Requires Heparan Sulfate Proteoglycans Research Articles

Antiviral activity of metal chelates of caffeic acid and similar compounds towards herpes simplex, VSV-Ebola pseudotyped and vaccinia viruses

Organic compounds with a caffeoyl moiety (e.g. caffeic acid, rosmarinic acid, chicoric acid, etc.) have antiviral properties towards herpes simplex (HSV), influenza and immunodeficiency viruses (HIV). This study evaluated the HSV antiviral properties of caffeic acid when paired with a variety of metal and other inorganic ions. The results demonstrated that the antiviral activity of caffeic acid increased upwards of 100-fold by the addition of cations, such as Fe3+, and anionic molecules, such as molybdate and phosphate. Cellular toxicity tests of the caffeic acid chelates showed that they have low toxicities with selectivity indices (TD50/EC50) for Fe3+, MoO42−, and PO43− chelates being 1700, >540, and >30, respectively. Caffeic acid paired with Fe3+ was tested against eight strains of viruses, including those from different families. The caffeic acid chelates were mostly effective against HSV1 and HSV2, but they also had moderate activity against vaccinia virus and a VSV-Ebola pseudotyped virus. All the viruses that were strongly impacted by the caffeic chelates require heparan sulfate proteoglycans for cellular attachment, so it is likely that caffeic chelates target and interfere with this mechanism. Since the caffeic acid chelates target an extra-cellular process, they might be able to be combined with existing medications, such as acyclovir, that target an intracellular process to achieve greater viral control.

Fractones: proteoglycan matrix for growth factor activity in health and disease

Growth factors, cytokines and chemokines are proteins that influence cell proliferation, differentiation, migration, apoptosis and other biological determinants that determine cell fate and cell functions, ultimately the phenomena of generation, regeneration, and degeneration throughout life. Most of these molecules diffuse throughout biological fluids and bind heparin. In vivo they require heparan sulfate proteoglycans (HSPG) located in the extracellular matrix (ECM) to target their cell‐surface receptors and ultimately induce specific biological effects. We have characterized by immunohistochemistry and electron microscopy a new structure found in some parts of the extracellular space as part of the ECM that we named fractones. Fractones are chemically similar to basement membranes and consist of laminin isoforms, collagen‐IV, nidogen and HSPG. However, fractones and basement membranes differ by their location. Fractones reside in the parenchyma of tissues and organs and are visualized as particles by light microscopy whereas they often display a fractal ultrastructure (a reference to their name). In contrast, basement membranes are located as sheaths at the connective tissue/parenchyma interface and in between the layers of the vascular wall. We have shown that fractone‐associated heparan sulfates bind, concentrate, and activate the growth factors FGF‐2, BMP‐4 and BMP‐7 to regulate stem cell proliferation in the neurogenic zone of the adult brain. We have also found fractones are abundantly expressed throughout mammalian development and distributed in a manner that reflects morphogenesis. Fractones were found in all animals that we investigated including vertebrates (human, mouse and rats) and invertebrates (cnidarians, sponges, copepods, decapodes, insects). HSPG are highly dynamic molecules formed by post‐translational processes in both the Golgi apparatus, and in the ECM where extracellular enzymes are also secreted. Our results strongly suggest that fractones are essential chemicals for constructing and maintaining biological structures. We are currently elaborating the model of tissue explants, i.e. miniature fragments of organs to study growth factors/fractone interactions that will ultimately provide insights into the mechanisms that control cell fate in a near‐physiological context. We anticipate that the model explants originating from physiological animals and from animal disease models will revolutionize our understanding of molecular interactions between proteoglycans and their ligands through tissues and organs and throughout life. This research will provide the basics for new paradigms that orchestrate biological structures and functions in health and disease. We are interested in establishing methods to identify specific carbohydrate sequences in the ECM structures that bind signaling proteins. This may allow elucidation of a signaling code used by tissues for growth and maintenance. Establishment of such a code would allow for discovery of disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transendothelial migration of effector T cells across inflamed endothelial barriers does not require heparan sulfate proteoglycans

Leukocyte diapedesis is a chemotactic multistep process that requires optimal chemoattractant presentation by the endothelial barrier. Recent studies have described a critical role for heparan sulfate glycosaminoglycans (HSGAGs) in the presentation and functions of chemokines essential for lymphocyte interactions with the lymph node vasculature. We wished to test whether HS expression by a prototypic endothelial cell type, i.e. human umbilical vein endothelial cells (HUVECs), is critical for their ability to support neutrophil and lymphocyte adhesion and transendothelial migration (TEM) under shear flow. We found that HUVECs deposit HS GAGs mainly at their basolateral compartments in both their resting and inflamed states. We next inactivated the key enzyme involved in HS biosynthesis, exostosin-1 (Ext1). Silencing Ext1 resulted in a complete loss of HS biosynthesis; nonetheless, TNF-α and IL-1β stimulation of key adhesion molecules and inflammatory chemokines necessary for neutrophil or lymphocyte adhesion and TEM remained intact. Ext1 silencing reduced neutrophil arrest and markedly impaired TEM, consistent with a role of basolateral HS GAGs in directing neutrophil crossing of inflamed endothelial barriers. Strikingly, however, the TEM of effector T cells across identically Ext1-silenced HUVECs remained normal. Importantly, the biosynthesis of the main promigratory chemokines for effector T cells and neutrophils, respectively, CCL2 and CXCL1, and their vesicle distributions were also Ext1 independent. These results suggest that transmigrating neutrophils must respond to chemokines transiently presented by apical and basolateral endothelial HS GAGs. In contrast, effector T cells can integrate chemotactic TEM signals directly from intra-endothelial chemokine stores rather than from externally deposited chemokines.

Heparin increases the infectivity of Human Papillomavirus Type 16 independent of cell surface proteoglycans and induces L1 epitope exposure

Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell-binding receptors for HPV-16, heparin-preincubated virus bound to the extracellular matrix (ECM) via laminin-332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S-domains of heparan sulfate (HS) chains of HSPGs, allowed HPV-16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope-specific antibody to the viral capsid after heparin binding suggested that initial conformational changes in the HPV-16 virion occur during infection by interaction with'heparin-like' domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV-16 infection.

Motoneurons Secrete Angiogenin to Induce RNA Cleavage in Astroglia

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder affecting motoneurons. Mutations in angiogenin, encoding a member of the pancreatic RNase A superfamily, segregate with ALS. We previously demonstrated that angiogenin administration shows promise as a neuroprotective therapeutic in studies using transgenic ALS mice and primary motoneuron cultures. Its mechanism of action and target cells in the spinal cord, however, are largely unknown. Using mixed motoneuron cultures, motoneuron-like NSC34 cells, and primary astroglia cultures as model systems, we here demonstrate that angiogenin is a neuronally secreted factor that is endocytosed by astroglia and mediates neuroprotection in paracrine. We show that wild-type angiogenin acts unidirectionally to induce RNA cleavage in astroglia, while the ALS-associated K40I mutant is also secreted and endocytosed, but fails to induce RNA cleavage. Angiogenin uptake into astroglia requires heparan sulfate proteoglycans, and engages clathrin-mediated endocytosis. We show that this uptake mechanism exists for mouse and human angiogenin, and delivers a functional RNase output. Moreover, we identify syndecan 4 as the angiogenin receptor mediating the selective uptake of angiogenin into astroglia. Our data provide new insights into the paracrine activities of angiogenin in the nervous system, and further highlight the critical role of non-neuronal cells in the pathogenesis of ALS.

Two Distinct Sites in Sonic Hedgehog Combine for Heparan Sulfate Interactions and Cell Signaling Functions

Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for their normal distribution and signaling activity. Here, we have used molecular modeling to examine the heparin-binding domain of sonic hedgehog (Shh). In biochemical and cell biological assays, the importance of specific residues of the putative heparin-binding domain for signaling was assessed. It was determined that key residues in human (h) Shh involved in heparin and HSPG syndecan-4 binding and biological activity included the well known cationic Cardin-Weintraub motif (lysines 32-38) but also a previously unidentified major role for lysine 178. The activity of Shh mutated in these residues was tested by quantitation of alkaline phosphatase activity in C3H10T1/2 cells differentiating into osteoblasts and hShh-inducible gene expression in PANC1 human pancreatic ductal adenocarcinoma cells. Mutated hShhs such as K37S/K38S, K178S, and particularly K37S/K38S/K178S that could not interact with heparin efficiently had reduced signaling activity compared with wild type hShh or a control mutation (K74S). In addition, the mutant hShh proteins supported reduced proliferation and invasion of PANC1 cells compared with control hShh proteins, following endogenous hShh depletion by RNAi knockdown. The data correlated with reduced Shh multimerization where the Lys-37/38 and/or Lys-178 mutations were examined. These studies provide a new insight into the functional roles of hShh interactions with HSPGs, which may allow targeting this aspect of hShh biology in, for example, pancreatic ductal adenocarcinoma.

HIV‐1 Infection of Trophoblasts Is Independent of gp120/CD4 Interactions but Relies on Heparan Sulfate Proteoglycans

Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) is the leading cause of HIV infection in infants. Direct infection of trophoblasts--cells forming the placental barrier--may cause this transmission. Entry of HIV-1 into trophoblasts is unusual for this retrovirus, because it is associated with endocytosis. However, given that trophoblasts express no or few receptors/coreceptors required for virus internalization, the mechanism underlying this event remains ambiguous. In the present study, we show that HIV-1 entry and infection of polarized trophoblasts are independent not only of CD4 but also of envelope (Env) glycoproteins gp120 and gp41. Virus internalization, cytoplasmic release, reverse transcription, integration, and HIV-1 gene expression occurred with both fusion-incompetent and Env-deficient viruses. Importantly, fusion-independent infection was observed when we used viruses produced in a natural cellular reservoir (i.e., primary human cells). Finally, HIV-1 requires heparan sulfate proteoglycans for uptake in trophoblasts. Together, our findings illustrate that HIV-1 utilizes an unusual pathway for entering human polarized trophoblasts.

Glycobiology of Neural Stem Cells

The mammalian central nervous system is organized by a variety of cells, such as neurons and glial cells, that are generated from a common progenitor, the neural stem cell (NSC). NSCs are defined as undifferentiated neural cells that are characterized by their high proliferative potential while retaining the capacity for self-renewal and multipotency. NSCs and their progeny may be distinguished by the expression of glycoconjugates (e.g., glycoproteins, glycolipids, and proteoglycans). The carbohydrate antigens carried by glycoconjugates are mainly localized on the plasma membrane surface of the cells and they serve as excellent biomarkers for various stages of cellular differentiation. Thus, they have been utilized as ligands for sorting NSCs or their progeny by cell cytometry. Methods have been established for utilizing polysialic acid-neural cell adhesion molecule (PSA-NCAM), stage-specific embryonic antigen-1 (SSEA-1), and gangliosides for cell sorting. Furthermore, glycoconjugates have also been suggested to have a wide range of receptor and signaling functions in NSCs. For example, basic fibroblast growth factor, an important mitogen of NSCs, requires heparan sulfate proteoglycans and glycosylated cystatin C for activity. Notch signaling, which regulates a wide variety of developmental processes in various cells including NSCs, is modulated by the O-fucose glycan modification. In peripheral nervous system (PNS), the human natural killer-1 (HNK-1) antigen regulates the migration of neural crest cells, cell populations containing the stem cells. Thus, glycoconjugates serve not only as marker molecules, but also as functional molecules as well. In the present review, we discuss the expression pattern and possible functions of glycoconjugates in NSCs.

Artemin Crystal Structure Reveals Insights into Heparan Sulfate Binding

Artemin (ART) promotes the growth of developing peripheral neurons by signaling through a multicomponent receptor complex comprised of a transmembrane tyrosine kinase receptor (cRET) and a specific glycosylphosphatidylinositol-linked co-receptor (GFRalpha3). Glial cell line-derived neurotrophic factor (GDNF) signals through a similar ternary complex but requires heparan sulfate proteoglycans (HSPGs) for full activity. HSPG has not been demonstrated as a requirement for ART signaling. We crystallized ART in the presence of sulfate and solved its structure by isomorphous replacement. The structure reveals ordered sulfate anions bound to arginine residues in the pre-helix and amino-terminal regions that were organized in a triad arrangement characteristic of heparan sulfate. Three residues in the pre-helix were singly or triply substituted with glutamic acid, and the resulting proteins were shown to have reduced heparin-binding affinity that is partly reflected in their ability to activate cRET. This study suggests that ART binds HSPGs and identifies residues that may be involved in HSPG binding.

Heparan Sulfate in trans Potentiates VEGFR-Mediated Angiogenesis

Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.

Glycobiology of neural stem cells: Functional implications

The mammalian central nervous system is organized by a variety of cells, such as neurons and glial cells, that are generated from a common progenitor, the neural stem cell (NSC). NSCs are defined as undifferentiated neural cells that are characterized by their high proliferative potential while retaining the capacity for self-renewal and multipotency. NSCs and their progeny may be distinguished by the expression of glycoconjugates (e.g., glycoproteins, glycolipids, and proteoglycans). The glycoconjugates are mainly localized on the plasma membrane surface of the cells and they serve as unique biomarkers for various stages of cellular differentiation. Thus, they have been utilized as ligands for sorting NSCs or their progeny by cell cytometry. Furthermore, glycoconjugates have also been suggested to have a wide range of receptor and signaling functions in NSCs. For example, basic fibroblast growth factor, an important mitogen of NSCs, requires heparan sulfate proteoglycans and glycosylated cystatin C for activity. Notch signaling, which regulates a wide variety of developmental processes in various cells including NSCs, is modulated by the O-fucose glycan modification. In PNS, the HNK-1 antigen regulates the cell migration of neural crest cells, cell populations containing the stem cells. Thus, glycoconjugates serves not only as marker molecules, but also as functional molecules as well. In the present presentation, we discuss the expression pattern and possible functions of several specific glycoconjugates, including GD3, O-acetyl GD3, and SSEA-1, in NSCs. Supported by NIH NS11853-31.

Genetic Alterations in Heparan Sulfate Disrupt MCP‐1 Binding in the Injured Vessel

Chemokine binding and distribution require heparan sulfate proteoglycans as co-receptors and are influenced by sulfation of the heparan sulfate (HS) side chains. The objective was to test the hypothesis that alterations in HS sulfation would disrupt chemokine binding and influence vascular remodeling in the setting of injury. We previously demonstrated that injury induced a 20 fold increase in mRNA encoding N-deactylase/N-sulfotransferase-1 (NDST1), an enzyme catalyzing the initial N-sulfation of HS side chains. We established a genetic mouse model in which NDST1 was deleted in smooth muscle (smMHCcre/NDST1flox). This model exhibited a significant decrease in NDST1 mRNA and in the ratio of mono-N-sulfated/unsulfated disaccharides (control 0.45±0.02 vs smMHCcre/NDST1flox 0.22±0.02, n=4, p<0.05). The macrovasculature develops normally in these mice. However, vascular injury induces a marked disruption in the gradient distribution of monocyte chemoattractant protein-1, a chemokine requiring HS and known to play a critical role in vascular remodeling. Similar results were observed in a second genetic model harboring a single allele deletion of NDST1. In sum, these findings provide early genetic evidence that HS sulfation is critical for chemokine distribution in the vessel in response to injury. This study is funded by American Heart Association.

Indian hedgehog and syndecans-3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis.

Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS-PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS-PGs for function. Because the HS-PG syndecan-3 is preferentially expressed by proliferating chondrocytes, we tested whether it mediates Ihh action. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh-N) in absence or presence of heparinase I or syndecan-3 neutralizing antibodies. While rIhh-N stimulated proliferation in control cultures, it failed to do so in heparinase- or antibody-treated cultures. In reciprocal gain-of-function studies, chondrocytes were made to overexpress syndecan-3 by an RCAS viral vector. Cells became more responsive to rIhh-N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4-5 chick wing buds and effects of syndecan-3 misexpression were monitored over time. Syndecan-3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan-3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan-3 and to identify a specific member of the syndecan family as mediator of hedgehog function.

Internalization but not binding of thrombospondin-1 to low density lipoprotein receptor-related protein-1 requires heparan sulfate proteoglycans.

The amino-terminal domain of the extracellular matrix (ECM) protein thrombospondin-1 (TSP-1) mediates binding to cell surface heparan sulfate proteoglycans (HSPG) as well as binding to the endocytic receptor, low density lipoprotein-related protein (LRP-1). We previously found that recombinant TSP-1 containing the amino-terminal residues 1-214, retained both of these interactions (Mikhailenko et al. [1997]: J Biol Chem 272:6784-6791). Here, we examined the activity of a recombinant protein containing amino-terminal residues 1-90 of TSP-1 and found that this domain did not retain high-affinity heparin-binding. The loss of heparin-binding correlated with decreased binding to the fibroblast cell surface. However, both ligand blotting and solid phase binding studies indicate that this truncated fragment of TSP-1 retained high-affinity binding to LRP-1. Consistent with this, it also retained the ability to block the uptake and degradation of (125)I-TSP-1. However, TSP-1(1-90) itself was poorly endocytosed and this truncated amino-terminal domain was considerably more effective than the full-length heparin-binding domain (HBD) of TSP-1 in blocking the catabolism of endogenously expressed TSP-1. These results indicate that TSP-1 binding to LRP-1 does not require prior or concomitant interaction with cell surface HSPG but suggest subsequent endocytosis requires high-affinity heparin-binding.

Low-density Lipoprotein Receptor-related Protein Mediates the Endocytosis of Anionic Liposomes in Neurons

We have recently demonstrated that anionic liposomes efficiently introduce foreign DNA into postmitotic neurons and other cell types (Lakkaraju, A., Dubinsky, J. M., Low, W. C., and Rahman, Y.-E. (2001) J. Biol. Chem. 276, 32000-32007). To investigate the mechanism of liposome uptake, we followed the internalization of anionic liposome-encapsulated Cy3-labeled oligonucleotides (AL-Cy3ONs) by hippocampal neurons using confocal microscopy. Uptake of AL-Cy3ONs was widespread and time- and temperature-dependent, indicative of receptor-mediated endocytosis. The low-density lipoprotein receptor-related protein (LRP) was crucial for anionic liposome endocytosis because the receptor-associated protein or an anti-LRP antibody inhibited internalization, and fibroblasts lacking LRP did not internalize AL-Cy3ONs. Using selective endocytosis inhibitors, we found that liposome endocytosis and intracellular transport required clathrin, dynamin, an intact cytoskeletal network, and phosphatidylinositol 3-kinase activity. Cy3ONs did not significantly colocalize with recycling endosomal/lysosomal markers and entered neuronal nuclei within 1-3 h of incubation. Approximately 50% of the internalized liposomal phospholipids were recycled back to the cell surface, in keeping with the fluidity of their acyl chains. Liposome endocytosis did not require heparan sulfate proteoglycans or cause calcium influx into neurons. Thus, constitutive endocytosis of anionic liposomes by LRP utilizes only one component, in contrast to the more involved heparan sulfate proteoglycan-LRP pathway implicated in the pathogenesis of Alzheimer's disease.

Inhibition of vascular smooth muscle cell proliferation by a novel fibroblast growth factor receptor antagonist.

One of the key events in post-angioplasty restenosis is the migration and proliferation of medial smooth muscle cells leading to neo-intima formation. This phase is mediated by several growth factors, mainly platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF2/bFGF) and heparin-binding epidermal growth factor (HB-EGF). In this study, we have focused on the role of FGF2, which requires heparan sulfate proteoglycans (HSPG) as cofactors for binding and activation of its cell surface tyrosine kinase receptor. The aim of this study was to identify and explore the effect of novel FGF antagonists on vascular smooth muscle cell (VSMC) proliferation. We have recently identified a novel class of small, positively charged molecules sharing a porphyrin core as inhibitors of FGF2 and vascular endothelial growth factor (VEGF) activity. Here we investigated the inhibitory effect of these compounds on VSMC proliferation and their effect on heparin-induced FGF receptor activity. We found that these molecules exert a marked inhibitory effect on FGF2-mediated smooth muscle cell (SMC) proliferation, manifested by reduced cell growth and DNA synthesis, which occurred in a dose-dependent manner with an IC(50) of approximately 1 microM of inhibitor. We demonstrate that the molecule, 5, 10, 15, 20-tetrakis (methyl-4-pyridyl)-21H, 23H-porphine tetra-p-tosylate salt (TMPP), inhibits binding of radiolabeled FGF2 to SMCs and to soluble FGF receptor 1 (FGFR1) in a manner that interferes with both ligand and receptor interactions with heparin, thereby blocking growth factor mediated SMC proliferation. We have identified an FGF antagonist, which may serve in clinical practice as a preventive measure of restenosis.

Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ.

Fibroblast growth factors (FGFs) require heparan sulfate proteoglycans (HSPGs) as cofactors for signaling. The heparan sulfate chains (HS) mediate stable high affinity binding of FGFs to their receptor tyrosine kinases (FR) and may specifically regulate FGF activity. A novel in situ binding assay was developed to examine the ability of HSPGs to promote FGF/FR binding using a soluble FR fusion construct (FR1-AP). This fusion protein probe forms a dimer in solution, simulating the dimerization or oligomerization that is thought to occur at the cell surface physiologically. In frozen sections of human skin, FGF-2 binds to keratinocytes and basement membranes of epidermis and dermal blood vessels. In contrast, in skin preincubated with FGF-2, FR1-AP binds avidly to FGF-2 immobilized on keratinocyte cell surfaces, but fails to bind to basement membranes at the dermo-epidermal junction or dermal microvessels despite the fact that these structures bind large amounts of FGF-2. Apparently, basement membrane and cell surface HSPGs differ in their ability to mediate the assembly of a FGF/FR signaling complex presumably due to structural differences of the heparan sulfate chains.

Transport of lipoprotein lipase across endothelial cells.

Lipoprotein lipase (LPL), synthesized in muscle and fat, hydrolyzes plasma triglycerides primarily while bound to luminal endothelial cell surfaces. To obtain information about the movement of LPL from the basal to the luminal endothelial cell surface, we studied the transport of purified bovine milk LPL across bovine aortic endothelial cell monolayers. 125I-labeled LPL (125I-LPL) added to the basal surface of the monolayers was detected on the apical side of the cells in two compartments: (i) in the medium of the upper chamber, and (ii) bound to the apical cell surface. The amount of 125I-LPL on the cell surface, but not in the medium, reached saturation with time and LPL dose. Catalytically active LPL was transported to the apical surface but very little LPL activity appeared in the medium. Heparinase treatment of the basal cell surface and addition of dextran sulfate (0.15 microM) to the lower chamber decreased the amount of 125I-LPL appearing on the apical surface. Similarly, the presence of increasing molar ratios of oleic acid/bovine serum albumin at the basal surface decreased the transport of active LPL across the monolayer. Thus, a saturable transport system, which requires heparan sulfate proteoglycans and is inhibited by high concentrations of free fatty acids on the basal side of the cells, appears to exist for passage of enzymatically active LPL across endothelial cells. We postulate that regulation of LPL transport to the endothelial luminal surface modulates the physiologically active pool of LPL in vivo.