Endothelial cell markers reflecting endothelial cell dysfunction in patients with mixed connective tissue disease

Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Open Access RESEARCH ARTICLE BioMed Cen al © 2010 Sol esz e al.; licensee BioMed Cen al L d. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Resea ch a icle Endo helial cell ma ke s e lec ing endo helial cell dys unc ion in pa ien s wi h mixed connec i e issue disease Pal Sol esz 1 , Daniel Be eczki 2 , Pe e Szodo ay 3 , Ma ia T Magya 4 , Hen ie a De 1 , Is an Csipo 1 , Ago a Hajas 1 , Gyo gy Pa agh 5 , Gyula Szegedi 1 and Edi Bodolay* 1 Abs ac In oduc ion: The aim o he p esen s udy was o in es iga e he associa ion be ween ca dio ascula isk ac o s and endo helial dys unc ion in pa ien s wi h mixed connec i e issue disease (MCTD) and o de e mine which bioma ke s a e associa ed wi h a he oscle o ic complica ions, such as ca dio ascula disease. Me hods: Fi y MCTD pa ien s and 38 heal hy age-ma ched and sex-ma ched con ols we e en olled in his s udy. In o de o desc ibe endo helial dys unc ion, we assessed low-media ed dila ion (FMD), ni a e-media ed dila ion (NMD) and ca o id a e y in ima-media hickness (IMT). We in es iga ed FMD o he b achial a e y a e eac i e hype emia and NMD a e sublingual ni oglyce in adminis a ion, while he IMT o he common ca o id a e y was de e mined by ul asound. An i-U1 ibonucleop o ein (an i-U1RNP) an ibodies, an i-ca diolipin (an i-CL) an ibodies, an i-endo helial cell an ibody (AECA) and endo helial cell ma ke s, such as soluble h ombomodulin (TM) and on Willeb and ac o an igen ( WFAg), we e assessed. Resul s: The endo helium-dependen asodila ion (FMD) was signi ican ly impai ed in pa ien s wi h MCTD, as compa ed wi h con ols (%FMD: 4.7 ± 4.2% s. 8.7 ± 5.0%; P < 0.001), while he pe cen age NMD did no di e (%NMD: 14.3 ± 6.6% s. 17.1 ± 6.7%; P = 0.073). Mean ca o id IMT alues we e highe in pa ien s han in con ols (IMT: MCTD, 0.64 ± 0.13 mm s. con ols, 0.53 ± 0.14 mm; P < 0.001). FMD nega i ely co ela ed wi h disease du a ion, he le els o apolipop o ein A1, he pa aoxonase-1 ac i i y, and sys olic blood p essu e in MCTD pa ien s. The pe cen age FMD was signi ican ly lowe in MCTD pa ien s wi h ca dio ascula diseases (CVD), han in hose wi hou CVD (%FMD: 3.5 ± 2.9 s. 5.8 ± 4.8, P < 0.0002), while pe cen age NMD did no di e be ween pa ien s wi h and wi hou CVDs. Se um le els o au oan ibodies (an i-U1RNP, AECA and an i-CL) we e signi ican ly highe in MCTD pa ien s and di e ed be ween MCTD pa ien s wi h and wi hou CVD. Endo helial cell ma ke s such as soluble TM (12.2 ± 8.1 ng/ml s. 3.2 ± 1.3 ng/ml; P < 0.001) and WFAg (224.1 ± 115% s. 89.4 ± 27.1%, P < 0.001) we e he highes in MCTD pa ien s wi h CVD. Conclusions: FMD is a eliable sensi i e ma ke o endo helial cell dys unc ion in MCTD. Beside he adi ional isk ac o s, an i-U1RNP, AECA and an i-CL an ibodies may be impo an no only in he pa hogenesis o MCTD bu in he induc ion o endo helial cell ac i a ion, and may play c ucial oles in he de elopmen o ea ly a he oscle osis in MCTD. In oduc ion Sys emic au oimmune diseases, such as sys emic lupus e y hema osus (SLE), heuma oid a h i is o sys emic scle osis, a e ch onic in lamma o y diso de s - signi ied by complex in e ac ions amongs adi ional and non a- di ional disease- ela ed phenomena, including in lamma- ion, dyslipidemia, h ombo ic e en s, and humo al au oimmune p ocesses [1-3]. Mixed connec i e issue disease (MCTD) is also a ch onic in lamma o y sys emic au oimmune disease, cha ac e ized by high i e s o an i- U1 ibonucleop o ein (an i-U1 RNP) an ibodies [4-7]. The ank issue in lamma ion and p oli e a ing ascu- la a e iopa hy is a speci ic ea u e o MCTD. P oli e a- i e asculopa hy in ol es he small and la ge a e ies in * Co espondence: edi [email protected] 1 3 d Depa men o Medicine, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Mo icz Zs. S . 22, Deb ecen 4032, Hunga y Full lis o au ho in o ma ion is a ailable a he end o he a icle Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Page 2 o 11 a ious o gans. The lung is he mos equen p edilec- ion place o he ascula damage, howe e , and may e en ually lead o pulmona y a e ial hype ension (PAH) [8]. Ou g oup and o he s ound ha PAH migh be asso- cia ed wi h coexis en an iphospholipid and an i- endo helial cell an ibodies (AECAs) [9,10]. In ou p e i- ous s udy we ound ha AECA p o okes he su ace exp ession o E-selec in and he ac i a ion o endo helial cells [11]. In se a o pa ien s wi h PAH, high concen a- ions o h ombomodulin (TM) and on Willeb and ac- o an igen ( WFAg) sec e ed om Weibel-Palade bodies imply an ac i a ed s a e o he endo helial cells. TM - an endo helial high-a ini y ecep o o h ombin - has an an icoagulan e ec , ac i a ing he p o ein C sys em [12]. Soluble TM can be measu ed in pe iphe al blood, and an ele a ed le el o soluble TM is a ma ke o endo helial inju y. P e iously we desc ibed high le els o o al se um cho- les e ol and educed pa aoxonase-1 (PON1) concen a- ions and ac i i y in pa ien s'se a [13]. PON1 has an an ioxidan unc ion, and i is a key ac o in a he oscle- o ic e en s [13]. These da a sugges ha pa ien s wi h MCTD ha e he adi ional isk ac o s o he ea ly de elopmen o a h- e oscle osis. The connec ion be ween endo helial cell damage and a he oscle osis in MCTD, howe e , has no been desc ibed p e iously. Endo helial dys unc ion is bo h an ea ly ma ke o as- cula diseases and a acili a ing ac o in he de elopmen o a he oscle osis [14-16]. Flow-media ed dila a ion (FMD) o he b achial a e y is a eliable and ep oducible non-in asi e ool o e alua e endo helial unc ion [17- 19]. The adminis a ion o sublingual ni a es is a good es o examine he asodila a o y e ec o an exogenous sou ce o ni ic oxide. The inc ease in ca o id in ima- media hickness (IMT) is a use ul ma ke o sys emic sub- clinical a he oscle osis and a s ong p edic o o subse- quen myoca dial in a c ion and s oke [20-22]. Since endo helial dys unc ion ep esen s an ea ly s age o a he ogenesis, he aim o his s udy was o de e mine whe he impai ed FMD amongs o he bioma ke s o endo helial dys unc ion, is cha ac e is ic o pa ien s wi h MCTD. We aimed o de e mine which ac o s a e associa ed wi h ca dio ascula e en s in MCTD. We also in es i- ga ed he endo helial cell unc ions, especially FMD, oge he wi h he ci cula ing endo helial cell ma ke s, soluble TM and WFAg, e lec ing he s a e o ac i a ion/ damage o he endo helium. Finally, we in es iga ed he ela ionship be ween pe iphe al endo helial dys unc ion and ca o id IMT. Ma e ials and me hods Pa ien s Fi y women wi h MCTD, ea ed and ollowed-up a he 3 d Depa men o In e nal Medicine, Uni e si y o Deb ecen, we e en olled in he p esen s udy. O hese, 23 women (46%) had a his o y o ca dio ascula diseases (CVDs). CVD was diagnosed i : he pa ien had acu e myoca dial in a c ion, o had ECG signs o myoca dial in a c ion, eco ded semi-annually; he co ona y disease was ea ed wi h co ona y bypass ope a ion o angio- plas y; o angina was e i ied by angiog aphy and/o he ischemic al e a ions we e e i ied by a non-in asi e es o issue Dopple 's examina ion. Twen y-se en emale MCTD pa ien s wi hou CVD we e included and deno ed as he MCTD/CVD-nega i e g oup. All pa ien s ul illed he c i e ia o MCTD acco ding o Ala con-Sego ia and Villa eal [23]. Clinical disease ac i i y was assessed by he sys emic lupus ac i i y mea- su e (SLAM) e ospec i ely om he pa ien s' epo s [24,25]. A SLAM alue >6 was conside ed high disease ac i i y. MCTD pa ien s we e ollowed-up e e y 4 mon hs a he ou pa ien clinic. Diagnos ic p ocedu es o MCTD included X- ay scan, lung unc ion es s, elec omyog a- phy and elec oneu og aphy. Esophageal in ol emen was de ec ed by adionuclid esophageal ansi scin ig a- phy and adiog aphic passage using gas og aphin, o ba ium. Myosi is was con i med by muscle biopsy and/o elec omyog am and c ea inine kinase ele a ion. Ray- naud's phenomenon was assessed by a posi i e colo cha o cold es , and mo phological abno mali ies we e assessed by nail old capilla y mic oscopy (Nikon Co p., Vienna, Aus ia). Abno mal indings we e assessed, as ea lie desc ibed by Ma icq [26]. Abno mal indings we e as ollows: num- be o loops in a linea 1 mm wid h, enla gemen o capil- la y loops, o p esence o bushy capilla ies and a ascula a eas. A ascula iza ion was assessed by me hods desc ibed by Lee and colleagues [27]. The scle ode ma pa e n was cha ac e ized by nail old mic ohemo hages, enla ged loops, and a ascula a eas. In labo a o y analyses, we assessed he e y h ocy e sed- imen a ion a e (ESR), high-sensi i i y C- eac i e p o ein (hs-CRP), he ou ine blood coun , and he enal and li e unc ion; u ine analysis was also pe o med. T adi ional isk ac o s o ca dio ascula disease such as age, body mass index (BMI), as ing plasma glucose, plasma lipid le els, as well as blood p essu e we e eco ded. Thi y-eigh age-ma ched and BMI-ma ched emale con ol subjec s we e also en olled in he s udy. Exclusion c i e ia included known CVD, diabe es melli us, obesi y (BMI >30), and in ec ion. Pa ien s and con ols had no consumed alcohol o aken asoac i e d ugs wi hin he Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Page 3 o 11 pas 24 hou s. No pa ien s ecei ed lipid-lowe ing he a- pies 48 hou s be o e he s udy. Cumula i e li e ime co icos e oid dose (p ednisone equi alen /g ams) was calcula ed om hospi al eco ds. Twen y- wo ou o 50 pa ien s wi h MCTD ecei ed non- s e oidal an i-in lamma o y d ugs, 10 pa ien s had he combina ion o co icos e oids (CS) and salazopy ine, and 11 pa ien s ecei ed a combina ion he apy o CS and me ho exa e, while se en pa ien s ecei ed CS he apy alone. The p o ocol was in ull compliance wi h he Good Clinical P ac ices, he Decla a ion o Helsinki, and he guidelines o he Medical and Heal h Science Cen e o he Uni e si y o Deb ecen. The p o ocol has been app o ed by he ins i u ional e hics commi ee (Regional and Ins i u ional E hics Commi ee, Medical and Heal h Science Cen e, Uni e si y o Deb ecen). W i en in o med consen was ob ained om all pa ien s and heal hy con ols. Immunose ological in es iga ions The de ec ion o an inuclea an ibodies on he HEp2 cell line was ca ied ou by indi ec immuno luo escence. The se um concen a ions o au oan ibodies we e ana- lyzed by ELISA acco ding o he manu ac u e 's ins uc- ions: an i-Sjög en synd ome-associa ed an igen A an ibodies, an i-Sjög en synd ome-associa ed an igen B an ibodies, an i-Sm an ibodies, an i-Sc170 an ibodies, an i-dsDNA an ibodies, an i-ca diolipin (an i-CL) an i- bodies (Cogen Diagnos ic, Edinbu gh, UK), and an i- U1RNP an ibodies (Pha macia and Upjohn, F eibu g, Ge many). Abso bances we e measu ed by a Labsys ems Mul iscan MS ELISA eade a 492 nm (Labsys ems Oy, Helsinki, Finland). The concen a ions o samples we e de e mined using a s anda d cu e ob ained om he op ical densi ies o s anda ds wi h known concen a ion. Endo helial cell ma ke s Th ombomodulin le els we e measu ed by ELISA using comme cial eagen s acco ding o he manu ac u e 's ins uc ions (Diagnos ic S ago, Asnie es, F ance). All assays we e pe o med in duplica e. The in a-assay and in e assay coe icien s o a ia ion o all ELISA assays we e <5% and <10%, espec i ely. The assessmen o AECAs was pe o med on endo he- lial cells om human umbilical co d eins, employing an ELISA me hod, desc ibed p e iously in de ail [11]. Labo a o y examina ions The o al choles e ol concen a ion was de e mined spec- opho ome ically. T iglyce ide and high-densi y lipo- p o ein choles e ol (HDL-C) was measu ed wi h he immune u bidime ic me hod. Low-densi y lipop o ein choles e ol (LDL-C) was measu ed by homogenous, enzyma ic, colo ime ic assay (Roche LDL-C plus 2nd gene a ion; Roche, Basel, Swi ze land). Apolipop o ein A1 (ApoA1) and apolipop o ein B we e assessed wi h he O ion Diagnos ica ki (O ion Diagnos- ica, Espoo, Finland), which employs an immune-neph- elome ic me hod. PON1 ac i i y was measu ed spec opho ome ically [13]. Endo helium-dependen ( low-media ed) and endo helium-independen (ni a e-media ed) asodila ion Endo helium-dependen asodila ion was assessed wi h a 7.5-MHz linea a ay ansduce (Hewle -Packa d Sonos 5500; Soma Technology Inc., Bloom ield, CT, USA) by scanning he b achial a e y in longi udinal sec ions, as published p e iously [28]. Endo helial unc ion es ing was pe o med by he same pe son (HD) and he e alua- ion was ca ied ou o line by a digi al so wa e ech- nique (AVITA; G ech In o ma ion Sys ems, Nape ille, IL, USA), as desc ibed p e iously in de ail [29]. The in e -obse e analysis ound he a iabili y on 20 pa ien s o be 8.95%. The in a-obse e analysis was pe - o med on 10 heal hy indi iduals h ee imes, wi h a 30- minu e in e al be ween he analyses. The in a-obse e a iabili y was 4.6%. We pe o med he a ia ion coe i- cien o baseline diame e in 20 cases, and i was 0.86%; he accu acy o he me hod is he e o e app op ia e acco ding o he in e na ional ecommenda ion [30]. Ca o id duplex ul asound in es iga ions; measu emen o ca o id a e y in ima-media hickness Ul asound examina ions we e pe o med immedia ely a e bloodsampling wi h he colo -coded Hewle Pack- a d SONOS 5500 (Soma Technology Inc.) ca o id duplex equipmen wi h a 7.5 MHz linea ansduce . The in es i- ga ion included longi udinal and ans e se examina ion- so he ca o id a e ies. Measu emen s o IMT we e pe o med a abou 10 mm p oximal o he ca o id bulb, o a 20 mm p oximal o he low di ide . The IMTwas measu ed as he dis ance be ween he leading edge o he i s echogenicline (lumen-in ima in e ace) and he sec- ond echogenic line(uppe laye o he ad en i ia) in he a (deepe ) a e y wall. All measu emen s we e pe - o med a he end o he hea cycle, when he ansduc- e was in he mediola e al di ec ion [31]. O line analysis was pe o med by digi al ideo images (AVITA; G ech In o ma ion Sys ems). Measu emen s we e pe o medin bo h ca o id a e ies and he la ge o he wo alues was used o analysis. S a is ical analysis No mali y o con inuous a iables was e alua ed by he Shapi o-Wilk es . As mos a iables we e no no mally dis ibu ed, he Mann-Whi ney es was used o compa e con ols and MCTD pa ien s. Fac o s ha di e ed signi - Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Page 4 o 11 ican ly be ween pa ien s and con ols in uni a ia e es s we e en e ed in a gene al linea model o es whe he hese ac o s a e independen p edic o s o FMD, ni a e- media ed dila ion (NMD) and IMT. S a is ica o Win- dows (S a So , Tulsa, OK, USA) was used o da a analy- sis. Co ela ions we e de e mined using Spea man co ela ion coe icien . S a is ical signi icance was assumed when P < 0.05. Resul s In he pa ien g oup he mean ± s anda d de ia ion age a he ime o in es iga ion was 50.2 ± 10.0 yea s ( ange: 17 o 69 yea s) and he disease du a ion was 9.56 ± 6.8 yea s ( ange: 3 o 26 yea s). Pa ien s wi h MCTD and he con- ol g oup we e simila wi h ega ds o age, sys olic and dias olic blood p essu es, iglyce ide, LDL-C, HDL-C, as well as BMI (Table 1). The o al choles e ol (P < 0.047), he PON1 ac i i y (P < 0.001) and ApoA1 le els (P < 0.001) we e signi ican ly lowe in MCTD pa ien s han in he con ols, while he e was no di e ence in apolipop o- ein B concen a ions be ween pa ien s wi h MCTD and con ols (P = 0.693). The ApoA1 and PON1 ac i i ies we e lowe in he MCTD/CVD+ and MCTD/CVD- pa ien g oups compa ed wi h con ols. The ESR and hs- CRP le els we e signi ican ly highe in pa ien s wi h MCTD and in he MCTD/CVD+ and MCTD/CVD- g oups compa ed wi h heal hy con ols. The in ol emen o a ious o gans is summa ized in Table 2. The p esence o Raynaud's phenomenon (P < 0.006), PAH (P = 0.0141), and seconda y an iphospho- lipid synd ome (P = 0.015) we e signi ican ly mo e e- quen in MCTD/CVD+ pa ien s han in he MCTD/CVD- pa ien g oup. Conce ning au oan ibody p o iles, all o he pa ien s we e posi i e o an inuclea an ibodies and an i-U1RNP an ibodies, 19 pa ien s (38%) had an i-CL an ibodies (IgG o IgM an i-CL an ibodies), and 22 pa ien s (44%) we e posi i e o AECAs. The equency o an i-CL-posi i e and AECA-posi i e pa ien s was signi ican ly highe in he MCTD/CVD+ g oup (an i-CL, P = 0.0195; AECA, P < 0.001). The a io o pa ien s aking CS a he ime o he s udy, he cumula i e median dose o CS, and o he medica ions a e also p esen ed in Table 2. Raynaud's phenomenon was de ec ed in 39 ou o 50 pa ien s wi h MCTD. In he MCTD/CVD+ g oup, 22 Table 1: Baseline cha ac e is ics o pa ien s Fea u e MCTDa Con ols P alue, MCTD s. con ols MCTDb CVD+ MCTD CVD- P alue, MCTD/ CVD+ s. MCTD/ CVD- n50 38 23 27 Age a in es iga ion (yea s) 50.2 ± 10.0 50.4 ± 10.3 0.936 50.1 ± 10.7 50.2 ± 9.5 0.99 Disease du a ion (yea s) 9.56 ± 6.8 (3 o 26) - - 10.3 ± 6.6 (3 o 26) 8.9 ± 6.9 (3 o 25) 0.481 SBP (mmHg) 137.4 ± 21.1 132.9 ± 19.6 0.367 144.7 ± 19.5 131.1 ± 20.6 0.037 DBP (mmHg) 90.9 ± 15.9 86.6 ± 15.3 0.215 88.5 ± 14.9 92.9 ± 16.8 0.299 BMI (kg/m2) 24.2 ± 1.3 23.2 ± 1.1 0.77 23.9 ± 1.1 24.81.9 0.383 Smoking Fo me smoke s 5 (10%) 5 (13.1) 0.7401 2 (8.6) 3 (11.1) 1.0 Nonsmoke s 45 (90%) 33 (86.8%) 0.7401 21 (91.3) 24 (88.8) 1.0 Se um iglyce ide (mmol/l) 1.611 ± 0.76 1.6 ± 0.79 0.755 1.5 ± 0.82 1.72 ± 0.70 0.3367 To al choles e ol (mmol/l) 5.94 ± 1.18 5.47 ± 1.03 0.047 5.8 ± 1.2 6.06 ± 1.13 0.108 HDL-C (mmol/l) 1.69 ± 0.5 1.68 ± 0.49 0.943 1.56 ± 0.48 1.8 ± 0.5 0.22 LDL-C (mmol/l) 3.62 ± 1.15 3.23 ± 0.88 0.148 3.52 ± 1.19 3.7 ± 1.12 0.333 ApoA1 (g/l) 1.31 ± 0.36 1.72 ± 0.47 <0.001 1.27 ± 0.3 1.33 ± 0.4 0.003 ApoB (g/l) 0.95 ± 0.31 0.91 ± 0.13 0.693 0.98 ± 0.38 0.89 ± 0.15 0.92 Pa aoxonase-1 ac i i y 113.6 ± 70.6 187.4 ± 68.3 <0.001 84.32 ± 69.66 138.7 ± 62.3 <0.001 hs-CRP (mg/l) 15.2 ± 9.62 1.44 ± 0.99 <0.001 22.1 ± 9.78 9.36 ± 3.96 <0.001 ESR (mm/hou ) 20.2 ± 17.8 8.3 ± 3.9 <0.001 26.5 ± 17.6 14.4 ± 16.2 0.014 ApoA1, apolipop o ein A1; ApoB, apolipop o ein B; BMI, body mass index; CVD, ca dio ascula disease; DBP, dias olic blood p essu e; ESR: e y h ocy e sedimen a ion a e; HDL-C, high-densi y lipop o ein-choles e ol; hs-CRP: high-sensi i i y C- eac i e p o ein; LDL-C, low-densi y lipop o ein-choles e ol; MCTD, mixed connec i e issue disease; SBP, sys olic blood p essu e. aMann-Whi ney U es . bK uskal-Wallis analysis o a iance. Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Page 5 o 11 Table 2: Clinical symp oms o mixed connec i e issue disease pa ien s wi h and wi hou ca dio ascula diseases MCTD mani es a ion MCTD (n = 50) MCTD/CVD+ (n = 23) MCTD/CVD- (n = 27) P aluea, CVD+ s. CVD- Disease du a ion (yea s) 9.56 ± 6.8 (3 o 26) 10.3 ± 6.6 (3 o 26) 8.9 ± 6.9 (3 o 25) 0.481 Polya h i is 46 (92) 22 (95.6) 24 (88.8) 0.6123 Raynaud's phenomenon 39 (78) 22 (95.6) 17 (62.9) 0.006 Myosi is 30 (60) 13 (56.5) 17 (62.9) 0.8621 In e s i ial lung disease 40 (80) 19 (82.6) 21 (77.7) 0.7356 Pho osensi i i y 12 (24) 7 (30.4) 5 (18.5) 0.5077 Esophageal dysmo ili y 35 (70) 15 (65.2) 20 (74.0) 0.5480 Pulmona y a e ial hype ension 12 (24) 9 (39.1) 3 (11.1) 0.0141 Lymphadenomegaly 13 (0.26) 5 (21.7) 8 (29.6) 0.7474 Se osi is 19 (38) 11 (47.8) 8 (29.6) 0.2465 Seconda y an iphospholipid synd ome 15 (30) 11 (47.8) 4 (14.8) 0.015 P e ious enous h ombosis 13 (26.0) 9 (39.1) 4 (14.8) 0.618 P e ious a e ial occlusion 2 (4.0) 2 (8.69) 0 An i-U1RNP (no mal: <10 U/ml) 50 (100) 23 (100) 27 (100) An i-CL IgG/IgM (no mal: <10 U/ml) 19 (38) 13 (56) 6 (22) 0.0195 AECA (no mal: <5 U/ml) 22 (44) 15 (65) 7 (26) 0.001 SLAM (median) 5 5 4 SLAM (mean ± SD) 6.28 ± 4.04 6.95 ± 4.08 5.44 ± 4.0 0.199 SLAM (n) 18 (36) 11 (47.8) 7 (25.9) 0.1438 Medica ion Nons e oidal an i-in lamma o y d ugs 22 (44) 10 (43.4) 12 (44.4) 1.0 Las 2 mon hs a e age dose o co icos e oids (mg/day) 3.36 ± 4.8 4.1 ± 3.4 3.1 ± 5.2 0.5306 Cumula i e li e ime dose (p ednisone equi alen ) 14.21 (4 o 58.965) 13.0 (4 o 59.432) 15.2 (4 o 57.437) 0.674 Co icos e oids alone o combina ion (n) 28 (56) 11 (47.8) 17 (62.9) 0.3926 Pa ien s cu en ly co icos e oids alone n (%) 7 (14) 2 (8.6) 5 (18.5) 0.4295 Pa ien s cu en ly co icos e oids plus salazopy ine 10 (20) 5 (21.7) 5 (18.5) 1.0 Pa ien s cu en ly co icos e oids plus me ho exa e 11 (22) 5 (21.7) 6 (22.2) 1.00 Cyclophosphamide ea men e e 37 (74) 21 (91.3) 16 (59.2) 0.0119 An imala ic d ugs ea men e e 39 (78) 19 (82.6) 20 (74.0) 0.515 Cyclospo in-A ea men e e 10 (20) 7 (30.4) 3 (11.1) 0.4804 Da a p esen ed as mean ± s anda d de ia ion, n (%) o median (25 h o 75 h pe cen ile). AECA, an i-endo helial cell an ibody; an i-CL, an i- ca diolipin; an i-U1RNP, an i-U1 ibonucleop o ein; CVD, ca dio ascula disease; MCTD, mixed connec i e issue disease. The sys emic lupus e y hema osus disease ac i i y measu e (SLAM) is a measu e o disease ac i i y [24,25]. aFische 's exac es and S uden es . pa ien s had Raynaud's phenomenon. Among hese, 10 pa ien s' se a con ained an i-CL IgG/IgM an ibodies, while 13 pa ien s' se a we e AECA-posi i e. An associa- ion was ound be ween Raynaud's phenomenon and an i-CL IgG/IgM (P < 0.03), and be ween Raynaud's phe- nomenon and he p esence o AECA (P < 0.02). Pe cen age FMD, pe cen age NMD, IMT, au oan ibodies and endo helial cell ma ke s An i-U1RNP au oan ibodies, an i-CL IgG/IgM ype au oan ibodies, AECA and bo h endo helial cell ma ke s (TM and WFAg) we e signi ican inc eased in MCTD Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Page 6 o 11 pa ien s e sus con ols and in he MCTD/CVD+ e sus MCTD/CVD- pa ien g oups (Table 3). Pe cen age FMD was signi ican lowe in MCTD pa ien s compa ed wi h con ols and in MCTD/CVD+ pa ien s e sus MCTD/CVD- pa ien s (%FMD: MCTD, 4.7 ± 4.2% s. con ols, 8.7 ± 5.05%, P < 0.001; MCTD/ CVD+ s. MCTD/CVD-, P < 0.0002). Pe cen age NMD did no show a di e ence be ween MCTD and con ols and be ween he MCTD/CVD+ e sus MCTD/CVD- pa ien g oups. The IMT was signi ican ly highe bo h in he MCTD pa ien s and in he MCTD/CVD+ s. MCTD/CVD- pa ien s (IMT: MCTD, 0.64 ± 0.13 mm s. con ols, 0.53 ± 0.14 mm, P < 0.001; MCTD/CVD+ s. MCTD/CVD-, P < 0.001). Co ela ion be ween low-and ni a e media ed asodila ion, IMT and o he measu ed pa ame e s in MCTD pa ien s A signi ican nega i e co ela ion was ound be ween pe cen age FMD and he disease du a ion ( = -0.6468, P < 0.001), and be ween pe cen age FMD and sys olic blood p essu e ( = -0.5423, P < 0.001) (Table 4). The e was a posi i e co ela ion be ween pe cen age FMD and ApoA1 le els, and pe cen age FMD and PON1 ac i i y (ApoA1: = 0.6203, P < 0.001; PON1 ac i i y: = 0.5957, P < 0.001). Signi ican co ela ion was ound be ween pe - cen age FMD and AECA ( = -0.3075, P = 0.029), and be ween pe cen age FMD and in lamma o y pa ame e s such as he ESR ( = -0.4283, P < 0.001) and hs-CRP ( = - 0.4057, P = 0.003). We ound a nega i e co ela ion be ween SLAM sco e and pe cen age FMD ( = -0.488, P < 0.001) (Figu e 1). NMD showed a nega i e associa ion wi h hs-CRP ( = - 0.3207, P = 0.023) and he ESR ( = -0.3981, P < 0.001). No o he co ela ions we e de ec ed. The IMT was signi ican ly highe in MCTD pa ien s compa ed wi h con ols (0.64 mm s. 0.53 mm; P < 0.001). The IMT was age dependen ( = 0.5468, P < 0.001), and showed an associa ion wi h se um le els o an i-U1RNP au oan ibodies (P = 0.0025) and AECA (P = 0.0417), he endo helial cell ma ke s TM (P <0.001) and WFAg (P < 0.001), and he ESR (P < 0.001) and hs-CRP (P < 0.001). In ou pa ien s we ound a co ela ion be ween he ESR and hs-CRP ( = 0.511, P < 0.001). In he MCTD/CVD+ g oup, 22 pa ien s had Raynaud's phenomenon. E alua- ion o he ela ionship be ween MCTD pa ien s wi h Raynaud's phenomenon and FMD is p esen ed in Table 5. The e was a signi ican associa ion in MCTD pa ien s wi h and wi hou Raynaud's phenomenon and FMD. The e was a nega i e associa ion be ween FMD and MCTD pa ien s wi h Raynaud's phenomenon in he CVD+ and CVD- g oups. Discussion Ca dio ascula diseases ha e ecen ly been shown o be he leading causes o mo bidi y and mo ali y in pa ien s wi h sys emic au oimmune diseases. Some e idence shows ha in au oimmune diso de s, such as SLE, heu- ma oid a h i is and an iphospholipid synd ome, he sys- emic in lamma o y s a e i sel p edisposes o a he oscle o ic diseases [32-35]. MCTD is a sys emic au oimmune disease, in ol ing many o gans, while he mos equen , se ious ou come is he de elopmen o p oli e a i e ascula lesions in he lungs and o he o gans. An i-U1RNP au oan ibodies may Table 3: Au oan ibodies, endo helial cell pa ame e s, FMD, NMD and IMT in MCTD pa ien s wi h/wi hou ca dio ascula diseases Fea u e MCTD (n = 50) Con ols (n = 38) P alue, MCTD s. con olsa MCTD/CVD+ (n = 23) MCTD/CVD- (n = 27) P alue, MCTD/ CVD+ s. MCTD/CVD-b An i-U1RNP (U/ml) 20.18 ± 14.6 8.02 ± 3.5 <0.001 30.3 ± 14.6 11.51 ± 7.28 <0.001 An i-CL IgG/IgM (U/ml) 13.98 ± 12.2 6.06 ± 2.93 <0.001 21.02 ± 15.04 7.98 ± 2.95 <0.001 AECA (IU/ml) 50.1 ± 34.5 17.1 ± 8.48 <0.001 62.9 ± 26.3 39.1 ± 37.2 <0.001 Th ombomodulin (ng/ml) 12.2 ± 8.1 3.2 ± 1.3 <0.001 15.9 ± 5.2 9.0 ± 8.8 <0.001 WFAg (%) 224.1 ± 115 89.4 ± 27.1 <0.001 311.5 ± 72.0 149.7 ± 90.0 <0.001 FMD (%) 4.76 ± 4.2 8.74 ± 5.05 <0.001 3.54 ± 2.9 5.81 ± 4.87 0.0002 NMD (%) 14.35 ± 6.67 17.16 ± 6.7 0.073 13.54 ± 6.06 15.03 ± 7.15 0.1283 IMT (mm) 0.64 ± 0.13 0.53 ± 0.14 <0.001 0.72 ± 0.11 0.57 ± 0.1 <0.001 AECA, an i-endo helial cell an ibody; an i-CL, an i-ca diolipin; an i-U1RNP, an i-U1 ibonucleop o ein; CVD, ca dio ascula disease; FMD, low- media ed dila ion; IMT, in ima-media hickness; MCTD, mixed connec i e issue disease; NMD, ni a e-media ed dila ion; WFAg, on Willeb and ac o an igen. aMCTD and con ols, Mann-Whi ney U es . bMCTD/CVD+ and MCTD/CVD-, analysis o a iance es . Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Page 7 o 11 ha e an e ec on endo helial cells; in some s udies on MCTD, howe e , he p esence o an iphospholipid o an i-endo helial an ibodies, in addi ion o an i-RNP an i- bodies, showed unc ional p ope ies such as endo helial cell ac i a ion - changing he pheno ype o endo helial cells, which become p oin lamma o y/p ocoagulan [36]. No p e ious s udies, howe e , ha e been ca ied ou o assess he a he oscle o ic isk ac o s in MCTD pa ien s. In ou s udy popula ion, we in es iga ed he adi ional isk ac o s, in associa ion wi h clinical symp oms and au oan ibodies, in MCTD pa ien s wi h and wi hou ca - dio ascula e en s. This is he i s s udy whe e endo he- lial s i ness ma ke s a e measu ed, including FMD, NMD and ca o id IMT. In ou pa ien s wi h MCTD, se um iglyce ides, HDL- C and LDL-C did no di e om heal hy subjec s, while o al choles e ol and he ApoA1 le els and se um PON1 ac i i y wi hin he lipo po ein ac ion we e lowe com- pa ed wi h con ols. Dec eased PON1 ac i i y and mild ele a ion in hs-CRP ha e d awn conside able in e es , in ela ion o he de el- opmen o a he oscle osis ha exempli ies a low-g ade ch onic in lamma o y p ocess [37,38]. Dec eased pe cen age FMD and inc eased IMT was ound in pa ien s wi h MCTD. Ou esul s indica ed ha educed FMD can clea ly dis inguish MCTD pa ien s om con ols and, mo eo e , MCTD pa ien s wi h and wi hou ca dio ascula e en s. Dec eased pe cen age FMD showed a close co ela ion wi h he disease du a- ion, sys olic blood p essu e, and in lama o y pa ame e s (hs-CRP and ESR). High se um le els o hs-CRP ha e been shown o ha e a close associa ion wi h CVDs, ep esen ing a link be ween ch onic in lamma ion and hs-CRP wi h a h e oscle osis [39]. A s ong co ela ion was desc ibed be ween hs-CRP and CVD e en s, when hs-CRP exceeded 3 mg/l [40]. In ou se ies, bo h hs-CRP and he ESR we e ele a ed in MCTD pa ien s, escpecially in he MCTD/CVD+ g oup, and pe cen age FMD showed a close nega i e co ela ion wi h ele a ed ESR. These esul s may sugges ha MCTD pa ien s ha e ongoing low-g ade in lamma ion, and hese pa ien s ha e an inc eased isk o se e e CV e en s. A close associa ion be ween SLAM sco e and pe cen age FMD shows ha he disease ac i i y in ol es endo helial cell in lamma ion, causing endo helial cell dys unc ion. Se um concen a ion o an i-U1RNP au oan ibodies and le els o AECA we e ele a ed in he pa ien s'se a, and bo h an ibodies we e highe in he MCTD/CVD+ pa ien s compa ed wi h he MCTD/CVD- g oup. Fu he mo e, we showed ha he ma ke s o endo helial cell dys unc ion, WFAg and soluble TM we e highe in pa ien s wi h MCTD han in he heal hy indi iduals. In MCTD, high se um le els o WFAg imply an ac i- a ed s a e o endo helial cells and can play a pa hogenic Table 4: FMD, NMD and IMT in MCTD pa ien s wi h/wi hou ca dio ascula disease and con ols %FMDa P alue %NMDa P alue IMTa P alue Age a in es iga ion (yea s) -0.0221 0.878 -0.6700 0.001 0.5468 0.001 Disease du a ion (yea s) -0.6468 0.001 -0.1314 0.3628 0.1968 0.1706 Sys olic blood p essu e (mmHg) -0.5423 0.001 -0.1094 0.4492 0.2220 0.1211 Dias olic blood p essu e (mmHg) 0.0946 0.5133 0.1005 0.4872 -0.1597 0.2676 Se um iglyce ides (mmol/l) -0.3396 0.0158 0.2165 0.1309 -0.0411 0.7765 Se um choles e ol -0.0459 0.7514 0.0508 0.7255 -0.0209 0.8851 Apolipop o ein A1 (g/l) 0.6203 0.001 -0.0805 0.5782 -0.0248 0.8640 Apolipop o ein B (g/l) -0.3102 0.061 -0.0891 0.5997 0.0767 0.6517 Pa aoxonase-1 ac i i y 0.5957 0.001 0.1317 0.3619 -0.2789 0.0497 hs-CRP -0.4057 0.003 -0.3207 0.023 0.7164 0.001 ESR -0.4283 0.001 -0.3981 0.001 0.5467 0.001 An i-U1RNP (U/ml) -0.0486 0.7371 -0.0694 0.6316 0.4182 0.0025 An i-CL IgG (U/ml) -0.0717 0.6206 -0.0411 0.776 0.2236 0.1184 AECA (IU/ml) -0.3075 0.029 -0.2686 0.0592 0.2890 0.0417 Th ombomodulin (ng/ml) -0.0642 0.6577 -0.0165 0.9089 0.4823 0.00038 WFAg (%) -0.0122 0.9329 -0.2692 0.058 0.5443 0.001 AECA, an i-endo helial cell an ibody; an i-CL, an i-ca diolipin; an i-U1RNP, an i-U1 ibonucleop o ein; ESR, e y h ocy e sedimen a ion a e; FMD, low-media ed dila ion; hs-CRP, high-sensi i i y C- eac i e p o ein; IMT, in ima-media hickness; MCTD, mixed connec i e issue disease; NMD, ni a e-media ed dila ion; WFAg, on Willeb and ac o an igen. aSpea man ank co ela ion. Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Page 8 o 11 ole in he de elopmen o a he o h ombo ic e en s [41]. Acco dingly, in MCTD/CVD+ pa ien s he soluble TM le els we e signi ican highe han in he MCTD/CVD- g oup. TM is an endo helial cell ac i a ion ma ke , and i s shedding om he endo helial cell inc eases he isk o ca dio ascula and h ombo ic e en s [12,42]. In pa ien s wi h a he oscle o ic diseases, soluble TM was ele a ed and i s le el showed co ela ion wi h he se e i y o co ona y a e y disease, and also an associa- ion wi h wo se ou come in su i als a e acu e myoca - dial in a c ion [42]. Mo eo e , soluble TM is a good ma ke o disease ac i i y in SLE wi h lupus neph i is [43]. WFAg is a ci cula ing glycop o ein, syn hesized by endo helial cells - and an inc eased se um concen a ion o WFAg has been shown o be a ma ke o endo helial dys unc ion in scle ode ma, SLE and MCTD pa ien s wi h PAH [10,44]. In ou pa ien s wi h MCTD, pe cen age FMD s ongly co ela ed wi h disease du a ion, au oan ibodies o an i- U1RNP, AECA and an i-CL, and endo helial cell ma ke s, such as TM and WFAg. These da a indica e ha he educed FMD is a good ma ke o moni o ing ca dio as- cula complica ions in MCTD. One o he ea lies s ages o a he oscle osis is he endo helial cell dys unc ion [45]. Endo helium-depen- den FMD was desc ibed p e iously o be signi ican ly impai ed in SLE [34], in heuma oid a h i is [35], and in an iphospholipid synd ome [29]. In ou ea lie s udy we also ound dec eased FMD in pa ien s wi h undi e en i- a ed connec i e issue disease (UCTD), which is an ea ly s age o well-es ablished connec i e issue diseases [46]. Mosca and colleagues also in es iga ed he ascula eac- i i y in UCTD, and ound ha FMD and he esponse o glyce yl ini a e-media ed asodila ion we e simila in UCTD pa ien s and heal hy subjec s. UCTD pa ien s we e cha ac e ized, howe e , by ha ing educed esponse o bo h ace hylcholine and sodium ni op usside in he o ea m mic oci cula ion, indi ec ly indica ing ha educed endo helium-dependen asodila ion, a pe iph- e al mic o ascula isk ac o , signi ies UCTD [47]. In he p esen s udy we ound ha FMD dec eased and he IMT was ele a ed in he MCTD/CVD+ pa ien g oup, bu he e was no signi ican di e ence in NMD be ween he MCTD/CVD+ and MCTD/CVD- pa ien g oups. We u he desc ibed he p esence o an i-CL an ibodies and AECA besides an i-U1RNP an ibodies in MCTD; mo e- o e , se um le els o an i-U1RNP an ibodies, an i-CL IgG and AECA we e highe in he MCTD/CVD+ g oup com- pa ed wi h MCTD/CVD- pa ien s. We assume ha AECA in MCTD pa ien s could con- ibu e o endo helial cell dys unc ion. AECAs a e o en associa ed wi h phospholipid eac i i y, p esen in SLE and in p ima y an iphospholipid synd ome. In con as o he da a by Lima and colleagues, we ound ha FMD showed a co ela ion wi h he ac i i y sco e [48]. Among he clinical symp oms, Raynaud's phe- nomenon was mo e equen in he MCTD/CVD+ g oup - ou da a being simila o he indings o Aize and col- leagues [49]. In e es ingly, an i-CL an ibodies and AECAs we e mo e equen in pa ien s wi h Raynaud's phenomenon, and hese an ibodies could p o oke Figu e 1 Ac i i y sco e and low-media ed dila ion in pa ien s wi h mixed connec i e issue disease. Y axis: low-media ed dila ion (%). X axis: sys emic lupus ac i i y measu e sco e (numbe ). 5.00 10.00 15.00 20.00 =-0.488 p<0.001 Sol esz e al. A h i is Resea ch & The apy 2010, 12:R78 h p://a h i is- esea ch.com/con en /12/3/R78 Page 9 o 11 endo helial cell damage. We assume ha dec eased FMD may occu as a esul o con inuous endo helial cell ac i- a ion and impai men . The daily doses o CS and o he immunosupp essi e ea men we e simila in he CVD+ and CVD- g oups. Based on ou da a we belie e ha he ascula p o ec ion is essen ial in pa ien s wi h MCTD, especially in Ray- naud's penomenon and PAH, and also aspi in and s a in he apy is impo an om he ea ly s age o MCTD. Ca o id IMT was signi ican highe in MCTD pa ien s and showed an associa ion wi h age, wi h disease du a- ion and wi h adi ional isk ac o s such as o al choles- e ol le els, sys olic blood p essu e and dias olic blood p essu e. Ul asound measu emen s o he ca o id a e y IMT iden i y ea ly s uc u al ascula abno mali ies. An in e se co ela ion has been desc ibed p e iously be ween he IMT and b achial a e ial FMD. Endo helial dys unc ion showed a co ela ion wi h he IMT, sugges - ing ha he ca o id IMT alone was a alid su oga e ma ke o ea ly a he oscle osis and had an impo an p edic ing ea u e in ca dio ascula e en s in he gene al popula ion [50-52]. Conclusions We s a e ha , besides adi ional a he oscle o ic ac o s, au oan ibodies play a c ucial ole in ea ly a he oscle osis in MCTD. We belie e ha an i-U1RNP an ibodies and AECA, as well as he up egula ion o p oin lamma o y cy okines associa ed wi h ascula endo helial cell dam- age, may play a pi o al ole in he ea ly a he oscle o ic e en s in MCTD. We assume ha WFAg and TM a e he mos impo - an ma ke s o endo helial cell ac i a ion, and hey impai endo helial cell unc ions. Finally, we ound a clea dec ease o pe cen age FMD in MCTD pa ien s wi h CVD, ce eb o ascula e en s, and pe iphe al a e ial dis- ease. Ou indings suppo he idea ha he immune-medi- a ed endo helial dys unc ion, accele a ed a he oscle osis p esen in pa ien s wi h MCTD. We assume ha he u ili- za ion o hese se um ma ke s and non-in asi e ca dio- ascula measu emen s in he diagnosis o ea ly a he oscle osis in MCTD p o ides a su icien back- g ound o he ea ly in oduc ion o endo helial p o ec- ion ( o example, aspi in, s a in) in he u u e managemen o he disease, be o e he de elopmen o se ious ca dio ascula symp oms. Abb e ia ions AECA: an i-endo helial cell an ibody; an i-CL: an i-ca diolipin; an i-U1 RNP: an i- U1 ibonucleop o ein; ApoA1: apolipop o ein A; BMI: body mass index; CVD: ca dio ascula disease; CS: co icos e oids; dsDNA: double-single DNA; ELISA: enzyme-linked immunoso ben assay; ESR: e y h ocy e sedimen a ion a e; FMD: low-media ed dila ion; HDL-C: high-densi y lipop o ein-choles e ol; hs- CRP: high-sensi i i y C- eac i e p o ein; IMT: in ima-media hickness; LDL-C: low-densi y lipop o ein-choles e ol; MCTD: mixed connec i e issue disease; NMD: ni a e-media ed dila ion; PAH: pulmona y a e ial hype ension; PON1: pa aoxonase-1; SLAM: sys emic lupus ac i i y measu e; SLE: sys emic lupus e y- hema osus; TM: h ombomodulin; UCTD: undi e en ia ed connec i e issue disease; WFAg: on Willeb and ac o an igen. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Au ho s' con ibu ions PSo pe o med acquisi ion and analysis o he da a. DB and PSz pe o med in e p e a ion o he da a and manusc ip p epa a ion. MTM, HD, IC and AH pe o med in e p e a ion o he da a and d a ed he manusc ip . GP and GS pe o med analysis and in e p e a ion o he da a. EB ga e inal app o al o he e sion o be published. All au ho s ead and app o ed he inal manusc ip . Acknowledgemen s The p esen wo k was suppo ed by esea ch G an Numbe s ETT 260/2009, ETT 197-05/2010 and ETT 158/2009 om he Minis y o Heal h, Republic o Hunga y. Au ho De ails 13 d Depa men o Medicine, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Mo icz Zs. S . 22, Deb ecen 4032, Hunga y, 2Depa men o Neu ology, Semmelweis Uni e si y o Budapes , Balassa S . 6, Budapes 1083, Hunga y, 3Ins i u e o Immunology, Rikshospi ale , Uni e si y o Oslo, Sogns anns eien S . 20, Oslo 0027, No way, 4Depa men o Neu ology, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Mo icz Zs. S . 22, Deb ecen 4032, Hunga y and 51s Depa men o Medicine, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Nagye dei S . 98, Deb ecen 4032, Hunga y Re e ences 1. Shee e Y, Shoen eld Y: Mechanism o disease: a he oscle osis in au oimmune diseases. Rheuma ology 2006, 2:99-106. 2. Bijl M: Endo helial ac i a ion ,endo helial dys unc ion and p ema u e a he oscle osis in sys emic au oimmune diseases. Ne h J Med 2003, 61:273-277. 3. Szucs G, Tima O, Szekanecz Z, De H, Ke ekes G, Szamosi S, Shoen eld Y, Szegedi G, Sol esz P: Endo helial dys unc ion p ecedes a he oscle osis in sys emic scle osis - ele ance o p e en ion o ascula complica ions. Rheuma ology 2007, 46:759-762. Recei ed: 28 Sep embe 2009 Re ised: 22 Feb ua y 2010 Accep ed: 6 May 2010 Published: 6 May 2010 This a icle is a ailable om: h p://a h i is- esea ch.com/con en /12/3/R78© 2010 Sol esz e al.; licensee BioMed Cen al L d. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.A h i i s Resea ch & Th e apy 2010, 12:R78 Table 5: Co ela ion be ween low-media ed dila ion and Raynaud's phenomenon nCo ela iona P alue MCTD wi h Raynaud's phenomenon 39/50 0.768 0.001 MCTD wi hou Raynaud's phenomenon 11/50 0.25 0.235 Raynaud's phenomenon in MCTD/CVD+ and MCTD/CVD- pa ien s 22/17 0.522 0.001 CVD, ca dio ascula disease; MCTD, mixed connec i e issue disease. aSpea man co ela ion coe icien .