Modifiers of inflammatory angiogenesis in a murine model 2

Thus, understanding the mechanisms of tissue repair, new vessel formation in the context of the immune response will help lay the foundation for better treatment of pathologies related t

CHAPTER I INTRODUCTION

1.1 Introduction

Wound healing is the body’s response to injury, in which angiogenesis is one of the key elements Immune cells have been shown to play roles in both angiogenesis and wound healing Angiogenesis is important in a number of normal physiological processes,

including embryogenesis, reproduction and wound healing Uncontrolled angiogenesis contributes to a variety of pathologies Wound healing is a complex chain of cellular and biochemical events designed to restore tissue integrity and function under tight regulation However, there are instances whereby the process is less well regulated and injury can lead

to a chronic wound (non-healing) or fibrosis (excessive scar formation), which remain common medical problems Thus, understanding the mechanisms of tissue repair, new vessel formation in the context of the immune response will help lay the foundation for better treatment of pathologies related to aberrant angiogenesis and wound healing

Neutrophils are the main components in innate immunity Traditionally, neutrophils are regarded mainly as phagocytic cells, providing the first line of defense against invading pathogens However, there is increasing evidence indicating that neutrophils may have more functions beyond that A previous study showed that neutrophils could release VEGF, one of the most potent angiogenic factors, and MIP-1α and MIP-2, the potent chemokines for neutrophil, macrophage and lymphocytes Furthermore, it has been shown that angiogenesis is dependent on the neutrophil in an in vitro model and matrigel model

However, there is no direct in vivo evidence to relate the neutrophil to the natural

inflammatory angiogenesis Similarly, there is evidence showing that lymphocytes may contribute to angiogenesis in a number of pathologic settings; their effects on the

inflammation-induced angiogenesis are still unclear

The role of the neutrophil in wound healing was first addressed in 1972 The exact

mechanism was uncertain at that time It is well accepted that lymphocytes also play roles

in skin wound healing, but the mechanisms also need to be further elucidated Although it

is well known that the foetal scarless healing follows a minimized inflammatory response, the effects of immune cells (neutrophils and lymphocytes) in adult scar formation are unknown

In this section, the basic knowledge of angiogenesis, wound healing and scar formation will be introduced Some important cytokines will be also reviewed, including MIP-1α, MIP-2, MCP-1, TNF-α, VEGF, and TGF-β1 Subsequently, the role of neutrophil in angiogenesis and wound healing is reviewed after a brief introduction of neutrophil biology Finally, the roles of lymphocytes and monocyte/macrophages in angiogenesis and wound healing are also reviewed

1.2 General introduction of angiogenesis and wound healing

1.2.1 Angiogenesis

Angiogenesis is defined as the formation of new vessels from the existing vessels This process is characterized by a combination of sprouting of new vessels from the sides and ends of pre-existing ones, or by longitudinal division of existing vessels with

periendothelial cells, either of which may then split and branch into precapillary arterioles and capillaries (Conaway et al., 2001) The classical angiogenesis process consists of the following three overlapping phases: the initiation of the angiogenic response, endothelial cells migration and proliferation, and maturation of neovasculature (Griffioen et al., 2000) Angiogenesis is a complicated and highly regulated process In adult, angiogenesis under tight control are found in the female reproductive system and during wound healing (Liekens et al., 2001) Unregulated angiogenesis can be divided into insufficient

angiogenesis and excessive angiogenesis Insufficient angiogenesis may result in tissue ischemia and delayed wound healing; excessive angiogenesis may result in pathologies that include cancer (both solid and hematologictumors), chronic inflammation(rheumatoid arthritis, Crohn's disease), diabetes (diabetic retinopathy) and psoriasis With increasing insight of the role of angiogenesis in these pathologies, modulation of angiogenesis is now regarded as a therapeutic target in these diseases (Griffioen et al., 2000) Further

delineating the regulation of angiogenesis will lead hopefully to more effective angiogenic and anti-angiogenic treatment approaches to some diseases

I Angiogenesis process

i) Initiation of angiogenesis

To initiate the formation of new capillaries, endothelial cells of existing blood vessels must degrade the underlying basement membrane and invade into the stroma of the

neighbouring tissue These processes of endothelial cell invasion and migration require the cooperative activity of the plasminogen activator (PA) system and the matrix

metalloproteinases (MMPs) (Conaway et al., 2001; Liekens et al., 2001)

Angiogenesis is rapidly initiated by hypoxic or ischemic conditions vascular endothelial growth factor (VEGF), transcriptionally upregulated in part by hypoxia, mediates an increase in vascular permeability and extravasation of plasma proteins including

plasminogen, which can be converted to plasmin by plaminogen activators- urokinase plasminogen activators (uPAs) and tissue plasminogen activators (tPAs) (Conaway et al., 2001) Plasmin has a broad trypsin-like specificity and degrades fibronectin, laminin, and the protein core of proteoglycans In addition, plasmin activates certain metalloproteinases Plasmin is believed to be the most important protease for the mobilization of fibroblast growth factor-2 (FGF-2 or basic FGF) from the extracellular matrix (ECM) pool MMPs play a central role in degrading extracellular membranes and basement membrane

structures, allowing endothelial cells to migrate (Conaway et al., 2001; Liekens et al., 2001)

ii) Endothelial cell migration and proliferation

Following proteolytic degradation of the ECM, “leader” endothelial cells start to migrate through the degraded matrix They are followed by proliferating endothelial cells, which

are stimulated by a variety of growth factors, some of which are released from the degraded ECM At this step, interplay between VEGF, angiopoeitin, FGFs and their receptors are responsible for mediating the process of angiogenesis Additionally, there are other factors that have also been implicated in the process, such as tumor necrosis factor alpha (TNF-α) and some chemokines (Conaway et al., 2001; Liekens et al., 2001)

iii) Maturation of neovasculature

As endothelial cell migrate into the extracellular matrix, they assemble into solid cord, and subsequently acquire a lumen To form a stable vasculature, the interaction between endothelial cell with ECM and mesenchymal cells occurs, regulated by platelet derived growth factor (PDGF), Transforming growth factor-beta ( TGF-β), and the interaction of angiopoeitin with its receptors, tyrosine kinase Tie1 and Tie2 (Conaway et al., 2001; Griffioen and Molema, 2000)

II Angiogenesis and inflammatory diseases

Rheumatoid arthritis (RA) is a chronic systemic disease characterised by an inflammatory erosive synovitis Early changes in the synovium are marked by revascularization,

inflammatory cell infiltration, and associated synoviocyte hyperplasia, which produce a pannus of inflammatory vascular tissue This pannus covers and erodes articular cartilage, eventually leading to joint destruction Angiogenesis is now recognized as a key event in the formation and maintenance of the pannus in RA This suggests that targeting blood

vessels in RA may be an effective future therapeutic strategy Disruption of the formation

of new blood vessels would not only prevent delivery of nutrients to the inflammatory site, but could also lead to vessel regression and possibly reversal of disease (Pandya et al., 2006)

Psoriasis is a common chronic dermatosis occurring in 2% of the population and associated with an inflammatory arthritis–psoriatic arthritis (PsA)–in up to 40% of cases PsA

accounts for approximately 15% of patients attending early synovitis clinics, therefore it represents the second most common diagnostic category after rheumatoid arthritis

Significant abnormalities of vascular morphology and angiogenic growth factors have been described in psoriasis and PsA Angiogenesis appears to be a fundamental

inflammatory response early in the pathogenesis (Pandya et al., 2006)

1.2.2 Wound healing

Wound healing is a highly dynamic process and involves complex interactions of ECM molecules, soluble mediators, various resident cells, and infiltrating leukocyte subtypes The ultimate goal in repair is to restore tissue integrity and function The healing process consists of three phases that overlap in time and space: inflammation, proliferation, and tissue remodeling (Diegelmann et al., 2004; Harding et al., 2002) Unregulated wound healing results in chronic wound (non-healing wound) or fibrosis (excessive scar

formation) Chronic wound and fibrosis represent the major health burden and threat (Bayat et al., 2003; Harding et al., 2002) To fully understand the mechanisms underlying

the wound healing and scar formation will shed light on the treatments of chronic wound and fibrosis

I Wound healing process

Wound healing generally involves the initiation and integration of a biological response, coordinating the migration, proliferation, and differentiation of a heterogeneous group of cells to achieve restoration of tissue integrity and function In skin full length injury, progress toward healing follows a complex series of events, characterized by:

inflammation, proliferation and tissue remodeling (Williams et al., 2003) Although inflammation, proliferation and maturation have been described as separate processes, in reality the phases of repair can overlap so that all three can be observed in different regions

of a large dead space wound (Arnold and West, 1991)

i) Inflammation phase

Inflammation phase occurs immediately after injury In the first 24 hours, this

commences as the formation of the hemostatic primary platelet plug stimulated by

thrombin and exposed fibrilla collagen and the influx of neutrophils from the blood (Williams et al., 2003) Neutrophils are attracted to wound sites within a few hours after injury by chemotactic mediators generated at the site Within a day or two of injury, tissue monocytes enter the wound and differentiate into mature tissue macrophages

Macrophages are thought to play an integral role in a successful outcome of wound healing

through the generation of growth factors that promote not only cell proliferation and protein synthesis but also components of the extracellular matrix Macrophages also stimulate lymphocyte proliferation and cytokine release in response to specific antigens In the late inflammatory phase of wound repair, T lymphocytes (T cells) appear in the wound

bed (Szpaderska and DiPietro, 2005)

The migrating epidermal part is characterized by the loss of tight binding between these epidermal cells and the basement membrane and underlying dermis Fibronectin appears to

be instrumental in allowing continual migration of these cells Fibronectin produced from plasma initially, and from plasma and fibroblast later, may also be derived from migrating

keratinocytes This suggests that the migrating epithelial cells may provide its own lattice for continual migration (Williams et al., 2003; Kirsner et al., 1993)

The basement membrane zone changes in other ways after wound formation Two

important basement membrane proteins, laminin and type IV collagen, which normally mediate epidermal-dermal adhesion, disappear Within 7-9 days after the reformation of the functional barrier of the skin, the basement membrane zone returns to normal It has been shown that growth factors also play a role in signaling cell migration, such as TGF-β (Williams et al., 2003; Kirsner et al., 1993)

While the migrating epithelial cells continue their journey across the wound to reestablish the functional barrier of the skin, cells just proximal to these are actively proliferating Until the wound is closed, a zone of proliferating cells remains between the migrating epithelium and the normal cells at the wound edge Although the stimulus for cells

undergoing rapid proliferation is not known, several growth factors may be involved, including EGF (Williams et al., 2003; Kirsner et al., 1993)

Angiogenesis

Wound healing cannot occur without angiogenesis The vasculature comprises up to 60%

of repair tissue (Dyson et al., 1991), and the original name of granulation tissue is derived from the prominence of its vessels (Arnold and West, 1991) An abundant blood supply is necessary to meet the enormous local metabolic demands of debridement and fibroplasia (Arnold and West, 1991)

Fibroplasia

The increase in fibroblast numbers and the matrix they produce coincide with the

formation of granulation tissue They arise from both in situ cell proliferation and

migration from adjacent areas (Williams et al., 2003) Fibroblasts migrate into the wound between 48 and 72 hours (Kirsner et al., 1993) Their function is to synthesize structural proteins, reorganize the wound matrix and promote wound contraction In addition to collagen, they produce tenascin, fibronectin and glycosaminoglycans Fibroblasts migrate

to wound by pulling themselves along a fibronectin matrix This migration occurs by contraction of intracellualar microfilaments The loose extracellular matrix made of fibronectin is laid down by the migrating fibroblasts themselves (Kirsner et al., 1993) Fibroblasts differentiate into myofibroblasts by development of cytoskeletal protein, including actin, myosin heavy chain and MyoD (Williams et al., 2003) This affords these cells contractile properties that enable the cells to promote wound edge contraction (Williams et al., 2003)

Wound contraction

In full-thickness wounds, the wound contraction is an important part of wound healing, accounting for up to a 40% decrease in the size of wound (Kirsner et al., 1993) The contractile forces produced by granulation tissue in the wound are derived from

myofibroblasts that contain contractile proteins Myofibroblasts within the wound align along the lines of contaction and differ from the other cellular components, including

leukocytes and endothelial cells, which do not exhibit such organized orientation Collagen and fibronectin have been shown to assist in wound contraction (Kirsner et al., 1993)

iii) Tissue remodeling

The remodeling phase consists of the deposition of matrix materials and their subsequent change over time Dermal macromolecules such as fibronectin, hyaluronic acid,

proteoglycans and collagen are deposited during repair and serve as a scaffold for cellular migration and tissue support (Kirsner et al., 1993) The total amount of collagen increases early in the repair process, reaching a maximum between 2 and 3 weeks after injury (Kirsner et al., 1993) Tensile strength increase to 40% 1 month after injury and may continue to increase for as long as a year after injury (Kirsner et al., 1993) Changes in the types of collagen occur in the remodeling phase Type III collagen, as mentioned before, is the major type of collagen synthesized by wound fibroblast during wound repair Over the period of a year or more, the dermis in the wound must return to its more stable preinjury phenotype, consisting largely of type 1 collagen The conversion from type III collagen to type 1 collagen is accomplished through tightly controlled interaction between synthesis of new collagen and lyses of old collagen (Kirsner et al., 1993) As wounds mature, more collagen is deposited, cross-linked and organized Over time, most of the vessels,

fibroblasts, and inflammatory cells slowly disappear leaving a relatively acellualr scar (Kirsner et al., 1993)

II Scar formation

Even when it occurs with optimal efficiency, wound repair in most vertebrate organs is dominated by a fibroproliferative response that produces a fibrotic scar (Harty et al., 2002) This is most obvious in cutaneous wound Microscopically, the hallmarks of the cutaneous scar include disorganized dermal collagen architecture, failure of hair follicles and other dermal appendages to regenerate Wounds that heal with scar also never reach the same tensile strength as that of the normal surrounding tissue (Beanes et al., 2003; Harty et al., 2002) In the past 20 years, tremendous progress has been achieved in understanding the cellular and molecular events of wound repair, but the tendency among vertebrates for scarring rather than regeneration remains unexplained (Harty et al., 2002)

In contrast to the adult repair process, skin wounds in first- and second-trimester fetuses heal in a scarless manner, regenerating normal skin, including the re-growth of hair follicles and other subepidermal appendages, without excessive, disorganized collagen production (Rowlatt, 1979; Longaker et al., 1990; Chin et al., 2000) Importantly, this phenomenon of scarless healing occurs associated with the minimal inflammatory

response Although the exact mechanism by which inflammation promotes scarring in this model is not known, it is believed that the inflammatory phase of wound repair drives the production of scar tissue and influences the quality of the new skin in the wound area (Wilgus et al., 2003)

III Abnormal wound healing

Chronic wound and fibrosis still remain the serious health burden to human kind The management of chronic wound places an enormous drain on healthcare resources Each year in the developed world 100 million patients acquire scars, some of which cause considerable problems There are an estimated 11 million keloid scars and four million burn scars People with abnormal scarring may face physical, aesthetic, psychological, and social consequences that may be associated with substantial emotional and financial costs (Bayat et al., 2003; Diegelman and Evans, 2004)

i) Chronic wound

Many factors can impair wound healing Reduction in tissue growth factors, an imbalance

between proteolytic enzymes and their inhibitors, and the presence of senescent cells seem

to be particularly important in chronic wounds Chronic ulcers are known to have reduced levels of platelet derived growth factor, basic fibroblast growth factor, epidermal growth factor, and TGF β compared with acute wounds (Higley et al., 1995) Excessive proteinase activity in chronic wounds, probably from overexpression of matrix metalloproteins, results in abnormal degradation of the extracellular matrix Dermal fibroblasts have an age related decrease in proliferation potential, called senescence Fibroblasts in chronic wounds have impaired responsiveness to growth hormone, which may be due to an increased number of senescent cells (Harding et al., 2002; Diegelman and Evans, 2004)

ii) Fibrosis

Fibrosis can be defined as the replacement of the normal structure elements of the tissue by distorted, non-functional and excessive accumulation of scar tissue Many clinical

problems are associated with excessive scar formation In skin, keloids and hypertrophic scars are the most common types related to excessive scar formation ((Muir, 1990) Keloids and hypertrophic scars are characterized by excessive accumulation of scar collagen The classical feature of a keloid scar is that the scar tissue progressively invade surrounding normal skin whereas a hypertrophic scar is confined to tissue damaged by the original injury (Muir, 1990)

These pathological scars form as a result of the protracted presence of inflammatory cells, which prolongs the presence of the myofibroblast population in the scar and therefore the increase in contraction of the wound and synthesis of the matrix Many cytokines are implicated in these processes but certain molecules, notably the TGF-β family are

particularly important (Muir, 1990)

1.3 Cytokines in angiogenesis and wound healing

1.3.1 Chemokines in angiogenesis and wound healing

Chemokines are a family of small secreted proteins that, depending on the spacing or presence of four conserved cysteine residues, are classified into CC, CXC, CX3C and C chemokines CXC chemokines can be further divided into two groups of molecules (ELR+ and non-ELR) according to the presence or absence of an ELR (Glu–Leu–Arg) motif

located immediately before the first cysteine residue In the hematopoietic system, the presence of an ELR sequence often correlates with a biological activity on neutrophils

(Bernardini et al., 2003)

I MIP-1α

Macrophage inflammatory protein-1α (MIP-1α) is an LPS-inducible chemokine of the CC

family that is strong chemoattractant for monocytes The human and murine MIP-1α gene

is inducible in most mature hematopoietic cells Although it appears that low levels of MIP-1 are expressed constitutively, induction is most often required for production of easily detectable amounts of MIP-1 protein (Davatelis et al., 1988)

MIP-1α proteins mediate their biological effects by binding to cell surface CC chemokine receptors, which belong to the G-protein-coupled receptor superfamily Receptor binding involves high affinity interactions and a subsequent cascade of intracellular events that rapidly leads to a wide range of target cell functions including chemotaxis, degranulation, phagocytosis, and mediator synthesis (Maurer and Stebut, 2004)

MIP-1 family members orchestrate acute and chronic inflammatory host responses at sites

of injury or infection mainly by recruiting proinflammatory cells In wounds, murine MIP-1α, produced by platelets and macrophages, was shown to be an effective

macrophage chemoattractant (DiPietro et al., 1998) Studies using MIP-1α-/-mice

demonstrated the requirement of murine MIP-1α for the induction of monocyte recruitment

to injured sites However, the wound healing of MIP-1α-/- mice showed no difference from control mice indicating that MIP-1α is not important in wound healing (DiPietro et al., 1998) It has been shown that MIP-1α also plays a role in T cell and neutrophil chemotaxis (Maurer and Stebut, 2004)

II CXCL1/MIP-2

CXCL1/MIP-2, a member of the ELR CXC chemokine family, is a functional homolog of CXCL8/IL-8 in mice (Endlich et al., 2002; Armstrong et al., 2004) Although in vitro study indicate that CXCL8/IL-8 is produced by a variety of cells in in vitro studies, an in vivo study indicates that MIP-2 is mainly produced by neutrophils and macrophages in the skin injury model (Baggiolini et al., 1994; Enelhardt et al., 1998) IL-8 is a potent

chemoattractant for neutrophils, basophils, and T-lymphocytes MIP-2 is also an extremely potent chemotactic agent for murine neutrophils (Baggiolini et al., 1994; Scapini et al.,

2004) It has been shown that CXCL8/IL-8 and CXCL1/MIP-2 induce angiogenesis in vitro and in vivo ( Belperio et al., 2000; Scapini et al., 2004) Furthermore, CXCL1/MIP-2

has been shown to induce angiogenesis in vivo mediated by neutrophil-derived VEGF (Benelli et al., 2002; Scapini et al., 2004)

III MCP-1

MCP-1 is a member of the CC chemokine family, which acts through a seven membrane spanning G-protein coupled receptor CCR2 (Charo et al., 1994) CCR2 is expressed on

neutrophils, monocytes, basophils, and a subset of T lymphocytes This chemokine plays

an important role in the inflammatory response, and has been implicated as an important factor in mediating monocytic infiltration of tissues in wounds and inflammatory diseases (Charo et al., 1994; DiPietro et al., 2001; Gibran et al., 1997)

MCP-1 is produced by macrophages, fibroblasts, endothelial cells, keratinocytes and smooth muscle cells in response to inflammatory stimuli (Gibran et al., 1997) MCP-1 mRNA and protein is found in burn and excisional wounds and correlates with macrophage infiltration (DiPietro et al., 2001;Gibran et al., 1997; Engelhardt et al., 1998)

Recently, it was demonstrated that MCP-1 induced endothelial cell chemotaxis in vitro as well as angiogenesis in the chick chorioallantoic membrane assay in vivo MCP-1 induced angiogenesis was associated with the expression of CCR2 on endothelial cell (Szekanec, et al., 2001) It was also demonstrated that MCP-1 play an important role in wound healing, probably by influencing gene expression /protein synthesis in murine macrophages (Low

et al., 2001)

1.3.2 TNF-α: proinflammatory cytokine

Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine produced by a variety of cell types, including macrophages, neutrophils, lymphocytes, and endothelial cells (Tracey, 1994) TNF signaling has been shown to proceed via two distinct cell surface receptors, TNFR1 (TNFRp55–60) and TNFR2 (TNFRp75–80), and the specific intracellular

signaling pathways that are activated by TNF determine the specificity of cellular

responses in a given cell (Lewis et al., 1991) It has been shown that TNFR1 initiates the majority of the biological activities of TNF-α, including cell growth and death,

oncogenesis, and inflammatory responses (Tartaglia et al., 1991)

There are no consistent reports on the role of TNF-α in angiogenesis TNF-α has been shown to inhibit growth and induce endothelial cell apoptosis in vitro (Fujita et al., 2007) However, this cytokine may also induce endothelial cell migration in vitro and

angiogenesis in vivo when implanted into corneas, chorioallantoic membranes, or sponge implants (Fujita et al., 2007) These apparently contrasting effects are likely secondary to the ability of TNF-α to release proangiogenic and antiangiogenic factors, depending on the local concentration or duration of exposure to TNF-α (Fujita et al., 2007)

1.3.3 VEGF: angiogenic factor

I VEGF and VEGF receptor

The VEGF family currently comprises seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF All members have a common VEGF homology domain VEGF-A is a 34- to 42-kDa, dimeric, disulfide-bound glycoprotein In normal tissues, the highest levels of VEGF-A mRNA are found in adult lung, kidney, heart, and adrenal gland Lower, but still readily detectable, quantities of VEGF-A transcript levels occur in liver, spleen, and gastric mucosa VEGF-A exists in at least seven

homodimeric isoforms The monomers consist of 121, 145, 148, 165, 183, 189, or 206 amino acids (Hoeben et al., 2004)

Transcription of VEGF mRNA is induced by different growth factors and cytokines, including PDGF, EGF, TNF-α, TGF-β, and IL-1β VEGF levels are also regulated by tissue oxygen tension Exposure to hypoxia induces VEGF expression rapidly and

reversibly, through both increased transcription and stabilization of the mRNA (Hoeben et al., 2004)

Three VEGF tyrosine kinase receptors have been identified: The fms-like tyrosine kinase Flt-1 (VEGFR-1/Flt-1), the kinase domain region, also referred to as fetal liver kinase (VEGFR-2/KDR/Flk-1), and Flt-4 (VEGFR-3) Each receptor has seven immunoglobulin like domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence inserted by a kinase insert domain VEGFR-2 appears to be the most important receptor in VEGF-induced mitogenesis and permeability The role of VEGFR-1 in endothelial cell function is less clear VEGFR-1 and VEGFR-2 are expressed predominantly by vascular endothelial cells, while VEGFR-3 binds only VEGF-C and VEGF-D Since the latter is generally restricted to lymphatic endothelial cells, activation stimulates mitosis, migration, differentiation, and survival of these cells (Hoeben et al., 2004; Liekens et al., 2002; Bao et al., 2008)

II VEGF and angiogenesis

VEGF (VEGF-A) has been shown to play an essential role in embryonic vasculogenesis and angiogenesis Inactivation of a single VEGF allele in mice resulted in embryonic lethality Besides its function during embryogenesis, VEGF also plays a crucial role in angiogenesis in the adult VEGF was detected in the ovary during corpus luteum formation and in the uterus during growth of endometrial vessels and at the site of embryo

implantation VEGF therefore plays a pivotal role in angiogenesis by contributing the multiple steps of angiogenesis (Bao et al., 2008)

Vasodilation

VEGF has the ability to increase vascular permeability and induce vascular leakage It binds to the KDR receptor, stimulating nitric oxide synthase and cyclooxygenase activities The resulting products, nitric oxide (NO) and prostacyclin, have been shown to promote simultaneous vasodilation and vascular permeability (Bao et al., 2008)

Degradation of basement membrane

VEGF directly increases endothelial cell secretion of interstitial collagenase (matrix metalloproteinase [MMP]-1), tissue inhibitor of metalloproteinases, and gelatinase A (MMP-2) VEGF also induces dose-dependent expression of urokinase-type and

tissue-type plasminogen activator (uPA and tPA) as well as plasminogen activator

inhibitor-1 In addition, VEGF stimulates vascular smooth muscle cells to express MMP-1, MMP-3, and MMP-9 (Bao et al., 2008)

Endothelial cell migration

VEGF induces endothelial cell migration through 2 primary mechanisms, chemotaxis and vasodilation Chemo taxis is a highly regulated process involving cell adhesion molecules’ interaction with the extracellular matrix VEGF induces the expression of αvβ5 integrin, uPA, as well as αvβ3 integrin and its receptor, all of which can promote the endothelial cell migration Another mechanism by which VEGF induces endothelial cell migration is related to the increase in vascular permeability mediated by NO and prostacyclin Leakage

of the plasma protein fibrinogen and its subsequent conversion in the extracellular space to

a fibrin gel stimulates endothelial migration

Endothelial Cell Proliferation

VEGF is described as a mitogen specific for endothelial cells VEGF induces endothelial cells grown on the surface of a collagen matrix to invade the underlying matrix and stimulates their proliferative response Furthermore, VEGF delays senescence and restores proliferative capacity to endothelial cells It lengthens the lifespan of endothelial cells and prevents apoptosis in human endothelial cells (Bao et al., 2008)

III VEGF and wound healing

The role of VEGF in wound healing is mainly associated with promoting angiogenesis During wound healing, various cellular responses to a wound involve the release of VEGF The platelet is the first vascular component to appear in the wound site, followed by neutrophils, and then macrophages Activated platelets release VEGF, particularly after thrombin stimulation (Bao et al., 2008) Monocytes play both a direct and an indirect role

in the angiogenic effects during wound healing Monocytes express the VEGF receptor Flt-1 and respond chemotactically to VEGF (Bao et al., 2008) Once recruited to the tissue, macrophages induce angiogenesis, in part by releasing TNF-α, which may in turn induce VEGF expression in keratinocytes and fibroblasts (Bao et al., 2008) Furthermore, cells involved in healing release cytokines and growth factors that may act as paracrine factors for further VEGF expression Recently, neutrophils have been shown to be another main source of VEGF (Gaudry et al., 1997)

1.3.4 TGF-β1

I TGF-β1 regulation and receptors

The TGF-β superfamily encompasses a diverse range of proteins, many of which play important roles during development, homeostasis, disease, and repair The structurally related but functionally distinct mammalian members of this family include TGF-β1–3, bone morphogenetic proteins, mullerian inhibiting substance, nodals, inhibins, and activins (Beans et al., 2003; Brown et al., 1995) The three mammalian TGF-β isoforms (TGF-β1, -β2, and -β3) are synthesized as latent precursors, which will be activated via proteolic cleavage extracellularly Active TGF-β then exert their biological functions via binding to

a heteromeric receptor complex, consisting of one type I and one type II receptor, both of which are serine-threonine kinases In addition, they bind with high affinity to a

non-signaling type III receptor, which functions mainly to present TGF-β to the type II receptor (Beans et al., 2003; Brown et al., 1995) The three TGF-β isoforms have both

distinct and overlapping functions The role of TGF-β1 in wound healing will be reviewed

in the following section

II TGF-β1 in wound healing

Immediately after wounding, TGF-β1 is released in large amounts from platelets (Beans et al., 2003) This initial upregulation of active TGF-β1 from platelets serves as a

chemoattractant for neutrophils, macrophages, and fibroblast; these cell types further enhance TGF-β1 levels in various cell types Aside from their active forms, latent TGF-βs are also produced and sequestered within the wound matrix, allowing sustained release by proteolytic enzymes This combination of different cellular sources and temporary storage ensures a continuous supply of TGF-β throughout the repair process Several publications report on the presence of TGF-βs in wound fluid of different species (Beans et al., 2003)

To further clarify the role of the TGF-β1 isoforms in wound repair, Brown et al (2003)

wounded transgenic mice deficient in TGF-β1 due to a targeted disruption of the tgf-β1 gene They found that histological analysis of the wounds at day 10 after injury revealed a

thinner, less vascular granulation tissue in the TGF-β1 deficient mice, associated with a marked inflammatory cell infiltrate Furthermore, decreased reepithelialization and

collagen deposition were observed in mutant animals when compared with control mice (Brown et al., 2003) This finding implies that TGF-β1 plays a crucial role later in the repair process

TGF-β1 can also control two important activities of granulation tissue: tissue contraction and ECM remodeling It is known that TGF-β1 can induce a-SMA expression in

fibroblasts both in vitro and in vivo (Desmouliere et al., 1993; Roy et al., 2001) TGF-β1 also stimulates ECM synthesis, especially collagen I, and inhibits the activity of matrix metalloproteinase by decreasing their expression and upregulating tissue inhibitor of metalloproteinase expression (Gabbiani, 2003)

Several studies support an important role of TGF-βs in cutaneous scarring (Beans et al., 2003; O’Kane and Ferguson, 1997) First of all, a reduced and/or more transient expression

of TGF-βs and their receptors was observed in non-scarring fetal wounds compared with adult wounds In addition, a strong and persistent expression of TGF-βs and their receptors was detected in fibroblasts of human postburn hypertrophic scars (Beans et al., 2003; O’Kane and Ferguson, 1997), and overexpression of TGF-β1 and -β2 was found in keloid tissues and keloid derived fibroblasts Furthermore, the activity of TGF-β appears to be increased in scar tissue (Beans et al., 2003; O’Kane and Ferguson, 1997)

Interestingly, treatment of fetal wounds with different concentrations of TGF-β1 caused marked scarring of these wounds, demonstrating a direct involvement of TGF-β1 in cutaneous scarring (Beans et al., 2003; O’Kane and Ferguson, 1997) This finding was further supported by studies from Shah et al (Beans et al., 2003; O’Kane and Ferguson, 1997) In those experiments, incisional wounds in rat were treated with neutralizing antibodies to TGF-β1 or to a combination of TGF-β1 and -β2 This treatment caused a significant reduction in extracellular matrix deposition and subsequent scarring, suggesting

that endogenous TGF-β1 and -β2 induce cutaneous scarring in adult animals A reduced scarring response was also observed in mouse wounds that were topically treated with antisense TGF-β1 oligodeoxynucleotides (Beans et al., 2003; O’Kane and Ferguson, 1997) Finally, topical application of a synthetic TGF-β antagonist reduced scarring in porcine burn and excisional wounds as well as in rabbit skin excisions (Beans et al., 2003; O’Kane and Ferguson, 1997)

1.4 Cellular response in angiogenesis and wound healing

1.4.1 Roles of neutrophils in angiogenesis and wound healing

I Neutrophil Biology

Since neutrophilic cells (neutrophils) were discovered by Paul Ehrlich in 1900, there has been vast literature regarding the subject (a brief search in PubMed finds over 76,000 papers on neutrophils) (Nathan, 2006; Borregaard and Cowland, 1997) Traditionally, the neutrophil is regarded as a major effect cell of innate immunity, mainly providing the first line of defense against invading pathogens through oxidative and non-oxidative killing mechanisms Recently, there is accumulating evidence indicating that neutrophils are also involving in angiogenesis and wound healing To better understand the role of neutrophil

in angiogenesis and wound healing, the biology of neutrophils is reviewed in this section

i) Neutrophil development

Hematopoietic stem cells are multipotent and have the capacity to differentiate into the cells of all 10 blood lineages — erythrocytes, platelets, neutrophils, eosinophils, basophils, monocytes, T and B lymphocytes, natural killer cells, and dendritic cells (Golde et al., 1992) There are 6 stages in the development of hematopoietic stem cell to neutrophils: myeloblast stage, promyelocyte stage, myelocyte stage, metamyelocyte stage, band form stage, and neutrophil stage

The polymorphonuclear neutrophil (PMN) or neutrophil is the end-stage of full maturation

of the granulocyte The mature PMN is of uniform size (13 microns in diameter) The nucleus is segmented into two to five (mean equals three) lobes connected by thin

chromatin strands The nuclear chromatin is coarse, clumped and stains deep purple with Wright-Giemsa The cytoplasm contains both smaller peroxidase-negative granules (secondary specific granules, tertiary gelatinase granules) and larger dense

peroxidase-positive granules (primary granules) (Golde et al., 1992)

ii) Neutrophil kinetics

Neutrophils are distributed within the bone marrow, the circulating blood, and in various tissues throughout the body depending upon specific need The bone marrow is the largest compartment, containing approximately 2.3 x 109 PMNs per kg body weight (Golde et al., 1992) The peripheral blood compartment is one-third the size of the bone marrow

compartment (0.7 x 109 cells per kg) One-half of these PMNs are in the main stream of the

circulation, while the other half are tumbling or loosely adhering to the endothelium of the microvasculature, referred as the circulating granulocyte pool (CGP) and marginating granulocyte pool (MGP) respectively; the two pools are in a constant state of dynamic equilibrium (Golde et al., 1992) The size of the extravascular tissue compartment has not been quantified but it varies depending on the body's need for PMNs at sites of infection or inflammation (Golde et al., 1992)

iii) Neutrophil granules

Neutrophils contain three subsets of granules (azurophilic, specific, and gelatinase) and secretory vesicles Azurophilic granules are also referred to peroxidase-positive granules, which is defined by the high content of myeoloperoxidase (MPO) Azurophilic granules are formed in early promylocytes The matrix of azurophil granules contains microbicidal proteins and acid hydrolase involved in oxidative and nonoxidative killing of bacteria and fungi (Borregaard and Coeland, 1997; Faurschou and Borregaard, 2003)

Specific (secondary) and gelatinase (tertiary) granules are part of the family of peroxidase negative granules Specific granules and gelatinase granules differ significantly from each other with respect to protein content and secretory properties Specific granules are larger and rich in antibiotic substances, participating mainly in the antimicrobial activities of neutrophils Gelatinase granules are smaller and easily exocytosed, acting primarily as a reservoir of matrix degrading enzymes and membrane receptors needed during neutrophil extravasation and diapedesis.Peroxidase-negative granules also contain three

metalloproteases with great physiological and pathophysiological significance, namely collagenase (matrix metalloproteinase-8 (MMP-8)), gelatinase (MMP-9), and the recently discovered leukolysin (MMP-25) (Borregaard and Coeland, 1997; Faurschou and

Borregaard, 2003)

Secretary vesicles are regulated exocytic vesicles that appear in segmented neutrophils and are formed by endocytosis (Faurschou and Borregaard, 2003) Secretary vesicles contain maily membrane-associated receptors needed at the earliest phases of the

neutrophil-mediated inflammatory response The membranes of secretary vesicles are rich

in β2-integrin CD11b/CD18 (Mac-1,CR3), the complement receptor 1, receptors for formylated bacterial peptides (formylmethionyl-leucylphenylalanine (fMLP)-receptors), the LPS/lipoteichoic acid-receptor CD14, the FccIII receptor CD16and the

metalloprotease leukolysin, all of which are incorporated in the plasma membrane after exocytosis The combined surface changes induced by incorporation of secretory vesicles allow the neutrophil to establish firm contact with activated vascular endothelium in vivo (Faurschou and Borregaard, 2003)

iv) Neutrophil migration

Inflammation is characterized histologically by the accumulation of leukocytes at the inflamed site due to the directional migration of circulating leukocytes Neutrophils always infiltrate the inflamed sites within 24 hours To reach the inflamed sites, neutrophils first

need to migrate out of the vasculature through the following steps: margination, rolling, attachment, and migration (Golde et al., 1992)

Margination — Hemoconcentration in the microvasculature due to vascular leakage

(edema formation) increases the contact of neutrophils with the endothelial lining

Rolling — Under regular conditions, neutrophils move freely in plasma as blood courses

over endothelial surfaces With infection or inflammation, endothelial cells are activated to express E-selectin and to release interleukin-8 (IL-8), granulocyte macrophage colony stimulating factor (GM-CSF), and platelet-activating factor (PAF), which induce

neutrophils to roll on activated endothelium via the loose binding of SleX and L-selectin on neutrophil surfaces to E-selectin on endothelial cells

Attachment —With the further release of inflammatory cytokines (from endothelial cells)

and/or chemoattractants (from endothelium and extravascular sources), the expression of CD18 β-2 integrins (Mac-1 and LFA-1) on the surface of neutrophils is upregulated, and the expression of L-selectin and SleX are downregulated As a result, neutrophils become firmly attached to the endothelial cells via the binding of β 2 integrins to intercellular adhesion molecule-1 (ICAM-1) on activated endothelial cells

Neutrophils also adhere to each other (via Mac-1) when exposed to chemotactic factors Aggregates of PMNs therefore slow blood flow, thereby allowing the recruitment of large numbers of neutrophils to the inflamed sites

Migration — Supported by the attachment of Mac-1 and LFA-1 to ICAM-1, neutrophils

crawl on endothelium; they subsequently migrate out of the vasculature by squeezing between endothelial cells via integrin attachments and adherence to platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31), a protein constitutively found at endothelial cell margins (and expressed on neutrophils) Neutrophil migration across subendothelial basement membranes appears to be dependent upon the degradation of membrane

constituents by proteinases released from neutrophil granules

v) Neutrophil function: the classical view of neutrophil function

Traditionally, the major role of recruited neutrophils in the inflamed sites is to phagocytose and destroy infectious agents (Golde et al., 1992) During the process, opsonization is usually required for neutrophil to exert its function Opsonization is a process, in which opsonins adsorb to the surface of bacteria or other particles, and this facilitates their adherence to the cytoplasmic membrane of phagocytes through opsonin receptors, thereby resulting in their recognition by specific receptors located on neutrophils This recognition results in immune adherence, which is commonly followed by phagocytosis of the

with lysosomes), as well as killing and degradation of ingested cells or other material proceed Simultaneously with the recognition and particle binding, a dramatic increase in

oxygen consumption (the respiratory burst) is observed It is responsible for the

production of superoxide and other oxygen radicals, and also for the secretion of a variety

of enzymes and biologically active substances that control inflammatory and cytotoxic reactions During phagocytosis, cytosolic granules (lysosomes) fuse with the invaginating plasma membrane (around the engulfing microorganism) to form a phagolysosome into which into which they release their contents, thereby creating a highly toxic

microenvironment This step is of importance because during the process two categories of cytotoxic substances, present in the preformed state in azurophil and specific granules and

synthesized de novo during the respiratory burst, arrive at the same cell compartment

However, some targets may be too large to be fully phagocytosed or they may manage to avoid engulfment, resulting in frustrated phagocytosis in which no phagosome is formed These may be killed extracellularly by the degranulation of neutrophils Degranulation is a cellular process that releases antimicrobial cytotoxic molecules from granules found inside neutrophils; this begins simultaneously with the onset of phagocytosis The intracellular granules that are available for mobilization in the neutrophil differ in availability for degranulation Secretary vesicles have the highest propensity for extracellular release, followed by gelatinase granules, specific granules, and azurophil granules Thus, in vitro stimulation with nanomolar concentrations of inflammatory mediators such as fMLP leads

to a rapid and almost complete discharge of secretory vesicles without significant release

of granules Stimulation with more powerful agonists like phorbol myristate acetate (PMA)

induces exhaustive release of gelatinase granules, moderate release of specific granules, and low-grade exocytosis of azurophil granules Importantly, a similar hierarchy of exocytosis has been demonstrated following in vivo migration of neutrophils into skin chambers (Faurschou and Borregaard,2003)

II Neutrophil derived cytokines

Neutrophils, as terminally differentiated cells, are generally not thought of as an important

source of de novo synthesis of polypeptide mediators; their role in host defense is thought

to be related to their phagocytic activity and to their production and release of enzymes and reactive oxygen species (ROI) However, recent data has shown that neutrophils are biosynthetically active and can produce a variety of cytokines known to play important roles in inflammatory processes

i) TNF-α: Pro-inflammatory cytokine

TNF-α is centrally involved in acute immune responses to infection and injury, as well as

in autoimmune and chronic inflammatory responses Neutrophils have the capacity to produce TNF-α in response to LPS, IL-2 and microorganisms TNF-α exerts its pleiotropic

effects in an autocrine/paracrine manner via two specific cell surface receptors: p55 and

p75 It is not yet known precisely how production and release of soluble TNF receptor are regulated in PMNs (Kasama et al., 2005)

ii) Chemokines

It has been clearly established that under appropriate in vitro experimental conditions,

human neutrophils can synthesize and secrete a number of chemokines, including IL-8, GROα, MIP-1α, MIP-1β, IP-10 and MIG The capacity of neutrophils to express the genes for I-TAC and GROβ has also been reported, but no information on their secretion is available yet In contrast, expression of the MCP-2, MCP-3, RANTES or I-309 gene product was undetectable in stimulated PMN Finally, contradictory findings have been

reported on the ability of neutrophils to express MCP-1 in vitro, a situation that clearly

requires further investigation (Kasama et al., 2005)

IL-8

IL-8 is the first chemokine shown to be produced by neutrophils, and is the most studied among the neutrophil-derived chemokines Neutrophils are not only producers, but also the primary targets of IL-8 Neutrophil can be attracted and activated by IL-8 Two receptors for IL-8 are expressed in neutrophils, namely CXCR1 and CXCR2, the latter being also used by NAP-2 and GRO family members (Kasama et al., 2005)

MIP-1α

MIP-1α, a member of CC chemokines, is released by neutrophils in response to various agonists, including LPS, TNF-α, several bacteria, fungi, protozoa, viruses and related microorganism products MIP-1α acts as the potent chemotactic/activating factor for monocytes, macrophages, natural killer (NK) cells and immature dendritic cells (1–3)

Moreover, it was recently reported that MIP-1α serve as a chemoattractant for Th1 but not Th2 cells (Kasama et al., 2005)

MCP-1

There are contradictory findings about the ability of neutrophils expressing MCP-1 in vitro

It was reported that neither MCP-1 mRNA nor the protein was detected in neutrophils treated with optimal doses of LPS, IL-1β, TNF-α or GM-SCF However, Yamashiro et al (1999) demonstrated that MCP-1mRNA was selectively upregulated after the stimulation with phytohemagglutinin

iii) Angiogenic factors

VEGF

VEGF is one of the most potent angiogenic factors, acting as an endothelial cell-specific mitogen Neutrophils have been identified as an important source of VEGF (Kasama et al., 2005) Of the several known alternative splice variants of VEGF (VEGF121, VEGF165, VEGF189 and VEGF206), the major transcript forms of VEGF expressed in neutrophils

are VEGF121 and VEGF165 (Kasama et al., 2005) A recent study by Gaudry et al (1997)

showed VEGF to be localized to specific PMN granules and that the nutrophil secrete significant amount of VEGF into the supernatant from a pre-formed pool unpon

stimulation by PMA ( a potent inducer of degranulation)(Gaudry et al., 1997)

MMPs

Three metalloproteases (MMPs) have been identified in neutrophils: neutrophil

collagenase (MMP-8, ~75 kDa), which is located in specific granules; gelatinase

(MMP-9, 92 kDa), which resides predominantly in gelatinase granules (Faurschou and Borreggard, 2003); and leukolysin (MT6-MMP/MMP-25, 56 kDa), which is distributed among specific granules, gelatinase granules, secretory vesicles and the plasma membrane

of resting neutrophils (Faurschou and Borreggard, 2003) The MMPs are stored as inactive proforms that undergo proteolytic activation following exocytosis Together, the MMPs are capable of degrading major structural components of the extracellular matrix including collagens, fibronectin, proteoglycans, laminin and gelatin, which is required for the initiation of angiogenesis (Liekens et al., 2001)

III Roles of neutrophils in angiogenesis

Neutrophils are traditionally regarded as rapidly recruited sacrificial cells that, upon stimulation by generic pathogen pattern recognition, degranulate to release proteases, activate phagocytosis and oxidative burst generation before dying However, the concept that alternative activation and plasticity of neutrophils can be a significant source of cytokines and chemokines to orchestrate physiological and pathological processes is now becoming more appreciated (Kasama et al., 2005)

Recent studies have indicated that neutrophils may play an important role in angiogenesis Firstly, isolated human neutrophils are known to release VEGF from preformed stores upon activation (Gaudry et al 1997); neutrophils are also shown to release MMP-9, which

also promotes angiogenesis by degrading ECM Secondly, neutrophils are associated with angiogenesis in a Matrigel sponge model in vivo induced by laminin peptides (Kibbey et al 1994) The induction of angiogenesis by IL-8/CXCL8 is also neutrophil dependent ― the angiogenic response is completely abrogated upon neutrophil depletion (Benelli et al 2002) Deletion of CD18, which results in defective neutrophil recruitment, slows

angiogenesis in wound healing models in vivo However, there is no direct in vivo evidence

to relate the neutrophil to the natural inflammatory angiogenesis

IV Roles of neutrophils in wound healing

Although the role of neutrophil in wound healing has been studied from 1970s, a proper conclusion has yet to be made Experiments in the 1970s showed that depletion of

neutrophils by antiserum did not significantly perturb tissue repair of incisional wounds under sterile conditions (Simpson and Ross 1972) A recent study by Dovi et al (2003), using a similar approach of neutrophil depletion, partially confirmed these early studies Although dermal repair parameters were not affected by neutropenia, reepithelialization was significantly accelerated (Dovi et al 2003) But there are limitations in these

experiments: the mouse model used in Simpson’s experiment is a incision wound model in which wound contraction is not involved Comparatively, the experiments carried out in

2003, neutrophils were only depleted for 2 days More importantly, the author did not present their data showing whether neutrophil recruitment had been impaired by the neutrophil depletion