Steven E. Brooks

VEGF differentially activates STAT3 in microvascular endothelial cells

Increased VEGF expression is found in several pathologies characterized by abnormal angiogenesis. Previous studies have shown that the transcription factor STAT3 mediates VEGF gene transcription and its activation. In this study, Western analysis and confocal immunocytochemistry were used to examine STAT3 activation in retinal microvascular endothelial cells (BREC). We found that VEGF rapidly induces STAT3 tyrosine phosphorylation and nuclear translocation. Immunoprecipitation studies also showed that VEGF forms a complex with VEGFR2 only in BREC and not in aortic macrovascular endothelial cells (BAEC). In addition, quantitative real‐time RT‐PCR analysis of VEGF‐induced VEGF expression showed a significant increase in specific mRNA formation only in BREC and not in BAEC, and this effect was significantly reduced by antisense‐mediated reduction of STAT3 expression. Furthermore, studies conducted in human dermal microvascular endothelial cells (HDMEC) showed that, in this endothelial cell type, VEGF autocrine expression is also accompanied by STAT3 activation as in BREC. In this study we showed that VEGF can differentially induce STAT3 activation in micro‐ versus macro‐vascular endothelial cells and that this effect is linked to VEGFR2/STAT3 complex formation, which correlates with VEGF autocrine ability to stimulate its own gene expression.

Vascular Endothelial Growth Factor Activates STAT Proteins in Aortic Endothelial Cells

Vascular endothelial growth factor (VEGF) intracellular signaling in endothelial cells is initiated by the activation of distinct tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR). Because the tyrosine kinase-dependent transcription factors known as STAT (signal transducers and activators of transcription) proteins are important modulators of cell growth responses induced by other growth factor receptors, we have determined the effects VEGF of on STAT activation in BAEC (bovine aortic endothelial cells). Here, we show that VEGF induces tyrosine phosphorylation and nuclear translocation of STAT1 and STAT6. VEGF also stimulates STAT3 tyrosine phosphorylation, but nuclear translocation does not occur. We found that placenta growth factor, which selectively activates VEGFR1, has no effect on the STATs. However, upon VEGF stimulation, STAT1 associates with the VEGFR2 in a tyrosine kinase-dependent manner, indicating that VEGF-induced STAT1 activation is mediated primarily by VEGFR2. Thus, our study shows for the first time that VEGF activates the STAT pathway through VEGFR2. Because the growth-promoting activity of VEGF depends upon VEGFR2 activation, these findings suggest a role for the STATs in the regulation of gene expression associated with the angiogenic effects of VEGF.

The effect of blood transfusion protocol on retinopathy of prematurity: A prospective, randomized study.

Objective. Controversy exists regarding the potential influence of anemia and blood transfusions on the rate of retinopathy of prematurity (ROP) in premature infants. A prospective, randomized, masked trial was performed to determine the influence of red blood cell transfusion protocol on ROP incidence and severity in a population of high-risk infants. Methods. A total of 50 infants with birth weights <1251 g were divided randomly into two groups beginning on day of life 29. Group 1 (n = 24) received red cell transfusions during the 6-week study period, only if certain symptom-based guidelines were met. Group 2 (n = 26) received red cell transfusions to maintain the hematocrit level above 40% for the entire 6 weeks. Infants were monitored for ROP, growth, and associated morbidity. Serial measurements of serum glucose, lactate, ferritin, total iron-binding capacity, and iron were performed. Results. ROP occurred in 83% of infants in group 1, and 73% of infants in group 2. There were no statistically significant differences in ROP severity, intraventricular hemorrhage, bronchopulmonary dysplasia, necrotizing enterocolitis, or any of the laboratory values except hemoglobin (10.8 vs 13.2 g/dL) and hematocrit (33.9% vs 41.8%) between the groups. Combining data from both groups, there was no association between hemoglobin or hematocrit ratios and ROP incidence or severity. Conclusions. A transfusion policy aimed at limiting the amount of blood given to premature infants (symptom-based) during the neonatal period does not impart a significantly different risk for ROP or other associated conditions, than does a policy in which transfusions are given more liberally for replacement purposes.

Anisometropia and Binocularity

PURPOSE To determine the effects of experimentally induced anisometropia on binocular function in healthy adults as a means of assessing the potentially detrimental effects of uncorrected anisometropia on binocular development in childhood. METHODS Nineteen adults with normal binocularity, ranging in age from 26 to 59 years, were studied. Unilateral myopia, hyperopia, or astigmatism (at 90 degrees or 45 degrees) was induced in each subject using trial lenses. Sensory status then was assessed by measuring stereoacuity, Worth four-dot fusion, and Bagolini lens response. RESULTS All subjects showed a decline in binocular function with increasing levels of anisometropia. Foveal suppression was evident on the Worth four-dot test, and increased in proportion to the anisometropia. Stereoacuity was similarly degraded by the induced anisometropia, with some subjects showing significant loss of stereoacuity with as little as 1 diopter of spherical anisometropia. Bagolini lens responses were binocular in almost all patients, although occasional abnormalities were found. CONCLUSIONS Relatively low degrees of anisometropia may cause significant abnormalities in high-grade binocular visual functions in adults. The potential effects of uncorrected anisometropia on binocularity in children require further investigation, but should be considered in developing guidelines for the empiric correction of refractive errors.

Telemedicine diagnosis of eye disorders by direct ophthalmoscopy ☆: A pilot study

OBJECTIVE To report a pilot study of telemedical direct ophthalmoscopy in the diagnosis of acquired immune deficiency syndrome (AIDS)-related retinopathy in a human immunodeficiency virus (HIV)-positive population and in the diagnosis of glaucoma, cataract, and retinopathy in a diabetic population. DESIGN Prospective comparative case series. PARTICIPANTS Seventeen HIV-positive and 20 diabetic patients. METHODS A direct ophthalmoscope custom-fitted with a digital microcamera capable of transmitting images from any of 61 sites within the Georgia Statewide Telemedicine Program was used by a nonophthalmologist to examine 34 eyes of 17 HIV-positive patients and 39 eyes of 20 patients with diabetes. Fundus images were transmitted in real-time to a reviewing ophthalmologist. An in-person, comprehensive examination including indirect ophthalmoscopy, was performed by a second ophthalmologist. Telemedical examination was compared to the in-person comprehensive examination. RESULTS For the HIV study, 21 eyes did not show HIV retinopathy (noninfectious retinopathy with cotton-wool spots) by in-person examination. Telemedical examination correctly identified 20 of these eyes as disease-free (specificity = 95%). HIV retinopathy was present in 12 of the 34 eyes by in-person evaluation with telemedical examination correctly diagnosing 10 of these eyes (sensitivity = 83%). One eye with dense cataract and retinal detachment was unable to be evaluated ophthalmoscopically by either in-person or telemedical examination. Telemedical and in-person assessments for HIV retinopathy were identical in 100% of eyes without cataract. Disagreement in diagnosis between telemedical and in-person examination was associated with cataract (P < 0.0007). For the diabetes study, because of an inadequate image, telemedical examination was unable to classify 46% and 36% of eyes for glaucoma and diabetic retinopathy, respectively. Inability to make a telemedical determination for glaucoma (P < 0.011), nonproliferative (P < 0.064) and proliferative (P < 0.064) diabetic retinopathy was associated with cataract. Of the eyes that were able to be assessed by telemedical examination for diabetic retinopathy (n = 25), glaucoma (n = 21), and cataract (n = 39), the accuracy was poor (sensitivity = 29%, 50%, and 41%, respectively). Telemedical examination for diabetic retinopathy and glaucoma was more likely to agree with in-person examination in eyes without cataract as compared to eyes with cataract (not statistically significant). CONCLUSION Telemedical direct ophthalmoscopic, real-time fundus imaging may provide a valuable means for providing ophthalmic consultation to the primary care physician in younger patients without lens or media opacity, but is inadequate for eyes with any degree of lens or media opacity.

Augmented Hummelsheim Procedure for Paralytic Strabismus

PURPOSE To report a modification of the Hummelsheim procedure for use in the management of paralytic strabismus. METHODS Eight patients with paralytic strabismus secondary to third nerve palsy (n=1), sixth nerve palsy (n=3), combined cranial nerve palsy (n=1), or extraocular muscle damage (n=3) were treated using a modification of the Hummelsheim transposition procedure. The procedure involves half-tendon transpositions of the adjacent rectus muscles to the insertion of the paralyzed muscle, coupled with resection of the transposed halves. Further augmentation was achieved by surgical or pharmacologic weakening of the ipsilateral (n=6) or contralateral (n=1) antagonist. One patient underwent the procedure bilaterally. All patients underwent at least 6 weeks of follow-up. RESULTS The mean preoperative primary position deviation in the seven unilateral cases was 54 prism diopters (delta) (range: 25-85 delta). Preoperative forced ductions were positive in four cases. Resections varied from 4-8 mm. Ipsilateral antagonist recession varied from 0-14 mm. The mean change was 52 delta (range: 25-85 delta). Five cases were aligned within 15 delta of orthotropia at 6 weeks. No cases of anterior segment ischemia or induced vertical deviation were noted. CONCLUSION The modified Hummelsheim procedure appears capable of correcting large angles of strabismus associated with muscle palsy of various etiologies. It is safe, amenable to adjustable sutures, and relatively tissue- and vessel-sparing. Additional study is required to understand more fully the procedures component effects and its interaction with ocular rotation.

Fat adherence syndrome treated with intraoperative mitomycin-C: a rabbit model.

We used an animal model of restrictive strabismus analogous to the fat adherence syndrome in humans to test the efficacy of topical intraoperative mitomycin-C (MMC) in preventing the development of restrictive scar tissue. A cicatricial adhesion was created between the inferior rectus muscle and the inferior orbital rim of each eye in eight rabbits, and passive forced ductions were quantitatively measured with a spring scale. Eight eyes were treated intraoperatively with topical MMC 0.5 mg/mL, the other eight with sterile water. Passive forced ductions were again measured 4 weeks postoperatively and representative orbits were exenterated for histopathologic examination. Significant restriction of motility was produced in six of the eight control eyes. Though prophylactic treatment with MMC may have been beneficial in some cases, on average, the restriction developing in these eyes did not significantly differ from that in the control eyes. In addition, longer exposure times to MMC led to marked orbital inflammation and severe restriction of ocular motility. Finally, histopathologic evaluation of the orbits of the MMC-treated eyes revealed marked fibrosis of perimuscular connective tissues. Although MMC may have a role in the management of fat adherence syndrome, further study is needed to establish safe and efficacious methods of delivery.

Neuroprotection from retinal ischemia/reperfusion injury by NOX2 NADPH oxidase deletion

PURPOSE The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. METHODS Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2(-/-) mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor κB (NF-κΒ) was measured by Western blot analysis. RESULTS NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60% decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-κB was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2(-/-) retina (P < 0.01). CONCLUSIONS These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.

Detection and Localization of Steel Intraocular Foreign Bodies Using Computed Tomography: A Comparison of Helical and Conventional Axial Scanning

PURPOSE To compare the sensitivity and specificity of detection, and accuracy of localization, of small steel intraocular and episcleral foreign bodies, using conventional axial and helical computed tomographic scanning in an experimental model. METHODS Small steel foreign bodies ranging in size from 0.048 to 0.179 mm3 were placed in intraocular and episcleral locations in eye bank eyes mounted in the orbits of a human skull and scanned using helical and conventional axial techniques. Helical scanning was performed using 1-mm and 3-mm thick sections. Conventional axial scanning was performed using 3-mm thick sections. Images were reviewed by masked observers to determine sensitivity, specificity, and accuracy of localization for each imaging method. RESULTS Steel foreign bodies as small as 0.048 mm3 were detectable with each scanning protocol. Although the helical scans appeared to provide higher levels of sensitivity compared to conventional axial scanning, the difference in outcome between the scan types was not statistically significant. Sensitivity was dependent on the size of the foreign body and ranged from 45% to 65% for the smaller ones (< 0.06 mm3) to 100% for the larger ones (> 0.06 mm3). Multiplanar reformatting of images was helpful in achieving optimal accuracy. CONCLUSION In an experimental model of steel intraocular foreign body, helical computed tomographic scanning provided images of high quality similar to that of conventional axial scanning.

Inflammatory Opacities of the Vitreous in Rifabutin-associated Uveitis

PURPOSE To describe rifabutin-associated uveitis with opacities in the inferior and posterior vitreous in three patients with acquired immunodeficiency syndrome. METHOD Case reports of the three patients are presented. RESULTS The patients, who were being treated with rifabutin and fluconazole, developed anterior and posterior uveitis. The posterior uveitis was characterized by white-yellow inflammatory opacities located in the inferior and posterior vitreous. Discontinuation of rifabutin and the start of topical corticosteroid therapy resulted in improvement of the uveitis and visual acuity. CONCLUSION Recognition of rifabutin-associated uveitis with opacities in the inferior and posterior vitreous may prevent unnecessary invasive diagnostic and therapeutic procedures.