Mikrovezikli kot pokazatelji vnetnega in koagulantnega odziva človeških endotelijskih celic koronarnih arterij na serumski amiliod A : diplomska naloga

Jazbar, Janja (author), Božič, Borut (mentor), Sodin-Šemrl, Snežna (comentor)

Ateroskleroza je vnetna bolezen arterij, ki predstavlja vodilni vzrok za razvoj številnih srčno-žilnih bolezni. Akutno-fazni protein serumski amiloid A (SAA) je napovedni dejavnik akutnih srčno-žilnih dogodkov, povečane vrednosti SAA pa so značilne za bolnike z aterosklerozo in so povezane z disfunkcijo endotelija. Endotelijski mikrovezikli so pokazatelji endotelijske disfunkcije pri srčno-žilnih boleznih in se kopičijo v aterosklerotičnih lehah. Naš namen je bil raziskati, kako SAA vpliva na izločanje mikroveziklov v endotelijskih celicah. Ker je za mikrovezikle znano, da sodelujejo pri vnetju in koagulaciji, nas je zanimalo, ali so endotelijski mikrovezikli pokazatelji vnetne in koagulantne aktivnosti endotelijskih celic po stimulaciji s SAA. Koncentracija protiteles proti SAA v serumu je znižana pri arterijski trombozi in nekaterih avtoimunskih boleznih s pospešeno aterosklerozo, zato smo raziskali tudi vpliv protiteles proti SAA na izločanje mikroveziklov. Človeške endotelijske celice koronarnih arterij smo stimulirali s človeškim rekombinantnim SAA in po različnih časih stimulacije izolirali mikrovezikle z metodo diferencialnega centrifugiranja. S pretočno citometrijo smo določili število mikroveziklov, vezavo aneksina A5 in prisotnost CD31, CD62E in tkivnega faktorja na mikroveziklih. Vzporedno smo določili vnetno aktivnost celic z določanjem interlevkina-6 in interlevkina-8 ter koagulantno aktivnost celic z določanjem izražanja in aktivnosti tkivnega faktorja. Rezultati so pokazali, da stimulacija endotelijskih celic s SAA povzroči izločanje mikroveziklov. Število mikroveziklov je naraščalo s časom stimulacije celic in se je povečevalo vzporedno s količino interlevkina-6 in interlevkina-8. Število mikroveziklov, pozitivnih na aneksin A5, CD31 in CD62E, ki so zvišani v različnih srčno-žilnih boleznih, kot je na primer koronarna arterijska bolezen, je naraščalo s časom stimulacije endotelijskih celic s SAA. SAA je sprožil tudi izražanje tkivnega faktorja na mikrovezklih. Izražanje tkivnega faktorja na mikroveziklih je bilo največje po 4 urah stimulacije in je sledilo izražanju in aktivnosti tkivnega faktorja na endotelijskih celicah. Pokazali smo, da SAA sproži mikrovezikulacijo endotelijskih celic, ki sledi vnetni in koagulantni aktivnosti celic. Protitelesa proti SAA so močno zmanjšala izločanje mikroveziklov tako po 4 kot po 24 urah stimulacije endotelijskih celic s SAA. Endotelijski mikrovezikli so tako lahko pokazatelji in/ali sodejavniki pri endotelijski disfunkciji, ki jo povzroči SAA.

• Language: Slovenian
• Keywords: ateroskleroza amiliod A tromboza mikrovezikli odziv celic
• Work type: Undergraduate thesis
• Typology: 2.11 - Undergraduate Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [J. Trček]
• Year: 2011
• Number of pages: 66 f.
• PID: 20.500.12556/RUL-71201
• UDC: 616.1
• COBISS.SI-ID: 3049073
• Publication date in RUL: 10.07.2015

Secondary language

• Language: English
• Title: Microvesicles as inflammatory and coagulant markers of human coronary artery endothelial cells following their stimulation with serum amyloid A

Abstract

Atherosclerosis is an inflammatory arterial disease, which is the leading cause of several cardiovascular diseases. Elevated serum amyloid A (SAA), a positive acute-phase reactant, predicts future acute cardiovascular events and is associated with increased progression of atherosclerosis and endothelial dysfunction. Endothelial microvesicles are biomarkers of endothelial dysfunction in cardiovascular diseases and accumulate in atherosclerotic lesions. The aim of our study was to investigate the influence of SAA on microvesicle release in endothelial cells. Because microvesicles are known to participate in inflammation and coagulation, we determined, whether endothelial microvesicles could reflect SAA-induced proinflammatory and procoagulant response in endothelial cells. Since serum levels of anti-SAA antibodies are significantly lower in arterial thrombosis and some autoimmune diseases with accelerated atherosclerosis than in healthy blood donors, we investigated the influence of anti-SAA antibodies on endothelial microvesicle release. Human coronary artery endothelial cells were stimulated with human recombinant SAA and microvesicles were isolated by differential centrifugation following different times of stimulation. Flow cytometry was used to measure endothelial microvesicle number, annexin A5 binding and CD31, CD62E and tissue factor exposure. The inflammatory response was measured using interleukin-6 and interleukin-8 protein levels and the procoagulant response was measured with tissue factor exposure and activity. We demonstrated that SAA stimulation causes microvesicle release from endothelial cells. A time-dependent rise in microvesicle number correlated with interleukin-6 end interleukin-8 levels. The number of annexin A5, CD31 and CD62E-positive microvesicles (elevated in different cardiovascular diseases, such as coronary artery disease) increased with the time of stimulation. SAA caused tissue factor expression on microvesicles to peak at 4 hours and this increase correlated with tissue factor expression and activity in endothelial cells. Altogether, we showed that SAA causes microvesiculation that reflects inflammatory and procoagulant responses in endothelial cells. Anti-SAA antibodies decreased SAA-induced microvesicle release following 4 hour and 24 hour stimulation. Endothelial microvesicles may serve as biomarkers and/or mediators of SAA-induced changes of endothelial function.