Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.

Recombinant human FGF18 preserves depth-dependent mechanical inhomogeneity in articular cartilage.

Articular cartilage is a specialised tissue that has a relatively homogenous endogenous cell population but a diverse extracellular matrix (ECM), with depth-dependent mechanical properties. Repair of this tissue remains an elusive clinical goal, with biological interventions preferred to arthroplasty in younger patients. Osteochondral transplantation (OCT) has emerged for the treatment of cartilage defects and osteoarthritis. Fresh allografts stored at 4 °C have been utilised, though matrix and cell viability loss remains an issue. To address this, several studies have developed media formulations to maintain cartilage explants in vitro. One promising factor for these applications is sprifermin, a human-recombinant fibroblast growth factor-18, which stimulates chondrocyte proliferation and matrix synthesis and is in clinical trials for the treatment of osteoarthritis. The study hypothesis was that addition of sprifermin during storage would maintain the unique depth-dependent mechanical profile of articular cartilage explants, a feature not often evaluated. Explants were maintained for up to 6 weeks with or without a weekly 24 h exposure to sprifermin (100 ng/mL) and the compressive modulus was assessed. Results showed that sprifermin-treated samples maintained their depth-dependent mechanical profile through 3 weeks, whereas untreated samples lost their mechanical integrity over 1 week of culture. Sprifermin also affected ECM balance by maintaining the levels of extracellular collagen and suppressing matrix metalloproteinase production. These findings support the use of sprifermin as a medium additive for OCT allografts during in vitro storage and present a potential mechanism where sprifermin may impact a functional characteristic of articular cartilage in repair strategies.

A large animal model that recapitulates the spectrum of human intervertebral disc degeneration.

OBJECTIVE: The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics. DESIGN: Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (µCT), and measurement of disc biomechanical properties. RESULTS: Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact. CONCLUSIONS: A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration.

A high-throughput model of post-traumatic osteoarthritis using engineered cartilage tissue analogs.

OBJECTIVE: A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. METHOD: We utilized a high-throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. RESULTS: Engineered cartilage response to injury was strain dependent, with a 2-fold increase in glycosaminoglycan (GAG) loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in lactate dehydrogenase (LDH) release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor [Z-VAD-FMK (ZVF)] was effective at reducing cell death, while the amphiphilic polymer [Poloxamer 188 (P188)] and the free-radical scavenger [N-Acetyl-L-cysteine (NAC)] reduced GAG loss as compared to injury alone. CONCLUSIONS: The injury response in this engineered cartilage model replicated key features of the response of cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA.

Prostaglandin E inhibits the production of human interleukin 2.

Prostaglandins of the E type specifically inhibited the production of interleukin 2 (IL-2) by normal human lymphocytes, whereas PG synthetase inhibitors such as indomethacin and fentiazac raised IL-2 production above normal levels. Removal of adherent cells from mononuclear cell populations also resulted in enhanced IL-2 production. The resultant nonadherent cell population lost sensitivity to the enhancement effect of PG synthetase inhibitors, suggesting that a PGE-producing adherent cell plays a major role in the regulation of IL-2.

Immunohistochemical detection and immunochemical analysis of type II collagen degradation in human normal, rheumatoid, and osteoarthritic articular cartilages and in explants of bovine articular cartilage cultured with interleukin 1.

Articular cartilage destruction and loss of function in arthritic diseases involves proteolytic degradation of the connective tissue matrix. We have investigated the degradation of cartilage collagen by developing immunochemical methods that permit the identification and analysis of type II collagen degradation in situ. Previously, a technique to specifically identify type II collagen degradation in situ in articular cartilage did not exist. These methods utilize a polyclonal antiserum (R181) that specifically reacts with unwound alpha-chains and CNBr-derived peptides, alpha 1(II)CB11 and alpha 1(II)CB8, of human and bovine type II collagens. The experimental approach is based on the fact that when fibrillar collagens are cleaved the helical collagen molecule unwinds, exposing hidden epitopes. Here we demonstrate the use of R181 in studying type II collagen degradation in bovine articular cartilage that has been cultured with or without IL-1 and in human normal, rheumatoid, and osteoarthritic articular cartilages. Compared to cartilages either freshly isolated or cultured without IL-1, bovine cartilage cultured with IL-1 for 3-5 d showed an increase in both pericellular and intercellular immunohistochemical staining. Extracts of these cartilages contained type II collagen alpha chains that were increased in amount after culture with IL-1 for 11 d. In addition, culture with IL-1 resulted in the appearance of alpha chain fragments of lower molecular weight. All human arthritic tissues examined showed areas of pronounced pericellular and territorial staining for collagen degradation as compared with non-diseased tissues, indicating that chondrocytes are responsible in part for this degradation as compared with non-diseased tissues. In most cases rheumatoid cartilage was stained most intensely at the articular surface and in the deep and mid-zones, whereas osteoarthritic cartilage usually stained more in the superficial and mid-zones, but less intensely. Distinct patterns of sites of collagen degradation reflect differences in collagen destruction in these diseases, suggesting possible different sources of chondrocyte activation. These experiments demonstrate the application of immunological methods to detect collagen degradation and demonstrate an increase of collagen degradation in human arthritides and in IL-1-treated viable bovine cartilage.

Stimulation of protein synthesis in isolated pancreatic acini from chronically ethanol-fed rats is due to alterations in post-transcriptional regulation.

In an earlier study, we reported that isolation of acini from the pancreas of rats fed ethanol chronically, led to a 2- to 3-fold increase in the rate of protein synthesis compared to acini from rats fed the control diet. In the present study, we wanted to investigate whether the enhanced rate of protein synthesis was due to an increased rate of degranulation, reflecting a stimulation of cellular signal transduction processes, and/or to changes at the level of transcription/translation. The rate of degranulation was monitored by initially prelabelling the secretory proteins in vivo with [3H]leucine followed by determination of their fate in the intact tissue as well as in the subsequently isolated acini. The recovery of the label in isolated acini as a fraction of that incorporated into the tissue was similar for control and ethanol-fed groups, suggesting that ethanol feeding had no effect on the rate of degranulation during the isolation of acini. The rate of incorporation of [3H]uridine into total RNA was about 70% higher in acini from the ethanol-fed group as compared to the control group, suggesting a higher rate of transcription. However, the steady-state level of mRNA for trypsinogen, a representative digestive enzyme mRNA, showed only a moderate increase of 20% in acini from the ethanol-fed group compared to those from the intact tissue. These results suggest that the increased rate of protein synthesis in isolated acini from ethanol-fed rat pancreas is primarily due to post-transcriptional modifications.

Immunochemical and immunohistochemical evidence of estrogen-mediated collagenolysis as a mechanism of cervical dilatation in the guinea pig at parturition.

The mechanism of dilatation of the uterine cervix at birth is poorly understood. Several indirect lines of evidence have suggested that cervical ripening is accompanied by collagen degradation. In this study, immunochemical methods have been developed to identify and analyze type I collage degradation in the cervix of the pregnant guinea pig. Using cyanogen bromide-derived peptides of purified guinea pig type I collagen as an immunogen, a polyclonal rabbit antiserum was prepared that recognizes epitopes on the denatured and degraded alpha 2 chain of type I collagen as demonstrated by enzyme-linked immunosorbant assay and immunoblotting. This antibody was used to demonstrate degradation of type I collagen in the extracellular matrix of the dilated cervix at parturition. Moreover, physiological concentrations of 17 beta-estradiol stimulated degradation of type I collagen in the nonpregnant cervix in organ culture. Collagenase degradation products were detected in the extracellular matrix and in the culture media. The effect of 17 beta-estradiol (10(-6) M) was completely blocked by progesterone (10(-4) M). These studies suggest that dilatation of the guinea pig cervix at parturition may be associated with estrogen-mediated degradation of type I collagen.

Cloning of equine type II procollagen and the modulation of its expression in cultured equine articular chondrocytes.

The complete nucleotide sequence of equine type II procollagen has not been previously reported, and equine-specific probes have not been available. We report the complete sequence and discuss the molecular characteristics of equine type II procollagen mRNA which was cloned from a cDNA library prepared from mRNA isolated from equine articular chondrocytes. The coding sequence (4257 bp) was 92.4% homologous to the cDNA of the human sequence, and the propeptide was 97% identical to the human sequence. We demonstrated that when equine chondrocytes are grown in phenotypically-maintained cultures, the expression of type II procollagen is linked to the age of the animal. Additionally, in cultures of young equine chondrocytes, IL1-beta and TNF-alpha both reduced the expression of type II procollagen mRNA in a dose responsive manner.

Characterization of human type II procollagen and collagen-specific antibodies and their application to the study of human type II collagen processing and ultrastructure.

Type II collagen is the most abundant collagen in articular cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shock-absorbing properties and its resiliency to stress. Specific antibodies which recognize various collagen types have been very useful in the study of collagen biosynthesis, structure and metabolism in normal and pathological conditions. Antibodies which recognize epitopes of type II collagen have been described previously; however, many of these antibodies display cross-reactivity with other collagens or with type II collagen from other species, reflecting the high degree of homology of the helical domains of fibrillar collagens. In this study, we prepared antibodies to sequential determinants of human type II procollagen employing synthetic peptides with sequences deduced from the nucleotide sequence of the human alpha 1 (II) procollagen cDNA. The antibodies were highly specific for epitopes in either the C-terminal propeptide or the telopeptide of the human type II collagen and did not cross-react with other human interstitial collagens or with murine type II collagen. These antibodies were used in conjunction with biosynthetic labeling to study the secretion and processing of human type II procollagen and collagen in human chondrocytes in vitro. The results indicated that a lag period of about 90 min was required for the secretion of newly synthesized type II procollagen. Conversion of the secreted procollagen into fully processed alpha-chains and their deposition in the cell layer were first apparent 240 min following the initiation of biosynthetic labeling. The antibodies were also used to examine, by immunoelectron microscopy, the structure of the extracellular matrix produced by human chondrocytes maintained in long-term cultures under conditions which permit the preservation of the cartilage-specific phenotype. These highly specific antibodies provide valuable tools to study the metabolism and structure of human type II procollagen and collagen in normal and pathologic conditions.

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes.

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.

Molecular characteristics of equine stromelysin and the tissue inhibitor of metalloproteinase 1.

OBJECTIVE: To clone the entire coding sequence of equine matrix metalloproteinase-3 (MMP-3, stromelysin) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and compare their nucleotide and amino acid sequences with those of MMP-3 and TIMP-1 from other species. SAMPLES: Articular cartilage harvested from the joints of 4 foals, 2 yearlings, and 3 adult horses. PROCEDURE: A cDNA library was constructed from mRNA extracted from equine chondrocytes. The library was screened and clones selected that contained the cDNA for MMP-3 and TIMP-1. The cDNA was sequenced and the nucleotide and deduced amino acid sequences compared with known sequences in other species. Northern blot analysis was performed, using the resulting cDNA clones. RESULTS: An 1803-bp cDNA for MMP-3 including the entire coding sequence of 1434 bases was cloned and sequenced. A 744-bp cDNA for TIMP-1 including the entire coding sequence of 624 bases was cloned and sequenced. Northern analysis revealed MMP-3 to hybridize to a single mRNA species at approximately 2.1 kb. TIMP-1 hybridized to a single mRNA species at approximately 0.8 kb. CONCLUSIONS: MMP-3 and TIMP-1 were highly homologous to that of other species at the nucleotide and amino acid level although each had unique residues in part of the peptide that is generally conserved. CLINICAL RELEVANCE: Understanding the molecular structure of MMP-3 and TIMP-1 and the availability of their cDNA should allow a more detailed understanding of their balance in cartilage and the degradative processes in joint disease.

Dose-dependent effects of corticosteroids on the expression of matrix-related genes in normal and cytokine-treated articular chondrocytes.

OBJECTIVE AND DESIGN: To assess the effects of glucocorticoids on the expression of multiple matrix-related genes in normal and cytokine-treated cultured equine articular chondrocytes in a phenotypically correct suspension culture. MATERIAL OR SUBJECTS: Articular cartilage harvested from the joints of 15 foals, 7 yearling horses, and 16 adult horses. TREATMENT: Glucocorticoids (dexamethasone, prednisolone, triamcinolone) at 10(-10) to 10(-4) M. METHODS: Equine articular chondrocytes maintained in suspension cultures were treated with glucocorticoids with and without human recombinant interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Northern blots of total RNA from the treated cells were probed with equine specific cDNA probes for a number of cartilage matrix-related genes. Zymography, Western blotting, and fluorography were also performed to study the effects on protein synthesis. RESULTS: The glucocorticoids, dexamethasone, triamcinolone, and prednisolone, markedly decreased MMP1, MMP3, MMP13, TIMPI, and ferritin steady-state mRNA levels. There were no qualitative differences seen among the tested corticosteroids although dexamethasone and triamcinolone appeared to be slightly more potent than prednisolone. The effects of the glucocorticoids on MMP transcription occurred consistently at lower doses than those required to similarly downregulate type II collagen and aggrecan. Link protein and fibronectin mRNA were increased by the glucocorticoids, and biglycan and decorin were minimally affected. Fluorography of [14-C] proline-labeled media demonstrated that the decrease in type II collagen transcription (mRNA levels) was paralleled at the protein level. Zymography and Western blotting confirmed the decrease in functional metalloproteinases found in chondrocyte cultures following glucocorticoid treatment. CONCLUSIONS: The effects of glucocorticoids are complex inasmuch as they differentially affect numerous genes involved in the composition of cartilage matrix and the degradation of that matrix. This study provides new insight into the effects of glucocorticoids on the regulation of extra-cellular matrix and matrix-related genes by demonstrating that low doses of glucocorticoids can inhibit the degradative metalloproteinases with minimal negative effects on the transcription of extracellular matrix genes.

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes.

OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.

The degradation of type II collagen in rheumatoid arthritis: an immunoelectron microscopic study.

Rabbit antibodies were prepared that react only with denatured type II collagen alpha-chains and cleavage products. The epitopes that these antibodies recognize reside in cyanogen bromide peptides CB8 and CB11. The antibodies do not react with triple helical collagen nor with any other collagen or protein present in hyaline cartilage (Dodge and Poole, J. Clin. Invest. 83:647-661, 1989). These antibodies can therefore be used to detect denatured type II collagen produced, for example, by enzymatic cleavage. In this study they were used to determine, at the ultrastructural level, using immunogold staining, type II collagen fibril cleavage in articular cartilages remote from synovium and pannus of patients with rheumatoid arthritis. Comparisons were made with site- and age-matched healthy articular cartilages. Antibody binding was detected in the extracellular matrix, at the articular surface and in the deep zone, usually on visibly damaged collagen fibrils which exhibited a loss of the normal banding pattern of staining produced by lead citrate and uranyl acetate: binding was also observed in disrupted fibrils, sometimes at their ends. Binding was commonly associated with amorphous-looking material (and occasionally unstained sites) in the extracellular matrix which, because of the specificity of the antibody, can be identified as containing denatured or fragmented type II collagen, stained (and unstained) with heavy metals. In both rheumatoid and healthy articular cartilages, there was no antibody binding to intact well stained fibrils which exhibited a regular banding pattern. Little or no staining was detected at the ultrastructural level in normal cartilages.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the basement membrane heparan sulfate proteoglycan (perlecan) in human synovium and in cultured human synovial cells.

BACKGROUND: Perlecan is a heparan sulfate proteoglycan that has a core protein of 466 kDa and is composed of five modules, four of which share a high degree of homology with a variety of biologically important molecules including the low-density lipoprotein receptor, laminin, and the neural cell adhesion molecule. Previously, this specialized proteoglycan, characteristic of the basement membrane, was not identified as a constitutive extracellular component of human synovium. EXPERIMENTAL DESIGN: Using human synovium and cultured human synovial cells, we examined perlecan gene expression in human synovium, a tissue lacking a classic basement membrane. The modulation of gene expression of the perlecan core protein by transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (FGF) was examined in cultures of normal human synovial cells, and by metabolic labeling using radioactive sodium sulfate, the question of whether this specialized protein is produced by synovial cells as a proteoglycan was addressed. RESULTS: Cultures of normal human synovial cells were shown to contain the large 14.5 kb perlecan mRNA and produced substantial amounts of perlecan core protein as shown by immunohistochemistry employing specific human perlecan Ab. Immunohistochemical detection showed intense staining in the intimal and subintimal layers of human synovial membrane. Moreover, the perlecan core protein was shown to be up-regulated by TGF-beta and down-regulated by basic FGF. In addition, a sizable portion (approximately 25%) of the synovial cell-produced proteoglycan was shown to contain heparan sulfate, providing evidence that synovial cell perlecan is produced as a proteoglycan. CONCLUSIONS: The presence of perlecan in human synovium and the modulation of the biosynthesis of its core protein by TGF-beta and FGF suggest that, in addition to its structural role, this unique heparan sulfate proteoglycan may be involved in normal synovial membrane function and in the pathogenesis of arthritis.

Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor.

We have determined the complete nucleotide and deduced amino acid sequence of the major protein core of the human heparan sulfate proteoglycan HSPG2/perlecan of basement membranes. Eighteen overlapping cDNA clones comprise 14.35 kilobase pairs (kb) of contiguous sequence with an open reading frame of 13.2 kb. The mature protein core, without the signal peptide of 21 amino acids, has a M(r) of 466,564. This large protein is composed of multiple modules homologous to the receptor of low density lipoprotein, laminin, neural cell adhesion molecules, and epidermal growth factor. Domain I, near the amino terminus, appears unique for the proteoglycan since it shares no significant homology with any other proteins. It contains three Ser-Gly-Asp sequences that could act as attachment sites for heparan sulfate glycosaminoglycans. Domain II is highly homologous to the LDL receptor and contains four repeats with perfect conservation of all 6 consecutive cysteines. Next is domain III which shares homology to the short arm of laminin A chain and contains four cysteine-rich regions intercalated among three globular domains. Domain IV, the largest module with greater than 2000 residues, contains 21 repeats of the immunoglobulin type as found in neural cell adhesion molecule. Near the beginning of this domain, there is a stretch of 29 hydrophobic amino acids which could allow the molecule to interact with the plasma membrane. Domain V, similar to the carboxyl-terminal globular G-domain of laminin A and to the related protein merosin, contains three globular regions and four EGF-like repeats. In situ hybridization and immunoenzymatic studies show a close association of this gene product with a variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Our studies show that this proteoglycan is present in all vascularized tissues and suggest that this unique molecule has evolved from the utilization of modular structures with adhesive and growth regulatory properties.