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

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ObjectiveAlong 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
   ARTHRITIS & RHEUMATISMVol. 58, No. 1, January 2008, pp 292–301DOI 10.1002/art.23168© 2008, American College of Rheumatology Investigation of Sensory Neurogenic Components in aBleomycin-Induced Scleroderma Model UsingTransient Receptor Potential Vanilloid 1 Receptor– andCalcitonin Gene-Related Peptide–Knockout Mice  A ´rpa´d Szabo´, 1 La´szlo´ Czirja´k, 1 Zolta´n Sa´ndor, 1 Zsuzsanna Helyes, 1 Tere´zia La´szlo´, 1 Krisztia´n Elekes, 1 Tama´s Czo¨mpo¨ly, 1  Anna Starr, 2 Susan Brain, 2 Ja´nos Szolcsa´nyi, 1 and Erika Pinte´r 1 Objective.  Along with their classic afferent func-tion (nociception), capsaicin-sensitive transient recep-tor potential vanilloid 1 (TRPV1) receptor–expressingsensory nerve terminals exert local and systemic effer-ent activities. Activation of TRPV1 causes sensory neu-ropeptide release, which modulates the inflammationprocess. The aim of the present study was to examinethe role of this modulatory role of TRPV1 receptor andthat of calcitonin gene-related peptide (CGRP) inbleomycin-induced scleroderma, using transgenic mice.  Methods.  Cutaneous sclerosis was induced withdaily subcutaneous injections of bleomycin for 30 days.Control groups were treated with phosphate bufferedsaline (PBS). TRPV1 receptor gene–deficient(TRPV1   /  ) mice and CGRP-knockout (CGRP   /  )mice and their wild-type (WT) counterparts were inves-tigated. A composite sclerosis score was calculated onthe basis of thickening, leukocyte infiltration, and theamount/orientation of collagen bundles. Dermal thick-ness and the number of    -smooth muscle actin (  -SMA)–positive cells were also determined. The quantityof the collagen-specific amino acid hydroxyproline wasmeasured by spectrophotometry.  Results.  Bleomycin treatment induced markedcutaneous thickening and fibrosis compared with thatobserved in control mice treated with PBS. The compos-ite sclerosis score was 18% higher, dermal thickness was19% higher, the number of    -SMA–positive cells was47% higher, and the amount of hydroxyproline was 57%higher in TRPV1   /  mice than in their WT counter-parts. Similarly, the composite sclerosis score was 47%higher, dermal thickness was 29% higher, the number of   -SMA–positive cells was 76% higher, and the amountof hydroxyproline was 30% higher in CGRP   /  micethan in the respective WT groups. Conclusion.  These results suggest that activationof the TRPV1 receptor by mediators of inflammationinduces sensory neuropeptide release, which might exertprotective action against fibrosis. We confirmed theprotective role of CGRP in the development of cutane-ous sclerosis. Scleroderma (systemic sclerosis [SSc]) is a com-plex autoimmune disease. It is characterized by excessivecollagen production by activated fibroblasts, pathologicremodeling of connective tissue resulting in collagendeposition in the dermis, as well as vascular injury andimmune abnormalities (1). In localized scleroderma,these pathologic changes are limited to the skin andsubcutaneous tissue, but various internal organs are alsoaffected in SSc (2). Although several studies have beenperformed, the pathogenesis of scleroderma is complex and remains largely unknown. Supported by the Wellcome Trust International ResearchDevelopment Award, Hungarian Research grants (OTKA-T-046729and ETT 05-598/2003), and the Pe´ter Pa´zma´ny Programme of the Hungarian National Office of Research and Technology. Dr. Starr’s work was supported by the Biotechnology and Biological SciencesResearch Council, UK. 1  A ´rpa´d Szabo´, MD, La´szlo´ Czirja´k, MD, PhD, DSc, Zolta´n Sa´ndor, MD, PhD, Zsuzsanna Helyes, MD, PhD, Tere´zia La´szlo´, MD, PhD, Krisztia´n Elekes, MSc, Tama´s Czo¨mpo¨ly, MD, PhD, Ja´nos Szolcsa´nyi, MD, PhD, DSc, Erika Pinte´r, MD, PhD, DSc: University of Pe´cs, Pe´cs, Hungary;  2  Anna Starr, PhD, Susan Brain, PhD: King’sCollege London, London, UK. Address correspondence and reprint requests to Erika Pinte´r,MD, PhD, DSc, Department of Pharmacology and Pharmacotherapy,University of Pe´cs, Faculty of Medicine, Szigeti u. 12, H-7624 Pe´cs,Hungary. E-mail: for publication February 15, 2007; accepted inrevised form September 21, 2007.292  Potent profibrotic cytokines (soluble factors)such as transforming growth factor   , interleukin-4,platelet-derived growth factor, monocyte chemoattrac-tant protein 1, and connective tissue growth factor areup-regulated in SSc (3). Earlier studies have shown thatscleroderma fibroblasts express   -smooth muscle actin(  -SMA), and their number and distribution coincide with the localization and progression of the scleroticprocess (4). In SSc, the vasculopathy includes fibrointi-mal proliferation and episodes of vasospasms that leadto ischemia and consequent obliterative fibrosis (1). The vasospastic episodes, called Raynaud’s phenomenon,occur following exposure to cold or stress and veryfrequently are the first manifestation of the disease (5).Previous studies suggested that activation of the immunesystem plays an important role in pathogenesis of SSc;however, it is not clear how autoimmunity and tissuefibrosis interact with each other (2). Although numerous animal models of SSc havebeen developed, murine models are used most exten-sively. No animal model has been described that repro-duces all manifestations of SSc precisely (6). In thisstudy, we used the bleomycin-induced model developedby Yamamoto et al in 1999 (7). Bleomycin is an antibi-otic obtained from  Streptomyces venticillus . It possessesantitumor activity and is frequently used to treat variouscancers (8). Bleomycin binds to DNA through its amino-terminal peptide, and the activated complex generatesfree radicals (by interacting with O 2  and Fe 2  ) that areresponsible for scission of the deoxyribose backbone of the DNA (9). In vitro studies indicate that bleomycincauses accumulation of cells in the G 2  phase of the cellcycle (10). Bleomycin is degraded by a specific hydrolasethat is found in a variety of normal tissue, and hydrolaseactivity is low in the skin and lung (11). Lung fibrosis isa well-known side effect of bleomycin treatment (12);therefore, bleomycin is frequently used to induce exper-imental pulmonary fibrosis in rodents. In addition,scleroderma has been reported in patients with cancerafter they received bleomycin therapy (13), andYamamoto et al observed that local injection of bleo-mycin causes skin fibrosis (7).Capsaicin, the active ingredient in hot peppers,selectively excites and then desensitizes a major sub-population of nociceptive sensory nerve fibers, whichcontain the above-mentioned sensory neuropeptidesand thus are classified as “capsaicin-sensitive afferents”(14). In recent studies, the receptor for capsaicin, firstcalled vanilloid receptor 1 and now called transientreceptor potential vanilloid 1 receptor (TRPV1), wasidentified and cloned (15). The TRPV1 receptor isassociated with a nonselective cation channel that can beactivated by noxious heat, protons, vanilloids such ascapsaicin, and mediators of inflammation, e.g., the5-lipoxygenase product 12-hydroperoxyeicosatetraenoicacid; however, its endogenous ligand has not yet beenidentified (16).Besides causing the classic afferent function (no-ciception), activation of TRPV1 receptors causes therelease of sensory neuropeptides. Among these, calcito-nin gene-related peptide (CGRP) mediates vasodilata-tion, and tachykinins, for example substance P, evokeplasma protein extravasation. Furthermore, somatosta-tin with antiinflammatory and antinociceptive action isalso released. It is known that TRPV1 receptor–expressing sensory neurons play an important modula-tory 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 inflam-mation, the TRPV1 receptor (24) may play a role inrepair mechanisms and the chronic fibrotic phase of inflammatory processes.Previous studies have established that the num-ber of CGRP-immunoreactive C fibers is significantlydecreased in skin samples from patients with sclero-derma (25–27). The importance of vasculopathy andsubsequent obliterative fibrosis in the pathogenesis of SSc is well known (1). Based on these facts, we proposethat the vasodilator neuropeptide CGRP (28) may exerta protective action in scleroderma. The aim of thepresent study was to examine the potential modulatoryrole of the TRPV1 receptor and CGRP in an experi-mental animal model of bleomycin-induced sclero-derma, using genetically manipulated mice. MATERIALS and METHODS  Animals.  Experiments were performed on 4–6-week-old female TRPV1 receptor gene–knockout mice (TRPV1   /  )and their wild-type (WT) counterparts (TRPV1   /   ); all mice weighed 20–25 gm. The mice were successfully bred at theLaboratory Animal Centre of the University of Pecs, understandard pathogen-free conditions at 24–25°C, and had hadaccess 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 homologousrecombination in embryonic stem cells (129 ES) to generate amouse lacking transmembrane domains 2–4 of the murineTRPV1 gene. Germline chimeras were crossed onto femaleC57BL/6 mice to generate heterozygotes, which were inter-crossed, giving rise to healthy homozygous mutant offspring in ROLE OF TRPV1 RECEPTOR IN SCLERODERMA 293  the expected Mendelian ratio, as described by Davis et al (29).TRPV1 receptor–knockout mice were fully backcrossed ontoC57BL/6 mice, and these mice were used to generate WT andTRPV1 receptor–knockout colonies. Alpha CGRP–knockout mice were created by disrup-tion of exon 5 (specific to   CGRP) of the calcitonin/   CGRPgene, using a cassette containing  lac Z/cytomegalovirus/ neomycin resistance genes (30). We received a pair of mice (1WT mouse and 1 CGRP-knockout mouse) that had previouslybeen fully backcrossed with C57BL/6 mice. We then used thesemice to generate WT and CGRP-knockout colonies. Induction of cutaneous sclerosis with bleomycin.  Cu-taneous sclerosis was induced by daily 0.1-ml subcutaneousinjections of bleomycin (100   g/ml; Pharmachemise, Haarlem,The Netherlands) for 30 days, with a 27-gauge needle on thedorsal skin of the animals. The control group was treated withthe solvent (phosphate buffered saline [PBS]). Mice werekilled by cervical dislocation, under anesthesia with ketamine(100 mg/kg intraperitoneally; Richter Gedeon, Budapest, Hun-gary) and xylazine (5 mg/kg intramuscularly; Lavet Ltd.,Budapest, Hungary). The excised skin samples were investi-gated by histologic, biochemical, and molecular biologic meth-ods (7). Groupings of mice.  For each experiment, 4 groups of mice were used, as follows: for the TRPV1 study, PBS-treatedTRPV1   /   mice, PBS-treated TRPV1   /  mice, bleomycin-treated TRPV1   /   mice, and bleomycin-treated TRPV1   /  mice (n    10–12 animals/group); for the CGRP study, PBS-treated CGRP   /   mice, PBS-treated CGRP   /  mice,bleomycin-treated CGRP   /   mice, and bleomycin-treatedCGRP   /  mice (n  8 animals/group). Histologic analysis.  The day after mice received thefinal injections, the shaved dorsal skin was removed, fixed in4% paraformaldehyde, and embedded in paraffin. The generalhistologic appearance of the tissue was examined by hematoxy-lin and eosin and collagen-specific picrosyrius staining. Skinspecimens were assessed and scored using a semiquantitativecomposite sclerosis score. The composite histologic sclerosisscore 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 com-pared 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 thicknessat the injection sites was analyzed with an Olympus BX-51microscope (Tokyo, Japan) at 40   magnification using theSoft Imaging system (Olympus). The distance between theepidermal–dermal junction and the dermal–subcutaneous fat junction was measured in 3 consecutive skin sections from eachanimal. In each group of mice, dermal thickness was expressedin micrometers. Detection of myofibroblasts.  Paraffin-embedded tissuesections from injected skin were used to quantify the numberof myofibroblasts, by staining for   -SMA. After deparaffiniza-tion, skin sections were immunostained with monoclonal anti-body against   -SMA (clone 1A4; DakoCytomation, Carpinte-ria, CA), according to the manufacturer’s instructions, usingthe Dako Autostainer Universal Staining System. Sections were visualized with diaminobenzidine and counterstained with hematoxylin. In each section,   -SMA–positive cells werecounted in 3 randomly chosen high-power fields (32). Measurement of hydroxyproline content.  The aminoacid hydroxyproline is a major component of the proteincollagen; therefore, it can be used as an indicator to determinethe amount of collagen. Full-thickness, 6-mm–diameter punchbiopsy 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 totalcontent of hydroxyproline in the skin. The stored skin pieces were hydrolyzed with 6  M   hydrochloric acid at 130°C for 3hours, according to the method previously described (33). After neutralization with sodium hydroxide, the hydrolysates were diluted with distilled water and oxidated with chloramineT (Sigma, Munich, Germany), and staining was performed with  p -dimethylaminobenzaldehyde (Ehrlich’s reagent;Sigma). The absorbance at 557 nm was determined spectro-photometrically, and the quantity of hydroxyproline was cal-culated from a standard curve. Results were expressed asmicrograms of hydroxyproline per 6-mm–diameter skin pieces. Reverse transcriptase–polymerase chain reaction (RT-PCR).  Shaved dorsal skin was incised and stored in 1 ml of RNAlater solution (Ambion, Cambridge, UK) at  20°C untilprocessed further. Total RNA was isolated using a GenEluteMammalian Total RNA Kit (Sigma) with proteinase K (Fluka,Buchs, Switzerland), according to the manufacturer’s instruc-tions. RNA yield and purity were determined by spectropho-tometry (NanoDrop Technologies, Wilmington, DE) and werealso analyzed by electrophoresis on 1% agarose gels. Specificmessenger RNA (mRNA) levels were quantified by the Light-Cycler RNA Master SYBR Green I quantitative real-timeRT-PCR assay on a LightCycler system (Roche, Mannheim,Germany).Type I collagen   1 chain mRNA (GenBank accessionno. NM007742.2) was amplified by sense primer 5  -TCTACTGCAACATGGAGACAG-3   at position 3932 andantisense 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  -GCAGTGGCAAAG-TGGAGATT-3   at position 122 and antisense primer 5  -TCTCCATGGTGGTGAAGACA-3   at position 370. The20-  l reaction mixture contained 250 ng total RNA, 5 m  M  MgCl 2 , 0.5    M   primers, plus reaction buffers, according to themanufacturer’s recommendation. The LightCycler PCR pro-gram consisted of the initial reverse transcription step at 55°Cfor 20 minutes, followed by a denaturation step at 95°C for 30seconds 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, meltingcurve analysis was performed at the end of the experiment.Quantification of results was accepted only when a singledominant peak was present in the melting analyses. In order tofurther confirm the purity and size of the PCR products, thereactions were also analyzed by electrophoresis on 1% agarosegels. The results were evaluated using LightCycler3 Data Analysis software version 3.5.28 (Roche).To compare the different RNA transcription levels,threshold cycle (C t ) values were compared directly. The C t  isdefined as the number of cycles needed for the fluorescencesignal to reach a specific threshold level of detection and is 294 SZAB´O ET AL   inversely correlated with the amount of template nucleic acidpresent in the reaction (34). First, expression of type I collagen  1 chain mRNA was quantified relative to that of the house-keeping gene GAPDH mRNA of the same sample, by calcu-lating the corrected difference in C t  (  C t ) value according tothe following formula:  C t  C t collagen  C t GAPDH  (35). Next,the differences between the variously treated animals wereanalyzed by calculating the x-fold difference compared with thePBS-treated WT control animals, according to the formula x-fold    2.7    (  C t treated    C t PBS-treated WT control ),because a 1-cycle   C t  difference corresponded to a 2.7-foldchange in the mRNA level (see Results). Ethics considerations.  All experimental procedures were carried out according to the 1998/XXVIII Act of theHungarian Parliament on Animal Protection and Consider-ation Decree of Scientific Procedures of Animal Experiments(243/1988) and the Animals (Scientific Procedures) Act 1986(Great Britain). The studies were approved by the EthicsCommittee on Animal Research of Pecs University accordingto the Ethical Codex of Animal Experiments, and a license wasgiven (license no. BA 02/200-6-2001). Statistical analysis.  Results are expressed as themean    SEM. Statistical analysis was carried out with thenonparametric Mann-Whitney U test to determine significantdifferences between histologic scores, hydroxyproline content,and type I collagen mRNA levels in different groups.  P   valuesless than 0.05 were considered significant. RESULTSEstablishment of bleomycin-induced dermalsclerosis in TRPV1   /  mice.  In the initial studies previ-ously performed, subcutaneous injections of 0.01–1.0 mg/ml bleomycin for 18–24 days induced markeddermal sclerosis around the injection site in CH3 andBALB/c mice, but not in PBS-treated mice (15,36). 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 significantlyincreased the composite histologic score, dermal thickness, and the number of    -SMA–positive cells in bothTRPV1   /   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 thanthat 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 significantincrease in the hydroxyproline content in the 6-mm skin patches compared with the respective PBS-treatedcontrol groups. Although there was no difference between the PBS-injected TRPV1   /   and TRPV1   /   mice, thebleomycin-induced increase in hydroxyproline content was significantly greater in TRPV1   /   mice than inTRPV1   /   mice. Values are the mean and SEM (n  10–12 mice per group).     P   0.05, TRPV1   /    versusWT, by Mann-Whitney U test.ROLE OF TRPV1 RECEPTOR IN SCLERODERMA 295  Because TRPV1 receptor–knockout mice were gener-ated from a C57BL/6 mouse strain, we first validated thesclerotic effect of bleomycin treatment in TRPV1   /   animals. Histologic analysis showed thickened and ho-mogeneous collagen bundles, thickening of the dermis,replacement of subcutaneous fat by collagen bundles,and moderate inflammatory infiltrates in bleomycin-treated TRPV1   /   mice compared with PBS-treatedmice (Figures 1a and 2).The composite sclerosis score was 58% higher inbleomycin-treated TRPV1   /   mice compared with thatin 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   /   micecompared with that in PBS-treated TRPV1   /   mice(393.05  15.41   m versus 278.62  11.38   m) (Figure1b). The number of    -SMA–positive cells was increasedby 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, thelevel of the collagen-specific amino acid hydroxyproline was 47.5% higher in bleomycin-treated WT mice com-pared 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 dermalsclerotic changes compared with the naive skin of un-treated TRPV1   /   mice. There was no detectable dif-ference in the microscopic structure of the skin betweennaive TRPV1   /   mice and TRPV1   /  mice (results notshown). Involvement of TRPV1 receptors in bleomycin-induced dermal sclerosis.  In TRPV1   /  mice, the his-tologic changes were more pronounced, and the com-posite sclerosis score was 18% higher than that inbleomycin-treated TRPV1   /   mice (mean    SEM7.46    0.20 versus 6.33    0.19) (Figure 1a). Dermalthickness was increased 19% in bleomycin-treatedTRPV1   /  mice compared with TRPV1   /   mice(464.86  10.15   m versus 393.05  15.41   m) (Figure1b). The number of    -SMA–positive cells in bleomycin-treated TRPV1   /  mice was 47% higher than that inbleomycin-treated TRPV1   /   mice (24.03    3.07 cells/ field versus 16.33    3.31 cells/field) (Figure 1c). Simi-larly, the hydroxyproline content after bleomycin treat-ment was 57% greater in the knockout animals than inthe 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 scle-rosis scores, dermal thickness, hydroxyproline content, Figure 2.  Histopathologic evaluation of dermal sclerosis inTRPV1   /   mice and their WT counterparts.  a  and  b,  Hematoxylinand eosin (H&E)–stained sections from a TRPV1   /   mouse ( a ) anda TRPV1   /   mouse ( b ) treated with PBS for 4 weeks. Neither fi-brosis nor sclerosis was noted.  c,  H&E-stained section from aTRPV1   /   mouse treated with bleomycin (100   g/ml) for 4 weeks.Dermal sclerosis was induced in the subcutaneous tissue.  d,  H&E-stained section from a TRPV1   /   mouse treated with bleomycin(100   g/ml) for 4 weeks. Homogeneous collagen bundles, thickeningof the dermis, and total replacement of subcutaneous fat by collagenbundles were observed.  e  and  f,  Collagen-specific picrosyrius–stainedsections 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 withanti–  -SMA antibody (visualized with diaminobenzidine and counter-stained with hematoxylin). An increased number of    -SMA–positivecells was observed. (Original magnification    40 in  a – f  ;    100 in  g and  f  ). See Figure 1 for other definitions.296 SZAB´O ET AL 
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