Connective tissues: matrix composition and its relevance to physical therapy.
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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. (+info)
Distinct secondary structures of the leucine-rich repeat proteoglycans decorin and biglycan. Glycosylation-dependent conformational stability.
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Biglycan and decorin have been overexpressed in eukaryotic cells and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans (Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the final form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that in this specific domain the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provide further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1 and 2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of stable unfolding intermediates. (+info)
Decorin is a Zn2+ metalloprotein.
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Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by leucine-rich repeat motifs. We show here that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells has a high affinity for Zn2+ as demonstrated by equilibrium dialyses. The Zn2+-binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in a spacing reminiscent of a zinc finger. A recombinant 41-amino acid long peptide representing the N-terminal domain of decorin has full Zn2+ binding activity and binds two Zn2+ ions with an average KD of 3 x 10(-7) M. Binding of Zn2+ to this peptide results in a change in secondary structure as shown by circular dichroism spectroscopy. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn2+-binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, do not bind Zn2+ with high affinity. (+info)
Resistance of small leucine-rich repeat proteoglycans to proteolytic degradation during interleukin-1-stimulated cartilage catabolism.
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A bovine nasal-cartilage culture system has been utilized to analyse the catabolic events occurring in response to interleukin-1beta over a 14-day period. An early event following the start of interleukin-1 treatment was the release of glycosaminoglycan into the culture medium. This release was accompanied by the appearance in the tissue, and shortly thereafter also in the culture media, of a globular domain (G1)-containing aggrecan degradation product generated by the action of aggrecanase. Link protein was also released from the cartilage with a similar timeframe to that of the G1 fragment, although there was no evidence of its proteolytic degradation. By comparison with aggrecan, the small leucine-rich repeat proteoglycans decorin, biglycan and lumican showed a resistance to both proteolytic cleavage and release throughout the culture period. In contrast, fibromodulin exhibited a marked decrease in size after day 4, presumably due to proteolytic modification, but the major degradation product was retained throughout the culture period. Also in contrast with the early changes in the components of the proteoglycan aggregate, type II collagen did not display signs of extensive degradation until much later in the culture period. Collagen degradation products compatible with collagenase action first appeared in the medium by day 10 and increased thereafter. These data demonstrate that the leucine-rich repeat proteoglycans are resistant to proteolytic action during interleukin-1-stimulated cartilage catabolism, compared with aggrecan. This resistance and continued interaction with the surface of the collagen fibrils may help to stabilize the collagen fibrillar network and protect it from extensive proteolytic attack during the early phases of cartilage degeneration. (+info)
Differential regulation of extracellular matrix molecules by mechanical strain of fetal lung cells.
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We have previously shown that an intermittent mechanical strain regimen (5% elongation, 60 cycles/min, 15 min/h) that simulates fetal breathing movements stimulated fetal rat lung cell proliferation. Because normal lung growth requires proper coordination between cell proliferation and extracellular matrix (ECM) remodeling, we subjected organotypic cultures of fetal rat lung cells (day 19 of gestation, term = 22 days) to this strain regimen and examined alterations in ECM gene and protein expression. Northern analysis revealed that mechanical strain reduced messages for procollagen-alpha1(I) and biglycan and increased the levels of mRNA for collagen-alpha1(IV) and -alpha2(IV), whereas laminin beta-chain mRNA levels remained constant. Regardless of mRNA changes, mechanical strain increased the protein content of type I and type IV collagen as well as of biglycan in the medium. Mechanical strain did not affect gene expression of several matrix metalloproteinases (MMPs), such as MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), and MMP-3 (stromelysin-1). Neither collagenase nor gelatinase (A and B) activities in conditioned medium were affected by mechanical strain. Tissue inhibitor of metalloproteinase activities in conditioned medium remained unchanged during the 48-h intermittent mechanical stretching. These data suggest that an intermittent mechanical strain differentially regulates gene and protein expression of ECM molecules in fetal lung cells. The observed increase in matrix accumulation appears to be mainly a result of an increased synthesis of ECM molecules and not of decreasing activity of degradative enzymes. (+info)
The macromolecular characteristics of cartilage proteoglycans do not change when synthesis is up-regulated by link protein peptide.
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Previous studies have shown that a synthetic, unglycosylated analogue of the N-terminal peptide from link protein can function as a growth factor and up-regulate proteoglycan biosynthesis in explant cultures of normal human articular cartilage from a wide age range of subjects (McKenna et al., Arthritis Rheum. 41 (1998) 157-162). The present work further shows that link peptide increased proteoglycan synthesis by cartilage cultured in both the presence and absence of serum, suggesting that the mechanism of up-regulation may be different from that of insulin-like growth factors. The proteoglycans synthesised during stimulation with link peptide were of normal hydrodynamic size and the ratio of core protein to glycosaminoglycan side chains and the proportions of the large proteoglycan aggrecan to the small proteoglycans, decorin and biglycan, remained constant. Aggrecan molecules were equally capable of forming aggregates as those from control tissues and the relative proportions of decorin and biglycan were unchanged showing that both were co-ordinately up-regulated. These results confirmed that this novel peptide is a potent stimulator of proteoglycan synthesis by articular cartilage and showed that the newly synthesised proteoglycans were of normal composition. (+info)
External beam radiation after stent implantation increases neointimal hyperplasia by augmenting smooth muscle cell proliferation and extracellular matrix accumulation.
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OBJECTIVES: We sought to examine the effects of high volume external beam radiation (EBR) after stent implantation on neointimal hyperplasia, smooth muscle cell (SMC) proliferation, presence of inflammatory cells and expression of extracellular matrix (ECM). BACKGROUND: Endovascular irradiation has been shown to reduce restenosis rates after angioplasty in preliminary trials, but conflicting results have been reported for the effects of external beam irradiation. METHODS: Forty-three Palmaz-Schatz stents were implanted into iliac arteries of New Zealand White rabbits. The arteries were externally irradiated after stent implantation with a single dose of 8 Gy (at day 3) or 16 Gy in two fractions (8 Gy at days 3 and 4) by means of a linear accelerator. In the control rabbits, no radiation was applied after stent implantation. Smooth muscle cells, macrophages and ECM were studied by immunohistochemistry at one and 12 weeks after stent implantation. Collagen type I and biglycan messenger ribonucleic acid (mRNA) levels were assessed by Northern blot analysis at one week. Neointimal cell densities and arterial lumen stenosis were measured by histomorphometry at 12 weeks. RESULTS: At 1 week, SMC proliferation at the site of stent implantation was increased after EBR with 8 and 16 Gy (26 +/- 5%, 32 +/- 3% vs. 17 +/- 8%; p < 0.01, 16 Gy vs. control). External beam radiation with 8 and 16 Gy augmented SMC proliferation proximal and distal to the angioplasty site (11 +/- 3%, 14 +/- 3 vs. 6 +/- 1%; p < 0.01, 16 Gy vs. control). Collagen type I and biglycan mRNA levels were elevated in stented arteries after EBR with 16 Gy. At 12 weeks, a marked decrease in neointimal cell density (248 +/- 97 vs. 498 +/- 117 SMCs/0.1 mm2 neointima; p < 0.005 vs. control) was noted after EBR with 16 Gy. Irradiation with 8 and 16 Gy increased arterial lumen stenosis compared with nonirradiated control rabbits (45 +/- 7%, 55 +/- 9% vs. 33 +/- 7%; p < 0.05, 8 Gy and p < 0.001, 16 Gy vs. control). CONCLUSIONS: High volume external beam radiation at doses of 8 or 16 Gy causes restenosis by augmenting proliferative activity at and adjacent to the site of stent implantation, and by dose-dependent up-regulation of extracellular matrix expression. The study suggests that excessive matrix accumulation is an important determinant of failure of radiation therapy to prevent restenosis. (+info)
Cyclic expression of mRNA transcripts for connective tissue components in the mouse ovary.
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In the ovary, differentiation of germinal cells into primordial follicles, functional ovulatory follicles and corpus luteum, all take place in a connective tissue matrix. We postulated that extracellular matrix (ECM) of the ovary participates actively in ovarian functions. To test this, the mRNA levels for several ECM components were determined in the mouse ovary at six distinct stages of the 4-day oestrous cycle. Northern analysis revealed statistically significant cyclic expression patterns for the mRNAs coding for type III, IV and VI collagens as well as for the small proteoglycan, biglycan, and for syndecan-1 and osteonectin. The cyclic changes observed in the mRNAs for these structural components exceeded those for matrix metalloproteinases (MMP)-2, -9 and -13, and for tissue inhibitors of matrix metalloproteinases (TIMP)-1, -2 and -3, where the changes were not statistically significant, despite their apparent role in ECM remodelling in the ovary. These observations support the hypothesis that cyclic changes in the production and degradation of ECM are part of normal ovarian function connected with follicular maturation, rupture and corpus luteum formation. (+info)