Growth Factors and Wound

Historical overview of Growth Factors, Cytokines, and themkines

In 1962 a small polypeptide was isolated from the mouse submaxillary gland that promoted early
eruption of incisors and eyelid opening in neonatal mice. Further studies demonstrated that this
polypeptide stimulated epidermal cells to proliferate continuously in chemically defined culture
medium containing essential nutritional factors (i.e., adequate levels of vitamins, essential amino
acids, and cofactors). Prior to these discoveries, no single, biochemically defined agent was known
that could promote continuous cycles of cell division in vitro under conditions of complete
nutritional sufficiency. Based on these properties, the polypeptide was named epidermal growth
factor (EGF). These results firmly established the concept of the regulation of cell proliferation and
differentiation by a specific protein. The field of cell growth and differentiation factors has grown
to include offer classes of regulatory molecules including cytokines and chemokines. Collectively,
their actions include essentially all types of cells and span from the earliest phases of embryonic
development to regulation of. the most highly specialized, terminally differentiated tissues. Today,
several hundred growth factors, cytokines, and chemokines have been identified, and their impact
on biology and medicine was recognized by the 1986 Nobel Prize in Physioloy or Medicine
awarded to Dr. Rita Levi-Montalcini and Dr. Stanley Cohen for their pioneering work in the
discovery and characterization of growth factors and their receptors.

The nomenclature of growth factors arid cytokines evolved in an inconsistent manner, often
•resulting in confusing and misleading terminology. Historically, growth factors were named based
on the type of cell that was stimulated (e.g., fibroblast growth factor (FCF) or EGF), the source of
the growth factor (e.g., platelet-derived growth factor, PDCF), the structure of the protein (e.g.,
insulin-like growth factor-I, ICF-I), or the action of protein on cultured cells (e.g., transforming
growth factor-ß, TCF-ß). Six major fåmilies of growth factors are presented in Table 2—1. Proteins
that were identified by their ability to regulate leukocyte and lymphocyte proliferation and
differentiation were initially named interleukins (e.g., interleukin-2, ILA), and other proteins were
named based on their regulation of the differentiation of precursor inflammatory cells (e.g.,
granulocyte/macrophage - colony-stimulating factoroGM-CSF) or their äbility to suppress viral
infections (e.g., interferon-alfa, IFNO). Further research on these regulatory proteins, however,
invalidated the initial concept that growth factors and cytokines were produced by, or acted on, a
narrow range of target cells. This led to the adoption of the more general term, cytokine (Latin:
cyto meaning cell and kine tneaning action or motion) to describe these regulatory proteins, as
they shared the ability to regulate the proliferation and differentiation of cells.

In several cases, there has been a trend to revise the nomenclature for many of these growth
factors and cytokines. One such example is the effort to standardize the nomenclature for
members of the tumor necrosis factor (TNF) and TNF receptor superfamilies (ligands being call
TNFS and a numberi and the receptors called TNFSR and a number). In this chapter, however, we
use the more historical basis for grouping these regulatory proteins, term growth factor is used to
describe the mitogenic proteins listed in Table 2—.1 and the term cytokine to indicate protein
mediators listed in Table 2—2 that have major regulatory effects on inflammatory cells. A third
important group of small regulatory proteins, listed in Table 2—3, has been identified; they are
collectively named chemokines based on a contraction of "chemoattractive cytokine(s)." The
structural and functional similarities among chemokines were not initially appreciated, and this
has led to an idiosyncratic nomenclature consisting of many acronyms that were based on their
biologic functions (e.g., monocyte chemoattractant protein I (MCP-I), macrophage inflammatory
protein l , MIP-Il, their source for isolation (platelet factor 4, PF-4), or their biochemical properties
[interferon-inducible protein of 10 kDa (IP-IO) or regulated upon activation normal T-cell
expressed and secreted, RANTESI.

As their biochemical properties were established, it was recognized that the approximately 40
chemokines could be grouped into four major classes based on the pattern of cysteine residues
located near the N-terminus. In fact, there has been a recent trend to reestablish a tnore
organized nornenclature system based on these (our tnaior classes. In general, chemokines have
two pritnary functions: (l) they regulate the trafficking of leukocyte populations during normal
health and development, and (2) they direct the recruitment and activation of neutrophils,
lytnphocytes, Inacrophages, eosinophils, and basophils during inflatnmation.

Receptors and Signal

Transduction Pathways for Growth Factors, Cytokines, and Chernokines

All growth factors, cytokines, and chemokines affect cells by activating receptors that are integral
membrane proteins. In general, both the specificity and affinity of the receptors for their ligands is
high, with Kd values in the range of 10-9 to 10-12 M. A physiologically significant effect is often
generated by occupancy of a low percentage of receptors (< 10%), which can cause cells to
respond to very low levels of growth factors, cytokines, and chemokines. Also, the expression of
many receptors for growth factors, cytokines, and chemokines may be regulated by another
cytokine or monomeric EGF-R molecules in their plasma membrane. After binding EGF, the EGF-R
monomers rapidly dimerize,

and the kinase domain autophosphorylates selected tyrosine side chains in the cytoplasmic
segment of the companion EGF-R protein. Adaptor proteins in the cytoplasm (e.g., GRB2)
bind the specific phosphotyrosine residues that are generated on the activated EGF-R. Guanine
nucleotideexchange (GEF) proteins (e.g., sos) then bind to specific (lotnains on the adaptor
protein, generating a trimeric com-

plex of the EGF-R, the adaptor protein, and the guanine nucleotide-exchange protein. The prinvary
function of the GEF protein is to induce an exchange of GTP with GDP bound on GDP-Ras protein.
The activated GTP-Ras protein then initiates a kinase cascade by activating two kinases (Raf and
Mek), which culminates in the phosphorylation and activation of the serine/threonine kinase
activity of a key regulatory protein, mitogen-activated protein (MAP) kinase. After dimerizing,
activated MAP kinase translocates to the nucleus, where it phosphorylates specific sites on various
transcription factors (e.g., serum response factor, SRF; ternary complex factor, TCF), which then
associate and bind to specific DNA sequences (e.g., serum response element, SRE) that regulate
expression of specific genes. Phosphorylation of various transcription factors by MAP kinase can
produce multiple effects on gene expression, thereby producing distinct effects by different
growth factors on different cells.

Several mechanisms act to limit activation of an RTK signal transduction system. Protein
phosphatase enzymes in the cytosol rapidly inactivate signal transduction proteins by removing
phosphate groups that were added to tyrosine, even the same cytokine, which permits positive
amplification or negative feedback inhibition.

The principal function of all receptor proteins is to convert an extracellular signal (i.e., the specific
binding of the growth factor, cytokine, and chemokine by the receptor) into intracellular signals,
which then trigger physiologic responses in the target cell. However, receptors for growth factors,
cytokines, and chemokines utilize different transduction pathways to generate their intracellular
signals. Receptors for growth factors typically have intrinsic tyrosine kinase activity that is
activated when a specific ligand is bound. This initiates a cascade of signal-transduction steps that
eventually causes changes in the target cell's physiology (e.g., mitosis, migration, apoptosis, or
synthesis of specialized proteins such as collagen). These changes result frotn alterations in basic
processes in the responding cell, such as ion fluxes, tnicrofibril polymerization, or transcription of
genes.

The EGF receptor (EGF-R) and its signal-fransduction system have been studied extensively and
provide an excellent example of how receptor tyrosine kinases (RTKs) generate changes in cells. As
shown in Figure 2—1A, target cells typically have about 10,000 specific, high-affinity, serine, and
threonine side chains by the kinase enzymes. Many growth factor—receptor complexes are rapidly
internalized and degraded in lysozomes, which limits the duration of the initiating step of the
signal transduction pathway. Also, the key transduction protein Ras has intrinsic-GTPase activity,
which -limits the duration of the GTP—Ras complex. It remains active by limiting the time the GTP
—Ras persists before the GTP is hydrolyzed to CDP.

Cytokine receptor proteins differ substantially from RTK proteins (Fig. 2—1B). They typically
consist of homodimers, heterodimers, or heterotrimers of 01-, ß-, and At-type transmembrane
proteins, and all lack intrinsic kinase activity. A general model for cytokine receptor signaling
proposes that signaling is initiated when ligand binding causes aggregation of receptor subunits.
These receptor aggregates become the structural framework for the interaction with either
docking proteins or kinases. The receptors for many families of cytokines, such as the IL-2, ILr6,
and IL-IO families, interact noncovalently with a family of tyrosine kinases called Jak kinases (Janus
kinases) through specialized regions of the cytoplasmic segments of receptor subunits. The Jaks
phosphorylate each other when they are brought into close apposition upon aggregation of the
receptor subunits. Phosphorylation of Jaks greatly increases their catalytic activity, and they
further phosphorylate themselves and tyrosines in the cytoplasmic tails of the aggregated cytokine
receptor subunits. These receptor phosphotyrosines form sites where latent cytoplasmic
transcription factors, called Stats (signal transducers and activators of transcription), dock and are
activated by phosphorylation of a single tyrosine by the Jaks. The phosphorylation step releases
the Stats from the receptor—Jak complex, and the Stats dimerize and translocate to the nucleus,
where they bind to specific sequences of DNA and initiate gene transcription: Thus the Jaks
provide the cytokine receptors with tyrosine kinase activity that generates all the downstream
signaling of cytokines.

In the IL-2 and IL-6 superfamilies, specificity for cytokine signaling is determined by the
composition of the subunits comprising the receptor complex, the four known Jaks that bind the
receptor, and the seven known Stats that are phosphorylated. The activity of the cytokine receptor
Jak/Stat signaling system is limited by pr8tein phosphatase enzymes, as noted for the growth
factor RTK signaling system. In addition, the concentration of biologically active protein for some
cytokines in blood is modulated by the-presence of soluble receptors (e.g., TNF-a, ILA, IL-6) that
are generated by proteolytic cleavage of membrane receptors and act as sinks, or by natural
competitive inhibitors (e.g., IL-Ira).

Chemokine receptors (Fig. 2—1C) and their signal transduction mechanism comprise a third type
of system. All chemokine signaling receptors are 7-transmembrane spanning proteins, and they
generate intracellular signals by interacting with trimeric G protein-coupled systems that are
functionally linked to phosphölipases. Although most chemokine receptors bind more than One
chemokine, the four known CXC rece+tors (CXCR 1—4) bind only CXC chemokines (a family of
chemokines), the eight known CC receptors (CCR 1—8) bind only CC chemokines (ß family of
chemokines), and the one known receptor (CX3CRl) binds the only member of the CX3C family
member, fractalkine (6 family of chemokines).

Many cell-surface receptors are coupled to trimeric, signal-transducing, G protein complexes

including the chemokines, epinephrine, glucagon, ACTH, and prostaglandin El (PCEi), and all utilize
a similar post receptor signaling pathway. Binding of a chemokine to its receptor produces
conformational changes in the cytöplasmicsegments of the receptor, which generate a binding site
for the GDP-Gs subunit of the trimeric C protein complex. Interaction of the CDP-Cs subunit with
the chemokine—receptor complex promotes displacement of CDP by GTP in the Cs subunit and
dissociation of the GTP-Cs subunit from the Cß and C subunits.

The GTP-GS subunit then docks with and activates phospholipase-C (PLC), a key enzyme in the
cytoplasmic leaflet of the plasma membrane. Active PLC cleaves a phosphate ester bond of the
membrane lipid phosphoinositol 4,5-bisphosphate (PIP2), which generates two important signal
transduction molecules: the water-solubl< molecule inositol trisphosphate (IPO and the lipid-
soluble molecule diacylglycerol (DAG). DAG molecules activate the membrane-bound enzyme
protein kinase-C (PKC), which phosphorylates and activates various transcription factors that
influence gene expression. IP3 molecules diffuse through_the cytosol and interact with In-sensitive
Ca2+ channels in the membrane of the endoplasmic reticulum, causing release of stored Ca2+
ions. The localized increase in the Ca2+ ion concentration triggers various responses, including
activation ofthe small cytosolic protein called calmodulin. The Ca2+-calmodulin molecule can bind
and activate several kinases that phosphorylate and activate various transcription factors, leading
to changes ill the profile of mRNAs and proteins synthesized by target cells.

Although the three receptor models present distinct signal transduction pathways for prototypical
growth factor, cytokine, and chemokine receptor systems, the situation is more complex because
interaction (cross-talk) occurs between the pathways. For example, the cytoplasmic regions of RTK
receptors can interact directly with Jak proteins leading to phosphorylation and activation of Stat
transcription factors. The C-protein-coupled receptors of the chemokine receptors also can
activate adenylate cyclase, the enzyme that generates cyclic AMP (cAMP), which is a major second
messenger that influences multiple cellular processes. Although most of the major effects of
growth factors, cytokines, and chemokines result from the signal transduction pathways that are
presented, other signal transduction pathways, such as the Smads, are involved in mediating the
effects of growth factors, especially transforming growth factor-ß (TCF-ß). Local Jersus Systemic
Actions of Growth Factors, Cytokines, and Chemokines
The hallmark property of growth factors, cytokines, and chemokines is their ability to regulate the
migration, differentiation, and proliferation of cell populations during inflammation and tissue
repair. Their effect on cells depends to a certain extent on their mode of production. Some growth
factors, cytokines, and chemokines are released into the bloodstream and produce a systemic
effect, resembling classic endocrine hormones that are synthesized in specialized tissues and
released into the bloodstream to act on distant target organs. Interleukins IL-6 and IL-IO are
examples of cytokines that appear systemically in the circulation and regulate the hepatic acute-
phase and acquired immune responses, respectively.

In contrast, cytokines such as tumor necrosis factor-a (TNF-a) and IL.rla are primarily membrane-
associated and are released locally to induce tissue inflammation. Their appearance in the
bloodstream is infrequent, but when it does occur they can mediate the systemic inflammat01Y
response syndrome (SIRS) response. However, Inost growth factors, cytokines, and chemokines
exert their physiologic effects locally on neighboring cells (a parachnc action) or on the same cell
that synthesized and released it (an autocrine action).

It has been recognized that some growth factors and cytokines are present in the plasma
membrane of cells as integral membrane pro-proteins [e.g., TNF-a and transforming growth
factor-a (TCF-a)) and are able to bind receptors on adjacent cells through a iuxtacrine interaction.
Futhermore, some growth factors and cytokines produce their effects on cells while still within the
cytoplasm after synthesis by a process called intracrine action. Additionally, many growth factors,
cytokines, and chemokines have the ability to enhance or suppress their own production by a
direct autoregulatory feedback mechanism. In summary, growth factors, cytokines, and
chemokines typically act locally through Paracrine, autocrine, juxtacrine, and
intracrinemechanisms and are key regulators in inflammation and surgical wound healing.

Key Growth Factors

Growth factors share the important characteristic of acting as a direct mitogen for target cells.
Although they play essential roles in embryonic development, their importance for surgeons
focuses more on their key roles in normal cell turnover and in the response of tissues to injury.
They are synthesized and secreted by many differentiated cells involved in wound healing,
including platelets, inflammatory cells, fibroblasts, epithelial cells, and vascular endothelial cells.
Growth factors typically are grouped into major families based on their biochemical structure:
epidermal growth factor (EGF), TCF-ß, insulin-like growth factor (ICF), platelet-derived growth
factor (PDCF), fibroblast growth factor (FCF), and recently connective tissue growth factor (CTCF).

Epidermal Growth Factor Family

The epidermal growth factor family comprises four mammalian proteins: EGF, TCF-a,
amphiregulin, heparin-binding epidermal growth factor (HB-ECF), and betacellulin. These peptides
are similar in structure, bind to the same cell membrane receptor, and have similar but

not identical biologic effects. EGF, the most thoroughly studied member of the family, is
synthesized as a large transmembrane glycoprotein precursor molecule that is proteolytically
cleaved to release a small biologically active 53-amino-acid fragment. It is folded into a triple loop
structure that is stabilized by disulfide bonds, is required for biologic activity, and distinguishes all
members of the EGF family from other families of growth factors.
Platelets contain a substantial amount of EGF (approximately 500 pmol/10 12 platelets). After
clotting, the local concentration of EGF in the interstitial fluid rises to levels sufficient to induce
mitosis and migration of cells, suggesting a role for EGF during the early phase of wound healing.
EGF is secreted by keratinocytes and, in an autocrine fashion, directs epithelialization. EGF is
synthesized by several tissues, including kidney, lacrimal gland, submandibular gland, Brunner's
gland, and megakaryocytes; it is found in many secretions such as saliva, tears, and urine.

Reports on clinical trials with EGF have noted that repeated topical application of EGF accelerated
the rate of epidermal regeneration of partial-thickness dermatome wounds, enhanced healing of
chronic wounds, and promoted epithelial regeneration of corneal injuries. EGF promotes healing
in these wounds presumably by stimulating migration and division of epithelial cells and by
increasing synthesis of proteins such as fibronectin, which aid in cell attachment and migration.
Although EGF does not efficiently induce synthesis of mRNA for extracellular matrix proteins such
as collagen, EGF presumably increases the numbers of fibroblasts in wounds through chemotaxis
and mitosis, resulting in more total collagen production.

Transforming growth factor-a is synthesized by a large variety of normal cells including activated
macrophages, eosinophils, hepatocytes, keratinocytes, gastrointestinal cells, brain cells, and
placental cells. TCF-a and EGF have many similar effects on cells. Both factors stimulate mitosis of
keratinocytes and fibroblasts, suppress gastric acid secretion, and accelerate healing of epidermal
injuries. Although the role of heparin-binding EGF in wound healing is not known, its production by
macrophages and its ability to bind a component of the extracellular matrix (heparin sulfate)
suggest that it may be involved in wound healing. Both heparin-binding EGF and EGF are mitogen:
for keratinocytes and fibroblasts, but heparin-binding EGF unlike EGF, is not a mitogen for vascular
endothelial cells. The mitogens amphiregul\n qnd betacellulin were isolated from tumor cells'
(human colon carcinoma cells and mouse pancreatic beta cell tumors) and share the three-
disulfide, triple-loop structure characteristic of EG family members. They also bind, albeit more
weakly, to the ECF receptor (ECF-R).

Transforming Growth Factor-ß Family

Three distinct TGF-ß varieties have been identified in humans: TGF-ßl, TGF-ß2, and TGF-ß3. All are
sized as latent 28-kDa dimers that appear to be activated by proteolytic cleavage in a complex
process involving the binding of latent TGF-ß by the mannose-6-phosphate receptor and cleavage
by plasmin. TGF-ßl was initially isolated and sequenced from platelets; and like TCF-a, it was
named for its ability to stimulate reversibly the growth of normal fibroblasts in soft agar. The TGF-
ßs are synthesized by a wide variety of cell types, including platelets, macrophages, lymphocytes,
fibroblasts, bone cells, and keratinocytes. Nearly all cells have TCF-ß receptors. Thus, TGF-ßs are
probably the most broadly acting of all the families of growth factors. A distinguishing
characteristic of the TGF-ß family of proteins is their ability to inhibit the growth of a number of
cell types, particularly cells derived from the ectoderm such as keratinocytes and leukocytes.
There are three distinct TGF-ß receptor proteins, designated types I, Il, and Ill, that interact in a
complex fashion to generate the cellular signal transduction pathway. The type Il receptor appears
to bind TCF-ß and then aggregate with the type I receptor protein, which activates the
serine/threonine kinase domain in the cytoplasmic region of the type I receptor, leading to
phosphorylation of Smad transcription factors present in the cytoplasm. A balance of Smad-3,
Smad-4, and Smad-7 actions leads to transcription of TCF-ß-responsive genes. The type Ill TGF-ß
receptor probably does not participate in signal transduction directly but may act as a reservoir for
TCF-ß.

The TGF-ßs have similar but not identical biologic actions on cells. All three of the TCF-ß isomers
can stimulate chemotaxis of inflammatory cells and stimulate production of extracellular matrix
through increåsed synthesis of collagens and proteoglycans. In addition, TCF-ßs downregulate
synthesis of the matrix metalloproteinases (MMPs) in fibroblasts and upregulate synthesis of the
natural inhibitors of MMPs, the tissue inhibitors of metalloproteinases (TIMPs). These properties
make TCF-ßs important regulators of the deposition and removal of extracellular matrix. It is not
surprising that incisional wound healing can be accelerated by addition of exogenous TCF-ß
protein or by transfection of the wound incision by plasmids expressing the TCF-ß gene. However,
excess or prolonged action ofTGF-ßs have been implicated in several fibroproliferative diseases,
such as scleroderma, hepatic sclerosis, and interstitial pulmonary fibrosis. Studies on keloid and
hypertrophic scars showed

increased expression of TCF-ßl mRNA in the tissues. Experiments in animal models suggest that
blocking TCF-ßl and TCF-ß2 with inje9tions of neutralizing antibodies _reduces scarring in skin
•incisions; and TGF-ß3, in some animal models, suppresses scarring. Other important members of
the TCF-ß superfamily include the bone morphogenic proteins (BMPs) and the activin and inhibin
hormones. Although this chapter focuses primarily on the role of growth factors, cytokines, and
chemokines in soft tissue, BMPs clearly play key roles in the normal maintenance and repair of
cartilage and bone injuries.

Insulin-like Growth Factor Family

Insulin-like growth factor I (IGF-I) and insulin-like growth factor Il (IGF-II) have substantial amino
acid sequence homology to proinsulin. Both IGF-I and IGF-II are synthesized as large precursor
molecules (195 and 156 amino acids) that are proteolytically cleaved to release the biologically
active monomeric proteins (70 and 67 amino acids). Although IGF-I and IGF-II are synthesized by a
wide range of adult tissues, IGF-II is synthesized predominantly during fetal development,
whereas IGF-I synthesis remains high in a wide range of adult tissues including liver, heart, lung,
kidney, pancreas, cartilage, brain, and muscle. Many of the biologic acti6ns originally attributed to
growth hormone are mediated in part by IGF-I, which is also known as somatomedin C. IGF-I
stimulates skeletal, cartilage, and bone growth. However, combinations of growth hormone and
ICE-I are more effective than either hormone alone.

This led to the dual effector theov of growth hormone and ICE-I action: Growth hormone initiates
cell differentiation and increases the production of IGF-I, which then promotes cell division. The
TCF-I receptor is a tyrosine kinase, as is the insulin receptor. However, the IGF-II receptor is not a
kinase; it is also called the mannose-6phosphate (M6P) receptor because of its ability to bind
mannose-6-phosphate groups that are part of the carbohydrate structures of glycoproteins. The
signal transduction pathway for the ICF-II receptor is not understood.'

Unlike other peptide growth factors, plasma contains substantial levels of IGF-I, which primarily
reflects hepatic synthesis. ICF-I is reversibly bound by highaffinity ICF-binding proteins in plasma.
At least six ICF binding proteins have been identified, and most tissues that synthesize ICFs also
synthesize ICF-binding proteins. Because the IGFs are inactive while bound to their binding
proteins, the dynamic balance between free and bound IGFs has a substantial influence on the
effects of IGFs during wound healing. ICF-I is found in substantial levels in platelets and is released
during clotting along with the other growth factors present in platelets. IGF-I is a chemotactic
agent for vascular endothelial cells and may stimulate angiogenesis under certain conditions. LCF-I
also stimulates mitosis of many cells in vitro, such as fibroblasts, osteocytes, and chondrocytes. It
may also act synergistically with PDCF to enhance epidermal and dermal regeneration.

Platelet-Derived Growth Factor Family

The PDGF family comprises two major proteins of interest for surgeons: PDGF and vascular
endothelial growth factor (VEGF). PDGF and VEGF have similar structures, but they act through
different receptors and stimulate different actions. PDGF is primarily mitogenic for mesenchymal
cells, whereas VEGF is almost exclusively a mitogen for vascular endothelial cells. PDGF is
composed of two polypeptide chains (A and B) combined in three disulfide-linked dimeric isoforms
(AA, AB, BB). It is secreted by a variety of cells important for wound healing, including placental
cells, fibroblasts, vascular smooth muscle cells, and vascular endothelial cells. Macrophages also
synthesize a PDGF-like protein that binds to PDGF receptors. The macrophage-derived PDCF-like
protein together with PDCF-AA isoform are present in human wound fluids for several days after
surgery. The PDC,F receptor is a tyrosine kinase formed by two subunits, designated a and B. The
01 receptor subunit recognizes both the A and B subunits of PDCF, whereas the ß receptor subunit
recognizes only the B subunit of PDGF. Because all three PDGF receptor types (aa, ocß, ßß) are
expressed by different cell types, treatment with the PDCF-BB isoform generally has the broadest
effect.

Significantly decreased levels of all three PDCF isomers and its receptors were demonstrated in
chronic, nonhealing wounds. The treatment of acute and chronic wounds with PDGF-BB resulted
in elevation of PDCF-AAL in both types of wounds. The topical application of recombinant PDCF-BB
to acute wounds in animal models resulted in improved wound-breaking strength and healing
times. Clinical studies using topical recombinant PDGF-BB on chronic, nonhealing diabetic ulcers
have also demoristrated decreased healing times and an increased percentage of wounds that go
on to heal compared to untreated controls. The recombinant human PDCF-BB isoform (Regranex)
is the only recombinant growth factor medication approved by the U.S. Food and Drug
Administration (FDA) for the treatment of chronic wounds, specifically diabetic foot ulcers.

A heparin-binding, disulfide-linked, homodimeric protein, VECF was originally isolated from

pituitary cell cultures, It is secreted mainly by keratinocytes but is also produced by macrophages
and fibroblasts. It is a potent mitogen for endothelial cells and has minimal mitogenic effect on
cells that are responsive to PDCF, including fibroblasts and vascular smooth muscle cells. •In
addition to its ability to stimulate mitosis of cultured endothelial cells, VECF was found to be
angiogenic in vivo. Although little is known about the role VECF may play in wound healing, its
selective mitogenic action on vascular endothelial cells, its ability of bind to heparin, and its
angiogenic action in vivo suggests that it may be an important factor in wound healing.

Fibroblast Growth Factor Family

The FGF family consists of 14 homogeneous, heparinbinding, 28 kDa proteins that appear to play
important roles in regulating key aspects of embryonic development and wound healing. Two
members, named acidic FGF (aFGF) and basic FGF (bFGF) are closely related proteins with
isoelectric points of pH 5.6 and 9.6, which are reflected in their names. Neither the aFGF nor the
bFGF precursor proteins contain a typical leader sequence, which is found in essentially all
secreted proteins, so it is unclear how they are released from cells. Nevertheless, substantial
amounts of FCF are found bound to proteoglycans such as heparan sulfate in the extracellular
matrix, especially in the basement membrane of capillaries. Their association with the extracellular
matrix may serve to protect FGF from proteolytic degradation by functioning as a storage depot,
with the liberation of FGF through matrixdegrading enzymes such as heparinase or cathepsin D
following injury. FGF receptors are tyrosine kinases, and binding of FGFs require the combined
action of heparan sulfate proteoglycans and the FGF receptors, of which there are multiple forms
that have varying specificity for the FCFs.

The FGFs appear to play a major role in wound healing. They stimulate proliferation of all the
major cell types involved in wound healing including vascular endothelial cells, fibroblasts,
keratinocytes, and other specialized cell types such as chondrocytes and myoblasts.• Many of the
cells that respond to FGF also synthesize the peptide, including fibroblasts, astrocytes,
endothelium, smooth muscle cells, chondrocytes and osteoblasts. The FCFs may have their most
important effect in wound healing by stimulating angiogenesis through inducing endothelial cell
migration and proliferation. Studies using a topical application of recombinant bFCF on diabetic
Ulcers demonstrated improved healing. In a phase Ill trial in China using a topical application of
recombinant bFCF on bums, surgical incisions and chronic dermal ulcers showed an accelerated
rate of wound closure; moreover, healing time decreased by an average of 3 to 4 days, and the
wound closure rate was higher than 90% for all of the groups. Recombinant bFCF was also shown
to improve healing of chronic pressure sores when given alone or in sequential treatment with
granulocyte/macrophage colony-stimulating factor (CM-CSF).

Keratinocyte growth factor (KCF or FCF-7) is another important member of the FCF family. KCF is a
22.5 kDa glycoprotein that shares about 40% amino acid sequence identity with aFCF and bFCF; it
contains a hydrophobic N-terminal secretory signal sequence and has two •major •isoforms, KCF-I
and KCF-2. In contrast to FCFs, synthesis of KCF'is restricted to fibroblasts; and, most importantly,
KCF stimulates mitosis almost exclusively of keratinocytes. has led to the concept that KGF is a
paracrine effector of epithelial cell growth. role of KGF in wound healing is not fully-
understood, but it is highly upregulated in the dermis during wound healing.

Because it selectively stimulates mitosis of keratinocytes, it is logical to assume that KGF would be
useful for treating partial-thickness burns or meshed skin grafts. Topical KGF treatment of
explanted meshed human skin grafts in athymic animals bas been shown to accelerate closure of
interstices; and studies using topical recombinant KGF-2 on chronic venous stasis ulcers have
demonstrated improved wound closure. Phase 'Ill studies are currently in progress. The KGF
receptor is a tyrosine kinase that is an isoform of the FCFR-2 gene.

Connective Tissue Growth Factor

Connective tissue growth factor (CTGF), a cysteine-rich,

38 kDa protein, is a member of the recently described CCN family, which includes CTGF, cysteine-
rich 61 (cyr61), neuroblastoma overexpressed (nov), elm l, Cop-I, and WTP-3. CTGF was first
identified in medium conditioned by cultured human vascular endothelial cells due to a fortuitous
cross-reactivity of a polyclonal antiserum to PDCF with CTCF. CTGF is produced by a variety of cells
including fibroblasts, vascular endothelial cells, vascular smooth muscle cells, chondrocytes, and
glioblastoma cells. Interestingly, physiologic levels of the coagulation cascade protease thrombin
upregulates expression of CTGF mRNA in fibroblasts, suggesting that the coagulation process
promotes CTGF formation during normal tissue repair. CTGF receptor has not been biochemically
identified.

From a surgical viewpoint, the most important biologic effect of CTGF is its ability to promote
formation of scar tissue. CTGF is mitogenic and chemotactic for fibroblasts, and it strongly
stimulates synthesis of type I collagen and fibronectin. It has been implicated in a variety of fibrotic
disorders, such as systemic and localized sclerosis, keloids, and Dupuytren's contractures.

Furthermore, studies have shown important links beKveen TCF-ß and CTGF. TGF-ß directly
upregulates expression of CTCF mRNA and protein in cultured fibroblasts; levels of TCF-ß and CTGF
mRNAs are coordinately expressed during wound repair; and CTGF transcripts are induced in skin
fibroblasts at the site of epidermal injections of TCF-ß in neonatal mice. Most importantly,
neutralizing antibodies to CTGF or antisense oligonucleotides targét• ing CTCF block the induction
of collagen synthesis by TCF-ß, which indicates that CTGF functions as a downstream mediator of
TCF-ß action on fibroblasts. nerapies friat selectively inhibit CTGF action may be useful for reducing
fibrosis in numerous pathologic conditions.

Kev Cytokines

Historically, the term cytokine was used to describe proteins that regulated mitosis and
differentiated the functions of immune system cells (e.g., T and B lymphocytes, macrophages,
polymorphonuclear leukocytes, and tumor cell lines). However, as it became clear that cytokines
could also influence nonimmune system cells (e.g., fibroblasts, endothelial cells, epidermal cells)
and that some of the classic growth factors could influence

important functions of immune system cells (e.g., migration, proliferation), separation of the
proteins into cytokines and growth factors became blurred. Hence the more generic term cytokine
was frequently used to describe both groups of regulatory proteins. In this chapter, we use the
term cytokine in its more original context to describe proteins that are involved in the immune
response to inflammation and/or tissue injury, and we emphasize their importance in processes
that are important for surgeons.

Pro-inflammatory Cytokines

tumoral necrosis factor-a

The activity of most cytokines can often the categorized as being proinflammatory or anti-
inflammatory depending on their role in the host immune response and their involvement in local
and systemic inflammation. One of the earliest proinflammatory cytokines identified was TNF-ct.
TNF-a was induced unintentionally during the 1880s when a New York surgeon, William Coley,
injected a preparation of gram-positive and •gram-negative bacteria, "Coley's toxins", into
patients with inoperable neoplastic disease. In some of the patients treated with the bacterial
cocktail, hemorrhagic necrosis developed in the tumors, which in certain cases progressed to
complete tumor resolution.

Historically called cachectin, TNF-a is produced by macrophages and other cells from the
monocyte lineage in response to microbial pathogens, tumors, hemorrhagic shock, and tissue
injury. It acts in a paracrine fashion to initiate the innate immune response by stimulating
production of other proinflammatory cytokines and by stimulating the influx of inflammatory cells.
TNF-a also plays a significant role in the development of a Thi responx and serves as a
communication link bewveen the innate and acquired immune responses. Another significant role
of TNF-O. is in early wound healing, where it modulates angiogenesis, suppresses proliferation of
vascular endothe lial cells, and induces the production of IL-I, IL-3, C-CSI and GM-CSF. TNF-O( also
stimulates fibroblast proliferation and inhibits their synthesis of collagen; and it indu fibroblasts to
produce matrix metalloproteinases (MMPs), IL-I, IL-6, and leukocyte inhibitory factor (LIE).

Normal wound healing requires the release of TNF-O( at the appropriate times and in adequate
levels. Excessive activation of immune cells can lead to tissue damage through the production of
reactive oxygen species, proteolytic enzymes, and arachidonic acid metabolites. Studies have
shown that rats with chronically elevated TNF-u levels had impaired wound healing compared to
controls. More recently, studies have reported elevated levels of TNF-O( in nonhealing versus
healing chronic venous ulcers, although no cause-and-effect relation has been established.
Systemically, excessive levels of circulating TNF-a has been associated with multisystem organ
failure and increased morbidity and mortality during inflammatory disease states.

Interleukin- T

The general term interleukin was originally used to refer to the molecules that signaled between
cells in the immune system. It was later determined that interleukins have many more functions.
They are currently designated by the order in which they are identified. They are used to define
the class of mediators that regulate specific components of immune signaling between leukocytes
and somatic cell subpopulations.

Interleukin-I, originally known as lymphocyte-activating factor and as an endogenous pyrogen, is

actually a family of at least three proteins that are primarily secreted by activated macrophages.
Two forms of IL-I (IL-la and Il-rlß)

are agonists; the third (IL-I receptor antagonist, or IL-Ira), is a pure receptor antagonist. IL-lß is
processed from an inactive precursor and is readily secreted, whereas Il-rlo. exists primarily as a
membrane-associated form. Members of the Il-rl superfamily are produced by a variety of other It
has the ability to initiate and sustain the production of MMPs and suppress the production of
tissue inhibitor metalloproteinases (TIMPs), thereby modifying the extracellular matrix to aid in
cell migration. Another important proinflammatory property of IL-I is its ability to increase the
expression of adhesion molecules such as intercellular adhesion molecule-I (ICAM-I) on endothelial
and other cell surfaces. This property promotes the infiltration of inflammatory and
immunocompetent cells into the extravascular space.
YlnterIeukin-2

Interleukin-2 is primarily a T cell growth factor. It induces growth and differentiation of T-cells, NK
cells, some B lymphocytes, and lymphokine-activated killer cells.

Activation of NK cells by IL-2 results in increased interferon-Y (IFN-Y) production and increased
cytotoxic effects against tumor targets. Clinical trials have studied the use of IL-2 to treat some
human cancers. Although IL-2 has high toxicity (in part through the induction of TNF-u), it has been
approved for the treatment of patients with metastatic melanoma. Patients infected with human
immunodeficiency virus type I (HIV-I) with low CD4 T cell counts following IL-2 therapy developed
increased numbers of circulating T cells.

Interleukin-2 appears to be essential for sustaining the postinjury repair response during wound
healing. IL-2 activates monocytes to produce TNF-ot. In a study involving rats treated with
doxorubicin, 11.2 administration increased the infiltration of inflammatory cells and fibroblasts
into the wounds, resulting in increased wound-breaking strength. This action is most important
in wounds in immunocompromised hosts, as ILr2 did not increase wound étrength in normal
control rats.

cells, including B and T lymphocytes, neutrophils, natural killer (NK) cells, fibroblasts, endothelial
cells, epithelial

Elfitérleukin-6

Interleukin-6 (11.6), a pleiotropic cytokine, is unique in cells, smooth muscle cells, and vascular
tissues in response to trauma, infection, or exposure to antigens.

Interleukin-I is involved in the escalation of local and systemic inflammatory responses. As an

inducer of the innate immune response, IL-la and IL-lß directly stimulate fibroblasts and vascular
endothelial cells. IL-I enhances local inflammation through secondary cytokine production and acts
in an endocrine fashion to induce liver hepatocytes to produce acute-phase proteins, fibrinogen,
C-reactive protein, and haptoglobin, which are mediators of systemic inflammation. IL-I •is also
ihvolved in acquired immunity, primarily as an adjuvant and a co-mitogen for proliferating T cells.

Interleukin-la is found in large quantities in the skin, where it is presumed to serve as a regulator
of growth an proliferation. Il-rl is important in wound healing because it increases the expression
of ptoinflammatory genes. that it has both proinflammatory and anti-inflammatory characteristics.
IL-6 has numerous biologic activities that can be divided into effects on regulation of the
hematopoietic system and those involved in activation of the innate or acute-phase immune
response. Similar to other proinflammatory cytokines, ILc6 is predominantly produced by
macrophages and cells from the monocyte lineage. Locally, within the wound, it is secreted by
polymorphonuclear neutrophils (PMNs) and fibroblasts. Its concentration in the wound directly
parallels the number of PMNs. IL-6 has been shown to be a potent inducer of fibroblast
proliferation. It is also able to suppress proinflammatory responses by down-regulating TNF-u and
IL-I, which are potent inducers of IL-6 expression. It reduces chemokine expression as well.
Unlike most cytokines, IL-6 has a significant endocrir component and is rapidly released into the
circulation in response to inflammation. The systemic effects of 11.6 on nonimmune cells are
closely tied to activation of the innate immune system, activation of the hypothalamic- pituitary-
adrenal axis, and induction of the hepatic acutephase protein response. IL-6 has also been shown
to be pyrogenic. Burn patients and patients with acute bacterial infections have elevated plasma
IL-6 concentrations, which have been shown to be occasionally predictive of the outcome of
patients with sepsis or SIRS. Studies have documented that IL-6 is produced by synovial fibroblasts
in the presence of rheumatoid arthritis, and several studies have shown elevated serum 11.6
concentrations in patients with this disease, frequently correlating with disease severity.

EThterleukin-8

Interleukin-8 (IL-8), a proinflammatory cytokine, is a member of the CXC chemokine family. It

appears to exert its effects early in acute wounds. IL-8 is primarily secreted by macrophages and
fibroblasts and is chemotactic for neutrophils and monocytes. It has been shown to increase
neutrophil degranulation and the expression of endothelial cell adhesion molecules. IL-8 is found
in highest concentrations during the first day of an injury and appears to be important in
promoting keratinocyte maturation and margination. Elevated levels of IL,-8 have been identified
in fibroblasts from patients with psoriasis compared to normal controls, implicating it in the
pathogenesis of the psoriatic wound-healing phenotype. The expression of IL-8 mRNA in fetal
fibroblasts is significantly less than in adult control fibroblasts, indicating that the diminished
proinflammatory response in fetal tissue may contribute to the scarless healing seen in utero.

Interferon-y (IFN-Y), primarily produced by T lymphocytes and macrophages, activates

macrophages and PMNs and increases their cytotoxicity. IFN-Y is involved in• tissue remodeling
during healing. It has been shown to reduce wound contraction by retarding collagen production
and lattice cross-linking while increasing collagenase production. In experimental wound models,
treatment with IFN-Y has been shown to impair reepithelialization and wound disruption strength
in a dose-dependent fashion when applied locally or given systemically.

PJnterJeukin-4

Interleukin-4, predominantly produced by T lymphocytes and to a lesser extent by mast cells and
basophils, activates B-lymphocyte proliferation and immunoglobulin E (IgE) antibody-mediated
immunity. Its most significant role in wound healing is its anti-inflammatory effects: its ability to
inhibit macrophage production of the proinflammatory cytokines TNF-cL, 11.1, and 11.6. It
promotes healing through stimulating fibroblast proliferation and collagen synthesis, and it is able
to stimulate fibroblasts directly to synthesize proteoglycans

Interleukin-IO is an important anti-inflammatory cytokine produced by T lymphocytes, monocytes,

and epithelial cells. 11.10 has been shown to suppress a spectrum of cytokines: the
proinflammatory cytokines, Il-rl, TNF-0t, IL-6, 11.8, and ILA 2; the hematopoietic growth factors
GM-CSF, a-CSF, and VI-CSF; and IL-IO itself. Additionally, it inhibits the synthesis of MMPs,
gelatinase, and collagenase. Using neutralizing antibodies, Sato et al.

were able to demonstrate the inhibitory effects of 11310 on PMN and macrophage infiltration and
on proinflammatory cytokine expression. Elevated 11310 levels were identified in chronic venous
stasis ulcers, indicating a contribution to the nonhealing state.
Keg Chemokines

Chemokines are a complex family of at least 40 lowmolecular-weight (6 to 14 kDa) secreted

proteins with varying cellular targets and biologic responses. Four major chemokine families have
been identified at the present time. The families are distinguished by the pattern of conserved
cysteine residues located near the N-terminus.

The CXC family members (also known as a-chemokines) have a single noncysteine amino acid
situated between the two N-terminal cysteine disulfide residues. The CXC chemokines can be
further di+ided into those that contain the sequence glutamic acid-leucine-arginine (E-L-R) near
the N-terminal preceding the CXC sequence and those that do not. The CXC chemokines (e.g., IL-
8) that contain the E-R-L sequence are generally chemotactic for neutrophils, whereas those that
do not contain the sequence (e.g., interferon-inducible protein 10, or IPIO, and monokine induced
by IFN-Y, or MIC) act on lymphocytes. The CC family members (also knowns ß-chemokines) have
Evo adjacent cysteine disulfide residues near the N-terminus (no amino acids intervene). The C
family members (also known as Y-chemokines) have a single N-terminal cysteine disulfide bond.
The single member of the family (also known as a 6-chemokine), fractalkine, has three amino acids
residues intervening between the two N-terminal cysteine disulfide bonds.

Chemokines can also be defined as being either homeostatic or inflammatory. Homeostatic

chemokines are those that are constitutively expressed during development or other normal
physiologic processes, whereas inflammatory chemokines are those that are strongly upregulated
in response to inflammatory or immune stimuli. An example of a homeostatic chemokine is
secondary lymphoid organ cytokine (SLC, CCL21). Mice with spontaneous mutations preventing
expression of functional SLC were observed to have a severe depletion of lymph node T cells.

There is growing appreciation that a large number of chemokines and chemokine receptors are
essential for fine-tuning the trafficking and recruitment of leukocyte populations to lymphoid
tissues and to the sites of tissue injury and inflammation. Chemokines are the dominant factors
that control the attraction of leukocytes to tissues during inflammation. They play a significant role
in sepsis and particularly in the development of adult respiratory distress syndrome. In addition,
chemokines also play key roles in regulating normal T-cell, B-cell, and NK-cell development. The
coordinated synthesis of chemokines together with changes in the expression of cell surface
adhesion molecules is primarily responsible for the selective trafficking Of leukocyte populations
from intravascular to extravascular compartments. In addition, chemokines play a predominant
role in. the activation of these leukocyte populations upon their extravasation into extravascular
compartments and provide paracrine signaling for angiogenesis. whereas inflammatory
chemokines are those that are strongly upregulated in response to inflammatory or immune
stimuli. An example of a homeostatic chemokine is secondary lymphoid organ cytokine (SLC,
CCL21). Mice with spontaneous mutations preventing expression of functional SLC were observed
to have a severe depletion of lymph node T cells.

There is growing appreciation that a large number of chemokines and chemokine receptors are
essential for fine-tuning the trafficking and recruitment of leukocyte populations to lymphoid
tissues and to the sites of tissue injury and inflammation. Chemokines are the dominant factors
that control the attraction of leukocytes to tissues during inflammation. They play a significant role
in sepsis and particularly in the development of adult respiratory distress syndrome. In addition,
chemokines also play key roles in regulating normal T-cell, B-cell, and NK-cell development. The
coordinated synthesis of chemokines together with changes in the expression of cell surface
adhesion molecules is primarily responsible for the selective trafficking Of leukocyte populations
from intravascular to extravascular compartments. In addition, chemokines play a predominant
role in. the activation of these leukocyte populations upon their extravasation into extravascular
compartments and provide paracrine signaling for angiogenesis.

The primary inducers of chemokine synthesis are the proinflammatory cytokines IL-I and TNF-a.
There is also growing evidence that cellular adhesion per se can activate the expression of several
chemokines. In particular, recruited leukocytes interacting with adhesion molecules on endothelial
cells, basement membranes, extracellular matrix, and fibroblasts often induce the expression of
various chemokines, including IL-8 (CXCL8). This augmentation of chemokine synthesis by
recruited leukocyte populations as they traverse cell-to-cell or cell-to-matrix contact may promote
autocrine feedback loops that stimulate a chemokine cascade in the local microenvironment.

Several members of this superfamily are mitogenic for keratinocytes, which is of obvious
importance in wound healing, Similarly, chemokines can be both angiogenic and antiangiogenic.
As becomes readily evident, the biologic properties of chemokines far exceed their chemotactic or
chemokinetic properties. Only during the past few years has the role of chemokines in lymphoid
development and in T-helper (Thl, Th2) cell responses been identified. Much of the interest in
chemokines and lymphocyte function resulted from the discovery that CD4+ cells express
chemokine receptors and that the entry of HIV-I is dependent on the chemokine receptors CXCR4
and CCR5, which serve as co-receptors (with CD4) for HIV-I entry. This observation .has resulted in
an explosion of interest in chemokines and their receptors in T-cell biology distinct from their role
in the recruitment andactivation of inflammatory cells. More importaråtly, these findings have led
to greater exploration for the role of chemokines in directing the acquired immune response to
bacterial and viral pathogens.

Role of Growth Factors, Cytokines, and Chernokines in Normal IJound Healing

Wound healing is a precisely orchestrated sequence of events that includes migration, maturation,
and activation of various cell populations during the three phases of wound healing: inflammation,
fibroplasia, and scar maturation. Although it is not within the scope of this chapter to discuss
wound healing in depth, it is important for the surgeon to have a general understanding of the
role of growth factors, cytokines, and chemokines in the regulation of each phase of healing.

Inflammation

Tissue repair begins after an injury has occurred whether the result of physical trauma, chemical
exposure, or an antigen—antibody response. The sequence and magnitudeof cellular events that
occur after ary injury depend to a large extent on the wound conditions and the presence of tissue
and cellular debris, infection, and foreign bodies. Tissue debris and bacterial agents, in particular,
induce a more severe inflammatory response.

A skin laceration illustrates the early events leading to inflammation. The body's initial response,
to stop the bleeding involves the synergistic effects of platelet aggregation and activation of the
clotting cascade. Once platelet aggregation has developed, the preformed a-granules in the
cytoplasm degranulate, releasing PDCF, TCF-ß, TCF-a, platelet-derived epidermal growth factor
(PDEGF), and platelet-derived endothelial cell growth factor (PDECCF). In addition to these
proteins, a-granules release factor V, fibrinogen, fibronectin, plasminogen, and platelet factor-4
(PF-4, or CXCL4). These cytokines and proteins are involved early in coagulation and in inflam
mation. The intrinsic clotting cascade, initiated by exposur< to damaged tissue, together with
platelet aggregation accelerates the conversion of prothrombin to thrombin in the presence of
factor V. Thrombin participates in the conversion of fibrinogen to fibrin to provide a network for
clot formation and a significant component for the provisional matrix important for healing to
continue. As coagulation progresses, the inflammatory response continues.to escalate. Local
factors, histamine, prostagland E2, PDCF, and PF-4 are chemotactic for neutrophils.

Monocytes are also stimulated to migrate from the capillaries into the extravascular space by
chemotactic factors, including collagen fragments, fibronectin fragments, elastin from damaged
matrix, PF-4, and TGF-ß. Once in the wound, neutrophils and macrophages serve. as scavengers to
remove foreign material, infectious agents, and cellular debris. In addition to its phagocytic
activity, neutrophils have been found to produce proinflammatory cytokines, which provide the
early chemotactic signals for local fibroblasts and keratinocytes.

Repair and Collagen Synthesis

MacrOphages are a mmor sounce ot cytokines that regulate fibroblast proliferation, collagen
production, and scar remodeling. As inflammation progresses, macrophages further increase their
numbers through their secretion of

colony-stimulating factor I (CSF-I). Macrophages also secrete collagenases and elastase to remove
the damaged matrix while cell' migration, proliferation, and extracellular matrix (ECM) production
continue. Fibroblasts. then migrate into the provisional matrix under the influence of chemotactic
cytokines such as PDGF, TGF-ß, EGF, and lymphokines. movement of cells through the wounded
area is made possible by their ability to bind and release fibronectin, fibrin, and vitronectin
molecules via their cell surface integrin receptors. Both PDCF'.and TCF-ß have been demonstrated
to upregulate integrin receptors on fibroblasts. As they migrate, one pole of the fibroblast remains
fixed, whereas at the opposite pole lamellipodia extend in search of the correct ECM molecule to
which they can attach. Once the initial fixed pole releases, the cell moves while attached to the
new site. The direction of movement is guided by the presence of integrin receptors and by the
alignment of the fibrils in the preliminary wound matrix, where fibroblasts have been
demonstrated to migrate along, not across, matrix fibers. Migration is facilitated by large
quantities of hyaluronic acid in the provisional matrix, permitting easier penetration by migratory
cells.

Matrix metalloproteinases are a group of enzymes that can cleave a path through the lattice to
further facilitate fibroblast migration through the matrix. Enzymes involved in this process include
MMP-I, gelatin•ase (MMP-2), stromelysin (MMP-3), and plasminogen activator, which generates
plasmin through the breakdown of plasminogen. These enzymes share a common catalytic domain
con taining a zinc ion, and they target matrix proteins (collagen, fibronectin, laminin, gelatin) as
their substrates! The induction and expression of MMPs is mainly under the regulatory Control of
proinflammatory cytokines such as 11=1, TNF-a, and EGF, but they can be induced by cellular
debris and antigen-antibqdy complexes as well. MMPs also contribute to scar remodeling during
the final stage of healing.

Transforming growth factor-ß may be the most potent stimulator of collagen synthesis. By
decreasing protease activity, it contributes to collagen accumulation. Specific antibodies to TGF-ß
have been demonstrated to limit collagen accumulation in wounds. As previously mentioned in
the growth factor section, CTGF is the downstream mediator for TCF-ß production of collagen and
proteoglycans in the extracellular matrix. The PDGF isoforms have different effects on collagen
synthesis. PDGF-AB has been shown to increase expression of procollagen mRNA, whereas PDCF-
BB downregulates it. FGF and EGF stimulate collagen synthesis, and glucocorticoids inhibit collagen
production.

Angiogenesis

Endothelial cell migration and capillary tube formation are facilitated by changes in the matrix in
the capillary wall induced by collagenases. The advancing endothelial cells synthesize fibronectin
to modify the matrix to facilitate their own migration. The cells also must express various integrins
to allow their migration. Many of the macrophage-derived cytokines directly and indirectly
stimulate the endothelial cell migration and proliferation required for angiogenesis. Basic
fibroblast growth factor (bFCF, FGF2) is a potent angiogenic stimulant. Endothelial cells can
synthesize and respond to bFCF (FCF2). Multiple growth factors and cytokines have been identified
as angiogenic stimulants, including TCF-a, EGF, TCF-ß, TNF-a, PDECGF, VECF, and angiogenin.

Epithelialization

Epithelialization begins as early as 24 to 48 hours after injury. For injuries that include a damaged
basement membrane, the cells migrate over a provisional matrix tha includes fibrin, fibronectin,
and vitronectin. The epithelial cells themselves add components to the provisional matrix if the
substances are not already present. Cells continue to •migrate until contact inhibition occurs; then
the cell monolayer differentiates into more basal-like cell: Migration and proliferation of epithelial
cells are stimulated by EGF. It is found in a wide variety of tissues, and

most cells have receptors for EGF. TCF-a is also a poten stimulant of epithelial cell migration and
proliferation. Other factors that stimulate epithelial cell proliferation include heparin-binding
epidermal growth factor (HB-EGF IGF, KGF, and bFGF. The processes, cell migration and
proliferation, are regulated at least to some degree independently; cytokines such as TCF-ß can
stimulate migration but not proliferation. Epithelial cells are an important source of PDCFÆ, TCF-ß,
and TCF-a. Keratinocytes at•the advancing wound edge have the ability to secrete MMPs, which
facilitate their migration and penetration through eschar in a wound.

In open wounds, wound contraction proceeds until complete epithelization has occurred or the
defect has been surgically closed. Contraction is a cell-directed process that requires cell division
but not collagen synthesis. TGF-ß stimulates collagen lattice contraction and is a mediator of
wound contraction. It also facilitates the
transition of fibroblasts to myofibroblasts. Myofibroblasts attached to the collagen lattice are
stimulated to shorten their actin and myosin-like filaments to contract. PDGF can also stimulate
contraction of matrices by a TGF-ß independent mechanism.

Scar Maturation

Scar remodeling is the hallmark of the final period of healing. At approximately 21 days following
injury the net accumulation of wound collagen becomes stable. Collagen synthesis is most likely
downregulated through a mechanism involving IFN-Y and collagen matrix. TNF-u also
downregulates collagen synthesis, possibly by downregulating TGF-ß expression. This occurs
despite the fact that TGF-ß may still be present. As more collagen is synthesized, fibronectin is
broken down. IFN-Y downregulates synthesis of fibronectin as well as collagen.

During this remodéling phase, there is a continual turnover of collagen molecules as old collagen is
broken down and new collagen is synthesized in a denser, more organized fashion along lines of
stress. Several of the MMPs that degrade collagen and proteoglycans have been identified: MMP-I
(interstitial collagenase); MMP-2 (gelatinase); MMP-3 (stromelysin). They are found in both scar
tissue and normal connective tissues. In their role as regulators of healing, macrophages are able
to control both the synthesis of collagen and proteoglycans and their degradation through release
of metalloproteinase tissue inhibitors (TIMPs). Other enzymes, such as hyaluronidase, are also
involved in scar remodeling. The activities of these enzymes and their inhibitors are under the
influence of cytokines such as TGF-ß, PDGF, IL-I, and EGF.

Abnormal Wound Healing

The healing process in chronic wounds is generally prolonged or incomplete and uncoordinated,
resulting in a poor anatomic and functional outcome. In clinical

practice, the most common chronic wounds are chronic venous stasis ulcers, diabetic ulcers, and
chronic wounds resulting from pressure. Chronic nonhealing wounds are a prime clinical example
of the importance of the wound cytokine profile and the critical balance necessary for normal
healing to proceed.

Several studies have demonstrated a difference in the microenvironment of acute versus chronic
wounds. Falanga and colleagues (Bucalo et al.) demonstrated stimulation of the proliferation of
human dermal fibroblasts and endothelial cells in exudates from healing wounds (partial-thickness
skin donor sites). In contrast, an inhibitory effect on cell proliferation was noted in fluids obtained
from chronic wounds (venous ulcers). This difference in acute and chronic wounds was also noted
by Bennett and Schultz, who reported that mastectomy drainage fluids exhibited cell proliferation
enhancement, whereas exudates from chronic wounds showed cell proliferation inhibition.

To determine why these differences occurred, studies were done to determine the levels of
certain growth factors and their receptors. Using dextranomer beads to recover endogenous
growth factors in wounds, levels of PDGF and TGF-ß were determined to be significantly lower in
chronic wounds than in acute wounds. Using a rat model, both PDGF and PDGF receptors were
found to be downregulated in impaired wounds.
In general, most chronic wounds exhibit a decrease in growth factor expression and, in some
cases, growth factor receptors as well. The precise pattern of growth factor expression in the
various types of chronic wound is not yet known. The absolute •levels of growth factors may not
be as important as the relative concentrations necessary to replace thespecific deficiencies in the
tissue repair processes. For the treatment of chronic wounds, Robson proposed that growth
factor therapy be tailored to the deficiency in the repair process. Effectiveness of therapy is
predicated on adequate growth factor levels and the expression of their receptors. Excessive
degradation of receptors and binding of growth factors by macromolecules such as macroglobulin
and albumin would reduce the effectiveness of therapy.

The mechanisms involved in the creation and perpetuation of chronic wounds are varied and
depend on the individual wounds. In general, the inability of chronic venous stasis ulcers to heal
appears to be related to impaired wound epithelialization. Microscopically, the wound edges show
hyperproliferative epidermis, even though further immunohistochemical studies revealed optimal
conditions for keratinocyte recruitment, proliferation, and differentiation. The ECM and the
expression of integrin receptors by keratinocytes that allow it to translocate play an important
regulatory role in epithelialization. After receiving the signal to migrate, epidermal cells begin by
disassembling its attachments from the basement membrane and neighboring cells. They then
travel over a provisional matrix containing fibrinogen, fibronectin, vitronectin, and.tenascin; they
stop when they encounter laminin, During this process, keratinocytes are producing fibronectin
and continue to do so until the epithelial cells establish contact, at which time they again begin
manufacturing laminin to regenerate the basement membrane.

There is evidence that the interaction between the integrin receptors on keratinocytes and the
ECM transforms resting cells to a migratory phenotype. Integral to this transformation is an
alteration in the pattern of the integrin receptors expressed. After epithelialization is complete,
integrin expression reverts back to the resting pattern. To complicate this process further, growth
factors are involved in mediating keratinocyte activation and integrin expression and in alterations
in the matrix. Growth factors are able to affect these processes differentially; for example, TGF-ß is
able to promote epithelial migration while inhibiting proliferation. Although TGF-ß induces

the integrin expression necessary for migration, the cells behind those at the leading edge have
little proliferative ability, so epithelial coverage of the wound is inhibited. Some chronic wounds
may be deficient in TGF-ß and its receptors.

Chronic wounds have also been demonstrated to have elevated matrix-degrading enzymes and
decreased levels of

inhibitors for these enzymes. Unlike chronic venous stasis

ulcers, pressure ulcers appear to have difficulty healing, which is likely related to impaired ECM
production. Studies have indicated that the neutrophil elastase present in chronic wounds can
degrade peptide growth factors and is responsible for degrading fibronectin. Pressure ulcers have
also shown an increase in MMPs and plasminogen activators in tissue, Chronic wound fluids
demonstrate increased levels of the gelatinases MMP-2 and MMP-9. Levels of MMP-I and MMP-8
were also found to be higher in pressure ulcers and yenous stasis ulcers than in acute healing
wounds. In addition, several of the endogenous proteinase inhibitors were shown to be decreased
in chronic wounds. Proteinase inhibitors serve a regulatory role in matrix degradation by
containing matrix-degrading enzymes. Factors that promote MMP production or activation could
counteract the effectiveness of proteinase inhibitors (e.g., the destruction of TIMP by neutrophil
elastase). The MMP-9rr1MP-1 levels ratio may indicate an imbalance, which contributes to the
chronic nature of the wound. Conditions that promote the chronicity of wounds are repeated
trauma, foreign bodies, pressure necrosis, infection, ischemia, and tissue hypoxia. These wounds
share a chronic inflammatory state characterized by an increased number of neutrophils,
macrophages, and lymphocytes, which produce inflammatory cytokines (e.g., TNF-ayand the
chemokines IL-I and IL-6. In vitro studies have shown these cytokines to induce the expression of
MMPs in a variety of cells including macrophages, fibroblasts, keratinocytes, and endothelial cells.
They are also involved in the downregulation of TIMP expression. Although there may be a relative
excess of MMPs in the wound, they are secreted as proenzymes, which require activation for
matrix degradation to occur. Serine proteinases degrade matrix components and activate MMPs.
Neutrophil elastase, also present in increased concentrations in chronic wounds, is important for
orchestrating matrix-degrading events. Although the inflammatory profile differs for the various
types of chronic ulcer, the general relation is an increase in inflammatory cytokines, which leads to
the activation of proteinases and MMPs• and a decrease in tissue inhibitors, resulting in a
degradative state and wound chronicity. Nwomeh and colleagues described this common pathway
as a self-perpetuating environment of oxygen metabolites and degradative enzymes that
overwhelm the equilibrium to destroy the endogenous protease inhibitors, thereby establishing a
cronic wound