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Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis

Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis Introduction: Systemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin. Methods: Ang II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-b1, TGF-b2, TGF-b3, CTGF, aSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, aSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1. Results: Ang II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-b2 and TGF-b3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of aSMA-positive cells, as well as CD163B macrophages in the upper dermis. This correlated with significantly increased mRNA levels of aSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only. Conclusions: This work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies. Introduction SSc patients and can precede definite diagnosis of SSc by Systemic sclerosis (SSc) is a complex autoimmune years or even decades. In patients with SSc, RP is asso- inflammatory disorder of unknown etiology characterized ciated with structural abnormalities of the microvascula- by vascular alterations, activation of the immune system, ture and the presence of SSc-specific autoantibodies [2], indicating an early link between immune system activa- and fibrosis of the skin and internal organs [1]. Vascular insufficiency and immune dysfunction manifest early in tion and vascular injury. Fibrosis results from excessive the disease and are followed by increased extracellular production and accumulation of ECM components pro- matrix (ECM) production as the disease progresses. duced by activated fibroblasts, which might be triggered Raynaud phenomenon (RP) is present in the majority of by cytokines and growth factors released from the infil- trating immune cells during the inflammatory stage [3]. * Correspondence: [email protected] However, interrelations between the key pathologic com- Arthritis Center, Boston University School of Medicine, Boston, MA 02118, ponents of the disease are still poorly understood. USA © 2012 Stawski et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 2 of 12 http://arthritis-research.com/content/14/4/R194 Angiotensin II (Ang II), a main component of the skin surrounding the pump outlet was collected by using rennin-angiotensin system (RAS), is a vasoactive peptide an 8-mm-diameter punch biopsy device. that induces vascular constriction, salt and water reten- tion, and increased blood pressure [4]. Ang II has been Gomori Trichrome staining reported to play a critical role in renal and heart fibrosis Gomori Trichrome staining was used to detect collagen through inflammation and upregulation of matrix deposi- fibers and collagen deposition in the mouse skin. The skin tion [5,6]. Previous studies also suggest that Ang II may samples were fixed in 4% paraformaldehyde for 24 hours be involved in the pathogenesis of skin fibrosis in SSc. It and then processed for paraffin embedding. Staining was has been shown that Ang II levels are increased in the performed on 8-μm-thick paraffin sections by following blood of SSc patients and that, in contrast to healthy the manufacturer’s instructions (Chromaview, Dublin, skin, the Ang II precursor angiotensinogen is expressed OH, USA; Gomori Trichrome blue collagen Kit S7440- in SSc skin [7]. Furthermore, the profibrotic effects of 19). Collagen fibers were stained blue, nuclei were stained Ang II are mediated via the AT1a receptor in cultured black, and the background was stained red. human and mouse skin fibroblasts [8]. In addition, dysre- gulation of RAS components was shown in patients with Hydroxyproline assay SSc, with a prevalence of the vasoconstricting Ang II Collagen deposition was quantified by measuring total over the vasodilating Ang-(1-7), suggesting inhibition of hydroxyproline content in 4-mm skin-punch biopsies endothelium-dependent vasodilatation and increased obtained from PBS and Ang II infusion sites by using a pre- vasoconstriction in SSc vessels [9]. viously described method with some modifications [17]. In Utilization of animal models has been instrumental in brief, the skin samples were hydrolyzed with 6 M sodium delineating complex pathologic features of SSc. In the last hydroxide at 110°C for 12 hours. The hydrolyzate was then decade, a number of new animal models became available oxidized with oxidation buffer (one part 7% chloramine T to study mechanisms of SSc fibrosis [10-13]. The inducible and four parts of acetate citrate buffer) for 4 minutes at models of SSc include the widely studied bleomycin and room temperature. Ehrlich aldehyde reagent was added to more recently established hypochlorous acid (HOCH) each sample, and the chromophore was developed by incu- injection models, as well as the immune-based scleroder- bating the samples at 65°C for 25 minutes. Absorbance of matous graft-versus-host model. A growing number of each sample was read at 560 nm by using a spectro- genetic models are very valuable for investigating specific phometer. Results were expressed as total hydroxyproline signaling pathways involved in fibrosis [14,15]. Whereas content (in micrograms) per 0.1 g of tissue. A standard none of the currently available models recapitulate the curve was performed for all hydroxyproline measurements complex features of SSc, they provide important insights by using known quantities of hydroxyproline. into selected aspects of SSc pathogenesis and allow precli- nical testing of antifibrotic compounds. Quantitative RT-PCR analysis Angiotensin II has been widely used to investigate kidney Total RNA was isolated by using the RNeasy Fibrous Tis- [5], heart [6], and liver [16] fibrosis by using mouse models. sue Mini Kit (Qiagen, Valencia, CA, USA). Then, 1 μgof However, the profibrotic potential of Ang II has not been total RNA was reverse transcribed with random hexam- evaluated in any model of dermal fibrosis. Given the poten- ers by using the Transcriptor First Strand Complemen- tial involvement of Ang II in the pathogenesis of SSc, the tary DNA Synthesis kit (Roche Applied Science, goal of this study was to investigate the effect of Ang II on Indianapolis, IN, USA) according to the manufacturer’s dermal fibrosis in a mouse model. protocol. Real-time PCR assays were performed by using the StepOnePlus Real-Time PCR system (Applied Biosys- Materials and methods tems, Foster City, CA, USA). The amplification mixture Subcutaneous infusion of angiotensin II using ALZET (10 μl) contained 1 μl of complementary DNA, 0.5 μM of osmotic minipumps each primer, and 5 μlofSYBRGreen PCRMasterMix. C57BL/6 mice were purchased from The Jackson Labora- The primers are listed in Supplementary Table 1. Relative tory. All of the experiments were performed under the change in the levels of genes of interest was determined -ΔΔCT guidelines of the Boston University Institutional Animal by the 2 method. Care and Use Committee (protocol AN-15037). Alzet osmotic miniature pumps (model 2002) delivering angio- Immunofluorescence staining on frozen sections tensin II (Sigma-Aldrich, St. Louis, MO, USA) at a rate of For all immunofluorescence staining, skin samples were 1,000 ng/kg/min (pressor dose) or 2,000 ng/kg/min, or directly embedded in O.C.T. compound, flash frozen, PBS, were implanted subcutaneously on the backs of and stored at -80°C. Staining was performed on 8-μm 8-week-old mice. After 14 days, mice were killed, and the cryosections of mouse skin. In brief, slides were blocked Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 3 of 12 http://arthritis-research.com/content/14/4/R194 Table 1 Primers for quantitative real-time polymerase described [18]. Cells were cultured on bovine collagen- chain reaction coated six-well plates in EBM medium supplemented with m-B2MG Forward: 5’-TCGCTCGGTGACCCTAGTCTTT-3’ 10% FBS and EC growth supplement mix at 37°C with 5% CO in air. The culture medium was changed every other Reverse: 5’-ATGTTCGGCTTCCCATTCTCC-3’ 2 day. For immunofluorescence, cultured HDMECs grown m-Fli1 Forward: 5’-ACTTGGCCAAATGGACGGGACTAT-3’ on collagen-coated coverslips were treated with Ang II Reverse: 5’-CCCGTAGTCAGGACTCCCG-3’ (1,000 ng/ml) for 96 hours. Control and Ang II-treated m-Col1a1 Forward: 5’-GCCAAGAAGACATCCCTGAAG-3’ cells were fixed with 4% paraformaldehyde for 15 minutes Reverse: 5’-TGTGGCAGATACAGATCAAGC-3’ followed by incubation with 0.15 M glycine for 30 min- m-Col1a2 Forward: 5’-GCCACCATTGATAGTCTCTCC-3’ utes. Nonspecific protein binding was blocked with 3% Reverse: 5’-CACCCCAGCGAAGAACTCATA-3’ BSA for 1 hour. Next, cells were incubated at 4°C over- m-COL3a1 Forward: 5’-GCCAAGAAGACATCCCTGAAG-3’ night with primary antibodies: goat anti-mouse VE-cad- Reverse: 5’-TGGACTGCTGTGCCAAAATA-3’ herin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-mouse FSP1 (Abcam, Cambridge, MA, m-Col5a1 Forward: 5’-GGACTAGTCCGCTTTCCCTGTCAACTTG-3’ USA). After washing, cell cultures were incubated with Reverse: 5’-GTGGTCACTGCGGCTGAGGAACTTC-3’ Alexa fluor 488 donkey anti-goat (Invitrogen, Grand m-CTGF Forward: 5’-CTGCAGACTGGAGAAGCAGA-3’ Island, NY, USA) and Alexa fluor 594 donkey anti-rabbit Reverse: 5’-GATGCACTTTTTGCCCTTCTT-3’ (Invitrogen) antibodies for 1.5 hour. Cells were mounted m-aSMA Forward: 5’-CCCACCCAGAGTGGAGAA-3’ on slides by using Vectashield with DAPI (Vector Labora- Reverse: 5’-ACATAGCTGGAGCAGCGTCT-3’ tories) and examined by using a FluoView FV10i confocal m-FSP1 Forward: 5’-GGAGCTGCCTAGCTTCCTG-3’ microscope system (Olympus, Center Valley, PA, USA) at Reverse: 5’-TCCTGGAAGTCAACTTCATTGTC-3’ 488 nm (green), 594 nm (red), and 405 nm (blue). m-MCP1 Forward: 5’-CATCCACGTGTTGGCTCA-3’ Reverse: 5’-GATCATCTTGCTGGTGAATGAGT-3’ Immunohistochemistry Immunohistochemistry was performed on formalin- m-CD3 Forward: 5’-AACACGTACTTGTACCTGAAAGCTC-3’ fixed, paraffin-embedded skin tissue sections by using Reverse: 5’-GATGATTATGGCTACTGCTGTCA-3’ the Vectastain ABC kit (Vector Laboratories) according m-Emr1 Forward: 5’-CCTGGACGAATCCTGTGAAG-3’ to the manufacturer’s instructions. In brief, sections (8- Reverse: 5’-GGTGGGACCACAGAGAGTTG-3’ μm thick) were mounted on APES (aminopropyl- m-CD45/B220 Forward: 5’-AATGGCTCTTCAGAGACCACATA-3’ triethoxy silane solution)-coated slides, deparaffinized Reverse: 5’-AGTCAGGCTGTGGGGACA-3’ with Histo-Clear (National Diagnostics, Atlanta, GA, TGF-b1 Forward: 5’-GCAGCACGTGGAGCTGTA-3’ USA), and rehydrated through a graded series of etha- Reverse: 5’-CAGCCGGTTGCTGAGGTA-3 nol. Endogenous peroxidase was blocked by incubation TGF-b2 Forward: 5’-CCTTCTTCCCCTCCGAAAC-3’ in 3% hydrogen peroxide for 30 minutes, followed by incubation with 0.15 M glycine for 45 minutes, and nor- Reverse: 5’-AGAGCACCTGGGACTGTCTG-3’ mal blocking serum for 1 hour. The sections were then TGF-b3 Forward: 5’-AAGAAGCGGGCTTTGGAC-3’ incubated overnight at 4°C with antibodies against CD3 Reverse: 5’-CGCACACAGCAGTTCTCC-3’ (Abcam, Cambridge, MA, USA), Mac3 (BD Bioscience, San Jose, CA, USA), CD45R (AbD Serotec, Raleigh, NC, USA), or CD163B (Epitomics, Burlingame, CA, USA), with a blocking solution (3% BSA (Sigma-Aldrich), and diluted 1:100 in blocking buffer, followed by incubation 0.3% Triton X-100 in PBS) for 2 hours. After washing, for 30 minutes with a biotinylated secondary antibody tissue sections were incubated at 4°C overnight with pri- mary antibodies (Table 2). Tissue sections were then solution. A solution containing avidin:biotin:peroxidase washed and incubated with secondary Ab (Table 2) at complexes was applied to the sections subsequently. room temperature for 2 hours. Coverslips were mounted Immunoreactivity was visualized with diaminobenzidine by using Vectashield with DAPI (Vector Laboratories, (Vector Laboratories), and the sections were counter- Burlingame, CA, USA), and staining was examined by stained with hematoxylin. Images were collected by using a FluoView FV10i confocal microscope system using a microscope (BH-2; Olympus, Center Valley, PA, (Olympus, Center Valley, PA, USA) at 488 nm (green), USA). 594 nm (red), and 405 nm (blue). Statistical analyses Immunofluorescence staining on adherent cell cultures All data were analyzed with the Student paired t test. Human dermal microvascular endothelial cells (HDMECs) The level for statistical significance was set at P ≤ were isolated from human foreskin, as previously 0.05. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 4 of 12 http://arthritis-research.com/content/14/4/R194 Table 2 Primary and secondary antibodies for immunofluorescence staining Primary antibodies Secondary antibodies Myofibroblasts Rabbit anti-mouse aSMA Ab (Novus Biologicals, Littleton, CO); 1:100 Alexa fluor 488 donkey anti-rabbit IgG (Invitrogen, Grand Island, NY); 1:1,000 Fibrocytes Rat anti-mouse CD45 Ab (BD Rabbit anti-mouse FSP1 Ab Alexa fluor 594 donkey anti- Alexa fluor 488 donkey anti- Pharmingen, San Diego, CA); 1:50 (Abcam, Cambridge, MA); rat IgG (Invitrogen); 1:1,000 rabbit IgG (Invitrogen); 1:100 1:1,000 EndoMT Goat anti-mouse VE-cadherin Ab (Santa Rabbit anti-mouse FSP1 Ab Alexa fluor 488 donkey anti- Alexa fluor 594 donkey anti- Cruz Biotechnology, Santa Cruz, CA); (Abcam); 1:100 goat IgG (Invitrogen); rabbit IgG (Invitrogen); 1:300 1:1,000 1:1,000 addition, the fat layer of the reticular dermis was partially Results replaced with extracellular matrix (Figure 1A). Real-time Angiotensin II increases collagen synthesis and PCR analysis showed a statistically significant increase in deposition in mouse skin mRNA levels of the collagen-encoding genes: Col1a1, To determine whether angiotensin II can induce dermal Col1a2, Col3a1,and Col5a1,aswellas CTGF (2.4-fold, fibrosis, Ang II at a rate of 1,000 ng/kg/min was adminis- 1.9-fold, and 3.4-fold, 1.9-fold, and 2.1-fold, respectively; tered continuously with subcutaneous osmotic minipumps *P ≤ 0.05) in the skin of Ang II-infused mice when com- implanted under the shaved back skin of 8-week-old pared with control mice (Figure 1B). Total hydroxyproline C57BL/6 male mice. Treated skin was harvested at day 14, content from the injected sites was significantly higher in and histologic examination and collagen-content measure- Ang II (1,000 ng/kg/min)-infused skin (2,481 ± 920 μgper ment assays were performed. Gomori Trichrome staining 0.1 g of skin) compared with control PBS-infused skin showed that collagen fibers were more closely packed in (1,534 ± 430 μg per 0.1 g of skin; *P ≤ 0.05) (Figure 1C). angiotensin II-treated mice compared with control mice, A higher dose of Ang II (2,000 ng/kg/min) showed a larger reflecting increased collagen deposition (Figure 1A). In Figure 1 Angiotensin II increases collagen synthesis and deposition in mouse skin. (A) Gomori Trichrome staining shows a histologic evaluation of Ang II-induced lesions in C57BL/6 mice. (B) Real-time PCR analysis of collagen-encoding genes: Col1a1, Col1a2, Col3a1, Col5a1, along with CTGF (*P ≤ 0.05) in PBS-and Ang II-treated mice. (C) Total hydroxyproline content in PBS- and Ang II-treated mice. Values are the mean of six mice in each group; *P ≤ 0.05. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 5 of 12 http://arthritis-research.com/content/14/4/R194 increase in the total hydroxyproline content of local skin the mRNA expression of TGF-b1 (Figure 2A). Immuno- (5,272 ± 1,260 μg per 0.1 g of Ang II-infused skin versus histochemical staining of pSmad2 was performed on par- 1,748 ± 531 μg per 0.1 g of PBS-infused skin; *P ≤ 0.05) affin sections. We observed increased numbers of (Figure 1C). The profibrotic effects of Ang II were local, as pSmad2-positive cells distributed throughout all dermal no significant differences were observed in hydroxyproline layers in Ang II-treated mice (Figure 2B). These data sug- content in distal skin with either concentration of Ang II gest that Ang II potently activates TGF-b signaling in this (data not shown). Because a dose of 1,000 ng/kg/min was model. sufficient to induce significant dermal fibrosis, this dose was selected for further analyses. Angiotensin II increases the number of myofibroblasts in mouse skin Angiotensin II activates the TGF-b pathway in mouse skin Fibrosis is associated with accumulation of activated Transforming growth factor-b (TGF-b) plays a pivotal role fibroblasts/myofibroblasts in the affected tissues. To in the pathogenesis of scleroderma by activating fibroblasts determine whether Ang II treatment increases myofibro- and stimulating the production of ECM components [19]. blast presence, skin from infused sites was examined with To determine whether Ang II treatment activates the real-time PCR and immunostaining for aSMA. Real-time TGF-b pathway, we examined skin samples from the PCR analysis showed a statistically significant increase in infused sites with real-time PCR and immunostaining. the mRNA level of aSMA (1.9-fold, *P ≤ 0.05) in Ang II- Real-time PCR analysis showed statistically significant treated mouse skin (Figure 3A). aSMA staining was per- increases in the mRNA levels of TGF-b2 and TGF-b3 (3.2- formed on cryosections (immunofluorescence, Figure 3B fold and 4.8-fold, respectively; *P ≤ 0.05) in Ang II-treated and 3D) and paraffin sections (immunohistochemistry, mouse skin (Figure 2A). No differences were observed in Figure 3C) from PBS- and Ang II-treated mouse skin. Figure 2 Angiotensin II activates TGF-b signaling. (A) Real-time PCR analysis of TGF-b1, TGF-b2,and TGF-b3 (*P ≤ 0.05)inPBS- and Ang II- treated mice. (B) pSmad2 staining on paraffin sections from PBS- and Ang II-infused mice. Representative images are shown from four animals per group. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 6 of 12 http://arthritis-research.com/content/14/4/R194 Figure 3 Detection of activated fibroblasts in angiotensin II-treated skin. (A) Real-time PCR analysis of aSMA in PBS- and Ang II-treated mice (*P ≤ 0.05). aSMA staining in the upper dermis (B) by immunofluorescence and (C) by immunohistochemistry, and in the lower dermis (D) by immunofluorescence, on sections from PBS- and Ang II-treated mouse skin. Arrows indicate positive staining of myofibroblasts. Representative images are shown from five animals per group. Affected sites from PBS-treated mice contained few of CD3, CD45R, and two macrophage markers, a general aSMA-positive cells, observed exclusively around the marker, Mac3 [20], and a marker identifying M2 macro- blood vessels and the arrector pili muscles. In contrast, phages, CD163B [21,22] was performed on paraffin sec- affected sites from Ang II-treated mice showed an tions. We observed increased infiltration of CD3, CD45R, increased number of cells expressing aSMA distributed and Mac3-positive cells only in the hypodermis of Ang mainly throughout the upper dermis (Figure 3B and 3C), II-treated mice (Figure 4B and 5B). Interestingly, we whereas in the lower dermis, positive cells were observed observed an increased presence of CD163B-positive cells only around the blood vessels (Figure 3D). These data in the upper dermis of Ang II-treated mice, whereas no suggest that Ang II-induced myofibroblast differentiation increase was found in the lower dermis or hypodermis contributes to the development of fibrosis in this model. (Figure 5C). Angiotensin II increases inflammation in mouse skin Angiotensin II increases infiltration of fibrocytes in the Previous studies have shown that Ang II promotes upper dermis inflammation in kidney and heart models of fibrosis [5,6]. Circulating mesenchymal cells, fibrocytes, have been To evaluate the recruitment of inflammatory cells, skin shown to infiltrate areas of inflammation and tissue samples from the Ang II-infused sites were examined damage and contribute to the fibrotic process [23]. To with real-time PCR and immunostaining of immune cell determine whether fibrocytes are involved in the dermal markers. Real-time PCR analysis showed statistically sig- fibrosis induced by Ang II, skin cryosections were stained nificant increases in the mRNA levels of the T-cell mar- for the immune cell marker, CD45, and mesenchymal ker, CD3, B-cell marker, CD45/B220,andthe markers, FSP1 and P4H. Skin samples from the Ang II- macrophage marker Emr1 (2.0-fold, 3.4-fold, and 3.6- injected mice showed increased numbers of the CD45/ fold, respectively; *P ≤ 0.05), in Ang II-treated mouse FSP1 double-positive cells (3.83% ± 0.49% versus 0.94% ± skin (Figure 4A and 5A). Immunohistochemical staining 0.37% of total number of cells/HPF; *P ≤ 0.05) distributed Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 7 of 12 http://arthritis-research.com/content/14/4/R194 Figure 4 Effect of angiotensin II on inflammation in mouse skin. (A) Real-time PCR analysis of CD3 and CD45/B220 in PBS- and Ang II- treated mice (*P ≤ 0.05). (B) CD3 and CD45R staining on paraffin sections from PBS- and Ang II-infused mice. Representative images are shown from four animals per group. mostly around small vessels in the lower dermis and Angiotensin II increases endothelial-to-mesenchymal around the sclerotic collagen bundles in the upper dermis transition (Figure 6A). Similar results were observed for the CD45/ Recent studies have indicated that EndoMT can contri- P4H double-positive cells (5.11% ± 0.6% versus 2.85% ± bute to the progression of fibrotic diseases, and Ang II 0.75% of cells per HPF; *P ≤ 0.05; Figure 6A). In addition, has been implicated in this process [24-27]. To deter- although only a few FSP1-positive cells were seen in PBS- mine whether EndoMT could serve as a source of acti- infused skin, a significant increase in FSP1 cells was vated fibroblasts, we performed double-fluorescence observed in the upper dermis of Ang II-infused mice staining for VE-cadherin and FSP1 in the skin samples from Ang II- and PBS-treated mice (Figure 7A). Ang (Figure 6A). We also observed a 1.55-fold increase in II-treated mice showed increased numbers of VE-cad- FSP1 mRNA levels in circulating cells isolated from Ang herin/FSP1 double-positive cells distributed around II-treated mice; however, this difference was not statisti- small vessels in the lower dermis. No VE-cadherin/ cally significant (data not shown). However, mRNA levels of FSP1 and of the inflammatory chemokine MCP1,a FSP1 double-positive cells were detected in the upper chemoattractant for fibrocytes, were significantly elevated dermis. In a complementary experiment, cultured (1.94-fold, 2.5-fold, respectively; *P < 0.05) in Ang II-trea- HDMECs treated with Ang II (Figure 7B) demon- ted mouse skin (Figure 6B). No differences were observed strated decreased expression of the endothelial marker, in the mRNA expression of another fibrocyte chemoat- VE-cadherin, and increased expression of the mesench- tractant, SDF1/CXCL12 (data not shown). ymal marker, FSP1. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 8 of 12 http://arthritis-research.com/content/14/4/R194 Figure 5 Effect of angiotensin II on macrophage phenotype and localization in mouse skin. (A) Real-time PCR analysis of Emr1 in PBS- and Ang II-treated mice (*P ≤ 0.05). (B) Mac3 and (C) CD163B staining on paraffin sections from PBS- and Ang II-infused mice. Arrows indicate CD163-positive cells in the upper dermis. Representative images are shown from four animals per group. kidney and heart [5,30]. Ang II is known to act both inde- Discussion pendently and synergistically with TGF-b in promoting The renin-angiotensin system plays a key role in organ excessive ECM production [5,30,31]. We also observed an fibrosis, including heart, kidney, lung, and liver [5,6,28,29]. upregulation of CTGF, a well-known downstream media- In this study, we show that Ang II is also a potent inducer tor of the profibrotic effects of TGF-b [32]. Our data sug- of dermal fibrosis in a mouse model. Consistent with its gest that activation of resident fibroblasts is the primary role in other organs, we provide the evidence that Ang II induces dermal fibrosis through diverse pathogenic mechanism responsible for extracellular matrix accumula- tion. Consistent with this notion, we observed increased mechanisms, including stimulation of collagen and CTGF numbers of FSP1-positive cells and increased numbers of synthesis, myofibroblast differentiation, activation of M2 myofibroblasts, mainly throughout the upper dermis, sug- macrophages, recruitment of fibrocytes, and induction of gesting activation of resident fibroblasts, as well as their EndoMT. differentiation to myofibroblasts. Ang II may act directly Ang II infusion resulted in a dose-dependent deposition on resident fibroblasts, as Ang II can upregulate both of collagen in all dermal layers that was correlated with a aSMA and FSP1 in cultured dermal fibroblasts (Lukasz significant upregulation of interstitial collagen genes. Con- Stawski and Maria Trojanowska, unpublished data). An sistent with other studies, we observed activation of the increased number of myofibroblasts, correlating with TGF-b signaling pathway in response to Ang II treatment. increased expression of CTGF, was also observed in the The interaction between Ang II and TGF-b in the context bleomycin-induced model of skin fibrosis [33]. of fibrosis is well characterized in many organs, including Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 9 of 12 http://arthritis-research.com/content/14/4/R194 Figure 6 Recruitment of fibrocytes after angiotensin II treatment. (A) Immunofluorescence staining ofCD45(red) andFSP1(green) on cryosections of skin samples from PBS- and Ang II-treated mice. Arrows indicate CD45/FSP1 and CD45/P4H double-positive cells. The bar graphs represent quantification of percentage of total positive cells/HPF (high-power field) for CD45, FSP1, CD45/FSP1, and CD45/P4H in PBS- and Ang II-treated mice (*P ≤ 0.05). Representative images are shown from four animals per group. (B) Real-time PCR for MCP1 and FSP1 in PBS (white bars) and Ang II (black bars)-treated mice (*P ≤ 0.05). In renal and heart fibrosis models, Ang II contributes also called M1) and alternatively activated macrophages to increased infiltration of immune cells by activating (AAMs, also called M2) are known to play important the expression of the proinflammatory chemokine, roles in wound repair and fibrosis, either by directly MCP1 [5]. Inflammation was also shown to be closely releasing profibrotic cytokines or by recruiting other associated with fibrosis in a bleomycin-induced model cell types that regulate extracellular matrix turnover of skin fibrosis [34]. Similarly, in our study, we [36]. In our study, M2 macrophages and myofibroblasts observed increased local infiltration of T cells, B cells, showed similar distribution throughout the upper der- and macrophages in the hypodermis of mouse skin, mis, suggesting a potential contribution of M2 macro- which correlated with increased expression of MCP1 in phages to myofibroblast differentiation. Interestingly, + + response to Ang II. Recruitment of inflammatory cells colocalization of CD163B macrophages with aSMA by MCP1 plays an important role in skin fibrosis, as myofibroblasts was recently demonstrated in the areas MCP1-deficient mice showed reduced fibrosis com- of glomerular and interstitial fibrosis in a model of IgA pared with WT mice in the bleomycin-induced skin nephropathy [37]. Furthermore, CD163B macrophages fibrosis model because of the decreased recruitment of displayed strong staining for CTGF, suggesting produc- immune cells to the affected sites [35]. Importantly, we tion of this profibrotic factor [37]. Activated M2 observed an increased presence of CD163B-positive macrophages (CD163 positive) have also been found in cells representing a population of alternatively activated the skin of patients with localized scleroderma and M2 macrophages in the upper dermis of Ang II-infused have been implicated as a possible source of profibrotic mice. Both classically activated macrophages (CAMs, cytokines [38]. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 10 of 12 http://arthritis-research.com/content/14/4/R194 Figure 7 Effect of angiotensin II on EndoMT. Immunofluorescence staining of VE-cadherin (green) and FSP1 (red) in skin samples from PBS- and Ang II-treated mice (left panel) and HDMECs treated with 1,000 ng/ml of Ang II and control (right panel). Arrows indicate Ve-cadherin/FSP1 double-positive cells. Representative images are shown from five animals per group. Accumulating evidence mainly from animal models of It was previously reported that Ang II plays an impor- organ fibrosis suggests that lesional myofibroblasts not tant role in inducing EndoMT in early stages of cardiac only may originate from resident fibroblasts, but also fibrosis [45]. Increased numbers of activated fibroblasts may arise from circulating mesenchymal cells or from originating from capillary endothelial cells also were endothelial cells through endothelial-to-mesenchymal shown in a bleomycin-induced lung-fibrosis model [46]. transitions. Fibrocytes have been implicated in the patho- Our results show increased number of cells co-expressing genesis of various experimental fibrotic conditions, the endothelial cell marker, VE-cadherin, and a mesenchy- including bleomycin-induced pulmonary fibrosis [39], mal cell marker, FSP1, in Ang II-infused mouse skin, suggesting increased EndoMT in response to Ang II. renal fibrosis [40], liver fibrosis [41], and heart fibrosis [42]. In our study, we showed an increased number of However, we have observed the presence of such cells infiltrating fibrocytes (CD45/FSP1 or CD45/P4H double- only in the lower dermis, suggesting that they may have a positive cells) in the skin of Ang II-infused mice, which limited role in this model because the majority of myofi- may contribute to the development of fibrosis in broblasts were present in the upper dermis. However, response to Ang II. In scleroderma, higher numbers of determination of the precise contribution of the process of cells described as “collagen-producing monocytes” was EndoMT to this model of dermal fibrosis will require observed in the peripheral blood of patients with intersti- reporter mice that would allow the lineage tracing of tial lung disease [43,44]. endothelial cells. EndoMT is induced in response to Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 11 of 12 http://arthritis-research.com/content/14/4/R194 year prospective study of 586 patients, with validation of proposed abnormal fibrillin-1 expression and chronic oxidative criteria for early systemic sclerosis. Arthritis Rheum 2008, 58:3902-3912. +/- stress in the Tsk mouse, another model of SSc [47]. 3. Meyer M, Muller AK, Yang J, Sulcova J, Werner S: The role of chronic inflammation in cutaneous fibrosis: fibroblast growth factor receptor deficiency in keratinocytes as an example. J Invest Dermatol Symp Proc Conclusions 2011, 15:48-52. This work demonstrates that Ang II infusion induces 4. Zaman MA, Oparil S, Calhoun DA: Drugs targeting the renin-angiotensin- both inflammation and fibrosis in the skin via MCP1 aldosterone system. Nat Rev Drug Discov 2002, 1:621-636. 5. Mezzano SA, Ruiz-Ortega M, Egido J: Angiotensin II and renal fibrosis. upregulation and accumulation of activated fibroblasts. Hypertension 2001, 38:635-638. Additionally, our data suggest that populations of these 6. Brecher P: Angiotensin II and cardiac fibrosis. Trends Cardiovasc Med 1996, fibroblasts originate from circulating blood cells. Ele- 6:193-198. 7. Kawaguchi Y, Takagi K, Hara M, Fukasawa C, Sugiura T, Nishimagi E, vated serum levels of Ang II found in a subset of Harigai M, Kamatani N: Angiotensin II in the lesional skin of systemic patients with dcSS in the early stage of the disease sug- sclerosis patients contributes to tissue fibrosis via angiotensin II type 1 gest that Ang II may contribute to the pathogenesis of receptors. Arthritis Rheum 2004, 50:216-226. 8. Min LJ, Cui TX, Yahata Y, Yamasaki K, Shiuchi T, Liu HW, Chen R, Li JM, dcSSc, at least in a subset of patients [7]. The patho- Okumura M, Jinno T, Wu L, Iwai M, Nahmias C, Hashimoto K, Horiuchi M: genic features observed in the Ang II model of dermal Regulation of collagen synthesis in mouse skin fibroblasts by distinct fibrosis, such as infiltration of fibrocytes and a colocali- angiotensin II receptor subtypes. Endocrinology 2004, 145:253-260. 9. Pignone A, Rosso AD, Brosnihan KB, Perfetto F, Livi R, Fiori G, Guiducci S, zation of myofibroblasts with the M2 macrophages, are Cinelli M, Rogai V, Tempestini A, Bartoli F, Generini S, Ferrario CM, particularly interesting, because they may help to eluci- Cerinic MM: Reduced circulating levels of angiotensin-(1-7) in systemic date similar processes occurring during the pathogenesis sclerosis: a new pathway in the dysregulation of endothelial-dependent vascular tone control. Ann Rheum Dis 2007, 66:1305-1310. of SSc. We believe that the Ang II model of dermal 10. Batteux F, Kavian N, Servettaz A: New insights on chemically induced fibrosis will be very useful for future mechanistic studies animal models of systemic sclerosis. Curr Opin Rheumatol 2011, of SSc pathogenesis and for the evaluation of novel anti- 23:511-518. 11. Wu M, Varga J: In perspective: murine models of scleroderma. Curr fibrotic treatments. Rheumatol Rep 2008, 10:173-182. 12. Artlett CM: Animal models of scleroderma: fresh insights. Curr Opin Rheumatol 2010, 22:677-682. Abbreviations 13. Derrett-Smith EC, Denton CP, Sonnylal S: Animal models of scleroderma: αSMA: alpha-smooth muscle actin; Ab: antibody; Ang II: angiotensin II; APES: lessons from transgenic and knockout mice. Curr Opin Rheumatol 2009, aminopropyltriethoxysilane; BSA: bovine serum albumin; CTGF: connective 21:630-635. tissue growth factor; dcSS: diffuse cutaneous systemic sclerosis; EC: 14. Smith GP, Chan ES: Molecular pathogenesis of skin fibrosis: insight from endothelial cell; ECM: extracellular matrix; EndoMT: endothelial-to animal models. Curr Rheumatol Rep 2010, 12:26-33. mesenchymal transition; FBS: fetal bovine serum; FSP1: fibroblast-specific 15. Beyer C, Schett G, Distler O, Distler JH: Animal models of systemic protein 1; HDMEC: human dermal microvascular endothelial cell; HPF: high- sclerosis: prospects and limitations. Arthritis Rheum 2010, 62:2831-2844. power field; MCP1: monocyte chemotactic protein-1; PBS: phosphate- 16. Bataller R, Sancho-Bru P, Gines P, Brenner DA: Liver fibrogenesis: a new buffered saline; PCR: polymerase chain reaction; RAS: renin-angiotensin role for the renin-angiotensin system. Antioxid Redox Signal 2005, system; RP: Raynaud phenomenon; SSc: systemic sclerosis; TGF-β: 7:1346-1355. transforming growth factor β; WT: wild type. 17. Samuel CS: Determination of collagen content, concentration, and sub- types in kidney tissue. Methods Mol Biol 2009, 466:223-235. Acknowledgements 18. Richard L, Velasco P, Detmar M: Isolation and culture of microvascular This study was supported by the National Institutes of Health (NIAMS) grant endothelial cells. Methods Mol Med 1999, 18:261-269. RO1 AR42334-18 to M. Trojanowska and by the Scleroderma Foundation 19. Ihn H: Scleroderma, fibroblasts, signaling, and excessive extracellular Young Investigator Grant to Andreea Bujor. matrix. Curr Rheumatol Rep 2005, 7:156-162. We thank Dr. Giuseppina Stifano for help with CD163B immunostaining and 20. Ho MK, Springer TA: Tissue distribution, structural characterization, and Paul Haines for help with editing the manuscript. biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity. J Biol Chem 1983, 258:636-642. Authors’ contributions 21. Sulahian TH, Högger P, Wahner AE, Wardwell K, Goulding NJ, Sorg C, LS performed all experiments and wrote the manuscript. RH performed Droste A, Stehling M, Wallace PK, Morganelli PM, Guyre PM: Human blood analyses and contributed to manuscript writing. AB contributed to monocytes express CD163, which is upregulated by IL-10 and identical experimental design and manuscript writing. MT was the principal to p155. Cytokine 2000, 12:1312-1321. investigator and was involved in conception and design of the study, data 22. Komohara Y, Hirahara J, Horikawa T, Kawamura K, Kiyota E, Sakashita N, analysis, and manuscript writing. All authors read and approved the Araki N, Takeya M: AM-3K, an anti-macrophage antibody, recognizes manuscript for publication. CD163, a molecule associated with an anti-inflammatory macrophage phenotype. J Histochem Cytochem 2006, 54:763-771. Competing interests 23. Quan TE, Cowper SE, Bucala R: The role of circulating fibrocytes in No conflict of interest was identified for all authors. fibrosis. Curr Rheumatol Rep 2006, 8:145-150. 24. Kizu A, Medici D, Kalluri R: Endothelial-mesenchymal transition as a novel Received: 25 April 2012 Revised: 6 July 2012 Accepted: 20 August 2012 mechanism for generating myofibroblasts during diabetic nephropathy. Published: 22 August 2012 Am J Pathol 2009, 175:1371-1373. 25. Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, McMullen JR, References Gustafsson E, Chandraker A, Yuan X, Pu WT, Roberts AB, Neilson EG, 1. Trojanowska M: Cellular and molecular aspects of vascular dysfunction in Sayegh MH, Izumo S, Kalluri R: Endothelial-to-mesenchymal transition systemic sclerosis. Nat Rev Rheumatol 2010, 6:453-460. contributes to cardiac fibrosis. Nat Med 2007, 13:952-961. 2. Koenig M, Joyal F, Fritzler MJ, Roussin A, Abrahamowicz M, Boire G, 26. Li J, Qu X, Bertram JF: Endothelial-myofibroblast transition contributes to Goulet JR, Rich E, Grodzicky T, Raymond Y, Senécal JL: Autoantibodies and the early development of diabetic renal interstitial fibrosis in microvascular damage are independent predictive factors for the streptozotocin-induced diabetic mice. Am J Pathol 2009, 175:1380-1388. progression of Raynaud’s phenomenon to systemic sclerosis: a twenty- Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 12 of 12 http://arthritis-research.com/content/14/4/R194 27. Tang R, Li Q, Lv L, Dai H, Zheng M, Ma K, Liu B: Angiotensin II mediates the high-glucose-induced endothelial-to-mesenchymal transition in human aortic endothelial cells. Cardiovasc Diabetol 2010, 9:31. 28. Pereira RM, dos Santos RA, da Costa Dias FL, Teixeira MM, Simoes e Silva AC: Renin-angiotensin system in the pathogenesis of liver fibrosis. World J Gastroenterol 2009, 15:2579-2586. 29. Kuba K, Imai Y, Penninger JM: Angiotensin-converting enzyme 2 in lung diseases. Curr Opin Pharmacol 2006, 6:271-276. 30. Rosenkranz S: TGF-beta1 and angiotensin networking in cardiac remodeling. Cardiovasc Res 2004, 63:423-432. 31. Gabriel VA: Transforming growth factor-beta and angiotensin in fibrosis and burn injuries. J Burn Care Res 2009, 30:471-481. 32. Blom IE, Goldschmeding R, Leask A: Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy? Matrix Biol 2002, 21:473-482. 33. Liu S, Taghavi R, Leask A: Connective tissue growth factor is induced in bleomycin-induced skin scleroderma. J Cell Commun Signal 2010, 4:25-30. 34. Yamamoto T, Nishioka K: Cellular and molecular mechanisms of bleomycin-induced murine scleroderma: current update and future perspective. Exp Dermatol 2005, 14:81-95. 35. Ferreira AM, Takagawa S, Fresco R, Zhu X, Varga J, DiPietro LA: Diminished induction of skin fibrosis in mice with MCP-1 deficiency. J Invest Dermatol 2006, 126:1900-1908. 36. Murray PJ, Wynn TA: Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 2011, 11:723-737. 37. Ikezumi Y, Suzuki T, Karasawa T, Hasegawa H, Yamada T, Imai N, Narita I, Kawachi H, Polkinghorne KR, Nikolic-Paterson DJ, Uchiyama M: Identification of alternatively activated macrophages in new-onset paediatric and adult immunoglobulin A nephropathy: potential role in mesangial matrix expansion. Histopathology 2011, 58:198-210. 38. Higashi-Kuwata N, Makino T, Inoue Y, Takeya M, Ihn H: Alternatively activated macrophages (M2 macrophages) in the skin of patient with localized scleroderma. Exp Dermatol 2009, 18:727-729. 39. Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM: Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 2004, 114:438-446. 40. Okada H, Kalluri R: Cellular and molecular pathways that lead to progression and regression of renal fibrogenesis. Curr Mol Med 2005, 5:467-474. 41. Kisseleva T, Uchinami H, Feirt N, Quintana-Bustamante O, Segovia JC, Schwabe RF, Brenner DA: Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis. J Hepatol 2006, 45:429-438. 42. Krenning G, Zeisberg EM, Kalluri R: The origin of fibroblasts and mechanism of cardiac fibrosis. J Cell Physiol 2010, 225:631-637. 43. Mathai SK, Gulati M, Peng X, Russell TR, Shaw AC, Rubinowitz AN, Murray LA, Siner JM, Antin-Ozerkis DE, Montgomery RR, Reilkoff RA, Bucala RJ, Herzog EL: Circulating monocytes from systemic sclerosis patients with interstitial lung disease show an enhanced profibrotic phenotype. Lab Invest 2010, 90:812-823. 44. Tourkina E, Bonner M, Oates J, Hofbauer A, Richard M, Znoyko S, Visconti RP, Zhang J, Hatfield CM, Silver RM, Hoffman S: Altered monocyte and fibrocyte phenotype and function in scleroderma interstitial lung disease: reversal by caveolin-1 scaffolding domain peptide. Fibrogenesis Tissue Repair 2011, 4:15. 45. Tang RN, Lv LL, Zhang JD, Dai HY, Li Q, Zheng M, Ni J, Ma KL, Liu BC: Effects of angiotensin II receptor blocker on myocardial endothelial-to- mesenchymal transition in diabetic rats. Int J Cardiol 2011. 46. Hashimoto N, Phan SH, Imaizumi K, Matsuo M, Nakashima H, Kawabe T, Shimokata K, Hasegawa Y: Endothelial-mesenchymal transition in Submit your next manuscript to BioMed Central bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 2010, and take full advantage of: 43:161-172. 47. Xu H, Zaidi M, Struve J, Jones DW, Krolikowski JG, Nandedkar S, Lohr NL, • Convenient online submission Gadicherla A, Pagel PS, Csuka ME, Pritchard KA, Weihrauch D: Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial • Thorough peer review mesenchymal transition in a murine model of systemic sclerosis. Am J • No space constraints or color figure charges Physiol Cell Physiol 2011, 300:C550-C556. • Immediate publication on acceptance doi:10.1186/ar4028 • Inclusion in PubMed, CAS, Scopus and Google Scholar Cite this article as: Stawski et al.: Angiotensin II induces skin fibrosis: a • Research which is freely available for redistribution novel mouse model of dermal fibrosis. Arthritis Research & Therapy 2012 14:R194. Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arthritis Research & Therapy Springer Journals

Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis

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Copyright © 2012 by Stawski et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Rheumatology; Orthopedics
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Abstract

Introduction: Systemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin. Methods: Ang II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-b1, TGF-b2, TGF-b3, CTGF, aSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, aSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1. Results: Ang II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-b2 and TGF-b3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of aSMA-positive cells, as well as CD163B macrophages in the upper dermis. This correlated with significantly increased mRNA levels of aSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only. Conclusions: This work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies. Introduction SSc patients and can precede definite diagnosis of SSc by Systemic sclerosis (SSc) is a complex autoimmune years or even decades. In patients with SSc, RP is asso- inflammatory disorder of unknown etiology characterized ciated with structural abnormalities of the microvascula- by vascular alterations, activation of the immune system, ture and the presence of SSc-specific autoantibodies [2], indicating an early link between immune system activa- and fibrosis of the skin and internal organs [1]. Vascular insufficiency and immune dysfunction manifest early in tion and vascular injury. Fibrosis results from excessive the disease and are followed by increased extracellular production and accumulation of ECM components pro- matrix (ECM) production as the disease progresses. duced by activated fibroblasts, which might be triggered Raynaud phenomenon (RP) is present in the majority of by cytokines and growth factors released from the infil- trating immune cells during the inflammatory stage [3]. * Correspondence: [email protected] However, interrelations between the key pathologic com- Arthritis Center, Boston University School of Medicine, Boston, MA 02118, ponents of the disease are still poorly understood. USA © 2012 Stawski et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 2 of 12 http://arthritis-research.com/content/14/4/R194 Angiotensin II (Ang II), a main component of the skin surrounding the pump outlet was collected by using rennin-angiotensin system (RAS), is a vasoactive peptide an 8-mm-diameter punch biopsy device. that induces vascular constriction, salt and water reten- tion, and increased blood pressure [4]. Ang II has been Gomori Trichrome staining reported to play a critical role in renal and heart fibrosis Gomori Trichrome staining was used to detect collagen through inflammation and upregulation of matrix deposi- fibers and collagen deposition in the mouse skin. The skin tion [5,6]. Previous studies also suggest that Ang II may samples were fixed in 4% paraformaldehyde for 24 hours be involved in the pathogenesis of skin fibrosis in SSc. It and then processed for paraffin embedding. Staining was has been shown that Ang II levels are increased in the performed on 8-μm-thick paraffin sections by following blood of SSc patients and that, in contrast to healthy the manufacturer’s instructions (Chromaview, Dublin, skin, the Ang II precursor angiotensinogen is expressed OH, USA; Gomori Trichrome blue collagen Kit S7440- in SSc skin [7]. Furthermore, the profibrotic effects of 19). Collagen fibers were stained blue, nuclei were stained Ang II are mediated via the AT1a receptor in cultured black, and the background was stained red. human and mouse skin fibroblasts [8]. In addition, dysre- gulation of RAS components was shown in patients with Hydroxyproline assay SSc, with a prevalence of the vasoconstricting Ang II Collagen deposition was quantified by measuring total over the vasodilating Ang-(1-7), suggesting inhibition of hydroxyproline content in 4-mm skin-punch biopsies endothelium-dependent vasodilatation and increased obtained from PBS and Ang II infusion sites by using a pre- vasoconstriction in SSc vessels [9]. viously described method with some modifications [17]. In Utilization of animal models has been instrumental in brief, the skin samples were hydrolyzed with 6 M sodium delineating complex pathologic features of SSc. In the last hydroxide at 110°C for 12 hours. The hydrolyzate was then decade, a number of new animal models became available oxidized with oxidation buffer (one part 7% chloramine T to study mechanisms of SSc fibrosis [10-13]. The inducible and four parts of acetate citrate buffer) for 4 minutes at models of SSc include the widely studied bleomycin and room temperature. Ehrlich aldehyde reagent was added to more recently established hypochlorous acid (HOCH) each sample, and the chromophore was developed by incu- injection models, as well as the immune-based scleroder- bating the samples at 65°C for 25 minutes. Absorbance of matous graft-versus-host model. A growing number of each sample was read at 560 nm by using a spectro- genetic models are very valuable for investigating specific phometer. Results were expressed as total hydroxyproline signaling pathways involved in fibrosis [14,15]. Whereas content (in micrograms) per 0.1 g of tissue. A standard none of the currently available models recapitulate the curve was performed for all hydroxyproline measurements complex features of SSc, they provide important insights by using known quantities of hydroxyproline. into selected aspects of SSc pathogenesis and allow precli- nical testing of antifibrotic compounds. Quantitative RT-PCR analysis Angiotensin II has been widely used to investigate kidney Total RNA was isolated by using the RNeasy Fibrous Tis- [5], heart [6], and liver [16] fibrosis by using mouse models. sue Mini Kit (Qiagen, Valencia, CA, USA). Then, 1 μgof However, the profibrotic potential of Ang II has not been total RNA was reverse transcribed with random hexam- evaluated in any model of dermal fibrosis. Given the poten- ers by using the Transcriptor First Strand Complemen- tial involvement of Ang II in the pathogenesis of SSc, the tary DNA Synthesis kit (Roche Applied Science, goal of this study was to investigate the effect of Ang II on Indianapolis, IN, USA) according to the manufacturer’s dermal fibrosis in a mouse model. protocol. Real-time PCR assays were performed by using the StepOnePlus Real-Time PCR system (Applied Biosys- Materials and methods tems, Foster City, CA, USA). The amplification mixture Subcutaneous infusion of angiotensin II using ALZET (10 μl) contained 1 μl of complementary DNA, 0.5 μM of osmotic minipumps each primer, and 5 μlofSYBRGreen PCRMasterMix. C57BL/6 mice were purchased from The Jackson Labora- The primers are listed in Supplementary Table 1. Relative tory. All of the experiments were performed under the change in the levels of genes of interest was determined -ΔΔCT guidelines of the Boston University Institutional Animal by the 2 method. Care and Use Committee (protocol AN-15037). Alzet osmotic miniature pumps (model 2002) delivering angio- Immunofluorescence staining on frozen sections tensin II (Sigma-Aldrich, St. Louis, MO, USA) at a rate of For all immunofluorescence staining, skin samples were 1,000 ng/kg/min (pressor dose) or 2,000 ng/kg/min, or directly embedded in O.C.T. compound, flash frozen, PBS, were implanted subcutaneously on the backs of and stored at -80°C. Staining was performed on 8-μm 8-week-old mice. After 14 days, mice were killed, and the cryosections of mouse skin. In brief, slides were blocked Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 3 of 12 http://arthritis-research.com/content/14/4/R194 Table 1 Primers for quantitative real-time polymerase described [18]. Cells were cultured on bovine collagen- chain reaction coated six-well plates in EBM medium supplemented with m-B2MG Forward: 5’-TCGCTCGGTGACCCTAGTCTTT-3’ 10% FBS and EC growth supplement mix at 37°C with 5% CO in air. The culture medium was changed every other Reverse: 5’-ATGTTCGGCTTCCCATTCTCC-3’ 2 day. For immunofluorescence, cultured HDMECs grown m-Fli1 Forward: 5’-ACTTGGCCAAATGGACGGGACTAT-3’ on collagen-coated coverslips were treated with Ang II Reverse: 5’-CCCGTAGTCAGGACTCCCG-3’ (1,000 ng/ml) for 96 hours. Control and Ang II-treated m-Col1a1 Forward: 5’-GCCAAGAAGACATCCCTGAAG-3’ cells were fixed with 4% paraformaldehyde for 15 minutes Reverse: 5’-TGTGGCAGATACAGATCAAGC-3’ followed by incubation with 0.15 M glycine for 30 min- m-Col1a2 Forward: 5’-GCCACCATTGATAGTCTCTCC-3’ utes. Nonspecific protein binding was blocked with 3% Reverse: 5’-CACCCCAGCGAAGAACTCATA-3’ BSA for 1 hour. Next, cells were incubated at 4°C over- m-COL3a1 Forward: 5’-GCCAAGAAGACATCCCTGAAG-3’ night with primary antibodies: goat anti-mouse VE-cad- Reverse: 5’-TGGACTGCTGTGCCAAAATA-3’ herin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-mouse FSP1 (Abcam, Cambridge, MA, m-Col5a1 Forward: 5’-GGACTAGTCCGCTTTCCCTGTCAACTTG-3’ USA). After washing, cell cultures were incubated with Reverse: 5’-GTGGTCACTGCGGCTGAGGAACTTC-3’ Alexa fluor 488 donkey anti-goat (Invitrogen, Grand m-CTGF Forward: 5’-CTGCAGACTGGAGAAGCAGA-3’ Island, NY, USA) and Alexa fluor 594 donkey anti-rabbit Reverse: 5’-GATGCACTTTTTGCCCTTCTT-3’ (Invitrogen) antibodies for 1.5 hour. Cells were mounted m-aSMA Forward: 5’-CCCACCCAGAGTGGAGAA-3’ on slides by using Vectashield with DAPI (Vector Labora- Reverse: 5’-ACATAGCTGGAGCAGCGTCT-3’ tories) and examined by using a FluoView FV10i confocal m-FSP1 Forward: 5’-GGAGCTGCCTAGCTTCCTG-3’ microscope system (Olympus, Center Valley, PA, USA) at Reverse: 5’-TCCTGGAAGTCAACTTCATTGTC-3’ 488 nm (green), 594 nm (red), and 405 nm (blue). m-MCP1 Forward: 5’-CATCCACGTGTTGGCTCA-3’ Reverse: 5’-GATCATCTTGCTGGTGAATGAGT-3’ Immunohistochemistry Immunohistochemistry was performed on formalin- m-CD3 Forward: 5’-AACACGTACTTGTACCTGAAAGCTC-3’ fixed, paraffin-embedded skin tissue sections by using Reverse: 5’-GATGATTATGGCTACTGCTGTCA-3’ the Vectastain ABC kit (Vector Laboratories) according m-Emr1 Forward: 5’-CCTGGACGAATCCTGTGAAG-3’ to the manufacturer’s instructions. In brief, sections (8- Reverse: 5’-GGTGGGACCACAGAGAGTTG-3’ μm thick) were mounted on APES (aminopropyl- m-CD45/B220 Forward: 5’-AATGGCTCTTCAGAGACCACATA-3’ triethoxy silane solution)-coated slides, deparaffinized Reverse: 5’-AGTCAGGCTGTGGGGACA-3’ with Histo-Clear (National Diagnostics, Atlanta, GA, TGF-b1 Forward: 5’-GCAGCACGTGGAGCTGTA-3’ USA), and rehydrated through a graded series of etha- Reverse: 5’-CAGCCGGTTGCTGAGGTA-3 nol. Endogenous peroxidase was blocked by incubation TGF-b2 Forward: 5’-CCTTCTTCCCCTCCGAAAC-3’ in 3% hydrogen peroxide for 30 minutes, followed by incubation with 0.15 M glycine for 45 minutes, and nor- Reverse: 5’-AGAGCACCTGGGACTGTCTG-3’ mal blocking serum for 1 hour. The sections were then TGF-b3 Forward: 5’-AAGAAGCGGGCTTTGGAC-3’ incubated overnight at 4°C with antibodies against CD3 Reverse: 5’-CGCACACAGCAGTTCTCC-3’ (Abcam, Cambridge, MA, USA), Mac3 (BD Bioscience, San Jose, CA, USA), CD45R (AbD Serotec, Raleigh, NC, USA), or CD163B (Epitomics, Burlingame, CA, USA), with a blocking solution (3% BSA (Sigma-Aldrich), and diluted 1:100 in blocking buffer, followed by incubation 0.3% Triton X-100 in PBS) for 2 hours. After washing, for 30 minutes with a biotinylated secondary antibody tissue sections were incubated at 4°C overnight with pri- mary antibodies (Table 2). Tissue sections were then solution. A solution containing avidin:biotin:peroxidase washed and incubated with secondary Ab (Table 2) at complexes was applied to the sections subsequently. room temperature for 2 hours. Coverslips were mounted Immunoreactivity was visualized with diaminobenzidine by using Vectashield with DAPI (Vector Laboratories, (Vector Laboratories), and the sections were counter- Burlingame, CA, USA), and staining was examined by stained with hematoxylin. Images were collected by using a FluoView FV10i confocal microscope system using a microscope (BH-2; Olympus, Center Valley, PA, (Olympus, Center Valley, PA, USA) at 488 nm (green), USA). 594 nm (red), and 405 nm (blue). Statistical analyses Immunofluorescence staining on adherent cell cultures All data were analyzed with the Student paired t test. Human dermal microvascular endothelial cells (HDMECs) The level for statistical significance was set at P ≤ were isolated from human foreskin, as previously 0.05. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 4 of 12 http://arthritis-research.com/content/14/4/R194 Table 2 Primary and secondary antibodies for immunofluorescence staining Primary antibodies Secondary antibodies Myofibroblasts Rabbit anti-mouse aSMA Ab (Novus Biologicals, Littleton, CO); 1:100 Alexa fluor 488 donkey anti-rabbit IgG (Invitrogen, Grand Island, NY); 1:1,000 Fibrocytes Rat anti-mouse CD45 Ab (BD Rabbit anti-mouse FSP1 Ab Alexa fluor 594 donkey anti- Alexa fluor 488 donkey anti- Pharmingen, San Diego, CA); 1:50 (Abcam, Cambridge, MA); rat IgG (Invitrogen); 1:1,000 rabbit IgG (Invitrogen); 1:100 1:1,000 EndoMT Goat anti-mouse VE-cadherin Ab (Santa Rabbit anti-mouse FSP1 Ab Alexa fluor 488 donkey anti- Alexa fluor 594 donkey anti- Cruz Biotechnology, Santa Cruz, CA); (Abcam); 1:100 goat IgG (Invitrogen); rabbit IgG (Invitrogen); 1:300 1:1,000 1:1,000 addition, the fat layer of the reticular dermis was partially Results replaced with extracellular matrix (Figure 1A). Real-time Angiotensin II increases collagen synthesis and PCR analysis showed a statistically significant increase in deposition in mouse skin mRNA levels of the collagen-encoding genes: Col1a1, To determine whether angiotensin II can induce dermal Col1a2, Col3a1,and Col5a1,aswellas CTGF (2.4-fold, fibrosis, Ang II at a rate of 1,000 ng/kg/min was adminis- 1.9-fold, and 3.4-fold, 1.9-fold, and 2.1-fold, respectively; tered continuously with subcutaneous osmotic minipumps *P ≤ 0.05) in the skin of Ang II-infused mice when com- implanted under the shaved back skin of 8-week-old pared with control mice (Figure 1B). Total hydroxyproline C57BL/6 male mice. Treated skin was harvested at day 14, content from the injected sites was significantly higher in and histologic examination and collagen-content measure- Ang II (1,000 ng/kg/min)-infused skin (2,481 ± 920 μgper ment assays were performed. Gomori Trichrome staining 0.1 g of skin) compared with control PBS-infused skin showed that collagen fibers were more closely packed in (1,534 ± 430 μg per 0.1 g of skin; *P ≤ 0.05) (Figure 1C). angiotensin II-treated mice compared with control mice, A higher dose of Ang II (2,000 ng/kg/min) showed a larger reflecting increased collagen deposition (Figure 1A). In Figure 1 Angiotensin II increases collagen synthesis and deposition in mouse skin. (A) Gomori Trichrome staining shows a histologic evaluation of Ang II-induced lesions in C57BL/6 mice. (B) Real-time PCR analysis of collagen-encoding genes: Col1a1, Col1a2, Col3a1, Col5a1, along with CTGF (*P ≤ 0.05) in PBS-and Ang II-treated mice. (C) Total hydroxyproline content in PBS- and Ang II-treated mice. Values are the mean of six mice in each group; *P ≤ 0.05. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 5 of 12 http://arthritis-research.com/content/14/4/R194 increase in the total hydroxyproline content of local skin the mRNA expression of TGF-b1 (Figure 2A). Immuno- (5,272 ± 1,260 μg per 0.1 g of Ang II-infused skin versus histochemical staining of pSmad2 was performed on par- 1,748 ± 531 μg per 0.1 g of PBS-infused skin; *P ≤ 0.05) affin sections. We observed increased numbers of (Figure 1C). The profibrotic effects of Ang II were local, as pSmad2-positive cells distributed throughout all dermal no significant differences were observed in hydroxyproline layers in Ang II-treated mice (Figure 2B). These data sug- content in distal skin with either concentration of Ang II gest that Ang II potently activates TGF-b signaling in this (data not shown). Because a dose of 1,000 ng/kg/min was model. sufficient to induce significant dermal fibrosis, this dose was selected for further analyses. Angiotensin II increases the number of myofibroblasts in mouse skin Angiotensin II activates the TGF-b pathway in mouse skin Fibrosis is associated with accumulation of activated Transforming growth factor-b (TGF-b) plays a pivotal role fibroblasts/myofibroblasts in the affected tissues. To in the pathogenesis of scleroderma by activating fibroblasts determine whether Ang II treatment increases myofibro- and stimulating the production of ECM components [19]. blast presence, skin from infused sites was examined with To determine whether Ang II treatment activates the real-time PCR and immunostaining for aSMA. Real-time TGF-b pathway, we examined skin samples from the PCR analysis showed a statistically significant increase in infused sites with real-time PCR and immunostaining. the mRNA level of aSMA (1.9-fold, *P ≤ 0.05) in Ang II- Real-time PCR analysis showed statistically significant treated mouse skin (Figure 3A). aSMA staining was per- increases in the mRNA levels of TGF-b2 and TGF-b3 (3.2- formed on cryosections (immunofluorescence, Figure 3B fold and 4.8-fold, respectively; *P ≤ 0.05) in Ang II-treated and 3D) and paraffin sections (immunohistochemistry, mouse skin (Figure 2A). No differences were observed in Figure 3C) from PBS- and Ang II-treated mouse skin. Figure 2 Angiotensin II activates TGF-b signaling. (A) Real-time PCR analysis of TGF-b1, TGF-b2,and TGF-b3 (*P ≤ 0.05)inPBS- and Ang II- treated mice. (B) pSmad2 staining on paraffin sections from PBS- and Ang II-infused mice. Representative images are shown from four animals per group. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 6 of 12 http://arthritis-research.com/content/14/4/R194 Figure 3 Detection of activated fibroblasts in angiotensin II-treated skin. (A) Real-time PCR analysis of aSMA in PBS- and Ang II-treated mice (*P ≤ 0.05). aSMA staining in the upper dermis (B) by immunofluorescence and (C) by immunohistochemistry, and in the lower dermis (D) by immunofluorescence, on sections from PBS- and Ang II-treated mouse skin. Arrows indicate positive staining of myofibroblasts. Representative images are shown from five animals per group. Affected sites from PBS-treated mice contained few of CD3, CD45R, and two macrophage markers, a general aSMA-positive cells, observed exclusively around the marker, Mac3 [20], and a marker identifying M2 macro- blood vessels and the arrector pili muscles. In contrast, phages, CD163B [21,22] was performed on paraffin sec- affected sites from Ang II-treated mice showed an tions. We observed increased infiltration of CD3, CD45R, increased number of cells expressing aSMA distributed and Mac3-positive cells only in the hypodermis of Ang mainly throughout the upper dermis (Figure 3B and 3C), II-treated mice (Figure 4B and 5B). Interestingly, we whereas in the lower dermis, positive cells were observed observed an increased presence of CD163B-positive cells only around the blood vessels (Figure 3D). These data in the upper dermis of Ang II-treated mice, whereas no suggest that Ang II-induced myofibroblast differentiation increase was found in the lower dermis or hypodermis contributes to the development of fibrosis in this model. (Figure 5C). Angiotensin II increases inflammation in mouse skin Angiotensin II increases infiltration of fibrocytes in the Previous studies have shown that Ang II promotes upper dermis inflammation in kidney and heart models of fibrosis [5,6]. Circulating mesenchymal cells, fibrocytes, have been To evaluate the recruitment of inflammatory cells, skin shown to infiltrate areas of inflammation and tissue samples from the Ang II-infused sites were examined damage and contribute to the fibrotic process [23]. To with real-time PCR and immunostaining of immune cell determine whether fibrocytes are involved in the dermal markers. Real-time PCR analysis showed statistically sig- fibrosis induced by Ang II, skin cryosections were stained nificant increases in the mRNA levels of the T-cell mar- for the immune cell marker, CD45, and mesenchymal ker, CD3, B-cell marker, CD45/B220,andthe markers, FSP1 and P4H. Skin samples from the Ang II- macrophage marker Emr1 (2.0-fold, 3.4-fold, and 3.6- injected mice showed increased numbers of the CD45/ fold, respectively; *P ≤ 0.05), in Ang II-treated mouse FSP1 double-positive cells (3.83% ± 0.49% versus 0.94% ± skin (Figure 4A and 5A). Immunohistochemical staining 0.37% of total number of cells/HPF; *P ≤ 0.05) distributed Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 7 of 12 http://arthritis-research.com/content/14/4/R194 Figure 4 Effect of angiotensin II on inflammation in mouse skin. (A) Real-time PCR analysis of CD3 and CD45/B220 in PBS- and Ang II- treated mice (*P ≤ 0.05). (B) CD3 and CD45R staining on paraffin sections from PBS- and Ang II-infused mice. Representative images are shown from four animals per group. mostly around small vessels in the lower dermis and Angiotensin II increases endothelial-to-mesenchymal around the sclerotic collagen bundles in the upper dermis transition (Figure 6A). Similar results were observed for the CD45/ Recent studies have indicated that EndoMT can contri- P4H double-positive cells (5.11% ± 0.6% versus 2.85% ± bute to the progression of fibrotic diseases, and Ang II 0.75% of cells per HPF; *P ≤ 0.05; Figure 6A). In addition, has been implicated in this process [24-27]. To deter- although only a few FSP1-positive cells were seen in PBS- mine whether EndoMT could serve as a source of acti- infused skin, a significant increase in FSP1 cells was vated fibroblasts, we performed double-fluorescence observed in the upper dermis of Ang II-infused mice staining for VE-cadherin and FSP1 in the skin samples from Ang II- and PBS-treated mice (Figure 7A). Ang (Figure 6A). We also observed a 1.55-fold increase in II-treated mice showed increased numbers of VE-cad- FSP1 mRNA levels in circulating cells isolated from Ang herin/FSP1 double-positive cells distributed around II-treated mice; however, this difference was not statisti- small vessels in the lower dermis. No VE-cadherin/ cally significant (data not shown). However, mRNA levels of FSP1 and of the inflammatory chemokine MCP1,a FSP1 double-positive cells were detected in the upper chemoattractant for fibrocytes, were significantly elevated dermis. In a complementary experiment, cultured (1.94-fold, 2.5-fold, respectively; *P < 0.05) in Ang II-trea- HDMECs treated with Ang II (Figure 7B) demon- ted mouse skin (Figure 6B). No differences were observed strated decreased expression of the endothelial marker, in the mRNA expression of another fibrocyte chemoat- VE-cadherin, and increased expression of the mesench- tractant, SDF1/CXCL12 (data not shown). ymal marker, FSP1. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 8 of 12 http://arthritis-research.com/content/14/4/R194 Figure 5 Effect of angiotensin II on macrophage phenotype and localization in mouse skin. (A) Real-time PCR analysis of Emr1 in PBS- and Ang II-treated mice (*P ≤ 0.05). (B) Mac3 and (C) CD163B staining on paraffin sections from PBS- and Ang II-infused mice. Arrows indicate CD163-positive cells in the upper dermis. Representative images are shown from four animals per group. kidney and heart [5,30]. Ang II is known to act both inde- Discussion pendently and synergistically with TGF-b in promoting The renin-angiotensin system plays a key role in organ excessive ECM production [5,30,31]. We also observed an fibrosis, including heart, kidney, lung, and liver [5,6,28,29]. upregulation of CTGF, a well-known downstream media- In this study, we show that Ang II is also a potent inducer tor of the profibrotic effects of TGF-b [32]. Our data sug- of dermal fibrosis in a mouse model. Consistent with its gest that activation of resident fibroblasts is the primary role in other organs, we provide the evidence that Ang II induces dermal fibrosis through diverse pathogenic mechanism responsible for extracellular matrix accumula- tion. Consistent with this notion, we observed increased mechanisms, including stimulation of collagen and CTGF numbers of FSP1-positive cells and increased numbers of synthesis, myofibroblast differentiation, activation of M2 myofibroblasts, mainly throughout the upper dermis, sug- macrophages, recruitment of fibrocytes, and induction of gesting activation of resident fibroblasts, as well as their EndoMT. differentiation to myofibroblasts. Ang II may act directly Ang II infusion resulted in a dose-dependent deposition on resident fibroblasts, as Ang II can upregulate both of collagen in all dermal layers that was correlated with a aSMA and FSP1 in cultured dermal fibroblasts (Lukasz significant upregulation of interstitial collagen genes. Con- Stawski and Maria Trojanowska, unpublished data). An sistent with other studies, we observed activation of the increased number of myofibroblasts, correlating with TGF-b signaling pathway in response to Ang II treatment. increased expression of CTGF, was also observed in the The interaction between Ang II and TGF-b in the context bleomycin-induced model of skin fibrosis [33]. of fibrosis is well characterized in many organs, including Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 9 of 12 http://arthritis-research.com/content/14/4/R194 Figure 6 Recruitment of fibrocytes after angiotensin II treatment. (A) Immunofluorescence staining ofCD45(red) andFSP1(green) on cryosections of skin samples from PBS- and Ang II-treated mice. Arrows indicate CD45/FSP1 and CD45/P4H double-positive cells. The bar graphs represent quantification of percentage of total positive cells/HPF (high-power field) for CD45, FSP1, CD45/FSP1, and CD45/P4H in PBS- and Ang II-treated mice (*P ≤ 0.05). Representative images are shown from four animals per group. (B) Real-time PCR for MCP1 and FSP1 in PBS (white bars) and Ang II (black bars)-treated mice (*P ≤ 0.05). In renal and heart fibrosis models, Ang II contributes also called M1) and alternatively activated macrophages to increased infiltration of immune cells by activating (AAMs, also called M2) are known to play important the expression of the proinflammatory chemokine, roles in wound repair and fibrosis, either by directly MCP1 [5]. Inflammation was also shown to be closely releasing profibrotic cytokines or by recruiting other associated with fibrosis in a bleomycin-induced model cell types that regulate extracellular matrix turnover of skin fibrosis [34]. Similarly, in our study, we [36]. In our study, M2 macrophages and myofibroblasts observed increased local infiltration of T cells, B cells, showed similar distribution throughout the upper der- and macrophages in the hypodermis of mouse skin, mis, suggesting a potential contribution of M2 macro- which correlated with increased expression of MCP1 in phages to myofibroblast differentiation. Interestingly, + + response to Ang II. Recruitment of inflammatory cells colocalization of CD163B macrophages with aSMA by MCP1 plays an important role in skin fibrosis, as myofibroblasts was recently demonstrated in the areas MCP1-deficient mice showed reduced fibrosis com- of glomerular and interstitial fibrosis in a model of IgA pared with WT mice in the bleomycin-induced skin nephropathy [37]. Furthermore, CD163B macrophages fibrosis model because of the decreased recruitment of displayed strong staining for CTGF, suggesting produc- immune cells to the affected sites [35]. Importantly, we tion of this profibrotic factor [37]. Activated M2 observed an increased presence of CD163B-positive macrophages (CD163 positive) have also been found in cells representing a population of alternatively activated the skin of patients with localized scleroderma and M2 macrophages in the upper dermis of Ang II-infused have been implicated as a possible source of profibrotic mice. Both classically activated macrophages (CAMs, cytokines [38]. Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 10 of 12 http://arthritis-research.com/content/14/4/R194 Figure 7 Effect of angiotensin II on EndoMT. Immunofluorescence staining of VE-cadherin (green) and FSP1 (red) in skin samples from PBS- and Ang II-treated mice (left panel) and HDMECs treated with 1,000 ng/ml of Ang II and control (right panel). Arrows indicate Ve-cadherin/FSP1 double-positive cells. Representative images are shown from five animals per group. Accumulating evidence mainly from animal models of It was previously reported that Ang II plays an impor- organ fibrosis suggests that lesional myofibroblasts not tant role in inducing EndoMT in early stages of cardiac only may originate from resident fibroblasts, but also fibrosis [45]. Increased numbers of activated fibroblasts may arise from circulating mesenchymal cells or from originating from capillary endothelial cells also were endothelial cells through endothelial-to-mesenchymal shown in a bleomycin-induced lung-fibrosis model [46]. transitions. Fibrocytes have been implicated in the patho- Our results show increased number of cells co-expressing genesis of various experimental fibrotic conditions, the endothelial cell marker, VE-cadherin, and a mesenchy- including bleomycin-induced pulmonary fibrosis [39], mal cell marker, FSP1, in Ang II-infused mouse skin, suggesting increased EndoMT in response to Ang II. renal fibrosis [40], liver fibrosis [41], and heart fibrosis [42]. In our study, we showed an increased number of However, we have observed the presence of such cells infiltrating fibrocytes (CD45/FSP1 or CD45/P4H double- only in the lower dermis, suggesting that they may have a positive cells) in the skin of Ang II-infused mice, which limited role in this model because the majority of myofi- may contribute to the development of fibrosis in broblasts were present in the upper dermis. However, response to Ang II. In scleroderma, higher numbers of determination of the precise contribution of the process of cells described as “collagen-producing monocytes” was EndoMT to this model of dermal fibrosis will require observed in the peripheral blood of patients with intersti- reporter mice that would allow the lineage tracing of tial lung disease [43,44]. endothelial cells. EndoMT is induced in response to Stawski et al. Arthritis Research & Therapy 2012, 14:R194 Page 11 of 12 http://arthritis-research.com/content/14/4/R194 year prospective study of 586 patients, with validation of proposed abnormal fibrillin-1 expression and chronic oxidative criteria for early systemic sclerosis. Arthritis Rheum 2008, 58:3902-3912. +/- stress in the Tsk mouse, another model of SSc [47]. 3. Meyer M, Muller AK, Yang J, Sulcova J, Werner S: The role of chronic inflammation in cutaneous fibrosis: fibroblast growth factor receptor deficiency in keratinocytes as an example. J Invest Dermatol Symp Proc Conclusions 2011, 15:48-52. This work demonstrates that Ang II infusion induces 4. Zaman MA, Oparil S, Calhoun DA: Drugs targeting the renin-angiotensin- both inflammation and fibrosis in the skin via MCP1 aldosterone system. Nat Rev Drug Discov 2002, 1:621-636. 5. Mezzano SA, Ruiz-Ortega M, Egido J: Angiotensin II and renal fibrosis. upregulation and accumulation of activated fibroblasts. Hypertension 2001, 38:635-638. Additionally, our data suggest that populations of these 6. Brecher P: Angiotensin II and cardiac fibrosis. Trends Cardiovasc Med 1996, fibroblasts originate from circulating blood cells. Ele- 6:193-198. 7. Kawaguchi Y, Takagi K, Hara M, Fukasawa C, Sugiura T, Nishimagi E, vated serum levels of Ang II found in a subset of Harigai M, Kamatani N: Angiotensin II in the lesional skin of systemic patients with dcSS in the early stage of the disease sug- sclerosis patients contributes to tissue fibrosis via angiotensin II type 1 gest that Ang II may contribute to the pathogenesis of receptors. Arthritis Rheum 2004, 50:216-226. 8. Min LJ, Cui TX, Yahata Y, Yamasaki K, Shiuchi T, Liu HW, Chen R, Li JM, dcSSc, at least in a subset of patients [7]. The patho- Okumura M, Jinno T, Wu L, Iwai M, Nahmias C, Hashimoto K, Horiuchi M: genic features observed in the Ang II model of dermal Regulation of collagen synthesis in mouse skin fibroblasts by distinct fibrosis, such as infiltration of fibrocytes and a colocali- angiotensin II receptor subtypes. Endocrinology 2004, 145:253-260. 9. Pignone A, Rosso AD, Brosnihan KB, Perfetto F, Livi R, Fiori G, Guiducci S, zation of myofibroblasts with the M2 macrophages, are Cinelli M, Rogai V, Tempestini A, Bartoli F, Generini S, Ferrario CM, particularly interesting, because they may help to eluci- Cerinic MM: Reduced circulating levels of angiotensin-(1-7) in systemic date similar processes occurring during the pathogenesis sclerosis: a new pathway in the dysregulation of endothelial-dependent vascular tone control. Ann Rheum Dis 2007, 66:1305-1310. of SSc. We believe that the Ang II model of dermal 10. Batteux F, Kavian N, Servettaz A: New insights on chemically induced fibrosis will be very useful for future mechanistic studies animal models of systemic sclerosis. Curr Opin Rheumatol 2011, of SSc pathogenesis and for the evaluation of novel anti- 23:511-518. 11. Wu M, Varga J: In perspective: murine models of scleroderma. Curr fibrotic treatments. Rheumatol Rep 2008, 10:173-182. 12. Artlett CM: Animal models of scleroderma: fresh insights. Curr Opin Rheumatol 2010, 22:677-682. Abbreviations 13. Derrett-Smith EC, Denton CP, Sonnylal S: Animal models of scleroderma: αSMA: alpha-smooth muscle actin; Ab: antibody; Ang II: angiotensin II; APES: lessons from transgenic and knockout mice. Curr Opin Rheumatol 2009, aminopropyltriethoxysilane; BSA: bovine serum albumin; CTGF: connective 21:630-635. tissue growth factor; dcSS: diffuse cutaneous systemic sclerosis; EC: 14. Smith GP, Chan ES: Molecular pathogenesis of skin fibrosis: insight from endothelial cell; ECM: extracellular matrix; EndoMT: endothelial-to animal models. Curr Rheumatol Rep 2010, 12:26-33. mesenchymal transition; FBS: fetal bovine serum; FSP1: fibroblast-specific 15. Beyer C, Schett G, Distler O, Distler JH: Animal models of systemic protein 1; HDMEC: human dermal microvascular endothelial cell; HPF: high- sclerosis: prospects and limitations. Arthritis Rheum 2010, 62:2831-2844. power field; MCP1: monocyte chemotactic protein-1; PBS: phosphate- 16. Bataller R, Sancho-Bru P, Gines P, Brenner DA: Liver fibrogenesis: a new buffered saline; PCR: polymerase chain reaction; RAS: renin-angiotensin role for the renin-angiotensin system. Antioxid Redox Signal 2005, system; RP: Raynaud phenomenon; SSc: systemic sclerosis; TGF-β: 7:1346-1355. transforming growth factor β; WT: wild type. 17. Samuel CS: Determination of collagen content, concentration, and sub- types in kidney tissue. Methods Mol Biol 2009, 466:223-235. Acknowledgements 18. Richard L, Velasco P, Detmar M: Isolation and culture of microvascular This study was supported by the National Institutes of Health (NIAMS) grant endothelial cells. Methods Mol Med 1999, 18:261-269. RO1 AR42334-18 to M. Trojanowska and by the Scleroderma Foundation 19. Ihn H: Scleroderma, fibroblasts, signaling, and excessive extracellular Young Investigator Grant to Andreea Bujor. matrix. Curr Rheumatol Rep 2005, 7:156-162. We thank Dr. Giuseppina Stifano for help with CD163B immunostaining and 20. Ho MK, Springer TA: Tissue distribution, structural characterization, and Paul Haines for help with editing the manuscript. biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity. J Biol Chem 1983, 258:636-642. Authors’ contributions 21. Sulahian TH, Högger P, Wahner AE, Wardwell K, Goulding NJ, Sorg C, LS performed all experiments and wrote the manuscript. 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Xu H, Zaidi M, Struve J, Jones DW, Krolikowski JG, Nandedkar S, Lohr NL, • Convenient online submission Gadicherla A, Pagel PS, Csuka ME, Pritchard KA, Weihrauch D: Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial • Thorough peer review mesenchymal transition in a murine model of systemic sclerosis. Am J • No space constraints or color figure charges Physiol Cell Physiol 2011, 300:C550-C556. • Immediate publication on acceptance doi:10.1186/ar4028 • Inclusion in PubMed, CAS, Scopus and Google Scholar Cite this article as: Stawski et al.: Angiotensin II induces skin fibrosis: a • Research which is freely available for redistribution novel mouse model of dermal fibrosis. Arthritis Research & Therapy 2012 14:R194. Submit your manuscript at www.biomedcentral.com/submit

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