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Fibroblast Responses to Exogenous and Autocrine Growth FactorsRelevant to Tissue Repair

The Effect of Aging

DIMITRIS KLETSAS,a,b HARRIS PRATSINIS,b IRENE ZERVOLEA,b PANAGIOTIS HANDRIS,b ELENI SEVASLIDOU,b ENZO OTTAVIANI,c ANDDIMITRI STATHAKOSb

bInstitute of Biology, National Centre for Scientific Research “Demokritos,” 153 10, Athens, GreececDepartment of Animal Biology, via Berengario 14, University of Modena, 41100 Modena, Italy

ABSTRACT: The aging process is often associated with impaired wound healing,but the cellular and molecular mechanisms implicated are not completely un-derstood. Accordingly, we have investigated the response of human fibroblastsfrom donors of various ages to platelet-derived and autocrine growth factors,in terms of mitogenicity as well as extracellular matrix synthesis and degrada-tion. Our data indicate that fibroblast responses persist during aging, suggest-ing the involvement of systemic factors in the regulation of the healing process.In this context, we have found that neutral endopeptidase-24.11, a metallopro-teinase controlling the action of neuroendocrine peptides and also of immuno-cyte chemotaxis, is overexpressed during aging. Finally, the connectionbetween these data and those from in vitro aging studies is discussed.

INTRODUCTION

A prominent demonstration of homeostasis in the adult tissue is the regulation ofthe tissue repair process. Wound repair is a complex procedure, involving an intri-cate interplay among a variety of cells, growth factors, as well as extracellular matrixproteins and the proteases that degrade them, and is characterized by an orderly se-quence of events that can be temporally categorized into three major phases: inflam-mation, tissue formation, and remodeling. The three stages, however, are notmutually exclusive but rather overlap in time.1

Immediately after wound formation, blood platelets aggregate and subsequentlysecrete a plethora of growth factors that (a) chemotactically attract several cell typesinto the wound area, (b) stimulate their proliferation, and (c) induce the secretion of

aAddress for correspondence: Dr. D. Kletsas, Institute of Biology, National Centre for Scien-tific Research “Demokritos,” 153 10, Athens, Greece. Phone: +30 1 6503565; fax: +30 16511767.

dkletsas@mail.demokritos.gr

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extracellular matrix components. On the other hand, in the last phases of the repairprocess, as well as in the intact tissue, platelet-derived mitogens are obviously ab-sent. However, the cells still secrete growth factors that regulate the above processesand, ultimately, tissue homeostasis in an autocrine and paracrine manner.

Of the various cell types implicated in wound repair in the connective tissue, fi-broblasts are among the most important ones, because they migrate and proliferatein the wound area, they secrete autocrine growth factors, and they synthesize and de-posit extracellular matrix (ECM) components—critical for wound closure andcontraction—and, finally, also secrete proteases that regulate tissue remodeling.

Aging is one of the major events that affects the wound repair process, often lead-ing to impaired and/or delayed healing. Diminished and delayed connective tissuedeposition, reduced fibroblastic activity and growth, as well as less efficient woundcontraction characterize the response to tissue injury in the elderly, in comparison tothe response in younger individuals. However, the cellular and molecular mecha-nisms underlying these changes are not completely delineated and are under intenseinvestigation.2

The aim of this study was to address the question of whether the differences inthe wound repair process between young and aged individuals is also expressed atthe cellular level or if they are the consequence of a generalized, systemic failure ofhomeostasis. In this context, previous studies have shown that in vitro senescent fi-broblasts are unable to respond to mitogenic stimuli3 and, furthermore, that they pro-duce increased amounts of collagenases,4 leading to the hypothesis that theaccumulation of senescent cells in the tissues of old individuals could affect local tis-sue homeostasis.

Here, an alternative approach has been followed: the study of the features of fi-broblasts aged in vivo, that is, cells derived from donors of different ages. In partic-ular, several important parameters concerning tissue repair have been investigated,namely the ability of these cells to proliferate after mitogenic stimulation, the secre-tion of autologous growth factors, and the production and secretion of ECM constit-uents, as well as of collagenases. Finally, since the role of the neuroendocrine systemon the repair of the connective tissue has recently received special focus, the involve-ment of neutral endopeptidase-24.11 (NEP), which regulates the action of the com-ponents of this system, is also discussed in this paper.

METHODS

Cell Strains

Ten strains of human fibroblasts were developed in our laboratory from skin ex-plants, one from a neonatal donor and nine from healthy, consenting volunteers ofvarious ages, that is, 7 (two donors), 8, 23, 60, 65, 74, 75, and 100 years old. Thesecells have been used at early passages.5 Furthermore, the following commerciallyavailable human fibroblast cell strains were used as controls: (a) human lung fibro-blasts, one fetal strain (Flow 2002) and one from a 20-year-old adult donor (CCD19Lu, ECACC, U.K.); (b) human skin fibroblasts, one fetal strain (Detroit 551,ATCC, USA), and one from a 23-year-old adult donor (1.BR3, ECACC).

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Conditioned Medium Collection

Confluent cultures of human fibroblasts were incubated with serum-free MEMfor 48-hour periods and the conditioned medium (CM) was clarified from cell debrisby centrifugation.6

DNA Synthesis Assay

Cells were grown until confluency, arrested in MEM/0.1% fetal calf serum(FCS), and stimulated with growth factors (human recombinant (h.r.) PDGF-BB andh.r.TGF-β1) or conditioned media (1:1 with fresh medium) in the presence of[methyl-3H]thymidine. After 48 hours of incubation, the radioactivity incorporatedinto newly synthesized DNA was estimated as previously described.7

Inhibition of CM Mitogenicity with Neutralizing Antibodies

Neutralizing antibodies were preincubated with CM for 3 hours at 37°C, at a con-centration that totally inhibits the respective growth factors. Inhibition was estimatedby DNA synthesis assay, as described above.

Collagen Synthesis Assay

Human fibroblast cultures were rendered quiescent by serum deprivation for 48hours, as described above (see DNA synthesis assay). Then they were incubated for48 hours with medium supplemented with L-[3H]proline, β-aminopropionitrile(βAPN), and ascorbic acid, in the presence or absence of TGF-β1. Collagen syn-thesis was measured by a modification of the protease-free collagenase method.8

Triplicate samples were measured, and the results were normalized to the number ofcells recovered.

Zymography

Gelatin zymography was performed by using a modification of the procedure ofHerron et al.9 Human fibroblast–conditioned media were separated on nonreducing10% SDS-polyacrylamide gels that contained 0.1% gelatin. After removal of SDS,the gels were incubated in substrate buffer and stained with Coomassie brilliant blueR250. Zones containing proteases appeared as clear bands on a blue background.

Analysis of the NEP Effect on Immunocyte Migration

Invertebate immunocytes were incubated with growth factors in the presence orabsence of NEP, and their migration was estimated after computer-assisted micro-scopic image analysis by using a shape factor (SF) formula.10

Analysis of NEP Activity

Confluent cultures of human fibroblasts were scraped, sonicated, and subjectedto spectrofluorimetric analysis of NEP activity, as described.5

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RESULTS AND DISCUSSION

Fibroblast Response to Platelet-Derived Mitogens Persists during Aging

One of the initial events after tissue injury is the aggregation of blood platelets,which leads to the secretion of several growth factors from their α-granules. Mostprominent among these factors are platelet-derived growth factor (PDGF), a potentmitogen for cells of mesenchymal origin,11 and transforming growth factor-β (TGF-β), a true multifunctional factor.12 Both PDGF and TGF-β control several key func-tions in the wound site, such as cell migration and proliferation and ECM formationand remodeling.

Concerning cell proliferation, we have previously shown that the response of hu-man fibroblasts to PDGF and TGF-β—as well as to other growth factors such as epi-dermal growth factor (EGF) and fibroblast growth factor (FGF)—declines sharplyas a result of in vitro aging after serial passaging.13 Here, we present evidence on theeffect of growth factors on human fibroblasts aged in vivo, that is, from donors ofvarious ages. As can be seen in FIGURE 1, the response of adult fibroblasts to PDGFdoes not decline as an effect of aging. Interestingly, similar responses have also beenfound regardless of the developmental state of the donor (fetal, neonatal, or adult)

FIGURE 1. Effect of PDGF on DNA synthesis of human fibroblasts. Quiescent cul-tures of human fetal, neonatal, or adult fibroblasts (numbers indicate the age of the donor) ofskin (open bars) and lung (hatched bars) origin were stimulated with PDGF-BB (3 ng/ml),and DNA synthesis was estimated as described under MATERIALS AND METHODS. Values rep-resent stimulation in comparison to the respective unstimulated culture (mean of three indi-vidual experiments, with each experiment performed in triplicate dishes).

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and of the tissue of origin (skin or lung). As far as neonatal and adult fibroblasts areconcerned, the stimulatory effect of TGF-β does not seem to decline with the age ofthe donor (not shown here). The only change observed was on the action of TGF-βon fibroblasts from fetal donors, which were strongly inhibited, reflecting the differ-ent repair strategies between fetuses and adults (Kletsas et al., submitted forpublication).

The above data are in accordance with those of Freedland et al.,14 who also foundno evidence for an age-dependent decline of the response of human adult fibroblaststo several growth factors, such as EGF, TNF-α, PDGF, and fetal bovine serum. In thesame context, Tesco et al.15 have reported that the growth characteristics of skin fi-broblasts from centenarian donors are remarkably similar to those of young controls.

The Effect of Aging on Extracellular Matrix Formation

Growth factors released by platelets, beyond their chemotactic and mitogenic ac-tivity, also induce the synthesis of ECM components. Collagen is the most prominentECM constituent, at least for the connective tissue, and it is mainly secreted by fi-broblasts in the wound area.1 Accordingly, we have studied collagen secretion by hu-man fibroblasts, before (basal levels) and after stimulation with the most potentinducer of ECM synthesis, that is, TGF-β.12

As can be seen in FIGURE 2A, beyond some extreme cases due to an expected in-terdonor variability, collagen secretion by human adult fibroblasts does not seem tochange considerably with the age of the donor. Furthermore, the fetal and neonatalcells strains that have been used in this study secrete only slightly higher collagenconcentrations from the average secretion by adult-donor fibroblasts. In FIGURE 2B,the stimulation of collagen synthesis and secretion after treatment with TGF-β incomparison to the respective control— that is, unstimulated cultures— is depicted.From these data also there is no evidence for an age-related decline in the secretionof collagen from human fibroblasts.

Another important determinant of tissue repair and remodeling is the local levelsof matrix metalloproteinases, and in particular collagenases, that regulate the transi-tion from the final stages of the wound— the granulation tissue— to the maturescar.1 Accordingly, we have investigated the levels of collagenases secreted by hu-man fibroblasts derived from donors of various ages. By the use of gelatin zymogra-phy, we have observed that fibroblasts secrete a major metalloproteinase that inSDS-PAGE migrates at 53 kDa, corresponding to the MMP-1, that is, the interstitialcollagenase (FIG. 3). As can be seen, beyond interdonor variabilities, no age-relatedchange was observed in the secretion of MMP-1. Similar levels of MMP-1 werefound to be secreted by the fetal and neonatal cell strains studied. These data, at vari-ance with the strong upregulation of collagenase production by human fibroblastsaged in vitro after serial passaging,4 indicate a difference between in vivo and in vitroaging at this level.

Autocrine Growth Regulation during Aging

At the early, acute phase of the wound, the healing process is mainly dependenton the bulk of growth factors released by blood platelets and other immunocytes,such as activated macrophages. However, at the late stages of the wound and during

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A

FIGURE 2. Basal and TGF-�–stimulated collagen synthesis of human fibroblasts.In (A) the secreted collagenous protein by quiescent cultures of human fetal, neonatal, oradult skin fibroblasts (numbers indicate the age of the donor) labeled with [3H]proline wasestimated as described under MATERIALS AND METHODS. Values represent the mean of twoindividual experiments, with each experiment performed in triplicate dishes. In (B) valuesrepresent collagen synthesis after stimulation of the above cultures with TGF-β (5 ng/l) incomparison to the respective unstimulated culture (mean of two individual experiments,with each experiment performed in triplicate dishes).

B

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normal tissue turnover, this “potential” of exogenous growth factors is not available.Consequently, at this stage, it is the sum of the factors released by the cells, whichact locally in an autocrine or paracrine manner, that is more significant for the ho-meostatic regulation.

As previously reported, we have shown that human fibroblasts secrete autocrinemitogens that, in the absence of exogenous growth factors, provide them with a cer-tain degree of autonomy in the maintenance of tissue repair and regeneration.6 Here,we have investigated the possibility that alterations of this autocrine mechanism dur-ing the aging process could be involved in the decline of homeostasis in the elderly.Accordingly, we have studied the mitogenic potential of media conditioned by fibro-blasts, which contain, by definition, all the secreted growth factors. As can be seenin FIGURE 4A, the mitogenic potential secreted by fibroblasts originating from do-nors of various ages does not decline as a consequence of aging, indicating that thisautocrine mechanism persists during aging in vivo.

PDGF and TGF-β, beyond being released by platelets and other immunocytes,have also been observed to be secreted by fibroblasts.6 In order to test whether themitogenic action of the conditioned media is due to the presence of one of these twofactors (or both), we have treated the conditioned media with pan-specific, neutral-izing antibodies against PDGF, TGF-β, or both. As can be seen in FIGURE 4B, bothantibodies only partly inhibit the effect of the conditioned medium, indicating (a)that both factors are included in this medium and contribute to its mitogenicity and(b) that additional autocrine mitogens are also secreted by human fibroblasts.

Furthermore, in order to estimate whether the levels of TGF-β change with theage of the donor, we have used the most sensitive bioassay for TGF-β, based on thespecific inhibition of mink lung epithelial cells by this factor. Interestingly, our pre-liminary observations indicate that the levels of TGF-β secreted by human fibro-blasts remain relatively constant, regardless of the aging level of the donors (data notshown).

FIGURE 3. Secretion of collagenase-1 by human skin fibroblasts. The collagenase-1 (MMP-1) activity secreted by quiescent cultures of human fibroblasts (strains as in FIG. 2)was estimated by gelatin zymography (see MATERIALS AND METHODS). One of two similarexperiments is depicted.

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A

B

FIGURE 4. Mitogenic activity of human fibroblast-conditioned media. (A) Quies-cent cultures of human skin fibroblasts from a 7-year-old donor were stimulated with mediaconditioned by fetal, neonatal, and adult fibroblast strains (numbers indicate the age of thedonor), and DNA synthesis was estimated as described under MATERIALS AND METHODS.Values represent stimulation in comparison to the control, that is, MEM/0.1% FCS (mean oftwo individual experiments, with each experiment performed in triplicate dishes). (B) Qui-escent cultures (C) of human fibroblasts were stimulated with autologous conditioned medi-um (CM) preincubated with anti-TGF-β (AbT) or anti-PDGF (AbP) or both pan-specificantibodies (10 µg/ml), and DNA synthesis was estimated as above. The * corresponds to sta-tistically significant inhibition with reference to CM ( p < 0.05 after Student’s t test analysis).

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Neutral Endopeptidase-24.11 Function and Activity during Aging

All the data presented above indicate that there are no major differences, as aneffect of the aging of the donor, in the response of human fibroblasts to growth fac-tors that could possibly account for impaired healing in the elderly. It has to be men-tioned that only a few, although important, parameters have been studied here. Thisis becoming increasingly problematic with the growing understanding of the multi-plicity of the systems involved in tissue repair. As an example, the interaction be-tween the neuroendocrine and the skin immune systems has been recently receivingincreasing attention.16 One of the important components of the neuroendocrine sys-tem is neutral endopeptidase-24.11 (NEP), which regulates the action of small bio-active peptides.5 As can be seen in FIGURE 5A, we have found that NEP is able tocompletely inhibit the chemotactic activity of PDGF and TGF-β on invertebrate im-munocytes.17 Because the chemotactic attraction of immunocytes is a key event inthe early phases of wound repair, we have studied the activity of NEP in human fi-broblasts during development and aging. The results from these studies, depicted inFIGURE 5B, show an increase in NEP activity during the fetal-to-adult transition.Furthermore, an increase in the activity has been found in cells from aged donors,whereas fibroblasts from a centenarian donor display an activity at the level of youngcells.5 Although these data originate from a few donors, Solmi et al.18 provided sim-ilar data from a large number of young and aged individuals. The physiological sig-nificance of the increased expression of NEP with aging on the regulation ofhomeostasis obviously requires further investigation.

CONCLUSIONS, EMERGING QUESTIONS, AND FUTURE PROSPECTS

In this report we have studied several important parameters of the wound repairprocess focusing on the ability of human fibroblasts to respond to platelet-derivedand autocrine mitogens as well as the secretion of ECM components and the metal-loproteinases that degrade them. Interestingly, when we have used cells from donorsof various ages, no age-related decline in any of these aspects has been found thatcould possibly account for impairments of the wound-healing process in the elderly.Although several parameters have to be further investigated in future studies, thesedata point to the importance of systemic factors, such as hormone levels, that regu-late body homeostasis in general.

As mentioned above, studies on cells aged in vitro after serial passaging haveshown that these cells are unable to respond to exogenous stimuli and, moreover, se-crete large amounts of collagenases. However, the results presented here and by oth-er laboratories (see above in RESULTS AND DISCUSSION) indicate a major differenceat this level between cells aged in vivo and those aged in vitro. Several hypotheses,not mutually exclusive, could be raised to explain this discrepancy:

(a) in vivo and in vitro aging processes are, in part or in general, two differentmechanisms;

(b) the percentage of senescent cells in the tissues of old individuals is negligi-ble; and

(c) the development of the primary culture from tissue explants represents a se-lection process in favor of the “young” cells.

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The increase in NEP activity in fibroblasts from old donors requires further stud-ies in order to be properly evaluated. These, along with experiments on human im-munocytes, could possibly provide information concerning the interplay of theneuroendocrine and the immune system in the processes of tissue repair and of agingin general.

FIGURE 5. NEP function and activity. (A) Effect of NEP (0.25 ng/ml) on the chemo-tactic activity of PDGF (P) and TGF-β (T) on molluscan immunocytes, expressed as shapefactor, compared to unstimulated cells (C). (B) NEP activity in human skin fibroblasts fromfetal and adult donors (numbers indicate the age of the donor) expressed as pmol/106 cells.In both A and B, the mean and standard deviation of three experiments is shown.

A

B

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Finally, the in vitro study of a complex process such as this of wound repair re-quires cell assay systems that simulate the conditions of the tissue in vivo as closelyas possible. Accordingly, we are currently studying the response of fibroblasts fromyoung and aged donors when cultured in three-dimensional gels of polymerized col-lagen (Zervolea et al., in preparation), as these gels represent the closest in vitro ap-proximation of the granulation tissue, that is, one of the last phases of tissue repair.Our preliminary observations indicate qualitative differences in the response of fi-broblasts under these conditions, in comparison to the remote from the in vivo statecultures on plastic surfaces.

ACKNOWLEDGMENT

This work has been partially supported by the Greek General Secretary of Re-search and Technology (PENED’95 Programme).

REFERENCES

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