Complement Membrane Attack Complex Endothelial Cell Stimulating Effect And Its Activation Mechanism

The sublytic effects of membrane attack complexes ofcomplement on Human Umbilical Vein endothelial cellsand its possible mechanism of signal transdutionAbstractThe best established function of C5b-9 is the ability to lyse cells after assembly in the plasma membrane. In recent years,increasing evidence suggests that MAC also stimulate a variety of metabolic activities in cells in vitro. Several signal molecules have been indicated to be involved in this process, but how does MAC activate signal messengers inside the cell is still unclear. Because no molecule have been demonstrated interacting directly with MAC except two complement regulatory protein CD59 and C8bp, both of which are GPI-anchored protein. As a well known hydrophobic protein complex, MAC can interact with lipid molecules in membrane intensively. Is there anything special for this complex on signaling?In recent years, particular attention has been paid to cell membrane domains with peculiar lipid/protein compositions. It is demonstrated that gangliosides, glycospbingolipids and GPI-anchored proteins clustered with together to form GSD domain. A particular type of domain,with morphologically character of flask-shaped invaginations in the plasma membrane and characteristically enriched in caveolin, terms caveolae. A number of different functions have been proposed for caveolae including signal transduction, potocytosis, calcium regulation and non-clatbrin-dependent endocytosis and transcytosis.Analysis of the mechanism of MAC signaling, we found that most of the known signal messengers related to MAC have been demonstrated located to caveolae. Combining with the clue that MAC is a hydrophobic proteinC9mplex Which can bind lipid molecules on membrane, We ask whether MACcan transduce stimulatory signals though illteracting with the caveo1ae andother membrane microdomains. To demonstrae this opinion, we establishsublytic MAC attack model on endothelial cells and detect the stimulatoryeffects of MAC on endothelia1 cells. Low-density caveo1ae-rich fraction waspurified and the association of MAC with it was detected. The role of caveolaein mediate signailing after MAC dePosition was further evaluated wtthcholesterol binding reagent. Through these research,we detect the possiblephysical association and hationa relation~ beteewen MAC and caveolae.The main colltent and results of our research including:Firstly, the fifth comPonent of human complement C5 was activated bynon-enzymical chemical oxidation with chloramine T The products is C5b-likeWhich are caPable of binding C6 and form the nuc1eus fOr the cytotoxiccomPlex C5b-9. With assembly of sublytic the MAC through this C5b-like andother purified comPonent of terminal comPlement, we established the sublytictwk system on HU'VECs.Secondly, we examined the functional caPacity of sublytic concendations ofMAC to induce the secretion of two differeat kinds of molecules: one is vWFwhich have been synthesized and stored in the W-P body and can be secreteddirectly after stimulation; the other one is interlechn(IL)-8, a neutrophilchemotactic cytokine which must be synthesize starting with mRNAtranscription. Analysis of conditioned medium from MAC-bearing HUVECsrevealed that the IL-8 and vWF rise definitely but in different pattem.Thirdly, cytosolic-free calcium changes in cultured HUVECs ancckedwith sublytic MAC were measured using laser scanning confocal microscope(LSCM) processing of fura-4-loaded cells. We find that [Ca2+1i in HUVECsnot only raise after MAC attack but also have a heterogeneous pallern, rangingfrom small transient increases in cytosolic ca1ciurn to raPid higher [Ca2+]i. IV.hoges.FoUrthly, extreds of endothe1ial ce1ls line ECV304 in l% Trton X-l00 at4'C resulted in a detergent-insoluble pellet that contained most of caveolin andGPI-anchored proteins. Lipid-linked signal transduction molecules such as Srcfamily non-receptor tyrosine kinases and heterotrimeric G protein have also...