Institutionen för medicin / Department of Medicine
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
