Araştırma makalesi Abstract Backgrund: Allgeneic stem cell transplantatin (SCT) is an imprtant treatment mdality used in hematlgical malignant disrders. Graft-versus-hst disease (GVHD) is the main cmplicatin after allgeneic SCT. It is suggested that endthelial cell damage has an imprtant rle in develpment f GVHD. In this study we investigated the effect f adrenmedullin (AMD) in TNF-α stimulated in-vitr GVHD mdel since AMD has a prtective effect n cells via the regulatin inflammatry pathway. Materials and Methds: Umbilical crd derived endthelial cells were incubated with TNF-α fr 2 hurs in grup1 and with TNF-α+AMD in grup2 in rder t make in-vitr GVHD mdel and see the effect f AMD. Micrparticles (CD62, CD146, CD31, CD51/61) riginated frm endthelial cells that are the markers f injury r apptsis were measured. Results: We fund n significant difference between the micrparticle levels in bth grups. Althugh ur hypthesis was t find decrement in micrparticle levels in grup2 there were increases and decreases in micrparticle levels in bth grups (p>0,05). Cnclusin: T detect whether there is a prtective effect f AMD n develpment f in-vitr acute GVHD mdel extensive studies with multiple measurements are needed. Key wrds: Adrenmedullin, graft versus hst disease, micrparticle Özet Giriş: Alljeneik kök hücre nakli (KHN) özellikle hematljik malignitelerin tedavisinde önemli bir yer tutmaktadır. Greft versus hst hastalığı (GVHH) alljeneik KHN'nin en sık görülen kmplikasynlarından biridir. Nakil snrası GVHH'da temel patljik mekanizmalardan birinin inflamatuar ylak üzerinden endtel hücre hasarı lduğu düşünülmektedir. İnflamatuar ylak üzerinden kruyucu etkisi lan adrenmedullin (AMD)'nin TNF-α ile luşturulmuş in-vitr GVHH mdelindeki sitprtektif etkisi araştırıldı. İnsan Umblikal Ven Endtel Hücre Kültüründe TNF- lfa İle Oluşturulmuş İn Vitr Greft Versus Hst Hastalığı Mdelinde Adrenmedullinin Sitprtektif Etkisi Effects Of Adrenmedullin On Endthelial Micrparticle Release In TNF-Αlpha Stimulated In-Vitr Graft Versus Hst Disease Mdel 1 2 3 4 3 3 Fatih Kurnaz, Yasemin Trun, Çiğdem Pala, Esma Kaya, Serdar Şıvgın, Leylagül Kaynar, Bülent 3 3 5 3 Eser, Fatih Şahin, Ahmet Öztürk, Mustafa Çetin 1 Harran Üniversitesi Tıp Fakültesi, İç Hastalıkları Anabilim Dalı, Hematlji BD 2 Kayseri Eğitim Araştırma Hastanesi, Çcuk Ruh Sağlığı ve Hastalıkları Anabilim Dalı, Hematlji BD 3 Erciyes Üniversitesi Tıp Fakültesi, İç Hastalıkları Anabilim Dalı, Hematlji BD 4 Erciyes Üniversitesi Tıp Fakültesi, Mikrbiylji Anabilim Dalı, 5 Erciyes Üniversitesi Tıp Fakültesi, Biyistatistik Anabilim Dalı, Yazışma adresi: Fatih Kurnaz, Harran Üniversitesi Tıp Fakültesi, İç Hastalıkları Anabilim dalı, Hematlji Bilim Dalı, Şanlıurfa, Tel: (0414) 3183000, E-mail: kurnazfatih@gmail.cm Geliş tarihi / Received: 31.03.2014 Kabul tarihi / Accepted: 09.04.2014 Harran Üniversitesi Tıp Fakültesi Dergisi (Jurnal f Harran University Medical Faculty) Cilt 11. Sayı 3, 2014 210
Methds: İnsan umblikal veninden elde edilen endtel hücreleri TNF-α (grup1) ve TNF-α+AMD (grup2) ile iki saat inkübe edildi ve endtel hücre kaynaklı mikrpartiküller (CD62, CD146, CD31, CD51/61) ve apptzis belirteci lan Anneksin-V ölçüldü. Bulgular: Her iki grupta endtelyal mikrpartikül seviyeleri arasında fark yktu. AMD snrası bazı ölçümlerde artış tespit edilirken bazı ölçümlerde düşüş tespit edildi (p>0,05). Snuç: In vitr akut GVHH mdelinde AMD'in kruyucu etkisi lup lmadığıni tespit etmek için çk ölçümlü ve büyük çalışmalara ihtiyaç vardır. Anahtar Kelimeler: Adrenmedullin, greft versus hst hastalığı, mikrpartikül Intrductin Allgeneic stem cell transplantatin (SCT) is an intensive therapy used t treat high-risk hematlgical malignant disrders and ther lifethreatening hematlgical and genetic diseases. The main cmplicatin after allgeneic SCT is graft-versus-hst disease (GVHD). There is a hst tissue damage in GVHD and secreted prinflammatry cytkines, [such as tumr necrsis factr alpha (TNF-α), interleukin-1 (IL- 1)] play a rle in the develpment f GVHD (1). It is suggested that endthelial cell damage als has an imprtant rle in GVHD after allgeneic SCT (2-5). TNF-α is ne f the majr mediatrs that stimulates micrparticle (MP) secretin frm endthelial cells (6,7). In a recent study it was shwn that endthelial MPs increased in patients with acute GVHD and this increment in endthelial MP levels suggested that GVHD may be assciated with severe endthelial cell injury (2). Adrenmedullin (AMD) is an adrenal medulla derived prtein and acts as a regulatry prtein in the feed-back mechanism between prinflammatry and anti-inflammatry cytkines in inflammatry respnse and als has a cytprtective effect by inhibiting ver prductin f prinflammatry cytkines like TNF-α, IL-1β (8-11). In this study we aimed t investigate the cytprtective effect f AMD n the endthelial cells that were stimulated with TNF-α. We investigated the MP levels after administratin f TNF-α and TNFα+AMD n endthelial cells. Material and Methd This study was cnducted at Hakan Çetinsaya Clinical Research Center Cell Culture Labaratry, Erciyes University Schl f Medicine. Prir t subject recruitment, the study prtcl was reviewed and apprved by the university ethics cmmittee, in accrdance with the ethical principles fr human investigatins, as utlined by the Secnd Declaratin f Helsinki and written infrmed cnsents were btained frm all the patients. Tw umbilical crds f tw pregnant wmen were taken after birth. Preparatin f cells and cell culture was dne with a mdified prcedure cnducted by Jaffe et al. (12). Endthelial cells were btained frm human umbilical crd veins. A sterile technique was utilized in all manipulatins f the crd. The crd was severed frm the placenta sn after birth, placed in a sterile cntainer filled with HANK'S slutin, and held at 4 C until prcessing. Strage time averaged abut 30 minutes, and crds were discarded if held mre than 2 h. The crd was inspected, and all areas with clamp marks were cut ff. The umbilical vein was cannulated with a blunt 8 Fr feeding tube, 3 cm 211 Harran Üniversitesi Tıp Fakültesi Dergisi (Jurnal f Harran University Medical Faculty) Cilt 11. Sayı 3, 2014
lng, and the feeding catheter was fixed with an umbilical crd tie. The vein was perfused with 50 ml f PBS t wash ut the bld and clean the lumen. The ther end f the umbilical vein was then cannulated with a blunt, hubless, 8 Fr feeding tube 3 cm lng and fixed. 10 ml f 0.2% cllagenase (gibc 17101-015 PAA, N: K.21-240) was infused int the umbilical vein lumen, the bth ends/edges f plyethylene tubing was clamped and incubated in a petri dish cntaining HNAK'S slutin at 37 C fr 15 minutes. After incubatin, the cllagenase slutin cntaining the endthelial cells was flushed frm the crd by perfusin with 30 ml f PBS. The effluent was cllected in a sterile 50 ml cnical centrifuge tube (2070, Falcn Plastics, Oxnard, Calif.) cntaining 10 ml f Medium 199 (TC 199) ' with 20% fetal calf serum (FCS). The cells were centrifuged at 1200 rpm fr 10 min and supernatant were remved with a sterile pipet. Endthelial cell medium was added int the centrifuge tube and after this the suspensin was vrtexed. After having a hmgenus mixture the medium was recentrifuged at a 1200 rpm fr 10 min. This prcedure was dne twice. After these prcedures 2 ml endthelial cell medium was added n the cells at bttm f the tube and cell were cultured in TC 199 cntaining 20% FCS, penicillin (200 U/ml), streptmycin (200,xg/ml), and L- glutamine n petri dishes. Petri dishes were incubated at 37 C under 5% C0 2. 24 hurs after incubatin endthelial cells were washed after with PBS and endthelial cell medium was added. The cells were fed with cell medium every 48 hurs. On the fifth day endthelial cells were washed with PBS and 3 ml tripsin-edta was added int each flask and incubated at 37 C fr 3-4 minutes. After it was shwn with invert micrscpe that the cells leaved the flask they were cllected int a centrifuge tube ECM (PAA Endtel Cell Medium U15-002/Millipre supplement 02-102) was added and suspensin was centrifuged at a 1200 rpm fr 5 min. The cell suspensin at the bttm f the tube were taken and used fr analyzes. By this methd ten tubes were prepared. Every 2 tubes were prepared frm ne cell culture medium. 1. 10ng/ml TNF-α +0,5ml cell suspensin 2. 10 M AMD and 10ng/ml TNF-α +0,5ml cell suspensin 3. 10ng/ml TNF-α +0,5ml cell suspensin 4. 10 M AMD and 10ng/ml TNF-α +0,5ml cell suspensin 5. 10ng/ml TNF-α +0,5ml cell suspensin 6. 10 M AMD and 10ng/ml TNF-α +0,5ml cell suspensin 7. 10ng/ml TNF-α +0,5ml cell suspensin 8. 10 M AMD and 10ng/ml TNF-α +0,5ml cell suspensin 9. 10ng/ml TNF-α +0,5ml cell suspensin (24 hurs) 10. 10 M AMD and 10ng/ml TNF-α +0,5ml cell suspensin (24 hurs) T see the effect f AMD and TNF-α n endthelial cells, endthelial cells were incubated with AMD+TNF-α and with nly TNF-α fr 2 hurs. In ne analyze incubatin perid/duratin was 24 hurs. Dsage f AMD was 10 M and TNF-α was 10 ng/ml. After the incubatin f endthelial cells with TNF-α and AMD, MP (CD31, CD51/61, CD62 and CD146) levels were analyzed by flwcytmetric studies. After btaining five endthelial cell mediums 0.5 ml endthelial cells were taken frm each mediums and by this way/methd each medium was divided int tw equal amunts. It was fllwed by incubating the endthelial cells with nly AMD r with AMD+TNF-α. Harran Üniversitesi Tıp Fakültesi Dergisi (Jurnal f Harran University Medical Faculty) Cilt 11. Sayı 3, 2014 212
Statistical analysis All statistical analyses were perfrmed using SPSS 16.0 fr Windws (SPSS, Chicag, IL, USA). Independent sample T-test and Mann- Whitney U tests were respectively used in nrmally and nn-nrmally distributed cntinuus variables between grups. A tw-sided p value < 0.05 was cnsidered t be statistically significant. Results Five endthelial cell medium was prepared t analyze the MP levels after incubatin f the endthelial cells with TNF-α and TNF-α+AMD fr tw hurs. MP levels were measured by using anti CD62, anti CD31, anti CD51/61 and anti CD146 antibdies with flwcytmetric methd. There were difference in MP levels after incubatin with 10ng/ml TNF-α and 10ng/ml TNF-α+10 M AMD. While MP levels were high in sme measurements after incubatin with TNFα MP levels were als high in sme measurements after incubatin with TNF-α+AMD. It was seen that Annexin-V psitive MP levels related with apptsis were higher with 24 hurs incubatin than incubatin with 2 hurs. There were n difference in MP levels between the grups after incubatin with TNF-α and TNF-α+AMD fr tw hurs. Results were shwn in graphics. Discussin T ur knwledge this is the first in the literature. It was nt shwn that AMD has a prtective rle against TNF-α stimulated MP increase. Althugh cytprtective effect f AMD is knwn (8-11) it was nt shwn against tissue/cell injury in artificial GVHD mdel. Endthelial cell injury develped after preparatin f the patient t allgeneic SCT with raditherapy and/r chemtherapy suggested t play an essential rle in pathgenesis f acute GVHD (2-4). In a study vascular endthelial cell activatin and tissue/cell damage was evaluated and seen that sluble vascular adhesin mlecule levels increased this suggested that endthelial cell injury r endthelial cell activity may play a rle in develpment f GVHD (13). TNF-α and several ther cytkines plays an imprtant rle in the develpment f GVHD after allgeneic SCT (1, 5, 14). And TNF-α is ne f the majr mediatrs makes endthelial cells MP release. It is seen that endthelial MPs als increase in sme autimmune and systemic diseases (6, 15-18). AMD plays an anti-inflammatry rle by inhibiting the verprductin f inhibiting the TNF-α and IL-1β r by inflammatin pathway via TNF-α cytkine cascade, this mechanism may have prtective effect in GVHD (19-21). AMD reduces the tissue damage with the same mechanism in animal mdels f sepsis, arthritis and ischemic bwel (22-26). Dsage may be imprtant in prtective effect f AMD; in a study it was shwn that AMD has a mre -3-5 prtective effect with 10 M and10 M dsage than -8-10 10 M and 10 M dsage (27-29). In ur study we used the dsage f 10 M AMD and reprted that there were n difference between EMP levels in bth AMD and nn-amd grups. In ur study it was detected that MP levels were high in sme measurements after incubatin with TNF-α and it was als high in sme measurements after incubatin with AMD+TNF-α. Cytprtective effect f AMD may als be related with duratin f incubatin; in sme studies it was reprted that AMD has mre cytprtective effect with lng incubatin perid (9, 13, 30). In ur study we incubated the cells fr tw hurs and have seen that there were nt a standard between MP levels in AMD administrated grup and withut AMD administratin grup. Only 213 Harran Üniversitesi Tıp Fakültesi Dergisi (Jurnal f Harran University Medical Faculty) Cilt 11. Sayı 3, 2014
in ne analyze endthelial cells were incubated with AMD fr 24 hurs and seen that Annexin-V psitive MPs were high in CD62, CD146 and CD51/61 grups after incubatin with AMD+TNF-α than incubatin with nly TNF-α. This may suggest that AMD des nt have a prtective effect against apptsis. Cnclusin, as t ur knwledge; there is n study revealed abut the prtective effect f AMD n MP release in allgeneic SCT. It is necessary t make an extensive study cnsist f multiple analysis t detect whether there is a prtective effect f AMD n develpment f acute GVHD. Acknwledgements This study was funded by Turkish Sciety f Hematlgy. Disclsure f interests All the authrs declare that they have n cnflict f interest Figure 1: CD62 psitive MP levels p>0,05, Q1 shws the particles psitive nly fr CD62, Q2 shws the particles psitive bth fr Annexin-V and CD62 and Q4 shws the particles psitive nly fr Annexin-V. Figure 2: CD 146 psitive MP levels p>0,05, Q1 shws the particles psitive nly fr CD146, Q2 shws the particles psitive bth fr Annexin-V and CD146 and Q4 shws the particles psitive nly fr Annexin-V. Harran Üniversitesi Tıp Fakültesi Dergisi (Jurnal f Harran University Medical Faculty) Cilt 11. Sayı 3, 2014 214
Figure 3: CD31 psitive MP levels p>0,05, Q1 shws the particles psitive nly fr CD31, Q2 shws the particles psitive bth fr Annexin-V and CD31 and Q4 shws the particles psitive nly fr Annexin-V. Figure 4: CD51/61 psitive MP levels p>0,05, Q1 shws the particles psitive nly fr CD51/61, Q2 shws the particles psitive bth fr Annexin- V and CD51/61 and Q4 shws the particles psitive nly fr Annexin-V. 215 Harran Üniversitesi Tıp Fakültesi Dergisi (Jurnal f Harran University Medical Faculty) Cilt 11. Sayı 3, 2014
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