- 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.
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.
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.