Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation

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1 ORIGINAL ARTICLE Endothelil Cells Promote Pigmenttion through Endothelin Reeptor B Ativtion Clire Regzzetti, Gin Mro De Dontis, Houd Hmmmi Ghorel 2, Nthlie Crdot-Lei 3, Dmien Amrosetti 3, Philippe Bhdorn 2,4, Bérengère Chignon-Sird 5, Jen-Philippe Lour 2, Roert Bllotti, Andre Mhns 6 nd Thierry Psseron,2 Findings of inresed vsulriztion in melsm lesions nd hyperpigmenttion in quired ilterl telngietti mules suggested link etween pigmenttion nd vsulriztion. Using high-mgnifition digitl epiluminesene dermtosopy, lser onfol mirosopy, nd histologil exmintion, we showed tht enign vsulr lesions of the skin hve restrited ut signifint hyperpigmenttion ompred with the surrounding skin. We then studied the role of mirovsulr endothelil ells in regulting skin pigmenttion using n in vitro o-ulture model using endothelil ells nd melnoytes. These experiments showed tht endothelin relesed y mirovsulr endothelil ells indues inresed melnogenesis signling, hrterized y mirophthlmi-ssoited trnsription ftor phosphoryltion, nd inresed tyrosinse nd dophrome tutomerse levels. Immunostining for endothelin in vsulr lesions onfirmed the inresed expression on the sl lyer of the epidermis ove smll vessels ompred with perilesionl skin. Endothelin ts through the tivtion of endothelin reeptor B nd the mitogen-tivted protein kinse, extrellulr signlregulted kinse (ERK)/2, nd p38, to indue melnogenesis. Finlly, ulturing of reonstruted skin with mirovsulr endothelil ells led to inresed skin pigmenttion tht ould e prevented y inhiiting EDNRB. Tken together these results demonstrted the role of underlying mirovsulriztion in skin pigmenttion, finding tht ould open new fields of reserh for regulting physiologil pigmenttion nd for treting pigmenttion disorders suh s melsm. Journl of Investigtive Dermtology (205) 35, ; doi:0.038/jid ; pulished online 24 Septemer 205 INTRODUCTION Pigmenttion is omplex nd tightly regulted proess. Mirophthlmi-ssoited trnsription ftor () is the mster gene of pigmenttion nd ontrols severl key mehnisms in melnoytes suh s melnogenesis, dendriity, nd prolifertion in response to environmentl ftors inluding UV rdition nd to moleules produed y other ells in the skin. Ativtion of indues expression of the key enzymes of melnogenesis, whih re tyrosinse, C3M, INSERM U065, tem 2, Nie, Frne; 2 Deprtment of Dermtology, University Hospitl Center of Nie, Nie, Frne; 3 Deprtment of Pthology, University Hospitl Center of Nie, Nie, Frne; 4 Centre de Reherhe Clinique (CRC), University Hospitl of Nie, Nie, Frne; 5 Deprtment of Plsti Surgery, University Hospitl Center of Nie, Nie, Frne nd 6 Beiersdorf AG, Front End Innovtion, Hmurg, Germny Correspondene: Thierry Psseron, Deprtment of Dermtology, Hôpitl Arhet 2, University Hospitl Center of Nie, 5, Route de St Antoine de Ginestière, Nie, Frne. E-mil: psseron@unie.fr Arevitions:, dophrome tutomerse; Edn, endothelin ; ERK, extrellulr signlregulted kinse; HMVEC, humn derml mirovsulr endothelil ell;, mirophthlmi-ssoited trnsription ftor; NHK, norml humn kertinoyte; NHM, norml humn melnoyte; NO, nitri oxide Reeived 23 Deemer 204; revised 22 June 205; epted 29 June 205; epted rtile preview online 26 August 205; pulished online 24 Septemer 205 dophrome tutomerse (), nd tyrosinse-relted protein, leding to the prodution of melnin. Numerous ftors dditionlly provide the finer regultion of melnin pigment prodution nd/or melnoyte growth nd differentition. Alph-melnoyte-stimulting hormone nd ACTH re the most potent tivtors of melnogenesis, wheres nitri oxide (NO) nd some growth ftors present in the irultion or sereted y kertinoytes t to vrying degrees on melnogenesis nd melnoyte growth, inluding si firolst growth ftor, KIT lignd, heptoyte growth ftor, endothelin (Edn), nd some prostglndins (Hiroe, 2005; Plonk et l., 2009). Firolsts lso hve key role in melnoytogenesis nd melnogenesis. Plmoplntr firolsts express high levels of Dikkopf, whih redues melnoyte prolifertion nd differentition y ting on, explining (t lest prtilly) the lower pigmenttion generlly oserved on humn plms nd soles (Ymguhi et l., 2007 nd 2008). Firolsts lso produe melnogenesis-ssoited ftors tht differ ording to the skin type of the individul. One of these ftors, known s neuregulin-, is sereted y firolsts in lk skin (skin type VI) nd signifintly inreses the pigmenttion of humn melnoytes in ulture (Choi et l., 200). Interestingly, firolsts lso seem to e involved in melsm pthophysiology vi their seretion of Wnt inhiitor ftor- (Kng et l., 20; Kim et l., 203;Prket l., 204) Journl of Investigtive Dermtology (205), Volume The Soiety for Investigtive Dermtology

2 In ddition to kertinoytes nd firolsts, growing evidene lso implites endothelil ells in pigmenttion (Plonk et l., 2009). Indeed, melnoytes express reeptors tht n potentilly e regulted y severl ftors sereted y endothelil ells suh s vsulr endothelil growth ftor (VEGF), Edn, NO, nd leukotrienes (Kim et l., 2005; Ymguhi nd Hering, 2009). In ddition, histologil studies hve lerly shown signifint inrese in vsulriztion within melsm lesions ompred with tht in the surrounding helthy skin (Kim et l., 2007). These results were susequently onfirmed y lser onfol mirosopy exmintion (Kng et l., 200), lthough the signifine of this inresed vsulriztion in melsm remins poorly understood. However, reent linil report ited ses presenting with quired telngietti nd hyperpigmented mules (Prk et l., 204), for whih the linil nd histologil findings of skin hyperpigmenttion lolized ove telngietsis suggested lose reltionship etween melnoytes nd endothelil ells. In the urrent study, we therefore exmined the role of derml mirovsulr endothelil ells in regulting skin pigmenttion. RESULTS Vsulriztion influenes skin pigmenttion in vivo A totl of 00 enign vsulr skin lesions were ssessed, omprising herry ngioms, otriomyoms, spider ngioms, involutive infntile hemngioms, pillro-venous mlformtion, quired ilterl telngietti mules, nd leg telngietsis. Using high-mgnifition digitl dermtosopy, we oserved mild to mrked hyperpigmenttion within nd surrounding the vsulr lesions ompred with the surrounding skin in 89% of ses (Figure ; Supplementry tle online). The hyperpigmenttion ws mrked in 22% of ses. The ssoition etween the vsulr lesions nd the hyperpigmenttion of the overlying skin ws signifintly more frequent in drk skin types (III to V) ompred with light ones (I nd II; P = 0.02) nd in photoexposed res ompred with those loted in photoproteted res (Po0.00) (Supplementry Tles nd 2 online). No orreltion ws found etween the size of vsulr lesions nd the overlying pigmenttion (Supplementry Tle 3 online). Anlysis of the vsulr lesions using lser onfol mirosopy onfirmed the inresed pigmenttion of the skin ove the vsulr prolifertion ompred with the surrounding skin (Supplementry Figure online). We next nlyzed tissue smples of enign vsulr lesions of the skin, ville in our ionk. Ten smples from herry ngioms, otriomyoms, pillro-venous mlformtion, nd quired ilterl telngietsi were stined with hemtoxylin eosin nd FontnMsson stining to ssess the vsulriztion nd pigmenttion, respetively. stining ws lso performed to identify the melnoytes. For eh smple, histologil setions were nlyzed in the enter of the lesion nd in the orders on the immeditely surrounding, non-ffeted skin. The lesion setions showed n inresed melnin ontent in the epidermis ompred with the surrounding skin. However, there ws no signifint inrese in melnoyte numer with men rtio of melnoytes/kertinoytes of 0.28 nd 0.22 in lesionl nd perilesionl res, respetively (P = 0.4; Figure 2). Mirovsulr endothelil ells inrese the melnogenesis pthwy in melnoytes To further investigte the role of vsulriztion in skin pigmenttion, we studied the effets of humn derml mirovsulr endothelil ells (HMVECs) on the melnogenesis pthwy in norml humn melnoytes (NHMs). In o-ulture experiments with HMVECs nd NHMs, we oserved upregultion of the phosphorylted form of in melnoytes fter 30 minutes of o-ulture (Figure 3). After 3 dys of o-ulture, the expression level of tyrosinse nd ws inresed y pproximtely twofold (Figure 3). This effet ws lso speifi to the endothelil ells, euse o-ulturing of the NHMs with norml humn kertinoytes (NHKs) under the sme onditions produed no suh effet (Supplementry Figure 2 online). We then investigted the potentil role of endothelil ells in melnoyte prolifertion. After plting the sme numer of NHMs for 7 dys in o-ulture with HMVECs or lone, the melnoytes were ounted. After 7 dys of o-ulture, the HMVECs indued slight ut signifint inrese in melnoytes numer (Figure 3). Tken together, these results showed tht the mirovsulr endothelil ells ould promote melnogenesis nd, to lesser extent, enhne melnoyte prolifertion. Endothelin relesed y HMVECs upregultes melnogenesis signling in NHMs To understnd how endothelil ells upregulte the melnogenesis pthwy in melnoytes, we next investigted the ftors relesed y endotheli y inuting NHMs with HMVEC-onditioned medium for 30 minutes nd 3 dys under the o-ulture experiment onditions. The NHM strvtion medium onditioned in the presene of endothelil ells (CM. HMVEC) indued similr phosphoryltion of fter 30 minutes to tht oserved in the o-ulture experiment with HMVECs (Figure 4) nd similrly inresed tyrosinse nd expression fter 3 dys (Figure 4). Mny ftors re produed nd sereted y endothelil ells, ut herein we foused on moleules tht ould potentilly t on melnogenesis nd melnoyte prolifertion suh s NO, VEGF, endothelin, nd leukotriene. In our o-ulture experiments, inhiition of NO with L-N G -Nitrorginine methyl ester nd 2-Phenyl-4,4,5,5-tetrmethylimidzoline--oxyl 3-oxide (PTIO; Supplementry Figure 3 online), VEGF reeptor with VEGF reeptor inhiitor IV nd CBO-P (Supplementry Figure 3 online), nd leukotriene with ilein (Supplementry Figure 3 online) did not impir the phosphoryltion of. In ontrst, inhiition of endothelin reeptor with PD42893 inhiited the phosphoryltion of under our o-ulture onditions (Figure 4). Furthermore, we oserved the sme effets, with the sme kinetis, on the phosphoryltion of nd the levels of nd tyrosinse in melnoytes stimulted with Edn (Supplementry Figure 4 online). Anlyzes of Edn seretion lso showed tht the HMVECs ultured in NHM strvtion medium sereted fourfold more Edn thn NHK, nd no seretion ws deteted

3 d e f g h i j k l m n o Figure. Clinil nd epiluminesene dermtosopy pttern of hyperpigmenttion ssoited with vsulriztion. () Aquired ilterl telngietti mules. () Digitl epiluminesene dermtosopy of the lesions ( 50). () Digitl epiluminesene dermtosopy of the lesions ( 200). (d) Superfiil ongenitl hemngiom in n infnt of 2 months of ge. (e) Clinil presenttion t the ge of 5 yers with regression of the vsulr omponent nd hyperpigmenttion lolized only on the re of the hemngiom. (f) Digitl epiluminesene dermtosopy of the lesions ( 0). Digitl epiluminesene dermtosopy ( 200) of herry ngioms (gm) nd leg telngietsis (n nd o). for NHM (Figure 4d). Finlly, to orrelte these results in vivo, we ssessed the expression of Edn in skin smples of vsulr lesions ompred with the perilesionl skin nd found inresed Edn t the sl lyers of the epidermis ove vsulr lesions (Figure 4e nd h) Journl of Investigtive Dermtology (205), Volume 35 HMVECs indue melnogenesis vi the tivtion of endothelin reeptor B nd the MAPK pthwy vi ERK/2 nd p38 in melnoytes There re two sutypes of the endothelin reeptor: EDNRA nd EDNRB. In order to identify the reeptor involved in this

4 g h i d e f j k l m n o s t u p q r v w x Figure 2. Histologil nlysis of vsulr lesions. Cherry ngiom with hemtoxylin nd eosin (HE) (), FontnMsson () nd mirophthlmi-ssoited trnsription ftor () () stining. Perilesionl norml skin of the sme herry ngiom with HE (d), FontnMsson, (e) nd (f) stining. Cpillry venous mlformtion with HE (g), FontnMsson, (h) nd (i) stining. Perilesionl norml skin of the sme pillry venous mlformtion with HE (j), FontnMsson, (k) nd (l) stining. Botriomyom with HE (m), FontnMsson, (n) nd (o) stining. Perilesionl norml skin of the sme otriomyom with HE (p), FontnMsson, (q) nd (r) stining. Aquired ilterl telngietti mules with HE (s), FontnMsson, (t) nd (u) stinings. Perilesionl norml skin of the sme lesion with HES (v), FontnMsson, (w) nd (x) stining. All the pitures were tken with 200 mgnifition. Arrows designte -positive ells. Sle r = 50 μm. proess, we used speifi inhiitors of EDNRA (BQ23) nd EDNRB (BQ788). The inhiition of EDNRA showed no effet on phosphoryltion indued y endothelil ells, wheres the inhiition of EDNRB inhiited the effets of HMVECs on NHMs (Figure 5). To determine whether Edn diretly stimultes the melnoytes, we repeted the experiment using onditioned medium of HMVECs with or without BQ788. Tretment with BQ788 inhiited the phosphoryltion of indued y HMVEC-onditioned medium s oserved for NHMs nd HMVECs o-ultured in the presene of BQ788, showing tht Edn relesed y HMVECs diretly stimultes NHM (Figure 5). The effet of EDNRB in this mehnism ws further onfirmed using sirna knokdown of EDNRA nd EDNRB expression. Speifilly, deresing EDRNB expression in NHMs inhiited the phosphoryltion of, ut lso the upregultion of tyrosinse indued y HMVECs, wheres silening EDNRA hd no effet on these proesses (Figure 5 nd d, Supplementry Figure 5 online). In melnoytes, the stimultion of EDNRB tivtes protein kinse C, whih, in turn, stimultes extrellulr signl regulted kinse (ERK)/2 nd p38, mitogen-tivted protein kinses tht re implited in the phosphoryltion of nd the upregultion of tyrosinse, respetively. We therefore lso investigted the sttus of ERK/2 nd p38 in NHMs in o-ulture with HMVECs nd found tht, fter 30 minutes, ERK/2 nd p38 were phosphorylted (Figure 5e), with the phosphoryltion of ERK lso inhiited y sirna EDNRB (Supplementry Figure 5 online). Furthermore, inhiition of the ERK pthwy with U026 inhiited the phosphoryltion of in NHMs o-ultured with HMVECs (Figure 5f), wheres the inhiition of p38 with SB did not prevent this phosphoryltion (Figure 5g). The inresed tyrosinse nd fter 3 dys of o-ulture ws inhiited y U026 nd prtilly inhiited y SB203580, suggesting role for oth ERK nd p38 tivtion in this mehnism (Figure 5h nd i). Tken together these results show tht the mirovsulr endothelil ells serete endothelin, whih tivtes EDNRB on melnoytes nd stimultes the ERK nd p38 pthwys. Ativtion of ERK leds first to the phosphoryltion of, wheres the tivtion of oth ERK nd p38 indues the susequent upregultion of tyrosinse nd. Mirovsulr endothelil ells inrese the pigmenttion of reonstruted epidermis through endothelin seretion On the sis of the stimultion of melnogenesis y endothelil ells under o-ulture onditions, we then ssessed

5 Atin Co. HMVEC: GAPDH Co. HMVEC: Reltive NHM numer Co. HMVEC: ** Phospho Reltive protein level Reltive phospho level their effet on melnin synthesis in reonstruted epidermis omposed of humn kertinoytes nd melnoytes ultured with or without HMVECs. After 3 weeks, the o-ulture with HMVECs indued n inresed pigmenttion of the reonstruted epidermis tht ould e oserved linilly (Figure 6 nd ) nd using FontnMsson stining (Figure 6). This HMVEC-indued hyperpigmenttion of the epidermis ws prevented y the inhiition of EDNRB with BQ788 (Figure 6). DISCUSSION In 963 Fitzptrik nd Brethnh formulted the onept of the epiderml melnin unit, with melnoytes nd kertinoytes working together to produe skin olor. Forty yers lter, Jim Nordlund expnded this onept to utneous troik involving kertinoytes, Lngerhns ells, nd melnoytes (Nordlund, 2007), with firolsts lso reently implited in regulting skin pigmenttion (Ymguhi et l., 2007 nd 2008; Choi et l., 200). Here, we demonstrted tht the derml mirovsulr endothelil ells lso hve role in the omplex regultion of skin pigmenttion. In our experiments, endothelil ells, ut not kertinoytes, ould stimulte melnogenesis in melnoytes ** Co. HMVEC: * ** Co. HMVEC Figure 3. Mirovsulr endothelil ells inrese the melnogenesis pthwy in melnoytes. Norml humn melnoytes (NHMs) re inuted with humn derml mirovsulr endothelil ells (HMVECs) in the trnswell hmer during 30 minutes () or 3 dys (). The lyste of NHMs is nlyzed y western lot with indited ntiodies nd the reltive protein level quntified (n = 6 nd 8, respetively). The sme numer of NHMs is plted nd inuted with HMVECs in the trnswell hmer for 7 dys (). The HMVECs re hnged every seond dy. After 7 dys of o-ulture, the NHMs re trypsinized nd ounted on Mlssez ell (n = 3). *P 0.05; **P without UV stimultion. The sene or low stimultion of melnogenesis in our o-ultures with kertinoytes is not so surprising, nd indeed ll studies demonstrting stimulting effet of kertinoytes on melnogenesis were onduted in the presene of UV stimultion (Duvl et l., 200) or nother pigmenttion induer suh s Alph-melnoyte-stimulting hormone (Lei et l., 2002). We now show tht endothelil ells hve role, through the seretion of endothelin, in upregulting key gene regultors of melnogenesis,, tyrosinse, nd, without ny UV stimultion. Ativtion of melnogenesis through tivtion of the MAPK pthwy nd phosphoryltion of is in ordne with previous reports (Sto-Jin et l., 2008). However, the inresed ontrst in pigmenttion etween photo-exposed res of the epidermis ove vsulr skin lesions nd the perilesionl skin led us to hypothesize tht the stimultion of melnogenesis y endothelil ells might e even stronger fter UV rdition. Further studies re wrrnted to onfirm this hypothesis. Interestingly, in smples of the enign vsulr lesions, the melnin ontin ws inresed in the epidermis loted ove the mirovsulriztion long with n inresed expression of Edn in the sl lyers of the epidermis, further onfirming tht Edn is produed in vivo y endothelil ells, rehes the sl lyers of the epidermis, nd is ple of stimulting melnogenesis. We dditionlly oserved in vitro slight inrese in melnoyte prolifertion when o-ultured with endothelil ells, lthough the numer of melnoytes ws not signifintly inresed in either reonstruted epidermis experiments or skin smples of enign vsulr lesions. Thus, the prolifertive effet of Edn ppers limited, t lest in vivo. Interestingly, it ws reently reported tht horoidl melnoytes ould regulte uvel vsulriztion through the seretion of firomodulin (Adini et l., 204), emphsizing the onstnt ross-tlk etween melnoytes nd endothelil ells. Edn is lso produed y proliferting endothelil ells or y ells involved in inflmmtory proesses. Thus, on the sis of the urrent study, it ould e hypothesized tht Edn lso hs role in post-inflmmtory hyperpigmenttion. Although more pronouned in drker skin types nd in photo-exposed re, our linil nd histologil dt show tht the vsulr omponent of the dermis influenes the pigmenttion of the skin in vivo. Clerly, the physiologil funtion tht endothelil ells hve in skin pigmenttion, eyond their role in pigmenttion disorders, wrrnts further investigtions. Indeed, the potentil impt of vsulriztion on hyperpigmented lesions oserved in quired ilterl telngietti mules ws suspeted y the uthors who reported this entity (Prk et l., 204). Now, our results onfirm role for endothelil ells ut lso rule out the implited role of VEGF s initilly hypothesized nd show insted the key role of the relesed endothelin. The vsulr omponent my lso hve key role in melsm. Histologil studies nd onfol lser mirosopy studies hve lerly shown signifint inrese in vsulriztion within melsm lesions ompred with tht in the surrounding helthy skin (Kim et l., 2007; Kng et l., 200). Although the signifition of this inresed vsulriztion ws unknown, studies using different therpeuti 300 Journl of Investigtive Dermtology (205), Volume 35

6 Atin Atin HMVEC CM: Co. HMVEC: d pg Edn sereted/µg protein Atin HMVEC CM: e f NHK NHM Lesionl Perilesionl Edn Edn Edn g PD HMVEC Lesionl Perilesionl Edn h Edn Edn Edn Edn Figure 4. Endothelil ells serete endothelin implited in the upregultion of the melnogenesis pthwy in melnoytes. Humn derml mirovsulr endothelil ells (HMVECs) re inuted 24 hours with the norml humn melnoytes (NHMs) strvtion medium. NHMs re inuted with HMVEConditioned medium (HMVEC CM.) for 30 minutes () or 3 dys nd hnged every dy with new HMVEC CM. (). NHMs nd HMVECs re treted with the endothelin reeptor ntgonist PD42893 (5 μm) (), 2 hours efore the strt of the o-ulture with HMVECs for 30 minutes.the lyste of NHMs is nlyzed y western lot with indited ntiodies. Numers ove the gels indite levels of intensity ompred with tin. The seretion of endothelin y HMVECs, norml humn kertinoytes (NHKs), nd NHM in NHM strvtion medium is mesured using the ELISA method (d). The expression of endothelin nd mirophthlmi-ssoited trnsription ftor () is nlyzed in mirovsulr lesionl skin setions ompred with perilesionl skin (e) (herry ngiom), (f) (otriomyom), (g) (pillry venous mlformtion), (h) (quired ilterl telngietti mules)). Arrows designte -positive ells. Sle r = 50 μm. pprohes, ut shring the sme im of trgeting the vsulr omponent of melsm, hve een reently reported. A prospetive omprtive split fe rndomized study showed tht the omintion of stilized Kligmn s trio nd pulsed dye lser (PDL) ws signifintly more effetive thn the stilized Kligmn s trio lone (Psseron et l., 20). Interestingly, the omintion pproh prevented, t lest prtilly, the typil relpses fter the summer period, wheres the rem lone did not. In support of these findings, dditionl dt suggest preventive role of trgeting vessels in the relpses of melsm (Psseron, 203). A different kind of pproh lso ssessed the effet on melsm of trnexmi id, n nti-firinolyti used to prevent nd to tret some hemorrhgi events. The omined use of this gent topilly nd orlly during 8 weeks deresed hyperpigmenttion in melsm lesions, wheres histologil exmintions onfirmed derese in melnin ontent nd in vsulriztion (N et l., 203). These pilot studies underline the potentil interest in trgeting the vsulr omponent for treting melsm. However, using lser pprohes to remove the vessels my promote postinflmmtory hyperpigmenttion, espeilly in drker skin 30

7 Atin Co. HMVEC: BQ 23 BQ 788 Atin BQ 788 HMVEC CM. HMVEC CM. BQ788 (NHM) HMVEC CM. BQ788 (NHMHMVEC) d EDNRA EDNRB Atin EDNRA EDNRB Atin Co. HMVEC: Co. HMVEC: sirna: trl EDNRA EDNRB sirna: trl EDNRA EDNRB e f g perk/2 pp38 GAPDH Co. HMVEC: perk/2 GAPDH Co. HMVEC: UO26 pp38 GAPDH Co. HMVEC: SB h i perk/2 Atin pp38 Atin Co. HMVEC: Co. HMVEC: UO26 SB Figure 5. Endothelin ts through endothelin reeptor B nd mitogen-tivted protein kinse (MAPK) extrellulr signlregulted kinse (ERK)/2 nd p38 to inrese the melnogenesis pthwy in melnoytes. Norml humn melnoytes (NHMs) nd humn derml mirovsulr endothelil ells (HMVECs) re pre-treted 2 hours with EDNRA nd EDNRB inhiitors, respetively, BQ23 nd BQ788 (2 μm), nd put in o-ulture for 30 minutes (). NHMs re pre-treted 2 hours with EDNRB inhiitor (2μM) nd inuted with HMVECs treted or not with BQ788-onditioned medium for 30 minutes (). NHMs re trnsfeted with sirna direted ginst EDNR, EDNR, or sirna-negtive ontrol. One ()or4(d) dys lter, NHMs re inuted with HMVECs during 30 minutes () or 3 dys (d). NHMs re inuted with HMVECs in the trnswell hmer during 30 minutes (e). NHMs nd HMVECs re stimulted with ERK/2 inhiitor UO26 (0 μm) or p38 inhiitor SB (0 μm) 2 hours efore the ddition of the HMVEC trnswell hmer in the well for 30 minutes (f nd g) or 3 dys (h nd i). The lyste of NHM is nlyzed y western lot with indited ntiodies. Numers ove the gels indite the levels of intensity ompred with tin. types (Psseron et l., 20), nd the effiy of trnexmi id hs still to e onfirmed in prospetive omprtive tril. Moreover, its effets re nonspeifi nd my indue side effets. By disseting the pthwy involved in the pigmenttion ssoited with vsulriztion, we demonstrte here the key role of the EDNRB. Thus, developing topil gents to inhiit ENDRB tivtion on melnoytes my limit the impt of the underlying vsulriztion nd provide, in omintion with lssi depigmenting gents, powerful pproh to tret melsm nd prevent relpses. MATERIALS AND METHODS Ptients Conseutive ptients presenting t lest one enign vsulr prolifertion on the skin were inluded. Exlusion riteri were inflmmtory skin disorders, photodermtoses, melsm, quired rhil utneous dyshromi, post-inflmmtory hyperpigmenttion, 302 Journl of Investigtive Dermtology (205), Volume 35

8 Ctrl BQ788 HMVEC HMVEC Ctrl ginst irulr nd fixed-shpe models. We thus deteted optilly hyperrefletive kertinoytes surrounding n ngiom vity nd less-pigmented kertinoytes loted on the perilesionl skin for the quntifition of pigmenttion. Regent nd ntiodies We otined ntiodies ginst nd β-tin from Am (Cmridge, MA), perk, nd pp38 from Cell Signling Tehnology (Beverly, MA), EDNRA, EDNAB, nd GAPDH from Snt Cruz Biotehnology (Snt Cruz, CA), nd tyrosinse nd from V. Hering. U026 nd SB203580, ilein, VEGF reeptor inhiitor IV, CBO-P, nd PTIO were purhsed from Millipore (Billeri, MA), PD42893 from Enzo Life Siene (Frmingdle, NY), nd LNAME, endothelin, BQ23, nd BQ788 from Sigm Aldrih (Sint Quentin Fllvier, Frne). BQ788 HMVEC Figure 6. Endothelin relesed y endothelil ells inreses the pigmenttion in reonstruted epidermis. Reonstruted humn pigmented epidermis in trnswell hmer is stimulted with EDNR inhiitor BQ788 (2 μm) nd inuted with humn derml mirovsulr endothelil ells (HMVECs) t the ottom of the well for 3 weeks. HMVECs re hnged every other dys, nd BQ788 dded every dy. Reonstruted humn pigmented epidermis is photogrphed in full size () or in 20 mgnifition (). Melnin quntity is determined y FontnMsson stining nd oserved t 40 (). nd onomitnt medition with photosensitizing drugs. Cherry ngioms, otriomyoms, spider ngioms, involutive infntile hemngioms, pillro-venous mlformtion, quired ilterl telngietti mules, nd leg telngietsis were inluded fter written informed onsent ws grnted. The ptient ge, gender, nd skin type were noted together with the loliztion nd size of involved lesions. As only noninvsive exmintion (epiluminesene dermosopy) ws performed nd ll the dt were definitively nonymized, institutionl review ord pprovl ws wived. Assessment of pigmenttion A digitl epiluminesene dermtosope t 200 mgnifition (Dinolite, Nrden, The Netherlnds) ws used for evluting the vsulr lesions nd the pigmenttion ove the lesions. The sme physiin performed (HHG) ll the evlutions. Pigmenttion ws ssessed using physiin glol ssessment sore tht grdes the differene in pigmenttion within nd in the infr millimeter order of vsulr lesions ompred with tht in the surrounding skin. The ontrst in pigmenttion ws sored s none, mild, moderte, or mrked. Vsulr lesions were lso ssessed using lser onfol mirosopy (Vivsope 500, Clier ID, Rohester, NY). Optil setions (Vivlok) of 4-mm width were quired every 20 mirons depth from the strtum orneum up to the superfiil dermis. Confol imges were exported from the VivSn Dtse to nlyze using Confosn to disern high- (right) or low- (drk) intensity ojets Cell ulture NHMs nd NHKs were otined from the foreskin of skin type IV hildren s desried previously. Briefly, epiderml ells re otined y overnight digestion of the skin in dispse solution t 4 C followed y inution of the epidermis in trypsin/edta solution for 20 minutes t 37 C. NHMs were isolted in MCDB 53 medium (Sigm Aldrih) supplemented with 2% FBS (Perio; Helsingorg, Sweden), 5 μgml insulin (Sigm Aldrih), 0.5 μgml hydroortisone (Sigm Aldrih), 6 nm TPA (Sigm Aldrih), ng ml FGF (Promeg; Mdison, WI), 5 μgml ovine pituitry extrt (Invitrogen; Crlsd, CA), 0 μm forskolin (Sigm Aldrih), nd 20 μgml genetiin (Invitrogen) over 2 weeks. NHKs were isolted in KGM 2 medium (Promoell, Heidelerg, Germny). HMVECs were otined from Invitrogen nd grown in Csde 3 medium supplemented with mirovsulr growth supplement on tthment ftor-oted pltes (Invitrogen). All ells were mintined t 37 C in 5% CO2 tmosphere. Co-ulture experiments For the o-ulture experiments, NHMs were seeded in six-well pltes, wheres HMVECs nd NHKs were seeded seprtely on 0.4 μm Trnswell inserts(beton Dikinson; Est Rutherford, NJ). Two dys efore experiments, NHMs were strved in MCDB 53 medium supplemented with 5 μgml insulin, 5 μgml ovine pituitry extrt, nd 2% fetl ovine serum to remove propigmenting gents. HMVECs nd NHKs were lso inuted for 2 dys in the NHM strvtion medium to void medium-dependent effets on pigmenttion. The o-ultures were initited when Trnswell oted with HMVECs or NHKs ws pled in well ontining ultured NHM. Reonstruted humn pigmented epidermis (RHPE) experiment RHPE (skin type IV), otined from SkinEthi (Lyon, Frne), is hrterized y kertinoytes nd melnoytes 3-D ulture from foreskin disposed on 0.4 μm trnswell hmer tht llows n irliquid interfe. Aording to SkinEthi proedure, RHPE re inuted 24 hours in RHPE growth medium efore experiment. For the HMVEC/RHPE o-ulture experiment, HMVECs re seeded in 24-well pltes nd inuted 24 hours with RHPE growth ftor efore inution with the RHPE. HMVECs re hnged every seond or third dys during the 3 weeks of o-ulture

9 Histopthology Eh iopsy ws fixed in 4% formlin nd prffin emedded. Morphologil exmintion ws rried out on setions (2-μm thikness) stined y hemtoxylin nd eosin or FontnMsson. Setions were heted for 20 minutes t 97 C in low ph uffer solution for ntigen retrievl (PT-link Dko devie, Glostrup, Denmrk). immunoleling (mouse ma produt ode: M362, Clone:D5, dilution /00, immunogen: Histidine fusion protein of the mino-terminl Tq-S frgment of humn DNA, Dko Frne SAS, les Ulis,) nd endothelin immunoleling (got polylonl ntiody, produt ode: ET- (N-8) s-2625, dilution /200, Snt Cruz Biotehnology) were performed using Dko utostiner ording to the mnufturer s reommendtions. After wshing with phosphte-uffered sline, the setions were inuted with Envision FLEXmouse (Linker)/HRP (DAKO) for 5 minutes t room temperture. After wshing with phosphte-uffered sline, the setions were inuted with Envision FLEX/HRP (DAKO) for 20 minutes t room temperture, followed y reveltion using the Envision System ording to the mnufturer s reommendtions, with DAB (diminoenzidine) for 8 minutes s hromogen. Finlly, the setions were ounterstined in Myer s hemtoxylin nd mounted in glyergel mounting medium (Dko). Melnoytes, identified y Mitf immunohistohemil stining, nd sl kertinoytes were ounted long the sement memrne, eside nd wy from the vsulr lesion, within the entire length of eh iopsy. We then set the rtio of kertinoytes/melnoytes. For eh smple, t lest 0 fields t 400 mgnifition were studied. Trnsient sirna trnsfetion A totl of 500,000 NHMs re used for reverse trnsfetion. NHMs re trypsinized, resuspended in strvtion medium, nd trnsfeted using INTERFER in (Polyplus Trnsfetion; Illkirh, Frne) ording to the mnufturer instrutions. Briefly, sirna-negtive ontrol, EDNRA nd EDNRB (50 pmol) (#-Thermo Fisher Sientifi; Wlthm, MA), (#2-Amion; Foster City, CA) re inuted with INTERFER in 5 minutes nd ly into the well efore the ddition of the ells. The experiments of 30 minutes of o-ulture were onduted 4 dys fter the trnsfetion, nd the experiments of 3 dys of o-ulture were performed dy ve the trnsfetion. Sttistil nlysis Sttistil differenes etween groups were nlyzed y Student's t- test. They were onsidered signifint t Po0.05. CONFLICT OF INTEREST AM is n employee of Beiersdorf. The remining uthors stte no onflit of interest. ACKNOWLEDGMENTS Light mirosopy ws performed on the C3M Imging Core Fility (prt of Mirosopy nd Imging pltform Côte d Azur, MICA). SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t REFERENCES Adini I, Ghosh K, Adini A et l. (204) Melnoyte-sereted firomodulin promotes n ngiogeni miroenvironment. JClinInvest24: Choi W, Woler R, Gerwt W et l. (200) The firolst-derived prrine ftor neuregulin- hs novel role in regulting the onstitutive olor nd melnoyte funtion in humn skin. JCellSi23:302 Duvl C, Regnier M, Shmidt R (200) Distint melnogeni response of humn melnoytes in mono-ulture, in o-ulture with kertinoytes nd in reonstruted epidermis, to UV exposure. Pigment Cell Res 4:34855 Fitzptrik TB, Brethnh AS (963) The epiderml melnin unit system. Dermtologishe Wohenshrift 47:489 Hiroe T (2005) Role of kertinoyte-derived ftors involved in regulting the prolifertion nd differentition of mmmlin epiderml melnoytes. Pigment Cell Res 8:22 Kng HY, Bhdorn P, Suzuki I et l. (200) In vivo refletne onfol mirosopy detets pigmentry hnges in melsm t ellulr level resolution. Exp Dermtol 9:e22833 Kng HY, Suzuki I, Lee DJ et l. (20) Trnsriptionl profiling shows ltered expression of wnt pthwy- nd lipid metolism-relted genes s well s melnogenesis-relted genes in melsm. J Investig Dermtol Symp Pro 3: Kim EH, Kim YC, Lee ES et l. (2007) The vsulr hrteristis of melsm. J Dermtol Si 46:6 Kim EJ, Prk HY, Yr M et l. (2005) Modultion of vsulr endothelil growth ftor reeptors in melnoytes. Exp Dermtol 4:62533 Kim JY, Lee TR, Lee AY (203) Redued WIF- expression stimultes skin hyperpigmenttion in ptients with melsm. J Investig Dermtol Symp Pro 33:9200 Lei TC, Virdor VM, Vieir WD et l. (2002) A melnoyte-kertinoyte oulture model to ssess regultors of pigmenttion in vitro. Anl Biohem 305:2608 N JI, Choi SY, Yng SH et l. (203) Effet of trnexmi id on melsm: linil tril with histologil evlution. J Eur Ad Dermtol Venereol 27: 0359 Nordlund JJ (2007) The melnoyte nd the epiderml melnin unit: n expnded onept. Dermtol Clin 25:278 Prk JH, Lee DJ, Lee YJ et l. (204) Aquired ilterl telngietti mules: distint linil entity. JAMA Dermtol 50:9747 Prk TJ, Kim M, Kim H et l. (204) Wnt inhiitory ftor (WIF)- promotes melnogenesis in norml humn melnoytes. 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