ellagic and tannic acids protect newly synthesized

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Ellagic and Tannic Acids Protect Newly Synthesized

Elastic Fibers from Premature Enzymatic Degradationin Dermal Fibroblast CulturesFelipe Jimenez1, Thomas F. Mitts1,2, Kela Liu3, Yanting Wang3 and Aleksander Hinek1,3,4

Progressive proteolytic degradation of cutaneous elastic fibers, that cannot be adequately replaced or repairedby adult dermal fibroblasts, constitutes a major feature of aging skin. Our present investigations, employingmonolayer cultures of human dermal fibroblasts and organ cultures of skin biopsies, were aimed at testingwhether the hydrophilic tannic acid (TA) and lipophilic ellagic acid (EA) would protect dermal elastin fromexogenous and endogenous enzymatic degradation. Results from both culture systems indicated that dermalfibroblasts, maintained with TA or EA, deposit significantly more elastic fibers than untreated control cultures

despite the fact that neither polyphenol enhanced transcription of elastin mRNA or cellular proliferation.Results of a pulse and chase experiment showed that pretreatment with both polyphenols enhancedbiostability of tropoelastin and newly deposited elastin. Results of in vitro assays indicated that bothpolyphenols bound to purified elastin and significantly decreased its proteolytic degradation by elastolyticenzymes belonging to the serine proteinase, cysteine proteinase, and metallo-proteinase families. Importantly,both polyphenols also synergistically enhanced elastogenesis induced by selected elastogenic compounds incultures of dermal fibroblasts. We propose that EA and TA may be useful for preventing proteolytic degradationof existing dermal elastic fibers and for enhancing more efficient elastogenesis in aged skin.

Journal of Investigative Dermatology (2006) 126, 1272–1280. doi:10.1038/sj.jid.5700285; published online 6 April 2006

INTRODUCTION

Children with inherited diseases, characterized by impairedprimary deposition of elastic fibers (i.e. Costello Syndromeor Cutis Laxa), develop wrinkles and deep dermal creases.Similar, but steadily developing signs of premature skinaging can also be observed in individuals with pseudox-anthoma elasticum and in normal persons after prolongedexposure to sun. Histological analysis of wrinkledskin demonstrates disappearance and altered organizationof elastic fibers due to premature proteolytic degradationand impaired remodeling (solar elastosis) of thesecomponents of dermal extracellular matrix. This observedloss of physiologically relevant elastic fibers is also affectedby the fact that fully differentiated (adult) dermal fibroblasts

lose their ability to synthesize elastin and thus cannot

replace damaged elastic fibers. As elastic fibers are solely

responsible for cutaneous elasticity/resilience, there is anobvious need for development of methods that mightprotect existing elastic fibers from premature degradationby elastolytic proteinases and facilitate new elastogenesisin skin.

We have recently described that a proteolytic digest of elastin (ProK-60) (Hinek et al ., 2005) and trivalent iron (ferricammonium citrate) (Bunda et al ., 2005) can stimulateproduction of new tropoelastin and its effective assemblyinto new elastic fibers in primary cultures of human dermalfibroblasts and in organ cultures of skin explants obtainedfrom adult individuals. We have also established that even inoptimal conditions the elastogenic process is not 100%

efficient. Therefore, a significant fraction (30–40%) of newlyproduced tropoelastin is not assembled into extracellularelastic fibers and instead interact with the cell surface elastinreceptor (Hinek, 1996) to further enhance new elastogenesis,stimulate promitogenic and promigratory signaling pathways(Mochizuki et al ., 2002), and stimulate secretion of elastolyticmetalloproteinases (Brassart et al ., 2001; Huet et al ., 2001).Whereas stimulation of dermal fibroblast proliferation andmigration can contribute to an overall antiaging effect,induced by factors initially triggering new elastogenesis, thesimultaneous upregulation of elastolytic enzymes may causerapid degradation of newly produced elastin and existingelastic fibers. Hence there is a need to protect existing and

ORIGINAL ARTICLE

272 Journal of Investigative Dermatology (2006), Volume 126 & 2006 The Society for Investigative Dermatology

Received 15 September 2005; revised 13 February 2006; accepted 17 February 2006; published online 6 April 2006

1Research Department, Human Matrix Sciences, LLC, Visalia, California,USA; 2Department of Surgery, Loma Linda University School of Medicine,Loma Linda, California, USA; 3Division of Cardiovascular Research, The Hospital for Sick Children, Toronto, Ontario, Canada and 4Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto,Ontario, Canada

Correspondence: Dr Thomas F. Mitts, Research Department, Human Matrix Sciences, LLC, 205 S West Street, Suite A, Visalia, California 93291,USA. E-mail: [email protected]

Abbreviations: EA, ellagic acid; FBS, fetal bovine serum; TA, tannic acid



new elastic fibers from premature enzymatic proteolysis inorder to achieve a ‘‘true’’ antiaging effect.

Ellagic acid (EA) and tannic acid (TA) are polyphenolsfound in a wide variety of fruits and nuts such as raspberries,

strawberries, walnuts, grapes, and black currants (de Ancoset al ., 2000; Priyadarsini et al ., 2002). These moleculespossess potent ability to scavenge reactive oxygen speciesand reactive nitrogen species (Priyadarsini et al ., 2002;Ignatowicz et al ., 2003). Recently, EA was shown to decreaseexpression of prometalloproteinase (MMP)-2 and pro-MMP-9,precursors of two elastolytic enzymes (Losso et al ., 2004).Moreover, TA has been shown to bind to insoluble bovineand porcine elastin and inhibit their degradation by porcinepancreatic elastase (Isenburg et al ., 2004). In this report, wepresent novel data indicating that treatment of culturedhuman dermal fibroblasts and organ cultures of human skinbiopsies with EA or TA significantly enhances their net

deposition of elastic fibers. We provide evidence that thiseffect is due to the fact that these reagents prevent prematureproteolytic degradation of tropoelastin and fully polymerizedelastin (major component of mature elastic fibers) thusfacilitating more efficient elastogenesis.

RESULTS

EA and TA enhance deposition of elastin by dermal fibroblastsResults of immunohistochemical analysis (Figure 1a and b)and quantitative assessment of metabolically labeled inso-luble elastin (Figure 1c) indicated that 7-day-old monolayercultures of dermal fibroblasts maintained with EA or TAcontain thicker elastic fibers and a higher net content of NaOH-insoluble elastin than untreated control cultures.Moreover, results of morphometric analysis demonstratedthat both EA and TA caused a significant (P o0.005) increase(6776 and 96712%, respectively) in net elastogenesisobserved in organ cultures of human skin explants main-tained for 10 days with 5% fetal bovine serum (FBS). Therepresentative micrographs presented in Figure 2 additionally

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Figure 1. Assessment of immunodetected and insoluble elastic fibers in

dermal fibroblast cultures. (a) Micrographs of immunodetected tropoelastinin cultures maintained in the presence and absence of EA and TA (1 mg/ml

each). (b) Results of morphometric evaluation of tropoelastin levels in

fibroblast cultures. (c) Evaluation of metabolically labeled insoluble elastin in

treated and control cultures. All results were obtained in 7-day-old cultures of

dermal fibroblasts derived from a 36-year-old Caucasian female. Data

demonstrate that treatment with EA or TA significantly increases deposition of

extracellular elastic fibers as compared to respective untreated controls.

a b

c d

Figure 2. Movat pentachrome-stained transverse sections of skin biopsy

explants derived from abdominal skin of a 30-year-old woman. The

presented micrographs show that skin explants maintained for 10 days in

culture medium with TA (c) contain thicker and longer elastic fibers than

those present in respective explants treated either with control medium

(a) or with medium containing ProK-60 (b). Panel (d) shows that TA alsosignificantly enhanced the elastogenic effect of ProK-60. Movat’s

pentachrome stain shows elastin as black, collagen as yellow, cells red,

and nuclei as dark blue (original magnification Â200).

www.jidonline.org 12

F Jimenez et al.Polyphenols Enhance Elastin Biostability



demonstrate that explants maintained for 10 days in culturemedia containing TA (Figure 2c) contain thicker and longerelastic fibers than those present in explants maintained onlyin control medium (Figure 2a) or medium with ProK-60(Figure 2b). Interestingly, the presence of TA seems toparticularly enhance elastogenesis in cells protruding from

the stratum basale, toward the papillary dermis, and in cellssurrounding small capillaries. Results of semiquantitativePCR and Northern blotting indicated, however, that treatmentof cultured dermal fibroblasts with EA or TA did not induceany increase in the transcription of their elastin gene (data notshown) nor change their proliferation rate, as assessed byincorporation of radioactive thymidine and total DNAcontent (data not shown). Despite this finding, results of Western blotting, with anti-tropoelastin antibody, showedthat both cell extracts and conditioned media, of dermalfibroblasts incubated with EA or TA, contained more intact70 kDa tropoelastin and less immuno-detectable degradationproducts of lower molecular weight than untreated counter-

parts (Figure 3). This finding gave evidence that bothpolyphenols protected newly produced tropoelastin frompremature intracellular and pericellular degradation byendogenous proteinases.

EA and TA protect elastin from proteolytic degradationResults of a pulse and chase experiment (Figure 4a and b)demonstrated that cultures of dermal fibroblasts, exposed for7 days to EA and TA, sustain their high net content of insoluble elastin (metabolically pulsed with [3H]valinebetween days 4 and 7) when maintained for an additional7 days (chase period) in media containing only 1% FBS (noEA or TA), which did not stimulate proliferation (Figure 4c)

and new elastogenesis. In contrast, 14-day-old control

(untreated) cultures demonstrated a significant decrease intheir net content of metabolically labeled insoluble elastin(detected at the end of pulse period, at day 7). Moreover,results of an in vitro elastolytic assay demonstrated thatsamples of purified insoluble elastin (purity confirmed byamino-acid analysis), pretreated with EA or TA, were more

resistant to proteolytic degradation by all tested elastolyticenzymes belonging to the serine proteinase (human leuko-cyte elastase and porcine pancreatic elastase), MMP-2, andcysteine proteinase (papaine) families (Figure 5). These datasuggested that EA and TA protected newly deposited and

Cell layer

extracts

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70 kDa Tropoelastin

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Figure 3. Representative Western blot of tropoelastin in cell extracts and

media. Western blotting with anti-tropoelastin antibody demonstrates that

both cell extracts and conditioned media, of dermal fibroblasts incubated for

24 hours with EA or TA (1 mg/ml each), contain more intact 70 kDa

tropoelastin and less immunodetectable degradation products of lower

molecular weight than untreated counterparts. The results depicted represent

a single experiment, but identical beneficial influence of TA and EA on the

integrity of intracellular and secreted tropoelastin has been confirmed in

triplicate cultures of fibroblasts derived from three different donors.

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Figure 4. Pulse and chase experiment to evaluate pretreated tropoelastin/

elastin stability against nonspecific enzymatic degradation. (a) Results of morphometric assessment of immunodetectable elastin and (b) content of

metabolically labeled insoluble elastin, detected at the respective ends of the

indicated pulse and chase periods, demonstrate that cultures of dermal

fibroblasts derived from a 26-year-old female, while were incubated the first

7 days in the presence of EA and TA (1 mg/ml each), sustain their high net

content of insoluble elastin (metabolically pulsed with [3H]valine between

days 4 and 7) even when maintained for an additional 7 days (chase period) in

media containing only 1% FBS and no polyphenols. In contrast, 14-day-old

control (untreated) cultures demonstrate a significant decrease in their net

content of metabolically labeled insoluble elastin (initially deposited at the

end of pulse period, at day 7). (c) The assessment of [3H]thymidine

incorporation and assay of total DNA indicate that conditions of the chase

period in which cells were maintained in 1% FBS caused inhibition of cellular

proliferation.

274 Journal of Investigative Dermatology (2006), Volume 126




purified insoluble elastin against degradation by both endo-genous and exogenous elastolytic enzymes, respectively,through association with elastin.

EA and TA bind to elastin and tropoelastinResults of a spectrophotometric assay (displaying a linear

concentration curve for both EA and TA at an absorbance of 280nm), comparing concentrations of both polyphenolsbefore and after incubation with insoluble elastin, demon-strated that 1 mg of pure insoluble elastin, isolated fromligamentum nuchae, absorbed 8773% of the TA, and8172% of the EA in solutions having had initial concentra-tions of 20 mg/ml of each polyphenol. This finding impliedthat both polyphenols bind to insoluble elastin. Additionalresults showed that preincubation of a [3H]valine-labeledrecombinant peptide, containing the most characteristic hydro-phobic and crosslink generating domains of tropoelastin, withEA and TA did not preclude its effective (practically identical)immunoprecipitation with respective anti-VGVAPG and

anti-AKAAAKAAAKA antibodies (data not shown). Thisindicated that these polyphenols associate with and protecttropoelastin in a way which does not obscure hydrophobicdomains (e.g. VGVAPG), necessary for self-aggregation, norKAAAK sequences participating in crosslinking.

EA and TA enhance elastogenic effect of selected stimulators of elastogenesisResults of immunostaining and metabolic labeling estab-lished that addition of EA or TA to fibroblast culturessimultaneously treated with known stimulators of elastin

gene expression, ProK-60 (Hinek et al ., 2005) or ferricammonium chloride (Bunda et al ., 2005), significantlyenhanced their net deposition of insoluble elastin asestimated in 7-day-old monolayer cultures (Figure 6). Mor-phometric analysis additionally demonstrated that bothpolyphenols significantly (P o0.02) enhanced elastogenic

effect of ProK-60 observed in 10-day-old organ culturesof human skin explants (EA¼2274 and TA¼3576%).

*P <0.001

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Human leukocyte elastase Porcine pancreatic elastase

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Figure 5. Evaluation of the protective effect of polyphenols against

elastolytic degradation of insoluble elastin. Results of in vitro assay

demonstrate that samples of insoluble [3H]elastin from bovine ligamentum

nuchae, pretreated with EA or TA (1 and 10 mg/ml each), demonstrate higher

resistance to proteolytic degradation by indicated enzymes belonging to three

different classes of proteinases (elastases) capable of elastin degradation.

*P <0.001

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Figure 6. Assessment of the effect of polyphenols on elastogenesis induced

by known elastogenic compounds. (a) Results of the quantitative assessmentof newly deposited insoluble elastin (metabolically labeled with [3H]valine)

detected in 7-day-old cultures of dermal fibroblasts derived from a healthy

50-year-old Caucasian female. Fibroblasts maintained in the presence of

stimulators of elastin synthesis, mixture of small elastin-derived peptides

(ProK-60, 25mg/ml) or ferric ammonium citrate (FAC, 20mM), significantly

increased their net deposition of insoluble elastin as compared with the

untreated control. Additional treatment either with EA (1 mg/ml) or TA (1 mg/

ml) caused further proportional increase in net elastin content in all tested

experimental groups. (b) Representative micrographs of 7-day-old cultures of

dermal fibroblasts (derived from 50-year-old Caucasian female) immunos-

tained with anti-tropoelastin antibody. Cultures treated with ProK-60 (25mg/

ml) produced more elastic fibers than untreated control cultures. Additional

treatment either EA or with TA (both in 1 mg/ml concentration) caused a

further increase in net deposition of immunodetectable elastic fibers.





Representative micrographs depicting synergistic effect of ProK-60 and TA are presented in Figure 2d.

DISCUSSIONOur results demonstrate that the two polyphenols, EA and TA,used in concentration of 1 mg/ml, did not modulate cellular

proliferation of normal human dermal fibroblasts, despite thefact that antiproliferative properties of both of these com-pounds were reported in cultures of various normal andmalignant cell lines when used in higher doses (Ramanathanet al ., 1992; Devi and Das, 1993; Losso et al ., 2004). Dermalfibroblasts treated with both acids did not demonstrate anyincrease in levels of elastin mRNA yet facilitated a significantincrease in net elastic fiber content detected by immuno-chemistry and metabolic labeling of insoluble elastin. Wetherefore speculated that both EA and TA might bind tointracellular tropoelastin and to newly assembled crosslinkedelastin and protect them from proteolytic degradation byfibroblast-secreted proteolytic enzymes engaged in early

remodeling of extacellular matrix. Our hypothesis regardingpotential preferential binding of both polyphenols to intra-cellular tropoelastin and extracellular elastin polymer wasbased on a previously described observation that addition of 0.25% TA to glutaraldehyde fixative dramatically enhancedcontrast of intracellular secretory vesicles containing tropo-elastin and contrast of extracellular elastic fibers in tissuesobserved under the electron microscope (Hinek et al ., 1976;Hinek and Thyberg, 1977, 1981). In fact, in the preimmuno-staining era, treatment with TA became a widely acceptedmethod for ultrastructural identification of elastic fibers thatwere previously described as ‘‘electron lucent and amor-phous’’ under electron microscopy (Kageyama et al ., 1985).

This hypothesis was further supported by results of our pulse-and-chase experiment where EA and TA pretreated tropo-elastin and insoluble elastin, deposited by dermal fibroblasts,remained resistant against nonspecific endogenous degrada-tion in the absence of EA or TA in culture medium. We alsoshowed that preincubation of 3H-labeled pure insolubleelastin, with either EA or TA, significantly reduced its rate of degradation (in the absence of either polyphenol in the digestbuffer) by several exogenous elastolytic enzymes includingporcine pancreatic elastase, human leukocyte elastase,papaine, and the UVB-inducible MMP-2. Results of thisprotection assay are consistent with recent observations of Isenburg et al . (2004, 2005), who described that addition of

TA to the glutaraldehyde fixation process increased thestability of porcine aortic explants exposed to pancreaticelastase digestion. Moreover, our spectrophotometric studydemonstrated that pure insoluble elastin, isolated fromligamentum nuchae, binds both TA and EA, sequesteringthem from their solvent solutions. Results of this study are infurther agreement with those of Isenburg et al . (2004) whodemonstrated similar binding of TA by pure aortic elastinover time.

Our studies did not estimate how much of the describedprotective effect of EA and TA could also be due to directinhibition of elastolytic proteinases. It has previously beenshown that plant-derived polyphenols, tannins, and their

synthetic derivatives can inhibit human leukocyte elastase(Mrowietz et al ., 1991) and MMP-2/-9 activity in severaltumor cells (Vayalil et al ., 2004; Tanimura et al ., 2005).Other studies directly demonstrated that TA specificallyinhibits the chymotrypsin-like activity of purified 20Sproteasomes (Nam et al ., 2001) and the activity of tissue-

type plasminogen activator, urokinase-type plasminogenactivator, and plasmin activity (Taitzoglou et al ., 2000,2001). We therefore, speculate that a certain fraction of thedescribed protective effect may be due to a direct inhibitionof proteolytic enzymes by both polyphenols absorbed by orreleased from the elastin substrate.

The practical biological significance of our observation isthat EA or TA added to cultures of living cells facilitatenormal secretion of tropoelastin and its assembly into elasticfibers by protecting intra- and extracellular tropoelastin fromdegradation by unspecific proteinases. As EA and TA did notblock domains responsible for self-aggregation and subse-quent crosslinking of this protein, we speculate that EA and

TA may act in concert with the 67-kDa elastin bindingprotein that acts as a protective molecular chaperone forintracellular tropoelastin. Moreover, the fact that EA and TAsignificantly decreased degradation of newly producedelastin, in dermal fibroblast cultures, and fully crosslinkedelastin, from ligamentum nuchae, indicates that both poly-phenols may enhance longevity of elastic fibers.

Given the presented beneficial effects of both testedpolyphenols, we hypothesize that both may be used intopical preparations aimed at prevention of elastin degrada-tion characteristic of normal aging and after chronic exposureto sunlight (photoaging). As EA and TA did not negativelyinterfere with the action of two known stimulators of new

elastogenesis, but rather enhanced their net effect, we furtherpropose their use in combination with compounds aimed atrestoring cutaneous elastic fibers, not only in aged skin, butalso in the skin of patients afflicted by diseases caused byelastin gene insufficiency (i.e. Williams–Beuren syndrome andCutis Laxa). Results of our recent experiments (data not shown)indicated that TA and EA prevented rapid, MMP-dependentdegradation of tropoelastin produced by dermal fibroblastsderived from three Williams–Beuren syndrome patients,yielding a significant (21–26%) net increase in deposition of insoluble elastin by these cells. However, further in vivo studies must be undertaken to determine if topical treatmentwith EA or TA will result in deposition of functionally

competent elastic fibers that will lead to enhanced elasticityand aesthetic qualities of aged and diseased skin. Being awareof studies showing that higher dosages (200–300mg) of EAinitiated blood coagulation (Bock et al ., 1981; Chino et al .,2003) and thrombosis in experimental animals after its intra-arterial injection (Uzunova et al ., 1978; Hara et al ., 1994), weconclude that of the two tested polyphenols, TA is the morepromising therapeutic reagent for future translational studies.Thus, results of our in vitro studies, presented herein, and lackof previously described clinical side effects of TA fullyencourage its use in clinical studies aimed at demonstratingin vivo protection of existing elastic fibers and more efficientelastogenesis in skin.





MATERIALS AND METHODSMaterialsAll chemical-grade reagents were obtained from Sigma (St Louis,

MO). aMEM medium, FBS, 0.2% trypsin–0.02% EDTA, and other

cell culture products were obtained from GIBCO Life Technologies

(Burlington, Canada). Polyclonal antibody to tropoelastin and BA4

mAb to VGVAPG were purchased from Elastin Products CompanyInc. (Owensville, MO). Monospecific polyclonal anti-AKAAAKAAA-

KA antibody was a generous gift of Dr Barry Starcher from the

University of Texas. Secondary antibody fluorescein-conjugated goat

anti-rabbit (GAR-FITC) was purchased from Sigma (St Louis, MO).

DNeasy Tissue system for DNA assay and RNeasy Mini Kit for

isolation of total RNA were purchased from Qiagen (Mississauga,

Canada). Expression probe for elastin was purchased from Applied

Biosystems (Foster City, CA, USA). The radiolabeled reagents,

[3H]valine, and [3H]thymidine were purchased from Amersham

Canada Ltd (Oakville, Canada).

Cell cultures

Biological effects of EA and TA were tested in cultures of dermalfibroblasts derived from punch biopsies of healthy skin from

Caucasian female subjects of different ages ranging from 4 to 52

years old. All fibroblasts were originally isolated by allowing them to

migrate out of skin explants and then passaged by trypsinization and

maintained in alpha-minimum essential medium supplemented with

20 mM 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid

(HEPES), 1% antibiotics and antimycotics, 1% L-glutamate, and 2%

FBS as previously described (Hinek et al ., 2000b). In all experiments,

consecutive passages 3–5 were tested. Cells were densely plated

(50Â105 cells/dish) to reach confluency and then cultured for 7 days

in the presence and absence of EA (dissolved in DMSO) and TA

(dissolved in water), both in concentration of 1 mg/ml. This optimal

concentration was chosen after a series of pilot experimentsindicated that 1mg/ml of EA and TA induced optimal effect on net

deposition of elastin and did not trigger any change in cellular

proliferation rate nor affect basic metabolic performance.

In a parallel series of experiments, cultures of dermal fibroblasts

were treated with well-established stimulators of tropoelastin

synthesis, ProK-60 (25mg/ml) (Hinek et al ., 2005) and ferric

ammonium citrate (20mM) (Bunda et al ., 2005), and simultenously

incubated in the presence and absence of EA or TA (both in

concentration 1mg/ml).

Institutional Review Board approval and patient informed

consent were obtained for this study. Guidelines for the protection

of human subjects of the Department of Health and Human Services

and of the Declaration of Helsinki Principles were strictly followedin obtaining tissues for this investigation.

Assessment of cellular proliferationCellular proliferation rates of control and EA- and TA-treated

fibroblasts were assessed at the end point by counting of trypsinized

cells, by total DNA assay using the DNeasy Tissue System from

Qiagen and by assessment of [3H]thymidine incorporation, which

was added to all cultures (2mCi/well) for the last 24hours as

described previously (Hinek and Wilson, 2000; Hinek et al ., 2000a).

Assessment of elastin mRNA levelsFibroblasts were cultured to confluency in medium with 2% FBS and

then in serum-free medium for 24 hours. The medium was changed

again and cells were incubated for the next 24 hours in the presence

and absence of EA or TA (both in 1mg/ml concentration). At the end

of the incubation period, total RNA was extracted using TRIs

Reagent (Sigma, St Louis, MO). Steady-state levels of elastin mRNA

were analyzed by semiquantitative PCR and by Northern blot using a

human elastin cDNA recombinant H-11 probe, as described

previously (Urban et al ., 2002). In all experiments, performed intriplicate, the loading control was routinely performed.

Assessment of elastic fiber content by immunohistochemistryCultures of fibroblasts, 7- and 14-day-old, which produce abundant

extracellular matrix, were assessed. All cultures were fixed in cold

100% methanol at À201C for 30 min, and then incubated for 1 hour

with 2mg/ml of polyclonal antibody to tropoelastin. Cultures were

then incubated for an additional hour with appropriate fluorescein-

conjugated secondary antibody (GAR-FITC). Nuclei were counter-

stained with propidium iodide (Hinek et al ., 2000a). Morphometric

analysis of five separate cultures in each experimental group,

immunostained with antibodies recognizing extracellular matrix

components, was performed using a computerized video analysissystem (Image-Pro Plus software 3.0, Media Cybernetics, Silver

Spring, MD) as described previously (Hinek and Wilson, 2000;

Hinek et al ., 2000a).

Radioactive metabolic labeling and quantification of newlydeposited insoluble elastinQuintuplicate, 4-day-old cultures of dermal fibroblasts maintained in

the presence and absence of 1 mg/ml of EA or TA were additionally

exposed for the 3 following days to 20 mCi [3H]valine. At the end of

the incubation period, the contents of radioactive, NaOH-insoluble,

elastin was assessed separately in each culture by scintillation

counting, as described previously (Hinek and Wilson, 2000; Hinek

et al ., 2000a). Final results reflecting amounts of metabolicallylabeled, insoluble elastin were expressed as c.p.m./ mg DNA. DNA

was determined with the DNeasy Tissue System from Qiagen.

Pulse and chase experiments aimed at the assessment of durability of newly deposited metabolically labeled elastinIn this series of experiments, dermal fibroblasts, plated as described

above in media with 10% FBS, were maintained in the presence and

absence of 1mg/ml of EA or TA for the first 7 days and pulsed with

20mCi [3H]valine between days 4 and 7. Whereas quadruplicate

7-day-old cultures from each experimental group were directly

processed for the assessment of radioactive NaOH-insoluble elastin,

parallel quadruplicate cultures from each experimental group were

transferred to media containing only 1% FBS, which did notstimulate proliferation and new elastogenesis, and maintained for

the next 7 days (chase period) in the absence of EA or TA. At day 14,

these cultures were terminated and the net content of the radioactive

NaOH-insoluble elastin was assessed as described above.

Organ cultures of explants derived from surgical biopsies of human skinIn order to further test whether EA and TA would penetrate into skin

tissue and enhance elastogenesis, fragments of normal skin (from

30- and 34-year-old women) obtained during plastic surgery

procedures were tested in organ culture system. Informed patient

consent and local Institutional Review Board approval were





obtained and guidelines of the Declaration of Helsinki Principles for

the protection of human subjects were strictly followed in collecting

excess skin fragments. Skin fragments were cut into multiple 1 mm2

pieces and placed on top of metal grids immersed in culture medium

containing 5% FBS and maintained for 10 days in the presence and

absence of 1 mg/ml of EA or TA alone or combined with 25 mg/ml of

ProK-60. The media were changed every second day. All organcultures were fixed in 1% buffered formalin and their transversal

serial histological sections were stained with Movat’s pentachrome

as described previously (Hinek et al ., 2005). Morphometric analysis

was performed as described above. In each analyzed group (three

explants from each patient), low-power fields (1 mm2) of 20 serial

sections stained with Movat’s pentachrome were analyzed and all

structures stained black (elastic fibers) were counted.

Assessement of tropoelastin integrity by Western blotsTo determine the influence of TA and EA on the integrity of soluble

tropoelastin, dermal fibroblasts obtained from three different donors

(initially plated at 50,000 cells/dish) were cultured to confluency in

medium with 5% FBS and then triplicate cultures were incubatedfor the next 24 hours in the presence and absence of EA or TA (both

in 1mg/ml concentration). At the end of the incubation period,

conditioned media were collected and then the soluble proteins

present in the intracellular compartments were extracted with

0.5 M acetic acid in the presence of proteinase inhibitors in the

following final concentrations: 2 mM benzamidine, 2 mM E-amino-

caproic acid, 2 mM phenylmethylsulfonyl fiuoride, 1 mM EDTA,

and 1 mg/ml Trasylol, as previously described (Hinek and Rabino-

vitch, 1994). Extraction was carried out for 6 hours at 41C and the

insoluble material was pelleted by centrifugation. The supernatant

was dialyzed exhaustively (4,000kDa cutoff membrane) at 41C

against water containing proteinase inhibitors, then lyophilized.

Concentrated preparations of the conditioned media and cellextracts from all analyzed cultures were analyzed for their protein

content, and then samples containing equal amounts of protein

(20mg/sample) were suspended in 2ÂSDS sample buffer with

dithiothreitol, resolved by SDS-PAGE, routinely transferred to

nitrocellulose and immunoblotted with specific anti-tropoelastin

antibody.

Immunoprecipitation of radioactive tropoelastin-like peptidesThis experiment was aimed at elucidating whether binding of EA or

TA to tropoelastin molecules would block two characteristic elastin

domains responsible for orderly self-aggregation and crosslinking,

respectively. Instead of very unstable tropoelastin, we used triplicate

samples of a [3H]valine-labeled recombinant polypeptide containinglinear amino-acid sequences encoded by human tropoelastin gene

exons 20-(21-23-24)2 (generous gift of Dr Fred Keeley from the

Hospital for Sick Children in Toronto) that have been proven as a

convenient tool for studies of tropoelastin self-aggregation and

crosslinking (Miao et al ., 2003). Samples (100 ml) of this radioactive

recombinant polypeptide modeled after human elastin dissolved in

PBS (specific radioactivity 1,000c.p.m./sample) were incubated in

the presence and absence of 1 mg/ml of EA or TA, at room tempe-

rature for 6 hours. Aliquots of all control and experimental samples

were then immunoprecipitated with (BA4) mAb (recognizing

VGVAPG and other similar domains, encoded by exons 20 and

24, responsible for self-aggregation of tropoelastin) and with

monospecific polyclonal anti-AKAAAKAAAKA antibody recognizing

the exon 21- and 23-encoded crosslinking sequences (Starcher et al .,

1999). We anticipated that in case that binding of EA or TA would

block one or both of these crucial domains present in the tested

radioactive recombinant peptide, it could not be immunoprecipi-

tated with anti-VGVAPG or anti-AKAAAKAAAKA antibodies.

Proteolytic degradation protection assay of insoluble elastinTo determine whether EA or TA may directly protect fully

crosslinked ‘‘insoluble elastin’’ against elastolytic activity of several

elastases, an in vitro assay measuring degradation of an insoluble

[3H]elastin substrate was used as described previously (Hinek and

Rabinovitch, 1994). Briefly, insoluble elastin was purified from

bovine ligamentum nuchae using a modification of the hot alkali

technique as previously described (Mecham et al ., 1997) and was

shown by amino-acid analysis to be free of microfibrillar protein and

other contaminants. Sequencing of insoluble, digested elastin was

performed using an Applied Biosystems model 473A protein

sequencer equipped with a model 610A data analysis program.

The stock of this pure insoluble elastin preparation was washedtwice with water and twice with acetonitrile and then labeled with

sodium [3H]borohydride as previously described (Banda and Werb,

1981) and stored at À201C. Before each experiment, the [3H]elastin

substrate suspended in PBS was boiled for 5 minutes and extensively

washed to remove all unbound radioactivity. Then, its 100mg

aliquots (specific activity 300 c.p.m./1mg) were suspended in serum-

free culture medium and preincubated for 1 hour in the presence and

absence of 1 or 10 mg/ml of EA or TA. All samples of radioactive

elastin were then submitted to three 5 minutes washes in serum-free

culture medium before their 18 hours incubation at 371C with

aliquots (50 ng) of human leukocyte elastase, porcine pancreatic

elastase, MMP-2, or papaine dissolved in the assay buffer (50 mM

Tris-HCl, pH 7.5, containing 150 mM NaCl, 10mM CaCl2, 0.02%Brij, and 0.02% sodium azide). Each treatment was tested in

quadruplicate samples. At the end of the incubation, all samples

were microcentrifuged (8,000Âg for 5 minutes) and 100ml aliquots

of supernatant containing the solubilized degradation products were

mixed with 4 ml scintillation fluid and counted in triplicate in liquid

scintillation counter. The radioactivity (c.p.m./sample) released into

the supernatant reflecting the degradation of [3H]elastin substrate

was assessed and the mean and standard deviations (mean7SD)

were calculated from sixtiplicate assessments from three different

experiments

Assessement of TA and EA binding to insoluble elastin

In order to directly prove that both polyphenols bind to elastin,triplicate (1 mg) aliquots of our above-mentioned preparation of pure

insoluble elastin were incubated with 20mg/ml of TA or EA for

2hours at 371C. The initial concentration of both polyphenols was

confirmed by a direct spectrophotometric reading at 280 nm. This

method adopted from Gori et al . (2005) demonstrated a dose-

dependent linear increase in absorbancy. At the end of the

incubation period, the insoluble elastin slurries were separated by

centrifugation and the concentrations of polyphenols in supernatants

were determined again. The detected differences between the initial

and final concentration of polyphenols in supernatants from

particular samples directly indicated that both TA and EA bound to

elastin slurries during the incubation period. In each experimental





group, means7SD were calculated and values obtained were stati-

stically compared with initial concentrations of both polyphenols.

Statistical analysisIn all above-mentioned quantitative assays, mean7SD were

calculated and statistical analyses were carried out by analysis of

variance (ANOVA) to establish whether detected differences werestatistically significant.

CONFLICT OF INTERESTHuman Matrix Science, LLC (HMS) funded this project. Felipe Jimenez, Ph.D.is an employee of HMS. Thomas F. Mitts, M.D. is the owner of HMS.Aleksander Hinek, M.D., Ph.D., D.Sc. is a consultant to HMS.

ACKNOWLEDGMENTSWe wish to thank Jeff Wheaton, R.N. for bringing ellagic acid to our attention.This work was funded entirely by Human Matrix Sciences, LLC.

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