Laminin isoforms in atherosclerotic arteries from mice and man.

The properties of the arterial vasculature depend to a large extent on the activities of smooth muscle cells, which, in turn, are determined by their extracellular environment. During pathological conditions, such as atherosclerosis, this interaction is altered. In close proximity to medial smooth muscle cells are basement membrane components, such as different isoforms of laminin. These proteins can have great impact on cellular function via interaction with cell surface integrins. However, knowledge of laminins in smooth muscle cell basement membranes during normal and pathological conditions is scarce. Therefore, we have analyzed the presence of laminin isoforms in atherosclerotic lesions of apolipoprotein E (ApoE)-deficient mice. Our study revealed that the laminin chain isotype composition within atherosclerotic plaque tissue was different from the chain composition in the media. In addition, obvious differences in laminin chain composition could be observed in areas of the media, which were or were not associated with plaque tissue. Our major findings demonstrate that laminin gamma3 was exclusively present in media associated with plaque tissue. Laminin alpha2 was also enriched in these medial areas. Plaque tissue was predominantly enriched in laminin alpha5 chains. This general distribution applied to lesions both with and without a fibrous cap-like structure. The differential distribution of laminin chains were partially accompanied by changes in the presence of the integrin alpha subunits 7 and V. The distribution of laminin chains in human atherosclerotic arteries, with different size and morphology, grossly resembled their distribution in mouse arteries.

Production of type VI collagen by human macrophages: a new dimension in macrophage functional heterogeneity.

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.

Laser microdissection-based analysis of mRNA expression in human coronary arteries with intimal thickening.

Intimal thickening is an early phase of atherosclerosis characterized by differentiation of plaque smooth muscle cells (SMCs) from a contractile to a synthetic phenotype. We used laser microdissection (LMD) plus real-time RT-PCR to quantify mRNAs for calponin-1 and smoothelin, markers of the contractile phenotype, and for serum response factor (SRF), a regulator of SMC differentiation, in intimal and medial SMCs of human coronary arteries with intimal thickening. RNA expression was also analyzed by ISH and protein expression was detected by IHC. LMD plus RT-PCR found similar levels of SRF mRNA in intimal and medial SMCs, while medial mRNA levels for calponin-1 and smoothelin were higher. ISH confirmed that smoothelin mRNA levels in media exceeded those in intima, whereas SRF mRNA levels were similar at both sites. For calponin-1 and smoothelin, protein levels mirrored respective mRNA levels. By contrast, more medial than intimal SRF protein was present. Our results indicate that intimal SMCs exhibit a largely synthetic phenotype, perhaps reflecting lower intimal levels of SRF protein; ISH and LMD plus real-time RT-PCR provide comparable results; as a valuable alternative to ISH, LMD plus RT-PCR allows parallel measurement of several transcripts; and tissue gene expression studies must measure both protein and mRNA levels.

Differential interactions of decorin and decorin mutants with type I and type VI collagens.

The small leucine-rich proteoglycan decorin can bind via its core protein to different types of collagens such as type I and type VI. To test whether decorin can act as a bridging molecule between these collagens, the binding properties of wild-type decorin, two full-length decorin species with single amino acid substitutions (DCN E180K, DCN E180Q), which previously showed reduced binding to collagen type I fibrils, and a truncated form of decorin (DCN Q153) to the these collagens were investigated. In a solid phase assay dissociation constants for wild-type decorin bound to methylated, therefore monomeric, triple helical type I collagen were in the order of 10(-10) m, while dissociation constants for fibrillar type I collagen were approximately 10(-9) m. The dissociation constant for type VI was approximately 10(-7) m. Using real-time analysis for a more detailed investigation DCN E180Q and DCN E180K exhibited lower association and higher dissociation constants to type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude. In contrast, the affinities of these mutants to type VI collagen were 10 times higher than the affinity of wild-type decorin (K(D) approximately 10(-8) m). Further investigations verified that complexes of type VI collagen and decorin bound type I collagen and that the affinity of collagen type VI to type I was increased by the presence of decorin. These data show that decorin not only can regulate collagen fibril formation but that it also can act as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites.

Expression of transcription factors and matrix genes in response to serum stimulus in vascular smooth muscle cells.

During atherogenesis vascular smooth muscle cells are converted from a contractile into a synthetic phenotype characterized by enhanced matrix production. The transcription factors Gax and GATA-6 are considered negative, and Oct-1 positive regulators of the synthetic phenotype. Since the phenotype transition can be induced by culturing the cells with serum, we followed the expression of Gax, GATA-6 and Oct-1, integrins and matrix genes in quiescent porcine vascular smooth muscle cells after serum application. Comparisons were made between enzymatically released primary smooth muscle cells and cells grown out from explants of the medial layer of porcine aorta. The serum-mediated down-regulation of Gax was more intense than that of GATA-6, and stronger in explant-derived than in primary cells. Serum was without influence on the expression of Oct-1. Changes in the expression of the transcription factors preceded the induction of integrin alpha2 and the down-regulation of decorin, while mRNAs for laminin beta1 and osteopontin rose immediately after serum stimulation. Primary cells reacted more rapidly than explant cells with respect to changes in laminin isoforms. Studies with a Gax-expressing adenovirus indicated that among all the gene products tested only the expression of integrin alpha2 responded to Gax induction. Thus, our data show that i) Gax should be considered a transcription factor being directly responsible for only few aspects of the phenotypic conversion of smooth muscle cells and that ii) explant cells may represent a subpopulation of smooth muscle cells, which differ from the total population of smooth muscle cells, as obtained in primary culture, in their response to serum stimuli.

Rupture of the atherosclerotic plaque: does a good animal model exist?

By its very nature, rupture of the atherosclerotic plaque is difficult to study directly in humans. A good animal model would help us not only to understand how rupture occurs but also to design and test treatments to prevent it from happening. However, several difficulties surround existing models of plaque rupture, including the need for radical interventions to produce the rupture, lack of direct evidence of rupture per se, and absence of convincing evidence of platelet- and fibrin-rich thrombus at the rupture site. At the present time, attention should therefore focus on the processes of plaque breakdown and thrombus formation in humans, whereas the use of animal models should probably be reserved for studying the function of particular genes and for investigating isolated features of plaques, such as the relationship between cap thickness and plaque stability.