\set{final}

\def\Author{Meij}
\def\author{meij}
\def\vol{13}
\def\year{2007}
\def\anum{226}
\def\pages{2012-2018}
\def\txt_title{Targeted expression of a lumican transgene rescues corneal deficiencies in lumican-null mice}
\def\txt_authors{Johanna T.A. Meij, Eric C. Carlson, Li Wang, Chia-Yang Liu, James V. Jester, David E. Birk, Winston W.-Y. Kao}

\def\rcvd{27 August 2007}
\def\accept{18 October 2007}
\def\publ{18 October 2007}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

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\def\graphpad{www.graphpad.com}

\| Internal defs


\article{

\title{Targeted expression of a lumican transgene rescues corneal
deficiencies in lumican-null mice}

\authors{\mailto{meijjt@uc.edu}{Johanna T.A. Meij},\sup{1}
\mailto{eric.carlson@case.edu}{Eric C. Carlson},\sup{1}
\mailto{liwang71@hotmail.com}{Li Wang},\sup{1}
\mailto{liucg@uc.edu}{Chia-Yang Liu},\sup{1}
\mailto{jjester@uci.edu}{James V. Jester},\sup{3}
\mailto{David.Birk@jefferson.edu}{David E. Birk},\sup{4}
\mailto{kaoww@uc.edu}{Winston W.Y. Kao}\sup{1,2}}

\institutions{Departments of \sup{1}Ophthalmology, and \sup{2}Cell and
Cancer Biology, University of Cincinnati, Cincinnati, OH; \sup{3}The Eye
Institute, University of California, Irvine, CA; \sup{4}Department of
Pathology, Anatomy \and\ Cell Biology, Thomas Jefferson University,
Philadelphia, PA}

\correspondence{Winston W.Y. Kao, Ph.D., 3223 Eden Ave, HPB Suite 350,
Cincinnati, OH, 45267-0838; Phone: (513) 558-2802; FAX: (513) 558-6021;
email: kaoww@uc.edu \br\sp\br Dr. Carlson is now at the Department of
Ophthalmology and Pathology, Visual Science Research Center, Case
Western Reserve University, Cleveland, OH.}

\abstract

\abs_purpose{To investigate whether targeted expression of lumican in
the mouse cornea rescued the \i{Lum}\sup{-/-} phenotype.}

\abs_methods{\i{Lum}\sup{-/-}/\i{Kera-Lum} mice were generated by
crossing \i{Lum}\sup{-/-} mice with \i{Kera-Lum} transgenic mice that
overexpressed lumican under the control of the keratocan promoter. Mouse
eyes were analyzed in vivo by confocal microscopy through focusing
(CMTF) to determine corneal sublayer thickness and haze. Subsequently,
one cornea from each mouse was processed for SDS-PAGE/western blotting
while the other was used for either electron microscopy (EM) or
real-time polymerase chain reaction (RT-PCR).}

\abs_results{Overall, corneas of \i{Lum}\sup{-/-}/\i{Kera-Lum} mice
showed significant improvement over \i{Lum}\sup{-/-} but were still
deficient when compared to wildtype (WT) mice. Specifically, analysis of
\i{Lum}\sup{-/-}/\i{Kera-Lum} mouse eyes by CMTF showed a similar
stromal but slightly increased epithelial thickness compared to matching
\i{Lum}\sup{-/-} mice. Analysis of the CMTF scans for light
backscattering revealed a small yet significant reduction in corneal
haze in \i{Lum}\sup{-/-}/\i{Kera-Lum} mice as compared to
\i{Lum}\sup{-/-} mice. At the EM level, the pronounced disarray of the
posterior fibrillar matrix seen in \i{Lum}\sup{-/-} mice was not
observed in \i{Lum}\sup{-/-}/\i{Kera-Lum} mice. Moreover, analyses of
collagen fibril diameter distributions showed a significant reduction in
the number of large-diameter (\gt 40 nm) fibrils in
\i{Lum}\sup{-/-}/\i{Kera-Lum} mice as compared to \i{Lum}\sup{-/-} mice.
No significant differences in keratocan expression were found at the
mRNA level, but western blot analysis detected an approximately two fold
increase in keratocan protein levels in \i{Lum}\sup{-/-}/\i{Kera-Lum}
over \i{Lum}\sup{-/-} mice.}

\abs_conclusions{Together these data suggest that despite the low
keratocan promoter activity driving the transgene in \i{Lum}\sup{-/-}
cornea, transgenic lumican expression was sufficient to partially rescue
corneal phenotypic deficiencies.}

\introduction

\p{The vertebrate cornea is a transparent and avascular tissue that
covers the anterior chamber of the eye and provides most of the eye's
refractive power. It consists of three functionally and structurally
different cellular layers, the corneal epithelium, stroma, and
endothelium. The mechanical and optical properties of the cornea are
determined by its extracellular matrix (ECM) organization such as
collagens and proteoglycans in the stroma. The stromal ECM is abundant
in collagen fibrils and two small class II leucine-rich proteoglycans,
lumican and keratocan, which are synthesized and deposited by stromal
keratocytes [1]. The functional importance of these keratan sulfate
proteoglycans for corneal structure is most evident in knockout mouse
lines. Mice lacking the gene for keratocan display a thinner and flatter
but functionally normal cornea [2]. Mice deficient in lumican
(\i{Lum}\sup{-/-}) have a disorganized corneal posterior stroma,
characterized by the presence of thickened and irregular collagen
fibrils [3-5]. As a consequence, corneal transparency is dramatically
reduced. In addition, \i{Lum}\sup{-/-} mice have reduced corneal
keratocan levels, consistent with the finding that lumican regulates
keratocan transcription [6].}

\p{Unlike keratocan, lumican is present in a wide range of connective
tissues. Lumican-null mice exhibit a phenotype that includes severe skin
fragility as well as cloudy corneas [3-5,7]. Therefore, it remains to be
determined whether the corneal deficiencies in \i{Lum}\sup{-/-} mice
result solely from a cornea-intrinsic defect or also from a systemic
defect. In the present study, we investigated whether corneal
reexpression of lumican in a \i{Lum}\sup{-/-} background rescued the
phenotypic deficits. To this end, \i{Kera-Lum} transgenic mice carrying
a minigene with lumican cDNA under the control of the 3.2 kb keratocan
promoter [6] were crossed with \i{Lum}\sup{-/-} mice [3] to generate
\i{Lum}\sup{-/-}/\i{Kera-Lum} mice. Our findings show a significant
improvement of corneal properties in \i{Lum}\sup{-/-}/\i{Kera-Lum} as
compared to \i{Lum}\sup{-/-} mice, indicating that reexpression of
corneal lumican in a lumican-null background is able to rescue
phenotypic deficits.}

\methods

\subsection{Animals}

\p{Animal care and use conformed to the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research. All animal protocols were
approved by the Institutional Animal Care and Use Committee (IACUC) of
the University of Cincinnati. \i{Lum}\sup{-/-} mice [3] of a mixed
J129/B6 background strain were cross-bred with \i{Kera-Lum} transgenic
mice (FVB/B6) that overexpressed lumican under control of the keratocan
promoter [6]. After genotyping, the bitransgenic
\i{Lum}\sup{-/-}/\i{Kera-Lum} mice from one line were used in this study
and compared to age-matched and strain-matched \i{Lum}\sup{-/-} mice.}

\subsection{In vivo confocal microscopy}

\p{In vivo confocal microscopy through focusing (CMTF) was used to
measure corneal epithelial thickness, stromal thickness, and stromal
light scattering. Mice were anesthetized by intraperitoneal injection of
ketamine HCl (100 mg/kg body weight; Dodge Animal Health, Fort Dodge,
IA) and xylazine (10 mg/kg body weight; Akorn Inc., Decatur, IL), and
the central cornea was scanned using a tandem scanning confocal
microscope (Tandem Scanning Corp., Reston, VA). Thickness and light
scattering measurements were obtained using previously described
techniques [8-10]. Three confocal microscopy through focus (CMTF) scans,
each comprised of a sequential series of 200 images extending from the
corneal epithelial surface through the corneal endothelium, were
obtained from each mouse eye. Depth intensity profiles were generated,
and thickness measurements for the epithelium and stroma as well as
stromal light scattering were obtained using previously published
equations [9-11]. An average of the epithelial thickness, stromal
thickness, and stromal light scattering from the three separate z-scans
taken from the same eye were recorded. The value per mouse was
calculated from the average of both eyes and used for statistical
analysis. After in vivo confocal microscopy, mice were sacrificed and
the eyes enucleated and either immediately frozen for biochemical
analysis or fixed for electron microscopy.}

\subsection{Transmission electron microscopy}

\p{Corneas from three to four mice per group were analyzed by
transmission electron microscopy. The corneas were processed as
previously described [4,12]. Briefly, fixation was with 4%
paraformaldehyde, 2.5% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.4,
and 8.0 mM CaCl\sub{2} followed by postfixation with 1% osmium
tetraoxide and \i{en bloc} stained with uranyl acetate/50% ethanol.
After dehydration in an ethanol series followed by propylene oxide, the
corneas were infiltrated and embedded in a mixture of EMbed 812, nadic
methyl anhydride, dodecenyl succinic anhydride, and DMP-30 (Electron
Microscopy Sciences, Hatfield, PA). Thin sections were cut using a
Reichert UCT ultramicrotome equipped with a diamond knife and stained
with 2% aqueous uranyl acetate, 1% phosphotungstic acid, pH 3.2.
Sections taken from the central cornea and the anterior and posterior
stroma were analyzed independently using electron microscopy. Corneas
were examined and photographed at 80 kV using a Tecnai 12 transmission
electron microscope with a Gatan 2K Ultrascan bottom mount CCD camera.}

\subsection{Fibril diameter analyses}

\p{Corneas from two to three different animals were analyzed for each
transgenic line. Digital images were taken from nonoverlapping regions
of the central portion of anterior and posterior areas of the cornea at
28,610X. Images (10-15 anterior and 15-20 posterior/group) were
randomized and fibril diameters were measured using a RM
Biometrics-Bioquant Image Analysis System (Nashville,TN) in a masked
manner. A total area of 0.211 \mu m\sup{2} per image at a final
magnification of 161,990X was analyzed. For each group, the number of
animals and number of different images (animals/images [fibril count,
min-max diameter]) was as follows: wildtype (WT) anterior (3/15 [2,379,
8.2 - 41.9 nm]); WT posterior (3/15 [1,893, 8.2 - 43.2 nm]);
\i{Lum}\sup{-/-} anterior (2/10 [1,496, 8.2 - 38.7 nm]);
\i{Lum}\sup{-/-} posterior (3/15 [1,815, 5.8 - 59.9 nm]);
\i{Lum}\sup{-/-}/\i{Kera-Lum} anterior (3/15 [2,135, 9.2 - 50.0 nm]);
\i{Lum}\sup{-/-}/\i{Kera-Lum} posterior (4/20 [2,224, 5.8 - 51.4 nm]).
The smallest diameter measurements (5-9 nm) represent the tapered ends
of corneal collagen fibrils near their termination [12-14]. Although the
corneal stroma also contains fibrillin-containing microfibrils in this
diameter range, those have a distinctive structure and organization and
were excluded from the measurements.}

\subsection{Real-time polymerase chain reaction}

\p{Corneas were excised from frozen eyes of a subset of animals (11
\i{Lum}\sup{-/-} and 10 \i{Lum}\sup{-/-}/\i{Kera-Lum}), and RNA was
prepared using the FastRNA Pro Green kit (QBiogene, Morgan Irvine, CA).
Briefly, each cornea was placed in 0.8 ml RNA\i{pro} solution and
homogenized with Lysing Matrix D in a Fastprep instrument (QBiogene) at
setting 6.0 for 40 s. After cooling on ice, supernatants were
transferred and the lysing matrices rinsed with 0.2 ml RNA\i{pro}
solution. Combined supernatants were chloroform-extracted, and RNA was
precipitated from the upper phase with an equal volume of isopropanol
overnight at -20 \deg C. Pellets were rinsed with 70% ethanol,
air-dried, and resuspended in 10 \mu l/cornea DEPC-treated H\sub{2}O at
55-60 \deg C for 10 min. RNA quality was checked by agarose gel
electrophoresis.}

\p{To obtain cDNA, RNA was reverse-transcribed using a RETROScript kit
with random primers (Ambion, Austin, TX). Real-time polymerase chain
reaction (RT-PCR) was performed on a SmartCycler system (Cepheid,
Sunnyvale, CA) using \beta-actin as the reference gene. The primer pairs
were as follows: Keratocan: Kerac-F2: 5'-AAT GCT AAC CTG CAG CAC CTT
CAC-3' and Kerac-R2: 5'-TTC ATT CCC ATC CAG ACG CAG GTA-3'; \beta-actin:
bact-F1: 5'-TGG CTC CTA GCA CCA TGA AGA TCA-3' and bact-R2: 5'-ACT CAT
CGT ACT CCT GCT TGC TGA-3'. For the reaction mix, a LightCycler DNA
Master SYBR Green I kit (Roche Applied Science, Indianapolis, IN) was
employed. Optimum conditions were determined by PCR using the kit
components and agarose gel analysis. Also, a cDNA dilution series was
run to determine the RT-PCR efficiency of each primer pair. One
\i{Lum}\sup{-/-} sample was randomly chosen as the "control" and
included in all subsequent runs. Crossing point differences between
samples and the "control" and the calculated efficiencies were applied
to compute the expression ratio of keratocan in the corneal extracts
according to the formula by Pfaffl [15].}

\subsection{SDS-PAGE/western blotting}

\p{Corneas were excised from frozen eyes and solubilized in 50 mM
Tris-NaOH, pH 12.0, containing 0.25 U/\mu l Benzonase nuclease (Novagen,
Madison, WI) and protease inhibitor cocktail (Roche Applied Science,
Indianapolis, IN), overnight at 4 \deg C in a rotator [16]. The samples
were centrifuged at 14,000x g for 15 min, and the collected supernatants
were neutralized with 1/10 volume of 500 mM Tris-HCl, pH 6.0. To
estimate protein concentration, absorbance was measured with a
spectrophotometer at 280 nm. Extracts were digested with 0.1 U/ml
endo-\beta-galactosidase (Sigma, St Louis, MO) at 37 \deg C overnight,
and the reaction was terminated by the addition of 1 vol 2x SDS-PAGE
sample buffer. SDS-PAGE and western blotting were performed as described
previously [16], using goat anti-Keratocan [2] as the primary antibody
and IRDye 800-conjugated donkey anti-goat IgG (Rockland Immunochemicals,
Gilbertsville, PA) as the secondary antibody. Immunopositive bands were
quantified with an Odyssey Infrared Imaging system (LI-COR Biosciences,
Lincoln, NE). One \i{Lum}\sup{-/-}/\i{Kera-Lum} sample was chosen for
normalization and included in all assays.}

\subsection{Statistical analysis}

\p{Data were analyzed using GraphPad Prism version 5.00 for Windows
(\hot{\graphpad}{GraphPad Software}, San Diego, CA). Data were compared
by either ANOVA followed by Newman-Keuls Multiple Comparison Test or
\i{t}-test with Welch's correction for unequal variances where stated.
Differences were considered significant when p\lt 0.05.}

\results

\p{Absence of lumican in the mouse cornea causes corneal thinning and
severe opacity associated with disorganization of and aberrations in
collagen fibrils most prominently in the posterior stroma [3-5,17]. In
addition, \i{Lum}\sup{-/-} mice have reduced corneal keratocan levels
[6]. In the present study, we investigated whether reexpression of
lumican in the \i{Lum}\sup{-/-} cornea could rescue these phenotypic
deficits by using \i{Lum}\sup{-/-}/\i{Kera-Lum} mice carrying a
keratocan promoter-driven lumican transgene [6] in a \i{Lum}\sup{-/-}
background.}

\p{As shown in \figref{1}, not only corneal stromal but also epithelial
thickness was reduced in \i{Lum}\sup{-/-} mice as compared to WT
(\i{Lum}\sup{+/+} and \i{Lum}\sup{+/-} combined) mice. This effect on
epithelium was not observed in previous knockout models of CD-1
background [5,8]. The presence of the lumican transgene did not increase
either corneal stromal or epithelial thickness to WT levels.
Nevertheless, epithelial thickness was increased in
\i{Lum}\sup{-/-}/\i{Kera-Lum} as compared to \i{Lum}\sup{-/-} mice
(\figref{1}{A}). The slightly higher stromal thickness in bitransgenic
\i{Lum}\sup{-/-}/\i{Kera-Lum} corneas did not reach statistical
significance (\figref{1}{B}). Corneal opacity was reduced from 1,116\pom
55.2 in the \i{Lum}\sup{-/-} mice to 927.6\pom 43.7 in
\i{Lum}\sup{-/-}/\i{Kera-Lum} mice (\figref{2}). Although the latter was
still higher than the value in WT mice in this study of 489.1\pom 28.6
units (n=7; not shown), the improvement was significant (p\lt 0.01).}

\p{Ultrastructural analyses of the corneal stroma demonstrated a
remarkable recovery in the fibrillar organization in the posterior
stroma of \i{Lum}\sup{-/-}/\i{Kera-Lum} compared to \i{Lum}\sup{-/-}
mice (\figref{3}{A}). The anterior phenotype was comparable to that seen
in \i{Lum}\sup{-/-} and WT mice. The recovery of the posterior stroma
was confirmed after examination of fibril diameter distributions. The
number of large-diameter (\gt 40 nm) fibrils was significantly lower
(p=0.0013, \chi\sup{2}-test) in \i{Lum}\sup{-/-}/\i{Kera-Lum} than in
\i{Lum}\sup{-/-} mouse posterior stroma (\figref{3}{B}).}

\p{Finally, the levels of keratocan protein and message were determined
by western blotting and real-time RT-PCR, respectively (\figref{4}). On
average, the keratocan protein level was increased (p\lt 0.05, Welch's
\i{t}-test) in \i{Lum}\sup{-/-}/\i{Kera-Lum} as compared to
\i{Lum}\sup{-/-} mice (\figref{4}{B}) whereas no significant difference
was detected at the mRNA level (\figref{4}{C}). It should be noted that
even with the different means, the \i{Lum}\sup{-/-} and
\i{Lum}\sup{-/-}/\i{Kera-Lum} groups had similar median keratocan levels
(\figref{4}{B}, dotted line), reflecting the broad variation in the
\i{Lum}\sup{-/-}/\i{Kera-Lum} mice as is also evident when comparing
\figref{4}{A}, top row, lanes 1 and 2. A wide variation was not found
for keratocan mRNA values, but it should be pointed out that the number
of samples for RT-PCR was limited. As shown in \figref{4}{A}, lumican
expression was detectable in \i{Lum}\sup{-/-}\i{/Kera-Lum} cornea
(bottom row, lanes 1 and 2), but overall, the changes were too subtle
for accurate quantification. Quantifying changes in keratocan levels was
feasible probably because those were amplified.}

\discussion

\p{The data presented here demonstrate that reexpression of corneal
lumican in a lumican-null background is able to rescue phenotypic
deficits. In particular, significant improvements in corneal epithelial
thickness, transparency, collagen matrix organization, and keratocan
protein levels were observed. However, the \i{Lum}\sup{-/-}/\i{Kera-Lum}
corneas were still deficient in most of these parameters when compared
to WT mice. Furthermore, some other parameters examined were not
affected by the lumican transgene at all. The reduced thickness of the
corneal stromal layers in \i{Lum}\sup{-/-} mice was unchanged in the
presence of the transgene. Also, no differences in keratocan mRNA
expression were detected between \i{Lum}\sup{-/-} and
\i{Lum}\sup{-/-}/\i{Kera-Lum} mice, albeit that there was a two-fold
increase in keratocan protein. Thus, the rescue of phenotypic deficits
was not complete.}

\p{Lumican is present in a wide range of connective tissues, and its
absence results in multiple deficiencies [4]. Since it is shown here
that the corneal stroma-specific lumican transgene is only partially
effective, it can not be absolutely excluded that a systemic lack of
lumican may contribute to the corneal deficiencies in \i{Lum}\sup{-/-}
mice. This notion of indirect actions of lumican is supported by the
observation that the corneal epithelium, a tissue that does not contain
lumican, is thinner in \i{Lum}\sup{-/-} mice of this background strain
and was partially rescued by reexpression of lumican in the adjacent
stroma in \i{Lum}\sup{-/-}\i{/Kera-Lum} mice. It should be noted that
previous studies by Chakravarti and associates [5,8] showed no
significant difference in corneal epithelium thickness between wild type
and \i{Lum}\sup{-/-} mice. The different outcomes in epithelium
thickness can be explained in part by the dissimilarity in genetic
background of the experimental mice. In the present studies, our
experimental mice are in a mixed 129/J and C57BL genetic background
whereas the \i{Lum}\sup{-/-} mice used by Chakravarti et al. [3-5] were
in a CD-1 genetic background. In any case, the absence of lumican from
surrounding ocular surface tissues such as the periocular mesenchyme and
eyelid stroma may have contributed to the incomplete rescue of the
corneal phenotype by the solely stromal presence of lumican in
\i{Lum}\sup{-/-}\i{/Kera-Lum} mice.}

\p{On the other hand, low keratocan promoter activity in the
\i{Lum}\sup{-/-} genetic background as evident from \figref{4} may
provide the foremost explanation for the incomplete rescue seen in this
study. In a WT background, endogenous keratocan is abundant and as our
previous study shows, the \i{Kera-Lum} transgene induces robust
overexpression of lumican as well as keratocan [6]. That same study also
showed that keratocan expression is dependent on lumican and that in a
\i{Lum}\sup{-/-} background, keratocan levels are very low. Therefore,
it may be assumed that in the \i{Lum}\sup{-/-}/\i{Kera-Lum} mice,
transcriptional activity of the \i{Kera-Lum} transgene is sluggish.
Thus, it is likely that low levels of both transgenic lumican and
keratocan fail to completely restore corneal morphology and functions.}

\p{Aside from the restricted presence and low level of transgenic
lumican, another factor contributing to its limited rescue effect could
be the developmental timing of its expression. In the WT mouse cornea,
lumican is expressed by E12 [18] whereas keratocan is detectable and
presumably the keratocan promoter is active by E13.5 [19]. Hence, in
\i{Lum}\sup{-/-}/\i{Kera-Lum} cornea, lumican expression is delayed
until E13.5. Moreover, it is conceivable that in a \i{Lum}\sup{-/-}
background, keratocan is not expressed until well beyond E13.5 due to
the absence of endogenous lumican to power up its promoter. It is worthy
to mention that there is a surge of keratan sulfate proteoglycan (KSPG)
synthesis during neonatal stromal development before eye opening. It has
been suggested that such an increase in KSPG synthesis is crucial for a
normal growth of stroma [20]. Therefore, a lack of any stromal lumican
expression in the proper developmental window (in this case between E12
and at least E13.5) and during the neonatal stages before eye opening
may have also hampered a complete rescue of corneal deficiencies.}

\p{Regardless of the low initial keratocan promoter activity, if
sufficient transgenic lumican accumulated in the
\i{Lum}\sup{-/-}/\i{Kera-Lum} cornea over time, the activity of
\i{Kera-Lum} transgene might gradually increase thereby further raising
transgene and keratocan expression. The outlying values in the
measurements of corneal haze and keratocan expression could be
interpreted to suggest that such a feed-forward loop was generated in a
few cases. Of note, there was no discernible correlation between low
corneal haze and high keratocan expression (data not shown). Even so,
the outcome of transgenic lumican expression may have differed per eye
depending on its intrastromal site of accumulation.}

\p{On the whole, it appears that some corneal properties require much
earlier and/or higher expression of lumican for their normal function
than others. The modest level of lumican expressed by the transgene and
the concurrent subtle increase in keratocan expression were enough to
restore collagen fibril assembly and matrix organization in the
posterior stroma of \i{Lum}\sup{-/-}/\i{Kera-Lum} corneas. Normal
stromal fibril diameter results from the interaction of fibrils with
fibril-associated molecules such as lumican in the posterior stroma. In
the absence of lumican, the fibrils associate laterally and give rise to
larger diameter fibrils not characteristic of cornea but the norm in
tissues like sclera. This lateral fusion disrupts fibril spacing and
leads to a less organized posterior stroma. In addition, lumican and
other small leucine-rich proteoglycans (SLRPs) may be involved in the
regular packing, and thus, the absence of lumican would result in the
less ordered packing. In the \i{Lum}\sup{-/-}/\i{Kera-Lum} mice, there
is apparently sufficient lumican to stabilize the fibrils and prevent
lateral association and fusion. Furthermore, the availability of lumican
to associate with and organize fibrils may account for the regular
packing of the posterior corneal stroma in \i{Lum}\sup{-/-}/\i{Kera-Lum}
mice. In contrast, corneal thickness and transparency were not raised to
wild type levels. This suggests that lumican has multiple mechanisms of
action. It has been shown that lumican regulates collagen fibril
assembly by direct interaction in the ECM [21]. Furthermore, lumican is
able to affect cell function by binding to cell surface receptors
[22,23]. Intracellular actions of lumican can also not be excluded.}

\p{In summary, the present study shows that reexpression of lumican in
the cornea in a \i{Lum}\sup{-/-} background only partially rescued
corneal phenotypic deficiencies. We propose that the absence of lumican
in surrounding tissues, the delay in lumican transgene expression,
and/or the low level of its expression are factors contributing to the
incomplete recovery. Nonetheless, the lumican transgene resulted in
significant improvements in corneal morphology and function.}

\acknowledgements

\p{This study was supported by NEI EY011845 (W.W.K.), NEI EY005129
(D.E.B.), Research to Prevent Blindness (W.W.K.), Ohio Lions Eye
Research Foundation (W.W.K.).}

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\endreferences

}

\beginfigures

\figfile{1}{
\figtitle{1}{Corneal thickness in \i{Lum}\sup{-/-}, \i{Lum}\sup{-/-}/\i{Kera-Lum}
(Lum\sup{-/-}/TG), and WT mice}

\p{Thickness of corneal epithelial (\panel{A}) and stromal (\panel{B})
layers in \i{Lum}\sup{-/-}, \i{Lum}\sup{-/-}/\i{Kera-Lum}
(Lum\sup{-/-}/TG), and WT mice are shown. Mice were anesthetized and the
central corneas were scanned by confocal microscopy through focusing
(CMTF) as described in Methods. Data represent the mean\pom SEM of six
scans per mouse (three per eye) of 14 \i{Lum}\sup{-/-}, 21
\i{Lum}\sup{-/-}/\i{Kera-Lum}, and seven WT mice. Reexpression of
lumican in \i{Lum}\sup{-/-} partially rescued the epithelial thickness
in comparison to WT and \i{Lum}\sup{-/-}/\i{Kera-Lum} versus
\i{Lum}\sup{-/-}; however, no significant improvement of stromal
thickness could be determined comparing \i{Lum}\sup{-/-}/\i{Kera-Lum} to
\i{Lum}\sup{-/-}. Both epithelial and stromal thickness are more reduced
in \i{Lum}\sup{-/-} and \i{Lum}\sup{-/-}/\i{Kera-Lum} than in wild type
mice. The asterisk indictates significannce (p\lt 0.05) versus WT; The
hash mark indicates significannce (p\lt 0.05) versus \i{Lum}\sup{-/-}
(ANOVA, Newman-Keuls test).}

\ctr{\gifimage{1}{700}{371}{22}}

}


\figfile{2}{
\figtitle{2}{Corneal haze in \i{Lum}\sup{-/-} and \i{Lum}\sup{-/-}/\i{Kera-Lum} (\i{Lum}\sup{-/-}/TG) mice}

\p{The same CMTF scans as used for \figref{1} were analyzed for stromal
light scattering as described in Methods. Symbols represent the average
of six scans (three per eye) per individual \i{Lum}\sup{-/-} mouse (open
symbol) and \i{Lum}\sup{-/-}/\i{Kera-Lum} mouse (closed symbol).
Horizontal lines respresent the mean values per group: 1,116\pom 55.2
nm, \i{Lum}\sup{-/-}; 927.6\pom 43.7 nm, \i{Lum}\sup{-/-}/\i{Kera-Lum};
489.1\pom 28.6 nm, WT (not shown). The hash mark indicates significance
(p\lt 0.02) versus \i{Lum}\sup{-/-} (\i{t}-test with Welch's
correction).}

\ctr{\gifimage{2}{600}{582}{26}}

}


\figfile{3}{
\figtitle{3}{Ultrastructural analysis of stromal collagen matrix
organization in corneas from \i{Lum}\sup{-/-}, \i{Lum}\sup{-/-}/\i{Kera-Lum}, and WT mice}

\p{After experiments represented in \figref{1} and \figref{2}, mouse
eyes were processed for transmission EM as described in Methods.
\panel{A}-\panel{F}: EM images of anterior stroma (\panel{A}, \panel{C},
\panel{E}) show little difference between groups whereas in EM images of
posterior stroma (\panel{B}, \panel{D}, \panel{F}), the matrix in
\i{Lum}\sup{-/-}/\i{Kera-Lum} (\panel{D}) appears normal like in WT
(\panel{F}) and lacks the disarray and large-diameter fibrils (arrows)
of \i{Lum}\sup{-/-} (\panel{B}). \panel{G}: The distribution of fibril
diameters (5 nm bins) in posterior corneal stroma is shown. Inset:
frequency of fibrils larger than 40 nm. Data represent percent of 1815,
2224, and 1893 fibrils measured in \i{Lum}\sup{-/-},
\i{Lum}\sup{-/-}/\i{Kera-Lum}, and WT, respectively.}

\ctr{\jpgimage{3}{600}{1219}{366}}

}


\figfile{4}{
\figtitle{4}{Relative keratocan levels in \i{Lum}\sup{-/-} and
\i{Lum}\sup{-/-}/\i{Kera-Lum} (\i{Lum}\sup{-/-}/TG) mice}

\p{\panel{A},\panel{B}: After SDS-PAGE/western blotting of cornea
extracts, immunopositive bands were visualized on a LI-COR Odyssey
Infrared Imaging system. \panel{A}: Examples of keratocan (top row) and
lumican (bottom row) protein bands in two \i{Lum}\sup{-/-}\i{/Kera-Lum}
(left two lanes) and one \i{Lum}\sup{-/-} (right lane) cornea. Note the
variation in band intensity between the individual
\i{Lum}\sup{-/-}/\i{Kera-Lum} samples. \panel{B}:
Keratocan-immunopositive band intensities were determined and normalized
against one \i{Lum}\sup{-/-} value. Symbols represent the relative
keratocan protein level in each sample (one cornea/mouse), solid lines
represent the mean values and the dotted lines represent the medians of
13 \i{Lum}\sup{-/-} and 21 \i{Lum}\sup{-/-}/\i{Kera-Lum} mice.
\panel{C}: From the remaining corneas (one/mouse), RNA was extracted and
real-time PCR for keratocan was performed with \beta-actin as the
reference gene. Relative keratocan levels were computed as described in
Methods as the ratios over the same \i{Lum}\sup{-/-} value. Bars
represent mean\pom SEM of 11 \i{Lum}\sup{-/-} mice and 10
\i{Lum}\sup{-/-}/\i{Kera-Lum} mice. The hash mark represents the
significance (p\lt 0.05) versus \i{Lum}\sup{-/-} (\i{t}-test with
Welch's correction).}

\ctr{\jpgimage{4}{800}{419}{104}}

}