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From Surgical Services,*
Massachusetts General
Hospital/Shriners Burns Hospital/Harvard Medical School, Boston,
Massachusetts, and the Department of
Dermatology,
University of Utah Health
Sciences Center, Salt Lake City, Utah
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PDGF acts predominantly on the cells of the mesenchyme. Most cells of the dermis express a receptor for PDGF, and in addition to stimulating the proliferation of fibroblasts, smooth muscle cells, and microvascular endothelial cells, PDGF is chemotactic for fibroblasts, macrophages, neutrophils, and smooth muscle cells. PDGF isoforms are synthesized by numerous cell types, including keratinocytes, the principal cell type of the epidermis, which synthesize both PDGF-A and B isoforms.5 Recently, we demonstrated that when keratinocytes genetically modified to overexpress the released isoform of PDGF-A were transplanted to the athymic mouse, these PDGF-A-secreting grafts formed a normal epidermal structure and induced an increase in the cellularity and vascularity of the connective tissue that formed subjacent to these grafts.6 These data suggest that the released isoform of PDGF-A produced by the epidermis acts as a paracrine mediator that controls distant cellular events in the adjacent dermis.
In the present study, we have sought to understand the role of PDGF-B produced by the epidermis, a PDGF isoform that is not released but is predominantly cell associated. Retroviral-mediated gene transfer was used to introduce into human keratinocytes the gene encoding either wild-type PDGF-B or a truncation mutant of PDGF-B that is released. In vitro, modified cells synthesized increased levels of PDGF and released 20-fold more of the mutant PDGF-B isoform than wild-type PDGF-B. When modified cells were grafted as epithelial sheets onto athymic mice, distinct changes occurred in the adjacent connective tissue. Grafts overexpressing the released mutant of PDGF-B induced an increase in the cellularity and vascularity throughout the subjacent connective tissue, similar to our previous results with PDGF-A.6 In contrast, the cellularity of the connective tissue subjacent to grafts overexpressing wild-type PDGF-B was also increased, but this cellularity was confined to a zone at the interface of the epidermis and the connective tissue. These data demonstrate that the released and cell-associated isoforms of PDGF both act as paracrine mediators that control cellular events in the adjacent dermis but that the released isoforms control more distal events, whereas the cell-associated isoforms control more proximal events.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A cDNA encoding human PDGF-B was amplified by
polymerase chain reaction (PCR) using 5'-GACGATCATGAATCGCTGCTGGGCG-3'
and 5'-CCGATGGATCCCTAGGCTCCAAGGGTCTC-3' as forward and reverse primers,
respectively. After PCR, the primers were removed and the product
digested with BspHI and BamH, gel purified, and
ligated into the NcoI/BamHI sites of the
retroviral vector MFG.7
The retroviral vector expressing
the truncation mutant of PDGF-B with a stop codon at
position 211 (PDGF-B211) as originally
described2
was prepared in a similar manner except that the
sequence 5'-CTGCTGGATCCTAAATGGTCACCCGAGTTTGGG-3' was used as the
reverse primer. The fidelity of the inserts were verified by DNA
sequencing. To generate cell lines producing recombinant retrovirus,
plasmid DNAs encoding MFG-PDGF-B and
MFG-PDGF-B211 were transfected into the 
-CRIP packaging
cell line.8
Clones of transfectants were isolated and
screened for ones producing the highest titer.
Cell Culture
Human keratinocytes derived from neonatal foreskins (strains A to
E) were grown on a mitomycin-C-treated (15 µg/ml) feeder layer of
mouse 3T3-J2 cells (originally obtained from H. Green, Harvard Medical
School, Boston, MA) in a mixture of the Dulbecco-Vogt modification of
Eagle's medium (DMEM) and Ham's F12 medium. Supplements were as
described.9
Swiss mouse 3T3-J2 and virus-producing cells
(-CRIP) were grown in DMEM supplemented with 10% calf serum.
Genetic Modification
Keratinocytes were genetically modified as previously described.10 Briefly, preconfluent primary cultures were dissociated, and cells were passed to a dish containing mitomycin-C-treated virus-producing cells (2.5 x 104 cells per cm2). After 4 to 6 days of co-cultivation, modified cells were dissociated and parallel cultures were prepared for protein analysis, grafting, and plating efficiency. Unmodified control cells were cultured in parallel on 3T3-J2 cells.
Assays for PDGF
Synthesis of PDGF proteins by transduced cells was assayed by ELISA, specific for human PDGF-BB. Briefly, 96-well plates were coated with an affinity-isolated polyclonal rabbit anti-hPDGF antibody (10 µg/ml in 0.1 mol/L NaHCO3, pH 9.6; R&D Systems, Minneapolis, MN) for 16 hours at 4°C. The wells were blocked with PBS, containing 1% bovine serum albumin, 0.5% Tween 80 for 2 hours at room temperature. Test samples or known amounts of recombinant human PDGF-BB (Boehringer Mannheim, Indianapolis, IN) were added to the wells for 3 hours at room temperature. The wells were washed, and affinity-isolated polyclonal goat anti-hPDGF-BB antibody conjugated to horseradish peroxidase (R&D Systems) was added for 3 hours at room temperature, followed by tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO). The limit of detection of PDGF-BB was 30 pg/ml.
To measure the levels of PDGF proteins released into the medium, modified keratinocytes were grown to confluence (107 cells/10-cm dish), fresh medium was added (30 ml), and portions (1 ml) of the culture medium were removed over a 4-day period. The rate of PDGF synthesis was determined by averaging the daily rate of PDGF synthesis over a 4-day period.
To measure the amount of PDGF that was cell associated, secondary cultures of MFG-PDGF-B or MFG-PDGF-B211 transduced keratinocytes were grown to confluence in serum-free medium (Clonetics MCDB 153). The cells were washed with serum-free medium and exposed three times to 1 mol/L NaCl for 30 minutes at 4°C. After each treatment, the NaCl solution was harvested, and the amount of PDGF-BB protein was measured by ELISA.
The bioactivity of PDGF released by modified keratinocytes was determined using a modification of a cell proliferation assay that measures the stimulation of [3H]thymidine incorporation.11 Quiescent Swiss 3T3 cells were incubated for 12 hours with medium (5% platelet-poor plasma; PPP) conditioned for 30 hours by modified or unmodified keratinocytes. The 3T3 cells were washed with serum-free DMEM and incubated for 20 hours with [3H]thymidine (1 µCi/well with 5% PPP in DMEM). Cells were washed in serum-free DMEM, DNA was precipitated with two washes of 5% trichloroacetic acid (TCA) at 4°C, and cells were lysed with 0.6% sodium dodecyl sulfate (SDS). TCA-insoluble material was harvested on a glass fiber filter and counted in a scintillation counter (Packard Instrument Co., Meriden, CT). A standard curve was established by incubating quiescent Swiss 3T3 cells with DMEM 5% PPP supplemented with recombinant human PDGF-BB (Boehringer Mannheim).
Grafting
Grafting was performed using 6- to 8-week-old NIH swiss nu/nu mice (Taconic Farms, Germantown, NY) anesthetized with an intraperitoneal injection of 2,2,2,-tribromoethanol (0.58 mg/g body weight; Aldrich, Milwaukee, WI).
Cultures of confluent keratinocytes (35-mm dishes) were detached as intact epithelia with Dispase II solution (1.2 U/ml for 1 hour at 37°C; Boehringer Mannheim) and grafted to athymic mice as described.12 Briefly, the detached epithelium was placed basal side up on a small piece of Silastic membrane (thickness, 0.005 inches; Dow Corning, Arlington, TN). This graft was inserted under a full-thickness skin flap on the back of the mouse so that when the flap was closed, the basal side of the epithelium came in contact with the inner side of the mouse skin. Before inserting the epithelium, the loose connective tissue covering the inner side of the mouse skin was removed by dissection. In this way, the basal side of the epithelium was placed directly on the epimysium of the panniculus carnosus. The incision was closed with surgical clips, and grafts were harvested after 7 days.
Histology
The human epithelium and subjacent mouse connective tissue were fixed in buffered formaldehyde, and paraffin-embedded sections (5 µm) were stained for hematoxylin and eosin (H&E; control, n = 8; MFG-PDGF-B, n = 8; MFG-PDGF-B211, n = 7). The thickness of the mouse connective tissue layer was determined by image analysis using the Image Analysis/Acquisition Software system (Universal Image Corp., West Chester, PA) coupled to a Nikon Diaphon 300 microscope (Nikon, Melville, NY). The edges of the epithelial grafts were avoided as these showed signs of hyperproliferation. The cross-sectional area of the mouse connective tissue layer was determined for 10 fields of view per graft, and the thickness was calculated by dividing the area by the length of the sample field of view (1.06 mm). Thickness measurements for control (n = 8), PDGF-B-modified (n = 8), and PDGF-B211-modified grafts (n = 6) were made from a total of 80-mm, 80-mm, and 60-mm lengths of graft, respectively. Statistical comparisons were done using analysis of variance and the Tukey honestly significant difference test.
For immunostaining, cryostat sections (6 µm) were fixed in ice-cold acetone for 5 minutes, treated with blocking solution (3% bovine serum albumin, 1% normal goat serum, 0.02% sodium azide in PBS) for 1 hour at room temperature, and incubated with the following antibodies: affinity-isolated polyclonal rabbit anti-laminin antibody (1:400; Sigma), an affinity-isolated monoclonal mouse anti-laminin antibody (1:400; Sigma), an affinity-isolated monoclonal mouse anti-human collagen IV antibody (1:10; Biodesign, Kennebunk, ME), an affinity-isolated polyclonal rabbit anti-human PDGF-BB antibody (1:50; Genzyme, Cambridge, MA), adsorbed with an acetone powder of mouse skin or an affinity-isolated polyclonal rabbit anti-human PDGF-BB antibody (1:50; R&D Systems) for 2 hours at room temperature. Slides were washed four times for 15 minutes each in PBS and incubated with fluorescein-conjugated affinity-purified goat anti-rabbit IgG antibody (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA) or with lissamine rhodamine-conjugated affinity-isolated goat anti-mouse IgG antibody (1:100) for 1 hour at room temperature. When using mouse monoclonal antibodies, endogenous mouse IgG was blocked with an affinity-isolated goat anti-mouse IgG Fab fragment (1:100; Jackson ImmunoResearch Laboratories). The slides were washed four times for 15 minutes each in PBS and coverslipped using 1% n-propyl-gallate mounting solution (Sigma). All antibodies were diluted in blocking solution. Control sections were not exposed to primary antibody. Fluorescence due to immunoreactive PDGF-BB was viewed in a laser scanning confocal microscope (model MRC 500, BioRad Laboratories, Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of cell-associated versus released isoforms of PDGF, we prepared recombinant retroviral vectors expressing cDNAs encoding either wild-type PDGF-B, (MFG-PDGF-B), which is predominantly cell associated, or a truncation mutant of PDGF-B211 (MFG-PDGF-B211), which is predominantly released. This mutant has a stop codon inserted at position 211, and the resulting protein lacks exon 6 sequences.2 La Rochelle et al showed that comparable amounts of the truncated and wild-type proteins were cell associated but that the release of the mutant protein was significantly higher than the wild-type protein.
Secondary cultures of human keratinocytes were genetically modified by
co-cultivation with mitomycin-C-treated -CRIP cells producing the
MFG-PDGF-B or MFG-PDGF-B211 virus. After 4 days
of co-cultivation, the virus-producing cells were removed and the
keratinocytes were dissociated and passed onto plates containing a
normal 3T3 fibroblast feeder layer. Control cultures of unmodified
cells were carried in parallel. To measure the release of PDGF protein,
aliquots of culture medium from modified cells were harvested over
time, and PDGF levels were determined by ELISA. Cells expressing the
MFG-PDGF-B211 retrovirus synthesized and released PDGF
protein into the medium over a 4-day period at an average daily rate of
112 ng/107
cells/hour (strain A) or 145 ng/107
cells/hour (strain B) (Figure 1A)
. In
contrast, cells modified with MFG-PDGF-B released relatively
little PDGF protein into the medium, with an average daily rate of 7
ng/107
cells/hour (strain A) or 6 ng/107
cells/hour (strain B). The rate of PDGF release by cells modified with
the mutant gene was 20 times higher than the cells modified with
the wild-type gene. The level of PDGF-BB released by unmodified cells
under these conditions was undetectable.
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. Levels of cell-associated molecules were similar for cells
modified with either the wild-type or mutant PDGF genes, consistent
with the previous results.2
Treatment of unmodified control
cells did not release detectable amounts of PDGF-BB protein. Wild-Type and Mutant PDGF Isoforms Have Comparable Biological Activity
The biological activity of the wild-type and mutant PDGF proteins
produced by modified keratinocytes was measured using a bioassay.
Conditioned medium from modified keratinocytes was harvested after 30
hours and tested in a bioassay that measures the incorporation of
[3H]thymidine into Swiss 3T3 cells (Figure 2)
. As a control, conditioned medium was
harvested from unmodified cells. This control conditioned medium had
significant levels of stimulatory activity, presumably due to other
growth factors normally secreted by unmodified keratinocytes. As
measured by ELISA, the level of PDGF-BB was only 0.25 ng/ml. When
compared with this control, conditioned medium from modified cells had
significantly increased levels of PDGF-BB and showed a dose-dependent
stimulation of [3H]thymidine uptake that was greater than
control levels and correlated with PDGF-BB levels. When equated with
PDGF-BB levels, conditioned medium from cells producing the wild-type
molecule had comparable stimulatory activity to conditioned medium from
cells producing the truncation mutant.
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Keratinocytes expressing wild-type or mutant PDGF-B genes were tested for their ability to form an epidermis when transplanted to athymic mice. Normal or modified keratinocytes were grown to confluence in 35-mm dishes, detached as epithelial sheets using Dispase, and grafted to the underside of a dorsal skin flap. The basal side of the sheet was placed directly on the epimysium of the panniculus carnosus, and 7 days after grafting the human epithelium and the subjacent mouse tissue were fixed and stained.
Both control and modified keratinocytes developed a stratified
epidermal structure several cell layers thick, which contained stratum
granulosum and stratum corneum (Figure 3)
. The stratum granulosum of the
epithelia expressing wild-type PDGF-B was less developed and
thinner, compared with that of control and grafts expressing
PDGF-B211.
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. This increased cellularity and
vascularization of the connective tissue subjacent to the
PDGF-B211-expressing grafts was uniformly present throughout
the entire connective tissue layer. These results are similar to our
previous results with grafts overexpressing PDGF-A, a PDGF isoform that
is also released.6
In contrast, the cellularity in the
connective tissue subjacent to the grafts expressing wild-type
PDGF-B was increased but was predominantly localized in a
zone at the epidermal-dermal junction, often resulting in the
disruption of the integrity and continuity of the epidermal-dermal
border in wide areas of the graft (Figure 3B)
and most obvious under
high magnification (Figure 3E)
. Vascularization of the connective
tissue was also less prominent (eight of eight grafts) when compared
with the connective tissue subjacent to PDGF-B211-expressing
grafts.
To identify the basement membrane of the epidermal-dermal junction and
blood vessels in the connective tissue, cryosections of control and
modified grafts were stained for laminin (Figure 3, G–I)
. Laminin
staining was abundant throughout the connective tissue layer subjacent
to the grafts expressing PDGF-B211, identifying the presence
of capillaries and/or small blood vessels. In contrast, laminin
staining in the connective tissue layer subjacent to control and grafts
expressing wild-type PDGF-B was nearly absent. Furthermore,
laminin staining was continuous along the basement membrane zone of the
epidermal-dermal junction of grafts of unmodified cells (four of four
grafts; Figure 3G
) and grafts expressing PDGF-B211 (four of
four; Figure 3I
). In contrast, grafts expressing PDGF-B
(three of four) showed a rather disjointed and discontinuous staining
pattern in wide areas of the epidermal-dermal region (Figure 3H)
.
Similar staining patterns were seen with antibodies to other basement
membrane components, including collagen types IV and VII (data not
shown).
The cellular and vascular response of PDGF-B211-modified
grafts was accompanied by a thickening of the connective tissue layer
developed adjacent to these grafts. The thickness of the subjacent
connective tissue of control grafts and grafts expressing PDGF-B
versus grafts expressing PDGF-B211 was measured
by image analysis from three different sets of grafts, using three
different strains of keratinocytes (Figure 4)
. In each experiment, the connective
tissue subjacent to the grafts expressing PDGF-B211 was
significantly thicker than control grafts or grafts expressing
wild-type PDGF-B, with an average increase in thickness of
54% and 50%, respectively.
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To localize PDGF protein in vivo, cryosections of
unmodified and modified epithelia 7 days after grafting were stained
with an affinity-isolated polyclonal antibody against human recombinant
PDGF-BB (Figures 3, J–L, and 5)
.
Staining of endogenous PDGF-BB protein in control grafts and
PDGF-B211-modified grafts was relatively weak and diffuse
(Figure 3, J and L)
. In contrast, strong and localized staining for
PDGF-BB was evident in the region of the epidermal-dermal junction in
those grafts expressing wild-type PDGF-B (Figures 3K and 5
,
B and D). Laminin staining had shown that the basement membrane region
in these grafts was often disrupted and discontinuous. Thus, to
determine whether PDGF-BB protein co-localized with proteins of the
basement membrane, we simultaneously stained cryosections for PDGF-BB
and laminin or PDGF-BB and human collagen type IV (Figure 5)
. In both
cases, the staining for PDGF-BB co-localized with staining for laminin
and collagen type IV in short stretches where the basement membrane was
contiguous. This staining pattern for PDGF-BB was unique to the grafts
expressing wild-type PDGF-B and was confirmed using a
different affinity-isolated polyclonal against human PDGF-BB (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These results have similarities with our previous experiments in which PDGF-A was overexpressed by epithelial grafts and suggest that PDGF isoforms produced by the epidermis are paracrine regulators of the dermis.6 Grafts overexpressing PDGF-A, a released isoform, induced the formation of a thicker and more cellular connective tissue similar to the connective tissue induced by overexpression of PDGF-B211. Both grafts also induced connective tissues that were highly vascular. Any direct in vivo roles for either PDGF-BB or especially PDGF-AA on vascular endothelial cells remains controversial.13 The increased vascularity is more likely mediated by indirect actions of PDGF, such as the stimulation of other cells, probably microvascular pericytes and/or fibroblasts, to produce factors that promote angiogenesis.14-16
In the present study, vascularization was one significant difference between the connective tissues induced by grafts expressing wild-type PDGF-B versus PDGF-B211. Overexpression of PDGF-B211 induced a highly vascular connective tissue, whereas overexpression of wild-type PDGF-B did not. Both molecules bind the same receptor, and so the different biological responses are probably not at the receptor level, an argument that would be relevant to comparisons between PDGF-A and wild-type PDGF-B, which bind different receptors.1 Rather, the different vascular responses are most likely due to differences in the bioavailability of the two molecules and or the accessibility of endothelial cells to the mitogenic signal. Released PDGF-B211 molecules can diffuse away from the graft and stimulate endothelial cell proliferation distal to the graft, whereas diffusion of wild-type PDGF-B deposited in the basement membrane zone is limited, and these molecules act on those cells proximal to the graft. Both grafts are placed on the epimysium of the panniculus carnosus, a thin layer of predominantly fibroblasts surrounding the muscle layer; thus, few endothelial cells would be accessible to PDGF-B deposited in the basement membrane.
Our experiments provide additional information on the role of exon 6 in PDGF function and suggest exon 6 is required for the deposition of PDGF-B in the basement membrane zone in vivo. Exon 6 of PDGF-B encodes a highly basic domain of 14 amino acids, and PDGF-B211 lacks only this domain. PDGF-B211 was originally described by LaRochelle et al, who transfected 3T3 fibroblasts with a series of truncation mutants and showed that both wild-type and PDGF-B211 were cell associated and could be released by high-salt treatment. However, very little wild-type PDGF-B was released, whereas most of PDGF-B211 was released.2 Using keratinocytes, we have similar results in vitro that show comparable cell-associated levels of wild-type and mutant molecules and high-level release of PDGF-B211. However, when these cells were transplanted, we detected only wild-type PDGF-B in the basement membrane zone in vivo. Thus, exon 6 is required for deposition of PDGF-B in the basement membrane zone. Exon 6 has been shown to be necessary for binding of PDGF isoforms to heparan-sulfate proteoglycans and SPARC (secreted protein, rich in cysteine) in vitro and both are components of the basement membrane.17 The removal of the cationic domain in PDGF-B211 results in a significant decrease in the theoretical isoelectric point of the secreted molecule and potentially its binding properties. Wild-type PDGF-B (residues 21 to 241) has a pI of 9.42, whereas PDGF-B-211 (residues 21 to 211) has a pI of 8.21.
PDGF-A also has two natural isoforms that arise by alternate splicing of exon 6. PDGF-AS, the predominant form that lacks exon 6 is released, and PDGF-AL, which contains exon 6, is cell associated.3 In our previous studies, we overexpressed PDGF-AS, which was released and promoted vascularization and formation of a thicker connective tissue.3 Exon 6 in PDGF-A might also serve a similar function and direct the deposition of PDGF-AL to the basement membrane zone. It would be interesting to use our system to determine the in vivo tissue response to overexpression of PDGF-A with and without exon 6 and compare this response to our results with PDGF-B.
Vascular endothelial growth factor (VEGF), an endothelial cell mitogen and chemoattractant for monocytes, shares significant homology with PDGF and also occurs as multiple isoforms through alternate splicing.18 Two short forms, VEGF121 and VEGF165, lack exon 6 and are released, whereas two longer forms, VEGF189 and VEGF206, contain exon 6 and are cell associated. Like PDGF, exon 6 of VEGF also encodes a domain of basic amino acids. Keratinocytes have been shown to synthesize at least three of the splice variants, VEGF121,165,189,19,20 raising the possibility that the bioavailability of VEGF produced by the epidermis is controlled in a manner similar to PDGF isoforms.
Other growth factors have been shown to be associated with the extracellular matrix, including granulocyte/macrophage colony-stimulating factor,21 transforming growth factor-ß,22 and differentiation-inhibiting activity.23 A notable example is basic fibroblast growth factor (bFGF), an endothelial cell mitogen that binds glycosaminoglycans in the basement membrane of cornea.24 It has been suggested that sequestered bFGF serves to control angiogenesis and can act as a signal for repair when released after injury.24 In normal skin, such a role is also possible for sequestered PDGF-B in the basement membrane.
Conversely, over-accumulation of growth factors in the matrix and or basement membrane could underlie some pathologies. It has been widely suggested that PDGF, a potent mitogen for smooth muscle cells, is involved in the neointimal hyperplasia associated with atherosclerosis.25 The association of PDGF-B isoforms with the basement membrane zone as seen in our studies could be one mechanism by which PDGF contributes to localized hyperproliferative responses.
In our grafting model, overexpression of wild-type PDGF-B resulted in disruption of the basement membrane zone. Lichenoid reactions of the skin and mucosa are also characterized by a degeneration and disruption of the basement membrane zone. Growth factors, such as PDGF-B, that are associated with components of the basement membrane zone could play a role in the pathogenesis. Disruption of the basement membrane might also be an important event in the multistep process of tumorigenesis and metastasis. PDGF-B is structurally related to the v-sis transforming gene of simian sarcoma virus, and like wild-type PDGF-B, the v-sis gene product is also cell associated after secretion.1 Typically, autocrine pathways have been implicated in oncogenesis, and several tumors, such as sarcomas and gliomas, express PDGF and receptors for PDGF.26,27 However, some tumors expressing PDGF do not express receptors for PDGF, suggesting an alternative role for PDGF in oncogenesis.28 Overproduction by a tumor cell of a matrix-associated growth factor, such as PDGF-B, might stimulate the proliferation of neighboring nontransformed cells that disrupt the extracellular matrix and or basement membrane and allow further proliferation and migration of the tumorigenic cells.
Lastly, our experiments demonstrate that changes in the concentration/bioavailability of a growth factor such as PDGF can profoundly influence the type of biological response in vivo as well as its spatial organization. For example, PDGF-BB deposited in the basement membrane promoted a proximal cellular response but failed to elicit either a distal or proximal vascular response, whereas the same growth factor released as PDGF-B211 promoted distal vascularization but failed to promote a proximal cellular response. Such compartments or gradients of growth factors could be a means by which cellular events are spatially controlled during tissue regeneration as well as during development.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Shriners Hospitals for Children, the National Institutes of Health, and the Fritz Thyssen and German Research Foundation.
S.A. Eming's present address: Department of Dermatology, University of Cologne, Cologne, Germany.
Accepted for publication September 24, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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