- 2006 -

Inhibition of VEGF Expression and Corneal Neovascularization by siRNA Targeting the Cytochrome P450 (CYP) 4B1

M.L. Schwartzman, F. Seta, L. Bellner, A. Mezentsev, M.W. Dunn. Pharmacology, New York Medical College, Valhalla, NY.

Purpose: Injury to the cornea leads to formation of distinct classes of mediators that initiate and amplify inflammatory and neovascular responses including the CYP4B1-derived 12-HETrE, a potent inflammatory and angiogenic eicosanoid acting via induction of VEGF. We used small interfering RNA (siRNA) targeting CYP4B1 in a rabbit model of corneal neovascularization to substantiate the cause/effect relationship between CYP4B1 expression, 12-HETrE production and neovascularization. Methods: siRNA sequences were derived from the coding sequence of the rabbit CYP4B1. The efficacy of each siRNA duplex to inhibit CYP4B1 catalytic activity, i.e., 12-HETrE production, was assayed using RCE cells expressing the CYP4B1. Corneal neovascularization was induced by placing a single 7.0 silk suture at midstromal depth approximately 2 mm from the limbus in anesthetized rabbits. CYP4B1 or scrambled siRNAs (20 µM, 20µl) were administered by subconjunctival injection adjacent to the suture location. Injections of siRNA were repeated at day 2 and 4 after suture placement. Corneal neovascularization was examined by slit lamp microscopy and quantified by image analysis. Corneas were dissected and processed for measurement of CYP4B1 and VEGF expression by real time PCR and for determination of 12-HETrE levels by GC/MS. Results: 12-HETrE production in CYP4B1-expressing RCE cells was markedly inhibited (81 and 85%) by CYP4B1-specific siRNAs but was unaltered by CYP4B1-scrambled siRNA, Corneal neovascularization was markedly reduced in eyes treated with the CYP4B1-specific siRNA as compared to eyes treated with the control siRNA. Inhibition of neovascularization by CYP4B1-specific siRNA was 50 and 60% at day 4 and 7 after suture placement, respectively. Endogenous levels of 12-HETrE were barely detected in corneal homogenates from eyes injected with CYP4B1-specific siRNA Relative expression of VEGF mRNA measured by real time PCR (ΔCP ratio VEGF/28S) in suture-containing corneal limbal wedges was 0.12±0.08 indicating inhibition of VEGF expression in CYP4B1 siRNA-treated eyes. Conclusions: The results further substantiate the role of CYP4B1 as an angiogenic pathway in the cornea. CYP4B1 through its catalytic activity produces the angiogenic eicosanoid 12-HETrE, which contributes to corneal neovascularization by mechanisms that include VEGF induction. Inhibition of CYP4B1 may constitute part of therapeutic strategies that target angiogenic factors such as VEGF.

- 2005 -

 

The Corneal Epithelial CYP4B1 Produces Angiogenic Eicosanoids and Induces Inflammation of the Ocular Surface

A.V. Mezentsev, F. Seta, R. Kemp, M.W. Dunn, M. Laniado-Schwartzman. Pharmacology, New York Medical College, Valhalla, NY.

 

Purpose: We have previously shown that inflammation and neovascularization in response to injury to the ocular surface is mediated by 12-hydroxyeicosanoids (12-HETE and 12-HETrE) produced in the corneal epithelium by cytochrome P450 monooxygenase. This enzyme has been isolated and identified as the corneal CYP4B1, however its relationship to the production of 12-hydroxyeicosanoids and the inflammatory response has not been demonstrated. We have constructed expression plasmids containing the corneal CYP4B1 cDNA and evaluated its catalytic activity in vitro and in vivo and its role in ocular surface inflammation and neovascularization.
Methods: Rabbit corneal epithelial (RCE) cells were transfected with the expression vectors pIRES2-EGFP or pIRES2-EGFP-CYP4B1. Arachidonic acid metabolism by transfected cells was determined using HPLC and GC/MS analyses. Plasmids were also administered in vivo by repeated injection into the limbus of the rabbit eye. Transfection efficiency was monitored by following GFP fluorescence. Inflammatory response was examined by pachymetry and slit lamp microscopy over a 6-day period. Angiogenic response was determined ex vivo by measuring the length of the neovessel sprouts formed by corneal-limbal explants placed in Matrigel-coated culture dishes. The angiogenic response was correlated with the levels of 12-hydroxyeicosanoids. 
Results: RCE cells transfected with the CYP4B1 cDNA metabolized arachidonic acid to a product that had the HPLC elution profile of authentic 12-HETrE. 12-HETrE constituted the main metabolite in cell transfected with the CYP4B1 and was formed at a rate of 5.33 nmol/107 cells/h as compared to a rate of 1.22 nmol/107 cells/h in cells transfected with the control plasmid, pIERS2-EGFP. Eyes transfected with pIRES2-EGFP-CYP4B1 showed significant inflammation. Corneal-limbal explants from eyes transfected with pIRES2-EGFP-CYP4B1 demonstrated marked angiogenic activity which was correlated with increased levels of 12-HETrE. 
Conclusions
: The results indicate that the corneal CYP4B1 is the enzyme catalyzing the synthesis of the pro-inflammatory and angiogenic eicosanoid, 12-HETrE and further implicate it as a component of the inflammatory cascade initiated by injury of the ocular surface.

 

The Corneal-Derived 12(R)-Hydroxyeicosatrienoic Acid (12-Hetre) Stimulates IL-8 Production in Microvessel Endothelial Cells

F. Seta, A. Mezentsev, M. Dunn, M. Laniado-Schwartzman. Pharmacology, New York Medical College, Valhalla, NY.

 

Purpose: 12(R)-HETrE is an arachidonic acid metabolite synthesized by and released from the corneal epithelium in response to injury. 12(R)-HETrE stimulates corneal neovascularization both in vitro and in vivo. We have previously shown that VEGF expression is increased in microvessel endothelial cells (MEC) treated with 12(R)-HETrE and this effect is mediated in part by ERK1/2 activation. In the present study, we further evaluate the role of Akt and PKC in 12-HETrE signal transduction and use microarrays to identify genes that may contribute to its inflammatory and angiogenic activity.
Methods: MEC where grown to 70% confluent and starved for 48hr in absence of serum before treatment with 12(R)-HETrE (0.1, 1 and 10nM). In some experiments cells were incubated with the AKT specific inhibitor (LY294002 20µM) or PKC inhibitors (Calphostin C, 500 nM; Bisindolylmeleimide I, 10 nM) prior to the addition of 12-HETrE. Akt phosphorylation and PKC activation was measured by Western Blot analysis. Gene expression profile was performed on mRNA from cells treated with 12(R)-HETrE (1nM) using a Superarray gene profiling system.
Results: 12(R)-HETrE induced ERK1/2 phosphorylation in a time- and concentration-dependent manner as previously described. AKT was not phosphorylated by 12(R)-HETrE treatment at any of the concentrations tested. PKC α, βΙ, βΙΙ and δ were not activated in response to 12(R)-HETrE (5 min) since cellular protein extract did not immunoreact with a pan antibody which recognizes α, βΙ, βΙΙ and δ PKC isoforms. DNA microarray analysis of 12(R)-HETrE-treated cells showed a significant increase in IL-8 gene expression compared to control cells. The IL-8 protein increase in cells in response to 12(R)-HETrE was confirmed by ELISA.
Conclusions: 12(R)-HETrE-induced ERK1/2 activation in RLME is not mediated by AKT or PKC activation. PKC isoforms other than α, βΙ, βΙΙ and δ may be involved. Interestingly, 12(R)-HETrE stimulated both IL-8 gene and protein expression. These results suggest that IL-8 may play an important role in the angiogenic response of limbal vasculature to mitogenic and inflammatory 12(R)-HETrE

 

Heme Oxygenase-2 (HO-2) Knockout Mice Display Impaired Corneal Wound Healing and Marked Corneal Neovascularization

M. Laniado-Schwartzman1A, A. Mezentsev1A, F. Seta1A, M.W. Dunn1B, N.G. Abraham1A, K.Gronert1A. APharmacology, BOphthalmology, 1New York Medical College, Valhalla, NY.

Purpose:Heme oxygenases (HO-1 and HO-2) constitute an intrinsic cytoprotective and anti-inflammatory system in cells and tissues. This cytoprotection is attributed to the ability of HO to inhibit expression of inflammatory cytokines and proteins as well as to its products bilirubin (a powerful antioxidant) and CO (a vasodilator and anti-apoptotic factor). Coexisting with the HO system in the corneal epithelium is the cytochrome P450 (CYP) dependent pathway that converts arachidonic acid into two potent inflammatory mediators that constitute a critical part of the ocular surface inflammatory response. Induction of HO activity substantially attenuates the ocular surface inflammatory response and the associated corneal changes while enhancing cell survival by decreasing CYP activity. The present study was set to examine whether a deficiency in the ability to express this system impairs corneal recovery following injury.
Methods:We used HO-2 knockout mice. Injury was performed by removing the epithelium from corneas using an Algerbrush. Wound healing, re-epithelialization, opacity and neovascularization were assessed by slit lamp vital microscopy. Corneas were collected at different time after injury (1-14 days) and inflammatory markers quantified by ELISA, mass spectrometry, and RT-PCR.
Results:This injury produced a consistent and predictable inflammatory response including injection, neutrophil infiltration and neovascularization and a time dependent re-epithelization with full wound closure by 4 days. The results clearly indicate that HO-2 deficiency results in an aberrant inflammatory response including delayed wound closure, ulceration, persistent neovascularization and perforation. This response was associated with inability to upregulate HO-1, increased expression of inflammatory genes including CYP, COX-2 and IL-12, and increased production of inflammatory mediators such as PGE2 and the angiogenic CYP-derived eicosanoid, 12-HETrE.
Conclusions:These results demonstrate that the HO-2 knockout mice experience an exaggerated uncontrolled chronic inflammation and suggest a novel role for HO-2 in providing cytoprotection, a role previously assigned primarily to the HO inducible form, HO-1. Better understanding of the complex heme-heme oxygenase system may result in novel tools to combat diverse conditions, such as inflammation.

 

2004 -

Transfection of CYP4B1 into the cornea includes angiogenic activity in the limbal vessels

A.V. Mezentsev, F. Seta, M.W. Dunn, M. Laniado-Schwartzman. Pharmacology, New York Medical College, Valhalla, NY.

 

Purpose: Injury to the ocular surface provokes an inflammatory response that is mediated, at least in part, by corneal epithelial-derived 12-hydroxyeicosanoids. These eicosanoids, which exhibit potent inflammatory and angiogenic properties, are formed by a cytochrome P450 enzyme, presumably CYP4B1. We have isolated and cloned rabbit corneal CYP4B1 and examined the effect of CYP4B1 overexpression on corneal inflammation and neovascularization in vivo and in vitro.
Methods:
The expression vectors pIRES2-EGFP or pIRES2-CYP4B1 (2 µg/4 µl) were administered to the left eye using a 30G1/2in. needle dipped in the plasmid solution and inserted repeatedly into the limbus over 360o. Inflammatory response was examined by slit lamp microscopy over a 6-day period. Eyes were excised and corneas processed for GFP detection by fluorescence and immunohistochemistry 3 and 6 days after treatment. Corneal-limbal explants were placed in Matrigel-coated 24-well plates and their angiogenic capacity was determined over time (1-7 days) by measuring the length of the neovascular sprouts; the lectin Ulex europeus was used to identify the sprouts as endothelial cells. The culture medium was collected for determination of 12-hydroxyeicosanoid levels.
Results:
Plasmid inserted into the limbus effectively transduced GFP expression in the corneal epithelial cells primarily at the limbus and in the corneal endothelial cell layer. Slit lamp microscopy in vivo revealed a significant inflammatory response that included limbal vasodilation and corneal neovascularization in eyes transfected with the pIRES2-CYP4B1 but not with pIRES2-EGFP. Corneal limbal explants from eyes transfected with pIRES2-CYP4B1 exhibited a marked 3-fold increase in neovascular sprouting composed of GFP positive endothelial cells. This angiogenic activity was associated with increased levels of 12-HETrE, the angiogenic eicosanoid formed by CYP4B1.
Conclusions:
The results further implicate corneal CYP4B1 as a component of the inflammatory cascade initiated by injury to the ocular surface. Neovascularization was associated with increased levels of the angiogenic eicosanoid 12-HETrE. These finding suggest a novel strategy for control of ocular surface inflammation and neovascularization.

 

CORNEAL-DERIVED 12(R)-HYDROXYEICOSATRIENOIC ACID (12-HETrE), STIMULATES LIMBAL ENDOTHELIAL ANGIOGENESIS VIA ERK1/2 INDEPENDENT OF RAS/RAF ACTIVATION

 

F. Seta, A.Mezentsev, M.W. Dunn, M.Laniado-Schwartzman. Pharmacology, New York Medical College, Valhalla, NY

This presentation won travel award from Fondazione GB Bietti per l'Oftalmologia 

Purpose: 12(R)-HETrE is a corneal epithelial-derived angiogenic factor whose synthesis is induced in response to injury in vitro and in vivo; it acts in a paracrine manner on the limbal vessels to activate endothelial cells via a specific receptor/binding site and stimulates angiogenesis. We have identified the MAPK-ERK1/2 pathway as one mechanism mediating 12(R)-HETrE-induced VEGF production and angiogenesis. We further examined the cellular mechanisms upstream of ERK1/2 activation using endothelial cells derived from rabbit limbal microvessels (RLMVE cells).
Methods: RLMVE cells (passages 3-10) were grown until 70% confluent and then quiesced for 36 hours. The cells were treated with 12(R)-HETrE (0.1-1 nM) in the presence and absence of Calphostin (a PKC inhibitor), Wortmannin (PI3K inhibitor), Gentistein (a tyrosine kinase inhibitor), PD98059 (a MEK inhibitor), and FTSA and Manumycin (inhibitors of Ras activation). RLMVE cells were transfected with plasmid expressing the dominant negative form of Ras or Raf proteins 24 h before treatment with 12(R)-HETrE; cells were harvested 5-30 min after 12(R)-HETrE addition and Western blot analysis was used to determine MAPK activation. In vitro capillary formation assay using Matrigel was used to assess the functional relationship between 12(R)-HETrE and ERK1/2 activation.
Results: 12(R)-HETrE induced phosphorylation of ERK1/2 in a time- and concentration- dependent manner. The maximal response occurred within 5 min of addition of 0.1 nM 12(R)-HETrE. 12(R)-HETrE-induced ERK1/2 phosphorylation was inhibited by pretreatment of cells with Calphostin, Wortmannin, Gentistein and PD98059 but not with inhibitors of Ras activation. ERK1/2 activation in response to 12(R)-HETrE was not altered in cells expressing Ras or Raf dominant negative proteins. Inhibition of ERK1/2 activation attenuated 12(R)-HETrE-stimulated capillary-like tube formation in RLMVE cells.
Conclusions: The results indicate that 12(R)-HETrE induces ERK1/2 phosphorylation in a mechanism that involves activation of PI3K, tyrosine kinase and PKC but not Ras and Raf. 12-(R)-HETrE is produced by the corneal epithelium in response to injury, displays potent inflammatory properties, is a mitogen for microvessel endothelial cells, and is angiogenic in vitro and in vivo. Identifying signaling molecules and processes linking its receptor binding to effect is critical for elucidating the pathophysiological ramification of this eicosanoid.

 

- 2003 -

 

Promoter Activity and Regulation of the Corneal CYP4B1 Gene by Hypoxia

A.V. Mezentsev, W.-X. Zhang, V. Mastyugin, M.W. Dunn, M.Laniado-Schwartzman. Pharmacology, New York Medical College, Valhalla, NY

 

Purpose: Hypoxic injury to the ocular surface provokes an inflammatory response that is mediated, at least in part, by corneal epithelial-derived 12-hydroxyeicosanoids. These eicosanoids which exhibit potent inflammatory and angiogenic properties are formed by a cytochrome P450 enzyme, presumably CYP4B1. We have isolated and cloned the promoter region of the corneal epithelial CYP4B1 and studied its transcriptional regulation by hypoxia.
Methods: GenomeWalker libraries were constructed from rabbit corneal epithelial genomic DNA and used to isolate the promoter region with gene- and adaptor-specific primers. DNA fragments of different lengths were cloned into the luciferase reporter vector (pGL3-Basic) and used for promoter analysis in the rabbit epithelial cell line (RCE).
Results: A 3.4-kb DNA fragment of the 5'-flanking region of the corneal CYP4B1 promoter was isolated and cloned. Analysis of the promoter sequence revealed the presence of DNA binding motifs for hypoxia-sensitive transcription factors including HIF-1, NFkB, AP-1, HSF-1, Egr-1 and Sp1. Incubation of RCE cells transfected with luciferase reporter vectors containing different lengths of the promoter fragment under hypoxic condition resulted in a marked transcriptional activation of CYP4B1 gene that was correlated primarily with the presence of the HIF-1 binding motif. Promoter activity was also increased in response to CYP inducers including 3-MC and clofibrate suggesting the involvement of XRE and ARNT (HIF-1b) as well as PPAR in hypoxic activation of CYP4B1 gene.
Conclusions: The findings of sequences in the promoter region of the corneal CYP4B1 gene that are recognized by transcription factors whose activity is regulated by hypoxia provide a molecular mechanistic explanation for the induction of CYP4B1 and, thereby, the production of inflammatory eicosanoids by the corneal epithelium in response to hypoxic injury.

 

The Angiogenic Properties of the Corneal Epithelial Metabolite 12(R)-Hydroxyeicosatrienoic Acid (12(R)-HETrE) in Human Microvessel Endothelial Cells

F. Seta, A.Mezentsev, S. Ashkar, M.W. Dunn, M.Laniado-Schwartzman. Pharmacology, New York Medical College, Valhalla, NY.

 

Purpose: We have shown a strong correlation between the inflammatory/neovascular response in injured cornea and an enhanced production of the arachidonic acid metabolite 12(R)-HETrE which possesses biological activities indicative of a pro-inflammatory factor (vasodilation, increased capillary permeability, neutrophil chemotaxis and angiogenesis). It is readily released from the injured epithelium and its levels increased dramatically in tears from inflamed human eyes suggesting a paracrine role for 12(R)-HETrE. We studied whether human microvessel endothelial cells respond to 12(R)-HETrE.
Methods: Endothelial cell cultures from human microvessel endothelial cells (HMVE) were grown until 70% confluent and then quiesced for 24-36 hours. The cells were treated with 12(R)-HETrE (1nM to 10 nM) for 24-72 h. Cell proliferation and capillary like tube formation in cells grown on Matrigel were measured using standard methods. Intracellular Ca was measured using Fura 2AM.
Results: 12(R)-HETrE (1 nM) caused a rapid and marked increase in intracellular calcium that was inhibited by the PLC inhibitor U73122. 12(R)-HETrE (1 nM) increased HMVE cell number by 3 fold; this increase was also abolished by U73122. Addition of VEGF antibody to HMVE grown in Matrigel-coated plates inhibited 12(R)-HETrE-induced capillary formation.
Conclusions: 12(R)-HETrE is mitogenic and angiogenic in human endothelial cells; these effects include the activation of intracellular Ca coupled to PLC-IP3 signaling and are mediated, at least in part, by activation of VEGF. The findings that 12(R)-HETrE is present in inflamed human tears and that it potently activates human endothelial cells to form capillaries implicate its clinical relevance in human disease.

 

Retinoic Acid Induces Corneal Epithelial CYP4B1 Gene Expression and Stimulates the Synthesis of Inflammatory 12-Hydroxyeicosanoids

S.C. Ashkar, W.-X. Zhang, A.V. Mezentsev, V. Mastyugin, M.W. Dunn, M.Laniado-Schwartzman. Pharmacology, New York Med College, Valhalla, NY

 

Purpose: Injury to the ocular surface provokes an inflammatory response that is mediated, at least in part, by corneal epithelial derived 12-hydroxyeicosanoids (HETE) including 12-HETE and 12-HETrE; both metabolites exhibit potent inflammatory and angiogenic properties and are formed by a cytochrome P450 enzyme, namely CYP4B1. Retinoids are known to mediate wound healing processes in many tissues and as such are integral components of the inflammatory response. We studied the effect of various retinoids on corneal synthesis of 12-hydroxyeicosanoids and on activation of CYP4B1 gene expression.
Methods: Corneal organ cultures were used to assess the effect of retinoic acid on epithelial metabolism of arachidonic acid to 12-hydroxyeicosanoids. Lucifrease reporter vectors containing different lengths of the CYP4B1 3.4 kb-5'-untranslated region were used to examine the effect of retinoids (vitamin D, 9-cis-retinoic acid and all trans retinoic acid) on transcriptional activation of CYP4B1 in transient transfection experiments with HepG2 cells.
Results: Vitamin D had no effect on CYP4B1 promoter activity, whereas, 9-cis and all trans retinoic acids increased promoter activity by up to 70% over control. Addition of both 9-cis and all trans retinoic acids resulted in an additive effect increasing promoter activity by 2-fold. The increased promoter activity correlated with the presence of RXR/RAR binding motifs RXR/RAR. Incubation of corneal organ culture for 24 h in the presence of 9-cis and all trans retinoic acids increased the synthesis of 12-HETE and 12-HETrE by 2 fold.
Conclusions:The finding that retinoic acid increases the expression of the CYP4B1 gene and enhances production of the inflammatory 12-hydroxyeicosanoids in the corneal epithelium may provide a linkage between wound healing and inflammation in the ocular surface.

 

 

Closed Eye Tear Fluid Contains a Potent High Molecular Weight Angiogenic Factor

R.A. Sack1, S. Sathe1, M.Laniado-Schwartzman2, A.R. Beaton1, A. Mesentsev2, C. Morris3, B.I. Bogart4. 1Biological Sciences, SUNY College of Optometry, New York, NY; 2Pharmacology, New York Medical College, Valhalla, NY; 3CIBA Vision, Deluth, GA; 4Cell Biology, New York University Medical College, New York, NY

 

Purpose: Overnight eye closure is known to induce a sub-clinical inflammation and result in the accumulation in closed eye tear fluid (CTF) of a wide range of bioactive proteins. This study was designed to characterize the principal angiogenic factors in CTF.
Methods: Capillary tube collected CTF samples were recovered from 10 donors over a several week period and centrifuged. The resultant supernatants were pooled and on occasion individually separated into 24 molecular weight fractions using a preparative molecular sieve TSK 3000 HPLC. These fractions ranged in size from > 500 kDa to <10 kDa. Samples were tested for angiogenic activity based on the capacity to induce capillary-like tubes of endothelial cells grown on MatrigelTM. Fractions that exhibited bioactivity were subjected to 2-D electrophoretic characterization and western blot probing. Active fractions were further purified using isoelectric focusing and/or affinity chromatography. Results: Neat open eye and CTF samples on bioassay exhibit negligible to very low levels of net angiogenic activity. After HPLC separation, high levels of previously masked angiogenic activity could be detected in the CTF eluent in two distinct fractions. Minor activity eluted in a post-lysozyme fraction (<14,000 kDa). 2-D electrophoresis and western blot probing reveals this fraction to be complex and to contain IL-8. This angiogenic factor is known to accumulate in CTF. The majority of the angiogenic activity, however, elutes in a protein-sparse >200 kDa fraction. 2D gel analysis reveals a highly complex mixture of proteins. Comparative analysis of this and adjacent eluting non-active HPLC fractions allows the exclusion of the vast majority of proteins and reveals trace levels of several protein species that are endemic only to the bioactive fraction. Results suggest a bioactivity activity in the ng to sub ng range.
Conclusions: CTF contains a yet to be identified highly potent high molecular weight (> 200 kDa) protein or complex that appears to be the principal angiogenic factor in CTF.

 

- 2002 -

12(R)-HYDROXYEICOSATRIENOIC ACID, A CORNEAL EPITHELIAL DERIVED EICOSANOID, STIMULATES ANGIOGENESIS VIA ERK1/2 ACTIVATION OF VEGF INDUCTION F. Seta, A. Mezentsev, M.W. Dunn, M. Laniado Schwartzman Departments of Pharmacology and Ophthalmology, New York Medical College, Valhalla, NY.

 

Purpose. In response to hypoxic injury, corneal epithelial cells synthesize an eicosanoid via cytochrome P450. This eicosanoid, 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], then induces limbal vascular endothelial cells to proliferate and migrate into the cornea and begin neovascularization. We further investigate cellular mechanisms underlying its angiogenic activity using endothelial cells derived from rabbit limbal microvessels (RLMV cells). Methods. Endothelial cell cultures from rabbit limbal microvessels (RLMV) were grown until 70% confluent and then quiesced for 36 hours. The cells were treated with 12(R)-HETrE (0.1 nM) for various times. Northern or slot blot hybridization was used to determine VEGF mRNA levels and ELISA was used for measuring VEGF protein. Western blot analysis and in vitro kinase assays were used to determine MAPK activation. In vitro capillary formation assay using Matrigel was used to access functional relationship between 12(R)-HETrE and VEGF. Results. 12(R)-HETrE increased VEGF mRNA and protein in time and concentration dependent manners. Both transcriptional and posttranscriptional mechanisms accounted for the increase in VEGF mRNA. 12(R)-HETrE activated Erk1/2 as indicated by rapid increases in the level of phosphorylated Elk. 12(R)-HETrE- stimulated Erk1/2 activity as well as 12(R)-HETrE- induced VEGF expression was inhibited by the MEK selective inhibitor, PD98059. Addition of VEGF antibody to endothelial cells grown in Matrigel-coated plates inhibited 12(R)-HETrE- induced capillary formation. Conclusions. The results indicate that in microvessel endothelial cells, 12(R)-HETrE induces VEGF expression via activation Erk1/2 and that VEGF mediates at least in part the angiogenic activity of 12(R)-HETrE. Given the fact that both VEGF and 12(R)-HETrE are produced in the cornea following hypoxic injury their interaction may be an important determinant in the development of neovascularized cornea.

- 2001 -

CORNEAL EPITHELIAL VEGF AND CYP4B1 EXPRESSION IN A RABBIT MODEL OF CLOSED EYE CONTACT LENS WEAR. V. Mastyugin, S. Mosad, A. Bonazzi, A. Mezentsev, M.W. Dunn, M. Laniado-Schwartzman. Departments of Pharmacology and Ophthalmology, New York Medical College, Valhalla, NY.

 

Purpose. The similar and overlapping activity of VEGF and the potent corneal-derived angiogenic eicosanoid 12(R)-HETrE calls for a study of the temporal relationship in the expression of these two autocoids. Since recent evidence suggests that hypoxia induces the expression of a CYP4B1 mRNA which might be involved in the conversion of arachidonic acid to 12(R)-HETrE, we determined its time-dependent expression and correlated it to that of VEGF mRNA in the rabbit model of closed eye contact lens-induced injury. Methods. Rabbit eyes were fitted with contact lenses followed by a silk suture tarsorrhaphy. The anterior surface was analyzed at 2-, 4- and 7-days by slit lamp biomicroscopy, subjective inflammatory scoring and corneal pachymetry. Corneal epithelium was scraped and CYP4B1 and VEGF mRNA levels were measured by Southern hybridization of RT-PCR products amplified from a single cornea with specific primers. Results. Corneal thickness and inflammatory scores increased in a time dependent manner in the model of closed eye contact lens induced hypoxic injury. Corneal epithelial CYP4B1 and VEGF mRNAs, as well as the production of the angiogenic eicosanoid, 12(R)-HETrE, increased in a time-dependent manner and correlated with the in situ inflammatory response. Conclusions. The present study documents the increased expression of CYP4B1 isoform in the corneal epithelium during hypoxic injury in vivo. It also demonstrates the presence of VEGF mRNA in the corneal epithelium and its increased expression in this model of hypoxic injury. All together, the results of this study raise the possibility of interaction between these autocoids, VEGF and CYP4B1-12(R)-HETrE, in mediating the neovascularization response induced by the prolonged hypoxic state brought about by closed eye contact lens wear.

 

THE ANGIOGENIC ACTIVITY OF 12(R)-HYDROXYEICOSATRIENOIC ACID IS MEDIATED BY VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF).

A. Mezentsev, V. Mastyugin, A. Bonazzi, M.W. Dunn, M. Laniado Schwartzman. Departments of Pharmacology and Ophthalmology, New York Medical College, Valhalla, NY.

 

Purpose. In response to hypoxic injury, corneal epithelial cells synthesize an eicosanoid via cytochrome P450. This eicosanoid, 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], then induces limbal vascular endothelial cells to proliferate and migrate into the cornea and begin neovascularization. It is well documented that VEGF is a major mediator of angiogenesis in other tissues under hypoxic circumstances. We hypothesize that 12(R)-HETrE mediates its angiogenic actions, at least in part, through the induction of VEGF in vascular endothelial cells. Methods. Endothelial cell cultures from rabbit limbal microvessels (RLMVE) or immortalized rabbit corneal epithelial cells (RCE) were grown until 80% confluent and then quiesced for 24 hours. The cells were treated with 12(R)-HETrE (0.1nM to 10 nM) for 4-24 h. Northern Blot analysis and ELISA were used to determine VEGF mRNA and protein levels, respectively, and an in vitro capillary formation assay using Matrigel was used to assess functional relationship between 12(R)-HETrE and VEGF. Results. In RLMVE cells, 12(R)-HETrE (1 nM) time-dependently, increased VEGF mRNA. The increase in mRNA was followed by an increase in immunoreactive VEGF protein in the culture medium and this increase was concentration-dependent. 12(R)-HETrE-stimulated VEGF expression was attenuated by the PD 98059, a selective inhibitor of MEK-1. Addition of VEGF antibody to endothelial cells grown in Matrigel-coated plates inhibited 12(R)-HETrE-induced capillary formation. In RCE cells, hypoxia-induced VEGF mRNA and protein; addition of 12(R)-HETrE (1nM) increased VEGF mRNA by 5-fold. Conclusions. The ability of 12(R)-HETrE to stimulate corneal epithelial VEGF expression suggests that this eicosanoid can act within the corneal epithelium to activate and augment the angiogenic response. Moreover, the results also suggest that VEGF mediates, at least in part, the angiogenic response to 12(R)-HETrE.1