Biomedical subjects

Carol Merges

Publications and source records attributed to Carol Merges.

7 recordsLinked to original sources

PubMed2006

Nanoparticle tethered antioxidant response element as a biosensor for oxygen induced toxicity in retinal endothelial cells.

PURPOSE: A novel system, based on biosensor DNA tethered to a nanoparticle, was developed for the treatment of retinopathy of prematurity. METHODS: The construction of a five-layered nanoparticle was visualized with gel electrophoresis. Transcriptionally active PCR products (TAP) containing the biosensor sequence, were bioconjugated to the surface of magnetic nanoparticles yielding biosensor tethered magnetic nanoparticles (MNP). The biosensor was based on an enhanced green fluorescent protein (EGFP) reporter gene driven by an enhanced antioxidant response element (ARE). Image analysis and flow cytometry were used to characterize MNP delivery and biosensor activity. RESULTS: The MNP penetrated dividing and migrating cells more often than quiescent endothelial cells in a wound-healing in vitro assay. Prussian blue staining demonstrated that more cells have nanoparticle cores than are transfected. When compared to naked TAP alone, MNP transfected more cells in a dose dependent manner. Both the biosensor alone and MNP induce gene expression in the presence of hyperoxia, greater than 1.5 fold over normoxic controls. These data also show that the MNP had a signal to noise ratio of 0.5 greater than the plasmid form of the biosensor as demonstrated by flow cytometry. CONCLUSIONS: This approach has the potential to allow the endothelial cells of the retinal vasculature to prevent or treat themselves after hyperoxic insult, rather than systemic treatment to protect or treat only the retina.

Animals↗

PubMed2006

Canine retinal angioblasts are multipotent.

The purpose of this study was to culture and characterize endothelial cells and angioblasts, vascular precursors, from adult and neonatal dog retina and determine if angioblasts are committed to endothelial cell lineage or have the potential to be multipotent, i.e. express phenotypic characteristics of other vascular cell types. Endothelial cells were established from adult dog retina (ADREC) by the technique of Gitlin and D'Amore. For angioblasts, pieces of neonatal day 2 (P2) avascular peripheral retina were placed under coverslips until sufficient cells had explanted. All cells were maintained initially on hyaluronic acid (HA)/fibronectin (FN) substratum. Neonatal canine retinal angioblasts (NCRA) were maintained initially on retinal-derived growth factor with alpha-amino adipic acid to inhibit growth of Muller cells. Cell lines were characterized by enzyme histochemistry [menadione-dependent alpha glycerophosphate dehydrogenase (alphaGPDH), marker for angioblasts] and immunocytochemistry. Once characterized, cells were grown on FN, or collagens I or IV substrata and fed platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF-2). The phenotypic expression of a marker for endothelial cells [acetylated LDL (acLDL) uptake] or a marker for pericytes and smooth muscle cells, production of alpha smooth muscle actin (alphaSMA), was evaluated under those conditions. The canine retinal cell lines that were established had the following characteristics when maintained on serum and a retinal extract. Angioblasts had low expression of vWf and VEGF-R2 (two markers for canine endothelial cells), and very low uptake of acLDL but high expression of alphaGPDH and adenosine A2a receptors (A2aR) (two markers for canine angioblasts in vivo). ADREC had high expression of endothelial cell markers (vWf, VEGF-R2, and acLDL uptake) but minimal expression of alphaGPDH and A2aR. Both angioblasts and endothelial cells expressed CXCR4, a marker for hemangioblasts. Angioblasts grown on any of the substrata in the presence of FGF-2 had high uptake of acLDL and low expression of alphaSMA, while those grown in the presence of PDGF-BB had high expression of alphaSMA and low uptake of acLDL. In conclusion, angioblasts cultured from peripheral vascular retina have low expression of endothelial cell markers and high alphaGPDH and A2aR, markers for canine angioblasts in vivo. Angioblasts will internalize acLDL when maintained on FGF-2 and express alphaSMA when maintained on PDGF-BB, suggesting that they have the potential to become endothelial cells or pericytes, i.e. are multipotent.

Actins↗

PubMed2006

The initial fetal human retinal vasculature develops by vasculogenesis.

There is increasing evidence that the hemangioblast, a common progenitor for hematopoietic cells and endothelial cells, participates in embryonic and extra-embryonic vasculogenesis in some organs. Whether resident angioblasts or endothelial progenitor cells (EPCs) contribute to human retinal vasculogenesis is still a matter of controversy. To address this controversy, fetal human retinas of 6-23 weeks gestation (WG) were examined using immunohistochemistry and a panel of antibodies against endothelial cell markers (CD34, CD31), a marker for retinal angioblasts and endothelium (CD39/ecto-ADPase), and a marker for precursors and hemangioblasts (CXCR4). Confocal microscopic spectral analysis and double labeling with Ki67 was used to identify the proliferating cell types. In the inner neuroblastic layer of the 6-8 WG retina and in the putative ganglion cell layer in avascular regions of older eyes (14 WG-20 WG), scattered CD39+ angioblasts were well in advance of forming vasculature. There was a layer of CXCR4+ cells in the inner retina that was reduced in size with development. As blood vessels formed, CD39+ cells were always well in advance of the vascular front and they expressed CXCR4. This demonstrates that a pool of resident angioblasts express CD39 and CXCR4 as they differentiate and participate in vasculogenesis in the fetal human. They retain expression of CD39 as endothelial cells in the newly formed retinal vasculature but they down-regulate CXCR4 expression.

Antigens, CD↗

PubMed2005

Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in aged human choroid and eyes with age-related macular degeneration.

The purpose of this study was to examine the localization and relative levels of vascular endothelial growth factor (VEGF; an angiogenic factor) and pigment epithelium-derived factor (PEDF; an antiangiogenic factor) in aged human choroid and to determine if the localization or their relative levels changed in age-related macular degeneration (AMD). Ocular tissues were obtained from eight aged control donors (age range, 75-86 years; mean age, 79.8 years) with no evidence or history of chorioretinal disease and from 12 donors diagnosed with AMD (age range, 61-105 years; mean age, 83.9 years). Tissues were cryopreserved and streptavidin alkaline phosphatase immunohistochemistry was performed with rabbit polyclonal anti-human VEGF and rabbit polyclonal anti-human PEDF antibodies. Binding of the antibodies was blocked by preincubation of the antibody with an excess of recombinant human PEDF or VEGF peptide. Choroidal blood vessels were identified with mouse anti-human CD-34 antibody in adjacent tissue sections. Three independent observers graded the immunohistochemical reaction product. The most prominent sites of VEGF and PEDF localization in aged control choroid were RPE-Bruch's membrane-choriocapillaris complex including RPE basal lamina, intercapillary septa, and choroidal stroma. There was no significant difference in immunostaining intensity and localization of VEGF and PEDF in aged control choroids. The most intense VEGF immunoreactivity was observed in leukocytes within blood vessels. AMD choroid had a similar pattern and intensity of VEGF immunostaining to that observed in aged controls. However, PEDF immunoreactivity was significantly lower in RPE cells (p=0.0073), RPE basal lamina (p=0.0141), Bruch's membrane (p<0.0001), and choroidal stroma (p=0.0161) of AMD choroids. The most intense PEDF immunoreactivity was observed in disciform scars. Drusen and basal laminar deposits (BLDs) were positive for VEGF and PEDF. In aged control subjects, VEGF and PEDF immunostaining was the most intense in RPE-Bruch's membrane-choriocapillaris complex. In AMD, PEDF was significantly lower in RPE cells, RPE basal lamina, Bruch's membrane and choroidal stroma. These data suggest that a critical balance exists between PEDF and VEGF, and PEDF may counteract the angiogenic potential of VEGF. The decrease in PEDF may disrupt the balance and be permissive for the formation of choroidal neovascularization (CNV) in AMD.

Aged↗

PubMed2004

Localization of collagen XVIII and the endostatin portion of collagen XVIII in aged human control eyes and eyes with age-related macular degeneration.

PURPOSE: Endostatin, a C-terminal fragment of collagen XVIII (coll XVIII) formed by proteolysis, specifically inhibits endothelial cell migration and proliferation in vitro and potently inhibits angiogenesis and tumor growth in vivo. The purpose of this study was to examine the immunolocalization of endostatin and coll XVIII in the retina and choroid of human donor tissue sections from aged control donor eyes and to determine whether the localization or relative levels are changed in age-related macular degeneration (AMD). METHODS: Ocular tissues were obtained from six aged control donors (age range, 75-86 years; mean age, 80.5 years) without evidence or history of chorioretinal disease and from nine donors with AMD (age range, 74-105 years; mean age, 88.6 years). Tissues were cryopreserved, and streptavidin alkaline phosphatase immunohistochemistry was performed with goat anti-human and mouse anti-human endostatin antibodies and rabbit anti-mouse coll XVIII. Blood vessels were identified with mouse anti-human CD-34 antibody in adjacent sections. Pigment in RPE and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In aged control eyes, coll XVIII and endostatin (the endostatin portion of coll XVIII) immunoreactivity was observed in large retinal blood vessels and in capillaries in some individuals, but the internal limiting membrane (ILM) had the most intense retinal immunostaining. There was no significant difference in immunoreactivity to both antibodies in retinal blood vessels in aged control eyes. In the choroid, endostatin and coll XVIII were localized to blood vessels, Bruch's membrane, and RPE basal lamina. AMD retina and choroid had a similar pattern and intensity of coll XVIII immunostaining, as observed in control eyes but reaction product was more diffuse in the choroid. Endostatin immunoreactivity was significantly higher in ILM (P = 0.037) in AMD retina and significantly lower in the choriocapillaris, Bruch's membrane, and RPE basal lamina of AMD choroids (P < 0.05) and completely negative in some areas of AMD choroids. CONCLUSIONS: These data suggest that reduced levels of the endostatin portion of coll XVIII in Bruch's membrane, RPE basal lamina, intercapillary septa, and choriocapillaris in eyes with AMD may be permissive for choroidal neovascularization.

Aged↗

PubMed2003

Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid.

Pigment epithelium-derived factor (PEDF) has been shown to be an inhibitor of angiogenesis as well as a multipotent neurotrophic factor in the mammalian eye. Changes in PEDF levels have been correlated with development of retinal neovascularization in oxygen-induced retinopathy. The purpose of this study was to determine the localization and relative level of PEDF in human retinas and choroids using immunohistochemistry and evaluate the changes in PEDF and vascular endothelial growth factor (VEGF) localization and their relation to the progression of proliferative sickle cell retinopathy. Cryopreserved tissues from eyes of normal subjects and subjects with non-proliferative or proliferative sickle cell retinopathy were used with streptavidin peroxidase immunohistochemistry. A rabbit polyclonal antibody was made against recombinant human PEDF. Binding of the antibody was blocked by preincubation of the antibody with excess human recombinant PEDF. Relative levels of immunoreactivity were scored with a seven-point grading system and by microdensitometric analysis.The most prominent sites of PEDF localization in the normal eye were the vitreous condensed at the internal limiting membrane and RPE-Bruch's membrane-choriocapillaris complex. PEDF was also prominent in choroidal stroma. There was limited immunoreactivity in some cells of the neural retinas, in blood vessels and in the interphotoreceptor matrix (IPM). There was no difference in ratio (1.47 vs. 1.44) of PEDF/VEGF or the relative levels of either growth factor in the retinal vasculatures of the control subjects and perfused area of non-proliferative sickle cell retinas. The ratio was increased in the non-perfused area of the non-proliferative sickle cell retinas (2.24). In eyes with proliferative sickle cell retinopathy, elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations as well as feeder vessels of sea fans. The PEDF/VEGF ratio in sea fans was 1.0. Immunoreactivity for PEDF was prominent in retinal vessels in non-perfused regions and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these structures. In conclusion, PEDF and VEGF were both significantly elevated in viable sea fan formations in sickle cell disease (p<0.05) but only PEDF was present in non-viable sea fans. The highest levels of PEDF in all eyes were associated with extracellular matrices (vitreous, choroidal stroma, IPM, and walls of blood vessels). PEDF might play an important role in inhibiting angiogenesis and inducing the regression of sea fans. Progression of angiogenesis may be dependent on the ratio of PEDF/VEGF.

Adolescent↗

PubMed2002

Mechanisms for sickle red blood cell retention in choroid.

PURPOSE: Although sickle (SS) red cell-mediated vaso-occlusion in retina and resultant retinopathy is well documented, the effects of SS red cells on choroidal vasculature are poorly understood. The intent of this study was to determine, using a rat model, the conditions under which retention of sickle erythrocytes in choroid occur and if that retention can be inhibited. METHODS: Sickle red cells were density separated into high density (SS4) or normal density, reticulocyte-enriched fractions (SS2). Red cells were labeled with FITC and administered IV to anesthetized Sprague Dawley rats. Rats were made either hypoxic or were given TNF-alpha intraperitoneally 5 hours before intravenous administration of red cells. Five minutes after administration of red cells, rats were exsanguinated, the retinas removed, and choroids prepared as flatmounts. The number of red cells retained in five high power fields of choroid was then determined. In other experiments, SS red cells were preincubated with the cyclic peptide TBC772 [inhibits binding of alpha4beta1 (VLA-4) and alpha4beta7 to their ligands], a control peptide TBC1194, or a VLA-4 neutralizing antibody before administration to the rat or antibodies against VLA-4 ligands were delivered IV before administration of SS red cells. RESULTS: Hypoxic conditions before administration of SS red cells significantly stimulated retention of SS4 cells (P = 0.0003), but did not significantly increase retention of SS2 cells. Administration of TNF-alpha significantly increased retention of all types of SS red cells (P < 0.001). Preincubation of cells with anti-VLA-4 or TBC 772 inhibited retention of SS red cells in choriocapillaris of TNF-alpha-treated rats (P < 0.0001). Complete inhibition of cytokine-stimulated retention was also accomplished by IV administration of monoclonal antibodies against fibronectin or its CS-1 domain, a ligand for VLA-4. CONCLUSIONS: The mechanisms for retention of SS red cells in retina and choroid appear identical: hypoxia-mediated retention of dense red cells and adherence of red cells in reticulocyte-rich fractions after cytokine stimulation. TNF-alpha-stimulated retention of SS red cells in choroid appears to be mediated by VLA-4, presumably on the surface of some reticulocytes. This increased retention was inhibited by a VLA-4 antagonist (TBC772), a VLA-4 neutralizing antibody or by blocking one of VLA-4's ligands, the CS-1 portion of fibronectin.

Anemia, Sickle Cell↗