Basement Membrane Heparan Sulfate 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.

MASP-2 Is a Heparin-Binding Protease; Identification of Blocking Oligosaccharides.

It is well-known that heparin and other glycosaminoglycans (GAGs) inhibit complement activation. It is however not known whether fractionation and/or modification of GAGs might deliver pathway-specific inhibition of the complement system. Therefore, we evaluated a library of GAGs and their derivatives for their functional pathway specific complement inhibition, including the MASP-specific C4 deposition assay. Interaction of human MASP-2 with heparan sulfate/heparin was evaluated by surface plasmon resonance, ELISA and in renal tissue. In vitro pathway-specific complement assays showed that highly sulfated GAGs inhibited all three pathways of complement. Small heparin- and heparan sulfate-derived oligosaccharides were selective inhibitors of the lectin pathway (LP). These small oligosaccharides showed identical inhibition of the ficolin-3 mediated LP activation, failed to inhibit the binding of MBL to mannan, but inhibited C4 cleavage by MASPs. Hexa- and pentasulfated tetrasaccharides represent the smallest MASP inhibitors both in the functional LP assay as well in the MASP-mediated C4 assay. Surface plasmon resonance showed MASP-2 binding with heparin and heparan sulfate, revealing high Kon and Koff rates resulted in a Kd of ~2 μM and confirmed inhibition by heparin-derived tetrasaccharide. In renal tissue, MASP-2 partially colocalized with agrin and heparan sulfate, but not with activated C3, suggesting docking, storage, and potential inactivation of MASP-2 by heparan sulfate in basement membranes. Our data show that highly sulfated GAGs mediated inhibition of all three complement pathways, whereas short heparin- and heparan sulfate-derived oligosaccharides selectively blocked the lectin pathway via MASP-2 inhibition. Binding of MASP-2 to immobilized heparan sulfate/heparin and partial co-localization of agrin/heparan sulfate with MASP, but not C3b, might suggest that in vivo heparan sulfate proteoglycans act as a docking platform for MASP-2 and possibly prevent the lectin pathway from activation.

HGSNAT enzyme deficiency results in accumulation of heparan sulfate in podocytes and basement membranes.

Mucopolysaccharidosis III type C is a lysosomal storage disorder caused by the accumulation of heparan sulfate in lysosomes. The disorder occurs due to Heparan Acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT) deficiency, an enzyme which typically catalyzes the transmembrane acetylation of heparan sulfate, a basement membrane component. When the gene encoding this enzyme is mutated, it cannot perform the processing of heparan sulfate, leading to un-acetylated heparan sulfate build-up in the lysosomes of cells, causing a storage disorder. This defect has been studied primarily in brain and liver cells, but its effect on the structural integrity of the glomerulus is poorly known. The present study focuses on the effect of Hgsnat gene inactivation and heparan sulfate toxicity on the integrity of the renal corpuscle. This cortical structure was chosen because of its abundance of basement membranes and heparan sulfate as well as the renal corpuscle's physiological importance in glomerular filtration. Light microscopy, electron microscopy, and immunocytochemistry of genetically modified mice revealed a buildup of lysosomes in the podocytes, suggesting that these cells are responsible for the processing of glomerular basement membranes.

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.

An iminosugar-based heparanase inhibitor heparastatin (SF4) suppresses infiltration of neutrophils and monocytes into inflamed dorsal air pouches

Local infiltration of inflammatory cells is regulated by a number of biological steps during which the cells likely penetrate through subendothelial basement membranes that contain heparan sulfate proteoglycans. In the present study, we examined whether administration of heparastatin (SF4), an iminosugar-based inhibitor of heparanase, could suppress local inflammation and degradation of heparan sulfate proteoglycans in basement membranes. In a carrageenan- or formyl peptide-induced dorsal air pouch inflammation model, the number of infiltrated neutrophils and monocytes was significantly lower in mice after topical administration of heparastatin (SF4). The concentration of chemokines MIP-2 and KC in pouch exudates of drug-treated mice was similar to control. In a zymosan-induced peritonitis model, the number of infiltrated cells was not altered in drug-treated mice. To further test how heparastatin (SF4) influences transmigration of inflammatory neutrophils, its suppressive effect on migration and matrix degradation was examined in vitro. In the presence of heparastatin (SF4), the number of neutrophils that infiltrated across a Matrigel-coated polycarbonate membrane was significantly lower, while the number of neutrophils passing through an uncoated membrane was not altered. Lysate of bone marrow-derived neutrophils released sulfate-radiolabeled macromolecules from basement membrane-like extracellular matrix, which was suppressed by heparastatin (SF4). Heparan sulfate degradation activity was almost completely abolished after incubation of lysate with protein G-conjugated anti-heparanase monoclonal antibody, strongly suggesting that the activity was due to heparanase-mediated degradation. Taken together, in a dorsal air pouch inflammation model heparastatin (SF4) potentially suppresses extravasation of inflammatory cells by impairing the degradation of basement membrane heparan sulfate.

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.

Podocyte-specific deletion of NDST1, a key enzyme in the sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo

Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to- and pedicel organization on- the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1-/-) was developed, having podocyte-specific deletion of NDST1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1-/- mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

光老化における基底膜ヘパラン硫酸の変化とその役割

表皮と真皮の間に存在する基底膜は，表皮と真皮の間で情報を適切にやり取りする機能「表皮真皮コミュニケーション」によって，皮膚全体を健やかな状態に保っている。紫外線を浴びることで基底膜の機能変化がどのように関与しているのかはわかっていなかった。基底膜に存在するヘパラン硫酸は，皮膚に重要な増殖因子と結合することで，表皮-真皮間の移動を制御していることから，われわれは基底膜のヘパラン硫酸に着目し，日常紫外線を浴びている光老化皮膚における基底膜のヘパラン硫酸の変化を検討した。紫外線を浴びた皮膚表皮では，ヘパラン硫酸を分解する酵素「ヘパラナーゼ」の発現増加，活性化に伴い，基底膜のヘパラン硫酸が著しく分解されていることを発見した。さらに，ヘパラナーゼの活性を抑えると，皮膚全体が良好な状態を維持できることを明らかにした。このことは，『紫外線によって酵素ヘパラナーゼが活性化すると，皮膚全体へダメージを与え，シワなどの光老化を促進する』ということを示唆している。この研究結果に基づき，酵素ヘパラナーゼの活性を制御する成分の探索を進め，「ムクロジエキス」を開発した。

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.

Heparanase and Autoimmune Diabetes

Heparanase (Hpse) is the only known mammalian endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS), found attached to the core proteins of heparan sulfate proteoglycans (HSPGs). Hpse plays a homeostatic role in regulating the turnover of cell-associated HS and also degrades extracellular HS in basement membranes (BMs) and the extracellular matrix (ECM), where HSPGs function as a barrier to cell migration. Secreted Hpse is harnessed by leukocytes to facilitate their migration from the blood to sites of inflammation. In the non-obese diabetic (NOD) model of autoimmune Type 1 diabetes (T1D), Hpse is also used by insulitis leukocytes to solubilize the islet BM to enable intra-islet entry of leukocytes and to degrade intracellular HS, an essential component for the survival of insulin-producing islet beta cells. Treatment of pre-diabetic adult NOD mice with the Hpse inhibitor PI-88 significantly reduced the incidence of T1D by ~50% and preserved islet HS. Hpse therefore acts as a novel immune effector mechanism in T1D. Our studies have identified T1D as a Hpse-dependent disease and Hpse inhibitors as novel therapeutics for preventing T1D progression and possibly the development of T1D vascular complications.

Heparanase localization during palatogenesis in mice.

Palatogenesis is directed by epithelial-mesenchymal interactions and results partly from remodeling of the extracellular matrix (ECM) of the palatal shelves. Here, we assessed heparanase distribution in developing mouse palates. No heparanase was observed in the vertically oriented palatal shelves in early stages of palate formation. As palate formation progressed, the palatal shelves were reorganized and arranged horizontally above the tongue, and heparanase localized to the epithelial cells of these shelves. When the palatal bilateral shelves first made contact, the heparanase localized to epithelial cells at the tips of shelves. Later in fusing palatal shelves, the cells of the medial epithelial seam (MES) were labeled with intense heparanase signal. In contrast, the basement membrane heparan sulfate (HS) was scarcely observed in the palatal shelves in contact. Moreover, perlecan labeling was sparse in the basement membrane of the MES, on which laminin and type IV collagen were observed. Moreover, we assessed the distribution of matrix metalloproteinase- (MMP-) 9, MMP-2, and MMP-3 in developing mouse palates and these MMPs were observed in the MES. Our findings indicated that heparanase was important for palate formation because it mediated degradation of the ECM of palatal shelves. Heparanase may, in concert with other proteases, participate in the regression of the MES.

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.

Fibrinogen, Riboflavin, and UVA to Immobilize a Corneal Flap – Molecular Mechanisms

Tissue glue containing fibrinogen (FIB) and riboflavin (RF), upon exposure to long wavelength ultraviolet light (UVA, 365 nM) has been proposed potentially to solve long-standing problems presented by corneal wound and epithelial ingrowth side-effects from laser-assisted in situ keratomileuis (LASIK). Data presented in a previous study demonstrated an ability of FIB + RF + UVA to adhere two stromal surfaces; however, to our knowledge no molecular mechanisms have been proposed to account for interactions occurring between corneal extracellular matrix (ECM) and tissue glue molecules. Here, we document several covalent and noncovalent interactions between these classes of macromolecules. SDS-PAGE and Western blot techniques were used to identify covalent interactions between tissue glue molecules and corneal ECM molecules in either the presence or absence of RF and UVA, in vitro and ex vivo. Surface plasmon resonance (SPR) was used to characterize noncovalent interactions, and obtain k(a), k(d), and K(D) binding affinity values. SDS-PAGE and Western blot analyses indicated that covalent interactions occurred between neighboring FIB molecules, as well as between FIB and collagen type I (Coll-I) proteins (in vitro and ex vivo). These interactions occurred only in the presence of RF and UVA. SPR data demonstrated the ability of FIB to bind noncovalently to corneal stroma molecules, Coll-I, decorin, dermatan sulfate, and corneal basement membrane molecules, laminin and heparan sulfate--only in the presence of Zn(2+). Covalent and (zinc-mediated) noncovalent mechanisms involving FIB and stromal ECM molecules contribute to the adhesion created by FIB + RF + UVA.

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.

Minimal Change Disease: A CD80 podocytopathy?

Minimal change disease is the most common nephrotic syndrome in children. Although the etiology of minimal change disease remains to be elucidated, it has been postulated that it is the result of a circulating T-cell factor that causes podocyte cytoskeleton disorganization leading to increased glomerular capillary permeability and/or changes in glomerular basement membrane heparan sulfate glycosaminoglycans resulting in proteinuria. Minimal change disease has been associated with allergies and Hodgkin disease. Consistent with these associations, a role for interleukin-13 with minimal change disease has been proposed. Furthermore, studies evaluating podocytes also have evolved. Recently, increased expression of CD80 (also termed B7-1) on podocytes was identified as a mechanism for proteinuria. CD80 is inhibited by binding to CTLA-4, which is expressed on regulatory T cells. Recently, we showed that urinary CD80 is increased in minimal change disease patients and limited studies have suggested that it is not commonly present in the urine of patients with other glomerular diseases. Interleukin-13 or microbial products via Toll-like receptors could be factors that induce CD80 expression on podocytes. CTLA-4 appears to regulate CD80 expression in podocytes, and to be altered in minimal change disease patients. These findings lead us to suggest that proteinuria in minimal change disease is caused by persistent CD80 expression in podocytes, possibly initiated by stimulation of these cells by antigens or cytokines.

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.

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.