HS3ST2 expression induces the cell autonomous aggregation of tau.

Heparan sulfates have long been known to intracellularly accumulate in Alzheimer's disease neurons, where they colocalize with neurofibrillary tangles made of abnormally phosphorylated and aggregated tau protein. However, the reasons and consequences of the heparan sulfates accumulation in the Alzheimer's cells are not yet well understood. Previously, we showed that the neural heparan sulfate 3-O-sulfotransferase HS3ST2 is critical for the abnormal phosphorylation of tau in Alzheimer's disease-related tauopathy. Using cell models of tauopathy we showed that intracellular 3-O-sulfatated heparan sulfates interact with tau inducing its abnormal phosphorylation. However, it is unknown whether HS3ST2 expression induces the intracellular aggregation of tau in cells. Here, by using replicative pEBV plasmids, we engineered HEK293 cells to stably express HS3ST2 together with human tau carrying or not the P301S mutation. We show that HS3ST2 gain of function induces the cell autonomous aggregation of tau not only in cells expressing tauP301S, but also in cells expressing the wild type tau. Our engineered cells mimicked both the HS intracellular accumulation observed in neurons of Alzheimer's disease and the tau aggregation characteristic of tauopathy development and evolution. These results give evidence that the neural HS3ST2 plays a critical role in the cell autonomous self-aggregation of tau.

Real-Time Optical Vascular Imaging, a new method for the diagnosis and monitoring of oral diseases.

INTRODUCTION: Real-Time Optical Vascular Imaging (RTOVI) is a technology developed in the Centre for Oral Clinical & Translational Sciences, within the Faculty of Dentistry, Oral & Craniofacial Sciences, King's College London, that allows rapid and preparation free, in vivo imaging of the microvascular anatomy of the human oral cavity. Microvascular changes are known to be related to disease subtypes, in particular cancer. This makes in vivo microvascular examination clinically valuable. However, at present there is lack of any analytical method able to objectively assess microvascular morphology images. DISCUSSION: The assessment of microvascular morphology based on a subjective evaluation was proven to be unreliable. There was a need to develop a software-based analysis for in vivo microvascular images to support the validation of RTOVI. This paper reviews the authors work to develop and test an automated microvascular analysis method for RTOVI based on ImageJ, an open-source software. This allowed to determined which parameters offered a more robust mathematical representation of the microvascular anatomy of the gingival margin, such as the mean area per capillary and mean aspect ratio. However, in vivo microvascular images from elsewhere within the oral cavity posed a bigger challenge to the analysis procedure due to the microvascular architectural complexity and poorer contrast. Angiogenesis Analyzer, a well-known ImageJ plugin used for the quantification of in vitro microvascular images, is under development in collaboration with the University of Paris Est Créteil. The aim of this work is to obtain an automated analysis method for in vivo microvascular images able to offer a solid foundation for the diagnostic potential of RTOVI and subsequent clinical integration of this technology. CONCLUSION: An automated analysis method for in vivo microvascular images is paramount before any attempt to clinically validate RTOVI. Our initial work of testing a software-based analysis demonstrated the effectiveness of some parameters, which is valuable for future work, and led us to move into a more sophisticated method involving customising the Angiogenesis Analyzer plugin. This is an essential step, aiming to extend the potential of in vivo microscopy with the clinical integration of RTOVI. LAY DESCRIPTION: This article summarises the initial research work done in the field on in vivo microvascular imaging aiming to develop a technique for the diagnosis of oral diseases based on the shape of small blood vessels found just below the surface of the "skin" inside the mouth. This offers the potential to examine lesions without the need to take a sample (biopsy/cutting tissue) to observe it microscopically. This ultimately offers a potential to accelerate diagnostic decision making, avoid unpleasant and often deterrent surgical procedures and reducing diagnostic laboratory time and cost burdens. However, in order to assess images of small blood vessels obtained in clinic, we needed to develop and test a software-based analysis to avoid the subjective human interpretation, known not to work. This article describes the authors journey to achieve an automated and sophisticated analysis method unique in the world for in vivo microvascular images derived from real-time optical vascular imaging.

[Heparan sulphates, amyloidosis and neurodegeneration]. / Heparan sulfatos, amiloidosis y neurodegeneracion.

INTRODUCTION: A number of neurodegenerative disorders have been linked directly to the accumulation of amyloid fibres. These fibres are made up of proteins or peptides with altered structures and which join together in vivo in association with heparan sulphate-type polysaccharides. AIMS: To examine the most recent concepts in the biology of heparan sulphates and their role in the aggregation of the peptide Abeta, of tau protein, of alpha-synuclein and of prions. The study also seeks to analyse their implications in neurodegenerative disorders such as Alzheimer's and Parkinson's disease and prion diseases. DEVELOPMENT: In vitro, heparan sulphates have played an important role in the process of oligomerisation and fibrillation of amyloidogenic proteins or peptides, in the stabilisation of these bodies and their resistance to proteolysis, thereby participating in the formation of a wide range of amyloid fibres. Heparan sulphates have also been related to the internalisation of pro-amyloid fibres during the process of intercellular propagation (spreading), which is considered to be crucial in the development of proteinopathies, the best example of which is Alzheimer's disease. CONCLUSION: This study suggests that the fine structures of heparan sulphates, their localisation in cells and tissues, together with their local concentration, may regulate the amyloidosis processes. The advances made in the understanding of this area of glyconeurobiology will make it possible to improve the understanding of the cell and molecular mechanisms underlying the neurodegenerative process.

TITLE: Heparan sulfatos, amiloidosis y neurodegeneracion.Introduccion. Numerosos trastornos neurodegenerativos se han asociado directamente a la acumulacion de fibras amiloides. Estas fibras estan formadas por proteinas o peptidos con conformaciones alteradas y que se agregan in vivo en asociacion con polisacaridos de tipo heparan sulfatos. Objetivos. Examinar los conceptos mas recientes sobre la biologia de los heparan sulfatos y su papel en la agregacion del peptido Abeta, de la proteina tau, de la alfa-sinucleina y de los priones, y analizar sus implicaciones en trastornos neurodegenerativos como las enfermedades de Alzheimer y de Parkinson y las enfermedades prionicas. Desarrollo. In vitro, los heparan sulfatos han desempeñado un papel importante en el proceso de oligomerizacion y fibrilacion de proteinas o peptidos amiloidogenos, en la estabilizacion de estos cuerpos y su resistencia a la proteolisis, participando asi en la formacion de una gran variedad de fibras amiloides. Los heparan sulfatos se han relacionado tambien con el proceso de internalizacion de fibras proamiloides durante el proceso de propagacion intercelular (spreading) considerado como central en la evolucion de las proteinopatias, cuyo mejor ejemplo es la enfermedad de Alzheimer. Conclusion. Este trabajo sugiere que las estructuras finas de los heparan sulfatos, sus localizaciones celulares y tisulares, asi como sus concentraciones locales, pueden regular los procesos de amiloidosis. Avances en la comprension de esta area de la gliconeurobiologia permitiran mejorar la comprension de los mecanismos celulares y moleculares del proceso neurodegenerativo.

A fine structural modification of glycosaminoglycans is correlated with the progression of muscle regeneration after ischaemia: towards a matrix-based therapy?

Critical limb ischaemia often leads to amputation of the limb and potential mortality. Moreover, there are still significant problems with current therapeutic treatments, according to poor revascularisation of degenerated tissue probably due to modifications within the microenvironment. This study is focused on the changes of structure and bioactivity of glycosaminoglycans (GAGs), especially heparan sulphate (HS) and chondroitin sulphate (CS) in rat Extensor Digitorum Longus (EDL) muscle after ischaemia. Male Wistar rats were subjected to ischaemic-injury by ligation of the neurovascular trunk accompanying EDL-tendon. After 4, 8, 15, 21, 60 and 90 d, the rats were sacrificed and the muscles were collected and submitted to histological, biochemical and gene expression assays. We demonstrated that ischaemia induced modification of expression of enzymes involved in GAG biosynthesis which correlated with significant changes in HS and CS structural features such as size and sulphation pattern. These major structural changes are associated to modifications of GAG abilities to bind growth factors and to modulate cell activity. Moreover, a CS hallmark of injury is maintained as well after the regeneration process. Finally, we showed the relevance of the role of this glycanic matrix remodelling, since a GAG mimetic treatment accelerated muscle repair after ischaemia.

Insights on a new path of pre-mitochondrial apoptosis regulation by a glycosaminoglycan mimetic.

Lysosomal cathepsins have recently been reported to play crucial roles in the regulation of the mitochondrial death cascade by an unclear mechanism leading to mitochondrial membrane permeabilization. Glycosaminoglycans (GAG) are a family of ionic polysaccharides present at the lysosomal compartment and shown to inhibit lysosomal cathepsin activities. The implication of this family of polysaccharides in the regulation of the pre-mitochondrial death cascade has still not been considered. Here, we demonstrate in a model of skin fibroblasts submitted to oxidative stress that a GAG-mimetic protects the lysosome from membrane disruption, reduces intracellular ROS levels, and inhibits mitochondrial membrane potential collapse, cytochrome c release and caspases-9 and -3 activations without affecting the extrinsic pathway of apoptosis. Heparan sulfate and chondroitin sulfate, but not heparin, showed also protecting effects when assessing key points of the intrinsic pathway of apoptosis. We suggest the existence of molecular links between endogenous GAGs and the regulation of apoptosis.

Matrix therapy in regenerative medicine, a new approach to chronic wound healing.

Nonhealing wounds remain a major health problem whose treatment is challenging and costly. Treatments based on cells or growth factors are still not very effective. We developed an entirely novel strategy consisting in treatment of the wound-tissue matrix with biopolymers engineered to mimic heparan sulfates called OTR4120. This compound was dextran polymer with sulfated and carboxymethyl groupments. After binding to matrix proteins, the heparan-sulfate-mimicking polymer protects the microenvironment, maintaining the normal production of signals and growth factors needed for healing to occur. Here, we show that a specific biopolymer accelerates ulcer closure and improves re-epithelialization and dermal-matrix-component remodeling. OTR4120 treatment was associated with faster maturation of epidermal structures, most notably regarding the number of epithelial-cell layers, and with an appearance that more closely resembled normal skin. Treatment had also a main effect on collagen I and III expression. Necrotic skin ulcers induced in mice with doxorubicin recovered normal collagen levels and organization, with no evidence of fibrosis. Thus, appropriate polymer-based matrix therapy is a valid and simple alternative to regenerative medicine.

Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activity.

Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N-acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity.

Specific RGTA increases collagen V expression by cultured aortic smooth muscle cells via activation and protection of transforming growth factor-beta1.

Regenerating agents (RGTA) are defined as heparan sulfate mimics, which in vivo stimulate tissue repair. RGTA are obtained by controlled grafting of carboxymethyl and sulfate groups on dextran polymers. RGTA are selected in vitro, on their ability to protect heparin binding growth factors such as TGF-beta1 for example, as well as to alter extracellular matrix biosynthesis. We had reported that RGTA were able to modulate smooth muscle cell (SMC) collagen biosynthesis. Here, we demonstrated that a specific RGTA (RG-1503), altered differentially collagen type expression by post-confluent SMC and that this action involves TGF-beta1. RG-1503 decreased, by 50%, collagen I and III biosynthesis and stimulated specifically, by twofold, collagen V biosynthesis. TGF-beta1 stimulated collagen I and V by 1.5- and threefold, respectively. A synergic action for RGTA in association with TGF-beta1 was observed specifically for collagen V expression (eightfold increase). The stimulation of collagen V biosynthesis by RGTA was abolished by TGF-beta1 neutralizing antibodies. These modulations occurred at protein and mRNA levels. RG-1503 did not alter TGF-beta1 mRNA steady state level or total TGF-beta1 protein content (latent+active forms). However, RG-1503 significantly induced an elevated proportion of active TGF-beta1 form, which could result from the selective protection from proteolytic degradation of TGF-beta1 by RG-1503. These data open a rationale for understanding the stimulation of tissue repair induced by RGTA, and also, a new insight for developing drugs adapted to inhibit excess collagen deposition in smooth muscle cells associated vascular disorder, and in fibrotic diseases.

New agents for the treatment of infarcted myocardium.

Local delivery of angiogenic growth factors for the treatment of myocardial ischemia has been well documented in various animal models, and clinical trials are now in progress. Our strategy was radically different, based on selective protection of some of the growth factors naturally present within the injured tissue. This protection was obtained by applying a chemically defined substitute for Dextran called RGTA11 (for ReGeneraTing Agent). RGTA is a family of agents, which has properties mimicking those of heparan sulfates toward heparin-binding growth factors (HBGF) and which stimulate tissue repair and protection. Indeed, we have previously shown that RGTA prevents most of the damage resulting from acute skeletal muscle ischemia [FASEB J. (1999) 13, 761-766]. We now show that the same agent can be used for the treatment of myocardial infarction. Acute myocardial infarction was induced in pigs by ligation of the left circumflex artery. One hour later, a single injection of 10 mg of RGTA11 was made in the center of the infarcted area. Three weeks later we observed 1) recovery of 84% of the initial left ventricular ejection fraction (only 55% in saline-treated controls), 2) an almost 50% reduction in the infarct size, 3) a reduction in fibrotic tissue formation, 4) significant preservation of myocytes, and 5) an increase in the number of blood vessels. The treatment of ischemic heart disease with RGTA would have clear advantages over other therapies such as growth factor, gene, or cell transplants, based on a stable, simple, and easy-to-develop chemical product.

Human plasmin enzymatic activity is inhibited by chemically modified dextrans.

Some synthetic dextran derivatives that mimic the action of heparin/heparan sulfate were shown to promote in vivo tissue repair when added alone to wounds. These biofunctional mimetics were therefore designated as "regenerating agents" in regard to their in vivo properties. In vitro, these biopolymers were able to protect various heparin-binding growth factors against proteolytic degradation as well as to inhibit the enzymatic activity of neutrophil elastase. In the present work, different dextran derivatives were tested for their capacity to inhibit the enzymatic activity of human plasmin. We show that dextran containing carboxymethyl, sulfate as well as benzylamide groups (RG1192 compound), was the most efficient inhibitor of plasmin amidolytic activity. The inhibition of plasmin by RG1192 can be classified as tight binding hyperbolic noncompetitive. One molecule of RG1192 bound 20 molecules of plasmin with a K(i) of 2.8 x 10(-8) m. Analysis with an optical biosensor confirmed the high affinity of RG1192 for plasmin and revealed that this polymer equally binds plasminogen with a similar affinity (K(d) = 3 x 10(-8) m). Competitive experiments carried out with 6-aminohexanoic acid and kringle proteolytic fragments identified the lysine-binding site domains of plasmin as the RG1192 binding sites. In addition, RG1192 blocked the generation of plasmin from Glu-plasminogen and inhibited the plasmin-mediated proteolysis of fibronectin and laminin. Data from the present in vitro investigation thus indicated that specific dextran derivatives can contribute to the regulation of plasmin activity by impeding the plasmin generation, as a result of their binding to plasminogen and also by directly affecting the catalytic activity of the enzyme.