hepatocyte growth factor/scatter factor in the eye

Hepatocyte Growth Factor/Scatter Factor in the Eye

Ian Grierson*, Lisa Heathcote, Paul Hiscott, Penny Hogg, Mike Briggsand Suzanne Hagan

Unit of Ophthalmology, Department of Medicine, University of Liverpool, Duncan Building,Liverpool, L69 3GA, UK

CONTENTS

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780

2. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780

2.1. The factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780

2.2. Synthesis and activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782

2.3. The receptor(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784

2.4. Biological activities and its possible in vivo role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

2.4.1. Multifunctional role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

2.4.2. Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

2.4.3. Maintenance, injury and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

2.4.4. Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

3. The front of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789

3.1. The cornea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789

3.2. Aqueous humour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790

3.3. The out¯ow system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790

3.4. The lens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792

4. The back of the eye. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793

4.1. Vitreous humour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793

4.2. The neural retina and pigment epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794

4.3. Retinal detachment and the proliferative retinopathies . . . . . . . . . . . . . . . . . . . . . . . . . . 795

4.4. Uveal melanoma and ocular tumours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

5. Future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

AbstractÐHepatocyte growth factor, also known as scatter factor (HGF/SF) is a multipotential cytokine which can pro-duce a range of responses in target cells and its in¯uence in the eye in health and disease is just beginning to be appreci-ated. Usually HGF/SF is synthesised by mesenchymally derived cells and targets and signals epithelial cells in a paracrinemanner via their c-Met surface receptor. However, there is growing evidence for the existence of autocrine loops in a num-ber of cell systems prominent among which are ocular cells such as the corneal endothelium, the lens epithelium, the reti-nal pigment epithelium (RPE) and others. Marked cellular proliferation is stimulated when activated HGF/SF is exposedto hepatocytes, renal epithelium, melanocytes and vascular endothelial cells but it is often a poor mitogen for other celltypes. In target cells the cytokine promotes other bioactions such as junctional breakdown, shape change, cell scattering,

Progress in Retinal and Eye Research Vol. 19, No. 6, pp. 779 to 802, 20007 2000 Published by Elsevier Science LtdPrinted in Great Britain1350-9462/00/$ - see front matter

PII: S1350-9462(00)00015-X

*Corresponding author.

directional and nondirectional migration, cell survival, invasive behaviour and/or tubule formation. These activities seemto depend on HGF/SF linking with the c-Met receptor and pathways to stimulate the various types of cytokine/receptorresponse are being unravelled at the present time. In corneal wound healing, HGF/SF is produced by stromal keratocytesand targets the repairing epithelium. HGF/SF is a constituent of tears, aqueous humour and vitreous humour at levelsabove that found in plasma although it is not clear how much is activated. Aqueous HGF/SF may well in¯uence lens epi-thelial, corneal endothelial and trabecular meshwork cell survival. Vitreous levels of HGF/SF are elevated in proliferativevitreoretinopathy (PVR), where a target cell is the RPE and in proliferative diabetic retinopathy (PDR) where HGF/SFhas been shown to be a major angiogenesis factor. Finally HGF/SF may be involved in the metastatic spread of tumourcells from uveal melanomata and in the formation of vascular channels in these tumours. 7 2000 Published by ElsevierScience Ltd

1. INTRODUCTION

We know that cytokines in general, and growthfactors in particular, have a far wider range ofbioactivity than simply causing target cells toproliferate. Settlement, survival, adhesion, syn-thesis, motility, invasiveness, cytostasis, inductionof tubule formation and phenotypic alterationare among the many behavioural features whichare modulated by growth factors and other cyto-kines. One growth factor which is better knownfor its motility and shape-change stimulatingfunctions than for its proliferative action is hep-atocyte growth factor or scatter factor (HGF/SF). HGF/SF has been recognized for some time

as a factor of considerable importance to cell andtissue function and is now beginning to be ap-preciated for its role within the eye in health anddisease.

2. BACKGROUND

2.1. The factor

Stoker and Perryman (1985) found that cul-tured 3T3 ®broblasts secreted a substance thathad a marked e�ect on Madin-Darby canine kid-ney (MDCK) epithelium. MDCK cells formcompact colonies after plating in tissue culture

Fig. 1. Phase contrast micrographs of MDCK cells in the absence (A) or in the presence (B) of HGF/SF (�250).

780 I. Grierson et al.

and the ®broblast-derived factor has the intri-guing e�ect of causing the tightly packed coloniesto break up by inducing each individual cell tospread out (Fig. 1). The ®broblast-derived sub-stance was called scatter factor and subsequentlyit was shown to be secreted by a wide range ofmesenchymal cells and, in turn, produced scatter-ing in an equally wide rage of normal and trans-formed epithelia (Stoker et al., 1987; Rosen etal., 1994a,b). Scatter factor, when puri®ed frommedium conditioned by cultured human ®bro-blasts, is seen to be a heparin-binding 92 kD gly-coprotein which, by proteolysis, could beconverted into a heavier (termed a and about 62kD) and lighter (termed b and between 32 and 34kD) subunit. The subunits are linked together bya disulphide bond (Weidner et al., 1990) (Fig. 2).

Furthermore, scatter factor exhibits closesequence similarities to plasminogen and agrowth factor known as hepatocyte growth fac-tor. Hepatocyte growth factor was ®rst discov-ered in 1984 (Nakamura et al., 1984; Russell et

al., 1984), isolated from rat platelets (Russell etal., 1984) and later it was extracted from normalhuman plasma (Zarnegar and Michalopoulos,1989). It is also a 90 kD protein which can becleaved into approximately 60 and 30 kD disul-phide-linked subunits by proteolytic action(Nakamura et al., 1989). The cytokine not onlystimulates hepatocytes to proliferate but also hasa mitogenic e�ect on a wide range of epithelial,endothelial and melanocytic cells (Kan et al.,1991; Matsumoto et al., 1991a,b; Tamagnoneand Comoglio, 1997). In common with scatterfactor, hepatocyte growth factor has been shownto evoke numerous biological e�ects in targetcells including the loss of junctional communi-cation (Ikejima et al., 1995; Moorbey et al.,1995) and dissociation of epithelial sheets (Nal-dini et al., 1991a); properties not at all dissimilarto colony scattering. Moreover, both peptidescause individual epithelial cells to adopt a ®bro-blast-like phenotype: an alteration of consider-able importance to those who investigate``epithelial to mesenchymal transitions'' (seelater).

Scatter factor, hepatocyte growth factor andanother cytokine called macrophage stimulatingprotein are grouped together in the same familywith the structural similarity of having a 4-krin-gle domain and a serine protease-like domainthat is devoid of enzymic activity (Strain, 1993)(Fig. 2). It is the case that hepatocyte growth fac-tor and scatter factor are to all intents and pur-poses one in the same multifunctionalpolypeptide (Furlong et al., 1991; Bhargava etal., 1992; Warn, 1995). As a result, the nameshepatocyte growth factor and scatter factor areused interchangeably or they are denoted in com-bination as HGF/SF.

The HGF/SF gene has been localised tochromosome 5 in the mouse (Weidner et al.,1991) but it is on chromosome 7 in the human(Fukuayama, 1991). The human gene spansaround 70 kilobases, it is located at 7q21.1 and itis composed of 18 exons and 17 introns. Asmight be expected from the structural homologyin amino acid sequences produced by the twogenes, the HGF/SF gene and the plasminogengene have many features in common. The proteinproducts of the two genes, although remarkably

Fig. 2. Diagrams of (A) the inactive single chain form of(pro) HGF/SF and (B) the activated heterodimer. The 4-kringle domain is on the a chain whereas the serine pro-

tease-like domain is associated with the b chain.

Hepatocyte growth factor in the eye 781

similar, di�er in that plasminogen is an e�ectiveprotease whereas HGF/SF has no proteolytic ac-tivity although it retains the proteolytic acti-vation mechanism of proteases (Mars et al.,1993; Miyazawa et al., 1993: Trusolino et al.,1998a) which is necessary for its biological action(see later).

2.2. Synthesis and activation

Typically ®broblasts are considered to syn-thesise and secrete HGF/SF but they are farfrom being the only source; it would seem thatthe cells which produce this cytokine belong to afar broader church. In addition to ®broblasts,other cells that produce HGF/SF include forexample vascular smooth muscle cells, endo-thelium, glial cells, macrophages, activated Tlymphocytes and various tumour cells (Stoker etal., 1987; Rosen et al., 1994a). The list continues

to increase but essentially the cells which syn-thesise HGF/SF are of mesenchymal origin.

Producer cells release HGF/SF as an inactivesingle chain precursor (Nakamura et al., 1989;Tashiro et al., 1990). The chain consists of sixdomainsÐthe N domain (similar to the acti-vation peptide of plasminogen), four kringledomains and an inactive serine proteinasedomain (Fig. 2) (Chirgadze et al., 1998). In theextracellular environment of normal adult tissuethe dormant precursor predominates until HGF/SF is activated to a heterodimer by stimuli suchas injury and in¯ammation (Miyazawa et al.,1994) (Figs 2B and 3).

Understanding the regulation of the pro-duction of HGF/SF is an important but rela-tively recent area of research and as yet only afew of the players (promoters and inhibitors) areknown. Tumour necrosis factor alpha and inter-leukins such as IL-1 have been shown to increase

Fig. 3. Classically HGF/SF is produced by mesenchymal cells and there is a pool of inactive or pro HGF/SF within theextracellular matrix (ECM) of a tissue. The pro HGF/SF can be activated by, for example, serum fluids or tissue macro-phages prior to linking with the high affinity c-Met receptors of the target cells. Production of HGF/SF, activation ofHGF/SF and control of receptor density on the target cells are the three principal means of regulating of the cytokine.


the secretion of HGF/SF by cultured ®broblasts(Tamura et al., 1993). On the other side of thecoin, transforming growth factor beta (TGFb)has a clear dose dependent inhibitory action on®broblast production of HGF/SF (Gohda et al.,1992). A variety of substances in the extracellularenvironment are thought to have a retaininge�ect on secreted HGF/SF and act as a reservoirfor this growth factor. Among the extracellularmatrix components which bind to HGF/SF theones with greatest a�nity are thrombospondin,®bronectin and heparin sulphate (Lamaszus etal., 1996), although others like collagen, laminin,tenascin, vitronectin and chondroitin sulphate arereasonably e�ective.

A variety of mechanisms are involved in HGF/SF activation, most of which (particularly thosewhich operate in vivo) are not well understood atpresent. It is known, however, that proteolyticprocessing is an essential step for the conversionfrom the single chain form to the bioactive het-erodimer (Nakamura et al., 1989; Naka et al.,1992; Miyazawa et al., 1994) (Fig. 2). The pro-teolytic cleavage at an argenine-valine bond andestablishes a heterodimer disulphide linkage.``Hepatocyte growth factor converting enzyme''is a serine proteinase and is one of several serumfactors isolated from bovine serum that can acti-vate HGF/SF (Mizuno et al., 1994). The protein-ase itself needs to be activated before it becomesan e�ective converting enzyme. Another, onlyslightly less e�ective enzyme, has been called``hepatocyte growth factor activator'' and is hom-ologous with blood coagulation factor XIIa (Shi-momura et al., 1995). Again an activationprocess is needed for the converting enzyme tobecome functional and this activation is throm-bin dependent (Shimomura et al., 1995). Ad-ditional members of the coagulation system,including kallikrein and factor XIa, have someconversion activity at least in vitro (Wang et al.,1994).

Since part of the HGF/SF molecule is plasmi-nogen-like, it is a reasonable assumption thatsubstances like urokinase and tissue plasminogenactivators would be e�ective at activating thiscytokine. It turns out that both types of plasmi-nogen activators successfully cleave the singlechain to the heterodimer (Mars et al., 1993). The

reaction in vitro however is not very e�cient andsome researchers are not convinced that plasmi-nogen activators have any role to play in vivo.Such a view probably, on the balance of avail-able evidence, is too dismissive.

A reasonable alternative explanation is that thee�ciency of the HGF/SF conversion relates tothe nature of plasminogen activation. In an enzy-mic reaction all the substrate is converted givenenough time but the plasminogen activation ofsingle chain HGF/SF is a stoichiometric reactionwhere activation stops because of the formationof stable complexes. The reaction therefore islimited by the availability of free plasminogenactivator (Naldini et al., 1995); these authorsargue the case that monocytes, producing largeamounts of urokinase-type plasminogen activa-tor, would be one e�ective mechanism for the ac-tivation of HGF/SF in in¯amed tissue.

It would seem to be the case for normal tissueand ¯uids that, if they have any HGF/SF, theytend to contain predominantly the inactive formwhereas when there is tissue injury and the pre-sence of serine proteases, the active form beginsto predominate. Thus, as is the case with severalother cytokines including TGFb, measuring theamount of HGF/SF in a particular environmentis only functionally meaningful if the ratio ofactive to inactive growth factor also is known. Atleast three broad categories of modulation ofHGF/SF activity seem to exist: (1) regulation ofthe production of inactive HGF/SF from produ-

Fig. 4. Immunofluorescent staining of MDCK cells toshow the distribution of the c-Met receptor on these epi-

thelial cells (�350).


cer cells; (2) control of the activation of the cyto-kine in the extracellular environment; and (3) thenature and the regulation of receptors on the tar-get cells (Fig. 3; see Section 2.3).

2.3. The receptor(s)

A particular feature of target cells is that theyexpress a high a�nity receptor for HGF/SF intheir plasma membranes. Low a�nity receptorsalso can be found on the cell surface and arebelieved to be heparin sulphate proteoglycans(Trusolino et al., 1998a,b). The high a�nity SF/HGF receptor has been shown to be a proto-oncogene product called c-Met (Bottaro et al.,1991; Naldini et al., 1991a,b) found predomi-nately, but certainly not exclusively, on epithelialcells (Di Renzo et al., 1991; Prat et al., 1991)(Fig. 4). The gene for the receptor has beenshown to be located on a one thousand kilobaseregion of chromosome 7q31 (Lin et al., 1996).

The list of cell types expressing the c-Met recep-tor has grown steadily to include vascular endo-thelium, melanocytes and others but still excludesall varieties of ®broblast. Essentially the cellsexpressing the c-Met receptor are ectoderm,endoderm and, of considerable interest withrespect to the eye, neural crest-derived cells.

The receptor is a transmembrane glycoproteinof 190 kD which can be cleaved into an a chainof 50 kD and a b chain of 145 kD (Fig. 5). Thea chain and the NH2 terminal section of the bchain are exposed on the cell surface whereas theCOOH terminal part of the b chain is on thecytoplasmic side of the membrane and contains atyrosine kinase domain (Giordano et al., 1989;Comoglio and Vigna, 1995) (Fig. 6). As a resultthe c-Met receptor is classi®ed as belonging to abranch of the receptor protein tyrosine kinasefamily with two other close relatives called c-Seaand RON (Van der Geer et al., 1994). Althoughstructurally very similar to c-Met, neither c-Seaor RON have HGF/SF as their ligand.

The ligand binds with the extracellulardomains of c-Met and produces a conformationalchange in these domains. As with other growthfactor/receptor interactions of this type, the con-formational change probably acts as a signal forautophosphorylation events in the cytoplasmicdomain of the b chain including the tyrosinekinase site (Bottaro et al., 1991; Naldini et al.,1991a,b; Mark et al., 1992; Heldin and Ostman,1996). Ultimately the conformational changeinduced by ligand/receptor binding, results in theappropriate signals to promote proliferation, dis-sociation, shape change, motility, invasiveness,tube formation and a host of other HGF/SF cel-lular e�ects produced in the target cell (Komadaand Kitamura, 1993; Sachs et al., 1996).

It is hardly surprising that at one time it wasconsidered likely that HGF/SF must have severalhigh a�nity receptors to account for the bewil-dering array of di�erent bioactivities evoked bythis cytokine in its target cells. We know this notto be the case (among others, Weidner et al.,1993) but it leaves the question how does c-Metcope as the lone receptor type? Given that thereare multiple contact points between the ligandand receptor, and there is some evidence to thate�ect (Lokker et al., 1994), then this may have

Fig. 5. Cultured and confluent bovine RPE cells werelysed and total proteins extracted. The proteins wereresolved by 7.5% sodium dodecyl sulphate polyacryl-amide gel electrophoresis and then subjected to westernblotting with an anti c-met antibody. Immunoreactionwas at 145 kD (large arrow). This band coincides withthe molecular weight of the large transmembrane bchain of the receptor. Other arrows indicate molecular

weight markers.


an in¯uence on the activation of the appropriatesignal pathway route. On the other hand autop-hosphorylation creates a series of docking sitesfor signalling molecules which in turn facilitatesmultiple intracellular signalling pathways (Pon-zetto et al., 1994; Trusolino et al., 1998a).

The signal transduction mechanism of c-Met isnot entirely clear but some characteristic featuresare beginning to be appreciated. Key phosphoryl-ation sites on the cytoplasmic component of theb chain are outlined in the diagram (Fig. 6). It isthe case that the phosphorylation of two tyro-sines in the kinase catalytic domain stimulateskinase activity (Longati et al., 1994) whereasphosphorylation of a serine in the juxtamembra-nous segment has the opposite e�ect (Gandino etal., 1994). A most important phosphorylation siteis where the tyrosines numbered 1349 and 1356are located which is towards the cytoplasmic tailend of the b chain (Fig. 6).

The tyrosine 1349,1356 region acts as a dock-

ing site for various signal transduction and adap-tation systems (Ponzetto et al., 1994). HGF/SFinduced proliferative but not motile bioactivity isinhibited when the tyrosine 1356 is negated (Pon-zetto et al., 1994). Pathways involving Ras andP13 K (phosphatidylinositol 3 kinase) are stimu-lated from the tyrosine 1349,1356 region and thepathways seem to ®gure prominently in HGF/SFinduced stimulation of proliferation, motility as-sociated alterations and increased invasiveness(Ponzetto et al., 1994; Weidner et al., 1996). Agrowth factor receptor bound protein (GRB2) isan adaptor which links the site with the Ras andtriggers the MAP (mitogen activated protein)kinase cascade but also GRB2 links to anotherpathway involving Gab-1 (Weidner et al., 1996;Trusolino et al., 1998a). The Gab-1 pathway isone which is thought to mediate the HGF/SFshape change and morphogenic response (Weid-ner et al., 1996). These and other pathways aresubject to considerable current investigation but

Fig. 6. The c-Met receptor consists of a large transmembrane b chain linked to a small extracellular a chain. The variedbioresponses depend on phosphorylation events (P) at key sites within the cytoplasmic domain of the b chain including

the kinase domain and the 1349, 1356 docking region. Adapted from Trusolino et al. (1998a).


Table 1. In vitro e�ects produced by HGF/SF on target cells

Phenomenon Typical target cells Bioassay e�ect Selected authors

Cell±cell communication Keratinocyte and hepatocyte cells Passage of intracellular Lucifer Yellowthrough gap junctions is suppressed

Moorbey et al. (1995) and Ikejima etal. (1995)

Alteration to adhesionmolecules

Colon carcinoma cells Time-dependent reduction inimmunostaining of e.g. a-6 integrin, b-catenin and E-cadherin

Herrera (1998)

Adhesion to substrate Germinal center B, hematopoieticprogenitor, thyroid and papillarycarcinoma cells

Poor attachment to a ®bronectinsubstrate

van der Voort et al. (1997), Weimar etal. (1998) and Trusolino et al. (1998b)

Junctional breakdown Hepatocytes and MDCK cells Immunohistochemistry shows loss of gapjunctions and adhering junctions

Potempa and Ridley (1998)

Shape change MDCK, PtK2 kidney cells Cells exhibited increased ru�ingbecoming ®broblastic and elongatedbased on time-lapse and electronmicroscopy

Dowrick et al. (1993)

Scattering MDCK, cervical keratinocytes,mammary epithelial cells

Spreading of cells away from tightlypacked colonies

Stoker and Perryman (1985), Stoker etal. (1987) and Rosen et al. (1990)

Cytoskeletal change Hepatocyte cells Immunoblotting, phase contrastmicroscopy and immunolocalizationstudies show pronounced alteration incytoskeletal components

Pagan et al. (1997) and Dugina et al.(1995)

Migration MDCK and intestinal epithelium cells Chemoattraction assays using a Boydenchamber show increased directionalmovement of target cells

Stoker (1989) and Polk and Tong(1999)

Invasion MDCK, lung and pancreatic carcinomacells

Increases penetration of cells into acollagen matrix

Weidner et al. (1990), Weimar et al.,(1997)

Tubule formation MDCK, mammary gland epithelium Formation of branched duct-likestructures by cells within a collagenmatrix

Montesano et al. (1991) and Soriano etal. (1995)

Mitogenesis Keratinocytes, hepatocytes, mammaryepithelium and vascular endotheliumcells

Increased proliferation based oncounting cell numbers on tritiatedthymidine incorporation

Russell et al. (1984), Furlong et al.(1991), Kan et al. (1991) andTamagnone and Comoglio (1997)

Anti-apoptosis MLP-29 cells Staurosporin induced apoptosis seen bythe TUNEL reaction was partiallyinhibited

Giordano et al. (2000)

786

I.Grierso

net

al.

much remains to be determined. For that matternot all the HGF/SF bioe�ects require phos-phorylation of the c-Met receptor. The antiapop-totic action of HGF/SF (mediated via Bag)would seem to be phosphorylation independent(Trusolino et al., 1998a).

2.4. Biological activities and its possible in vivo role

2.4.1. Multifunctional role

As a result of HGF/SF's ability to promote awide range of biological activities in its varioustarget cells, the cytokine has been called a pleio-trophic growth factor (Boros and Miller 1995).In addition to scattering of colonies of culturedepithelial cells (Stoker et al., 1987; Rosen et al.,1990) and the promotion of the growth of a widerange of cells including hepatocytes (Kan et al.,1991; Matsumoto et al., 1991a,b; Tamagnoneand Comoglio, 1997), some of the other beha-vioural e�ects promoted by HGF/SF in vitro arelisted in Table 1. The question arises whetherthese various in vitro phenomena translate in anymeaningful way to give a signi®cant biologicalrole for HGF/SF in the body in health and dis-ease? The answer to the question, on the basis ofcurrent research, is a fairly emphatic ``yes''(Warn, 1995)! HGS/SF seems to be involved insuch diverse events as organ development, tissuemaintenance and homeostasis in the adult andwound healing particularly with respect to themodulation of the repair process. Cell survival bymeans of an anti-apoptosis action may be of sig-ni®cance in vivo and the factor may play a cru-cial part in some forms of tumorigenesis.

2.4.2. Embryogenesis

If the c-Met gene is inactivated in the develop-ing mouse then, among other adverse events inthe embryo, the limb bud myoblasts fail todetach from the somite blocks and the myoblastsdo not migrate (Bladt et al., 1995). The limb budmyoblasts normally highly express the c-Metreceptor and it is thought that locally producedHGF/SF is the necessary migration stimulantneeded to mobilise these cells prior to normal

muscle and limb formation. In addition manyorgans such as the liver are grossly undersized inthese c-Met de®cient embryos (Bladt et al., 1995).Presumably the failure can be explained byHGF/SFs marked proliferative e�ect on the epi-thelium of developing organs.

When segments of developing kidney are main-tained in organ culture conditions then kidneyconnective tissue cells will produce HGF/SF andthe uretic bud, whose cells express c-Met, willbranch. On the other hand if neutralising anti-bodies active against HGF/SF are introducedinto the organ culture system then the branchingprocess is constrained (Woolf et al., 1995). HGF/SF and c-Met also are important in the develop-ment of the mammary gland (Tsarfaty et al.,1994). HGF/SF is replete throughout the mam-mary tissue of virgin mice reaching its highestlevels when the mammary ducts are forming anddi�erentiating (Warn, 1995). It seems likely thatthe growth factor is involved in this processbecause mouse mammary gland epithelium, likeseveral other types of epithelium, forms tubulesin the presence of HGF/SF when embeddedwithin a collagen matrix in vitro (Soriano et al.,1995). Not surprisingly, expression of HGF/SFfalls precipitously following pregnancy and lacta-tion (Tsarfaty et al., 1994).

The blossoming literature on the function ofHGF/SF in embryogenesis indicates that thiscytokine plays an important role in tissue andorgan constructional events by means of an arrayof regulatory actions (Boros and Miller, 1995).HGF/SF in¯uences and modulates structuraldevelopment by triggering proliferative events,migratory activity, cell detachment, epithelial tomesenchymal transitions and tubule formation.The signalling is principally (but not exclusively)by a paracrine route.

2.4.3. Maintenance, injury and disease

A role for HGF/SF in the maintenance of nor-mal tissues in the body is suspected because it islocally produced in circumstances where no path-ology is evident (Okajima et al., 1990). Howeverthere is far stronger evidence that the cytokine isinvolved in wound repair processes and upregu-


lated in some forms of disease. The upregulationin various disease processes such as acute renalfailure (Santos et al., 1994), acute lung injury(Yanigita et al., 1993) and fulminant hepatic fail-ure (Tsubouchi et al., 1989) probably relates toan involvement in the regulation of regenerativeevents following organ damage. Further HGF/SF has been shown to have therapeutic possibili-ties in the treatment of peptic ulcers (Takahashiet al., 1995) and in stimulating liver regeneration(Trusolino et al., 1998a).

It appears that HGF/SF and the c-Met recep-tor protein are present in and around bloodvessels (Nakamura, 1991; Boros and Miller,1995) where the cytokine/receptor system isthought by some to be protective against endo-thelial disfunction (Nakamura et al., 1998). Cer-tainly HGF/SF is an extremely e�ectivestimulant for the proliferation of vascular endo-thelial cells (Rosen and Goldberg, 1995) and cir-culating HGF/SF has been shown to be higher inhypertensive than in normotensive people (Naka-mura et al., 1996). Indeed, a positive relationshipexists between the amount of circulating HGF/SF and the maximum systolic blood pressure.Therefore plasma HGF/SF levels might serve asan index of severity of hypertension (Nakamuraet al., 1998).

2.4.4. Tumorigenesis

An enthusiasm to implicate HGF/SF in atleast some forms of tumorigenesis was initiatedby the in vitro work which showed that it stimu-lated the migration and also promoted the inva-siveness of epithelial cells (Table 1). Indeed theassociation the cytokine has with junctionalbreakdown, shape change and cellular scatteringalso supports the supposition that HGF/SFmight be a metastasis-promoting agent (Stoker etal., 1987; Herrera, 1998; Trusolino et al., 1998b).Additional support comes from experimentswhich show that gastric carcinoma cells in cul-ture can be induced to scatter and migrate (Shi-bamoto et al., 1992). That the HGF/SF receptorc-Met is encoded by a proto-oncogene furthersupports a role for HGF/SF in cancer.

A series of investigations have found that

robust HGF/SF expression is associated with anumber of tumours including breast carcinomasand bladder carcinomas (Rosen et al., 1994a;Joseph et al., 1995; Yao et al., 1996). In additionthe c-Met receptor, as well as being present onnormal cells, can be identi®ed in solid tumoursincluding gastric tumours, colorectal carcinoma,ovarian carcinoma, breast carcinoma, leukaemiaand even some sarcomas (Di Renzo et al., 1991;Prat et al., 1991; Tuck et al., 1966; Nagy et al.,1996; Scotlandi et al., 1996).

The co-expression of HGF/SF and c-Metwithin the same breast tumour might be pre-dicted, but there is clear overexpression of bothproteins when malignant are compared withbenign breast lesions (Tuck et al., 1996; Jin et al.,1997). Over and above it does seem that HGF/SF (Yamashita et al., 1994; Yao et al., 1996) andc-Met (Ghoussoub et al., 1998) are closely associ-ated with the most aggressive tumours and theirpresence can be taken as e�ective indicators of apoor prognosis for human breast cancer.

Experimental studies show that culturedhuman breast cancer cells, transfected withHGF/SF cDNA, overexpress HGF/SF and havegreater tumour growth following their injectioninto mice compared to nontransfected cellsinjected into mice injected into (Lamaszus et al.,1997). It appears that, in this situation at least, amajor cause for the HGF/SF promotion oftumour growth is because the cytokine inducesstimulation of angiogenesis in the cancerous tis-sue. The HGF/SF transfected tumours havemore blood vessels that the nontransfected,extracts of the transfected tumours promoteangiogenesis. The angiogenic e�ect can benegated by treating the extract with neutralisingantibodies against HGF/SF (Lamaszus et al.,1997).

With the expansion of research in the area, evi-dence for a role for HGF/SF in tumour for-mation is increasing. None the less we are still along way short of knowing the full signi®cance ofthis cytokine in the pathobiology of a whole hostof eminent cancers. In many systems it remainsof considerable importance to be able to dissectdesirable from undesirable e�ects of this multi-functional growth and motility factor (Trusolinoet al., 1998a).


3. THE FRONT OF THE EYE

3.1. The cornea

Over the years research into bioactive agentswhich are relevant to the cornea has focused ona number of growth factors including EGF,members of the FGF family, TGFb and others(Kitazawa et al., 1990; Schultz et al., 1994;Wilson et al., 1994; Honma et al., 1997; Andre-sen and Ehlers, 1998). Each group in turn hashad its period of intense research but it is only inrecent times that the possible signi®cance ofHGF/SF to corneal welfare has started to be ap-preciated.

Corneal stromal ®broblasts, corneal endo-thelium and even corneal epithelial cells havebeen shown to produce messenger RNA codingfor HGF/SF although the levels expressed by theepithelium appeared to be extremely low (Wilsonet al., 1993). Finding even low levels of HGF/SFmessenger RNA in the corneal epithelium is sur-prising because the presence of HGF/SF messagein any epithelium is unusual (Stoker et al., 1987;Matsumoto et al., 1991a,b; Sonnenberg et al.,1993; Warn, 1995), and this ®nding needs furtherinvestigation and clari®cation. It is a well heldbelief, as mentioned earlier, that HGF/SF is pro-duced exclusively by non-epithelial cells and actsin a paracrine fashion on cells of an epithelialorigin (Gherardi et al., 1989; Sonnenberg et al.,1993) (Fig. 3). Overall however, the corneal ®nd-ings to date are consistent with stromal ®bro-blasts being a principal source of HGF/SF andthe corneal epithelium being the main target.

Cultures of corneal epithelium and endo-thelium are stimulated to proliferate by levels ofHGF/SF as low as 1 ng/ml but this mitogenice�ect is lost when concentrations reach 25 ng/ml(Wilson et al., 1993). Corneal stromal ®broblastson the other hand, as might be expected, areuna�ected by the presence of HGF/SF. The fac-tor in corneal epithelium at least, activates theRas-MAP kinase signal transduction pathway(presumably via the tyrosine 1349,1356 dockingregion of the a chain of c-Met) but the activationis rapid and transient. Receptor-ligand inter-action seems to initiate at least two pathways(Liang et al., 1998), one involves the receptor-

Grb2/Sos complex to Ras and the other is viaprotein kinase C. It should be borne in mind,however, that because of the multifunctionalnature of HGF/SF that, as with other epithelia(see earlier), a multiplicity of pathways may beinvolved depending on the circumstances.

It would seem likely that HGF/SF has somerole to play in corneal development and even themaintenance of normal structure in the adult cor-nea. As a result the possible capacity the cytokinehas in for example the repair of epithelial ero-sions, along with EGF and keratinocyte growthfactor (KGF) (Kitazawa et al., 1990; Wilson etal., 1994; Honma et al., 1997), is worthy of con-sideration. In addition, research into HGF/SF asa promotor of graft survival in vitro and in vivois not without some merit. Certainly, in cornealwound healing, HGF/SF may well be one of thekey players in stromal ®broblast modulation ofepithelial and endothelial behaviour during theirrepair activities since the expression of thisgrowth factor is upregulated in stromal ®bro-blasts following corneal injury (Wilson et al.,1999). HGF/SF e�ects on corneal epithelial beha-viour still require investigation but it does bringabout rapid dispersion of cells from cohesivesheets and has a marked e�ect on cell migrationrates (McBain et al., 1998).

There is however, a second source of HGF/SFthat reaches the epithelium and that is via thetears; tear levels go up from around 200 pg/ml toover 450 pg/ml following corneal injury in therabbit (Li et al., 1996). Further it is known thatthe ®rst 2 days following excimer laser photore-fractive procedures in human subjects that thebioavailability of HGF/SF from the tears to thecorneal epithelium is markedly increased. Theactual amounts of the growth factor decrease intears in the immediate postoperative period, butbecause tear output is extremely high during thistime frame, bioavailability and therefore, pro-duction must be enhanced (Tervo et al., 1997).Tear HGF/SF arises from the lachrymal glandand, on the basis of evidence provided by immu-nohistochemistry, it originates from the interal-veolar connective tissue cells (Li et al., 1996).

As has been stated in earlier sections, HGF/SFis an especially e�ective mitogen for vascular en-dothelium (Rosen and Goldberg, 1995) and


induces new vessel formation in injured and can-cerous tissues. It is likely therefore that endogen-ous HGF/SF has a role to play in cornealvascularisation but as yet we know of no litera-ture on the distribution and function of the cyto-kine in those corneal diseases which areassociated with new vessel formation. It has beenshown however that tens to hundreds of nano-gram levels of HGF/SF injected into the rat cor-nea induces corneal neovascularisation andtherefore may be considered as a potential indu-cer of angiogenesis, although it is not knownwhether these high concentrations are physiologi-cally relevant. (Rosen and Goldberg, 1995;Lamaszus et al., 1996).

We think of HGF/SF as being produced bymesenchymal cells which, after extracellular acti-vation, has its biological in¯uence on target epi-thelial cells which carry the transmembranous c-Met receptor protein. In addition to the classicalparacrine mechanism, there is growing evidenceof autocrine pathways. However, they remainpoorly understood and are only just beginning tobe appreciated. Earlier in this section evidence ofa very limited production of HGF/SF by c-Metexpressing corneal epithelium was suggested(Wilson et al., 1993). A better and more convin-cing example is the corneal endothelium whichseems to produce HGF/SF in respectableamounts and also is a target cell that expressesthe c-Met receptor (Wilson et al., 1993).

3.2. Aqueous humour

Aqueous humour contains numerous cytokinesincluding EGF, members of the FGF family,members of the TGFb family, insulin-like growthfactors, in addition to a number of others (Cas-tro et al., 1990; Parelman et al., 1990; Tripathi etal., 1994) but none are present in exceptionallylarge amounts although in some cases, (e.g.TGFb2) there is su�cient active growth factor toproduce biological e�ects (Tripathi et al., 1994).The level of HGF/SF in aqueous humour takenfrom cataract patients at surgery is variable inthe literature being in the range 0.020±0.194 ng/ml from the work of Araki-Sasaki et al. (1997),averaging 0.556 ng/ml from Ritch et al. (1999) orbetween 0.133 and 0.93 ng/ml from the work of

Shinoda et al. (1999). The quantities in aqueousdo not correlate with blood serum HGF/SFlevels suggesting that it is likely that there is localintraocular production of this cytokine (Araki-Sasaki et al., 1997).

Local production may well come from the cellswhich line the anterior chamber and chief amongthese are the corneal endothelial cells. Cornealendothelial cells, according to Wilson et al.(1993), are producers of HGF/SF and that pro-duction in tissue culture experiments is notdependent on whether the cells are rapidly divid-ing or quiescent. Since corneal endothelial cellshave a low or even marginal turnover in vivo(Waring et al., 1982) the quiescent results in theprevious study are of some importance to poss-ible in vivo production of HGF/SF. It should besaid that there is no direct evidence that cornealendothelium contributes to aqueous HGF/SFlevels and the possible contribution made by forexample the mesenchymal tissue of the irisremains to be evaluated.

HGF/SF is thought by some to contribute tothe maintenance of normal vascular endothelialcells (see previous Section 2.4 on biological ac-tivities). It has been suggested that it has a simi-lar trophic action on the corneal endothelium. Insupport of the argument is the fact that endo-thelial cell numbers correlate positively with theaqueous concentrations of HGF/SF (Araki-Sasaki et al., 1997). Conversely, it is possible thatthe correlation is due to the ability of corneal en-dothelial cells to make HGF/SF and that is anequally valid explanation why aqueous humourconcentrations of HGF/SF are linked with thenumber of corneal endothelial cells. Indeed it isnot known how much of the HGF/SF in aqueousis activated or how HGF/SF compares to theother cytokines in aqueous such as FGF andEGF (Tripathi et al., 1994). These growth factorsare just as likely to be survival factors for cornealendothelium as HGF/SF.

3.3. The out¯ow system

The aqueous humour passes out of the eyemainly via the conventional out¯ow route whichconsists of the trabecular meshwork, Schlemm'scanal and a series of aqueous out¯ow vessels


which connect to the episcleral venous system.The trabecular meshwork is made up of a com-plex arrangement of perforated connective tissueplates lined by meshwork cells. These meshworkcells, like corneal endothelial cells, are in intimatecontact with the aqueous humour. They also areneural crest derived and have an extremely lim-ited turnover in normal circumstances in vivo(Grierson and Hogg, 1995). However they can bemade to proliferate in tissue culture conditionsand also in some experimental and pathologicalcircumstances (Grierson and Hogg, 1995). In thisrespect they are very similar to corneal endo-thelium.

There is evidence for the presence of the c-Metreceptor on meshwork cells based on the identi®-cation of the appropriate mRNA (Wordinger etal., 1998). Functional studies have shown thatHGF/SF is an competent mitogen for meshworkcells with a maximal e�ect at around 5 ng/mland signi®cant activity at 1 ng/ml; the lower con-centration is close to the upper limit of HGF/SFlevels in aqueous humour (see earlier).

Meshwork cells exhibit a slow decrease innumbers with ageing (see Grierson and Hogg,1995 for review) which is also a feature of thecorneal endothelial cell population. In primaryopen angle glaucoma the loss of meshwork cellsis particularly severe and the loss has been con-sidered by some to precipitate the decrease inmeshwork drainage function. In turn, compro-mised drainage leads to the pathologically elev-ated intraocular pressure which commonly isassociated with glaucoma (Alvarado et al., 1984;Grierson and Hogg, 1995).

Compromised meshwork cells die by apoptosis(Sibayan et al., 1998; Agarwal et al., 1999) and areasonable assumption is that apoptotic death isa means by which at least some of the age andglaucoma related loss of these cells is broughtabout. As a result, it is important to know whichconstituents of the normal and the diseased aqu-eous have either apoptotic or anti-apoptotice�ects to modulate meshwork cell survival.Obviously HGF/SF ®ts the bill as a potentialmeshwork cell survival factor but there is no ex-perimental evidence to support this hypothesis.

In addition to apoptosis, there are other mech-anisms operating to deplete the ageing and glau-

comatous meshwork cell population. The mostimportant of these is thought to be active cell mi-gration (Grierson and Hogg, 1995; Hogg et al.,1995). Meshwork cells have properties in com-mon with macrophages in that they are phagocy-tic and migratory and, as such, they can detachfrom the trabeculae, move away from their nor-mal location and be lost from the tissue (Grier-son and Hogg, 1995). Some aqueousconstituents, including ®bronectin and laminin,have been shown to stimulate directional andnon-directional migration of meshwork cells.

On the other hand from the published litera-ture, the only growth factor which so far hasbeen shown to be an e�ective migratory stimu-lant to meshwork cells is platelet derived growthfactor (PDGF) (Hogg et al., 1995) but it is onlyfound in trace amounts in aqueous in normal cir-cumstances (Tripathi et al., 1994). AlthoughHGF/SF is a known migratory stimulant for arange of cell types nothing to this point in timehas been reported on its action on meshworkcells. One of us (P.H.) recently has examined theability of HGF/SF to stimulate the migration ofcultured human meshwork cells in vitro by usingmicrochemotaxis chambers (Neuro Probe, CabinJohn, USA).

These chambers consist of two sections whichwhen ®tted together form a number of wells(usually 48 or 96) with an upper lower division inregister. Between the two divisions there is a por-ous membrane through which cells can migrate.The cells are placed in the upper well and settleon the permeable membrane whereas the poten-tial attractant is in solution in the lower well. Ifthe cells are stimulated to migrate they will passthrough the membrane and spread out on itslower surface. After the experiment is over themembrane is removed, ®xed, stained and themigrated cells on the lower membrane surfacecan then be counted.

It can be seen that HGF/SF is a reasonablygood chemoattractant for meshwork cells secondonly to PDGF among the growth factors testedand being almost half as powerful as soluble®bronectin (Fig. 7). For e�ective motile re-sponses, HGF/SF needs to be in the nanogramrange, picogram and microgram levels are non-responsive but signi®cant migration is produced


by 1 and 100 ng/ml (P < 0.05) with a responseoptimum around 10 ng/ml (P < 0.01). The opti-mum response level is way outside normal aqu-eous levels of HGF/SF but the highest aqueousconcentration given by Shinoda et al., 1991comes very close to 1.0 ng/ml level which pro-duces a migratory response from bovine mesh-work cells nearing 20 times background (Fig. 7).

As yet estimates for HGF/SF levels are avail-able for normal aqueous humour (see earlier) butthey are still spartan for the various forms ofglaucoma. It will be of considerable value toknown whether or not aqueous levels areincreased or decreased in primary open angleglaucoma and in some of the key secondary glau-comas like neovascular glaucoma. It seems fromrecently published work in abstract form (Ritchet al., 1999) suggests that HGF/SF levels areincreased by over 50% in POAG patients and

300% in patients with pseudoexfoliation glau-coma. Speculation that high levels of HGF/SFmight help preserve the meshwork cell popu-lation in glaucoma is counteracted by the veryclear migratory action of HGF/SF on these cellswhich would tend to deplete the population. Thee�ects of HGF/SF in these circumstances is intri-guingly ambiguous and future studies shouldyield interesting ®ndings.

3.4. The lens

The lens is a unique epithelial system of amaz-ing regulatory and proliferative precision butwithout a vascular supply for nutrition and hor-monal control and without a nerve system input/output for appropriate signalling. Biostimulationand paracrine contact with other ocular tissuesdepends on the lens' own nutritional ¯uid, the

Fig. 7. Migration of cultured human meshwork cells to recombinant HGF/SF. Each column is a mean plus SEM and n= 4 or greater. Soluble fibronectin (sFn) was used as a positive control and HGF/SF free buffer provided backgroundmigratory levels. An optimum migratory response was produced by 10.0 ng/ml of around 20 times greater than back-

ground.


aqueous humour. In addition to a great extent

the lens relies on its own resources and rely on

autocrine and juxtacrine signals.

HGF/SF, being present in the aqueous

humour, is a reasonable candidate for a growth

factor which may modulate the behaviour of lens

epithelium and lens ®bres in health and disease.

Certainly established cultures of human lens epi-

thelium express messenger RNA for c-Met

(Fleming et al., 1998) as do lens epithelium in

primary culture from rabbits and humans and

freshly removed rabbit lens epithelium not sub-

jected to culturing (Weng et al., 1997). Although

there is good evidence that the receptor for

HGF/SF is present within the epithelium, no in-

formation currently is available about the topo-

graphy of the receptor distribution in the lens

nor whether the receptor is expressed by lens

®bres.

It remains to be seen whether or not HGF/SF

has a part to play with other growth factors in

epithelial growth, lens ®bre di�erentiation and ul-

timately the maintenance of transparency. As yet,

it is seen to produce only a modest proliferative

response when given to lens epithelial cells in tis-

sue culture (Reddan et al., 1996) despite the pre-

sence of the appropriate receptor. HGF/SF may

well be more involved in the control of lens cell

shape and lens maintenance.

On the down side, it is also worth remember-

ing that activated HGF/SF has a negative e�ect

on gap junction communication (Moorbey et al.,

1995) and the lens has the highest density of

these structures in the body. There is some sug-

gestion that negating gap junction function is a

necessary prerequisite before the induction of

exuberant cell growth. The common complication

of cataract surgery is the development of a sec-

ondary cataract which consists of a scar-like

membrane on the lens capsule and the newly

implanted lens. The membrane consists of lens

epithelium that have undergone an epithelial to

mesenchymal transformation; the change is in¯u-

enced by HGF/SF (George Duncan and Michael

Wormstone, University of East Anglia; personal

communication). It would seem at least that

there is some evidence of HGF/SF being

involved in the shape change of lens cells albeit

in pathological rather than normal circum-stances.

Evidence is available which shows that lens isnot a typical epithelium with respect to HGF/SF.It has been said before that with few exceptions,epithelia which respond to HGF/SF through thec-Met surface receptor system are not producersof HGF/SF (Stoker et al., 1987, Matsumoto etal., 1991a,b, Sonnenberg et al., 1993, Warn1995). Lens epithelium is distinctive because bothcultured and noncultured cells can be shown toexpress HGF/SF (Weng et al., 1997). Thus it ispossible that the lens epithelial cells contribute toaqueous humour HGF/SF levels in addition tothe corneal endothelium. Whether or not theHGF/SF acts locally in the lens itself in an auto-crine manner or has a paracrine action at aremote site depends on where HGF/SF activationtakes place. The possibility exists that the tissuesin contact with the aqueous humour, such as thelens, iris stroma, corneal endothelium and theout¯ow system, in¯uence the survival and beha-vioural activities of each other by secreting po-tentially bioactive peptides like HGF/SF into thecollective aqueous lake.

4. THE BACK OF THE EYE

4.1. Vitreous humour

The vitreous content of soluble protein is lowand has been estimated to be around 0.01% ofthe ¯uid compartment (Swann and Constable,1972) which compares poorly with a soluble pro-tein value of 7.0% for blood. Even although theoverall protein concentration is low, the vitreousis still thought to act as a reservoir (Bito, 1977)or holding pen for some key substances like pep-tide growth factors. In times of crisis for adjacenttissues like the retina and lens, the vitreous canprovide a limited supply of growth factors andalso of sugars like glucose. On the other handthe vitreous can serve as a sponge by soaking upharmful metabolites like pyruvate and lactate.

In the context of the vitreous as a reservoir forgrowth factors, it has been shown that the levelof HGF/SF in vitreous is 1.5 ng/ml (Nishimuraet al., 1999) which is over seven times the upper


limit for normal serum HGF/SF (usually lessthan 0.2 ng/ml) and somewhat higher than aqu-eous humour levels (highest estimates are notmore than 1 ng/ml; see Section 2.2). Of coursecare has to be taken over the interpretation ofthese data taken from di�erent laboratories withdi�ering assaying procedures. Further, thehuman ``normals'' are not normal as suchbecause the vitreous samples are taken frompatients with di�ering pathologies; albeit thatthese pathologies are thought likely to have mini-mal e�ects on the concentration of the targetgrowth factor. Nishimura et al. (1999) had astheir control ``normals'' (our word not theirs)vitreous from patients mostly with macular holeswhereas the aqueous humour samples (Araki-Sasaki, 1997; Ritch et al., 1999; Shinoda et al.,1999) originated from cataract patients.

Even when the previous reservations are takeninto consideration it does seem likely that thevitreous is a reservoir for HGF/SF and it is heldat levels which, if activated would be su�cient tohave biological in¯uence on target cells. Unfortu-nately the activation status of the HGF/SF innormal vitreous is not known at this time. In ad-dition, the origins of the vitreal HGF/SF has notbeen determined. It is unlikely to have comefrom blood serum, and the lens and retina standout as the most probable sources.

4.2. The neural retina and pigment epithelium

Little work has been done to date on c-Met ex-pression in, HGF/SF e�ects on or HGF/SF pro-duction by the cells of the neural retina. It ishowever reasonable to assume if the neural retinais anything like the brain in this context, that thetissue would not be entirely barren ground.Microglia, which are neural tissue histiocytes de-rived from mesenchyme, do manufacture ratherlarge amounts of HGF/SF and astrocytes arerather more modest producers (Moriyama et al.,1995; Rosen et al., 1996). Retinal microglial,astrocytic and Muller cell production of HGF/SFstill has to be determined, but these glial cellsremain likely sources of any locally producedneuroretinal HGF/SF.

Retinal glial cells do not seem to express the c-Met receptor either in health or disease (Okada

et al., 1995). Limited evidence is available in the

literature on receptor distribution in the neural

retina (Lashkari et al., 1999). The neural retina

appears, on the basis of immunohistochemical

localization, to express marginal c-Met staining

associated with the photoreceptor cells and the

nerve ®bres of the ganglion cell layer. The stain-

ing is much less than can be found for example

in the adjacent choroid (Lashkari et al., 1999).

Such marginal staining can be interpreted either

way and the ``jury remains out'' awaiting future

more detailed investigations. It is however an

issue of some importance to establish whether or

not retinal neurones are responsive to HGF/SF

through the c-Met receptor because in other sys-

tems HGF/SF has been established as an import-

ant survival factor and neurotrophic agent

(Ebens et al., 1996).

One cell type that expresses the c-Met receptor

at high levels is the retinal pigment epithelium

(RPE) and it does so rather exuberantly accord-

ing to the available literature (He et al., 1998;

Lashkari et al., 1999) which also has been our

own experience (Figs 5 and 8). The receptor has

been shown to be functional by induced phos-

phorylation in the presence of HGF/SF (Lash-

kari et al., 1999). The RPE, in its crucial

nursemaid role to the photoreceptors (Grierson

et al., 1997), may require HGF/SF (as it does

other growth factors) to aid survival and help

overcome the day-to-day punishing e�ects of

photoreceptor disc digestion. Other e�ects of

HGF/SF on RPE are of particular relevance to

Fig. 8. Immunohistochemical staining of cultured humanRPE cells for the c-Met receptor, most of the cells stain

brown with the reaction product (�250).


retinal detachment and the proliferative retinopa-thies and will be dealt with in the next section.

A controversy exists about whether or notRPE cells also are producers of HGF/SF. Heand co-workers (He et al., 1998) consider, on thebasis of positive RT-PCR and ELISA assays onserum starved RPE in culture, that the cells pro-duce message for HGF/SF and secrete the pep-tide into the culture medium. They think thatRPE are one of the cell types, uncommon in thebody as a whole but strangely prevalent in theeye, that express both the receptor and thegrowth factor (perhaps indicating an autocrineloop). Alternatively others, Lashkari et al. (1999)have failed to identify HGF/SF production byRPE in culture down to the detection base of 125pg/ml for their ELISA assay. As a result of these®ndings these authors suggest that HGF/SFreaches RPE by a paracrine route. It remains tobe established which observation is correct.

4.3. Retinal detachment and the proliferative

retinopathies

Scar tissue formation complicates approxi-mately 10% of originally simple rhegmatogenousretinal detachments. The scar tissue develops asepiretinal and subretinal membranes and thepathological condition has been termed prolifera-tive vitreoretinopathy or PVR by the Retina So-ciety Terminology Committee (1983) andMachemer et al. (1991). RPE cells are a key com-ponent of epiretinal and subretinal membranes(see for review Grierson et al., 1997). In thoseeyes which develop PVR, the RPE cells beneaththe detached neural retina undergo an epithelialto mesenchymal alteration. The alterationinvolves numerous changes whereby the epi-thelium loses its hexagonal shape to becomeelongated and ®broblast-like and goes from beinga static non-dividing cell to become a migratoryand proliferating variant (Hiscott and Grierson,1994; Hiscott and Sheridan, 1998; Grierson et al.,1997).

It does not take much of a leap to think thatthe HGF/SF and c-Met system could play a keyrole in the RPE loss of communication, shapechange and mobilization so important to theearly pathogenesis of PVR. It turns out, not too

surprisingly, that HGF/SF may well be a keyplayer. Human RPE cells do not form particu-larly good colonies so scattering cannot be ap-preciated in culture. On the other hand, thesecells do undergo an impressive elongated shapechange to a more ®broblastic appearance in thepresence of HGF/SF (Fig. 9) (Briggs et al.,1995). In addition HGF/SF is a weak mitogenbut an e�ective motogen for cultured humanRPE cells (Briggs et al., 1995; He et al., 1998;Lashkari et al., 1999).

It might be expected that ¯uid removed fromthe subretinal space of PVR patients would con-tain HGF/SF. Our own in house ELISA determi-nations have been conducted on only one sampleso far and it was positive but we still await a sub-stantial series to determine the quantities ofHGF/SF present in the subretinal space afterretinal detachment. From the subretinal space,the detached and migratory RPE cells gain entryto the vitreous cavity by means of the retinalhole which characterises rhegmatogenous detach-ments. The RPE cells (along with ®broblasts andin¯ammatory cells) then come together with glialsheets on the surface of the retina to form epiret-inal membranes (Hiscott and Grierson, 1994;Hiscott and Sheridan, 1998). These membranesare contractile (see for background informationGrierson et al., 1996, 1997) and their traction dis-torts the retina with obvious visual consequences.

We have found positive immunohistochemicallocalisation of the c-Met receptor to some cells in6 out of 7 PVR epiretinal membranes examined(Briggs et al., 1995) (Fig. 10). Subsequent workin our laboratory (M.B.) has established a co-dis-tribution of c-Met and cytokeratin (epithelialmarker) in at least some of the epiretinal mem-branes. Further, work by others (Lashkari et al.,1999) found that 3 out of 3 PVR membranescontained cells positively immunostained for thec-Met receptor whereas only 1 out of 3 idiopathicepiretinal membranes were positive. That glialcells rather than RPE predominate in many idio-pathic membranes is the probable explanationfor the lack of positive cells seen by the authors.Based on current thinking it would be unlikelyfor any of the major constituent cells in idio-pathic epiretinal membranes (glia, ®broblasts andin¯ammatory cells) to carry the c-Met receptor


although they might well be HGF/SF producers(experimental evidence for this is lacking so far).

It does appear likely that the vitreous in PVR

patients contains more HGF/SF than patientswith pathologies associated with little prolifera-tive and migratory behaviour such as macularholes. However, the values for individual patientscan be extremely variable. Values published byNishimura et al. (1999) ranged between 1.10 and7.38 ng/ml whereas our own ®gures gave a rangevalue of 3 ng/ml going up to 250 ng/ml (Briggset al., 1995)! Such variation is intriguing but byno means exceptional for studies of cytokine dis-tribution in vitreoretinal diseases (Boulton, 1999).At least some of the HGF/SF is active andmature because PVR vitreous can induce scatter-ing of colonies of MDCK cells (Briggs et al.,1995).

Diabetic retinopathy has been the subject ofconsiderable growth factor research over theyears spurred on by the hypothesis of Michaelson(1948) who proposed that a di�usible agentcould induce the vascular changes associatedwith the disease. One by one growth factors withangiogenic properties, such as the insulin-likegrowth factors, TGFb, the FGF family havebeen greeted enthusiastically, studied and thenconsidered as being probably of secondary im-portance particularly in the later stage of the reti-nopathy called proliferative diabetic retinopathy(PDR) where neovascularisation is the hallmark.More recently vascular endothelial growth factor

Fig. 9. Normal cultured human RPE cells (A) become elongated and spindle shaped (B) in the presence of HGF/SF.Cells stained with haematoxylin and eosin (�200).

Fig. 10. Immunohistochemical staining of RPE cells inepiretinal membranes for the c-Met receptor. The mem-brane is counter stained with haematoxylin and the posi-tive staining is seen as the brown reaction product where

there is a prominent layer of cells (�250).


(VEGF), placenta growth factor and HGF/SFhave been implicated as important proliferativestimuli in PDR and these are the subjects ofintensive current research (Boulton, 1999).

It is with some caution that HGF/SF has beenintroduced as a potential key angiogenic factor inPDR (Boulton, 1999). As pointed out, however,HGF/SF has got several features that make it anattractive candidate. For one thing it is a potentstimulator of new vessel formation and is forexample far more e�ective at stimulating vascularendothelial proliferation than FGF or evenVEGF (Bussolino et al., 1992; Nakamura et al.,1996). HGF/SF is found at higher levels in thevitreous of patients with PDR than those with-out; particularly if the PDR is in an advanced oractive phase (Katsura et al., 1998; Nishimura etal., 1999). Highest levels of vitreal HGF/SF arefound in patients with advanced disease and newvessel formation on the iris (average 7.33 ng/ml).Those patients without iris neovascularisationhave lower values (average 4.49 ng/ml) but theirlevels are still far higher than diabetics withoutPDR (average 1.29 ng/ml) which seem to be atnormal vitreal levels (Nishimura et al., 1999). Inaddition aqueous humour levels of HGF/SF areelevated in PDR patients but not in diabeticswithout proliferative changes (Shinoda et al.,1999).

4.4. Uveal melanoma and ocular tumours

The behaviour of choroidal melanoma rangefrom being relatively benign to being highlymalignant. Immunohistological information atleast indicates that tumour cells expressing the c-Met receptor could be found only in sectionsdominated by the more malignant phenotype(Hendrix et al., 1998). In vitro studies indicatethat melanoma cells which invade collagenmatrices most e�ectively and these exhibiteddirectional migration to HGF/SF in a chemotaxischamber were c-Met positive (Hendrix et al.,1998).

The proposal under some scrutiny at present isthat disseminating ocular melanoma cells are par-ticularly responsive to HGF/SF as a motogenand therefore growth factor may be a particularlyrobust signal for metastatic behaviour. It appears

that secondary tumours, which form preferen-tially in the liver, are rich in HGF/SF whereasprimary uveal tumours are far less so. It hasbeen established that a major adverse histopatho-logical feature of uveal melanoma is the presenceof complex microvascular patterns (Makitie etal., 1999) presumably by aiding the disseminationof tumour cells. The role of HGF/SF in angio-genesis in other tumours (Rosen and Goldberg1995; Lamaszus et al., 1997) and in other diseases(Nakamura et al., 1998) has been mentioned ear-lier and is of obvious relevance to uveal mela-noma.

5. FUTURE DIRECTIONS

HGF/SF produces a range of biological e�ectsin target cells and it is to be found at varioussites within the eye. The research conducted onthe activities of this cytokine on ocular cells andtissues still is limited but it will be the subject ofconsiderable ocular research interest in the nearfuture. HGF/SF may have a key role in suchdiverse activities as ocular development, the regu-lation of normal adult tissue and it may beinvolved in pathologies ranging from to PDR tothe development and spread of uveal melanoma.Only time will tell whether this cytokine, in termsof ophthalmic eye disease, will be of passinginterest or turn out to be a key player.

AcknowledgementsÐThe authors acknowledge the support ofthe Sir Jules Thorn Charitable Trust, Guide Dogs for theBlind Association, The Dunhill Medical Trust and TheFoundation for Prevention of Blindness.

REFERENCES

Agarwal, R., Talati, M., Lambert, W., Clark, A. F., Wilson,S. E., Agarwal, N. and Wordinger, R. J. (1999) Fas-activated apoptosis and apoptosis mediators in humantrabecular meshwork cells. Exp. Eye Res. 68, 583±590.

Alvarado, J., Murphy, C. and Juster, R. (1984) Trabecularmeshwork cellularity in primary open angle glaucomaand non glaucomatous normals. Ophthalmology 91,564±579.

Andresen, J. L. and Ehlers, N. (1998) Chemotaxis of humankeratocytes is increased by platelet-derived growth fac-tor-BB, epidermal growth factor, transforming growthfactor-alpha, acidic ®broblast growth factor, insulin-like


growth factor-I, and transforming growth factor-beta.Curr. Eye Res. 17, 79±87.

Araki-Sasaki, K., Danjo, S., Kawaguchi, S., Hosohata, J. andTano, Y. (1997) Human hepatocyte growth factor(HGF) in the aqueous humor. Jap. J. Ophthalmol. 41,409±413.

Bhargava, M., Joseph, A., Knesel, J., Halaban, R., Li, Y.,Pang, S., Goldberg, I., Setter, E., Donovan, M. A.,Zarnegar, R., Michalopoulos, G. A., Nakamura, T.,Faletto, D. and Rosen, E. M. (1992) Scatter factor andhepatocyte growth factor: activities, properties andmechanism. Cell. Growth Di�eren. 3, 11±20.

Bito, L. Z. (1977) The physiology and pathophysiology ofintraocular ¯uids. Exp. Eye Res. 25(Suppl), 273±289.

Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, S. andBirchmeier, C. (1995) Essential role for the c-Met recep-tor in the migration of myogenic precursor cells to thelimb bud. Nature 376, 768±771.

Boros, P. and Miller, C. M. (1995) Hepatocyte growth factor:a multifunctional cytokine. Lancet 345, 293±295.

Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M.,Kmiecik, T. E., Van de Woude, G. F. and Aaronson,S. A. (1991) Identi®cation of the hepatocyte growth fac-tor receptor as the c-met proto-oncogene product.Science 251, 802±804.

Boulton, M. (1999) A role for hepatocyte growth factor indiabetic retinopathy. Br. J. Ophthalmol. 83, 763±764.

Briggs, M. C., Grierson, I., Hiscott, P., Sheridan, C. M.,Wong, S. H. D., McGalliard, J. N. and Hunt, J. (1995)Scatter factor: a possible role in proliferative vitreoret-inal disease. Invest. Ophthalmol. Vis. Sci. 36(Suppl),751.

Bussolino, F., Di Renzo, M. F., Ziche, M., Bocchietto, E.,Olivero, M., Naldini, L., Gaudino, G., Tamagnone, L.,Co�er, A. and Comoglio, P. M. (1992) Hepatocytegrowth factor is a potent angiogenic factor whichstimulates endothelial cell motility and growth. J. Cell.Biol. 119, 629±641.

Castro, J., Vigo, J. F., Larranaga Cordido, J., Sabugal, J. andDiaz, J. A. (1990) Insulin-like growth factor (IGF-1) inaqueous humour. Study in non diabetic and diabeticpatients. Invest. Ophthalmol. Vis. Sci. 31(Suppl), 196.

Chirgadze, D. Y., Hepple, J., Byrd, R. A., Sowdhamini, R.,Blundell, T. L. and Gherardi, E. (1998) Insights intothe structure of the hepatocyte growth factor/scatterfactor (HGF/SF) and implications for receptor acti-vation. FEBS Lett. 430, 126±129.

Comoglio, P. M. and Vigna, E. (1995) Structure and func-tions of the HGF receptor (c-Met). In LiverRegeneration and Carcinogenesis (ed. R. L. Jirtle) pp.51±70. Academic Press, San Diego.

Di Renzo, M. F., Narsimhan, R. P., Olivero, M., Bretti, S.,Giordano, S., Medico, E., Gaglia, P., Zara, P. andComoglio, P. M. (1991) Expression of the Met/HGFreceptor in normal and neoplastic human tissues.Oncogene 6, 1997±2003.

Dowrick, P., Kenworthy, P., McCann, B. and Warn, R.(1993) Circular ru�e formation and closure lead tomacropinocytosis in hepatocyte growth factor/scatterfactor-treated cells. Eur. J. Cell. Biol. 61, 44±53.

Dugina, V. B., Alexandrova, A. Y., Lane, K., Bulanova, E.and Vasiliev, J. M. (1995) The role of the microtubularsystem in the cell response to HGF/SF. J. Cell. Sci.108, 1659±1667.

Ebens, A., Brose, K., Leonardo, E. D., Hanson, M. G. Jr,

Bladt, F., Barres, B. A. and Tessier-Lavigne, M. (1996)Hepatocyte growth factor/scatter factor is an axonalchemoattractant and a neurotrophic factor for spinalmotor neurons. Neuron 17, 1157±1172.

Fleming, T. P., Song, Z. and Andley, U. P. (1998) Expressionof growth control and di�erentiation genes in humanlens epithelial cells with extended life span. IOVS 39,1387±1398.

Fukuayama, R. (1991) Regional localisation of the hepatocytegrowth factor (HGF) gene to human chromosome 7band q21.1. Genomics 11, 410±415.

Furlong, R. A., Takehara, T., Taylor, W. G., Nakamura, T.and Rubin, J. S. (1991) Comparison of biological andimmunochemical properties indicates that scatter factorand hepatocyte growth factor are indistinguishable. J.Cell. Sci. 100, 173±177.

Gandino, L., Longati, P., Medico, E., Prat, M. andComoglio, P. M. (1994) Phosphorylation of serine 985negatively regulates the hepatocyte growth factor recep-tor kinase. J. Biol. Chem. 269, 1815±1820.

Gherardi, E., Gray, J., Stoker, M., Perryman, M. andFurlong, R. (1989) Puri®cation of scatter factor, a®broblast-derived basic protein that modulates epi-thelial interactions and movement. Proc, Natl. Acad.Sci. USA 86, 5844±5848.

Ghoussoub, R. A. D., Dillon, D. A., D'Aquila, T., Rimm, E.B., Fearon, E. R. and Rimm, D. L. (1998) Expressionof c-met is a strong independent prognostic factor inbreast carcinoma. Cancer 82, 1513±1520.

Giordano, S., Ma�e, A., Williams, T. A., Artigiani, S., Gual,P., Bardelli, A., Basilico, C., Michaeli, P. andComoglio, P. M. (2000) Di�erent point mutations inthe met oncogene elicit distinct biological properties.FASEB J. 14, 399±405.

Giordano, S., Ponzetto, C., Di Renzo, M. F., Cooper, C. S.and Comoglio, P. M. (1989) Tyrosine kinase receptor isindistinguishable from the c-met protein. Nature 339,155±156.

Gohda, E., Matsunaga, T., Kataoka, H. and Yamamoto, I.(1992) TGF-b is a potent inhibitor of hepatocytegrowth factor secretion by human ®broblasts. Cell.Biol. Int. Rep. 16, 917±926.

Grierson, I., Hiscott, P., Sheridan, C. and Tugulu, I. (1997)The pigment epithelium: friend and foe of the retina.Proc. Royal Mic. Soc. 32, 161±170.

Grierson, I. and Hogg, P. (1995) The proliferative and mi-gratory activities of trabecular meshwork cells. Prog.Ret. Eye Res. 15, 33±67.

Grierson, I., Mazure, A., Hogg, P., Hiscott, P. and Wong, D.(1996) Non-vascular vitreoretinopathy: the cells and cel-lular basis of contraction. Eye 10, 671±684.

He, P. M., He, S., Garner, J. A., Ryan, S. J. and Hinton, D.R. (1998) Retinal pigment epithelial cells secrete andrespond to hepatocyte growth factor. Bioch. Biophys.Res. Com. 249, 253±257.

Heldin, C. H. and Ostman, A. (1996) Ligand-induced dimeri-zation of growth factor receptors: variations on thetheme. Cytokine Growth Factor Rev. 7, 3±10.

Hendrix, M. J., Seftor, E. A., Seftor, R. E., Kirschmann, D.A., Gardner, L. M., Bolt, H. C., Meyer, M., Pe'er, J.and Folberg, R. (1998) Regulation of uveal melanomainterconverted phenotype by hepatocyte growth factor/scatter factor (HGF/SF). Am. J. Path. 152, 855±863.

Herrera, R. (1998) Modulation of hepatocyte growth factor-


induced scattering of HT29 colon carcinoma cells. J.Cell. Sci. 111, 1039±1049.

Hiscott, P. and Grierson, I. (1994) Retinal detachment. InPathobiology of Ocular Disease: A Dynamic Approach,Second ed. (eds. A. Garner and G. K. Klintworth) pp.675±700. Marcel Dekker Inc, New York.

Hiscott, P. and Sheridan, C. (1998) The retinal pigment epi-thelium, epiretinal membranes and proliferative vitreor-etinopathy. In Retinal Pigment Epithelium ± CurrentAspects of Function and Disease (eds. M. F. Marmorand T. J. Wolfensberger) pp. 478±491. OxfordUniversity Press, New York.

Hogg, P., Calthorpe, M., Ward, W. and Grierson, I. (1995)The migration of cultured bovine trabecular meshworkcells to cytokines and aqueous humor. Invest.Ophthalmol. Vis. Sci. 36, 2449±2460.

Honma, Y., Nishida, K., Sotozono, C. and Kinoshita, S.(1997) E�ect of transforming growth factor-b1 and -b2on in vitro rabbit corneal epithelial cell proliferationpromoted by epidermal growth factor, keratinocytegrowth factor, or hepatocyte growth factor. Exp. EyeRes. 65, 391±396.

Ikejima, K., Watanabe, S., Kitamura, T., Hirose, M.,Miyazaki, A. and Sato, N. (1995) Hepatocyte growthfactor inhibits intercellular communication via gapjunctions in rat hepatocytes. Biochem. Biophys. Res.Com. 214, 440±446.

Jin, L., Fuchs, A., Schnitt, S. J., Yao, Y., Joseph, A.,Lamaszus, K., Park, M., Goldberg, I. D. and Rosen, E.M. (1997) Expression of scatter factor and C-met recep-tor in benign and malignant breast tissue. Cancer 79,749±760.

Joseph, A., Weiss, G. H., Jin, L., Fuchs, A., Chowdhury, S.,O'Shaugnessy, P., Goldberg, I. D. and Rosen, E. M.(1995) Expression of scatter factor in human bladdercarcinoma. J. Natl Cancer Inst. 87, 372±377.

Kan, M., Zhang, G., Zarnegar, R., Michalopoulos, G.,Myoken, Y., McKeehan, W. L. and Stevens, J. T.(1991) Hepatocyte growth factor/hepatopoietin Astimulates the growth of rat kidney proximal tubuleepithelial cells (RPTE), rat non-parenchymal liver cells,human melanoma cells, mouse keratinocytes and stimu-lates anchorage dependent growth of SV-40 trans-formed RPTE. Biochem. Biophys. Res. Com. 174, 331±337.

Katsura, Y., Okano, T., Noritake, M., Kosano, H., Nishigori,H., Kado, S. and Matsouka, T. (1998) Hepatocytegrowth factor in vitreous ¯uid of patients with prolif-erative diabetic retinopathy and other retinal disorders.Diabetes Care 21, 1759±1763.

Kitazawa, T., Kinoshita, S., Fujita, K., Araki, K., Watanabe,H., Ohashi, Y. and Manabe, R. (1990) The mechanismof accelerated corneal epithelial healing by human epi-dermal growth factor. Invest. Ophthalmol. Vis. Sci. 31,1773±1778.

Komada, M. and Kitamura, N. (1993) The cell dissociationand motility triggered by scatter factor/hepatocytegrowth factor mediated through the cytoplasmicdomain of the c-Met receptor. Oncogene 8, 2381±2390.

Lamaszus, K., Jin, L., Fuchs, A., Shi, E., Chowdhury, S.,Yao, Y., Polverini, P. J., Laterra, J., Goldberg, I. D.and Rosen, E. M. (1997) Scatter factor stimulatestumor growth and tumor angiogenesis in human breastcancers in the mammary fat pads of nude mice. Lab.Invest. 76, 339±353.

Lamaszus, K., Joseph, A., Jin, L., Yao, Y., Chowdhury, S.,Fuchs, A., Polverini, P. J., Goldberg, I. D. and Rosen,E. M. (1996) Scatter factor binds to thrombospondinand other extracellular matrix components. Am. J.Path. 149, 805±819.

Lashkari, K., Rahimi, N. and Kazlauskas, A. (1999)Hepatocyte growth factor receptor in human RPE cells:implications in proliferative vitreoretinopathy. Invest.Ophthalmol. Vis. Sci. 40, 149±156.

Li, Q., Weng, J., Mohan, R. R., Bennett, G. L., Schwall, R.,Wang, Z. F., Tarbor, K., Kim, J., Hargrave, S.,Cuevas, K. H. and Wilson, S. E. (1996) Hepatocytegrowth factor and hepatocyte growth factor receptor inthe lacrimal gland, tears and cornea. Invest.Ophthalmol. Vis. Sci. 37, 727±739.

Liang, Q., Mohan, R. R., Chen, L. and Wilson, S. E. (1998)Signalling by HGF and KGF in corneal epithelial cells:Ras/MAP kinase and Jak-STAT pathways. IOVS 39,1329±1338.

Lin, J. C., Scherer, S. W., Tougas, L., Traverso, G., Tsui, L.C., Andrulis, I. L., Jothy, S. and Park, M. (1996)Detailed deletion mapping with a re®ned physical mapof 7q31 localizes a putative tumour suppressor gene forbreast cancer in the region of MET. Oncogene 13,2001±2008.

Lokker, N. A., Presta, L. G. and Godowski, P. J. (1994)Mutational analysis and molecular modelling of the N-terminal kringle-containing domain of hepatocytegrowth factor identi®es amino acid side chains import-ant for interaction with the c-Met receptor. ProteinEng. 7, 895±903.

Longati, P., Bardelli, A., Ponzetto, C., Naldini, L. andComoglio, P. M. (1994) Tyrosines 1234±1235 are criti-cal for the activation of the tyrosine kinase encoded bythe MET proto-oncogene (HGF receptor). Oncogene 9,49±57.

Machemer, R., Aaberg, T. M., Mackenzie, Freeman H.,Irvine, A. R., Lean, J. S. and Michels, R. M. (1991) Anupdated classi®cation of retinal detachment with prolif-erative vitreoretinopathy. Am. J. Ophthalmol. 112, 159±165.

Makitie, T., Summanen, P., Tarkkanen, A. and Kivela, T.(1999) Microvascular loops and networks as prognosticindicators in choroidal and ciliary body melanomas. J.Natl Cancer Inst. 91, 359±367.

Mark, M. R., Lokker, N. A., Zioncheck, T. F., Luis, E. A.and Godowski, P. J. (1992) Expression and characteriz-ation of hepatocyte growth factor receptor-IgG furionproteins. E�ects of mutations in the potential proteo-lytic cleavage site on processing and ligand binding. J.Biol. Chem. 267, 26166±26171.

Mars, W. M., Zarnegar, R. and Michaelopoulos, G. K.(1993) Activation of hepatocyte growth factor by theplasminogen activators UPA and TPA. Am. J. Path.143, 949±958.

Matsumoto, K., Hashimoto, K., Yoshikawa, K. andNakamura, T. (1991a) Marked stimulation of growth,motility of human keratinocytes by hepatocyte growthfactor. Exp. Cell. Res. 196, 114±120.

Matsumoto, K., Tajiama, H. and Nakamura, T. (1991b)Hepatocyte growth factor is a potent stimulator ofhuman melanocyte DNA synthesis and growth.Biochem. Biophys. Res. Com. 176, 45±51.

McBain, V. A., Forrester, J. V. and McCaig, C. D. (1998)E�ects of hepatocyte growth factor (HGF), keratino-


cyte growth factor (KGF) and electric ®elds on bovinecorneal epithelial cells. Invest. Ophthalmol. Vis. Sci.39(Suppl), 1039.

Michaelson, I. (1948) The mode of development of the vascu-lar system of the retina, with some observation for itssymptoms for certain retinal diseases. Trans.Ophthalmol. Soc. UK 68, 137±186.

Miyazawa, K., Shimomura, T., Naka, D. and Kitamura, N.(1994) Proteolitic activation of hepatocyte growth fac-tor in response to tissue injury. J. Biol. Chem. 269,8966±8970.

Mizuno, K., Tanoue, Y., Harano, T., Takada, K. andNakamura, T. (1994) Puri®cation and characterizationof hepatocyte growth factor (HGF)-converting enzyme:activation of pro-HGF. Biochem. Biophys. Res. Com.198, 1161±1169.

Montesano, R., Matsumoto, K., Nakamura, T. and Orci, L.(1991) Identi®cation of a ®broblast-derived epithelialmorphogen as hepatocyte growth factor. Cell 67, 901±908.

Moorbey, C. D., Stoker, M. and Gherardi, E. (1995) HGF/SF inhibits junctional communication. Exp. Cell. Res.219, 657±663.

Moriyama, T., Kataoka, H., Tsubouchi, H. and Koono, M.(1995) Concomitant expression of hepatocyte growthfactor (HGF), HGF activator and c-met genes inhuman glioma cells in vitro. FEBS Lett. 372, 78±81.

Nagy, J., Curry, G. W., Hillan, K. J., Mckay, I. C., Mallon,E., Purushotham, A. D. and George, W. D. (1996)Hepatocyte growth factor/scatter factor expression andc-met in primary breast cancer. Surg. Oncol. 5, 15±21.

Naka, D., Ishii, T., Yoshiyama, Y., Miyazawa, K., Hara, H.,Hishida, T. and Kitamura, N. (1992) Activation of hep-atocyte growth factor by proteolitic conversion of asingle chain form to a heterodimer. J. Biol. Chem. 267,20114±20119.

Nakamura, T., Nawa, K. and Ichihara, A. (1984) Partial puri-®cation and characterization of hepatocyte growth fac-tor from serum of hepatectomized rats. Biochem.Biophys. Res. Com. 122, 1450±1459.

Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T.,Shimonishi, M. and Shimizu, S. (1989) Molecular clon-ing and expression of human hepatocyte growth factor.Nature 342, 440±443.

Nakamura, T. (1991) Structure and function of hepatocytegrowth factor. Prog. Growth Factor Res. 3, 67±85.

Nakamura, Y., Morishita, R., Higaki, K., Kida, I., Aoki, M.,Moriguchi, A., Yamada, K., Hayashi, S., Yo, Y.,Nakano, H., Matsumoto, K., Nakamura, T. andOgihara, T. (1996) Hepatocyte growth factor is a novelmember of the endothelium-speci®c growth factors:additive stimulatory e�ect of hepatocyte growth factorwith basic ®broblast growth factor but not with vascu-lar endothelial growth factor. J. Hypertens. 14, 1067±1072.

Nakamura, S., Moriguchi, A., Morishita, R., Aoki, M., Yo,Y., Hayashi, S., Nakano, N., Katsuya, T., Nakata, S.,Takami, S., Matsumoto, K., Nakamura, T., Higaki, J.and Ogihara, T. (1998) A novel vascular modulator,hepatocyte growth factor (HGF), as a potential indexof the severity of hypertension. Biochem. Biophys. Res.Com. 242, 238±243.

Naldini, L., Vigna, E., Narsimhan, R. P., Gaudino, G.,Zarnegar, R., Michapoulis, G. K. and Comoglio, P. M.(1991a) Hepatocyte growth factor (HGF) stimulates the

tyrosine kinase activity of the receptor encoded by theproto-oncogene c-Met. Oncogene 6, 501±504.

Naldini, L., Weidner, K. M., Vigna, E., Gaudino, G.,Bardelli, A., Ponzetto, C., Narsimhan, R. P., Hartman,G. and Zarnegar, R. (1991b) Scatter factor and hepato-cyte growth factor are indistinguishable ligands for theMet receptor. EMBO J. 10, 2867±2878.

Naldini, L., Vigna, E., Bardelli, A., Follenzi, A., Galimi, F.and Comoglio, P. M. (1995) Biological activation ofpro-HGF (hepatocyte growth factor) by urokinase iscontrolled by a stoichiometric reaction. J. Biol. Chem.270, 603±611.

Nishimura, M., Ikeda, T., Ushiyama, M., Nanbu, A.,Kinoshita, S. and Yoshimura, M. (1999) Increased vitr-eous concentrations of hepatocyte growth factor in pro-liferative diabetic retinopathy. J. Clin. Endo. Metab. 84,659±662.

Okada, M., Ogino, N., Matsumura, M., Honda, Y. andNagai, Y. (1995) Histological and immunohistochem-ical study of idiopathic epiretinal membrane.Ophthalmic Res. 27, 118±128.

Okajima, A., Miyazawa, K. and Kitamura, N. (1990) Primarystructure of rat hepatocyte growth factor and inductionof its mRNA during liver regeneration following hepa-tic injury. Eur. J. Biochem. 193, 375±381.

Pagan, R., Martin, I., Llobera, M. and VilaroÂ , S. (1997)Growth and di�erentiation factors inhibit the migratoryphenotype of cultured neonatal rat hepatocytes inducedby HGF/SF. Exp. Cell. Res. 235, 170±179.

Parelman, J. J., Nicolson, M. and Pepose, J. S. (1990)Epidermal growth factor in human aqueous humor.Am. J. Ophthalmol. 109, 603±604.

Polk, B. D. and Tong, W. (1999) Epidermal and hepatocytegrowth factors stimulate chemotaxis in an intestinal cellline. Am. J. Physiol. 277, C1149±C1159.

Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., della Zonca,P., Giordano, S., Graziani, A., Panayotou, G. andComoglio, P. M. (1994) A multifunctional docking sitemediates signalling and transformation by the scatterfactor/hepatocyte growth factor receptor family. Cell77, 261±271.

Potempa, S. and Ridley, A. J. (1998) Activation of both MAPkinase and phosphatidylinositide-3-kinase is requiredfor hepatocyte growth factor/scatter factor-inducedadherens junction disassembly. Mol. Biol. Cell. 9, 2185±2200.

Prat, M., Narsimhan, R. P., Crepaldi, T., Nicotra, M. R.,Natali, P. G. and Comoglio, P. M. (1991) The receptorencoded by the human c-MET oncogene is expressed inhepatocytes, epithelial cells and solid tumors. Int. J.Cancer 49, 323±328.

Reddan, J. R., Dziedzic, D., Fleming, T. P., Smith, D. A. andAudley, U. P. (1996) Identi®cation of factors thatstimulate and inhibit growth of cultured human lensepithelial cells. Invest. Ophthalmol. Vis. Sci. 37(Suppl),145.

Retina Society Terminology Committee (1983) The classi®-cation of retinal detachment with proliferative vitreo-retinopathy. Ophthalmology 90, 121±125.

Ritch, R., Lippes, J., Hu, D. N., Fihlo, R. G. andSrivasthana, B. (1999) Hepatocyte growth factor(HGF) in human aqueous humor. Invest. Ophthalmol.Vis. Sci. 40(Suppl), S677.

Rosen, E. M. and Goldberg, I. D. (1995) Scatter factor andangiogenesis. Adv. Cancer Res. 67, 257±279.


Rosen, E. M., Meromsky, L., Goldberg, I., Bhargava, M. andSetter, E. (1990) Studies on the mechanism of scatterfactor. E�ects of agents that modulate intra-cellular sig-nal transduction, macromolecule synthesis and cytoske-leton assembly. J. Cell Sci. 96, 639±649.

Rosen, E. M., Knesel, J., Goldberg, I. D., Jin, L., Bhargava,M., Joseph, A., Zitnik, R., Wines, J., Kelley, M. andRockwell, S. (1994a) Scatter factor modulates the meta-static phenotype of the EMT6 mouse mammary tumor.Int. J. Cancer 57, 706±714.

Rosen, E. M., Laterra, J., Jin, L., Way, D., Witte, M.,Weinand, M. and Goldberg, I. D. (1996) Scatter factorexpression and regulation in human glial tumors. Int. J.Cancer 67, 248±255.

Rosen, E. M., Nigam, S. K. and Goldberg, I. D. (1994b)Scatter factor and the c-Met receptor: a paradigm formesenchymal/epithelial interaction. J. Cell. Biol. 127,1783±1787.

Russell, W. E., McGowan, J. A. and Butcher, N. L. (1984)Partial characterisation of a hepatocyte growth factorfrom rat platelets. J. Cell. Physiol. 119, 183±192.

Sachs, M., Weidner, K. M., Brinkmann, V., Walther, I.,Obermeier, A., Ullrich, A. and Birchmeier, W. (1996)Motogenic and morphogenic activity of epithelial recep-tor tyrosine kinases. J. Cell. Biol. 133, 1095±1107.

Santos, O. F., Barros, E. J., Yang, X. M., Matsumoto, K.,Park, M. and Nigam, S. K. (1994) Involvement of hep-atocyte growth factor in kidney development. Dev. Biol.163, 525±529.

Schultz, G., Khaw, P. T., Oxford, K., Macauley, S., VanSetten, G. and Chegini, N. (1994) Growth factors andocular wound healing. Eye 8, 184±187.

Scotlandi, K., Baldini, N., Oliviero, M., Di Renzo, M. F.,Martano, M., Serra, M., Manara, M. C., Cornoglio, P.M. and Ferracini, R. (1996) Expression of met/hepato-cyte growth factor receptor gene and malignant beha-viour of musculoskeletal tumours. Am. J. Pathol. 149,1209±1219.

Shibamoto, S., Hayakawa, M., Hori, T., Oku, N., Miyazawa,K., Kitamura, N. and Ito, F. (1992) Hepatocyte growthfactor and transforming growth factor beta stimulateboth cell growth and migration of human gastricadenocarcinoma cells. Cell. Struc. Funct. 17, 185±190.

Shimomura, T., Miyazawa, K., Komiyama, Y., Hiraoka, H.,Naka, D., Morimoto, Y. and Kitamura, N. (1995)Activation of hepatocyte growth factor by two homolo-gous proteases, blood-coagulation factor XIIa and hep-atocyte growth factor activator. Eur. J. Biochem. 229,257±261.

Shinoda, K., Ishida, S., Kawashima, S., Wakabayashi, T.,Matsuzaki, T., Takayama, M., Shinmura, K. andYamada, M. (1999) Comparison of the levels of hep-atocyte growth factor and vascular endothelial growthfactor in aqueous ¯uid and serum with grades of retino-pathy in patients with diabetes mellitus. Brit. J.Ophthalmol. 83, 834±837.

Sibayan, S. A. B., Latina, M. A., Sherwood, M. E., Flotte, T.J. and White, K. (1998) Apoptosis and morphologicchanges in drug-treated trabecular meshwork cells invitro. Exp. Eye Res. 66, 521±529.

Sonnenberg, E., Meyer, D., Weidner, K. M. and Birchmeier,C. (1993) Scatter factor/hepatocyte growth factor andits receptor, the c-met tyrosine kinase can mediate a sig-nal exchange between mesenchyme and epithelia duringmouse development. J. Cell. Biology 123, 223±235.

Soriano, J. V., Pepper, M. S., Nakamura, T. and Montesano,R. J. (1995) Hepatocyte growth factor stimulates exten-sive branching duct like structures by cloned mammarygland epithelial cells. J. Cell. Sci. 108, 413±430.

Stoker, M. (1989) E�ect of scatter factor on motility of epi-thelial cells and ®broblasts. J. Cell. Physiol. 139, 565±569.

Stoker, M. and Perryman, M. (1985) An epithelial scatter fac-tor released by embryo ®broblasts. J. Cell. Sci. 77, 209±223.

Stoker, M., Gherardi, E., Perryman, M. and Gray, J. (1987)Scatter factor is a ®broblast-derived modulator of epi-thelial cell mobility. Nature 327, 239±242.

Strain, A. J. (1993) Hepatocyte growth factor: another ubiqui-tous cytokine. J. Endocrinol. 137, 1±5.

Swann, D. A. and Constable, I. J. (1972) Vitreous structure:Distribution of hyaluronate and protein. Invest.Ophthalmol. 11, 159±163.

Takahashi, M., Ota, S., Shimada, T., Hamada, E., Kawabe,T., Okudaira, T., Matsumura, M., Kaneko, N., Terano,A. and Nakamura, T. (1995) Hepatocyte growth factoris the most potent endogenous stimulant of rabbit gas-tric epithelial cell proliferation and migration in pri-mary culture. J. Clin. Invest. 95, 1994±2003.

Tamagnone, L. and Comoglio, P. M. (1997) Control of inva-sive growth by hepatocyte growth factor (HGF) and re-lated scatter factors. Cytokine Growth Factor Rev. 8,129±142.

Tamura, M., Arakaki, N., Tsoubouchi, H., Takada, H. andDaikuhara, Y. (1993) Enhancement of human hepato-cyte growth factor production by interleukin-1 alphaand -1 beta and tumor necrosis factor-alpha by ®bro-blasts in culture. J. Biol. Chem. 268, 8140±8145.

Tashiro, K., Hagiya, M., Nishizawa, T., Seki, T., Shimonishi,M., Shimizu, S. and Nakamura, T. (1990) Deduced pri-mary structure of rat hepatocyte growth factor and ex-pression of the messenger RNA in rat tissues. Proc.Natl. Acad. Sci. USA 87, 3200±3204.

Tervo, T., Vesaluoma, M., Bennett, G. L., Schwall, R.,Helena, M., Liang, Q. and Wilson, S. E. (1997) Tearhepatocyte growth factor (HGF) availability increasesmarkedly after excimer laser surface ablation. Exp. EyeRes. 64, 501±504.

Tripathi, R. C., Borisuth, N. S. C., Li, J. and Tripathi, B. J.(1994) Growth factors in the aqueous humor and theirclinical signi®cance. J. Glau. 3, 248±258.

Trusolino, L., Pugliese, L. and Comoglio, P. M. (1998a)Interactions between scatter factors and their receptors:hints for therapeutic applications. FASEB J. 12, 1267±1280.

Trusolino, L., Serini, G., Cecchini, G., Besati, C., AbesiImpiombata, F. S., Marchisia, P. C. and De Filippi, R.(1998b) Growth factor dependent activation of avb3integrin in normal epithelial cells: implications fortumour invasion. J. Cell. Biol. 142, 1145±1156.

Tsarfaty, I., Rong, S., Resau, J. H., Rulong, S., da Silva, P.P. and Vande Woude, G. F. (1994) The met proto-oncogene mesenchymal to epithelial cell conversion.Science 263, 98±101.

Tsubouchi, H., Hirono, S., Gohda, E., Nakayama, H.,Takahashi, K., Sugihara, J., Tomita, E. and Muto, Y.(1989) Clinical signi®cance of human hepatocyte growthfactor in blood from patients with fulminant hepaticfailure. Hepatology 9, 875±881.

Tuck, A. B., Park, M., Sterns, E. E., Boag, A. and Elliott, B.


E. (1996) Coexpression of hepatocyte growth factorand receptor (Met) in human breast carcinoma. Am. J.Pathol. 148, 225±232.

van der Geer, P., Hunter, T. and Linberg, R. A. (1994)Receptor protein-tyrosine kinases and their signal trans-duction pathways. Ann. Rev. Cell. Biol. 10, 251±337.

van der Voort, R., Taher, E. I., Keehnen, M. J., Smit, L.,Groenink, M. and Pals, S. T. (1997) Paracrine regu-lation of germinal center B cell adhesion through c-Met-hepatocyte growth factor/scatter factor pathway.J. Exp. Med. 185, 2121±2131.

Wang, M. H., Yoshimura, T., Skeel, A. and Leonard, E. J.(1994) Proteolytic conversion of single chain precursormacrophage-stimulating protein to a biologically activeheterodimer by contact enzymes of the coagulation cas-cade. J. Biol. Chem. 269, 3436±3440.

Waring, G. O., Bourne, W. M., Edelhauser, H. F. andKenyon, K. R. (1982) The corneal endothelium:Normal and pathologic structure and function.Ophthalmol. 89, 531±590.

Warn, R. (1995) The scattered jigsaw. Nature 376, 723.Weidner, K. M., Behrens, J., Vandekerckhove, J. and

Birchmeier, W. (1990) Scatter factor: Molecular charac-teristics and e�ect on the invasiveness of epithelial cells.J. Cell. Biol. 111, 2097±2108.

Weidner, K. M., Di Cesare, S., Sach, M., Brinkmann, V.,Behrens, J. and Birchmeier, W. (1996) The interactionbetween Gab I and the c-met receptor tyrosine kinase isresponsible for epithelial morphogenesis. Nature 384,173±176.

Weidner, K. M., Arakaki, N., Hartmann, G.,Vandekerckhove, J., Reidner, H., Fonatsch, C. and tsu-bouchi, I. (1991) Evidence for the identity of humanscatter factor and human hepatocyte growth factor.Proc. Natl Acad. Sci. USA 88, 7001±7005.

Weidner, K. M., Sachs, M. and Birchmeier, W. (1993) Themet receptor tyrosine kinase transduces motility, pro-liferation and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells. J. Cell.Biol. 121, 145±154.

Weimar, I., de Jong, D., Muller, E. J., Nakamura, T., vanGorp, J. M. H. H., de Gast, G. C. and Gerritsen, W.R. (1997) Hepatocyte growth factor/scatter factor pro-motes adhesion of lymphoma cells to extracellularmatrix molecules via a4b1 and a5b1 integrins. Blood89, 990±1000.

Weimar, I., Miranda, N., Muller, E. J., Hekman, A., Kerst,M., de Gast, G. C. and Gerritsen, W. R. (1998)Hepatocyte growth factor/scatter factor (HGF/SF) isproduced by human bone marrow stromal cells andpromotes proliferation, adhesion and survival of human

hematopoietic progenitor cells (CD34+). Exp. Hematol.26, 885±894.

Weng, J., Mohan, R. R., Li, Q. and Wilson, S. E. (1997) IL-1upregulates IL-1 upregulates keratinocyte growth factorand hepatocyte growth factor mRNA and protein pro-duction by cultured stromal ®broblast cells : interleu-kin-1 beta expression in the cornea. Cornea 16, 465±471.

Wilson, S. E., Walker, J. W., Chwang, E. L. and He, Y. G.(1993) Hepatocyte growth factor, keratinocyte growthfactor, their receptors, ®broblast growth factor recep-tor-2, and the cells of the cornea. Invest. Ophthalmol.Vis. Sci. 34, 2544±2561.

Wilson, S. E., He, Y. G., Weng, J., Zieske, J. D., Jester, J. V.and Schultz, G. S. (1994) E�ect of epidermal growthfactor, hepatocyte growth factor, and keratinocytegrowth factor, on proliferation, motility and di�eren-tiation of human corneal epithelial cells. Exp. Eye Res.59, 665±678.

Wilson, S. E., Liu, J. J. and Mohan, R. R. (1999) Stromal-epithelial interactions in the cornea. Prog. Ret. EyeRes. 18, 293±309.

Woolf, A. S., Kolatsi-Joannou, M., Hardman, P.,Andermarcher, E., Moorbey, C., Fine, L. G., Jat, P. S.,Noble, M. D. and Gheradi, E. (1995) Roles of hepato-cyte growth factor/scatter factor and the met receptorin the early development of the metanephros. J. Cell.Biol. 128, 171±184.

Wordinger, R. J., Clark, A. F., Agarwal, R., Lambert, W.,McNatt, L., Wilson, S. E., Qu, Z. and Fung, B. K. K.(1998) Cultured human trabecular meshwork cellsexpress functional growth factor receptors. Invest.Ophthalmol. Vis. Sci. 39, 1575±1589.

Yamashita, J., Ogawa, M., Yamashita, S., Nomura, K.,Kuramoto, M., Saishoji, T. and Shin, S. (1994)Immunoreactive hepatocyte growth factor is a strongand independent predictor of recurrence and survival inhuman breast cancer. Cancer Res. 54, 1630±1633.

Yanigita, K., Matsumoto, K., Sekiguchi, K., Ishibashi, H.,Niho, Y. and Nakamura, T. (1993) Hepatocyte growthfactor may act as a pulmotrophic factor on lung regen-eration after acute lung injury. J. Biol. Chem. 268,21212±21217.

Yao, Y., Jin, L., Fuchs, A., Joseph, A., Hastings, H. M.,Goldberg, I. D. and Rosen, E. M. (1996) Scatter factorprotein levels in human breast cancers: clinicopathologi-cal and biological correlations. Am. J. Path. 149, 1707±1717.

Zarnegar, R. and Michalopoulos, G. K. (1989) Puri®cationand biological characterization of human hepatopoietinA, a polypeptide growth factor for hepatocytes. CancerRes. 49, 3314±3320.


hepatocyte growth factor/scatter factor in the eye

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