C1抑制剂在血管通透性调节和抑制白细胞内皮迁移的作用课件

1、C1 Inhibitor: Regulation of Vascular Permeability and Inhibition of Leukocyte Migration Across the Vascular EndotheliumAlvin E. Davis III, M.D.Senior Investigator, CBR Institute for Biomedical ResearchProfessor of Pediatrics, Harvard Medical SchoolBoston, Massachusetts, USA2020年10月2日1C1 Inhibitor: R

2、egulation of VA serpin (serine proteinase inhibitor).Serpins inactivate proteases by forming a stable complex with the protease.Protease recognizes a substrate-like region of the inhibitor that is referred to as the reactive center loop.C1 INHIBITOR FUNCTION2020年10月2日2A serpin (serine proteinase inP

3、roteaseSerpinReactive centerThe protease recognizesthe reactive center of theinhibitor. Cleavage of thereactive center loop isfollowed by:covalent bond formaton between the protease active site serine and the reactive center amino acid of the serpin.rearrangement of the serpin with insertion of the

4、reactive center loop (in yellow) into sheet A (in red), which results in movement of the protease from one pole of the molecule to the other. This results in structural distortion of the protease. 2020年10月2日3ProteaseSerpinReactive centerTProteases Inactivated byC1 InhibitorComplement SystemClassical

5、 pathway: C1r, C1sLectin pathway: MASP 1 & 2Intrinsic Coagulation/Contact SystemsFactor XIa, Plasma kallikrein, factor XIIa2020年10月2日4Proteases Inactivated byC1 InC4b2a3bC3bBb3bC5C5aC5b + C6, C7, C8, C9C5b-9Membrane Attack ComplexCLASSICAL PATHWAYAntigen-antibodycomplexmicrobeMBL + MASP 1,2LECTIN PA

6、THWAYALTERNATIVE PATHWAYmicrobe+ C3b, B, D, PC3b,Bb,PC1q, C1r, C1sC4C2C4b2aC3C3aC3b + C4b2aC3b + C3bBbPC1INHC1INHFactor IFactor HC1INH2020年10月2日5C4b2a3bC5C5aC5b + C6, C7, C8, Endothelial cell prolylcarboxypeptidaseFactor XIINegativelychargedsurfaceXIIaC1INHPrekallikreinHigh molecularweight kininogen

7、 KallikreinHigh molecularweight kininogenBradykininC1INHC1INHContact System Activation2020年10月2日6 Endothelial cell prolylcC1 Inhibitor is a MultifunctionalAnti-Inflammatory Protein1. Regulation of vascular permeabilityInhibition of contact system activation.C1 inhibitor knockout mouse.2. Modulation

8、of leukocyte transmigration.Inhibition of complement activation.Interaction with E- and P-selectins onendothelial cells.3. Protection from endotoxin shock.Inhibition of both complement and contactSystems.Interaction with gram negative endotoxinlipopolysaccharide (LPS).2020年10月2日7C1 Inhibitor is a Mu

9、ltifunctioRecurrent localized edema of skin or mucosa (larynx, gastrointestinal tract)Dominant inheritance: heterozygous for deficiency of C1 inhibitorPathophysiology:Decreased C1INH functionSpontaneous activation of the complement system (C1r, C1s) decreased C4, C2Spontaneous activation of the cont

10、act system (plasma kallikrein, factor XIIa) cleaved high molecular weight kininogenMediator?Hereditary Angioedema2020年10月2日8Recurrent localized edema of sPathophysiology of Hereditary AngioedemaBecause there is clearcut evidence for both complement and contact system activation, the question arises

11、as to which is responsible for generation of the mediator of angioedema.Until recently, some data suggested that the mediator was a product of complement system activation while other data suggested that it was derived from contact system activation.2020年10月2日9Pathophysiology of HereditaryDefine med

12、iator(s) of angioedemaTest new therapies and define their mechanism of actionPathophysiology of vascular permeabilityAnalysis of variability of symptoms in HAEC1 Inhibitor Knockout Mouse. Why?2020年10月2日10Define mediator(s) of angioedeC1 INHIBITOR KNOCKOUT MICEC1INH protein levels:-/- mice: C1INH not

13、 detectable.+/- mice: 50% of normal.Decreased C4 levels.Normal in size, appearance and growth.Reproduction, fetal growth and development are normal.Two +/- and six -/- mice intestinal wall edema with obstruction at 10-11 weeks.IV Evans blue dye in +/- & -/- mice revealed enhanced vascular permeabili

14、ty.2020年10月2日11C1 INHIBITOR KNOCKOUT MICEC1INEvans Blue Dye InjectionWild TypeC1INH +/-2020年10月2日12Evans Blue Dye InjectionWild TC1INH genotype +/+ +/+ -/- -/- Evans blue dye - + + + C1INH treatment - - - + Does intravenous C1INH reverse theincreased vascular permeability in the C1INH knockout mice?

15、2020年10月2日13C1INH genotype +/+ +/+C1INH genotype+/+ +/- -/- +/- -/- C1INH treatment 0 0 0 + + Mean .10 .15 .16 .11 .10 S.E. .005 .008 .012 .003 .006 Vascular Permeability - Footpads2020年10月2日14C1INH genotype+/+ +/- -/- C1INH genotype +/+ -/- -/-Bk2R genotype +/+ +/+ -/-Evans blue dye + + + Is Angioe

16、dema Mediated by Bradykinin?Bradykinin induces increased vascular permeability via the Bk type 2 receptor (Bk2R).C1INH -/- mice were mated with Bk2R -/- mice.Hypothesis: C1INH -/-, Bk2R -/- mice will be resistant to the development of increased vascular permeability.2020年10月2日15C1INH genotype +/+C1I

17、NH genotype+/+ +/- -/- -/- Bk2R genotype+/+ +/+ +/+ -/- Treatment 0 0 0 0 Mean .10 .15 .16 .10 S.E. .005 .008 .012 .003 Vascular Permeability - Footpads2020年10月2日16C1INH genotype+/+ +/- -/- C1INH genotype +/+ -/- -/- -/- -/-Treatment 0 0 Captopril Bk1RA Hoe140 Mean .2577 .4087 .8860 .4477 .0252Stand

18、ard error .0284 .0806 .0773 .0667 .0252Intestinal Vascular Permeability2020年10月2日17C1INH genotype +/+ Is Angioedema Mediated byBradykinin?In addition, the increased vascular permeability in C1INH -/- mice was reversed by treatment with the following:A recombinant variant Kunitz domain inhibitor, DX8

19、8 (Dyax), that is highly specific for plasma kallikrein.A recombinant C1INH Ala443 Val, that inhibits the contact system but not the complement system.A Bk2R antagonist, Hoe140 (Icatibant).These data all support the hypothesis that the mediator of vascular permeability in C1INH deficiency is bradyki

20、nin.2020年10月2日18Is Angioedema Mediated byIn adSelectins: adhesion molecules responsible for leukocyte rolling on the endothelium.E-selectin: endothelial cells; expression induced by TNF-, IL-1, LPS.P-selectin: platelets & endothelial cells; mobilized to cell surface from intracellular granules by hi

21、stamine, thrombin, etc.Selectins bind fucosylated mucin-like glycoproteins.Sialyl Lewisx is a fucose-containing tetrasaccharide expressed by a number of proteins; binds all three selectins.C1 Inhibitor and Selectins2020年10月2日19Selectins: adhesion moleculesC1INH is extremely heavily glycosylated (35%

22、 of its total mass).27% of the N-glycans are fucosylated.Human liver, the primary site of synthesis of C1INH, expresses 1,3-fucosyltransferase-VI which catalyzes attachment of the sialyl Lewisx tetrasaccharide (NeuNAc2-3Gal1-4(Fuc1-3)GlcNAc).Hypothesis: C1INH expresses the Lewisx moiety.C1 Inhibitor

23、 and Selectins2020年10月2日20C1INH is extremely heavily glyC1 Inhibitor Expresses Sialyl LewisxHECA452: monoclonal anti-sLewx and sLewaLanes 1-4: C1INH 8, 4, 2, 1 gLane 5: U937 lysateLane 6: LEC11 lysateLane 7: CHO-K1 lysateLane 8: BSACSLEX-1: monoclonal anti-sLewxLane 1: BSALanes 2-3: C1INH 5 & 2.5 gL

24、ane 4: U937 lysateDeglycosylation experiments: sLewx isexpressed on N-linked carbohydrate.2020年10月2日21C1 Inhibitor Expresses Sialyl Recombinant C1INH Expressed in LEC11 Cells Expresses the Sialyl Lewisx EpitopeLEC 11 cells express a (1,3)-fucosyltransferaseCHOK1 cells do not2020年10月2日22Recombinant C

25、1INH Expressed inEndothelial Cell E and P SelectinCo-precipitate with C1 InhibitorHUVEC treated with TNF- & H2O2, then incubated with C1 inhibitor.Cells lysed & immunoprecipitated with rabbit anti-C1 inhibitor antiserum.SDS, Western blot probed with antiserum to E or P selectin.E SelectinP selectinB

26、inding of C1INH to CHO cells thatexpress either E or P selectin alsowas demonstrated by FACS.2020年10月2日23Endothelial Cell E and P SelecC1INH Suppresses U937 Cell Adhesion to Endothelial CellsHUVEC monolayers treated with TNF- & H2O2, then incubated with or withoutC1INH (31.5 500 g/ml). BCECF-AM labe

27、lled U937 cells added & incubated for60 min at 37oC, then washed & bound cells quantitated by analysis of fluorescenceintensity.2020年10月2日24C1INH Suppresses U937 Cell AdhHUVEC (3x104) were seeded onto an insert and grown in 24-well plates for 4 days, then stimulated with TNF alpha (50 ng/ml, 18 h).

28、The integrity of the monolayer was tested with FITC-BSA. U937 cells were labeled with BCECF-AM, 5x105 cells (100l) in the absence or presence of either native or reactive center cleaved C1INH at the indicated concentrations were incubated with the HUVEC at 37oC for 45 min with 600l medium in the low

29、er chamber. The fluorescence intensity of 100 l medium from the lower chamber of each well was measured.C1INH Inhibits U937 Cell Transmigration Across the Endothelium2020年10月2日25HUVEC (3x104) were seeded ontoC1INH Blocks Carcinoembryonic AntigenBinding to E-selectin2020年10月2日26C1INH Blocks Carcinoem

30、bryonicC1INH Blocks HL-60 Cells from Rolling on E- and P-Selectin Under Flow Conditions In VitroImmobilizedE-selectinCHO-PCells2020年10月2日27C1INH Blocks HL-60 Cells fromC1INH Inhibits TNF- Induced Leukocyte Rolling In VivoC1INHiC1INHdN-C1INHPBS2020年10月2日28C1INH Inhibits TNF- Induced C1INH Blocks Leuk

31、ocyte Infiltration inThioglycollate Induced Peritonitis2020年10月2日29C1INH Blocks Leukocyte InfiltC1INH expresses the Lewisx moiety on one or more of its N-linked sugars.C1INH binds to both E- and P-selectins.Interaction with selectins does not require protease inhibitory function.This interaction inhibits adherence of U937 cells to endothelial cells, preve