Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors: strategies to improve neuronal regeneration

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a "regeneration cocktail." In this review, we describe the ability of peptides derived from tenascin- C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

The role of extracellular matrix in CNS regeneration

Chondroitin sulfate proteoglycans are the principal inhibitory component of glial scars, which form after damage to the adult central nervous system and act as a barrier to regenerating axons. Recent findings have furthered our understanding of the mechanisms that result in a failure of regeneration after spinal cord injury and suggest that a multipartite approach will be required to facilitate long-distance regeneration and functional recovery.     

Ependymins from the cerebrospinal fluid of salmonid fish: gene structure and molecular characterization.

So far, ependymins (Epds) have been sequenced only from cypriniform fish, and in the past all attempts have failed to characterize, on a molecular level, homologous Epd proteins in higher vertebrates. Therefore, rainbow trout (Oncorhynchus mykiss) Epds, which represent the predominant proteins of the cerebrospinal fluid, have been N-terminally sequenced and the encoding cDNA subsequently cloned using the polymerase chain reaction. Surprisingly, only 40-42% of the amino acids are identical with the corresponding sequences from goldfish (Carassius auratus), and no convincing immunological cross-reactivity is observed with an antiserum raised against purified Epds from C. auratus. O. mykiss possesses two highly homologous genes encoding Epds (Om-I, Om-II), a feature typical of a quasi-tetraploid species. Western analysis, using two specific antibodies against Epds from O. mykiss, revealed a variety of different glycosylation variants. In contrast to C. auratus, Epds from O. mykiss probably do not form disulfide-linked dimers. The structure of one Epd gene and its flanking regions have been determined for the Atlantic salmon (Salmo salar). Six exons were deduced by comparison with the corresponding cDNA sequence from O. mykiss (almost 98% homology with Om-II).     

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a “regeneration cocktail.” In this review, we describe the ability of peptides derived from tenascin-C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

The role of brain extracellular proteins in neuroplasticity and learning

Double labeling studies of the pattern of protein synthesis in goldfish and mouse brain identified a class of glycoproteins (the ependymins) whose turnover rate was enhanced after training. A variety of control experiments indicated that these macromolecules have an important role in the molecular and cell biology of learning. Antisera to the ependymins when injected into the brains of trained goldfish cause amnesia of a newly acquired behavior. Isolation and localization studies by immunocytochemical methods indicate that the ependymins are released into the brain extracellular fluid by a class of neurosecretory cells. In mammalian brain ependymin-containing cells are highly concentrated in the limibic system. The ependymins are constituted from two disulfide-linked acidic polypeptide chains (M.W.37K and 31K). They contain at least 5% covalently bound carbohydrate per chain with mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid as the predominant components. The highly soluble ependymins can rapidly polymerize to form an insoluble fibrous matrix if calcium is removed from solution by the addition of a Ca2+-chelating agent or dialysis. The self-aggregation property of the ependymins can be triggered by the depletion of Ca2+ from the extracellular space. Studies of the kinetics of the aggregation phenomenon by measurements of turbidity changes indicate that the process can be terminated but not reversed by restoring Ca2+ to its normal CSF level. Immunohistochemical studies of the brains of trained goldfish show the presence of punctate statining sites in the perimeter of certain cells located in specific brain regions. This suggests that ependymin aggregation might occur in vivo during learning. A molecular hypothesis relating the aggregation properties of the ependymins to neuroplasticity and learning is proposed.     

Laminin-like glycoproteins in extracellular matrix of endodermal cells

Extracellular matrix from a mouse endodermal cell line consisted mainly of two polypeptides with molecular weights of about 200,000 (200K) and 400,000 (400K). Both poly-peptides incorporated radioactivity from [³H]proline and [³H]glucosamine and were solubilized from the matrix by treatment with bacterial collagenase or 0.5 m sodium chloride. These polypeptides appeared similar to those of laminin (R. Timpl, H. Rohde, P. G. Robey, S. I. Rennard, J.-M. Foidart, and G. R. Martin, 1979, J. Biol. Chem., 254, 9933–9937) in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate but the laminin polypeptides seemed slightly larger than the 200K and 400K polypeptides, respectively. The amino acid compositions of the isolated 200K and 400K polypeptides resembled one another and the previously published amino acid composition of laminin. Antibodies prepared against the solubilized extracellular matrix protein (mixture of 200K and 400K components) as well as those against the isolated 400K component precipitated both the 400K component and the 200K component from culture media. These antisera and antisera to laminin showed identical reactivities in immunodiffusion and in immunofluorescence of tissue sections where they stained basement membranes. The immunofluorescent staining pattern was similar to that obtained with antifibronectin except in the liver where antifibronectin stained the biliary ducts and the liver sinusoids, while laminin-like immunoreactivity was not present in the sinusoidal areas. Such differences in distribution of matrix components could be involved in generation of signals for differentiation and growth of the adjacent cells.     

Regulation of autophagy by extracellular matrix glycoproteins in HeLa cells

Macroautophagy is a major lysosomal degradation pathway for cellular components in eukaryotic cells. Baseline macroautophagy is important for quality control of the cytoplasm in order to avoid the accumulation of cytotoxic products. Its stimulation by various stressful situations, including nutrient starvation, is important in maintaining cell survival. Here we demonstrate that macroautophagy is regulated differently depending on whether HeLa cells adhere to collagen I or collagen IV, proteins typical of connective tissue and basal membrane, respectively. We observed that the basal levels of macroautophagy were higher in cells plated on collagen IV than in cells plated on collagen I or on uncoated substrate. However, the stimulation of macroautophagy by nutrient starvation, as reflected by the buildup of autophagosomes and the increase in the autophagic flux, was higher in cells plated on collagen I than in cells plated on collagen IV. These contrasting results were not due to differences in the starvation-dependent inhibition of mTOR complex 1 signaling. Interestingly, cells plated on collagen IV formed numerous focal adhesions (FAs), whereas fewer FAs were observed in cells plated on the other substrates. This implies that focal adhesion kinase (FAK) was more robustly activated by collagen IV. Silencing the expression of FAK by siRNA in cells plated on collagen IV shifted the autophagic phenotype of these cells to an "uncoated substrate autophagic phenotype" under both basal and starvation-induced conditions. Moreover, cells plated on collagen IV were less dependent on autophagy to survive in the absence of nutrients. We conclude that extracellular matrix components can modulate macroautophagy and mitigate its role in cell survival.     

The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis

Atherosclerotic plaque forms in regions of the vasculature exposed to disturbed flow. NF-kappaB activation by fluid flow, leading to expression of target genes such as E-selectin, ICAM-1, and VCAM-1, may regulate early monocyte recruitment and fatty streak formation. Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling. Therefore, we examined the involvement of the extracellular matrix in this process. Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not. In vivo, fibronectin and fibrinogen are deposited at atherosclerosis-prone sites before other signs of atherosclerosis. Ligation of integrin alpha2beta1 on collagen prevents flow-induced NF-kappaB activation through a p38-dependent pathway that is activated locally at adhesion sites. Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.     

On the role of proteases, their inhibitors and the extracellular matrix in promoting neurite outgrowth

Using a novel method, a monoclonal antibody was produced which can directly block the activity of an extracellular matrix-associated neurite outgrowth promoting complex (Matthew and Patterson, 1983). Presumably binding at or near the active site, this antibody recognizes a determinant consisting of heparan sulfate and a larger molecule which is likely to be laminin (Matthew et al., in preparation). The antibody has been further used to localize this determinant in adult tissues in vivo. Extracellular binding is seen at sites known to promote axon regeneration in the peripheral nervous system and is not seen in the central nervous system (Matthew et al., in preparation). In investigating how neurons may modify their environment as they grow processes, we have recently found that sensory and sympathetic neurons spontaneously release a collagenase and a plasminogen activator from their distal processes and/or growth cones (Pittman, 1985). A 43 kD irreversible inhibitor of the plasminogen activator is secreted by cardiac myocytes and is found on the surfaces of cultured neurons (Pittman, 1984). This inhibitor is also released by nonneuronal cell cultures from peripheral, but not central, nerves (Pittman, unpublished). Of interest in relation to the proteoglycan neurite outgrowth promoting complex is the finding that the 43 kD inhibitor preparation binds heparin tightly and can displace laminin from its heparin binding site (Patterson and Pittman, unpublished). Thus it is possible that the protease/inhibitor system could affect outgrowth via interaction with the neurite outgrowth promoting complex in the extracellular matrix.     

Increased digitalis-like activity in human cerebrospinal fluid after expansion of the extracellular fluid volume

The present study was designed to determine whether acute expansion of the extracellular fluid volume influenced the digitalis-like activity of human cerebrospinal fluid (CSF), previously described by our laboratory. Human CSF samples, drawn before and 30 minutes after the intravenous infusion of 1 liter of either saline or glucose solutions, were assayed for digitalis-like activity by inhibition of either the 86Rb+ uptake into human erythrocytes or by the activity of a purified Na+ - K+ ATPase. The CSF inhibitory activity on both systems significantly increased after the infusion of sodium solutions but did not change after the infusion of glucose. These results indicate that the digitalis-like factor of human CSF might be involved in the regulation of the extracellular fluid volume and electrolyte content and thereby in some of the physiological responses to sodium loading.     

Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process.

Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans. Once established in this harsh ecological niche, it remains there virtually for the entire life of the host. To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium. Nevertheless, a critical feature of H. pylori is its ability to establish a long-lasting infection. In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection. A large body of evidence has shown that the interaction between H. pylori and the host is very complex. In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H. pylori as a chronically persisting pathogen. Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant. This review examines the binding of host proteins (serum and extracellular matrix proteins) to H. pylori and considers the significance of these interactions in the infectious process. A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H. pylori infections and could represent an alternative to antibiotic treatment.     

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche

The repair of injured tendons remains a great challenge, largely owing to a lack of in-depth characterization of tendon cells and their precursors. We show that human and mouse tendons harbor a unique cell population, termed tendon stem/progenitor cells (TSPCs), that has universal stem cell characteristics such as clonogenicity, multipotency and self-renewal capacity. The isolated TSPCs could regenerate tendon-like tissues after extended expansion in vitro and transplantation in vivo. Moreover, we show that TSPCs reside within a unique niche predominantly comprised of an extracellular matrix, and we identify biglycan (Bgn) and fibromodulin (Fmod) as two critical components that organize this niche. Depletion of Bgn and Fmod affects the differentiation of TSPCs by modulating bone morphogenetic protein signaling and impairs tendon formation in vivo. Our results, while offering new insights into the biology of tendon cells, may assist in future strategies to treat tendon diseases.