Letter to the Editor
References 1 Rao N N, Gomez-Garcia M R, Kornberg A. Annu Rev Biochem 2009: 78: 605–647. 2 Wurst H, Kornberg A. J Biol Chem 1994: 269: 10996–11001. 3 Kumble K D, Kornberg A. J Biol Chem 1995: 270: 5818–5822.
4 Crooke E, Akiyama M, Rao N N., et al. J Biol Chem 1994: 269: 6290–6295. 5 Abramov A Y, Fraley C, Diao C T., et al. Proc Natl Acad Sci U S A 2007: 104: 18091–18096. 6 Tammenkoski M, Koivula K, Cusanelli E., et al. Biochemistry 2008: 47: 9707–9713.
DOI: 10.1111/exd.12733
7 D’Angelo A, Garzia L, Andre A., et al. Cancer Cell 2004: 5: 137–149. 8 Seidlmayer L K, Gomez-Garcia M R, Blatter L A., et al. J Gen Physiol 2012: 139: 321–331. 9 DiVito K A, Trabosh V A, Chen Y S., et al. Pigment Cell Melanoma Res 2010: 23: 795–808.
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
High-glucose-cultivated peripheral blood mononuclear cells impaired keratinocyte function via reduced IL-22 expression: implications on impaired diabetic wound healing Shu-Mei Huang1,2, Ching-Shuang Wu3, David Chao1, Chin-Han Wu2,4, Ching-Chia Li5, Gwo-Shing Chen2 and Cheng-Che E. Lan2,4,6 1 Department of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan; 2Department of Dermatology, College of Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 3Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan; 4Center for Lipid and Glycomedicine Research, Kaohsiung Medical University, Kaohsiung, Taiwan; 5Department of Urology, College of Medicine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; 6Department of Dermatology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Correspondence: Professor Cheng-Che E. Lan, Department of Dermatology, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd, Kaohsiung, Taiwan, Tel.: +8867 3121101 (ext 6108), Fax: +8867 3216580, e-mail: [email protected]
Key words: high glucose – interleukin-22 – keratinocytes – MMP-3 – mononuclear cells
Accepted for publication 26 April 2015
Background
Results
Diabetes mellitus (DM) affects more than 170 million people worldwide with the number expected to increase (1). Impaired wound healing is a major complication associated with DM. Re-epithelialization following cutaneous injury involves keratinocyte migration and is essential for proper healing (2). Matrix metalloproteinases (MMPs) that degrade extracellular matrix proteins are critically involved in this process (3). Mononuclear cells have been recognized to play significant roles in re-epithelialization by producing various growth factors (4). Although diabetics showed impaired wound healing, diabetic wounds harbour significant amount of mononuclear cells at the wound edge (5).
The viability of PBMC cultivated at high-glucose (HG) environment for 2 days was not significantly altered. The viability and proliferation of keratinocytes 1 day after treatment with HG-cultivated PBMC CM were 1.02 0.08 and 0.96 0.04 folds, respectively, of their normal-glucose (NG)-cultivated PBMC CM-treated counterpart. No significant difference was found. To determine whether PBMC culture results in release of promigratory factors into CM, the migration of keratinocytes treated with media with or without prior PBMC cultivation was compared. The CM after PBMC cultivation significantly enhanced keratinocyte migration as compared to the control media without PBMC cultivation (Figure S1), indicating PBMC release active factors into the CM. Subsequently, the migration of keratinocytes treated with CM from different PBMC groups was compared. Keratinocyte migration showed significant reduction in HG-cultivated PBMC CM-treated group as compared to their NG-cultivated PBMC CM-treated counterpart (Fig. 1a,b). For osmolarity control, PBMC was cultivated under NG environment with addition of 20 mM mannitol. Similar migratory capacity was found between keratinocytes treated with CM from NG-cultivated PBMC and their osmolarity control-treated counterpart (Figure S2). This result indicated that reduced migration of keratinocytes treated with CM from HG-cultivated PBMC was due to elevated glucose level but not increased osmolarity. As keratinocyte
Questions addressed We hypothesized that mononuclear cells, although properly recruited to the diabetic wound area, were dysfunctional in facilitating the re-epithelialization process due to their suboptimal interaction with keratinocytes.
Experimental design Cultured keratinocytes were treated with conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) cultivated at various glucose environments, and the effects of indicated PBMC CM on keratinocyte migration were determined. A diabetic rat model was used to validate the results. Detailed methodology is described in the supplementary file.
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 630–641
639
Letter to the Editor
(a)
(c)
(a)
(d) (b)
(b) (e)
Figure 1. (a) Migration of cultured human keratinocytes (KC) after treatment with indicated peripheral blood mononuclear cell (PBMC)-conditioned media (CM) as evaluated by wound scratching assay. Left panel represented KC treated with CM from normal-glucose-cultured PBMC (PBMCN), and right panel represented KC treated with CM from 26 mM glucose-cultured PBMC (PBMCH). A representative photograph of three independent experiments is shown. (b) The per cent wound closure of (a) measured with Image J software from three independent experiments. *Indicates P < 0.05 as compared to PBMCN group. (c) The effect of IL-22 neutralizing binding protein (BP) on PBMC CM-treated keratinocytes in terms of metalloproteinase (MMP)-3 mRNA expression. Cultured human keratinocytes treated with CM from normal-glucose-cultivated PBMCN or high-glucose-cultivated PBMCH with or without IL-22 neutralizing BP. *Indicates P < 0.05 as compared to PBMCN group. (d) Migration of cultured KC after treatment with CM from normalglucose-cultivated PBMC with or without IL-22 BP. (e) The per cent wound closure of (d) measured with Image J software from three independent experiments. *Indicates P < 0.05 as compared to group without IL-22 BP.
migration is associated with MMP expressions, the effects of CM derived from different PBMC on MMP expression of keratinocytes were determined. The CM from HG-cultivated PBMC reduced MMP-3 mRNA expression of keratinocytes as compared to their NG-cultivated PBMC CM-treated counterpart, while the MMP-1, 2, -9, 10 and TIMP-1 and TIMP-2 expressions showed no significant difference between the two groups (Table S2). No significant difference in MMP-3 expression was found between keratinocytes treated with NG-cultivated PBMC CM and the osmolarity controltreated counterpart (Figure S3). IL-22 is a recognized regulatory factor for MMP-3 in keratinocytes (6), and HG cultivation significantly reduced IL-22 mRNA expression in PBMC to 65.76 2.24% of their NG-cultivated counterpart. To examine the functional roles of IL-22, relevant neutralizing binding protein was added to the CM derived from NG-cultivated PBMC. Keratinocytes treated with this experimental condition showed significantly reduced MMP-3 expression (Fig. 1c). In addition, migration of keratinocytes treated with NGcultivated PBMC CM was significantly reduced by the addition of IL-22-binding protein (Fig. 1d,e). To determine the functional relevance of MMP-3 on keratinocyte migration, a gene silencing approach was employed. The migration of MMP-3 silenced kerati-
640
Figure 2. (a) At 9 days after wounding, the healing process of normal rat was almost complete, while diabetic rat showed delayed wound healing. (b) The epidermal metalloproteinase (MMP)-3 mRNA expression of normal and diabetic rat skin on day 0 and day 1 after wounding. The increase in MMP-3 expression after wounding was documented. *Indicates p < 0.05 as compared to the normal rat.
nocyte was not significantly different from the non-silenced control (Figure S4). These results indicated that IL-22 in the PBMC CM regulated the migration and MMP-3 expression of keratinocytes distinctively. To confirm our in vitro results in vivo, a diabetic rat model was used. The diabetic rats showed impaired healing (11.8 1.9 days until wound closure) as compared to control (9.3 0.8 days) (Fig. 2a). The IL-22 mRNA expression from diabetic rat skin 1 day after wounding was 53.5 3.7% of control rats. Similar to results from cell model, the folds of epidermal MMP-3 increase after wounding was significantly lower in diabetic rats as compared to control rats (Fig. 2b).
Conclusions In this study, we found that the migration of keratinocytes treated with HG-cultivated PBMC CM was significantly reduced as compared to their NG-cultivated PBMC CM-treated counterpart. In addition, the keratinocytes treated with HG-cultivated PBMC CM showed significantly lower MMP-3 expression as compared to their NG-cultivated PBMC CM-treated counterpart. MMP-3 is a molecule recognized to regulate wound healing process. Physiologically, MMP-3 expression was negligible in normal skin but was observed in keratinocytes after injury (7). Additionally, mice that lacked MMP-3 demonstrated impaired skin healing due to inadequate wound contraction but not keratinocyte migration (8). In
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 630–641
Letter to the Editor
support with this notion, our gene silencing experiment indicated that MMP-3 has limited effect on keratinocyte migration in our experimental condition. Reduced IL-22 production from HG-cultivated PBMC contributed to hampered keratinocyte migration as keratinocyte migration induced by PBMC CM was significantly reduced after the addition of IL-22-binding protein. It has been shown that besides modulating inflammation, IL-22 plays a significant role during wound healing. Upon IL-22 stimulation, cultured keratinocyte showed enhanced migration through the activation of STAT3-related signalling (6). Therefore, reduced production of IL-22 in PBMC under HG environment likely contributed to the impaired wound healing observed in diabetic condition. It is noted that CM from HG-cultivated PBMC only reduced MMP-3 of keratinocytes as compared to their NG-cultivated PBMC CM-treated counterpart, while the effects on other MMPs were limited. A detailed analysis of the proteins contained in various CM is warranted to provide an explanation to this intriguing phenomenon. In summary, we demonstrated that HG cultivation reduced the IL-22 production in PBMCs that significantly reduced MMP3 expression and hampered the migration of treated keratinocytes. Clinically, downregulation of IL-22 and MMP-3 would result in impaired keratinocyte migration and inadequate wound contraction. Increasing IL-22 in the microenvironment of wound may present a novel therapeutic approach to promote diabetic healing.
Acknowledgement This study was supported by National Science Council, Taiwan (grants NSC 101- 2314-B-037-020-MY3), Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan (Grants KMUH1033R49), and Center for Lipid and Glycomedicine, Kaohsiung Medical University, Kaohsiung, Taiwan (KMU- TP103D10). S.M.H., C.S.W. and C.H.W. performed the experiments; D.C., C.C.L., G.S.C. and C.C.L designed the research study. All of the authors analysed the data, wrote the paper and approved the final version.
Conflict of interest The authors have declared no conflicting interests.
Supporting Information Additional supporting data may be found in the supplementary information of this article. Figure S1. (a) Keratinocyte migration after treatment with peripheral blood mononuclear cell (PBMC) culture media with or with out PBMC cultivation. (b) The percent wound closure of figure S1a measured with Image J software from three independent experiments. Figure S2. (a) For osmolarity control, PBMC was cultivated under normal glucose environment with (PBMC M) or without (PBMC N) addition of 20 mM mannitol. (b) The percent wound closure of S2a measured with Image J software from three independent experiments. Figure S3. MMP-3 expression of keratinocytes treated with CM from normal glucose cultivated (PBMC N) and its osmolarity control counterpart (PBMCM). Figure S4. (a) Migratory capacity of cultured human keratinocytes after MMP-3 gene silencing was evaluated by wound scratching assay. (b) The percent wound closure of S4a measured with Image J software from three independent experiments. Table S1. Primer sequences used for real-time PCR analyses. Table S2. Percent of metalloprotianse (MMP) and (TIMP) mRNA expression of human keratinocytes treated with conditioned media (CM) from high glucose (HG) cultivated PBMC as compared to CM from normal glucose (NG) cultivated PBMC. Data S1. Materials and methods.
References
1 Wild S, Roglic G, Green A et al. Diabetes Care 2004: 27: 1047–1053. 2 Yates S, Rayner T E. Wound Repair Regen 2002: 10: 5–15. 3 Legrand C, Gilles C, Zahm J M et al. J Cell Biol 1999: 146: 517–529.
4 Singer A J, Clark R A. N Engl J Med 1999: 341: 738–746. 5 Loots M A, Lamme E N, Zeegelaar J et al. J Invest Dermatol 1998: 111: 850–857. 6 Boniface K, Bernard F X, Garcia M et al. J Immunol 2005: 174: 3695–3702.
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 630–641
7 Pilcher B K, Wang M, Qin X J et al. Ann N Y Acad Sci 1999: 878: 12–24. 8 Bullard K M, Lund L, Mudgett J S et al. Ann Surg 1999: 230: 260–265.
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References 1 Rao N N, Gomez-Garcia M R, Kornberg A. Annu Rev Biochem 2009: 78: 605–647. 2 Wurst H, Kornberg A. J Biol Chem 1994: 269: 10996–11001. 3 Kumble K D, Kornberg A. J Biol Chem 1995: 270: 5818–5822.
4 Crooke E, Akiyama M, Rao N N., et al. J Biol Chem 1994: 269: 6290–6295. 5 Abramov A Y, Fraley C, Diao C T., et al. Proc Natl Acad Sci U S A 2007: 104: 18091–18096. 6 Tammenkoski M, Koivula K, Cusanelli E., et al. Biochemistry 2008: 47: 9707–9713.
DOI: 10.1111/exd.12733
7 D’Angelo A, Garzia L, Andre A., et al. Cancer Cell 2004: 5: 137–149. 8 Seidlmayer L K, Gomez-Garcia M R, Blatter L A., et al. J Gen Physiol 2012: 139: 321–331. 9 DiVito K A, Trabosh V A, Chen Y S., et al. Pigment Cell Melanoma Res 2010: 23: 795–808.
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
High-glucose-cultivated peripheral blood mononuclear cells impaired keratinocyte function via reduced IL-22 expression: implications on impaired diabetic wound healing Shu-Mei Huang1,2, Ching-Shuang Wu3, David Chao1, Chin-Han Wu2,4, Ching-Chia Li5, Gwo-Shing Chen2 and Cheng-Che E. Lan2,4,6 1 Department of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan; 2Department of Dermatology, College of Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 3Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan; 4Center for Lipid and Glycomedicine Research, Kaohsiung Medical University, Kaohsiung, Taiwan; 5Department of Urology, College of Medicine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; 6Department of Dermatology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Correspondence: Professor Cheng-Che E. Lan, Department of Dermatology, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd, Kaohsiung, Taiwan, Tel.: +8867 3121101 (ext 6108), Fax: +8867 3216580, e-mail: [email protected]
Key words: high glucose – interleukin-22 – keratinocytes – MMP-3 – mononuclear cells
Accepted for publication 26 April 2015
Background
Results
Diabetes mellitus (DM) affects more than 170 million people worldwide with the number expected to increase (1). Impaired wound healing is a major complication associated with DM. Re-epithelialization following cutaneous injury involves keratinocyte migration and is essential for proper healing (2). Matrix metalloproteinases (MMPs) that degrade extracellular matrix proteins are critically involved in this process (3). Mononuclear cells have been recognized to play significant roles in re-epithelialization by producing various growth factors (4). Although diabetics showed impaired wound healing, diabetic wounds harbour significant amount of mononuclear cells at the wound edge (5).
The viability of PBMC cultivated at high-glucose (HG) environment for 2 days was not significantly altered. The viability and proliferation of keratinocytes 1 day after treatment with HG-cultivated PBMC CM were 1.02 0.08 and 0.96 0.04 folds, respectively, of their normal-glucose (NG)-cultivated PBMC CM-treated counterpart. No significant difference was found. To determine whether PBMC culture results in release of promigratory factors into CM, the migration of keratinocytes treated with media with or without prior PBMC cultivation was compared. The CM after PBMC cultivation significantly enhanced keratinocyte migration as compared to the control media without PBMC cultivation (Figure S1), indicating PBMC release active factors into the CM. Subsequently, the migration of keratinocytes treated with CM from different PBMC groups was compared. Keratinocyte migration showed significant reduction in HG-cultivated PBMC CM-treated group as compared to their NG-cultivated PBMC CM-treated counterpart (Fig. 1a,b). For osmolarity control, PBMC was cultivated under NG environment with addition of 20 mM mannitol. Similar migratory capacity was found between keratinocytes treated with CM from NG-cultivated PBMC and their osmolarity control-treated counterpart (Figure S2). This result indicated that reduced migration of keratinocytes treated with CM from HG-cultivated PBMC was due to elevated glucose level but not increased osmolarity. As keratinocyte
Questions addressed We hypothesized that mononuclear cells, although properly recruited to the diabetic wound area, were dysfunctional in facilitating the re-epithelialization process due to their suboptimal interaction with keratinocytes.
Experimental design Cultured keratinocytes were treated with conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) cultivated at various glucose environments, and the effects of indicated PBMC CM on keratinocyte migration were determined. A diabetic rat model was used to validate the results. Detailed methodology is described in the supplementary file.
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 630–641
639
Letter to the Editor
(a)
(c)
(a)
(d) (b)
(b) (e)
Figure 1. (a) Migration of cultured human keratinocytes (KC) after treatment with indicated peripheral blood mononuclear cell (PBMC)-conditioned media (CM) as evaluated by wound scratching assay. Left panel represented KC treated with CM from normal-glucose-cultured PBMC (PBMCN), and right panel represented KC treated with CM from 26 mM glucose-cultured PBMC (PBMCH). A representative photograph of three independent experiments is shown. (b) The per cent wound closure of (a) measured with Image J software from three independent experiments. *Indicates P < 0.05 as compared to PBMCN group. (c) The effect of IL-22 neutralizing binding protein (BP) on PBMC CM-treated keratinocytes in terms of metalloproteinase (MMP)-3 mRNA expression. Cultured human keratinocytes treated with CM from normal-glucose-cultivated PBMCN or high-glucose-cultivated PBMCH with or without IL-22 neutralizing BP. *Indicates P < 0.05 as compared to PBMCN group. (d) Migration of cultured KC after treatment with CM from normalglucose-cultivated PBMC with or without IL-22 BP. (e) The per cent wound closure of (d) measured with Image J software from three independent experiments. *Indicates P < 0.05 as compared to group without IL-22 BP.
migration is associated with MMP expressions, the effects of CM derived from different PBMC on MMP expression of keratinocytes were determined. The CM from HG-cultivated PBMC reduced MMP-3 mRNA expression of keratinocytes as compared to their NG-cultivated PBMC CM-treated counterpart, while the MMP-1, 2, -9, 10 and TIMP-1 and TIMP-2 expressions showed no significant difference between the two groups (Table S2). No significant difference in MMP-3 expression was found between keratinocytes treated with NG-cultivated PBMC CM and the osmolarity controltreated counterpart (Figure S3). IL-22 is a recognized regulatory factor for MMP-3 in keratinocytes (6), and HG cultivation significantly reduced IL-22 mRNA expression in PBMC to 65.76 2.24% of their NG-cultivated counterpart. To examine the functional roles of IL-22, relevant neutralizing binding protein was added to the CM derived from NG-cultivated PBMC. Keratinocytes treated with this experimental condition showed significantly reduced MMP-3 expression (Fig. 1c). In addition, migration of keratinocytes treated with NGcultivated PBMC CM was significantly reduced by the addition of IL-22-binding protein (Fig. 1d,e). To determine the functional relevance of MMP-3 on keratinocyte migration, a gene silencing approach was employed. The migration of MMP-3 silenced kerati-
640
Figure 2. (a) At 9 days after wounding, the healing process of normal rat was almost complete, while diabetic rat showed delayed wound healing. (b) The epidermal metalloproteinase (MMP)-3 mRNA expression of normal and diabetic rat skin on day 0 and day 1 after wounding. The increase in MMP-3 expression after wounding was documented. *Indicates p < 0.05 as compared to the normal rat.
nocyte was not significantly different from the non-silenced control (Figure S4). These results indicated that IL-22 in the PBMC CM regulated the migration and MMP-3 expression of keratinocytes distinctively. To confirm our in vitro results in vivo, a diabetic rat model was used. The diabetic rats showed impaired healing (11.8 1.9 days until wound closure) as compared to control (9.3 0.8 days) (Fig. 2a). The IL-22 mRNA expression from diabetic rat skin 1 day after wounding was 53.5 3.7% of control rats. Similar to results from cell model, the folds of epidermal MMP-3 increase after wounding was significantly lower in diabetic rats as compared to control rats (Fig. 2b).
Conclusions In this study, we found that the migration of keratinocytes treated with HG-cultivated PBMC CM was significantly reduced as compared to their NG-cultivated PBMC CM-treated counterpart. In addition, the keratinocytes treated with HG-cultivated PBMC CM showed significantly lower MMP-3 expression as compared to their NG-cultivated PBMC CM-treated counterpart. MMP-3 is a molecule recognized to regulate wound healing process. Physiologically, MMP-3 expression was negligible in normal skin but was observed in keratinocytes after injury (7). Additionally, mice that lacked MMP-3 demonstrated impaired skin healing due to inadequate wound contraction but not keratinocyte migration (8). In
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 630–641
Letter to the Editor
support with this notion, our gene silencing experiment indicated that MMP-3 has limited effect on keratinocyte migration in our experimental condition. Reduced IL-22 production from HG-cultivated PBMC contributed to hampered keratinocyte migration as keratinocyte migration induced by PBMC CM was significantly reduced after the addition of IL-22-binding protein. It has been shown that besides modulating inflammation, IL-22 plays a significant role during wound healing. Upon IL-22 stimulation, cultured keratinocyte showed enhanced migration through the activation of STAT3-related signalling (6). Therefore, reduced production of IL-22 in PBMC under HG environment likely contributed to the impaired wound healing observed in diabetic condition. It is noted that CM from HG-cultivated PBMC only reduced MMP-3 of keratinocytes as compared to their NG-cultivated PBMC CM-treated counterpart, while the effects on other MMPs were limited. A detailed analysis of the proteins contained in various CM is warranted to provide an explanation to this intriguing phenomenon. In summary, we demonstrated that HG cultivation reduced the IL-22 production in PBMCs that significantly reduced MMP3 expression and hampered the migration of treated keratinocytes. Clinically, downregulation of IL-22 and MMP-3 would result in impaired keratinocyte migration and inadequate wound contraction. Increasing IL-22 in the microenvironment of wound may present a novel therapeutic approach to promote diabetic healing.
Acknowledgement This study was supported by National Science Council, Taiwan (grants NSC 101- 2314-B-037-020-MY3), Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan (Grants KMUH1033R49), and Center for Lipid and Glycomedicine, Kaohsiung Medical University, Kaohsiung, Taiwan (KMU- TP103D10). S.M.H., C.S.W. and C.H.W. performed the experiments; D.C., C.C.L., G.S.C. and C.C.L designed the research study. All of the authors analysed the data, wrote the paper and approved the final version.
Conflict of interest The authors have declared no conflicting interests.
Supporting Information Additional supporting data may be found in the supplementary information of this article. Figure S1. (a) Keratinocyte migration after treatment with peripheral blood mononuclear cell (PBMC) culture media with or with out PBMC cultivation. (b) The percent wound closure of figure S1a measured with Image J software from three independent experiments. Figure S2. (a) For osmolarity control, PBMC was cultivated under normal glucose environment with (PBMC M) or without (PBMC N) addition of 20 mM mannitol. (b) The percent wound closure of S2a measured with Image J software from three independent experiments. Figure S3. MMP-3 expression of keratinocytes treated with CM from normal glucose cultivated (PBMC N) and its osmolarity control counterpart (PBMCM). Figure S4. (a) Migratory capacity of cultured human keratinocytes after MMP-3 gene silencing was evaluated by wound scratching assay. (b) The percent wound closure of S4a measured with Image J software from three independent experiments. Table S1. Primer sequences used for real-time PCR analyses. Table S2. Percent of metalloprotianse (MMP) and (TIMP) mRNA expression of human keratinocytes treated with conditioned media (CM) from high glucose (HG) cultivated PBMC as compared to CM from normal glucose (NG) cultivated PBMC. Data S1. Materials and methods.
References
1 Wild S, Roglic G, Green A et al. Diabetes Care 2004: 27: 1047–1053. 2 Yates S, Rayner T E. Wound Repair Regen 2002: 10: 5–15. 3 Legrand C, Gilles C, Zahm J M et al. J Cell Biol 1999: 146: 517–529.
4 Singer A J, Clark R A. N Engl J Med 1999: 341: 738–746. 5 Loots M A, Lamme E N, Zeegelaar J et al. J Invest Dermatol 1998: 111: 850–857. 6 Boniface K, Bernard F X, Garcia M et al. J Immunol 2005: 174: 3695–3702.
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 630–641
7 Pilcher B K, Wang M, Qin X J et al. Ann N Y Acad Sci 1999: 878: 12–24. 8 Bullard K M, Lund L, Mudgett J S et al. Ann Surg 1999: 230: 260–265.
641
