Simultaneous Bilateral Open-Globe Repair and Vitreoretinal Surgery for Explosive-Related Ocular Injury.

Purpose: To report an approach to explosive injuries with simultaneous, co-surgeon bilateral ruptured globe repair and pars plana vitrectomy for bilateral intraocular foreign bodies (IOFBs). Methods: A case and its findings were analyzed. Results: A 31-year-old man had bilateral vision loss after an air compressor malfunction that caused a high-pressure explosion to his face. An examination showed bilateral open-globe injuries and IOFBs, necessitating urgent repair. Given the risk for endophthalmitis and the need for expeditious repair, open-globe repair surgery was performed in both eyes simultaneously by co-surgeons followed by pars plana lensectomy, vitrectomy with IOFB removal, and silicone oil placement. The final visual acuity after bilateral scleral-fixated intraocular lens implantation was 20/20 OD and 20/25 OS. Conclusions: This case of bilateral open-globe injuries and IOFBs required expeditious repair with bilateral, simultaneous surgery that ultimately resulted in excellent visual outcomes. Simultaneous surgery may be an option for bilateral ocular trauma.

Surgical management of acanthamoeba chorioretinitis.

PURPOSE: Acanthamoeba chorioretinitis is a rare manifestation of the parasitic infection, and reported cases often result in enucleation. Surgical removal of Acanthamoeba chorioretinitis has not been previously described. We report a surgical case of Acanthamoeba chorioretinitis spread from keratitis that ultimately resulted in a disease-free outcome. OBSERVATIONS: A healthy 80-year-old male with a history of keratoconus requiring a penetrating keratoplasty in the fellow eye presented with a severe corneal ulcer clinically consistent with Acanthamoeba keratitis. He ultimately required a penetrating keratoplasty and improved clinically until he developed vitritis on post-operative month 1 and was diagnosed with endophthalmitis. B-scan ultrasound demonstrated vitreous opacities and a large retinal mass that reduced in size following serial intravitreal injections of antibiotics, oral antibiotics, and a limited pars plana vitrectomy. He underwent a repeat pars plana vitrectomy 6 weeks later and a retinal mass in the mid-periphery with an associated tractional retinal detachment was noted. A localized retinectomy was performed around the lesion which was excised entirely, and silicone oil was instilled. Pathology of the lesion showed acute and chronic granulomatous necrotizing inflammation with the presence of several definitive amoebic organisms and numerous cells suspicious for amoebae. The patient was maintained on oral antibiotics by the Infectious Disease Service and was disease-free 1-year post-infection. CONCLUSIONS AND IMPORTANCE: Acanthamoeba chorioretinitis is a rare, devastating disease and often leads to enucleation. We present a surgical case showing control of the infection utilizing a surgical retinectomy. Aggressive local therapy and a multidisciplinary approach with the Infectious Disease Service may lead to a successful outcome.

Personalized Proteomics in Proliferative Vitreoretinopathy Implicate Hematopoietic Cell Recruitment and mTOR as a Therapeutic Target.

PURPOSE: To profile vitreous cytokine expression of proliferative vitreoretinopathy (PVR) patients. DESIGN: Case-control study. METHODS: Liquid biopsies were collected from 2 groups: control subjects (n = 3) undergoing pars plana vitrectomy to remove an epiretinal membrane (ERM), and test subjects (n = 7) with varying degrees of PVR. A high-throughput cytokine screen measured expression of 200 cytokines. Cytokine expression patterns were prospectively validated in separate cohorts of control patients and those with PVR-A, PVR-B, and PVR-C (n = 10 for each group). Expression changes were evaluated by analysis of variance (significant P value < .05), hierarchical cluster algorithm, and pathway analysis, to identify candidate pathways for prospective studies. RESULTS: In PVR vitreous, 29 cytokines were upregulated compared to controls. Early PVR vitreous showed upregulation of T-cell markers, profibrotic cytokines, and cytokines downstream of mTOR activation (IL-2, IL-6, and IL-13), whereas in late PVR vitreous, cytokines driving monocyte responses and stem-cell recruitment (SDF-1) prevailed. Prospective validation confirmed the differential expression of specific cytokines from PVR-A to C. CONCLUSIONS: Early PVR is characterized by activation of T cells and mTOR signaling, whereas advanced PVR is characterized by a chronic monocyte response. PVR might be treated by rational repositioning of existing drugs that target mTOR and IL-6. Our analysis demonstrates that successful therapeutic intervention will be highly dependent on the specific therapeutic target and the stage of PVR. This study provides insights into cytokines that will serve as biomarkers and therapeutic targets. These biomarkers will help design clinical trials that intervene at appropriate times.

Proteomic Analysis of Elevated Intraocular Pressure with Retinal Detachment.

PURPOSE: To report a case of elevated intraocular pressure with retinal detachment. OBSERVATIONS: Liquid chromatography and tandem mass spectrometry was performed on the patient aqueous biopsy. Protein levels were analyzed with 1-way analysis of variance (ANOVA) and unbiased clustering. High levels of rod outer segment proteins were not detected, suggesting that this was not a case of Schwartz-Matsuo syndrome. Instead, elevated levels of Hepcidin (HEPC) and Cystatin C (CYTC; candidate biomarkers for primary open angle glaucoma) were detected, suggesting a different, unknown etiology. CONCLUSIONS AND IMPORTANCE: Molecular diagnoses can differentiate between clinical diagnoses and point to common biomarkers or disease mechanisms.

Precision Medicine: Personalized Proteomics for the Diagnosis and Treatment of Idiopathic Inflammatory Disease.

IMPORTANCE: To better characterize posterior uveitis, vitreous samples from 15 patients were subjected to antibody arrays, and the expression levels of 200 human cytokines were evaluated. Expression was analyzed by 1-way analysis of variance (significance at P < .01), unsupervised cluster algorithm, and pathway analysis. OBSERVATIONS: Unbiased clustering of patients, based on their cytokine expression profile, suggested that particular protein networks and molecular pathways are altered in various forms of uveitis. Expression of interleukin 23 (IL-23), IL-1 receptor I (IL-1RI), IL-17R, tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2), insulinlike growth factor-binding protein 2 (IGFBP-2), nerve growth factor (b-NGF), platelet-derived growth factor receptor ß polypeptide (PDGFRb), bone morphogenic protein 4 (BMP-4), and stem cell factor (SCF) constituted a common cytokine signature in the vitreous of patients with uveitis. In 1 patient with progressive, idiopathic visual loss, this last-line analysis implicated retinal autoimmunity, a diagnosis that was validated when her serum sample was found to contain antibodies to S-arrestin, a retinal protein and potent cause of autoimmune retinal degeneration. CONCLUSIONS AND RELEVANCE: The analysis identifies a common cytokine signature for posterior uveitis and guides the diagnosis of a patient with idiopathic uveitis. Personalized treatment reversed the visual loss, illustrating how proteomic tools may individualize therapy.

Orbital Relapsing Polychondritis: A Unique Presentation, Complication, and Treatment.

An 87-year-old man with a history of relapsing polychondritis presented to the emergency department after 4 days of worsening left periorbital swelling and erythema. On examination, he demonstrated clinical features consistent with orbital cellulitis and was treated with a trial of intravenous antibiotics. His condition did not improve over the next 36 hours and intravenous methylprednisolone was initiated. This led to rapid improvement in orbital symptoms and signs, and a diagnosis of specific orbital inflammation secondary to relapsing polychondritis was made. The patient was discharged on a tapering dose of prednisone. As a steroid-sparing measure, adalimumab was initiated; however, the patient developed Sweet Syndrome. Adalimumab was subsequently discontinued, steroid dose was increased, and anakinra treatment was initiated. This therapeutic course led to significant clinical improvement. Since initiating anakinra, the patient has had no recurrences of Sweet Syndrome. Anakinra may be a useful adjunct therapy for ophthalmic manifestations of relapsing polychondritis.

TREATMENT OF RETINOPATHY AND MACULAR EDEMA SECONDARY TO A CAROTID-CAVERNOUS FISTULA.

PURPOSE: To present a case report on a patient with retinal complications from a carotid-cavernous fistula. METHODS: Observational case report. RESULTS: A 26-year-old patient sustained head trauma following a motorcycle accident. Examination and retinal imaging demonstrated a venous stasis retinopathy with cystoid macular edema. The edema resolved with aflibercept, but not with bevacizumab. CONCLUSION: The patient was diagnosed with venous stasis retinopathy secondary to carotid-cavernous fistula. Pathologic findings completely resolved with appropriate management.

MULTIMODAL IMAGING OF THE RETINA AND CHOROID IN SYSTEMIC AMYLOIDOSIS.

PURPOSE: To present the multimodal imaging findings of four patients with systemic amyloidosis, renal failure, and chorioretinopathy. METHODS: Retrospective analysis of four patients presenting to four institutions with evidence of amyloid induced chorioretinopathy. Fundus photography, autofluorescence, and spectral domain optical coherence tomography findings were studied and are presented. RESULTS: Four patients with biopsy-proven systemic amyloidosis demonstrated progressive chorioretinal degeneration with color fundus photography and autofluorescent imaging. With spectral domain optical coherence tomography analysis, amyloidosis-induced chorioretinopathy was characterized by a widened choriocapillaris band, choroidal infiltration, diffuse photoreceptor dysfunction, and thinning of the outer nuclear layer. CONCLUSION: Multimodal imaging including spectral domain optical coherence tomography analysis in eyes of patients with systemic amyloidosis shows deposition in the choroid. The deposition may cause a secondary toxic and or barrier effect resulting in diffuse retinal pigment epithelium and photoreceptor dysfunction.

Oral mineralocorticoid antagonists for recalcitrant central serous chorioretinopathy.

PURPOSE: To evaluate the effect and tolerance of oral mineralocorticoid antagonists, eplerenone and/or spironolactone, in recalcitrant central serous chorioretinopathy. METHODS: Retrospective consecutive observational case series. Primary outcome measures included central macular thickness (CMT, µm), macular volume (MV, mm(3)), Snellen visual acuity, and prior treatment failures. Secondary outcomes included duration of treatment, treatment dosage, and systemic side effects. RESULTS: A total of 120 patients with central serous chorioretinopathy were reviewed, of which 29 patients were treated with one or more mineralocorticoid antagonists. The average age of patients was 58.4 years. Sixteen patients (69.6%) were recalcitrant to other interventions prior to treatment with oral mineralocorticoid antagonists, with an average washout period of 15.3 months. The average duration of mineralocorticoid antagonist treatment was 3.9±2.3 months. Twelve patients (52.2%) showed decreased CMT and MV, six patients (26.1%) had increase in both, and five patients (21.7%) had negligible changes. The mean decrease in CMT of all patients was 42.4 µm (range, -136 to 255 µm): 100.7 µm among treatment-naïve patients, and 16.9 µm among recalcitrant patients. The mean decrease in MV of all patients was 0.20 mm(3) (range, -2.33 to 2.90 mm(3)): 0.6 mm(3) among treatment-naïve patients, and 0.0 mm(3) among recalcitrant patients. Median visual acuity at the start of therapy was 20/30 (range, 20/20-20/250), and at final follow-up it was 20/40 (range, 20/20-20/125). Nine patients (39.1%) experienced systemic side effects, of which three patients (13.0%) were unable to continue therapy. CONCLUSION: Mineralocorticoid antagonist treatment had a positive treatment effect in half of our patients. The decrease in CMT and MV was much less in the recalcitrant group compared to the treatment-naïve group. An improvement in vision was seen only in the treatment-naïve group. Systemic side effects, even at low doses, may limit its usage in some patients.

Proteomic insight into the molecular function of the vitreous.

The human vitreous contains primarily water, but also contains proteins which have yet to be fully characterized. To gain insight into the four vitreous substructures and their potential functions, we isolated and analyzed the vitreous protein profiles of three non-diseased human eyes. The four analyzed substructures were the anterior hyaloid, the vitreous cortex, the vitreous core, and the vitreous base. Proteins were separated by multidimensional liquid chromatography and identified by tandem mass spectrometry. Bioinformatics tools then extracted the expression profiles, signaling pathways, and interactomes unique to each tissue. From each substructure, a mean of 2,062 unique proteins were identified, with many being differentially expressed in a specific substructure: 278 proteins were unique to the anterior hyaloid, 322 to the vitreous cortex, 128 to the vitreous base, and 136 to the vitreous core. When the identified proteins were organized according to relevant functional pathways and networks, key patterns appeared. The blood coagulation pathway and extracellular matrix turnover networks were highly represented. Oxidative stress regulation and energy metabolism proteins were distributed throughout the vitreous. Immune functions were represented by high levels of immunoglobulin, the complement pathway, damage-associated molecular patterns (DAMPs), and evolutionarily conserved antimicrobial proteins. The majority of vitreous proteins detected were intracellular proteins, some of which originate from the retina, including rhodopsin (RHO), phosphodiesterase 6 (PDE6), and glial fibrillary acidic protein (GFAP). This comprehensive analysis uncovers a picture of the vitreous as a biologically active tissue, where proteins localize to distinct substructures to protect the intraocular tissues from infection, oxidative stress, and energy disequilibrium. It also reveals the retina as a potential source of inflammatory mediators. The vitreous proteome catalogues the dynamic interactions between the vitreous and surrounding tissues. It therefore could be an indirect and effective method for surveying vitreoretinal disease for specific biomarkers.

Scleral electrocautery and its effects on choroid vessels: implications for subretinal fluid drainage during scleral buckling surgery.

BACKGROUND AND OBJECTIVE: External drainage of subretinal fluid as part of a scleral buckling procedure rapidly restores the retinal pigment epithelium-neural retina interface in rhegmatogenous retinal detachments but carries the inherent risk of subretinal hemorrhage and retinal incarceration. The authors investigated variations to the technique to reduce the chance of subretinal hemorrhage originating from the choroid. MATERIALS AND METHODS: A novel method for needle drainage using electrocautery of the sclerochoroidal layers before puncture was employed. The effect of 0% to 50% scleral electrocautery in a porcine model was investigated. RESULTS: A significant decrease in choroidal vessel diameter and choroidal vessel density at 40% electrocautery was demonstrated. CONCLUSION: Electrocautery without scleral cut-down before external drainage of subretinal fluid likely decreases the chance of subretinal hemorrhage by decreasing choroidal vascularity.

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina.

Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternative pathway. DES1 is expressed in retinal Müller cells, where it coimmunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in Rpe65(-/-) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 had very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.

Motorcycle trauma in a St Lucian hospital / Traumas por motocicletas en un hospital de Santa Lucía

OBJECTIVE: A motorcycle is a single-track, two-wheeled motor vehicle that is used worldwide for transportation. The use of the motorcycle has resulted in trauma that is associated with significant morbidity and mortality. The aim of this study is to document the pattern of motorcycle accidents and the demographics of the cyclists in St Lucia. METHOD: This is a 15-month prospective study on all patients with motorcycle injuries that reported to the emergency room at the Victoria Hospital. Information on patients: age, gender, helmet use, intake of alcohol/drugs before the motorcycling and mechanism of injury were obtained and filled into a prepared proforma by the attending physician. Those admitted were followed-up to know the outcome and complications of treatment. RESULTS: Total number of patients studied was 136 in 115 accidents, males (M) were 127 while females (F) were 9, with M:F ratio of 14.1:1.0. There were 105, 28 and 3 riders, passengers and pedestrians respectively; 87.5 % of the patients were below the age of 35 years. Fifty-three per cent of the accidents occurred over the weekend. The limbs were mostly injured, constituting 81.9% of the parts of the body injured. CONCLUSION: The study revealed that young and productive males were mainly injured in motorcycle accidents and the injuries were more in the limbs. More than fifty per cent of the accidents were found to occur during the weekends and more than fifty per cent of the motorcyclists were not wearing crash helmets.

OBJETIVO: La motocicleta es un vehículo motor dos ruedas y vía única, usado mundialmente como medio de transporte. El uso de la motocicleta ha traído consigo traumas asociados con una morbilidad y una mortalidad significativas. El objetivo de este estudio es documentar el patrón de accidentes de motocicletas así como la demografía de los ciclistas en Santa Lucía. MÉTODO: Se trata de un estudio prospectivo de 15 meses en torno a pacientes con lesiones de motocicleta. Los heridos acudieron a la sala de emergencias del Hospital Victoria. Se obtuvo información sobre los pacientes - edad, género, uso del casco, consumo de alcohol/drogas antes de montar la moto, y el mecanismo de la lesión. Con la información obtenida una pro forma fue preparada por el médico asistente. A los ingresados se les hizo un seguimiento a fin de conocer la evolución clínica y las complicaciones del tratamiento. RESULTADOS: El número total de pacientes estudiados fue 136 en 115 accidentes. Los varones (M) fueron 127 mientras que las hembras (F) fueron 9, para una proporción M:F de 14.1: 1.0. Hubo 105, 28 y 3 motoristas, pasajeros y peatones respectivamente. El 87.5% de los pacientes estaban por debajo de los 35 años de edad. Cincuenta y tres por ciento de los accidentes ocurrieron durante el fin de semana. Las extremidades fueron principalmente dañadas, constituyendo el 81.9% de las partes del cuerpo lesionadas. CONCLUSIÓN: El estudio reveló que los lesionados en accidentes de motocicletas eran principalmente varones jóvenes y productivos, y que las lesiones eran mayormente en las extremidades. Se halló que más del cincuenta por ciento de los accidentes ocurrieron durante los fines de semana, y más del cincuenta por ciento de los motociclistas no llevaban el casco de protección.

Environmental enrichment produces a behavioral phenotype mediated by low cyclic adenosine monophosphate response element binding (CREB) activity in the nucleus accumbens.

BACKGROUND: Previous research has shown that rats reared in an enriched condition (EC) are more sensitive to the acute effects of amphetamine than rats reared in an isolated condition (IC); yet, EC rats self-administer less amphetamine than IC rats. The present study used cocaine to further explore this environmental enrichment behavioral phenotype, as well as the underlying molecular mechanisms involved. METHODS: Enriched condition and IC rats were studied in a broad battery of behavioral tests, including cocaine conditioned place preference (CPP) and self-administration and several measures of anxiety- and depression-related behavior. The involvement of the transcription factor, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), in mediating EC versus IC differences was investigated. RESULTS: Enriched condition rats exhibited less cocaine self-administration, despite showing enhanced cocaine CPP. Enriched condition rats also displayed less depression-like behavior but higher levels of anxiety-like behavior. This behavioral phenotype is consistent with low CREB activity in the nucleus accumbens, a key brain reward region. Indeed, EC rats have less phospho-CREB (the transcriptionally active form of the protein) in the nucleus accumbens than IC rats, and a selective knockdown of CREB in this brain region of normally reared rats, by use of a novel viral vector expressing a short hairpin RNA (shRNA) directed against CREB, reproduced the EC behavioral phenotype. CONCLUSIONS: These studies identify a potential molecular mechanism for how rearing environment-a nonpharmacological, nonsurgical manipulation-can modify a wide range of complex emotional behaviors.

Activation of NFkappaB is inhibited by curcumin and related enones.

The transcription factor NFkappaB (NFkappaB) is up-regulated in many cancer cells where it contributes to development of the pro-survival, anti-apoptotic state. The natural product curcumin is a known inhibitor of activation of NFkappaB. Enone analogues of curcumin were compared with curcumin for their abilities to inhibit the TNFalpha-induced activation of NFkappaB, using the Panomics' NFkappaB Reporter Stable Cell Line. The enones tested included curcumin analogues that retained the 7-carbon spacer between the aromatic rings, analogues with a 5-carbon spacer, and analogues with a 3-carbon spacer. Inhibitors of NFkappaB activation were identified in all three series, a number of which were more active than curcumin. Enone analogues in the series with the 5-carbon spacer were especially active, including members that contained heterocyclic rings. 1,5-Bis(3-pyridyl)-1,4-pentadien-3-one was the most active analogue, IC50 = 3.4 +/- 0.2 microM. The most active analogues retain the enone functionality, although some analogues devoid of the enone functionality exhibited activity. The activity of the analogues as inhibitors of the activation of NFkappaB did not correlate with their anti-oxidant activity. The data suggest that the abilities of curcumin and analogues to prevent the stress-induced activation of NFkappaB result from the inhibition of specific targets rather than from activity as anti-oxidants.

Aberrant accumulation of fibulin-3 in the endoplasmic reticulum leads to activation of the unfolded protein response and VEGF expression.

PURPOSE: The inherited early-onset macular degenerative disease known as malattia leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) have been linked to a missense mutation leading to production of a mutant fibulin-3 protein (R345W). R345W is poorly secreted and accumulates in the RPE of ML/DHRD retinas. Accumulation of misfolded proteins within the endoplasmic reticulum (ER) causes activation of unfolded protein response (UPR) signaling and expression of ER stress-responsive genes, including vascular endothelial growth factor (VEGF). Therefore, we hypothesized that the expression of R345W activates the UPR, leading to VEGF expression. METHODS: Adenoviral vectors were used to overexpress fibulin-3 wild-type (Wt) and R345W mutant proteins in ARPE-19 cells. Secretion and intracellular accumulation of Wt and R345W were compared by Western blot analysis and immunocytochemistry. Activation of the UPR was evaluated by measuring the expression of glucose-regulated protein 78 (GRP78 [BiP]) and editing of the X-box binding protein (XBP-1) mRNA. VEGF expression and transcriptional activation of the VEGF promoter were determined by Northern blot analysis, Western blot analysis, and use of a novel VEGF promoter-reporter construct containing 8.2 kb of the human VEGF gene. RESULTS: R345W was poorly secreted by ARPE-19 cells and accumulated in the ER, leading to UPR activation and increased VEGF expression. Compared with Wt mutant proteins, the expression of R345W was more effective at causing UPR activation, increasing VEGF expression, and stimulating transcription from the VEGF promoter. CONCLUSIONS: These findings demonstrated that the expression of mutated fibulin-3 caused UPR activation and increased VEGF expression. Expression of mutant fibulin proteins may contribute to macular degeneration and choroidal neovascularization by causing ER stress leading to RPE dysfunction and increased VEGF expression.

The oxidative stressor arsenite activates vascular endothelial growth factor mRNA transcription by an ATF4-dependent mechanism.

Aberrant retinal expression of vascular endothelial growth factor (VEGF) leading to neovascularization is a central feature of age-related macular degeneration and diabetic retinopathy, two leading causes of vision loss. Oxidative stress is suggested to occur in retinal tissue during age-related macular degeneration and diabetic retinopathy and is suspected in the mechanism of VEGF expression in these diseases. Arsenite, a thiol-reactive oxidative stressor, induces VEGF expression by a HIF-1alpha-independent mechanism. Previously, we demonstrated that homocysteine, an endoplasmic reticulum stressor, increases VEGF transcription by a mechanism dependent upon activating transcription factor ATF4. Because ATF4 is expressed in response to oxidative stress, we hypothesized that ATF4 was also responsible for increased VEGF transcription in response to arsenite. We now show that arsenite increased steady state levels of VEGF mRNA and activated transcription from a VEGF promoter construct. Arsenite induced eIF2alpha phosphorylation, resulting in increased ATF4 protein levels. Inactivation or loss of ATF4 greatly diminished the VEGF response to arsenite treatment. Overexpression of ATF4 was sufficient to activate the VEGF promoter, and arsenite cooperated with exogenous ATF4 to further activate the promoter. A complex containing ATF4 binds a DNA element at +1767 bp relative to the VEGF transcription start site, and DNA binding activity is increased by arsenite treatment. In addition, the ability of a thiol antioxidant, N-acetylcysteine, to inhibit the effect of arsenite on VEGF expression coincided with its ability to inhibit phosphorylation of eIF2alpha and ATF4 protein expression. Thus, arsenite-induced up-regulation of VEGF gene transcription occurs by an ATF4-dependent mechanism.

Homocysteine increases the expression of vascular endothelial growth factor by a mechanism involving endoplasmic reticulum stress and transcription factor ATF4.

Vascular endothelial growth factor (VEGF) plays a key role in the development and progression of diabetic retinopathy. We previously demonstrated that amino acid deprivation and other inducers of endoplasmic reticulum-stress (ER stress) up-regulate the expression of VEGF in the retinal-pigmented epithelial cell line ARPE-19. Because homocysteine causes ER stress, we hypothesized that VEGF expression is increased by ambient homocysteine. dl-Homocysteine-induced VEGF expression was investigated in confluent ARPE-19 cultures. Northern analysis showed that homocysteine increased steady state VEGF mRNA levels 4.4-fold. Other thiol-containing compounds, including l-homocysteine thiolactone and DTT, induced VEGF expression 7.9- and 8.8-fold. Transcriptional run-on assays and mRNA decay studies demonstrated that the increase in VEGF mRNA levels was caused by increased transcription rather than mRNA stabilization. VEGF mRNA induction paralleled that of the ER-stress gene GRP78. Homocysteine treatment caused transient phosphorylation of eIF2alpha and an increase in ATF4 protein level. Overexpression of a dominant-negative ATF4 abolished the VEGF response to homocysteine treatment and to amino acid deprivation. VEGF mRNA expression by ATF4-/- MEF did not respond to homocysteine treatment and the response was restored with expression of wild-type ATF4. These studies indicate that expression of the pro-angiogenic factor VEGF is increased by homocysteine and other thiol-containing reductive compounds via ATF4-dependent activation of VEGF transcription.