PubMed Health⌕ Search

Biomedical subjects

Janet R McColm

Publications and source records attributed to Janet R McColm.

14 recordsLinked to original sources
PubMed2006

Orientation of endothelial cell division is regulated by VEGF signaling during blood vessel formation.

New blood vessel formation requires the coordination of endothelial cell division and the morphogenetic movements of vessel expansion, but it is not known how this integration occurs. Here, we show that endothelial cells regulate division orientation during the earliest stages of blood vessel formation, in response to morphogenetic cues. In embryonic stem (ES) cell-derived vessels that do not experience flow, the plane of endothelial cytokinesis was oriented perpendicular to the vessel long axis. We also demonstrated regulated cleavage orientation in vivo, in flow-exposed forming retinal vessels. Daughter nuclei moved away from the cleavage plane after division, suggesting that regulation of endothelial division orientation effectively extends vessel length in these developing vascular beds. A gain-of-function mutation in VEGF signaling increased randomization of endothelial division orientation, and this effect was rescued by a transgene, indicating that regulation of division orientation is a novel mechanism whereby VEGF signaling affects vessel morphogenesis. Thus, our findings show that endothelial cell division and morphogenesis are integrated in developing vessels by flow-independent mechanisms that involve VEGF signaling, and this cross talk is likely to be critical to proper vessel morphogenesis.

Animals↗
PubMed2006

Exogenous leukemia inhibitory factor (LIF) attenuates retinal vascularization reducing cell proliferation not apoptosis.

To study the effect of leukemia inhibitory factor (LIF) on rat retinal vascular development, Sprague-Dawley rats at postnatal age 3 days (p3) were given intraperitoneal (IP) LIF and analysis performed at p6 (p3/6). p7 rats were given intravitreous (IV) LIF and analysis performed at p9 (p7/9). Control animals were PBS injected. At the time of analysis retinal flatmounts were prepared and stained with Griffonia lectin and activated caspase-3. The retinal peripheral avascular area was measured and number of apoptotic cells counted. In vitro, human retinal microvascular endothelial cells (RMVECs) were cultured in media containing LIF, with and without neutralizing antibody to LIF. Cells were stained with activated caspase-3 and apoptotic cells counted. Proliferation was measured by counting cell numbers, and cell cycle stage was determined using propidium iodide staining and FACS analysis. LIF injected either IP or IV had no effect on body weight or total retina area, but significantly increased the peripheral retinal avascular area. In both IP and IV injected groups there was no difference in the number of apoptotic cells between PBS- or LIF-injected groups; although in the p7/9 retinas, both injected groups had significantly more apoptotic cells than the non-injected group. In vitro, there was no effect of LIF on RMVEC apoptosis; however, cell counts were significantly lower in the LIF-treated group. Antibody to LIF restored the cell counts to untreated levels. LIF reduced the number of cells in S phase. LIF attenuates retinal vascular development in vivo through growth arrest, and not apoptosis, of endothelial cells.

Animals↗
PubMed2006

Characterization of barrier properties and inducible VEGF expression of several types of retinal pigment epithelium in medium-term culture.

PURPOSE: To investigate and compare the characteristics of four different types of retinal pigment epithelium (RPE) cells cultured for 2 to 5 weeks to provide guidance when choosing RPE cells for experimentation. METHODS: Human cell lines ARPE-19 (ARPE) and D407, primary RPE cells from C57Bl/6 mouse (mRPE), and primary human fetal RPE (hfRPE) cells were grown in respective media previously reported to be optimal for each cell type. Two methods to obtain hfRPE were used: one isolated outside and transported to our laboratory, and one isolated primarily within our laboratory from donor human fetal eyes. Barrier function was determined by transepithelial electrical resistance (TER) and permeability and structure by localization of Na+,K+-ATPase alpha-1, ZO-1, and actin. VEGF expression, determined by real-time polymerase chain reaction (PCR) for mRNA and ELISA for protein, was determined after exposure to 24 h of 1% oxygen. Madin-Darby canine kidney (MDCK) cells were compared as a non-RPE epithelial cell line. RESULTS: ARPE at passage 15, but not passage 32, maintained steady low TER measurements (up to 30 ohms x cm(2)) despite forming a monolayer with apical Na+,K+-ATPase alpha-1 labeling after 35 days. mRPE developed and maintained a TER of 30 ohms x cm(2) for 2 weeks but did not localize ATPase. hfRPE showed two phenotypes. hfRPE isolated remotely and sent to us appeared more mesenchymal and undifferentiated (hfRPE-U) and had a slow but steady increase in measured TER to approximately 25 ohms x cm(2), whereas hfRPE isolated from donor eyes in our laboratory showed well-differentiated monolayers (hfRPE-D) with TER measurements > 500 ohms x cm(2) within 1 month of culture. TER measurements reflected permeability determined by the measurement of paracellular movement of sodium fluorescein. All human RPE cell types showed expression of VEGF mRNA and protein, and expression was upregulated by hypoxia in hfRPE and D407, but not in ARPE, which had constitutively high expression. ARPE expressed high levels of VEGF protein in media and cell lysates (777.2; 54.4 pg/mg protein, respectively), whereas hfRPE and D407 produced significantly less (media: 5.7 [p = 0.001], 323.6 pg/mg protein [p = 0.01]; lysate: 0 [p < 0.001], 3.5 pg/mg protein [p < 0.001], respectively). CONCLUSIONS: Primary RPE cells and those from cell lines had different responses to medium-term culture or hypoxic stress. Primary isolation of hfRPE cells with careful control of culture conditions to assure adequate differentiation is recommended when using this cell as an example of a highly polarized epithelium. For disease, use of RPE cells that do not require long-term culture are more efficient and may be more relevant to study certain pathologies.

Actins↗
PubMed2006

Fibrovascular organization in the vitreous following laser for ROP: implications for prognosis.

PURPOSE: To study associations between surgical outcome and mean postmenstrual age (PMA) when fibrovascular organization is detected between vascular and avascular retina following laser for acute retinopathy of prematurity (ROP). METHODS: PMA at the time of detection of fibrovascular organization was determined in infants who had laser treatment for stage 3 ROP. Retinal features abstracted from examination sheets included zone, stage, and clock hours of fibrovascular organization, PMA at the times of first surgery and diagnosis of fibrovascular organization, and outcomes (retinal attachment after one surgery and retinal attachment at follow-up). Statistical analyses were performed to compare categorical data (Mann-Whitney U test, t-test, Fisher exact test) and determine correlations (Spearman rank test). RESULTS: Fibrovascular organization was diagnosed in 38/39 eyes that required surgery and 19/41 eyes that did not. In surgical eyes, older PMA at the time of detection of fibrovascular organization, zone II ROP, and stage 4 (versus stage 5) ROP were each associated with successful reattachment of the retina after one surgery and at the end of follow-up. PMA at diagnosis of fibrovascular organization was associated with zone, but not stage, of ROP when surgical intervention was performed. CONCLUSION: Fibrovascular organization between the vascular and avascular retina is important because it is associated with the development of retinal detachment after laser for acute ROP. Further study is required to determine if improved detection of fibrovascular organization in eyes of infants of early PMA will improve surgical outcomes for retinal detachment.

Disease Progression↗
PubMed2006

Triamcinolone reduces neovascularization, capillary density and IGF-1 receptor phosphorylation in a model of oxygen-induced retinopathy.

PURPOSE: To study the effects of intravitreous triamcinolone acetonide (TA) on neovascularization (NV), capillary density, and retinal endothelial cell (REC) viability in a model of oxygen-induced retinopathy (OIR). METHODS: Newborn rats exposed to OIR underwent intravitreous injections (right eye) at day 14 to achieve intravitreous concentrations of: dexamethasone (DEX) (0.3 mg/mL), triamcinolone (TA; 0.4-4 mg/mL), or PBS. Animals were removed to room air and at day 18, retinal flatmounts were assayed for clock hours of NV, percent peripheral avascular retina, capillary density, apoptosis, and VEGF protein. At day 15, retinas were assayed for insulin-like growth factor (IGF)-1 receptor phosphorylation (IGF-1Rphos). Human RECs exposed to TA were assayed for trypan blue exclusion or activated caspase-3. RESULTS: TA but not DEX or PBS reduced NV (ANOVA, P < 0.001), capillary density (ANOVA, P < 0.001), and systemic weight gain (ANOVA, P = 0.002). VEGF protein was not different between TA- and PBS-injected or noninjected groups. Apoptosis was not increased in vivo or in vitro between groups, but there was a dose-dependent toxic effect of TA on cultured RECs (P < 0.001). At day 15, retinas from the 4 mg/mL TA-injected OIR group had a trend toward reduced IGF-1Rphos compared with room air-raised PBS- or non-injected OIR groups. CONCLUSIONS: TA caused dose-dependent reductions in NV, retinal vascularization, and systemic weight gain associated with a reduction in IGF-1Rphos. Long-term studies are needed to assess TA toxicity in vivo. TA doses should be carefully considered before administering the drug in diseases with ongoing retinal vascular development, such as retinopathy of prematurity.

Animals↗
PubMed2005

Choroidal endothelial cells transmigrate across the retinal pigment epithelium but do not proliferate in response to soluble vascular endothelial growth factor.

The purpose of this study was to investigate the effects of soluble VEGF on human choroidal endothelial cell (CEC) transmigration across an RPE monolayer as it relates to choroidal neovascularization in AMD. In coculture assays, ARPE-19 (ARPE) was plated on the undersides of Transwell inserts having 0.4 microm pores. Primary human CECs were then plated into the insert. CECs in the Transwell inserts were counted after 72 hr of growth. CEC proliferation was also measured after culturing CECs in ARPE-CEC coculture-conditioned media or in media with exogenous VEGF121 and/or VEGF165 added. Transmigration assays were performed on Transwells with 8.0 microm pores: green-labelled CECs were plated in Transwell inserts with or without red-labelled ARPE plated on the undersides of the insert. In some transmigration assays, ARPE was plated into the wells to provide a chemotactic gradient for CEC transmigration. After 72 hr CECs were plated, green cells were counted either within the well media as CECs that transmigrated the epithelial monolayer, or on the underside of the insert as CECs that transmigrated the Transwell insert to but not beyond the ARPE monolayer. A neutralizing antibody to VEGF was added to the wells of Transwells at the time the CECs were plated in the insert and transmigrated CECs were counted. VEGF protein was measured in the conditioned media of ARPE and CEC coculture and in transmigration assays. Compared to control, CEC proliferation significantly increased when CECs were cultured in coculture conditioned media (p=0.001) or in coculture assays (p<0.001). However, there was no effect on CEC proliferation when VEGF121, VEGF165, or both were added to solo CECs. Antibody to VEGF did not reduce the proliferative effects of coculture conditioned media on CEC. ARPE plated in the well significantly increased CEC transmigration (p<0.001) compared to transmigration assays without ARPE in the well. VEGF protein measured in the well media of transmigration assays having ARPE within the well was significantly greater than in the assays without ARPE within the well (p<0.004). Exogenous neutralizing antibody to VEGF significantly reduced transmigration, and this effect was dose-dependent. VEGF provides a chemotactic gradient for human CECs to transmigrate across a monolayer of ARPE. Neutralization of VEGF in the media partially reduces transmigration. Whereas soluble VEGF does not increase proliferation of solo CECs, coculture conditioned media enhances proliferation, suggesting that growth factors other than VEGF cause CEC proliferation. These findings may have relevance to the transformation of occult CNV into CNV within the neurosensory retina in AMD.

Apoptosis↗
PubMed2004

VEGF isoforms and their expression after a single episode of hypoxia or repeated fluctuations between hyperoxia and hypoxia: relevance to clinical ROP.

PURPOSE: Fluctuations in oxygen are associated with the development of severe retinopathy of prematurity (ROP) in humans. However, the causal relationships between oxygen variability and severe ROP remain unknown. We investigated whether isoforms of vascular endothelial growth factor (VEGF) were differentially stimulated by hypoxia and by repeated fluctuations between hypoxia and hyperoxia, and whether isoforms were differentially expressed in association with intravitreous neovascularization. We also determined whether pigment epithelium-derived factor (PEDF) was dysregulated by oxygen fluctuations perhaps contributing to a delay in normal retinal vascular development. METHODS: We used the 50/10 oxygen-induced retinopathy (50/10 OIR) model that exposes newborn rat pups to repeated cycles of 24 h of 50% oxygen alternating with 24 h of 10% oxygen to cause a condition similar to human ROP. Animals were euthanized at postnatal day 2 (P2; after one cycle of 50/10% oxygen), P7 (after 3.5 cycles of 50/10% oxygen), and P14 (after seven cycles of 50/10% oxygen). Room air raised control rat pups were also exposed to a single episode of 24 h of hypoxia at P7 and P14 and assayed immediately afterwards. Retinal VEGF isoforms and PEDF were measured by RT-PCR. Total VEGF protein was measured by ELISA. RESULTS: We found that repeated cycles of hyperoxia and hypoxia caused greater expression of VEGF protein compared to control than did a single cycle of hyperoxia and hypoxia. VEGF164 mRNA had a greater fold change over control after repeated oxygen fluctuations than after a single episode of hypoxia. However, the other isoforms, VEGF188 and VEGF120, were expressed to a similar degree regardless of whether the stimulus was a single episode of hypoxia or repeated fluctuations in oxygen. VEGF164 was the predominant isoform expressed at the time of maximal intravitreous neovascularization. Retinal PEDF expression was elevated in pups in the 50/10 OIR model compared to control at P7, immediately after 50% oxygen. PEDF expression in the experimental group was similar to control at P18, when intravitreous neovascularization occurred. CONCLUSIONS: Repeated fluctuations in oxygen results in a greater expression of the pathologic isoform, VEGF164, than does hypoxia alone. However, the other isoforms were upregulated to an equivalent degree over control by repeated fluctuations in oxygen or a single episode of hypoxia. Total VEGF protein was increased to a greater degree by repeated fluctuations in oxygen compared to a single cycle of oxygen. PEDF was increased over control early in the 50/10 OIR model and may play a role in the observed delay in retinal vascularization. These findings provide insight into the effect of repeated oxygen fluctuations on the development of severe ROP in preterm infants.

Animals↗
PubMed2004

Retinal features predictive of progressive stage 4 retinopathy of prematurity.

PURPOSE: To determine the retinal features predictive of progressive stage 4 retinopathy of prematurity (ROP) after laser treatment for threshold ROP. METHODS: Retrospective review of 72 eyes of 37 infants after laser treatment for threshold ROP between 1993 and 2002. Retinal features were abstracted from examinations made within 1 week of development of stage 4A ROP or 2 weeks after laser treatment in eyes with regressed threshold disease. Predictive features of progressive stage 4 ROP were determined using a generalized estimating equation model to account for within-subject variability. RESULTS: The generalized estimating equation showed that vitreous state, ridge elevation of six or more clock hours, and two or more quadrants of plus disease predicted progressive retinal detachment, whereas two or more quadrants of neovascularization did not. CONCLUSIONS: Progressive stage 4 ROP requiring surgical intervention was predicted by the absence of clear vitreous, ridge elevation of six or more clock hours, and two or more quadrants of plus disease, but not by neovascularization. These results may be useful in the management of eyes after laser treatment for threshold ROP.

Disease Progression↗
PubMed2004

Comparison of retinal outcomes after scleral buckle or lens-sparing vitrectomy for stage 4 retinopathy of prematurity.

PURPOSE: To compare anatomic outcomes after lens-sparing vitrectomy (LSV) or scleral buckle (SB) for stage 4 retinopathy of prematurity (ROP). METHODS: Nonrandomized, retrospective study of infants consecutively treated for stage 4 ROP by LSV or SB. Outcomes were retinal attachment at 1 month of initial surgery and at the end of follow-up (6 months) and number of procedures to achieve retinal attachment. Exact chi2 methods determined significance, and student's t-test compared mean postmenstrual age and birthweight between the groups. RESULTS: After one procedure, LSV (72%) was associated with retinal attachment more often than was SB (31%). At the end of follow-up, after one or more procedures, there was no difference in retinal reattachment rate between LSV or SB as the first procedure. There were no differences between the surgical groups by mean postmenstrual age and birthweight or severity of ROP determined by zone, clock hours of ridge elevation, or quadrants of plus disease. CONCLUSION: This study supports the hypothesis that vitrectomy by LSV stops progressive stage 4 ROP. As an initial procedure, LSV was associated with retinal attachment more often than SB. Future prospective studies can determine the effects of LSV and SB on visual development in progressive stage 4 ROP.

Birth Weight↗
PubMed2003

Hypoxic oxygen fluctuations produce less severe retinopathy than hyperoxic fluctuations in a rat model of retinopathy of prematurity.

The aim of this study was to investigate whether the mean around which arterial oxygen fluctuations take place was important in a unique animal model of oxygen-induced retinopathy. Retinopathy of prematurity (ROP) is associated with fluctuating arterial oxygen. A recent retrospective study suggested that management of high-risk preterm infants at lower oxygen saturations was associated with less severe ROP. Rat pups were raised in a variable oxygen environment around a high (24%), normal (21%) or low (17%) mean inspired oxygen for 14 d. Rat pups raised in the high (24%) mean variable oxygen environment had more retarded retinal vascular development than did rats raised in an environment that fluctuated around 21% mean oxygen. In contrast, rats raised in a lower mean (17%) but still variable oxygen environment had no discernible retinal differences from controls raised in constant room air. Rats raised in a relatively hypoxic but variable oxygen environment develop less severe retinal vascular abnormalities than those raised in variable oxygen around higher oxygen means.

Animals↗
PubMed2003

Long-term vision results measured with Teller Acuity Cards and a new Light Perception/Projection Scale after management of late stages of retinopathy of prematurity.

OBJECTIVES: To report visual acuity (VA) measured by Teller Acuity Cards (TACs) and a new Light Perception/Projection (LPP) Scale in infants with regressed or treated stage 3, 4, or 5 retinopathy of prematurity (ROP), and to compare VA in eyes that underwent successful vitreoretinal surgery for stage 5 ROP with eyes with persistent retinal detachment. METHODS: Nineteen infants (35 eyes) underwent VA testing using TACs and the LPP scale. The correlation between the methods was determined. Comparisons in VA scores were made in eyes by stage of ROP at the first examination and retinal status at the end of follow-up and between eyes with successful surgical reattachment and persistent retinal detachment. RESULTS: Scores obtained with the LPP scale and TACs were highly correlated (Spearman rank-order correlation coefficient, 0.92; P<.001). Visual acuity was better in eyes with retinal attachment at the end of follow-up than in eyes with retinal detachment whether the ROP stage at first examination was 4A (n = 6), 4B (n = 16), or 5 (n = 6). In eyes that progressed to stage 5 ROP and had successful surgical retinal reattachment (n = 16), both methods of measurement yielded better visual function than in eyes with persistent retinal detachment. The LPP scale provided scores for eyes without quantifiable grating acuity determined with TAC. CONCLUSIONS: The LPP scale scores were correlated with TAC scores in infants with stages 3, 4, and 5 ROP. Surgical retinal reattachment in stage 5 ROP resulted in better visual function. The LPP scale may be useful in measuring low vision in infants without quantifiable grating acuity and with later stages of ROP.

Female↗
PubMed2003

Duration of effect of intravenous antibiotics on spirometry and sputum cytokines in children with cystic fibrosis.

Intravenous (IV) antibiotics are a mainstay of therapy in children with cystic fibrosis. It is unclear, however, over what period associated improvements in pulmonary function are maintained, and to what extent the underlying inflammatory process is impeded in children admitted for a course of IV antibiotics. This was a prospective, interventional study of 14 children (median age, 14 years; interquartile range, 10-14) with cystic fibrosis who were regular sputum producers and who required admission for a 2-week course of IV antibiotics. Children performed spirometry and provided a sputum sample prior to starting IV antibiotics and then weekly for 6 weeks, the first 2 weeks of which IV antibiotics were given. Sputum IL-8, TNF-alpha, IL-6, IL-10, MIP1-alpha, and elastase were measured. Seven children were asked to repeat the protocol in a subsequent exacerbation to assess similarities in response to therapy. Significant improvements were seen in forced expired volume in 1 sec (FEV(1)) in association with IV antibiotics (27% relative improvement in predicted from baseline to end of week 1, median FEV(1) 41.3% increasing to 52.2%), but this continued only 1 week following cessation of antibiotics. Although IL-8 demonstrated a trend for reduction in association with antibiotics, no significant profile was demonstrated for any of the cytokines assessed. IL-10 was detectable in 64% of samples (all <100 pg/ml). In children with two episodes assessed, although there was a close correlation of FEV(1) and FVC between exacerbations (before antibiotics), no significant correlation was seen for IL-8, TNF-alpha, or IL-10 measured in both sets of samples at any sample point (indeed, a discordant response was seen between sample points in the two exacerbations). Although FEV(1) temporarily improves in response to admission for IV antibiotics, no such response is seen in sputum cytokine values. In addition, assessment of cytokines in subsequent exacerbations does not show a similar pattern of response to treatment.

Adolescent↗
PubMed2003

Retinal pigment epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism.

PURPOSE: To investigate the effect of endothelial cells (EC) on the barrier function of the retinal pigment epithelium (RPE). METHODS: Primary bovine RPE were grown in solo culture or in coculture with bovine EC. Culture media of RPE were varied to develop a monolayer with stable barrier properties determined by transepithelial electrical resistance (TER) and permeability to sodium fluorescein. The effect of EC on the barrier properties of RPE was tested in contacting and non-contacting cocultures of RPE and EC. The conditioned media of cocultures were analysed for soluble vascular endothelial growth factor (VEGF) by ELISA. A neutralizing antibody to VEGF(165) was added to cocultures of RPE and EC and the TER was measured. RESULTS: RPE had maximal barrier properties (high TER, low permeability, positive staining for barrier proteins) at day 10 that persisted until day 20. Compared to solo RPE culture, cocultivation of RPE with EC reduced RPE barrier function significantly and led to a greater release of soluble VEGF into the conditioned media (p<0.05). Neutralizing VEGF with antibody led to partial recovery of barrier properties in the coculture conditions (p<0.03). CONCLUSIONS: Coculture of RPE with EC reduces RPE barrier properties and the reduction is, in part, mediated by soluble VEGF. EC-RPE contact-induced disruption of barrier properties occurs in ocular pathologies such as choroidal neovascularization, where EC move through Bruch's membrane and contact the RPE, leading to further exacerbation of the already compromised blood-retinal barrier.

Animals↗