Differential Activation of Migration by Hypoxia in Keratinocytes Isolated from Donors of Increasing Age: Implication for Chronic Wounds in the Elderly
2001; Elsevier BV; Volume: 116; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.2001.00209.x
ISSN1523-1747
AutoresYuping Xia, Yanan Zhao, John W. Tyrone, Alex Chen, Thomas A. Mustoe,
Tópico(s)Periodontal Regeneration and Treatments
ResumoChronic wound healing conditions are often observed in elderly patients with poor tissue oxygenation. Impaired re-epithelialization is a hallmark of these wounds, which is seen in both clinical studies and in our animal models of impaired healing. To investigate the pathogenic mechanism of chronic wounds, we studied the effect of hypoxia on migration of keratinocytes isolated from human donors of increasing age. Keratinocytes from elderly donors had depressed migratory activity when exposed to hypoxia, as opposed to an increase in migration in young cells. Analysis of underlying biochemical changes demonstrated a differential activation of matrix metalloproteinases by hypoxia in keratinocytes isolated from the young and the old. Matrix metalloproteinases-1 and -9 and tissue inhibitor of matrix metalloproteinase-1 were strongly upregulated by hypoxia in young cells, whereas no induction was observed in aged cells. Furthermore, transforming growth factor-β1 signaling appears to be involved in the keratinocyte differential response to hypoxia, as transforming growth factor-β type I receptor was upregulated by hypoxia in young cells, while there was no induction in aged cells. Transforming growth factor-β neutralizing reagents blocked hypoxia-induced matrix metalloproteinase-1, matrix metalloproteinase-9 expression, and hypoxia-induced cell migration as well. Our results suggest that an age-related decrease in response to hypoxia plays a crucial part in the pathogenesis of retarded re-epithelialization in wound. Chronic wound healing conditions are often observed in elderly patients with poor tissue oxygenation. Impaired re-epithelialization is a hallmark of these wounds, which is seen in both clinical studies and in our animal models of impaired healing. To investigate the pathogenic mechanism of chronic wounds, we studied the effect of hypoxia on migration of keratinocytes isolated from human donors of increasing age. Keratinocytes from elderly donors had depressed migratory activity when exposed to hypoxia, as opposed to an increase in migration in young cells. Analysis of underlying biochemical changes demonstrated a differential activation of matrix metalloproteinases by hypoxia in keratinocytes isolated from the young and the old. Matrix metalloproteinases-1 and -9 and tissue inhibitor of matrix metalloproteinase-1 were strongly upregulated by hypoxia in young cells, whereas no induction was observed in aged cells. Furthermore, transforming growth factor-β1 signaling appears to be involved in the keratinocyte differential response to hypoxia, as transforming growth factor-β type I receptor was upregulated by hypoxia in young cells, while there was no induction in aged cells. Transforming growth factor-β neutralizing reagents blocked hypoxia-induced matrix metalloproteinase-1, matrix metalloproteinase-9 expression, and hypoxia-induced cell migration as well. Our results suggest that an age-related decrease in response to hypoxia plays a crucial part in the pathogenesis of retarded re-epithelialization in wound. matrix metalloproteinase tissue inhibitor of matrix metalloproteinase Clinical observations suggest that the development of chronic wounds frequently associates with persistent low tissue oxygen supply (hypoxia). The prolonged tissue hypoxia exposes wounds to bacterial infection, a prolonged inflammatory response, and eventually tissue necrosis (Niinikoski et al., 1972Niinikoski J. Hunt T.K. Dunphy J.E. Oxygen supply in healing tissue.Am J Surg. 1972; 123: 247-252Abstract Full Text PDF PubMed Scopus (95) Google Scholar;Franklin and Poyton, 1996Franklin B. Poyton R.O. Oxygen sensing and molecular adaptation to hypoxia.Physiol Rev. 1996; 76: 839-885PubMed Google Scholar). The elderly population accounts for a large portion of this morbidity (Frantz and Gardner, 1994Frantz R.A. Gardner S. Elderly skin care: principles of chronic wound care.J Gerontol Nurs. 1994; 20: 35-44Crossref PubMed Scopus (3) Google Scholar;Van de Kerkhof et al., 1994Van de Kerkhof P.C. Bergen B. Spruijt K. Kuiper J.P. Age-related changes in wound healing.Clin Exp Dermatol. 1994; 19: 369-374Crossref PubMed Scopus (64) Google Scholar). Despite the debilitating effect of chronic wounds in the elderly, the pathogenesis of chronic wounds is poorly understood. Consistent with clinical observations, compelling evidence from laboratory studies have shown that age affects wound healing in several aspects: (i) sprouting of aged microvessels was significantly less than the sprouting of young microvessels (Arthur et al., 1998Arthur W.T. Vernon R.B. Sage E.H. Reed M.J. Growth factors reverse the impaired sprouting of microvessels from aged mice.Microvasc Res. 1998; 55: 260-270https://doi.org/10.1006/mvre.1998.2078Crossref PubMed Scopus (71) Google Scholar); (ii) increased gelatinase and collagenase levels in skin of aged donors (Ashcroft et al., 1997aAshcroft G.S. Horan M.A. Ferguson M.W. Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model.J Invest Dermatol. 1997; 108: 430-437Crossref PubMed Scopus (180) Google Scholar) and in wound fluid from chronic leg ulcers (Wysocki et al., 1993Wysocki A.B. Staiano-Coico L. Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9.J Invest Dermatol. 1993; 101: 64-68Abstract Full Text PDF PubMed Google Scholar;Weckroth et al., 1996Weckroth M. Vaheri A. Lauharanta J. Sorsa T. Konttinen Y.T. Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers.J Invest Dermatol. 1996; 106: 1119-1124Crossref PubMed Scopus (205) Google Scholar); (iii) decreased TIMP (tissue inhibitor of matrix metalloproteinase) levels in the skin of aged donors (Ashcroft et al., 1997bAshcroft G.S. Herrick S.E. Tarnuzzer R.W. Horan M.A. Schultz G.S. Ferguson M.W. Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP) -1 and -2 proteins and mRNA.J Pathol. 1997; 183: 169-176Crossref PubMed Scopus (94) Google Scholar); and (iv) reduced deposition of matrix components and re-epithelialization (Ashcroft et al., 1997cAshcroft G.S. Horan M.A. Herrick S.E. Tarnuzzer R.W. Schultz G.S. Ferguson M.W.J. Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMP) in normal skin and acute cutaneous wounds of healthy humans.Cell Tissue Res. 1997; 290: 581-591https://doi.org/10.1007/s004410050963Crossref PubMed Scopus (149) Google Scholar). Our study presents novel observations with respect to age contribution to the altered migration in response to hypoxia. This age-modulated hypoxia response causes imbalance of matrix metalloproteinase (MMP) and TIMP expression. Our data also indicate the transforming growth factor (TGF) -β signaling pathway as a potential mediator of downstream MMP/TIMP imbalance that ultimately impairs re-epithelialization. To investigate pathologic mechanism of chronic wounds, we studied the migration of skin keratinocytes and associated biochemical changes in a hypoxic in vitro wound healing environment. During normal process of wound healing, basal keratinocytes begin to migrate over the provisional wound bed within several hours after injury. Over the next few days, in the absence of complications, the migrating keratinocytes proliferate to re-establish the epithelium along with the basement membrane (Grinnell, 1992Grinnell F. Wound repair, keratinocyte activation and integrin modulation.J Cell Sci. 1992; 101: 1-5PubMed Google Scholar;Cavani et al., 1993Cavani A. Zambruno G. Marconi A. Manca V. Marchetti M. Giannetti A. Distinctive integrin expression in the newly forming epidermis during wound healing in humans.Invest Dermatol. 1993; 101: 600-604Crossref PubMed Scopus (170) Google Scholar). Studies have shown that migration of keratinocytes depends on the function of MMP (Pilcher et al., 1997Pilcher B.K. Dumin J.A. Sudbeck B.D. Krane S.M. Welgus H.G. Parks W.C. The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix.J Cell Biol. 1997; 137: 1445-1457Crossref PubMed Scopus (472) Google Scholar). MMP are a family of zinc-dependent endopeptidases that have previously been implicated in the pathobiology of chronic wounds, because a high level of MMP has been detected in exudate and wound tissue (Wysocki et al., 1993Wysocki A.B. Staiano-Coico L. Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9.J Invest Dermatol. 1993; 101: 64-68Abstract Full Text PDF PubMed Google Scholar;Moses et al., 1996Moses M.A. Marikovsky M. Harper J.W. Vogt P. Eriksson E. Klagsbrun M. Langer R. Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing.J Cell Biochem. 1996; 60: 379-386Crossref PubMed Scopus (108) Google Scholar;Vaalamo et al., 1996Vaalamo M. Weckroth M. Puolakkainen P. Kere J. Saarinen P. Lauharanta J. Saarialho-Kere U.K. Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds.Br J Dermatol. 1996; 135: 52-59Crossref PubMed Scopus (171) Google Scholar;Weckroth et al., 1996Weckroth M. Vaheri A. Lauharanta J. Sorsa T. Konttinen Y.T. Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers.J Invest Dermatol. 1996; 106: 1119-1124Crossref PubMed Scopus (205) Google Scholar;Ashcroft et al., 1997aAshcroft G.S. Horan M.A. Ferguson M.W. Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model.J Invest Dermatol. 1997; 108: 430-437Crossref PubMed Scopus (180) Google Scholar). These proteinases are needed for extracellular matrix cleavage during re-epithelialization (Pilcher et al., 1997Pilcher B.K. Dumin J.A. Sudbeck B.D. Krane S.M. Welgus H.G. Parks W.C. The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix.J Cell Biol. 1997; 137: 1445-1457Crossref PubMed Scopus (472) Google Scholar), as well as the remodeling phase of wound healing (Agren, 1994Agren M.S. Gelatinase activity during wound healing.Br J Dermatol. 1994; 131: 634-640Crossref PubMed Scopus (137) Google Scholar). Several forms of MMP have been implicated to play important parts in wound repair. Interstitial collagenase MMP-1, a protease that cleaves fibrillar collagens types I, II, and III at a specific locus in their triple helical domains (Welgus et al., 1981Welgus H.G. Jeffery J.J. Eisen A.Z. The collagen substrate specificity of human skin fibroblast collagenase.J Biol Chem. 1981; 256: 9511-9515Abstract Full Text PDF PubMed Google Scholar;Wu et al., 1990Wu H. Byrne M.H. Stacey A. Goldring M.B. Birkhead J.R. Jaenisch R. Krane S.M. Generation of collagenase-resistant collagen by site-directed mutagenesis of murine pro alpha 1 (I) collagen gene.Proc Natl Acad Sci USA. 1990; 87: 5888-5892Crossref PubMed Scopus (90) Google Scholar;Liu et al., 1995Liu X. Wu H. Byrne M. Jeffrey J. Krane S. Jaenisch R. A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling.J Cell Biol. 1995; 130: 227-237Crossref PubMed Scopus (235) Google Scholar), is expressed by migrating basal keratinocytes in all types of wounds with a breached basement membrane (Vaalamo et al., 1997Vaalamo M. Mattila L. Johansson N. Kariniemi A.L. Karjalainen-Lindsberg M.L. Kahari V.M. Saarialho-Kere U. Distinct populations of stromal cells express collagenase-3 (MMP-13) and collagenase-1 (MMP-1) in chronic ulcers but not in normally healing wounds.J Invest Dermatol. 1997; 109: 96-101Crossref PubMed Scopus (234) Google Scholar;Sudbeck et al., 1997Sudbeck B.D. Pilcher B.K. Welgus H.G. Parks W.C. Induction and repression of collagenase-1 by keratinocytes is controlled by distinct components of different extracellular matrix compartments.J Biol Chem. 1997; 272: 22103-22110Crossref PubMed Scopus (93) Google Scholar). Mutations that block the proteolytic activity of MMP-1 inhibit the migration process of keratinocytes on a provisional wound surface (Pilcher et al., 1997Pilcher B.K. Dumin J.A. Sudbeck B.D. Krane S.M. Welgus H.G. Parks W.C. The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix.J Cell Biol. 1997; 137: 1445-1457Crossref PubMed Scopus (472) Google Scholar;Sudbeck et al., 1997Sudbeck B.D. Pilcher B.K. Welgus H.G. Parks W.C. Induction and repression of collagenase-1 by keratinocytes is controlled by distinct components of different extracellular matrix compartments.J Biol Chem. 1997; 272: 22103-22110Crossref PubMed Scopus (93) Google Scholar). Other studies have shown that wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases, MMP-2 (72 kDa) and MMP-9 (92 kDa) (Wysocki et al., 1993Wysocki A.B. Staiano-Coico L. Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9.J Invest Dermatol. 1993; 101: 64-68Abstract Full Text PDF PubMed Google Scholar;Moses et al., 1996Moses M.A. Marikovsky M. Harper J.W. Vogt P. Eriksson E. Klagsbrun M. Langer R. Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing.J Cell Biochem. 1996; 60: 379-386Crossref PubMed Scopus (108) Google Scholar;Weckroth et al., 1996Weckroth M. Vaheri A. Lauharanta J. Sorsa T. Konttinen Y.T. Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers.J Invest Dermatol. 1996; 106: 1119-1124Crossref PubMed Scopus (205) Google Scholar;Ashcroft et al., 1997cAshcroft G.S. Horan M.A. Herrick S.E. Tarnuzzer R.W. Schultz G.S. Ferguson M.W.J. Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMP) in normal skin and acute cutaneous wounds of healthy humans.Cell Tissue Res. 1997; 290: 581-591https://doi.org/10.1007/s004410050963Crossref PubMed Scopus (149) Google Scholar). MMP-2 and MMP-9, also known as gelatinase A and B, respectively, cleave type I, type IV and V collagens, and elastin (Salo et al., 1994Salo T. Makela M. Kylmaniemi M. Autio-Harmainen H. Larjava H. Expression of matrix metalloproteinase-2 and -9 during early human wound healing.Lab Invest. 1994; 70: 176-182PubMed Google Scholar). MMP-2 was recently described specifically to cleave laminin-5 (Giannelli et al., 1997Giannelli G. Falk-Marzillier J. Schiraldi O. Stetler-Stevenson W.G. Quaranta V. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5.Science. 1997; 277: 225-228https://doi.org/10.1126/science.277.5323.225Crossref PubMed Scopus (1010) Google Scholar), a major epidermal basement membrane component in intact skin (Carter et al., 1991Carter W.G. Ryan M.C. Gahr P.J. Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes.Cell. 1991; 65: 599-610Abstract Full Text PDF PubMed Scopus (647) Google Scholar). In the extracellular environment, the activation of metalloenzymes is regulated, in part, by TIMP. TIMP-1 is a 29 kDa N-glycosylated protein that forms high-affinity, noncovalent complexes with pro-MMP-9 and active MMP-1, -3, and -9, and inhibits the catalytic activity of these MMP (Woessner, 1991Woessner Jr., J.F. Matrix metalloproteinases and their inhibitors in connective tissue remodelling.FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3009) Google Scholar;Murphy and Reynolds, 1993Murphy G. Reynolds J.J. Extracellular matrix degradation.in: Royce P.M. Steinmann B. Connective Tissue and its Heritable Disorders. Wiley, New York1993: 287-316Google Scholar). Another physiologic inhibitor of MMP, TIMP-2, is a 22 kDa protein that forms complexes with pro-MMP-2 and active MMP-2 (Murphy and Reynolds, 1993Murphy G. Reynolds J.J. Extracellular matrix degradation.in: Royce P.M. Steinmann B. Connective Tissue and its Heritable Disorders. Wiley, New York1993: 287-316Google Scholar;Howard and Banda, 1991Howard E.W. Banda M.J. Binding of tissue inhibitor of metalloproteinases 2 to two distinct sites on human 72-kDa gelatinase. Identification of a stabilization site.J Biol Chem. 1991; 266: 17972-17977Abstract Full Text PDF PubMed Google Scholar). Studies have shown that TIMP-1 expression was not detected in chronic wound biopsies (Vaalamo et al., 1996Vaalamo M. Weckroth M. Puolakkainen P. Kere J. Saarinen P. Lauharanta J. Saarialho-Kere U.K. Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds.Br J Dermatol. 1996; 135: 52-59Crossref PubMed Scopus (171) Google Scholar). In acute wounds, however, TIMP-1 was expressed at the epithelial edges, colocalizing with MMP-1 and MMP-9 (Vaalamo et al., 1996Vaalamo M. Weckroth M. Puolakkainen P. Kere J. Saarinen P. Lauharanta J. Saarialho-Kere U.K. Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds.Br J Dermatol. 1996; 135: 52-59Crossref PubMed Scopus (171) Google Scholar;Madlener et al., 1998Madlener M. Parks W.C. Werner S. Matrix metalloproteinases (MMP) and their physiological inhibitors (TIMP) are differentially expressed during excisional skin wound repair.Exp Cell Res. 1998; 242: 201-210https://doi.org/10.1006/excr.1998.4049Crossref PubMed Scopus (273) Google Scholar). Thus, the level of TIMP may have a profound effect on re-epithelialization of wounds. The potential involvement of TGF-β signaling in the hypoxic response was also tested. This is based on our previous study using an animal model of ischemia-impaired wound healing (Wu et al., 1999Wu L. Xia Y.-P. Siddiqui A. Roth S. Gruskin E. Mustoe T. TGF-beta 1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic ulcer model: the importance of oxygen and age.Am J Pathol. 1999; 154: 301-309Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We have documented (in rabbit wound healing) that the level of TGF-β1 mRNA increased significantly in ischemic over the nonischemic wounds of young animals. In contrast, there was no obvious TGF-β induction in ischemic wounds of aged animals (Wu et al., 1999Wu L. Xia Y.-P. Siddiqui A. Roth S. Gruskin E. Mustoe T. TGF-beta 1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic ulcer model: the importance of oxygen and age.Am J Pathol. 1999; 154: 301-309Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Other studies have shown that TGF-β1 regulates MMP mRNA expression in a cell-type-specific manner. In keratinocytes, TGF-β1 induces the expression of MMP-1 and MMP-9 (Mauviel et al., 1996Mauviel A. Chung K.Y. Agarwal A. Tamai K. Uitto J. Cell-specific induction of distinct oncogenes of the June family is responsible for differential regulation of collagenase gene expression by transforming growth factor-β in fibroblasts and keratinocytes.J Biol Chem. 1996; 271: 10917-10923Crossref PubMed Scopus (136) Google Scholar;Uria et al., 1998Uria J.A. Jimenez M.G. Balbin M. Freije J.M.P. Lopez-Otin C. Differential effects of transforming growth factor-beta on the expression of collagenase-1 and collagenase-3 in human fibroblasts.J Biol Chem. 1998; 273: 9769-9777Crossref PubMed Scopus (181) Google Scholar). Despite the debilitating effect of chronic wounds in elderly patients, the pathophysiologic factors that cause the nonhealing conditions are unclear. In this study, we examined the effect of age and hypoxia on keratinocyte migration. A reduced migration was observed under hypoxia in cultured keratinocytes isolated from aged donors. This finding was consistent with a previous observation of impaired epithelialization in an animal model of ischemic wounds (Wu et al., 1999Wu L. Xia Y.-P. Siddiqui A. Roth S. Gruskin E. Mustoe T. TGF-beta 1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic ulcer model: the importance of oxygen and age.Am J Pathol. 1999; 154: 301-309Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Here we examined the migratory response of aged keratinocytes to hypoxia, and a potential regulatory mechanism through the action of MMP and TIMP. We also found that TGF-β neutralizing reagents repressed hypoxia-induced MMP-1 and -9 expression and hypoxia-modulated keratinocyte migration. Primary human skin keratinocyte cultures were established from skin biopsy samples of healthy donors undergoing elective surgical procedures. All tissue was collected in accordance with the guidelines of the North-western University Human Subjects Review Committee. Skin tissue was first digested with dispase to remove the dermis. The remaining epidermis was subsequently digested with trypsin to release keratinocytes from tissue. To maintain cell phenotype in culture, low passage cells (passage 2) were used in our experiments. Cultures were maintained in keratinocyte-SFM medium (Gibco BRL, Gaithersburg, MD) and the medium was changed every other day. Age groups were classified as young (20–39 y old), middle (40–59 y old), and old age (≥ 60 y old). A total of seven different donors were used for each age group, and the results were uniformly consistent, although the magnitudes varied. The aged cells retained their ‘‘aged’' phenotype in vitro. They grew more slowly, quickly became senescent (after three to four passages), and adhered more slowly. To subject cell cultures to hypoxia, keratinocytes were placed in a large hypoxia incubator (Coy Laboratory Products, Glass Lake, MI), which allows precise oxygen and temperature regulation whereas permitting media change and other manipulations through a gloved box. The incubator was pre-equilibrated with a gas mixture of 5% CO2/95% N2 to achieve an O2 level of 1%. Culture medium was flushed with N2, then pre-equilibrated under 1% oxygen tension for 48 h prior to use and the hypoxia incubator is monitored and maintained at this oxygen level throughout the experiment. Keratinocyte migration was assessed using the method of Albrecht-Buehler as modified by Woodley (Woodley et al., 1988Woodley D.T. Bachmann P.M. O'keefe E.J. Laminin inhibits human keratinocyte migration.J Cell Physiol. 1988; 136: 140-146Crossref PubMed Scopus (137) Google Scholar). Briefly, colloidal gold salts were immobilized on coverslips and covered with extracellular matrix protein type I collagen (15 μg per ml). Early passage keratinocytes were plated on to the coverslips and allowed to migrate for 20 h. The cells were fixed in 0.1% formaldehyde in phosphate-buffered saline and examined under dark field optics with a video camera attached to a computer equipped with a frame grabber. The computer analyzed 15 nonoverlapping fields in each experimental condition with NIH Image 1.4 and determined the area of each field consumed by cell migration tracks as a percentage of the entire field, termed the migration index. The lengths of individual tracks were also calculated. All migration assays were performed at 1% oxygen tension for hypoxia and 21% oxygen tension for normoxia. Antibodies against MMP-1, -2, and -9 and TIMP-1 and -2 (Oncogene Research, Cambridge, MA) were used at 1:100 dilution in immunoblotting. Peroxidase-conjugated rabbit antimouse IgG (DAKO, Carpinteria, CA) was used at 1:5000 dilution as a secondary antibody. TGF-β RI and II antibodies (Santa Cruz, Santa Cruz, CA) were used at 1:200 dilution for immunoblotting. Recombinant human TGF-β sRII/Fc chimera is commercially available from R&D Systems (Minneapolis, MN). Neutralizing TGF-β antibody is a generous gift from Genzyme (Cambridge, MA). Expression levels of keratinocyte MMP and TIMP were quantitated by immunoblotting analysis. For the detection of MMP, aliquots of equal amounts of protein extracts were resolved by 10% sodium dodecyl sulfate–polyacrylamide gels and transferred to nitrocellulose membranes in 20 mM Tris–HCl, pH 8.0, 150 mM glycine, 20% (vol/vol) methanol. The membranes were blocked with 5% (vol/vol) nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20), incubated with MMP-1, -9, or -2 specific antibody (diluted 1:100), washed, incubated with horseradish peroxidase anti-immunoglobulin conjugate (diluted 1:5000) and extensively washed. Protein bands were visualized using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). For detection of TIMP, NuPAGE 10% Bis-Tris gel (Invitrogen, Carlsbad, CA) was used for electrophoresis and protein was transferred in NuPAGE transfer buffer (Novex) and immunoblotted under the same conditions as described above. Human keratinocytes were plated on type I collagen at a density of 17,500 cells per cm2. The cells were incubated under normoxic and hypoxic conditions for 20 h before collecting conditioned culture medium. Gelatinolytic proteinases were assayed by gelatin-substrate enzymography. Gelatin was melted at 50°C for 20 min and added at a 1.5% final concentration to a 10% acrylamide gel. The conditioned media was concentrated using a Centricon-10 microconcentrator (Millipore, Bedford, MA). Aliquots of the media were prepared for electrophoresis without heating or reducing reagents. After sodium dodecyl sulfate–polyacrylamide gel electrophoresis, sodium dodecyl sulfate was removed from the gels by 2.5% (vol/vol) Triton X-100 washes (2 × 20 min), the gels were incubated in assay buffer (15 mM Tris–HCl, pH 7.4, 5 mM CaCl2) at 37°C for 24 h. The reaction was stopped by staining the gels with Coomassie Brilliant Blue (BioRad, Hercules, CA). Gelatinolytic activity was detected as clear bands against the Aqua Blue-stained gelatin background. To quantitate the intensity of individual bands on autoradiographs, the optical density was measured by scanning the films with a Bio-Rad BLS-670 densitometer (Bio-Rad) and analyzed using the computer software program Molecular Analyst (BioRad). All data presented as mean ± SEM. Differences between means of two age groups were evaluated by a paired two-tailed Student's t test, with the aid of Excel version 5.0 (Microsoft, Redmond, WA). p ≤ 0.05 was considered statistically significant. Each test using cultured keratinocytes was repeated at least three times to include cells isolated from different donors. One of the crucial events in re-establishing the basement membrane after cutaneous injury is the migration of basal keratinocytes over the dermal matrices. To assess if the migration of aged keratinocytes is altered by hypoxia, we studied cell migration under hypoxia and normoxia using a primary culture of keratinocytes. Human keratinocytes isolated from healthy aged donors were plated on a type I collagen-coated surface and cultured under either hypoxia (1% oxygen) or normoxia (21% oxygen) for 20 h. The migration index was determined by gold salt migration assay (O'Toole et al., 1997O'Toole E.A. Marinkovich M.P. Peavey C.L. Amieva M.R. Furthmayr H. Mustoe T.A. Woodley D.T. Hypoxia increases human keratinocyte motility on connective tissue.J Clin Invest. 1997; 100: 2881-2891Crossref PubMed Scopus (110) Google Scholar, which allows the measurement of individual phagokinetic tracks of a single cell. Interestingly, the aged cells showed a significant reduced migration when exposed to hypoxia (Figure 1a,b), whereas the control young keratinocytes showed an upregulation of migration (Figure 1c,d) as previously reported (O'Toole et al., 1997O'Toole E.A. Marinkovich M.P. Peavey C.L. Amieva M.R. Furthmayr H. Mustoe T.A. Woodley D.T. Hypoxia increases human keratinocyte motility on connective tissue.J Clin Invest. 1997; 100: 2881-2891Crossref PubMed Scopus (110) Google Scholar). The reduced migration was not due to a general cytotoxic effect of hypoxia exerted on aged cells, as the aged cells seemed to have a normal degree of viability under hypoxia as assessed by Trypan Blue staining (data not shown). As MMP-1 activity is required for keratinocyte migration on type I collagen matrix (Pilcher et al., 1997Pilcher B.K. Dumin J.A. Sudbeck B.D. Krane S.M. Welgus H.G. Parks W.C. The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix.J Cell Biol. 1997; 137: 1445-1457Crossref PubMed Scopus (472) Google Scholar), we first investigated the MMP-1 expression in response to hypoxia in several different age groups. Keratinocytes from donors of increasing age were plated on a type I collagen surface, allowed sufficient time to attach, and transferred to either a hypoxic or normoxic condition. Conditioned culture supernatants were collected 20 h after exposure to hypoxia or normoxia and analyzed for MMP-1 expression by immunoblotting. Expression of both the active form (42–46 kDa) and inactive form (52–57 kDa) of MMP-1 increased with hypoxia in young cells, but not in aged cells under the same low oxygen tension (Figure 2). These results suggest that MMP-1 is likely to play an important part in hypoxia-modulated keratinocyte migration on collagen matrix. We also examined the hypoxia-modulated expression of MMP-2 and MMP-9, gelatinases that become activated in wound repair. Gelatin zymography was carried out to determine the protein level of active and inactive forms of MMP. The protocol involves in-gel activation of MMP by incubating in assay buffer as specified in Materials and Methods section. We found that the expression of the active form of MMP-9 (83 kDa) is predominant over the inactive form (92 kDa) on zymography (Figure 3a). Zymography demonstrated that the active form of MMP-9 was stimulated by hypoxia in young cells cultured on type I collagen. On the contrary, hypoxia repressed the expression of MMP-9 in aged keratinocytes (Figure 3a). We also noticed that there was a higher level of MMP-9 in aged keratinocytes compa
Referência(s)