All-Trans Retinoic Acid Antagonizes UV-Induced VEGF Production and Angiogenesis via the Inhibition of ERK Activation in Human Skin Keratinocytes

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1 ORIGINAL ARTICLE All-Trns Retinoic Acid Antgonizes UV-Induced VEGF Production nd Angiogenesis vi the Inhibition of ERK Activtion in Humn Skin Kertinocytes Mi-Sun Kim 1,2,3, Yeon K. Kim 1,2,3, Hee C. Eun 1,2,3, Kwng H. Cho 1,2,3 nd Jin H. Chung 1,2,3 Incident UV rdition leds to the upregultion of vsculr endothelil growth fctor (VEGF), potent ngiogenic fctor, in humn skin. However, the moleculr bsis of UV-induced ngiogenesis in skin remins to be elucidted. In this study, we investigted the roles of UV exposure on cutneous ngiogenesis, its ssocited signling mechnisms, nd the effect of ll-trns retinoic cid () on UV-induced vsculriztion, nd VEGF expression. Using humn epiderml cell line, HCT, we found tht UV induces VEGF mrna nd protein expression vi the MAPK/ERK kinse ERK1/2 (extrcellulr signl-regulted kinse 1/2) pthwy but not vi the phosphtidylinositol 3-kinse (PI3K)/Akt pthwy, nd tht pretretment significntly inhibits UV-induced VEGF overexpression nd ERK1/2 ctivtion. In humn skin in vivo, we confirmed tht skin vsculriztion significntly incresed fter single exposure to UV, s ws evidenced by prominent increse in vessel size, vsculr density, nd in the cutneous re occupied by vessels, nd we found tht these events re ssocited with VEGF upregultion. Topicl pretretment with under occlusion inhibited not only UV-induced VEGF upregultion nd ngiogenesis with significnt reduction of vessel density but lso UV-induced ERK1/2 ctivtion in humn skin. Collectively, our dt demonstrte tht inhibits the UV-induced ngiogenic switch vi downmodultion of ERK1/2 ctivtion nd consecutive VEGF overexpression. These findings my help us understnd the moleculr mechnisms tht regulte skin ngiogenesis due to UV exposure, nd provide evidence of the potentil of in terms of preventing ngiogenesis-ssocited skin dmge following exposure to UV irrdition. Journl of Investigtive Dermtology (26) 126, doi:1.138/sj.jid.57463; published online 29 June 26 INTRODUCTION Humn skin is exposed dily to solr UV rdition nd the epidermis is primry trget for UV rdition. Chronic exposure of skin to UV rdition leds to photoging nd photocrcinogenesis (Clydesdle et l., 21; Ichihshi et l., 23). Recently, it ws reported tht single exposure to UV rdition my induce vsculr hyperpermebility, derml blood vessel diltion, nd led to n imblnce between ngiogenic fctors nd inhibitors in humn skin (Yno et l., 25). Angiogenesis, the process of generting new cpillry blood vessels, is ccompnied with physiologicl nd 1 Deprtment of Dermtology, Seoul Ntionl University College of Medicine, Seoul, Kore; 2 Lbortory of Cutneous Aging Reserch, Clinicl Reserch Institute, Seoul Ntionl University Hospitl, Seoul, Kore nd 3 Institute of Dermtologicl Science, Medicl Reserch Center, Seoul Ntionl University, Seoul, Kore Correspondence: Dr Kwng Hyun Cho or Dr Jin Ho Chung, Deprtment of Dermtology, Seoul Ntionl University Hospitl, 28 Yongon-dong, Chongno-gu, Seoul , Kore. E-mils: khcho@snu.c.kr or jhchung@snu.c.kr Abbrevitions: ERK, extrcellulr signl-regulted kinse; MED, miniml erythem dose; MEK, MAPK/ERK kinse; PI3K, phosphtidylinositol 3-kinse;, ll-trns retinoic cid; VEGF, vsculr endothelil growth fctor Received 26 Jnury 26; revised 19 April 26; ccepted 22 My 26; published online 29 June 26 pthologicl conditions of skin (Detmr, 2; Velsco nd Lnge-Asschenfeldt, 22). However, the moleculr bsis nd regultion of ngiogenesis-ssocited skin dmges induced by cute UV exposure remin uncler. It is ccepted tht epiderml kertinocytes hve primry role in the physiology nd pthology of cutneous ngiogenesis (Mlhotr et l., 1989). Moreover, epidermis-derived vsculr endothelil growth fctor (VEGF) is regrded s potent ngiogenic fctor in mny cutneous diseses (Bowden et l., 22; Gir et l., 24). VEGF is homodimeric, heprin-binding glycoprotein tht lso binds to specific receptors, which re minly expressed in endothelil cells. Alterntive splicing of the originl mrna trnscript genertes t lest five different VEGF isoforms (Btes et l., 22), the 121, 165, nd 189 mino-cid isoforms of which predominte in humn kertinocytes (Bllun et l., 1995). The expression of VEGF in both immortlized nd cultured kertinocytes is incresed by severl stimulnts, for exmple, oxidnts, TNF-, nd kertinocyte growth fctor (Frnk et l., 1995; Bruchle et l., 1996). In prticulr, severl reports hve suggested tht VEGF is induced in humn kertinocytes fter UV exposure (Mildner et l., 1999; Bludschun et l., 2). The retinoids consist of vitmin A (retinol), nd its nturlly occurring nd synthetic derivtives, mny of which re potent gents for the tretment of different skin disorders, & 26 The Society for Investigtive Dermtology

2 such s cne, psorisis, photoging, nd cncer (Roos et l., 1998). They modulte cell growth nd differentition in skin (Fisher nd Voorhees, 1996), nd inhibit ngiogenesis, endothelil cell migrtion nd crcinogenesis in skin cncers (Lingen et l., 1998; Pl et l., 2). Moreover, recent studies hve demonstrted tht retinoids inhibit bsl VEGF expression in cultured kertinocytes nd humn skin grfted onto nude mice (Lchgr et l., 1999; Diz et l., 2). However, the effects nd moleculr mechnisms underlying the ction of ll-trns retinoic cid () on cute UVinduced ngiogenesis in humn skin hve not been studied. In this study, we exmined VEGF expression fter dministering by single dose of UV rdition to HCT cells nd to humn skin epidermis, nd sought to identify the signling molecules involved in UV-induced VEGF expression. In ddition, we investigted the effects of on VEGF expression nd cutneous vsculrity fter single UV exposure in vitro nd in vivo experimentl systems. RESULTS VEGF levels re incresed by UV irrdition in kertinocytes To investigte the effect of UV on VEGF expression in the humn kertinocyte cell line HCT, cells were irrdited with 5 mj/cm 2 of UV nd hrvested t different times (2 1 h) fter UV irrdition. To exmine UV-induced VEGF b VEGF (pg/ml) Time fter UV irrdition (h) c VEGF 189 VEGF VEGF GAPDH UV VEGF expression (%) Figure 1. UV induced VEGF expression in HCT cells. Cells were seeded t cells/well on 6-mm-well plte nd strved for 24 hours before UV irrdition (5 mj/cm 2 ). () VEGF levels were nlyzed using sndwich ELISA specific for humn VEGF in the superntnts of UV-irrdited or shm-irrdited HCT cells t vrious time points. Dt re presented s men7sem of four independent experiments. Po.5 versus nonirrdited cells (Mnn Whitney U-test). (b) VEGF mrna expression ws nlyzed by semiquntittive RT-PCR t 4 hours post-uv. VEGF products of 516 bp, 648 nd 73 bp for VEGF 121, VEGF 165, nd VEGF 189, respectively, were obtined. (c) Desitometry ws used to nlyze the bnds generted from VEGF nd GAPDH in five independent experiments. Po.5 versus nonirrdited cells (Mnn Whitney U-test). secretion, superntnts were checked for VEGF protein t different time points by ELISA (Figure 1). Relesed VEGF protein levels were significntly incresed by pproximtely 3.6-fold in UV-irrdited conditioned medium versus shmirrdited control medium 6 hours post-uv, nd this ws mintined t 8 hours post-uv. As shown in Figure 1b, low level of VEGF mrna ws detected in quiescent, nonirrdited cells nd VEGF mrna expression peked t 4 hours post-uv by 2.1-fold versus the shm-irrdited control cells. The MEK ERK1/2 pthwy involves UV-induced VEGF upregultion in kertinocytes To determine the mechnisms underlying VEGF regultion in response to UV exposure, we ttempted to identify the pthwys involved in the regultion of VEGF expression post- UV in HCT cells. Extrcellulr signl-regulted kinse 1/2 (ERK1/2) ctivtion nd Akt ctivtion were observed in previous study. We pretreted cells for 3 minutes with either specific MAPK/ERK kinse (MEK) inhibitor, U126 (Fvt et l., 1998), or specific phosphtidylinositol 3-kinse PI3K inhibitor, wortmnnin (Powis et l., 1994) prior to UV. Cells were hrvested t 2 hours post-uv. As shown in Figure 2, these pretretments were found to inhibit the downstrem trgets, ERK1/2 or Akt. U126 (2 mm) blocked ERK1/2 ctivtion, nd wortmnnin (5 nm) blocked Akt ctivtion. Wortmnnin hd no effect on ERK phosphoryltion nd U126 hd no effect on Akt phosphoryltion. To investigte the involvements of these signling pthwys on UV-induced VEGF expression, we exmined VEGF expressions in the presence nd bsence of specific inhibitors. VEGF levels secreted from cells pretreted with/ U126 Wortmnnin 5 p-erk1 U126 p-erk2 4 Wortmnnin ERK1 3 ERK2 2 p-akt Akt 1 c b VEGF (pg/ml) U126 Wortmnnin VEGF 189 VEGF 165 VEGF 121 GAPDH Figure 2. The MEK ERK1/2 pthwy involves UV-induced VEGF upregultion in HCT cells. () HCT cells were pretreted for 3 minutes with Vehicle (DMSO), 2 mm U126, or 5nM wortmnnin before UV irrdition. The ctivtions of ERK1/2 nd Akt were detected by Western blotting t 2 hours post-uv. Similr results were obtined for three independent experiments. (b) At 6 hours post-uv, VEGF levels were nlyzed by ELISA in superntnts of UV-irrdited (5 mj/cm 2 ) or shm-irrdited cells in the bsence or presence of inhibitors. The dt shown represent the mens7sem of four independent experiments. Po.5 versus nonirrdited cells in ech condition, nd w Po.5 versus vehicle pretreted UV-irrdited cells (Mnn Whitney U-test). (c) At 4 hours post-uv, the effects of inhibitors on VEGF mrna expression were exmined by semiquntittive RT-PCR. Similr results were obtined for three independent experiments Journl of Investigtive Dermtology (26), Volume 126

3 without U126 or wortmnnin for 3 minutes prior to UV irrdition were nlyzed by ELISA. The mounts of VEGF relesed into culture medi 6 hours post-uv from cells ws lmost 3.8-fold increse compred with cells not exposed to UV. This UV-induced VEGF relese ws significntly inhibited by U126 (81% inhibition), but not by wortmnnin (Figure 2b). The effect of ERK inhibition on VEGF mrna expressions ws similr to tht of protein expression t 4 hours post-uv (Figure 2c). U126 completely inhibited UVinduced VEGF mrna expression to the bsl mrna level, wheres wortmnnin hd no inhibitory effect on UV-induced VEGF upregultion. These findings demonstrte tht the MEK ERK pthwy positively modultes VEGF expression in UV-irrdited kertinocytes. inhibits UV-induced VEGF expression nd ERK1/2 ctivtion in kertinocytes We next investigted the effects of on UV-induced VEGF relese nd ERK ctivtion. HCT cells were treted with vehicle (.5% DMSO) or (1 9 M) for 24 hours prior to UV exposure (5 mj/cm 2 ), nd VEGF levels in superntnt were mesured by ELISA 6 hours post-uv (Figure 3). Bsl VEGF secretion ws reduced by 44.1% by in nonirrdited cells nd completely blocked UV-induced VEGF relese. We next exmined the effect of on UV-induced ERK1/2 phosphoryltion. Cells were treted with vehicle or (1 9 M) for 24 hours prior to UV exposure (5 mj/cm 2 ), nd hrvested t 2 hours post-uv (Figure 3b). Pretretment with obviously inhibited ERK1/2 ctivtion, in nonirrdited cells nd irrdited cells. These dt indicte cn inhibit UV-induced ctivtion of ERK1/2 protein following VEGF upregultion in humn kertinocytes. Derml ngiogenesis is induced by single dose of UV irrdition in humn skin in vivo To observe the effects of UV irrdition on ngiogenesis nd VEGF expression in humn skin in vivo, sun-protected buttock skin (n ¼ 3) ws exposed to single dose of UV rdition (2 MED). Skin smples were obtined fter 1, 2, nd 3 dys. Routine hemtoxylin nd eosin stining reveled VEGF (pg/ml) p-erk1 p-erk2 ERK1 ERK2 Figure 3. inhibited the induction of VEGF nd ERK1/2 protein by UV in HCT cells. Serum-strved cells were treted for 24 hours with (1 9 M) nd then irrdited with UV (5 mj/cm 2 ). () Superntnts were nlyzed using VEGF-specific ELISA t 6 hours post-uv. Dt represent the mens7sem of three independent experiments. Po.5 versus. vehiclepretreted nonirrdited cells, nd w Po.5 versus vehicle-pretreted cells in ech condition (Mnn Whitney U-test). (b) Cell lystes were nlyzed using Western blotting with nti-phospho ERK1/2 nd ERK1/2 ntibodies t 2 hours post-uv. Similr results were obtined for three independent experiments. b obvious epiderml hyperplsi nd n incresed epiderml thickness fter UV exposure. Moreover, inflmmtory cell recruitment ws observed in the upper dermis s compred with norml nonirrdited skin (Figure 4). Immunohistochemistry for endothelil cell dhesion molecule CD31 (PECAM-1) reveled progressive increse in the number of enlrged vessels in UV-irrdited skin compred with untreted control skin (Figure 4b). Computer-ssisted quntittive imge nlysis of CD31-stined cutneous vessels ws performed to quntify cutneous vessel numbers nd sizes. Three different fields per section were exmined, nd the number of vessels per mm 2, verge vessel size, nd the reltive re occupied by blood vessels were determined within 2 mm from the epiderml derml junction. Vsculr densities were found to be significntly incresed on dy 2 (52.6% increse) nd on dy 3 (5.5% increse) fter UV irrdition compred with nonirrdited skin (Figure 4c). In ddition, verge vessel size ws significntly incresed on dy 1 (45.3% increse), dy 2 (57.2% increse), nd dy 3 (9.2% increse) post-uv (Figure 4d). Consequently, the cutneous re covered by blood vessels locted within 2 mm from epiderml derml junction grdully incresed from 1.5% in norml skin to 4.3% in UV-irrdited skin on dy 3 (Figure 4e). These chnges in neovsculriztion were ccompnied by incresed endothelil cell prolifertion (dt not shown), nd were prominent in the ppillry dermis, which immeditely underlies the epidermis. VEGF is upregulted by single exposure to UV in humn skin epidermis The effects of cute UV irrdition on epiderml VEGF expression in sun-protected buttock skin were exmined by immunohistochemistry, Western blotting, nd semiquntittive RT-PCR. Wheres VEGF expression ws low in nonirrdited control skin, it ws strongly expressed in the grnulr lyer dy 1 post-uv (2 MED) irrdition (n ¼ 3). On dys 2 nd 3 post-uv, VEGF expression ws distributed throughout the epidermis (Figure 5). Western blot nlyses of epidermis showed tht VEGF expressions were significntly incresed on dys 2 nd 3 post-uv (n ¼ 5, Figure 5b). As shown in Figure 5c, VEGF mrna ws lso overexpressed on dy 1 post-uv versus nonirrdited skin (n ¼ 5). To investigte the involvement of the ERK pthwy, humn buttock skin ws irrdited with 2 MED of UV nd skin biopsies were obtined from ech subject t 2, 4, nd 8 h, nd on dys 1, nd 2 post-uv. A significnt increse in ERK phosphoryltion ws observed t 4 nd 8 hours post-uv nd this returned to its bsl level t 2-dy post-uv (n ¼ 4, Figure 5d). UV exposure did not lter totl ERK levels t these times, nd like HCT cells, the epidermis expressed considerbly more ERK2 thn ERK1. inhibits UV-induced ngiogenesis nd VEGF upregultion in humn skin in vivo We investigted whether pretreting humn skin with could inhibit UV-induced cutneous ngiogenesis. Humn buttock skin (n ¼ 4) ws treted with vehicle nd

4 N H&E b CD31 c 1 Vessels/mm N d1 d2 d3 d1 d2 d3 d Vessel re (%) Vessel size (μm 2 ) e Figure 4. Acute UV exposure induced epiderml hyperplsi nd ngiogenesis in humn skin. Sun-protected buttock skin ws exposed to single dose of UV (2 MED) nd smples were obtined by punch biopsy t 1, 2, nd 3 dys post-uv. () Hemtoxylin nd eosin stining nd (b) immunohistochemicl stining for CD31 were performed on skin sections (see Mterils nd Methods, n ¼ 3). Br ¼ 1 mm. (c) Vessel density, (d) vessel size, nd (e) vessel re within 2 mm from the epiderml derml junction were nlyzed using computer-ssisted morphometric nlysis progrm. Three different fields per section were exmined, nd the number of vessels per mm 2, the verge vessel size, nd the reltive re occupied by blood vessels were determined in the ppillry dermis. Dt re presented s mens7sem for three subjects. Po.5 versus nonirrdited norml skin (Wilcoxon Signed-Rnk test). (.25%) for 48 hours before UV irrdition (2 MED) under occlusion. Skin smples were obtined t 48 hours fter UV irrdition nd derml vsculriztion ws nlyzed by immunohistochemistry of CD31 (Figure 6). pplied for 4 dys to norml skin tended to increse the vsculrity of the dermis. Topicl ppliction of before UV irrdition significntly inhibited UV-induced ngiogenesis s determined by vessel density (Figure 6c, 86.8% inhibition) nd by vessel re (Figure 6d, 64.6% inhibition), s compred with skin pretreted with vehicle nd exposed to UV. However, pretretment did not ffect UV-induced vessel size increses (Figure 6b). We next exmined the effects of on UV-induced VEGF protein nd mrna expression, nd ERK1/2 phosphoryltion. Humn buttock skin ws pretreted with vehicle nd (.25%) for 48 hours before UV irrdition (2 MED) under occlusion, nd skin smples were obtined 48 hours post-uv. Immunohistology reveled tht UV induced VEGF protein throughout the epidermis nd tht pretretment substntilly inhibited UV-induced VEGF expression versus vehicletreted, UV-irrdited skin (n ¼ 4, Figure 7). Epidermis thickness increses fter UV irrdition nd slightly inhibited by pretretment. Moreover, the pretretment of humn skin with blocked UV-induced mrna overexpression of VEGF (n ¼ 3, Figure 7b). Owing to mximl ERK ctivtion ws observed 4 or 8 hours post-uv (Figure 5d), skin smples were obtined t 8 hours post-uv nd ERK phosphoryltion levels in epidermis were nlyzed (n ¼ 3, Figure 7c). Topicl pretretment with ws found to significntly inhibit UVinduced ERK1/2 ctivtion versus vehicle-treted, UV-irrdited skin. These results indicte tht topicl tretment with cn prevent cute UV response by inhibiting UV-induced ERK1/2 ctivtion, VEGF upregultion, nd ngiogenesis. DISCUSSION Angiogenesis hs remined mjor focus of cncer reserch for the pst two decdes. However, the vsculr chnges re lso the clinicl chrcteristics of noncncerous skin diseses like rosce nd psorisis (Bhushn et l., 22). A recent niml study suggested tht skin ngiogenesis plys criticl role in photoging, nd tht the inhibition of ngiogenesis diminishes UVB-induced wrinkle formtion (Yno et l., 22). Although neovsculriztion is typicl rection of UV-ssocited skin diseses, the mechnism underlying humn skin ngiogenesis remins uncler. The level of VEGF, the mjor ngiogenic fctor, is importnt prmeter in mintining blnced skin ngiogenesis. It hs been identified s mjor kertinocyte-derived 27 Journl of Investigtive Dermtology (26), Volume 126

5 b c d VEGF VEGF 189 VEGF 165 VEGF 121 N 2 h 4 h 8 h d1 d2 p-erk1 p-erk2 Actin GAPDH ERK1 ERK2 % of norml % of norml % of norml N d1 d2 d3 N 2 h 4 h 8 h d1 d2 Figure 5. Acute UV exposure induced VEGF overexpression nd ERK1/2 ctivtion in humn skin epidermis. Sun-protected buttock skin ws exposed to single dose of UV (2 MED). Smples were obtined by punch biopsy t the indicted times post-uv. () Immunostining for VEGF ws performed on frozen sections (n ¼ 3). Br ¼ 1 mm. (b) Protein extrcts from epidermis were nlyzed by Western blotting using ntibody for VEGF (n ¼ 5). Epidermis ws seprted from dermis with forceps fter incubting skin smples t 581C for 2 minutes in PBS. (c) Semiquntittive RT-PCR showed chnges in VEGF mrna expressions in the epidermis (n ¼ 5). The products of VEGF were of 516, 648, nd 73 bp, nd those of GAPDH were 565 bp, respectively. (d) The time-dependent effect of UV on ERK1/2 ctivtion ws investigted in epidermis (n ¼ 4). The results from ll subjects were nlyzed by densitometry (normliztion by ctin or GAPDH) in the lower pnels nd results re presented s mens7sem for ll subjects. Po.5 versus nonirrdited norml skin (Wilcoxon Signed-Rnk test). b Vessel size (μm 2 ) c Vessels/mm 2 d Vessel re (%) Figure 6. UV-induced derml ngiogenesis ws inhibited by in humn skin in vivo. Humn skin ws pretreted with vehicle nd.25% for 48 hours before UV irrdition (2 MED). Skin smples were obtined 48 hours fter UV exposure. () Immunohistologicl stining of CD31, (b) vessel size, (c) vessel density, nd (d) blood vessel re within 2 mm from the epiderml derml junction were nlyzed. Computer-ssisted imge nlysis of CD31 stined sections ws performed in three different fields per section. Dt re presented s mens7sem for four subjects. Po.5 versus vehicle-pretreted nonirrdited cells, nd w Po.5 versus vehicle-pretreted cells in ech condition (Wilcoxon Signed-Rnk test). Br ¼ 1 mm. vessel-specific growth fctor (Detmr et l., 1995), nd the expression hs been reported to be upregulted in the hyperplstic epidermis of psorisis (Detmr et l., 1994), in keloids (Gir et l., 24), in bsl cell crcinom (Oh et l., 23), squmous cell crcinom (Bowden et l., 22), melnom (Gorski et l., 23), nd in chronic wounds (Luer et l., 2). Recently, efforts hve being mde to reduce VEGF levels for ntingiogenic nd ntitumor therpy. Thus, studies on the regultion of VEGF in skin provide bsic understnding tht cn be used for the 271

6 H&E VEGF b c VEGF 189 VEGF 165 VEGF 121 GAPDH p-erk1 p-erk2 ERK1 ERK2 % of level % of level 4 3 Figure 7. inhibited UV-induced VEGF overexpression nd ERK1/2 ctivtion in humn skin in vivo. Humn skin ws pretreted with vehicle nd.25% for 48 hours before UV irrdition (2 MED). () Skin smples were obtined t 48 hours post-uv exposure. Hemtoxylin nd eosin stining nd immunostining for VEGF were performed (n ¼ 4). Br ¼ 1mm. (b) VEGF mrna expression in epidermis ws determined by semiquntittive RT-PCR t 48 hours post-uv. Densitometry vlues re mens7sem for three subjects. (c) Skin biopsies were tken t 8 hours post-uv nd epiderml extrcts were prepred nd nlyzed for phospho- nd totl ERK1/2 protein levels by Western blotting. Densitometric dt re presented s mens7sem for three subjects. Po.5 versus vehicle-pretreted nonirrdited cells, nd w Po.5 versus vehicle-pretreted cells (Mnn Whitney U-test). development of tretments for ngiogenesis-ssocited skin diseses. In this study, we found tht UV induces VEGF overexpression in HCT kertinocytes nd in the epidermis of humn skin. According to recent reports, the PI3K Akt pthwy nd the Rs-p42/p44 mitogen-ctivted protein kinse (ERK1/2) signling pthwy ply importnt regultory roles in ngiogenesis (Berr et l., 2; Segrelles et l., 24). Therefore, we investigted whether or not ERK1/2 nd Akt pthwys re involved in UV-induced VEGF production in kertinocytes. Our results show tht UV-induced VEGF overexpression ws suppressed by U126, specific MEK inhibitor, but not by wortmnnin, specific PI3K inhibitor, which suggests tht the MEK ERK1/2 pthwy is involved in UV-stimulted VEGF upregultion in kertinocytes. In ddition, remrkbly inhibited the UV-induced ERK1/2 ctivtion nd VEGF overexpression in kertinocytes. It hs been reported tht UV cn induce VEGF upregultion in immortlized HCT cells developed from humn dult skin kertinocytes (Fusenig nd Boukmp, 1998). It hs been demonstrted tht the first induction of VEGF by UVB is regulted in coordinted fshion by the ctivtion of epiderml growth fctor receptor nd subsequent delyed relese of TGF- (Bludschun et l., 2). It ws lso reported tht severl stimultors induced VEGF upregultion vi mitogen-ctivted protein kinse signl trnsduction pthwys in kertinocytes. Recently, tretment of HCT kertinocytes with insulin-like growth fctor-ii (IGF-II) ws reported to probbly ctivte single mitogen-ctivted protein kinse pthwy, tht is, n ERK pthwy, but not p38 or JNK pthwy, nd tht this incresesd VEGF expression (Kwon et l., 24). Moreover, it ws demonstrted tht IGF-II induces VEGF gene expression vi tyrosine kinse-src-erk1/2 nd PI3K pthwys in HCT kertinocytes (Kim nd Kim, 25). In ddition, VEGF secretion ws found to be regulted by tyrosine kinse-src-erk1/2, but this secretion ws independent of PI3K. Another stimultor, heptocyte growth fctor/sctter fctor, my functions s potent inducer of VEGF expression in kertinocytes (Gille et l., 1998). Heptocyte growth fctor/sctter fctor-induced specificity protein 1 phosphoryltion ws suggested to ctivte VEGF promoter ctivity, which requires contributions by 272 Journl of Investigtive Dermtology (26), Volume 126

7 distinct signling molecules, such s PI3K, ERK1/2, nd PKCz (Reisinger et l., 23). The results of the present study show tht UV-induced VEGF upregultion occurs vi MEK ERK1/2 ctivtion nd not vi PI3K-Akt ctivtion in humn kertinocytes, nd indicte tht signl trnsduction pthwys ssocited with VEGF regultion depend on specific stimultions in kertinocytes. It is well known tht retinoids regulte cell differentition, prolifertion nd poptosis. In ddition, they hve been shown to inhibit ngiogenesis in vitro nd in vivo experiments (Liudet-Coopmn et l., 1997; Pl et l., 2). Such ntingiogenic effects of retinoids my be the result of ltertions in the ctivities of endothelil cells, for exmple, the inhibition of endothelil cell migrtion, nd in the production of fctors tht stimulte or inhibit vessel growth. In this study, we observed the inhibition of bsl VEGF expression by in HCT kertinocytes. This inhibitory effect is consistent with other reports tht demonstrted tht RA inhibits spontneous VEGF production in primry cultured humn norml kertinocytes (Weninger et l., 1998; Lchgr et l., 1999). hs been reported to stimulte or suppress the ctivtion of mitogen-ctivted protein kinse signling pthwys in different cell models (Alsyed et l., 21; Kunisd et l., 25). Our results show tht inhibits UV-induced VEGF expression vi ERK1/2 downregultion in kertinocytes. However, in contrst to our results concerning the inhibitory effect of on VEGF expression, it ws suggested tht RA stimultes the relese of VEGF vi ERK1/2 ctivtion in ortic smooth muscle cells (Tnbe et l., 24). The resons of this discrepncy nd how RA ffects ERK ctivtion still remin to be investigted. Further studies re needed to understnd the moleculr mechnism of the inhibitory effects of on UV-induced ERK1/2 ctivtion. Topicl retinoids re widely used to tret psorisis nd rosce, which suggests tht VEGF hs pthogenic role (Rolewski, 23). Topicl retinoid therpy is lso used to reverse some of the effects of photoging (Strtigos nd Ktsmbs, 25). The long-term ppliction of drmticlly chnges the skin rchitecture; it increses epiderml thickness, fcilittes the depositions of extrcellulr mtrix proteins, nd increses derml vsculrity. The short-term ppliction of (for 4 dys) to buttock skin in this study lso tended to increse derml vsculrity nd epiderml VEGF expression. These findings contrdict the in vitro results concerning the inhibition of bsl VEGF expression by in kertinocytes. It is possible tht the effects of tretment in kertinocytes in vitro differ from its effects when pplied topiclly to epidermis in humn skin in vivo. Our results show tht (i) inhibits bsl VEGF expression in kertinocytes in vitro, but induces VEGF expression in humn skin epidermis in vivo; (ii) blocks UV-induced VEGF expression both in vitro nd in vivo; nd (iii) tht this inhibitory effect of depends on the inhibition of ERK1/2 phosphoryltion in signling pthwys. Thus, our results indicte tht hs n inhibitory effect on UVinduced ngiogenesis vi the downregultion of VEGF signling. These findings demonstrte tht is potent UV Humn skin epidermis (kertinocytes) MEK/ERK VEGF Derml ngiogenesis Figure 8. Schemtic representtion of UV-induced ngiogenesis in humn skin. UV upregultes kertinocyte-derived VEGF nd promotes derml ngiogenesis through the MEK ERK1/2 pthwy in humn skin. effectively inhibited UV-induced ERK1/2 ctivtion, consecutive VEGF overexpression, nd derml ngiogenesis in humn skin in vivo. inhibitor of the ngiogenesis cused by UV exposure, nd tht it might be useful therpeutic gent ginst ngiogenesis-ssocited photo-dmges. Despite recent dvnces in our understnding of ngiogenesis, mny questions remin unnswered. Further studies re needed to chrcterize the reltionship between skin ngiogenesis nd the skin dmge induced by UV. In ddition, we need to know whether RA cn regulte other ngiogenic fctors nd signling molecules tht lter the moleculr blnce for ngiogenesis in humn skin. In conclusion, our study demonstrtes for the first time tht cute exposure to UV rdition triggers ngiogenesis vi VEGF induction nd MEK ERK1/2 ctivtion, nd tht inhibits UV-induced ERK1/2 ctivtion, VEGF upregultion, nd ngiogenesis in humn skin (Figure 8). Thus, strtegy bsed on VEGF inhibition fter UV exposure my prevent or rrest the evolution of skin dmges ssocited with UV, which re chrcterized by incresed vsculr permebility nd ngiogenesis. An understnding of the cscde of moleculr signls in UV-induced signling pthwys my provide us with new insights nd therpeutic trget molecules tht llow the prevention of ngiogenesis-ssocited skin photo-dmges. MATERIALS AND METHODS Regents nd ntibodies Cell culture medium, DMEM, nd fetl bovine serum were purchsed from Gibco BRL (Grnd Islnd, NY), nd U126, wortmnnin nd (tretinoin) from Sigm-Aldrich, Inc. (St Louis, MO). For RT-PCR, VEGF, nd GAPDH primer sets were synthesized by Bioneer Co. (DeJeon, Kore). For immunohistochemistry nd Western blotting, nti-humn CD31 ntibody ws purchsed from PhrMingen (Sn Diego, CA); nti-humn VEGF ntibody from Snt Cruz Biotechnology (Snt Cruz, CA); phospho-specific ERK nd totl ERK ntibodies from Cell Signling Technology (Beverly, MA); nd nti-mouse IgG nd nti-rbbit IgG conjugted peroxidse from Amershm Phrmci Biotechnology (Brunschweig, Germny). For ELISA, nti-humn VEGF ntibody, biotinylted nti-humn VEGF 273

8 ntibody, nd recombinnt VEGF were obtined from R&D Systems (Minnepolis, MN). Cell culture nd UV exposure The humn epiderml cell line, HCT, ws cultured in DMEM supplemented with 1% fetl bovine serum, penicillin (4 U/ml), streptomycin (5 mg/ml), nd glutmine (2 mm), nd grown on plstic tissue culture dishes in humidified 5% CO 2 tmosphere t 371C. A Wldmnn brod-bnd UVB (Wldmnn, Villingen-Schwenningen, Germny) phototherpy device equipped with F85/1W/UV21 fluorescent sunlmps with n emission spectrum between 285 nd 35 nm (peking 313 nm) ws used s the UV source. A Kodcel filter (TA41/47, Kodk, Rochester, NY) ws mounted 5 cm in front of the UV lmps to remove wvelengths of less thn 29 nm (UVC). UV intensity ws mesured using Wldmnn UV meter (Model No. 5851). In order to void interference due to the cytotoxic effect of UV, MTT ssys were performed to determine the optiml noncytotoxic dose (4 1 mj/cm 2 ). Subconfluent monolyer cultures of serum-strved HCT cells (2 1 5 cells/ml) were rinsed with phosphte-buffered sline (PBS) nd therefter irrdited under thin lyer of PBS t UV dose of 5 mj/cm 2. Shm-irrdited control cells were hndled in n identicl mnner, but were not exposed to UV. After irrdition, medi were replenished with fresh DMEM medium without fetl bovine serum, nd cells were cultured for vrious times. In experiments involving tretment with inhibitors, cells were pretreted with 2 mm U126 ( specific MEK inhibitor) or 5 nm wortmnnin ( specific PI3K inhibitor) for 3 minutes before UV irrdition. In experiments involving, cells were preincubted with 1 9 M for 24 hours before UV irrdition. All regents were dissolved in DMSO, nd cells were treted with the sme mounts of DMSO in medium before UV irrdition. Mesurement of VEGF secretion After UV irrdition, cell-free superntnts were collected nd stored t 71C until required for cytokine ssy. Secreted VEGF levels in the culture medi of HCT cells were determined by sndwich ELISA, ccording to the mnufcture s protocol (R&D systems). Absorption of vidin-horserdish peroxidse color rection ws mesured t 45 nm nd VEGF concentrtions were determined using stndrd grph prepred using seril dilutions of humn recombinnt humn VEGF s stndrd. Humn skin smples Eighteen Koren volunteers (17 men nd 1 womn, men ge yer, ge rnge 2 36 yer) without current or prior skin disese were enrolled in the study. For UV exposure, we used the device described bove for in vitro study. The MED for ech subject ws determined 24 hours fter irrdition. Usully, the MED mesured for filtered UV ws round 7 9 mj/cm 2. The phototypes of the volunteers include types III, IV, nd V. Irrdited (2 MED of UV) nd nonirrdited buttock skin smples were obtined from ech subject by punch biopsy t the indicted time points fter UV irrdition. The smples were plced immeditely into cryomtrix (Shndon, Pittsburgh, PA) for immunohistochemicl stining nd into liquid nitrogen for Western blotting, nd ll smples were stored t 71C until required. This study ws conducted ccording to the Declrtion of Helsinki Principles. All procedures involving humn subjects were pproved by the Institutionl Review Bord t the Seoul Ntionl University Hospitl, nd ll subjects provided written informed consent. preprtion nd topicl ppliction ws freshly emulsified in polyethyleneglycol 4 (PEG 4) nd mintined t 41C in drk tubes. The buttock skins of young subjects were treted with.25% nd vehicle (62.5 ml/cm 2 ) under occlusion for 48 h, nd then treted skins were irrdited with 2 MED of UV. Skin smples were obtined t 8 hours (for ERK nlysis) or 48 hours (for VEGF nd vsculriztion nlysis) post-uv from ech subject by punch biopsy. Western blotting nlysis After irrdited nd non-irrdited buttock skin smples hd been obtined from ech subject by punch biopsy, epidermis nd dermis were seprted using forceps fter incubting skin smples t 581C for 2 minutes in PBS. Soluble protein ws extrcted from epidermis nd HCT cells using lysis buffer contining 5 mm Tris-HCl (ph 7.4), 1% Triton X-1, 75 mm NCl, 1 mm EDTA, 1 mm PMSF, 1 mm DTT, nd protese inhibitor cocktil (Sigm-Aldrich). The protein concentrtions of extrcts were mesured by BCA protein dye regent. VEGF levels were determined in protein extrcts from epidermis smples nd HCT cells. Smples were loded nd seprted on 1% SDS-polycrylmide gels, nd proteins were electrotrnsferred onto nitrocellulose membrnes (Amershm Phrmci) t 41C. Membrnes were then blocked with 5% nonft dry milk in PBS contining.1% Tween-2 (PBST) for 1 h, nd fter brief wsh with PBST, membrnes were probed with primry ntibody for 1 h, nd wshed three times with PBST. They were then treted with horserdish peroxidse-conjugted secondry ntibody for 45 minutes nd blots were detected using ECL detection regent (Amershm Biosciences, Pisctwy, NJ). Proteins were visulized by fluorogrphy using Agf X-ry film blue. Bnds were quntified using densitometric progrm (Bio 1D; Vilber Lourmt, Torcy Z.I., Frnce). Semiquntittive RT-PCR Totl RNA ws extrcted from tissues nd cells using TRI ZOL regent (Invitrogen Life Technologies, Crlsbd, CA), nd 1 mg of totl RNA ws converted to cdna using reverse trnscriptse t 371C for 1 hours (First Strnd cdna Synthesis Kit, Ferments, Hnover, MD). PCR rections were performed using.5 2 ml of cdna nd preliquoted ReddyMix TM PCR mster mix (Abgene, Surrey, UK). Optiml semiquntittive conditions were set to fll in the liner PCR product rnge (dt not shown). PCR consisted of 34 mplifiction cycles (941C, 3 seconds; 61C, 3 seconds; 721C, 1 min) for humn VEGF using oligonucleotide primer sets. In prllel, the GAPDH gene ws mplified in ech RNA smple s n internl control. The VEGF primers used were 5 -CCA TGA ACT TTC TGC TGT CTT-3 (forwrd) nd 5 -TCG ATC GTT CTG TAT CAG TCT-3 (reverse), nd the GAPDH primers used were 5 -ATT GTT GCC ATC AAT GAC CC-3 (forwrd) nd 5 -AGT AGA GGC AGG GAT GAT GT-3 (reverse). The primer sets used yielded PCR products of 516, 648 nd 73 bp for VEGF 121, VEGF 165, nd VEGF 189, respectively. PCR products were electrophoresed on 1.8% grose gels nd visulized with ethidium bromide. Signl strengths were quntified using densitometric progrm (Bio 1D; Vilber Lourmt, Torcy Z.I., Frnce). 274 Journl of Investigtive Dermtology (26), Volume 126

9 Immunohistochemistry Frozen humn skin tissues were exmined for VEGF nd CD31 expression s described previously (Chung et l., 22). Briefly, snp-frozen tissues were plced immeditely into cryomtrix (Shndon, Pittsburgh, PA), stored t 71C, sectioned t 4 mm, mounted onto silne-coted slides (Dko, Glostrup, Denmrk), nd cetone fixed t 21C for 15 min. The frozen sections were then rehydrted in DW nd endogenous peroxidse ctivity ws quenched using 3% hydrogen peroxide for 1 min. The sections were then blocked with blocking solution (Zymed, Sn Frncisco, CA) for 3 min, nd wshed nd incubted with primry ntibodies in humidified chmber t 41C for 18 h. After wshing in PBS, they were incubted with biotinylted secondry ntibody for 3 min, followed by horserdish-streptovidin conjugte for 15 min. After further wshing in PBS, color ws developed using AEC (3-mino-9- ethylcrbzole; Zymed) for 5 1 min. The sections were then wshed in running tp wter for 5 min, nd cell nuclei were counterstined with Myer s hemtoxylin (Dko). The cutneous blood vessel numbers nd dimeters were cptured nd nlyzed using n imge nlysis progrm (Vedio Test-Mster Morphology, Imge nd Microscope Technology, Kore). Vessel numbers per mm 2, verge vessel sizes, nd the reltive res occupied by vessels were determined in dermis. Three different 2 fields within 2 mm from the epiderml derml junction were exmined in the dermis of ech section. Sttistics Results re expressed s mens7sem of independent experiments, nd ll experiments were repeted t lest four times. The significnce of differences between independent two groups ws nlyzed using the Mnn Whitney U-test nd tht of differences between dependent two groups ws nlyzed using the Wilcoxon Signed-Rnk test. For ll tests, P-vlue of o.5 ws considered sttisticlly significnt. All dt were nlyzed using SPSS 11. for Windows (SPSS Inc., Chicgo, IL). CONFLICT OF INTEREST The uthors stte no conflict of interest. ACKNOWLEDGMENTS This work ws supported by Kore Reserch Foundtion Grnt ( E243) nd by reserch greement with Estee Luder (Melville, NY). We thnk Ye Kyung Woo, Ju Mi Shim, nd SeRh Lee for their excellent technicl ssistnce. REFERENCES Alsyed Y, Uddin S, Mhmud N, Lekmine F, Klvkolnu DV, Minuicci S et l. (21) Activtion of Rc1 nd the p38 mitogen-ctivted protein kinse pthwy in response to ll-trns-retinoic cid. J Biol Chem 276:412 9 Bllun C, Weninger W, Uthmn A, Weich H, Tschchler E (1995) Humn kertinocytes express the three mjor splice forms of vsculr endothelil growth fctor. J Invest Dermtol 14:7 1 Btes DO, Hillmn NJ, Willims B, Nel CR, Pocock TM (22) Regultion of microvsculr permebility by vsculr endothelil growth fctors. J Ant 2: Berr E, Pges G, Pouyssegur J (2) MAP kinses nd hypoxi in the control of VEGF expression. Cncer Metstsis Rev 19: Bhushn M, Young HS, Brenchley PE, Griffiths CE (22) Recent dvnces in cutneous ngiogenesis. 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10 Luer G, Sollberg S, Cole M, Flmme I, Sturzebecher J, Mnn K et l. (2) Expression nd proteolysis of vsculr endothelil growth fctor is incresed in chronic wounds. J Invest Dermtol 115:12 8 Liudet-Coopmn ED, Berchem GJ, Wellstein A (1997) In vivo inhibition of ngiogenesis nd induction of poptosis by retinoic cid in squmous cell crcinom. Clin Cncer Res 3: Lingen MW, Polverini PJ, Bouck NP (1998) Retinoic cid nd interferon lph ct synergisticlly s ntingiogenic nd ntitumor gents ginst humn hed nd neck squmous cell crcinom. Cncer Res 58: Mlhotr R, Stenn KS, Fernndez LA, Brvermn IM (1989) Angiogenic properties of norml nd psoritic skin ssocite with epidermis, not dermis. Lb Invest 61:162 5 Mildner M, Weninger W, Trutinger F, Bn J, Tschchler E (1999) UVA nd UVB rdition differentilly regulte vsculr endothelil growth fctor expression in kertinocyte-derived cell lines nd in humn kertinocytes. Photochem Photobiol 7:674 9 Oh CK, Kwon YW, Kim YS, Jng HS, Kwon KS (23) Expression of bsic fibroblst growth fctor, vsculr endothelil growth fctor, nd thrombospondin-1 relted to microvessel density in nonggressive nd ggressive bsl cell crcinoms. J Dermtol 3:36 13 Pl S, Iruel-Arispe ML, Hrvey VS, Zeng H, Ngy JA, Dvork HF et l. (2) Retinoic cid selectively inhibits the vsculr permebilizing effect of VPF/VEGF, n erly step in the ngiogenic cscde. Microvsc Res 6:112 2 Powis G, Bonjouklin R, Berggren MM, Gllegos A, Abrhm R, Ashendel C et l. (1994) Wortmnnin, potent nd selective inhibitor of phosphtidylinositol-3-kinse. Cncer Res 54: Reisinger K, Kufmnn R, Gille J (23) Incresed Sp1 phosphoryltion s mechnism of heptocyte growth fctor (HGF/SF)-induced vsculr endothelil growth fctor (VEGF/VPF) trnscription. J Cell Sci 116: Rolewski SL (23) Clinicl review: topicl retinoids. Dermtol Nurs 15: Roos TC, Jugert FK, Merk HF, Bickers DR (1998) Retinoid metbolism in the skin. Phrmcol Rev 5: Segrelles C, Ruiz S, Sntos M, Mrtinez-Plcio J, Lr MF, Prmio JM (24) Akt medites n ngiogenic switch in trnsformed kertinocytes. Crcinogenesis 25: Strtigos AJ, Ktsmbs AD (25) The role of topicl retinoids in the tretment of photoging. Drugs 65: Tnbe K, Hirde K, Ishiski A, Shu E, Sug H, Kitjim Y et l. (24) Possible involvement of p44/p42 MAP kinse in retinoic cid-stimulted vsculr endothelil growth fctor relese in ortic smooth muscle cells. Atherosclerosis 175: Velsco P, Lnge-Asschenfeldt B (22) Dermtologicl spects of ngiogenesis. Br J Dermtol 147: Weninger W, Rendl M, Mildner M, Tschchler E (1998) Retinoids downregulte vsculr endothelil growth fctor/vsculr permebility fctor production by norml humn kertinocytes. J Invest Dermtol 111:97 11 Yno K, Kdoy K, Kjiy K, Hong YK, Detmr M (25) Ultrviolet B irrdition of humn skin induces n ngiogenic switch tht is medited by upregultion of vsculr endothelil growth fctor nd by downregultion of thrombospondin-1. Br J Dermtol 152: Yno K, Our H, Detmr M (22) Trgeted overexpression of the ngiogenesis inhibitor thrombospondin-1 in the epidermis of trnsgenic mice prevents ultrviolet-b-induced ngiogenesis nd cutneous photodmge. J Invest Dermtol 118: Journl of Investigtive Dermtology (26), Volume 126