Willi Halfter

Collagen XVIII Is a Basement Membrane Heparan Sulfate Proteoglycan

The present study shows that collagen XVIII is, next to perlecan and agrin, the third basal lamina heparan sulfate proteoglycan (HSPG) and the first collagen/proteoglycan with heparan sulfate side chains. By using monoclonal antibodies to an unidentified HSPG in chick, 14 cDNA clones were isolated from a chick yolk sac library. All clones had a common nucleotide sequence that was homologous to the mRNA sequences of mouse and human collagen XVIII. The deduced amino acid sequence of the chick fragment shows an 83% overall homology with the human and mouse collagen XVIII. Similar to the human and mouse homologue, the chick collagen XVIII mRNA has a size of 4.5 kilobase pairs. In Western blots, collagen XVIII appeared as a smear with a molecular mass of 300 kDa. After treatment with heparitinase, the protein was reduced in molecular mass by 120 kDa to a protein core of 180 kDa. Collagen XVIII has typical features of a collagen, such as its existence, under non-denaturing conditions, as a non-covalently linked oligomer, and a sensitivity of the core protein to collagenase digestion. It also has characteristics of an HSPG, such as long heparitinase-sensitive carbohydrate chains and a highly negative net charge. Collagen XVIII is abundant in basal laminae of the retina, epidermis, pia, cardiac and striated muscle, kidney, blood vessels, and lung. In situ hybridization showed that the main expression of collagen XVIII HSPG in the chick embryo is in the kidney and the peripheral nervous system. As a substrate, collagen XVIII moderately promoted the adhesion of Schwann cells but had no such activity on peripheral nervous system neurons and axons.

Agrin binds to β-amyloid (Aβ), accelerates Aβ fibril formation and is localized to Aβ deposits in Alzheimer's disease brain

Abstract Agrin is an extracellular matrix heparan sulfate proteoglycan (HSPG) well known for its role in modulation of the neuromuscular junction during development. Although agrin is one of the major HSPGs of the brain, its function there remains elusive. Here we provide evidence suggesting a possible function for agrin in Alzheimers disease brain. Agrin protein binds the amyloidogenic peptide Aβ (1–40) in its fibrillar state via a mechanism that involves the heparan sulfate glycosaminoglycan chains of agrin. Furthermore, agrin is able to accelerate Aβ fibril formation and protect Aβ (1–40) from proteolysis, in vitro. Supporting a biological significance for these in vitro data, immunocytochemical studies demonstrate agrins presence within senile plaques and cerebrovascular amyloid deposits, and agrin immunostained capillaries exhibit pathological alterations in AD brain. These data therefore suggest that agrin may be an important factor in the progression of Aβ peptide aggregation and/or its persistence in Alzheimers disease brain.

Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches.

Biomechanical properties of native basement membranes

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal‐vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Youngs modulus from 0.95 MPa to 3.30 MPa. The apparent Youngs modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.

Collagen XVIII/endostatin is essential for vision and retinal pigment epithelial function

Age‐related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub‐RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age‐dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/endostatin is essential for RPE function, and suggest an important role of this collagen in Bruchs membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruchs membrane, shows that it is part of basement membrane molecular networks.

Distribution and substrate properties of agrin, a heparan sulfate proteoglycan of developing axonal pathways.

The distribution and substrate properties of agrin, an extracellular matrix heparan sulfate proteoglycan (HSPG), was investigated in the developing chick nervous system by immunocytochemistry, Western blotting, and in neurite outgrowth assays. By comparing the distribution of agrin with that of laminin‐1, merosin (laminin‐2), neurofilament, and neural cell adhesion molecule (NCAM), it was found that throughout development, agrin is a constituent of all basal laminae. From embryonic day (E) 4 onwards, agrin is also abundant in axonal pathways of the central nervous system, such as the optic nerve, the tectobulbar pathway, the white matter of the spinal cord, and the marginal and the molecular layers of the forebrain and the cerebellum. The abundance of agrin in brain decreases from E13 onwards. In the peripheral nervous system, agrin is present throughout development as a constituent of the Schwann cell basal laminae. Western blots confirmed the immunocytochemical data, showing maximum expression of agrin occurs during the early to medium stages of brain development. Western blots also showed that in mouse and human brain, agrin exists as an HSPG. Purified agrin did not support neurite outgrowth, rather it inhibited retinal neurite extension on mixed agrin/merosin substrates. Despite the fact that agrin, when used as a substrate inhibited neurite outgrowth, its temporal and spatial overlap with growing axons suggests that agrin has a supportive role in the development of axonal pathways, possibly as a binding component for growth factors and cell adhesion proteins. J. Comp. Neurol. 383:1–17, 1997.

Age-dependent changes in the structure, composition and biophysical properties of a human basement membrane.

Basement membranes (BMs) are considered to be uniform, approximately 100 nm-thin extracellular matrix sheets that serve as a substrate for epithelial cells, endothelial cells and myotubes. To find out whether BMs maintain their ultrastructure, protein composition and biophysical properties throughout life the natural aging history of the human inner limiting membranes (ILM) was investigated. The ILM is a BM at the vitreal surface of the retina that connects the retina with the vitreous. Transmission electron microscopy (TEM) showed that the ILM steadily increases in thickness from 70 nm at fetal stages to several microns at age 90. By the age of 20, the ILM loses its laminated structure to become an amorphous and very irregular extracellular matrix layer. Atomic force microscopy (AFM) showed that the native, hydrated ILMs are on average 4-fold thicker than the dehydrated ILMs as seen by TEM and that their thickness is prominently determined by its water-binding proteoglycans. The morphological changes are accompanied by age-related changes in the biochemical composition, whereby the relative concentrations of collagen IV and agrin increase, and the concentration of laminin decreases with age. Force-indentation measurements by AFM also showed that ILMs become increasingly stiffer with advancing age. The data suggest that BMs from other human tissues may undergo similar age-related changes.

Expression of collagen XVIII and localization of its glycosaminoglycan attachment sites

Collagen XVIII is the only currently known collagen that carries heparan sulfate glycosaminoglycan side chains. The number and location of the glycosaminoglycan attachment sites in the core protein were determined by eukaryotic expression of full-length chick collagen XVIII and site-directed mutagenesis. Three Ser-Gly consensus sequences carrying glycosaminoglycan side chains were detected in the middle and N-terminal part of the core protein. One of the Ser-Gly consensus sequences carried a heparan sulfate side chain, and the remaining two had mixed chondroitin and heparan sulfate side chains; thus, recombinant collagen XVIII was a hybrid of heparan sulfate and chondroitin proteoglycan. In contrast, collagen XVIII from all chick tissues so far assayed have exclusively heparan sulfate side chains, indicating that the posttranslational modification of proteins expressed in vitro is not entirely identical to the processing that occurs in a living embryo. Incubating the various mutated collagen XVIIIs with retinal basement membranes showed that the heparan sulfate glycosaminoglycan side chains mediate the binding of collagen XVIII to basement membranes.

A role of midkine in the development of the neuromuscular junction

Midkine (MK) is a member of a family of developmentally regulated neurotrophic and heparin-binding growth factors. It is expressed during the midgestation period in a retinoid-acid dependent manner during embryogenesis in the mouse. In vitro, it promotes neurite outgrowth from spinal cord neurons and cell migration. It expression is strongest in the central nervous system, thus suggesting a function for this protein in neural development. In this study, the role of MK in synaptogenesis was examined in the Xenopus system. A Xenopus MK cDNA was cloned from an embryonic library encompassing neurulation and synaptogenesis stages. By Northern blot analysis, MK mRNA was detected from the onset of neurulation and throughout the stages of synaptogenesis in the Xenopus embryo. This suggests that MK is also an important growth regulator in Xenopus embryogenesis. To study the function of MK in the development of the neuromuscular junction (NMJ), fusion proteins were made and their ability to induce the formation of acetylcholine receptor (AChR) clusters in cultured muscle cells was studied. Beads coated with MK strongly induce AChR clustering. When nerve-muscle cocultures were labeled with antibodies made against the MK fusion protein, MK immunoreactivity was detected at the NMJ. Unlike heparin-binding growth-associated molecule (HB-GAM), another member of this growth factor family, MK expression cannot be detected in the muscle but is present in spinal cord neurites. Consistent with these in vitro data is the observation that MK mRNA is only localized in the central nervous system but the protein is deposited at the intersomitic junction where the NMJ is located in vivo. Exogenously applied MK does bind to the heparan sulfate proteoglycan on the surface of Xenopus muscle cells. Agrin, a heparan-sulfate proteoglycan that induces the formation of AChR clusters in cultured muscle cells, binds strongly to MK. Bath application of MK in conjunction with agrin results in a change in the pattern of AChR clustering induced by agrin alone. These data suggest that MK is a neuron-derived factor that participates in the signal transduction process during NMJ development.

Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.