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From the Liver Center and Department of Medicine,*
University of California, San Francisco, California; and Biogen,
Inc.,
Cambridge, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important aspect of the injury response is the elaboration of ECM, including collagens I, III, IV, VI as well as fibronectin and other glycoproteins. ECM is elaborated presumably to contain the agent of injury, close the wound, and complete healing. When ECM deposition is excessive, however, it may disrupt the organization of the tissue and lead to impaired function. From numerous recent studies, it is clear that ECM in injury derives principally from stellate cells.12-19 A pivotal event in the response of the liver to injury is the activation of these cells, which is characterized by the de novo appearance of cytokine receptors, ECM production, proliferation, and contraction.18,20,21 Because of its importance to fibrosis and chronic liver injury, the regulation of stellate activation has been a subject of intensive study. Both soluble mediators and the ECM itself play a role.18
Fibronectin is one of the earliest of the ECM components to be expressed after injury. Its primary transcript has three regions, termed EIIIA, EIIIB, and V, that are variably spliced.22-24 The EIIIA and EIIIB domains are either completely included or excluded within the mature molecule. Splice variants containing EIIIA are expressed during development but are minimally present in normal adult tissue. They reappear, however, in the setting of epithelial repair; this has been studied extensively in cutaneous repair.25 It has been demonstrated that the EIIIA isoform is also expressed in various forms of liver injury.26,27 We have shown that this derives from hepatic sinusoidal endothelial cells (SEC) and that it critically modifies the local ECM so as to activate stellate cells.26 Moreover, increased expression of this fibronectin variant is detectable within 12 hours of an injury, preceding any increase in expression of collagen and other structural ECM proteins.26
The present study concerns the regulation of [EIIIA]Fn production, focusing on cytokines that are part of the injury milieu. Although TGFß is particularly prominent in the early repair process, its effects in vivo are not well understood with respect to cellular targets and quantitative changes in Fn isoform expression. We show that it modulates production of [EIIIA]Fn in primary cultures of hepatic sinusoidal endothelial cells and in vivo. The in vivo experiments take advantage of a novel recombinant TGFß-soluble receptor to block the action of TGFß during wound repair. The findings point to the central role of endothelial cells in wound repair and suggest that manipulation of [EIIIA]Fn expression in injury may be beneficial in reducing neomatrix formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pronase and DNase were purchased from Boehringer Mannheim (Indianapolis, IN), collagenase from Serva (Heidelburg, Germany), and Ham’s F-12, Medium 199, DME, and fetal calf and donor horse sera from Flow Laboratories (McLean, VA). Eagle’s MEM without calcium was prepared using amino acids purchased from Sigma Chemical Co. (St Louis, Mo). Accudenz was obtained from Accurate Chemicals (Westbury, NY). Collagen I from rat tail tendon was prepared in the laboratory.
TRI reagent was from Molecular Research Center, Inc., (Cincinnati, OH).
Acrylamide, bis-acrylamide and agarose were from Bio-Rad (Richmond,
CA). Ultrapure urea, Trypsin-EDTA, and RNaseT2 were from GIBCO BRL
(Gaithersburg, MD). T7 RNA polymerase, RQ1 DNase, and RNasin were from
Promega (Madison, WI). Radiolabeled cytidine-5'-triphosphate
([
-32P]CTP, >800 Ci/mmol)
and3
H-thymidine (>50 Ci/mmol) were from
Amersham Corp. (Arlington Heights, IL);
Trans35S-label was from ICN (Irvine, CA). Protein
A sepharose CL-4B was purchased from Pharmacia (Kalamazoo, MI).
Mink lung epithelial cells (CCL64) were obtained from the University of
California San Francisco Cell Culture Facility (San Francisco, CA).
The monoclonal antibody IST-9, which is specific for the EIIIA domain of fibronectin,28 was kindly provided by L. Zardi (Genoa, Italy). Biotinylated sheep anti-mouse IgG and streptavidin-linked Texas red were purchased from Amersham. Avidin-biotin complex (Vectastatin) was from Vector (Burlingame, CA). Purified TGFß and TGFß antibodies were purchased from R&D Systems (Minneapolis, MN). Other chemicals were from Sigma.
Soluble TGFß Type II Receptor (sTGFßR)
The TGFß receptor fusion protein was synthesized using the extracellular domain of the rabbit TGFß type II receptor, which was amplified by PCR from plasmid 3F11.29 The forward primer consisted of nucleotides 1–21 of the rabbit TGFß type II receptor, and the reverse primer was the complement of nucleotides 466–486. To prepare a plasmid encoding the desired fusion protein, DNA fragments encoding amino acids 1–160 of the rabbit TGFß type II receptor were ligated to a DNA fragment encoding the Fc domain of human IgG1 in the Biogen transient expression vector SAB132. DNA sequencing revealed a glycine-to-arginine mutation in the fourth amino acid of the signal peptide. The rabbit TGFß type II receptor-IgG fusion DNA fragment was cloned into the Biogen stable expression plasmid pMDR901 to generate plasmid pMSN001, which was used to stably transfect Chinese hamster ovary cells. Fusion protein secreted by the cells was purified on a protein A sepharose affinity column as previously described.30 The extracellular domain of the rabbit TGFß type II receptor is 83% homologous to that of the rat. The inhibitory activity of the protein was verified and titered in a standard bioassay for TGFß31 as described below.
Animal Model of Liver Injury
Injury was induced in male Sprague-Dawley rats (~400 g body weight) by laparotomy and high ligation of the bile duct.21,32 Controls underwent laparotomy and manipulation of the bile duct but no ligation. The effects of this injury are well documented.20 After 3 days, the liver exhibits cholestasis with periductular inflammation, and collagen I mRNA is markedly increased in stellate cells; after 5 to 7 days, periportal fibrosis is seen histologically. At the time of laparotomy, some animals received sTGFßR (5 mg/kg) or normal human IgG by slow infusion into the inferior vena cava. In preliminary experiments, this amount was found to be sufficient for maximal inhibition of TGFß. Unless otherwise stated, animals were sacrificed 24 hours later for cell isolation. In preliminary studies with varying doses of sTGFßR, 5 mg/kg was found to maximally reduce [EIIIA]Fn expression; in the experiments presented here, this dose was used.
Endothelial Cell Isolation and Culture
SECs were isolated by in situ perfusion with pronase and collagenase and fractionation by centrifugation on a discontinuous gradient of Accudenz (15.6 and 8.2%),33 then further purified by centrifugal elutriation.34 Purity was monitored by labeling the cells in vivo with fluorescent acetoacetylated low density lipoprotein, administered before cell isolation,34 and was >90%. SECs were plated at confluent density in 60 mm culture dishes precoated with a thin layer of type I collagen (~ 40 µg/60 mm dish) to facilitate cell attachment. The culture medium was a modified Medium 19935 with 20% serum (10% calf, 10% horse), insulin (4 mU/ml), and gentamicin (40 µg/ml). For comparison of treated and control cultures, cells from a single animal were used, initiated in culture under identical conditions.
Quantitation of mRNA
Total RNA was isolated from whole liver tissue or from purified SECs using TRI reagent. Its integrity was assessed routinely by visualization of 18S and 28S ribosomal bands on agarose/formaldehyde gel electrophoresis. Radiolabeled probe for [EIIIA]Fn was prepared as described26 and mRNA quantified by RNase protection. Fibronectin mRNA containing the EIIIA domain protected a 280-bp fragment of the labeled cRNA probe, and that lacking EIIIA protected 109 bp. Samples were assayed also for S-14 mRNA, which encodes a ribosomal protein.36 S-14 exhibits minimal variation after bile duct ligation (BDL)26 and was used to control for mRNA loading.
Probes were hybridized with either 20 µg (EIIIA) or 5 µg (S14) of total RNA at 55°C for 16 to 18 hours. Unhybridized RNA was digested with ribonuclease T2.20 The RNA-cRNA hybrids were extracted in phenol-chloroform, precipitated with ammonium acetate and ethanol, and then dissociated by boiling for 3 minutes in electrophoresis buffer containing 80% formamide. Samples were run on a 6% (EIIIA) or 5% (S-14) polyacrylamide/urea gel, dried, and visualized by autoradiography (Kodak X-OMat AR-5). Specific bands were quantitated by scanning densitometry (Hoefer Scientific Instruments, San Francisco, CA) with correction of the raw data for the size difference of the [EIIIA]-positive and [EIIIA]-negative fragments.
Nuclear Run-on Assay for Fibronectin Gene Transcription
Fibronectin transcription was measured by a modification of the nuclear run-on assay described by Kavanaugh et al.37 SECs were isolated and cultured for 24 hours in 20% serum growth medium. After a further overnight incubation in serum-free medium, the cells were incubated for 2 hours with either TGFß (7.5 ng/ml) or vehicle, then lysed in ice cold 10 mmol/L Tris-HCl, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2, and 0.5% NP-40. The lysate was centrifuged to pellet nuclei, which were washed and resuspended in storage buffer (20 mmol/L Tris-HCl, pH 8.0, 50% glycerol, 60 mmol/L NaCl, 100 µmol/L EDTA, 1 mmol/L DTT) to a concentration of 1 to 2 x 107 nuclei/100 µl. An aliquot was inspected by light microscopy and the remainder snap-frozen in liquid nitrogen until use.
Run-on analysis was performed in a total volume of 250 µl comprising
125 µl of nuclei brought to a final concentration of 50 mmol/L
Tris-HCl, pH 8.0, 5 mmol/L MgCl2, 0.5 mmol/L
MnCl2, 100 mmol/L KCl, 0.25 mg/ml BSA, 250 U/ml
RNasin, 50 µmol/L EDTA, 1 mmol/L DTT, 0.5 mmol/L each of rCTP, rGTP,
and rATP, and 125 µCi of -32P-labeled rUTP
(3000 mCi/mol, Amersham). Following incubation on a rotary shaker for
30 minutes at room temperature, the reaction was terminated with the
addition of 12.5 units of DNase I for 20 minutes. Proteinase K (200
µg/ml), SDS (0.2%), and EDTA (5 mmol/L) were added, and the mixture
was incubated at 37°C for 30 minutes. Nuclei were extracted using TRI
reagent, and the final RNA pellet was resuspended in RNase-free water.
An aliquot was counted, and an equal amount of radioactivity in each
sample was used for hybridization to denatured plasmid DNA (5 µg)
slot blotted on nitrocellulose filters. Hybridization was carried out
at 65°C for 40 hours in a buffer consisting of 50 mmol/L PIPES, pH
6.8, 10 mmol/L EDTA, 0.2% SDS, 2.5x Denhardt’s, 100 µg/ml herring
DNA, 100 µg/ml poly(A), and 600 mmol/L NaCl. The filters were then
washed serially in 2x SSC, 2x SSC containing RNase A (10 µg/ml,
37°C), and finally in 2x SSC. After air drying, they were examined
by autoradiography.
Immunohistochemistry of [EIIIA]Fn
After low pressure perfusion with buffered saline via the portal vein, the liver was cut into 0.5-cm2 pieces, which were snap-frozen in liquid nitrogen and stored at -70°C until use. Cryostat sections were stained for [EIIIA]Fn with IST-9 mouse anti-EIIIA antibody; the secondary antibody was sheep anti-mouse.26 Negative controls consisted of sections incubated with nonimmune mouse IgG and processed in parallel.
Metabolic Labeling and Immunoprecipitation of [EIIIA]Fn
SECs in culture (~80% confluent) were placed in serum free medium containing 0.5% bovine serum albumin for 16 hours before metabolic labeling. Cells were labeled for 6 hours with Trans35S-label (200 µCi/ml) in serum free medium without methionine or cysteine. Newly synthesized [EIIIA]Fn protein in conditioned medium and in cell lysates was immunoprecipitated as described,38 with modifications. Cells were lysed on ice for 30 minutes in buffer (0.1 mol/L Tris-HCl, pH 8.3, 0.1% SDS, 1 mmol/L N-ethylmaleimide, 1 mmol/L iodoacetic acid, 2 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L EDTA, 0.5% NP-40, 0.5% deoxycholate). The lysate DNA was then sheared by repeated passage through a 23 G needle, and the mixture was spun at 10,000 x g at 4°C for 10 minutes. The extent of radiolabel incorporation into cellular proteins was determined by trichloroacetic acid precipitation.
Before immunoprecipitation with specific antibody the supernatant was precleared by shaking for 45 minutes at 4°C with protein A sepharose beads previously equilibrated in lysis buffer containing 5 mg/ml ovalbumin. The beads were pelleted (14,000 rpm for 10 seconds), and the supernatant was incubated overnight at 4°C with the primary antibody (IST-9) or nonimmune mouse IgG. One hour before the end of the incubation, polyclonal rabbit anti-mouse IgG was added, and the mixture was incubated for 45 minutes at 4°C in a rotary shaker with 50 µl of protein A sepharose. The beads were washed four times in buffer, resuspended in an equal volume of 2x buffer (6% SDS, 125 mmol/L Tris-HCl, pH 6.8, 20% glycerol) containing 6% ß-mercaptoethanol, and boiled for 5 minutes to dissociate antigen-antibody complexes. After centrifugation at 14,000 rpm for 1 minute to sediment sepharose beads, the proteins in the supernatant were resolved on a 5% SDS-PAGE gel.39 The gel was dried and exposed to X-ray film for 2 to 7 days.
Quantitation of TGFß Secretion
Bioactive TGFß in conditioned medium was measured by its ability to inhibit the proliferation of mink lung epithelial cells.31 Mink lung cells (CCL64) were maintained in DMEM/10% FCS in 75-cm2 flasks. For the bioassay, subconfluent cultures between passages 3 and 10 were used. Cells were washed and trypsinized, and 1 x 104 cells were seeded onto 96-well microtiter plates in 190 µl of DMEM/0.2% FCS/10 mmol/L HEPES (pH 7.4). SECs from normal or BDL rats were isolated and plated directly in serum-free Medium 199. The conditioned medium was harvested 24 hours later and assayed directly for active TGFß. For determining total TGFß, samples were acidified for 10 minutes. (0.05 N HCl final concentration), followed by neutralization with 0.05 N NaOH and HEPES, pH 7.4 (0.025 mol/L final concentration); this treatment converts latent TGFß to the active form.31 Ten microliters of sample were then added to each well and incubated at 37°C for 24 hours. The cells were subsequently incubated with3 H-thymidine (0.1 µCi) for 4 hours, lysed at -70°C, and the incorporated radioactivity trapped onto glass fiber filters (Cambridge Technology Inc., Watertown, MA) using a cell harvester (Cambridge Technology). The radioactivity was measured in a liquid scintillation counter. Each assay was carried out in triplicate; the concentration of TGFß was determined by comparison with a standard curve generated using purified TGFß1.
Statistical Analysis
Data are presented as means ± SE of at least three independent experiments. Differences between groups were analyzed using the unpaired Student’s t-test (2 groups) or the one-factor analysis of variance (multiple comparisons). The significance level for all tests was P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One day after BDL, [EIIIA]Fn mRNA in fresh isolates of SECs was
increased eightfold compared to sham-operated controls (Figure 1
; P < 0.0001). Although
expression of [EIIIA]-negative Fn also was increased (Figure 1)
,
[EIIIA]-positive Fn mRNA as a percent of total Fn mRNA rose from 32%
in controls to 42% in SECs from BDL rats; this difference was
significant (P < 0.001). The stability of the
observed changes in primary culture was examined by placing freshly
isolated SECs from normal or BDL rats in culture for 24 hours under
serum-free conditions. Over this period of culture, [EIIIA]Fn mRNA
remained elevated about sixfold (Figure 2, A and B)
in SECs from BDL animals
compared to controls (P < 0.001). However, the
previously noted spontaneous change in splicing26
was
confirmed: after 24 hours in culture the proportion of [EIIIA]Fn was
similar in all cultures, whether derived from control or BDL rats (57%
[EIIIA]-positive in cells from controls, 61% in cells from BDL
animals).
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We isolated SECs and plated them for 24 hours in medium with 20%
serum to allow for recovery from the isolation procedures. After two
washes the medium was replaced with one without serum, containing
TGFß1 (from a stock in 4 mmol/L HCl and 1 mg/ml BSA) or vehicle.
TGFß1 produced a dose-dependent increase in [EIIIA]Fn mRNA up to
threefold (Figures 3, A and B
;
P < 0.05); expression of [EIIIB]Fn in the same
samples was minimal (data not shown). Concentrations greater than 7.5
ng/ml appeared to be cytotoxic as judged by cellular morphology.
TGFß1 also produced a dose-dependent increase in [EIIIA]Fn mRNA
splicing; at a concentration of 7.5 ng/ml TGFß1, [EIIIA]Fn, as a
proportion of total Fn, increased from 57% at baseline to an average
of 70% after incubation with TGFß1 (7.5 ng/ml). By nuclear run-on
analysis, the change in fibronectin expression was at least in part
transcriptional (Figure 4)
. Exogenous
TGFß1 also induced [EIIIA]Fn protein synthesis, as shown by
metabolic labeling and immunoprecipitation (Figure 5)
.
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, the inhibitor reduced [EIIIA]Fn mRNA
to the level in control cultures; there was relatively little effect on
[EIIIA]-Fn.
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We have postulated that effects of TGFß on hepatic stellate
cells involve predominantly autocrine stimulation.12
To
investigate this, we first measured release of the cytokine by SECs
from injured or normal rat liver. Freshly isolated SECs from BDL rats
were placed in serum-free medium, and TGFß protein secretion was
quantitated after 24 hours using the mink lung cell growth inhibition
assay. Endothelial cells from bile duct ligated animals produced
2.7-fold greater active (P < 0.05) and 3.2-fold
greater total (P < 0.01) TGFß protein than
did SECs isolated from sham-operated controls (Table 1)
.
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; the fraction of total Fn as [EIIIA]Fn also decreased (sTGFßR
32% versus IgG 39%; P < 0.001). The
soluble receptor was similarly effective in preventing the
spontaneous increase in [EIIIA]Fn expression exhibited by normal SEC
placed in culture.26
[EIIIA]Fn protein paralleled mRNA
in these experiments, as documented by studies in which SEC were
metabolically labeled with radiomethionine and [EIIIA]Fn isolated by
immunoprecipitation (data not shown).
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Five rats in each treatment group were subjected to either sham
operation, BDL with infusion of the sTGFßR, or BDL with infusion of
control human IgG. Whole liver tissue was extracted 24 hours later for
assay of fibronectin mRNA. [EIIIA]Fn mRNA in whole liver extracts
rose 2.3 ± 0.2-fold in bile duct-ligated rats receiving control
IgG compared to sham-operated controls (P <
0.02). This was similar to the increase observed in untreated BDL rats.
By contrast, the level in rats infused with the recombinant peptide
increased only 1.2 ± 0.1-fold. The difference between this and
the value from animals infused with control IgG was significant (Figure 8
; P < 0.01). Infusion
of sTGFßR also reduced [EIIIA]Fn mRNA splicing compared to rats
receiving control peptide (Table 2
;
6.2 ± 1.3% versus 10.4 ± 1.5%;
P < 0.01). The percent of [EIIIA]Fn in rats
receiving IgG was not different from that in sham-operated animals. We
next examined [EIIIA]Fn mRNA expression and splicing in SECs isolated
from from BDL rats that had been treated with sTGFßR or IgG (Figure 9A)
. In the animals receiving sTGFßR,
[EIIIA]Fn mRNA expression was less than one-half that in controls
(Figure 9B
; P < 0.0003); [EIIIA]Fn as a percent of
total fibronectin was also reduced by 16% (P <
0.05).
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. In animals receiving
sTGFßR after BDL, the intensity of perisinusoidal staining was
similar to that seen in control liver (Figure 10B)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This cytokine has been widely implicated in the wound repair response as a direct stimulus to ECM production by fibroblasts and myofibroblasts.7,40-44 Although it acts similarly on stellate cells in culture,15,19 its effects are modest relative to the change in ECM expression that occurs in liver injury in vivo.20 This suggests either that it acts indirectly or that other factors are at play in stellate activation. The present results provide both cell-culture and in vivo evidence that much of the effect of TGFß is indirect, through stimulation of [EIIIA]Fn production by sinusoidal endothelial cells. Previous studies of fibrosing injury to the kidney45 or lung46 have suggested that [EIIIA]Fn expression is modulated by TGFß but have provided no information as to the cellular source(s) or target(s) of this fibronectin variant.
The source of TGFß in liver injury is an interesting and important question. In the current paradigm, injury elicits inflammatory cells, which release TGFß among other cytokines and may locally stimulate SEC and stellate cells. This is a model of paracrine signaling. Although platelets are considered the richest source of this cytokine,26 mononuclear cells express it also.47 When TGFß is elevated in liver (for example, in liver infected with the hepatitis C virus48 ), it may be due in part to the mononuclear cell infiltrate that is characteristic of this injury. However, it is reasonable to consider also the possibility that TGFß in injury derives from resident liver cells. We have shown previously that TGFß is produced constitutively by SEC and Kupffer cells.11 Moreover, these same cells express type I and type II TGFß receptors.49 Thus, a potential pathway for autocrine signaling is present. As discussed elsewhere,11 autocrine signaling may be a mechanism for locally restricting the effects of TGFß within the intact tissue. The present results are consistent with autocrine effects of this cytokine on SEC production of [EIIIA]Fn, at least in the context of wound repair.
The findings do not preclude a role for other factors in the initiation of stellate activation. The direct effect of TGFß on sinusoidal endothelial cells involves a threefold increase in [EIIIA]Fn at the mRNA level. Although this is significant, it is less than the approximately eightfold increase exhibited by SECs isolated directly from the injured liver. This suggests that TGFß is responsible for only a portion of the change in [EIIIA]Fn expression. Consistent with this, the effect of inhibitory sTGFßR on the culture-induced increase, although significant, was incomplete. Though some TGFß may be inaccessible to the soluble inhibitor, the findings more likely reflect the presence of other regulators of stellate activation. From the perspective of using this kind of inhibitor as anti-fibrotic therapy, incomplete inhibition is desirable: total inhibition of the repair process would raise significant safety concerns.
Information from culture is valuable in that it is direct. Its confirmation in vivo, however, is required, given that culture can induce rapid change in the cell phenotype. Moreover, in autocrine or paracrine pathways, the actual (physiological) concentration of an agonist at the receptor is unknowable. Thus, the relevance of culture experiments in which agonist is added at an arbitrary concentration is necessarily uncertain. Some in vivo models have the same shortcoming, eg, infusion or transgenic overexpression of TGFß. In a transgenic model involving constitutive overproduction and secretion of TGFß by hepatocytes under the albumin promoter, liver fibrosis was surprisingly limited, suggesting barriers to paracrine stimulation of stellate cells. Rather, the principal site of damage was the kidneys, reflecting the high circulating levels of active TGFß.43
Modulation of the endogenous cytokine avoids some of the issues inherent in transgenic animals. This was accomplished by introducing a competitive inhibitor of the type II receptor, consisting of human IgG into which a peptide representing the ligand-binding extracellular domain of the receptor was inserted. The chimeric IgG competes for endogenous TGFß, preventing its binding to receptors on endothelial (and other) cells. As an IgG, it circulates with a half-life of several days. A single injection blocked the induction of [EIIIA]Fn by about 50%, which is entirely consistent with the culture data and confirms in vivo the role of TGFß in the early phase of the hepatic injury response. Because the [EIIIA]Fn elaborated by SEC is capable of directly stimulating stellate cells,15 the possibility exists that blockade of TGFß at the level of the SEC would have significant downstream effects on fibrogenesis. In ongoing work, we have found that administration of sTGFßR reduces markers of stellate cell activation including collagen I expression.50
The apparently transcriptional effect of TGFß and the fact that this cytokine causes both increased expression of [EIIIA]Fn and an altered pattern of splicing also are of interest. Although the change in splicing in response to TGFß appears small, it is statistically highly significant; moreover, it is similar to previously published data.7 The mechanisms for alternate splicing and its relationship to transcription and posttranscriptional processing are not clearly defined. Recent studies demonstrated that the extent of EIIIA splicing is dependent on promoter architecture.51 Our data support the postulate that regulation of transcription and splicing are intimately associated. A comparison of promoter architecture in SECs and hepatocytes, respectively, could prove informative, given the complete absence of a response to injury in hepatocytes. Such studies are anticipated.
Finally, it is worth noting that these data may be broadly applicable to epithelial fibrosis. Mechanisms of fibrogenesis in kidney appear to be very similar to those in liver, including an injury-related increase in [EIIIA]Fn expression.45,52 By analogy with the liver, it seems likely that the source of [EIIIA]Fn in the kidney is the microvascular endothelium, although this remains to be shown. Moreover, the demonstrated antifibrogenic effect of administered anti-TGFß in this tissue53 may well reflect inhibition of [EIIIA]Fn expression. In short, the regulatory effect of TGFß on [EIIIA]Fn production likely is of general relevance to epithelial injury.
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Acknowledgements |
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Footnotes |
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Supported by grants DK31198 and DK26743 from the U.S. National Institutes of Health and by RO3 TW00717 from the Fogarty International Center, NIH. J.G. was the recipient of a Neil-Hamilton-Fairley Fellowship (Australia) and a postdoctoral fellowship from the American Liver Foundation.
Accepted for publication September 16, 1999.
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References |
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1ß1 integrin in wound contraction A quantitative analysis of liver myofibroblasts in vivo and in primary culture. J Biol Chem 1997, 272:30911-30917 in chronic liver disease: effects of interferon alfa therapy. New Engl J Med 1991, 324:933-940[Abstract]
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