Defective perlecan-associated basement membrane regeneration and altered modulation of transforming growth factor beta in corneal fibrosis.

In the cornea, the epithelial basement membrane (EBM) and corneal endothelial Descemet's basement membrane (DBM) critically regulate the localization, availability and, therefore, the functions of transforming growth factor (TGF)ß1, TGFß2, and platelet-derived growth factors (PDGF) that modulate myofibroblast development. Defective regeneration of the EBM, and notably diminished perlecan incorporation, occurs via several mechanisms and results in excessive and prolonged penetration of pro-fibrotic growth factors into the stroma. These growth factors drive mature myofibroblast development from both corneal fibroblasts and bone marrow-derived fibrocytes, and then the persistence of these myofibroblasts and the disordered collagens and other matrix materials they produce to generate stromal scarring fibrosis. Corneal stromal fibrosis often resolves completely if the inciting factor is removed and the BM regenerates. Similar defects in BM regeneration are likely associated with the development of fibrosis in other organs where perlecan has a critical role in the modulation of signaling by TGFß1 and TGFß2. Other BM components, such as collagen type IV and collagen type XIII, are also critical regulators of TGF beta (and other growth factors) in the cornea and other organs. After injury, BM components are dynamically secreted and assembled through the cooperation of neighboring cells-for example, the epithelial cells and keratocytes for the corneal EBM and corneal endothelial cells and keratocytes for the corneal DBM. One of the most critical functions of these reassembled BMs in all organs is to modulate the pro-fibrotic effects of TGFßs, PDGFs and other growth factors between tissues that comprise the organ.

Increased Risk of Aortic Dissection with Perlecan Deficiency.

Perlecan (HSPG2), a basement membrane-type heparan sulfate proteoglycan, has been implicated in the development of aortic tissue. However, its role in the development and maintenance of the aortic wall remains unknown. Perlecan-deficient mice (Hspg2-/--Tg: Perl KO) have been found to show a high frequency (15-35%) of aortic dissection (AD). Herein, an analysis of the aortic wall of Perl KO mice revealed that perlecan deficiency caused thinner and partially torn elastic lamina. Compared to the control aortic tissue, perlecan-deficient aortic tissue showed a significant decrease in desmosine content and an increase in soluble tropoelastin levels, implying the presence of immature elastic fibers in Perl KO mice. Furthermore, the reduced expression of the smooth muscle cell contractile proteins actin and myosin in perlecan-deficient aortic tissue may explain the risk of AD. This study showed that a deficiency in perlecan, which is localized along the elastic lamina and at the interface between elastin and fibrillin-1, increased the risk of AD, largely due to the immaturity of extracellular matrix in the aortic tissue. Overall, we proposed a new model of AD that considers the deficiency of extracellular molecule perlecan as a risk factor.

Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development.

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y-Neo Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y-Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

Trabecular Bone Deficit and Enhanced Anabolic Response to Re-Ambulation after Disuse in Perlecan-Deficient Skeleton.

Perlecan/Hspg2, a large monomeric heparan sulfate proteoglycan, is found in the basement membrane and extracellular matrix, where it acts as a matrix scaffold, growth factor depot, and tissue barrier. Perlecan deficiency leads to skeletal dysplasia in Schwartz-Jampel Syndrome (SJS) and is a risk factor for osteoporosis. In the SJS-mimicking murine model (Hypo), inferior cortical bone quality and impaired mechanotransduction in osteocytes were reported. This study focused on trabecular bone, where perlecan deficiency was hypothesized to result in structural deficit and altered response to disuse and re-loading. We compared the Hypo versus WT trabecular bone in both axial and appendicular skeletons of 8-38-week-old male mice, and observed severe trabecular deficit in Hypo mice, approximately 50% reduction of Tb.BV/TV regardless of skeletal site and animal age. Defects in endochondral ossification (e.g., accelerated mineralization), increases in osteoclast activity, and altered differentiation of bone progenitor cells in marrow contributed to the Hypo phenotype. The Hypo trabecular bone deteriorated further under three-week hindlimb suspension as did the WT. Re-ambulation partially recovered the lost trabecular bone in Hypo, but not in WT mice. The novel finding that low-impact loading could counter detrimental disuse effects in the perlecan-deficient skeleton suggests a strategy to maintain skeletal health in SJS patients.

Loss of perlecan heparan sulfate glycosaminoglycans lowers body weight and decreases islet amyloid deposition in human islet amyloid polypeptide transgenic mice.

Islet amyloid is a pathologic feature of type 2 diabetes (T2D) that is associated with ß-cell loss and dysfunction. These amyloid deposits form via aggregation of the ß-cell secretory product islet amyloid polypeptide (IAPP) and contain other molecules including the heparan sulfate proteoglycan perlecan. Perlecan has been shown to bind amyloidogenic human IAPP (hIAPP) via its heparan sulfate glycosaminoglycan (HS GAG) chains and to enhance hIAPP aggregation in vitro. We postulated that reducing the HS GAG content of perlecan would also decrease islet amyloid deposition in vivo. hIAPP transgenic mice were crossed with Hspg2Δ3/Δ3 mice harboring a perlecan mutation that prevents HS GAG attachment (hIAPP;Hspg2Δ3/Δ3), and male offspring from this cross were fed a high fat diet for 12 months to induce islet amyloid deposition. At the end of the study body weight, islet amyloid area, ß-cell area, glucose tolerance and insulin secretion were analyzed. hIAPP;Hspg2Δ3/Δ3 mice exhibited significantly less islet amyloid deposition and greater ß-cell area compared to hIAPP mice expressing wild type perlecan. hIAPP;Hspg2Δ3/Δ3 mice also gained significantly less weight than other genotypes. When adjusted for differences in body weight using multiple linear regression modeling, we found no differences in islet amyloid deposition or ß-cell area between hIAPP transgenic and hIAPP;Hspg2Δ3/Δ3 mice. We conclude that loss of perlecan exon 3 reduces islet amyloid deposition in vivo through indirect effects on body weight and possibly also through direct effects on hIAPP aggregation. Both of these mechanisms may promote maintenance of glucose homeostasis in the setting of T2D.

Perlecan regulates pericyte dynamics in the maintenance and repair of the blood-brain barrier.

Ischemic stroke causes blood-brain barrier (BBB) breakdown due to significant damage to the integrity of BBB components. Recent studies have highlighted the importance of pericytes in the repair process of BBB functions triggered by PDGFRß up-regulation. Here, we show that perlecan, a major heparan sulfate proteoglycan of basement membranes, aids in BBB maintenance and repair through pericyte interactions. Using a transient middle cerebral artery occlusion model, we found larger infarct volumes and more BBB leakage in conditional perlecan (Hspg2)-deficient (Hspg2 - / - -TG) mice than in control mice. Control mice showed increased numbers of pericytes in the ischemic lesion, whereas Hspg2 - / - -TG mice did not. At the mechanistic level, pericytes attached to recombinant perlecan C-terminal domain V (perlecan DV, endorepellin). Perlecan DV enhanced the PDGF-BB-induced phosphorylation of PDGFRß, SHP-2, and FAK partially through integrin α5ß1 and promoted pericyte migration. Perlecan therefore appears to regulate pericyte recruitment through the cooperative functioning of PDGFRß and integrin α5ß1 to support BBB maintenance and repair following ischemic stroke.

Heparan sulfate proteoglycan deficiency up-regulates the intracellular production of nitric oxide in Chinese hamster ovary cell lines.

We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O2- ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.

The reduction of heparan sulphate in the glomerular basement membrane does not augment urinary albumin excretion.

BACKGROUND: Heparan sulphate proteoglycan (HSPG) is present in the glomerular basement membrane (GBM) and is thought to play a major role in the glomerular charge barrier. Reductions and structural alterations of HSPG are observed in different types of kidney diseases accompanied by proteinuria. However, their causal relations remain unknown. METHODS: We generated podocyte-specific exostosin-like 3 gene (Extl3) knockout mice (Extl3KO) using a Cre-loxP recombination approach. A reduction of HSPG was expected in the GBM of these mice, because EXTL3 is involved in its synthesis. Mice were separated into three groups, according to the loads on the glomeruli: a high-protein diet group, a high-protein and high-sodium diet group and a hyperglycaemic group induced by streptozotocin treatment in addition to maintenance on a high-protein and high-sodium diet. The urinary albumin:creatinine ratio was measured at 7, 11, 15 and 19 weeks of age. Renal histology was also investigated. RESULTS: Podocyte-specific expression of Cre recombinase was detected by immunohistochemistry. Moreover, immunofluorescent staining demonstrated a significant reduction of HSPG in the GBM. Electron microscopy showed irregularities in the GBM and effacement of the foot processes in Extl3KO. The values of the urinary albumin:creatinine ratio were within the range of microalbuminuria in all groups and did not significantly differ between the control mice and Extl3KO. CONCLUSIONS: The reduction of HSPG in the GBM did not augment urinary albumin excretion. HSPG's anionic charge appears to contribute little to the glomerular charge barrier.

Knockdown of the pericellular matrix molecule perlecan lowers in situ cell and matrix stiffness in developing cartilage.

The pericellular matrix (PCM) is a component of the extracellular matrix that is found immediately surrounding individual chondrocytes in developing and adult cartilage, and is rich in the proteoglycan perlecan. Mutations in perlecan are the basis of several developmental disorders, which are thought to arise from disruptions in the mechanical stability of the PCM. We tested the hypothesis that defects in PCM organization will reduce the stiffness of chondrocytes in developing cartilage by combining a murine model of Schwartz-Jampel syndrome, in which perlecan is knocked down, with our novel atomic force microscopy technique that can measure the stiffness of living cells and surrounding matrix in embryonic and postnatal tissues in situ. Perlecan knockdown altered matrix organization and significantly decreased the stiffness of both chondrocytes and interstitial matrix as a function of age and genotype. Our results demonstrate that the knockdown of a spatially restricted matrix molecule can have a profound influence on cell and tissue stiffness, implicating a role for outside-in mechanical signals from the PCM in regulating the intracellular mechanisms required for the overall development of cartilage.

The heparan sulphate deficient Hspg2 exon 3 null mouse displays reduced deposition of TGF-ß1 in skin compared to C57BL/6 wild type mice.

This was an observational study where we examined the role of perlecan HS on the deposition of TGF-ß1 in C57BL/6 and Hspg2(∆3-/∆3-) perlecan exon 3 null mouse skin. Despite its obvious importance in skin repair and tissue homeostasis no definitive studies have immunolocalised TGF-ß1 in skin in WT or Hspg2(∆3-/∆3-) perlecan exon 3 null mice. Vertical parasagittal murine dorsal skin from 3, 6 and 12 week old C57BL/6 and Hspg2(∆3-/∆3-) mice were fixed in neutral buffered formalin, paraffin embedded and 4 µm sections stained with Mayers haematoxylin and eosin (H & E). TGF-ß1 was immunolocalised using a rabbit polyclonal antibody, heat retrieval and the Envision NovaRED detection system. Immunolocalisation of TGF-ß1 differed markedly in C57BL/6 and Hspg2(∆3-/∆3-) mouse skin, ablation of exon 3 of Hspg2 resulted in a very severe reduction in the deposition of TGF-ß1 in skin 3-12 weeks postnatally. The reduced deposition of TGF-ß1 observed in the present study would be expected to impact detrimentally on the remodelling and healing capacity of skin in mutant mice compounding on the poor wound-healing properties already reported for perlecan exon 3 null mice due to an inability to signal with FGF-2 and promote angiogenic repair processes. TGF-ß1 also has cell mediated effects in tissue homeostasis and matrix stabilisation a reduction in TGF-ß1 deposition would therefore be expected to detrimentally impact on skin homeostasis in the perlecan mutant mice.

Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle.

The autophagy-lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.

Perlecan heparan sulfate deficiency impairs pulmonary vascular development and attenuates hypoxic pulmonary hypertension.

AIMS: Excessive vascular cell proliferation is an important component of pulmonary hypertension (PH). Perlecan is the major heparan sulfate (HS) proteoglycan in the vascular extracellular matrix. It binds growth factors, including FGF2, and either restricts or promotes cell proliferation. In this study, we have explored the effects of perlecan HS deficiency on pulmonary vascular development and in hypoxia-induced PH. METHODS AND RESULTS: In normoxia, Hspg2(Δ3/Δ3) mice, deficient in perlecan HS, had reduced pericytes and muscularization of intra-acinar vessels. Pulmonary angiography revealed a peripheral perfusion defect. Despite these abnormalities, right ventricular systolic pressure (RVSP) and myocardial mass remained normal. After 4 weeks of hypoxia, increases in the proportion of muscularized vessels, RVSP, and right ventricular hypertrophy were significantly less in Hspg2(Δ3/Δ3) compared with wild type. The early phase of hypoxia induced a significantly lower increase in fibroblast growth factor receptor-1 (FGFR1) protein level and receptor phosphorylation, and reduced pulmonary artery smooth muscle cell (PASMC) proliferation in Hspg2(Δ3/Δ3). At 4 weeks, FGF2 mRNA and protein were also significantly reduced in Hspg2(Δ3/Δ3) lungs. Ligand and carbohydrate engagement assay showed that perlecan HS is required for HS-FGF2-FGFR1 ternary complex formation. In vitro, proliferation assays showed that PASMC proliferation is reduced by selective FGFR1 inhibition. PASMC adhesion to fibronectin was higher in Hspg2(Δ3/Δ3) compared with wild type. CONCLUSIONS: Perlecan HS chains are important for normal vascular arborization and recruitment of pericytes to pulmonary vessels. Perlecan HS deficiency also attenuates hypoxia-induced PH, where the underlying mechanisms involve impaired FGF2/FGFR1 interaction, inhibition of PASMC growth, and altered cell-matrix interactions.

Deficiency in perlecan/HSPG2 during bone development enhances osteogenesis and decreases quality of adult bone in mice.

Perlecan/HSPG2 (Pln) is a large heparan sulfate proteoglycan abundant in the extracellular matrix of cartilage and the lacunocanalicular space of adult bones. Although Pln function during cartilage development is critical, evidenced by deficiency disorders including Schwartz-Jampel Syndrome and dyssegmental dysplasia Silverman-Handmaker type, little is known about its function in development of bone shape and quality. The purpose of this study was to understand the contribution of Pln to bone geometric and mechanical properties. We used hypomorph mutant mice that secrete negligible amount of Pln into skeletal tissues and analyzed their adult bone properties using micro-computed tomography and three-point-bending tests. Bone shortening and widening in Pln mutants was observed and could be attributed to loss of growth plate organization and accelerated osteogenesis that was reflected by elevated cortical thickness at older ages. This effect was more pronounced in Pln mutant females, indicating a sex-specific effect of Pln deficiency on bone geometry. Additionally, mutant females, and to a lesser extent mutant males, increased their elastic modulus and bone mineral densities to counteract changes in bone shape, but at the expense of increased brittleness. In summary, Pln deficiency alters cartilage matrix patterning and, as we now show, coordinately influences bone formation and calcification.

Perlecan is required for FGF-2 signaling in the neural stem cell niche.

In the adult subventricular zone (neurogenic niche), neural stem cells double-positive for two markers of subsets of neural stem cells in the adult central nervous system, glial fibrillary acidic protein and CD133, lie in proximity to fractones and to blood vessel basement membranes, which contain the heparan sulfate proteoglycan perlecan. Here, we demonstrate that perlecan deficiency reduces the number of both GFAP/CD133-positive neural stem cells in the subventricular zone and new neurons integrating into the olfactory bulb. We also show that FGF-2 treatment induces the expression of cyclin D2 through the activation of the Akt and Erk1/2 pathways and promotes neurosphere formation in vitro. However, in the absence of perlecan, FGF-2 fails to promote neurosphere formation. These results suggest that perlecan is a component of the neurogenic niche that regulates FGF-2 signaling and acts by promoting neural stem cell self-renewal and neurogenesis.

Perlecan maintains microvessel integrity in vivo and modulates their formation in vitro.

Perlecan is a heparan sulfate proteoglycan assembled into the vascular basement membranes (BMs) during vasculogenesis. In the present study we have investigated vessel formation in mice, teratomas and embryoid bodies (EBs) in the absence of perlecan. We found that perlecan was dispensable for blood vessel formation and maturation until embryonic day (E) 12.5. At later stages of development 40% of mutant embryos showed dilated microvessels in brain and skin, which ruptured and led to severe bleedings. Surprisingly, teratomas derived from perlecan-null ES cells showed efficient contribution of perlecan-deficient endothelial cells to an apparently normal tumor vasculature. However, in perlecan-deficient EBs the area occupied by an endothelial network and the number of vessel branches were significantly diminished. Addition of FGF-2 but not VEGF(165) rescued the in vitro deficiency of the mutant ES cells. Furthermore, in the absence of perlecan in the EB matrix lower levels of FGFs are bound, stored and available for cell surface presentation. Altogether these findings suggest that perlecan supports the maintenance of brain and skin subendothelial BMs and promotes vasculo- and angiogenesis by modulating FGF-2 function.

Comparative immunolocalisation of fibrillin-1 and perlecan in the human foetal, and HS-deficient hspg2 exon 3 null mutant mouse intervertebral disc.

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ³â»/Δ³â» exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ³â»/Δ³â» exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell-matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-ß a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.

A mouse model of Schwartz-Jampel syndrome reveals myelinating Schwann cell dysfunction with persistent axonal depolarization in vitro and distal peripheral nerve hyperexcitability when perlecan is lacking.

Congenital peripheral nerve hyperexcitability (PNH) is usually associated with impaired function of voltage-gated K(+) channels (VGKCs) in neuromyotonia and demyelination in peripheral neuropathies. Schwartz-Jampel syndrome (SJS) is a form of PNH that is due to hypomorphic mutations of perlecan, the major proteoglycan of basement membranes. Schwann cell basement membrane and its cell receptors are critical for the myelination and organization of the nodes of Ranvier. We therefore studied a mouse model of SJS to determine whether a role for perlecan in these functions could account for PNH when perlecan is lacking. We revealed a role for perlecan in the longitudinal elongation and organization of myelinating Schwann cells because perlecan-deficient mice had shorter internodes, more numerous Schmidt-Lanterman incisures, and increased amounts of internodal fast VGKCs. Perlecan-deficient mice did not display demyelination events along the nerve trunk but developed dysmyelination of the preterminal segment associated with denervation processes at the neuromuscular junction. Investigating the excitability properties of the peripheral nerve suggested a persistent axonal depolarization during nerve firing in vitro, most likely due to defective K(+) homeostasis, and excluded the nerve trunk as the original site for PNH. Altogether, our data shed light on perlecan function by revealing critical roles in Schwann cell physiology and suggest that PNH in SJS originates distally from synergistic actions of peripheral nerve and neuromuscular junction changes.

HSPG-deficient zebrafish uncovers dental aspect of multiple osteochondromas.

Multiple Osteochondromas (MO; previously known as multiple hereditary exostosis) is an autosomal dominant genetic condition that is characterized by the formation of cartilaginous bone tumours (osteochondromas) at multiple sites in the skeleton, secondary bursa formation and impingement of nerves, tendons and vessels, bone curving, and short stature. MO is also known to be associated with arthritis, general pain, scarring and occasional malignant transformation of osteochondroma into secondary peripheral chondrosarcoma. MO patients present additional complains but the relevance of those in relation to the syndromal background needs validation. Mutations in two enzymes that are required during heparan sulphate synthesis (EXT1 or EXT2) are known to cause MO. Previously, we have used zebrafish which harbour mutations in ext2 as a model for MO and shown that ext2⁻/⁻ fish have skeletal defects that resemble those seen in osteochondromas. Here we analyse dental defects present in ext2⁻/⁻ fish. Histological analysis reveals that ext2⁻/⁻ fish have very severe defects associated with the formation and the morphology of teeth. At 5 days post fertilization 100% of ext2⁻/⁻ fish have a single tooth at the end of the 5(th) pharyngeal arch, whereas wild-type fish develop three teeth, located in the middle of the pharyngeal arch. ext2⁻/⁻ teeth have abnormal morphology (they were shorter and thicker than in the WT) and patchy ossification at the tooth base. Deformities such as split crowns and enamel lesions were found in 20% of ext2⁺/⁻ adults. The tooth morphology in ext2⁻/⁻ was partially rescued by FGF8 administered locally (bead implants). Our findings from zebrafish model were validated in a dental survey that was conducted with assistance of the MHE Research Foundation. The presence of the malformed and/or displaced teeth with abnormal enamel was declared by half of the respondents indicating that MO might indeed be also associated with dental problems.

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

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

Perlecan/Hspg2 deficiency alters the pericellular space of the lacunocanalicular system surrounding osteocytic processes in cortical bone.

Osteocytes project long, slender processes throughout the mineralized matrix of bone, where they connect and communicate with effector cells. The interconnected cellular projections form the functional lacunocanalicular system, allowing fluid to pass for cell-to-cell communication and nutrient and waste exchange. Prevention of mineralization in the pericellular space of the lacunocanalicular pericellular space is crucial for uninhibited interstitial fluid movement. Factors contributing to the ability of the pericellular space of the lacunocanalicular system to remain open and unmineralized are unclear. Immunofluorescence and immunogold localization by transmission electron microscopy demonstrated perlecan/Hspg2 signal localized to the osteocyte lacunocanalicular system of cortical bone, and this proteoglycan was found in the pericellular space of the lacunocanalicular system. In this study we examined osteocyte lacunocanalicular morphology in mice deficient in the large heparan sulfate proteoglycan perlecan/Hspg2 in this tissue. Ultrastructural measurements with electron microscopy of perlecan/Hspg2-deficient mice demonstrated diminished osteocyte canalicular pericellular area, resulting from a reduction in the total canalicular area. Additionally, perlecan/Hspg2-deficient mice showed decreased canalicular density and a reduced number of transverse tethering elements per canaliculus. These data indicated that perlecan/Hspg2 contributed to the integrity of the osteocyte lacunocanalicular system by maintaining the size of the pericellular space, an essential task to promote uninhibited interstitial fluid movement in this mechanosensitive environment. This work thus identified a new barrier function for perlecan/Hspg2 in murine cortical bone.