*Sanja Schuller-Petrovic, *Susanne Siedler, *Thomas Kern, {Johann Meinhart, Kurt Schmidt & 1 Friedrich Brunner

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1 British Journl of Phrmcology (1997) 122, 772 ± Stockton Press All rights reserved 7 ± 1188/97 $12. Imlnce etween the endothelil cell-derived contrcting fctors prostcyclin nd ngiotensin II nd nitric oxide/cyclic GMP in humn primry vricosis *Snj Schuller-Petrovic, *Susnne Siedler, *Thoms Kern, {Johnn Meinhrt, Kurt Schmidt & 1 Friedrich Brunner Deprtment of Phrmcology nd Toxicology, University of Grz, *Deprtment of Dermtology, University Clinic Grz nd {Deprtment of Vsculr Surgery, Linz Hospitl, Vienn, Austri 1 The role of the endothelium in the vsomotor control of humn veins in the lower extremity is little understood. We tested the hypothesis tht the production of relxing nd contrcting fctors is ltered in endothelil cells from vricose sphenous veins which my predispose to the decresed vessel tone oserved in primry vricosis. 2 We determined the intrcellulr ccumultion of gunosine 3':5'-cyclic monophosphte cyclic GMP; mesure of nitric oxide production) nd the relese of endothelin nd prostcyclin (mesured s its stle metolite -keto-prostglndin F 1 ) from cultured cells derived from the long sphenous veins of ptients with primry vricosis (Vricose sphen group, n=27) or from ptients undergoing coronry rtery ypss surgery (Helthy sphen group, n=22). In ddition, levels of endothelin, ngiotensin II, rdykinin, cyclic GMP nd cyclic AMP in plsm from ptients with primry vricosis nd helthy volunteers (n=8 ± 11 in ech group) were determined. 3 Although sl cyclic GMP levels were similr, more cyclic GMP ccumulted in response to histmine (1 ± 1 mmol l 71 ) in cells from vricose sphenous veins ( pmol per well) thn in cells from veins without vricosis ( pmol per well). Furthermore, the relxnt potency of nitroprusside (1 nmol l 71 ± 3 mmol l 71 ) in vitro ws higher for vricose veins (men EC 5 =5.9 m mol l 71 ; n=8) thn helthy veins (men EC 5 =2. mmol l 71 ; n=7). The production of prostcyclin ws signi cntly less in cells from vricose thn helthy sphenous veins (+8.7 nd nmol g 71 protein), ut the production of endothelin ws similr in oth groups. Prostcyclin (3 nmol l 71 ±3 mmol l 71 ) consistently contrcted rings of vricose sphenous vein in vitro with men EC 5 vlue of 1 ± 2 mmol l 71 (n=7); the mximum tension generted ws *5% of tht of completely depolrizing solution of K + (12 mmol l 71 ). 5 In plsm from ptients with vricose veins, levels of cyclic GMP were higher thn in helthy controls ( nd nmol l 71 ), levels of ngiotensin II were lower ( nd pmol l 71 ), nd levels of endothelin, cyclic AMP, nd rdykinin were not di erent. It is concluded tht endothelil cells from disesed sphenous veins secrete less constrictor meditors thn cells from helthy veins nd tht in disesed veins the nitric oxide/cyclic GMP system is upregulted which my shift the lnce of vsoctive fctors towrds vsodilttion nd contriute to the development of primry vricosis. Keywords: Relxing nd contrcting fctors; sphenous vein (humn); vricosis (primry); vsculr tone; endothelil cell culture; umilicl vein (humn) Introduction 1 Author for correspondence t: Deprtment of Phrmcology nd Toxicology, University of Grz, UniversitÈ tspltz 2, A-81 Grz, Austri Vricose veins of lower lim occur in high prevlence, ecting *2% of the dult popultion (Tolins, 1983), nd some 25% of ptients with leg ulcers nd re ux in the deep veins hve primry disorder s result of decrese in the venous tone (Clrke et l., 1992). With the veins dilted, the cusp will not occlude the lumen, cusing vlve incompetence, re ux, nd high venous pressure (Wtts, 198). A numer of chnges occur in the microcircultion of the skin of lower lims, summrized under the heding of venous hypertensive microngiopthy. Phleoedem, indurtion of tissue in the distl leg, thromophleitis nd ulcertions re common complictions of untreted vricosity (Cornwll et l., 198). The etiology of primry vricosity is unknown. Severl hypotheses hve een proposed, including iochemicl defects in the collgen structure of the vein wll, seprtion of vsculr smooth muscle cells y rous in ltrtion (Rose, 198), nd presence of rteriovenous shunts (Himovici, 1987). Hypoxic-induced ctivtion of endothelil cells in venous stsis, followed y synthesis of pro-in mmtory fctors nd dhesion of polymorphonucler neutrophils hs lso een suggested (Michiels et l., 1993). -Adrenoceptor responsiveness is reduced in vricose sphenous veins (Thulesius et l., 1991; Lowell et l., 1992) nd hnd veins of ptients with vricosis (BloÈ chl-dum et l., 1991), which might represent primry defect resulting in decresed venous tone nd predisposing to development of vricose veins. Besides sympthetic ctivity, endotheliumderived vsoctive meditors re importnt in controlling locl vsculr tone (Furchgott & Zwdzki, 198; Furchgott & Vnhoutte, 1989), including venous tone (De Mey & Vnhoutte, 1982; Monos et l., 1995). Two importnt vsodiltors re prostcyclin nd nitric oxide, nd two vsoconstrictors re endothelin nd ngiotensin II (Vne et l., 199). Their locl levels re controlled y iosynthesis from inctive precursors, minly in vsculr endothelium. This study ws designed to test the hypothesis tht the lnce of the production of vsodiltors nd vsoconstrictors is distured in endothelil cells of humn vricose sphenous veins. We cultured endothelil

2 cells from helthy nd vricose sphenous veins nd determined their sl nd stimulted nitric oxide (mesured s gunosine 3' : 5'-cyclic monophosphte (cyclic GMP)), prostcyclin (mesured s -keto-prostglndin F 1 ) nd endothelin production. Furthermore, plsm levels of cyclic GMP, denosine 3' :5'-cyclic monophosphte (cyclic AMP), endothelin nd ngiotensin II were lso mesured. Methods Origin of endothelil cells We studied vsculr endothelil meditors from 27 ptients (21 women nd men, men ge 2+2 yers) with primry vricosis of the long sphenous vein s con rmed y phleogrphy, nd from control group (coronry ypss ptients; 1 men nd women, men ge 5+2 yers) without vricosis. Of the sujects with vricosis, 1 were smokers, took orl contrceptives nd thyroid hormones; fmily history showed vricosis in the prents of 1 sujects; 11 su ered from hypercholesterolemi. Of the control sujects, 9 were smokers, 7 hd hd myocrdil infrction, were dietics; they ll were tking long-term mediction, i.e. (numer of ptients) - drenoceptor lockers (11), isosoride mononitrte (1), cetylslicylic cid (9), ngiotensin converting enzyme inhiitors (8), nitroglycerin (7), molsidomine (5), nifedipin (), nd isosoride dinitrte (1). In ddition, we mesured plsm levels of vsoctive meditors in 11 helthy volunteers (7 women nd men, men ge +5 yers) who were free of ny signi cnt previous ngiologicl (phleologicl) or other disese, nd who were not tking ny mediction t the time of exmintion. Lstly, meditor relese from endothelil cells derived from umilicl cords of 18 full-term women (men ge 27+2 yers) with norml delivery ws lso studied. Orgn th experiments S. Schuller-Petrovic et l E ect of prostcyclin in vricose veins To determine the mechnicl e ect of prostcyclin, segments of *3 ± cm length of long sphenous vein were tken from the inguinl re nd elow the knee, respectively of 3 femle ptients (men ge 3+22 yers) undergoing vricose vein stripping, immeditely plced in chilled Kres-Henseleit u er nd rpidly trnsported to the lortory (time etween excision nd egin of experiment: 15 ± 9 min). Veins were super cilly clened nd cut into rings of * mm length nd the tissues suspended in 5 ml-orgn ths for isometric tension mesurement. The ths were lled with oxygented (95% O 2 /5% CO 2 ) wrmed (378C) Kres-Henseleit solution contining 2.5 mmol l 71 C 2+. After equilirtion (1 ± 1.5 h), the contrctile response of the rings ws tested with phenylephrine (1 mmol l 71 ; *8 min), the gonist ws removed y repeted wshes with Kres-Henseleit u er, nd mximum contrctile ctivity ws determined with depolrizing solution of K + (12 mmol l 71 ; the N + concentrtion ws reduced ccordingly). Rings which did not redily contrct to depolriztion y excess K + were considered to e dmged nd discrded. Following removl of K +, seline tone (lmost complete relxtion) ws reched within *3 min nd the experiment ws strted. Prostcyclin ws dded non-cumultively (due to its quick degrdtion in wrmed solution) t nl concentrtions rnging from 3 nmol l 71 to 3 mmol l 71 (*1 ngml 71 ±*1 mg ml 71 ), llowing 3 min or ttinment of equilirium for ech concentrtion. At the end of the experiment, K + ws dded once more to estlish the mximum contrctile response. E ects of nitroprusside in vricose nd non-vricose veins To determine whether responses to nitrovsodiltors re incresed in vricose sphenous veins, vricose vein segments from 2 femle ptients (ged 31 nd 38 yers) nd segment from mle ptient with helthy veins (ged 5 yers) undergoing coronry ypss surgery were prepred nd tested with K + s Endothelium nd primry vricosis 773 descried ove (time from opertion to strt of experiment: 5 ± 1 min) nd pre-contrcted with comintion of the thromoxne receptor gonist U 19 (5 ± 1 nmol l 71 ) (KuÈ herger et l., 199) nd ngiotensin II (5 ± 1 nmol l 71 ). Nitroprusside ws dded t nl concentrtions rnging from 1 nmol l 71 to 3 mmol l 71, llowing 3 min for ech concentrtion. At the end of the experiment, ppverine (2 mmol l 71 ) ws dded to determine mximum relxtion (Kukovetz et l., 1979). Endothelil cell culture Segments (* cm) of the inguinl sphenous vein or umilicl vein nd their endothelil cells were hrvested s descried previously (Zill et l., 199). Cells were suspended in Medium 199 contining humn serum (1%) nd endothelil cell growth supplement (75 mg l 71 ) nd divided mong three wells precoted with humn ronectin (*2 cm 2 ech). The cells were fed twice week nd llowed to grow to con uence (1 ± 15 dys). At this time, the medium ws removed from two of the wells nd fresh medium ws dded for 3 h nd 21 h, respectively. After this period the medium ws removed nd immeditely frozen t 778C until nlysis of endothelin (3 h incution) or -ketoprostglndin F 1 (21 h incution). Becuse rtes of meditor relese from con uent cells re known to vry with cell density, the two incutions were repeted on the next dy, nd conditioned medi nd cells (for determintion of cellulr protein) were frozen. At this time, the third well ws supssged, rst to one well of six well plte (*9.5 cm 2 per well) nd fter reching con uence ( dys), to ll 12 wells of two -well pltes. The cells were grown to con uence (1 ± 1 dys) nd used for cyclic GMP mesurements. Determintion of intrcellulr cyclic GMP ccumultion Intrcellulr cyclic GMP ccumultion ws determined s previously descried (Schmidt et l., 1989). Brie y, con uent monolyers were wshed nd equilirted in 5 mmol l 71 HEPES u er, ph 7., contining 3-isoutyl-1-methylxnthine (1 mmol l 71 ). After 15 min, histmine or nitroprusside ws dded to give the nl concentrtions s indicted nd rections were stopped 2 min lter y removl of the incution u er nd tretment for 1 h with 1 ml of.1 N HCl. Intrcellulr cyclic GMP ws mesured in the superntnts of the lysed cells y rdioimmunossy. Determintion of endothelin nd -keto-prostglndin F 1 in conditioned medium The concentrtion of endothelin ws determined essentilly s descried previously (Brunner, 1995). Brie y, conditioned medium ws pplied to C2 crtridges, endothelin ws eluted with cetonitrile (7%), eluted endothelin ws dried nd reconstituted for rdioimmunossy y use of commercil kit (Peninsul; Belmont, CA). The detection limit ws.5+.8 fmol per tue (n=3); the cross-rectivity of other endothelin isomers nd ig endothelin in this ssy ws less thn 5% nd 37%, respectively, ccording to the supplier. - Keto-prostglndin F 1 ws determined y use of commercil rdioimmunossy kit (Amershm; Vienn, Austri). The detection limit ws.5+.1 fmol per tue (n=3). The smples were ppropritely diluted with ssy u er nd ssyed directly. The cross-rectivity of four other prostglndins nd of thromoxne B 2 in this ssy ws less thn 1% in ech cse, ccording to the supplier. Determintion of cyclic GMP, cyclic AMP, ngiotensin II, endothelin nd rdykinin in plsm Blood (1 ml) ws drwn from the long sphenous vein of ptients undergoing vricose vein stripping (7 women nd men, ge 5+ yers; 3 smokers nd 8 non-smokers) nd of humn volunteers with pprently helthy sphenous veins (7

3 77 S. Schuller-Petrovic et l women nd men, ge +5 yers; smokers nd 7 nonsmokers), the plsm seprted nd immeditely frozen. Blood ws drwn from the sme ntomicl loction to void meditor metolism y peripherl orgns (De Nucci et l., 1988). For oth cyclic nucleotides, plsm (.3 ml) ws dded to 3 ml trichlorocetic cid (5% v/v), shken for 25 min, centrifuged, nd the superntnt extrcted times with 7 ml of diethyl ether for 5 min, the ether ws removed nd the queous smple sujected to rdioimmunossy. For ngiotensin II, plsm (1 ml) ws diluted 1 : 1 nd pplied to C2 crtridges, ngiotensin II ws eluted from the crtridge with cetonitrile (7%), eluted ngiotensin II ws dried nd reconstituted for rdioimmunossy y commercil kit (Amershm; Vienn, Austri). The recovery rte for the extrction procedure ws 89+.9% (n=3) s determined y the ddition of 3-[ 125 I]- iodotyrosyl -ngiotensin II. The detection limit ws.8+.2 fmol per tue (n=3). There ws complete crossrectivity of ngiotensin III nd less thn 2% cross-rectivity of ngiotensin I in this ssy, ccording to the supplier. For endothelin, plsm (1 ml) ws diluted 1 : 1 nd processed s conditioned medium descried ove. For determintion of rdykinin, 2 ml of lood were drwn from the rchil vein of sujects without vricosis (5 men, 3 women, men ge +3 yers) or sujects with vricosis (3 men, 5 women, men ge 5+. yers) into chilled syringes spiked with.2 ml of chilled inhiitor cocktil comprising protinin (1, kllikrein inhiitor units ml 71 ), soy en trypsin inhiitor (.8 mg ml 71, polyrene ( mg ml 71 ), 1,1-phennthroline (1 mg ml 71 ) nd EDTA (2 mg ml 71 ). The mixture (.8 ml) ws treted with cold ethnol (3 ml), centrifuged, nd the superntnt ws dried down in vcuum concentrtor. The sediment ws tken up in cetone (%,. ml), vortexed, petrol ether (1. ml) ws dded, vortexed gin, centrifuged, the upper lyer removed, nd the lower lyer dried down for determintion of rdykinin y rdioimmunossy s descried previously (Miki et l., 199). Dt nlysis All results re given s rithmetic mens+s.e.men. Concentrtion-e ect curves were tted individully to Hill type eqution yielding (EC 5 vlues y use of the GIPMAX curve- tting progrmme s descried previously (Brunner et l., 1991). All dt points were given equl weight. For ese of comprison, the dt re given s untrnsformed gonist concentrtion/tension plots. n refers to the numer of individuls from whom endothelil cells were tken for culture or, in orgn th experiments, the numer of vein rings used (the numer of individuls from which they were derived is given in the legends). Dt were nlysed y Student's t test for unpired oservtions. A P vlue.5 ws considered s signi cnt nd is indicted y n sterisk. Mterils Angiotensin II, U 19 (9,11-dideoxy-11,9-epoxymethnoprostglndin F 2 ), prostcyclin (N-slt), 3-isoutyl-1-methylxnthine, soy en trypsin inhiitor, polyrene, 1,1- phennthroline nd HEPES were purchsed from Sigm (Vienn, Austri). All other chemicls were otined from Merck (Drmstdt, Germny). Aprotinin (trsylol) ws gift from Byer (Vienn). Endothelil cell growth supplement ws from Collortive Reserch (Vienn, Austri) nd ll other tissue culture mterils from sources descried previously (KuÈ herger et l., 199). Results Vlidtion experiments Totl cellulr protein per culture well did not di er for the cells derived from volunteers with helthy (n=22) or vricose veins Endothelium nd primry vricosis (n=27), nor from endothelil cells derived from umilicl veins (n=15), indicting tht similr degree of con uence ws reched in ll cses. The production of meditors per well ws similr t con uence nd the dy fter; therefore, oth sets of dt were comined nd expressed s secretion rte sed on totl cellulr protein (dt not shown). Intrcellulr cyclic GMP ccumultion Formtion of nitric oxide y endothelil cells ws determined with cyclic GMP s iochemicl mrker. In cells from helthy sphenous veins (Figure 1) sl cyclic GMP level ws pmol per well nd histmine (1 ± 1 mmol l 71 ), n endothelium-dependent gonist, incresed ccumultion up to pmol per well (n=1). To determine the responsiveness of (endothelil) solule gunylyl cyclse, endothelil cells were stimulted with nitroprusside (1 mmol l 71 ) resulting in mximum cyclic GMP ccumultion of pmol per well. In cells from vricose veins (Figure 1), sl cyclic GMP content ws similr ( pmol per well), ut histmine nd nitroprusside incresed it to nd pmol per well, respectively (n=2) (P5.5 in ech cse vs cells from helthy veins). However, the sl levels of cyclic GMP nd the potency of histmine were similr in helthy vein (EC 5 = mmol l 71 ) nd vricose vein (EC 5 =2.+. mmol l 71 ; P.5). Cyclic GMP (pmol per well) Cyclic GMP (pmol per well) SNP. 7 5 SNP Histmine (log mol l 1 ) Figure 1 Accumultion of cycic GMP in endothelil cells from helthy () or vricose () veins in response to histmine or nitroprusside (SNP, 1 mmol l 71 ). The EC 5 vlue for histmine ws similr in oth cses (*3 mmol l 71 ), ut histmine nd nitroprusside produced greter mximum e ect in sphenous vein cells (indicted y the sterisk; P5.5). Men vlues+s.e.men of 1 () or 2 () cultures re given.

4 S. Schuller-Petrovic et l Endothelium nd primry vricosis 775 Decrese in tension (g) Pp -Keto-prostglndin F 1α (nmol g 1 protein) Decrese in tension (g) Pp SNP (log mol l 1 ) Figure 2 Nitroprusside (SNP)-induced relxnt e ects in rings of non-vricose (), or vricose () humn sphenous vein. Results re expressed s decrese in tension (g) strting from the tension reched t the end of pre-contrction with U 19 nd ngiotensin II (.8+.5 g). The gonist ws signi cntly more potent in vricose (men EC 5 =5.9 mmol l 71 ) thn non-vricose veins (men EC 5 =2. mmol l 71 ). For comprison, the relxnt e ect of ppverine (Pp; 2 mmol l 71 ) is lso given. Dt points represent the men+s.e.men of 7 rings derived from 1 ptient () or 8 rings derived from 2 ptients () (ll from distl vein segments). Relxnt ctivity of nitroprusside in helthy nd disesed sphenous veins The response to nitroprusside of sphenous vein rings derived from non-vricose long sphenous veins nd ptients with vricose veins is shown in Figure 2. The degree of pre-constriction with U 19 nd ngiotensin II ws the sme for helthy nd vricose tissues (.8+.5 g; n=15). Nitroprusside relxed ll tissues in concentrtion-dependent mnner ut with di erent potency, i.e. helthy sphenous vein with n EC 5 vlue of mmol l 71 (n=7; Figure 2), nd vricose vein with n EC 5 vlue of mmol l 71 (n=8; Figure 2; P5.5) (in oth cses distl vein segments were used). As to intrinsic ctivity, the highest concentrtion of nitroprusside used (.3 mmol l 71 ) decresed tension y g in helthy veins nd y g in vricose veins (P=.5). In contrst, ppverine, the most potent smooth muscle relxnt, ws similrly e ective in helthy nd disesed veins (Figure 2). Meditor secretion into conditioned medium The relese of -keto-prostglndin F 1 is shown in Figure 3. Cells from helthy sphenous veins secreted , Endothelin (nmol g 1 protein) Figure 3 Secretion of prostcyclin (mesured s -keto-prostglndin F 1 ) () or endothelin () y endothelil cells derived from helthy (solid columns; n=22) or vricose (open columns, n=27) sphenous veins, or umilicl cords (htched columns, n=15). Secretion ws monitored over 3 h () or 21 h () nd is given s men vlues+s.e.men. *Signi cntly di erent from helthy sphenous veins. nd cells from vricose veins secreted +8.7 nmol g 71 protein (P5.5). In comprison, cells from umilicl veins secreted 2. times s much -keto-prostglndin F 1 ( nmol g 71 protein) s cells from helthy sphenous veins (P5.5). The relese of endothelin is shown in Figure 3. Cells from helthy nd disesed veins produced similr mounts of the peptide (.3+.3 nd nmol g 71 protein; P.5), wheres production in umilicl vein endothelil cells ws gin 2. times higher ( nmol g 71, P5.5). Contrctile ctivity of vricose sphenous veins Figure shows the responsiveness of sphenous vein derived from ptients with vricosis towrds prostcyclin, phenylephrine nd depolrizing concentrtion of K +. Strting from seline tension of g, prostcyclin (3 nmol l 71 ±3 mmol l 71 ) contrcted rings of the vein, tken either proximlly or distlly, with similr potency (EC 5 vlues: nd mmol l 71, n= nd 8, respectively; P.5). However, the mximum force generted y 3 mmol l 71 prostcyclin ws higher in proximl ( g) thn distl tissues ( g; P5.5). Similrly, sumximum concentrtion of phenylephrine (1 mmol l 71 ) generted higher tension in proximl vein ( g) thn distl vein ( g; P5.5), nd similr rtio ws otined for K + (Figure ).

5 77 S. Schuller-Petrovic et l Endothelium nd primry vricosis Increse in tension (g) 12 8 Cyclic GMP (nmol l 1 ) 8 Endothelin (pmol l 1 ) 3 Increse in tension (g) 8 2 Meditor levels in plsm Figure 5 shows the plsm levels of cyclic nucleotides, endothelin, nd ngiotensin II in volunteers with non-vricose veins nd ptients with vricose sphenous veins (n=11 in ech cse). The cyclic GMP concentrtions were lower in sujects with helthy sphenous veins (7.1+. nmol l 71 ) thn in vricose ptients ( nmol l 71 ; P5.5); the endothelin nd cyclic AMP levels were similr in oth groups (7.+.3 nd pmol l 71, nd nmol l 71 ; P.5 in ech cse), nd the ngiotensin II concentrtion ws higher in sujects with helthy veins ( pmol l 71 ) thn in those with vricose veins ( pmol l 71 ; P5.5), ut the rdykinin levels (otined from the rchil vein) were not di erent ( vs pmol l 71, n=8; dt not shown). Discussion PE K PE K + PGI 2 (log mol l 1 ) Figure Prostcyclin (PGI 2 )-induced contrctile e ects in rings of humn proximl vricose () nd distl vricose () sphenous vein. All rings were stly relxed (seline tension: g) when prostcyclin ws dded. Results re expressed s increse in tension (g) ove seline. For comprison, the contrctile e ects of phenylephrine (PE, 1 mmol l 71 ) nd K + (12 mmol l 71 ) re lso given. Dt points represent the men+s.e.men of rings derived from 2 ptients (), or 8 rings derived from 2 ptients (). The present study is the rst demonstrtion of impired relese nd ltered plsm levels of endothelil relxing nd contrcting fctors in ptients with primry vricosis. We hve found tht conditioned medium exposed to primry culture endothelil cells derived from vricose veins ccumulted signi cntly less -keto-protglndin F 1 thn cells otined from Angiotensin II (pmol l 1 ) c non-vricose veins, nd tht the locl levels of cyclic GMP were higher, nd of ngiotensin II were lower in ptients with vricose veins, wheres secretion nd plsm level of endothelin were not ected. Collectively, these chnges my predispose to reduced venous tone in ptients with vricosis. The relxnt e ect of nitric oxide on smooth muscle re medited y increses in cyclic GMP (Moncd et l., 1991), nd in vsculr preprtions extrcellulr levels of cyclic GMP correlte well with intrcellulr levels (Rpoport & Murd, 1983). Like their rteril counterprts, venous endothelil cells re fully functionl with respect to nitric oxide synthesis nd relese (Ignrro et l., 1987), nd veins, including humn sphenous veins, show nitric oxide-dependent vsodilttion (Yng et l., 1991; Chu et l., 1993; Higmn et l., 1993). Little is known out the role of nitric oxide in humn primry vricosis. In the present study we hve found tht endothelil cells from vricose sphenous veins synthesized greter mounts of cyclic GMP when stimulted with histmine or high concentrtion of nitroprusside thn cells from non-vricose sujects (Figure 1). Similrly, gunylyl cyclse ctivity of sphenous vein smooth muscle ws higher in vricose thn helthy tissues, s judged y the four times lower EC 5 vlue for relxnt ctivity of nitroprusside in orgn th experiments (Figure 2). The ltter lso showed similr cpcity for relxtion, irrespective of ntomicl origin of the vein (proximl vs distl, not shown) nd the cellulr mechnism involved (cyclic GMP in the cse of nitroprusside, nd cyclic AMP for ppverine). Together, these dt support the view tht, in humn vricose veins, endothelil production of nitric oxide is ctivted nd the sensitivity of venous smooth muscle is incresed which, individully or in comintion, my then led to incresed synthesis of cyclic GMP in smooth muscle cells nd heightened vsculr relxtion. However, the ctul pro- Cyclic AMP (nmol l 1 ) d Figure 5 Plsm levels of cyclic GMP (), endothelin (), ngiotensin II (c) nd cyclic AMP (d) mesured in sujects with helthy (solid columns, n=11) or vricose (open columns, n=11) sphenous veins. Men vlues+s.e.men re given. *Signi cnt di erence etween groups

6 S. Schuller-Petrovic et l Endothelium nd primry vricosis 777 relxnt pthophysiologicl stimuli re unknown. We lso found tht levels of cyclic GMP in plsm, smpled directly from the sphenous vein, were higher in ptients with vricosity thn sujects with helthy veins, which grees with the higher iosynthetic cpcity for cyclic GMP of endothelil cells. In spite of this, the exct reltionship etween ltered plsm levels nd endothelil production of cyclic GMP in sujects with norml nd vricose sphenous veins cnnot e scertined from this study, ut would require smpling of lood from the proximl sphenous vein s well s the corresponding rteril loction. Like other vsculr tissues, sphenous veins produce prostnoids, of which prostcyclin ppers to e the most undnt (Monos et l., 1995). In the present study, we oserved smller rte of relese of -keto-prostglndin F 1 from cells derived from disesed veins compred to helthy veins. The signi cnce of this nding ws scertined y functionl experiments in vitro with isometric tension mesurements. Prostcyclin consistently contrcted vricose sphenous veins in concentrtion-dependent fshion, clerly supporting the present premise tht prostcyclin exerts constrictor ctivity in this vessel. The prostnoid ws similrly potent in proximl nd distl sphenous vein segments, ut the proximl tissues developed higher tension upon depolriztion with K + (Figure ). Our ndings corroorte nd extend previous results of one group in which prostcyclin ws found to contrct norml sphenous veins, lthough few quntittive dt were given (Sinzinger & Fitsch, 198; Sinzinger & Feigl, 1985). Moreover, cnine pulmonry nd femorl veins lso pper to e contrcted y prostcyclin (Miller & Vnhoutte, 1985). The prostcyclin production of segments of vricose veins hs een found to e reduced (Bigi et l., 1988), incresed (Hynes et l., 199), or similr (Sinzinger & Feigl, 1985) to tht of helthy veins. These inconsistencies my e due to di erent methods of tissue smpling nd hndling which my mssively lter in vitro prostnoid production nd re voided in the present study. We hve found no di erence in cyclic AMP levels in sphenous vein plsm from individuls with helthy nd vricose veins, indicting tht incresed cyclic AMP production my not e involved in vricosity. The peptide endothelin (Yngisw et l., 1988) is synthesized in rteril nd venous endothelil cells nd is potent vsoconstrictor nd pressor gent in nimls nd mn (Mski, 1995). Endothelin elicits its e ects vi speci c memrne receptors, ET A nd ET B, nd is more potent nd e ective s constrictor of isolted veins thn of isolted rteries. In view of the sensitivity of humn sphenous veins to exogenous endothelin nd the presence of ET A nd ET B receptors, oth mediting vsoconstriction in these vessels (White et l., 199), the ssessment of the relese of endothelin from sphenous veins ws of prticulr interest. Our ndings tht neither the rtes of endothelin synthesis nd relese y cultured cells nor the plsm levels di ered in the two study groups mke it unlikely tht this meditor plys role in the development of vricosity. Rther, ngiotensin II my e of importnce. Angiotensin converting enzyme ctivity (which results in ngiotensin II formtion) ws demonstrted in endothelil cells s well s the dventiti of di erent lrge vessels, including humn sphenous vein (Rogerson et l., 1992). This ctivity persisted fter removl of the endothelium nd ws mostly ssocited with the vs vsorum in these vessels (Rogerson et l., 1992). Therefore, we determined this meditor in the plsm of sphenous veins, which my etter represent its totl locl production. The predominnt venoctive e ect of ntiotensin II is constriction, either directly vi ngiotensin II receptors (Smith & Timmermns, 199), nd/or possily indirectly vi ctivtion of the sympthetic nd endothelin systems. In this light, the reduced ngiotensin II plsm level we found in the group with vricosity my predispose to lowered tone of the sphenous vein wll nd contriute to the development of the disese. Also, the lower production of ngiotensin II y vricose veins my e t the origin of the reduced, or lost, contrction-fcilitting e ect of the endothelium of vricose vein segments stimulted with nordrenline (Thulesius et l., 1991). On the other hnd, rdykinin levels were not incresed in the vricose vein group s would e expected when ngiotensin converting enzyme ctivity is reduced, which my e due to compenstory degrdtion y other mechnisms. Further studies of endothelil ngiotensin converting enzyme ctivity, ngiotensin II formtion, relese nd ction re wrrnted to sustntite cusl role of ngiotensin II in primry vricosity. Finlly, we would dvise cution in the use of umilicl vein endothelil cells s controls. We found much higher meditor relese in these cells thn in cells from helthy sphenous veins, s lso shown previously in comprison etween humn lte pregnncy decidul nd umilicl vein endothelil cells (Gllery et l., 1995). This csts dout on the vlidity of their widespred use s generl surrogte for endothlil cells. In conclusion, the present study shows tht cultured endothelil cells derived from humn vricose sphenous veins secrete less -keto-prostglndin F 1 nd ngiotensin II, two vsoconstrictor fctors in this vessel, thn cells derived from helthy sphenous veins. The imlnce my result in reduced ctive wll tension due to wek smooth muscle contrction, nd my constitute n importnt fctor in chronic venous insu ciency. These dt signi cntly extend previous evidence implicting endothelil dysfunction in primry vricosity (Bigi et l., 1988; BloÈ chl-dum et l., 1991; Thulesius et l., 1991; Lowell et l., 1992) nd my hve importnt therpeutic consequences for the tretment of this disese. 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