-2005-
Transfection
of CYP4B1 into the cornea increases angiogenic activity of the limbal vessels
(Travel
award)
Alexandre Mezentsev1,
1Department of
Pharmacology, New York Medical College, Valhalla, NY 10595 and 2Departments
of Biochemistry and Pharmacology, University of Texas Southwestern Medical
Center, Dallas, TX 75390
Injury to the ocular surface induces
the production of corneal epithelial-derived 12-hydroxyeicosanoids, which
exhibit potent inflammatory and angiogenic properties and are formed by a
cytochrome P450 enzyme, CYP4B1. We have cloned the rabbit corneal CYP4B1 into
the expression plasmid pIRES2-EGFP and examined the effect of CYP4B1 overexpression on corneal inflammation in vivo and limbal
vessel sprouting in vitro. Transfection
of pIRES2-EGFP or pIRES2-EGFP-CYP4B1 plasmids was performed in vitro in rabbit
corneal epithelial (RCE) cells and in vivo into rabbit eyes. The inflammatory
response was examined by slit lamp microscopy. Eyes were excised 6 days after
transfection and corneas processed for EGFP detection by fluorescence and immunohistochemistry.
Corneal-limbal explants were placed in Matrigel-coated plates and their
angiogenic capacity was determined by measuring the
length of the neovessel sprouts. The culture
medium was assessed for 12-hydroxyeicosanoid levels. RCE cells transfected with pIRES2-EGFP-CYP4B1 metabolized arachidonic
acid to 12-HETrE at a rate 5 times higher than that of pIRES2-EGFP-transfected
cells. Administration of either plasmid
into the rabbit eye resulted in EGFP fluorescence in the corneal epithelial
cells primarily at the limbus. Slit lamp microscopy revealed a significant
inflammatory response in eyes transfected with the
pIRES2-CYP4B1 but not with pIRES2-EGFP. Corneal limbal explants from eyes transfected with pIRES2-CYP4B1 demonstrated a marked 3-fold
increase in angiogenic activity, which correlated with increased levels of
12-HETrE, the CYP4B1-derived angiogenic 12-hydroxyeicosanoid and was inhibited
by the CYP inhibitor 17-ODYA. The results further implicate the corneal CYP4B1
as a component of the inflammatory cascade initiated by injury to the ocular
surface.
Heme oxygenase-2 (HO-2) null mice display impaired corneal wound
healing and marked neovascularization
(Travel award)
Francesca Seta, Alexandre Mesentsev,
Michal W. Dunn, karsten Gronert, Nader G. Abraham and Michal Laniado-Schwartzman
Department of
Pharmacology,
Heme oxygenases (HO-1 and HO-2)
represent an intrinsic cytoprotective and
anti-inflammatory system attributed to the ability of HO to inhibit expression
of inflammatory cytokines and proteins as well as to its products bilirubin (an 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 inflammatory mediators (12R-HETE
and 12R-HETrE) that constitute a
critical part of the ocular surface inflammatory response. Induction of HO
attenuates the ocular surface inflammatory response and the associated changes
while enhancing cell survival by decreasing CYP activity. The present study was
set to examine whether a deficiency in the ability to fully express the HO
system impairs corneal recovery following injury. Injury was performed by
removing the epithelium from corneas of wild type (wt) and HO-2 null mice. Wound healing
(re-epithelialization) and neovascularization were
assessed by slit lamp vital microscopy. Inflammatory markers were quantified by
ELISA, GC/MS, and RT-PCR. Epithelial removal produced a consistent inflammatory
response and a time-dependent re-epithelization with
wound closure by 5 days. HO-2 deficiency resulted in an aberrant inflammatory
response including impaired wound closure (49±5% wound closure at day 5),
ulceration, and persistent (14 days after injury) neovascularization (43.5±9.7 vs 2±1 mm corneal vessel length). This response was
associated with inability to upregulate HO-1,
increased expression of inflammatory genes (CYP4B1, COX-2 and IL-12), and
increased production of the inflammatory eicosanoids PGE2 (9.5±1.8 vs 6.2±0.9 ng/cornea, p<0.05) and 12-HETrE (0.38±0.06 vs 0.07±0.01 ng/cornea, p<0.05). These results demonstrate that the HO-2 null
mice experienced an exaggerated uncontrolled chronic inflammation and suggest a
novel role for HO-2 in providing cytoprotection, a
role previously assigned to the HO inducible form, HO-1. Better understanding
of the complex HO system and its role in regulating inflammatory eicosanoids
may result in novel tools to combat diverse conditions, such as inflammation.