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The multifunctional role of fibroblasts during wound healing

inHirudo medicinalis(Annelida, Hirudinea)

Gianluca Tettamanti a,*, Annalisa Grimaldia, Liliana Rinaldi a, Francesca Arnaboldi b,

Terenzio Congiu c, Roberto Valvassori a, Magda de Eguileor a

a Department of Structural and Functional Biology, University of Insubria - Via J.H. Dunant 3, 21100 Varese, Italyb Department of Human Morphology, University of Milano - Via Mangiagalli 31, 20133 Milan, Italyc Department of Human Morphology, University of Insubria - Via O. Rossi 9, 21100 Varese, Italy

Received 26 November 2003; accepted 15 April 2004

Available online 31 May 2004

Abstract

Extracellular matrix components play a key role during the angiogenic process for a correct development of blood vessels: fibroblasts are

the main cell type involved in the regulation of ECM protein production. In this study we characterize H. medicinalis fibroblasts and

demonstrate that they take part to the regulation of angiogenesis that occurs during wound healing process. Massive proliferation and

phenotypic modification are two distinctive markers of fibroblast activation. These cells, that are usually responsible for collagen production

and function as an energy reservoir, are recruited during wound healing to form a collagen scaffold through a direct mechanic action and

through secretion of specific proteoglycans. In addition we show that the activity of fibroblasts is modulated by EGF, a growth factor involved

in wound healing in vertebrates. The formation of bundles of collagen fibrils by fibroblasts is fundamental for the development and migration

of new blood vessels in lesioned areas during wound repair: administration of lovastatin in explanted leeches affects fibroblasts, damages

collagen scaffold and indirectly causes the reduction of neo-capillary formation.

2004 Elsevier SAS. All rights reserved.

Keywords: Angiogenesis; Collagen; Extracellular matrix; Fibroblast; Leech

1. Introduction

The extracellular matrix (ECM) acts as a scaffold dur-

ing tissue development and repair, providing structural sup-

port and attachment sites for cell surface receptors. More-over, it works as a regulated reservoir for signaling

molecules that modulate diverse processes such as angiogen-

esis, cell proliferation and migration, and inflammation

(Badylak, 2002).

In particular, ECM plays a crucial role during the angio-

genic process (Modlich and Bicknell, 2002).

Vertebrate angiogenesis is a complex process that relies

upon a precise signalling network which regulates pheno-

typic changes, mitogenesis and migration of endothelial cells

(ECs) (Carmeliet, 2003). The migration of ECs into the

surrounding tissue requires both the degradation of the base-

ment membrane of pre-existing vessels by proteolytic en-

zymes (Modlich and Bicknell, 2002), such as matrix metal-

loproteinases (MMPs), and the construction of a stable

matrix scaffold to support new vessel growth (Vernon and

Sage, 1996; Whelan and Senger, 2003).Subsequent migration of ECs in the ECM and vascular

cell survival depend on adhesion to the ECM, coordinated by

integrins such as a5b3, a1b1 and a2b1, which also trigger

specific intracellular signaling events (Senger et al., 1997;

Eliceiri et al., 1998).

The importance of the interaction between ECM and

newly forming vessels is evident in vertebrate wound heal-

ing: while integrins initially bind ligands such as vitronectin,

fibronectin, fibrin and osteopontin, that make a provisional

matrix for ECs, angiogenesis often proceeds in a microenvi-

ronment consisting predominantly of interstitial collagen

(Senger et al., 1997; Whelan and Senger, 2003). Defects in

collagen synthesis impair mechanical stability of the circula-

tory system (Lohler et al., 1984). During the granulation* Corresponding author.

E-mail address:[email protected] (G. Tettamanti).

Biology of the Cell 96 (2004) 443455

www.elsevier.com/locate/biocell

2004 Elsevier SAS. All rights reserved.

doi:10.1016/j.biolcel.2004.04.008

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tissue stage, fibroblasts greatly contribute to angiogenesis by

synthesizing fibronectin and proteoglycans; growth factor

secretion also contributes to the process (Moulin, 1995).

We have previously demonstrated that in Hirudo medici-

nalis the wound healing process and neo-angiogenesis fol-low the same sequence of events described in vertebrates (de

Eguileor et al., 2001a; de Eguileor et al., 2001b; Tettamanti et

al., 2003a; Tettamanti et al., 2003b). In addition, a massive

change in collagen organization has been observed during

formation of new vessels (de Eguileor et al., 2001a; Tetta-

manti et al., 2003a).

In the present work we characterize, in theH. medicinalis

model, fibroblasts actively involved in tissue repair by mor-

phological, immunohistochemical and biochemical proce-

dures. Moreover, we show that lovastatin, a molecule able to

alter fibroblast activity (Koch et al., 1997), can indirectly

inhibit angiogenesis during the wound healing process.Our data demonstrate an active involvement of fibroblasts

in the formation of the blood vessel network that takes place

during wound healing in leeches.

2. Results

2.1. Morphology of fibroblasts: optical microscopy,

Transmission Elecron Microscopy (TEM) and Scanning

Electron Microscopy (SEM)

In untreatedHirudoa thick layer of ECM filled the space

between circomyarian muscle fibers of the body wall: few

fibroblasts were observed in this collagenous matrix(Fig. 1A).

Fibroblasts appeared as elongated cells with cytoplasm

almost completely filled by spherical lipid droplets of about

1-2 m, which compressed the nucleus, confering it a flat-

tened shape (Fig. 1B). TEM pictures revealed also the pres-

ence of Golgi apparatus (Fig. 1C), centrioles (Figs. 1C, 1D)

and mitochondria (Fig. 1E), localized in the scanty cyto-

plasm and partially concealed by lipid droplets; vesicles of

irregular shape were located in the juxtamembrane area

(Fig. 1E).

Electron microscopy observations evidenced the exist-

ence of two different fibroblast cell phenotypes. While inunlesioned leeches the majority of fibroblasts were spindle-

shaped (Fig. 1F), after surgical stimulation of the animal,

fibroblasts appeared as more tapered cells with lateral fold-

ings on their surface: these structures run along the major

axis of the cell for its entire lenght (Fig. 1G). Accordingly,

cross-sections of cells were different in the two cases: in the

former situation, the cell presented an ovoidal shape with a

smooth membrane (Fig. 1B); in the latter, the presence of the

cytoplasmic membrane laminae gave to the cell a stellate

shape (Fig. 1H). These membrane expansions appeared to

bind few (Fig. 1I)or numerous extracellular collagen fibrils

(Figs. 1J, 1K). The presence of small bulges on the cell outer

surface due to the thick packaging of cytoplasmic lipids

(Figs. 1F, 1G)confirmed that both kinds of cells (i. e. those

spindle-shaped and those with membrane foldings) were

fibroblasts.

2.2. Characterization of fibroblasts

In order to characterize the cytosolic content of fibroblasts

and to evaluate the mitogenic activity of these cells, we

performed two immunohistochemical labelings, three sepa-

rate histochemical stainings and a BrdU proliferation assay

on unlesioned and surgically explanted H. medicinalis.

Collagen I and bFGF receptor/Flg were chosen as specific

markers to identify fibroblasts. Besides being massively ex-

pressed in the leech ECM, collagen I (red signal) was de-

tected in the fibroblast cytoplasm (Figs. 2A, 2B); staining for

Flg (brown signal) was specifically localized on fibroblasts,

recognizable by lipid droplets (Fig. 2C).

These cytoplasmic droplets were positive for the Oil Red

O (O.R.O) reaction: the red staining, shown in Figure 2D,confirmed the presence of lipids in these structures.

The Alcian Blue critical-electrolyte-concentration (CEC)

method was performed to stain fibroblast proteoglycans.

While in unlesioned animals positivity was practically

absent (Fig. 2E), in fibroblasts of surgically lesioned leeches

Alcian Blue staining, detectable at low concentrations of

MgCl2, persisted at higher concentrations of MgCl2(CECs > 0.5) and produced a localized reaction. In fact, after

surgical lesion, a strong signal was visible in the juxtamem-

brane area of fibroblast (Fig. 2F): in particular, the staining

was localized in vesicles (Figs. 2F, 2G), and the positivity

pattern showing the presence of proteoglycans overlappedthe vesicles seen inFig. 1E.

The potassium ferrocyanide technique allowed us to de-

tect the presence of cytoplasmic calcium ions either in un-

stimulated (Fig. 2H)and stimulated (Fig. 2I)leeches. How-

ever, the intensity of calcium signal increased in surgically

lesioned H. medicinalis (Fig. 2I). The specificity of the

reaction (positive control) was provided by the staining of

calcium in obliquely striated muscle fibers of the leech (Tet-

tamanti et al., 2003b).

A BrdU proliferation assay was performed to monitor the

mitogenic activity of fibroblasts in unlesioned (Fig. 2J)and

explant-stimulated (Fig. 2K)animals.

24 hours after surgery, fibroblasts were found to be posi-

tive to the BrdU reaction (Fig. 2K), while in untreated ani-

mals positivity was almost absent (Fig. 2J). Specificity of the

reaction was monitored by analyzing nuclei of leech muscle

fibers, as described at the end of the next section.

The increase in fibroblast number in explanted leeches,

compared to fibroblast amount in unlesionedHirudo, issum-

marized inFig. 3.

2.3. Effect of EGF administration onHirudo fibroblast

proliferation

We tested the effects of direct intramuscular injection of

epidermal growth factor (EGF) in unlesioned animals

(Fig. 4A), analyzing leeches at 6 hours (Fig. 4B) and 24 hours

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(Fig. 4C) after the stimulation (i. e. EGF injection). The

administration of EGF determined a massive increase of

fibroblasts, gathered in the spaces between muscle fibers, at

the injection site: as early as 6 hours after growth factor

administration, a huge number of cells, with the typicalelongated shape, was visible (Figs. 4B, 5). The number of

cells remained high 24 hours after treatment (Figs. 4C, 5).

BrdU analysis confirmed that, 6 hours after EGF injection,

cells were in active proliferation (Fig. 4D), while at 24 hours

(Fig. 4E) DNA synthesis was practically absent in cell nuclei.

An internal positive control used to evaluate the specificity

of the reaction was provided by the nucleus of muscle cells,

which rest in S-phase during the whole lifespan of the leech

(Fig. 4E)(Tettamanti et al., 2003b).

2.4. Expression of cathepsin B in fibroblasts

The migration of new vessels in the ECM during theangiogenic process is controlled by a finely regulated net-

work of molecules synthesized by cells surrounding newly-

forming vessels, such as fibroblasts. For this reason we ana-

lyzed in fibroblasts the presence of cathepsin B, a secreted

enzyme that favours endothelial cell migration by controlling

matrix metalloproteinase activity (Kostoulas et al., 1999).

Western blot analysis was used to show that the anti-

cathepsin B antibody reacts with the leech protein. Immuno-

blot analysis carried out 3 hours after surgical stimulation

revealed a band of about 35 kDa (Fig. 6A, lane e), while in

unlesionedHirudothe signal for cathepsin B was drastically

reduced (Fig. 6A, lane d). The same results were found inimmunohistochemical stainings. In fact, while in surgically-

stimulated leeches immunohistochemistry revealed that

3 hours after surgery the cytoplasm of fibroblasts was posi-

tively stained by the anti-cathepsin B antibody (Figs. 6B,

6C), in unlesioned animals immunoreactivity was virtually

absent (Figs. 6D, 6E).

2.5. Impairment of angiogenesis by lovastatin treatment

To evaluate the role of fibroblasts in neo-angiogenesis,

leeches were subjected to surgical stimulation (i. e. explant)

and subsequently treated with the compound lovastatin. In

vivo treatment with this drug was well tolerated and we didnot observe any sign of cytotoxicity.

In treated animals, lovastatin (5 M solution) caused sev-

eral changes in fibroblasts (Figs. 7A, 7B): cells became

roundish, showed a lack of cytoplasmic membrane laminae

and a reduction in packaging of lipid droplets (Fig. 7B).

Lovastatin affected not only cells but also the surroundingECM: collagen fibrils were randomly and irregularly ar-

ranged (Fig. 7C), in a pattern completely different from the

highly-packed distribution typical of collagen in wounded

leeches (Fig. 7D).

Neovascularization of the muscle tissue was markedly

reduced (Fig. 7E)when compared to wounded animals re-

ceiving lovastatin vehicle only (Fig. 7F).

After treatment with lovastatin, no fibroblasts resulted

positive in apoptosis assay (Figs. 7G, 7H), while BrdU posi-

tive fibroblasts were still detectable (Fig. 7I).

Alcian Blue staining showed that the distribution of pro-

teoglycans seen in fibroblasts of unlesioned leeches

(Figs. 2F, 2G) was lost in lovastatin-treatedHirudo (Figs. 7J).

In addition, while in lesioned, lovastatin-untreated

leeches, microfilament bundles were visible in the cytoplas-

mic membrane laminae of the fibroblast (Fig. 7K), after

lovastatin administration membrane foldings disappeared

and parallely filaments were localized in the juxtamembrane

area (Fig. 7L).

Botryoidal tissue cells (from which new vessels arise)

were not affected by lovastatin treatment (Fig. 7M) and

showed a morphology comparable to that one present in

lovastatin-untreatedHirudo (Fig. 7N); in addition, a prolif-

erative activity was detectable in their nuclei (Fig. 7O) and

clusters of botryoidal cells showed early stages of bloodvessel lumen formation (Figs. 7M, 7O).

3. Discussion

In the present work we have characterized the activity of

fibroblasts in the regulation of angiogenesis that takes place

during wound healing process in the leech H. medicinalis.

Leech fibroblasts have been described by Bradbury (Brad-

bury and Meek, 1958)as adipose reservoirs (Sawyer, 1986)

and fibrillar collagen producers (Bradbury, 1958; Bradbury

and Meek, 1958). For the identification of this cell type we

have used not only their peculiar morphology, but also sev-eral markers as the positivity for O.R.O. reaction, and the

Fig. 1.Morphology of fibroblasts: optical microscopy, TEM and SEM. A. Semithin section of the body wall of unlesioned Hirudo medicinalisstained with

crystal violet and basic fuchsin. Fibroblasts (arrows) are localized in the ECM. N: nucleus; L: lipid droplets. Scale bar: 10 m. B. TEM image of a leech

fibroblast. The cytoplasm of the cell is completely filled by lipid droplets (L) and the nucleus (N) is pushed toward theplasmamembrane. M: musclefiber. Scale

bar: 5 m. C,D. TEM. Details of fibroblast cytoplasm. In the cytoplasmic space, Golgi apparatus (arrowhead) and centrioles (arrow) are recognizable among

lipid droplets (L). N: nucleus. Scale bars: 0.25 m; 0.5 m. E. TEM. Densely-packed vesicles (arrows) are present under the cell membrane. L: lipid droplets;

ECM: extracellular matrix ; m: mitochondria. Scale bar: 1 m. F. SEM. In unlesioned leeches, fibroblasts arespindle-shaped cells with a smoothmembrane.The

small bulges (arrowheads) on the plasma membrane demonstrate the thick packaging of lipid droplets (L), which are better visible after breaking the membrane

(encircled area).Scalebar: 8 m. G. SEM. After surgical stimulation of theanimal, fibroblastsbecome tapered andshow cytoplasmic expansionson their surface

(arrows),which run along themajor axis of thecell.Arrowhead:bulgescaused by cytoplasmic lipids. Scale bar: 8 m. H. TEM. In surgically-stimulated leeches,

cross-sectioned fibroblasts present membrane foldings (arrows) along the entire cellular surface. N: nucleus; L: lipid droplets; c: collagen fibrils; m:

mitochondria. Scale bar: 2 m.I-K. TEM. Details of cross-sections of tapered cell tips of activated fibroblasts (F) showing cytoplasmic laminae present on thecell surface. These structures(arrows)are in close contactwith small (panelI) or large (panels J and K) bundles of collagen fibrils(c). Scale bars: 0.3m; 1.5 m;

0.5 m.


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expression of collagen I (Bloom and Fawcett, 1968) and

bFGF receptor/Flg (Hughes, 1997).

Surgical wounding induces proliferation of fibroblasts,

paralleled by a change in cell phenotype: in fact, the surgical

stimulus determines the formation of prominent cell surface

foldings that are aligned along the cell axis and are in inti-

mate association with bundles of collagen fibrils. Surpris-

ingly, these morpho-functional changes in activated fibro-

blasts are strikingly similar to those described in vertebrates

byTrelstad and Hayashi (1979)for tendon assembly and by

Fig. 2.Characterization of fibroblasts. A,B. Cryosections of 10 m. Immunolocalization of collagen I (red signal) reveals a positivity both in ECM and in

fibroblast cytoplasm (panel A). Panel A has been obtained by overlaying the fluorescent image onto transmission image (panel B).C. Paraffin section of 7 m.

Immunostaining of Flg. The positivity (brown staining) is detected on fibroblasts. arrows: lipid droplets. D. O. R. O. reaction. Cytoplasmic lipid droplets

(arrows) are markedly stained (positivity: red stain). M: muscle fiber. Scale bar: 10 m. E-G. TEM. Alcian Blue staining (MgCl2 concentration = 0.8 M) in

unlesioned (panel E) and explant-stimulated animals (panels F and G). Positivity (black staining) is detectable only in lesioned Hirudoand localizes in discrete

spots in proximity of the fibroblast membrane (panel F, arrows). These spots correspond to vesicles (panels F and G, arrowheads), within which Alcian staining

is located. L: lipid droplets; c: collagen fibrils. Scale bars: 1 m (E,F); 0.25 m (G).H,I. TEM. Calcium histochemistry (Forbes method). In control leeches

(panel H), the signal for calcium (positivity: black staining) in the fibroblast cytoplasm is weak (arrows); positivity increases markedly (arrows) after surgical

stimulation of the animal (panel I). L: lipid droplets. Scale bars: 1 m. J,K. BrdU proliferation assay. Positivity for BrdU incorporation (brown staining), which

is absent in unlesioned leech fibroblasts (panel J, arrow), is detectable in the nucleus of fibroblasts of surgically-wounded leeches (panel K, arrow). M: muscle

fiber. Scale bars: 10 m.


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Birk and Trelstad (1984)for corneal development. In verte-

brate cornea and in tendons, fibroblasts show a complex

topography that ensures a precise spatial deposition of neo-

synthetized ECM. Since inHirudo, following a tissue injury,

collagen fibrils rearrange according to a peculiar and geo-

metrical organization (de Eguileor et al., 2001a), it is con-

ceivable that in our animal model fibroblasts affect post-

translational arrangement of collagen through the control of

extracellular microenvironments by a mechanic action of the

laminae. Thus these surface recesses defined by the laminaeof leech fibroblast, within which collagen fibrils begin to

assemble into bundles, can be comparable to those described

byTrelstad and Hayashi (1979).An additional feature, strengthening the evidence that

fibroblasts play an important role in the organization of leech

collagen architecture, is the production of proteoglycans, as

demonstrated by Alcian Blue staining (Figs. 2F, 2G). These

macromolecules are visible up to critical-electrolyte-

concentration (CECs) of > 0.5 M, indicating an increase in

sulphation of proteoglycans (Scott and Bosworth, 1990).

This evidence indicates the presence of oversulfated pro-

teoglycans, such as heparan sulphate proteoglycans, chon-

droitin sulphate or keratansulphate (Scott and Bosworth,1990): all these molecules, after secretion in the ECM, can

take part in collagen bundle assembly (de Eguileor, unpub-

lished data), thus driving the formation of vascular tubes, as

previously seen byJackson and coworkers (1994)for human

ECs.

Fig. 3.Quantitative evaluation of fibroblast number in H. medicinalis.

Column 1: unlesioned leeches; column 2: surgically-stimulated leeches

analyzed 24 hours after explant. The data represent mean SEM; *p

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The numerous secreting vesicles located just beneath the

plasma membrane in activated fibroblasts (Fig. 1E) could

indeed be the consequence of proteoglycan secretion: this

hypothesis is supported by the strong Alcian Blue signal

detected within these vesicles. Alternatively, as in verte-

brates, these vesicles could be necessary to reseal local mem-

brane breaks that frequently occur in fibroblasts after tissue

injury, by a progressive fusion of these vescicular units to the

plasma membrane (McNeil and Steinhardt, 1997; Grinnell,

2003). Both suggested roles of these vesicles in stimulated

leeches are supported by an observation on calcium localiza-

tion. The concentration of this ion, as detected by Forbes

staining, changes during fibroblast activity. The intensity of

calcium staining increases in activated fibroblasts, thus indi-

cating a possible role both in regulation of cell secretion

(Rodriguez et al., 1997), and/or in restoration of plasma

membrane integrity, as previously shown by McNeil and

Steinhardt (1997).

In vertebrate wound healing, EGF is an important compo-

nent of the cytokine network regulating fibroplasia and an-

giogenesis through autocrine and paracrine mechanisms

(Schultz et al., 1991; Moulin, 1995). It is well known that in

mice and rats the administration of this growth factor in thelesioned region accelerates the wound healing process by

stimulating proliferation, differentiation and collagen-

proteoglycan production in fibroblasts (Niall et al., 1982;

Buckley et al., 1985; Grzybowski et al., 1999). We have

observed thatHirudofibroblasts are promptly responsive to

human EGF and it proves to be a potent mitogen: the admin-

istration of this growth factor in the animal promotes DNA

synthesis in leech fibroblasts with a consequent increase in

cell number. As early as 6 hours after EGF treatment, BrdU

positivity is high and the number of fibroblasts massively

increases: interestingly, staining is virtually undetect-

able 24 hours after EFG administration. Thus, it appears that

EGF is unable to maintain fibroblasts in S-phase after

24 hours: this may be related to the route of growth factor

administration, given that injected EGF is rapidly degraded

by tissue proteases (Flaumenhaft and Rifkin, 1991).Previously, we have demonstrated a change in the expres-

sion pattern of fibronectin and integrin aVb3, two molecules

that are necessary for the growth of new blood vessels (de

Eguileor et al., 2001a). Since the data reported in the present

study suggest that morpho-functional changes in activated-

secreting fibroblasts are closely related to ECM modifica-

tions, we have investigated the relationship between newlyforming vessels and ECM duringHirudowound healing, in

order to confirm the role of fibroblasts in neo-angiogenesis.

For this reason we treated animals with lovastatin, a drug not

only acting on vertebrate fibroblast functions essential for

normal cell homeostasis, but also able to affect cytoskeletal

organization in these cells (Koch et al., 1997; Tan et al.,

1999).Firstly, we have shown that lovastatin effects are recogniz-

able on leech fibroblasts themselves and, in turn, on the

ECM: cells do not show cytoplasmic laminae typical of

secreting fibroblasts and are characterized by a reduced

amount of proteoglycans within the cell; as a consequence,

ECM collagen fibrils are no longer organized by fibroblast

laminae in the geometrical ECM arrangement typical of

Fig. 5.EGF administration: quantitative evaluation of fibroblast num-

ber. Column 1: control leeches; column 2: leeches treated with EGF and

analyzed 6 hours after injection; column 3: leeches treated with EGF and

analyzed 24 hours after injection. The data represent mean SEM; *p0.05.

Fig. 6.Expression of cathepsin B in fibroblasts. A. Western blot analysis

of unlesioned and explanted H. medicinalis. 3 hours after surgical stimula-

tion the anti-cathepsin B antibody recognizes a band of about 35 kDa (Lane

e); the signal is reduced in unlesioned leeches (Lane d). Lane a: molecular

weight standard; Lane b: unlesioned leech extract; Lane c: explanted leech

extract. Lanes a, b and c are stained with Coomassie blue. B-E. Immunolo-

calization of cathepsin B. The positivity for cathepsin B is detected in the

cytoplasm of surgically-stimulated leech fibroblasts (panels B and C),among lipid droplets. No signal is revealed in unlesioned animal (panels D

and E). Panels B and D have been obtained by overlaying the fluorescent

images onto transmission images (panels C and E). The profile of the

fibroblast is outlined. L: lipid droplets; M: muscle fiber.


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Fig. 7.Impairment of angiogenesis by lovastatin treatment. A-D. Inhibition of angiogenesis by administration of lovastatin. Explanted leeches treated withlovastatin show changesin fibroblastmorphology (panelA, arrowheads). Comparing these cells with control fibroblasts(see Figures 1Aand 1B for comparison)

it is easy to notethat cells becomeroundish (panels A and B) and lipid droplets (L) arenot densely packed(panelB); fibroblastlaminae,typical of activated cells,

are no longer visible (seeFigure 1Hfor comparison). Collagen fibrils, surrounding fibroblasts, are disorganized (panel C) and are not arranged according to the

precise architecture peculiar of wounded leeches ECM (panel D). c: collagen fibrils. Scale bars: 15 m (A); 2 m (B); 1 m (C,D). E,F. Inhibition of

angiogenesisby administration of lovastatin (semithin sections stained with crystal violet and basic fuchsin).The result of the ECM damage caused by this drug

is visible on neo-angiogenesis: the growth of the new blood vessels (arrows) is drastically reduced (panel E) if compared to lesioned animals that have received

lovastatin vehicle only (panel F). M: muscle fibers. Scale bars: 100 m. G,H. Apoptosis assay on lovastatin-treated leeches. Nuclei of fibroblasts, revealed by

DAPI staining (panel G, arrow), are negative (panel H, arrows). I. BrdU proliferation assay performed on lovastatin-treated leeches (positivity: brown staining).

After topical administration of the drug, fibroblasts still remain in S-phase (arrows). Scale bar: 15 m. J. Alcian Blue staining on lovastatin-treated animals. The

positivityfor proteoglycans(black staining) is almostcompletely disappeared (see Figures 2Fand 2G forcomparison): a weak positivity (arrow) canbe detected

under the plasma membrane. L: lipid droplet. Scale bar: 0.5 m.K,L. TEM. Detail of a fibroblast in control (panel K) and lovastatin-treated (panel L) leeches.

While microfilaments form the scaffold of the cytoplasmic membrane laminae of fibroblasts (panel K, bracket), in lovastatin-treated Hirudothese cytoplasmic

expansions are absent and filaments are observable in the juxtamembrane area (panel L, bracket). Fibroblast membrane: arrowhead. c: collagen fibrils; L: lipid

droplet. Scale bars: 150 nm.M-O. Semithin sections of botryoidal tissue in lovastatin-treated (panels M and O) and in control animals (panel N) stained with

crystal violet and basic fuchsin. The morphology of botryoidal tissue cells after lovastatin treatment (panel M) is comparable to that one observed in untreatedHirudo(panel N). In both situations, a vessel cavity (v) is forming. BrdU assay (positivity: brown staining) performed in lovastatin-treated animals reveals a

proliferative activity in botryoidal cells (panel O, arrow). B: botryoidal cell; arrowhead: nucleus of botryoidal cell. Scale bars: 10 m.


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wounded animals. Secondly, topical administration of this

drug in surgically-stimulated leeches drastically reduces the

formation of new vessel networks if compared to lovastatin-

untreatedHirudo. Thirdly, it must be noticed that botryoidal

tissue cells, responsible for blood vessel production (deEguileor et al., 2001b), are not affected by lovastatin treat-

ment: these cells proliferate and early stages of angiogenesis

(i. e. vessel lumen formation) are visible.

Thus only fibroblasts, and not botryoidal tissue cells, are

affected by lovastatin treatment; the impairment of the

fibroblast-collagen system (but not of botryoidal tissue cells)

by lovastatin leads to a decrease in neo-angiogenesis rate:

botryoidal tissue is recruited in the formation of the new

vessels (de Eguileor et al., 2001a) initiating angiogenesis

(see beginning of vessel lumen formation), but the new ves-

sels are not able to grow and migrate given to the lack of

collagen scaffold, usually created by fibroblasts (Badylak,2002) (see diagram inFig. 8).

So lovastatin seems to work as an indirect anti-angiogenic

agent, not directly inhibiting botryoidal tissue cells, but act-

ing on the surrounding ECM used to drive vessel formation

and migration.

It is noteworthy to underline that, following lovastatin

treatment, some fibroblasts still persist in S-phase, while

their cell architecture is affected. These changes are due

neither to apoptosis (no positivity is found in apoptosis as-

say), nor to necrosis (although we could not perform any

direct assay on fibroblast cell culture, no sign related to early

or late stages of necrosis, such as generalized disintegration

of the cell, lysis, dispersal of cell contents into the ECM,nuclear pyknosis, inflammation events (Mosley et al., 2000;

Bhatia, 2004)was found).

Thus, although further evidence is needed to dissect how

lovastatin affects leech fibroblasts, it seems that lovastatin

neither affects completely DNA synthesis, nor induces cell

death (Tan et al., 1999): our hypothesis is that lovastatin is

responsible for an actin cytoskeleton rearrangement (Koch et

al., 1997; Kelynack et al., 2002; Maddala et al., 2003),

undermining in this way the efficiency of the collagen bundle

assembly performed by fibroblasts, but not completely im-

pairing DNA synthesis machinery.

The role of fibroblasts in the control of angiogenesis issupported by additional observations: the upregulation of

cathepsin B in the cytoplasm of activated fibroblasts is a

marker of the possible involvement of these cells in the

regulation of neovascularization through the control of in-

hibitors of matrix metalloproteases, enzymes required for

endothelial cell migration during development of blood cap-

illaries (Kostoulas et al., 1999).

In conclusion, we have described in Hirudo medicinalisa

direct involvement of fibroblasts in the rearrangement of

matrix collagen during wound healing. Fibroblasts, follow-

ing tissue injury, become responsive to growth factors such

as EGF, proliferate and, not only synthesize collagens and

proteoglycans, but also exert a mechanical action on collagen

packaging. These activities, together with the

synthesis/secretion of cathepsin B, ensure the creation of a

well-organized ECM scaffold, that, according to our prelimi-

nary evidences in lovastatin experiments, may be used by

endothelial cells to drive mature vessel formation and capil-

lary network extension.

4. Materials and methods

4.1. Animals and treatments

Leeches (Hirudo medicinalis, Annelida, Hirudinea, from

Ricarimpex, Eysines, France) measuring 10 X 1.00 cm were

kept in water at 22-23C in aerated tanks, and were fed

monthly with calf blood. Before each experiment leeches

were starved for 4 weeks.

Animals were randomly divided into separate experimen-

tal groups (10 animals/group).Injections or surgical lesions (explant) were performed at

the distal portion of the animal, about 2/3 from the oral side,

at about the 80th dorsal superficial metamere.

Before surgical procedures, treatments and fixation,

leeches were anaesthetised with a saturated solution of me-

phenesin (3-o-toloxy-1,2-propanediol).

4.1.1. Leeches: surgical stimulation

To stimulate wound healing response and angiogenesis,

leeches were subjected to lesions consisting of a tissue ex-

plant (2 mm X 2 mm X 2 mm) affecting the entire thickness

of body wall (including cuticle, epithelium and the three

muscle layers). The explanted tissue was surgically removedwith microdissecting scissors.

At different times after surgery, leeches were processed

for morphology, immunohistochemistry, histochemistry,

BrdU and western blot analyses, lovastatin treatments.

4.2. Light and Transmission Electron Microscopy (TEM)

Leeches were dissected and fixed in 2% glutaraldehyde

(in 0.1 M Na-cacodylate buffer, pH 7.2) for 2 h at room

temperature. Specimens were then postfixed for 2 h with 1%

osmic acid in 0.1 M Na-cacodylate buffer at room tempera-

ture, dehydrated in ethanol series and embedded in an Epon-

Araldite 812 mixture. Sections were cut with a ReichertUltracut S ultratome (Leica, Nussolch, Germany). Semithin

sections were stained by conventional methods (crystal violet

and basic fuchsin) and observed with a light microscope

(Olympus,Tokyo, Japan). Images were acquired with a Leica

DC 300F camera.

Thin sections were stained with uranyl acetate and lead

citrate, and observed with a Jeol 1010 EX electron micro-

scope (Jeol, Tokyo, Japan).

4.3. Scanning Electron Microscopy (SEM)

To obtain three-dimensional imaging by SEM, cross sec-

tions of the leech body (1 mm thick) from control and

wounded animals were submitted to osmic maceration that


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Fig. 8. Diagram of how lovastatin action on Hirudo fibroblasts determines a reduction in neo-angiogenesis. Left. In explanted leeches, botryoidal tissue cell

activation leads to vessel cavity formation, while the collagen scaffold created by fibroblasts is necessary for blood vessel formation/migration. Right. In

explanted leeches, treated with lovastatin, fibroblast action is altered: thus, botryoidal tissue is activated, but the new vessel is unable to growth, due to the lack

of collagen scaffold. V: vessel cavity.


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improves, removing cytosol, the visualization of cytoplasmic

ultrastructure. Afterward samples were fixed with 0.25%

glutaraldehyde and 0.25% paraformaldehyde in 0.1 M Na-

cacodylate buffer (pH 7.2) for 20 min at room temperature.

The specimens, washed in 0.1 M cacodylate buffer (pH 7.2),were postfixed in a solution of 1% osmium tetroxide and

potassium ferrocyanide for 2 h. Each specimen was cut into

slices about 0.2 mm thick and was post-fixed in 1% osmium

tetroxide and 1.25% potassium ferrocyanide for 1 h. Slices

were washed in phosphate buffered saline (PBS) (pH 7.2)

and then immersed in 0.1% osmium tetroxide in PBS for

48 h. Slices were dehydrated in an increasing series of etha-

nol, subjected to critical point drying with CO2. Dried slices

were mounted on stubs, gold coated with a Sputter K250

coater, and then observed with a SEM-FEG XL-30 micro-

scope (Philips, Eindhoven, The Netherlands).

4.4. Alcian Blue staining

Samples from control and explanted leeches were treated

according toRuggeri et al. (1975),to stain proteoglycans.

Tissue samples were fixed for 2-4 h at 4C in 3% glutaral-

dehyde (in PBS 0.1 M, pH 7.4) and were then reduced into

small blocks of about 100 m. After a preincubation for 1 h at

room temperature in 25 mM acetate buffer, pH 5.8, contain-

ing MgCl2at the same concentration used in the subsequent

staining solution, samples were incubated in the staining

solution (0.05 % w/v Alcian Blue 8 GX in 25 mM acetate

buffer and MgCl2at final concentrations of 0.05 M, 0.3 M,

0.8 M, 1 M, 1.2 M) for 7 h. After rinsing for 1 h in MgCl2-

acetate buffer, specimens were placed first in 0.01 N HCl for

4 h, then in distilled water for 1 h, and finally in PBS for

30 min. Dehydration in an ethanol series preceded inclusion

in Epon resin.

4.5. Histochemistry

4.5.1. Calcium

To reveal the presence of calcium in examined tissues,

specimens from control and explanted leeches were post-

fixed in the presence of 2% osmic acid/0.8% potassium

ferrocyanide for 2 h according to Forbes et al. (1977).

4.5.2. LipidsFrozen sections (10 m thick) were stained using a his-

toenzymatic kit (Bio-Optica, Milan, Italy). Positivity for

O.R.O. reaction was evidenced as a red stain (Dubowitz and

Brooke, 1973). Section were counterstained with crystal vio-

let.

4.6. Leeches: EGF treatment

Leeches were given an intramuscular injection of 10 l

human epithelial growth factor (EGF) solution (Pepro Tech,

London, U.K.) (5 ng/l dissolved in PBS, pH 7.2). After 3 h,

treatment with EGF was repeated at the same site.During treatments, animals were kept in moist chambers.

Unlesioned animals or animals injected with 20 l PBS were

used as controls.

6 and 24 h after the first injection, the treated area was

processed for electron microscopy as described above.Within this group, three leeches were used to monitor cell

proliferation at the explant site by the 5-Bromodeoxyuridine

(BrdU) labeling technique (see BrdU proliferation assay sec-tion).

4.7. Lovastatin treatments

Leeches were subjected to lesions consisting of a tissue

explant (2 mm X 2 mm X 2 mm) affecting the entire thick-

ness of body wall; subsequent lovastatin treatment was per-

formed by topical administration of 15 l of a 5 M solution

of the drug in the area of the explant.Each animal was treated twice at an interval of 24 h.

During treatment, leeches were kept in moist chambers, to

avoid dispersion of topically administered drugs in tank

water. Animals were analyzed 48 h after the first treatment:

they were fixed for routine microscopy, Alcian Blue staining,

BrdU and apoptosis assays. ExplantedHirudo, administered

with 30 l lovastatin vehicle were used as controls.Identification of new blood vessels in the muscle body

wall was accomplished by direct observation with an optical

microscope as described in de Eguileor et al. (2001a): it

should be noted that leeches do not have circulating red blood

cells.

4.8. 5-Bromodeoxyuridine (BrdU) proliferation assay

S-phase cells were detected by using a cell proliferation

kit (Amersham-Pharmacia, Buckinghamshire, UK). Three

leeches, previously injected with 100 ng EGF, or subjected totissue explant, or to lovastatin treatment, and a leech injected

with PBS as control, were immersed in tap water containing

BrdU at a final concentration of 0.05% for 6 h before fixation.

Fixation was performed 24 h after explant or 6-24 h after

EGF injection or 48 h after lovastatin administration. After

BrdU incubation leeches were washed in clean, non-labeled

running tap water and then prepared for electron microscopy

using routine techniques described above. Semi-thin sections

were treated for 2 min with a resin-removing mixture (2 g

KOH in 5 ml propylene oxide and 10 ml methanol) and then

rinsed with methanol and placed in PBS. Sections were

incubated overnight at 4C with a monoclonal antibodyagainst BrdU diluted 1:60 in PBS. A pretreatment of 20 min

with 0.3% H2O2in PBS was performed to quench the poten-

tial activity of endogenous peroxidase. Sections were incu-

bated for 3 h with peroxidase-conjugated anti-mouse IgG at

room temperature and subsequently incubated with 0.05 %

3,3-diaminobenzidine (DAB) and 0.03 % H2O2 in PBS.

They were then rinsed in distilled water. Sections were coun-

terstained with crystal violet. Controls were performed by

omitting the primary antibody.

4.9. Immunohistochemistry

4.9.1. bFGF receptor (Flg)

Unlesioned leeches were fixed in 4% paraformaldehyde

(in 0.1 M PBS,pH 7.2) for 3 h atroom temperature.After five


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washes in PBS, samples were dehydrated in an ethanol se-

ries, treated in Bioclear (BioOptica) and paraffin embedded.

For immunohistochemical labelling, paraffin sections

(7 m) were deparaffinized by a treatment with Bioclear,

rehydrated in an ethanol series and then washed 4 times in

PBS.

After incubation with a blocking solution (2% bovine

serum albumin (BSA), 0.01 Tween 20 in PBS) for 30 min the

sections were incubated with the primary antibody (anti-

human Flg polyclonal antibody (Santa Cruz Biotechnology,

Santa Cruz, CA, U.S.A.) (1:20)). Binding was visualized by

incubation for 1 h with a horseradish peroxidase-conjugated

antibody (diluted 1:50), (Santa Cruz). A DAB substrate was

used to detect secondary antibody. Sections were counter-

stained with Meyer hematoxylin and eosin. Controls were

obtained by omitting the primary antibody, and treating sec-

tions with BSA-containing PBS.

4.9.2. Cathepsin B - Collagen I

Unlesioned leeches and animals at different times after

lesion were anesthetized and dissected in a cold Ringer

solution into small blocks, that were immediately embedded

in PolyFreeze cryostat embedding medium (Polyscience Eu-

rope, Eppelheim, Germany), and stored in liquid nitrogen.

Cryosections (10 m thick) of unfixed leeches were obtained

with a Leica CM 1850; slides were immediately used or

stored at -20C.

Sections were incubated for 15 min with a 1% solution ofEvans blue to reduce autofluorescence (De la Lande and

Waterson, 1968), washed with PBS, and incubated for

30 min with a blocking solution (2% BSA, 0.01 Tween 20 in

PBS). Sections were then incubated for 1 h with the primary

antibody/antiserum (anti-human cathepsin B monoclonal an-

tibody (Calbiochem, San Diego, CA, U.S.A.) (1:20); anti-

human Collagen I (Santa Cruz (1:40)). After incubation with

primary antibody, specimens were washed and incubated

with an appropriate fluorescein isothiocyanate (FITC)-

conjugated or tetramethylrhodamine (TRITC)-conjugated

secondary antibodies (diluted 1:100), (Jackson, Immuno Re-

search Laboratories, West Grove, PA, U.S.A.) for 1 h in a

dark moist chamber. The PBS buffer used for washing steps

and antibody dilutions contained 2% BSA. In control

samples, antibodies were omitted and sections were treated

with BSA-containing PBS.

Coverslips were mounted in Vectashield Mounting Me-

dium for fluorescence (Vector Laboratories, Burlingame,

CA, U.S.A.), and slides were examined with a confocal laser

microscope (laser 492 nm for fluorescein, laser 568 nm for

rhodamine) (MRC 1024, Bio-Rad Laboratories, Hemel

Hempstead, UK) using X40 and X63 objectives (NA 1.30,

1.25). Confocal images were superimposed using the Photo-

shop 5.0 program: fluorescent images were overlayed onto

transmission images showing the corresponding tissue sec-

tions.

4.10. Apoptosis assay

Paraformaldehyde-fixed tissue sections of lovastatin-

treated leeches were deparaffinized, dehydrated in an ethanol

series and then washed 3 times in PBS. Detection of apopto-sis by the TUNEL assay was carried out using the Apoptosis

Detection System-Fluorescein (Promega, Madison, WI,

U.S.A.). DAPI (4,6-Diamidino-2-phenylindole dihydro-

chloride) was used for nuclear staining.

4.11. Biochemical procedures

Specimens of unlesioned and explantedHirudo medicina-

lis, were homogenized in liquid nitrogen with a mortar.For SDS-polyacrylamide gel electrophoresis (SDS-

PAGE), homogenates were suspended in extraction buffer

(Laemmlis Buffer 2X) in presence of protease inhibitor

cocktail (Sigma, Milan, Italy); particulate material was re-

moved by centrifugation at 13000 rpm for 10 min at 4C in a

refrigerated Eppendorf Minispin microcentrifuge. Superna-

tants were denatured at 100C for 10 min. Samples were

assayed for protein concentration with the Coomassie Bril-

liant Blue G-250 (Pierce, Rockford, IL, U.S.A.) protein as-

say, using bovine serum albumin as standard.

4.11.1. SDS-PAGE

Equal amounts of the solubilized proteins were separated

in analytical SDS-PAGE using 15% acrylamide minigels.

Molecular weights were determined by concurrently running

broad range standards from Bio-Rad (Bio-Rad, Richmond,

MA, USA). Gels were stained with 2.5% Coomassie blue(Bio-Rad) in methanol:acetic acid:water 5:1:5.

4.11.2. Western blot

Proteins separated by SDS-PAGE were transferred onto

Bio-Rad nitrocellulose filters. Before immunostaining,

membranes were saturated with 2% BSA in Tris-buffered

saline (TBS, 20mM Tris-HCl buffer, 500mM NaCl, pH 7.5)

at room temperature for 2 h. Nitrocellulose membranes were

incubated overnight at 4C with a mouse anti-human cathe-

psin B antibody (1:250 dilution in 2% TBS-BSA).After three

washes of the membrane with TBS, antigens were revealed

with an appropriate secondary antibody (1:1000) coupled

with alkaline phosphatase (Sigma). Immunoreactivity wasdetected with SIGMA FAST BCIP/NBT (Sigma).

4.12. Cell counting

The number of fibroblasts in various experimental condi-

tions (control, explant 24 hs, EGF injection 6 h, EGF injec-

tion 24 h) was determined according to the following proce-

dure.

Three leeches were used for each counting. For each leech

we randomly selected three sample body wall areas of 250 x

250 m (62500 m2) in the analysis region, and in each area

we counted the number of cells using three serial sections

(1 m thick) stained with crystal violet and basic fuchsin.

Fibroblasts were morphologically identified at optical micro-

scope.


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Counts were analyzed using the Students ttest.

It must be underlined that in BrdU experiments the count-

ing of the positive fibroblasts was not feasible, since not all

their nuclei were included in each plane of section: thus in

every experiment we could count only the overall number offibroblasts.

Acknowledgements

We are thankful to Dr Gianpaolo Perletti for critical dis-

cussion of the manuscript; we thank Luisa Guidali for tech-

nical help and Dr Roberto Ferrarese for graphical assistance.

This work has been supported in part by Progetto di

Eccellenza per la Ricerca di Ateneo, University of Insubria,

year 2003-2004.

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