What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles?

PURPOSE: The purpose of the study was to establish the mechanism by which the estrogen concentration difference between the follicular fluid and the serum is maintained. METHODS: We used dialysis membrane with a pore size of <3 KD to characterize the estrogen-binding capacity of the follicular fluid. We performed PCR, western blot, and ELISA on luteinized granulosa cells to determine if sex hormone-binding globulin (SHBG) is produced by granulosa cells, and finally we used affinity columns and mass spectrometry to identify the estrogen-binding protein in the follicular fluid. RESULTS: We found that a significant estrogen concentration difference is maintained in a cell-free system and is lost with proteolysis of the follicular fluid proteins. Luteinized granulosa cells are likely not a source of SHBG, as we were not able to detect expression of SHBG in these cells. Perlecan was the most highly enriched follicular fluid protein in the affinity columns. CONCLUSIONS: We were able to identify perlecan as the most likely candidate for the major estrogen-binding protein in the follicular fluid.

Mast cells produce novel shorter forms of perlecan that contain functional endorepellin: a role in angiogenesis and wound healing.

Mast cells are derived from hematopoietic progenitors that are known to migrate to and reside within connective and mucosal tissues, where they differentiate and respond to various stimuli by releasing pro-inflammatory mediators, including histamine, growth factors, and proteases. This study demonstrated that primary human mast cells as well as the rat and human mast cell lines, RBL-2H3 and HMC-1, produce the heparan sulfate proteoglycan, perlecan, with a molecular mass of 640 kDa as well as smaller molecular mass species of 300 and 130 kDa. Utilizing domain-specific antibodies coupled with N-terminal sequencing, it was confirmed that both forms contained the C-terminal module of the protein core known as endorepellin, which were generated by mast cell-derived proteases. Domain-specific RT-PCR experiments demonstrated that transcripts corresponding to domains I and V, including endorepellin, were present; however, mRNA transcripts corresponding to regions of domain III were not present, suggesting that these cells were capable of producing spliced forms of the protein core. Fractions from mast cell cultures that were enriched for these fragments were shown to bind endothelial cells via the α(2)ß(1) integrin and stimulate the migration of cells in "scratch assays," both activities of which were inhibited by incubation with either anti-endorepellin or anti-perlecan antibodies. This study shows for the first time that mast cells secrete and process the extracellular proteoglycan perlecan into fragments containing the endorepellin C-terminal region that regulate angiogenesis and matrix turnover, which are both key events in wound healing.

SHh activity and localization is regulated by perlecan.

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.

SHh activity and localization is regulated by perlecan

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.

Soluble perlecan domain I enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells.

BACKGROUND: Immobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro. RESULTS: In solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951), and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2. CONCLUSIONS: Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.

Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1.

BACKGROUND: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. RESULTS: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor. CONCLUSIONS: With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

Unique extracellular matrix heparan sulfate from the bivalve Nodipecten nodosus (Linnaeus, 1758) safely inhibits arterial thrombosis after photochemically induced endothelial lesion.

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing approximately 4.6 mg g(-1) of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional (1)H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg(-1), 5-fold lower than porcine heparin (180 IU mg(-1)), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC(50) = 0.835 microg ml(-1)) and thrombin (IC(50) = 9.3 microg ml(-1)) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg(-1), the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.

The modulation of platelet and endothelial cell adhesion to vascular graft materials by perlecan.

Controlled neo-endothelialisation is critical to the patency of small diameter vascular grafts. Endothelialisation and platelet adhesion to purified endothelial cell-derived perlecan, the major heparan sulfate (HS) proteoglycan in basement membranes, were investigated using in vivo and in vitro assays. Expanded polytetrafluoroethylene (ePTFE) vascular grafts were coated with perlecan and tested in an ovine carotid interposition model for a period of 6 weeks and assessed using light and scanning microscopy. Enhanced endothelial cell growth and reduced platelet adhesion were observed on the perlecan coated grafts when compared to uncoated controls implanted in the same sheep (n=5). Perlecan was also found to stimulate endothelial cell proliferation in vitro over a period of 6 days in the presence of plasma proteins and fibroblastic growth factor 2 (FGF-2), however in the absence of FGF-2 endothelial cell growth could not be maintained during this period. Perlecan was found to be anti-adhesive for platelets, however after removal of the HS chains attached to perlecan, platelet adhesion and aggregation were supported. These results suggest a role for HS chains of perlecan in improving graft patency by selectively promoting endothelial cell proliferation while modulating platelet adhesion.

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.

Spatiotemporal distribution of heparan sulfate epitopes during murine cartilage growth plate development.

Heparan sulfate proteoglycans (HSPGs) are abundant in the pericellular matrix of both developing and mature cartilage. Increasing evidence suggests the action of numerous chondroregulatory molecules depends on HSPGs. In addition to specific functions attributed to their core protein, the complexity of heparan sulfate (HS) synthesis provides extraordinary structural and functional heterogeneity. Understanding the interactions of chondroregulatory molecules with HSPGs and their subsequent outcomes has been limited by the absence of a detailed analysis of HS species in cartilage. In this study, we characterize the distribution and variety of HS species in developing cartilage of normal mice. Cryo-sections of femur and tibia from normal mouse embryos were evaluated using immunostaining techniques. A panel of unique phage display antibodies specific to particular HS species were employed and visualized with secondary antibodies conjugated to Alexa-fluor dyes. Confocal microscopy demonstrates that HS species are dynamic structures within developing growth plate cartilage and the perichondrium. GlcNS6S-IdoUA2S-GlcNS6S species are down regulated and localization of GlcNS6S-IdoUA-GlcNS6S species within the hypertrophic zone of the growth plate is lost during normal development. Regional differences in HS structures are present within developing growth plates, implying that interactions with and responses to HS-binding proteins also may display regional specialization.

Identification and distribution of heparan sulfate proteoglycans in the white muscle of Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor).

Heparan sulfate proteoglycans (HSPGs) were identified in pre-rigor muscle of two species of cold water fish, Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor) by biochemical and immunological methods. The distribution was described by immunohistology. Special emphasis was directed to the extracellular matrix (ECM) HSPGs perlecan and agrin. In vivo 35S-sulfate labeling combined with ultracentrifugation in CsCl2, DEAE chromatography and scintillation counting of the eluates, revealed that the content of 35S-labeled PGs was much higher in wolffish than in cod. A considerable proportion of the 35S-sulfated PGs in both species was HSPG, as judged by nitrous acid degradation. HSPG represented, however, a higher proportion of the 35S-sulfated PGs in cod compared to wolffish. Dot blot and electrophoresis/western blot using two different HS-mAbs, 10E4 and HepSS-1 indicated structural differences in the HS-chains of the PGs present. This observation was strengthened by immunohistochemistry, showing that both mAbs detected epitopes in the pericellular area, but the staining patterns were not superimposable. Two different agrin isoforms were identified in both species. Furthermore, in the white muscle of both cod and wolffish, perlecan mAb (A7L6) showed positive staining restricted to the transition between myocommata and myofibers.

Perlecan from human epithelial cells is a hybrid heparan/chondroitin/keratan sulfate proteoglycan.

Perlecan is a multidomain proteoglycan, usually substituted with heparan sulphate (HS), and sometimes substituted with both HS and chondroitin sulphate (CS). In this paper, we describe perlecan purified from HEK-293 cells substituted with HS, CS and keratan sulphate (KS). KS substitution was confirmed by immunoreactivity with antibody 5D4, sensitivity to keratanase treatment, and fluorophore-assisted carbohydrate electrophoresis. HEK-293 perlecan failed to promote FGF-dependent cell growth in an in vitro assay. This study is the first to report perlecan containing KS, and makes perlecan one of only a very few proteoglycans substituted with three distinct types of glycosaminoglycan chains.

Chondroitin sulfate proteoglycans are structural renewable constituents of the rabbit vitreous body.

PURPOSE: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. METHODS: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [35S]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. RESULTS: The electron microscopic study revealed a network with hyaluronic acid (HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous (e.g., one heparan- and two chondroitin-sulfate ones). CONCLUSIONS: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.

Normal labor associated with changes in uterine heparan sulfate proteoglycan expression and localization.

HYPOTHESIS: Proteoglycans are well-known modulators of intercellular communication and signaling. Remodeling of the proteoglycans in the human uterus occurs throughout pregnancy, and during labor. We therefore hypothesize that heparan sulfate proteoglycans (HSPGs) play an important role in establishing normal labor. In this study HSPGs were characterized and localized in human uterine tissue. METHODS: Uterine biopsies were obtained from four nonpregnant women, four women during elective cesarean section and four during emergency cesarean section. The biopsies were extracted using 4 m guanidinium hydrochloride (GuHCL). HSPGs were then purified by repeated ion-exchange chromatography on dehydroepiandrosterone (DEAE)-cellulose after digestion with chondroitinase ABC and finally precipitated with Alcian blue. HSPGs were identified by agarose gel electrophoresis and Western blotting. Controlled degradation of the heparan sulfate (HS) side-chains was performed using heparitinase or deglycosylation with trifluoromethanesulfonic acid (TFMS). The resulting core proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie staining. HSPGs were localized in uterine tissue by immunohistochemistry. RESULTS: SDS-PAGE after deglycosylation indicated the presence of multiple distinct core proteins tentatively identified as syndecans 1-4 and glypican 1. Western blots confirmed the presence of these proteoglycans and also perlecan. Immunohistochemistry revealed that the HSPGs were localized mainly in the smooth muscle with few in the extracellular matrix (ECM). Syndecan 3, the dominant proteoglycan, showed the most pronounced changes during pregnancy and labor. CONCLUSION: For the first time several heparan sulfate proteoglycans have been identified and localized in the human uterus and shown to vary in expression during pregnancy and labor. Syndecan 3 had the most outstanding features in this respect.

Structural and functional changes in heparan sulfate proteoglycan expression associated with the myofibroblastic phenotype.

The principal cells implicated as the source of the extracellular matrix in areas of progressive fibrosis are fibroblasts with the phenotypic appearance of myofibroblasts. This report describes differences in heparan sulfate proteoglycan expression between myofibroblasts and normal fibroblasts, associated with impaired responses to fibroblast growth factor-2 (FGF-2). Although both cell types responded to platelet-derived growth factor, myofibroblasts, unlike fibroblasts, did not proliferate to FGF-2. A response was acquired, however, when myofibroblasts were incubated with FGF-2 in the presence of heparan sulfate (HS) and heparin. Selective digestion with pronase, NaOH/NaBH(4), heparinase I, or low pH nitrous acid showed that each HS-glycosaminoglycan region comprised a pronase-resistant peptide separating two HS chains. The HS-glycosaminoglycan chains from myofibroblasts were larger (K(av), 0.32; molecular weight, 50 kd) than those from fibroblasts (K(av), 0.4; molecular weight, 33 kd), although their disaccharide composition was identical. The chains from myofibroblasts, however, contained three, compared to two, heparinase 1-resistant sequences separated by larger contiguous areas of low sulfation. Furthermore, although there was no difference in FGF-2-binding affinity between the two cell types, the chains secreted by myofibroblasts had twice the binding capacity of those from fibroblasts. Thus, it is likely that the difference in response to FGF-2 is because of a difference in FGF-2 sequestration and receptor interaction with FGF-2-HS complexes. A comparative investigation into HS fine structure is being undertaken to examine these findings in more detail.

Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan.

Perlecan, a ubiquitous basement membrane heparan sulfate proteoglycan, plays key roles in blood vessel growth and structural integrity. We discovered that the C terminus of perlecan potently inhibited four aspects of angiogenesis: endothelial cell migration, collagen-induced endothelial tube morphogenesis, and blood vessel growth in the chorioallantoic membrane and in Matrigel plug assays. The C terminus of perlecan was active at nanomolar concentrations and blocked endothelial cell adhesion to fibronectin and type I collagen, without directly binding to either protein; henceforth we have named it "endorepellin." We also found that endothelial cells possess a significant number of high affinity (K(d) of 11 nm) binding sites for endorepellin and that endorepellin binds endostatin and counteracts its anti-angiogenic effects. Thus, endorepellin represents a novel anti-angiogenic product, which may retard tumor neovascularization and hence tumor growth in vivo.

Not all perlecans are created equal: interactions with fibroblast growth factor (FGF) 2 and FGF receptors.

Human basement membrane heparan sulfate proteoglycan (HSPG) perlecan binds and activates fibroblast growth factor (FGF)-2 through its heparan sulfate (HS) chains. Here we show that perlecans immunopurified from three cellular sources possess different HS structures and subsequently different FGF-2 binding and activating capabilities. Perlecan isolated from human umbilical arterial endothelial cells (HUAEC) and a continuous endothelial cell line (C11 STH) bound similar amounts of FGF-2 either alone or complexed with FGFRalpha1-IIIc or FGFR3alpha-IIIc. Both perlecans stimulated the growth of BaF3 cell lines expressing FGFR1b/c; however, only HUAEC perlecan stimulated those cells expressing FGFR3c, suggesting that the source of perlecan confers FGF and FGFR binding specificity. Despite these differences in FGF-2 activation, the level of 2-O- and 6-O-sulfation was similar for both perlecans. Interestingly, perlecan isolated from a colon carcinoma cell line that was capable of binding FGF-2 was incapable of activating any BaF3 cell line unless the HS was removed from the protein core. The HS chains also exhibited greater bioactivity after digestion with heparinase III. Collectively, these data clearly demonstrate that the bioactivity of HS decorating a single PG is dependent on its cell source and that subtle changes in structure including secondary interactions have a profound effect on biological activity.

Electrophoretic, biosensor, and bioactivity analyses of perlecans of different cellular origins.

Three cellular sources of perlecan were examined in this study, namely human umbilical arterial endothelial cells (HUAEC), a transformed human umbilical venous endothelial cell line (C 1 1 STH) and a human colon carcinoma cell line (WiDr). Perlecans were immunopurified from conditioned media of the above cells and the purity of the perlecan preparations was examined by composite agarose polyacrylamide gel electrophoresis (CAPAGE) and semi-dry immunoblotting with monoclonal antibodies directed to either the perlecan core protein (mAb A76) or heparan sulphate (HS) side-chain (mAb10E4). The ability of each perlecan species to bind fibroblast growth factor-l (FGF-1) was examined using a biosensor (BIAcore). The bioactivity of perlecan FGF-1 interactions was also analysed using BaF3 cells transfected with fibroblast growth factor receptors FGFR1b and 1c. CAPAGE demonstrated subtle differences between the perlecans, indicating they had differing charge to mass ratios with C 11 STH perlecan being slightly more mobile in CAPAGE than the HUAEC and WiDr sample. BIAcore biosensor analysis demonstrated distinct differences in the ability of perlecan preparations to bind FGF-1; HUAEC and C 11 STH perlecan showed similar high binding responses as compared to WiDr perlecan, which bound FGF-1 very poorly. Binding of FGF-1 to endothelial perlecans was shown to be HS-dependent. Interestingly, HUAEC perlecan stimulated the growth of FGFR1b and FGFR1c expressing cells in the presence of FGF-1 comparable to heparin, whereas C 11 STH perlecan showed only very limited stimulation of FGFR 1b cells and was incapable of stimulating FGFR1c cells. WiDr perlecan exhibited no stimulation of growth in either cell line. Collectively the data presented herein indicate that. different cell types express perlecans which vary in the growth factor binding capabilities, which may suggest differences in their HS chain substructure. This may represent a subtle mechanism whereby cells can modulate the responsiveness of perlecan to a range of biologically important ligands and thus in a broader context may have important implications for cell signalling.