Return to Woundbiotech Home
Page
Return to Growth
Factors
I.
Introduction
Over the past several decades, the
discovery of growth factors has led to much hope and speculation about the use
of these potent peptides in the treatment of difficult to heal wounds,
particularly chronic wounds. In vitro experiments showed that growth factors
were very effective in regulating cell proliferation, chemotaxis, and
extracellular matrix formation. Animal experiments confirmed the notion that
growth factors could accelerate wound repair, although most such experiments
dealt with wounds created by acute injury. However, it was not until later, when
further advances in recombinant technology made it possible to obtain large
amounts of purified growth factors, that these agents could be tested in human
clinical trials. Over the last 10 to 15 years, a large number of trials have
been performed to evaluate the safety and effectiveness of growth factors in the
healing of chronic wounds due to pressure (decubitus ulcers), diabetic
neuropathy, and venous insufficiency (1). Platelet-derived growth factor (PDGF)
is now approved for topical treatment of diabetic neuropathic ulcers (2). In
this brief discussion, we will review growth factors, their mode of action, and
the experience from clinical trials, with particular emphasis on the use of PDGF
in diabetic foot ulcers. We will end our discussion by providing a perspective
on the future of growth factors in chronic wounds, including diabetic foot
ulcers.
2. General
aspects of growth factors
Although more easily
conceptualized by the division into three distinct phases (inflammation,
fibroplasia, and maturation), the process of wound repair is characterized by a
series of complex cellular and molecular events with a great degree of overlap
and interdependence (3). Growth factors play fundamental roles in this process,
by stimulating chemotaxis and cellular proliferation, by providing signaling
among cells of the same and different type, by controlling extracellular matrix
formation and angiogenesis, by regulating the process of contraction, and by
re-establishing tissue integrity. As soon as blood vessels are disrupted,
platelets enter the wound in great numbers and release several growth factors,
including platelet-derived growth factor (PDGF) and transforming growth
factor-ß1 (TGF-ß1). These and other growth factors are chemotactic for a number
of cell types critical to the repair process, such as macrophages, fibroblasts,
and endothelial cells. Later, during the proliferative phase of wound repair,
several growth factors, including vascular endothelial growth factor (VEGF),
fibroblast growth factors (FGFs) and PDGF and TGF-ß isoforms, provide a potent
stimulus for angiogenesis and for fibroblasts to synthesize key extracellular
components (i.e., collagens, proteoglycans, fibronectin, elastin). During the
later stages of wound repair, growth factors are important in tissue remodeling,
aided by the action of matrix-degrading metalloproteinases (MMPs). It is likely,
however, that the action of growth factors does not end with wound closure and
tissue remodeling, but that they are key players in the maintenance of tissue
integrity and in cell to cell communication.
Growth factors are
multifunctional peptides that are extremely potent in vitro, often in the
picogram range (4). Table
1 shows a list of growth factors, some of them grouped into families, which
have been tested for the treatment of chronic wounds. This list is not meant to
be inclusive, and many more clinical studies have been performed than suggested
by the number of published results. The nomenclature used to define growth
factors tends to be confusing to the clinician for a number of reasons. First of
all, the names of growth factors have more to do with the circumstances in which
they were identified than with their specific effects on cells. Thus, fibroblast
growth factors (FGFs) are very potent angiogenic factors, and transforming
growth factor-ß's (TGF-ß's) are not transforming to cells and actually appear to
be important in preventing cancer. Also, the term growth factor is generally
used in a broad sense, to indicate substances which increase cell proliferation,
mitogenic activity, and extracellular matrix formation. The actual category in
which such substances are placed often depends on the context in which they were
discovered. For example, chances are that the same substance identified by a
biochemist, an immunologist, and a hematologist would be called a growth factor,
an interleukin, and a colony stimulating factor, respectively.
Growth factors work by binding
to specific cell surface receptors and can target cells in a number of
recognized ways or modes. Release of these substances into the blood stream
allows them to get to distant targets (endocrine mode). From the cell of origin,
growth factors can diffuse over short distances to affect other cells
(juxtacrine mode), and to influence neighboring cells (paracrine mode). Growth
factors can also act on the cell in which they are produced (autocrine mode).
These different modes are all likely to be operative during tissue repair
(1,4).
After binding to receptors,
growth factors can have a profound influence on cell proliferation, chemotactic
activity, and extracellular matrix synthesis. Interestingly, not all of these
actions are stimulatory and not for all cell types. For example, transforming
growth factor-ß's (TGF-ß's) are potent inhibitors of keratinocyte and
endothelial cell proliferation. However, these same agents are a potent stimulus
for the deposition of collagen and other extracellular matrix proteins.
Harvesting the inhibitory activities of growth factors has great therapeutic
potential, i.e., using antibodies to TGF-ß's to decrease
scarring.
3. Mode of action of growth
factors
Much of the progress made
in the last ten years in the basic science aspects of growth factors has been in
identifying and cloning their specific cellular receptors and in elucidating the
complex signaling pathways leading from receptor binding to a biologic response
(reviewed in ref. 5). Tyrosine kinase receptors are membrane-spanning molecules
with kinase activity (ability to phosphorylate or add phosphate groups) on the
cytoplasmic domain. The kinase activity is activated upon binding of the growth
factor (ligand). Almost 60 tyrosine kinases have been described, and they have
been grouped in 14 families. Dimerization of several tyrosine kinase molecules
is brought about by ligand binding (i.e., PDGF). After activation, the receptor
can add phosphate groups to certain downstream targets or, by virtue of its
phosphotyrosine residues, can bring other molecules into the signaling complex.
There are other ways to transmit the initial signals. For example, the
downstream target phosphorylated by the insulin receptor tyrosine kinase is a
small protein called insulin receptor substrate-1 (IRS-1). Some receptors, i.e.,
those for interferons, do not have intrinsic tyrosine kinase activity. They
instead recruit molecules possessing this ability to phosphorylate. The
intracellular domain of these receptors is associated with a protein kinase
family of the Janus family (JAKs) which, once autophosphorylated, recruits STATs
(signal transducers and activators or transcription).
Receptors of the
TGF-ß superfamily of molecules have intrinsic serine/threonine kinase activity
(6). TGF-ß binds to a class II receptor, which is a constitutively active
kinase. Binding results in recruitment of a Type I receptor, which is
phosphorylated and can activate downstream targets, such as SMADs proteins,
which can move to the nucleus and interact with transcription factors. More than
10 SMADs proteins have been identified, with some causing activation (i.e., Smad
4) and others donwnregulating the signaling (i.e., Smad 6 and Smad 7).
Once
tyrosine kinase activation and initial targeting occurs, further signals are
generally transmitted by the Ras-Raf-MAP kinase pathway and the phospholipase C
second messenger system (7). These systems work in a cascade-like manner, and
often involve a series of phosphorylations, from one molecule to another. There
is a Ras pathway, consisting of Rac and Rho proteins and cycling between
GTP-associated (active) and GDP-associated (inactive) states. Another prominent
system for downstream activation is the MAP (mitogen-activated protein kinase)
pathway. Ras and Raf molecules are involved here, with Raf phosphorylating a MAP
kinase kinase (MAPKK) or Mek (MAPK/Erk) kinase).
4. Growth factors in
human acute wounds
The greatest potential of
growth factors is to accelerate the healing of chronic wounds. Still, there are
situations where accelerating the healing of acute wounds would be highly
desirable and perhaps cost effective. Moreover, acute wounds are in general less
complex than chronic wounds, and allow investigators to develop proof of
principle for certain parameters, such as dose of growth factors, development of
optimal vehicle and delivery system, and other important variables.
Split-thickness donor sites are in many ways the ideal acute wound for testing
growth factors in humans. These wounds are easily made in a reproducible way,
generally on the thighs. They offer a side to side comparison for testing
products, are shallow, and normally heal within 7 to 10 days, which is a
reasonable time period for testing effectiveness in acute wounds. Importantly,
there is great value in accelerating the healing of split-thickness graft donor
sites, for this would allow clinicians to re-harvest skin more quickly in burn
patients.
There have been four studies
of graft donor sites treated with growth factors. These have been reviewed in
more detail elsewhere (1). Topically applied EGF, bFGF, IL-1, and HGH have been
tested. bFGF was found not to accelerate the healing of these acute wounds. The
magnitude of the positive effect of growth factors in healing donor sites was
generally not very dramatic. However, the results of these studies do show that
growth factors can accelerate human healing when applied
topically.
4. Growth factors in
human chronic wounds
It is not possible to describe
in detail all the trials that have been reported with growth factors and chronic
wounds. Table
2 is a partial list; it must be recognized that the results of unsuccessful
trials are generally not published. As shown in Table 2, a number of growth
factors have been tested in more than one type of chronic wound. Although the
results were not statistically significant in most of these studies, overall
wounds treated with the growth factor seemed to do better than those treated
with the placebo. All of the studies listed in Table 2 were done with topically
applied growth factors, except for GMCSF; this peptide was injected into the
skin surrounding the wound (10). Particularly promising results were obtained
with PDGF (12) and FGF (17) in pressure (decubitus) ulcers, EGF (8) and TGF-ß2
(11) in venous ulcers, and PDGF in diabetic ulcers (14). The results obtained in
the treatment of diabetic ulcers with PDGF have been confirmed in larger trials
(2,15) and will be described in more detail in the next section. Predictably,
specific growth factors may be more effective in certain types of wounds. For
example, growth factors capable of stimulating extracellular matrix formation
and angiogenesis (i.e., PDGF and FGFs) are more likely to be useful in deep
wounds, such as pressure ulcers. EGF was promising in venous ulcers (8), where
failure of reepithelialization is the major clinical problem (19).
One
must be careful in evaluating the published literature on the use of growth
factors in chronic wounds, such as the information in Table
2. As shown in Table
2, except for the PDGF trials in diabetic ulcers, the rest were pilot
studies consisting of a small number of patients, sometimes from a single
center. However, it is highly likely that large, multicenter studies were
performed with most of the growth factors shown in Tables
1 and 2, but that the results were not satisfactory and were not
published.
Our discussion has dealt exclusively with purified or
recombinant growth factors. There has been a considerable amount of information
on the use of autologous platelet preparations for accelerating the healing of
chronic wounds, including diabetic ulcers (20,21). The results have been
promising, and the approach is based on the principle that more than one growth
factor is needed for accelerating wound repair and that platelets are a rich
source of growth factor peptides. However, these studies have been small, and it
remains unclear whether autologous platelet releasate preparations are reliably
active.
5. PDGF and diabetic
foot ulcers
The only growth factor
which has more convincingly been shown to stimulate healing of chronic wounds
and which is approved for use in diabetic neuropathic ulcers by the Food and
Drug Administration (FDA) is PDGF-BB (becaplermin). In a phase II clinical
trial, Steed and colleagues tested the effect of a recombinant human PDGF-BB
(rhPDGF-BB) gel preparation in the treatment of neuropathic diabetic ulcers of
at least 8 weeks= duration (14). A total of 118 patients were randomized to
receive either rhPDGF-BB gel or placebo gel until complete wound closure or 20
weeks, whichever came first. The gel preparation was spread over the wound, and
covered with a non-adherent saline-soaked gauze dressing. This primary dressing
was held in place with roll gauze. Dressings changes were done twice, twelve
hours apart, but the study or placebo gel preparation were applied only once
daily. The study was randomized, double-blind, and placebo controlled, and
patients were enrolled from 10 centers. There were no differences in the
patients receiving the study drug or the placebo, except that the patients
treated with rhPDGF-BB were on the average 5 years older (p=0.02). By
approximately 6 weeks of therapy, differences emerged between the active and
placebo group. Throughout the 20 weeks of the study, 29 (48%) of 61 patients
treated with rhPDGF-BB achieved complete wound closure, compared to 14 (25%) of
the 57 patients in the placebo group (p=0.01). Wounds also healed more quickly
in the rhPDGF-BB group, by about 30 to 40 days (p=0.01). No statistically
significant differences were present in the rate of ulcer recurrences between
the two groups, the mean time for recurrence being 8.5 weeks. The rhPDGF-BB gel
preparation proved to be safe.
A very interesting relationship between
wound debridement and the effect of rhPDGF-BB emerged in the trial just
described and published by Steed et al (14). Surgical debridement, with removal
of the callus around the ulcer, was performed at the beginning of the study and
throughout the trial, as required. However, there were differences in the rate
of wound debridement, depending on the study site. In general, a lower rate of
healing was observed in those centers performing less frequent debridement (22).
It appears that there may have been a synergistic effect of aggressive surgical
debridement and the use of rhPDGF-BB. The reasons behind this interesting
observation are not clear. At higher debridement rates in the placebo group,
there was no definite relationship between the healing rate and the frequency of
debridement. An attractive hypothesis is that debridement removes tissue
containing cells that are no longer responsive to the action of growth
factors.
The effectiveness of rhPDGF-BB in the treatment of diabetic
neuropathic ulcers have been confirmed in an additional and larger study,
although a higher dose of the peptide (100 µg/g) were required for optimal
efficacy (15). This was a multicenter double-blind placebo-controlled phase III
trial of 382 patients. Ulcers were treated once daily with either 30 or 100 µg/g
of rhPDGF-bb or placebo gel. As in the previous study, dressings were changed
twice daily, and consisted of saline-soaked gauze. Compared to placebo gel,
rhPDGF-BB in a dose of 100 µg/g increased the incidence of complete wound
closure by 43% (p=0.007) and decreased the time to complete healing by 32% (86
vs. 127 days; p=0.013). It remains unclear why the lower dose of rhPDGF-BB did
not prove as effective in this larger follow-up study. However, the safety of
the rhPDGF-BB preparation remains established (23).
6. Perspective on
growth factor therapy of diabetic ulcers
Substantial progress has
been made with regard to growth factors in the treatment of chronic wounds. It
appears that no serious safety issues have arisen; systemic absorption appears
to be minimal, and no ontoward local effects have been reported. These peptides
have not caused cancer at the site of application, they have not been absorbed
in substantial amounts and caused fibrosis, and they have not worsened diabetic
retinopathy. Of course, much still remains to be done. It may very well be that
the delivery systems used in these clinical trials were ineffective and did not
allow the peptides to reach their target cells and tissues. Another reason,
probably related to the first, is that the micro environment of these chronic
wounds is very hostile to proteins, and that breakdown of peptides by proteases
is very likely. The success of PDGF in diabetic ulcers may be due to the
persistance of biologic activity of the peptide in the wound micro environment
(24). A third reason is that the resident cells in chronic wounds have been
altered by the pathogenic mechanisms responsible for the wounds in the first
place. There is indeed evidence that fibroblasts from chronic wounds, including
diabetic ulcers, are not able to respond to certain growth factors (25). Removal
of tissue from around the wound, as has been advocated for the use of PDGF in
diabetic ulcers (22), may remove these unresponsive cells and allow peptides to
function as they should.
There are of course other ways to deliver growth
factors to wounds. Gene therapy may be ideal for wounds, because peptides would
only be needed for a short period of time. Bioengineered skin products and skin
substitutes represent another very exciting development and a major advance in
the treatment of chronic wounds. Some of these agents supply matrix materials
alone, while others contain living cells that are probably able to adjust to the
wound micro environment and provide growth factors and other substances that may
be lacking in chronic wounds (26,27). We still don't know whether the
transplanted cells survive in the wound, but we think that they remain there
long enough to stimulate and accelerate wound healing. These bioengineered
products may well provide growth factors in the right concentration and in the
right sequence, something that has proved difficult to achieve with the topical
application of recombinant growth factors. It is also likely that these
bioengineered skin products will be engineered to deliver certain growth factors
in large quantities, i.e., PDGF (28). This type of delivery may render growth
factor therapy more effective.
REFERENCES
Return to Woundbiotech Home
Page
Return to Growth
Factors
