Article |
Address correspondence to Dr. Manuela Martins-Green, Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521. Tel.: (909) 787-2585. Fax: (909) 787-4286. E-mail: mmgreen{at}ucrac1.ucr.edu
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Abstract |
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Key Words: chemokine; myofibroblast; wound healing; 9E3/cCAF; -smooth muscle actin
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Introduction |
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Chemokines are small, positively charged, secreted proteins that consist of an NH2-terminal region of variable conformation followed by a loop, three antiparallel ß strands, and a COOH-terminal helix (Clark-Lewis et al., 1995). They can be divided into four families based on the position of the first two cysteines. The two major families are the CXC family in which the two cysteines are separated by any single amino acid (e.g., cCAF, interleukin [IL]-8, gro
/melanocyte growthstimulating activity [MGSA], SDF-1, PF4, IP-10) and the CC family in which the two cysteines are adjacent (MCPs, RANTES, Eotaxin, MIPs) (Prieschl et al., 1995; Bazan et al., 1997; Zlotnik et al., 1999). These proteins have no modifications other than two disulfide bonds and are multifunctional. Chemokines function in a very tightly regulated dose- and time-dependent manner, strongly suggesting that their actions are affected by the microenvironmental conditions (Dunleavy and Couchman, 1995; Gharaee-Kermani et al., 1996; Rennekampff et al., 1997; Young et al., 1997).
The first evidence that chemokines are associated with healing was reported in 1990 when it was shown that the chemokine chicken chemotactic and angiogenic factor (cCAF) is overexpressed during wound healing (Martins-Green and Bissell, 1990). This chemokine is highly expressed in the first 2448 h after injury and remains elevated for at least 16 d after wounding (Martins-Green and Bissell, 1990; Martins-Green et al., 1992; Martins-Green and Hanafusa, 1997). It is primarily expressed by the fibroblasts of the granulation tissue, especially where interstitial collagen is abundant and by the endothelial cells of microvessels of the granulation tissue (Martins-Green and Bissell 1990; Martins-Green et al., 1996).
In the chicken chorioallantoic membrane (CAM) assay, at low doses cCAF is chemotactic for monocyte/macrophages and lymphocytes and after several days of exposure to this chemokine, the ectoderm of the CAM becomes thickened and a granulation-like tissue develops beneath the cCAF-containing pellet. In this granulation-like tissue, there is an increase in the amount of interstitial collagen and the fibroblasts in the mesoderm are consistently aligned with the collagen fibers and appear to cause tissue contraction (Martins-Green and Feugate, 1998). At high concentrations, however, cCAF stimulates blood vessel sprouting from the preexisting vessels of the CAM (angiogenesis) in the absence of leukocyte chemotaxis (Martins-Green and Feugate, 1998).
Other CXC chemokines have also been associated with wound-healing events. For example, in burn wounds, gro/MGSA is expressed by keratinocytes as they differentiate after reepithelialization of the wound. Furthermore, CXCR2, the receptor for MGSA, is present in migrating and proliferating keratinocytes (Nanney et al., 1995; Rennekampff et al., 1997). In the granulation tissue, MGSA expression is associated with fibroblasts, smooth muscle cells/myofibroblasts, and a subpopulation of macrophages (Nanney et al., 1995). This pattern of expression strongly suggests a role of this chemokine in healing of burn wounds. It also has been shown that in transgenic mice expressing IP-10, a CXC chemokine that inhibits angiogenesis, wounds heal poorly and exhibit defects in development of the granulation tissue (Luster et al., 1998).
It is becoming increasingly more evident that chemokines are expressed at the sites of injury and that they affect processes involving proper development of the granulation tissue. Fibroblasts are critical participants in the development of this healing tissue and they also express high levels of chemokines upon stimulation by stress-inducing agents such as those released upon wounding. Despite this correlative evidence, little is known about how these small cytokines affect wound fibroblast function. cCAF is highly expressed by the fibroblasts of healing tissue, stimulates the formation of granulation-like tissue in the CAM, and is highly homologous to several human chemokines (Stoeckle and Barker, 1990). Therefore, we investigated the effects of this chemokine on proliferation and differentiation of fibroblasts, both important processes during granulation tissue development. We find that cCAF stimulates fibroblasts to differentiate into myofibroblasts and accelerates wound closure in vivo.
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Results |
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When fibroblasts are cultured in the presence of serum, cCAF suppresses proliferation of these cells by 25% compared with untreated cells (Fig. 1; P < 0.01). This effect is dose dependent, with the greatest suppression occurring at 500750 ng cCAF/ml medium (Fig. 1 A), and at 2 d after plating (unpublished data). It is commonly observed that chemokines cause their effects only in a narrow range of concentrations; at concentrations higher than the optimal dose, their receptors are very quickly desensitized and/or downregulated (e.g., Zlotnik et al., 1999; Murdoch and Finn, 2000). Although chemokine concentrations of 10-210 ng/ml can chemoattract and activate leukocytes, chemokines acting on other cell types such as endothelial cells, smooth muscle cells and fibroblasts require concentrations in the range of 102 ng/ml (Gupta and Singh, 1994; Gharaee-Kermani et al., 1996; Luo et al., 1996). This is within physiological range; wound fluid from burn patients has MGSA concentrations of 102103 ng/ml at 67 d after injury (Rennekampff et al., 1997). Experiments using an antibody specific for cCAF showed that this antibody abrogates the effects of this chemokine on proliferation (Fig. 1 B).
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Discussion |
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Although little is known about chemokines and wound closure, expression of cCAF, IL-8, and MGSA is elevated until wound closure and then decreases to low but still elevated levels during granulation tissue formation (Martins-Green and Bissell, 1990; Engelhardt et al., 1998). In addition, knockout mice for CXCR2, a receptor for IL-8 and MGSA, exhibit delayed wound closure (Devalaraja et al., 2000). The results presented here shine light into these finding in vivo. Our observations suggesting that cCAF stimulates -SMA expression directly, that this elevation in expression leads to increased contraction of collagen gels and to more rapid wound contraction and closure, indicate that chemokines potentially play significant roles in formation of the granulation tissue of wounds. Furthermore, the small (15 amino acids) NH2-terminal peptide of the cCAF molecule has the same effects, suggesting that similar behavior in a human chemokine could be a promising target in designing drugs that affect the differentiation of myofibroblasts.
It has been shown that myofibroblasts can differentiate from fibroblasts when these cells are exposed to TGFß1 in culture (Desmouliere et al., 1993) and that, in vivo, this growth factor directly stimulates myofibroblast differentiation (Serini and Gabbiani, 1999). Furthermore, most previously known stimulators of myofibroblast differentiation appear to act indirectly through TGFß1 (Serini and Gabbiani, 1999). Whether cCAF can stimulate -SMA independently of TGFß is not known. However, the fact that this chemokine stimulates
-SMA expression shortly after treatment is initiated, and that it stimulates signals through G-proteincoupled receptors, whereas TGFß1 elicits its effects through serine kinase receptors, suggests that they stimulate different signal transduction pathways to activate
-SMA expression. Nevertheless, it is possible that these signal transduction pathways share common downstream signaling molecules. We are currently investigating the molecular mechanisms of cCAF-induced
-SMA production, whether the human homologues of cCAF function in the same manner, and how they relate to the signaling mechanisms stimulated by TGFß.
Cells containing -SMA (myofibroblasts, smooth muscle cells, and pericytes) play important functions in a variety of processes involved in wound healing, vasculogenesis/angiogenesis, and pathological conditions, especially in diseases that are characterized by excess scarring. In wound healing, myofibroblasts are particularly important in wound closure and contraction. For example, lack of myofibroblasts after corneal surgery leads to corneal flattening and widening of the wound, whereas stimulation of myofibroblast differentiation in noncontractile fetal wounds leads to contraction of the wounds (Jester et al., 1999; Lanning et al., 2000). In disease states characterized by an accumulation of myofibroblasts, such as pulmonary fibrosis and scleroderma, myofibroblasts are thought to contribute significantly to the pathology of the disease, primarily because myofibroblasts tend to be highly fibrogenic (Powell et al., 1999). In addition, myofibroblasts are present in the stroma of many tumors and appear to be important for the survival of these tumors (Coffin et al., 1998; Schürch et al., 1998).
In addition to stimulating wound closure through the differentiation of myofibroblasts, cCAF may also be acting to increase the stability of new blood vessels in the granulation tissue. Smooth muscle cells of the new vasculature are known to differentiate from mesenchymal cells in response to signals from the endothelial cells (e.g., Carmeliet, 2000). These smooth muscle cells are essential for vascular maturation in connective tissue (Carmeliet, 2000). CXC chemokines are produced by the endothelial cells and fibroblasts of the connective tissue and promote angiogenesis (Martins-Green and Bissell, 1990; Martins-Green et al., 1991, 1992; Martins-Green and Feugate, 1998; Belperio et al., 2000). These chemokines are known to affect endothelial cell migration, but part of their role in the formation of new blood vessels may be in stimulating fibroblasts to acquire -SMA and become the smooth muscle cells that stabilize the newly formed vasculature (unpublished data).
Because myofibroblast accumulation is prominent and high levels of chemokines are present in conditions characterized by excessive scarring, such as keloids, scleroderma, and pulmonary fibrosis (Zhang et al., 1996; Nedelec et al., 1998), it is possible that chemokines may participate in such diseases by stimulating myofibroblast differentiation. For example, levels of MCP-1, IL-8, and MIP-1 are high in pulmonary fibrosis (Keane et al., 1997; Hasegawa et al., 1999). These chemokines are also elevated in sclerotic tissue (Kadono et al., 1998; Hasegawa et al., 1999). Myofibroblasts in keloids express MGSA, whereas the cells of normal scars do not (Nirodi et al., 2000). Our results suggest that some of the problems in these conditions may be due to the high levels of chemokines, leading to an increase in myofibroblast numbers, excess deposition of matrix molecules, and contraction of the tissue.
Controlling the differentiation of myofibroblasts could mitigate the effects of fibrotic and other diseases. CXC chemokines could be suitable for this purpose because they are not constitutively expressed and do not have the broad-ranging effects that TGFß1 does. In addition, chemokines are very small molecules with no modification other than disulfide bonds, therefore they can be produced using recombinant approaches without much difficulty and they bind to 7-transmembrane receptors, which are highly amenable to pharmacological manipulations. As a consequence, once the mode of action of these proteins on -SMA expression is deciphered, they or their antagonists could be used to modulate the presence of myofibroblasts in both disease states and in abnormal wound healing. Furthermore, the ability of the 15 amino acid NH2-terminal peptide to stimulate effects similar to those stimulated by the whole cCAF molecule strongly suggests that the peptide itself or peptide mimetics could be used for treatment of wounds with impaired closure.
In conclusion, a major role of cCAF in the granulation tissue development may be the stimulation of proper wound closure through the stimulation of myofibroblast differentiation. This is a previously unknown function for chemokines and it could represent a novel mechanism for the induction of myofibroblast differentiation. In addition, our results may explain why chemokines contribute to the pathology of fibrotic diseases in which myofibroblasts play a significant part.
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Materials and methods |
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Cell cultures
Primary chicken embryonic connective tissue fibroblasts (CEFs) were cultured as described previously in Martins-Green and Feugate (1998). Briefly, these fibroblasts were isolated from 10-d-old chicken embryo body walls, plated at 6 x 106 cells/100-mm plate and cultured for 4 d in 199 medium with 5% donor calf serum, 1% chick serum, and 0.3% tryptose phosphate broth. These cultures were passaged once (secondary cultures); the cells were plated at 0.4 x 106 cells/35-mm plate in 199/tryptose phosphate broth and 2% donor calf serum.
Fibroblast growth assay
Secondary fibroblasts were plated at 0.4 x 106 cells/35-mm plate and cCAF was added to experimental plates. For each experiment, plating efficiency was determined by trypsinizing plates 4 h after plating. Cells were counted with a Coulter particle counter to confirm even plating of cells. The media was replaced approximately every 16 h with 1 ml of serum-free 199 medium containing 0.3% tryptose phosphate broth and 2% donor calf serum, and 1001,000 ng cCAF (990 nM) or C- or N-peptide (64640 nM) was added to the experimental plates, whereas control plates contained media only. On day 3, plates were trypsinized to remove all cells and cells were counted using a Coulter counter. To test for specificity, anti-cCAF rabbit serum was preincubated with cCAF (3 µl serum/750 ng cCAF [68 nM] or N-peptide [480 nM]) for 1 h at room temperature before being added to cells.
Trypan blue staining
The supernatant of treated and untreated CEFs was collected and the cells centrifuged to a pellet and resuspended in a small volume of medium. 1% trypan blue was added to a final concentration of 0.5% trypan blue. Cells were stained for 5 min and then counted in a hemocytometer. Necrotic cells stained blue.
Apoptosis assay
To detect cCAF-induced apoptosis we used the DNA Laddering Detection System from Roche Biochem. Fibroblasts were plated, treated as described above, and the DNA was prepared as suggested by the manufacturer. Briefly, at the end of each treatment, cells were trypsinized, pelleted, resuspended in binding buffer, and incubated for 10 min at room temperature. After this incubation period, isopropanol was added and the preparation vortexed and passed through a filter tube with glass binding fleece. The samples were then centrifuged for 1 min at 8,000 rpm, the flow through was discarded, and the DNA retained in the fleece was washed two times with wash buffer and then eluted with prewarmed elution buffer and used for agarose gel electrophoresis.
Immunostaining for -SMA
Plates of fibroblasts were treated with cCAF as described previously. After 4 d, the cells were rinsed with PBS, fixed in 4% paraformaldehyde, permeabilized with 0.15% Triton X-100, and incubated with PBS containing 0.1 M glycine for 10 min. Cells were blocked for 30 min with 10% goat serum in PBS, incubated with mouse anti-SMA to a final IgG concentration of 20 µg/ml in 1% BSA/PBS for 1 h at room temperature, and washed three times with 0.1% BSA/PBS for 10 min each. The cells were then incubated in To-Pro3 (1:1,000) and goat antimouse FITC or sheep antimouse Texas red (1:100) in 1% BSA/PBS for 1 h at room temperature, washed three times for 10 min with 0.1% BSA in PBS, and mounted with Vectashield. Confocal fluorescence microscopy was performed on a ZEISS LSM510. Collagen gels were rinsed with PBS, fixed in 4% paraformaldehyde for 2 h, washed three times 30 min each with PBS, and incubated for an additional 30 min in PBS containing 0.1 M glycine. This treatment was followed by incubation at 4°C O/N in 15% sucrose and then incubation under the same conditions in 30% sucrose. After a brief rinse with PBS, the gels were frozen in OCT, sections were prepared and collected on gelatin-coated slides, rinsed with PBS, fixed in 4% paraformaldehyde for 10 min, and then incubated again in PBS containing 0.1 M glycine for 10 min. This was followed by blocking for 30 min with 10% goat serum in PBS, incubation in primary and secondary antibody as described above, and mounting with Vectashield.
Immunoblotting
Plates of fibroblasts were treated as described previously with 750 ng/ml cCAF or 2.5 ng/ml TGFß. To block TGFß or cCAF activity, anti-cCAF (3 µl) or anti-TGF (1 µl) antibody was preincubated in 1 ml media for 1 h at room temperature before being added to cells. After 4 d of treatment, protein extracts were prepared in 1 ml 150 mM RIPA buffer containing protease inhibitors. Protein concentrations were determined using the DC protein assay kit and samples were adjusted to contain equal amounts of protein. SDS-PAGE was performed on 7.5% separating Doucet gels (Doucet and Trifaro, 1988). Protein transfer to nitrocellulose was performed using a wet-transfer apparatus (Bio-Rad Laboratories) at 100 V for 45 min. The membranes were blocked for 1 h in 5% milk in TTBS and then incubated overnight at 4°C in anti
-SMA (1:1,500), antivimentin (1:100), antidesmin (1:100), antimyosin heavy chain (1:100), or anti-myoD (1:200) in 1% milk in TTBS. The membranes were washed three times for 20 min each with TTBS, incubated in anti-mouse HRP at 1:10,000 in 1% milk for 1 h, and washed as above; and the bands were visualized using the ECL.
Collagen gel contraction
1.5 ml Vitrogen 100 collagen gels were made in 35-mm plates. Secondary fibroblasts were plated at 0.4 x 106 cells/35-mm plate on top of the gels. After the cells had adhered to the collagen, cCAF was added to experimental plates. The medium was replaced approximately every 16 h with 1 ml of 199 supplemented with 0.3% tryptose phosphate broth and 2% donor calf serum, and 750 ng cCAF (68 nM) or N-peptide (480 nM) was added to the experimental plates. Control plates contained media only. After 4 d, gels were released. Treatment was continued for two more days and gels were photographed every 12 h. The photographs were used for evaluation of gel contraction by determining the area of the gel using NIH image analysis. For the experiments involving inhibition of -SMA expression, we used phosphorothioate antisense ODN, which was synthesized and HPLC purified by Sigma-Aldrich Genosys, to block the
-SMA production. The sequence of ODN is specific for the 3'-untranslated region of
-SMA (5'-CACAGTAATATGCTAAAAAGAC-3') as described previously (Ronnov-Jessen and Petersen, 1996; Nakajima et al., 1999). The sense strand of this ODN was used as control (5'-GTCTTTTTAGCATATTACTGTG-3'). When preparing the collagen gels, 2 µM of antisense or sense ODN were incorporated into the gel before the seeding of CEFs. The treatments and measurement of gel contraction were performed as described above, except that 2 µM of antisense or sense ODN were applied each time the chemokine or peptide was applied.
RT-PCR
Total RNA for -SMA was extracted using TRIzol reagent from untreated fibroblasts, fibroblasts treated with 750 ng/ml cCAF, or fibroblasts treated for varying periods of time with cCAF. The RT-PCR procedure was performed using the Promega Access RT-PCR System, which is designed to finish RT and PCR in one tube and following the protocol recommended by Promega, except that 1.5 times the recommended amount of dNTP, reverse transcriptase, and Tfl DNA polymerase were used to ensure strong synthesis of 18S ribosomal RNA. The reaction conditions included: 1 µg total RNA, first strand synthesis at 48°C for 45 min, then 95°C for 5 min to inactivate the reverse transcriptase, followed by DNA amplification at 95°C for 45 s, 58°C for 60 s, 68°C for 90 s for 40 cycles. Finally, 68°C for 7 min to extend the strands. 3 µl of Quantum mRNA classic 18S primers (Ambion) were added to the reaction to produce the control band. The primers used for the amplification of
-SMA were: sense primer 5'-GGAGCACCTGAGGACATTGAC-3' and antisense primer 5'-GCTTCAGTCAGCAGAGTTGGG-3'. RT-PCR products were analyzed by electrophoresis in 1.5% agarose and the density of the bands was measured by densitometry using Glyko BandScan.
Wounding experiment
Full-thickness excision wounds (0.5 x 0.5 cm) were made using a scalpel blade on the underside of the wings of 2-wk-old chicks. The left wing was treated with vehicle alone (water) and the right wing with 1 µg cCAF (90 nM). The wounds were photographed immediately after wounding and then covered with Biocclusive bandage. 50 µl vehicle (water) or cCAF was deposited through the bandage onto the wound using a 30-gauge needle. This procedure was repeated the next day and every other day thereafter. On days 3, 5, and 7 the bandages were removed and the wounds were photographed before replacing the bandages and applying the treatment again. Wings were placed on a flat surface at a distance of 25 cm from the digital camera and were always photographed with the same camera settings. All images were printed at the same magnification.
Preparation and staining of wing sections
At the specified time points, chickens were killed with sodium pentobarbitol. The wounded wings were collected and fixed for 18 h in 4% paraformaldehyde and decalcified for 3 d in 5% formic acid, 2.5% formaldehyde at 4°C. The tissue was embedded in paraffin and sectioned. Sections were stained with Masson Trichrome to visualize interstitial collagen. Other sections were immunolabeled for -SMA. Sections were deparaffinized three times with 15 min washes in Hemo-De and rehydrated in 5 min washes of ethanol (100, 95, 70, 50, and 30%). After rinsing with PBS, sections were fixed in 2% paraformaldehyde 1 h. Autofluoresence and nonspecific staining was blocked by 30 min in 0.1 M glycine in PBS, followed by 30 min in 1% Evans Blue in PBS to quench red blood cell autofluorescence. Sections were incubated with
-SMA antibody in 1% BSA in PBS (1:50) for 2 h at room temperature. After three 10 min washes in 0.1% BSA in PBS, the sections were incubated for 40 min with antimouse Alexa antibody in 1% BSA in PBS (1:200). They were then washed and mounted with Vectashield. To quantify the number of myofibroblasts in the wounds, the number of fluorescently labeled cells in four high power fields of each wound was counted and statistical analysis was applied.
Statistical methods
Significance was determined using Student's t test for comparison between two means and ANOVA for comparison between more than two means. All data were examined to assure homogeneity of variance. Means were considered significantly different when P < 0.05.
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Footnotes |
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Acknowledgments |
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This work was supported in part by a Dissertation Research Grant from the University of California, Riverside, and a GAANN fellowship (US Department of Education) to J.E. Feugate and by National Institutes of Health grant GM48436 to M. Martins-Green.
Submitted: 13 March 2001
Revised: 27 November 2001
Accepted: 27 November 2001
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