fibrin and wound healing

13
355 Fibrin and Wound Healing RICHARD A.F. CLARK Department of Dermatology, SUNY at Stony Brook, Stony Brook, New York, USA KEYWORDS: Wounds; Fibrinogen; Fibronectin; Epidermal injury; Angiogen- esis; Fibroplasia; Epidermal cells; Fibroblasts; Endothelial cells. INTRODUCTION During the past two decades advances in the molecular and cellular biology of fibrin(ogen) have greatly expanded our comprehension of the role of fibrin in wound healing. Clearly, recent scientific breakthroughs in the understanding of these basic processes will lead to future therapeutic successes of fibrinogen preparations in wound care and tissue engineering. In this brief treatise the molecular and cellular biology of fibrin(ogen) are reviewed in the context of cutaneous wound repair. The reader is referred to The Molecular and Cellular Biology of Wound Repair 1 for a more detailed discussion of the many processes involved in wound healing. INFLAMMATION Severe tissue injury causes blood vessel disruption with concomitant extravasa- tion of blood constituents. Blood coagulation and platelet aggregation generate a fibrin rich clot that plugs severed vessels and fills any discontinuity in the wounded tissue. Although the blood clot within vessel lumen reestablishes hemostasis, the clot within wound space provides a provisional matrix for cell migration. Clearly, hemostasis is a major function of coagulation, however, blood clotting is also a part of the inflammatory response. For example, Hageman factor activation leads to generation of its fragments and bradykinin, potent vasoactive agents, 2 and to the initiation of classical and alternative complement cascades 3 with the resultant generation of the anaphylatoxins C3a and C5a. The anaphylatoxins directly increase blood vessel permeability and attract neutrophils and monocytes to sites of tissue injury. 4 In addition, these substances stimulate the release of other vasoactive medi- ators, such as histamine and leukotriene C4 and D4 from mast cells, 5 and the release of granule constituents and biologically active oxygen products from neutrophils and macrophages. 6 The clot also provides a matrix scaffold for the recruitment of tissue cells to an injured site. Specifically, fibrin in conjunction with fibronectin act as a provisional matrix 7 for the influx of monocytes, 8,9 fibroblasts, 10–12 and endothelial cells. 13 These migrating cells use integrin receptors (see TABLE I), 14,15 that recognize fibrin, Address for correspondence: Richard A.F. Clark, M.D., Department of Dermatology, SUNY at Stony Brook, Stony Brook, NY 11794-8165, USA. Voice: 613-444-3843; fax: 613- 444-3844.

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Page 1: Fibrin and Wound Healing

355

Fibrin and Wound Healing

RICHARD A.F. CLARK

Department of Dermatology, SUNY at Stony Brook, Stony Brook, New York, USA

K

EYWORDS

: Wounds; Fibrinogen; Fibronectin; Epidermal injury; Angiogen-esis; Fibroplasia; Epidermal cells; Fibroblasts; Endothelial cells.

INTRODUCTION

During the past two decades advances in the molecular and cellular biology offibrin(ogen) have greatly expanded our comprehension of the role of fibrin in woundhealing. Clearly, recent scientific breakthroughs in the understanding of these basicprocesses will lead to future therapeutic successes of fibrinogen preparations inwound care and tissue engineering. In this brief treatise the molecular and cellularbiology of fibrin(ogen) are reviewed in the context of cutaneous wound repair. Thereader is referred to

The Molecular and Cellular Biology of Wound Repair

1

for amore detailed discussion of the many processes involved in wound healing.

INFLAMMATION

Severe tissue injury causes blood vessel disruption with concomitant extravasa-tion of blood constituents. Blood coagulation and platelet aggregation generate afibrin rich clot that plugs severed vessels and fills any discontinuity in the woundedtissue. Although the blood clot within vessel lumen reestablishes hemostasis, the clotwithin wound space provides a provisional matrix for cell migration.

Clearly, hemostasis is a major function of coagulation, however, blood clotting isalso a part of the inflammatory response. For example, Hageman factor activationleads to generation of its fragments and bradykinin, potent vasoactive agents,

2

andto the initiation of classical and alternative complement cascades

3

with the resultantgeneration of the anaphylatoxins C3a and C5a. The anaphylatoxins directly increaseblood vessel permeability and attract neutrophils and monocytes to sites of tissueinjury.

4

In addition, these substances stimulate the release of other vasoactive medi-ators, such as histamine and leukotriene C4 and D4 from mast cells,

5

and the releaseof granule constituents and biologically active oxygen products from neutrophilsand macrophages.

6

The clot also provides a matrix scaffold for the recruitment of tissue cells to aninjured site. Specifically, fibrin in conjunction with fibronectin act as a provisionalmatrix

7

for the influx of monocytes,

8,9

fibroblasts,

10–12

and endothelial cells.

13

These migrating cells use integrin receptors (see T

ABLE

I),

14,15

that recognize fibrin,

Address for correspondence: Richard A.F. Clark, M.D., Department of Dermatology,SUNY at Stony Brook, Stony Brook, NY 11794-8165, USA. Voice: 613-444-3843; fax: 613-444-3844.

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356 ANNALS NEW YORK ACADEMY OF SCIENCES

fibronectin, and vitronectin to interact with the clot matrix.

16–19

Since extracellularmatrix molecules can provide signals for gene expression through integrin recep-tors,

20

the interaction of these tissue cells with the provisional matrix might beexpected to alter cell phenotype and function. In fact, Damskey and Werb

21

haveshown that a fibronectin-rich extracellular matrix can control the expression of col-lagenase (matrix metalloproteinase I, MMP-1) and our laboratory has recently foundthat fibronectin or fibrin matrix can modulate fibroblast response to cytokines

22

andendothelial cell expression of integrins.

23

Proper clearance of the clot provisional matrix appears just as important as itsdeposition. The major proteolytic enzymes, plasminogen activators and plasmin,escape inactivation by fluid phase protease inhibitors, like plasminogen activatorinhibitor and

α

2

-antiplasmin, through binding to the fibrin clot

24

and cell surfaces.

25

Although plasminogen activator and plasmin have the ability to degrade a wide vari-ety of extracellular matrix proteins, a specific inhibitor of plasminogen activator(PAI-1) binds to the extracellular matrix

26

and limits matrix degradation to themicroenvironment around cell surfaces. However, inadequate removal of the provi-sional matrix may lead to fibrosis.

27

For example, fibrin deposits and suppressedfibrinolysis are found in pulmonary fibrosis.

28,29

Furthermore, transgenic mice that

T

ABLE

1. Integrin superfamily

β

1 Integrins Ligands

α

v Integrins Ligands

α

1

β

1 fibrillar collagen,laminin-1

α

v

β

1 fibronectin (RGD),vitronectin

α

2

β

1 fibrillar collagen,laminin-1

α

v

β

3 vitronectin (RGD),fibronectin, fibrinogen,von Willebrand factor,thrombospondin,denatured collagen

α

3

β

1 fibronectin (RGD),laminin-5,entactin,denatured collagens

α

4

β

1 fibronectin (LEDV),VCAM-1

α

v

β

5 fibronectin (RGD),vitronectin

α

5

β

1 fibronectin (RGD)

α

v

β

6 fibronectin,tenascin

α

6

β

1 laminin

α

7

β

1 laminin

β

2 Integrins Ligands

α

8

β

1 fibronectin,vitronectin

α

M

β

2 ICAM-1,iC3b, fibrinogen, factor X

α

9

β

1 tenascin

α

L

β

2 ICAM-1, 2, and 3

Other ECM Integrins Ligands

α

X

β

2 iC3b, fibrinogen

α

IIb

β

3 Same as

α

v

β

3

α

6

β

4 laminin

Page 3: Fibrin and Wound Healing

357CLARK: FIBRIN AND WOUND HEALING

overexpress plasminogen activator inhibitor accumulate significantly more collagenin their lungs after bleomycin injury than their normal littermates.

30

In addition,inadequate removal of fibrin may impede the normal wound healing processes. Forexample, transgenic mice that have the no plasminogen demonstrate a marked delayin cutaneous wound repair.

31

Interestingly, crossbreeding these mice with transgenicafibrinogenemic mice abrogates the delay in wound healing. Interestingly, afibrino-genemic mice do not have impaired wound healing as long as adequate hemostasisis maintained. This study strongly suggests that the major role of fibrin in wounds ishemostasis. However, once present in the wound, complex interactions must occurbetween fibrin and migrating leukocytes and tissue cells.

NEUTROPHILS

Neutrophils and monocytes begin to emigrate into injured tissue concurrently, butneutrophils arrive first in great numbers partly due to their abundance in the circula-tion. A variety of chemotactic factors attract both cell types to the site of injury.

32,33

General leukocyte chemoattractants include fibrinopeptides cleaved from fibrinogenby thrombin

34

and fibrin degradation products produced by plasmin degradation offibrin.

35

As well as providing the stimulus for directed migration, chemotactic fac-tors also increase CD11/CD18 expression on the neutrophil surface.

36

These het-erodimeric complexes, in conjunction with

L

-selectin and sialyl Lewis factor

χ

,mediate adherence of neutrophils to blood vessel endothelium and thereby facilitatetransmigration of leukocytes through the endothelium.

37

In addition, CD11c/CD18mediates adhesion of neutrophils to fibrinogen through a domain at the N terminusof the A alpha chain.

38

Neutrophil activation by chemoattractants, including fibrin-ogen degradation products,

39

also stimulates release of neutrophil proteases. Theseenzymes facilitate cell penetration through blood vessel basement membranes aswell as degradation of the fibrin clot.

40–42

Neutrophils at the wound site destroy con-taminating bacteria via phagocytosis coupled with toxic oxygen radical generationand enzyme digestion.

43–45

One report

46

has demonstrated that these phagocytic anddigestive processes can be modulated by the fibrinogen degradation products Dand E.

MONOCYTES

Whether neutrophil infiltrates resolve or persist, monocyte accumulation contin-ues, stimulated by selective monocyte chemoattractants, such as fragments of col-lagen,

47

elastin,

48

fibronectin,

49

enzymatically active thrombin,

50

TGF-

β

,

51

andfibrin(ogen) degradation peptides.

46

Similar to neutrophil recruitment, chemoattrac-tants stimulate circulating monocytes to attach to the endothelium of blood vesselsat the site of injury and to migrate through the blood vessel wall into the extracellularmatrix.

52

Physicochemical properties of the three-dimensional fibrin matrix, as wellas the presence of chemoattractants and fibronectin, control macrophage invasion ofthe wound clot.

8,9

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358 ANNALS NEW YORK ACADEMY OF SCIENCES

Binding of monocytes or macrophages to specific extracellular matrix proteinsthrough integrin receptors stimulates extracellular matrix phagocytosis, and Fc- andC3b-mediated phagocytosis.

17

Fibrin interaction with the monocyte/macrophagescan modulate these processes through the integrin receptor Mac-1 (CD11b/CD18;CR3).

53,54

Cultured macrophages, and presumably wound macrophages, releaseenzymes, such as collagenase, elastase, and plasminogen activator.

55

Fibrin frag-ment D-dimer has been reported to induce the secretion of interleukin-1, urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in a humanpromonocytic leukemia cell line, and may do so in human monocytes.

56

These pro-teases facilitate tissue debridement including the removal of fibrin itself. In fact,Mac-1 (CD11b/CD18) and the urokinase receptor (CD87) may form a functionalunit on monocytic cells to digest fibrin(ogen).

57

As well as promoting phagocytosis and debridement, adherence to extracellularmatrix molecules also stimulates monocytes to undergo metamorphosis into inflam-matory or reparative macrophages. Adherence induces selective mRNA expressionof colony stimulating factor-1, a cytokine necessary for monocyte/macrophage sur-vival; tumor necrosis factor-

α

(TNF-

α

), a potent inflammatory cytokine; and PDGF,a potent chemoattractant and mitogen for fibroblasts; as well as c-fos and c-jun,transactivating factors necessary for many activation signals.

58,59

mRNAs for otherimportant macrophage cytokines are adherence-independent; for example, TGF-

β

isconstitutively expressed; interleukin-l (IL-1) mRNA is stimulated by bacterialendotoxin; and HLA-DR is stimulated by gamma-interferon (

γ

-IFN).

58

Thus, mac-rophages appear to play a pivotal role in the transition between wound inflammationand repair

60

and fibrin(ogen) can modulate macrophage activity and, thereby, therate of this transition.

EPITHELIALIZATION

Reepithelialization of a wound begins within hours after injury. It is clear thatrapid reestablishment of any epithelial barrier decreases victim morbidity and mor-tality. Epithelial cells from residual epithelial structures move quickly to dissect clotand damaged stroma from the wound space and repave the surface of viable tissue.The epithelial cells at the wound edge loose their apical-basal polarity and extendpseudopodia from their free baso-lateral sides into the wound.

If the epidermal basement membrane is damaged, provisional matrix, composedof fibronectin,

61

tenasin,

62

vitronectin,

63

and fibrinogen,

64

accumulates among stro-mal type I collagen bundles beneath the epidermis at the wound margin.

65

In contrastto normal epidermal cells, wound keratinocytes express integrin receptors forfibronectin, tenasin, and vitronectin,

66–69

which allows them to interact with thesesubstrates. In addition,

α

2b1 type 1 collagen receptors, that are normally disposedalong the lateral sides of basal keratinocytes, redistribute to the basal membrane ofwound keratinocytes that have come in contact with dermal type 1 collagen fibers.

In vitro

experiments suggest that these receptors are required for keratinocyte migra-tion on type 1 collagen.

70

The migrating wound epidermis does not simply transit over a wound surfacecoated with provisional matrix but rather dissects through the wound, separating

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359CLARK: FIBRIN AND WOUND HEALING

desiccated eschar from viable tissue.

61

The path of dissection appears to be deter-mined by the array of integrins that the migrating epidermal cells express on theircell membranes, as already described. Importantly,

α

v

β

3, the receptor for fibrino-gen/fibrin

16

and denatured collagen,

71

is not expressed on keratinocytes

in vitro

or

in vivo.

73

Hence, keratinocytes lack the capacity to interact with these matrix pro-teins.

74

Furthermore, epidermal cells fail to bind fibronectin in the presence of fibrin,but can bind in the presence of fibrinogen, whereas neither fibrin nor fibrinogeninhibit cell interaction with type 1 collagen.

74

Since the fibrinogen beneath the epi-dermis at the wound margin is not clotted,

64

the migrating wound epidermis avoidsthe fibrin/fibronectin-rich clot, migrating over a matrix composed of fibrinogen,fibronectin, and type 1 collagen.

Extracellular matrix degradation is clearly required for the dissection of migrat-ing wound epidermis between the collagenous dermis the fibrin eschar

31

and proba-bly depends on epidermal cell production of both collagenase

70,75 and plasminogenactivator.76 Plasminogen activator activates collagenase as well as plasminogen77

and, therefore, facilitates the degradation of interstitial collagen as well as the fibrineschar. These proteases would enzymatically digest the extracellular matrix in theplane of epidermal migration. Epidermal migration and dissection between viableand nonviable tissue ultimately results in sloughing the eschar.

As reepithelialization ensues, basement membrane proteins reappear in a veryordered sequence from the margin of the wound inward in a zipper-like fashion.61

Epidermal cells revert to their normal phenotype, once again firmly attaching to rees-tablished basement membrane.

GRANULATION TISSUE

New stroma, often called granulation tissue, begins to invade the wound spaceapproximately four days after injury. The name granulation tissue derives from thegranular appearance of newly forming tissue when it is incised and visually exam-ined. Numerous new capillaries endow the neostroma with its granular appearance.Macrophages, fibroblasts and blood vessels move into the wound space as a unit78

that correlates well with the proposed biologic interdependence of these cells duringtissue repair. The macrophages provide a continuing source of growth factors neces-sary to stimulate fibroplasia and angiogenesis, fibroblasts construct newextracellular matrix necessary to support cell ingrowth, and blood vessels carry oxy-gen and nutrients necessary to sustain cell metabolism.

Fibroplasia

Fibroblasts and the extracellular matrix that they synthesize are collectivelyknown as fibroplasia. Growth factors, particularly PDGF79 and TGF-β,80 in concertwith the clot matrix proteins fibrin and fibronectin,81–83 presumably stimulate fibro-blasts of the periwound tissue to proliferate, express appropriate integrin receptorsand migrate into the wound space.

The early wound extracellular matrix was coined provisional matrix.61 It is ini-tially composed of plasma derived fibrin, fibronectin, and vitronectin and later com-posed of in situ produced hyaluronan and fibronectin. These provisional matrix

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360 ANNALS NEW YORK ACADEMY OF SCIENCES

constituents contribute to tissue formation by providing a scaffold or conduit for cellmigration (fibronectin),12 low impedance for cell invasion (hyaluronic acid),84 a res-ervoir for cytokines (fibrinogen),85 and direct signals to the cells through integrinreceptors.20

Fibroblasts presumably require fibronectin for movement through the wound clotas they do for migration through fibrin matrices in vitro.12 Nevertheless, fibroblastscan interact directly with fibrin and vitronectin at Arg-Gly-Asp-Ser (RGDS) sites viathe integrin receptors α3β1, α5β1, αvβ1, αvβ3, and αvβ5. The RGD-dependent,fibronectin receptors α3β1 and α5β1 are upregulated on periwound fibroblasts theday prior to moving into the wound clot and on early granulation tissue fibroblasts.83

In contrast, α1β1 and α2β1 collagen receptors on these fibroblasts were either sup-pressed or did not appear to change appreciably.83,86 Thus, periwound fibroblastsspecifically upregulate integrins that can interaction with the provisional matrix justprior to their migration into the wound space.

Interestingly, fibrin and fibronectin begin to appear in the periwound environmenttwo days after wounding (Greiling and Clark, unpublished observations). In thepresence of PDGF these provisional matrix proteins are known to support anincrease expression of their integrin receptors 24 hours later.83 Thus, the appearanceof provisional matrix proteins in the periwound stroma at day two may be causallyrelated to the appearance of provisional matrix integrins on the fibroblasts at daythree. Together, we believe that these phenomena are rate limiting for the com-mencement of granulation tissue formation.12,13,83,86,87

Fibroblast movement into a crosslinked fibrin blood clot or any tightly wovenextracellular matrix may necessitate an active proteolytic system that can cleave apath for migration. A variety of fibroblast derived enzymes in conjunction withserum derived plasmin are potential candidates for this task, including plasminogenactivator, interstitial collagenase-1 and -3 (MMP-1 and MMP-13, respectively), the72-kDa gelatinase A (MMP-2), and stromelysin (MMP-3).77,88 In vitro both plasmi-nogen activator12,89 and collagenase activity (Greiling and Clark, unpublishedobservations) are required for fibroblast movement from a collagen matrix into fibringel.

Once the fibroblasts have migrated into the wound they gradually switch theirmajor function to collagen production.90 After an abundant collagen matrix is depos-ited in the wound, fibroblasts cease collagen production. The process of fibroplasiacreation from a fibrin clot context has been modeled in vitro.91

NEOVASCULARIZATION

Fibroplasia would halt if neovascularization failed to accompany the newly form-ing complex of fibroblasts and extracellular matrix. The process of new blood vesselformation is called angiogenesis.92 Angiogenesis is a complex process that relies onan appropriate extracellular matrix in the wound bed as well as endothelial cell phe-notype alteration, stimulated migration and mitogenic stimulation of endothelialcells.92

Soluble factors that may be responsible for wound angiogenesis include basicfibroblast growth factor (bFGF),93 vascular endothelial growth factor (VEGF),94

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361CLARK: FIBRIN AND WOUND HEALING

angiopoietin,95 PDGF,96 and many other.97 Several isoforms of VEGF98 andangiopoietins99 have been identified that effect endothelial cell growth and angio-genesis differentially.

Besides growth factors and chemotactic factors, an appropriate extracellularmatrix is also necessary for angiogenesis. Fibrin has been reported to induce angio-genesis directly.100–102 If fibrin itself is sufficient to induce angiogenesis, it mustperform at least two functions: (1) provide a three-dimensional matrix that supportscell migration, and (2) express selective chemotactic and/or chemokinetic activitysuch that endothelial cells migrate into fibrin clot. Given that bFGF and PDGF canbind to fibrin with a fairly high affinity85 (Galanakis and Clark, unpublished obser-vations), it is quite possible that one or more angiogenic (chemotactic) factors werepresent in the fibrin preparations previously found to induce angiogenesis directly.Recently we have developed an in vitro angiogenesis assay using human dermalmicrovascular endothelial cells that are cultured on microcarrier beads and suspend-ed in an extracellular matrix. Our assay was derived from an assay previouslydescribed by Nehls and Drenckhahn.103 When purified human fibrin is used as theextracellular matrix no angiogenesis occurs. However, either VEGF, VEGF-C, orbFGF added to this assay stimulant angiogenesis in a dose dependent manner (Feng,Clark, Galanakis, and Tonnesen, unpublished observations). Using a similar micro-carrier based angiogenesis assay, Nehls and Herrmann demonstrated that fibrinstructure plays an important role in bovine pulmonary artery endothelial cell migra-tion and capillary morphogenesis. They showed that the degree of rigidity of fibringel strongly influences tube formation by bovine endothelial cells in response tobFGF or VEGF.104

In support of the finding that fibrin can support growth factor stimulated angio-genesis, angiogenesis in the chick chorioallantoic membrane is dependent on theexpression of the αvβ3 integrin that recognizes fibrin as well as fibronectin and vit-ronectin.105 Furthermore, in porcine cutaneous wounds αvβ3 is only expressed oncapillary sprouts as they invade the fibrin clot.13 In vitro studies in fact demonstratethat αvβ3 can promote endothelial cell migration on provisional matrix proteins.106

Tight regulation of αvβ3 by fibrin matrix is supported by the finding that fibrin, butnot collagen, gels induce αvβ3 on cultured human microvascular endothelial cells.23

Thus, endothelial cell expression of provisional matrix integrins is regulated by theprovisional matrix proteins in a positive feedback fashion in a manner similar to thepositive feedback between integrins and their ligands observed in fibroblasts.22

Within a day or two after removal of angiogenic stimuli, capillaries undergoregression as characterized by mitochondria swelling in endothelial cells, plateletadherence to the blood vessel wall, vascular stasis, and endothelial cell apoptosisand ingestion of by macrophages. Although αvβ3 has been shown to regulate apop-tosis of endothelial cells in culture and in tumors,107 αvβ3 is not present on woundendothelial cells as they undergo programmed cell death, indicating another path-way of apoptosis in healing wound blood vessels (Feng, Clark, and Tonnesen,unpublished observations). Thrombospondin appears to be a good candidate for thisphenomenon.108

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362 ANNALS NEW YORK ACADEMY OF SCIENCES

SUMMARY

Although hemostasis is the major role of fibrin in wound repair,31 once the clotis present the wound cells must deal with it. The invasion and clearing of fibrin bythese cells involves multiple complex processes that may go array XXX and delaywound repair. A good example, of the latter is leg ulcers. These chronic wounds con-tain a plethora of proteases that digest fibronectin and growth factors in the fibrinclot109,110 resulting in a corrupt provisional matrix that no longer supports reepithe-lialization or granulation tissue formation. Every good wound care provider knowsthat these wounds will not heal unless the corrupt matrix is removed by vigorousdebridement that stimulates the accumulation of a competent provisional matrix.

ACKNOWLEDGMENTS

The original data reported in this paper was supported by NIH grants AG 101143-14 and AR 42987-05 from the National Institute of Aging and the National Instituteof Arthritis, Musculoskeletal and Skin Disease, respectively.

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