Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant's Superior Transmission.

Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more transmissible than the original virus, the SARS-CoV-2 B.1.617 lineage, named the Delta variant, swept through the world in 2021. The mutations in the Delta's spike protein shift the protein towards a net positive electrostatic potential. To understand the key molecular drivers of the Delta infection, we investigate the cellular uptake of the Delta spike protein and Delta spike-bearing SARS-CoV-2 pseudoviruses. Specific in vitro modification of ACE2 and syndecan expression enabled us to demonstrate that syndecan-4, the syndecan isoform abundant in the lung, enhances the transmission of the Delta variant by attaching its mutated spike glycoprotein and facilitating its cellular entry. Compared to the wild-type spike, the Delta one shows a higher affinity towards heparan sulfate proteoglycans than towards ACE2. In addition to attachment to the polyanionic heparan sulfate chains, the Delta spike's molecular interactions with syndecan-4 also involve syndecan-4's cell-binding domain that mediates cell-to-cell adhesion. Regardless of the complexity of these interactions, exogenously added heparin blocks Delta's cellular entry as efficiently as syndecan-4 knockdown. Therefore, a profound understanding of the molecular mechanisms underlying Delta infections enables the development of molecularly targeted yet simple strategies to reduce the Delta variant's spread.

Dispirotripiperazine-core compounds, their biological activity with a focus on broad antiviral property, and perspectives in drug design (mini-review).

Viruses are obligate intracellular parasites and have evolved to enter the host cell. To gain access they come into contact with the host cell through an initial adhesion, and some viruses from different genus may use heparan sulfate proteoglycans for it. The successful inhibition of this early event of the infection by synthetic molecules has always been an attractive target for medicinal chemists. Numerous reports have yielded insights into the function of compounds based on the dispirotripiperazine scaffold. Analysis suggests that this is a structural requirement for inhibiting the interactions between viruses and cell-surface heparan sulfate proteoglycans, thus preventing virus entry and replication. This review summarizes our current knowledge about the early history of development, synthesis, structure-activity relationships and antiviral evaluation of dispirotripiperazine-based compounds and where they are going in the future.

Lactoferrin as potential preventative and adjunct treatment for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is rapidly advancing across the globe despite drastic public and personal health measures. Antivirals and nutritional supplements have been proposed as potentially useful against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that causes COVID-19, but few have been clinically established. Lactoferrin (Lf) is a naturally occurring, non-toxic glycoprotein that is orally available as a nutritional supplement and has established in vitro antiviral efficacy against a wide range of viruses, including SARS-CoV, a closely related coronavirus to SARS-CoV-2. Furthermore, Lf possesses unique immunomodulatory and anti-inflammatory effects that may be especially relevant to the pathophysiology of severe COVID-19 cases. Here we review the underlying biological mechanisms of Lf as an antiviral and immune regulator, and propose its unique potential as a preventative and adjunct treatment for COVID-19. We hope that further research and development of Lf nutritional supplementation would establish its role for COVID-19.

Receptor tyrosine kinases and heparan sulfate proteoglycans: Interplay providing anticancer targeting strategies and new therapeutic opportunities.

The development of pharmacological and biological inhibitors of receptor tyrosine kinases (RTKs) has changed the treatment paradigm of several neoplastic diseases. However, the occurrence of intrinsic and acquired resistance represents a limit to the efficacy of these drugs even in RTK-addicted cancers. The identification of innovative therapeutic approaches and rationale-based drug combinations remains a primary need to improve patients' outcome. Heparan sulfate proteoglycans (HSPGs) at the cell surface and in the extracellular matrix bind to and modulate the biological activity of a great number of heparan sulfate (HS) binding proteins. The participation of HSPGs as accessory molecules in the growth factor-receptor interactions and mechanism of activation of several RTKs provides the basis for developing alternative therapeutic strategies based on targeting HSPGs by antibodies or HS mimetics to interfere with the aberrant oncogenic signaling implicated in the pathobiology of several tumors. Here, we focus on the FGF-FGFR-HSPG and HGF-Met-HSPG axes as paradigmatic examples of the multiple-level interconnections between RTKs and HSPGs influencing cell signaling, gene expression, drug sensitivity, and promoting a permissive microenvironment for tumor growth and progression. In these reciprocal regulations, the HS degrading enzymes heparanase and endosulfatases play key roles contributing to the high structural complexity and heterogeneity of HS chains as well as to the specificity of their interaction with proteins. Actually, heparanase and endosulfatases represent, in turn, promising therapeutic targets. We also report some studies describing the effects of FGFR and Met inhibitors on the expression of genes encoding HSPGs and related enzymes, and discuss about the potential impact of these effects on drug response. Finally, we argue about the need of in-depth investigation of the role of HSPGs and their modifying enzymes in specific tumor pathologies to exploit the opportunity of combination treatments including HS mimetics or HSPG directed antibodies to improve efficacy of RTK inhibitors and overcome drug resistance.

Internalization mechanisms of brain-derived tau oligomers from patients with Alzheimer's disease, progressive supranuclear palsy and dementia with Lewy bodies.

Tau aggregates propagate in brain cells and transmit to neighboring cells as well as anatomically connected brain regions by prion-like mechanisms. Soluble tau aggregates (tau oligomers) are the most toxic species that initiate neurodegeneration in tauopathies, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Exogenous tau aggregates have been shown to be internalized by brain cells; however, the precise cellular and molecular mechanisms that underlie the internalization of tau oligomers (TauO) remain elusive. Using brain-derived tau oligomers (BDTOs) from AD, PSP, and DLB patients, we investigated neuronal internalization mechanisms of BDTOs, including the heparan sulfate proteoglycan (HSPG)-mediated pathway, clathrin-mediated pathway, and caveolae-mediated pathway. Here, we demonstrated that the HSPG-mediated pathway regulates internalization of BDTOs from AD and DLB, while HSPG-mediated and other alternative pathways are involved in the internalization of PSP-derived tau oligomers. HSPG antagonism significantly reduced the internalization of TauO, prevented tau translocation to the endosomal-lysosomal system, and decreased levels of hyperphosphorylated tau in neurons, the well-known contributor for neurofibrillary tangles (NFT) accumulation, degeneration of neurons, and cognitive decline. Furthermore, siRNA-mediated silencing of heparan sulfate (HS)-synthesizing enzyme, exostosin-2, leads to decreased internalization of BDTOs, prevented tau-induced autophagy-lysosomal pathway impairment, and decreased hyperphosphorylated tau levels. Collectively, these findings suggest that HSPG-mediated endocytosis and exostsin-2 are involved in neuronal internalization of TauO and subsequent tau-dependent neuropathology in AD and DLB.

Phagocyte Transcriptomic Analysis Reveals Focal Adhesion Kinase (FAK) and Heparan Sulfate Proteoglycans (HSPGs) as Major Regulators in Anti-bacterial Defense of Crassostrea hongkongensis.

Invertebrates generally lack adaptive immunity and compensate for this with highly efficient innate immune machineries such as phagocytosis by hemocytes to eradicate invading pathogens. However, how extrinsically cued hemocytes marshal internal signals to accomplish phagocytosis is not yet fully understood. To this end, we established a facile magnetic cell sorting method to enrich professional phagocytes from hemocytes of the Hong Kong oyster (Crassostrea hongkongensis), an ecologically and commercially valuable marine invertebrate. Transcriptomic analysis on presorted cells shows that phagocytes maintain a remarkable array of differentially expressed genes that distinguish them from non-phagocytes, including 352 significantly upregulated genes and 479 downregulated genes. Pathway annotations reveal that focal adhesion and extracellular matrix-receptor interactions were the most conspicuously enriched pathways in phagocytes. Phagocytosis rate dramatically declined in the presence of an FAK inhibitor, confirming importance of the focal adhesion pathway in regulating phagocytosis. In addition, we also found that heparan sulfate proteoglycan (HSPG) families were lineage-specifically expanded in C. hongkongensis and abundantly expressed in phagocytes. Efficiency of phagocytosis and hemocytes aggregation was markedly reduced upon blockage of endogenous synthesis of HSPGs, thus implicating these proteins as key surface receptors in pathogen recognition and initiation of phagocytosis.

Targeting Heparan Sulfate Proteoglycans and their Modifying Enzymes to Enhance Anticancer Chemotherapy Efficacy and Overcome Drug Resistance.

Targeting heparan sulfate proteoglycans (HSPGs) and enzymes involved in heparan sulfate (HS) chain editing is emerging as a new anticancer strategy. The involvement of HSPGs in tumor cell signaling, inflammation, angiogenesis and metastasis indicates that agents able to inhibit aberrant HSPG functions can potentially act as multitarget drugs affecting both tumor cell growth and the supportive boost provided by the microenvironment. Moreover, accumulating evidence supports that an altered expression or function of HSPGs, or of the complex enzyme system regulating their activities, can also depress the tumor response to anticancer treatments in several tumor types. Thereby, targeting HSPGs or HSPG modifying enzymes appears an appealing approach to enhance chemotherapy efficacy. A great deal of effort from academia and industry has led to the development of agents mimicking HS, and/or inhibiting HSPG modifying enzymes. Inhibitors of Sulf-2, an endosulfatase that edits the HS sulfation pattern, and inhibitors of heparanase, the endoglycosidase that produces functional HS fragments, appear particularly promising. In fact, a Sulf-2 inhibitor (OKN-007), and two heparanase inhibitors/HS mimics (roneparstat, PG545) are currently under early clinical investigation. In this review, we summarized preclinical studies in experimental tumor models of the main chemical classes of Sulf-2 and heparanase inhibitors. We described examples of different mechanisms through which heparanase and HSPGs, often in cooperation, may impact tumor sensitivity to various antitumor agents. Finally, we reported a few preclinical studies showing increased antitumor efficacy obtained with the use of candidate clinical HS mimics in combination regimens.

Disruption of endogenous perlecan function improves differentiation of rat articular chondrocytes in vitro.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) are necessary for normal cartilage development and chondrocyte differentiation. However, recent studies demonstrated that HSPG accelerate dedifferentiation and catabolism in chondrocytes from degenerative cartilage. In this study, we investigated the inhibitory effect of HSPG on chondrocyte differentiation in vitro. Rat articular chondrocytes were cultured at low (0.3 × 10(4) cells/cm(2) ) and high (1.5 × 10(5) cells/cm(2) ) density in the presence or absence of heparitinase I, an HS degrading enzyme. Cells cultured at low density dedifferentiated and exhibited an elongated morphology, and treatment with heparitinase I precluded cell elongation. Conversely, populations of chondrocytes cultured at high density exhibited either a dedifferentiated or differentiated phenotype. Glycosaminoglycan accumulation increased in heparitinase I-treated cells. To determine the function of perlecan, an important HSPG for cartilage development, in chondrocyte differentiation, rat chondrocyte cultures were exposed to an anti-perlecan antiserum to inhibit perlecan function. Western blotting analysis indicated that preventing perlecan activity increased type II collagen synthesis. Our results suggest that HSPG are negative regulators of chondrocyte differentiation in vitro and that perlecan contributes to chondrocyte dedifferentiation in vitro.

Contrasting effect of perlecan on adipogenic and osteogenic differentiation of mesenchymal stem cells in vitro.

Perlecan, a basement membrane component, shows diverse functions in different organs and tissues. However, the role of perlecan in differentiation of mesenchymal stem cells (MSCs) has been barely investigated. In this study, we examined the effect of perlecan on adipogenic and osteogenic differentiation of MSCs in vitro by adding extrinsic perlecan to culture media or blocking the function of intrinsic perlecan expressed into culture media by differentiating MSCs. Extrinsic perlecan suppressed adipogenic differentiation; however, it promoted osteogenic differentiation. These functions were further confirmed by a study of blocking intrinsic perlecan. Perlecan treated with heparitinase-I also showed the suppressive effect on adipogenic differentiation. In contrast, the promotive effect on osteogenic differentiation was found to be heparan sulfate-dependent. Intrinsic perlecan was suggested to be effective at the late stage of adipogenic differentiation by a study of perlecan-blocking performed at distinct periods, but was suggested to be effective at the early stage of osteogenic differentiation. Our results showed perlecan has contrasting effect on adipogenic and osteogenic differentiation of MSCs due to its diverse actions. Based on these outcomes, we recognized that employing extrinsic perlecan or blocking intrinsic perlecan is effective for regulating adipogenic and osteogenic differentiation of MSCs by restricting its direction.

The function of heparanase in diabetes and its complications.

Heparan sulfate proteoglycans are ubiquitous glycoproteins that contain several heparan sulfate polysaccharide side chains attached to a core protein. They function not only as a primary structural component of the extracellular matrix, but also provide a storage depot for bioactive molecules, such as basic fibroblast growth factor, vascular endothelial growth factor and lipoprotein lipase. Heparanase is an endoglycosidase that specifically hydrolyzes heparan sulfate into oligosaccharides. Recent studies have indicated that heparanase is engaged in the initiation and progression of diabetes, in addition to its associated complications. This review focuses on the participation of heparanase in the cleavage of heparan sulfate proteoglycans in pancreatic islets promoting beta cell death, promotion of atherosclerosis, and its role in cardiac metabolic switching in the early stage of cardiomyopathy during diabetes. Understanding the mechanisms by which heparanase is regulated in diabetes could provide a drug target to prevent diabetes and its complications.

Direct and allosteric inhibition of the FGF2/HSPGs/FGFR1 ternary complex formation by an antiangiogenic, thrombospondin-1-mimic small molecule.

Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

A spoonful of sugar makes the melanoma go: the role of heparan sulfate proteoglycans in melanoma metastasis.

Heparan sulfate proteoglycans (HSPGs) have been shown to regulate signaling in many systems and are of increasing interest in cancer. While these are not the only sugars to drive melanoma metastasis, HSPGs play important roles in driving metastatic signaling cascades in melanoma. The ability of these proteins to modulate ligand-receptor interactions in melanoma has been quite understudied. Recent data from several groups indicate the importance of these ligands in modulating key signaling pathways including Wnt and fibroblast growth factor (FGF) signaling. In this review, we summarize the current knowledge regarding the structure and function of these proteoglycans and their role in melanoma. Understanding how HSPGs modulate signaling in melanoma could lead to new therapeutic approaches via the dampening or heightening of key signaling pathways.

Heparan sulphate proteoglycan and the low-density lipoprotein receptor-related protein 1 constitute major pathways for neuronal amyloid-beta uptake.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder in which the aggregation and deposition of amyloid-ß (Aß) peptides in the brain are central to its pathogenesis. In healthy brains, Aß is effectively metabolized with little accumulation. Cellular uptake and subsequent degradation of Aß is one of the major pathways for its clearance in the brain. Increasing evidence has demonstrated significant roles for the low-density lipoprotein receptor-related protein 1 (LRP1) in the metabolism of Aß in neurons, glia cells, and along the brain vasculatures. Heparan sulfate proteoglycan (HSPG) has also been implicated in several pathogenic features of AD, including its colocalization with amyloid plaques. Here, we demonstrate that HSPG and LRP1 cooperatively mediate cellular Aß uptake. Fluorescence-activated cell sorter and confocal microscopy revealed that knockdown of LRP1 suppresses Aß uptake, whereas overexpression of LRP1 enhances this process in neuronal cells. Heparin, which antagonizes HSPG, significantly inhibited cellular Aß uptake. Importantly, treatment with heparin or heparinase blocked LRP1-mediated cellular uptake of Aß. We further showed that HSPG is more important for the binding of Aß to the cell surface than LRP1. The critical roles of HSPG in cellular Aß binding and uptake were confirmed in Chinese hamster ovary cells genetically deficient in HSPG. We also showed that heparin and a neutralizing antibody to LRP1 suppressed Aß uptake in primary neurons. Our findings demonstrate that LRP1 and HSPG function in a cooperative manner to mediate cellular Aß uptake and define a major pathway through which Aß gains entry to neuronal cells.

Novel glycosaminoglycan biosynthetic inhibitors affect tumor-associated angiogenesis.

Heparan sulfate proteoglycans (HSPGs) are essential players in several steps of tumor-associated angiogenesis. As co-receptors for several pro-angiogenic factors such as VEGF and FGF, HSPGs regulate receptor-ligand interactions and play a vital role in signal transduction. Previously, we have employed an enzymatic strategy to show the importance of cell surface HSPGs in endothelial tube formation in vitro. We have recently found several fluoro-xylosides that can selectively inhibit proteoglycan synthesis in endothelial cells. The current study demonstrates that these fluoro-xylosides are effective inhibitors of endothelial tube formation in vitro using a matrigel based assay to simulate tumor-associated angiogenesis. These first generation scaffolds offer a promising stepping-stone to the discovery of more potent fluoro-xylosides that can effectively neutralize tumor growth.

Heparan sulfate proteoglycans in extravasation: assisting leukocyte guidance.

Heparan sulfate proteoglycans (HSPGs) are glycoconjugates that are implicated in various biological processes including development, inflammation and repair, which is based on their capacity to bind and present several proteins via their carbohydrate side chains (glycosaminoglycans; GAGs). Well-known HSPGs include the family of syndecans and glypicans, which are expressed on the plasma membrane and perlecan, agrin and collagen type XVIII, which are present in basement membranes. In this review, we provide an overview of the current knowledge on the role and regulation of HSPGs in leukocyte extravasation. In the non-inflamed endothelial glycocalyx HSPGs are anti-adhesive, and there are several indications that active regulation of HSPG core protein expression and/or GAG modification occurs upon inflammation. We address the current evidence for the role of HSPGs in leukocyte extravasation through interaction with the leukocyte adhesion molecule L-selectin, chemokines and other binding partners. Finally, a number of possibilities to use HSPGs as therapeutics or targets in anti-inflammatory strategies are discussed.

Novel interactions of perlecan: unraveling perlecan's role in angiogenesis.

Perlecan, a highly conserved and ubiquitous basement membrane heparan sulfate proteoglycan, is essential for life, inasmuch as its absence results in embryonic lethality in mice and C. elegans, and neonatal lethality in humans. Perlecan plays an essential role in vasculogenesis and chondrogenesis, as well as in pathological states where these processes are maladapted. Although a large body of evidence supports a pro-angiogenic role for perlecan, recent findings suggests that portions of the perlecan protein core can be antiangiogenic, requiring a further evaluation of the functioning of this complex molecule. This review is focused on the genetics of mammalian and nonmammalian perlecan, the elucidation of its novel interacting partners and its role in angiogenesis. By more fully understanding perlecan's functioning in angiogenesis, we may gain invaluable insight that could lead to therapeutic interventions in cancer and other pathologic states.

Non-anticoagulant heparins and inhibition of cancer.

Low-molecular-weight heparins (LMWH) appear to prolong survival of patients with cancer. Such a beneficial effect is thought to be associated with interruption of molecular mechanisms involving the heparan sulfate (HS) chains of cell surface and extracellular matrix proteoglycans (HSPGs), growth factors and their receptors, heparanase, and selectins. The beneficial effects of heparin species could also be associated with their ability to release tissue factor pathway inhibitor from endothelium. The utility of heparin and LMWH as anticancer drugs is limited due to their anticoagulant properties. Non-anticoagulant heparins can be obtained either by removing chains containing the antithrombin-binding sequence, or by inactivating critical functional groups or units of this sequence. The non-anticoagulant heparins most extensively studied are regioselectively desulfated heparins and 'glycol-split' heparins. Some modified heparins of both types are potent inhibitors of heparanase. A number of them also attenuate metastasis in experimental models. With cancer cells overexpressing selectins, heparin-mediated inhibition of tumor cells-platelets aggregation and tumor cell interaction with the vascular endothelium appears to be the prevalent mechanism of attenuation of early stages of metastasis. The structural requirements for inhibition of growth factors, heparanase, and selectins by heparin derivatives are somewhat different for the different activities. An N-acetylated, glycol-split heparin provides an example of application of a non-anticoagulant heparin that inhibits cancer in animal models without unwanted side effects. Delivery of this compound to mice bearing established myeloma tumors dramatically blocked tumor growth and progression.

The syndecan-1 heparan sulfate proteoglycan is a viable target for myeloma therapy.

The heparan sulfate proteoglycan syndecan-1 is expressed by myeloma cells and shed into the myeloma microenvironment. High levels of shed syndecan-1 in myeloma patient sera correlate with poor prognosis and studies in animal models indicate that shed syndecan-1 is a potent stimulator of myeloma tumor growth and metastasis. Overexpression of extracellular endosulfatases, enzymes which remove 6-O sulfate groups from heparan sulfate chains, diminishes myeloma tumor growth in vivo. Together, these findings identify syndecan-1 as a potential target for myeloma therapy. Here, 3 different strategies were tested in animal models of myeloma with the following results: (1) treatment with bacterial heparinase III, an enzyme that degrades heparan sulfate chains, dramatically inhibited the growth of primary tumors in the human severe combined immunodeficient (SCID-hu) model of myeloma; (2) treatment with an inhibitor of human heparanase, an enzyme that synergizes with syndecan-1 in promoting myeloma progression, blocked the growth of myeloma in vivo; and (3) knockdown of syndecan-1 expression by RNAi diminished and delayed myeloma tumor development in vivo. These results confirm the importance of syndecan-1 in myeloma pathobiology and provide strong evidence that disruption of the normal function or amount of syndecan-1 or its heparan sulfate chains is a valid therapeutic approach for this cancer.

Mechanisms of heparin induced anti-cancer activity in experimental cancer models.

BACKGROUND: Retrospective analyses of clinical trials and prospective clinical studies have suggested that heparins may have an effect on cancer survival. This putative anti-cancer activity of heparins is supported by data from studies in animal tumour models. OBJECTIVE: To clarify the various potential mechanisms of heparin anti-cancer activity we evaluated the data from pre-clinical studies in which heparins have been tested as anti-cancer therapy. METHODS: Pre-clinical studies, published between 1960 and 2005 were assessed. Data were collected on the type and dose of heparin used, duration of exposure to heparin, interval between heparin administration and cancer cell inoculation, and the animal tumour model used. In addition, a distinction was made in the analysis between heparin effects on the primary tumour or on established metastases and effects on the metastatic potential of infused cells. RESULTS: Heparins seemed to affect the formation of metastasis rather than the growth of primary tumours. Chemically modified heparins with no or limited anticoagulant activity also showed anti-metastatic properties. Possible mechanisms to explain the effects on the process of metastases include inhibition of blood coagulation, inhibition of cancer cell-platelet and -endothelial interactions by selectin inhibition and inhibition of cell invasion and angiogenesis. CONCLUSION: The anti-cancer activity of heparins depends more on inhibition of metastasis formation than on the effects on primary tumour growth. These effects are probably related to both coagulation and non-coagulation dependent factors. For a definitive proof of the anti-cancer activity of heparins in the clinic, prospective randomized trials especially in patients with early metastatic disease or in the adjuvant setting are urgently needed.

Ribozyme-mediated perlecan knockdown impairs chondrogenic differentiation of C3H10T1/2 fibroblasts.

Perlecan (Pln) is an abundant heparan sulfate (HS) proteoglycan in the pericellular matrix of developing cartilage, and its absence dramatically disrupts endochondral bone formation. This study examined two previously unexamined aspects of the function of Pln in mesenchymal chondrogenesis in vitro. Using the well-established high-density micromass model of chondrogenic differentiation, we first examined the requirement for endogenous Pln synthesis and secretion through the use of Pln-targeted ribozymes in murine C3H10T1/2 embryonic fibroblasts. Second, we examined the ability of the unique N-terminal, HS-bearing Pln domain I (PlnDI) to synergize with exogenous bone morphogenetic protein-2 (BMP-2) to support later stage chondrogenic maturation of cellular condensations. The results provide clear evidence that the function of Pln in late stage chondrogenesis requires Pln biosynthesis and secretion, because 60%-70% reductions in Pln greatly diminish chondrogenic marker expression in micromass culture. Additionally, these data support the idea that while early chondrocyte differentiation can be supported by exogenous HS-decorated PlnDI, efficient late stage PlnDI-supported chondrogenesis requires both BMP-2 and Pln biosynthesis.