The Loss of MCP-1 Attenuates Cutaneous Ischemia Reperfusion Injury in a Mouse Model of Pressure Ulcer

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1 ORIGINAL ARTICLE The Loss of MCP- Attenuates Cutaneous Ishemia Reperfusion Injury in a Mouse Moel of Pressure Uler Yuki Saito, Minoru Hasegawa, Manabu Fujimoto, Takashi Matsushita, Mayuka Horikawa, Motoi Takenaka, Fumihie Ogawa, Junko Sugama, Douglas A. Steeber, Shinihi Sato an Kazuhiko Takehara The formation of pressure ulers is epenent on multiple fators inluing ishemia reperfusion (IR). This stuy assesse the mehanism of a previously reporte murine moel of utaneous IR injury. Three yles of IR (ays ) by external appliation of two magneti plates were performe to inue pressure uler formation. Inrease infiltration of neutrophils an marophages, an augmente expression of proinflammatory ytokines an inuible nitri oxie synthase (inos), were observe uring IR yles. In this moel, monoyte hemoattratant protein- (MCP-) was remarkably inrease at ay in the skin followe by inflammatory ell infiltration. Therefore, IR yles were performe in MCP--efiient (MCP- / ) mie to evaluate the role of this hemokine in pressure uler evelopment. MCP- / mie showe reue marophage infiltration an expression of tumor-nerosis fator-a (TNF)-a an inos uring IR yles leaing to attenuate apoptosis an skin injury. Importantly, MCP- playe a role in apoptosis an injury via inuing inos uring the reperfusion rather than the ishemi perio. These finings iniate that MCP- may be a ritial fator for marophage reruitment an subsequent skin inflammation an injury uring IR yles. We propose that this is a useful moel for investigating the mehanism of pressure uler formation using various transgeni mie. Journal of Investigative Dermatology (), 5; oi:./sj.ji.575; publishe online January INTRODUCTION Chroni pressure ulers have beome a muh larger linial problem ue to the inreasing population of senior itizens aroun the worl. Although the etiology of pressure ulers is multifatorial an remains unlear, tissue ishemia has long been onsiere the main fator (Kosiak, 959; Daniel et al., 9). However, inreasing eviene emonstrates a prinipal role of ishemia reperfusion (IR) in the formation of Department of Dermatology, Kanazawa University Grauate Shool of Meial Siene, Kanazawa, Ishikawa, Japan; Department of Dermatology, Nagasaki University Grauate Shool of Biomeial Sienes, Nagasaki, Japan; Department of Clinial Nursing, Division of Health Sienes, Kanazawa University Grauate Shool of Meial Siene, Kanazawa, Ishikawa, Japan an Department of Biologial Sienes, University of Wisonsin-Milwaukee, Milwaukee, Wisonsin, USA Corresponene: Dr Minoru Hasegawa, Department of Dermatology, Kanazawa University Grauate Shool of Meial Siene, -, Takara-mahi, Kanazawa 9-, Japan. minoruha@erma.m.kanazawa-u.a.jp Abbreviations: W, N-(-aminomethyl) benzylaetamiine; CCR, CC hemokine reeptor ; C t, threshol yle; HIF, hypoxia-inue fator; HPF, high-power mirosopi fiel; HSP, heat-shok protein; inos, inuible nitri oxie synthase; IR, ishemia reperfusion; KC, keratinoyte-erive hemokine; MCP, monoyte hemoattratant protein; MCP- /, MCP-- efiient; MIP, marophage inflammatory protein; NO, nitri oxie; RT-PCR, reverse transription-pcr; TNF, tumor-nerosis fator; TUNEL, terminal eoxynuleotiyl transferase-meiate UTP nik-en labeling Reeive July 7; revise November 7; aepte November 7; publishe online January hroni skin wouns, inluing pressure ulers (Salio et al., 99; Peire et al., ) as well as other organs (Quinones-Balrih an Caswell, 99; Kubes, 995). Nonetheless, limitations of urrent animal moels of pressure ulers have mae it iffiult to larify the unerlying mehanism of their formation. Peire et al. () reate an exellent reprouible pressure uler moel by surgially implanting a stainless-steel plate in the orsal rat skin an using ylial magnet ompression. In their stuy, repeate IR yles were more amaging to skin tissues than prolonge ishemia alone, suggesting that the reperfusion phase of the IR yle was ruial for skin injury. However, this moel requires a surgial proeure to initiate the proess an leaves a foreign material uner the woun. Although a similar moel was also generate in the mouse (Rei et al., ), it ha the same problems. Tsuji et al. (5) establishe a novel utaneous IR injury moel using their original skin fol hamber. Uner their system, the miroirulatory response following IR injury an be visualize. However, this system is not simple an requires immobilization of mie uring the ompression treatments. Reently, Staler et al. () has reporte a very simple, reprouible, an noninvasive IR moel using the external appliation of two roun magneti plates without long-term immobilization of the mie. However, the preise linial or histopathologial ourse an the mehanism of pressure uler formation in this moel remain unknown. This may be one reason Journal of Investigative Dermatology (), Volume & The Soiety for Investigative Dermatology

2 Y Saito et al. MCP- in Ishemia Reperfusion Skin Injury why reports using this moel have not been publishe thus far. IR injury has been efine as ellular injury resulting from bloo reperfusion to previously ishemi sites (MCor, 95; Pretto, 99; Grae, 99; Woolfson et al., 99; Kubes, 995). IR inues more severe tissue injury ompare with ishemia alone (Parks an Granger, 9). It is thought that ishemi tissue reues its metabolism to preserve funtion. However, subsequent reperfusion of bloo to the nutrient- an oxygen-eplete tissue an initiate a series of harmful events ue to an inrease in oxygen-erive free raials, whih exee the apaity of the normal freeraial-savenging mehanisms. Free raial oxiative injury an ause inflammation an severe erangement of the ell mm mm μm μm mm mm μm μm Neutrophil Marophage Figure. Short-term hanges in skin tissue an inflammation uring IR yles. (a) Representative photographs of wouns uring IR yles (ays ) in wiltype mie. Marosopi uler was unlear uring this perio. (b) Representative histologial tissue setions (hematoxylin an eosin staining) showing eema, inflammatory ell infiltration, an neroti hange in the woune area (original magnifiation.5). Re arrowheas iniate the ege of the wouns treate with IR yles. The areas magnifie in () an () are iniate by the letter an in (b). () Higher magnifiation of epiermis an ermis at the woun ege (original magnifiation ). () Higher magnifiation of hypoermis at the entral part of the woun be (original magnifiation ). (e) IR yleinue reruitment of neutrophils an marophages in the skin. Numbers of neutrophils an marophages per setion were etermine by ounting myeloperoxiase- an F/-staine skin setions, respetively. Eight of HPFs (.7 mm) in five serial setions were ounte an average. All values represent the mean±sem of results obtaine from more than six mie for eah time point of examination. Representative histologial tissue setions showing the infiltration of neutrophils an marophages are also shown (original magnifiations ). 9

3 MCP- in Ishemia Reperfusion Skin Injury reruitment proess to the site of injury (Robson et al., ). Furthermore, altere enothelial ell funtion an limit ytokine proution an elay woun healing (Robson et al., ). Nitri oxie (NO) is one suh free raial an exessive NO or its synthase, inuible NO synthase (inos), is onsiere to play pathogeni roles in IR-inue apoptosis an tissue injury of some organs, inluing skin, myoarium, an kiney (Lipton et al., 99; Nathan an Xie, 99; Kubes, ; Furuihi et al., ; Rei et al., ; Shulz et al., ; Hayasaki et al., ). Leukoyti infiltration plays a ruial role in IR injury, suh as erebrovasular aient, myoarial ishemia, an ishemi renal injury. Chemokines are small heparin-bining proteins that iret the migration of irulating leukoytes to sites of injury or inflammation (Gerar an Rollins, ; Rot an von Anrian, ). The prototypial CC hemokine is monoyte hemoattratant protein- (MCP-/CCL), a key moleule for hemotaxis an ativation of marophages (Matsushima et al., 99; Leonar an Yoshimura, 99). MCP- is serete by various ell types suh as marophages, T lymphoytes, enothelial ells, epiermal ells, an fibroblasts. MCP- an its reeptor, CC hemokine reeptor (CCR), have been shown to ontribute to IR injury of some organs, inluing myoarium an the kiney (Furuihi et al., ; Hayasaki et al., ). Other members of the CC family inlue marophage inflammatory protein-a (MIP- a/ccl), MIP-b/CCL, an RANTES/CCL5. The main members of the CXC hemokines that an attrat an ativate neutrophils are keratinoyte-erive hemokine (KC/CXCL) an MIP-/CXCL in mie. The thir family, the CX C family, has only one known member, fratalkine/cx CL. In soluble form, fratalkine is a potent hemoattratant for CX CR- expressing marophages, T ells, an natural killer ells (Imai et al., 997). Importantly, in an inisional skin woun healing moel, MCP- an MIP-a are foun in high levels in the early phase (Fahey et al., 99; DiPietro et al., 995). Furthermore, MCP--efiient (MCP- / ), but not MIP-a / mie, isplay signifiantly elaye woun repair in the early phase of healing, although the ultimate enpoints are similar to that of wil-type mie (Low et al., ). However, the iret role of MCP- or other hemokines remains unknown in the pathogenesis of IR injury in the skin. In this stuy, we investigate the preise features an mehanism of IR injury, using the mouse pressure uler moel reporte by Staler et al. (). Our finings suggest that reruitment of marophages an subsequent release of proinflammatory ytokines an toxi oxygen-erive free raials inue the apoptosis of skin fibroblasts an skin injury uring IR yles. Furthermore, using this moel in MCP- / mie emonstrate that MCP- may play a ritial role in this proess. RESULTS Marosopi an histologial finings uring IR yles A total of wil-type mie were treate with magneti plates. Among these, magnets isloge in only one mouse (.%) uring the treatment. The mie were observe to be quite ative an i not appear to be hinere by the aitional weight. Marosopi skin ulers were not apparent uring IR yles (ays ), although faint epressions were observe after ay (Figure a). Histopathologially, marke eema was foun with sparse inflammatory ell infiltration in the hypoermis after the first IR yle (ay ; Figures b ). After IR yles, reue eema, ense inflammatory ell infiltration, an early signs of nerosis of the epiermis, ermis an a part of the hypoermis were observe. Reue eema an inflammatory ell infiltration were foun after IR yles (ay ). Thus, remarkable eema an inflammatory ell infiltration peake at ays an, respetively, leaing to nerosis of the skin uring IR yles. Infiltrating neutrophils an marophages uring IR yles We assesse the types of leukoytes that were infiltrating the skin uring IR yles. Speially, numbers of infiltrate neutrophils in the hypoermis were assesse in the woun tissues by immunohistohemial analysis using anti-myeloperoxiase mab (Figure e). At ay, neutrophil numbers were signifiantly inrease ompare with ontrol tissue (Po.). However, neutrophil numbers were further inrease at ay (Po. vs ay ). Although neutrophil numbers were slightly erease at ay, the number was still higher ompare with that at ay (Po.). Marophage infiltration was assesse by immunohistohemistry using the F/ mab (Figure e). Marophage numbers rapily inrease at ays an ompare with ontrol tissues (Po.). Although the number of marophages was reue at ay ompare with that at ays an (Po. an Po., respetively), it was still higher than ontrol tissues (Po.). In ontrast to neutrophils an marophages, CD þ T-ell infiltration was sparse uring IR injury (ata not shown). Thus, the kinetis of neutrophil an marophage infiltration showe a peak at ay uring IR yles. Figure. Long-term hanges in skin tissue an inflammation after IR yles. (a) Representative photographs of wouns after IR yles in wil-type mie. Marosopi skin ulers beame apparent at ay, followe by a progressive reution in size. (b) Representative histologial tissue setions showing the formation of uler an granulation tissue after IR yles (original magnifiation.5). The istane between re arrowheas iniates the epithelial gap that is the istane between the migrating eges of keratinoytes uner the eshar. In (b), the areas magnifie in () an () are iniate by the letter an, respetively. () Higher magnifiation of epiermis an ermis at the woun ege (original magnifiation ). () Higher magnifiation of hypoermis at the entral part of the woun be (original magnifiation ). (e) Time ourse of marosopi uler size in wil-type mie. The skin ulers were heale by ay in all mie. (f) Time ourse of mirosopi uler size was assesse as the epithelial gap. (g) Numbers of neutrophils an marophages per HPF were etermine by ounting myeloperoxiase- an F/-staine skin setions, respetively. Eight of HPFs in five serial setions were ounte an average for eah time point. All values represent the mean±sem of results obtaine from more than six mie for eah time point of examination. Representative histologial tissue setions showing inflammatory ell infiltration are also shown (original magnifiation ). Journal of Investigative Dermatology (), Volume

4 Y Saito et al. MCP- in Ishemia Reperfusion Skin Injury Formation an healing of skin uler after IR yles analyze sine the attahe eshar makes the woun size unlear. Speifially, migration of keratinoytes uner the eshar was assesse by mirosopially measuring the epithelial gap that is the istane between the migrating eges of keratinoytes. An epithelial gap beame apparent at ay an isappeare by ay in all mie (Figures b,, Marosopi skin ulers with thik eshar beame apparent ays following the IR yles (ay ; Figure a). From this point on, the size of the marosopi skin uler graually erease an was ompletely heale by ay in all mie (Figures a an e). Histologial woun losure was also mm mm mm mm μm μm μm μm Epithelial gap (mm) Neutrophil Marophage Size (mm )

5 MCP- in Ishemia Reperfusion Skin Injury an f). Histopathologially, the epiermis, ermis, panniulus arnosus, an a part of the hypoermis showe neroti hange with moest inflammatory ell infiltration at ay (Figures b ). The epth of the skin uler was greatest aroun ay or. At ay, granulation tissue formation evelope an this fining was more prominent at ay. Thus, peak size of the marosopi or mirosopi skin uler was aroun ays after the start of IR yles. Infiltrating neutrophils an marophages after IR yles While infiltrating neutrophils an marophages were most remarkable at ay (Figure e), the numbers of these leukoytes reue graually until ay (Figure g). By ontrast, fibroblasts, ientifie by their morphologial harateristis, an newly forme apillaries were inrease in assoiation with inrease granulation tissue formation uring this proess (Figure ). mrna expression of inos an proinflammatory ytokines uring IR yles Sine inos is important for tissue injury uring IR injury, mrna expression levels of inos were assesse by real-time reverse transription-pcr (RT-PCR) in the skin uring IR yles. Relative levels of inos mrna were progressively inrease by IR yles until ay (Figure a). Sine proinflammatory ytokines may also have roles in the evelopment of IR-inue skin injury, mrna expression of IL-b, IL-, IFN-g, an tumor-nerosis fator (TNF)-a was assesse in the woune skin (Figure b). The relative mrna expression of IL-b an IL- were highest at ay, an then graually erease uring subsequent IR yles. By ontrast, mrna expression of TNF-a was highest at ay uring IR yles. However, mrna expression of IFN-g was not etete in the skin uring any of the IR yles (ata not shown). Thus, augmente expression of some proinflammatory ytokines an inos were foun uring the proess of IR-inue skin injury. Proution of hemokines uring IR yles Leukoyte infiltration an subsequent proution of ytokines an inos are likely ritial for IR-inue skin injury. Therefore, protein levels of hemokines known to be ritial for leukoyte reruitment (MCP- an MIP-a for marophages an T ells; fratalkine for marophages an natural killer ells; KC for neutrophils) were measure in supernatants from homogenize woune skin by enzyme-linke immunosorbent assay (Figure ). Protein levels of MCP- were remarkably inrease, with peak levels being foun at ay uring IR yles. Aitionally, protein levels of KC were signifiantly elevate uring all IR yles. However, the levels of MIP-a an fratalkine were not signifiantly inrease uring IR yles. Thus, proution of MCP- an KC was signifiantly inrease in the ompresse skin uring IR yles. MCP- efiieny reue the IR yle-inue skin injury In general, marophages, rather than neutrophils, are more likely to be the major soure of inos or proinflammatory ytokines suh as IL-b, IL-, an TNF-a. In aition, MCP- is ritial for the reruitment an ativation of marophages, an MCP- proution was inrease early uring IR yles (Figure ). Therefore, we assesse the role of MCP- in IR-inue skin injury using MCP- / mie. Sine the quantitative evaluation of skin nerosis was iffiult uring IR yles, the egree of tissue amage was estimate by assessing woun healing. Marosopi skin ulers were etete in MCP- / mie at ay, an the size of the uler was omparable with that of wil-type mie (Figures a, 5a an e). However, woun losure was signifiantly Relative levels of inos mrna Relative levels of TNF-α mrna Relative levels of IL-β mrna Relative levels of IL- mrna MCP-/protein (ng ml ) MCP-α/protein (pg mg ) KC/protein (pg mg ) Fratalkine/protein (ng mg ) Figure. Expression or proution of inos, ytokines, an hemokines uring IR yles. mrna expression of (a) inos, (b) TNF-a, IL-b, an IL- in skin wouns of wil-type mie. Relative mrna expression was quantifie by real-time RT-PCR. () Protein levels of MCP-, MIP-a, KC, an fratalkine were measure in supernatants of woune skin homogenates by ELISA. The ata were expresse as ytokine or growth fator (pg ml or ng ml )/total protein (mg ml ) for eah sample. All values represent the mean±sem. Signifiant ifferenes between sample means are iniate, *Po.5; **Po.. These results were obtaine from six mie for eah time point of examination. Journal of Investigative Dermatology (), Volume

6 Y Saito et al. MCP- in Ishemia Reperfusion Skin Injury wouns of the same size was examine in MCP- / mie. However, MCP- / mie showe elaye inisional woun healing ompare with wil-type mie, as previously reporte (ata not shown an Low et al., ). Furthermore, we also assesse skin woun healing following a single ishemi treatment of hours in both wil-type an MCP / mie (Figures 5h an i). In wil-type mie, skin ulers ue to this treatment were repaire at least ays earlier than aelerate in the absene of MCP-, leaing to omplete healing by ay in all mie (Figures 5a an e). The aelerate woun losure was also onfirme by measuring the epithelial gap in these mie (Figures 5b,, an f). Speifially, the epithelial gap isappeare ays earlier in MCP- / mie than in wil-type mie (Figure 5f). To larify whether this was ue to reue tissue injury or improve woun healing in MCP- / mie, repair of inisional mm mm mm mm μm μm μm μm Neutrophil Marophage 5 Figure. Skin injury uring IR yles in MCP- / mie. (a) Representative photographs of wouns uring IR yles (ays ) in MCP- / mie. Marosopi uler was unlear uring this perio. (b) Representative histologial tissue setions (hematoxylin an eosin staining) showing eema, inflammatory ell infiltration, an neroti hange in the woune area (original magnifiation.5). Re arrowheas iniate the ege of the wouns treate with IR yles. The areas magnifie in () an () are iniate by the letter an, respetively, in (b). () Higher magnifiation of epiermis an ermis at the woun ege (original magnifiation ). () Higher magnifiation of hypoermis at the entral part of the woun be (original magnifiation ). (e) IR yle-inue reruitment of neutrophils an marophages in the skin of MCP- / mie. Numbers of neutrophils an marophages per HPF were etermine by ounting myeloperoxiase- an F/-staine skin setions, respetively. Eight HPFs in five serial setions were ounte an average. All values represent the mean±sem of results obtaine from more than six mie for eah time point of examination. Representative histologial tissue setions showing the infiltration of neutrophils an marophages are also shown for eah time point (original magnifiations ).

7 Y Saito et al. MCP- in Ishemia Reperfusion Skin Injury ishemi treatment of hours in wil-type an MCP- / mie (Figures 5h an i). Therefore, our finings iniate that IR tissue injury was reue espeially uring the reperfusion proess but not the ishemi proess by the loss of MCP-. IR yle-inue skin ulers espite the same total ishemi time (total repair perio, ays vs ays, respetively; Figures 5e, f, h, an i). However, the total perio of woun losure was similar in both treatments of MCP- / mie ( ays; Figures 5e, f, h, an i). These finings iniate that MCP- efiieny improves woun losure by reuing reperfusion-inue injury rather than ishemia-inue injury or affeting woun healing. Consistent with this, the kinetis of woun losure was omparable after a single MCP- efiieny reue marophage infiltration MCP- / mie showe reue numbers of skin-infiltrating ells uring an following IR yles, ompare with wiltype mie (Figures an 5). Speifially, the numbers of mm mm mm mm μm μm μm μm Size (mm) MCP- / Epithelial gap (mm) ays MCP- / ays MCP- / ays / MCP- ays Figure 5. Skin hanges following IR yles in MCP- / mie. (a) Representative photographs of wouns after IR yles in MCP- / mie. Marosopi skin ulers beame apparent at ay, followe by a progressive reution in size. (b) Representative histologial tissue setions showing the formation of uler an granulation tissue after IR yles (original magnifiation.5). In (b), the areas magnifie in () an () are iniate by the letter an, respetively. () Higher magnifiation of epiermis an ermis at the woun ege (original magnifiation ). () Higher magnifiation of hypoermis at the entral part of the woun be (original magnifiation ). (e) Time ourse of marosopi uler size in MCP- / mie. The skin ulers were heale by ay in all MCP- / mie. (f) Time ourse of epithelial gap uring skin woun healing. (g) Numbers of neutrophils an marophages per HPF were etermine by ounting myeloperoxiase- an F/-staine skin setions, respetively. Eight HPFs in five serial setions were ounte an average. Representative histologial tissue setions showing inflammatory ell infiltration are also shown (original magnifiation ). (h) Time ourse of marosopi uler size in wil-type an MCP- / mie following a treatment with hours of ishemia. (i) Time ourse of epithelial gap size in wil-type an MCP- / mie following a treatment with hours of ishemia. All values represent the mean±sem of results obtaine from more than six mie for eah time point of examination in eah genotype. Signifiant ifferenes between sample means are iniate, *Po.5; **Po.. Journal of Investigative Dermatology (), Volume

8 MCP- in Ishemia Reperfusion Skin Injury Neutrophil Marophage Size (mm ) -hour ishemia MCP- / -hour ishemia Epithelial gap (mm) -hour ishemia MCP- / -hour ishemia -hour ishemia ays -hour ishemia ays MCP- / ays MCP- / ays -hour ishemia -hour ishemia Figure 5. Continue. infiltrating marophages but not neutrophils were signifiantly reue in MCP- / mie ompare with wil-type mie uring an following IR yles (Figures e an 5g). Thus, MCP- is likely ritial for marophage infiltration in IR-inue skin injury an subsequent woun healing. MCP- efiieny inhibite the expression of inos an proinflammatory ytokines The role of MCP- in the expression of inos an proinflammatory ytokines was examine in the skin of MCP- / mie treate with IR yles. mrna expression of inos was signifiantly inhibite in MCP- / mie ompare with wil-type mie at ays an (Figure a). Similarly, the mrna expression of TNF-a was signifiantly reue in the lesional skin from MCP- / mie ompare with wil-type mie uring IR yles at ay (Figure a). By ontrast, mrna levels of IL-b an IL- were not signifiantly hange by the loss of MCP- (Figure a). Thus, the loss of MCP- inhibite the expression of inos an TNF-a in the skin uring IR yles. Cirulating protein levels of TNF-a an MCP- uring IR yles Serum levels of TNF-a an MCP- uring IR yles were assesse in wil-type an MCP- / mie (Figure b). Serum TNF-a levels were not signifiantly hange uring IR yles in wil-type mie. Aitionally, serum TNF-a levels in MCP- / mie were omparable with that in wil-type mie. Similarly, serum MCP- levels were not signifiantly hange uring IR yles in wil-type mie. Thus, lesional but not irulating TNF-a an MCP- are likely important for IRinue skin injury. MCP- efiieny i not affet the expression of HIF-a an HSP9 Cytoprotetive fators suh as hypoxia-inue fators (HIFs) an heat-shok proteins (HSPs) are known to funtion uring ishemi onition. HIF-a is a heteroimeri transription fator onsisting of HIF-a an HIF-b (Jiang et al., 99). HIF-a protein is ubiquitinate an egrae in normoxia, but stabilize in hypoxia an upregulates genes that promote survival (Huang et al., 99; Kallio et al., 999). Aitionally, it has been emonstrate that several HSPs, inluing HSP9, have antiapoptoti roles in IR-inue tissue injury (Shen et al., 7; Siso et al., 7). Therefore, the formation of IR yle-epenent skin injury may be the result of an IR ylerelate erease of ytoprotetive genes. Furthermore, inhibite skin injury in MCP- / mie oul be ue to inrease expression of ytoprotetive genes. Therefore, we assesse the gene expression of HIF-a an HSP9 uring IR yles in wil-type an MCP- / mie (Figure ). Although HIF-a expression tene to erease uring IR yles in wil-type mie, this was not signifiant. By ontrast, HSP9 expression graually inrease uring IR yles in wil-type mie, although it i not reah statistial signifiane. The 5

9 MCP- in Ishemia Reperfusion Skin Injury Relative levels of inos mrna MCP- / Relative levels of TNF-α mrna MCP- / Relative levels of IL-β mrna Relative levels of IL- mrna 5 μm 5 μm TNF-α (pg ml ) 5 MCP- / MCP- (pg ml ) 5 μm 5 μm Relative levels of HIF-α mrna MCP- / Relative levels of HSP9 mrna Figure. Levels of inos, ytokines, an ytoprotetive genes uring IR yles in MCP- / mie. Relative mrna expression of (a) inos, TNF-a, IL- b, an IL- in skin wouns of MCP- / mie was quantifie by real-time RT-PCR. (b) Serum levels of TNF-a an MCP- were measure by ELISA. () Relative mrna expression of HIF-a an HSP9 was assesse by real-time RT-PCR. All values represent the mean±sem. These results were obtaine from six mie for eah time point of examination in eah genotype. Signifiant ifferenes between sample means are iniate, *Po.5; **Po MCP- / 5 μm 5 μm 5 μm μm IR ay -hour MCP- / IR ishemia ay MCP- / -hour ishemia + W loss of MCP- i not signifiantly affet the expression levels of either of these genes. Thus, yle-epenent skin injury an suppresse skin injury in MCP- / mie are not explaine by the hanges in the expression levels of HIF-a an HSP9. Apoptosis uring IR yles Nulei of apoptoti ells were learly ientifie in skin setions using the terminal eoxynuleotiyl transferasemeiate UTP nik-en-labeling (TUNEL) assay. The number of fibroblasti apoptoti ells was etermine by ounting the number of TUNEL-positive ells that ha the harateristi mirosopi appearane of fibroblasts. The number of apoptoti ells in the hypoermis of the entral part of the woun be uring IR yles was inrease epening on the number of yles (Figures 7a an b). However, the number of apoptoti ells was signifiantly reue uring IR yles in MCP- / mie ompare with wil-type mie at ay (Po.). Skin tissue from wil-type mie that ha been subjete to ishemia for hours without any reperfusion (immeiately harveste after hours of ishemia) showe signifiantly erease apoptoti ells ompare with the usual three yles of IR Figure 7. Detetion of apoptosis in MCP- / mie. (a) Representative rosssetions of hypoermis at the entral part of the woun be staine by the TUNEL tehnique uring IR yles in wil-type an MCP- / mie. Fibroblasti TUNEL-positive ells with blue-staine nulei are iniate by arrows (original magnifiation ). The stuie region was stanarize between skin slies. (b) The number of the fibroblasti apoptoti ells was ounte in HPFs of serial setions, an average uring IR yles in wil-type an MCP- / mie. () The inution of apoptosis following - hour ishemia was ompare with that of the usual IR yles. The hange in apoptosis by injetion of a speifi inos inhibitor, W, is also shown. Signifiant ifferenes between sample means are iniate, **Po.. treatment in spite of the same ishemi perio (Figure 7, Po.). Both wil-type mie an MCP- / mie treate with hours of ishemia showe moest levels of apoptosis that was similar with that of MCP- / mie treate with IR yles (Figure 7). Thus, reperfusion rather than ishemia is likely important for MCP--epenent apoptosis inution. inos blokae attenuate IR yle-inue apoptosis Treatment with N-(-aminomethyl) benzylaetamiine (W), a seletive inos inhibitor, signifiantly reue the number of IR-inue apoptoti ells at ay Journal of Investigative Dermatology (), Volume

10 MCP- in Ishemia Reperfusion Skin Injury in wil-type mie (Figure 7, Po.). By ontrast, W i not signifiantly affet IR-inue apoptosis in MCP- / mie, refleting the role of MCP- in inos inution. Aitionally, inos inhibition i not hange the number of apoptoti ells following hours ishemia in either wiltype or MCP- / mie, iniating that inos funtion in apoptosis inution is espeially important in the reperfusion perio. Thus, MCP- likely has a role in apoptosis inution via inos expression uring the reperfusion perio of IR yles. DISCUSSION In this stuy, we examine the preise proess an mehanism of a reprouible murine moel of utaneous IR injury by the external appliation of magnets. Infiltration of marophages an neutrophils was inrease in the treate sites, with peak infiltration ourring at ay uring IR yles (Figure e). Proinflammatory ytokines suh as TNF-a, IL-b, an IL- were also inrease in the skin showing peak expression at ay or (Figure b). inos expression progressively inrease until ay, in parallel with the inrease in apoptoti ells (Figures a an 7b). Therefore, IRinue apoptosis an skin injury an be inue by inflammatory ell infiltration in ooperation with proinflammatory ytokine proution an inos. Importantly, protein levels of MCP- were remarkably inrease at ay in the skin, followe by inflammatory ell infiltration (Figure ), refleting the role of this hemokine in initiating the IR injury. Consistent with this, MCP- / mie showe miler skin inflammation an injury uring an following IR yles (Figures an 5). Skin expression of TNF-a an inos were also signifiantly reue by the loss of MCP- (Figure a). Importantly, MCP- was foun to be ritial for apoptosis inution an skin injury uring the reperfusion proess rather than the ishemi perio (Figure 7). Furthermore, inos-bloking stuies larifie the funtional role of inos in this proess (Figure 7). These finings iniate that MCP- plays a ritial role in the evelopment of IR-inue skin injury. We first assesse the marosopi an histopathologial hanges that ourre uring an following IR yles in this moel. Marosopially, the formation of skin ulers was foun at ay an these were repaire by ay in all mie (Figures a, a an e). However, in the original report of this moel, skin ulers were not ompletely repaire in all mie at ay (Staler et al., ). Aelerate woun repair in our stuy may be ue to the mouse bakgroun (C57BL/) an the environment (speifi pathogen-free barrier faility) that are ifferent from the original report (BALB/ an ontrolle environment, respetively). Histopathologially, skin eema an inflammatory ell infiltration showe peak responses at ays an, respetively (Figures b ). Interestingly, skin eema an inflammatory ell infiltration onsisting primarily of neutrophils an marophages were reue after three IR yles (ay ) ompare with reution after two IR yles (ay ; Figures b e). Proinflammatory ytokines suh as TNF-a, IL-b, an IL- were signifiantly inrease uring the early stage (ays ) of the IR yles (Figure b), in parallel with the inflammatory ell infiltration. Sine these proinflammatory ytokines are primarily proue by marophages, these effeter moleules proue by infiltrate marophages may ontribute to the skin injury. Aumulating eviene iniates that the exessive proution of NO plays a pathogeni role in inflammation (Nathan an Xie, 99; Christopherson an Bret, 997; Kubes, ). NO is a free raial proue from L-arginine by three istint enzymes. Among those enzymes, inos is not normally etete in tissues, but its expression is inue in inflammatory onitions. The expression of inos in various ells, inluing enothelium, epithelium, an inflammatory ells, is inue by proinflammatory ytokines an lipopolysaharie, an proues signifiant amounts of NO for extene perios of time (Nathan an Xie, 99). inos has been shown to have multiple biologial effets. Although small amounts of inos are essential for normal healing in the skin an intestinal muosa (Kubes, ), exessive inos an ontribute to IR tissue injury via promoting over proution of NO from enothelial ells (Kubes, ; Furuihi et al., ; Rei et al., ; Hayasaki et al., ). In fat, inos / mie exhibit reue IR injury in multiple organs (Ling et al., 999; Suzuki et al., ; Qi et al., ). In this stuy, infiltrating ells an proinflammatory ytokine expression were reue at ay (after the thir IR yle) ompare to ay (Figures b e an b). By ontrast, inos expression appeare to be yleresponsive, sine a onsistent aily progression was seen (Figure a). Sine the number of IR yles is ritial for skin tissue injury (Peire et al., ), inos expression may strongly assoiate with the extent of IR-inue skin injury. Sine inos is proue by various ells inluing marophages stimulate with proinflammatory ytokines (Nathan an Xie, 99), marophage infiltration an proinflammatory ytokine expression may ooperatively ontribute to the subsequent inos expression uring IR yles. These finings suggest that the marophage is an important meiator for the inution of IR injury an strategies that inhibit initial marophage infiltration or ativation may be useful in the treatment of pressure ulers. As far as we know, investigation of IR skin injury has not been reporte in any transgeni mie, probably ue to the lak of an establishe mouse moel. Our results using the murine moel propose by Staler et al. () in wil-type mie suggest that MCP-, a prototype hemotati fator for marophages, playe an important role in utaneous IRinue injury. Speifially, protein levels of MCP- were foun to peak early at ay (Figure ), followe by inflammatory ell infiltration, whih peake at ay (Figures an e). Aitionally, reent stuies have emonstrate that the MCP-/CCR pathway has important roles for IR injury via reruiting marophages an subsequent proution of ytokines an inos in some organs inluing myoarium an kiney (Furuihi et al., ; Hayasaki et al., ). Therefore, we examine the role of MCP- in pressure uler formation by generating a utaneous IR injury using MCP- / mie. The loss of MCP- signifiantly reue the infiltration of marophages but not neutrophils (Figure e), onsistent 7

11 MCP- in Ishemia Reperfusion Skin Injury with its ritial role for the reruitment of marophages. Furthermore, MCP- efiieny signifiantly inhibite the expression of TNF-a an inos uring IR yles (Figure a). Unfortunately, it was iffiult to quantitatively evaluate the severity of tissue injury uring IR yles in this moel. However, woun healing may be aelerate or elaye epening on the prior tissue injury. As expete, woun repair was aelerate an the ulers were ompletely heale ays earlier in all MCP- / mie ompare to wil-type mie (Figures 5e an f). Sine the repair of inisional skin wouns was rather elaye by the loss of MCP- as previously reporte (ata not shown an Low et al., ), this further supports the fining of miler utaneous IR injury in MCP- / mie. Aitionally, ontinuous -hour ishemia treatment evelope skin uler that heale earlier than treatment with IR yles, in spite of the same perio of ishemia in wil-type mie (Figures 5h an i). By ontrast, both -hour ishemia an IR yles showe similar skin injury in MCP- / mie (Figures 5h an i). These finings suggest that MCP- has important roles in IR-inue skin injury via reruiting marophages an subsequent proution of TNF-a or inos, espeially uring the reperfusion proess. MCP- may also play a role in IR injury through the ativation of CCR-expressing marophages, as emonstrate in renal IR injury (Furuihi et al., ). Furthermore, MCP- has been emonstrate to inue the release of lysosomal enzymes an the generation of inos an superoxie anions, resulting in tissue estrution (Baggiolini et al., 99; Ikea et al., 99; Christopherson an Bret, 997). Thus, the interation between MCP- an CCR may be ritial for the generation of utaneous IR injury. Most therapeuti investigations of pressure uler formation have been performe using inision skin wouning. However, an inision woun healing moel annot larify the mehanism of pressure uler evelopment an thus may not ientify important moleules for therapeuti targeting. Furthermore, the ourse of woun healing of inision skin may be ifferent from IR-inue skin uler or pressure uler. MCP- / mie emonstrate elaye healing of inision skin wouns ompare with wil-type mie (ata not shown an Low et al., ). However, the IR-inue skin uler was heale earlier in MCP- / mie than in wil-type mie, likely as a result of suppresse IR-skin injury in this moel (Figures 5a e). Similarly, wil-type mie treate with an inos inhibitor an inos / mie show elaye healing of utaneous exisional wouns espite the ritial role of inos for eveloping IR injury (Yamasaki et al., 99). Therefore, a utaneous IR injury moel is more appropriate for the investigation of woun healing in pressure ulers than an inision woun-healing moel. Thus, this mouse moel may be useful for investigating the mehanism or therapeuti strategy of both IR injury an woun healing in pressure ulers. In the urrent stuy, apoptosis was signifiantly inrease yle epenently uring IR yles (Figures 7a an b). Furthermore, reperfusion rather than the ishemi perio was ritial for the inution of apoptosis (Figure 7). This is onsistent with an experimental IR moel of myoarium (Eefting et al., ). Interestingly, apoptosis was inhibite by the loss of MCP- (Figures 7a an b). Furthermore, our finings iniate that MCP- has a role in apoptosis inution uring reperfusion rather than ishemia (Figure 7). Inreasing eviene has emonstrate that ytoprotetive genes suh as HIF-a an several HSPs an protet organs from IRinue apoptosis (Marber et al., 995; Plumier et al., 995). A previous rat moel of skin IR injury emonstrate that genes with ytoprotetive effets are initially upregulate uring the first yle, but their upregulation was subsequently reue uring the seon yle (Siso et al., 7). These finings may explain the yle-epenent evelopment of skin injury. However, in this stuy, expression levels of ytoprotetive genes suh as HIF-a an HSP9 were not signifiantly hange uring IR yles (Figure ). Furthermore, these expression levels were not signifiantly hange by the loss of MCP- (Figure ). Therefore, yle-epenent apoptosis or reue apoptosis by MCP- efiieny is not ue to hanges in the expression levels of these ytoprotetive genes. Although the effet of NO on apoptosis is omplex, it is generally aepte that high onentration of NO inues apoptosis (Lipton et al., 99; Shulz et al., ). In our experiments, inos expression was signifiantly inrease by IR yles (Figure a). Aitionally, the augmente inos expression was remarkably reue by the loss of MCP- (Figure a). Furthermore, bloking of inos expression signifiantly reue the apoptosis uring the reperfusion proess of IR yles in wil type but not MCP- / mie (Figure 7). Taken together, these observations suggest that MCP- has a role in inos expression an subsequent apoptosis an skin injury uring the reperfusion proess of IR yles. Despite the absene of MCP-, skin inflammation an uler formation were not ompletely inhibite (Figures an 5). Although MCP- is the entral hemokine for reruiting marophages, other hemokines, inluing fratalkine, MIP-a, an MCPs, also ontribute to the hemotaxis of marophages. Therefore, other hemokines may ompensate for the role of MCP- to some extent in this moel. Aitionally, MCP--inepenent mehanisms likely exist. For example, neutrophil infiltration was inepenent of MCP- in this moel (Figure e), although several stuies have reporte that the interation between MCP- an CCR promotes the hemotaxis of neutrophils uring inflammation (Tessier et al., 99; Johnston et al., 999). Sine the expression of KC, a CXC hemokine, was inrease uring IR yles in wil-type mie (Figure ), CXC hemokines, inluing KC, may also have a role in IR-inue skin injury via reruiting neutrophils. Future stuies using transgeni mie will be neee to larify the role of various hemokines, ytokines, or ahesion moleules in IR-inue skin injury. Appliations of this moel on the vast number of transgeni mie will be benefiial to exten our unerstaning of the mehanism of pressure uler evelopment. MATERIALS AND METHODS Animals C57BL/ wil-type an MCP- / mie (C57BL/ bakgroun; Lu et al., 99) were obtaine from The Jakson Laboratory (Bar Journal of Investigative Dermatology (), Volume

12 MCP- in Ishemia Reperfusion Skin Injury Harbor, ME). All mie were healthy, fertile, an i not isplay any eviene of infetion or isease. Female mie (- to -week-ol) were use for all the experiments. All mie were house in speifi pathogen-free barrier faility an sreene regularly for pathogens. All mie were iniviually house in stainless ages to prevent aiental isloation of the magnets an to prevent tampering with the resultant uler by other mie. All stuies an proeures were approve by the Committee on Animal Experimentation of Kanazawa University Grauate Shool of Meial Siene. IR yles The IR yle moel that has been previously reporte was use (Staler et al., ). Briefly, mie were anesthetize with iethyl ether an their baks were shave an leane with 7% alohol. A template was use to mark the loation of the magneti plates to assure a onsistent plaement on eah mouse. It has been emonstrate that an external pressure of 5 mm Hg is suffiient to inue IR skin nerosis by reuing bloo flow by % (Peire et al., ). The skin was gently pulle up an plae between two roun erami magneti plates that ha a -mm iameter ( mm ) an were 5 mm thik, with an average weight of. g an, G magneti fores (Seiko Sangyo Co., Ihikawa, Japan). Epiermis, ermis, subutaneous fat layer, panniulus arnosus, an subutaneous loose onnetive tissue layer (hypoermis), but not musles, were pinhe with the magnet plates. This proess reate a ompressive pressure of 5 mm Hg between the two magnets (Peire et al., ; Staler et al., ). Three IR yles were performe in eah mouse to initiate eubitus uler formation. A single IR yle onsiste of a -hour perio of magnet plaement, followe by a release or rest perio of hours. Mie were not immobilize, anesthetize, or otherwise treate uring IR yles. All of the mie evelope two irular ulers separate by a brige of normal skin, as reporte previously (Staler et al., ). For analysis, mie were anesthetize an the areas of the skin uler were measure by traing the woun openings onto a transpareny. The mean area of the two skin ulers was efine as the size of the skin uler in eah mouse. Skin tissues uring or after IR yles were harveste an were use for subsequent analysis. In some experiments, a single treatment of -hour ishemia by magnet plaement was performe instea of IR yles. Histologial examination an immunohistohemistry After the mie were kille, wouns were harveste with a -mm rim of unwoune skin tissue. The wouns were ut into laterally halves, fixe in.5% paraformalehye, an embee in paraffin. Setions ( mm) were staine with hematoxylin an eosin or proesse for immunostaining. For immunohistohemistry, eparaffinize setions were treate with enogenous peroxiase-bloking reagent (DAKO Cytomation A/S, Copenhagen, Denmark) an proteinase K (DAKO Cytomation A/S) for minutes at room temperature. Setions were then inubate with rat mabs speifi for marophages (lone F/; Amerian Type Culture Colletion, Rokville, MD), rabbit antimyeloperoxiase polylonal antiboy (Neomarkers, Fremont, CA), or rat anti-mouse CD mab (Dainippon Pharmaeutial Company, Osaka, Japan). Rat IgG (Southern Biotehnology Assoiates In., Birmingham, AL) was use as a ontrol for nonspeifi staining. Setions were then inubate sequentially ( minutes, 7 C) with a biotinylate rabbit anti-rat IgG seonary antiboy (Vetastain ABC metho; Vetor Laboratories, Burlingame, CA) or a biotinylate goat anti-rabbit IgG seonary antiboy (BD Biosienes, San Jose, CA), an then with horseraish peroxiase-onjugate aviin biotin omplexes. Setions were washe three times with phosphatebuffere saline between inubations. Setions were evelope with, -iaminobenziine tetrahyrohlorie an hyrogen peroxie, an then ounterstaine with methyl green. The number of neutrophils, marophages, an T lymphoytes etermine with immunostaining was ounte an average in eight high-power mirosopi fiels (HPFs,.7 mm ) of five serial setions. The number of mie use for eah time point of examination was more than in eah genotype. Real-time RT-PCR Total RNAs were extrate from injure skin samples using QIAGEN RNeasy spin olumns (QIAGEN Lt., Crawley, UK) an igeste with DNase I (QIAGEN Lt.) to remove hromosomal DNA in aorane with the manufaturer s protools. Total RNA was reverse transribe to DNA using a Reverse Transription System with ranom hexamers (Promega, Maison, WI). Real-time RT-PCR was performe using the TaqMan system (Applie Biosystems, Foster City, CA) on an ABI Prism 7 Sequene Detetor (Applie Biosystems) aoring to the manufaturer s instrutions. TaqMan probes an primers for inos, TNF-a, IFN-g, IL-b, IL-, HIF-a, HSP9, an glyeralehye--phosphate ehyrogenase (GAPDH) were purhase from Applie Biosystems. Relative expression of real-time PCR prouts was etermine using the DDC t tehnique. Briefly, eah set of samples was normalize using the ifferene in threshol yle (C t ) between the target gene an housekeeping gene: DC t ¼ (C t target gene C t GAPDH ). Relative mrna levels were alulate by the expression DDCt, where DDC t ¼ DC t sample DC t alibrator. Eah reation was performe in, at least, tripliate. The number of mie use for eah time point of examination was more than six in eah genotype. ELISA Woun tissues were homogenize with. ml of lysis buffer ( mm phosphate-buffere saline,.% SDS, % Noniet P-, an 5 mm EDTA) ontaining Complete Protease Inhibitor mixture (Rohe Diagnostis, Tokyo, Japan), as esribe previously (Mori et al., ). The homogenates were entrifuge at, r.p.m. for 5 minutes, with the supernatants being analyze by ELISA. Protein levels of MCP-, MIP-a, KC, fratalkine, an TNF-a in tissue extrat or serum were measure with ommerial ELISA kits (R&D Systems In., Minneapolis, MN), aoring to the manufaturers reommenation, an the olor intensity was measure at 5 nm. The etetion limits for eah hemokine were as follows: MCP-, 5. pg ml ; MIP-a,.7 pg ml ; KC, 5. pg ml ; fratalkine,. ng ml ; TNF-a,.7 pg ml. Total protein in the supernatant was measure with a ommerial kit (BCA Protein Assay kit; Piere, Rokfor, IL). The ata were expresse as hemokine (pg ml or ng ml )/total protein (mg ml ) for eah sample. The number of mie use for eah time point of examination was more than six in eah genotype. Detetion of apoptosis To loalize an assess apoptoti ells, a TUNEL kit (DermaTACS; R&D Systems In.) was use. The TUNEL assay was performe 9

13 MCP- in Ishemia Reperfusion Skin Injury aoring to the protool supplie by the manufaturer. The number of fibroblasti apoptoti ells was etermine by ounting the number of TUNEL-positive ells that ha the harateristi mirosopi appearane of fibroblasts. Two inepenent observers unaware of either treatment or genotype assesse the number of apoptoti ells present in HPFs (.7 mm ) of serial setions, with the average results being shown. The stuie region (hypoermis) was stanarize between skin slies. Seletive inhibition of inos ativity In some experiments, we use an inhibitor of inos ativity, W (BIOMOL Researh Laboratories In., Plymouth Meeting, PA) (Garvey et al., 997). Mie unerwent the same proeure of IR yles or -hour ishemia, but were also treate with W ( mg kg, intravenous) aministere 5 minutes before an eah hours uring the proeure. Statistial analysis The Mann Whitney U-test was use to etermine the level of signifiane of ifferenes between the sample means, an Bonferroni test was use for multiple omparisons. A P-value o.5 was onsiere statistially signifiant. CONFLICT OF INTEREST The authors state no onflit of interest. ACKNOWLEDGMENTS We thank Ms M Matsubara an Y Yamaa for tehnial assistane. This work was supporte by a grant-in-ai from the Ministry of Euation, Siene, an Culture of Japan (to M Hasegawa, S Sato, an K Takehara). REFERENCES Baggiolini M, Dewal B, Moser B (99) Interleukin- an relate hemotati ytokines CXC an CC hemokines. Av Immunol 55:97 79 Christopherson KS, Bret DS (997) Nitri oxie in exitable tissues: physiologial roles an isease. J Clin Invest : 9 Daniel RK, Priest DL, Wheatley DC (9) Etiologi fators in pressure sores: an experimental moel. Arh Phys Me Rehabil :9 DiPietro LA, Polverini PJ, Rahbe SM, Kovas EJ (995) Moulation of JE/MCP- expression in ermal woun repair. 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