- Email: [email protected]
Hypoxic conditions decrease the mRNA expression of proα1(I) and (III) collagens and increase matrix metalloproteinases-1 of dermal fibroblasts in three-dimensional cultures
Journal of Dermatological Science 24 (2000) 99 – 104 www.elsevier.com/locate/jdermsci
Hypoxic conditions decrease the mRNA expression of proa1(I) and (III) collagens and increase matrix metalloproteinases-1 of dermal fibroblasts in three-dimensional cultures Masayoshi Yamanaka *, Osamu Ishikawa Department of Dermatology, Gunma Uni6ersity School of Medicine, 3 -39 -22, Showa-machi, Maebashi, Gunma 371 -8511, Japan Received 25 October 1999; received in revised form 19 January 2000; accepted 20 January 2000
Abstract The effect of hypoxia on the expression of extracellular matrix-related genes by human dermal fibroblasts was investigated using a novel three-dimensional culture supplemented with L-ascorbic acid 2-phosphate. Experiments were performed by placing replicate dishes in either hypoxic (2%) or in normoxic (20%) condition for various periods of time ranging up to 72 h. The mRNA expression levels of proa1(I), proa1(III) collagens and MMP-1 were analyzed using Northern blotting. Hypoxia transiently increased proa1(I) and proa1(III) collagen gene expression at 24 h, but a prolonged exposure to hypoxia decreased them. A slight increase in MMP-1 mRNA was observed at 24 h and prolonged exposure for up 72 h resulted in significantly increased expression of MMP-1 gene. Our results suggest that enhanced degradation as well as decreased synthesis of collagens induced by hypoxia may account for the delayed wound healing associated with circulatory disturbances. © 2000 Published by Elsevier Science Ireland Ltd. Keywords: Hypoxia; Fibroblasts; Three-dimensional culture; Collagens; Matrix metalloproteinases
1. Introduction Low oxygen tension (hypoxia) plays a pivotal role in many physiologic or pathologic conditions such as wound healing [1,2], fibrosis [3], and tumors [4]. Although the pathogenesis of chronic wounds recalcitrant to treatments is multifacto* Corresponding author. Tel.: +81-27-2208284; fax: + 8127-2208285. E-mail address: [email protected] (M. Yamanaka).
rial, the significance of hypoxia is well establised in chronic skin ulcers [1,2,5], especially in venous ulceration or in ulcers due to atherosclerosis and diabetes [6]. Accumulation of extracelluler matrices (ECM) occurs as a result of an imbalance between synthesis and degradation of ECM [7]. Previous in vitro studies showed that during hypoxic conditions the proliferation and longevity of fibroblasts were stimulated [8] and collagen gene expression was increased [9–11]. Cellular synthetic activity was also enhanced during hypoxia
0923-1811/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 0 8 6 - 4
100
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
[8,9,12–14]. The above results lead to the hypothesis that hypoxia may stimulate the fibrotic response in chronic wounds, and fibrotic tissue may interfere with proper tissue repair [15]. Alternative hypothesis focused on excessive proteinase production as a contributing factor for delayed healing [16,17]. Indeed, some authors reported that wound fluid from chronic ulcers contained elevated levels of metalloproteinases [16 – 19]. However, the effects of hypoxia on the degradative aspect of ECM have been studied only in proximal tubular cell [10]. In this study, we investigated the mRNA expression of the genes for proa1(I), proa1(III) collagens and matrix metalloproteinase-1 (MMP-1) in normal human dermal fibroblasts during hypoxic conditions. In our study of the fibroblast culture system, we introduced a novel three-dimensional culture, which provided more physiological environments for cultured fibroblasts [20 – 23].
2. Materials and methods
2.1. Cell culture Skin samples were taken from five normal individuals at operation after obtaining informed consent. Cells were initiated from explant cultures and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) in an atmosphere of 37°C humidified air and 5% CO2. At the second passage, fibroblasts were subcultured into three flasks (75 cm2), and experiments were conducted using cells between three and five passages. Fibroblasts were seeded at 5× 105 cells/10 cm dish in DMEM supplemented with 10% FCS and 1 mM magnesium salt of L-ascorbic acid 2-phosphate (Asc-2p, Wako Pure Chemical Industries, Osaka, Japan) for 2 weeks. The medium was changed twice a week. Asc-2p used in this experiment is stable and active for up to 7 days under conventional culture conditions. The supplementation of Asc-2p can render cells to the organization of the self-produced three-dimensional structure during this incubation period [20 – 22].
2.2. Hypoxic culture conditions To achieve hypoxic condition, culture dishes were transferred to a N2 –CO2 –O2 controllable incubator (APMW-36, ASTEC Co., Ltd., Fukuoka, Japan). Experiments were performed by placing replicate dishes in either hypoxic (2%) or in normoxic (20%) condition for up to 72 h. No obvious morphological changes were observed by phasecontrast light microscopy in fibroblasts in monolayer or three-dimensional cultures exposed to hypoxic condition for 72 h.
2.3. RNA extraction and Northern blot analysis The cell layer was harvested at 24, 48 and 72 h and rinsed three times with cold phosphate buffer saline. Total RNA was isolated from the cell layer by the guanidine thiocyanate–phenol–chloroform method using ISOGEN (Nippon gene, Toyama, Japan). Aliquot of 10 mg of total RNA denatured in formaldehyde were electrophoresed in 1.2% agarose–1.1 mol/l formaldehyde gels. The RNA was then transferred to a nylon membrane and cross-linked by exposure to 120 mJ/cm2 of 312 nm UV radiation in a spectro UV cross-linker (Spectronics Corporation, Westburg, NY). Filters were hybridized to probes labeled with [a-32P] dCTP by the random priming method (Gibco BRL, Gaithersburg, MD). Hybridization was performed at 42°C. After hybridization, the filters were washed and exposed to autoradiography film (Kodak XAR, Eastman Kodak Co., Rochester, NY) at − 80°C. The intensity was quantified using a Fujix BAS 2000 bioimage analyzer (Fuji Photo Film Co., Tokyo, Japan). The following human sequence-specific cDNAs were used for hybridization: a 1.4 kb cDNA, Hf677-6, for proa1(I) collagen mRNA, a 0.9 kb cDNA, pH, for proa1(III) collagen mRNA, a 0.7 kb cDNA, K4, for MMP-1 mRNA (kindly provided by Dr A.Hatamochi, Chiba University), 1.1 kb cDNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA purchased from Clontech (Palo Alto, CA), and a 0.5 kb cDNA for b-actin mRNA purchased from Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
101
2.4. Statistical analysis Results of the experiments using five fibroblast cell lines from different donors were expressed as mean 9S.D. Mean values were statistically analyzed by analysis of variance followed by Student’s t-test.
3. Results
3.1. The analysis of mRNA expression of GAPDH and b-actin In hypoxic conditions, the expression of GAPDH mRNA increased gradually, however, b-actin mRNA expression was unchanged (Fig. 1). Therefore, we used b-actin as an internal control instead of GAPDH in the present study.
Fig. 2. Northern blot of proa1(I) collagen, proa1(III) collagen, MMP-1, and b-actin mRNA expression in three-dimensional cultures during hypoxic conditions for 24, 48 and 72 h. The representative results are depicted.
3.2. The analyses of mRNA expression of proa1(I) collagen, proa1(III) collagen and MMP-1 in a hypoxic condition The representative results of Northern blot are shown in Fig. 2. Hypoxic conditions slightly increased mRNA expression of proa1(I) collagen at 24, 48 h and decreased significantly at 72 h (* PB 0.05 vs. 24 h, Fig. 3a). Similarly, hypoxic conditions significantly increased mRNA expression of proa1(III) collagen at 24 h (* PB 0.05 vs. control) and significantly decreased mRNA expression at 72 h (** PB 0.05 vs. 24, 48 h, Fig. 3b). By contrast, hypoxic conditions significantly enhanced the mRNA expression of MMP-1 at 48 h (* PB 0.01 vs. control, 24 h, Fig. 3c).
4. Discussion Fig. 1. Changes in GAPDH and b-actin mRNA expression in three-dimensional culture during hypoxic conditions. (a) The representative results of northern blot during hypoxic conditions for 24, 48 and 72 h. (b) The intensity of GAPDH mRNA expression normalized by the intensity of b-actin expression. Data are expressed as mean 9 S.D. of five samples. * PB 0.05 vs. control; ** P B 0.01 vs. control.
The significance of hypoxia in the occurrence of chronic skin ulcers has been extensively investigated [1,2,5]. For example, the dermal diffusion of oxygen measured by transcutaneous oxygen pressure decreased in limbs affected by venous ulcers
102
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
that are usually refractory to conservative treatments [2]. This is thought to be a consequence of the barrier function of fibrin deposition around the capillaries [2]. Although molecular oxygen is required for collagen synthesis and many cellular functions [24], accumulating evidence suggests that hypoxia has a stimulatory effect on angiogenesis [12], fibroblast proliferation [8], and the transcription and secretion of several growth factors [13,14]. The above results support the hypothesis that hypoxia may stimulate the fibrotic response in chronic wounds, and fibrotic tissue may delay the tissue repair process [15]. With respect to
Fig. 3. The changes in mRNA expression of proa1(I) collagen, proa1(III) collagen and MMP-1 during hypoxic conditions. The intensity of proa1(I) collagen mRNA, proa1(III) collagen mRNA and MMP-1 mRNA level was normalized by the intensity of b-actin expression, respectively. Data are expressed as mean 9S.D. of five samples. (a) * PB 0.05 vs. 24 h; (b) * PB 0.05 vs. control, ** PB 0.05 vs. 24, 48 h (c) * PB 0.01 vs. control 24 h.
collagens, increased mRNA expression of type I and III collagen(s) was reported in human dermal fibroblasts during hypoxic conditions for 96 h [9] and in rat cardiac fibroblasts for 48 h. It is of note that these experiments were performed using a monolayer culture system. By contrast, Herrick et al. [25] demonstrated the opposite result in human dermal fibroblasts. They found that the mRNA expression of type I collagen decreased during hypoxic conditions both in a monolayer and in a collagen gel culture. Furthermore, the decrease was more prominent in a collagen gel culture than in a monolayer culture. In our study, the experimental conditions, included both a three-dimensional culture and prolonged exposure to hypoxia. These conditions might have been critical in the decreasing mRNA expression of proa1(I) and proa1(III) collagens. Since in vivo fibroblasts are surrounded by ECM and hypoxic microenvironments are persistently present in chronic wounds, our experimental conditions may more closely reflect the in vivo conditions. It was reported that wound fluids from chronic skin ulcers contained elevated levels of MMPs: MMP-1, MMP-8, MMP-2 and MMP-9 [16–19]. The effect of hypoxia on the degradative aspect of ECM was studied only in proximal tubular cells [10], and have not been examined in dermal fibroblasts. The matrix metalloproteinases (MMPs) are a family of at least 14 distinct but structurally related neutral proteinases that can coordinately degrade ECM components [26]. At present, four separate mammalian enzymes are known to possess the ability to degrade fibrillar collagen: interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8), collagenase-3 (MMP-13), and MMP-2. Our results support the hypothesis that excessive proteinase production may bring about a delay of wound healing. It was previously reported that the expression of MMP-1 mRNA was minimal in monolayer conditions, but the level of MMP-1 mRNA was markedly enhanced in a collagen gel [17] or in our three-dimensional culture system [23]. Recently, it was reported that fibroblasts in a collagen gel produced less collagen during hypoxic conditions than during normoxic conditions [25]. These observations strongly suggest that cell to matrix
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
interactions are an important regulatory mechanism for MMP-1 production. It should be noted, however, that most previous studies were carried out in a monolayer culture system [8 – 11]. In order to examine the MMP gene expression and accumulation of ECM in fibroblasts culture, we believe a three-dimensional culture is more appropriate than a monolayer culture. In this study, we used a novel fibroblast culture system supplemented with L-ascorbic acid 2-phosphate (Asc-2p) [20 – 23]. The addition of Asc-2p enables dermal fibroblasts to organize a dermis-like structure by accumulating self-produced ECM around fibroblasts. This three-dimensional culture system can provide fibroblasts with close cell – matrix interactions more similar to in vivo conditions than a monolayer or collagen gel culture system. We demonstrated that hypoxia enhanced the expression of GAPDH mRNA. Previous studies had reported that the expression of GAPDH mRNA was enhanced by hypoxia in endothelial cells [27]. Our experiment is the first report of evidence that hypoxia also up-regulates GAPDH gene expression in dermal fibroblasts. In conclusion, we demonstrated that hypoxia increased the mRNA expression of MMP-1 and decreased mRNA expression of type I and III collagens in a novel three-dimensional culture. These results indicate that both the enhanced collagen degradation and the suppressed collagen synthesis induced by hypoxia may lead to a delay in wound healing associated with circulatory disturbances. Acknowledgements This study was supported in part by the Ministry of Health Welfare Scleroderma Research Committee (1999) and a research grant from the Ministry of Education, Science and Culture of Japan (90168188). References [1] Falanga V. Chronic wounds: pathophysiologic and experimental considerations. J Invest Dermatol 1993;100:721– 5.
103
[2] Falanga V, Mckenzie A, Eaglstein WM. Heterogeneity in oxygen around venous ulcers. J Dermatol Surg Oncol 1991;17:336 – 9. [3] Silverstein JL, Steen VD, Medsger TA, Falanga V. Cutaneous hypoxia in patients with systemic sclerosis (scleroderma). Arch Dermatol 1988;124:1379– 82. [4] Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply and metabolic environment of human tumors. Cancer Res 1989;49:6449 – 65. [5] Clyne CAC, Ramsden WH, Chant ADB, Webster JH. Oxygen tension on the skin of the gaiter area of limbs with venous disease. Br J Surg 1985;72:644 – 7. [6] Pecoraro RE. The nonhealing diabetic ulcer-major cause for limb loss. Prog Clin Biol Res 1991;365:27 – 43. [7] Woessner JF, Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145 – 54. [8] Falanga V, Kirsner RS. Low oxygen stimulates proliferation of fibroblasts seeded as single cells. J Cell Physiol 1993;154:506 – 10. [9] Falanga V, Martin TA, Takagi H, Kirsner RS, Helfman T, Pardes J, Ochoa MF. Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts. J Cell Physiol 1993;157:408 – 12. [10] Orphanides C, Fine LG, Norman JT. Hypoxia stimulates proximal tubular cell matrix production via a TGF-b1-independent mechanism. Kidney Int 1997;52:637 – 47. [11] Nunoyama H, Kusachi S, Ninoyama Y, Oohashi T, Kondo J, Murakami M, Murakami T, Ohnishi H, Doi M, Tsuji T. Hypoxia increases transforming growth factor-b1 concomitantly with types I and III collagen without further enhancement by reoxygenation in cultured rat cardiac fibroblasts. Connect Tissue 1998;30:207 – 11. [12] Knighton DR, Hunt TK, Scheunstuhl H, Halliday BJ. Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 1983;221:1283– 5. [13] Falanga V, Qian SW, Danielpour D, Katz MH, Roberts AB, Sporn MB. Hypoxia upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts. J Invest Dermatol 1991;97:634 – 7. [14] Kourembanas S, Hannan RL, Faller DV. Oxygen tension regulates the expression of the platelat-derived growth factor-B chain gene in human endothelial cells. J Clin Invest 1990;86:670 – 4. [15] Falanga V, Grinnell F, Gilchrest B, Maddox YT, Moshe A. Workshop on the pathogenesis of chronic wounds. J Invest Dermatol 1994;102:125 – 7. [16] Wysocki AB, Coico LS, Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9. J Invest Dermatol 1993;101:64 – 8. [17] Vaalamo M, Mattila L, Johansson N, Kariniemi AL, Lindsberg MLK, Khri VM, Kere US. Distinct population of stromal cells express collagenase-3 (MMP-1) and collagenase-1 (MMP-1) in chronic ulcers but not in normally healing wounds. J Invest Dermatol 1997;109:96 – 101.
104
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
[18] Weckroth M, Vaheri A, Lauharanta J, Sorsa T, Konttinen YT. Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers. J Invest Dermatol 1996;106:1119–24. [19] Yager DR, Zhang LY, Liang HX, Diegelmann RF, Cohen IK. Wound fluids from human pressure ulcers contain elevated matrix metalloproteinase levels and activity compared to surgical wound fluids. J Invest Dermatol 1996;107:743 – 8. [20] Hata R, Senoo H. L-Ascorbic acid 2-phosphate stimulates collagen accumlation, cell proliferation, and formation of three-dimensional tissue like substance by skin fibroblasts. J Cell Physiol 1989;138:8–16. [21] Ishikawa O, Kondo A, Okada K, Miyachi Y, Furumura M. Morphological and biochemical analyses on fibroblasts and self-produced collagens in a novel three-dimensional culture. Br J Dermatol 1997;136:6–11. [22] Ishikawa O, Yokoyama Y, Miyachi Y. Disaccharide analysis of dermal fibroblast-derived glycosaminoglycans in the three-dimensional culture. J Dermatol Sci 1994;8:203–7.
.
[23] Sato M, Ishikawa O, Abe M, Miyachi Y. Opposite effects of tumour necrosis factor-a on type I and III collagen gene expression by human dermal fibroblasts in monolayer and three-dimensional cultures. Br J Dermatol 1998;138:118 – 21. [24] Prokop DJ, Kivirikko KI, Tuderman L, Guzman NA. Biosynthesis of collagen and its disorders. N Engl J Med 1979;301:13 – 23. [25] Herrick SE, Ireland GW, Simon D, McCollum CN, Ferguson MWJ. Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions. J Invest Dermatol 1996;106:187 – 93. [26] Birkedal HH. Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol 1995;7:728 – 35. [27] Graven KK, Mcdonald RJ, Farber HW. Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase. Am J Physiol 1998;274:C347– 55.
Hypoxic conditions decrease the mRNA expression of proa1(I) and (III) collagens and increase matrix metalloproteinases-1 of dermal fibroblasts in three-dimensional cultures Masayoshi Yamanaka *, Osamu Ishikawa Department of Dermatology, Gunma Uni6ersity School of Medicine, 3 -39 -22, Showa-machi, Maebashi, Gunma 371 -8511, Japan Received 25 October 1999; received in revised form 19 January 2000; accepted 20 January 2000
Abstract The effect of hypoxia on the expression of extracellular matrix-related genes by human dermal fibroblasts was investigated using a novel three-dimensional culture supplemented with L-ascorbic acid 2-phosphate. Experiments were performed by placing replicate dishes in either hypoxic (2%) or in normoxic (20%) condition for various periods of time ranging up to 72 h. The mRNA expression levels of proa1(I), proa1(III) collagens and MMP-1 were analyzed using Northern blotting. Hypoxia transiently increased proa1(I) and proa1(III) collagen gene expression at 24 h, but a prolonged exposure to hypoxia decreased them. A slight increase in MMP-1 mRNA was observed at 24 h and prolonged exposure for up 72 h resulted in significantly increased expression of MMP-1 gene. Our results suggest that enhanced degradation as well as decreased synthesis of collagens induced by hypoxia may account for the delayed wound healing associated with circulatory disturbances. © 2000 Published by Elsevier Science Ireland Ltd. Keywords: Hypoxia; Fibroblasts; Three-dimensional culture; Collagens; Matrix metalloproteinases
1. Introduction Low oxygen tension (hypoxia) plays a pivotal role in many physiologic or pathologic conditions such as wound healing [1,2], fibrosis [3], and tumors [4]. Although the pathogenesis of chronic wounds recalcitrant to treatments is multifacto* Corresponding author. Tel.: +81-27-2208284; fax: + 8127-2208285. E-mail address: [email protected] (M. Yamanaka).
rial, the significance of hypoxia is well establised in chronic skin ulcers [1,2,5], especially in venous ulceration or in ulcers due to atherosclerosis and diabetes [6]. Accumulation of extracelluler matrices (ECM) occurs as a result of an imbalance between synthesis and degradation of ECM [7]. Previous in vitro studies showed that during hypoxic conditions the proliferation and longevity of fibroblasts were stimulated [8] and collagen gene expression was increased [9–11]. Cellular synthetic activity was also enhanced during hypoxia
0923-1811/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 0 8 6 - 4
100
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
[8,9,12–14]. The above results lead to the hypothesis that hypoxia may stimulate the fibrotic response in chronic wounds, and fibrotic tissue may interfere with proper tissue repair [15]. Alternative hypothesis focused on excessive proteinase production as a contributing factor for delayed healing [16,17]. Indeed, some authors reported that wound fluid from chronic ulcers contained elevated levels of metalloproteinases [16 – 19]. However, the effects of hypoxia on the degradative aspect of ECM have been studied only in proximal tubular cell [10]. In this study, we investigated the mRNA expression of the genes for proa1(I), proa1(III) collagens and matrix metalloproteinase-1 (MMP-1) in normal human dermal fibroblasts during hypoxic conditions. In our study of the fibroblast culture system, we introduced a novel three-dimensional culture, which provided more physiological environments for cultured fibroblasts [20 – 23].
2. Materials and methods
2.1. Cell culture Skin samples were taken from five normal individuals at operation after obtaining informed consent. Cells were initiated from explant cultures and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) in an atmosphere of 37°C humidified air and 5% CO2. At the second passage, fibroblasts were subcultured into three flasks (75 cm2), and experiments were conducted using cells between three and five passages. Fibroblasts were seeded at 5× 105 cells/10 cm dish in DMEM supplemented with 10% FCS and 1 mM magnesium salt of L-ascorbic acid 2-phosphate (Asc-2p, Wako Pure Chemical Industries, Osaka, Japan) for 2 weeks. The medium was changed twice a week. Asc-2p used in this experiment is stable and active for up to 7 days under conventional culture conditions. The supplementation of Asc-2p can render cells to the organization of the self-produced three-dimensional structure during this incubation period [20 – 22].
2.2. Hypoxic culture conditions To achieve hypoxic condition, culture dishes were transferred to a N2 –CO2 –O2 controllable incubator (APMW-36, ASTEC Co., Ltd., Fukuoka, Japan). Experiments were performed by placing replicate dishes in either hypoxic (2%) or in normoxic (20%) condition for up to 72 h. No obvious morphological changes were observed by phasecontrast light microscopy in fibroblasts in monolayer or three-dimensional cultures exposed to hypoxic condition for 72 h.
2.3. RNA extraction and Northern blot analysis The cell layer was harvested at 24, 48 and 72 h and rinsed three times with cold phosphate buffer saline. Total RNA was isolated from the cell layer by the guanidine thiocyanate–phenol–chloroform method using ISOGEN (Nippon gene, Toyama, Japan). Aliquot of 10 mg of total RNA denatured in formaldehyde were electrophoresed in 1.2% agarose–1.1 mol/l formaldehyde gels. The RNA was then transferred to a nylon membrane and cross-linked by exposure to 120 mJ/cm2 of 312 nm UV radiation in a spectro UV cross-linker (Spectronics Corporation, Westburg, NY). Filters were hybridized to probes labeled with [a-32P] dCTP by the random priming method (Gibco BRL, Gaithersburg, MD). Hybridization was performed at 42°C. After hybridization, the filters were washed and exposed to autoradiography film (Kodak XAR, Eastman Kodak Co., Rochester, NY) at − 80°C. The intensity was quantified using a Fujix BAS 2000 bioimage analyzer (Fuji Photo Film Co., Tokyo, Japan). The following human sequence-specific cDNAs were used for hybridization: a 1.4 kb cDNA, Hf677-6, for proa1(I) collagen mRNA, a 0.9 kb cDNA, pH, for proa1(III) collagen mRNA, a 0.7 kb cDNA, K4, for MMP-1 mRNA (kindly provided by Dr A.Hatamochi, Chiba University), 1.1 kb cDNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA purchased from Clontech (Palo Alto, CA), and a 0.5 kb cDNA for b-actin mRNA purchased from Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
101
2.4. Statistical analysis Results of the experiments using five fibroblast cell lines from different donors were expressed as mean 9S.D. Mean values were statistically analyzed by analysis of variance followed by Student’s t-test.
3. Results
3.1. The analysis of mRNA expression of GAPDH and b-actin In hypoxic conditions, the expression of GAPDH mRNA increased gradually, however, b-actin mRNA expression was unchanged (Fig. 1). Therefore, we used b-actin as an internal control instead of GAPDH in the present study.
Fig. 2. Northern blot of proa1(I) collagen, proa1(III) collagen, MMP-1, and b-actin mRNA expression in three-dimensional cultures during hypoxic conditions for 24, 48 and 72 h. The representative results are depicted.
3.2. The analyses of mRNA expression of proa1(I) collagen, proa1(III) collagen and MMP-1 in a hypoxic condition The representative results of Northern blot are shown in Fig. 2. Hypoxic conditions slightly increased mRNA expression of proa1(I) collagen at 24, 48 h and decreased significantly at 72 h (* PB 0.05 vs. 24 h, Fig. 3a). Similarly, hypoxic conditions significantly increased mRNA expression of proa1(III) collagen at 24 h (* PB 0.05 vs. control) and significantly decreased mRNA expression at 72 h (** PB 0.05 vs. 24, 48 h, Fig. 3b). By contrast, hypoxic conditions significantly enhanced the mRNA expression of MMP-1 at 48 h (* PB 0.01 vs. control, 24 h, Fig. 3c).
4. Discussion Fig. 1. Changes in GAPDH and b-actin mRNA expression in three-dimensional culture during hypoxic conditions. (a) The representative results of northern blot during hypoxic conditions for 24, 48 and 72 h. (b) The intensity of GAPDH mRNA expression normalized by the intensity of b-actin expression. Data are expressed as mean 9 S.D. of five samples. * PB 0.05 vs. control; ** P B 0.01 vs. control.
The significance of hypoxia in the occurrence of chronic skin ulcers has been extensively investigated [1,2,5]. For example, the dermal diffusion of oxygen measured by transcutaneous oxygen pressure decreased in limbs affected by venous ulcers
102
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
that are usually refractory to conservative treatments [2]. This is thought to be a consequence of the barrier function of fibrin deposition around the capillaries [2]. Although molecular oxygen is required for collagen synthesis and many cellular functions [24], accumulating evidence suggests that hypoxia has a stimulatory effect on angiogenesis [12], fibroblast proliferation [8], and the transcription and secretion of several growth factors [13,14]. The above results support the hypothesis that hypoxia may stimulate the fibrotic response in chronic wounds, and fibrotic tissue may delay the tissue repair process [15]. With respect to
Fig. 3. The changes in mRNA expression of proa1(I) collagen, proa1(III) collagen and MMP-1 during hypoxic conditions. The intensity of proa1(I) collagen mRNA, proa1(III) collagen mRNA and MMP-1 mRNA level was normalized by the intensity of b-actin expression, respectively. Data are expressed as mean 9S.D. of five samples. (a) * PB 0.05 vs. 24 h; (b) * PB 0.05 vs. control, ** PB 0.05 vs. 24, 48 h (c) * PB 0.01 vs. control 24 h.
collagens, increased mRNA expression of type I and III collagen(s) was reported in human dermal fibroblasts during hypoxic conditions for 96 h [9] and in rat cardiac fibroblasts for 48 h. It is of note that these experiments were performed using a monolayer culture system. By contrast, Herrick et al. [25] demonstrated the opposite result in human dermal fibroblasts. They found that the mRNA expression of type I collagen decreased during hypoxic conditions both in a monolayer and in a collagen gel culture. Furthermore, the decrease was more prominent in a collagen gel culture than in a monolayer culture. In our study, the experimental conditions, included both a three-dimensional culture and prolonged exposure to hypoxia. These conditions might have been critical in the decreasing mRNA expression of proa1(I) and proa1(III) collagens. Since in vivo fibroblasts are surrounded by ECM and hypoxic microenvironments are persistently present in chronic wounds, our experimental conditions may more closely reflect the in vivo conditions. It was reported that wound fluids from chronic skin ulcers contained elevated levels of MMPs: MMP-1, MMP-8, MMP-2 and MMP-9 [16–19]. The effect of hypoxia on the degradative aspect of ECM was studied only in proximal tubular cells [10], and have not been examined in dermal fibroblasts. The matrix metalloproteinases (MMPs) are a family of at least 14 distinct but structurally related neutral proteinases that can coordinately degrade ECM components [26]. At present, four separate mammalian enzymes are known to possess the ability to degrade fibrillar collagen: interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8), collagenase-3 (MMP-13), and MMP-2. Our results support the hypothesis that excessive proteinase production may bring about a delay of wound healing. It was previously reported that the expression of MMP-1 mRNA was minimal in monolayer conditions, but the level of MMP-1 mRNA was markedly enhanced in a collagen gel [17] or in our three-dimensional culture system [23]. Recently, it was reported that fibroblasts in a collagen gel produced less collagen during hypoxic conditions than during normoxic conditions [25]. These observations strongly suggest that cell to matrix
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
interactions are an important regulatory mechanism for MMP-1 production. It should be noted, however, that most previous studies were carried out in a monolayer culture system [8 – 11]. In order to examine the MMP gene expression and accumulation of ECM in fibroblasts culture, we believe a three-dimensional culture is more appropriate than a monolayer culture. In this study, we used a novel fibroblast culture system supplemented with L-ascorbic acid 2-phosphate (Asc-2p) [20 – 23]. The addition of Asc-2p enables dermal fibroblasts to organize a dermis-like structure by accumulating self-produced ECM around fibroblasts. This three-dimensional culture system can provide fibroblasts with close cell – matrix interactions more similar to in vivo conditions than a monolayer or collagen gel culture system. We demonstrated that hypoxia enhanced the expression of GAPDH mRNA. Previous studies had reported that the expression of GAPDH mRNA was enhanced by hypoxia in endothelial cells [27]. Our experiment is the first report of evidence that hypoxia also up-regulates GAPDH gene expression in dermal fibroblasts. In conclusion, we demonstrated that hypoxia increased the mRNA expression of MMP-1 and decreased mRNA expression of type I and III collagens in a novel three-dimensional culture. These results indicate that both the enhanced collagen degradation and the suppressed collagen synthesis induced by hypoxia may lead to a delay in wound healing associated with circulatory disturbances. Acknowledgements This study was supported in part by the Ministry of Health Welfare Scleroderma Research Committee (1999) and a research grant from the Ministry of Education, Science and Culture of Japan (90168188). References [1] Falanga V. Chronic wounds: pathophysiologic and experimental considerations. J Invest Dermatol 1993;100:721– 5.
103
[2] Falanga V, Mckenzie A, Eaglstein WM. Heterogeneity in oxygen around venous ulcers. J Dermatol Surg Oncol 1991;17:336 – 9. [3] Silverstein JL, Steen VD, Medsger TA, Falanga V. Cutaneous hypoxia in patients with systemic sclerosis (scleroderma). Arch Dermatol 1988;124:1379– 82. [4] Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply and metabolic environment of human tumors. Cancer Res 1989;49:6449 – 65. [5] Clyne CAC, Ramsden WH, Chant ADB, Webster JH. Oxygen tension on the skin of the gaiter area of limbs with venous disease. Br J Surg 1985;72:644 – 7. [6] Pecoraro RE. The nonhealing diabetic ulcer-major cause for limb loss. Prog Clin Biol Res 1991;365:27 – 43. [7] Woessner JF, Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145 – 54. [8] Falanga V, Kirsner RS. Low oxygen stimulates proliferation of fibroblasts seeded as single cells. J Cell Physiol 1993;154:506 – 10. [9] Falanga V, Martin TA, Takagi H, Kirsner RS, Helfman T, Pardes J, Ochoa MF. Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts. J Cell Physiol 1993;157:408 – 12. [10] Orphanides C, Fine LG, Norman JT. Hypoxia stimulates proximal tubular cell matrix production via a TGF-b1-independent mechanism. Kidney Int 1997;52:637 – 47. [11] Nunoyama H, Kusachi S, Ninoyama Y, Oohashi T, Kondo J, Murakami M, Murakami T, Ohnishi H, Doi M, Tsuji T. Hypoxia increases transforming growth factor-b1 concomitantly with types I and III collagen without further enhancement by reoxygenation in cultured rat cardiac fibroblasts. Connect Tissue 1998;30:207 – 11. [12] Knighton DR, Hunt TK, Scheunstuhl H, Halliday BJ. Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 1983;221:1283– 5. [13] Falanga V, Qian SW, Danielpour D, Katz MH, Roberts AB, Sporn MB. Hypoxia upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts. J Invest Dermatol 1991;97:634 – 7. [14] Kourembanas S, Hannan RL, Faller DV. Oxygen tension regulates the expression of the platelat-derived growth factor-B chain gene in human endothelial cells. J Clin Invest 1990;86:670 – 4. [15] Falanga V, Grinnell F, Gilchrest B, Maddox YT, Moshe A. Workshop on the pathogenesis of chronic wounds. J Invest Dermatol 1994;102:125 – 7. [16] Wysocki AB, Coico LS, Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9. J Invest Dermatol 1993;101:64 – 8. [17] Vaalamo M, Mattila L, Johansson N, Kariniemi AL, Lindsberg MLK, Khri VM, Kere US. Distinct population of stromal cells express collagenase-3 (MMP-1) and collagenase-1 (MMP-1) in chronic ulcers but not in normally healing wounds. J Invest Dermatol 1997;109:96 – 101.
104
M. Yamanaka, O. Ishikawa / Journal of Dermatological Science 24 (2000) 99–104
[18] Weckroth M, Vaheri A, Lauharanta J, Sorsa T, Konttinen YT. Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers. J Invest Dermatol 1996;106:1119–24. [19] Yager DR, Zhang LY, Liang HX, Diegelmann RF, Cohen IK. Wound fluids from human pressure ulcers contain elevated matrix metalloproteinase levels and activity compared to surgical wound fluids. J Invest Dermatol 1996;107:743 – 8. [20] Hata R, Senoo H. L-Ascorbic acid 2-phosphate stimulates collagen accumlation, cell proliferation, and formation of three-dimensional tissue like substance by skin fibroblasts. J Cell Physiol 1989;138:8–16. [21] Ishikawa O, Kondo A, Okada K, Miyachi Y, Furumura M. Morphological and biochemical analyses on fibroblasts and self-produced collagens in a novel three-dimensional culture. Br J Dermatol 1997;136:6–11. [22] Ishikawa O, Yokoyama Y, Miyachi Y. Disaccharide analysis of dermal fibroblast-derived glycosaminoglycans in the three-dimensional culture. J Dermatol Sci 1994;8:203–7.
.
[23] Sato M, Ishikawa O, Abe M, Miyachi Y. Opposite effects of tumour necrosis factor-a on type I and III collagen gene expression by human dermal fibroblasts in monolayer and three-dimensional cultures. Br J Dermatol 1998;138:118 – 21. [24] Prokop DJ, Kivirikko KI, Tuderman L, Guzman NA. Biosynthesis of collagen and its disorders. N Engl J Med 1979;301:13 – 23. [25] Herrick SE, Ireland GW, Simon D, McCollum CN, Ferguson MWJ. Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions. J Invest Dermatol 1996;106:187 – 93. [26] Birkedal HH. Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol 1995;7:728 – 35. [27] Graven KK, Mcdonald RJ, Farber HW. Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase. Am J Physiol 1998;274:C347– 55.