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Macrophage induced proteolysis zorina galis- emory u

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Macrophage induced proteolysis zorina galis- emory u

  1. 1. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. “Macrophage-induced proteolysis: how many MMPs and non-MMPs are involved?”? Zorina S. Galis, Ph.D.Zorina S. Galis, Ph.D. Division of Cardiology , Emory UniversityDivision of Cardiology , Emory University School of MedicineSchool of Medicine Department of Biomedical EngineeringDepartment of Biomedical Engineering Emory/Georgia Tech,Emory/Georgia Tech, Atlanta GAAtlanta GA March 16, 2002March 16, 2002 33rdrd Vulnerable Plaque SymposiumVulnerable Plaque Symposium March 16, 2002March 16, 2002 33rdrd Vulnerable Plaque SymposiumVulnerable Plaque Symposium
  2. 2. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. LipidLipid corecore ThrombusThrombus Natural history of human atherosclerosisNatural history of human atherosclerosisNatural history of human atherosclerosisNatural history of human atherosclerosis M. Davies, 1998M. Davies, 1998 Acute cardiovascular events representAcute cardiovascular events represent a late stage of arterial remodelinga late stage of arterial remodeling Acute cardiovascular events representAcute cardiovascular events represent a late stage of arterial remodelinga late stage of arterial remodeling adaptationadaptation sustainedsustained adaptationadaptation and repairand repair destructiondestruction Culprit = ruptureCulprit = ruptureCulprit = ruptureCulprit = rupture
  3. 3. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Selected MMPSelected MMP Selected substratesSelected substrates StromelysinStromelysin (SL / MMP-3)(SL / MMP-3) StromelysinStromelysin (SL / MMP-3)(SL / MMP-3) Proteoglycans, fibronectin, lamininProteoglycans, fibronectin, laminin pro-MMP-1, pro-MMP-9pro-MMP-1, pro-MMP-9 Proteoglycans, fibronectin, lamininProteoglycans, fibronectin, laminin pro-MMP-1, pro-MMP-9pro-MMP-1, pro-MMP-9 Gelatinases (GL)Gelatinases (GL) 72 kD GL, GL a (MMP-2)72 kD GL, GL a (MMP-2) 92 kD GL, GL b (MMP-9)92 kD GL, GL b (MMP-9) Gelatinases (GL)Gelatinases (GL) 72 kD GL, GL a (MMP-2)72 kD GL, GL a (MMP-2) 92 kD GL, GL b (MMP-9)92 kD GL, GL b (MMP-9) Collagen type IV / VCollagen type IV / V degraded collagen, elastindegraded collagen, elastin Collagen type IV / VCollagen type IV / V degraded collagen, elastindegraded collagen, elastin Interstitial collagenaseInterstitial collagenase (CL / MMP-1)(CL / MMP-1) Interstitial collagenaseInterstitial collagenase (CL / MMP-1)(CL / MMP-1) Fibrillar collagenFibrillar collagenFibrillar collagenFibrillar collagen membrane-type MMP-1membrane-type MMP-1 (MT-MMP)(MT-MMP) membrane-type MMP-1membrane-type MMP-1 (MT-MMP)(MT-MMP) pro-MMP-2, pro-MMP-13,pro-MMP-2, pro-MMP-13, collagen, fibronectin, laminincollagen, fibronectin, laminin pro-MMP-2, pro-MMP-13,pro-MMP-2, pro-MMP-13, collagen, fibronectin, laminincollagen, fibronectin, laminin The matrix metalloproteinase (MMP) family ofThe matrix metalloproteinase (MMP) family of enzymes can break-down matrix componentsenzymes can break-down matrix components The matrix metalloproteinase (MMP) family ofThe matrix metalloproteinase (MMP) family of enzymes can break-down matrix componentsenzymes can break-down matrix components MatrilysinMatrilysin (MMP-7)(MMP-7) MatrilysinMatrilysin (MMP-7)(MMP-7) fibronectin, collagen type IV,fibronectin, collagen type IV, laminin, elastinlaminin, elastin fibronectin, collagen type IV,fibronectin, collagen type IV, laminin, elastinlaminin, elastin Could MMPs beCould MMPs be responsible for theresponsible for the weakening ofweakening of atherosclerotic plaques?atherosclerotic plaques?
  4. 4. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Galis et al. 1994, JCIGalis et al. 1994, JCI Normal coronary arteryNormal coronary artery Coronary atheromaCoronary atheroma Immunohistochemistry: MMP-3Immunohistochemistry: MMP-3 Immuhistochemistry:Immuhistochemistry: MMP-3MMP-3 LumenLumen fibrous capfibrous cap In situIn situ zymography (activity assay)zymography (activity assay)In situIn situ zymography (activity assay)zymography (activity assay) Lysis ofLysis of fluorescentfluorescent substratesubstrate First…First… are MMPs expressed in human atheroma?are MMPs expressed in human atheroma? First…First… are MMPs expressed in human atheroma?are MMPs expressed in human atheroma? The shoulders of human atherosclerotic plaques contain active MMPs MMP proteins are overexpressed in the vulnerable shoulders, but are they enzymaticaly active ? MMP proteins are overexpressed in the vulnerable shoulders, but are they enzymaticaly active ?
  5. 5. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Vulnerable plaques have a high percentage of macrophage-foam cells Farb &VirmaniFarb &VirmaniFarb &VirmaniFarb &Virmani LumenLumenMacrophageMacrophage foam cellsfoam cells
  6. 6. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Macrophage (MΦ) foam cells in the shoulders of human atheroma express MMPs Immunohistology: Detection of MMP-3Immunohistology: Detection of MMP-3 Double immunohistology: Detection of MMP-1 and MΦ Double immunohistology: Detection of MMP-1 and MΦ (Galis et al., 1994, J Clin Invest)(Galis et al., 1994, J Clin Invest)
  7. 7. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Other MMP sightings in the atheroma… Other MMP sightings in the atheroma… • Messenger RNA for MMP-3 colocalizes with macrophageMessenger RNA for MMP-3 colocalizes with macrophage foam cells (Henney et al., 1991)foam cells (Henney et al., 1991) • Active MMP-9 synthesis in atherectomy specimens fromActive MMP-9 synthesis in atherectomy specimens from patients with unstable angina and MMP-13 colocalizepatients with unstable angina and MMP-13 colocalize with degraded collagen (Brown et al, 1995)with degraded collagen (Brown et al, 1995) • MMP-7 is expressed by macrophage foam cells at sitesMMP-7 is expressed by macrophage foam cells at sites of potential plaque rupture (Halpert et al. 1996)of potential plaque rupture (Halpert et al. 1996) • Macrophages can also express the elastolytic MMP-8Macrophages can also express the elastolytic MMP-8 (Herman, 2001)(Herman, 2001) Macrophage foam cells are associated with increased MMP expression and activity Macrophage foam cells are associated with increased MMP expression and activity
  8. 8. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Do MMPs degrade the collagen of the fibrous cap? Do MMPs degrade the collagen of the fibrous cap? • Macrophage MMP-2 and MMP-9 degrade ex vivoMacrophage MMP-2 and MMP-9 degrade ex vivo the collagen of the plaque’s fibrous cap (Shah etthe collagen of the plaque’s fibrous cap (Shah et al, 1995)al, 1995) • MMP-1 and MMP-13 colocalize in the plaque withMMP-1 and MMP-13 colocalize in the plaque with epitopes expressed by degraded collagenepitopes expressed by degraded collagen (immunohistochemistry, Sukhova et al, 1999)(immunohistochemistry, Sukhova et al, 1999) • MMP-8 colocalizes with epitopes expressed byMMP-8 colocalizes with epitopes expressed by cleaved type I collagen in the shoulders ofcleaved type I collagen in the shoulders of human plaque (immunohistochemistry, Hermanhuman plaque (immunohistochemistry, Herman et al., 2001)et al., 2001)
  9. 9. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Overexpression of interstitialOverexpression of interstitial collagenase (MMP-1 ) coincidescollagenase (MMP-1 ) coincides with the places subjected to thewith the places subjected to the highest tensile stress within thehighest tensile stress within the vulnerable shoulders (Leevulnerable shoulders (Lee et alet al.. 1996)1996) Bad luck?!?Bad luck?!?Bad luck?!?Bad luck?!?
  10. 10. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Determinants of atherosclerotic plaque stabilityDeterminants of atherosclerotic plaque stability Tissue characteristicsTissue characteristics Mechanical stressMechanical stress Active degradation of matrixActive degradation of matrix scaffold in the vulnerablescaffold in the vulnerable shoulders by MMPsshoulders by MMPs (Galis(Galis et alet al 1994, Galis1994, Galis et al.et al. 1995)1995) Tissue characteristicsTissue characteristics Mechanical stressMechanical stress Rupture of atherosclerotic plaqueRupture of atherosclerotic plaqueRupture of atherosclerotic plaqueRupture of atherosclerotic plaque thin fibrous capthin fibrous cap large lipid corelarge lipid core ((Cheng et al. 1993)Cheng et al. 1993) inflammationinflammation ((LendonLendon et al.et al. 1991,1991, van der Walvan der Wal et alet al.,., 1994)1994) mechanical “hotmechanical “hot spots”coincide with thespots”coincide with the weak pointsweak points ((Lee et al. 1996)Lee et al. 1996)
  11. 11. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. • Cytokine stimulation: human EC (Hanemaaijeret al. 1993), human SMC (Galis et al. 1994) • mechanical stretch: shoulders (Lee et al, 1996) • engagement of cell surface receptors: VCAM-1 (Romanic & Madri 1994), CD40 (Malik 1996, Schonbeck, Mach et al 1997), ICAM-1 (Aoudjit et al 1998) • modified lipoproteins: vascular cells (Rajavashist et al. 1999), macrophages (Xu et al 1999) • proteases: thrombin (Galis et al. 1995), plasmin/uPA (Carmeliet et al. 1997), cathepsins (Sukhova et al 1997), MT-MMP (Wang et al.,1998) • oxidative stress: • increased by superoxide, hydrogen peroxide, peroxynitrite (Rajagopalan et al. 1996), • Inhibited by N-acetyl cysteine (Galis et al. 1998), nitric oxide (Gurjar et al. 1999) • matrix composition: collagen I increases macrophage MMP(Wesley et al. 1997) • Infections: Chlamydia (Kol et al, 1998)Kol et al, 1998) • growth factors: FGF-2 (Pickering et al 1997); VEGF (Wang& Keiser 1998) Potential modulators of MMP expression and activity in atheroma Expression of pro-MMPs Activation of MMP enzymatic activity
  12. 12. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Experimental modelExperimental model for investigation offor investigation of macrophage foam cellmacrophage foam cell MMPsMMPs Subcutaneous granulomaSubcutaneous granuloma Balloon angioplastyBalloon angioplasty HypercholesterolemicHypercholesterolemic dietdiet MacrophageMacrophage (anti-RAM 11)(anti-RAM 11) Intracellular lipid (Nile red)
  13. 13. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. 0 200 400 600 MΦ FC x103 counts/min/106 cells PMA - + - + Macrophage (MMacrophage (MΦΦ)-derived)-derived foam cells (FC) producefoam cells (FC) produce reactive oxygen speciesreactive oxygen species (Rajagopalan et al. 1996 JCI)(Rajagopalan et al. 1996 JCI) Macrophage-derived FCMacrophage-derived FC activate the zymogen ofactivate the zymogen of MMP-9MMP-9 in vitroin vitro (Galis et al., 1998 Circulation)(Galis et al., 1998 Circulation) Macrophage-derived FCMacrophage-derived FC activate the zymogen ofactivate the zymogen of MMP-9MMP-9 in vitroin vitro (Galis et al., 1998 Circulation)(Galis et al., 1998 Circulation) SuperoxideSuperoxide PeroxidesPeroxides MΦMΦ FCFC 66 -66 - 97 -97 - 46 -46 - pro-MMP-9pro-MMP-9 MMP-9MMP-9 MMΦΦ FCFC MMP-9MMP-9
  14. 14. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Reactive oxygen species activate latent MMPsReactive oxygen species activate latent MMPs produced by human vascular smooth muscle cellsproduced by human vascular smooth muscle cells ++ Pro-MMP-2 Pro-MMP-9 MMP-9 MMP-2 Pro-MMP-2 MMP-2 In vivo?In vivo?In vivo?In vivo? + Xanthine/ Xanthine Oxidase + Xanthine/ Xanthine Oxidase (Rajagopalan et al. 1996, JCI)(Rajagopalan et al. 1996, JCI)
  15. 15. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. N-acetyl cysteine (NAC) treatment decreasesN-acetyl cysteine (NAC) treatment decreases in situin situ MMP-9 expression and activity in experimental rabbitMMP-9 expression and activity in experimental rabbit atheromaatheroma MMP-9 Macrophages MMP-9 + NAC Macrophages + NAC100100 µµmm IELIEL Pro-MMP-2 - MWM (kDa) Abdominal aorta Thoracic aorta + NAC + NAC0 0 MMP-2 - Pro-MMP-9 - MMP-9 - (Galis et al., 1998 Circulation)(Galis et al., 1998 Circulation)
  16. 16. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Lipid lowering therapy may increase plaque stability by decreasing the oxidative stress •improvement of endothelialimprovement of endothelial functionfunction •decreased plaque lipiddecreased plaque lipid •decreased MMP productiondecreased MMP production increased plaque stabilityincreased plaque stability •improvement of endothelialimprovement of endothelial functionfunction •decreased MMP productiondecreased MMP production increased plaque stabilityincreased plaque stability OxidativeOxidative stressstress LipidLipid Therapeutic interventions and plaque stabilityTherapeutic interventions and plaque stabilityTherapeutic interventions and plaque stabilityTherapeutic interventions and plaque stability
  17. 17. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Experimental macrophage-rich arterial lesions (ApoE KO mouse carotid artery ligation) Macrophage-Macrophage- rich neointimarich neointima Normal carotid arteryNormal carotid arteryNormal carotid arteryNormal carotid artery Atherosclerotic carotid arteryAtherosclerotic carotid arteryAtherosclerotic carotid arteryAtherosclerotic carotid artery (Lessner et al., unpublished)(Lessner et al., unpublished)
  18. 18. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Our studies indicate that arteries withOur studies indicate that arteries with macrophage-rich lesions undergomacrophage-rich lesions undergo enhanced positive remodelingenhanced positive remodeling Our studies indicate that arteries withOur studies indicate that arteries with macrophage-rich lesions undergomacrophage-rich lesions undergo enhanced positive remodelingenhanced positive remodeling 55 1010 1515 2020 2525 303055 1010 1515 2020 2525 3030 Macrophage areaMacrophage area 10001000 20002000 Outer perimeterOuter perimeter Time (days) Time (days) 1010 2020 3030 4040 AreaArea ((µµmm22 )) LengthLength ((µµm)m) - - Pro MMP-9 Pro MMP-2 98 kDa 72 kDa - - Days 0 14 28 WT WT WTKO KO KOST Arteries with macrophage-rich lesions have enhanced MMP activity Arteries with macrophage-rich lesions have enhanced MMP activity
  19. 19. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Positively remodeling isPositively remodeling is associated with plaque instabilityassociated with plaque instability Positively remodeling isPositively remodeling is associated with plaque instabilityassociated with plaque instability Schoenhagen et al., 2000 Circulation 101Schoenhagen et al., 2000 Circulation 101Schoenhagen et al., 2000 Circulation 101Schoenhagen et al., 2000 Circulation 101 UnstableUnstable StableStable PositivePositive NegativeNegativeAbsentAbsent 2020 3030 4040 5050 1010 RemodelingRemodeling %cohort%cohort
  20. 20. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. FoamFoam cell/macrophage-cell/macrophage- drivendriven Smooth muscleSmooth muscle cell-drivencell-driven UnstableUnstable plaqueplaque StableStable plaqueplaque Constrictive arterial remodelingConstrictive arterial remodeling Outward arterial remodelingOutward arterial remodeling(Galis and(Galis and Khatri, 2002)Khatri, 2002) Normal arteryNormal artery
  21. 21. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Macrophage-foam cells are key regulators of MMP-dependent degradation of vascular matrix Macrophage-foam cells are key regulators of MMP-dependent degradation of vascular matrix ROSROS TFTF ThrombinThrombin ModulateModulate MMPMMP activityactivity ExpressExpress pro-pro- MMPsMMPs CytokinesCytokines UpregulateUpregulate vascular cellvascular cell MMPsMMPs Macrophage foam cellsMacrophage foam cells
  22. 22. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Macrophage non-MMP proteases?Macrophage non-MMP proteases?Macrophage non-MMP proteases?Macrophage non-MMP proteases? • CathepsinsCathepsins K and S – have elastolytic activityhave elastolytic activity – Expressed by macrophages in atheroma (Sukhova etExpressed by macrophages in atheroma (Sukhova et al. 1998)al. 1998) • ThrombinThrombin – generated at sites of disruption, can be generated ingenerated at sites of disruption, can be generated in the atherosclerotic plaques via tissue factorthe atherosclerotic plaques via tissue factor expressed by macrophage foam cells (Wilcox et al.expressed by macrophage foam cells (Wilcox et al. 1989)1989) – can activate latent MMP-2 (Galis et al., 1997)can activate latent MMP-2 (Galis et al., 1997)
  23. 23. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Potential protease networksPotential protease networksPotential protease networksPotential protease networks • Macrophage MMPs may increase the activity of otherMacrophage MMPs may increase the activity of other proteases through:proteases through: – Activation of MMP zymogens - e.g., MMP-3 can activate pro-Activation of MMP zymogens - e.g., MMP-3 can activate pro- MMP-1MMP-1 – inactivation of inhibitors - e.g., MMP-1, MMP-3, and MMP-9caninactivation of inhibitors - e.g., MMP-1, MMP-3, and MMP-9can inactivate alpha 1-antitrypsin, the primary physiologic inhibitorinactivate alpha 1-antitrypsin, the primary physiologic inhibitor of human leukocyte elastase (Sires et al. 1994of human leukocyte elastase (Sires et al. 1994); MMP-1, MMP-7,); MMP-1, MMP-7, MMP-9, and MMP-12 cleave tissue factor pathway inhibitor (BelaaouajMMP-9, and MMP-12 cleave tissue factor pathway inhibitor (Belaaouaj et al, 2000)et al, 2000) • Macrophage non-MMPs may increase the activity ofMacrophage non-MMPs may increase the activity of other proteases through:other proteases through: – Activation of MMP zymogens -- e.g., uPA can activate pro-MMP-3,Activation of MMP zymogens -- e.g., uPA can activate pro-MMP-3, thrombin can activate pro-MMP-2thrombin can activate pro-MMP-2 – inactivation of inhibitors ?inactivation of inhibitors ?
  24. 24. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. RupturedRuptured plaqueplaque The mutual stimulation of generation of activeThe mutual stimulation of generation of active MMPs and thrombin may the basis of sustainedMMPs and thrombin may the basis of sustained plaque instability, with recurring episodes ofplaque instability, with recurring episodes of plaque disruption and thrombosisplaque disruption and thrombosis MMPsMMPs ThrombinThrombin MMPsMMPs ThrombinThrombin ThrombinThrombin activatesactivates latent MMPslatent MMPs (Galis et al.,(Galis et al., 1997)1997) Active MMPsActive MMPs stimulatestimulate generation ofgeneration of thrombinthrombin (Sawicki et(Sawicki et al., 1997)al., 1997) Proteases and the unstable atherosclerotic plaqueProteases and the unstable atherosclerotic plaqueProteases and the unstable atherosclerotic plaqueProteases and the unstable atherosclerotic plaque
  25. 25. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. Are these proteases redundant?Are these proteases redundant? What are the potential actions of allWhat are the potential actions of all these in relation to plaque rupture?these in relation to plaque rupture? Are these proteases redundant?Are these proteases redundant? What are the potential actions of allWhat are the potential actions of all these in relation to plaque rupture?these in relation to plaque rupture? • MMPs can degrade all the components of theMMPs can degrade all the components of the extracellular matrixextracellular matrix • uPAuPA activatesactivates MMP zymogens (MMP zymogens (Carmeliet et al. 1997) • Thrombin can activate MMP zymogens (Galis et al. 1997)Thrombin can activate MMP zymogens (Galis et al. 1997) Do MMPs provide a common pathway forDo MMPs provide a common pathway for plaque weakening by other proteases?!?plaque weakening by other proteases?!? Do MMPs provide a common pathway forDo MMPs provide a common pathway for plaque weakening by other proteases?!?plaque weakening by other proteases?!?
  26. 26. Content and GraphicsContent and Graphics Zorina Galis, Ph.D.Zorina Galis, Ph.D. ConclusionsConclusionsConclusionsConclusions • Macrophages provide several essential components thatMacrophages provide several essential components that can increase proteolytic activity within atheroscleroticcan increase proteolytic activity within atherosclerotic plaquesplaques – Produce proteases: MMPs, cathepsins, thrombin via TFProduce proteases: MMPs, cathepsins, thrombin via TF – Provide activators for proteases: ROS, MMPsProvide activators for proteases: ROS, MMPs – Provide means to inactivate protease inhibitors: MMPsProvide means to inactivate protease inhibitors: MMPs – Stimulate production of proteases in vascular cellsStimulate production of proteases in vascular cells All circumstantial evidence supports degradation of matrixAll circumstantial evidence supports degradation of matrix due to an increased macrophage-related proteolytic activitydue to an increased macrophage-related proteolytic activity as a mechanism thorough which macrophage infiltratesas a mechanism thorough which macrophage infiltrates precipitate plaque destabilization, however…..precipitate plaque destabilization, however….. All circumstantial evidence supports degradation of matrixAll circumstantial evidence supports degradation of matrix due to an increased macrophage-related proteolytic activitydue to an increased macrophage-related proteolytic activity as a mechanism thorough which macrophage infiltratesas a mechanism thorough which macrophage infiltrates precipitate plaque destabilization, however…..precipitate plaque destabilization, however….. The direct evidence is still missing!The direct evidence is still missing!The direct evidence is still missing!The direct evidence is still missing!

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