Mervyn J. Merrilees

Proteoglycans in Atherosclerosis and Restenosis: Key Roles for Versican

The proteoglycan versican is one of several extracellular matrix (ECM) molecules that accumulate in lesions of atherosclerosis and restenosis. Its unique structural features create a highly interactive molecule that binds growth factors, enzymes, lipoproteins, and a variety of other ECM components to influence fundamental events involved in vascular disease. Versican is one of the principal genes that is upregulated after vascular injury and is a prominent component in stented and nonstented restenotic lesions. The synthesis of versican is highly regulated by specific growth factors and cytokines and the principal source of versican is the smooth muscle cell. Versican interacts with hyaluronan, a long chain glycosaminoglycan, to create expanded viscoelastic pericellular matrices that are required for arterial smooth muscle cell (ASMC) proliferation and migration. Versican is also prominent in advanced lesions of atherosclerosis, at the borders of lipid-filled necrotic cores as well as at the plaque-thrombus interface, suggesting roles in lipid accumulation, inflammation, and thrombosis. Versican influences the assembly of ECM and controls elastic fiber fibrillogenesis, which is of fundamental importance in ECM remodeling during vascular disease. Collectively, these studies highlight the critical importance of this specific ECM component in atherosclerosis and restenosis.

Connective tissues: matrix composition and its relevance to physical therapy.

In the last 2 decades, the understanding of CT structure and function has increased enormously. It is now clear that the cells of the various CTs synthesize a variety of ECM components that act not only to underpin the specific biomechanical and functional properties of tissues, but also to regulate a variety of cellular functions. Importantly for the physical therapist, and as discussed above, CTs are responsive to changes in the mechanical environment, both naturally occurring and applied. The relative proportions of collagens and PGs largely determine the mechanical properties of CTs. The relationship between the fibril-forming collagens and PG concentration is reciprocal. Connective tissues designed to resist high tensile forces are high in collagen and low in total PG content (mostly dermatan sulphate PGs), whereas CTs subjected to compressive forces have a greater PG content (mostly chondroitin sulphate PGs). Hyaluronan has multiple roles and not only provides tissue hydration and facilitation of gliding and sliding movements but also forms an integral component of large PG aggregates in pressure-resisting tissues. The smaller glycoproteins help to stabilize and link collagens and PGs to the cell surface. The result is a complex interacting network of matrix molecules, which determines both the mechanical properties and the metabolic responses of tissues. Patients with CT problems affecting movement are frequently examined and treated by physical therapists. A knowledge of the CT matrix composition and its relationship to the biomechanical properties of these tissues, particularly the predictable responses to changing mechanical forces, offers an opportunity to provide a rational basis for treatments. The complexity of the interplay among the components, however, requires that further research be undertaken to determine more precisely the effects of treatments on the structure and function of CTs.

B-mode ultrasound images of the carotid artery wall: correlation of ultrasound with histological measurements

B-mode ultrasound is being used to assess carotid atherosclerosis in epidemiological studies and clinical trials. Recently the interpretation of measurements made from ultrasound images has been questioned. This study examines the anatomical correlates of B-mode ultrasound of carotid arteries in vitro and in situ in cadavers. Twenty-seven segments of human carotid artery were collected at autopsy, pressure perfusion fixed in buffered 2.5% glutaraldehyde and 4% paraformaldehyde and imaged using an ATL UM-8 (10 MHz single crystal mechanical probe). Each artery was then frozen, sectioned and stained with van Gieson or elastin van Gieson. The thickness of the intima, media and adventitia were measured to an accuracy of 0.01 mm from histological sections using a calibrated eye graticule on a light microscope. Shrinkage artifact induced by histological preparation was determined to be 7.8%. Digitised ultrasound images of the artery wall were analysed off-line. The distance from the leading edge of the first interface (LE1) to the leading edge of the second interface (LE2) was measured using a dedicated programme. LE1-LE2 measurements were correlated against histological measurements corrected for shrinkage. Mean values for the far wall were: ultrasound LE1-LE2 (0.97 mm, S.D. 0.26), total wall thickness (1.05 mm, S.D. 0.37), adventitia (0.35 mm, S.D. 0.16), media (0.61 mm, S.D. 0.18), intima (0.09 mm, S.D. 0.13). Ultrasound measurements corresponded best with total wall thickness, rather than elastin or the intima-media complex. Excision of part of the intima plus media or removal of the adventitia resulted in a corresponding decrease in the LE1-LE2 distance of the B-mode image. Furthermore, increased wall thickness due to intimal atherosclerotic thickening correlated well with LE1-LE2 distance of the B-mode images. B-mode images obtained from the carotid arteries in situ in four cadavers also corresponded best with total wall thickness measured from histological sections and not with the thickness of the intima plus media. In conclusion, the LE1-LE2 distance measured on B-mode images of the carotid artery best represents total wall thickness of intima plus media plus adventitia and not intima plus media alone.

Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro

Versican is an extracellular matrix proteoglycan produced by many cells. Although versican is generally known as a large chondroitin sulfate proteoglycan (CSPG), the smallest splice variant, V3, consists only of the amino‐ and carboxy‐terminal globular domains and is therefore predicted to be a small glycoprotein, lacking CS chains. The large size, negative charge, and ability of versican variants to form pericellular coats with hyaluronan are responsible for many of its effects. V3, lacking the large size and high charge density, but retaining the hyaluronan‐binding domain of the larger isoforms, may have different effects on cell phenotype. To determine whether V3 alters cell phenotype, Fisher rat arterial smooth muscle cells (ASMCs), which express the larger CSPG versican splice forms (V0 and V1) were retrovirally transduced with the rat V3 cDNA. Northern analysis for versican RNAs confirmed that cells transduced with V3 retrovirus, but not cells tranduced with the empty vector, expressed RNA of the size expected for V3/neor bicistronic RNA. V3 overexpressing cells were more spread on tissue culture plastic, had a smaller length‐to‐breadth ratio and were more resistant to release from the culture dish by trypsin. Interference reflection microscopy of sparsely plated cells showed larger areas of close contact between the V3 expressing cells and the coverslip, in comparison to control cells. Focal contacts in the periphery of V3 expressing cells were larger. Growth and migration studies revealed that V3 transduced cells grow slower and migrate a shorter distance in a scratch wound assay. The increased adhesion and the inhibition of migration and proliferation resulting from V3 overexpression are the opposites of the known and predicted effects of the other variants of versican. V3 may exert these effects through changes in pericellular coat formation, either by competing with larger isoforms for hyaluronan‐binding, or by altering other components of the pericellular matrix. J. Cell. Physiol. 190: 38–45, 2002.

Changes in elastic fibres in the small airways and alveoli in COPD

Small airways are the major site of airflow obstruction in chronic obstructive pulmonary disease (COPD). This is attributed to loss of elastin in alveoli and fibrosis in small airways. In the present study, it was hypothesised that changes to elastic fibres in alveoli might be paralleled by a similar reduction in elastic fibres in small airways. Tissue blocks from patients who had lobectomy for bronchial carcinoma were studied. Patients were classified as COPD (forced expiratory volume in one second (FEV1) <80% predicted, FEV1/forced vital capacity (FVC) <0.7) or controls (FEV1 ≥80% pred, FEV1/FVC ≥0.7). Elastic fibres were visualised using Elastic van Gieson staining and the volume fraction (v/f) of elastic fibres was determined as a percentage of tissue volume using point counting. Elastic fibre networks were also visualised by confocal microscopy. The v/f for elastic fibres in alveoli was 18.6% for COPD and 32.8% in controls. In the airways the v/f was 14.6% for COPD and 25.5% in controls. FEV1% predicted was correlated with v/f in both alveoli and small airways. The volume fraction of elastic fibres was reduced to a similar extent in small airways and alveoli in chronic obstructive pulmonary disease and both were correlated with the extent of airflow obstruction. Loss of elastic fibres in small airways may contribute to the development of airflow obstruction in chronic obstructive pulmonary disease.

Versican and the control of inflammation.

Versican is an extracellular matrix (ECM) proteoglycan that interacts with cells by binding to non-integrin and integrin receptors and to other ECM components that associate with the cell surface. Recent studies have shown also that versican interacts with myeloid and lymphoid cells promoting their adhesion and production of inflammatory cytokines. Versican is produced by stromal cells, as well as leukocytes, and is markedly increased in inflammation. Inflammatory agonists, such as double-stranded RNA mimetics (e.g., poly I:C), stimulate stromal cells, smooth muscle cells and fibroblasts, to produce fibrillar ECMs enriched in versican and hyaluronan (HA) that interact with leukocytes promoting their adhesion. Interference with the incorporation of versican into this ECM blocks monocyte adhesion and dampens the inflammatory response. Tumor cells also express elevated levels of versican which interact with myeloid cells to promote an inflammatory response, through stimulating cytokine release, and metastasis. In addition, myeloid cells, such as macrophages in tumors, synthesize versican which affects tumor cell phenotypes, inflammation, and subsequent metastasis. Versican, by binding to hyaluronan, influences T lymphocyte phenotypes and in part controls the ability of these cells to synthesize and secrete cytokines that influence the immune response. Collectively, these studies indicate that versican as an ECM molecule plays a central role in inflammation and as a result it is emerging as a potential target promising wide therapeutic benefits.

Inhibition of Versican Synthesis by Antisense Alters Smooth Muscle Cell Phenotype and Induces Elastic Fiber Formation In Vitro and in Neointima After Vessel Injury

The proteoglycan versican is implicated in several atherogenic events, including stimulation of vascular smooth muscle cell (VSMC) growth and migration, retention of lipoproteins, and promotion of thrombogenesis. A high content of intimal versican also correlates with a low content of elastin, suggesting an inhibitory role for versican in elastogenesis. To determine whether reduced production of versican can be used to enhance elastogenesis, we transduced Fischer rat VSMC (FRSMC) with a versican antisense sequence using the retroviral vector LXSN. Stable expression of versican antisense (LVaSN) significantly reduced versican production, induced a flattened morphology, reduced cell proliferation and migration, increased tropoelastin synthesis, increased elastin binding protein (S-Gal/EBP), and increased deposition of elastic fibers in long-term cultures. Add-back of chondroitin sulfate chains, or versican, decreased S-Gal/EBP and elastic fiber formation. LVaSN cells seeded into balloon catheter-injured rat carotid arteries formed neointimae containing low levels versican, increased amounts of S-Gal/EBP, and increased elastin deposits 7 days postinjury. At 4 weeks, neointimae formed from LVaSN cells were highly structured and contained multiple layers of elastic fibers and lamellae. These results indicate a central role for versican and its constituent chondroitin sulfate chains in controlling cell phenotype, elastogenesis, and intimal structure.

Changes in elastin, elastin binding protein and versican in alveoli in chronic obstructive pulmonary disease

BackgroundCOPD is characterised by loss of alveolar elastic fibers and by lack of effective repair. Elastic fibers are assembled at cell surfaces by elastin binding protein (EBP), a molecular chaperone whose function can be reversibility inhibited by chondroitin sulphate of matrix proteoglycans such as versican. This study aimed to determine if alveoli of patients with mild to moderate COPD contained increased amounts of versican and a corresponding decrease in EBP, and if these changes were correlated with decreases in elastin and FEV1.MethodsLung samples were obtained from 26 control (FEV1 ≥ 80% predicted, FEV1/VC >0.7) and 17 COPD patients (FEV1 ≥ 40% – <80% predicted, FEV1/VC ≤ 0.7) who had undergone a lobectomy for bronchial carcinoma. Samples were processed for histological and immuno-staining. Volume fractions (Vv) of elastin in alveolar walls and alveolar rims were determined by point counting, and versican and EBP assessed by grading of staining intensities.ResultsElastin Vv was positively correlated with FEV1 for both the alveolar walls (r = 0.66, p < 0.001) and rims (r = 0.41, p < 0.01). Versican was negatively correlated with FEV1 in both regions (r = 0.30 and 0.32 respectively, p < 0.05), with the highest staining intensities found in patients with the lowest values for FEV1. Conversely, staining intensities for EBP in alveolar walls and rims and were positively correlated with FEV1 (r = 0.43 and 0.46, p < 0.01).ConclusionPatients with mild to moderate COPD show progressively increased immuno-staining for versican and correspondingly decreased immuno-staining for EBP, with decreasing values of FEV1. These findings may explain the lack of repair of elastic fibers in the lungs of patients with moderate COPD. Removal of versican may offer a strategy for effective repair.

Versican and the regulation of cell phenotype in disease.

BACKGROUND Versican is an extracellular matrix (ECM) proteoglycan that is present in the pericellular environment of most tissues and increases in many different diseases. Versican interacts with cells to influence the ability of cells to proliferate, migrate, adhere and assemble an ECM. SCOPE OF REVIEW The structure of the versican molecule is briefly reviewed and studies highlighting those factors that promote versican synthesis and degradation and their impact on cell phenotype in disease are discussed. Particular attention is given to vascular disease, but other diseases where versican is important are covered as well, most notably different forms of cancers. Attention is given to mechanisms(s) by which versican influences cell behaviors through either direct or indirect processes. Versican produced by either stromal cells or myeloid cells can have a major impact influencing immunity and inflammation. Finally, studies controlling versican accumulation that either delay or inhibit the progression of disease will be highlighted. MAJOR CONCLUSIONS Versican is one component of the ECM that can influence the ability of cells to proliferate, migrate, adhere, and remodel the ECM. Targeting versican as a way to control cell phenotype offers a novel approach in the treatment of disease. SIGNIFICANCE ECM molecules such as versican contribute to the structural integrity of tissues and interact with cells through direct and indirect means to regulate, in part, cellular events that form the basis of disease. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Changes in Collagen Fibril Diameters Across Artery Walls Including a Correlation with Glycosaminoglycan Content

Collagen fibril diameters were measured at regular intervals across the walls of numerous arteries from human, pig and rat. In all vessels the smallest fibrils (mean-fibril diameters of 30-40 nm) occurred in the intima and inner media and the largest fibrils (MFDs 50-100 nm) in the outer adventitia. Between these two regions fibrils progressively increased in size. Circumferential and axial fibrils were of similar size and showed similar patterns of increase. At each sample site there was a range of diameters and frequency distributions were often multimodal with peaks 8 nm, or multiples of 8 nm, apart. Amounts of total and individual glycosaminoglycans (GAG) were determined at regular intervals across the wall of pig aorta and total and sulphated GAG levels were also determined at intervals across rat carotid artery using autoradiographic detection of incorporated [3H] glucosamine and 35S. In both vessels there was a strong correlation between decreasing GAG and increasing MFDs and over a narrow MFD range of 40 to 60 nm. These results demonstrate that collagen fibril diameters are excellent indicators to GAG levels and may be useful for making predictions about GAG levels in areas too small to sample biochemically.