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Investigation of sensory neurogenic components in a bleomycin-induced scleroderma model using transient receptor potential vanilloid 1 receptor and calcitonin gene-related peptideknockout mice.

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ARTHRITIS & RHEUMATISM
Vol. 58, No. 1, January 2008, pp 292–301
DOI 10.1002/art.23168
© 2008, American College of Rheumatology
Investigation of Sensory Neurogenic Components in a
Bleomycin-Induced Scleroderma Model Using
Transient Receptor Potential Vanilloid 1 Receptor– and
Calcitonin Gene-Related Peptide–Knockout Mice
Árpád Szabó,1 László Czirják,1 Zoltán Sándor,1 Zsuzsanna Helyes,1 Terézia László,1
Krisztián Elekes,1 Tamás Czömpöly,1 Anna Starr,2 Susan Brain,2
János Szolcsányi,1 and Erika Pintér1
ness and the number of ␣-smooth muscle actin (␣SMA)–positive cells were also determined. The quantity
of the collagen-specific amino acid hydroxyproline was
measured by spectrophotometry.
Results. Bleomycin treatment induced marked
cutaneous thickening and fibrosis compared with that
observed in control mice treated with PBS. The composite sclerosis score was 18% higher, dermal thickness was
19% higher, the number of ␣-SMA–positive cells was
47% higher, and the amount of hydroxyproline was 57%
higher in TRPV1ⴚ/ⴚ mice than in their WT counterparts. Similarly, the composite sclerosis score was 47%
higher, dermal thickness was 29% higher, the number of
␣-SMA–positive cells was 76% higher, and the amount
of hydroxyproline was 30% higher in CGRPⴚ/ⴚ mice
than in the respective WT groups.
Conclusion. These results suggest that activation
of the TRPV1 receptor by mediators of inflammation
induces sensory neuropeptide release, which might exert
protective action against fibrosis. We confirmed the
protective role of CGRP in the development of cutaneous sclerosis.
Objective. Along with their classic afferent function (nociception), capsaicin-sensitive transient receptor potential vanilloid 1 (TRPV1) receptor–expressing
sensory nerve terminals exert local and systemic efferent activities. Activation of TRPV1 causes sensory neuropeptide release, which modulates the inflammation
process. The aim of the present study was to examine
the role of this modulatory role of TRPV1 receptor and
that of calcitonin gene-related peptide (CGRP) in
bleomycin-induced scleroderma, using transgenic mice.
Methods. Cutaneous sclerosis was induced with
daily subcutaneous injections of bleomycin for 30 days.
Control groups were treated with phosphate buffered
saline (PBS). TRPV1 receptor gene–deficient
(TRPV1ⴚ/ⴚ) mice and CGRP-knockout (CGRPⴚ/ⴚ)
mice and their wild-type (WT) counterparts were investigated. A composite sclerosis score was calculated on
the basis of thickening, leukocyte infiltration, and the
amount/orientation of collagen bundles. Dermal thickSupported by the Wellcome Trust International Research
Development Award, Hungarian Research grants (OTKA-T-046729
and ETT 05-598/2003), and the Péter Pázmány Programme of the
Hungarian National Office of Research and Technology. Dr. Starr’s
work was supported by the Biotechnology and Biological Sciences
Research Council, UK.
1
Árpád Szabó, MD, László Czirják, MD, PhD, DSc, Zoltán
Sándor, MD, PhD, Zsuzsanna Helyes, MD, PhD, Terézia László, MD,
PhD, Krisztián Elekes, MSc, Tamás Czömpöly, MD, PhD, János
Szolcsányi, MD, PhD, DSc, Erika Pintér, MD, PhD, DSc: University of
Pécs, Pécs, Hungary; 2Anna Starr, PhD, Susan Brain, PhD: King’s
College London, London, UK.
Address correspondence and reprint requests to Erika Pintér,
MD, PhD, DSc, Department of Pharmacology and Pharmacotherapy,
University of Pécs, Faculty of Medicine, Szigeti u. 12, H-7624 Pécs,
Hungary. E-mail: erika.pinter@aok.pte.hu.
Submitted for publication February 15, 2007; accepted in
revised form September 21, 2007.
Scleroderma (systemic sclerosis [SSc]) is a complex autoimmune disease. It is characterized by excessive
collagen production by activated fibroblasts, pathologic
remodeling of connective tissue resulting in collagen
deposition in the dermis, as well as vascular injury and
immune abnormalities (1). In localized scleroderma,
these pathologic changes are limited to the skin and
subcutaneous tissue, but various internal organs are also
affected in SSc (2). Although several studies have been
performed, the pathogenesis of scleroderma is complex
and remains largely unknown.
292
ROLE OF TRPV1 RECEPTOR IN SCLERODERMA
Potent profibrotic cytokines (soluble factors)
such as transforming growth factor ␤, interleukin-4,
platelet-derived growth factor, monocyte chemoattractant protein 1, and connective tissue growth factor are
up-regulated in SSc (3). Earlier studies have shown that
scleroderma fibroblasts express ␣-smooth muscle actin
(␣-SMA), and their number and distribution coincide
with the localization and progression of the sclerotic
process (4). In SSc, the vasculopathy includes fibrointimal proliferation and episodes of vasospasms that lead
to ischemia and consequent obliterative fibrosis (1). The
vasospastic episodes, called Raynaud’s phenomenon,
occur following exposure to cold or stress and very
frequently are the first manifestation of the disease (5).
Previous studies suggested that activation of the immune
system plays an important role in pathogenesis of SSc;
however, it is not clear how autoimmunity and tissue
fibrosis interact with each other (2).
Although numerous animal models of SSc have
been developed, murine models are used most extensively. No animal model has been described that reproduces all manifestations of SSc precisely (6). In this
study, we used the bleomycin-induced model developed
by Yamamoto et al in 1999 (7). Bleomycin is an antibiotic obtained from Streptomyces venticillus. It possesses
antitumor activity and is frequently used to treat various
cancers (8). Bleomycin binds to DNA through its aminoterminal peptide, and the activated complex generates
free radicals (by interacting with O2 and Fe2⫹) that are
responsible for scission of the deoxyribose backbone of
the DNA (9). In vitro studies indicate that bleomycin
causes accumulation of cells in the G2 phase of the cell
cycle (10). Bleomycin is degraded by a specific hydrolase
that is found in a variety of normal tissue, and hydrolase
activity is low in the skin and lung (11). Lung fibrosis is
a well-known side effect of bleomycin treatment (12);
therefore, bleomycin is frequently used to induce experimental pulmonary fibrosis in rodents. In addition,
scleroderma has been reported in patients with cancer
after they received bleomycin therapy (13), and
Yamamoto et al observed that local injection of bleomycin causes skin fibrosis (7).
Capsaicin, the active ingredient in hot peppers,
selectively excites and then desensitizes a major subpopulation of nociceptive sensory nerve fibers, which
contain the above-mentioned sensory neuropeptides
and thus are classified as “capsaicin-sensitive afferents”
(14). In recent studies, the receptor for capsaicin, first
called vanilloid receptor 1 and now called transient
receptor potential vanilloid 1 receptor (TRPV1), was
identified and cloned (15). The TRPV1 receptor is
293
associated with a nonselective cation channel that can be
activated by noxious heat, protons, vanilloids such as
capsaicin, and mediators of inflammation, e.g., the
5-lipoxygenase product 12-hydroperoxyeicosatetraenoic
acid; however, its endogenous ligand has not yet been
identified (16).
Besides causing the classic afferent function (nociception), activation of TRPV1 receptors causes the
release of sensory neuropeptides. Among these, calcitonin gene-related peptide (CGRP) mediates vasodilatation, and tachykinins, for example substance P, evoke
plasma protein extravasation. Furthermore, somatostatin with antiinflammatory and antinociceptive action is
also released. It is known that TRPV1 receptor–
expressing sensory neurons play an important modulatory role in the pathomechanism of several diseases,
such as bronchial asthma (17), rheumatoid arthritis
(18–20), eczema (21), dermatitis (22), and migraine (23).
As an important integrator molecule of pain and inflammation, the TRPV1 receptor (24) may play a role in
repair mechanisms and the chronic fibrotic phase of
inflammatory processes.
Previous studies have established that the number of CGRP-immunoreactive C fibers is significantly
decreased in skin samples from patients with scleroderma (25–27). The importance of vasculopathy and
subsequent obliterative fibrosis in the pathogenesis of
SSc is well known (1). Based on these facts, we propose
that the vasodilator neuropeptide CGRP (28) may exert
a protective action in scleroderma. The aim of the
present study was to examine the potential modulatory
role of the TRPV1 receptor and CGRP in an experimental animal model of bleomycin-induced scleroderma, using genetically manipulated mice.
MATERIALS and METHODS
Animals. Experiments were performed on 4–6-weekold female TRPV1 receptor gene–knockout mice (TRPV1ⴚ/ⴚ)
and their wild-type (WT) counterparts (TRPV1⫹/⫹); all mice
weighed 20–25 gm. The mice were successfully bred at the
Laboratory Animal Centre of the University of Pecs, under
standard pathogen-free conditions at 24–25°C, and had had
access to standard chow and water ad libitum. Alpha CGRP–
knockout and WT mice were bred at the animal house of
King’s College London.
Generation of transgenic mice. The generation of
TRPV1 receptor–knockout mice was achieved by homologous
recombination in embryonic stem cells (129 ES) to generate a
mouse lacking transmembrane domains 2–4 of the murine
TRPV1 gene. Germline chimeras were crossed onto female
C57BL/6 mice to generate heterozygotes, which were intercrossed, giving rise to healthy homozygous mutant offspring in
294
the expected Mendelian ratio, as described by Davis et al (29).
TRPV1 receptor–knockout mice were fully backcrossed onto
C57BL/6 mice, and these mice were used to generate WT and
TRPV1 receptor–knockout colonies.
Alpha CGRP–knockout mice were created by disruption of exon 5 (specific to ␣CGRP) of the calcitonin/␣CGRP
gene, using a cassette containing lacZ/cytomegalovirus/
neomycin resistance genes (30). We received a pair of mice (1
WT mouse and 1 CGRP-knockout mouse) that had previously
been fully backcrossed with C57BL/6 mice. We then used these
mice to generate WT and CGRP-knockout colonies.
Induction of cutaneous sclerosis with bleomycin. Cutaneous sclerosis was induced by daily 0.1-ml subcutaneous
injections of bleomycin (100 ␮g/ml; Pharmachemise, Haarlem,
The Netherlands) for 30 days, with a 27-gauge needle on the
dorsal skin of the animals. The control group was treated with
the solvent (phosphate buffered saline [PBS]). Mice were
killed by cervical dislocation, under anesthesia with ketamine
(100 mg/kg intraperitoneally; Richter Gedeon, Budapest, Hungary) and xylazine (5 mg/kg intramuscularly; Lavet Ltd.,
Budapest, Hungary). The excised skin samples were investigated by histologic, biochemical, and molecular biologic methods (7).
Groupings of mice. For each experiment, 4 groups of
mice were used, as follows: for the TRPV1 study, PBS-treated
TRPV1⫹/⫹ mice, PBS-treated TRPV1ⴚ/ⴚ mice, bleomycintreated TRPV1⫹/⫹ mice, and bleomycin-treated TRPV1ⴚ/ⴚ
mice (n ⫽ 10–12 animals/group); for the CGRP study, PBStreated CGRP ⫹/⫹ mice, PBS-treated CGRP ⴚ/ⴚ mice,
bleomycin-treated CGRP⫹/⫹ mice, and bleomycin-treated
CGRPⴚ/ⴚ mice (n ⫽ 8 animals/group).
Histologic analysis. The day after mice received the
final injections, the shaved dorsal skin was removed, fixed in
4% paraformaldehyde, and embedded in paraffin. The general
histologic appearance of the tissue was examined by hematoxylin and eosin and collagen-specific picrosyrius staining. Skin
specimens were assessed and scored using a semiquantitative
composite sclerosis score. The composite histologic sclerosis
score was calculated on the basis of dermal inflammation (0 ⫽
none, 1 ⫽ little, 2 ⫽ mild, 3 ⫽ moderate, and 4 ⫽ severe),
thickened collagen bundles (0 ⫽ normal, 1 ⫽ little, 2 ⫽ mild,
3 ⫽ moderate, and 4 ⫽ severe), and dermal thickness compared with normal skin (0 ⫽ ⬍125%, 1 ⫽ 125–149%, 2 ⫽
150–174%, 3 ⫽ 175–200%, and 4 ⫽ ⬎200%). All parameters
were scored on a scale of 0 to 4, and the values were added (31).
Measurement of dermal thickness. Dermal thickness
at the injection sites was analyzed with an Olympus BX-51
microscope (Tokyo, Japan) at 40⫻ magnification using the
Soft Imaging system (Olympus). The distance between the
epidermal–dermal junction and the dermal–subcutaneous fat
junction was measured in 3 consecutive skin sections from each
animal. In each group of mice, dermal thickness was expressed
in micrometers.
Detection of myofibroblasts. Paraffin-embedded tissue
sections from injected skin were used to quantify the number
of myofibroblasts, by staining for ␣-SMA. After deparaffinization, skin sections were immunostained with monoclonal antibody against ␣-SMA (clone 1A4; DakoCytomation, Carpinteria, CA), according to the manufacturer’s instructions, using
the Dako Autostainer Universal Staining System. Sections
were visualized with diaminobenzidine and counterstained
SZABÓ ET AL
with hematoxylin. In each section, ␣-SMA–positive cells were
counted in 3 randomly chosen high-power fields (32).
Measurement of hydroxyproline content. The amino
acid hydroxyproline is a major component of the protein
collagen; therefore, it can be used as an indicator to determine
the amount of collagen. Full-thickness, 6-mm–diameter punch
biopsy specimens were obtained from the shaved dorsal skin of
each animal after the 4-week treatment and stored at –80°C.
Collagen deposition was estimated by determining the total
content of hydroxyproline in the skin. The stored skin pieces
were hydrolyzed with 6M hydrochloric acid at 130°C for 3
hours, according to the method previously described (33).
After neutralization with sodium hydroxide, the hydrolysates
were diluted with distilled water and oxidated with chloramine
T (Sigma, Munich, Germany), and staining was performed
with p-dimethylaminobenzaldehyde (Ehrlich’s reagent;
Sigma). The absorbance at 557 nm was determined spectrophotometrically, and the quantity of hydroxyproline was calculated from a standard curve. Results were expressed as
micrograms of hydroxyproline per 6-mm–diameter skin pieces.
Reverse transcriptase–polymerase chain reaction (RTPCR). Shaved dorsal skin was incised and stored in 1 ml of
RNAlater solution (Ambion, Cambridge, UK) at ⫺20°C until
processed further. Total RNA was isolated using a GenElute
Mammalian Total RNA Kit (Sigma) with proteinase K (Fluka,
Buchs, Switzerland), according to the manufacturer’s instructions. RNA yield and purity were determined by spectrophotometry (NanoDrop Technologies, Wilmington, DE) and were
also analyzed by electrophoresis on 1% agarose gels. Specific
messenger RNA (mRNA) levels were quantified by the LightCycler RNA Master SYBR Green I quantitative real-time
RT-PCR assay on a LightCycler system (Roche, Mannheim,
Germany).
Type I collagen ␣1 chain mRNA (GenBank accession
no. NM007742.2) was amplified by sense primer 5⬘TCTACTGCAACATGGAGACAG-3⬘ at position 3932 and
antisense primer 5⬘-GCTGTTCTTGCAGTGATAGGTG-3⬘
at position 4185. The housekeeping gene GAPDH mRNA
(GenBank accession no. BC083080) was used as a control,
amplified with sense primer 5⬘-GCAGTGGCAAAGTGGAGATT-3⬘ at position 122 and antisense primer 5⬘TCTCCATGGTGGTGAAGACA-3⬘ at position 370. The
20-␮l reaction mixture contained 250 ng total RNA, 5 mM
MgCl2, 0.5 ␮M primers, plus reaction buffers, according to the
manufacturer’s recommendation. The LightCycler PCR program consisted of the initial reverse transcription step at 55°C
for 20 minutes, followed by a denaturation step at 95°C for 30
seconds and 45 cycles of amplification for 10 seconds at 95°C,
5 seconds at 58°C, and 15 seconds at 72°C.
In order to verify the purity of the products, melting
curve analysis was performed at the end of the experiment.
Quantification of results was accepted only when a single
dominant peak was present in the melting analyses. In order to
further confirm the purity and size of the PCR products, the
reactions were also analyzed by electrophoresis on 1% agarose
gels. The results were evaluated using LightCycler3 Data
Analysis software version 3.5.28 (Roche).
To compare the different RNA transcription levels,
threshold cycle (Ct) values were compared directly. The Ct is
defined as the number of cycles needed for the fluorescence
signal to reach a specific threshold level of detection and is
ROLE OF TRPV1 RECEPTOR IN SCLERODERMA
295
Figure 1. Composite histologic sclerosis score, dermal thickness, number of ␣-smooth muscle actin (␣-SMA)–
positive cells, and collagen-specific hydroxyproline content in transient receptor potential vanilloid 1 receptor–
knockout (KO) (TRPV1⫺/⫺) and TRPV1⫹/⫹ (wild-type [WT]) mice. a–c, Bleomycin treatment significantly
increased the composite histologic score, dermal thickness, and the number of ␣-SMA–positive cells in both
TRPV1⫹/⫹ and TRPV1⫺/⫺ mice compared with the respective phosphate buffered saline (PBS)–treated groups.
Furthermore, each measured histologic value for bleomycin-treated TRPV1⫺/⫺ mice was significantly higher than
that for bleomycin-injected TRPV1⫹/⫹ animals, while no differences were observed between the groups of
PBS-treated mice. d, In both TRPV1⫹/⫹ and TRPV1⫺/⫺ mice, bleomycin treatment resulted in a significant
increase in the hydroxyproline content in the 6-mm skin patches compared with the respective PBS-treated
control groups. Although there was no difference between the PBS-injected TRPV1⫹/⫹ and TRPV1⫺/⫺ mice, the
bleomycin-induced increase in hydroxyproline content was significantly greater in TRPV1⫺/⫺ mice than in
TRPV1⫹/⫹ mice. Values are the mean and SEM (n ⫽ 10–12 mice per group). ⴱ ⫽ P ⬍ 0.05, TRPV1⫺/⫺ versus
WT, by Mann-Whitney U test.
inversely correlated with the amount of template nucleic acid
present in the reaction (34). First, expression of type I collagen
␣1 chain mRNA was quantified relative to that of the housekeeping gene GAPDH mRNA of the same sample, by calculating the corrected difference in Ct (⌬Ct) value according to
the following formula: ⌬Ct ⫽ Ct collagen ⫺ Ct GAPDH (35). Next,
the differences between the variously treated animals were
analyzed by calculating the x-fold difference compared with the
PBS-treated WT control animals, according to the formula
x-fold ⫽ ⫺2.7 ⫻ (⌬Ct treated ⫺ ⌬Ct PBS-treated WT control),
because a 1-cycle ⌬Ct difference corresponded to a 2.7-fold
change in the mRNA level (see Results).
Ethics considerations. All experimental procedures
were carried out according to the 1998/XXVIII Act of the
Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments
(243/1988) and the Animals (Scientific Procedures) Act 1986
(Great Britain). The studies were approved by the Ethics
Committee on Animal Research of Pecs University according
to the Ethical Codex of Animal Experiments, and a license was
given (license no. BA 02/200-6-2001).
Statistical analysis. Results are expressed as the
mean ⫾ SEM. Statistical analysis was carried out with the
nonparametric Mann-Whitney U test to determine significant
differences between histologic scores, hydroxyproline content,
and type I collagen mRNA levels in different groups. P values
less than 0.05 were considered significant.
RESULTS
Establishment of bleomycin-induced dermal
sclerosis in TRPV1ⴙ/ⴙ mice. In the initial studies previously performed, subcutaneous injections of 0.01–
1.0 mg/ml bleomycin for 18–24 days induced marked
dermal sclerosis around the injection site in CH3 and
BALB/c mice, but not in PBS-treated mice (15,36).
296
Figure 2. Histopathologic evaluation of dermal sclerosis in
TRPV1⫺/⫺ mice and their WT counterparts. a and b, Hematoxylin
and eosin (H&E)–stained sections from a TRPV1⫹/⫹ mouse (a) and
a TRPV1⫺/⫺ mouse (b) treated with PBS for 4 weeks. Neither fibrosis nor sclerosis was noted. c, H&E-stained section from a
TRPV1⫹/⫹ mouse treated with bleomycin (100 ␮g/ml) for 4 weeks.
Dermal sclerosis was induced in the subcutaneous tissue. d, H&Estained section from a TRPV1⫺/⫺ mouse treated with bleomycin
(100 ␮g/ml) for 4 weeks. Homogeneous collagen bundles, thickening
of the dermis, and total replacement of subcutaneous fat by collagen
bundles were observed. e and f, Collagen-specific picrosyrius–stained
sections from a TRPV1⫹/⫹ mouse (e) and a TRPV1⫺/⫺ mouse (f)
treated with bleomycin. g and h, Sections from a TRPV1⫹/⫹ mouse (g)
and a TRPV1⫺/⫺ mouse (h) treated with bleomycin labeled with
anti–␣-SMA antibody (visualized with diaminobenzidine and counterstained with hematoxylin). An increased number of ␣-SMA–positive
cells was observed. (Original magnification ⫻ 40 in a–f; ⫻ 100 in g
and f). See Figure 1 for other definitions.
SZABÓ ET AL
Because TRPV1 receptor–knockout mice were generated from a C57BL/6 mouse strain, we first validated the
sclerotic effect of bleomycin treatment in TRPV1⫹/⫹
animals. Histologic analysis showed thickened and homogeneous collagen bundles, thickening of the dermis,
replacement of subcutaneous fat by collagen bundles,
and moderate inflammatory infiltrates in bleomycintreated TRPV1⫹/⫹ mice compared with PBS-treated
mice (Figures 1a and 2).
The composite sclerosis score was 58% higher in
bleomycin-treated TRPV1⫹/⫹ mice compared with that
in PBS-treated TRPV1⫹/⫹ mice (mean ⫾ SEM 6.33 ⫾
0.19 versus 4.00 ⫾ 0.31) (Figure 1a). Dermal thickness
was 42% higher in bleomycin-treated TRPV1⫹/⫹ mice
compared with that in PBS-treated TRPV1⫹/⫹ mice
(393.05 ⫾ 15.41 ␮m versus 278.62 ⫾ 11.38 ␮m) (Figure
1b). The number of ␣-SMA–positive cells was increased
by 75% in bleomycin-treated TRPV1⫹/⫹ mice compared
with that in PBS-treated TRPV1⫹/⫹ control mice
(16.33 ⫾ 3.31 cells/field versus 9.3 ⫾ 1.34 cells/field)
(Figure 1c). Consistent with the histologic changes, the
level of the collagen-specific amino acid hydroxyproline
was 47.5% higher in bleomycin-treated WT mice compared with that in PBS-treated WT mice (118.5 ⫾ 6.70
␮g/skin site versus 80.3 ⫾ 10.20 ␮g/skin site) (Figure 1d).
PBS treatment itself did not induce significant dermal
sclerotic changes compared with the naive skin of untreated TRPV1⫹/⫹ mice. There was no detectable difference in the microscopic structure of the skin between
naive TRPV1⫹/⫹ mice and TRPV1ⴚ/ⴚ mice (results not
shown).
Involvement of TRPV1 receptors in bleomycininduced dermal sclerosis. In TRPV1ⴚ/ⴚ mice, the histologic changes were more pronounced, and the composite sclerosis score was 18% higher than that in
bleomycin-treated TRPV1⫹/⫹ mice (mean ⫾ SEM
7.46 ⫾ 0.20 versus 6.33 ⫾ 0.19) (Figure 1a). Dermal
thickness was increased 19% in bleomycin-treated
TRPV1ⴚ/ⴚ mice compared with TRPV1⫹/⫹ mice
(464.86 ⫾ 10.15 ␮m versus 393.05 ⫾ 15.41 ␮m) (Figure
1b). The number of ␣-SMA–positive cells in bleomycintreated TRPV1ⴚ/ⴚ mice was 47% higher than that in
bleomycin-treated TRPV1⫹/⫹ mice (24.03 ⫾ 3.07 cells/
field versus 16.33 ⫾ 3.31 cells/field) (Figure 1c). Similarly, the hydroxyproline content after bleomycin treatment was 57% greater in the knockout animals than in
the respective WT group (186.60 ⫾ 8.40 ␮g/skin site
versus 118.50 ⫾ 6.70 ␮g/skin site) (Figure 1d). There
were no significant differences between composite sclerosis scores, dermal thickness, hydroxyproline content,
ROLE OF TRPV1 RECEPTOR IN SCLERODERMA
297
Figure 3. Measurement of the type I collagen ␣1 chain mRNA level by quantitative reverse transcription–polymerase chain
reaction (PCR). a, The quantitative PCR was calibrated on a 2-fold dilution series of total mRNA isolated from skin biopsy
specimens. The raw threshold cycle (Ct collagen) values are presented as the mean and SEM results from 4 independent
measurements. b, Type I collagen mRNA levels were measured in dorsal skin specimens from bleomycin (bleo)–treated
animals. The amounts of mRNA in different samples relative to those in skin samples from PBS-treated WT mice (control
group) are shown. No significant differences were found between the different groups. n ⫽ 4–6 mice/group. See Figure 1 for
other definitions.
and numbers of ␣-SMA–positive cells in the PBS-treated
TRPV1⫹/⫹ mice and the PBS-treated TRPV1ⴚ/ⴚ mice
(Figures 1a–d).
Analysis of type I collagen ␣1 chain mRNA
expression in sclerotic skin, using quantitative RT-PCR.
The quantitative PCR was calibrated by amplifying
collagen mRNA, using a 2-fold serial dilution of total
RNA (33 ng, 66.5 ng, 125 ng, and 250 ng per reaction)
isolated from mouse skin as starting material, and
presented as the raw Ct collagen values (Figure 3a). Linear
regression analysis showed a good fit (r ⫽ 0.956) with a
slope of 2.7, indicating that a 1-unit decrease in the level
of Ct collagen corresponds to a 2.7-fold increase in the
concentration of collagen mRNA. Alternatively, 2-fold
more collagen mRNA is represented by a 0.74-unit
lower Ct collagen value.
The results of quantitative PCR measurements of
type I collagen ␣1 mRNA levels in skin biopsy specimens are presented in Figure 3b. Two groups of skin
samples (obtained 2 weeks and 4 weeks after initial
treatment) were examined. The mRNA levels of PBStreated TRPV1ⴚ/ⴚ, bleomycin-treated TRPV1⫹/⫹, and
bleomycin-treated TRPV1ⴚ/ⴚ mouse skin samples were
calculated relative to the levels in corresponding PBStreated WT control samples. Surprisingly, in most of the
samples, the level of collagen mRNA was slightly decreased compared with control samples. However, the
differences were not statistically significant in any of the
groups examined. These results show that bleomycin
treatment did not significantly alter the type I collagen
␣1 mRNA level compared with the control GAPDH
mRNA level.
Involvement of CGRP in bleomycin-induced
scleroderma model. Initially, the effect of bleomycin
injection in the C57BL/6-derived CGRP⫹/⫹ mice was
characterized, and histologic investigations showed
marked dermal sclerosis in the bleomycin-treated mice
(Figures 4a–c and Figure 5). Among WT mice, the
composite sclerosis score was 42% higher (mean ⫾ SEM
4.25 ⫾ 0.38 versus 2.98 ⫾ 0.51), dermal thickness was
increased 52% (434.49 ⫾ 16.41 ␮m versus 285.85 ⫾
17.36 ␮m), and the number of ␣-SMA–positive cells was
augmented by 62% (15.83 ⫾ 3.60 cells/field versus
9.75 ⫾ 0.88 cells/field) in those treated with bleomycin
compared with those that received PBS (Figures 4a–c).
In bleomycin-treated WT mice, hydroxyproline content
increased by 47% compared with that in PBS-treated
WT mice (137.60 ⫾ 14.58 ␮g/skin site versus 93.40 ⫾
10.65 ␮g/skin site) (Figure 4d). Lack of the CGRP gene
caused increased sclerotic changes, whereby the composite sclerosis score in bleomycin-treated CGRPⴚ/ⴚ
mice was 47% higher than that in the respective WT
mice (6.21 ⫾ 0.58 versus 4.25 ⫾ 0.47), dermal thickness
was increased by 29% (557.23 ⫾ 18.38 ␮m versus
434.49 ⫾ 16.41 ␮m), and the number of ␣-SMA–positive
cells was augmented by 76% (27.83 ⫾ 4.58 cells/field
versus 15.83 ⫾ 3.60 cells/field) (Figures 4a–c).
In accord with the histologic findings, the hy-
298
SZABÓ ET AL
Figure 4. Composite histologic sclerosis score, dermal thickness, number of ␣-smooth muscle actin (␣-SMA)–
positive cells, and collagen-specific hydroxyproline content in calcitonin gene-related peptide–knockout (KO)
(CGRP⫺/⫺) and CGRP⫹/⫹ (wild-type [WT]) mice. a–c, The composite histologic score, dermal thickness, and
number of ␣-SMA–positive cells were significantly increased in bleomycin-treated CGRP⫺/⫺ mice compared with
bleomycin-treated CGRP⫹/⫹ mice. However, these histologic parameters were similarly increased in the
phosphate buffered saline (PBS)–treated CGRP⫺/⫺ animals compared with their WT counterparts. d, The
hydroxyproline content in bleomycin-treated CGRP⫺/⫺ mice was significantly higher than that in bleomycintreated CGRP⫹/⫹ mice. However, in accordance with histologic findings, the hydroxyproline content in
PBS-treated CGRP⫺/⫺ animals was significantly greater than the content in PBS-treated CGRP⫹/⫹ mice. Values
are the mean and SEM (n ⫽ 4–6 mice per group in a–c and 8 mice per group in d). ⴱ ⫽ P ⬍ 0.05 versus WT
mice, by Mann-Whitney U test.
droxyproline content increased 30% (mean ⫾ SEM
179.30 ⫾ 18.90 ␮g/skin site versus 137.60 ⫾ 14.58
␮g/skin site) in bleomycin-treated CGRPⴚ/ⴚ animals
(Figure 4d). Surprisingly, we observed that the composite sclerosis score (3.96 ⫾ 0.49 versus 2.98 ⫾ 0.51),
dermal thickness (386.25 ⫾ 10.26 ␮m versus 285.85 ⫾
17.36 ␮m), the number of ␣-SMA–positive cells
(15.04 ⫾ 2.39 cells/field versus 9.75 ⫾ 0.88 cells/field),
and the hydroxyproline content (126.56 ⫾ 13.10 ␮g/skin
site versus 93.40 ⫾ 10.65 ␮g/skin site) were elevated in
the PBS-treated CGRPⴚ/ⴚ mice compared with PBStreated CGRP⫹/⫹ mice (Figures 4a–d).
The severity of sclerotic changes in PBS-treated
CGRPⴚ/ⴚ animals was similar to changes observed in
bleomycin-treated CGRP⫹/⫹ animals. Therefore, we
histologically analyzed naive dorsal skin samples from
CGRP-knockout and WT mice, and also measured the
hydroxyproline content. The histologic structures of the
naive skin samples obtained from CGRPⴚ/ⴚ mice and
CGRP⫹/⫹ mice did not differ. In addition, the hydroxyproline concentration in samples of naive skin
from CGRPⴚ/ⴚ and CGRP⫹/⫹ mice was not different
(80.05 ⫾ 12.66 ␮g/skin site versus 100.59 ⫾ 15.41 ␮g/skin
site) (results not shown).
DISCUSSION
In this study, we demonstrated that a genetic
deficit of TRPV1 receptors or CGRP peptide increases
the severity of sclerotic changes in bleomycin-induced
dermal sclerosis in mice. On the basis of these results, we
presume that activation of TRPV1 receptors and subse-
ROLE OF TRPV1 RECEPTOR IN SCLERODERMA
Figure 5. Histopathologic evaluation of dermal sclerosis in transient
receptor potential vanilloid 1 receptor–knockout (TRPV1⫺/⫺) and
CGRP⫺/⫺ mice and their WT counterparts. a and b, Hematoxylin and
eosin (H&E)–stained sections from an untreated (naive) CGRP⫹/⫹
mouse (a) and a naive CGRP⫺/⫺ mouse (b). Neither fibrosis nor
sclerosis was noted. c, H&E-stained section from a CGRP⫹/⫹ mouse
treated with PBS for 4 weeks. Dermal sclerosis was not observed. d,
H&E-stained section from a CGRP⫺/⫺ mouse treated with PBS for 4
weeks. Surprisingly, detectable sclerosis had developed. e and f,
H&E-stained sections from a CGRP⫹/⫹ mouse (e) and a CGRP⫺/⫺
mouse (f) treated with bleomycin (100 ␮g/ml) for 4 weeks. Sclerosis
developed in the subcutaneous tissue of both mice and was marked in
the knockout mouse. g and h, Picrosyrius-stained sections from a
CGRP⫹/⫹ mouse (g) and a CGRP⫺/⫺ mouse (h) treated with bleomycin (100 ı̀g/ml) for 4 weeks. Sclerosis developed in the subcutaneous
tissue of both mice and was marked in the knockout mouse. (Original
magnification ⫻ 40.) See Figure 4 for other definitions.
299
quent neuropeptide release exert a protective modulatory role in the pathogenesis of bleomycin-induced
scleroderma. Our experiments using CGRPⴚ/ⴚ mice
have provided the first direct evidence that a genetic
deficit of this peptide results in augmentation of the
pathologic alterations in dermal sclerosis.
Over the last 5 years, the modulatory function of
the TRPV1 receptor has been investigated in several
inflammatory diseases. Recent studies by our group and
other investigators demonstrated the pronociceptive and
proinflammatory roles that TRPV1 receptors play in the
mediation of acute pain and inflammation; however,
their function in chronic inflammatory conditions has
not yet been elucidated (36,37). Results of experiments
using TRPV1 receptor–knockout mice established that
the lack of TRPV1 receptors diminishes the symptoms
of experimental arthritis (38). An accumulating number
of reports support the protective role of TRPV1 receptors in immune-mediated inflammatory diseases, such as
oxazolone-induced allergic contact dermatitis (39), dinitrobenzene sulfonic acid–induced colitis (40), and
endotoxin-induced shock (41).
Our study focused on the role of TRPV1 receptors in chronic fibrotic–sclerotic conditions. In the
course of model validation, we measured ⬃150% skin
thickening, marked dermal sclerosis, an increased number of ␣-SMA–positive cells, and a 148.5% increase in
hydroxyproline content in bleomycin-treated TRPV1⫹/⫹
C57BL/6 mice compared with the PBS-treated controls,
which is consistent with previous data concerning the
C57BL/6 strain. PBS treatment itself did not induce
significant sclerotic changes compared with the naive
skin of untreated mice (42,43). However, in contrast to
previous studies (44,45), we were not able to demonstrate increased type I collagen ␣1 chain mRNA levels
after bleomycin treatment in TRPV1⫹/⫹ mice. The basis
of this observation currently is not clear, but the elevated
level of collagen protein along with normal mRNA
levels could be the result of diverse processes, such as
increased mRNA translation, increased procollagen protein processing, or decreased collagen turnover.
In the present study, we established that a genetic
deficiency of TRPV1 receptors increases the bleomycininduced dermal sclerosis detected by histologic and
biochemical methods. The absence of TRPV1 receptors
exacerbated histologic parameters of sclerotic changes,
such as dermal thickness, collagen bundles, inflammation, and number of ␣-SMA–positive cells. In addition,
after bleomycin treatment, the level of hydroxyproline
was elevated in the knockout animals compared with
that in their WT counterparts. Because there was no
300
detectable difference in the microscopic structure of the
skin between the naive TRPV1⫹/⫹ and the TRPV1ⴚ/ⴚ
mice (i.e., those that did not receive bleomycin), we
concluded that TRPV1 deficiency itself does not induce
fibrotic changes. The development of fibrotic reactions
requires the presence of a well-defined profibrotic agent,
such as bleomycin. Presumably, mediators of inflammation (e.g., bradykinin, prostanoids, or lipoxygenase products) released during the development of dermal sclerosis may activate or sensitize TRPV1 receptors (16).
Activation of TRPV1 causes sensory neuropeptide release, which subsequently exerts a protective influence
on pathologic alterations. The tachykinin substance P
mediates plasma protein extravasation (46), CGRP possesses well-known, long-lasting vasodilatory effects (28),
and somatostatin reduces the release of other neuropeptides and directly inhibits inflammatory and immune
cells (47).
Among neuropeptides released by TRPV1 receptor activation, only CGRP has been described as a
potential factor in the pathogenesis of scleroderma and
Raynaud’s phenomenon. Normal skin of the digits is
richly innervated by CGRP-containing nerve fibers that
play a role in nociception (48). A significant reduction in
the number of CGRP-immunoreactive neurons has been
observed in the skin of patients with primary Raynaud’s
phenomenon and those with SSc (25–27). In Raynaud’s
disease, a deficiency of CGRP-containing nerves may
limit the manifestation of cold vasodilatation (49). These
morphologic findings have actuated investigation of the
functional role of CGRP in in vivo sclerotic conditions,
using genetically manipulated CGRP-knockout mice in
a model of bleomycin-induced scleroderma. To our
knowledge, our results provide the first evidence that a
lack of CGRP exacerbates both histopathologic signs of
dermal sclerosis and elevates the collagen-specific hydroxyproline content of skin after long-term treatment
with bleomycin.
In the background of these findings, the absent
vasodilator effect of CGRP can be supposed. The consequent increase in vasospasm leads to ischemia. Periods
of ischemia alternating with reperfusion cause endothelial dysfunction, formation of free radicals, and activation of immune cells, which eventually provoke vasculopathy and obliterative fibrosis. We presume that
TRPV1 receptor–dependent CGRP release is responsible for the protective role of TRPV1 receptor. Based on
our findings, the role of TRPV1 receptor–dependent
release of other neuropeptides (substance P, somatostatin) cannot be excluded.
Unexpectedly, we observed significant sclerotic
SZABÓ ET AL
changes in the dorsal skin of PBS-treated CGRPⴚ/ⴚ
mice compared with that in the skin of PBS-treated
CGRP⫹/⫹ mice. Because there was no difference in the
skin structure of naive CGRPⴚ/ⴚ mice and CGRP⫹/⫹
mice, we assume that CGRPⴚ/ⴚ mice are more sensitive
to physical injury induced by needles. Evidence exists
that physical injury such as that induced by vibration,
trauma, or radiation therapy can provoke factors involved in idiopathic SSc (50). However, the exact mechanism of increased fibrotic response in PBS-treated
CGRPⴚ/ⴚ mice has not been elucidated.
According to the present findings, we can suppose that either TRPV1 receptor–mediated or TRPV1
receptor–independent release of CGRP has a protective
role against sclerotic changes, by acting on vascular
pathophysiologic factors. CGRP receptor would be a
potential target for the development of novel drugs to
treat scleroderma.
AUTHOR CONTRIBUTIONS
Dr. Pintér had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Szabó, Czirják, Brain, Pintér.
Acquisition of data. Szabó, Sándor, László, Elekes, Czömpöly, Starr,
Pintér.
Analysis and interpretation of data. Szabó, Czirják, Sándor, Helyes,
Brain, Szolcsányi, Pintér.
Manuscript preparation. Szabó, Czirják, Sándor, Helyes, Starr, Brain,
Pintér.
Statistical analysis. Szabó, Sándor.
Breeding experimental procedures relevant to CGRP-knockout mice.
Brain.
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