Angiogenesis in wound healing.

Pharmac. Ther. Vol. 52, pp. 407--422,1991 Printed in Great Britain. All rights reserved

0163-7258/91 $0.00+ 0.50 © 1992Pcrpmon Pro, Ltd

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ANGIOGENESIS IN W O U N D HEALING FRANK ARNOLD* a n d DAVID C. WEST? *Department of Cell and Structural Biology, University of Manchester M I3 9PL, U.K. t Department of ,tmmunology, University of Liverpool, P.O. Box 147, L69 3BX, U.K. Abstraet--Angiogenesis is an essential component of wound healing. Vessel growth is controlled by the local actions of chemical mediators, the extracellular matrix, metabolic gradients, and physical forces. Manipulation of some of these factors can improve healing in experimental wounds. The clinical potential and specific application of 'angiomodulatory' strategies arc discussed.

CONTENTS 1. Overview 2. Biology of Vessel Growth in Wounds 2.1. Angiogenesis 2.2. 'Generalized mesenchymal repair' 2.2.1. Inflammation 2.2.2. Proliferation 2.2.3. Maturation 2.3. Modifications of this schema 2.3.1. Specialized tissues 2.3.2. Types of wound 2.4. Regulation of angiogenesis in repair 2.4.1. Soluble angiogenic factors 2.4.2. Extracellular matrix: (ECM) 2.4.3. Metabolic gradients 2.4.4. Physical forces 2.4.5. Negative controls on angiogenesis 2.5. Regulation of repair by angiogenesis 2.6. Integration 3. Clinical Implications 3.1. Problems of methodology 3.2. Normal repair 3.3. Wound failure 3.4. Overhealing 4. A Pharmacology of Angiomodulation? 5. Conclusions Acknowledgements References

407 408 408 408 408 409 409 409 409 410 410 410 412 412 412 412 413 413 413 413 414 415 416 416 417 417 417

1. OVERVIEW The search for specific agents to improve healing is as old as the practice of medicine. Everything from fresh meat (mentioned in the Edwin Smith papyrus) to 'dragon's blood' (favored by Guy de Chaillac) has Abbreviations: AF(s), angiogenic factor(s); CAM, (chick) chorio-allantoic membrane; ECM, extracellular matrix; EGF, epidermal growth factor; ESAF, endothelial cell angiogenesis factor; FGF, flbroblast growth factor; bFGF, basic FGF; GH, growth hormone; IGF, insulin-like growth factor; NAD, nicotinamide adenine dinucleotide; pADPR, poly adenosine diphosphate-ribos¢; PDECGF, platelebderived endothelial cell growth factor; PDGF, platelet derived growth factor; PGE2, prostaglandin E2; TGF-, transforming growth factor alpha; TGF,#, transforming growth factor beta; TNF, tumour necrosis factor.

been applied to wounds (Cope, 1958). The efficacy of these maneuvers was summarized by Pare (ca. 1550, cited in Haeger, 1988) in his famous dictum: "I dressed the wound but God healed it". Florey (1970) reached similar conclusions: "We have at present, no knowledge of any substance or condition that can be used to accelerate healing above the normal rate." Wound healing cannot occur without angiogenesis, The vasculature comprises up to 60% of repair tissue (Dyson et aI., 1991), and the original name for the temporary organ of repair, 'granulation tissue' (Hunter, 1787), is derived from the prominence of its vessels. An abundant blood supply is obviously necessary to meet the enormous local metabolic demands of debridement and fibroplasia; the basal

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vasculature is seldom, if ever, sufficient support. Angiogenesis plays more than a nutritive role; the endothelial cell is also an organizer and a regulator of healing. Practical stratagems to modulate vessel growth have been exploited, consciously and otherwise, by surgeons since the ancient Hindu invention of flap repairs (Prakash, 1978). But recent advances in our understanding of neovascularization have made angiogenesis a prime target for therapeutic manipulation in wound healing. To reap these benefits will require a synthesis of answers from several disciplines. The key questions will be discussed in turn. (1) Cell biology. How do wound vessels grow and what are the signals which control them? How is angiogenesis coordinated with the rest of healing? (2) Surgery. In what pathological situations is neovascularization rate-limiting, absolutely insufficient, or excessive for repair? (3) Applied pharmacology. How can 'angiogenic modulators'--which may be scarce, expensive, or toxic--be safely and effectively delivered when and where they are needed?

2. BIOLOGY OF VESSEL GROWTH IN WOUNDS 2.1. ANGIOGENESIS The 'sprouting' model of angiogenesis (Ausprunk, 1979) originates from studies in various transparent, essentially two-dimensional, experimental wounds, from frog's webs (Wharton-Jones, 1850) to the rabbit ear chamber (Clark and Clark, 1939). These observations were supplemented more recently by histology and electron microscopy of repair in muscle and the cornea (Schoeffl, 1963; Cliff, 1963; Yamagami, 1970). According to this 'central dogma', angiogenesis is thought to initiate predominantly from venules. Endothelial cells are considered as the key actors; they degrade their underlying basement membrane, are mobilized, and migrate into surrounding dead tissue and clot. Here they form a hollow cord, in which cell division and tubular rearrangement occur proximally to a moving blind end. This conceptual model of sprouting has led to in vitro methods for studying components of endothelial cell behavior, such as enzyme secretion, motility, proliferation, and tubular organization. Early reports of the effects of specific stimuli on these activities are given *A classic experiment, often cited. The authors used the best techniques available in 1975 to abrogate macrophage function--anti-macrophage anti-sera and steroids. The relatively non-specific methods may have had effects on other repair processes. Repetition of these studies using monoclonal antibodies would be valuable. It is also still uncertain what contribution circulating monocytes and tissue-resident precursors make to the eventual population of wound macrophages. Studies of monocyte traffic using the techniques developed for lymphocyte recirculation might be informative.

in Gross et al. (1983), Banda et aL (1982), Gospodarowicz et aL (1978) and Folkman et al. (1979). However, it has also been shown that the endothelial cells isolated from different tissues show important heterogeneities. Procedures for isolating endothelium from healing wounds (Knighton et al., 1991) may make it necessary to reassess older findings. For all its virtues, the sprouting model is incomplete. It does not include initial effects of trauma, such as contraction of, and thrombosis within vessels. It is still fairly imprecise about how endothelial cells arrange themselves as conduits and anastomose to form loops which can carry blood. It says little about the commencement and effects of blood flow. During angiogenesis, there is an orderly deposition of a basement membrane containing glycosaminoglycans, type IV collagen and laminin (Ausprunk et al., 1981; Whitby and Ferguson, 1991). Primitive endothelial tubes become associated with pericytes which may inhibit further growth at their sites of contact (Wakui et al., 1989; Antonelli-Orlidge et al., 1989). Some new capillaries develop further to become arterioles and venules by addition of smooth muscle cells and more extracellular matrix (ECM). The vast majority of new vessels eventually regress; mature scars are relatively avascular. Although vascular regression is probably central to the maturation of scars, and has been examined by in vivo and electron microscopy (Clark and Clark, 1939; Ausprunk et aL, 1978), the mechanisms by which it occurs in healing are virtually unknown. Wound vascularization must be coordinated with the many other events which are essential for the successful formation of a scar. Space prohibits a full discussion of these, which is given in several excellent articles and books (see for example Hunt et al., 1984; Clark and Henson, 1988) but a brief summary is needed to relate angiogenesis to the behavior of other cells during the three classic phases of healing: Inflammation, fibroplasia and maturation. Most of the experimental data are from incised or excised wounds of skin or dead spaces beneath it; their validity in other types of injury or tissue is uncertain. 2.2. 'GENERALIZEDMESENCHYMALREPAIR'

2.2.1. Inflammation A clot forms, plasma extravasates, and platelets degranulate at the site of injury. Subsequently, neutrophils adhere to endothelium and migrate into the wound in large numbers. Surprisingly, depletiorl of these cells with anti-neutrophil serum has not been shown to affect subsequent repair in the absence of infection (Simpson and Ross, 1972). Macrophages, whether derived from circulating monocytes or tissue-resident precursors or both, are probably the key cell controlling repair. Macrophag¢ depletion reduces the rate of angiogenesis and fibroplasia during healing (Leibovitch and Ross, 1975).* Activated

Angiogenesis in wound healing macrophages make and secrete a vast array of enzymes, growth factors, and other macromolecules and are central to both debridement and the regulation of wound metabolism (Nathan 1987; Caldwell, 1988), while their addition to wound models accelerates repair (Thakral et al., 1979; Danon et al., 1989). The role of lymphocytes in repair has only recently been investigated. Depletion of all T cell populations diminishes both the breaking strength and collagen deposition; reduction of T helper cells has no apparent effect, while T suppressor depletion enhances healing in standardized mode~ (Regan and Barbul, 1991). At the same time, the extracellular environment is being modified by phagocytosis and secretion by inflammatory cells. Dead tissue is debrided and bacteria are killed largely within the lysosomes of neutrophils and macrophages. Some degradation is extracellular; wounds contain metalloproteases (collagenase, gelatinase, and stromelysin), plasminogen activator, and hyaluronidase (Mignatti et al., 1988; Bertolamin and Donoff, 1978). The enzymes are counterbalanced by inhibitors which may be locally or systemically produced. Hyaluronan, tenascin, and fibronectin are secreted; some of the latter may be plasma-derived (Alexander and Donoff, 1980; Mackie et al., 1988; Clark, 1990). The initial thrombus is modified to become a 'provisional wound matrix', which is rich in cytokines, extracellular ligands for cell adhesion, their degradation products and other regulatory signals.

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2.2.3. Maturation Granulation tissue is a contractile organ that responds to agonists which stimulate smooth muscle (Gabbiani et al., 1972). Wound contraction orientates an initially random collagen matrix and may make a major contribution to the early strength of wounds (Forrest, 1983). The importance of contraction to closure of full thickness defects varies between sites and species. In rat skin wounds, for example, it can make a far greater contribution relative to proliferative repair than in man. Contraction is an important cause of strictures, contractures, and other complications of healing. 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 acellular scar. Whether their absence is due to emigration, apoptosis, or other mechanisms of cell death is unknown. Persistent wound cellularity occurs in hypertrophic and keloid scarring. 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. Attempts to rigidly define their boundaries may be fruitless. 2.3. MODIFICATIONSOF THIS SCHEMA 2.3.1. Specialised Tissues

2.2.2. Proliferation Other than vessels, fibroblasts are the major component of granulation tissue. They are thought to migrate from surrounding tissue into the matrix, to divide, to produce and organize the majority of wound collagen and other definitive stromal elements. In vitro, they can also secrete regulatory molecules including platelet derived growth factor (PDGF), transforming growth factor/~ (TGF/~) and insulin-like growth factor 1 (IGF1) (Raines et al., 1989; Anzano et al., 1986; Ciemmons and Shaw, 1986). As myofibroblasts, they provide the force of wound contraction (Roche, 1990). It is conventional to refer to 'the fibroblast' as a single entity, but it is increasingly apparent that they consist of functionally, if not anatomically distinct sets ("a fibroblast is not a fibroblast is not a fibroblast"). Fibroblasts from wounds of different ages vary in their expression of enzymes, their ability to contract collagen gels, and their response to growth factors (Buckley-Sturrock et al., 1989; Finesmith et al., 1990). This heterogeneity is masked by the selection of particular clones in long-term tissue culture; such cells contain large amounts of f-actin, and may correspond more closely to myofibroblasts (Gabbiani et al., 1973).

The hypothetical entity described above, generalized mesenchymal repair, rarely takes place in its pure form, or in isolation. The pre-morbid vasculature (Wiedeman, 1981) and the response to injury vary between organs. The participation of angiogenesis in healing responses of various tissues has been documented by several techniques (Table 1). There is preliminary evidence of tissue specific influences on endothelial cell behaviour (Auerbach, 1991), which may account for some of the differences of repair seen at different sites. Mesodermal derivatives such as muscle or tendon heal almost exclusively by deposition of a fibrous scar. This can be associated with loss of function as in hearts undergoing fibrosis due to recurrent small infarctions, while glial scarring occurs in the central nervous system. In contrast, true regeneration can occur in bone. In liver both processes are possible--regeneration after partial excision, and fibrosis (cirrhosis) after other kinds of injury. Skin or gut are intermediate examples: reepithelialization accompanies repair, and interacts with it. Wound angiogenesis is adapted by the healing environment which it serves, and in which it occurs. In bone, for example, two distinct forms of ossification can occur simultaneously. In membranous ossification, osteoblasts directly secrete matrix into

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TABLE1. Demonstrations o f Wound-related Angiogenesis in Various Tissues and Implants Tissue Stomach Colon

Species rat rat

Heart Nerve Brain Retina Tendon Cartilage Bone Synthetics: vascular graft materials polyurethane prostheses

man rat rat man dog dog rabbit hamster rat

Injury acetic acid ulcer division and

Method dye uptake microangiography

Reference 1 2,3

anastomosis

immunohistology

4

infarction ischemia penetrating wound various division and suture incision bone chamber

histology histology; isotope uptake histology fluorescence ansiography histology histology vital microscopy

5 6 7 8 9 10 I1

subcutaneous implant subcutaneous implant

vital microscopy histology

12 13

References: (I) Hase et al. (1989); (2) Leaper (1983); (3) Houdart et al. (1985); (4) Brasken et aL (1990); (5) Fishbein et al. (1978) (6) Nukada and Dyck (1986); (7) Giulian et aL (1989); (8) Routine clinical use; (9) Gelberman et aL (1985); (10) Gershuni et aL (1989); (I1) Winet et al. (1990); (12) Menger et al. (1990); (13) Picha et al. (1990).

the granulation tissue which develops in the fracture hematoma. The sequence of enchondral ossification is more complex, and requires two cycles of angiogenesis. First, granulation tissue proliferates and is replaced by chondrocytes which deposit cartilage. Chondrocytes then divide, hypertrophy, and begin to deposit osteoid. Endothelial cells and osteoblasts invade this mineralizing milieu, and participate in remodelling. 2.3.2. Types o f W o u n d The mechanism of wounding can also influence neovascularization. In contrast with incised wounds, crush injury and infarction leave large volumes of dead tissue. Even when this does not act as a nidus for infection, its debridement prolongs the inflammatory phase and delays proliferation. Ischemic injury can be relative rather than absolute. Recovery of damaged but viable tissue depends on another form of vessel growth: Collateralization. The success of split skin grafts depends virtually exclusively on angiogenesis, but whether this occurs by inosculation, invasion, or both, is still debated (Smahel, 1977). 2.4. REGULATIONOF ANGIOGENESISIN REPAIR 2.4.1. Soluble Angiogenic Factors The concept of angiogenic factors (AFs) has its roots in embryology,* but the demonstration of

* " . . . the behavior and character of capillaries is even from the first intimately influenced by the tissues into which they grow. A new set of problems confronts us... questions concerning the differences in chemical nature of the tissues and a closer determination of the real stimulant for vessel growth." (Evans, 1912).

a diffusible stimulus and the isolation of the first agent responsible for neovascularization are products of tumor biology (Greenblatt and Shubik, 1968; Folkman et al., 1971). Since angiogenesis occurs in diverse physiological and pathological events, evidence for A F s was also sought in embryonic development, graft vs host responses, arthritis, wound healing, and myocardial infarction. (Risau and Ekblom, 1986; Auerbach and Sidky, 1979; Brown et al., 1980; Arnold et al., 1987; Kumar et al., 1983). Many attempts have been made to purify and determine the chemical structure of AFs. It proved far easier to show evidence for their existence in tissue than to characterize the agents extracted. This was due to the low yield of isolation procedures, the laborious and largely qualitative nature of in vivo assays for vessel growth stimulation, and the uncertain meaning of results obtained in vitro.

In contrast, many pure substances give positive angiogenic responses in rive, or suitably modify endothelial cell behavior in vitro. But the observation that a substance can cause vessel growth on a chick chorioallantoic membrane or in a rabbit cornea does not prove that it is operative in wound healing, or elucidate its role. We now have a plethora of putative angiogenic factors which have been characterized to varying extents. Many of these have been extensively reviewed in the present series, and elsewhere. Table 2 summarizes some (but not all) of the diverse agents which have been reported to be angiogenic, and which are, or might reasonably be expected to be found in wounds. The task of discovering meaning in the mass of available data is daunting. What would allow us to state with confidence that basic fibroblast growth factor, (bFGF), for example, is a physiologically relevant angiogenic agent in wounds? We need to establish a set of 'criteria of relevance' (Arnold, 1991) and having done so, demonstrate that they are satisfied by what we know of b F G F . We either know or need to show that:

Angiogenesis

in w o u n d

411

healing

TABLE2. Candidate Angiogenic Factors in Wound Healing Effects

Effects in vitro

Mitosis

Migration

Growth factors FGFs (~t and fl) 1 2 5 6 EGF 7 -TGFct 8 8 PDGF BB 10(b) -TGFfl IGF 1 14 -16(b) 16 TNF ECM macromolecules Hyaluronan oligosaccharides 18 18 Fibrin degradation products --Heparin 26(c) 27 Low molecular weight agents ESAF 30 31 -34(d) PGE2 Nicotinamide 38(g) -Unidentified cellular products of Macrophages 40(g) 40 Lymphocytes --Other recently discovered angiogenic cytokines: (reference only) Platelet derived endothelial cell growth factor (PDEFG) 42 Vascular endothelial growth factor (VEGF) 43 Heparin affinity regulatory peptide (HARP) 44

Other

in vivo

Presence in wounds

2(a)

3

---ll(c) -16(c)

3 7 -12,13 -16

--9 9 15 17

19 23,24 28(h),29

20,21(e) 25 (e)

32(t) ---

33 35,36 38,39

-37(e) (e)

---

40 41

(e) (e)

-22 --

4(e)

(a) Induces protease production; (b) Inhibitory; (c) Promotes tube formation; (d) Indirect action?; (e) Probable, unproven; (f) Activates procollagenase; (g) No effect; (h) Cofactor. References: (1) Duthu and Smith (1980); (2) Moscatelli et al. (1986); (3) Gospodarowicz et al. (1979); (4) Folkman et al. (1988); (5) Gospodarowicz et al. (1978); (6) McAuslan et al. (1985); (7) Schreiber et al. (1986); (8) Knighton et al. (1991); (9) Grotendorst et al. (1987); (10) Takehara et al. (1987); (11) Madri et al. (1988); (12) Roberts et al. (1986); (13) Dugan et al. (1988); (14) King et al. (1985); (15) Spencer et aL (1988); (16) Leibovitch et al. (1987); (17) Ford et aL (1989); (18) West and Kumar (1989); (19) West et al. (1985); (20) Bertolami and Donuff (1978); (21) Maurer and Hudack (1952); (22) Nicosia et al. (1982); (23) Dvorak et al. (1987); (24) Thompson et al. (1985); (25) Aberg et al. (1976); (26) Maciag et al. (1984); (27) Azizkham et al. (1980); (28) Taylor and Folkman (1982); (29) Fraser and Simpson (1983); (30) Keegan et al. (1982); (31) Odedra and Weiss (1991); (32) Weiss et aL (1984); (33) Weiss et al. (1979); (34) Alessandri et al. (1983); (35) BenEzra (1979); (36) Barnhill and Ryan (1983); (37) Humes et aL (1977); (38) Morris et al. (1989); (39) Kull et al. (1987); (40) Banda et al. (1982); (41) Auerbach and Sidky (1979); (42) Miyazono and Heldin (1990); (43) Ferrara et al. (1991); (44) Courty et al. (1991).

(1) It can cause vessel growth or appropriately modify endothelial cell behaviour. Many workers have produced evidence to this effect both in bioassay and in vitro (Gospodarowicz et al., 1978, 1979; Duthu and Smith, 1980; Lobb et al., 1985; Schreiber et al., 1986; Moscatelli et al., 1986). (2) Means for its production exist within wounds. Although macrophages and endothelial cells have been reported to produce b F G F in vitro, (Baird et al., 1985; Vlodavsky et al., 1987) there is no proof as yet that they do so in repairing tissue, b F G F is sequestered in normal extracellular matrix, and may be released by heparin or other agents during repair (Folkman et al., 1988). Although this mechanism fits readily with a role for b F G F in initiating wound angiogenesis, it is ill-suited to direct, sustained stimulation. Furthermore, b F G F lacks a signal peptide (Abraham et al., 1986) so the means by which its release from cells is effected or controlled (other than by cell death) is unknown. (3) b F G F and its receptor(s) are available when and where they are required, and that appropriate means exist to localize and terminate their expression or effect. Immunochemistry has been used to demonstrate their evolution in experimental ulcers over time

(Hanssen and Norstrom, 1991) and will undoubtedly be applied to other models of repair. (4) Addition of b F G F to wounds accelerates vascularization. This has been shown in a number of models, although it is uncertain whether the doses required are physiological. (Buntrock et al., 1982; Davidson et al., 1985, 1988). The converse---that inhibitors of F G F reduce angiogenesis--has been demonstrated by using anti-serum in a sponge wound model in rats (Broadley et al., 1989). Further studies using competitive analogs or other inhibitors are both feasible and desirable. These criteria (activity in assay, means of production, location, concentration, time course, effects of exogenous agonists and inhibitors) will be familiar to pharmacologists. They are derived from those used to demonstrate, for example, that acetyl choline is the transmitter at the neuromuscular synapse (Paton, 1958). They can usefully be applied to other putative 'wound hormones'. However, it will be necessary to determine whether results obtained in one particular model or species can be applied to others. Many, but perhaps not all, of the agents in Table 2 will emerge from this analysis as genuinely relevant angiogenic factors. It will then be desirable

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to show the relationship between different signals. Do they act in parallel or in cascade? Which are the rate-limiting steps? Which ones are deranged in defective human repair? 2.4.2. Extracellular Matrix: ( E C M )

systemic levels of oxygenation can alter the pattern of vessel growth in this system (Knighton et al., 1981). An elegant model has been described in which: (1) Increased lactate production causes a fall in intra-cell levels of NAD. (2) This lowers cellular production of poly ADP ribose (pADPR), a negative regulator of prolyl hydroxylase, thereby promoting collagen secretion (Hussain et al., 1989). (3) Nicotinamide is a putative angiogenic factor (Kull et al., 1987). It is produced and released in the same reaction which synthesizes pADPR. This mechanism could directly coordinate collagen deposition, vessel growth, and metabolic demand. The resulting metabolic gradients have pleiotropic effects. They stimulate macrophage secretion of an endothelial-chemotactic, angiogenic peptide agent (Knighton et al., 1983). The proliferative response of fibroblasts in vitro to EGF and PDGF is increased when oxygen concentration is reduced to 2.5%. Similar results are obtained even when oxygen tension is returned to normal for several hours before growth factor stimulation. (Storch and Talley, 1988). The bioavailability of angiogenic growth factors such as IGFI, TGFfl, and PDGF may be increased by falling pH (Wakefield et al., 1989; Spencer et al., 1988).

Vessel growth occurs into an environment which, by its differential adhesive properties, strongly influences cell movement. Interaction via integrins and other receptors with fibrin, fibroneetin, hyaluronan and collagen can alter angiogenesis and/or indices of endothelial cell behavior in vitro (Thompson et al., 1985; Dvorak et al., 1987; Alessandri et al., 1986; Feinberg and Beebe, 1983). Fibronectin appears to play an important part since both anti-fibronectin antibodies (Britsch et al., 1989) and RGD peptides/polypeptides (Nicosia and Bonanno, 1991; Saiki et al., 1990) inhibit angiogenesis. The concentration of flbronectin in substrata determines whether addition of FGF to endothelial cells in vitro will result in proliferation or differentiation into tubular structures (Ingber and Folkman, 1989). Thus the environment can determine the responses of endothelial cells to other signals they receive. Conversely, breakdown products of matrix components fulfil at least some of the criteria described above for wound angiogenic factors. For example, 2.4.4. Physical Forces degradation of macromolecular hyaluronan by Sheer stresses and pressure acting from within enzymes (Nakamura et al., 1990) or free radicals-new-formed vessels have long been held to modulate both of which are present in wounds--generates oligosaccharides which are angiogenic in vivo (West neovascularization, (Thoma, 1893) but their effects in wound angiogenesis have never been directly et al., 1985) and induce endothelial migration and mitosis (West and Kumar, 1989). Addition of these demonstrated. The transduction of extracellular fragments to granulation tissue accelerates vessel forces into intracellular biosynthetic events (Bissell growth (Andrade et al., 1987). Inhibition of endo- et al., 1982) is an area of active study. There is thelial cell proliferation by macromolecular hyaluro- evidence for a direct connection between physical nan can not be overcome by addition of FGF (West forces acting on new vessels and endothelial morphogenetic and secretory activity (Ingber and Folkman, and Kumar, 1991). 1989). ECM components can alter the activity or availability of angiogenic cytokines in interactions such as that between heparan sulphates and fibroblast 2.4.5. Negative Controls on Angiogenesis growth factors (Folkman et al., 1988) or between transforming growth factor fll and collagen IV Some means must exist whereby the effects of (Paralkar et al., 1991) or glycosaminoglycans. They angiogenic stimuli are limited in time and space to the may also modulate the expression of receptors by sites where they are observed. Concentration gradiendothelial cells. It is probable that the synthesis of ents are presumably important, but true negative basement membrane is tightly linked with vessel regulators may also play a part. There is indirect growth since inhibitors of the former also abrogate evidence for an inhibitor of vessel growth in serum, the latter (Ingber and Folkman, 1988; Maragoudakis and in animal and human wound fluids (Folkman et al., 1988). et al., 1971; Hunt et al., 1981; Arnold et al., 1987). Platelet factor IV, thrombospondin, and macromolecular hyaluronan can all inhibit angiogenesis 2.4.3. Metabolic Gradients (Folkman et al., 1983; Bouck et al., 1991; Feinberg The central area of a wound which has not yet and Beebe, 1983), and are almost certainly present in obtained a blood supply is intensely hypoxic, and wounds. Other naturally occurring inhibitors such as acidotic, and contains high levels of lactate, as shown a collagenase inhibitor in cartilage (Eisenstein et al., by micro-electrode studies in rabbit ear chambers 1975) may have parallels within wound tissue. The (Silver, 1973; Hunt and van Winkle, 1979). Changing stimuli which elicit regression during the maturation

413

Angiogenesis in wound healing of scars have not yet been investigated, perhaps because assays for them are difficult to design. 2.5. REGULATIONOF REPAIR BY ANGIOGENESIS

Not only the form, but also the functions of vessels change as healing proceeds. In inflammation, they regulate the access of leukocytes to the thrombus and provisional matrix. The orderly influx of neutrophils, macrophages and lymphocytes is usually explained with reference to gradients of chemo-tactic and -kinetic factors, but this picture is incomplete. The timing and rates of appearance and disappearance of inflammatory cells will also depend upon: (1) Availability. This is a product of local flow and the concentration of leukocyte populations in peripheral blood. (2) Cell adhesion molecules (CAMs) on wound endothelium. Sequential expression of CAMs by endothelium during angiogenesis could provide a mechanism for selection and regulation. Some of these molecules interact with complementary pairs on a spectrum of white cell types, but a relatively monocyte-specific CAM has recently been described (Gimbrone et al., 1990). Given the critical role of macrophages in repair, this molecule may be of considerable importance. (3) Leukocyte emigration and/or death in situ. Fibroplasia requires energy, oxygen and nutrients, and results in production of CO2 and lactate. The stoichiometry between angiogenesis, blood flow and collagen deposition is difficult to quantify directly in wounds, but proxy indices (mean oxygen tension and hydroxyproline incorporation) are directly correlated (Ehrlich et al., 1972). Diffusion distances between the most distal perfused vessel and the site of matrix synthesis will govern rates of transport, and are therefore potentially rate-limiting. These relationships are graphically illustrated in rabbit ear chambers: gradients of pH, lactate and oxygen correlate precisely with tissue architecture (Silver, 1973). These results have been summarized in a model, the 'wound module' (Hunt and van Winkle, 1979) in which macrophages form a leading front for granulation tissue. Behind them, fibroblasts divide, differentiate, and secrete collagen in waves. Vessel growth is directed toward the hypoxic front, advancing its supply line as it progresses across the surface of the dead space. The final link in this chain is the regulation of macrophage production of angiogenic stimuli by hypoxia and lactate (Knighton et al., 1983). Angiogenesis may also organize maturation. A newly discovered cytokine in the conditioned media of large vessel endothelial cells promotes the contraction of collagen matrices by skin fibroblasts (Chen et al., 1991). Repetition of the experiment using wound endothelial cells and fibroblasts would strongly support the physiological relevance of this mechanism.

Under some circumstances angiogenesis occurs in relation to stimulatory gradients, and fibroblasts are orientated perpendicular to this. In full thickness skin defects in pigs, especially under a semipermeable dressing as used in human wounds, vessels grow toward the denuded surface; fibroblasts lie parallel to it, and at right angles to the new capillaries (Dyson et al., 1988). In incised rat skin wounds, collagen fibres are at 90 ° to the epidermis, while vasculature is parallel to its axis. Thus vessel growth may coordinate both the extent and the direction of fibroplasia. 2.6. INTEGRATION Cytokines, matrix components, metabolic gradients, and physical forces are all important regulators of wound angiogenesis. Many 'wound angiogenic factors' also modify the behaviour of fibroblasts and inflammatory cells, both directly and through their effects on vessels. Angiogenesis can not be considered in isolation. Its mechanisms and controls are the products of evolutionary selection acting simultaneously on all other aspects of repair. Efforts to modulate wound angiogenesis will inevitably tend to alter the entire process of healing. The converse--that manipulating fibroblast or leukocyte activities will have effects on vessel growth--is also probable. This suggests that the number of pathways by which we can influence repair is large. It also makes interpretation of the results of treatment problematical.

3. CLINICAL IMPLICATIONS 3.1. PROBLEMSOF METHODOLOGY

The majority of wounds that patients suffer or surgeons make will heal satisfactorily. If this were not so, neither we nor our craft would exist. But several classical strategies used in everyday surgery depend on the promotion of angiogenesis. Timing is one variable we can sometimes control. 'Delay' in placing a skin graft or in dividing a flap depends upon allowing time for the growth of vessels in the recipient site or pedicle respectively. Natural sources of angiogenic stimulation have also been applied to wounds. Omentum and amnion induce vessel growth and have been transposed or transplanted to improve healing of critical anastomoses, and chronic ulcers (Silverman et al., 1988; Bennett et aL, 1980; Burgos, 1986). These treatments may also have beneficial effects on other, non-angiogenic aspects of repair. The measurement of outcome is a major problem in all human studies on repair. When a chronic wound heals rapidly on treatment, even if it is a venous ulcer which has resisted previous therapies for many years, how are we to prove that the relationship is causal? Optimal conventional care in the 'placebo' arm of trials (or even simply hospital admission) can produce excellent results in patients who have

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F. ARNOLI)and D. C. WEST

never before experienced them. We need to look at other indices, both scientifically and clinically. These include: Speed of closure of the defect, recurrence rates, and relief of pain. Acute wound failure is more awkward to study. It can present catastrophically as a leaking oesophageal anastomosis or distressingly in a pretibial laceration. While some cases are undoubtedly due to technical errors, there are also groups of patients whose intrinsic ability to heal is inadequate. Causes include well recognized systemic factors like steroids, previous irradiation, and malnutrition, (see Table 3) as well as subtler individual variations which may be due to impairment of one or more cellular activities or their regulatory signals. Because of the relative rarity of each specific problem within a given surgical series, the size of any human trial would be prohibitive unless limited by accurate patient selection. Hunt and coworkers (Goodson and Hunt, 1982; Jonsson et al., 1986) have developed an elegant and relatively non-invasive technique for assessing the acute healing potential of individual patients. It consists of a fine-bore, porous synthetic tube inserted subcutaneously. After seven days, the implant is withdrawn, and the granulation tissue response is assessed. They have used this method to show, for example, that variations in local oxygenation can cause up to a four-fold variation in collagen deposition. Since tissue oxygen tensions can be measured and improved in many cases, the clinical implications are substantial. The technique may also be useful in guiding operative decisions: Intrinsic healing potential could predict the need for a temporary colostomy to protect selected colonic anastomoses, or for nutritional support (Haydock and Hill, 1987; Barbul et al., 1990). At the same time, it provides a sample of human healing tissue, in which defects of angioTABLE3. Some Causes of Defective Repair (a) Local factors: Sepsis Prior irradiation Recurrent trauma (neuropathy, decubitus ulcers) Poor oxygenation or perfusion during healing Arterial insufficiency Venous insufficiency/hypertension Lymphoedema (b) Systemic factors: Hypoxia Collagen disorders Diabetes Uremia Jaundice Malignancy Malnutrition Auto-immune disorders: Rheumatoid arthritis Scleroderma Drugs: Cytotoxics Immunosuppressants Steroids ? others in common use.

genesis and of healing could be compared, although no method for quantifying vessel growth has yet been validated in this system. Animal studies on the effect of exogenous growth factors on healing have been reviewed by Ksander (1989). The design of these experiments and interpretation of their results are problematical. At least 7 growth factors have been tested in 8 different wound models in 5 species, using 8 vehicles. A minimum of 5 different parameters of effect have been employed, including extent of granulation tissue, contraction, epithelialization, rate of closure, collagen synthesis, and tensile strength. Combinations of growth factors have also been tested (Lynch et al., 1989). These figures are out of date, and take no account of unpublished negative studies. False negative results could arise if a true hormone is presented in the wrong dose, by the wrong method or at the wrong time, or if the optimum level of that factor is already present. Many human trials are underway, although only a few have yet been published. Despite many difficulties, real progress has been achieved. The prospect of new 'angiomodulatory' strategies makes it relevant to examine the objectives to which they can be applied. Should we aim to accelerate healing where it is progressing 'normally', to increase inadequate repair, or to inhibit overhealing? 3.2. NORMALREPAIR Local addition of EGF, FGF, TGFfl, PDGF, angiogenin, and hyaluronate have been shown to increase the amount of granulation tissue formed early in various models of repair (Table 4). It is highly probable (but still unproven) that some or all of their effects on healing may be due to the direct promotion of vessel growth. However, a lack of measurements is common to most of these studies. A more quantitative model, employing the measurement of blood flow by xenon m washout from healing sponge implants (Andrade et al., 1987) has been used to demonstrate that interleukin l~t, hyaluronan oligosaccharides, and several neuropeptides (bradykinin, vasoactive intestinal peptide, angiotensin II, and substance P) can promote angiogenesis at least in this system. Specific inhibitors of the neuropeptides abolished the responses to them (Fan and Hu, 1991). Preliminary evidence of accelerated repair of human wounds has been reported. Local EGF and systemic growth hormone (GH) treatment have been claimed to speed the closure of skin graft donor sites in burn patients (Brown et al., 1989; Herndon et al., 1990). No effect was seen when EGF was used in a virtually identical experiment in healthy volunteers (R. F. Diegelmann, personal communication). GH raises circulating levels of IGFI, which has angiogenic activity in bioassays. Other human trials have shown encouraging results but are not yet in print. The eventual cost of some of the new therapies

Angiogenesis in wound healing

GF aFGF bFGF EGF

PDGF

TGFb

IGF1 and PDGF TNF Angiogenin Heparin

415

TABLE4. Effect o f Exogenous Factors on Angiogenesis in Experimental Wounds Species Model Vehicle Results Reference rabbit rat rat rat rat pig rat rat rabbit pig pig guinea pig mouse rat pig pig mouse rabbit rat

island flap sponge implant sponge implant sponge implant sponge implant gut FT incision sponge implant SC tube FT defect PT defect incision incision incision sponge implant FT defect PT defect incision meniscuscut synth skin

topical heparin inj inj slow release heparin i.p. inj collagen collagen CMC gel collagen collagen collagen heparin slow release CMC gel collagen slow release soaking

Tviabihty (Q) Tgran Tgran Tgran nil 'rgran Tblood flow (Q) Tgran Tgran nil Tgran Tgran Tgran Tgran Tbloodflow (Q) Tgran nil Tvascularity vase volume (Q)

Homet al. (1988) Sprugel et al. (1987) Davidson et aL (1985) Buckley et al. (1985) Sprugel et al. (1987) Kingsnorth et al. (1990) Laato (1986) Sprugel et al. (1987) Mustoe et al. (1991) Lynch et al. (1989) Ksander et al. (1990) Ksander et al. (1990) Ksander et al. (1990) Sprugel et al. (1987) Beck et al. (1991) Lynch et al. (1989) Mooney et al. (1990) King and Vallee (1991) Ehrlichet al. (1988)

Hyaluronic acid hamster cheek pouch gelatin Tvessel density King et al. (1991) (macromolecular) rabbit ligament cut inj Tvascularity Wiig et al. (1990) There are many other reports on the effect of exogenous agents on healing, but few of these have examined vessel growth. Abbreviations: FT=full thickness; PT=partial thickness; inj=local injection; i.p.=intraperitoneal; CMC= carboxymethyl cellulose; gran = granulation tissue; (Q) - quantitative measure.

currently under investigation are totally unknown at this time. Because these are presently produced in small quantities, they appear to be expensive. However, if the benefit that they may confer is high, it may be that the cost will be fairly insignificant (M. C. Robson, personal communication). Certain types of repair pose special problems, whose solution could greatly improve the treatment options available. In burn patients whose area of remaining normal skin is small, grafts are sometimes taken repeatedly from the same site, and treatments to reduce the interval between harvests could be important. The healing of decubitus ulcers (pressure sores) may be difficult or even impossible to achieve by conventional means. Topical recombinant PDGF BB has resulted in a significant improvement in the rate of closure (Robson et al., 1992). When anastomoses of the colon or oesophagus fail to heal expeditiously, the resulting leak of gut contents can lead to serious complications or death. Inadequate blood supply is a limiting factor in bowel healing after colostomy closure in man, and of experimental anastomoses performed in obstructed gut (Forrester et al., 1981; Leaper, 1983). Local inhibitors of vessel growth are present in cartilage (Eisenstein et al., 1975; Moses et al., 1990) and may cause healing problems in surgery to the bronchus, and skin grafts onto denuded cartilage. In lung transplants, failure of angiogenesis at the bronchial anastomosis may be the key factor preventing progress. Likewise, prolonged immobilization is still required for the healing of

some fractures. Methods to accelerate angiogenesis in these situations would be particularly valuable and might justify costly treatments. Recently, it has been shown that intraperitoneal infusion of EGF can increase the tensile strength of colonic anastomoses (Kingsnorth et al., 1990), while TGFfl accelerates bone repair in primates (Beck et al., 1991). However, in some of these cases, the distinction between normal and defective repair is problematical.

3.3. WOUNDFAILURE

Some local and systemic conditions associated with impaired healing are listed in Table 3. Obviously, these can coexist, as in the classic problem of the elderly patient with rheumatoid arthritis treated with steroids, who sustains a flap laceration over the lower tibia, or the leg ulcers of diabetic renal transplant recipients. The relative contributions of each of these factors to clinical defects of repair, and the extent to which they exert their deleterious effects through alterations of angiogenesis are difficult to determine. Thus steroids impair a range of inflammatory responses, and (presumably) alter the availability of wound cytokines; they may also be 'angiostatic' (Folkman and Ingber, 1987). Cytotoxic drugs cause myelosuppression, thereby reducing the availability of leukocytes within wounds, but may also directly inhibit fihroblast or endothelial cell growth. Other commonly used drugs and their analogs, including

416


heparin, tetracyclines, diuretics and angiotensin converting enzyme inhibitors modify angiogenesis in experimental models. It is therefore conceivable that we may be altering vessel growth in wounds, beneficially or otherwise, without being aware of the fact. Local radiotherapy alters the subsequent proliferative and synthetic capacities of both cell types. The mechanisms by which infection can alter the component processes of repair have not yet been adequately dissected. Experimental models of some of these problems have been developed, but their comparability to the human situation needs critical assessment. Diabetic rats can be produced either by breeding or with the drug streptozocin; the impairment of healing in these two models may be different. Despite the development of a new rabbit ear model of full thickness skin repair in the presence of relative ischemia (Ahn and Mustoe, 1990) there is still no wholly adequate animal equivalent of some of the commonest healing problems in man--venous, arterial, and decubitus ulcers. Addition of some growth factors to certain animal models of defective repair has been shown to increase indices of healing towards that found in normal controls. Other agents which may modulate angiogenesis have also been reported to have restorative effects: Vitamin A reverses some of the consequences of radiotherapy (Levenson et al., 1984) and steroid treatment (Ehrlich et al., 1973), while application of hyaluronan to abdominal incisions may reduce the rate of abdominal wound dehiscence in obese surgical patients (Trabucchi et al., 1989). As we become better at dissecting the precise cellular mechanisms of defective healing and identifying the role of vessel growth amongst them, it is likely that angiogenic strategies will find wide clinical applications.

3.4. OVERHEALING Excessive repair can be detrimental in several contexts; vessel growth may contribute to the pathology of some of these. Adhesions are the commonest cause of small bowel obstruction. They are the 'fibrous ghosts' of vascular connections made to damaged or ischemic gut and are common sequelae of abdominal surgery (Ellis, 1971). Hypertrophic scarring and keloid formation are commonly held to be due to excess production of collagen with an abnormal organization. However, the underlying cause may also be related to defective angiogenesis giving rise to hypoxia (Kischer et al., 1982). Excessive contraction of some scars can lead to contractures and severe disability, as in burns to skin which overlies joints. Inappropriate repair, as in a transplanted cornea, a cirrhotic liver, or in reactions such as retroperitoneal fibrosis and fibrosing alveolitis, can destroy function. The formation of capsules around implants, such as those used after mastectomy, can cause discomfort

and deformity. Finally, some scars are cosmetically unacceptable. Despite a great deal of work, much of it by 'trial and error', little progress has been made in controlling overhealing, and there are few adequate animal models. However, the recent development of intra-uterine surgery has yielded a fascinating clue: Foetal wounds can heal without scarring, and the regeneration proceeds without evidence of angiogenesis (Longaker et al., 1990). However, when TGFp is applied to these wounds, they revert to an adult-like method of repair (Krummel et al., 1988). The relative contributions of cellular immaturity and the biochemical environment of the fetus to this important difference are uncertain.

4. A PHARMACOLOGY OF ANGIOMODULATION? Problems in the selection of patients who might benefit from improving repair by angiomodulation and the choice of agent are matched by difficulties in drug delivery. It is obviously simpler to give vitamin A by mouth than to repeatedly apply recombinant human EGF ointment directly to a wound. But choices of route, dose, frequency and vehicle are determined by many variables. Some agents are scarce, not absorbed orally, and may be ineffective or toxic when given systemically. Ingenuity will be required in the presentation of agents to wounds by local delivery systems. For example, some growth factors may promote wound repair at one dose, but inhibit it at a higher one. An increase in our understanding of pharmacokinetics within wounds, and improvements in the controlled release of drugs are necessary (Sawada et aL, 1990). In situ degradation of the agent may require blockade by coadministration of enzyme inhibitors (Okamura et al., 1990). Local delivery systems can mean frequent dressings, which carry the risk of infection or damage to delicate granulation tissue. Systemic absorption may also be a hazard. Furthermore, some important wounds are deeply buried. During every gut resection, incisions are made in skin, muscle, fascia, peritoneum, mesentery, and the bowel itself. The last is critical, since failure of healing here can be fatal; it is also the least accessible wound. Growth factors can be introduced intraperitoneally--EGF given in this route increases tensile strength in pig colonic wounds (Kingsnorth et al., 1990)---but undesirable angiogenesis, in the shape of adhesion formation may be a significant hazard. Alternatively, agents could be incorporated into biodegradable sutures and staples as a mechanism for their controlled release into the environment where they are needed. The removal of growth factors may also be beneficial. Opacification of a transplanted cornea can be caused by vessel and fibroblast invasion of the graft.

417

Angiogenesis in wound healing Large amounts of b F G F are sequestered in the ECM of the cornea, and are a major stimulus to these processes. Presoaking of grafts in heparin, which avidly binds and removes F G F , has been successfully used to prevent opacification. (J. Folkman, personal communication.) Although there is considerable interest in the application of pure growth factors and matrix macromolecules to wounds, a simpler alternative is already in use. The degranulation of platelets releases angiogenic stimuli (Knighton et al., 1982). These include P D G F , PDECGF, and TGFfl. Platelet lysates improve repair in animal models. Xenogeneic, allogeneic, and autologous preparations have been used in patients with chronic leg ulcers of various kinds. The results were encouraging but difficult to interpret because of patient heterogeneity and the use of other treatments including arterial grafting (Knighton et al., 1986). A double-blind crossover trial has been reported recently--81% of treated patients, but only 15% of the controls, obtained complete epithelialization by eight weeks. After crossover, all the control patients obtained healing (Knighton et al., 1990). Long-term follow up is not yet available. Eight weeks of treatment required the products of platelets from about one unit of blood, which is logistically feasible, and the use of autologous material should minimize infection hazards. Another option is to promote the local availability of angiogenic factors and other agents in impaired wounds indirectly, using other stimuli. It has been known for many years that moderate (but not overwhelming) bacterial infection can increase both vessel growth and fibroplasia. Thus cell wall mucopeptides of Staphylococcus aureus increase breaking strength and collagen production in rat wounds (Levenson et al., 1991). Yeast extracts cause neovascularization in biological assays and increase hydroxyproline and D N A content in wound chambers (Bentley et al., 1990); they have been reported to increase angiogenesis and epithelialization of human skin graft donor sites (Kaplan, 1984). Adding wound macrophages to an ear chamber can decrease the latency of vascularization, presumably by virtue of their production of angiogenic agents (Thakral et al., 1979). The local availability of these cells may be enhanced by microbial products. A similar effect might be achievable using pre-operative systemic agents such as monocyte colony stimulating factors, or by introducing monocyte-specific chemoattractants into wounds. Physical methods may also prove useful. The simplest of these is to protect gradients of regulatory molecules within wounds from the effects of dessication or trauma (Dyson et al., 1988). This may be one mechanism of the beneficial effects of occlusive dressings. The effects of infrared irradiation, ultrasound, and electromagnetic fields on wound vascularization and repair are also being studied.

5. CONCLUSIONS Until recently, surgical advances have depended primarily on developments in bacteriology, organic chemistry, and materials technology. The discipline of cell biology now promises to leave an imprint on the whole of medicine; angiogenic modulation is one of the most attractive proposals it offers. But the potential benefits of these strategies will also depend upon classical clinical questions: The choice of patient, in which wounds they can best be used and the consequences and mechanisms of failure of that particular example of repair. It is obviously more important, and probably more feasible, to attempt to improve defective healing or to inhibit excessive fibroplasia than to obtain relatively minor advantages in wounds which would heal acceptably without intervention. The traditional armamentarium of pharmacology (dose-response curves, drug kinetics, and delivery system studies) are essential tools for achieving this goal. Acknowledgements--We are grateful to Dr T. K. Hunt for advice and inspiration, Drs M. C. Robson, J. Folkman and R. Diegelman for permission to mention their (as yet unpublished) results, and to Dr M. Nassim, Mr D. Marsh and Professor M. W. J. Ferguson for helpful discussions.

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