Basement Membrane Heparan Sulfate Proteoglycan Research Articles

Proteoglycans of basement membranes: Crucial controllers of angiogenesis, neurogenesis, and autophagy.

Anti-angiogenic therapy is an established method for the treatment of several cancers and vascular-related diseases. Most of the agents employed target the vascular endothelial growth factor A, the major cytokine stimulating angiogenesis. However, the efficacy of these treatments is limited by the onset of drug resistance. Therefore, it is of fundamental importance to better understand the mechanisms that regulate angiogenesis and the microenvironmental cues that play significant role and influence patient treatment and outcome. In this context, here we review the importance of the three basement membrane heparan sulfate proteoglycans (HSPGs), namely perlecan, agrin and collagen XVIII. These HSPGs are abundantly expressed in the vasculature and, due to their complex molecular architecture, they interact with multiple endothelial cell receptors, deeply affecting their function. Under normal conditions, these proteoglycans exert pro-angiogenic functions. However, in pathological conditions such as cancer and inflammation, extracellular matrix remodeling leads to the degradation of these large precursor molecules and the liberation of bioactive processed fragments displaying potent angiostatic activity. These unexpected functions have been demonstrated for the C-terminal fragments of perlecan and collagen XVIII, endorepellin and endostatin. These bioactive fragments can also induce autophagy in vascular endothelial cells which contributes to angiostasis. Overall, basement membrane proteoglycans deeply affect angiogenesis counterbalancing pro-angiogenic signals during tumor progression, and represent possible means to develop new prognostic biomarkers and novel therapeutic approaches for the treatment of solid tumors.

Matrigel-Free Laminin-Entactin Matrix to Induce Human Renal Proximal Tubule Structure Formation In Vitro.

A successful in vitro tissue model must recapitulate the native tissue features while also being reproducible. Currently, Matrigel is the principal biomaterial used to induce the formation of proximal convoluted tubules (PCTs) in vitro, because of its similar composition and structure with the kidney tubular basement membrane and the presence of critical growth factors. However, Matrigel is not well-defined, and batch-to-batch variability is a significant issue. Here, we define a Matrigel-free method, using a laminin-entactin (L-E) matrix to support the formation of proximal tubular-like structures in vitro using immortalized human renal epithelial cells (RPTEC/TERT1) cocultured with murine fibroblast stromal cells (FOXD1lacZ+). The matrix supports the presence of specific components of the tubular basement membrane (laminin, entactin/nidogen, and heparan sulfate proteoglycan) in addition to fibroblast growth factor 8a (FGF-8a). The matrix also induces tubulogenesis, leading to the formation of PCTs based on several key markers, including E-cadherin, aquaporin-1, and Na+/K+ ATPase. Moreover, these PCT structures displayed cell polarity and a well-defined lumen after 18 days in culture. This laminin-entactin (L-E) matrix constitutes a defined and consistent biomaterial that can be used in kidney tissue engineering for understanding in vitro proximal tubule development and for nephrotoxicity studies.

Proteoglycan-driven Autophagy: A Nutrient-independent Mechanism to Control Intracellular Catabolism.

Proteoglycans are rapidly emerging as versatile regulators of intracellular catabolic pathways. This is predominantly achieved via the non-canonical induction of autophagy, a fundamentally and evolutionarily conserved eukaryotic pathway necessary for maintaining organismal homeostasis. Autophagy facilitated by either decorin, a small leucine-rich proteoglycan, or perlecan, a basement membrane heparan sulfate proteoglycan, proceeds independently of ambient nutrient conditions. We found that soluble decorin evokes endothelial cell autophagy and breast carcinoma cell mitophagy by directly interacting with vascular endothelial growth factor receptor 2 (VEGFR2) or the Met receptor tyrosine kinase, respectively. Endorepellin, a soluble, proteolytic fragment of perlecan, induces autophagy and endoplasmic reticulum stress within the vasculature, downstream of VEGFR2. These potent matrix-derived cues transduce key biological information via receptor binding to converge upon a newly discovered nexus of core autophagic machinery comprised of Peg3 (paternally expressed gene 3) for autophagy or mitostatin for mitophagy. Here, we give a mechanistic overview of the nutrient-independent, proteoglycan-driven programs utilized for autophagic or mitophagic progression. We propose that catabolic control of cell behavior is an underlying basis for proteoglycan versatility and may provide novel therapeutic targets for the treatment of human disease.

Cancer Metastasis: The Role of the Extracellular Matrix and the Heparan Sulfate Proteoglycan Perlecan.

Cancer metastasis is the dissemination of tumor cells to new sites, resulting in the formation of secondary tumors. This process is complex and is spatially and temporally regulated by intrinsic and extrinsic factors. One important extrinsic factor is the extracellular matrix, the non-cellular component of tissues. Heparan sulfate proteoglycans (HSPGs) are constituents of the extracellular matrix, and through their heparan sulfate chains and protein core, modulate multiple events that occur during the metastatic cascade. This review will provide an overview of the role of the extracellular matrix in the events that occur during cancer metastasis, primarily focusing on perlecan. Perlecan, a basement membrane HSPG is a key component of the vascular extracellular matrix and is commonly associated with events that occur during the metastatic cascade. Its contradictory role in these events will be discussed and we will highlight the recent advances in cancer therapies that target HSPGs and their modifying enzymes.

Cell surface chondroitin sulphate proteoglycan 4 (CSPG4) binds to the basement membrane heparan sulphate proteoglycan, perlecan, and is involved in cell adhesion.

Chondroitin sulphate proteoglycan 4 (CSPG4) is a cell surface proteoglycan highly expressed by tumour, perivascular and oligodendrocyte cells and known to be involved cell adhesion and migration. This study showed that CSPG4 was present as a proteoglycan on the cell surface of two melanoma cell lines, MM200 and Me1007, as well as shed into the conditioned medium. CSPG4 from the two melanoma cell lines differed in the amount of chondroitin sulphate (CS) decoration, as well as the way the protein core was fragmented. In contrast, the CSPG4 expressed by a colon carcinoma cell line, WiDr, was predominantly as a protein core on the cell surface lacking glycosaminoglycan (GAG) chains. This study demonstrated that CSPG4 immunopurified from the melanoma cell lines formed a complex with perlecan synthesized by the same cultured cells. Mechanistic studies showed that CSPG4 bound to perlecan via hydrophobic protein-protein interactions involving multiple sites on perlecan including the C-terminal region. Furthermore, this study revealed that CSPG4 interacted with perlecan to support cell adhesion and actin polymerization. Together these data suggest a novel mechanism by which CSPG4 expressing cells might attach to perlecan-rich matrices so as those found in connective tissues and basement membranes.

Abstract 234: Interleukin 2 Promotes Proliferation and Migration in Vascular Smooth Muscle Cells

Interleukin-2 (IL-2) is primarily known as a soluble cytokine that regulates T cell responses. We previously reported, however, that IL-2 is retained in the extracellular matrix by association with perlecan, a heparan sulfate proteoglycan (HSPG). Perlecan is the main HSPG in vascular basement membranes, and previous studies from our laboratory demonstrated that, in human arteries, vascular smooth muscle cells (VSMC) are surrounded by perlecan-bound IL-2. We also noted that IL-2 deficient mice lose SMCs with age, leading to widened esophagi and aortic aneurysms. Given this information, we hypothesized that IL-2 has a direct impact on VSMC, and that VSMC express functional IL-2 receptors (IL-2R). We therefore examined both protein and mRNA expression of each of the three IL-2R subunits (alpha, beta, gamma) on human VSMC grown from arterial explants. These VSMC expressed SMC actin, smooth muscle myosin heavy chain, and when quiescent, smoothelin. Protein expression was assessed by in cell Western and by Western blot analysis. Receptor expression was evaluated under distinct culture conditions, which yielded highly proliferative, intermediate, or quiescent VSMC. Contractile protein expression was low, intermediate, or high, respectively, consistent with the characteristics of proliferating vs quiescent SMCs. Each phenotype expressed all 3 subunits of the IL-2R. IL-2 subunits appeared to follow a cytoskeletal pattern in cells expressing high levels of contractile proteins. Western blot analysis of VSMC lysates revealed expression of all 3 receptors at molecular weights identical to lysates from a T cell line. VSMCs also expressed mRNA for each receptor subunit. Functionally, IL-2 promoted migration (using a Boyden chamber assay) and proliferation in a dose dependent fashion. Because excess proliferation and migration are critical to intimal hyperplasia, we asked whether IL-2 levels change under conditions known to generate intimal hyperplasia. In a rabbit model, IL-2 mRNA increased in venous grafts exposed to high flow for 2h. IL-2 levels, by Western blot, were also increased in human hyperplastic veins. In conclusion, these data show that VSMC have functional IL-2R, and suggest that IL-2 may contribute to the development of intimal hyperplasia.

Proteoglycans regulate autophagy via outside-in signaling: an emerging new concept

The importance of proteoglycans as regulators of key cellular processes such as angiogenesis, adhesion, and inflammation has long been appreciated. However, a new era appears to be dawning for the role of proteoglycans in the regulation of autophagy, a homeostatic mechanism whereby organelles and other cytoplasmic contents are degraded and recycled via lysosomal processing [1]. Depending on the context, autophagy has been regarded as either a cell-survival or cell-death pathway. For example, following starvation, a cell will undergo autophagy in order to break down cellular components for re-utilization, allowing the cell to survive under nutrient-poor conditions [2]. In contrast, when programmed cell death pathways are impaired, autophagy may substitute for apoptosis leading to cell death [3,4]. While basal autophagy is required for normal cell maintenance and contributes to standard cell signaling, aberrant autophagy has been implicated in a range of disease processes including diabetes, inflammation, and neurodegenerative disorders [5–8]. In the setting of cancer, the function of autophagy has been difficult to discern. Where some studies indicate that autophagy is beneficial for tumor cell survival [9,10], others suggest that the self-degradative process may prevent tumor growth during early stages of tumorigenesis by shielding healthy cells from damage due to protein aggregation and build-up of other by-products of metabolic turnover [5,11]. The complexity of this pathway lends itself to scrutiny in order to gain a better understanding of its regulation as well as its overall function in normal cell signaling and disease. Recent studies illustrate that select proteoglycans and other matrix constituents dynamically contribute to autophagic regulation [12–19]. In this commentary we will consider the noteworthy role of the following three matrix components in the regulation of the autophagic process: 1) The small leucine-rich proteoglycan, decorin, 2) The basement membrane heparan sulfate proteoglycan, perlecan, and 3) The C-terminal fragment of perlecan, endorepellin.

Abstract 033: Cardio-Cerebro-Vascular Diseases: Prevention in Metabolic Syndrome Patients

Background: ALP and GGT are indispensable constituents of brain microvessel endothelial cells, expressed and induced by IL-6. High values are entangled in affecting blood-brain barrier functionality. Since SOD bound to brain basement membrane and matrix heparan sulfate proteoglycan, is implicated in β-amyloid efflux, we examined in a pilot study of 11 patients with metabolic syndrome (MS) whether these biomarkers can be beneficially influenced through an antioxidant phytochemical approach with Ginkgo biloba (EGb 761, 2 × 120 mg/d). Also, MS is associated with an increased risk of developing diabetes and cardio-cerebro-vascular diseases (CCVD) (Figure). Methods: Photometric methods, ELISAs, EIAs and laser ellipsometry were applied. Results: A 2-month medication led to brain/liver biomarker changes: ALP -14.8% (p < 0.004), GGT -11.3% (p < 0.010). Antioxidative biomarker changes: SOD +17.7% (p < 0.009), GPx +11.6% (p <0.001), oxLDL/LDL -21.0% (p < 0.002), 8- iso -PGF 2α -39.8% (p < 0.003), MPO -29.6% (p < 0.014). Antiinflammatory biomarker changes: IL-6 -12.9% (p < 0.041), Lp(a) -26.3% (< 0.001), hs-CRP -39.3% (< 0.005), WBC -6.3% (< 0.023), MMP-9 -32.9% (< 0.042). Vasodilator biomarker changes: cAMP +43.5% (< 0.001), cGMP +32.9% (< 0.001), nanoplaque formation -14.3% (p < 0.008), nanoplaque size -23.4% (p < 0.0004). Conclusion: Since under ginkgo ALP and GGT were downregulated and SOD upregulated, and plasma biomarkers of oxidative stress, plaque stability and progression, inflammation, vasodilatory second messengers and lipid composition beneficially altered, this phytochemical may be estimated as complementary drug with potentially preventive character for CCVD.

Agrin expression is downregulated in OA and is required for chondrocyte differentiation

Purpose: Background: Agrin is a large basement membrane heparan sulphate proteoglycan best known for its function at the neuromuscular junction. It is responsible for synapse formation via binding to the MuSK and LRP4 receptor complex, leading to Acetylcholine receptor aggregation. Mice deficient of Agrin die perinatally as a result of respiratory failure. Agrin is also expressed in other cell types including chondrocytes of the developing growth plate, and agrin-deficient embryos in which agrin expression had been rescued at the neuromuscular junction develop skeletal abnormalities 1. Therefore it is possible that Agrin may play a role in cartilage homeostasis. Aims: The aim of this project is to determine if agrin plays a functional role in the homeostasis of the articular cartilage and in osteoarthritis. Methods: Human adult articular cartilage explants were obtained from preserved or damaged cartilage from individuals undergoing joint replacement for osteoarthritis. Experimental osteoarthritis was induced in 10 week old mice by destabilization of the medial meniscus2. Sham-operated controlateral knees were used as controls. Knees were collected 8weeks after surgery, decalcified and embedded in paraffin. Bovine primary articular chondrocytes (BPAC) were isolated by pronase/collagenase digestion from the articular cartilage of the metatarso-phalangeal joints of 18 month old calves from a local slaughterhouse. The BPAC and the human chondrogenic cell line C28/I2 were cultured in micromass to promote differentiation and extracellular matrix production. For gain- and loss-of-function experiments, chondrocytes were transfected with a mammalian expression vector encoding for full-length Agrin (FL-Agrin) or with Agrin siRNA's respectively. Accumulation of cartilage-specific extracellular matrix production, rich in highly sulphated glycosaminoglycans (GAGs), was quantified by alcian blue staining at pH 0.2 and alizarin red staining. Chondrogenic potential was measured using pellet analysis as previously described3, BPAC pellets were cultured for 14 days, weighed, sectioned and stained with alizarin red and safranin o. Gene expression analysis of Agrin, Col2A1, Aggrecan, Sox9 was performed by real time PCR. Immunofluorescence was used to determine the expression levels of Agrin at protein level in chondrocytes cultured in monoloayer and in paraffin-embedded tissue sections. Results: Agrin was expressed in healthy adult human and bovine articular cartilage. Agrin was signifcantly downregulated in human osteoarthritic cartilage. This downregulation was also replicated in experimental murine osteoarthritis, indicating that it is a consequence rather than a cause of osteoarthritis. Agrin expression was partially retained in the articular cartilage of sham operated knees, indicating that inflammation alone (present both in DMM and in sham) is not sufficient to induce Agrin downregulation, but cartilage damage is necessary. Endogenous expression of Agrin was confirmed in C28/I2 and in BPAC. Overexpression of FL-Agrin resulted in enhanced differentiation as demonstrated by upregulated the cartilage key transcription factor Sox9. . Knock-down of Agrin by siRNA resulted in reduced GAG production in C28/I2 and chondrocyte de-differentiation as documented by decreased expression Sox9, Col2A1 and Aggrecan mRNA. Conclusions: Agrin expressed in healthy adult articular cartilage and is downregulated in osteoarthritis; - Agrin is anabolic in cultured articular chondrocytes; - Agrin is required for chondrocyte differentiation in vitro.

Nephronectin binds to heparan sulfate proteoglycans via its MAM domain

Nephronectin is a basement membrane protein comprising five N-terminal epidermal growth factor (EGF)-like repeats, a central linker segment containing an Arg-Gly-Asp (RGD) motif and a C-terminal meprin-A5 protein-receptor protein tyrosine phosphatase μ (MAM) domain. Nephronectin has been shown to interact with α8β1 integrin through the central linker segment, but its interactions with other molecules remain to be elucidated. Here, we examined the binding of nephronectin to a panel of glycosaminoglycan (GAG) chains. Nephronectin bound strongly to heparin and chondroitin sulfate (CS)-E and moderately to heparan sulfate (HS), but failed to bind to CS-A, CS-C, CS-D, dermatan sulfate and hyaluronic acid. Deletion of the MAM domain severely impaired the binding of nephronectin to heparin but not CS-E, whereas deletion of the EGF-like repeats reduced its binding to CS-E but not heparin, suggesting that nephronectin interacts with CS-E and heparin through the EGF-like repeats and MAM domain, respectively. Consistent with these results, nephronectin bound to agrin and perlecan, which are heparan sulfate proteoglycans (HSPGs) in basement membranes, in HS-dependent manners. Site-directed mutagenesis of the MAM domain revealed that multiple basic amino acid residues in the putative loop regions were involved in the binding of the MAM domain to agrin. The binding of nephronectin to basement membrane HSPGs was further confirmed by in situ nephronectin overlay assays using mouse frozen tissue sections. Taken together, these findings indicate that nephronectin is capable of binding to HSPGs in basement membranes via the MAM domain, and thereby raise the possibility that interactions with basement membrane HSPGs may be involved in the deposition of nephronectin onto basement membranes.

An immunohistochemical study of basement membrane heparan sulfate proteoglycan (perlecan) in oral epithelial dysplasia and squamous cell carcinoma

Background:Basement membrane heparan sulfate proteoglycan (perlecan) has been demonstrated in precancer lesions and carcinomas of oral cavity. It helps in malignant transformation of epithelial cells. The aim of our study was to understand the immuno-localization of perlecan in oral dysplastic epithelium and oral carcinomas.Materials and Methods:A total of 50 cases comprising 10 normal mucosa, 20 dysplastic mucosa, and 20 oral squamous cell carcinomas (OSCC) were included in the retrospective study. They were examined for the presence of perlecan protein core by immunohistochemistry using monoclonal antibody. Interpretation of the pattern of staining was done, and majority of the observations were taken for statistical analysis.Results:In normal epithelium, perlecan was found to be present in basal layer at the cell border. In dysplastic epithelium, it was present in suprabasal layers also. With the increase in severity of dysplasia, its expression was more in suprabasal layers, and the immuno-localization was found to be at cell border and cytoplasm. In OSCC cases, perlecan was present in stroma and tumor islands.Conclusion:It was deduced from the above results that perlecan helps potentially in dysplastic changes of epithelial cells. It gets accumulated within the cell and intercellular spaces and serves as a reservoir for various growth factors. In OSCC, it breaks down and releases growth factors, which help in tumor progression, angiogenesis, and metastasis of the carcinoma.

Overall Sulfation of Heparan Sulfate from Pancreatic Islet β-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.

Perlecan (basement membrane heparan sulfate proteoglycan) and its role in oral malignancies: An overview

Perlecan means pearl-like structures. Perlecan is a large proteoglycan (400-500 kDa) present in virtually all vascularized tissues with a distribution that is primarily confined to basement membranes including those of oral mucosa. It is a basement membrane-type heparan sulfate proteoglycan. Perlecan is synthesized by basal cells and fibroblasts adjacent to the basal lamina . Perlecan is also synthesized by vascular endothelial and smooth muscle cells present in the extracellular matrix. It has been demonstrated in recent years that perlecan is distributed in the stromal space of various pathophysiological conditions. The complex pleiotropy of perlecan suggests that this gene product is involved in several developmental processes, at both early and late stages of embryogenesis, as well as in cancer and diabetes. In the oral cavity, perlecan expression is reported to basal cells in normal mucosa and its expression increases in precancer and cancerous conditions. It is also expressed in various odontogenic tumors such as ameloblastoma, keratocyst odontogenic tumor, and also salivary gland tumors such as adenoid cystic carcinoma, mucoepidermoid carcinoma, etc.

Podocytes require the engagement of cell surface heparan sulfate proteoglycans for adhesion to extracellular matrices

Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Cα, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity.

Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity

Activin A, a member of the transforming growth factor-β family, plays important roles in hormonal homeostasis and embryogenesis. In this study, we produced recombinant human activin A and examined its abilities to bind to extracellular matrix proteins. Recombinant activin A expressed in 293-F cells was purified as complexes of mature dimeric activin A with its pro-region. Among a panel of extracellular matrix proteins tested, recombinant activin A bound to perlecan and agrin, but not to laminins, nidogens, collagens I and IV, fibronectin, and nephronectin. The binding of recombinant activin A to perlecan was inhibited by heparin and high concentrations of NaCl and abolished by heparitinase treatment of perlecan, suggesting that activin A binds to the heparan sulfate chains of perlecan. In support of this possibility, recombinant activin A was capable of directly binding to heparin and heparan sulfate chains. Site-directed mutagenesis of recombinant activin A revealed that clusters of basic amino acid residues, Lys(259)-Lys(263) and Lys(270)-Lys(272), in the pro-region were required for binding to perlecan. Interestingly, deletion of the peptide segment Lys(259)-Gly(277) containing both basic amino acid clusters from the pro-region did not impair the activity of activin A to stimulate Smad-dependent gene expressions, although it completely ablated the perlecan-binding activity. The binding of activin A to basement membrane heparan sulfate proteoglycans through the basic residues in the pro-region was further confirmed by in situ activin A overlay assays using frozen tissue sections. Taken together, the present results indicate that activin A binds to heparan sulfate proteoglycans through its pro-region and thereby regulates its localization within tissues.

LTBP-2 has multiple heparin/heparan sulfate binding sites

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca 2+ ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca 2+ dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG–LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.

Revisiting the glomerular charge barrier in the molecular era

The glomerular filtration barrier consists of fenestrated glomerular endothelium, podocyte foot processes/slit diaphragms, and intervening glomerular basement membrane. Its characterization as both a size and charge-selective barrier emerged from studies conducted decades ago. The charge selectivity phenomenon is receiving renewed attention now that the identities and mechanisms of synthesis of relevant molecules are known. Here we summarize studies employing genetic or other in-vivo strategies to investigate glomerular charge. Attention has focused on glomerular basement membrane heparan sulfate proteoglycans, long considered primary charge barrier components. Agrin contributes significantly to glomerular basement membrane charge but, like perlecan and collagen XVIII, is dispensable for glomerular structure and function. Disruption of glomerular heparan sulfate through transgenic methods or administration of heparanase in vivo provides further evidence against a role for heparan sulfate in glomerular function. Disruption of glomerular sialoproteins, however, causes proteinuria and indicates a critical role for these cell-associated glycoproteins in glomerular filtration. Recent in-vivo manipulations of glomerular heparan sulfate proteoglycans fail to reveal a crucial role for either them or their anionic charge in glomerular filtration. In contrast, cell-associated sialoproteins are clearly important, but whether their functions actually involve contributions to the charge barrier is unknown.

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.

The extracellular matrix protein WARP is a novel component of a distinct subset of basement membranes

WARP is a recently described member of the von Willebrand factor A domain superfamily of extracellular matrix proteins, and is encoded by the Vwa1 gene. We have previously shown that WARP is a multimeric component of the chondrocyte pericellular matrix in articular cartilage and intervertebral disc, where it interacts with the basement membrane heparan sulfate proteoglycan perlecan. However, the tissue-specific expression of WARP in non-cartilaginous tissues and its localization in the extracellular matrix of other perlecan-containing tissues have not been analyzed in detail. To visualize WARP-expressing cells, we generated a reporter gene knock-in mouse by targeted replacement of the Vwa1 gene with β-galactosidase. Analysis of reporter gene expression and WARP protein localization by immunostaining demonstrates that WARP is a component of a limited number of distinct basement membranes. WARP is expressed in the vasculature of neural tissues and in basement membrane structures of the peripheral nervous system. Furthermore, WARP is also expressed in the apical ectodermal ridge of developing limb buds, and in skeletal and cardiac muscle. These findings are the first evidence for WARP expression in non-cartilaginous tissues, and the identification of WARP as a component of a limited range of specialized basement membranes provides further evidence for the heterogeneous composition of basement membranes between different tissues.

Comparison of the expression of agrin, a basement membrane heparan sulfate proteoglycan, in cholangiocarcinoma and hepatocellular carcinoma

Heparan sulfate proteoglycans mediate cell adhesion and control the activities of numerous growth and motility factors. They play a critical role in carcinogenesis and tumor progression. Agrin is a large multidomain heparan sulfate proteoglycan associated with basement membranes in several tissues. The expression of agrin in the liver has recently been described under physiologic and pathologic conditions. However, little is known about its role in malignancies. We aimed to study the mRNA and protein expression of agrin in cholangiocarcinoma (CC) and focused on the differences between CC and hepatocellular carcinoma (HCC). Eighty surgically removed liver specimens were studied by immunohistochemistry. Representative samples were used for immunoblotting. mRNA expression was measured in 32 samples by real-time polymerase chain reaction. By immunohistochemistry, agrin was seen around bile ducts and blood vessels within the portal areas in the normal liver. Although no expression was found within the hepatic lobules, agrin was deposited in the neovascular basement membrane in HCCs. Agrin was abundant in the tumor-specific basement membrane in well-differentiated areas of CCs, whereas with immunostaining, it was fragmented, decreased, or it even disappeared in less differentiated areas and sites of infiltration. By real-time polymerase chain reaction, up-regulation of agrin expression was measured in HCCs compared with that in the normal liver. CC samples showed an even higher expression of agrin. Immunoblotting confirmed these findings. Our results indicate that agrin might play an important role in neoangiogenesis in human HCC, being a part of the newly formed vasculature. In CC, however, agrin might be involved in tumor progression.