| Sažetak | Poznato je da su koncentracije kolesterola u lipoproteinu visoke gustoće (HDL) inverzno povezane s kardiovaskularnim rizikom. Farmakološki postupci s ciljem povećanja HDL-a, kojima bi se rizik smanjio, do sada nisu bili uspješni (inhibicija kolesterol ester transfer proteina; CETP). Nadalje, studije o HDL-u na pacijentima s dijabetesom, koronarnom arterijskom bolesti (CAD) te drugim upalnim bolestima povezanim s funkcionalnošću HDL-a dale su jasan dokaz da:
a) koncentracija HDL-a nije pouzdan prediktor kardiovaskularnog rizika
b) brojnost i veličina čestica HDL-a te
c) funkcionalnost HDL-a snažni su i nezavisni prediktori kardiovaskularnog rizika i smrtnosti.
Prijašnji i sadašnji eksperimenti s endotelnom lipazom (EL) jasno pokazuju da enzim svojom fosfolipaznom aktivnošću utječe na fosfolipide HDL-a. Primarno, na fosfatidillkolin (PC) čime dolazi do promjena u strukturi i funkcionalnosti HDL-a. Taj HDL nazvan je EL-HDL. EL- HDL je veličinom manji od kontrolnog HDL-a. Sadrži manje fosfatidilkolina ali više lizofosfatidilkolina (LPC). EL-HDL pokazuje slabiji afinitet vezanja za receptore (SR-BI; scavenger receptor class B type I ), smanjenu mogućnost posredovanja pri transportu kolesterola iz makrofaga (efflux), smanjenu količinu i aktivnost enzima paraoksonaza-1 (PON-1) te promijenjenu aktivnost endotelne dušik oksid sintetaze (e-NOS) koja utječe na vazodilataciju posredovanu dušikovim monoksidom (NO).
Učinak endotelne lipaze na HDL remodeliranje proučavan je u in vitro modelu stanične kulture HepG2 stanica inficiranih adenovirusima za endotelnu lipazu (EL), kolesterol ester transfer protein (CETP) te lecitin-kolesterol-acil-transferazu (LCAT), uz pripadajuće kontrole, kako bismo dobili ekspresiju spomenutih enzima. Nakon inkubacije seruma zdravih dobrovoljaca s HepG2 stanicama uz odsutsvo/prisutstvo inhibitora CETP-a (torcetrapib) i LCAT-a (DTNB-a (5,5-ditiobis-2-nitrobenzoična kiselina)), HDL je izoliran iz svakog uzorka seruma. Uzorci HDL-a su promatrani s obzirom na veličinu i sastav (sveukupni kolesterol, trigliceridi, fosfolipidi, sadržaj PON1) te funkcionalnost (PON-1 aktivnost, eNOS inducibilnu aktivnost, kapacitet prijenosa kolesterola iz makrofaga). Metode korištene u eksperimentu su bazirane na radu sa staničnim kulturama, gel elektroforezi, Western blotu, dostupnim kitovima i metodama bojenja lipida i proteina.
Cilj eksperimenta je bio ispitati učinak CETP inhibitora (torcetrapiba) i LCAT inhibitora (DTNB-a) na veličinu, sastav i funkcionalnost EL-HDL-a.
Rezultati pokazuju da je EL-HDL dobar supstrat za oba enzima – CETP, LCAT. Modulacija enzimima i inhibitori utječu na lipidni sastav EL-HDL-a, dok je veličina ostala ista, ali i dalje manja od kontrolnog HDL-a. Funkcionalnost EL-HDL-a je uvijek smanjena u usporedbi s kontrolnim HDL-om. Zaključno, farmakološkom modulacijom in vitro nije došlo do poboljšanja, spoznato narušenih kardioprotektivnih mehanizama EL-HDL-a u usporedbi s kontrolnim HDL-om. |
| Sažetak (hrvatski) | It is well established that HDL concentrations in plasma estimated by measuring cholesterol in HDL (HDL-cholesterol in clinical laboratory results, HDL-C = good cholesterol) is inversely related to cardiovascular risk. However, the failure of recent pharmacological attempts (inhibition of cholesterol ester transfer protein; CETP) to decrease cardiovascular events by increasing HDL-C levels, together with additional studies showing that HDL from patients with various pathologies (diabetes, CAD, inflammation) is dysfunctional, provided clear evidence that:
a) HDL-C levels are not reliable predictors of cardiovascular risk
b) HDL particle number and size as well as
c) HDL- functionality are strong and independent predictors of cardiovascular events and mortality.
Previous and current work clearly demonstrated that EL (endothelial lipase) by its phospholipase activity very potently cleaves HDL phospholipids, primarily phosphatidylcholine (PC), thereby generating structurally and functionally altered HDL, referred to as EL-HDL. EL-HDL is smaller in size, depleted in PC but enriched in lysophosphatidylcholine (LPC). It exhibits diminished binding to SR-BI (scavenger receptor class B type I), a reduced capacity to mediate cholesterol efflux from macrophages, decreased paraoxonase 1 (PON1)-content and altered capacity to induce endothelial NO (nitric oxide) production and to promote vasorelaxation.
Generation of modified HDL was achieved by incubation of healthy human pool serum with HepG2 cells transduced with adenoviruses encoding EL, CETP or LCAT. After isolation of modified HDL from serum (without and with inhibitors, after overexpression) following steps and methods were performed:
- Structural/compositional analysis of HDL (native gradient electrophoresis followed by Sudan staining and Coomassie staining, lipid composition analysis using kit measurements, PON1 Western Blots).
- Functionality assessment of HDL (HDL-associated PON1-activity, capacity of HDL to induce endothelial e-NOS activity and HDL cholesterol efflux capacity).
The aim was to examine the impact of CETP inhibitor – torcetrapib and LCAT inhibitor -DTNB, on EL-induced alterations in structural and functional properties of HDL.
Results showed that EL-modified HDL is a good substrate for CETP and LCAT. In vitro, both Torcetrapib and DTNB affected EL-HDL composition with no effect on the EL-HDL size. Furthermore, functionality of EL-HDL was decreased when compared to control HDL. In vitro pharmacological inhibition of CETP or LCAT activity failed to affect the impact of these enzymes on the functionality of EL-HDL. |
