- Email: [email protected]
1.W01.2 Proteoglycan-lipoprotein interactions
Monday 6 October 1997: Workshops WOl
I_
1 WOl
EXTRACELLULAR MATRIX OF THE VESSEL CELL-MATRIX INTERACTIONS DURING ATHEROGENESIS 1
Collagens
and atherosclerosis:
M.J. Barnes, C.G. Knight, Cambridge, UK
Collagen-cell
R.W. Farndale.
Sfrangeways
AND
interaction Laboratory,
Collagens in the vessel wall fulfil a mechanical function and provide a support for the cellular elements present. Their deposition in the extracellular matrix by vascular smooth muscle cells (SMCs) is partly responsible for the occlusion of the vessel lumen in vascular disease. Their proteolytic degradation may be a contributary factor in rupture of the atherosclerotic plaque associated with heart attack. Collagen interaction with SMCs mediated by specific integrins may play an important role in the regulation of SMC phenotype and the migratory behaviour of these cells. Most importantly, activation of blood platelets by collagens in the vessel wall may play a role in atherogenesis, may be a cause of thrombosis, especially that occurring with plaque rupture, and may be responsible, in part, for restenosis following angioplasty. We are investigating platelet-reactive sequences in collagen, their receptor recognition and the signals they induce, with the ultimate aim of obtaining specific inhibitors of collagen-platelet interaction. Our studies indicate a two-step mechanism of interaction involving initially recognition by specific collagen sequences of an adhesive receptor, integrin cz2@ 1, as a result of which platelets are able to attach to collagen fibres under conditions of blood flow. Subsequently, other sequences in collagen interact with platelet glycoprotein (GP) VI, inducing signalling leading to platelet activation and thrombus formation. We have identified Gly-Pro-Hyp (GPP*) as the sequence reacting with the activatory receptor, GP VI. A peptide comprising of a repeat GPP* structure exhibits potent cr2#Il-independent platelet reactivity but, like collagen, does not activate GP VI-deficient platelets. Signalling by the peptide involves tyrosine phosphorylation of FcR y chain, the kinases syk and fak, and of phospholipase Cy2, all characteristic features of collagen-induced platelet signalling associated with activation. Using synthetic peptides, we have also identified, in collagen III, an cr2filmcognition sequence involving residues 522-528 of the 011 (III) chain.
I
1 W01.2
Proteaglycan-lipoprotein
interactions
M. Bihari-Varga, I. Haynal. Univ. of Health Dept. of Pediatrics, Budapest, Hungary
Sci., Postgrad.
Med. School,
1st.
Proteoglycans (PG-s), biopolymers containing proteins and polysaccharide subunits (glycosaminoglycans, GAG-s) are enriched in me arterial wall and accumulate during the early phases of atherosclerosis. This accumulation predisposes the intima to lipid deposition, due to the specific ability of PG-s to interact with serum lipoproteins (LP-s). Altered length or molecular weight of the GAG chain or modification of core protein structure, induced by TGF-Beta 1 and PDGF, may exhibit different affinities for LP-s. Among the several forms of LDL, - the “small-dense-TG rich particles,” oxidised-LDL, and Lp(a) - are supposed to be the most atherogenic, partially because of their stronger interactions with PG-s. Complex formation with PG-s, induces further changes in the physical structure, mobility and susceptibility to oxidation of LP molecules, enhancing their deposition into the extracellular space. The complexes, taken up by endothelial and smooth muscle cells, via the small PG receptor, the LDL receptor. and/or the scavenger receptor, influence the proliferation and differentiation of the cells. Interaction of modified LP-s with monocytes, and their transformation into the foam cells, may also depend on interactions with PG-s.
I1 .WOl
3
Elastin-lambdn
M.P. Jacob. INSERM 75018 Paris, France
receptor
in atberogenesis
(I 460, UFR de Medecine
X, Bichat,
16, rue H. Huchard
During the formation and evolution of atherosclerotic plaques, interaction of smooth muscle cells with its surrounding extracellular matrix and especially elastic fibers is modified. Furthermore, degradation of elastic fibers is observed. The produced elastin peptides can act on adjacent cells or on 11th international
Symposium
blood cells as elastin peptides are detectable in blood. In fact, a high-affinity receptor for elastin peptides is present on smooth muscle cells, endothelial cells, monocytes, PMN. Recently, this receptor has also been detected on lymphocytes. This receptor also binds laminin. The addition of elastin peptides to the cells induced an increase of intracellular free calcium concentration through the activation of a G-protein and phospholipase C. This triggers the release of lytic enzymes and reactive oxygen species from cells. All these effects of elastin peptides are inhibited in presence of lactose. The sensitivity of this receptor also changes with age and atherosclerosis. More recently, the effect of elastin peptides on vascular tone has been demonstrated: elastin peptides induce an endothelium-dependent vasodilation of rat aortic rings. Elastin peptides act through the synthesis of prostanoids and the production of NO. The progressive degradation of elastic fibers, the liberation of elastin peptides and their action on the elastin receptor may well be involved in the progressive alteration of the vascular wall observed during atherogenesis. 1 .WOl 4 L-2J Marlene Canada
Elastase coronary
Rabinovitch.
and elastase inhibitors artery disease University
of Toronto,
and pulmonary Hospital
for
and
Sick Children,
Endogenous vascular elastase (EVE) appears to play a pivotal role in the pathophysiology of pulmonary vascular disease. Serine elastase inhibitors prevent or retard the progression of vascular disease in experimental animals. Studies in cultured smooth muscle cells (SMC) have shown that a serum factor can induce the release of EVE by a beta integrin signalling mechanism involving tyrosine phosphorylation of focal adhesion kinase and extracellular regulated kinase (erk). The latter transactivates and induces production of the transcription factor AML-1, which is necessary for induction of EVE. Further studies established that EVE induces release of SMC mitogens from the extracellular matrix. There is evidence that EVE also regulates the production of the matrix glycoprotein tenascin (TN) which amplifies the proliferative response by clustering growth factor receptors thereby facilitating their activation (phosphorylation) upon ligation. Withdrawal of TN will induce apoptosis, and this appears to correlate with regression of medial hypertrophy. The degradation products of elastin produced by EVE induce the production of fibronectin (IN) which stimulates SMC migration. A post-transcriptional mechanism has been elucidated which involves binding of a microtubule associated protein to the 3’ untranslated region (UTR) of the FN mRNA. The balance between EVE and its naturally-occurring inhibitor elafin is currently being addressed in studies in which we are overexpressing elafin under the regulation of a preproendothelin promoter in the vasculature of transgenic mice.
lzzl 1 WOl
5
Physicocbemical and endothelial proteoglycans
interaction between plasma cell membrane and vascular
lipoproteins matrix
G. Siegel’, M. Malmstet?, D. Kliigendorf’, W. Leonhardt3, W.G. Wood4, ‘Institute of Physiology, Biophysical Research Group, The Free University of Berlin, D-14195 Berlin; 31nstitute and Policlinic of Clinical Metabolic Research, Medical Faculty Carl Gustav Carus, D-01307 Dresden; 41nstitute of Clinical Laboratory Diagnostics, Clinic of the Hanse City Stralsund, D-18410 Stralsund, Germany; 21nstitute of Sutjace Chemistry, S-l 1486 Stockholm, Sweden The polyanionic and hydrophilic glycosaminoglycan (GAG) chains dominate the physical properties of proteoglycans (PGs) such as endothelial cell membrane syndecan and vascular matrix perlecan. They have a strong influence not only on tissue hydration and elasticity, but also on countercation attraction by their negative fixed charges. Thus, pericellular compartments with concentrated PGs and ions can influence spatiotemporally controlled reaction rates and concentration-dependent interactive physical processes. Under physiological ion concentrations and pH, the specific binding of GAGS to proteins such as proteases and antiproteases, growth factors and other cytokines, matrix proteins and glycoconjugates, cell adhesion molecules, lipoproteins and lipases allows PGs to intervene in cell and tissue development in numerous ways. Under pathophysiological conditions, chondroitin sulfate (CS)/heparan sulfate (HS) proteoglycans may initiate increased LDL/oxLDL binding and form an extracellular preatheromatous lipid deposition in the intima and inner media.
on Atherosclerosis,
Paris, October
1997
I_
1 WOl
EXTRACELLULAR MATRIX OF THE VESSEL CELL-MATRIX INTERACTIONS DURING ATHEROGENESIS 1
Collagens
and atherosclerosis:
M.J. Barnes, C.G. Knight, Cambridge, UK
Collagen-cell
R.W. Farndale.
Sfrangeways
AND
interaction Laboratory,
Collagens in the vessel wall fulfil a mechanical function and provide a support for the cellular elements present. Their deposition in the extracellular matrix by vascular smooth muscle cells (SMCs) is partly responsible for the occlusion of the vessel lumen in vascular disease. Their proteolytic degradation may be a contributary factor in rupture of the atherosclerotic plaque associated with heart attack. Collagen interaction with SMCs mediated by specific integrins may play an important role in the regulation of SMC phenotype and the migratory behaviour of these cells. Most importantly, activation of blood platelets by collagens in the vessel wall may play a role in atherogenesis, may be a cause of thrombosis, especially that occurring with plaque rupture, and may be responsible, in part, for restenosis following angioplasty. We are investigating platelet-reactive sequences in collagen, their receptor recognition and the signals they induce, with the ultimate aim of obtaining specific inhibitors of collagen-platelet interaction. Our studies indicate a two-step mechanism of interaction involving initially recognition by specific collagen sequences of an adhesive receptor, integrin cz2@ 1, as a result of which platelets are able to attach to collagen fibres under conditions of blood flow. Subsequently, other sequences in collagen interact with platelet glycoprotein (GP) VI, inducing signalling leading to platelet activation and thrombus formation. We have identified Gly-Pro-Hyp (GPP*) as the sequence reacting with the activatory receptor, GP VI. A peptide comprising of a repeat GPP* structure exhibits potent cr2#Il-independent platelet reactivity but, like collagen, does not activate GP VI-deficient platelets. Signalling by the peptide involves tyrosine phosphorylation of FcR y chain, the kinases syk and fak, and of phospholipase Cy2, all characteristic features of collagen-induced platelet signalling associated with activation. Using synthetic peptides, we have also identified, in collagen III, an cr2filmcognition sequence involving residues 522-528 of the 011 (III) chain.
I
1 W01.2
Proteaglycan-lipoprotein
interactions
M. Bihari-Varga, I. Haynal. Univ. of Health Dept. of Pediatrics, Budapest, Hungary
Sci., Postgrad.
Med. School,
1st.
Proteoglycans (PG-s), biopolymers containing proteins and polysaccharide subunits (glycosaminoglycans, GAG-s) are enriched in me arterial wall and accumulate during the early phases of atherosclerosis. This accumulation predisposes the intima to lipid deposition, due to the specific ability of PG-s to interact with serum lipoproteins (LP-s). Altered length or molecular weight of the GAG chain or modification of core protein structure, induced by TGF-Beta 1 and PDGF, may exhibit different affinities for LP-s. Among the several forms of LDL, - the “small-dense-TG rich particles,” oxidised-LDL, and Lp(a) - are supposed to be the most atherogenic, partially because of their stronger interactions with PG-s. Complex formation with PG-s, induces further changes in the physical structure, mobility and susceptibility to oxidation of LP molecules, enhancing their deposition into the extracellular space. The complexes, taken up by endothelial and smooth muscle cells, via the small PG receptor, the LDL receptor. and/or the scavenger receptor, influence the proliferation and differentiation of the cells. Interaction of modified LP-s with monocytes, and their transformation into the foam cells, may also depend on interactions with PG-s.
I1 .WOl
3
Elastin-lambdn
M.P. Jacob. INSERM 75018 Paris, France
receptor
in atberogenesis
(I 460, UFR de Medecine
X, Bichat,
16, rue H. Huchard
During the formation and evolution of atherosclerotic plaques, interaction of smooth muscle cells with its surrounding extracellular matrix and especially elastic fibers is modified. Furthermore, degradation of elastic fibers is observed. The produced elastin peptides can act on adjacent cells or on 11th international
Symposium
blood cells as elastin peptides are detectable in blood. In fact, a high-affinity receptor for elastin peptides is present on smooth muscle cells, endothelial cells, monocytes, PMN. Recently, this receptor has also been detected on lymphocytes. This receptor also binds laminin. The addition of elastin peptides to the cells induced an increase of intracellular free calcium concentration through the activation of a G-protein and phospholipase C. This triggers the release of lytic enzymes and reactive oxygen species from cells. All these effects of elastin peptides are inhibited in presence of lactose. The sensitivity of this receptor also changes with age and atherosclerosis. More recently, the effect of elastin peptides on vascular tone has been demonstrated: elastin peptides induce an endothelium-dependent vasodilation of rat aortic rings. Elastin peptides act through the synthesis of prostanoids and the production of NO. The progressive degradation of elastic fibers, the liberation of elastin peptides and their action on the elastin receptor may well be involved in the progressive alteration of the vascular wall observed during atherogenesis. 1 .WOl 4 L-2J Marlene Canada
Elastase coronary
Rabinovitch.
and elastase inhibitors artery disease University
of Toronto,
and pulmonary Hospital
for
and
Sick Children,
Endogenous vascular elastase (EVE) appears to play a pivotal role in the pathophysiology of pulmonary vascular disease. Serine elastase inhibitors prevent or retard the progression of vascular disease in experimental animals. Studies in cultured smooth muscle cells (SMC) have shown that a serum factor can induce the release of EVE by a beta integrin signalling mechanism involving tyrosine phosphorylation of focal adhesion kinase and extracellular regulated kinase (erk). The latter transactivates and induces production of the transcription factor AML-1, which is necessary for induction of EVE. Further studies established that EVE induces release of SMC mitogens from the extracellular matrix. There is evidence that EVE also regulates the production of the matrix glycoprotein tenascin (TN) which amplifies the proliferative response by clustering growth factor receptors thereby facilitating their activation (phosphorylation) upon ligation. Withdrawal of TN will induce apoptosis, and this appears to correlate with regression of medial hypertrophy. The degradation products of elastin produced by EVE induce the production of fibronectin (IN) which stimulates SMC migration. A post-transcriptional mechanism has been elucidated which involves binding of a microtubule associated protein to the 3’ untranslated region (UTR) of the FN mRNA. The balance between EVE and its naturally-occurring inhibitor elafin is currently being addressed in studies in which we are overexpressing elafin under the regulation of a preproendothelin promoter in the vasculature of transgenic mice.
lzzl 1 WOl
5
Physicocbemical and endothelial proteoglycans
interaction between plasma cell membrane and vascular
lipoproteins matrix
G. Siegel’, M. Malmstet?, D. Kliigendorf’, W. Leonhardt3, W.G. Wood4, ‘Institute of Physiology, Biophysical Research Group, The Free University of Berlin, D-14195 Berlin; 31nstitute and Policlinic of Clinical Metabolic Research, Medical Faculty Carl Gustav Carus, D-01307 Dresden; 41nstitute of Clinical Laboratory Diagnostics, Clinic of the Hanse City Stralsund, D-18410 Stralsund, Germany; 21nstitute of Sutjace Chemistry, S-l 1486 Stockholm, Sweden The polyanionic and hydrophilic glycosaminoglycan (GAG) chains dominate the physical properties of proteoglycans (PGs) such as endothelial cell membrane syndecan and vascular matrix perlecan. They have a strong influence not only on tissue hydration and elasticity, but also on countercation attraction by their negative fixed charges. Thus, pericellular compartments with concentrated PGs and ions can influence spatiotemporally controlled reaction rates and concentration-dependent interactive physical processes. Under physiological ion concentrations and pH, the specific binding of GAGS to proteins such as proteases and antiproteases, growth factors and other cytokines, matrix proteins and glycoconjugates, cell adhesion molecules, lipoproteins and lipases allows PGs to intervene in cell and tissue development in numerous ways. Under pathophysiological conditions, chondroitin sulfate (CS)/heparan sulfate (HS) proteoglycans may initiate increased LDL/oxLDL binding and form an extracellular preatheromatous lipid deposition in the intima and inner media.
on Atherosclerosis,
Paris, October
1997