Kameran Lashkari

Identification of Tyrosine Residues in Vascular Endothelial Growth Factor Receptor-2/FLK-1 Involved in Activation of Phosphatidylinositol 3-Kinase and Cell Proliferation

Activation of vascular endothelial growth factor receptor-2 (VEGFR-2) plays a critical role in vasculogenesis and angiogenesis. However, the mechanism by which VEGFR-2 activation elicits these cellular events is not fully understood. We recently constructed a chimeric receptor containing the extracellular domain of human CSF-1R/c-fms, fused with the entire transmembrane and cytoplasmic domains of murine VEGFR-2 (Rahimi, N., Dayanir, V., and Lashkari, K. (2000) J. Biol. Chem. 275, 16986–16992). In this study we used VEGFR-2 chimera (herein named CKR) to elucidate the signal transduction relay of VEGFR-2 in porcine aortic endothelial (PAE) cells. Mutation of tyrosines 799 and 1173 individually on CKR resulted in partial loss of CKRs ability to stimulate cell growth. Double mutation of these sites caused total loss of CKRs ability to stimulate cell growth. Interestingly, mutation of these sites had no effect on the ability of CKR to stimulate cell migration. Further analysis revealed that tyrosines 799 and 1173 are docking sites for p85 of phosphatidylinositol 3-kinase (PI3K). Pretreatment of cells with wortmannin, an inhibitor of PI3K, and rapamycin, a potent inhibitor of S6 kinase, abrogated CKR-mediated cell growth. However, expression of a dominant negative form of ras (N17ras) and inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD98059 did not attenuate CKR-stimulated cell growth. Altogether, these results demonstrate that activation of VEGFR-2 results in activation of PI3K and that activation of PI3K/S6kinase pathway, but not Ras/MAPK, is responsible for VEGFR-2-mediated cell growth.

Vascular Endothelial Growth Factor and Hepatocyte Growth Factor Levels Are Differentially Elevated in Patients with Advanced Retinopathy of Prematurity

Although the roles of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in angiogenesis are well described, the putative roles of these factors in retinopathy of prematurity (ROP) remain unknown. We evaluated VEGF and HGF protein levels in subretinal fluid of eyes with ROP, and expression of their corresponding receptors in retrolental membranes associated with stage 5 ROP. We examined subretinal fluid samples from eyes using rhegmatogenous retinal detachment as a control. VEGF and HGF were differentially elevated in eyes with ROP. In Stage 5 ROP (n = 22), the mean VEGF and HGF levels were 14.77 +/- 14.01 ng/ml and 16.56 +/- 9.62 ng/ml, respectively. Interestingly, in patients with active stage 4 ROP, mean VEGF levels were highly elevated (44.16 +/- 18.72 ng/ml), whereas mean HGF levels remained very low (4.77 +/- 2.50 ng/ml). Next, we investigated in vivo expression of VEGF receptor-2 and HGF receptor in retrolental membranes from 16 patients with stage 5 ROP. Both VEGF receptor-2 and HGF receptor proteins were detected mainly in posterior portions of the membrane as well as in vessel walls and along the retinal interface where angiogenesis was active. These findings together suggest that VEGF and HGF play important roles in the pathogenesis of ROP.

Characterization of Functional Changes in Macular Holes and Cysts

Precise characterization of functional loss in small retinal lesions is difficult with conventional techniques. Using the scanning laser ophthalmoscope, the authors evaluated functional changes and fixation behavior in 26 eyes with macular holes and 15 eyes with macular cysts. A dense scotoma was present over all macular holes; 24 had no detectable functional alteration at the margins of the hole, and fixation was above the horizontal meridian in all eyes. Nine eyes with cysts had no detectable functional loss over the cyst. Only two eyes had small areas of dense scotoma within the cyst area, and four had areas of relative scotoma. Fixation was central in all eyes. Characterization of functional changes is helpful in differentiating holes from cysts. Photocoagulation at the margin of the holes may result in further functional damage.

Conjunctival Goblet Cell Secretion Stimulated by Leukotrienes Is Reduced by Resolvins D1 and E1 To Promote Resolution of Inflammation

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT1 and CysLT2, were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB4, LTC4, LTD4, and LTE4, as well as PGD2, stimulated goblet cell secretion in rat goblet cells. LTD4 and LTE4 increased the intracellular Ca2+ concentration ([Ca2+]i), and LTD4 activated ERK1/2. The CysLT1 receptor antagonist MK571 significantly decreased LTD4-stimulated rat goblet cell secretion and the increase in [Ca2+]i. Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD4-stimulated goblet cell secretion in cultured rat goblet cells. LTD4-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD4- and LTE4-stimulated increases in [Ca2+]i, as well as LTD4 activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD4 using the CysLT1 receptor. Stimulated secretion is terminated by preventing the increase in [Ca2+]i and activation of ERK1/2 by RvD1 and RvE1.

Interferon γ–Inducible Protein-10 (IP-10) and Eotaxin as Biomarkers in Age-Related Macular Degeneration

PURPOSE To analyze serum cytokine levels in subjects with different stages of AMD and to study the expression of salient cytokines in postmortem eyes with AMD. METHODS A suspension array system was used to analyze sera (n = 18 to 20/group) from control subjects and those with early AMD (AREDS stage 1), intermediate dry AMD (AREDS stage 3), advanced AMD with geographic atrophy (GA), or neovascular AMD (CNV). Postmortem eyes with AMD or control eyes were examined immunohistochemically for expression of IP-10 and eotaxin (n = 4 to 8/group). RESULTS Serum eotaxin and IP-10 levels were significantly elevated in all stages of AMD, except for eotaxin levels in neovascular AMD (P < 0.07). The peak of serum IP-10 concentration was at intermediate dry AMD. In donor eyes, IP-10 and eotaxin expressions were increased in the RPE of eyes with early AMD, GA, and CNV. Eotaxin accumulated within the layer of basal linear/laminar deposits in all stages of AMD, while IP-10 was mainly in eyes with GA and CNV. IP-10 was abundant in the connective tissue matrix associated with CNV, and eotaxin was usually present but more focally and with less intense staining. Both IP-10 and eotaxin were expressed by neovascular endothelial cells. Both IP-10 and eotaxin were expressed in the neurosensory retina, but there was no detectable difference in staining between eyes with or without AMD. CONCLUSIONS IP-10 and eotaxin may be early biomarkers in AMD. The authors hypothesize that the relative balance between levels of IP-10 and eotaxin is critical in regulating the neovascular response.

Erosion and intrusion of silicone rubber scleral buckle. Presentation and management.

Objective To describe the clinical presentation and management of erosion and intrusion of silicone rubber implants that are used in scleral buckling procedures for the treatment of retinal detachment. Methods The authors identified four patients from their practices during the last 20 years (1978–1998) who had erosion or intrusion of silicone rubber scleral buckles that were used to manage retinal detachment. Approximately 4400 scleral buckling procedures were performed during this period. A retrospective review of the medical records of all patients was performed. Factors that influenced management decisions concerning the intruding buckle are emphasized. Results All four patients had myopia. The interval between placement of the scleral buckle and development of intrusion ranged from 1 to 20 years. The buckles were intrascleral in three cases and episcleral in one. Recurrent detachment and vitreous hemorrhage were indications for surgical intervention in three cases. After the surgical removal of buckling elements, visual acuity stabilized in all patients and the retina remained attached in all cases. Conclusions Erosion and intrusion of scleral buckle are rare complications of scleral buckling procedures. The intruding buckle may be left intact unless there is significant threat to the integrity of ocular structures, recurrent detachment, or hemorrhage. Manipulation of the encircling band or buckle does not necessarily alter the visual acuity or the status of the retina.

Biostatistical analysis of pseudophakic and aphakic retinal detachments

Removal of the crystalline lens increases the risk of rhegmatogenous retinal detachment (RD) by creating changes in the ocular environment that predispose to development of retinal breaks. The evolution of cataract surgery from intracapsular cataract extraction (ICCE) to extracapsular cataract extraction (ECCE) and phacoemulsification has reduced the incidence of RD, while advances in vitreoretinal surgery have resulted in improved outcomes when retinal detachment does occur. The incidence of RD varies between 0.4–3.6% for ICCE and between 0.55–1.65% for ECCE. In eyes having undergone phacoemulsification the incidence is similar to those of ECCE and ranges between 0.75–1.65%. In this article the authors review the incidence and risk factors associated with pseudophakic and aphakic RD. The risk factors discussed include pre-operative risk factors such as age, status of the fellow eye and myopia, and surgical risk factors such as vitreous loss, posterior capsular integrity and Nd : YAG capsulotomy.

Angiogenic and Inflammatory Vitreous Biomarkers Associated With Increasing Levels of Retinal Ischemia.

PURPOSE To characterize the angiogenic and inflammatory vitreous biomarker profiles in a spectrum of ischemic retinopathies, including neovascular glaucoma. METHODS This institutional review board-approved study retrospectively analyzed 80 undiluted vitreous samples obtained during pars vitrectomy. The specimens were frozen (-80°C) and sent for concentration analysis of 34 proteins by Bio-Plex Pro assays. Specimens were divided into four groups: patients undergoing epiretinal membrane (ERM) peeling and/or macular hole (MH) surgery with no history of diabetes (non-DM group), patients undergoing ERM peeling, and/or MH surgery with a history of diabetes (DM group), patients with proliferative diabetic retinopathy (PDR group), and patients with neovascular glaucoma (NVG group). Parametric and nonparametric analyses of demographics and cytokine levels were performed using SPSS. RESULTS There were no significant differences in demographics among cohorts. Numerous proteins were significantly elevated between non-DM and DM (G-CSF, sCD40L, Endoglin, IL-6, placental growth factor [PlGF], VEGF-D), DM and PDR (leptin, IL-8, PlGF, VEGF-A), and PDR and NVG (G-CSF, leptin, TIE-2, sCD40L, EGF, HB-EGF, IL-6, IL-8, PlGF, TNF-α). Only PlGF was significantly elevated between each successive cohort. The most potent drivers of NVG were PlGF, VEGF-A, IL-6, and IL-8. CONCLUSIONS While the role of angioproliferative growth factors is well documented in ischemic retinopathy, our study delineates the importance of inflammatory and previously underreported angiogenic proteins. It also demonstrates a significant incremental increase in certain factors with increasing levels of ischemia. Both of these findings may guide the development of future therapies for ischemic retinopathies.

ERK/p44p42 Mitogen-Activated Protein Kinase Mediates EGF-Stimulated Proliferation of Conjunctival Goblet Cells in Culture

PURPOSE To determine whether activation of the ERK pathway by EGF leads to rat and human goblet cell proliferation. METHODS The conjunctiva was removed from male Sprague-Dawley rats. Human conjunctiva was removed during ocular surgery. The tissue was minced and goblet cells were grown. The cells were stimulated with EGF (10(-7) M) for 1 and 5 minutes and Western blot analysis was performed with an antibody against phosphorylated EGFR, to measure the activation of the EGF receptor (EGFR). The cells were incubated with EGF (10(-7) M) for 24 hours, and cell proliferation was measured by WST-8. Inhibitors were added either 20 minutes before EGF or 2 hours after. The cells were stimulated with EGF (10(-7) M) for 1 minute to 24 hours. The number of cells expressing phosphorylated ERK (pERK) in the nucleus and Ki-67 was determined by immunofluorescence. RESULTS EGF increased the activation of EGFR in rat conjunctival goblet cells. EGF-stimulated proliferation was inhibited by the EGFR inhibitor AG1478 and the MEK inhibitor U0126 in rat and human cultured goblet cells. EGF caused the translocation of pERK to the nucleus in a biphasic manner. Inhibition of the second peak with U0126 prevented proliferation. EGF-stimulated goblet cells progressed through the cell cycle expressing pERK in the nucleus. CONCLUSIONS EGF stimulated human and rat conjunctival goblet cell proliferation by activating the EGFR. EGFR stimulated ERK causing its biphasic translocation to the nucleus. The second peak response is responsible for cell proliferation, but the role of the first peak is not known.

Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation

VEGFR-1 is devoid of ligand-dependent tyrosine autophosphorylation and its activation is not associated with proliferation of endothelial cells. The molecular mechanism responsible for this characteristic of VEGFR-1 is not known. In this study, we show that VEGFR-1 is devoid of ligand-dependent downregulation and failed to stimulate intracellular calcium release, cell migration and angiogenesis in vitro. To understand the molecular mechanisms responsible for the poor tyrosine autophosphorylation of VEGFR-1, we have either deleted the carboxyl terminus of VEGFR-1 or exchanged it with the carboxyl terminus of VEGFR-2. The deletion of carboxyl terminus of VEGFR-1 did not reverse its defective ligand-dependent autophosphorylation. The carboxyl terminus-swapped VEGFR-1, however, displayed ligand-dependent autophosphorylation, downregulation and also conveyed strong mitogenic responses. Thus, the carboxyl tail of VEGFR-1 restrains the ligand-dependent kinase activation and downregulation of VEGFR-1 and its ability to convey the angiogenic responses in endothelial cells.