Immune mechanisms behind plaque vulnerability : experimental and clinical studies
Author: Ovchinnikova, Olga
Date: 2010-11-11
Location: Hillarpssalen, Retzius väg 8, Karolinska institutet
Time: 09.00
Department: Institutionen för medicin / Department of Medicine
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thesis.pdf (3.415Mb)
Abstract
Physical disruption of atherosclerotic plaques causes many acute
thrombotic complications such as myocardial infarction and stroke. The
resistance of the atherosclerotic plaque to disruption depends in part on
the integrity of its fibrous cap, which prevents contact between the
highly thrombogenic lipid core and the circulating blood. The fibrillar
collagens types I and III synthesized by smooth muscle cells (SMCs)
largely determine the tensile strength of the cap.
Sites of plaque rupture display signs of active inflammation that can impair plaque stability. Macrophages and mast cells release a set of collagen-degrading enzymes. Additional possible mechanisms include inhibited expression of procollagen genes and induction of death or reduced renewal of the collagen-producing SMC population, both phenomena promoted by T cell-derived interferon-? (IFN?). However, little attention has been given to the post-translational modification of collagen fibers in the fibrous caps. It is known that efficient extracellular cross-linking of collagen catalyzed by the enzyme lysyl oxidase (LOX) confers biomechanical properties and proteolytic resistance of the mature collagen fiber. Thus, failure of collagen maturation may lead to a defective extracellular matrix in the fibrous cap.
Using atherosclerosis-prone mice and samples of human carotid endarterectomies, we investigated whether pro- and anti-inflammatory mediators can affect the LOX-dependent collagen maturation in atherosclerotic lesions, thus leading to plaque weakening.
To study the effect of T cell-driven inflammation, we used genetically modified mice with hypercholesterolemia and disrupted TGFß signaling in T cells (Apoe-/- x CD4dnTßRII). These mice developed larger atherosclerotic lesions with augmented levels of IFN?, increased numbers of activated macrophages and, importantly, impaired maturation of collagen fibers, consistent with a vulnerable phenotype (Paper I). Analysis of mRNA and protein content showed a significant decrease of LOX in aortae of Apoe-/- x CD4dnTßRII mice. T cell-driven inflammation in these mice provoked a limited selective increase in the expression of proteinases that degrade the extracellular matrix, but no increase in collagen fragmentation was detected. Therefore, we concluded that exaggerated T cell-driven inflammation limits the extracellular maturation of collagen in the atherosclerotic plaque.
The stability of atherosclerotic lesions was investigated in Apoe-/- mice after treatment with osteoprotegerin (OPG), a cytokine of the TNFR superfamily and a circulating decoy receptor for the receptor activator of nuclear factor ?B ligand (RANKL) (Paper II). Treatment with OPG facilitated accumulation of SMCs and increased formation of mature collagen fibers within the lesions of Apoe-/- mice. Aortic mRNA level of LOX was also upregulated in treated animals. In cell culture studies, OPG promoted proliferation of rat aortic SMCs. Therefore, we suggested that osteoprotegerin may be a possible mediator of lesion stabilization.
We further investigated if a similar pattern as that obtained in the animal experiments could also be found in the human disease (Paper III). We were able to detect LOX protein in SMC- and collagen-rich areas of human carotid lesions. A higher LOX mRNA and protein were associated with a more stable phenotype of the plaques. Examination of gene expression in plaques revealed a positive correlation between mRNA expression of LOX and mRNA for OPG, and a negative correlation between LOX mRNA and markers of inflammation. This data suggests that LOX may contribute to the stabilization of human atherosclerotic lesions and that its expression is controlled by inflammation.
In paper IV we reported that mRNA and protein content of 5-lipoxygenase activating protein (FLAP) were highly upregulated in aortae of Apoe-/- x CD4dnTßRII mice compared with Apoe-/- littermates. FLAP immunoreactive protein co-localized with CD68+ macrophages. Augmented ex vivo formation of leukotriene B4 in aortae of transgenic mice further supported functional significance of the increased level of FLAP. Treatment with the FLAP-inhibitor MK-886 not only decreased the number of CD3+ cells in lesions and IFN? mRNA levels in aortae of Apoe-/- x CD4dnTßRII mice, but, most importantly, significantly reduced atherosclerotic lesion size. Although FLAP inhibition did not have any significant effect on collagen synthesis, it can be considered as a possible therapeutic tool to stabilize the plaque by reducing the degree of local inflammation.
In summary, the findings of this thesis identify extracellular maturation of collagen, catalyzed by LOX, as important in maintaining the stability of the fibrous cap in the atherosclerotic lesion. The process of collagen maturation is regulated by pro- and anti-inflammatory mediators within the plaque, and it may serve as a target for development of new diagnostic and therapeutic tools.
Sites of plaque rupture display signs of active inflammation that can impair plaque stability. Macrophages and mast cells release a set of collagen-degrading enzymes. Additional possible mechanisms include inhibited expression of procollagen genes and induction of death or reduced renewal of the collagen-producing SMC population, both phenomena promoted by T cell-derived interferon-? (IFN?). However, little attention has been given to the post-translational modification of collagen fibers in the fibrous caps. It is known that efficient extracellular cross-linking of collagen catalyzed by the enzyme lysyl oxidase (LOX) confers biomechanical properties and proteolytic resistance of the mature collagen fiber. Thus, failure of collagen maturation may lead to a defective extracellular matrix in the fibrous cap.
Using atherosclerosis-prone mice and samples of human carotid endarterectomies, we investigated whether pro- and anti-inflammatory mediators can affect the LOX-dependent collagen maturation in atherosclerotic lesions, thus leading to plaque weakening.
To study the effect of T cell-driven inflammation, we used genetically modified mice with hypercholesterolemia and disrupted TGFß signaling in T cells (Apoe-/- x CD4dnTßRII). These mice developed larger atherosclerotic lesions with augmented levels of IFN?, increased numbers of activated macrophages and, importantly, impaired maturation of collagen fibers, consistent with a vulnerable phenotype (Paper I). Analysis of mRNA and protein content showed a significant decrease of LOX in aortae of Apoe-/- x CD4dnTßRII mice. T cell-driven inflammation in these mice provoked a limited selective increase in the expression of proteinases that degrade the extracellular matrix, but no increase in collagen fragmentation was detected. Therefore, we concluded that exaggerated T cell-driven inflammation limits the extracellular maturation of collagen in the atherosclerotic plaque.
The stability of atherosclerotic lesions was investigated in Apoe-/- mice after treatment with osteoprotegerin (OPG), a cytokine of the TNFR superfamily and a circulating decoy receptor for the receptor activator of nuclear factor ?B ligand (RANKL) (Paper II). Treatment with OPG facilitated accumulation of SMCs and increased formation of mature collagen fibers within the lesions of Apoe-/- mice. Aortic mRNA level of LOX was also upregulated in treated animals. In cell culture studies, OPG promoted proliferation of rat aortic SMCs. Therefore, we suggested that osteoprotegerin may be a possible mediator of lesion stabilization.
We further investigated if a similar pattern as that obtained in the animal experiments could also be found in the human disease (Paper III). We were able to detect LOX protein in SMC- and collagen-rich areas of human carotid lesions. A higher LOX mRNA and protein were associated with a more stable phenotype of the plaques. Examination of gene expression in plaques revealed a positive correlation between mRNA expression of LOX and mRNA for OPG, and a negative correlation between LOX mRNA and markers of inflammation. This data suggests that LOX may contribute to the stabilization of human atherosclerotic lesions and that its expression is controlled by inflammation.
In paper IV we reported that mRNA and protein content of 5-lipoxygenase activating protein (FLAP) were highly upregulated in aortae of Apoe-/- x CD4dnTßRII mice compared with Apoe-/- littermates. FLAP immunoreactive protein co-localized with CD68+ macrophages. Augmented ex vivo formation of leukotriene B4 in aortae of transgenic mice further supported functional significance of the increased level of FLAP. Treatment with the FLAP-inhibitor MK-886 not only decreased the number of CD3+ cells in lesions and IFN? mRNA levels in aortae of Apoe-/- x CD4dnTßRII mice, but, most importantly, significantly reduced atherosclerotic lesion size. Although FLAP inhibition did not have any significant effect on collagen synthesis, it can be considered as a possible therapeutic tool to stabilize the plaque by reducing the degree of local inflammation.
In summary, the findings of this thesis identify extracellular maturation of collagen, catalyzed by LOX, as important in maintaining the stability of the fibrous cap in the atherosclerotic lesion. The process of collagen maturation is regulated by pro- and anti-inflammatory mediators within the plaque, and it may serve as a target for development of new diagnostic and therapeutic tools.
List of papers:
I. Ovchinnikova O, Robertson AK, Wågsäter D, Folco EJ, Hyry M, Myllyharju J, Eriksson P, Libby P, Hansson GK (2009). "T cell activation leads to reduced collagen maturation in atherosclerotic plaques of Apoe(-/-) mice" Am J Pathol 174: 693-700
Pubmed
II. Ovchinnikova O, Gylfe A, Bailey L, Nordström A, Rudling M, Jung C, Bergström S, Waldenström A, Hansson GK, Nordström P (2009). "Osteoprotegerin promotes fibrous cap formation in atherosclerotic lesions of ApoE-deficient mice--brief report" Arterioscler Thromb Vasc Biol 29: 478-1480
Pubmed
III. Ovchinnikova O, Folkersen L, Lindeman JHN, Ueland T, Aukrust P, Hedin U, Gavrisheva NA, Shlyakhto EV, Olofsson P, Hansson GK (2010). "The collagen cross-linking enzyme lysyl oxidase is associated with a stable phenotype of human atherosclerotic lesions." (Manuscript)
IV. Bäck M, Sultan A, Ovchinnikova O, Hansson GK (2007). "5-Lipoxygenase-activating protein: a potential link between innate and adaptive immunity in atherosclerosis and adipose tissue inflammation." Circ Res 100: 946-949
Pubmed
I. Ovchinnikova O, Robertson AK, Wågsäter D, Folco EJ, Hyry M, Myllyharju J, Eriksson P, Libby P, Hansson GK (2009). "T cell activation leads to reduced collagen maturation in atherosclerotic plaques of Apoe(-/-) mice" Am J Pathol 174: 693-700
Pubmed
II. Ovchinnikova O, Gylfe A, Bailey L, Nordström A, Rudling M, Jung C, Bergström S, Waldenström A, Hansson GK, Nordström P (2009). "Osteoprotegerin promotes fibrous cap formation in atherosclerotic lesions of ApoE-deficient mice--brief report" Arterioscler Thromb Vasc Biol 29: 478-1480
Pubmed
III. Ovchinnikova O, Folkersen L, Lindeman JHN, Ueland T, Aukrust P, Hedin U, Gavrisheva NA, Shlyakhto EV, Olofsson P, Hansson GK (2010). "The collagen cross-linking enzyme lysyl oxidase is associated with a stable phenotype of human atherosclerotic lesions." (Manuscript)
IV. Bäck M, Sultan A, Ovchinnikova O, Hansson GK (2007). "5-Lipoxygenase-activating protein: a potential link between innate and adaptive immunity in atherosclerosis and adipose tissue inflammation." Circ Res 100: 946-949
Pubmed
Issue date: 2010-10-21
Rights:
Publication year: 2010
ISBN: 978-91-7457-059-5
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