Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan

The potent oxidants hypochlorous acid (HOCl) and hypobromous acid (HOBr) are produced extracellularly by myeloperoxidase, following release of this enzyme from activated leukocytes. The subendothelial extracellular matrix is a key site for deposition of myeloperoxidase and damage by myeloperoxidase-derived oxidants, with this damage implicated in the impairment of vascular cell function during acute inflammatory responses and chronic inflammatory diseases such as atherosclerosis. The heparan sulfate proteoglycan perlecan, a key component of the subendothelial extracellular matrix, regulates important cellular processes and is a potential target for HOCl and HOBr. It is shown here that perlecan binds myeloperoxidase via its heparan sulfate side chains and that this enhances oxidative damage by myeloperoxidase-derived HOCl and HOBr. This damage involved selective degradation of the perlecan protein core without detectable alteration of its heparan sulfate side chains, despite the presence of reactive GlcNH₂ residing within this glycosaminoglycan. Modification of the protein core by HOCl and HOBr (measured by loss of immunological recognition of native protein epitopes and the appearance of oxidatively-modified protein epitopes) was associated with an impairment of its ability to support endothelial cell adhesion, with this observed at a pathologically-achievable oxidant dose of 425 nmol oxidant/mg protein. In contrast, the heparan sulfate chains of HOCl/HOBr-modified perlecan retained their ability to bind FGF-2 and collagen V and were able to promote FGF-2-dependent cellular proliferation. Collectively, these data highlight the potential role of perlecan oxidation, and consequent deregulation of cell function, in vascular injuries by myeloperoxidase-derived HOCl and HOBr.     

The leucine-rich repeat protein PRELP binds perlecan and collagens and may function as a basement membrane anchor

PRELP (proline arginine-rich end leucine-rich repeat protein) is a heparin-binding leucine-rich repeat protein in connective tissue extracellular matrix. In search of natural ligands and biological functions of this molecule, we found that PRELP binds the basement membrane heparan sulfate proteoglycan perlecan. Also, recombinant perlecan domains I and V carrying heparan sulfate bound PRELP, whereas other domains without glycosaminoglycan substitution did not. Heparin, but not chondroitin sulfate, inhibited the interactions. Glycosaminoglycan-free recombinant perlecan domain V and mutated domain I did not bind PRELP. The dissociation constants of the PRELP-perlecan interactions were in the range of 3-18 nm as determined by surface plasmon resonance. As expected, truncated PRELP, without the heparin-binding domain, did not bind perlecan. Confocal immunohistochemistry showed that PRELP outlines basement membranes with a location adjacent to perlecan. We also found that PRELP binds collagen type I and type II through its leucine-rich repeat domain. Electron microscopy visualized a complex with PRELP binding simultaneously to the triple helical region of procollagen I and the heparan sulfate chains of perlecan. Based on the location of PRELP and its interaction with perlecan heparan sulfate chains and collagen, we propose a function of PRELP as a molecule anchoring basement membranes to the underlying connective tissue.     

Heparin–protein interactions: From affinity and kinetics to biological roles. Application to an interaction network regulating angiogenesis

Numerous extracellular proteins, growth factors, chemokines, cytokines, enzymes, lipoproteins, involved in a variety of biological processes, interact with heparin and/or heparan sulfate at the cell surface and in the extracellular matrix (ECM). The goal of this study is to investigate the relationship(s) between affinity and kinetics of heparin–protein interactions and the localization of the proteins, their intrinsic disorder and their biological roles. Most proteins bind to heparin with a higher affinity than their fragments and form more stable complexes with heparin than with heparan sulfate. Lipoproteins and matrisome-associated proteins (e.g. growth factors and cytokines) bind to heparin with very high affinity. Matrisome-associated proteins form transient complexes with heparin. However they bind to this glycosaminoglycan with a higher affinity than the proteins of the core matrisome, which contribute to ECM assembly and organization, and than the secreted proteins which are not associated with the ECM. The association rate of proteins with heparin is related to the intrinsic disorder of heparin-binding sites. Enzyme inhibitor activity, protein dimerization, skeletal system development and pathways in cancer are functionally associated with proteins displaying a high or very high affinity for heparin (K_D < 100 nM). Besides their use in investigating molecular recognition and functions, kinetics and affinity are essential to prioritize interactions in networks and to build network models as discussed for the interaction network established at the surface of endothelial cells by endostatin, a heparin-binding protein regulating angiogenesis.     

Perlecan: a major IL-2-binding proteoglycan in murine spleen

Although interleukin-2 (IL-2) is typically considered a soluble cytokine, our laboratory has shown that the availability of IL-2 in lymphoid tissues is regulated, in part, by an association with heparan sulfate glycosaminoglycan. Heparan sulfate is usually found in proteoglycan form, in which the heparan sulfate chains are covalently linked to a specific core protein. We now show that perlecan is one of the major IL-2-binding heparan sulfate proteoglycans in murine spleen. IL-2 binds perlecan via heparan sulfate chains, as enzymatic removal of heparan sulfate from splenic perlecan abolishes its ability to bind IL-2. Furthermore, we demonstrate that perlecan-bound IL-2 supports the proliferation of an IL-2-dependent cell line. Identification of perlecan as a major heparan sulfate proteoglycan that binds IL-2 has implications for both the localization and regulation of IL-2 in vivo.     

The role of vascular-derived perlecan in modulating cell adhesion,proliferation and growth factor signaling

Smooth muscle cell proliferation can be inhibited by heparan sulfate proteoglycans whereas the removal or digestion of heparan sulfate from perlecan promotes their proliferation. In this study we characterized the glycosaminoglycan side chains of perlecan isolated from either primary human coronary artery smooth muscle or endothelial cells and determined their roles in mediating cell adhesion and proliferation, and in fibroblast growth factor (FGF) binding and signaling. Smooth muscle cell perlecan was decorated with both heparan sulfate and chondroitin sulfate, whereas endothelial perlecan contained exclusively heparan sulfate chains. Smooth muscle cells bound to the protein core of perlecan only when the glycosaminoglycans were removed, and this binding involved a novel site in domain III as well as domain V/endorepellin and the α₂β₁ integrin. In contrast, endothelial cells adhered to the protein core of perlecan in the presence of glycosaminoglycans. Smooth muscle cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains and promoted the signaling of FGF2 but not FGF1. Also endothelial cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains, but in contrast, promoted the signaling of both growth factors. Based on this differential bioactivity, we propose that perlecan synthesized by smooth muscle cells differs from that synthesized by endothelial cells by possessing different signaling capabilities, primarily, but not exclusively, due to a differential glycanation. The end result is a differential modulation of cell adhesion, proliferation and growth factor signaling in these two key cellular constituents of blood vessels.     

硫酸乙酰肝素蛋白聚糖的功能机制研究进展

硫酸乙酰肝素蛋白聚糖是由核心蛋白和与之相连的硫酸乙酰肝素糖链组成,广泛分布于细胞膜与细胞外基质中。其中多配体蛋白聚糖（syndecan）和糖基磷脂酰肌醇锚定蛋白聚糖（glypican）存在与细胞膜上,而串珠蛋白聚糖（perlecan）和组合蛋白聚糖（agrin）表达在细胞外基质中。该类蛋白在生理与病理历程中,如发育、伤口愈合、肿瘤发生发展、感染、免疫应答等过程中担任重要作用,这些功能是其核心蛋白和糖链共同作用的结果。概述硫酸乙酰肝素蛋白聚糖的功能及其机制研究进展,同时强调其在作为药物靶标和临床诊断研究中的应用。     

Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan

Perlecan, the predominant basement membrane proteoglycan, has previously been shown to contain glycosaminoglycans attached at serine residues, numbers 65, 71, and 76, in domain I. However, the C-terminal domains IV and V of this molecule may also be substituted with glycosaminoglycan chains, but the exact substitution sites were not identified. The amino acid sequence of mouse perlecan reveals many ser-gly sequences in these domains that are possible sites for glycosaminoglycan substitution. We expressed recombinant domain IV and/or V of mouse perlecan in COS-7 cells and analyzed glycosaminoglycan substitution. Both heparan sulfate and chondroitin sulfate chains could be detected on recombinant domain V. One site, ser-gly-glu (serine residue 3593), toward the C-terminal region of domain V is a substitution site for heparan sulfate. When this sequence was absent, chondroitin/dermatan sulfate substitution was deleted, and the likely site for this galactosaminoglycan substitution was ser-gly-ala-gly (serine residue 3250) on domain V.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

beta-D-xyloside-mediated alteration in the synthesis of basement membrane proteoglycan

The effect of nitrophenyl-beta-D-xyloside (xyloside), a synthetic initiator of glycosaminoglycan synthesis, on proteoglycan and glycosaminoglycan synthesis by a basement membrane producing tumor was studied. While xyloside markedly stimulated the formation of chondroitin sulfate chains, it depressed the formation of a basement membrane heparan sulfate proteoglycan and caused only little formation of free heparan sulfate chains. However, when the synthesis of the core protein of the proteoglycan was inhibited by cycloheximide, heparan sulfate chains were produced by xyloside treatment. These heparan sulfate chains had a sulfate content higher than that of heparan sulfate found on the proteoglycan. The data indicate that xyloside can substitute for the heparan sulfate initiation site on the core protein of the proteoglycan and that this initiation is enhanced in the absence of core protein. This suggests that under normal conditions the formation of heparan sulfate chains may be tightly linked to the production of the core protein.     

Transient tropoelastin nanoparticles are early-stage intermediates in the coacervation of human tropoelastin whose aggregation is facilitated by heparan sulfate and heparin decasaccharides

Tropoelastin assembly is a key step in the formation of elastin. We consider how nanoscale intracellular assemblies of tropoelastin can congregate in an extracellular environment to give microscale aggregates. We describe novel 200–300 nm spherical particles that serve as intermediates in the formation of the coacervate. Their aggregation gives 800 nm to 1 µm species. This process is facilitated by heparan sulfate and dermatan sulfate interactions which effectively lower the critical concentration to facilitate this transition. This coacervation process was examined using a panel of heparin chains of various lengths and showed greatest efficacy for the decasaccharide, followed by the octasaccharide, while the hexasaccharide displayed the shortest efficacious length. We propose that these oligosaccharide interactions enable the charge-mediated aggregation of positively charged tropoelastin. This biochemistry models glycosaminoglycan interactions on the cell surface during elastogenesis which is characterized by the clustering of nascent tropoelastin aggregates to form micron-sized spherules.     

Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions

Heparan sulfate proteoglycans are complex molecules composed of a core protein with covalently attached glycosaminoglycan chains. While the protein part determines localization of the proteoglycan on the cell surfaces or in the extracellular matrix, the glycosaminoglycan component, heparan sulfate, mediates interactions with a variety of extracellular ligands such as growth factors and adhesion molecules. Through these interactions, heparan sulfate proteoglycans participate in many events during cell adhesion, migration, proliferation and differentiation. We are determining the multitude of proteoglycan functions, as their intricate roles in many pathways are revealed. They act as coreceptors for growth factors, participate in signalling during cell adhesion, modulate the activity of a broad range of molecules, and partake in many developmental and pathological processes, including tumorigenesis and wound repair. This review concentrates on biological roles of cell surface heparan sulfate proteoglycans, namely syndecans and glypicans, and outlines the progress achieved during the last decade in unraveling the molecular interactions behind proteoglycan functions.     

The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans

The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.     

Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures

The effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix (ECM) formation by cultured bovine corneal endothelial (BCE) cells was investigated. BCE cells actively proliferating on plastic dishes produced in the absence of xyloside an ECM containing various proteoglycans. Heparan sulfate was the main 35S-labeled glycosaminoglycan component (83%). Dermatan sulfate (14%) and chondroitin sulfate (3%) were also present. Exposure of actively proliferating BCE cells to xyloside totally inhibited synthesis of proteoglycans containing dermatan sulfate or chondroitin sulfate and caused an 86% inhibition of heparan sulfate proteoglycan synthesis. The heparan sulfate proteoglycans that were extracted from the ECM produced by BCE cells exposed to xyloside had a smaller size and a reduced charge density compared to their counterparts extracted from the ECM of cultures not exposed to xyloside. In contrast to the inhibitory effect of the xyloside on proteoglycan synthesis, exposure of actively proliferating BCE cells to xyloside stimulated synthesis of free chondroitin sulfate and heparan sulfate chains. All of the xyloside-initiated glycosaminoglycan chains were secreted into the culture medium. The proteoglycan-depleted matrices produced by BCE cells exposed to xyloside were used to study the effect of these matrices on proteoglycan synthesis by BCE cells. BCE cells growing on proteoglycan-depleted ECM showed a considerable increase in the rate of proteoglycan synthesis compared to BCE cells growing on normal ECM. Moreover, the pattern of glycosaminoglycan synthesis by BCE cells growing on proteoglycan-depleted ECM was changed to one which resembled that of BCE cells actively proliferating on plastic dishes. It is postulated that BCE cells are able to recognize when an ECM is depleted of proteoglycan and to respond to it by increasing their rate of proteoglycan synthesis and incorporation into the ECM.     

Heparan sulfate chains from glypican and syndecans bind the Hep II domain of fibronectin similarly despite minor structural differences

Numerous functions of heparan sulfate proteoglycans are mediated through interactions between their heparan sulfate glycosaminoglycan chains and extracellular ligands. Ligand binding specificity for some molecules, including many growth factors, is determined by complex heparan sulfate fine structure, where highly sulfated, iduronate-rich domains alternate with N-acetylated domains. Syndecan-4, a cell surface heparan sulfate proteoglycan, has a distinct role in cell adhesion, suggesting its chains may differ from those of other cell surface proteoglycans. To determine whether the specific role of syndecan-4 correlates with a distinct heparan sulfate structure, we have analyzed heparan sulfate chains from the different surface proteoglycans of a single fibroblast strain and compared their ability to bind the Hep II domain of fibronectin, a ligand known to promote focal adhesion formation through syndecan-4. Despite distinct molecular masses of glypican and syndecan glycosaminoglycans and minor differences in disaccharide composition and sulfation pattern, the overall proportion and distribution of sulfated regions and the affinity for the Hep II domain were similar. Therefore, adhesion regulation requires core protein determinants of syndecan-4.     

LTBP-2 competes with tropoelastin for binding to fibulin-5 and heparin,and is a negative modulator of elastinogenesis

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of ill-defined function associated with elastic fibers during elastinogenesis. Although LTBP-2 binds fibrillin-1, fibulin-5, and heparin/heparan sulfate, molecules critical for normal elastic fiber assembly, it does not interact directly with elastin or its precursor, tropoelastin. We investigated the modulating effect of LTBP-2 on two key interactions of tropoelastin during elastinogenesis a) with fibulin-5 and b) with heparan sulfate (using heparin). Firstly, using solid phase assays we showed that LTBP-2 bound fibulin-5 (Kd = 26.47 ± 5.68 nM) with an affinity similar to that of the tropoelastin-fibulin-5 interaction (Kd = 24.66 ± 5.64 nM). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In parallel experiments heparin was shown to have minor inhibitory effects on fibulin-5 interactions with tropoelastin and LTBP-2. However, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with heparan sulfate proteoglycans, perlecan and syndecan-4. Also addition of exogenous LTBP-2 to ear cartilage chondrocyte cultures blocked elastinogenesis in a concentration-dependent manner. Overall the results indicate that LTBP-2 may have a negative regulatory role during elastic fiber assembly, perhaps in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulfate proteoglycans.     

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.     

Chondroitin sulfate perlecan enhances collagen fibril formation. Implications for perlecan chondrodysplasias

Inactivation of the perlecan gene leads to perinatal lethal chondrodysplasia. The similarity to the phenotypes of the Col2A1 knock-out and the disproportionate micromelia mutation suggests perlecan involvement in cartilage collagen matrix assembly. We now present a mechanism for the defect in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional to the content of the 4,6-disulfated disaccharide in the different cartilage extracts, with growth plate cartilage glycosaminoglycan being the most efficient enhancer. These findings demonstrate a role for perlecan chondroitin sulfate side chains in cartilage extracellular matrix assembly and provide an explanation for the perlecan-null chondrodysplasia.     

Syndecan-1 mediates cell spreading in transfected human lymphoblastoid (Raji) cells

Syndecan-1 is a cell surface proteoglycan containing a highly conserved transmembrane and cytoplasmic domain, and an extracellular domain bearing heparan sulfate glycosaminoglycans. Through these domains, syndecan-1 is proposed to have roles in growth factor action, extracellular matrix adhesion, and cytoskeletal organization that controls cell morphology. To study the role of syndecan-1 in cell adhesion and cytoskeleton reorganization, mouse syndecan-1 cDNA was transfected into human Raji cells, a lymphoblastoid cell line that grows as suspended cells and exhibits little or no endogenous cell surface heparan sulfate. High expressing transfectants (Raji-Sl cells) bind to and spread on immobilized thrombospondin or fibronectin, which are ligands for the heparan sulfate chains of the proteoglycan. This binding and spreading as not dependent on the cytoplasmic domain of the core protein, is mutants expressing core proteins with cytoplasmic deletions maintain the ability to spread. The spreading is mediated through engagement of the syndecan-1 core protein, as the Raji-S 1 cells also bind to and spread on immobilized mAb 281.2, an antibody specific for the ectodomain of the syndecan-1 core protein. Spreading on the antibody is independent of the heparan sulfate glycosaminoglycan chains and can be inhibited by competition with soluble mAb 281.2. The spreading can be inhibited by treatment with cytochalasin D or colchicine. These data suggest that the core protein of syndecan-1 mediates spreading through the formation of a multimolecular signaling complex at the cell surface that signals cytoskeleton reorganization. This complex may form via intramembrane or extracellular interactions with the syndecan core protein.     

Adult equine bone marrow stromal cells produce a cartilage-like ECM mechanically superior to animal-matched adult chondrocytes

Our objective was to evaluate the age-dependent mechanical phenotype of bone marrow stromal cell- (BMSC-) and chondrocyte-produced cartilage-like neo-tissue and to elucidate the matrix-associated mechanisms which generate this phenotype. Cells from both immature (2–4 month-old foals) and skeletally-mature (2–5 year-old adults) mixed-breed horses were isolated from animal-matched bone marrow and cartilage tissue, encapsulated in self-assembling-peptide hydrogels, and cultured with and without TGF-β1 supplementation. BMSCs and chondrocytes from both donor ages were encapsulated with high viability. BMSCs from both ages produced neo-tissue with higher mechanical stiffness than that produced by either young or adult chondrocytes. Young, but not adult, chondrocytes proliferated in response to TGF-β1 while BMSCs from both age groups proliferated with TGF-β1. Young chondrocytes stimulated by TGF-β1 accumulated ECM with 10-fold higher sulfated-glycosaminoglycan content than adult chondrocytes and 2–3-fold higher than BMSCs of either age. The opposite trend was observed for hydroxyproline content, with BMSCs accumulating 2–3-fold more than chondrocytes, independent of age. Size-exclusion chromatography of extracted proteoglycans showed that an aggrecan-like peak was the predominant sulfated proteoglycan for all cell types. Direct measurement of aggrecan core protein length and chondroitin sulfate chain length by single molecule atomic force microscopy imaging revealed that, independent of age, BMSCs produced longer core protein and longer chondroitin sulfate chains, and fewer short core protein molecules than chondrocytes, suggesting that the BMSC-produced aggrecan has a phenotype more characteristic of young tissue than chondrocyte-produced aggrecan. Aggrecan ultrastructure, ECM composition, and cellular proliferation combine to suggest a mechanism by which BMSCs produce a superior cartilage-like neo-tissue than either young or adult chondrocytes.