PARACONIOTHYRIUM ENDOPHTHALMITIS

Ten years after BaerveldtTM implantation, a woman underwent immunosuppressive therapy for necrotizing scleritis. She subsequently developed symptoms suggestive of endophthalmitis in the setting of tube exposure. Cultures isolated fungal Paraconiothyrium. She underwent two vitrectomies with anterior chamber washouts, BaerveldtTM device removal, and intraocular and systemic antifungal therapy. The endophthalmitis has since resolved.   Purpose: To report the first documented case of Paraconiothyrium fungal endophthalmitis associated with a glaucoma drainage device. Methods: A 74-year-old woman with a history of primary open angle glaucoma had undergone BaerveldtTM glaucoma implantation 10 years earlier. She presented with pain and injection, was diagnosed with necrotizing scleritis, and started on prednisone and methotrexate. She subsequently developed worsening pain, vision loss, and infiltrates suggestive of fungal endophthalmitis, in the setting of an exposed BaerveldtTM tube. Results: Surgical cultures were positive for fungal Paraconiothyrium. The patient underwent two pars plana vitrectomies, anterior chamber washouts, removal of the BaerveldtTM device and intraocular lens, and intraocular and systemic antifungal therapy. The fungal infiltrates and inflammation have since resolved. Conclusions: Fungal endophthalmitis is an exceedingly rare complication of glaucoma drainage device implantation, but must be considered, especially in an immunocompromised patient

Fibromyxoma masquerading as dacryocystitis

Primary tumors of the lacrimal sac are extremely rare and predominantly epithelial in origin. We report a unique case of fibromyxoma of the lacrimal sac in an 86-year-old Caucasian woman who presented with 3 months of lower eyelid edema and 1 week of purulent discharge, epiphora, and acute pain. Examination revealed right lower eyelid erythema/edema and a tender, firm, palpable mass of the right lacrimal sac, which self-expressed a yellow, purulent discharge. On surgical exploration, a firm, rubbery, yellow mass was encountered and excised. Histopathologic analysis identified a nonencapsulated mass composed of loose interlacing fascicles and bundles of spindle-shaped cells with prominent myxoid changes; immunostaining was strongly positive for vimentin, weakly for smooth muscle actin, and negative for S-100 and muscle-specific actin. These findings were consistent with the diagnosis of a fibromyxoma of the lacrimal drainage system. After excision, the patient did well, with resolution of dacryocystitis

Glaucoma Drainage Device Erosions in Patients Receiving Anti-Vascular Endothelial Growth Factor Therapy for Diabetic Retinopathy

Purpose: To determine if intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections are associated with an increased risk of glaucoma drainage device (GDD) erosions. Patients and Methods: A retrospective chart review was conducted including patients with diabetic retinopathy and had a GDD implanted at a large academic institution. The rate of GDD erosions was compared between eyes that did or did not receive intravitreal anti-VEGF injections. A subanalysis was also performed the relationship between diabetic macular edema (DME) and intravitreal steroid injections and GDD erosions. Results: A total of 677 eyes from 608 patients was included. A total of 447 eyes received at least one anti-VEGF injection; 230 eyes never received such therapy. Twenty eyes (4.5%) receiving anti-VEGF had at least one erosion event, compared to 7 eyes (3.0%) of patients not receiving anti-VEGF therapy (OR 1.49, p=0.37). Diabetic macular edema was associated with a significantly increased rate of erosion in eyes not receiving anti-VEGF (71.4% versus 31.4%, p=0.034), but not in eyes receiving anti-VEGF (30.0% versus 40.7%, p=0.34). Receiving more than one specific anti-VEGF agent, an increased frequency or total number of anti-VEGF injections, or receiving intravitreal steroids were not associated with an increased risk of erosion (p>0.05). Conclusion: In patients with diabetic retinopathy, the use of anti-VEGF does not result in an increased rate of GDD erosions or recurrent erosions. Further research is needed over a longer follow-up period to determine if longer or more frequent anti-VEGF treatment is a risk factor for recurrent erosions

TGF-? Is Required for Vascular Barrier Function, Endothelial Survival and Homeostasis of the Adult Microvasculature

Pericyte-endothelial cell (EC) interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-β signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-β signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-β signaling was inhibited by systemic expression of soluble endoglin (sEng) and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-β signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG). Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-β signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-β signaling. These results implicate constitutive TGF-β signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-β signaling in vasoproliferative and vascular degenerative retinal diseases

Inhibition of TGF-β decreases endothelial barrier function in vitro.

<p>(A) SEM analysis demonstrated a characteristic endothelial cobblestone morphology in EC monocultures. Co-culture enhanced the association between adjacent cells, which was reversed with addition of SB-431542. Scale bars = 100 µm (left panel); 10 µm (right panel). Cell size was calculated by tracing individual ECs using ImageJ. Morphology of 10T1/2 cells from cocultures with ECs was not visibly altered when compared to 10T1/2 monoculture or with the addition of SB431542. (B) Analysis of EC size in monoculture or coculture with 10T1/2 cells (+/−SB431542). Coculture did not significantly alter average cell size, however, EC size were more uniform when compared to EC mono-cultures. Addition of SB431542 led to larger and more heterogenous cell size.</p

Inhibition of TGF-β decreases retinal perfusion and vascular autoregulation.

<p>After fourteen days of Ad-sEng and Ad-null expression, mice were injected with h.m.w. FITC-dextran through the left ventricle to localize perfused vessels. (A) Confocal analysis of retinal flat-mounts revealed reduced perfusion of the retina in the sEng-expressing mice compared to the control (representative photo of n = 12 mice). Scale bar = 200 µm. (B) The perfused vessels were visualized on retinal flat mounts by comparing the co-localization of type IV collagen (Cy3-red) and FITC and quantified on cryosections by comparing the number of vessels in the innermost vascular plexus (arrowheads) positive for both type IV collagen- and FITC to the number of vessels positive for type IV collagen but negative for FITC (C). (D) The retinas of sEng expressing mice show a marked reduction in the number of perfused vessels (n = 5, ** p<0.01). (E) After seven days of adenoviral expression, blood flow rates in the tail were measured non-invasively in response to intravascular injection of ACh. Measurements were made over 5 cycles pre-injection of ACh, normalized to 1 for each animal, and averaged at 5-cycle intervals post ACh injection. In Ad-null control mice, ACh increased tail vein blood flow rates 6–10 cycles post-injection, whereas blood flow rates were unchanged in Ad-sEng expressing mice (Ad-null: 1.619 µl/cycle, n = 5; Ad-sEng: 0.960 µl/cycle, n = 4, * p<0.01). Injection of 100 µl of saline in Ad-null or Ad-sEng mice elicited no response.</p

Endogenous VEGF Is Required for Visual Function: Evidence for a Survival Role on Müller Cells and Photoreceptors

Background Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical. Methods and Findings Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective. Conclusions These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution