Investigation of sensory neurogenic components in a bleomycin-induced scleroderma model using transient receptor potential vanilloid 1 receptor and calcitonin gene-related peptideknockout mice.код для вставкиСкачать
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: firstname.lastname@example.org. 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. REFERENCES 1. Abraham DJ, Varga J. Scleroderma: from cell and molecular mechanisms to disease models [review]. Trends Immunol 2005;26: 587–95. 2. Denton CP, Black CM, Abraham DJ. Mechanisms and consequences of fibrosis in systemic sclerosis [review]. Nat Clin Pract Rheumatol 2006;2:134–44. 3. Trojanowska M. Molecular aspects of scleroderma. Front Biosci 2002;7:608–18. 4. Sappino AP, Masouye I, Saurat JH, Gabbiani G. Smooth muscle differentiation in scleroderma fibroblastic cells. Am J Pathol 1990;137:585–91. 5. Sakkas LI. New developments in the pathogenesis of systemic sclerosis [review]. Autoimmunity 2005;38:113–6. 6. Jimenez SA, Christner PJ. Murine animal models of systemic sclerosis [review]. Curr Opin Rheumatol 2002;14:671–80. 7. Yamamoto T, Takagawa S, Katayama I. Animal model of sclerotic skin. I. Local injections of bleomycin induce sclerotic skin mimicking scleroderma. J Invest Dermatol 1999;112:456–62. 8. Umezawa H, Maeda K, Takeuchi T, Okami Y. New antibiotics, bleomycin A and B. J Antibiot (Tokyo) 1966;19:200–9. 9. Grollman AP, Takeshita M, Pillai KM, Johnson F. Origin and cytotoxic properties of base propenals derived from DNA. Cancer Res 1985;45:1127–31. ROLE OF TRPV1 RECEPTOR IN SCLERODERMA 10. Twentyman PR. Bleomycin: mode of action with particular reference to the cell cycle. Pharmacol Ther 1983;23:417–41. 11. Sebti SM, Jani JP, Mistry JS, Gorelic E, Lazo JS. Metabolic inactivation: a mechanism of human tumor resistance to bleomycin. Cancer Res 1991;51:227–32. 12. Adamson IV, Bowden DH. The pathogenesis of bleomycininduced pulmonary fibrosis. Am J Pathol 1974;77:185–98. 13. Finch WR, Rodnan GP, Buckingham RB, Prince RK, Winkelstein A. Bleomycin-induced scleroderma. J Rheumatol 1980;7:651–9. 14. Szolcsanyi J. Capsaicin-sensitive sensory nerve terminals with local and systemic efferent functions: facts and scopes of an unorthodox neuroregulatory mechanism. Prog Brain Res 1996;113:343–9. 15. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389:816–24. 16. Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, et al. Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances. Proc Natl Acad Sci U S A 2000;97:6155–60. 17. Barnes PJ. Asthma as an axon reflex. Lancet 1986;1:242–5. 18. Ferrel WR, Lam FY. Sensory neuropeptides in arthritis. In: Geppetti P, Holzer P, editors. Neurogenic inflammation. Boca Raton (FL): CRC Press; 1996. p. 211–27. 19. Helyes Z, Szabo A, Nemeth J, Jakab B, Pinter E, Banvolgyi A, et al. Antiinflammatory and analgesic effects of somatostatin released from capsaicin-sensitive sensory nerve terminals in a Freund’s adjuvant–induced chronic arthritis model in the rat. Arthritis Rheum 2004;50:1677–85. 20. Keeble J, Russell F, Curtis B, Starr A, Pinter E, Brain SD. Involvement of transient receptor potential vanilloid 1 in the vascular and hyperalgesic components of joint inflammation. Arthritis Rheum 2005;52:3248–56. 21. Naukkarinen A, Jarvikallio A, Lakkaporpi J, Harvima IT, Harvima RJ, Horsmanheimo M. Quantitative histochemical analysis of mast cells and sensory nerves in psoriatic skin. J Pathol 1996;180: 200–5. 22. Gutwald J, Goebeler M, Sorg CJ. Neuropeptides enhance irritant and allergic contact dermatitis. J Invest Dermatol 1991;96:695–8. 23. Buzzi MG, Moskowitz MA. The antimigraine drug, sumatriptan (GR43175), selectively blocks neurogenic plasma extravasation from blood vessels in dura mater. Br J Pharmacol 1990;99:202–6. 24. Tominaga M, Tominaga T. Structure and function of TRPV1. Pflugers Arch 2005;451:143–50. 25. Bunker CB, Terenghi G, Springall DR, Polak JM, Dowd PM. Deficiency of calcitonin gene-related peptide in Raynaud’s phenomenon. Lancet 1990;336:1530–3. 26. Terenghi G, Bunker CB, Liu YF, Springall DR, Cowen T, Dowd PM, et al. Image analysis quantification of peptide-immunoreactive nerves in the skin of patients with Raynaud’s phenomenon and systemic sclerosis. J Pathol 1991;164:245–52. 27. Bunker CB, Goldsmith PC, Leslie TA, Hayes N, Foreman JC, Dowd PM. Calcitonin gene-related peptide, endothelin-1, the cutaneous microvasculature and Raynaud’s phenomenon. Br J Dermatol 1996;134:399–406. 28. Brain SD, Williams TJ, Tippins JR, Morris HJ, MacIntyre I. Calcitonin gene-related peptide is a potent vasodilator. Nature 1985;313:54–6. 29. Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, et al. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000;405:183–7. 30. Salmon AM, Damaj I, Sekine S, Picciotto MR, Marubio L, Changeux JP. Modulation of morphine analgesia in ␣CGRP mutant mice. Neuroreport 1999;10:849–54. 31. Yamamoto T, Takagawa S, Katayama I, Mizushima Y, Nishioka K. Effect of superoxide dismutase on bleomycin-induced dermal 301 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. sclerosis: implications for the treatment of systemic sclerosis. J Invest Dermatol 1999;113:843–7. Distler JH, Jungel A, Huber LC, Schulze-Horsel U, Zwerina J, Gay RE, et al. Imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal fibrosis. Arthritis Rheum 2007;56:311–22. Woessener JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1961;93:440–7. Walker NJ. A technique whose time has come. Science 2002;296: 557–9. Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 2004;313:856–62. Bolcskei K, Helyes Z, Szabo A, Sandor K, Elekes K, Nemeth J, et al. Investigation of the role of TRPV1 receptors in acute and chronic nociceptive processes using gene-deficient mice. Pain 2005;117:368–76. Szallasi A, Cruz F, Gepetti P. TRPV1: a therapeutic target for novel analgesic drugs? Trends Mol Med 2006;12:545–54. Szabo A, Helyes Z, Sandor K, Bite A, Pinter E, Nemeth J, et al. Role of transient receptor potential vanilloid 1 receptors in adjuvant-induced chronic arthritis: in vivo study using gene-deficient mice. J Pharmacol Exp Ther 2005;314:111–9. Banvolgyi A, Palinkas L, Berki T, Clark N, Grant AD, Helyes Z, et al. Evidence for a novel protective role of the vanilloid TRPV1 receptor in a cutaneous contact allergic dermatitis model. J Neuroimmunol 2005;169:86–96. Massa F, Sibaev A, Marsicano G, Blaudzun H, Storr M, Lutz B. Vanilloid receptor (TRPV1)-deficient mice show increased susceptibility to dinitrobenzene sulfonic acid induced colitis. J Mol Med 2006;84:142–6. Okajima K, Harada M. Regulation of inflammatory responses by sensory neurons: molecular mechanism(s) and possible therapeutic applications. Curr Med Chem 2006;13:2241–51. Yamamoto T, Kuroda M, Nishoika K. Animal model of sclerotic skin. III. Histopathological comparison of bleomycin-induced scleroderma in various mice strains. Arch Dermatol Res 2000;292: 535–41. Chan ES, Fernandez P, Merchant AA, Montesinos MC, Trzaska S, Desai A, et al. Adenosine A2A receptors in diffuse dermal fibrosis: pathogenic role in human dermal fibroblasts and in a murine model of scleroderma. Arthritis Rheum 2006;54:2632–42. Yamamoto T, Nisshioka K. Increased expression of p53 and p21 (Waf1/Cip1) in the lesional skin of bleomycin-induced scleroderma. Arch Dermatol Res 2005;296:509–13. Matsushita M, Yamamoto T, Nishioka K. Plasminogen activator inhibitor-1 is elevated, but not essential, in the development of bleomycin-induced murine scleroderma. Clin Exp Immunol 2004; 139:429–38. Lembeck F, Holzer P. Substance P as neurogenic mediator of antidromic vasodilation and neurogenic plasma extravasation. Naunyn Schmiedebergs Arch Pharmacol 1979;310:175–83. Pinter E, Helyes Z, Szolcsanyi J. Inhibitory effect of somatostatin on inflammation and nociception. Pharmacol Ther 2006;112:440–56. Dalsgaard CJ, Jernbeck J, Stains W, Kjartansson J, Haegerstrand A, Hokfelt T, et al. Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin: relation to fibers containing substance P-, somatostatin- and vasoactive intestinal polypeptide-like immunoreactivity. Histochemistry 1989;91:35–8. Cooke JP, Marshall JM. Mechanisms of Raynaud’s disease [review]. Vasc Med 2005;10:293–307. Komocsi A, Tovari E, Kovacs J, Czirjak L. Physical injury as provoking factor in three patients with scleroderma. Clin Exp Rheumatol 2000;18:622–4.