Download Effect of Smad7 Gene Overexpression on Transforming Growth

Expression of Smad7 inhibits fibrogenic responses of keratocytes to TGFβ2
Ti Wang1, MD; Xingtao Zhou1, MD; Yan Yu2, PhD; Jinhui Dai1, MD; Xiaomei Qu1, MD; Qihua Le,
MD1; Renyuan Chu1, MD*
1Department
China
2
of ophthalmology, Eye & ENT Hospital of Fudan University, 200031, 83 Fenyang Road, Shanghai,
Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania, USA
*Correspondence to: Professor Renyuan Chu, tel: +86-21-64377134-816
(e-mail: [email protected])
Key Words: cornea; proliferation; fibrosis; Smad7; transforming growth factor β
Purpose. To determine the effects of lentiviral-mediated Smad7 gene expression on keratocyte
proliferation and fibrosis induced by transforming growth factor (TGF) β2 in vitro. Methods.
Keratocytes were cultured from corneal tissue isolated from Sprague-Dawley (SD) rats and transfected
with Smad7 expressing lentiviral vector (Lv-Smad7) or non-functioning control vector (Lv-blank).
Following the exposure to TGFβ2, keratocytes were processed for immunoblotting to assess the
phosphorylation of Smad2 as down-stream event of TGFβ/Smad signaling. Expression of fibrotic
markers α-smooth muscle actin (α-SMA), type III collagen (collagen III) were measured by western
blotting and quantitative real time RT-PCR. Overall cell proliferation was determined by MTT assay
and the expression of cell cycle–related marker Ki67 at both mRNA and protein levels. Results. The
Smad7 gene transfer suppressed TGFβ/Smad signaling in keratocytes by down-regulating
phosphorylation of Smad2. Markers of cell proliferation and fibrosis including Ki67, α-SMA, collagen
III were inhibited by introduction of Smad 7 into TGFβ exposed keratocytes. Consequently, the rate of
cell proliferation was attenuated. Conclusion. Smad7 gene transfer inhibited fibrogenic responses of
keratocytes to TGFβ2.
Corneal stromal fibrosis and scarring reduces transparency of the cornea, leading to the
impairment of the patients’ vision. One of the greatest challenges in corneal biology is to promote
tissue repair via regeneration rather than fibrosis. During corneal wound healing, stromal keratocytes
are thought to be the major cell type contributing to tissue repair. TGFβ has been identified as one of
the most important growth factors in the development of fibrosis and scarring on cornea [1,2]. TGFβ is
expressed in corneal tissue after injury and activates corneal keratocytes, initiating conversion to
myofibroblasts[3,4]. Furthermore, TGFβ2 was confirmed as the major TGFβ mediator of fibrotic marker
expression[5,6].In the process of TGFβ signal transduction, a group of proteins, Smads, has been shown
to be specifically activated by TGFβ superfamily members[7] . Upon TGFβ binding to its receptor,
serine-threonine kinase receptors are activated and then bind to receptor-actived Smads (R-Smads),
Smad2 and Smad3. R-Smads are consequently phosphorylated and then form a complex with a
common Smad4. Smad complexes translocate into the nucleus, where they regulate transcription of
target genes. Smad7 is an inhibitory Smad, which interferes with the activation of Smad2 and Smad3
by competitive binding to TGFβ receptor, resulting in inhibition of TGFβ signal transduction[8,9].
Blocking unfavorable TGFβ activity by using Smad7 gene transfer technique could ameliorate
an excessive wound healing reaction from keratocyte acticvation, myofibroblasts formatiom, and
extracellular matrix deposition.
In the present report, we investigated whether lentivirus-mediated transfer of Smad7 suppresses
the fibrogenic response of keratocyte to exogenous TGFβ2 by assessing the markers of fibrosis and
proliferation including α-SMA, collagen III, and Ki67 at the mRNA and protein levels. In addition, the
effect of Smad7 gene transfer on TGFβ/Smad signaling was determined by western blotting analysis of
Smad 2 activation by TGFβ2.
Materials and Methods
Lentiviral vector construction and virus purification.
Smad7 cDNA was obtained by extracting total RNA from rat cerebral cortex and amplified using
the forward primer 5’-CGGAATTCGCCACCATGTTCAGGACCAAACGATC-3’ and the reverse
primer 5’-CGGGATCCACTACCGGCTGTTGAAGATG-3’ by reverse transcription polymerase chain
reaction (RT-PCR). The identity was confirmed by sequencing and inserted into green fluorescence
protein (GFP) carrying lentivector (pCDH-CMV-MCS-EF1-copGFP Cloning and Expression Vector,
System Biosciences, USA). Virus particles were collected after Lenti-copGFP-Smad7 plasmid
(Lv-Smad7) or pcDNA-copGFP Lentivector (Lv-blank) transfection with Lentivirus Package plasmids
mix (System Biosciences, USA) into 293TN cells (Shanghai Institute of Cell Biology, CHN). Viral
titers were determined by transduction of H1299 cells (Shanghai Institute of Cell Biology, CHN) with
serially diluted virus supernatants followed by cytofluorimetric analysis of GFP positive cells 72 hours
after transduction. Virus particles with Smad7 (Lv-Smad7) or without Smad7 (Lv-blank) were used at
the concentration of 1×104ifu/μl.
keratocyte establishment and infection with viruses
Syngeneic Sprague-Dawley (SD) rats, approximately 8 weeks of age and 200 to 250 g weight,
were used as a source of corneal tissue. The use of animals and study procedures were conducted
according to the guidelines of Helsinki Declaration and the Association for Research in Vision and
Ophthalmology, Inc. (ARVO) guidelines for care of animals in ophthalmic research. Corneas were
excised and epithelium and endothelial cells were removed using the corneal epithelial abrasor.
Corneas were cut into small pieces, allowed to adhere to tissue culture dishes. DMEM containing 10%
fetal calf serum (FCS) (Gibco Carlsbad, CA) was then added, stimulating keratocytes to proliferate and
migrate from the corneal explants onto the dish. Once confluence was reached, keratocytes were
further subcultured. Cells were infected with Lv-Smad7 or Lv-blank at multiplicity of infection (MOI)
of 5. And 72 hours after that, cells were observed under a phase-contrast fluorescence microscope to
evaluate GFP-expression. GFP positive cells were counted using a hemocytometer and the keratocytes
were subcultured to establish Lv-blank cell line and Lv-Smad7 cell line. Expression of Smad7 in three
passages of corneal keratocytes were detected using quantitative real-time RT-PCR and western
blotting.
Cell culture and treatment
For cell treatments, normal keratocytes (NK), Lv-blank keratocytes and Lv-Smad7 keratocytes
were plated and serum starved for 8 hours before treatment with 10ng/ml TGFβ2 (abcam, UK). Total
of four treatment groups were established. Group 1: control group, normal keratocytes and no TGFβ2
treatment; Group 2: TGFβ2 control group, normal keratocytes supplemented with TGFβ2; Group 3:
Lv-blank group, Lv-blank keratocytes, supplemented with TGFβ2; Group 4: Lv-Smad7 group,
Lv-Smad7 keratocytes, supplemented with TGFβ2. Two hours after treatment with or without TGFβ2,
keratocytes were collected in six-well plates. The specimens were processed for SDS-PAGE and
western blotting to assess the levels of phosphorylated Smad2 (p-Smad2) and Smad2 as previously
reported [10]. 48 hours after treatment, the levels of α-SMA、Ki67 and collagen III mRNA and protein
were detected using quantitative real-time RT-PCR and western blotting. The cell proliferation was
determined by the MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Quantitative real-time RT-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, USA). cDNA was synthesized with
RTase M-MLV (TaKaRa, Japan). Primers were designed according to the cDNA sequences in the
GenBank database using the Primers Express software Oligo6.6. Quantitative real-time RT-PCR were
performed according to the following conditions: RT
conditions: 10 minutes at 70°C, 2 minutes at
0°C, 1 hour at 42°C, 15 minutes at 70°C; PCR conditions of Smad7, α-SMA and collagen III α1 chain
were as follows: 10 seconds at 95°C, 20 seconds at 60°C, 20 seconds at 72°C, and then 40 cycles of
amplification; PCR conditions of Ki67 were as follows: 10 seconds at 95°C, 20 seconds at 56°C, 20
seconds at 72°C, and then 40 cycles of amplification. Results were normalized with housekeeping gene
beta-actin.
Western Blot Analysis
2 hours and 48 hours after supplementation with TGFβ2, Cells were harvested for 30 minutes in
lysis buffer and spinning them at 12,000 rpm for 15 minutes at 4°C, the supernatant was collected.
Supernatant was loaded and run on 10% SDS-PAGE and transferred to PVDF membrane (Millipore,
USA). After being blocked in 5% nonfat milk, membrane was incubated with rabbit anti-Smad7
polyclonal antibody (Santa Cruz Biotechnology, CA), or mouse anti-Smad2 monoclonal antibody, or
rabbit anti-phosphorylated Smad2 polyclonal antibody (Cell Signaling Technology, USA)，or rabbit
anti-α-SMA polyclonal antibody, or rabbit anti-Collagen III polyclonal antibody (abcam, UK), or goat
anti-Ki67 polyclonal antibody, or mouse anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase)
monoclonal
antibody
goat-antimouse-IgG-HRP
(Santa
Cruz
(horseradish
Biotechnology,
peroxidase),
CA).
After
extensive
washing,
asses-antigoat-IgG-HRP
or
goat-antirabbit-IgG-HRP (Santa Cruz Biotechnology, CA) was added to the system. Proteins were
detected using ECL reagents (Pierce, IL).
MTT assay [11]
Viability of corneal keratocytes cultured at 1×104 cells/well in 96-well plates were detected using
MTT assay before treatment (0 hr) and at 24 h, 48 h, 72 h after treatment
5mg/ml solution of MTT (Sigma, MO) in PBS was added to each well. The plates were then incubated
for 4 hours at 37oC. Medium was removed and 100% dimethylsulfoxide (Sigma, MO) was added at
100 μl/well. The plates were placed on a plate shaker for 10 minutes to enhance solubilization of the
precipitate. Immediately following agitation, absorbance of each well was measured on MultiSkan FC
microplate reader (Thermo, USA) at wavelength of 570 nm with the background subtracted at 630 nm.
Data Analysis
Data are summarized as mean ± SD. The statistical analysis of the results was performed using
one-way analysis of variance (ANOVA). p <0.05 was considered to be significant.
Results
Transfection of Smad7 carrying lentivector into rat corneal keratocytes
The efficacy of gene transfer was determined by co-transfection of green fluorescence protein
carrying lentivector. 98.3% and 98.1% transfection efficiency was reached in Lv-blank or Lv-Smad7
transfected cells (Fig 1). Marked fluorescence was consistent for all three passages of either Lv-Smad7
or Lv-blank transfected keratocytes, indicating successful transfer of lentiviral vector.
To assess the induction of Smad 7 expression upon exogenous Smad 7 gene transfer, both mRNA
and protein levels of Smad 7 in the transfected cells was measured by quantitative real-time RT-PCR
and west blotting. Figure 2A showed substantially increased expression of Smad7 protein in Lv-Smad7
keratocytes at various passages compared with Lv-blank cells and normal cells. Band density of Smad7
was normalized by GAPDH (Fig. 2B). Consistent with the western blot analysis results, quantitative
real-time PCR demonstrated 7.7, 6.3 and 8.8 fold increase in Lv-Smad7 keratocytes compared
Lv-blank cells at three passages (Fig 2C).
Inhibition of TGFβ/Smad signaling by exogenous Smad7 in keratocytes
To investigate whether treatment with Lv-Smad7 blocks TGFβ/Smad signaling in keratocytes
exposed to TGFβ2, specimens were processed for western blot analysis using anti-p-Smad2 antibody
and anti-Smad2 antibody as phosphorylation of Smad2 indicates the activation of Smad signaling.
Western blotting revealed the induction of phosphorylated Smad2 upon TGFβ treatment, however, the
induced level of p-Smad2 was markedly reduced in cells transfected with Lv-Smad7, but not with
Lv-blank. (Fig. 3A and B).
Suppression of Ki67, α-SMA and collagen III expression by exogenous Smad7 in keratocytes
Expression of fibrotic markers α-SMA, type III collagen and cell cycle related protein Ki67 were
assessed by western blotting and quantitative real-time RT-PCR in keratocytes upon TGFβ2 exposure.
Figure 4 showed marked increase of both mRNA and protein levels of Ki67 in TGFβ2 treated
keratocytes with or without transfection compared with control group cells. Exogenous expression of
Smad7 partially suppressed the induced Ki67 at both mRNA (reduced to 45.8%) and protein levels
(reduced to 55.7%) in TGFβ2 treated keratocytes, whereas Lv-blank transfected cells had similar levels
of TGFβ2 induced Ki67 compared with cells without lentiviral vector transfection but exposed to
TGFβ2 (Fig. 4A and B(a, b)).
After 48 hours incubation with TGFβ2, both mRNA and protein levels of α-SMA were markedly
increased in keratocytes with or without lentiviral vector transfection as compared with cells in the
absence of TGFβ2. However, the TGFβ2 induced α-SMA at both mRNA and protein levels were
inhibited by 64.2% and 43.3% through introduction of Smad7 in keratocytes in comparison with
Lv-blank group (Fig 4 A and B(c, d)).
Keratocyte fibrosis is mainly associated with up-regulation of matrix molecule expression. We
therefore examined mRNA expression of collagen III α1 chain and protein expression of collagen III .
Quantitative real-time RT-PCR and western blotting demonstrated significantly up-regulated collagen
III α1 chain mRNA and collagen III protein by TGFβ2 regardless of whether cells received lentiviral
vector transfection. Consistent with another marker of fibrosis α-SMA, the TGFβ2 induced collagen III
α1 chain mRNA and collagen III protein were remarkably suppressed (61.7% and 44.7% reduction)
although not completely abolished by exogenous expression of Smad7. On the other hand, the TGFβ2
induced collagen III level in Lv-blank group remained almost the same as the level in cells without any
transfection (Fig 4 A and B(e, f)). Thus, exogenous expression of Smad7 attenuates TGFβ2 induced
expression of fibrotic markers α-SMA, type III collagen and cell cycle marker Ki67.
Inhibition of keratocyte proliferation by exogenous Smad7.
MTT Assay was conducted to assess keratocytes viability at 0h (before treatment), 24h, 48h and
72h incubation with TGFβ2. Addition of TGFβ2 significantly promoted keratocyte proliferation.
Compared with Lv-blank keratocytes, expression of exogenous Smad7 caused 42.1%, 23.8% and
32.3% reduction of cell density at 24h, 48h and 72h respectively (Figure 5) .The rate of inhibition did
not change throughout the time course. This was consistent with the reduced level of cell cycle marker
Ki67 expression as result of Smad7 overexpression in keratocytes.
Discussion
The present study showed that partial blocking of TGFβ/Smad signaling by gene transfer of
Smad7 resulted in attenuation of keratocyte proliferation and fibrogenic response as demonstrated by
the reduction of TGFβ2 induced expression of Ki67, a-SMA and collagen III .
First, Smad7 gene was successfully transfected into the keratocytes using lentiviral-mediated
transfer. This is evidenced not only by GFP positive cells, but also by the marked induction of Smad7
expression at both mRNA and protein levels. As an attractive candidate for gene therapy, lentiviral
vector can efficiently transduce not only corneal cells in vitro, but also corneal keratocytes in vivo [12,13].
Furthermore, compared with adenoviral vectors achieving short-term transgene expression[14], lentiviral
vectors are capable of supporting long-term expression due to chromosomal integration. Expression of
exogenous gene can last for 20 weeks after lentiviral-mediated infection into some ocular cells [15].
TGFβ, especially TGFβ2, is the predominant cytokine that plays an important role in the
development of fibrosis on the ocular surface [1,2,5,6]. In contrast to the receptor-associated Smad2 which
mediates TGFβ signaling, Smad7 is an inhibitory Smad which functions as intracellular antagonist that
inhibits TGFβ signaling[16] . Smad7 inhibits signaling through stable binding to activated TGFβ type
I receptors and competition with Smad2 for receptor activation. In addition, Smad7 can recruit the E3
ubiquitin ligases Smurf1 and Smurf2 to the type I receptors, resulting in receptor ubiquitination,
degradation, and termination of signaling. Furthermore, Smad7 appears to act in the nucleus to disrupt
the formation of the TGFβ induced functional Smad-DNA complex
[17,18]
. Herein, we demonstrated
that exogenous expression of Smad7 partially blocked Smad2 activation in corneal keratocytes,
indicated by reduction of TGFβ2 induced phosphorylated Smad2, which is consistent with other reports
in trabecular meshwork cells and len epitheliums [19,20].
The corneal wound healing response is a remarkably complex cascade mediated by cells,
cytokines, and growth factors. Experimental models have been utilized to reproduce the sequence of
morphologic, cellular and molecular changes that keratocytes undergo following incision injury to the
corneal stroma
[3,21]
. The healing process is initiated immediately after corneal injury through the
release of multiple cytokines and growth factors. Keratocytes located at the margin of the cut edge
begin to fragment and undergo cell death, and adjacent keratocytes begin to lose their quiescence and
become activated. Chemokines released from the epithelium and keratocytes in response to cytokine
stimulation to attract inflammatory cells, such as macrophages/monocytes, T cells and
polymorphonuclear cells, into the stroma resulting in inflammatory reactions. Then keratocytes
trans-differentiate into α-SMA-positive myofibroblasts, which is one of the well-established hallmarks
of fibrosis
[3,22~24]
. The activated fibroblasts and myofibroblasts synthesis altered extracellular matrix
(ECM), such as collagens, fibronectin, and glycosaminoglycans, et, al. The deposition of ECM results
in fibrosis and scarring and ultimately leads to corneal stroma opacification
cytoskeleton
of
myofibroblasts
and
trans-differentiation
α-SMA-positive myofibroblasts is up-regulated by TGFβ
[23~24]
of
[3,21,25,26]
. α-SMA is the
keratocytes
into
. To draw a causative link between the
Smad mediated TGFβ2 signaling and myofibroblasts, levels of α-SMA in TGFβ2 treated keratocytes
were detected. The results showed a clear reduction of α-SMA at both mRNA and protein levels as
result of the suppressed TGFβ2/Smad signaling by Smad7.
Proliferation of the corneal keratocytes likely contributes to stromal fibrosis and opacification. To
estimate the rate of cell proliferation, a time course study was undertaken using MTT assay. The
induction of proliferation by TGFβ2 was significantly inhibited by Smad7 overexpression at about the
same level across 72 hours of incubation with TGFβ2. Consistent with reduced cell proliferation, Ki67,
a hallmark of cells in G1to M phase and cell activation, was also suppressed by exogenous Smad7.
In a normal cornea, the dominant types of collagen are type I and type V. Type III collagen is also
present, but only at a very small portion[27~28]. However, in the normal process of corneal wound
healing, especially at the early stage of inflammatory reactions, type III collagen synthesis is
up-regulated. It deposites and polymerizes at the site of injury[29~31]. In the present study, we
demonstrated that Smad7 overexpression inhibited about 61.7% and 44.7% of TGFβ2 induced collagen
III mRNA and protein in keratocytes.
The inhibition of fibrosis lies in the control of fibroblast activation and myofibroblasts formation
[3,32]
. Our findings demonstrated that the suppression of TGFβ/Smad signaling by gene transfer of
Smad7 resulted in reduced expression of Ki67, a-SMA and collagen III, which are the characteristics of
keratocyte proliferation and fibrosis. Thus, modulation of Smad7 expression in stroma keratocytes
could have therapeutic potentials for amelioration of excessive corneal fibrosis and scarring.
References
1. Saika S: TGF-beta signal transduction in corneal wound healing as a therapeutic target. Cornea
2004;23:S25–S30.
2. Jester JV, Ho-Chang J: Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines
control in vitro contractility and extracellular matrix contraction. Exp Eye Res 2003;77:581–592.
3. Fini ME: Keratocyte and fibroblast phenotypes in the repairing cornea. Prog Retin Eye Res 1999;18:529–551.
4. Jester JV, Petroll WM, Barry PA: Expression of alpha-smooth muscle (alpha-SM) actin during corneal stromal
wound healing. Invest Ophthalmol Vis Sci 1995;36:809–819.
5. Kingsley DM: The TGF-beta superfamily: new members，new receptors and new genetic tests of function in
different organisms. Genes Dev 1994;8:133-146
6. Carrington LM, Albon J, Anderson I: Differential regulation of key stages in early corneal wound healing by
TGF-beta isoforms and their inhibitors. Invest Ophthalmol Vis Sci 2006;47:1886-1894.
7. Massague J: How cells read TGF-βsignals. Nat Rev Mol Cell Biol 2000;1:169-178.
8. Derynck R, Zhang YE: Smad-dependent and Smad-independent pathways in TGFβ family signalling . Nature
2003;425:577-584.
9. Nakao A, Afrakhte M, Moren A: Indentification of Smad7, a TGF beta-inducible antagonist of TGF beta
signaling. Nature 1997;389:631-635
10. Saika S, Ikeda K, Yamanaka O: Adenoviral gene transfer of BMP-7, Id2, or Id3 suppresses injury-induced
epithelial-to-mesenchymal transition of lens epithelium in mice. Am J Physiol Cell Physiol 2006;290:C282-C289.
11. Xiaoying Zhang, Gael Le Pennec, Renate Steffen: Application of a MTT Assay for Screening Nutritional
Factors in Growth Media of Primary Sponge Cell Culture. Biotechnol Prog 2004;20:151-155
12. Wang X, Appukuttan B, Ott S: Efficient and sustained transgene expression in human corneal cells mediated
by a lentiviral vector, Gene Ther 2000;7:196–200.
13. Tsutomu Igarashi1, Koichi Miyake1, Noriko Suzuki: New strategy for in vivo transgene expression in corneal
epithelial progenitor cells. Current Eye Research 2002; 24:46–50.
14. Saika S, Ikeda K, Yamanaka O, et al. Expression of Smad7 in mouse eyes accelerates healing of corneal tissue
after exposure to alkali. Am J Pathol 2005;166: 1405-1418.
15. Takahashi K, Luo T, Saishin Y: Sustained transduction of ocular cells with a bovine immunodeficiency viral
vector, Hum. Gene Ther 2002;13:1305–1316.
16. Park SH: Fine tuning and cross-talking of TGF beta signal by inhibitory Smads. J Biochem Mol Biol
2005;38:9-16.
17. Zhang S, Fei T, Zhang L: Smad7 antagonizes transforming growth factor beta signaling in the nucleus by
interfering with functional Smad-DNA complex formation. Mol. Cell Biol 2007;27:4488-4499.
18. Ten Dijke P, Hill CS: New insights into TGF beta-Smad signalling. Trends Biochem Sci. 2004;29:265–273.
19. Picht G, Welge-Luessen U, Grehn F: Transforming growth factor beta 2 levels in the aqueous humor in
different types of glaucoma and the relation to filtering bleb development. Graefes Arch Clin Exp Ophthalmol
2001;239:199-207.
20. Meyer-Ter-Vehn T, Grehn F, Schlunck G: Localization of TGF-beta type II receptor and ED-A fibronectin in
normal conjunctiva and failed filtering blebs. Mol Vis 2008;14:136-141.
21. Fini ME, Cook JR, Mohan R: Proteolytic mechanisms in corneal ulceration and repair. Arch Dermatol Res
1998;290:S12–S23.
22. Musselmann K, Kane BP, Hassell JR: Isolation of a putative keratocyte activating factor from the corneal
stroma. Exp Eye Res 2003;77:273–279.
23. Jester JV, Huang J, Petroll WM: TGFβ induced myofibroblast differentiation of rabbit keratocytes requires
synergistic TGFβ, PDGF and integrin signaling. Exp Eye Res 2002;75:645–657.
24. Desmouliere A, Darby IA, Gabbiani G: Normal and pathologic soft tissue remodeling: role of the
myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest 2003;83:1689–1707.
25. Mohan RR, Hutcheon AE, Choi R: Apoptosis, necrosis, proliferation, and myofibroblast generation in the
stroma following LASIK and PRK. Exp Eye Res 2003;76:71–87.
26. Hong JW, Liu JJ, Lee JS: Proinflammatory chemokine induction in keratocytes and inflammatory cell
infiltration into the cornea. Invest Ophthalmol Vis Sci 2001;42:2795–2803.
27. Aya Nagayasu, Yoshinao Hosaka, Ayako Yamasaki: A Preliminary Study of Direct Application of
Atelocollagen into a Wound Lesion in the Dog Cornea. Current Eye Research 2008;33:727-735.
28. Cintron C, Hong BS, Covington HI: Heterogeneity of collagens in rabbit cornea: Type III collagen. Invest
Ophthalmol Vis Sci 1988;29:767–775.
29. Javier JA, Lee JB, Oliveira HB: Basement Membrane and Collagen Deposition After Laser Subepithelial
Keratomileusis and Photorefractive Keratectomy in the Leghorn Chick Eye. Arch Ophthalmol 2006;124:703-709
30. Kato T, Nakayasu K, Kanai A: Corneal wound healing: Immunohistological features of extracellular matrix
following penetrating keratoplasty in rabbits. Jpn J Ophthalmol 2000;44:334–341.
31. Inoue M, Woo SLY, Gomez MA: Effect of surgical treatment and immobilization on the healing of the medial
collateral ligament: A long-term multidisciplinary study. Connect Tissue Res 1990;25:13–26.
32. Stramer BM, Zieske JD, Jung JC: Molecular mechanisms controlling the fibrotic repair phenotype in cornea:
implications for surgical outcomes. Invest Ophthalmol Vis Sci 2003;44:4237–4246.
A
B
C
D
Fig 1. Phase-contrast fluorescence microscopic examination of corneal keratocytes harvested at 72 hours after
transfection (original magnification ×160). (A & B) Keratocytes transfected with Lv-blank; (C & D) Keratocytes
transfected with Lv- Smad7. A & C are bright field images corresponding to fluorescence images B & D.
A
NK(P1) Blank(P1) Smad7(P1) NK(P2) Blank(P2) Smad7(P2) NK(P3) Blank(P3) Smad7 (P3)
51KD
Smad7
37KD
GAPDH
Smad7/GAPDH band density ratio
B
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
*▲
*▲
*▲
NK
Lv-bland
Lv-Smad7
P1
P2
P3
various passages of keratocytes
－△ct
Smad7/beta-actin 2
relative mRNA expression
C
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
*▲
*▲
P1
*▲
NK
Lv-blank
Lv-Smad7
P2
various passages of keratocytes
P3
FIG 2. Expression of Smad7 in three passages of corneal keratocytes. Western blotting (A & B) and quantitative
real-time RT-PCR (C) showed that relative Smad7 mRNA and protein expression. Data are shown as mean ± s.d.
(n = 6). Means marked with an asterisk(*) differed significantly (P < 0.05) from means of normal keratocytes, and
with a triangel(▲)differed significantly (P < 0.05) from means of Lv-blank keratocytes.
A
Control
TGF-β2 control
Lv-blank
Lv-Smad7
60KD
p-Smad2
60KD
Smad2
p-Smad2/Smad2 band density ratio
B
0.8
*
0.7
*
0.6
0.5
0.4
0.3
* ▲
0.2
0.1
▲
0
Control
TGF-β2 control
Lv-blank
Lv-Smad7
FIG 3. Expression of p-Smad2 protein in keratocytes. (A) Western blotting analysis of p-Smad2 and Smad2
protein expression. (B) Band density was normalized by Smad2. Data are shown as mean ± s.d. (n = 6). Means
marked with an asterisk(*) differed significantly (P < 0.05) from means of control group keratocytes, and with a
triangel(▲)differed significantly (P < 0.05) from means of Lv-blank keratocytes.
A
Control TGF-β2 control Lv-blank
Lv-Smad7
395KD
Ki67
42KD
α-SMA
300KD
collagen Ⅲ
37KD
GAPDH
b
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
*
relative mRNA expression
－△ct
ki67/beta-actin 2
Ki67/GAPDH band density ratio
B
a
*
*▲
▲
Control
TGFβ2 control
Lv-blank
*
*
0.03
0.025
0.02
*▲
0.015
0.01
▲
0.005
0
Lv-Smad7
c
Control
TGFβ2 control
Lv-blank
*
*
Lv-Smad7
d
0.6
0.07
*
*
0.5
relative mRNA expression
α-SMA/beta-actin 2 －△ct
α-SMA/GAPDH band density ratio
0.04
0.035
0.4
*▲
0.3
0.2
▲
0.1
0
0.06
0.05
0.04
0.03
*▲
0.02
0.01
▲
0
Control
TGFβ2 control
Lv-blank
Lv-Smad7
TGFβ2 control
Lv-blank
Lv-Smad7
f
0.045
0.04
0.6
0.5
*
relative mRNA expression
*
0.4
*▲
0.3
0.2
▲
0.1
0
Control
TGFβ2 control
Lv-blank
Lv-Smad7
collagenⅢ/beta-actin 2－△ct
collagen III/GAPDH band density
ratio
e
Control
*
*
0.035
0.03
0.025
0.02
0.015
0.01
*▲
▲
0.005
0
Control
TGFβ2 control
Lv-blank
Lv-Smad7
FIG 4. Expression of Ki67, α-SMA and collagen Ⅲ in keratocytes at mRNA and protein level. (A) Western
blotting analysis of Ki67, α-SMA, collagen Ⅲ and GAPDH expression. (B) Band density was normalized by
GAPDH (a, c, e) and quantitative real-time RT-PCR analysis of Ki67, α-SMA, collagen Ⅲ α1 chain expression (b,
d, f). Data are shown as mean ± s.d. (n = 6).
Means marked with an asterisk(*) differed significantly (P < 0.05)
from means of control keratocytes, and with a triangel(▲)differed significantly (P < 0.05) from means of Lv-blank
keratocytes.
350
*
cell density
(% of control)
300
*
*
250
200
▲
* *
*▲
▲
150
*
▲
*▲
▲
100
group1
group2
group3
group4
50
0
0h
24h
48h
TGFβ2 incubation time (h)
72h
Figure 5. Keratocytes viability at 0h (before treatment), 24h, 48h and 72h incubation with TGFβ2 by MTT assay.
Data are shown as mean ± s.d. (n = 6).
Means marked with an asterisk(*) differed significantly (P < 0.05) from
means of control group keratocytes, and with a triangel(▲) differed significantly (P < 0.05) from means of
Lv-blank keratocytes.