Uticaj trombina na regulaciju proliferacije glatkih mišićnih ćelija aorte pacova
Effects of trombin in the regulation of rat's aortic smooth muscle cells proliferation
Докторанд
Smiljanić, KatarinaМентор
Isenović, EsmaЧланови комисије
Isenović, EsmaĐorđević, Jelena
Đorđević, Jelena
Putniković, Biljana
Метаподаци
Приказ свих података о дисертацијиСажетак
Kardiovaskularne bolesti predstavljaju najveći uzrok smrtnosti ljudske populacije, a njihova
glavna i osnovna patološka komponenta je ateroskleroza. Proliferacija ili deoba glatkih
mišićnih ćelija krvnog suda (VSMC) ključni je događaj u nastanku raznih vaskularnih
oboljenja, uključujući aterosklerozu i hipertenziju. U procesu diferencijacije i abnormalne
deobe VSMC povezanih sa hipertenzijom i aterosklerozom uključen je i trombin.
Stimulisanje VSMC trombinom dovodi do aktivacije ekstracelularnim signalima regulisanih
kinaza 1 i 2 (ERK1/2), preko transaktivacije receptora za epidermalni faktor rasta (EGFR). U
ranijim studijama Isenović i saradnici potvrdili su na osnovu inhibicije ERK1/2 od strane
PD9805 inhibitora, učešće ERK1/2 u regulaciji proliferacije VSMC izazvanoj trombinom. U
nastavku, faktor rasta sličan epidermalnom faktoru rasta koji vezuje heparin (HB-EGF),
protein kinaza C delta (PKCδ) i matriksne metaloproteinaze (MMP), nađene su u VSMC i
pokazano je da je i njihova aktivn...ost regulisana trombinom. ADAM (engl. “A Disintegrin
And Metalloproteinase”) transmembranske su metaloproteinaze koje pripadaju
adamalizinskoj grupi i razlikuju se od matriksnih metaloproteinaza po tome što imaju
vanćelijski dizintegrinski i citoplazmatski domen. C terminalni domen može da stupa u
interakciju sa unutarćelijskim proteinima. Uvidom u literaturu, do sada ne postoji istraživanje
koje ukazuje na prisustvo ADAM metaloproteinazne aktivnosti vezane za proliferaciju VSMC
stimulisane trombinom. Predmet ove doktorske teze jeste proučavanje uloge EGFR, ERK1/2, HB-EGF, PKCδ,
ukupnih metaloproteinaza, kao i specifičnih MMP-2 i ADAM 12, u posredovanju
proliferativnog efekta trombina na VSMC pacova. Inkubacija VSMC pacova sa trombinom (1
U/ml) u periodu od 5 minuta rezultirala je u značajnom povećanju: fosforilacije ERK1/2 od
8.7 ± 0.9 puta (p<0.001), fosforilacije EGFR od 8.5 ± 1.3 puta (p<0.001) i sinteze DNK od
3.6 ± 0.4 puta (p<0.001). Prethodni pojedinačni tretmani ovih ćelija u trajanju od 30 minuta sa
10 μM PD169540 (PD), ireverzibilnim inhibitorom EGFR, i 20 μM antitelom protiv HB-EGF
značajno su smanjili trombinom stimulisanu fosforilaciju EGFR za 81 % i 72 % i ERK1/2...
ardiovascular disease is the largest single cause of mortality and its major underlying
pathology is atherosclerosis. The proliferation of vascular smooth muscle cells (VSMC) is a
key event in the pathogenesis of various vascular diseases, including atherosclerosis and
hypertension. Thrombin is involved in the differentiation and abnormal proliferation of
VSMC associated with atherosclerosis and hypertension.
Thrombin stimulation results in extracellular signal-regulated kinase (ERK 1/2) activation
through transactivation of the epidermal growth factor receptor (EGFR). Based on the studies
in which PD98059 used to inhibit ERK1/2, we have shown previously that ERK1/2 was
involved in the regulation by thrombin of VSMC’s proliferation. In addition, heparin-binding
EGF-like growth factor (HB-EGF) and matrix metalloproteinases (MMPs) have also been
detected in VSMC and shown to be regulated by thrombin. ADAMs (A Disintegrin And
Metalloproteinase) are transmembrane metalloproteinases, belong...ing to adamalysin group,
that are distinct from matrix metalloproteinases (MMPs) in that, they have an extracellular
disintegrin domain and cytoplasmic domain that can associate with intracellular proteins. To
the present knowledge there is no study that indicates the activation of an ADAM member in
thrombin-induced VSMC proliferation.
In this dissertation, the role of EGFR, ERK1/2, HB-EGF, general metalloproteinases, MMP-2
and ADAM 12, as well as PKCδ in mediating the mitogenic action of Thrombin in rat VSMC
was investigated. Incubation of rat VSMC with Thrombin (1 U/ml) for 5 minutes resulted in significant increase of ERK1/2 phosphorylation by 8.7 ± 0.9 fold (p<0.001), EGFR
phosphorylation by 8.5 ± 1.3 fold (p<0.001) and DNA synthesis by 3.6 ± 0.4 fold (p<0.001).
Separate pretreatments for 30 minutes with EGFR tyrosine kinase irreversible inhibitor, 10
μM PD169540 (PD), and 20 μM anti-HB-EGF antibody, significantly reduced thrombinstimulated
EGFR and ERK1/2 phosphorylation by 81 %, 72 % and by 48 % and 61%,
respectively. Furthermore, same pretreatments with PD and anti-HB-EGF antibody reduced
Thrombin-induced VSMC’s proliferation by 44% and 45%, respectively. In addition,
pretreatments for 30 minutes with 10 μM KB-R7785 (KB), a specific ADAM 12 inhibitor or
10 μM specific MMP2 inhibitor significantly reduced thrombin-stimulated EGFR..