Harnessing chondroitin sulphate in composite scaffolds to direct progenitor and stem cell function for tissue repair.

The development of bioscaffolds that incorporate chondroitin sulphate (CS) and their applications with progenitor and stem cells in cartilage, bone, cornea, skin, and neural repair are reviewed. CS is a heterogeneous structure due to the organisation of multiple CS disaccharide sulphation motifs, giving rise to a vast range of CS chain structures, and hence the wide range of biological activity. The incorporation of this biological molecule represents a significant advance in bioscaffold design and performance in tissue repair strategies. The intrinsic stem-cell directive properties of CS are covered in the context of tissue development, and the differing CS disaccharide motifs, referred to as the 'glyco-code'. These structural motifs contribute to stem cell proliferation and differentiation in the scaffold environment and improve outcomes in terms of tissue repair or regeneration worthy of future research.

Pericellular colocalisation and interactive properties of type VI collagen and perlecan in the intervertebral disc.

The aim of this study was to immunolocalise type VI collagen and perlecan and determine their interactive properties in the intervertebral disc (IVD). Confocal laser scanning microscopy co-localised perlecan with type VI collagen as pericellular components of IVD cells and translamellar cross-bridges in ovine and murine IVDs. These cross-bridges were significantly less abundant in the heparin sulphate deficient Hspg2 exon 3 null mouse IVD than in wild type. This association of type VI collagen with elastic components provides clues as to its roles in conveying elastic recoil properties to annular tissues. Perlecan and type VI collagen were highly interactive in plasmon resonance studies. Pericellular colocalisation of perlecan and type VI collagen provides matrix stabilisation and cell-matrix communication which allows IVD cells to perceive and respond to perturbations in their biomechanical microenvironment. Perlecan, at the cell surface, provides an adhesive interface between the cell and its surrounding extracellular matrix. Elastic microfibrillar structures regulate tensional connective tissue development and function. The 2010 Global Burden of Disease study examined 291 disorders and identified disc degeneration and associated low back pain as the leading global musculoskeletal disorder emphasising its massive socioeconomic impact and the need for more effective treatment strategies. A greater understanding of how the IVD achieves its unique biomechanical functional properties is of great importance in the development of such therapeutic measures.

Altered decorin leads to disrupted endothelial cell function: a possible mechanism in the pathogenesis of fetal growth restriction?

OBJECTIVE: Fetal growth restriction (FGR) is a key cause of adverse pregnancy outcome where maternal and fetal factors are identified as contributing to this condition. Idiopathic FGR is associated with altered vascular endothelial cell functions. Decorin (DCN) has important roles in the regulation of endothelial cell functions in vascular environments. DCN expression is reduced in FGR. The objectives were to determine the functional consequences of reduced DCN in a human microvascular endothelial cell line model (HMVEC), and to determine downstream targets of DCN and their expression in primary placental microvascular endothelial cells (PLECs) from control and FGR-affected placentae. APPROACH: Short-interference RNA was used to reduce DCN expression in HMVECs and the effect on proliferation, angiogenesis and thrombin generation was determined. A Growth Factor PCR Array was used to identify downstream targets of DCN. The expression of target genes in control and FGR PLECs was performed. RESULTS: DCN reduction decreased proliferation and angiogenesis but increased thrombin generation with no effect on apoptosis. The array identified three targets of DCN: FGF17, IL18 and MSTN. Validation of target genes confirmed decreased expression of VEGFA, MMP9, EGFR1, IGFR1 and PLGF in HMVECs and PLECs from control and FGR pregnancies. CONCLUSIONS: Reduction of DCN in vascular endothelial cells leads to disrupted cell functions. The targets of DCN include genes that play important roles in angiogenesis and cellular growth. Therefore, differential expression of these may contribute to the pathogenesis of FGR and disease states in other microvascular circulations.

Characterization and purification of glycosaminoglycans from crude biological samples.

Chondroitin sulfate (CS) is a glycosaminoglycan derived from cartilage and commonly used to treat osteoarthritis, psoriasis, and other conditions. The dimethylmethylene blue (DMMB) assay has been used often to measure glycosaminoglycan levels in relatively pure samples. In this study, we verified the accuracy of the DMMB assay in measuring CS levels in unpurified extract from bovine trachea and shark cartilage, despite potential interference from salts, proteins, and DNA. We found that the glycosaminoglycan signal obtained was due to CS and not to other glycosaminoglycan species. This was confirmed using fluorophore-assisted carbohydrate electrophoresis, which also revealed that the majority of the CS was monosulfated at the C4 or C6 position. Finally, we used anion-exchange chromatography to purify the bovine extract and obtained complete recovery of the glycosaminoglycans, with no contaminating protein. The results of this study should be very useful for future purification and analysis of this common supplement.

Perlecan: how does one molecule do so many things?

Perlecan is a large multi-domain extracellular matrix proteoglycan that plays a crucial role in tissue development and organogenesis. In vertebrates, perlecan functions in a diverse range of developmental and biological processes, from the establishment of cartilage to the regulation of wound healing. How can a single molecule modulate such a wide variety of processes? We suggest that perlecan employs the same basic mechanism, based on interactions with growth factors, morphogens and matrix proteins, to regulate each of these processes and that the local extracellular environment determines the function of perlecan and consequently its downstream effects on the structure and function of the organ. We discuss this hypothesis in relation to its role in three major vertebrate developmental processes: angiogenesis, chondrogenesis and endochondral ossification.

The effect of silica nanoparticulate coatings on serum protein adsorption and cellular response.

Serum protein adsorption on colloidal silica surfaces was investigated using a quartz crystal microbalance with dissipation (QCM-D) monitoring. The amount of serum proteins adsorbed on colloidal silica-coated surfaces was not significantly different from the control silica surfaces, with the exception of 21nm colloidal silica which experienced significantly less (P<0.05) fibrinogen adsorption compared with control silica. The adhesion and proliferation of human endothelial cells (C11STH) on nano-scale colloidal silica surfaces were significantly reduced compared with control silica surfaces, suggesting that the conformation of adsorbed proteins on the colloidal silica surfaces plays a role in modulating the amount of cell binding. Fibronectin is one of the main extracellular matrix proteins involved in endothelial cell attachment to biomaterial surfaces. There was reduced binding of a monoclonal anti-fibronectin antibody, that reacted specifically with the cell-binding fragment, to fibronectin-coated colloidal silica surfaces compared with control silica surfaces. This suggests that the fibronectin adsorbed on the colloidal silica-coated surfaces was conformationally changed compared with control silica reducing the availability of the cell-binding domain of fibronectin.

The role of heparan sulfate and perlecan in bone-regenerative procedures.

Tissue engineering, grafting procedures, regeneration, and tissue remodeling are developing therapeutic modalities with great potential medical value, but these regenerative modalities are not as effective or predictable as clinicians and patients would like. Greater understanding of growth factors, cytokines, extracellular matrix molecules, and their roles in cell-mediated healing processes have made these regenerative therapies more clinically viable and will continue advancing the fields of tissue engineering and grafting. However, millions of oral and non-oral bone-grafting procedures are performed annually, and only a small percentage yield the most desirable results. Here we review the heparan-sulfate-decorated extracellular biomolecule named perlecan and the research relating to its potential as an adjunct in bone-regenerative procedures. The review includes an overview of bone graft substitutes and biological adjuncts to bone-regenerative procedures in medicine as they apply to periodontal disease, alveolar ridge augmentation, and barrier membrane therapy. Perlecan is discussed as a potential biological adjunct in terms of growth factor sequestration and delivery, and promoting cell adhesion, proliferation, differentiation, and angiogenesis. Further, we propose delivery and application schemes for perlecan and/or its domains in bone-regenerative procedures, with particular emphasis on its heparan-sulfate-decorated domain I. The perlecan molecule, with its heparan sulfate glycosylation, may provide a multi-faceted approach for the delivery of a more comprehensive stimulus than other single potential adjuncts currently available for bone-regenerative procedures.

Specific affinity depletion of cell adhesion molecules and growth factors from serum.

Serum is a common component of most in vitro cell culture media, particularly of primary cells. Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement. We describe a combined affinity protocol for removing vitronectin and fibronectin from serum. This protocol can also be used to purify these molecules. We also describe the removal of growth-promoting elements using heparin-Sepharose. As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence. This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems. It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors.

The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7.

Perlecan is a multifaceted heparan sulfate proteoglycan that is expressed not only as an intrinsic constituent of basement membranes but also as a cell-surface and pericellular proteoglycan. Perlecan functions as a ligand reservoir for various growth factors that become stabilized against misfolding or proteolysis and acts as a co-receptor for basic fibroblast growth factor by augmenting high affinity binding and receptor activation. These biological properties are mediated by the heparan sulfate moiety. Rather little is known about the protein core's mediation of functions. We have recently discovered that fibroblast growth factor-7 (FGF7) binds to perlecan protein core and that exogenous perlecan efficiently reconstitutes FGF7 mitogenic activity in perlecan-deficient cells. In this report we examined the specific binding of FGF7 to various domains and subdomains of perlecan protein core. Using several experimental approaches including overlay protein assays, radioligand binding experiments, and the yeast two-hybrid system, we demonstrate that FGF7 binds specifically to the N-terminal half of domain III and to a lesser extent to domain V, with affinity constants in the range of 60 nM. Thus, perlecan protein core should be considered a novel biological ligand for FGF7, an interaction that could influence cancer growth and tissue remodeling.

Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.

Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.

Expression of human perlecan domain I as a recombinant heparan sulfate proteoglycan with 20-kDa glycosaminoglycan chains.

Recombinant forms of human perlecan domain I were secreted as proteoglycans by stably transfected human 293 cells. A recombinant domain I-only proteoglycan spanned the 95- to 265-kDa region in SDS-PAGE and appeared to be 160 kDa in denaturing gel filtration. Its glycosaminoglycan (GAG) content was approximately 67% heparan sulfate, and its average GAG chain size of 20 kDa suggested that the true molecular mass of the proteoglycan was 90 kDa. Domain I with enhanced green fluorescent protein fused to its C-terminus had an apparent molecular mass of 210-220 kDa and contained approximately 100% heparan sulfate. Its average GAG chain size (also 20 kDa) suggested a true molecular mass of 117 kDa for this proteoglycan. Its sulfate content of 53-77 mol SO2-4 per mole of protein indicated the presence of one sulfate group per 4-7 GAG sugar residues.

Inhibition of endothelial cell adhesion and proliferation by extracellular matrix from vascular smooth muscle cells: role of type V collagen.

Endothelial cells recovering from damage due to disease or surgical procedures come into close contact with extracellular matrix (ECM) secreted by intimal vascular smooth muscle cells (VSMCs). We have investigated these relationships using human umbilical artery endothelial cells (HUAECs) and human mammary artery VSMC in vitro. HUAEC adhesion and proliferation were significantly lower on ECM secreted by VSMC compared with HUAEC ECM or surface-coated fibronectin. Characterisation of the ECM of both cell types with monoclonal antibodies showed that the ECM secreted by VSMC contained significantly more elastin, chondroitin sulphate and collagen types I, III and V than that from HUAECs. HUAECs adhered poorly to collagen type V coated on plastic and not at all to elastin. When these proteins were co-coated with fibronectin, elastin did not inhibit migration or proliferation compared to the response on fibronectin but collagen type V significantly inhibited both. Treatment of VSMC ECM with enzymes which selectively depleted the matrix of collagen types I, III and IV, or chondroitin sulphate, had no effect on HUAEC responses to the ECM, suggesting that these molecules did not contribute to the inhibition of HUAECs. Treatment of VSMC ECM with a mixture of collagenases, selectively depleted the matrix of collagen type V, as well as types I, III and IV. Such depleted ECMs supported increased proliferation of HUAECs compared to buffer controls. Overall these results suggest that collagen V secreted into the ECM of VSMC may inhibit the recovery of adjacent endothelium.

Heparin fails to inhibit the proliferation of human vascular smooth muscle cells in the presence of human serum.

The proliferation of vascular smooth muscle cells (VSMC) plays a significant part in both the developing atherosclerotic lesion and in restenosis. Heparin has been widely reported to inhibit the growth of VSMC in culture and intimal VSMC in some animal models of vascular hyperplasia. Clinical trials with heparin, however, have failed to inhibit restenosis following angioplasty. Bovine serum is normally used as a growth supplement in in vitro VSMC growth assays. We have compared the effects of human serum with those of bovine serum on the cellular response to heparin in human VSMC culture. While heparin inhibited the proliferation of human VSMC in the presence of bovine serum, it was totally ineffective in the presence of human serum. These observations were consistent over a wide range of serum and VSMC samples. Experiments utilizing neutralizing antibodies to a number of growth factors showed that cells in either serum were similarly dependent on platelet-derived growth factor for proliferation. In contrast, proliferation in the presence of bovine serum was shown to be dependent on extracellular basic fibroblast growth factor, whereas that in human serum was not. Direct binding of [3H]-heparin to VSMC was significantly reduced in the presence of human serum compared with bovine serum, and the former contained twice the concentration of heparin-binding factors of the latter. Removal of heparin-binding factors from either serum type significantly reduced the proliferation potential. Fractionation of heparin-binding factors from human serum showed that the major growth-promoting activity, together with heparin resistance, was contained within a fraction excluded by a 100,000 molecular weight membrane. We conclude that the mechanism of resistance to heparin in human serum is likely to be due to a combination of differential growth factor binding and interference with heparin interaction with cellular receptors by a high molecular weight heparin-binding factor. This phenomenon may significantly contribute to the lack of success of heparin as an antirestenotic agent in clinical trials.

Antisense targeting of perlecan blocks tumor growth and angiogenesis in vivo.

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide.

The testing of a 30-mer dG-rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC) in vitro and in vivo is described. LG4PS at 0.3 microM inhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited approximately 80% by 5 microM LG4PS. The growth of proliferating rabbit and pig VSMC was inhibited approximately 70% by 0.3 microM and 5 microM LG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMC in vitro were low. Despite these promising results, adventitial application of 2-200 nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon-injured rabbit carotid arteries, and the highest dose caused extensive mortality.

The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases.

Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing, and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was removed from perlecan by heparitinase treatment, and the approximately 450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of approximately 300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin, rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of the growth factor.

Interleukin-1 beta and transforming growth factor-alpha/epidermal growth factor induce expression of M(r) 95,000 type IV collagenase/gelatinase and interstitial fibroblast-type collagenase by rat mucosal keratinocytes.

Rat mucosal keratinocytes serially propagated under permanently serum-free conditions responded to interleukin (IL)-1 beta/IL-alpha and to transforming growth factor (TGF)-alpha/epidermal growth factor (EGF) (as well as to 12-O-tetradecanoylphorbol-13-acetate (TPA)) by upregulation of M(r) 95,000 gelatinase (MMP-9) (M(r) 95K GL) and fibroblast-type collagenase (MMP-1) (FIB-CL), whereas control cells expressed barely detectable levels of either of these enzymes. The cells secreted 8-10 micrograms/10(6) cells/day (M(r) 95K GL) and 2-3 micrograms/10(6) cells/day (FIB-CL) of enzyme protein for at least 24 h when maximally induced. This level was attained only after a 24-h lag period, and the earliest emergence of enzyme protein in the culture medium required 10-14 h. IL-1 beta was by far the most potent cytokine with maximal effect already at 10(-10) M, whereas IL-1 alpha, TGF-alpha, and EGF required 20-100-fold higher concentrations. Pretreatment of the cells with TPA (10(-7) M) abolished the subsequent response to IL-1 beta, TGF-alpha, and EGF and at the same time resulted in > 90% reduction of cytosolic protein kinase C activity. Surprisingly, staurosporine, a potent kinase inhibitor, not only failed to block growth factor/cytokine responses but itself stimulated expression of the enzymes at a magnitude comparable to TPA. The inducing effect of TGF-alpha/EGF was down-regulated by 70-85% by 10(-7) M dexamethasone. Dexamethasone was less effective in ablating the IL-1 beta response yielding 60% reduction M(r) 95K GL and little or no reduction of FIB-CL. Dexamethasone also failed to block the TPA response.

Multiple levels of post-transcriptional regulation of collagenase (matrix metalloproteinase 1) in an epithelial cell line.

Multiple levels of regulation of collagenase (matrix metalloproteinase 1; MMP-1), have been demonstrated in a clonal rat epithelial cell line (A5P/B10). Secreted enzyme could not be demonstrated in culture medium from A5P/B10 cells but, using antibodies specific for collagenase, the enzyme was detected within the cytoplasm and on the surface of the cells. A probe for rat collagenase could not detect a signal for mRNA in the cytoplasm while nuclear run-on data demonstrated that the gene for collagenase was being transcribed. Incubating the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly increased cytoplasmic mRNA levels and slightly increased the intensity of staining in permeabilized cells, but collagenase activity was still not detected in the conditioned medium. This indicated that the protein was being synthesized by the TPA-treated cells but was not being secreted into the medium. These data suggest that the secretion of collagenase may be regulated both following transcription and after the completion of translation and it is suggested that multiple levels of control may be operating to determine the rate of collagenase release and hence, the rate of collagen turnover.