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Ocular levels of IL-1β, TNFα, IL-8, and IL-6 correlate with progression of diabetic retinopathy (DR). Müller cells (MC), which are crucial to maintaining retinal homeostasis, are targets and sources of these cytokines. We explored the relative capacities of these four DR-associated cytokines to amplify inflammatory signal expression both in and between human MC (hMC) and retinal microvascular endothelial cells (hRMEC) and in the mouse retina. Of the four cytokines, IL-1β was the most potent stimulus of transcriptomic alterations in hMC and hRMEC in vitro, as well as in the mouse retina after intravitreal injection in vivo. Stimulation with IL-1β significantly induced expression of all four transcripts in hMC and hRMEC. TNFα significantly induced expression of some, but not all, of the four transcripts in each cell, while neither IL-8 nor IL-6 showed significant induction in either cell. Similarly, conditioned media (CM) derived from hMC or hRMEC treated with IL-1β, but not TNFα, upregulated inflammatory cytokine transcripts in the reciprocal cell type. hRMEC responses to hMC-derived CM were dependent on IL-1R activation. In addition, we observed a correlation between cytokine expression changes following direct and CM stimulation and NFκB-p65 nuclear translocation in both hMC and hRMEC. Finally, in mice, intravitreal injections of IL-1β, but not TNFα, induced retinal expression of Il1b and CXCL8 homologues Cxcl1, Cxcl2, Cxcl3, and Cxcl5, encoding pro-angiogenic chemokines. Our results suggest that expression of IL-1β, TNFα, IL-8, and IL-6 may be initiated, propagated, and sustained by autocrine and paracrine signals in hRMEC and hMC through a process involving IL-1β and NFκB. Targeting these signals may help thwart inflammatory amplification, preventing progression to vision-threatening stages and preserving sight., Competing Interests: Declaration of competing interest There are no financial or non-financial competing interests declared by any author., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To develop a reliable method to generate a mouse model of branch retinal artery occlusion (BRAO) using laser-induced thrombosis of a major artery in the mouse retina. Also, to develop a reliable method to detect retinal hypoxia as predictive biomarker for the risk of neuronal cell damage in BRAO., Methods: A reliable and reproducible model of laser-induced BRAO was developed in mouse retina using Rose Bengal. To characterize retinal hypoxia in BRAO, pimonidazole immunostaining and HYPOX-4 molecular imaging methods were used. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was used to characterize neuronal cell damage in the BRAO retina. Expression of mRNA in retinal tissues from BRAO and age-matched control retinas were analyzed using qRT-PCR., Results: Occlusion of a branch retinal artery near the optic nerve head (ONH) caused a pattern of retinal tissue hypoxia covering about 12.5% of the entire retina. TUNEL-positive cells were localized in all layers in BRAO retinal tissue cross sections. In addition, qRT-PCR data analysis suggests that BRAO is associated with both inflammation and hypoxia., Conclusions: This study provides a reliable method for BRAO in mouse retina and demonstrates the utility of molecular imaging method to detect retinal hypoxia as predictive biomarker for the risk of neuronal cell damage in BRAO. In addition, our data suggest that BRAO retinas are associated with inflammation and also associated with hypoxia-related neuronal cell damage., Perspectives: Imaging areas of retinal hypoxia may provide accurate diagnosis, evaluating retinal tissue injury from BRAO., (© 2024 The Author(s). Microcirculation published by John Wiley & Sons Ltd.)

Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1β protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1β mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1β., (Copyright © 2024 The Authors.)

Chronic low-grade retinal inflammation is an essential contributor to the pathogenesis of diabetic retinopathy (DR). It is characterized by increased retinal cell expression and secretion of a variety of inflammatory cytokines; among these, IL-1β has the reputation of being a major driver of cytokine-induced inflammation. IL-1β and other cytokines drive inflammatory changes that cause damage to retinal cells, leading to the hallmark vascular lesions of DR; these include increased leukocyte adherence, vascular permeability, and capillary cell death. Nuclear factor of activated T-cells (NFAT) is a transcriptional regulator of inflammatory cytokines and adhesion molecules and is expressed in retinal cells. Consequently, it may influence multiple pathogenic steps early in DR. We investigated the NFAT-dependency of IL-1β-induced inflammation in human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC). Our results show that an NFAT inhibitor, Inhibitor of NFAT-Calcineurin Association-6 (INCA-6), decreased IL-1β-induced expression of IL-1β and TNFα in hMC, while having no effect on VEGF, CCL2, or CCL5 expression. We also demonstrate that INCA-6 attenuated IL-1β-induced increases of IL-1β, TNFα, IL-6, CCL2, and CCL5 (inflammatory cytokines and chemokines), and ICAM-1 and E-selectin (leukocyte adhesion molecules) expression in hRMEC. INCA-6 similarly inhibited IL-1β-induced increases in leukocyte adhesion in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Finally, INCA-6 rescued IL-1β-induced permeability in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Taken together, these data demonstrate the potential of NFAT inhibition to mitigate retinal inflammation secondary to diabetes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Free fatty acid dysregulation in diabetics may elicit the release of inflammatory cytokines from Müller cells (MC), promoting the onset and progression of diabetic retinopathy (DR). Palmitic acid (PA) is elevated in the sera of diabetics and stimulates the production of the DR-relevant cytokines by MC, including IL-1β, which induces the production of itself and other inflammatory cytokines in the retina as well. In this study we propose that experimental elevation of cytochrome P450 epoxygenase (CYP)-derived epoxygenated fatty acids, epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acid (EDP), will reduce PA- and IL-1β-induced MC inflammation. Broad-spectrum CYP inhibition by SKF-525a increased MC expression of inflammatory cytokines. Exogenous 11,12-EET and 19,20-EDP significantly decreased PA- and IL-1β-induced MC expression of IL-1β and IL-6. Both epoxygenated fatty acids significantly decreased IL-8 expression in IL-1β-induced MC and TNFα in PA-induced MC. Interestingly, 11,12-EET and 19,20-EDP significantly increased TNFα in IL-1β-treated MC. GSK2256294, a soluble epoxide hydrolase (sEH) inhibitor, significantly reduced PA- and IL-1β-stimulated MC cytokine expression. 11,12-EET and 19,20-EDP were also found to decrease PA- and IL-1β-induced NFκB-dependent transcriptional activity. These data suggest that experimental elevation of 11,12-EET and 19,20-EDP decreases MC inflammation in part by blocking NFκB-dependent transcription and may represent a viable therapeutic strategy for inhibition of early retinal inflammation in DR.

Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARβ/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARβ/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARβ/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARβ/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARβ/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARβ/δ inhibition is a potential therapeutic strategy against early DR pathology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Exudative age-related macular degeneration (AMD), characterized by choroidal neovascularization (CNV), is the leading cause of irreversible blindness in developed countries. Anti-vascular endothelial growth factor (VEGF) drugs are the standard treatment for AMD, but they have limitations. Cell therapy is a promising approach for ocular diseases, and it is being developed in the clinic for the treatment of retinal degeneration, including AMD. We previously showed that subretinal injection of human umbilical tissue-derived cells (hUTCs) in a rodent model of retinal degeneration preserved photoreceptors and visual function through rescue of retinal pigment epithelial (RPE) cell phagocytosis. Here we investigated the effect of hUTCs on a rat model of laser-induced CNV and on a human RPE cell line, ARPE-19, for VEGF production. We demonstrate that subretinal injection of hUTCs significantly inhibited CNV and lowered choroidal VEGF in vivo . VEGF release from ARPE-19 decreased when co-cultured with hUTCs. Soluble VEGF receptor 1 (sVEGFR1) is identified as the only factor in hUTC conditioned medium (CM) that binds to VEGF. The level of exogenous recombinant VEGF in hUTC CM was dramatically reduced and could be recovered with sVEGFR1-neutralizing antibody. This suggests that hUTC inhibits angiogenesis through the secretion of sVEGFR1 and could serve as a novel treatment for angiogenic ocular diseases, including AMD.

Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.

Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: To demonstrate the utility of a novel in vivo molecular imaging probe, HYPOX-4, to detect and image retinal hypoxia in real time, in a mouse model of retinal vein occlusion (RVO)., Methods: Retinal vein occlusion was achieved in adult mice by photodynamic retinal vein thrombosis (PRVT). One or two major retinal vein(s) was/were occluded in close proximity to the optic nerve head (ONH). In vivo imaging of retinal hypoxia was performed using, HYPOX-4, an imaging probe developed by our laboratory. Pimonidazole-adduct immunostaining was performed and used as a standard ex vivo method for the detection of retinal hypoxia in this mouse RVO model. The retinal vasculature was imaged using fluorescein angiography (FA) and isolectin B4 staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) analysis., Results: By application of the standard ex vivo pimonidazole-adduct immunostaining technique, retinal hypoxia was observed within 2 hours post-PRVT. The observed hypoxic retinal areas depended on whether one or two retinal vein(s) was/were occluded. Similar areas of hypoxia were imaged in vivo using HYPOX-4. Using OCT, retinal edema was observed immediately post-PRVT induction, resolving 8 days later. Nominal preretinal neovascularization was observed at 10 to 14 days post-RVO., Conclusions: HYPOX-4 is an efficient probe capable of imaging retinal hypoxia in vivo, in RVO mice. Future studies will focus on its use in correlating retinal hypoxia to the onset and progression of ischemic vasculopathies.

The objective of the present study was to assess the effect of elevating epoxygenated fatty acids on retinal vascular inflammation. To stimulate inflammation we utilized TNFα, a potent pro-inflammatory mediator that is elevated in the serum and vitreous of diabetic patients. In TNFα-stimulated primary human retinal microvascular endothelial cells, total levels of epoxyeicosatrienoic acids (EETs), but not epoxydocosapentaenoic acids (EDPs), were significantly decreased. Exogenous addition of 11,12-EET or 19,20-EDP when combined with 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of epoxide hydrolysis, inhibited VCAM-1 and ICAM-1 expression and protein levels; conversely the diol product of 19,20-EDP hydrolysis, 19,20-DHDP, induced VCAM1 and ICAM1 expression. 11,12-EET and 19,20-EDP also inhibited leukocyte adherence to human retinal microvascular endothelial cell monolayers and leukostasis in an acute mouse model of retinal inflammation. Our results indicate that this inhibition may be mediated through an indirect effect on NFκB activation. This is the first study demonstrating a direct comparison of EET and EDP on vascular inflammatory endpoints, and we have confirmed a comparable efficacy from each isomer, suggesting a similar mechanism of action. Taken together, these data establish that epoxygenated fatty acid elevation will inhibit early pathology related to TNFα-induced inflammation in retinal vascular diseases.

Objective: To determine the effect of oleic acid and linoleic acid on the production and secretion of specific diabetic retinopathy- (DR-) related cytokines: vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) by human retinal glial cells, retinal endothelial cells, and retinal pigment epithelial cells. These expression profiles will be compared to those obtained by treatment of the same cell types with elevated D-glucose, a diabetes-relevant stimulus often used in retinal cell culture experiments., Methods: Primary cultures of human retinal Müller cells, astrocytes, and microvascular endothelial cells (RMEC) and a human retinal pigment epithelial cell line (ARPE-19) were treated with oleic acid, linoleic acid, elevated D-glucose, or L-glucose as an osmotic control. VEGF, IL-6, and IL-8 concentrations in conditioned media were determined by colorimetric ELISA and normalized to total cellular protein., Results: In the conditioned medium of human Müller cells, linoleic and oleic acid increased VEGF production by 6.4-fold and 9.9-fold, respectively. Linoleic acid also significantly increased IL-6 by 2.9-fold and IL-8 by 5.7-fold. L-glucose and D-glucose both increased VEGF by 3.1-fold in Müller cell conditioned medium. Linoleic acid increased IL-8 concentrations by 56% in human RMEC conditioned medium. Human retinal astrocytes and ARPE-19 were unaffected by all stimuli., Conclusions: Linoleic and oleic acid induce inflammatory mediators believed to be involved in the pathogenesis of diabetic retinopathy (DR). In culture, the free fatty acid insults, particularly linoleic acid, significantly increased cytokine production by Müller cells. In summary, these data identified Müller cells as the primary producer of these inflammatory mediators when treated with unsaturated fatty acids. This study also demonstrates that elevated glucose is an inadequate stimulus for assessing the production of inflammatory mediators. Therefore this study provides a novel in vitro model system of the dyslipidemia-induced inflammation occurring in DR.

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.

Diabetic retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive, and have limited effectiveness. Retinal pericyte death is an early pathologic feature of DR. Although it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in ocular tissues. In this article we examined the involvement of the GAPDH/Siah1 interaction in human retinal pericyte (hRP) apoptosis. HRP were cultured in 5 mm normal glucose, 25 mm l- or d-glucose for 48 h (osmotic control and high glucose treatments, respectively). Siah1 siRNA was used to down-regulate Siah1 expression. TAT-FLAG GAPDH and/or Siah1-directed peptides were used to block GAPDH and Siah1 interaction. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on the association of GAPDH and Siah1. Apoptosis was measured by Annexin V staining and caspase-3 enzymatic activity assay. High glucose increased Siah1 total protein levels, induced the association between GAPDH and Siah1, and led to GAPDH nuclear translocation. Our findings demonstrate that dissociation of the GAPDH/Siah1 pro-apoptotic complex can block high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Thus, the work presented in this article can provide a foundation to identify novel targets for early treatment of DR., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose: The peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is a transcription factor with roles in metabolism, angiogenesis, and inflammation. It has yet undefined roles in retinal inflammation and diabetic retinopathy (DR). We used RNA-seq to better understand the role of the antagonist and inverse agonist of PPARβ/δ, GSK0660, in TNFα-induced inflammation. Understanding the underlying mechanisms of vascular inflammation could lead to new treatments for DR., Methods: RNA was isolated from human retinal microvascular endothelial cells treated with a vehicle, TNFα, or TNFα plus GSK0660. RNA-seq was performed with a 50 bp single read protocol. The differential expression was determined using edgeR and gene ontology, and a pathway analysis was performed using DAVID. RNA-seq validation was performed using qRT-PCR using the primers for ANGPTL4, CCL8, NOV, CXCL10, and PDPK1., Results: TNFα differentially regulated 1,830 transcripts, many of which are involved in the cytokine-cytokine receptor interaction, chemokine signaling, and inflammatory response. Additionally, TNFα highly upregulated genes involved in leukocyte recruitment, including CCL5, CX3CL1, and CXCL10. GSK0660 differentially regulated 273 transcripts in TNFα-treated cells compared to TNFα alone. A pathway analysis revealed the enrichment of cytokine-cytokine receptor signaling. In particular, GSK0660 blocks the TNFα-induced upregulation of CCL8, a chemokine involved in leukocyte recruitment., Conclusions: TNFα regulates several genes related to retinal leukostasis in retinal endothelial cells. GSK0660 blocks the effect of TNFα on the expressions of cytokines involved in leukocyte recruitment, including CCL8, CCL17, and CXCL10 and it may therefore block TNFα-induced retinal leukostasis.

Purpose: Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability contributes to diabetic macular edema (DME), a serious vision-threatening condition. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) antagonist/reverse agonist, GSK0660, inhibits VEGF-induced human retinal microvascular endothelial cell (HRMEC) proliferation, tubulogenesis, and oxygen-induced retinal vasculopathy in newborn rats. These VEGF-induced HRMEC behaviors and VEGF-induced disruption of endothelial cell junctional complexes may well share molecular signaling events. Thus, we sought to examine the role of PPARβ/δ in VEGF-induced retinal hyperpermeability., Methods: Transendothelial electrical resistance (TEER) measurements were performed on HRMEC monolayers to assess permeability. Claudin-1/Claudin-5 localization in HRMEC monolayers was determined by immunocytochemistry. Extracellular signal-regulated protein kinases 1 and 2 (Erk 1/2) phosphorylation, VEGF receptor 1 (VEGFR1) and R2 were assayed by Western blot analysis. Expression of VEGFR1 and R2 was measured by quantitative RT-PCR. Last, retinal vascular permeability was assayed in vivo by Evans blue extravasation., Results: Human retinal microvascular endothelial cell monolayers treated with VEGF for 24 hours showed decreased TEER values that were completely reversed by the highest concentration of GSK0660 (10 μM) and PPARβ/δ-directed siRNA (20 μM). In HRMEC treated with VEGF, GSK0660 stabilized tight-junctions as evidenced by Claudin-1 staining, reduced phosphorylation of Erk1/2, and reduced VEGFR1/2 expression. Peroxisome proliferator-activated receptor β/δ siRNA had a similar effect on VEGFR expression and Claudin-1, supporting the specificity of GSK0660 in our experiments. Last, GSK0660 significantly inhibited VEGF-induced retinal vascular permeability and reduced retinal VEGFR1and R2 levels in C57BL/6 mice., Conclusions: These data suggest a protective effect for PPARβ/δ antagonism against VEGF-induced vascular permeability, possibly through reduced VEGFR expression. Therefore, antagonism/reverse agonism of PPARβ/δ siRNA may represent a novel therapeutic methodology against retinal hyperpermeability and is worthy of future investigation., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: We investigated endoglin expression in hypoxic microvascular endothelial cells and retinal endoglin expression in rats that develop experimental oxygen-induced retinopathy (OIR). We also tested neutralizing antibodies (Abs) against endoglin (anti-CD105 Ab) and VEGF (anti-VEGF Ab) either alone or in combination for efficacy against serum-induced retinal microvascular endothelial cell proliferation and retinal neovascularization (NV) in OIR rats. To our knowledge, this marks the first time that a biologic agent has been used to target retinal endoglin and modulate retinal neovascularization., Methods: Induction of endoglin by hypoxia was measured by immunohistochemical analysis and ELISA. Proliferation was quantified using a colorimetric 5-bromo-2-deoxyuridine ELISA. Western blots were used to measure endoglin levels in retinas of OIR rats. Immunohistochemical staining was also preformed in OIR rats using anti-CD105 and fluorescein isothiocyanate-conjugated isolectin B4 antibodies., Results: Anti-CD105 Ab and Anti-VEGF Ab, administered alone or in combination, reduced serum-induced retinal microvascular endothelial cell proliferation. Additionally, in a rat model of oxygen-induced retinopathy, retinal endoglin was significantly increased at 14(2), 14(3), 14(4) and 14(6) compared with retinal levels in control rats. At 14(2), immunohistochemical analysis demonstrated that endoglin was elevated in newly developed vessels at the peripheral extent of major veins, precisely where NV is expected to develop in OIR rats. Neutralizing anti-CD105 reduced retinal NV in OIR rats., Conclusions: Our data support other studies showing that reduction of endoglin expression inhibits retinal NV. Our findings demonstrate that retinal endoglin immunolocalization overlaps with nascent neovascular structures in OIR rats. Therefore, endoglin may serve as a useful predictor of incipient neovascular disease., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: The purpose of this study was to investigate the role(s) of cytochrome P450 epoxygenases (CYPs) and their products, the epoxyeicosatrienoic acids (EETs), in hypoxia-induced VEGF production and pathologic retinal angiogenesis., Methods: Human retinal astrocytes, Müller cells, and retinal microvascular endothelial cells (HRMEC) were exposed to hypoxia, and relative CYP2C expression was measured by RT-PCR. Astrocyte and Müller cell VEGF production was measured by ELISA after exposure to hypoxia and treatment with the general CYP inhibitor, SKF-525a. Human retinal microvascular endothelial cells were treated with the CYP product, 11,12-epoxyeicosatrienoic acid [EET], or SKF-525a in the presence or absence of VEGF. Proliferation of HRMEC and tube formation were assayed. Oxygen-induced retinopathy (OIR) was induced in newborn rats. Retinal CYP2C11 and CYP2C23 expression were measured by RT-PCR. The OIR rats received SKF-525a by intravitreal injection and preretinal neovascularization (NV) was quantified. Retinal VEGF protein levels were measured by ELISA., Results: Human retinal astrocytes were the only cells to exhibit significant induction of CYP2C8 and CYP2C9 mRNA expression by hypoxia. Astrocytes, but not Müller cells, exhibited reduced hypoxia-induced VEGF production when treated with SKF-525a. 11,12-EET induced HRMEC proliferation and tube formation, and SKF-525a inhibited VEGF-induced proliferation. Oxygen-induced retinopathy induced expression of CYP2C23, but had no effect on CYP2C11. SKF-525a inhibited retinal NV and reduced retinal VEGF levels in OIR rats., Conclusions: The CYP-derived 11,12-EET may exhibit a proangiogenic biological function in the retina following stimulation by hypoxia in astrocytes. Inhibition of CYP may provide a rational therapy against retinal NV, because it can reduce VEGF production and VEGF-induced angiogenic responses in endothelial cells., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

The purpose of this study was to investigate the hypoxia-induced Vegf120, Vegf164 and Vegf188 mRNA expression profiles in rat Müller cells (MC), astrocytes, retinal pigmented epithelial cells (RPE) and retinal microvascular endothelial cells (RMEC) and correlate these findings to VEGF secreted protein. Cultured cells were exposed to normoxia or hypoxia. Total RNA was isolated from cell lysates and Vegf splice variant mRNA copy numbers were assayed by a validated qRT-PCR external calibration curve method. mRNA copy numbers were normalized to input total RNA. Conditioned medium was collected from cells and assayed for total VEGF protein by ELISA. Hypoxia increased total Vegf mRNA and secreted protein in all the retinal cell types, with the highest levels observed in MC and astrocytes ranking second. Total Vegf mRNA levels in hypoxic RPE and RMEC were comparable; however, the greatest hypoxic induction of each Vegf splice variant mRNA was observed in RMEC. RPE and RMEC ranked 3rd and 4th respectively, in terms of secreted total VEGF protein in hypoxia. The Vegf120, Vegf164 and Vegf188 mRNA splice variants were all increased in hypoxic cells compared to normoxic controls. In normoxia, the relative Vegf splice variant mRNA levels ranked from highest to lowest for each cell type were Vegf164 > Vegf120 > Vegf188. Hypoxic induction did not alter this ranking, although it did favor an increased stoichiometry of Vegf164 mRNA over the other two splice variants. MC and astrocytes are likely to be the major sources of total Vegf, Vegf164 splice variant mRNAs, and VEGF protein in retinal hypoxia., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To develop new therapies against ocular neovascularization (NV), we tested the effect of peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ) agonism and antagonism on angiogenic behaviors and in human retinal microvascular endothelial cells (HRMEC) and on preretinal NV in rat oxygen-induced retinopathy (OIR)., Methods: HRMECs were treated with the PPAR-β/δ agonist GW0742 and the antagonist GSK0660. Messenger RNA levels of a PPAR-β/δ target gene, angiopoietin-like-4 (angptl4) were assayed by qRT-PCR. HRMEC proliferation and tube formation were assayed according to standard protocols. OIR was induced in newborn rats by exposing them to alternating 24-hour episodes of 50% and 10% oxygen for 14 days. OIR rats were treated with GW0742 or GSK0660. Angptl4 protein levels were assessed by ELISA and preretinal NV was quantified by adenosine diphosphatase staining., Results: GW0742 significantly increased angptl4 mRNA, and GSK0660 significantly decreased angptl4 mRNA. GW0742 had no effect on HRMEC proliferation, but caused a significant and dose-responsive increase in tube formation. GSK0660 significantly reduced serum-induced HRMEC proliferation and tube formation in a dose-dependent manner. Intravitreal injection of GW0742 significantly increased total retinal Angptl4 protein, but intravitreal injection of GSK0660 had no effect. Intravitreal injection of GW0742 significantly increased retinal NV, as did GW0742 administered by oral gavage. Conversely, both intravitreal injection and intraperitoneal injection of GSK0660 significantly reduced retinal NV., Conclusions: PPAR-β/δ activation exacerbates, and its inhibition reduces, preretinal NV. PPAR-β/δ may regulate preretinal NV through a prodifferentiation/maturation mechanism that depends on Angptl4. Pharmacologic inhibition of PPAR-β/δ may provide a rational basis for therapeutic targeting of ocular NV.

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX activity, reduce the production of retinal VEGF and neovascularization in relevant models of ocular disease. We hypothesized that COX-2 mediates VEGF production in retinal Müller cells, one of its primary sources in retinal neovascular disease. The purpose of this study was to determine the role of COX-2 and its products in VEGF expression and secretion. These studies have more clearly defined the role of COX-2 and COX-2-derived prostanoids in retinal angiogenesis. Müller cells derived from wild-type and COX-2 null mice were exposed to hypoxia for 0-24 h. COX-2 protein and activity were assessed by western blot analysis and GC-MS, respectively. VEGF production was assessed by ELISA. Wild-type mouse Müller cells were treated with vehicle (0.1% DMSO), 10 microM PGE(2), or PGE(2) + 5 microM H-89 (a PKA inhibitor), for 12 h. VEGF production was assessed by ELISA. Hypoxia significantly increased COX-2 protein (p < 0.05) and activity (p < 0.05), and VEGF production (p < 0.0003). COX-2 null Müller cells produced significantly less VEGF in response to hypoxia (p < 0.05). Of the prostanoids, PGE(2) was significantly increased by hypoxia (p < 0.02). Exogenous PGE(2) significantly increased VEGF production by Müller cells (p < 0.0039), and this effect was inhibited by H-89 (p < 0.055). These data demonstrate that hypoxia induces COX-2, prostanoid production, and VEGF synthesis in Müller cells, and that VEGF production is at least partially COX-2-dependent. Our study suggests that PGE(2), signaling through the EP(2) and/or EP(4) receptor and PKA, mediates the VEGF response of Müller cells., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Purpose: To identify and characterize the role of cytosolic phospholipase A(2) (cPLA(2)) in retinal angiogenesis using relevant cell-based assays and a rodent model of retinopathy of prematurity., Methods: The phosphorylation states of cPLA(2) and p38 MAP kinase and the expression of COX-2 were assessed by Western blot analysis in rat Müller cells. The activities of PLA(2) enzymes in rat retinal lysates were assessed using a commercially available assay. Prostaglandin E(2) (PGE(2)) and VEGF levels in Müller cell-conditioned medium and in retinal tissue samples were measured by ELISA. Rat retinal microvascular endothelial cell proliferation was measured using a BrdU assay. Efficacy of the cPLA(2) inhibitor CAY10502 was tested using the rat model of oxygen-induced retinopathy (OIR) in which neovascularization (NV) was assessed by computer-assisted image analysis., Results: In Müller cells, hypoxia increased the phosphorylation of cPLA(2) and p38 MAP kinase by 4-fold and 3-fold respectively. The cPLA(2) inhibitor CAY10502 decreased hypoxia-induced PGE(2) and VEGF levels in Müller cell-conditioned medium by 68.6% (P < 0.001) and 46.6% (P < 0.001), respectively. Retinal cPLA(2) activity peaked 1 day after oxygen exposure in OIR rats. CAY10502 (250 nM) decreased OIR-induced retinal PGE(2) and VEGF levels by 69% (P < 0.001) and 40.2% (P < 0.01), respectively. Intravitreal injection of 100 nM CAY10502 decreased retinal NV by 53.1% (P < 0.0001)., Conclusions: cPLA(2) liberates arachidonic acid, the substrate for prostaglandin (PG) production by the cyclooxygenase enzymes. PGs can exert a proangiogenic influence by inducing VEGF production and by stimulating angiogenic behaviors in vascular endothelial cells. Inhibition of cPLA(2) inhibits the production of proangiogenic PGs. Thus, cPLA(2) inhibition has a significant influence on pathologic retinal angiogenesis.

Purpose: The efficacy of three matrix metalloproteinase (MMP) inhibitors with various selectivities (Ro-31-9790, AG3340, and DPC-A37668) was investigated in a rat model of retinopathy of prematurity, to examine the roles of MMP-2 and -9 in retinal neovascularization. The susceptibilities of MMP-2(-/-) and -9(-/-) mice to preretinal neovascularization were investigated in a mouse model of oxygen-induced retinopathy., Methods: Sprague-Dawley newborn rats were exposed to alternating episodes of 50% and 10% oxygen (variable oxygen exposure) to induce retinal neovascularization. Three MMP inhibitors with various selectivity profiles were administered to variable oxygen-exposed rats via local or systemic routes. Antineovascular efficacy was determined in drug-treated versus vehicle-treated rat pups by computerized imaging of adenosine diphosphatase (ADPase)-stained retinal flatmounts. Wild-type C57BL/6J and isogenic MMP-2(-/-) and -9(-/-) mice were exposed to 75% oxygen followed by normoxia. The mice were killed immediately before or after the normoxic exposure, and eyes were either harvested for retinal dissection and flatmounting or were paraffin embedded and sectioned. Retinal vascular area and retinal neovascularization were assessed by adenosine diphosphatase staining of retinal flatmounts and by counting preretinal nuclei of hematoxylin and eosin-stained retinal sections, respectively., Results: Ro-31-9790, AG3340, and DPC-A37668 had no effect on normal development of the rat retinal vasculature, regardless of dose or route of administration. Intravitreal injection of Ro-31-9790 (broad-spectrum) immediately after variable-oxygen exposure and 2 days after exposure resulted in 78% and 82% inhibition of retinal neovascularization, respectively. AG3340 (MMP-2- and -9-selective inhibitor) and DPC-A37668 (MMP-2-selective inhibitor) resulted in 65% and 52% inhibition, respectively, when administered by intravitreal injection immediately after variable-oxygen exposure. Intraperitoneal injection of 5, 15, and 50 mg/mL AG3340 or DPC-A37668 for 6 days after variable oxygen exposure resulted in 22% to 39% and 0% to 31% inhibition of neovascularization, respectively. AG3340 and DPC-A37668 administered by oral gavage at doses of 3, 10, or 30 mg/mL provided up to 42% and 86% inhibition of neovascularization, respectively. The average vascular areas of retinas from MMP-2(-/-) or -9(-/-) mice at postnatal day 12 were not significantly different from the wild-type control. There was a 75% (P < 0.001) and 44% (P < 0.01) reduction in preretinal neovascularization in oxygen-exposed MMP-2(-/-) and -9(-/-) mice at postnatal day 19, respectively, compared with wild-type control mice., Conclusions: The results of this study suggest that MMP-2 plays a predominant role in retinal angiogenesis in both the mouse and rat models of oxygen-induced retinopathy. Furthermore, MMP-2 inhibition may be a viable therapeutic approach for ocular diseases characterized by retinal neovascularization.

Eph receptor tyrosine kinases (RTKs) and their ligands, known as ephrins, play an important role in vascular remodeling during embryogenesis, but their functions in adult angiogenesis are just beginning to be investigated. In this report, we investigated the effect of blocking EphA receptor activation on VEGF-induced angiogenic responses of cultured retinal endothelial cells and on retinal neovascularization in a rodent model of retinopathy of prematurity (ROP). Soluble EphA2-Fc receptors inhibited ephrin-A1 ligand or VEGF-induced BRMEC migration and tube formation without affecting proliferation in vitro. Since EphA2-Fc receptors can inhibit activation of multiple EphA receptors, the specific role of EphA2 receptor in angiogenesis was further investigated in EphA2-deficient endothelial cells. Loss of EphA2 in endothelial cells leads to defective cell migration and assembly in response to either ephrin-A1 or VEGF. Finally, a significant reduction in the severity of abnormal retinal neovascularization was observed in the eyes treated with soluble EphA2-Fc receptors, yet the normal total retinal vascular area was not significantly changed. Because soluble Eph receptor significantly inhibited pathologic retinal angiogenesis without affecting normal intraretinal vessels, it may be a promising agent for treatment of retinal angiogenesis in a number of human ocular diseases.

Purpose: To characterize the angiostatic effect of penetrating ocular injury and to begin to explore its mechanism, with an emphasis on the role of pigment epithelium-derived factor (PEDF)., Methods: Using the rat model of oxygen-induced retinopathy (OIR), single or multiple dry needle injuries were made, penetrating the globe of one eye; the opposite eye served as a control. Eyes were harvested from rats killed 1, 3, and 6 days after injury, and retinas were dissected and processed for assessment of neovascularization and microglial activation or were processed for genetic and proteomic analysis. Temporal and spatial expression patterns of PEDF were analyzed by in situ hybridization., Results: Penetrating ocular injury resulted in a 30% decrease in neovascular area in the retinas of OIR rats. At day 1 after injury, needle insertion caused a 4.1-fold increase in retinal PEDF mRNA and a 1.5-fold increase in retinal PEDF protein. Vitreous PEDF protein increased 3.4-fold in injured eyes compared with noninjured eyes. In situ hybridization showed an increase in PEDF mRNA in areas surrounding the puncture site. Concentrated vitreous protein from injured eyes caused a 60% decrease in retinal neovascularization when injected into the vitreous cavity of OIR rats. Preincubation of vitreous samples with anti-PEDF partially abolished this efficacy., Conclusions: The pattern of angiostasis resulting from penetrating ocular injury is consistent with the release of an endogenous antiangiogenic factor from the wound site. Preliminary studies show a possible role for PEDF in this effect. Further characterization of this role and the identification of other factors may lead to new therapeutic strategies for angiogenic eye conditions.

Retinal capillary quiescence is regulated by a delicate balance between proangiogenic and anti-angiogenic factors. Pathological angiogenesis is the result of a shift in this balance towards proangiogenic influences. Pathological angiogenesis is produced in a rat model of oxygen-induced retinopathy (OIR) by exposing newborn rat pups to alternating periods of hyperoxia and hypoxia. Based upon previous work, two similar exposure paradigms were investigated and compared, exposure of rat pups to alternating periods of 45 and 12.5% oxygen, and to alternating periods of 40 and 15% oxygen. The resulting retinal pathology was assessed by measurement of retinal clock hours with pathological blood vessel growth and the percentage of the retina that is avascular. The 45 and 12.5% exposure produced significantly greater incidence and severity of pathology than the 40 and 15% protocol. To explain the difference in pathology between these two very similar exposure protocols, retinal levels of proangiogenic vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) and anti-angiogenic pigment epithelium-derived factor (PEDF) were measured by ELISA and western blot analysis at 0, 2, and 6 days post-exposure. In whole retinal lysates, there were no significant differences in VEGFR2 and PEDF levels. However, VEGF levels were approximately 48 and 78% higher on post-oxygen exposure day 0 and 2, respectively, in the group treated with alternating periods of 45 and 12.5% oxygen compared to the group treated with alternating periods of 40 and 15% oxygen. There was no significant difference in VEGF levels between these two groups on day 6 post-exposure. Therefore, the difference in pathology observed between these two experimental paradigms is associated with differences in whole retinal VEGF levels, but not changes in whole retinal VEGFR2 or PEDF levels. The results of this study suggest the existence of a threshold in the rat model of OIR, such that a small change in blood oxygen profile triggers a disproportionate increase in subsequent neovascularization, which is accompanied by more dramatic changes of retinal VEGF level than VEGFR2 or PEDF level. If a similar threshold exists for humans, it could explain why some oxygen-treated premature infants develop retinopathy and others do not, despite similar gestational ages, birth weights and clinical courses.

The pathogenesis of retinopathy of prematurity involves dysregulated angiogenesis resulting in pre-retinal growth of new vessels. Inhibition of tyrosine kinase-dependent pro-angiogenic signals may provide a rational therapeutic approach to the reduction of pre-retinal neovascularization. Vascular endothelial growth factor stimulates endothelial cell mitogenesis, differentiation and migration, by binding and activating the receptor tyrosine kinases vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2. One of the vascular endothelial growth factor receptor substrates implicated in vascular endothelial growth factor signal transduction is c-Src. The ability of herbimycin A, a c-Src-selective tyrosine kinase inhibitor, to inhibit vascular endothelial growth factor-induced bovine retinal microvascular endothelial cell proliferation and tube formation was investigated. The ability of the compound to inhibit pathologic angiogenesis was tested in a rat model of retinopathy of prematurity. Exposure of neonatal rats to oxygen concentrations cycling between 10 and 50% induced severe pre-retinal neovascularization in all rats. Some of the eyes of these variable oxygen-exposed rats were herbimycin A-injected or vehicle-injected 1 or 3 days post-oxygen exposure while some eyes were non-injected. All rats were sacrificed for assessment 6 days post-exposure. Herbimycin A inhibited both vascular endothelial growth factor-induced bovine retinal microvascular endothelial cell proliferation and capillary tube formation in a dose-dependent manner. Injection of herbimycin A into oxygen-treated rats 1 day post-oxygen exposure produced a 63% decrease in pre-retinal neovascularization relative to vehicle (P = 0.0029). There was a 41% decrease in pre-retinal neovascularization in herbimycin-injected eyes relative to vehicle-injected eyes 3 days post-oxygen (P = 0.031). Pre-retinal neovascularization was reduced in vehicle-injected eyes relative to non-injected eyes at both injection times. There were no significant differences in retinal vascular area between any of the experimental groups. Based on the results of this study, herbimycin A inhibits endothelial cell proliferation and tube formation at non-toxic concentrations and reduces pre-retinal neovascularization in a rat model of retinopathy of prematurity. Reduction of angiogenesis by the inhibition of tyrosine kinase activity may be a viable route to the development of effective chemotherapies applicable to eye disease.

Purpose: To study the differences in disease caused by two wild-type strains of Candida albicans in a model of contact lens-facilitated keratitis in rabbits., Methods: Two strains, SC5314 and VE175, were examined. Standardized inocula were placed on the debrided corneal surface of one eye in Dutch belted rabbits and covered with a contact lens. A temporary tarsorrhaphy was opened after 24 hours with removal of the contact lens. Six days later, corneas were photographed and animals killed. Corneas were bisected with one half for quantitative isolate recovery and the other for stromal penetration by hyphae., Results: Strain SC5314 was significantly more virulent. The mean hyphal penetration into the cornea was 24.4% +/- 8.5% of the corneal thickness, and in three of six corneas hyphae penetrated through the entire cornea. In contrast, for VE175, the mean hyphal penetration was 2.6% +/- 1.2%. The difference between these two strains was statistically significant (P = 0.0297). Hyphae did not penetrate into the deep layers of the cornea in any of the six rabbits infected with VE175. The grading of clinical disease was consistent with histology, in that strain SC5314 caused more severe infection than VE175 and the difference was statistically significant (P = 0.0048). There was no difference in isolate recovery., Conclusions: Wild-type strains of C. albicans can differ significantly in virulence as measured by depth of fungal invasion into corneas and clinical evaluation of infection. Further characterization of the intrinsic genetic differences between such strains may help identify factors responsible for fungal virulence.

Animal thioredoxin reductase is a selenoprotein. In this study, thioredoxin reductase activities in liver, kidney, and brain have been compared in rats fed selenium-deficient and control diets for 14 weeks following weaning. Selenium deficiency caused a decrease in thioredoxin reductase activity from control to 4.5% in liver and 11% in kidney. However, brain thioredoxin reductase activity was not affected by selenium deficiency of this severity. Gold inhibited thioredoxin reductase activity in the liver in a manner typical of its effect on selenoenzymes. Repletion of selenium-deficient rats with injections of selenium caused thioredoxin reductase activity to increase more rapidly in the liver than glutathione peroxidase activity but more slowly than selenoprotein P. These results indicate that thioredoxin reductase activity in liver and kidney is sensitive to selenium nutritional status but that brain thioredoxin reductase activity is less sensitive.

Administration of epidermal growth factor (EGF) to rats has been shown to induce both mitogenic and nonmitogenic responses in the intestine. The mechanisms to describe a multiplicity of hormonal responses within a single tissue are unclear but likely involve selectivity among receptor substrates. A nontransformed rat jejunal crypt intestinal epithelial cell line (IEC-6) was studied to determine if the regulation of receptor tyrosine kinase substrates is affected by cell population physiology. EGF stimulated a rapid increase in inositol trisphosphate in confluent but not subconfluent cells. Similarly, treatment of confluent IEC-6 cells with EGF provoked a significant increase in the hydrolysis of PtdIns 4,5-P2 by immunoisolated PLC gamma 1. The tyrosine phosphorylation state of PLC gamma 1 and the association of PLC gamma 1 with the EGF receptor were increased by EGF in confluent cells only. In contrast, the autophosphorylation state of the EGF receptor and the tyrosine phosphorylation state of another SH2-containing EGF receptor substrate SHC were increased by EGF regardless of cell density. Western blot analysis revealed equal protein expression of PLC gamma 1 in confluent and subconfluent cells. EGF receptor protein expression and ligand binding capacity were slightly increased in confluent compared to subconfluent cells. EGF regulation of PLC gamma 1, therefore, is regulated by physiological factors dependent on cell density in IEC-6 cells.

Oxygen-15 labeled 1-butanol and 2-butanol were prepared by the reaction of O2-15O with tri-n-butylborane and tri-sec-butylborane in tetrahydrofuran. The reaction products were isolated and identified via reversed-phase high performance column chromatography and gas chromatography. Radiochemical yields of 50% (EOB) were obtained. The use of 15O-labeled butanol is suggested for cerebral blood flow measurements in conjunction with PET.