Targeting Myofibroblasts in Model Systems of Fibrosis
by an Artificial a-Smooth Muscle-Actin Promoter Hybrid
Julia Hirschfeld Æ Æ Julia Maurer Æ Æ Diana Jung Æ Æ
Monika Kwiecinski Æ Æ Al Karim Khimji Æ Æ Hans Peter Dienes Æ Æ
Jochen W. U. Fries Æ Æ Margarete Odenthal
? Humana Press 2009
ducing extracellular matrix proteins in a variety of fibrotic
diseases. Therefore, they are useful targets for studies of
approaches in scarring diseases. An artificial promoter
containing the -702 bp regulatory sequence of the
a-smooth muscle actin (SMA) gene linked to the first
intron enhancer sequence of the b-actin gene and the
b-globin intron-exon junction was constructed and tested
for myofibroblast-dependent gene expression using the
green fluorescent protein as a reporter. Reporter expression
revealed myofibroblast-specific function in hepatic and
Myofibroblasts are the main cell types pro-
renal myofibroblasts, in vitro. In addition, differentiation-
dependent activation of the SMA-b-actin promoter hybrid
was shown after induction of myofibroblastic features in
mesangial cells by stretching treatment. Furthermore,
wound healing experiments with SMA-b-actin promoter
reporter mice demonstrated myofibroblast-specific action,
in vivo. In conclusion, the -702 bp regulatory region of
the SMA promoter linked to enhancing b-actin and
b-globin sequences benefits from its small size and is
suggested as a promising tool to target myofibroblasts as
the crucial cell type in various scarring processes.
Fibrosis ? Wound healing ? Gene therapy
Artificial promoter ? Myofibroblasts ?
The myofibroblast is a mesenchymal cell type with func-
tional and structural characteristics in common with both
fibroblasts and smooth muscle cells (SMC). In wound
healing of the skin, myofibroblasts participate in wound
closure by wound contraction and synthesis of extracellular
matrix (ECM) proteins. In several fibrotic diseases, such as
hepatitis, glomerulonephritis, or chronic pulmonary dis-
eases, myofibroblasts are even the dominant matrix pro-
ducing cells [1, 2].
Although myofibroblasts are regarded as modulated
fibroblasts, their origin can vary between different cell
types . Thus, during liver fibrogenesis, myofibroblastic
cells develop out of portal fibroblasts  and hepatic
stellate cells (HSC), a pericyte-like cell type [5, 6]. In
scarring diseases of the kidney, however, mesangial cells
transdifferentiate into myofibroblastic cells, after initial
mesangial cell proliferation, induced by inflammatory or
The first authors J. Hirschfeld and J. Maurer contributed equally to this
study as well as the senior authors J. W. U. Fries and M. Odenthal.
Electronic supplementary material
article (doi:10.1007/s12033-009-9186-4) contains supplementary
material, which is available to authorized users.
The online version of this
J. Hirschfeld ? J. Maurer ? D. Jung ? M. Kwiecinski ?
A. K. Khimji ? H. P. Dienes ? J. W. U. Fries (&) ?
M. Odenthal (&)
Institute of Pathology, University Hospital Cologne,
Department of Pharmacology, Howard Hughes Medical
Institute, University of Texas Southwestern Medical Center,
Dallas, TX, USA
A. K. Khimji
Internal Medicine, Digestive and Liver Diseases, University
of Texas Southwestern Medical Center, Dallas, TX, USA
metabolic causes or mechanical stress such as IgA, dia-
betic, or hypertension-based nephropathy. During tubulo-
interstitial fibrosis of the kidney, however, mainly
fibroblasts and also to much lesser extent, bone marrow
cells and tubular epithelial cells develop into myofibro-
blasts and participate in fibrotic ECM accumulation .
Besides wound healing and fibrosis, during the conversion
of in situ carcinomas into invasive cancer, myofibroblasts
with fibrogenic and contractile properties appear, which are
suggested to origin from epithelial cells undergoing epi-
thelial mesenchymal transition .
a-Smooth muscle actin (SMA) is presently the only
known unique immunological marker protein for myofi-
broblasts . SMA is a main cytoskeletal member of the
cytoskeleton of differentiated smooth muscle cells (SMC)
and is also transiently expressed in skeletal and cardiac
myotubes during embryogenesis . Expression in SMC
depends on several conserved cis-elements including three
CArG-elements designated CArG-A, B, and C [10–12].
CArG-boxes consist of an A/T repeat flanked by CC/GG
and are suggested to be involved in muscle-specific regu-
lation. The CArG-C is an inversed CArG-box and the
CArG-boxes B and A, mediating core promoter activity
, are conserved and bind SRF . The more upstream
located sequence comprises two E-box consensus sequen-
ces (CANNTG), which are binding sites for class I basic
helix-loop-helix proteins such as E12, E47, E2-2 [14, 15].
Since gene regulation studied in culture systems does not
represent the in vivo environmental cues, a transgenic
mouse reporter model for accurate investigation of pro-
moter activity in SMC has been performed [16–18]. Wang
et al.  reported that the 1100 bp 50-flanking region of
SMA, in addition to its entire first intron, could drive
expression of an IGF-I transgene in many smooth muscle
tissues. This study has been focused on analysis of IGF-I
expression in SMC and not on promoter activity and
expression pattern, which has been performed in further
study by Mack and Owens . They could show that a
547-bp promoter fragment leads to an embryonic SMA-
specific transgene expression in heart and in skeletal
muscle, but not in SMC. An extended 2560 bp fragment in
combination with the first intron, however, results in
smooth muscle-specific gene expression in embryonic and
adult mice . Kawada et al., and recently Tomasek et al.,
suggest that the transgenic gene expression in myofibro-
blasts also depends on the extended fragment of a
-2560 bp upstream region, in addition to the CArG-box of
the first ?2764 bp intron sequence, resulting in a regula-
tory unit of more than 5.5 kb [17, 19]. Since long and high
molecular weight constructs are difficult to handle and in
particular not suitable to be integrated into adenoassociated
adenovirus (AAV) vectors , we created and charac-
terized an artificial promoter of only 1595 bp length in total
as a most specific means for genetically engineering
myofibroblastic cells. In this artificial promoter construct,
the regulatory sequence of the -702 bp upstream region of
the SMA gene, consisting of the E1,2-boxes, the CArG-
boxes, and a TGFb-responsive element (TCE), was com-
bined to the first intron of the b-actin gene, which was in
turn linked to the b-globin splicing site for intron termi-
nation. Myofibroblast-specific and differentiation-depen-
dent regulation of the artificial promoter construct was
tested in myofibroblast-specific cell culture systems of liver
and kidney in vitro and after induction of wound healing
processes in a green fluorescent protein (GFP)-reporter
mouse model in vivo.
Materials and Methods
Plasmids and Adenoviral Vectors
A 720 bp fragment of 50-region of the rat SMA gene (-702
to ?18) was amplified from genomic rat DNA by PCR
using the SMA-for and SMA-rev primers (Table 1) and
cloned in pB-SKII ? (Stratagene, La Jolla, CA, USA).
Different deletion constructs were generated either by PCR
amplification, using the SMA-190 or SMA-230 primer
(Table 1),respectively, or
sequences of different length (Fig. 1a) were cloned into a
pRL-zero vector (Promega, Mannheim, Germany).
To obtain a construct for transgenic expression in
myofibroblasts, the pCX-EGFP plasmid  (kindly
provided by Dr. J. Hescheler, University of Cologne,
Germany) was modified by replacing the chicken b-actin
promoter with the -720 bp fragment (?18 to -702) of the
SMA gene. The resulting HindIII/HindIII expression cas-
sette was used for mice oocyte microinjection and for
further subcloning into the pShuttle vector (Stratagene)
producing the pShuttle-SMA-GFP vector.
The pShuttle-SMA-GFP and the pAdtrackCMV-GFP
(www.coloncancer.org) were recombined with the adeno-
virus backbone, resulting in pAd-SMA-GFP and in pAd-
Easy-GFP, respectively. Then, the PacI-linearized plasmids
were used for transfection and virus production in HEK293
cells. Adenoviral titration was carried out by immunologi-
cal detection of viral proteins using Adeno-XTMRapid Titer
Kit (BD Biosciences, Clontech, Palo Alto, USA). The viral
stock was stored at -70?C for future use. Depending on the
cell types, 5–80 MOI were used for viral transduction.
A7R5, L6, and Rat2 cell lines were purchased from ATCC
(Rockville, MD, USA) and LFCl2A cells from DSMZ
(Braunschweig, Germany). Immortalized myofibroblastic
HSC, Pav-1, were donated by Dr. J. Rosenbaum (Univer-
sity of Bordeaux, France) .
Primary SMC were isolated from the thoracic aorta from
adult rats. Isolation and plastic-induced in vitro activation
of primary HSC were performed as previously described
[23, 24]. Primary mesangial cells used for experiments in
passage 6–8 were purchased from Cell Systems (St. Kat-
harinen, Germany) and maintained in culture according to
the recommendations of the distributor. The other cell types
were cultured in DMEM (Sigma, Dreisendorf, Germany).
All cultured cells were characterized for SMA expres-
sion by immunofluorescence using FITC-conjugated 1A4
anti-SMA antibodies (Sigma) (1:400). Table 2 summarizes
the results of SMA immunostaining of various cell types
used in our study.
For transfection assays, FuGENE
manufacturer’s instructions (Roche, Mannheim, Germany).
All transfection experiments were performed in triplicates
two times, each with two different preparations of the pRL-
reporter and the pGL3-CMV reference plasmids. The
luciferase assays were carried out by means of the dual-
luciferase reporter-assay-system (Promega, Mannheim,
Germany) according to manufacturer’s instructions and the
pGL-CMV reporter activity was used for normalization.
TM6 was used according to
Preparation of Nuclear Extracts and Electrophoretic
Mobility Shift Assays
Nuclear extracts were prepared by the method of Dignam
et al. . Double-stranded oligonucleotide probes were
obtained by hybridizing single-stranded oligonucleotides
(Eurofins MWG, Ebersberg, Germany). The sense oligo-
nucleotide sequences of the regulatory elements are listed in
Table 1.Forgelmobilityassays,5 lgofnuclearextract was
incubated in a 20-ll reaction mix with 2–5 fmol c[32P] ATP
(Amersham Biosciences, Braunschweig, Germany) end-
labeled double-stranded oligonucleotides for 15 min on ice.
Stretching of Mesangial Cells
Human mesangial cells were plated on collagen-coated
elastic membranes (Bioflex, Dunn Labortechnik, Asbach,
Germany). Stretching was performed as described by
Banes et al.  and Harris et al.  in a defined rhythm
of 15 or 30 cycles per minute for 48 h.
Microfluidic-Based Flow Cytometry for GFP
pAdSMA-GFP transduced mesangial cells were collected
after stretching for 48 h and GFP expression was analyzed
quantitatively with the Bioanalyzer 2001 (Agilent, Palo
Alto, USA) using a Cell Assay Chip and the Fluorescence
LabChip kit. The procedure was performed according to
GFP Transgenic Reporter Mice and Wound Healing
Experimental animals were treated in accordance with the
criteria outlined in the PHS Policy on Humane Care and
Use of Laboratory Animals. Transgenic mice were gener-
ated as previously described . In brief, transgenic
mouse lines were established by injecting the purified
HindIII–HindIII fragment (Fig. 2a) into the pronuclei of
FVB mice fertilized eggs. Integration of the transgene was
tested by PCR in 16 newborns and 4 founder generations
were further tested.
Male GFP-reporter mice weighing 22–28 g and approx-
was collected (2–3 mm border was excised around the
wound), formalin fixed, and paraffin embedded.
Table 1 Oligonucleotides used
for cloning or EMSA
E-boxes and CArG elements are
shown in bold letters, small
letters of B-mut1,2 indicate
substituted bases of the
underlined CArG region B
Immunostaining of Formalin-Fixed
and Paraffin-Embedded Wound Biopsies
After blocking of endogenous biotin by the avidin-biotin
blocking kit (Vector Laboratories, Peterborough, UK) each
for 30 min, 3-lm sections of formalin-fixed and paraffin-
embedded tissues were treated with normal goat serum, and
then incubated with either rabbit anti-GFP antibody (1:200,
Abcam, UK) or anti-SMA-POX (1A4, 1:200; Sigma)
overnight. GFP antibodies were recognized by the avidin-
biotin complex using the alkaline phosphatase-coupled
Vectastain ABC AP-kit (Vector Laboratories, Peterbor-
ough, UK) followed by chromogen development of the
alkaline phosphatase with Fast Red as the substrate or DAB
as a substrate of peroxidase. Sections were then counter-
stained with hematoxylin.
Results and Discussion
-702 bp Upstream Region of the a-SMA Promoter
Drives Gene Expression in Myofibroblasts
The reporter activities of the deletion mutants of the SMA
regulatory sequence (Fig. 1a) revealed that in SMA
expressing A7R5 and primary SMC, all reporter constructs
showed 3–20-fold higher activities in comparison with
reporter activities of non-expressing Rat2 fibroblasts
Fig. 1 Regulatory capacities of the upstream sequences of the SMA
gene in fibroblasts, SMC, and hepatic myofibroblasts. a Reporter
constructs containing various upstream sequences of the SMA gene
(-125, -190, -215, -245, -480, -702 bp). The HLH-binding sites
(E1 and E2-box) are located at position -214[-219 and -252[
-257. Four CArG-boxes (A, B, C, and D) are present in the upstream
region of the SMA gene. The CArG-C (-162[-172) is an inversed
CArG box and the boxes-B and A, shown to bind the SRF , are
located in the core promoter region. In addition, a TCE (TGFb control
element) at position -43[-53 in the core promoter region is
suggested to mediate TGFb-induced transcription by Sp1/3 binding
[31, 32]. b Luciferase activity of reporter constructs of the upstream
region of the SMA gene (a) in SMA-negative Rat2 fibroblasts in
comparison to SMA-positive A7R5 and in primary SMC. c Reporter
activity of deletion constructs of SMA gene in early (HSC d3) and in
late stages of in vitro induced myofibroblastic differentiation of
primary HSC. d Binding of nuclear extracts from in vitro activated
myofibroblasts (HSC) compared to SMA-negative cells and SMA-
positive L6 cells to the two HLH consensus sequences E1 and E2, to
the inversed CArG-C element and to two CArG-boxes A and B shown
by EMSA. Binding to the Ap1 element was used as loading control.
Arrows indicate a similar pattern in binding of nuclear extracts from
myofibroblasts and SMA-positive cells compared to SMA-negative
(Fig. 1b). The SMA-190 reporter construct, containing the
conserved and inversed CArG-boxes A, B, C, was shown to
result in the highest activity. Since the CArG boxes bind
the non-smooth muscle-specific transcription factor SRF
, SMC-specific expression requires additional flanking
sequences, as previously postulated [14, 29]. Thus, only the
extended fragments of -480 bp, -702 bp showed no
reporter activity in fibroblasts, but moderate expression in
SMC. In agreement with former findings on embryonic
skeletal muscle cells, the -702 bp promoter fragment also
mimics transient SMA-expression in myotubes (Supple-
mental Figure S1) [9, 29, 30].
To determine the responsiveness of the deletion mutants
of the 50-flanking SMA gene sequences in myofibroblasts,
we studied reporter activity during in vitro induced myo-
fibroblastic transition of primary HSC. Myofibroblastic
differentiation of HSC is a key event of liver fibrogenesis
resulting in increased matrix deposition and hepatic
hypertension by induced contractility of these liver cells [5,
6]. In vitro, myofibroblastic differentiation is induced by
culturing the cells on plastic (Table 2), mimicking the
phenotypical alterations observed in vivo [23, 24]. In
agreement with the data of SMC versus fibroblasts shown
above (Fig. 2c), all different deletion constructs resulted in
high reporter activity in SMA-positive myofibroblasts, but
in only moderate activity in quiescent cells, expressing
only sparse amounts of SMA (Fig. 2c). Furthermore, these
transfection studies showed that extension of the upstream
sequence (-245 up to -703 bp) caused reporter repression
in all cell types. However, whereas the responsiveness of
reporter activities was remarkably repressed in SMA-
expressing cell types, the activities were almost completely
abolished in cells of an SMA-negative phenotype. Thus,
SMA-promoter activity in myofibroblasts depends on
additional sequences containing the E-boxes of the
-702 bp construct.
Next, we compared the binding pattern of myofibro-
blasts with SMA-positive SMC and skeletal myotubes. The
positive acting CARG-boxes  driving the high pro-
moter activity of the -190 bp fragment (Fig. 1a–c, and
Supplemental Figure S1) and the E1- and the E2-box of the
extended -702 bp promoter sequence [14, 15, 30] were
analyzed. The complexes exhibited identical electropho-
retic mobilities, but varying intensities in different cell
types. A prominent binding to the E1-box in non-SMA-
expressing cells, but moderate binding in SMA-expressing
cells occurred. In contrast, nuclear extracts of all SMA-
expressing cells, such as myotubes, SMC, and myofibro-
blasts, prominently bound to the CArG-boxes, especially to
the CArG-box C, as shown in Fig. 1a.
Thus, the mobility shift assays of the E-boxes and the
CArG-elements of the -702 bp SMA-promoter fragment
revealed an equivalent protein interaction in myofibroblasts
as observed in other SMA-positive cells.
Table 2 SMA Immunostaining of cell types, used in this study
Species Cell type/originTreatment/supplierFigure SMA
A7R5 RatAorta SMC Immortalized (ATCC)Figure 1b?
Rat2Rat Embryonic fibroblasts Immortalized (ATCC)Figure 1b-
Pav1 RatMyofibroblastic HSC, liver Immortalized Figure 2b?
LFCl2A Rat Hepatocarcinoma, liverImmortalized (DSMZ) Figure 2b-
L6 myoblasts RatEmbryonic skeletal muscle cells Immortalized (ATCC) Figure 1d,
L6 myotubesRat Embryonic skeletal muscle cellsImmortalized, FCS deprivation
SMCRatPrimary cells, rat aorta
HSCRatPrimary HSC, liver Freshly isolated, 0-36 h culture,
quiescent [23, 24]
HSCRatPrimary HSC, liverFreshly isolated, plastic-dish
activated by culture [23, 24]
Mesangial cells HumanPrimary mesangial cells, kidney Activated by high glucose
(4.5 g/l) in DMEMb
Mesangial cellsHumanPrimary mesangial cells, kidneyActivated by stretching [22, 26] Figure 2c?
SMC smooth muscle cells, HSC hepatic stellate cells, FCS fetal calf serum
aAccording to Hultgardh-Nilsson et al., Cardiovascular Research 34, 418–430, 1997
bAccording to Ayo et al. American Journal of Pathology 136, 1339–1348, 1990
Construction of an SMA-b-actin Promoter Hybrid
Our present studies have demonstrated that the -702 reg-
ulatory sequence, which is responsible for SMC-specific
gene expression in vitro, is also able to drive gene
expression in myofibroblasts. Besides the E-boxes and
CArG-elements, this fragment contains a TCE at position
-43, shown to contribute to transcriptional activation by
TGFb . Furthermore, a single-stranded transcription
factor binding sequence, which also regulates SMC-specific
Fig. 2 Promoter specific GFP-reporter fluorescence in myofibroblasts
and non-myofibroblasts. a GFP reporter expression cassette driven by
an artificial SMA promoter composed of the -702 bp upstream
regulatory region of the rat SMA gene (Accession no. S76011 from
position 10 to 730), the enhancing sequence of first intron from the
chicken b-actin gene (Accession no. X00182 from position 750 to
1503), and the intron/exon junction (IE) of the rabbit b-globin
sequence (intron 2/exon 3 Accession no. V00882 from position 1250
to 1343). b Hepatic Pav-1 myofibroblasts (b, d, f, h) and hepatic non-
myofibroblastic carcinoma LFCL2A cells (a, c, e, g) 2 days after
infection with five MOI pAdEasy-GFP (a–d) or pAdSMA-GFP (e–h).
Whereas the CMV promoter of the pAdEasy-GFP drives GFP-
reporter expression in both cell types, the artificial SMA promoter (a)
drives expression only in Pav-1 myofibroblasts. Bright field (a, b, e, f)
and dark field under GFP-exciting light (c, d, g, h) (magnifications
9100). c Reporter expression of mesangial cells infected with
pAdSMA-GFP after myofibroblastic activation by 48 h stretching
with 15 or 30 cycles per minute. GFP-positive cells (green bordered
window) were determined by microfluidic-based flow-cytometry.
GFP-reporter fluorescence increases after more intensive stretching
treatment (30 cycles per minute). (Color figure online)
connexin-40 expression, is located at position -192 .
These two elements regulating SMC-specific gene expres-
sion [31, 32] might be also important for myofibroblast-
specific transcription. However, transgenic reporter studies
revealed that extended SMA promoter fragments are nec-
essary to drive SMC-specific gene expression in vivo .
In addition to the extended promoter of -2560 bp including
the TCE at position -43, transcriptional control in SMC
and myofibroblastic cells was shown to depend on the first
intron of the SMA gene containing a conserved CArG-box
[17–19]. Therefore, we linked the -702 bp regulatory
SMA-fragment to 754 bp of the first intron of the chicken
b-actin gene, which includes a conserved CArG-box at
position ?1029, known to enhance transcriptional activity
. For proper intron termination, the b-globin intron
II/exon III splicing site was included (Fig. 2a).
Myofibroblast-Specific Gene Expression by the
SMA-b-Actin Promoter Hybrid, In Vitro and In Vivo
The promoter hybrid shown in Fig. 2a was inserted in an
adenoviral GFP vector type 5, which guarantees high
transduction efficiency of primary cells, and tested for
myofibroblast-specific gene regulation in hepatic and renal
cell systems (Table 2). Compared analyses of reporter
expression in myofibroblasts with several non-myofibrob-
lastic cells, such as fibroblasts, renal, and hepatic epithelial
cells, proved high myofibroblast-specific activity of the
artificial SMA-promoter construct (Fig. 2b; Supplemental
Figure S2). In an in vitro model of diabetic nephropathy
we have shown that glucosylated medium results in com-
plete myofibroblastic differentiation of mesangial cells
accompanied by SMA-promoter-driven GFP reporter
expression within 7 days (Supplemental Figure S2). Pre-
liminary attempts to reverse this process by continued
cultivation of transdifferentiated cells under normal med-
ium for three additional weeks did not succeed. Thus, we
next addressed myofibroblast-induced promoter activation
using pathophysiological conversion of primary mesangial
cells into myofibroblasts by mechanical stress [27, 33].
Stretching-induced activation of myofibroblastic transition
is believed to be based on autocrine TGFb stimulation ,
which was previously shown to be an important feature
of myofibroblastic transdifferentiation and induction of
SMA-expression [2, 31]. As shown in Fig. 2c, elevated
mechanical stress increased GFP reporter expression more
than twofold in mesangial cells.
myofibroblast-specific and differentiation-dependent gene
expression in various cell systems representing the myofi-
broblast occurrence in wound healing and scarring diseases.
However, promoter activity often differs when the
environmental cues are missing. In order to demonstrate
myofibroblast-specific gene regulation by the SMA-pro-
moter hybrid in vivo, we created a transgenic reporter
mouse model, carrying the expression cassette used for the
preceding in vitro studies and shown in Fig. 2a. Myofib-
roblastic transition of skin fibroblasts was induced by skin
injury. SMA-positive myofibroblasts occur after 5 days
with increasing number after 7 days. Immunochemical
GFP staining of serial sections revealed reporter expression
in spindle-typed SMA-positive myofibrobasts (Fig. 3).
Thus, the -702-SMA regulatory sequence combined
with a fragment of the first b-actin intron is able to control
also gene expression in myofibroblasts in vivo. In contrast
to the regulatory unit of previous study ([5.5 kb), this
artificial promoter hybrid benefits from the small size (ca.
1.5 kb). In this way, it is applicable to different viral vector
systems including AAV vectors, which are limited in the
size of the transgene . Vectors, carrying the transgene
under the control of the SMA-promoter construct, are
useful to target and manipulate gene expression of
Fig. 3 GFP expression in myofibroblasts of 7-day-old wounds in
reporter transgenic mice containing the SMA-b-actin promoter hybrid
as controlling sequence. Pattern of SMA-expression by spindle-
shaped cells (brown) clearly identified myofibroblasts in the scarring
wound. Myofibroblasts also express the reporter GFP (red), whose
expression was driven by the artificial SMA promoter hybrid in the
reporter transgenic mice. Immunostaining of developed with the
peroxidase substrate, DAB (brown), and of GFP developed by the
phosphatase substrate, New Fuchsin (red). (Color figure online)
myofibroblasts, in order to study not only different fibrotic
diseases, such as hepatic or renal scarring injuries of var-
ious causes, but also pulmonary chronic diseases, e.g.,
asthma. Furthermore, reporters driven by this promoter
hybrid allow both pharmaceutical screening and the fol-
low-up of different therapeutical strategies of various dis-
eases mentioned above.
Therefore, the linkage of the -702 bp regulatory part of
the SMA-promoter to the enhancing sequence of the b-
actin gene is a promising tool to address myofibroblasts
that have taken center stage as targets of molecular and
therapeutical approaches in different scarring diseases after
chronic organ damage.
Roth, and Melanie Scheffler for excellent technical support. This
study was supported by the German Competence Network for Viral
Hepatitis (HepNet), funded by the German Ministry of Education and
We would like to thank Daniela Lohfink, Tanja
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