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Connective tissue growth factor is induced in bleomycin-induced skin scleroderma

Connective tissue growth factor is induced in bleomycin-induced skin scleroderma J. Cell Commun. Signal. (2010) 4:25–30 DOI 10.1007/s12079-009-0081-3 RESEARCH ARTICLE Connective tissue growth factor is induced in bleomycin-induced skin scleroderma Shangxi Liu & Reza Taghavi & Andrew Leask Received: 28 July 2009 /Accepted: 22 October 2009 /Published online: 15 November 2009 The Author(s) 2009. This article is published with open access at Springerlink.com Abstract The origin of fibrotic cells within connective Introduction tissue is unclear. For example, the extent to which microvascular pericytes contribute to the number of myofi- Tissue repair involves the reconstitution of connective broblasts present in dermal fibrosis in uncertain. Connective tissue by a specialized from of fibroblast, termed the tissue growth factor (CTGF/CCN2) is a marker and mediator myofibroblast (Tomasek et al. 2002). This cell type is of fibrosis. In this report, we use an antibody recognizing characterized by the expression of the pro-contractile CCN2 to assess the cell types in mouse dermis which protein α-smooth muscle actin (α-SMA). Fibrosis can be express CCN2 in the bleomycin model of skin scleroderma. considered to arise due to a persistence of the tissue repair Control (PBS injected) and fibrotic (bleomycin-injected) program. Indeed, fibrotic lesions are populated large dermis was examined for CCN2, α-smooth muscle actin numbers of myofibroblasts (Desmouliere et al. 2005). (α-SMA) (to detect myofibroblasts), and NG2 (to detect Moreover, fibroblasts isolated from lesions of scleroderma pericytes) expression. Consistent with previously published patients show a persistently activated myofibroblast data, CCN2 expression was largely absent in the dermis of phenotype (Chen et al. 2005). The exact origin of control mice. However, upon exposure to bleomycin, CCN2 myofibroblasts in tissue repair and fibrosis is unclear, was observed in the dermis. Cells that expressed CCN2 were but a significant percentage of may derive from α−SMA-expressing myofibroblasts. Approximately 85% pericytes surrounding blood vessels or from local of myofibroblasts were NG2-positive, CCN2-expressing recruitment of fibroblasts (Hinz et al. 2007;Rajkumar pericytes, indicating that pericytes significantly contributed et al. 2006). For example, recently we found that, in to the presence of myofibroblasts in sclerotic dermis. Thus cutaneous wounds in mice, approximately 30% of the CCN2 is induced in fibrotic skin, correlating with the myofibroblasts were also pericytes (Kapoor et al. 2008). induction of myofibroblast induction. Moreover, CCN2- However, the extent to which pericytes contribute to skin expressing pericytes significantly contribute to the appearance fibrosis is unclear. of myofibroblasts in bleomycin-induced skin scleroderma. CCN2 (Connective tissue growth factor/CCN2) is a member of the CCN family of proteins (Leask and . . . Keywords CTGF CCN2 Connective tissue growth factor Abraham 2006). CCN2 is an adhesive protein which acts . . Scleroderma Fibrosis Pericyte through integrins and heparan sulfate-containing proteo- glycans (HSPGs) (Lau and Lam 1999; Gao and Brigstock 2004). CCN2 is expressed in mesenchymal cells during : : development and wound healing, and is characteristically S. Liu R. Taghavi A. Leask (*) CIHR Group in Skeletal Development and Remodeling, over-expressed in fibrotic diseases (Blom et al. 2002; Division of Oral Biology and Department of Physiology Leask and Abraham 2006). The majority of studies on and Pharmacology, Schulich School of Medicine and Dentistry, CCN2 gene regulation have been conducted in cell Dental Sciences Building, University of Western Ontario, culture; for example, in fibroblasts, CCN2 is induced by London, ON N6A 5C1, Canada transforming growth factor β through Smads, ets-1 and e-mail: [email protected] 26 S. Liu et al. ras/MEK/ERK (Holmes et al. 2001; Leask et al. 2003;van Varga 2008). CCN2 mRNA is induced in bleomycin- Beek et al. 2006). CCN2 overexpression in dermal induced lung fibrosis (Lasky et al. 1998; Ponticos et al. fibroblasts isolated from scleroderma patients, in contrast, 2009), but whether CCN2 protein is induced in response is independent of TGFβ signaling and dependent on to bleomycin-induced dermal fibrosis is unclear. More- endothelin-1 and Sp1 (Holmes et al. 2003; Shi-Wen et over, the cell types within the dermis that express al. 2007). In vivo, CCN2 is not normally expressed in CCN2 in response to bleomycin is unknown. adult mouse dermis, but is induced in myofibroblasts In this study, we subject mice to the bleomycin-induced post-wounding (Kapoor et al. 2008). However, the model of skin scleroderma. We investigate the expression of whether the appearance of CCN2 correlates with myofi- CCN2 using an anti-CCN2 antibody. We also detect the broblast induction in skin fibrosis is unknown. presence of myofibroblasts and pericytes using appropriate Although mouse model perfectly recapitulates the markers. Hence, we provide a first careful analysis of the cell characteristics of scleroderma, the bleomycin model skin types expressing CCN2 in skin and generate new insights into fibrosis is often used as a model of scleroderma (Wu and the origin of myofibroblasts during skin fibrosis. CCN2 α-SMA Merge PBS BLM PBS BLM * ** *p<0.001 α− SMA CCN2 Merge Fig. 1 CCN2 promoter is expressed in myofibroblasts in response to magnification of dermal tissue). The percentage of fibroblasts within bleomycin. Skin of mice treated with PBS or bleomycin was fixed, the wound that were α−SMA positive, CCN2- positive, and CCN2/ sectioned, and stained with DAPI to detect nuclei, anti-α-SMA antibody α-SMA positive was calculated as described in Methods. Representative to detect myofibroblasts and anti-CCN2 promoter antibody (10× data from n=4 wounds from 4 separate animals are shown Percentage of positive staining CCN2 expression in bleomycin-induced skin fibrosis 27 Results sion was strongly induced in the dermis (Fig. 1, CCN2, bleo). Similar patterns of expression were observed when tissue CCN2 is induced in myofibroblasts in response sections were stained with anti-α-SMA antibody to detect to bleomycin the presence of myofibroblasts (Fig. 1, α-SMA). Cells expressing CCN2 were also α−SMA positive (Fig. 1, The cell types expressing CCN2 in fibrosis are unclear. To merge). Collectively, these data indicate that CCN2 is address this issue, C57/BL6 mice were subjected to subcuta- expressed in myofibroblasts in response to bleomycin. neous injections of PBS or bleomycin over 28 days. As we were interested in the expression of CCN2 in connective tissue CCN2 is expressed in pericytes post-bleomycin treatment and in the origin of myofibroblasts within connective tissue, we focused our studies specifically on the dermis. When To assess the precise contribution of pericytes to the control PBS-injected skin was examined, a few CCN2- activated fibroblasts present in skin in response to bleomycin positive cells were detected in the dermis (Fig. 1, CCN2, and to investigate whether CCN2 promoter was expressed PBS). Conversely, in response to bleomycin, CCN2 expres- pericytes, we performed double immunolabeling of tissue CCN2 NG2 Merge PBS BLM PB S BLM *p<0.001 CCN2 NG2 Merge Fig. 2 CCN2 promoter is expressed in pericytes in response to of dermal tissue). The percentage of fibroblasts within the wound that bleomycin. Skin of mice treated with PBS or bleomycin was fixed, were CCN2 positive, NG2 positive, and both NG2/CCN2-positive sectioned, and stained with DAPI to detect nuclei, anti-NG2 antibody was calculated as described in Methods. Representative data from n=4 to detect myofibroblasts and anti-CCN2 antibody (10× magnification wounds from 4 separate animals per time point is shown Percentage of positive staining 28 S. Liu et al. sections with an anti-CCN2 antibody and an anti-NG2 The majority of the myofibroblasts in bleomycin-induced antibody to detect pericytes. NG2 is routinely used a skin scleroderma are pericytes pericyte-specific marker in the literature (Ozerdem and Stallcup 2004; Salvucci et al., 2009 and references To assess whether pericytes contributed to the number of therein). We found that 60% of the cells in the tissue myofibroblasts in bleomycin-induced fibrotic tissue, tissue were NG2-expressing pericytes (Fig. 2). Double-labeling sections were stained with both anti-NG2 and anti-α-SMA experiments revealed that CCN2 was expressed in antibodies. In response to bleomycin, ~75% of the cells in pericytes (Fig. 2). the tissue were both NG2-positive and α−SMA positive α-SMA NG2 Merge PBS BLM PBS BLM *p<0.001 α− SMA NG2 Merge Fig. 3 A subset of myofibroblasts are perictyes in response to the wound that were α−SMA positive, NG2 positive, and both NG2/ bleomycin. Skin of mice treated with PBS or bleomycin was fixed, α-SMA positive was calculated as described in Methods. Note that sectioned, and stained with DAPI to detect nuclei, anti-α-SMA essentially all cells in the wound area are myofibroblasts. Represen- antibody to detect myofibroblasts and anti-CCN2 antibody (10× tative data from n=4 wounds from 4 separate animals per time point is magnification of dermal tissue). The percentage of fibroblasts within shown Percentage of positive staining CCN2 expression in bleomycin-induced skin fibrosis 29 (Fig. 3). Collectively, these results not only support the used for this experiment were generated in a previously notion that CCN2 is an excellent marker of myofibro- published study (Liu et al. 2009). blast activation in response to fibrotic stimuli, but also suggest that pericytes can significantly contribute to the Immunofluorescence presence of myofibroblasts within fibrotic lesions. Tissue sections (0.5 µm) were cut using a microtome (Leica), collected on Superfrost Plus slides (Fisher Scientific), Discussion dewaxed in xylene and rehydrated by successive immersion in descending concentrations of alcohol. Tissue sections Elevated, constitutive CCN2 expression is a hallmark of were then incubated with mouse serum for 30 min, washed fibrotic disease and can be considered a surrogate marker of with PBS and incubated with primary antibodies for 1 hour fibrosis (Blom et al. 2002; Leask and Abraham 2006). In at room temperature. Primary antibodies used alone (single response to tissue injury, CCN2 is induced in myofibroblasts immunofluorescence) or in combination (double immuno- (Kapoor et al. 2008). In this report, we extend these studies fluorescence) were: rabbit anti-CCN2 (anti-CCN2; 1:100 and show that, in response to the bleomycin-induced model dilution, Abcam), mouse anti-NG2 (pericyte marker, 1:100 of skin fibrosis, CCN2 is expressed in myofibroblasts. dilution, Chemicon), mouse anti-alpha-smooth muscle Moreover, pericytes, which are believed to contribute to the actin (α-SMA, 1:100 dilution, Sigma). Double immuno- total number of myofibroblasts in fibrotic tissue (Hinz et al. fluorescence for α-SMA and NG2 was performed using 2007; Rajkumar et al. 2006), also express CCN2 and rabbit polyclonal antibody for α-SMA (Abcam) and mouse comprise the majority of fibroblasts activated in response antibody for NG2. Sections were washed with PBS, incubated to bleomycin. Our data are consistent with previously with appropriate fluorescent secondary antibodies (Jackson published data showing that CCN2 is expressed in pericytes Immunoresearch) for one hour at room temperature, washed in culture as well as in retinas of diabetic patients (Shiwen et with PBS, mounted using DAPI and photographed using al. 2009; Kuiper et al. 2004). Our data are also consistent Zeiss fluorescence microscope and Northern Eclipse software with a previous study from our group showing that (Empix). Six independent fields were examined per data CCN2, type I collagen protein and mRNAs encoding point. other fibrotic proteins such as fibronectin are upregulated Sections undergoing double immunofluorescence were in cultured pericytes (Shiwen et al. 2009). Our results photographed at 10× magnification. To detect number of also support the notion that CCN2 is a faithful marker of α-SMA, CCN2 or NG2 positive cells, total number of cells/ fibrogenesis in vivo (Blom et al. 2002; Leask and mm were counted. The percentage α-SMA, CCN2 and Abraham 2006). It is interesting to note that recently it NG2 positive cells/mm were then calculated. was shown that an anti-CCN2 antibody alleviated Results are expressed as the mean ± SEM. P<0.05 was bleomycin-induced lung fibrosis (Ponticos et al. 2009) considered statistically significant (*; Student’s t test). and siRNA against CCN2 reversed CCl -induced liver fibrosis (Brigstock 2009). Thus CCN2 may be both a Acknowledgements Our work is funded by the Canadian Institute of Heath Research and the Canadian Foundation for Innovation. A.L marker and a mediator of fibrosis (Grotendorst 1997; is an Arthritis Society (Scleroderma Society of Ontario) New Leask and Abraham 2004, 2006). Finally, our data suggest Investigator and a recipient of Early Researcher Award. RT was that pericytes are a significant source of myofibroblasts in supported by a Summer Studentship of the Canadian Scleroderma skin fibrosis. Collectively, our results may be useful in the Research Group. future for uncovering new strategies aimed at modulating Open Access This article is distributed under the terms of the (myo)fibroblast biology during fibrosis. Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Materials and methods Bleomycin-induced skin fibrosis References Bleomycin (Sigma) was diluted to 0.1 U/ml with PBS, and Blom IE, Goldschmeding R, Leask A (2002) Gene regulation of connective tissue growth factor: new targets for antifibrotic filter sterilized. 100 µl of bleomycin or PBS was injected therapy? Matrix Biol 21:473–482 subcutaneously into a single location on the shaved back of Brigstock DR (2009) Strategies for blocking the fibrogenic actions of C57/BL6 mice once daily for 4 weeks (Yamamoto et al. connective tissue growth factor (CCN2): from pharmacological 1999) Mice were the killed using CO , and skin was 2 inhibition in vitro to targeted siRNA therapy in vivo. J Cell Commun Signal 3:5–18 collected for immunohistochemical analysis. Tissue samples 30 S. Liu et al. Chen Y, Shiwen X, van Beek J, Kennedy L, McLeod M, Renzoni EA, Leask A, Holmes A, Black CM, Abraham DJ (2003) Connective Bou-Gharios G, Wilcox-Adelman S, Goetinck PF, Eastwood M, tissue growth factor gene regulation. Requirements for its Black CM, Abraham DJ, Leask A (2005) Matrix contraction by induction by transforming growth factor-beta 2 in fibroblasts. J dermal fibroblasts requires TGFbeta/ALK5, heparan sulfate Biol Chem 278:13008–13015 containing proteoglycans and MEK/ERK: Insights into patho- Liu S, Kapoor M, Denton CP, Abraham DJ, Leask A (2009) Loss of logical scarring in chronic fibrotic disease. Am J Pathol β1 integrin in mouse fibroblasts results in resistance to a mouse 167:1699–1711 model of skin scleroderma. Arthritis Rheum 60:2817–2821 Desmouliere A, Chaponnier C, Gabbiani G (2005) Tissue repair, Ozerdem U, Stallcup WB (2004) Pathological angiogenesis is reduced contraction, and the myofibroblast. Wound Repair Regen 13:7– by targeting pericytes via the NG2 proteoglycan. Angiogenesis 12 7:269–276 Gao R, Brigstock DR (2004) Connective tissue growth factor (CCN2) Ponticos M, Holmes AM, Shi-Wen X, Leoni P, Khan K, Rajkumar induces adhesion of rat activated hepatic stellate cells by binding VS, Hoyles RK, Bou-Gharios G, Black CM, Denton CP, of its C-terminal domain to integrin alpha(v)beta(3) and heparan Abraham DJ, Leask A, Lindahl GE (2009) Pivotal role of sulfate proteoglycan. J Biol Chem 279:8848–8855 connective tissue growth factor in lung fibrosis: MAPK- Grotendorst GR (1997) Connective tissue growth factor: a mediator of dependent transcriptional activation of type I collagen. Arthritis TGF-beta action on fibroblasts. Cytokine Growth Factor Rev Rheum 60:2142–2155 8:171–179 Rajkumar VS, Shiwen X, Bostrom M, Leoni P, Muddle J, Ivarsson M, Hinz B, Phan SH, Thannickal VJ, Galli A, Bochaton-Piallat ML, Gerdin B, Denton CP, Bou-Gharios G, Black CM, Abraham DJ Gabbiani G (2007) The myofibroblast: one function, multiple (2006) Platelet-derived growth factor-beta receptor activation is origins. Am J Pathol 170:1807–1816 essential for fibroblast and pericyte recruitment during cutaneous Holmes A, Abraham DJ, Sa S, Shiwen X, Black CM, Leask A (2001) wound healing. Am J Pathol 169:2254–2265 CCN2 and SMADs, maintenance of scleroderma phenotype is Salvucci O, Maric D, Economopoulou M, Sakakibara S, Merlin S, independent of SMAD signaling. J Biol Chem 276:10594– Follenzi A, Tosato G (2009) EphrinB reverse signaling contributes 10601 to endothelial and mural cell assembly into vascular structures. Holmes A, Abraham DJ, Chen Y, Shi-wen X, Denton C, Black CM, Blood 114:1707–1716 Leask A (2003) Sp1 is required for elevated CTGF expression in Shiwen X, Rajkumar V, Denton CP, Leask A, Abraham DJ (2009) scleroderma fibroblasts. J Biol Chem 278:41728–41733 Pericytes display increased CCN2 expression upon culturing. J Kapoor M, Liu S, Huh K, Parapuram S, Kennedy L, Leask A (2008) Cell Commun Signal 3:61–64 Connective tissue growth factor promoter activity in normal and Shi-Wen X, Renzoni EA, Kennedy L, Howat S, Chen Y, Pearson JD, wounded skin. Fibrogenesis Tissue Repair 1(1):3 Bou-Gharios G, Dashwood MR, du Bois RM, Black CM, Denton Kuiper EJ, Witmer AN, Klaassen I, Oliver N, Goldschmeding R, CP, Abraham DJ, Leask A (2007) Endogenous endothelin-1 Schlingemann RO (2004) Differential expression of connective signaling contributes to type I collagen and CCN2 overexpres- tissue growth factor in microglia and pericytes in the human sion in fibrotic fibroblasts. Matrix Biol 26:625–632 diabetic retina. Br J Ophthalmol 88:1082–1087 Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Lasky JA, Ortiz LA, Tonthat B, Hoyle GW, Corti M, Athas G, Myofibroblasts and mechano-regulation of connective tissue Lungarella G, Brody A, Friedman M (1998) Connective tissue remodelling. Nat Rev Mol Cell Biol 3(5):349–363 growth factor mRNA expression is upregulated in bleomycin- Van Beek JP, Kennedy L, Rockel JS, Bernier SM, Leask A (2006) The induced lung fibrosis. Am J Physiol 275:L365–L371 induction of CCN2 by TGFbeta1 involves Ets-1. Arthritis Res Lau LF, Lam SC (1999) The CCN family of angiogenic regulators: the Ther 8(2):R36 integrin connection. Exp Cell Res 248:44–57 Wu M, Varga J (2008) In perspective: murine models of scleroderma. Leask A, Abraham DJ (2004) TGF-beta signaling and the fibrotic Curr Rheumatol Rep 10:173–182 response. FASEB J 18:816–827 Yamamoto T, Takagawa S, Katayama I, Yamazaki K, Hamazaki Y, Leask A, Abraham DJ (2006) All in the CCN family: essential Shinkai H, Nishioka K (1999) Animal model of sclerotic skin. I: matricellular signaling modulators emerge from the bunker. J Local injections of bleomycin induce sclerotic skin mimicking Cell Sci 119:4803–4810 scleroderma. J Invest Dermatol 112:456–462 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Cell Communication and Signaling Pubmed Central

Connective tissue growth factor is induced in bleomycin-induced skin scleroderma

Liu, Shangxi; Taghavi, Reza; Leask, Andrew
Journal of Cell Communication and Signaling , Volume 4 (1) – Nov 15, 2009
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10.1007/s12079-009-0081-3
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Abstract

J. Cell Commun. Signal. (2010) 4:25–30 DOI 10.1007/s12079-009-0081-3 RESEARCH ARTICLE Connective tissue growth factor is induced in bleomycin-induced skin scleroderma Shangxi Liu & Reza Taghavi & Andrew Leask Received: 28 July 2009 /Accepted: 22 October 2009 /Published online: 15 November 2009 The Author(s) 2009. This article is published with open access at Springerlink.com Abstract The origin of fibrotic cells within connective Introduction tissue is unclear. For example, the extent to which microvascular pericytes contribute to the number of myofi- Tissue repair involves the reconstitution of connective broblasts present in dermal fibrosis in uncertain. Connective tissue by a specialized from of fibroblast, termed the tissue growth factor (CTGF/CCN2) is a marker and mediator myofibroblast (Tomasek et al. 2002). This cell type is of fibrosis. In this report, we use an antibody recognizing characterized by the expression of the pro-contractile CCN2 to assess the cell types in mouse dermis which protein α-smooth muscle actin (α-SMA). Fibrosis can be express CCN2 in the bleomycin model of skin scleroderma. considered to arise due to a persistence of the tissue repair Control (PBS injected) and fibrotic (bleomycin-injected) program. Indeed, fibrotic lesions are populated large dermis was examined for CCN2, α-smooth muscle actin numbers of myofibroblasts (Desmouliere et al. 2005). (α-SMA) (to detect myofibroblasts), and NG2 (to detect Moreover, fibroblasts isolated from lesions of scleroderma pericytes) expression. Consistent with previously published patients show a persistently activated myofibroblast data, CCN2 expression was largely absent in the dermis of phenotype (Chen et al. 2005). The exact origin of control mice. However, upon exposure to bleomycin, CCN2 myofibroblasts in tissue repair and fibrosis is unclear, was observed in the dermis. Cells that expressed CCN2 were but a significant percentage of may derive from α−SMA-expressing myofibroblasts. Approximately 85% pericytes surrounding blood vessels or from local of myofibroblasts were NG2-positive, CCN2-expressing recruitment of fibroblasts (Hinz et al. 2007;Rajkumar pericytes, indicating that pericytes significantly contributed et al. 2006). For example, recently we found that, in to the presence of myofibroblasts in sclerotic dermis. Thus cutaneous wounds in mice, approximately 30% of the CCN2 is induced in fibrotic skin, correlating with the myofibroblasts were also pericytes (Kapoor et al. 2008). induction of myofibroblast induction. Moreover, CCN2- However, the extent to which pericytes contribute to skin expressing pericytes significantly contribute to the appearance fibrosis is unclear. of myofibroblasts in bleomycin-induced skin scleroderma. CCN2 (Connective tissue growth factor/CCN2) is a member of the CCN family of proteins (Leask and . . . Keywords CTGF CCN2 Connective tissue growth factor Abraham 2006). CCN2 is an adhesive protein which acts . . Scleroderma Fibrosis Pericyte through integrins and heparan sulfate-containing proteo- glycans (HSPGs) (Lau and Lam 1999; Gao and Brigstock 2004). CCN2 is expressed in mesenchymal cells during : : development and wound healing, and is characteristically S. Liu R. Taghavi A. Leask (*) CIHR Group in Skeletal Development and Remodeling, over-expressed in fibrotic diseases (Blom et al. 2002; Division of Oral Biology and Department of Physiology Leask and Abraham 2006). The majority of studies on and Pharmacology, Schulich School of Medicine and Dentistry, CCN2 gene regulation have been conducted in cell Dental Sciences Building, University of Western Ontario, culture; for example, in fibroblasts, CCN2 is induced by London, ON N6A 5C1, Canada transforming growth factor β through Smads, ets-1 and e-mail: [email protected] 26 S. Liu et al. ras/MEK/ERK (Holmes et al. 2001; Leask et al. 2003;van Varga 2008). CCN2 mRNA is induced in bleomycin- Beek et al. 2006). CCN2 overexpression in dermal induced lung fibrosis (Lasky et al. 1998; Ponticos et al. fibroblasts isolated from scleroderma patients, in contrast, 2009), but whether CCN2 protein is induced in response is independent of TGFβ signaling and dependent on to bleomycin-induced dermal fibrosis is unclear. More- endothelin-1 and Sp1 (Holmes et al. 2003; Shi-Wen et over, the cell types within the dermis that express al. 2007). In vivo, CCN2 is not normally expressed in CCN2 in response to bleomycin is unknown. adult mouse dermis, but is induced in myofibroblasts In this study, we subject mice to the bleomycin-induced post-wounding (Kapoor et al. 2008). However, the model of skin scleroderma. We investigate the expression of whether the appearance of CCN2 correlates with myofi- CCN2 using an anti-CCN2 antibody. We also detect the broblast induction in skin fibrosis is unknown. presence of myofibroblasts and pericytes using appropriate Although mouse model perfectly recapitulates the markers. Hence, we provide a first careful analysis of the cell characteristics of scleroderma, the bleomycin model skin types expressing CCN2 in skin and generate new insights into fibrosis is often used as a model of scleroderma (Wu and the origin of myofibroblasts during skin fibrosis. CCN2 α-SMA Merge PBS BLM PBS BLM * ** *p<0.001 α− SMA CCN2 Merge Fig. 1 CCN2 promoter is expressed in myofibroblasts in response to magnification of dermal tissue). The percentage of fibroblasts within bleomycin. Skin of mice treated with PBS or bleomycin was fixed, the wound that were α−SMA positive, CCN2- positive, and CCN2/ sectioned, and stained with DAPI to detect nuclei, anti-α-SMA antibody α-SMA positive was calculated as described in Methods. Representative to detect myofibroblasts and anti-CCN2 promoter antibody (10× data from n=4 wounds from 4 separate animals are shown Percentage of positive staining CCN2 expression in bleomycin-induced skin fibrosis 27 Results sion was strongly induced in the dermis (Fig. 1, CCN2, bleo). Similar patterns of expression were observed when tissue CCN2 is induced in myofibroblasts in response sections were stained with anti-α-SMA antibody to detect to bleomycin the presence of myofibroblasts (Fig. 1, α-SMA). Cells expressing CCN2 were also α−SMA positive (Fig. 1, The cell types expressing CCN2 in fibrosis are unclear. To merge). Collectively, these data indicate that CCN2 is address this issue, C57/BL6 mice were subjected to subcuta- expressed in myofibroblasts in response to bleomycin. neous injections of PBS or bleomycin over 28 days. As we were interested in the expression of CCN2 in connective tissue CCN2 is expressed in pericytes post-bleomycin treatment and in the origin of myofibroblasts within connective tissue, we focused our studies specifically on the dermis. When To assess the precise contribution of pericytes to the control PBS-injected skin was examined, a few CCN2- activated fibroblasts present in skin in response to bleomycin positive cells were detected in the dermis (Fig. 1, CCN2, and to investigate whether CCN2 promoter was expressed PBS). Conversely, in response to bleomycin, CCN2 expres- pericytes, we performed double immunolabeling of tissue CCN2 NG2 Merge PBS BLM PB S BLM *p<0.001 CCN2 NG2 Merge Fig. 2 CCN2 promoter is expressed in pericytes in response to of dermal tissue). The percentage of fibroblasts within the wound that bleomycin. Skin of mice treated with PBS or bleomycin was fixed, were CCN2 positive, NG2 positive, and both NG2/CCN2-positive sectioned, and stained with DAPI to detect nuclei, anti-NG2 antibody was calculated as described in Methods. Representative data from n=4 to detect myofibroblasts and anti-CCN2 antibody (10× magnification wounds from 4 separate animals per time point is shown Percentage of positive staining 28 S. Liu et al. sections with an anti-CCN2 antibody and an anti-NG2 The majority of the myofibroblasts in bleomycin-induced antibody to detect pericytes. NG2 is routinely used a skin scleroderma are pericytes pericyte-specific marker in the literature (Ozerdem and Stallcup 2004; Salvucci et al., 2009 and references To assess whether pericytes contributed to the number of therein). We found that 60% of the cells in the tissue myofibroblasts in bleomycin-induced fibrotic tissue, tissue were NG2-expressing pericytes (Fig. 2). Double-labeling sections were stained with both anti-NG2 and anti-α-SMA experiments revealed that CCN2 was expressed in antibodies. In response to bleomycin, ~75% of the cells in pericytes (Fig. 2). the tissue were both NG2-positive and α−SMA positive α-SMA NG2 Merge PBS BLM PBS BLM *p<0.001 α− SMA NG2 Merge Fig. 3 A subset of myofibroblasts are perictyes in response to the wound that were α−SMA positive, NG2 positive, and both NG2/ bleomycin. Skin of mice treated with PBS or bleomycin was fixed, α-SMA positive was calculated as described in Methods. Note that sectioned, and stained with DAPI to detect nuclei, anti-α-SMA essentially all cells in the wound area are myofibroblasts. Represen- antibody to detect myofibroblasts and anti-CCN2 antibody (10× tative data from n=4 wounds from 4 separate animals per time point is magnification of dermal tissue). The percentage of fibroblasts within shown Percentage of positive staining CCN2 expression in bleomycin-induced skin fibrosis 29 (Fig. 3). Collectively, these results not only support the used for this experiment were generated in a previously notion that CCN2 is an excellent marker of myofibro- published study (Liu et al. 2009). blast activation in response to fibrotic stimuli, but also suggest that pericytes can significantly contribute to the Immunofluorescence presence of myofibroblasts within fibrotic lesions. Tissue sections (0.5 µm) were cut using a microtome (Leica), collected on Superfrost Plus slides (Fisher Scientific), Discussion dewaxed in xylene and rehydrated by successive immersion in descending concentrations of alcohol. Tissue sections Elevated, constitutive CCN2 expression is a hallmark of were then incubated with mouse serum for 30 min, washed fibrotic disease and can be considered a surrogate marker of with PBS and incubated with primary antibodies for 1 hour fibrosis (Blom et al. 2002; Leask and Abraham 2006). In at room temperature. Primary antibodies used alone (single response to tissue injury, CCN2 is induced in myofibroblasts immunofluorescence) or in combination (double immuno- (Kapoor et al. 2008). In this report, we extend these studies fluorescence) were: rabbit anti-CCN2 (anti-CCN2; 1:100 and show that, in response to the bleomycin-induced model dilution, Abcam), mouse anti-NG2 (pericyte marker, 1:100 of skin fibrosis, CCN2 is expressed in myofibroblasts. dilution, Chemicon), mouse anti-alpha-smooth muscle Moreover, pericytes, which are believed to contribute to the actin (α-SMA, 1:100 dilution, Sigma). Double immuno- total number of myofibroblasts in fibrotic tissue (Hinz et al. fluorescence for α-SMA and NG2 was performed using 2007; Rajkumar et al. 2006), also express CCN2 and rabbit polyclonal antibody for α-SMA (Abcam) and mouse comprise the majority of fibroblasts activated in response antibody for NG2. Sections were washed with PBS, incubated to bleomycin. Our data are consistent with previously with appropriate fluorescent secondary antibodies (Jackson published data showing that CCN2 is expressed in pericytes Immunoresearch) for one hour at room temperature, washed in culture as well as in retinas of diabetic patients (Shiwen et with PBS, mounted using DAPI and photographed using al. 2009; Kuiper et al. 2004). Our data are also consistent Zeiss fluorescence microscope and Northern Eclipse software with a previous study from our group showing that (Empix). Six independent fields were examined per data CCN2, type I collagen protein and mRNAs encoding point. other fibrotic proteins such as fibronectin are upregulated Sections undergoing double immunofluorescence were in cultured pericytes (Shiwen et al. 2009). Our results photographed at 10× magnification. To detect number of also support the notion that CCN2 is a faithful marker of α-SMA, CCN2 or NG2 positive cells, total number of cells/ fibrogenesis in vivo (Blom et al. 2002; Leask and mm were counted. The percentage α-SMA, CCN2 and Abraham 2006). It is interesting to note that recently it NG2 positive cells/mm were then calculated. was shown that an anti-CCN2 antibody alleviated Results are expressed as the mean ± SEM. P<0.05 was bleomycin-induced lung fibrosis (Ponticos et al. 2009) considered statistically significant (*; Student’s t test). and siRNA against CCN2 reversed CCl -induced liver fibrosis (Brigstock 2009). Thus CCN2 may be both a Acknowledgements Our work is funded by the Canadian Institute of Heath Research and the Canadian Foundation for Innovation. A.L marker and a mediator of fibrosis (Grotendorst 1997; is an Arthritis Society (Scleroderma Society of Ontario) New Leask and Abraham 2004, 2006). Finally, our data suggest Investigator and a recipient of Early Researcher Award. RT was that pericytes are a significant source of myofibroblasts in supported by a Summer Studentship of the Canadian Scleroderma skin fibrosis. Collectively, our results may be useful in the Research Group. future for uncovering new strategies aimed at modulating Open Access This article is distributed under the terms of the (myo)fibroblast biology during fibrosis. Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Materials and methods Bleomycin-induced skin fibrosis References Bleomycin (Sigma) was diluted to 0.1 U/ml with PBS, and Blom IE, Goldschmeding R, Leask A (2002) Gene regulation of connective tissue growth factor: new targets for antifibrotic filter sterilized. 100 µl of bleomycin or PBS was injected therapy? 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Journal

Journal of Cell Communication and Signaling – Pubmed Central

Published: Nov 15, 2009

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