1Intestinal Diseases Research Programme and 2Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada; and 3Department of Cardiovascular Medicine, Kyushu University, Fukuoka, Japan
Submitted 26 April 2004 ; accepted in final form 29 July 2004
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ABSTRACT |
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inflammation; adhesion; placental growth factor; monocyte chemoattractant protein-1; anti-inflammatory cytokine
However, because TGF- has counterinflammatory properties, inhibition of its actions may result in the promotion of inflammation. For example, the targeted disruption of TGF-
in mice resulted in a multifocal inflammatory response and caused early death (18, 40). Dominant negative TGF-
type II receptor (TGF
RII) transgenic mice expressing the mutant gene in the intestinal epithelium developed spontaneous colitis (12). These findings indicate that blockade of TGF-
signaling could result in an inflammatory response, offsetting any potentially beneficial antifibrotic effects.
VEGF and placental growth factor (PlGF) are potent angiogenic factors (10, 25, 34). They also have proinflammatory properties and promote monocyte activation and migration through VEGF type 1 receptor fms-like tyrosine kinase (Flt-1) (1), monocyte chemoattractant protein-1 (MCP-1) (28), and adhesion molecules (16). TGF- upregulates VEGF expression in rabbit pleural mesothelial tissue (11), rat peritoneum (27), and several types of cell cultures (3, 6, 7, 9, 33, 38). VEGF has been implicated in a carbon tetrachloride-induced model of murine hepatic fibrosis (51) and is produced in large quantities by fibroblasts from intestinal strictures in patients with Crohn's disease (2). Thus inhibition of VEGF may be of therapeutic use in the treatment of fibrotic conditions. TGF-
also induces the expression of PlGF in human umbilical vein endothelial cells (32). PlGF is upregulated during various pathological conditions (8) and may contribute to TGF-
-induced pathophysiology.
The soluble VEGF receptor-1 (sFlt-1), which lacks both the membrane-spanning and intracellular tyrosine kinase domains (14), has been used to inhibit tumor or inflammation-associated angiogenesis (19, 30). Recently, Zhao et al. (52) reported that blocking VEGF by electrogene transfer using an sFlt-1 plasmid attenuated early vascular inflammation and prevented subsequent arteriosclerosis. Thus, whereas the role of VEGF or PlGF in fibrotic disorders remains unclear, these findings raise the possibility that blockade of these angiogenic growth factors might prevent fibrosis through suppressing inflammation.
In this study, we used an adenovirus-mediated TGF--induced model of peritoneal fibrosis (27) to evaluate the therapeutic potential of blocking TGF-
or VEGF/PlGF. Blockade of TGF-
and VEGF/PlGF was achieved by intramuscular plasmids gene transfer of soluble TGF
RII (sTGF
RII) and soluble Flt-1 (sFlt-1), respectively. Our results show that transfection of the sFlt-1 gene into skeletal muscle inhibited TGF-
-induced inflammation and fibrosis in the peritoneum, whereas sTGF
RII gene transfer enhanced inflammation and failed to prevent fibrosis. The inhibition of fibrosis by sFlt-1 was associated with marked attenuation of serum PlGF level, ICAM-1, and MCP-1 mRNA expression. These observations indicate that sFlt-1 gene transfer might be of therapeutic benefit in preventing fibrosis.
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MATERIALS AND METHODS |
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Expression vector.
The 3.3-kb mouse sFlt-1 gene, originally obtained from the mouse lung DNA library (17), was cloned into the BamH1 (5') and Not1 (3') sites of the eukaryotic expression vector plasmid cDNA3 (Invitrogen, Burlington, ON, Canada) (52). The entire extracellular domain of the type II human TGF- receptor fused to the Fc portion of human IgG1 (44) was cloned into the Xho1 (5') and Xba1 (3') sites of the eukaryotic expression vector pCDM. Empty vectors cDNA3 and CDM were also used.
Animal model of peritoneal fibrosis and treatment protocol. C57BL/6 mice were obtained from Taconic (Germantown, NY), kept in sterilized, filter-topped cages, and fed autoclaved food in the animal facilities of McMaster University. Only 8- to 10-wk-old male mice were used. The protocols employed were in direct accordance with guidelines drafted by the McMaster University Animal Care Committee and the Canadian Council on the Use of Laboratory Animals.
Mice were divided into four groups, anesthetized with Enflurane (Abbott Laboratories, Quebec, Canada), and were injected with sTGFRII plasmid (100 µg DNA/100 µl PBS; group 1) or sFlt-1 plasmid (100 µg DNA/100 µl PBS; group 2) or PBS (100 µl; groups 3 and 4) into both of the posterior tibial muscles (50 µl per muscle; day 4). To enhance transgene expression, the animals including PBS group received electroporation at the injected site immediately after injection. Commercially available two-needle array electrodes with a 5-mm gap (model 532, BTX, San Diego, CA) were used for electroporation. Square-wave electrical pulses were administered six times using an ECM830 pulse generator (BTX) at 100 V and a rate of 1 pulse/s, with each pulse being 50 ms in duration. Four days later (day 0), each group was injected 1.5 x 108 plaque-forming unit (pfu) of AdTGF
diluted in 100 µl PBS or PBS alone intraperitoneally. Thus the four groups were 1) AdTGF
+ sTGF
RII plasmid (sTGF
RII treatment group), 2) AdTGF
+ sFlt-1 plasmid (sFlt-1 treatment group), 3) AdTGF
PBS (AdTGF
-alone group), and 4) PBS + PBS (control). PBS was used as control for both the intraperitoneal and intramuscular injections in this study, because there was no significant difference between PBS treatment and intraperitoneal injection of control virus AdDL70 or intramuscular injection of empty plasmids (data not shown). We also did not observe any significant effect in mice that received sTGF
RII plasmid alone or sFlt-1 plasmid alone compared with the control (data not shown).
At various time points, body weight was measured. Blood was taken at days 5 and 10 after intraperitoneal injection under anesthesia. Mice were euthanized at day 14 after AdTGF administration, and blood and tissues were collected for histological and biochemical analysis.
Histology.
Cytochemical staining for -galactosidase was performed on samples obtained from animals 1 day after intraperitoneal infection with AdLacZ. Fresh tissue samples were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in PBS for 3 h at 4°C, washed two times with PBS, and then reacted with X-gal staining solution [0.5 mg/ml 5-bromo-4-chloro-3-indoyl-
-D-galactopyranoside, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 2 mM MgCl2 in 100 mM Tris buffer, pH 8.0] for 6 h at 37°C. The samples were stored in 10% formaldehyde and then paraffin-processed and embedded, and 4-µm sections were cut. These sections were counterstained with nuclear fast red.
Tissue samples were obtained from the anterior abdominal wall of AdTGF-treated and control animals on day 14, embedded in OCT compound and frozen immediately. Sections were cut at 4 µm and stained with hematoxylin and eosin and with Masson's trichrome stain.
Hydroxyproline assay. Portions of mesentery and anterior abdominal wall were frozen for a hydroxyproline assay. Hydroxyproline is a component of collagen, and its concentration is directly correlated with collagen content. Total mesentery was harvested for the assay. An equal area of tissue was obtained from the abdominal wall from an identical position in all samples. The assay was modified from the method described by Reddy and Enwemeka (36). Tissues were weighed, homogenized in water using a polytron homogenizer, and centrifuged at 1,000 rpm for 5 min, and the superficial fatty material was removed by vacuum suction. After resuspension, aliquots of samples were mixed gently with sodium hydroxide (2N final concentration) in a total volume of 50 µl. The samples were hydrolyzed by autoclaving at 120°C for 20 min. Chloramine-T (450 µl) was added to the hydrolyzate, and the oxidation was allowed to proceed for 25 min at room temperature. Ehrlich's aldehyde reagent (500 µl) was added to each sample, and the chromophore was developed by incubating the samples at 65°C for 20 min. Absorbance of each samples was read at 550 nm using a spectrophotometer. Standard hydroxyproline samples were used to create a standard curve.
Measurement of serum amyloid-P component.
Serum amyloid-P (SAP), a major acute-phase protein in mice (42), was measured by an ELISA as an acute inflammatory marker. A 96-well microplate (MaxiSorp, Nunc, Naperville, IL) was coated with a sheep anti-mouse SAP polyclonal antibody (Calbiochem, San Diego, CA) overnight. One-hundred microliters of either SAP standard or serum samples from mice were added and then incubated for 1.5 h at room temperature. A rabbit anti-mouse SAP polyclonal antibody (Calbiochem) was added, and the plate was incubated for another 1.5 h at room temperature. A horseradish peroxidase-conjugated mouse anti-rabbit IgG (-chain specific) monoclonal antibody (Sigma, Oakville, ON, Canada) was added, and the plate was incubated for another 1.5 h at room temperature. Finally, substrate solution containing o-phenylenediamine (Sigma) was added to each well and incubated 30 min at room temperature. The absorbance at 492 nm was measured with a microplate reader. Standard SAP samples were used to create a standard curve.
Measurement of soluble TGF- receptor and Flt-1.
The concentration of the exogenous soluble TGF-
receptor protein in mouse serum was measured by ELISA. Human IgG concentration was measured instead of soluble TGF-
receptor, because it is fused to human sTGF
RII in the expression vector. A 96-well microplate (MaxiSorp, Nunc) was coated with a rabbit anti-human IgG (
-chain specific) polyclonal antibody (Sigma) overnight. One-hundred microliters of either human IgG standard (Sigma) or serum samples from mice were added and then incubated for 1 h at room temperature. A horseradish peroxidase-conjugated goat anti-human IgG (
-chain specific) polyclonal antibody (MBL, Nagoya, Japan) was added, and the plate was incubated for another 1 h at room temperature. Finally, substrate solution containing o-phenylenediamine (Sigma) was added to each well and incubated 30 min at room temperature. The absorbance at 492 nm was measured with a microplate reader. Standard human IgG1 samples were used to create a standard curve.
The concentration of the soluble Flt-1 protein in mouse serum was assayed by ELISA, using a kit (R&D Systems, Minneapolis, MN) according to the manufacturer instructions.
Cytokine analysis.
The serum samples were analyzed using a human TGF-1, a mouse VEGF, TNF-
, IL-12, or PlGF-2 ELISA kit (R&D Systems) according to the manufacturer instructions.
RT-PCR.
Total cellular RNA was isolated from the anterior abdominal wall obtained 14 days after AdTGF administration using RNeasy Mini Kits (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. An equal area of tissue was obtained from the abdominal wall from an identical position in all samples. The concentration of RNA was determined measuring absorbance at 260 nm, and its purity was assessed using the ratio of absorbency at 260 nm to that at 280 nm. RNA was stored at 70°C until used for RT-PCR. Messenger RNA was reverse transcribed as described previously (15) to yield cDNA; the cDNA was amplified by PCR using gene-specific primers.
PCR reactions were performed in a total volume of 50 µl in the presence of 2.5 U Taq DNA polymerase (GIBCO-Invitrogen, Burlington, ON, Canada), 10 nM 2-deoxynucleotide 5'-triphosphate (GIBCO-Invitrogen), and 15 pg of 5' and 3' primers. Amplification was performed by 30 cycles for the housekeeping gene -glucuronidase, 32 cycles for ICAM-1, 33 cycles for VCAM-1, 34 cycles for MCP-1, consisting of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s with the use of a Perkin-Elmer thermal cycler 480 (Branchburg, NJ). The following primers specific for
-glucuronidase and cytokines were used:
-glucuronidase: sense 5'-ATCCGAGGGAAAGGCTTCGAC-3', antisense 5'-GAGCAGAGGAAGGCTCATTGG-3' (13); ICAM-1: sense 5'-GTAGAGGTGACTGAGGAGTT-3', antisense 5'-ATACAGCACGTGCAGTTCCA-3' (45); VCAM-1: sense 5'-CACTGTCAACTGCACAGTCC-3', antisense 5'-AGAGGCTGTACACTCTGCCT-3' (GI: 20988694); MCP-1: sense 5'-AGCCAACTCTCACTGAAGCCA-3', antisense 5'-CTACAGAAGTGCTTGAGGTGGT-3' (37).
To exclude sample contamination by amplification of genomic DNA, experiments were also performed using RNA as the substrate for PCR. PCR products (-glucuronidase: 301 bp; ICAM-1: 360 bp; VCAM-1: 252 bp; MCP-1: 463 bp) were separated electrophoretically in 2% agarose gel, visualized by ethidium bromide staining, and photographed using a Polaroid Land film type 55 (Kodak, Rochester, NY). The negatives were used for densitometrical quantification of band intensity using the Kodak Digital Science 1D 2.0 Image-Analysis software. Results were normalized to the housekeeping gene and expressed as ratio of cytokines to
-glucuronidase mRNA expression.
Statistical analysis. Data were analyzed using one-way ANOVA with P < 0.05 considered to be significant. All results are expressed as means ± SE. The correlation between different parameters was analyzed by Spearman rank correlation coefficient with P < 0.05 considered to be significant.
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RESULTS |
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The gene transfer of sTGFRII failed to prevent fibrosis formation and aggravated inflammation.
Serum sTGF
RII protein in the mice that received sTGF
RII plasmid was elevated at the peak of day 10 after plasmid injection (20.3 ± 3.9 nM). The gene transfer of sTGF
RII decreased the level of TGF-
significantly compared with the AdTGF
-alone group, and it was significantly lower than even control group at day 14 (Fig. 2A). However, the treatment had no effect on the increased hydroxyproline contents in both the mesentery and anterior abdominal wall (Fig. 3, A and B).
Treatment with sTGFRII plasmid was accompanied by similar weight loss but delayed recovery (Fig. 4) and a significant increase of SAP level at day 10 compared with AdTGF
-alone group (Fig. 5). Moreover, this group of mice showed slight but significant increases of serum TNF-
at days 5, 10, and 14 (Fig. 6). Serum IL-12 level was also higher than AdTGF
-alone group, which was significant at day 14 (200.21 ± 4.77 vs. 250.2 ± 9.98 pg/ml, P = 0.001; AdTGF
-alone group vs. sTGF
RII treatment group, respectively). The expression of MCP-1 was higher but not significant compared with AdTGF
-alone group (Fig. 7A).
Serum sFlt-1, VEGF, and PlGF-2 levels were not significantly changed compared with the AdTGF-alone group (data not shown). However, ICAM-1 expression was upregulated significantly compared with AdTGF
-alone group (Fig. 7B).
The gene transfer of sFlt-1 attenuated fibrosis formation and inflammation.
The gene transfer of sFlt-1 decreased the TGF- levels significantly at day 14 compared with the AdTGF
-alone group (Fig. 2). It also reduced the AdTGF
-induced increase in collagen deposition in the mesentery by
81% (Fig. 3A).
Treatment with sFlt-1 plasmid showed a trend for less weight loss and more rapid weight gain compared with the AdTGF-alone group (Fig. 4) and a trend for lower level of SAP compared with AdTGF
-alone group at day 5 (Fig. 5). There was no significant increase of serum TNF-
(Fig. 6). Although we observed a trend for lower levels of IL-12 than the AdTGF
-alone group at days 5 and 10, this did not reach statistical significance (day 5: 270.84 ± 16.31 vs. 247.99 ± 30.44 pg/ml; day 10: 422.55 ± 26.93 vs. 334.41 ± 51.51 pg/ml, AdTGF
-alone group vs. sFlt-1 treatment group, respectively).
Treatment with sFlt-1 increased the level of serum Flt-1 (14.3 ± 5.5 ng/ml), but this was not significant compared with the AdTGF-alone group (11.4 ± 1.9 ng/ml). Although serum VEGF was elevated in all groups except the control group, there was no significant difference among them at any time point (data not shown). Whereas there was also no significant difference of serum PlGF-2 among these three groups (AdTGF
-alone group and plasmid treatment groups) at each time point (data not shown), a significant negative correlation (r = 0.548, P = 0.0143) between serum sFlt-1 and PlGF-2 in the Flt-1 treatment group at various time points was observed (Fig. 8), whereas there was a slight positive correlation between sFlt-1 and VEGF (r = 0.247, P = 0.0473).
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DISCUSSION |
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The model of peritoneal fibrosis induced by AdTGF- was also accompanied by an increase in serum levels of endogenous sFlt-1, which peaked at day 10 after administration of the adenovirus-TGF-
construct. This is in keeping with other reports showing induction of endogenous sFlt-1 during inflammatory conditions (8).
We used the model to compare two potential therapeutic strategies for preventing fibrosis: blockade of TGF- by sTGF
RII gene transfer or by administration of the anti-angiogenic sFlt-1 plasmid. Our results show that in this model of inflammation and fibrosis, blockade of TGF-
enhanced the inflammatory response and did not attenuate fibrosis. In contrast, sFlt-1 plasmid attenuated the inflammatory response and the fibrotic reaction.
Administration of sFlt-1 plasmid to AdTGF-treated mice was associated with reduced expression of MCP-1, attenuation of SAP and IL-12 increases, and the associated weight loss. Additionally, a reduction of TGF-
was observed in the treated mice; we suggest that this reflects a reduction in endogenous TGF-
secondary to the attenuated inflammatory response.
Soluble Flt-1 treatment also reduced PlGF- in AdTGF-treated mice. It has been shown that PlGF recruits and activates monocytes to produce increased amounts of MCP-1 and TNF-
(23). It has been also reported that PlGF could stimulate pathological angiogenesis by mobilizing bone marrow-derived myeloid progenitors into the peripheral blood, because anti-Flt-1 therapy inhibited the mobilization and neoangiogenesis (8, 24). Soluble Flt-1 gene transfer suppressed VEGF-induced neovascularization (52). Moreover, PlGF could enhance the angiogenic response to VEGF by forming VEGF/PlGF heterodimers (8). In the AdTGF
-induced peritoneal fibrosis model, upregulation of VEGF and neovascularization has been shown (27). Taken in conjunction with the previous studies, our findings are consistent with the interpretation that MCP-1 mRNA expression was downregulated by sFlt-1 gene transfer via a reduction of circulating free PlGF. This may have contributed to neoangiogenesis through Flt-1 and by enhancing the angiogenic effect of VEGF. The negative correlation observed between serum sFlt-1 and PlGF also supports this theory.
The important finding, however, was that administration of sFlt-1 plasmid resulted in an 81% reduction in collagen deposition in AdTGF-treated mice. Taken together, these results suggest that gene transfer of sFlt-1, which binds PlGF, may inhibit the proinflammatory and angiogenic effect of PlGF and may have reduced fibrosis in our model by downregulating MCP-1 and adhesion molecule expression. Reduction of the serum active TGF-
level in the sFlt-1 plasmid-treated group likely reflects the attenuation of inflammation.
The gene transfer of sTGFRII plasmid decreased the level of TGF-
significantly compared with mice that received AdTGF
alone. Interestingly, TGF-
levels were also significantly lower than the control group at day 14. Serum sTGF
RII protein in mice that received sTGF
RII plasmid was elevated day 10 after plasmid injection (20.3 ± 3.9 nM). These results suggest that exogenous sTGF
RII bound sufficient active TGF-
to inhibit its action. TGF
RII administration to AdTGF
-treated mice was accompanied by significant increases in SAP and TNF-
, and the mice showed slower recovery from weight loss compared with mice treated only with AdTGF
. In addition, ICAM-1 mRNA expression was increased. These results clearly indicate that the inflammation was enhanced in the sTGF
RII-treated mice and presumably reflects blockade of the anti-inflammatory effect of TGF-
. Furthermore, there was no significant change in collagen deposition in mice that received AdTGF
plus TGF
RII compared with the group that received AdTGF
alone, despite the reduction of serum active TGF-
. This discrepancy might be explained by the increased expression of MCP-1, which has been also shown to induce expression of both matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Thus MCP-1 may contribute to fibrosis formation through an imbalance of MMP-1 and TIMP-1 (50).
Our results do not support those of previous studies that demonstrated that blockade of TGF- attenuates fibrosis formation (20, 22, 29, 35, 39, 54). This apparent discrepancy may be due to the fact that the TGF-
-induced fibrosis was accompanied by an inflammatory response in the present study, and this was not evident in the previous studies. It is possible that had TGF-
blockade been delayed until resolution of the inflammatory response, and before the establishment of fibrosis, attenuation of fibrogenesis might have occurred.
In conclusion, this study demonstrates that sFlt-1 gene transfer attenuated AdTGF-induced inflammation and prevented late fibrosis formation in the peritoneum. We suggest that the inhibition of PlGF signaling and the subsequent downregulation of both MCP-1 and ICAM-1 expression were partly responsible for fibrosis attenuation in our model. In contrast, sTGF
RII plasmid administration aggravated AdTGF
-induced inflammation and failed to prevent fibrosis formation. From a clinical standpoint, sFlt-1 might be a useful therapeutic option to prevent fibrosis formation in an inflammatory setting. Our findings suggest that in such a setting, blockade of TGF-
is not a therapeutic preference, because it would likely enhance the inflammatory response.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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