Vascular Endothelial Growth Factor 165-Binding Heparan Sulfate Promotes Functional Recovery From Cerebral Ischemia.

BACKGROUND AND PURPOSE: Although VEGF165 (vascular endothelial growth factor-165) is able to enhance both angiogenesis and neurogenesis, it also increases vascular permeability through the blood-brain barrier. Heparan sulfate (HS) sugars play important roles in regulating VEGF bioactivity in the pericellular compartment. Here we asked whether an affinity-purified VEGF165-binding HS (HS7) could augment endogenous VEGF activity during stroke recovery without affecting blood-brain barrier function. METHODS: Both rat brain endothelial cell line 4 and primary rat neural progenitor cells were used to evaluate the potential angiogenic and neurogenic effects of HS7 in vitro. For in vivo experiments, male Sprague-Dawley rats were subjected to 100 minutes of transient focal cerebral ischemia, then treated after 4 days with either PBS or HS7. One week later, infarct volume, behavioral sequelae, immunohistochemical markers of angiogenesis and neural stem cell proliferation were assessed. RESULTS: HS7 significantly enhanced VEGF165-mediated angiogenesis in rat brain endothelial cell line 4 brain endothelial cells, and increased the proliferation and differentiation of primary neural progenitor cells, both via the VEGFR2 (vascular endothelial growth factor receptor 2) pathway. Intracerebroventricular injection of HS7 improved neurological outcome in ischemic rats without changing infarct volumes. Immunostaining of the compromised cerebrum demonstrated increases in collagen IV/Ki67 and nestin/Ki67 after HS7 exposure, consistent with its ability to promote angiogenesis and neurogenesis, without compromising blood-brain barrier integrity. CONCLUSIONS: A VEGF-activating glycosaminoglycan sugar, by itself, is able to enhance endogenous VEGF165 activity during the post-ischemic recovery phase of stroke.

Bringing Heparan Sulfate Glycomics Together with Proteomics for the Design of Novel Therapeutics: A Historical Perspective.

Increasing knowledge of how peptides bind saccharides, and of how saccharides bind peptides, is starting to revolutionize understanding of cell-extracellular matrix relationships. Here, a historical perspective is taken of the relationship between heparan sulfate glycosaminoglycans and how they interact with peptide growth factors in order to both drive and modulate signaling through the appropriate cognate receptors. Such knowledge is guiding the preparation of targeted sugar mimetics that will impact the treatment of many different kinds of diseases, including cancer.

Small Molecule Antagonist of Cell Surface Glycosaminoglycans Restricts Mouse Embryonic Stem Cells in a Pluripotent State.

Recently, the field of stem cell-based regeneration has turned its attention toward chemical approaches for controlling the pluripotency and differentiation of embryonic stem cells (ESCs) using drug-like small molecule modulators. Growth factor receptors or their associated downstream kinases that regulate intracellular signaling pathways during differentiation are typically the targets for these molecules. The glycocalyx, which plays an essential role in actuating responses to growth factors at the cellular boundary, offers an underexplored opportunity for intervention using small molecules to influence differentiation. Here, we show that surfen, an antagonist of cell-surface glycosaminoglycans required for growth factor association with cognate receptors, acts as a potent and general inhibitor of differentiation and promoter of pluripotency in mouse ESCs. This finding shows that drugging the stem cell Glycome with small molecules to silence differentiation cues can provide a powerful new alternative to existing techniques for controlling stem cell fate. Stem Cells 2018;36:45-54.

Improved recovery from limb ischaemia by delivery of an affinity-isolated heparan sulphate.

Peripheral arterial disease is a major cause of limb loss and its prevalence is increasing worldwide. As most standard-of-care therapies yield only unsatisfactory outcomes, more options are needed. Recent cell- and molecular-based therapies that have aimed to modulate vascular endothelial growth factor-165 (VEGF165) levels have not yet been approved for clinical use due to their uncertain side effects. We have previously reported a heparan sulphate (termed HS7) tuned to avidly bind VEGF165. Here, we investigated the ability of HS7 to promote vascular recovery in a murine hindlimb vascular ischaemia model. HS7 stabilised VEGF165 against thermal and enzyme degradation in vitro, and isolated VEGF165 from serum via affinity-chromatography. C57BL6 mice subjected to unilateral hindlimb ischaemia injury received daily intramuscular injections of respective treatments (n = 8) and were assessed over 3 weeks by laser Doppler perfusion, magnetic resonance angiography, histology and the regain of function. Mice receiving HS7 showed improved blood reperfusion in the footpad by day 7. In addition, they recovered hindlimb blood volume two- to fourfold faster compared to the saline group; the greatest rate of recovery was observed in the first week. Notably, 17% of HS7-treated animals recovered full hindlimb function by day 7, a number that grew to 58% and 100% by days 14 and 21, respectively. This was in contrast to only 38% in the control animals. These results highlight the potential of purified glycosaminoglycan fractions for clinical use following vascular insult, and confirm the importance of harnessing the activity of endogenous pro-healing factors generated at injury sites.

Monitoring the sustainable development goals through human rights accountability reviews.

The Universal Periodic Review is a comprehensive, state-to-state peer-review mechanism of the United Nations (UN) Human Rights Council. Created in 2006, the mechanism scrutinizes the human rights record of all UN Member States, including their efforts to realize the right to health. However, the mechanism is relatively under-used in global health governance compared to treaty-based procedures, such as those overseen by the Committee on the Rights of Persons with Disabilities or the Committee on the Elimination of Discrimination against Women. We suggest that the Universal Periodic Review could be used to support the monitoring and review processes of the sustainable development goals (SDGs). The review could offer a unique perspective for other actors on how to ensure accountability for the complex and intertwined SDGs, including their commitments for health. This article provides an overview of how health-related rights have been addressed in the Universal Periodic Review process and how the review can contribute to advancing global commitments to health, including those embodied in the SDGs. We present some of the current limitations in the way health is addressed in the Universal Periodic Review. We also consider what role specialized UN agencies, such as the World Health Organization, might play during the Universal Periodic Review process and how this involvement can contribute towards the comprehensive realization of health and wellbeing for all.

L'Examen périodique universel est un mécanisme complet d'évaluation entre États du Conseil des droits de l'homme des Nations Unies (ONU). Créé en 2006, ce mécanisme passe en revue les réalisations de l'ensemble des États membres de l'ONU dans le domaine des droits de l'homme, et notamment leurs efforts en faveur de l'application du droit à la santé. Ce mécanisme est néanmoins relativement sous-utilisé dans la gouvernance de la santé mondiale par rapport aux procédures fondées sur des traités comme celles supervisées par le Comité des droits des personnes handicapées ou le Comité pour l'élimination de la discrimination à l'égard des femmes. Nous suggérons d'utiliser l'Examen périodique universel pour soutenir les processus de suivi et d'examen des objectifs de développement durable (ODD). L'examen pourrait offrir une perspective unique à d'autres acteurs sur la façon de garantir le principe de responsabilité pour les ODD, complexes et interdépendants, et notamment leurs engagements en matière de santé. Cet article fournit un aperçu de la façon dont les droits liés à la santé sont traités dans le cadre de l'Examen périodique universel et de la façon dont l'examen peut contribuer à faire avancer les engagements mondiaux en faveur de la santé, et notamment ceux inclus dans les ODD. Nous présentons quelques-unes des limites actuelles de l'Examen périodique universel concernant la façon dont il traite de la santé. Nous avons également étudié le rôle que peuvent jouer certaines institutions spécialisées des Nations Unies, telles que l'Organisation mondiale de la Santé, dans le cadre de l'Examen périodique universel, et en quoi ce rôle peut contribuer à l'atteinte de l'objectif de la santé et du bien-être pour tous.

La Revisión periódica universal es un mecanismo integral de revisión entre pares de estado a estado del Consejo de Derechos Humanos de las Naciones Unidas (ONU). Creado en 2006, el mecanismo examina el historial relativo a los derechos humanos de todos los Estados Miembros de las Naciones Unidas, incluidos sus esfuerzos por cumplir el derecho a la salud. Sin embargo, el mecanismo está relativamente infrautilizado en la gobernanza de la salud mundial en comparación con los procedimientos basados en tratados, como los supervisados por el Comité sobre los Derechos de las Personas con Discapacidad o el Comité para la Eliminación de la Discriminación contra la Mujer. Se sugiere que la Revisión periódica universal se utilice para apoyar los procesos de seguimiento y revisión de los objetivos de desarrollo sostenible (ODS). La revisión podría ofrecer una perspectiva única para otros participantes sobre cómo asegurar la responsabilidad de los complejos y vinculados ODS, incluyendo sus compromisos con la salud. Este artículo ofrece una visión general de cómo se han abordado los derechos relacionados con la salud en el proceso de la Revisión periódica universal y cómo la misma puede contribuir al avance de los compromisos mundiales con la salud, incluidos los incorporados en los ODS. Se presentan algunas de las limitaciones actuales en la forma en que se aborda la salud en la Revisión periódica universal. También se valora qué papel podrían desempeñar los organismos especializados de las Naciones Unidas, como la Organización Mundial de la Salud, durante el proceso de la Revisión periódica universal y cómo esta participación puede contribuir a la realización integral de la salud y el bienestar para todos.

Structural determinants of heparin-transforming growth factor-ß1 interactions and their effects on signaling.

Transforming growth factor-ß1 (TGF-ß1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-ß1's activity on hMSCs. Using a proteomics "Protect and Label" approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin (Data are available via ProteomeXchange with identifier PXD002772). Competition assays in an optical biosensor demonstrated that TGF-ß1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-ß1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14-24 saccharides (dp14-24) in length all increased the phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) after 6 h of stimulation with TGF-ß1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-ß1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-ß1 in hMSCs.

Glycocalyx remodeling with proteoglycan mimetics promotes neural specification in embryonic stem cells.

Growth factor (GF) signaling is a key determinant of stem cell fate. Interactions of GFs with their receptors are often mediated by heparan sulfate proteoglycans (HSPGs). Here, we report a cell surface engineering strategy that exploits the function of HSPGs to promote differentiation in embryonic stem cells (ESCs). We have generated synthetic neoproteoglycans (neoPGs) with affinity for the fibroblast growth factor 2 (FGF2) and introduced them into plasma membranes of ESCs deficient in HS biosynthesis. There, the neoPGs assumed the function of native HSPGs, rescued FGF2-mediated kinase activity, and promoted neural specification. This glycocalyx remodeling strategy is versatile and may be applicable to other types of differentiation.

Rational synthesis of a heparan sulfate saccharide that promotes the activity of BMP2.

Heparan sulfate (HS) is a glycosaminoglycan (GAG) found throughout nature and is involved in a wide range of functions including modulation of cell signalling via sequestration of growth factors. Current consensus is that the specificity of HS motifs for protein binding are individual for each protein. Given the structural complexity of HS the synthesis of libraries of these compounds to probe this is not trivial. Herein we present the synthesis of an HS decamer, the design of which was undertaken rationally from previously published data for HS binding to the growth factor BMP-2. The biological activity of this HS decamer was assessed in vitro, showing that it had the ability to both bind BMP-2 and increase its thermal stability as well as enhancing the bioactivity of BMP-2 in vitro in C2C12 cells. At the same time no undesired anticoagulant effect was observed. This decamer was then analysed in vivo in a rabbit model where higher bone formation, bone mineral density (BMD) and trabecular thickness were observed over an empty defect or collagen implant alone. This indicated that the HS decamer was effective in promoting bone regeneration in vivo.

Variability in the composition of porcine mucosal heparan sulfates.

Commercial porcine intestinal mucosal heparan sulfate (HS) is a valuable material for research into its biological functions. As it is usually produced as a side-stream of pharmaceutical heparin manufacture, its chemical composition may vary from batch to batch. We analysed the composition and structure of nine batches of HS from the same manufacturer. Statistical analysis of the disaccharide compositions placed these batches in three categories: group A had high GlcNAc and GlcNS, and low GlcN typical of HS; group B had high GlcN and GlcNS, and low GlcNAc; group C had high di- and trisulfated, and low unsulfated and monosulfated disaccharide repeats. These batches could be placed in the same categories based on their 1H NMR spectra and molecular weights. Anticoagulant and growth factor binding activities of these HS batches did not fit within these same groups but were related to the proportions of more highly sulfated disaccharide repeats.

Glycosaminoglycans as regulators of stem cell differentiation.

ES (embryonic stem) cell differentiation is dependent on the presence of HS (heparan sulfate). We have demonstrated that, during differentiation, the evolution of specific cell lineages is associated with particular patterns of GAG (glycosaminoglycan) expression. For example, different HS epitopes are synthesized during neural or mesodermal lineage formation. Cell lines mutant for various components of the HS biosynthetic pathway are selectively impaired in their differentiation, with lineage-specific effects observed for some lines. We have also observed that the addition of soluble GAG saccharides to cells, with or without cell-surface HS, can influence the pace and outcome of differentiation, again highlighting specific pattern requirements for particular lineages. We are combining this work with ongoing studies into the design of artificial cell environments where we have optimized three-dimensional scaffolds, generated by electrospinning or by the formation of hydrogels, for the culture of ES cells. By permeating these scaffolds with defined GAG oligosaccharides, we intend to control the mechanical environment of the cells (via the scaffold architecture) as well as their biological signalling environment (using the oligosaccharides). We predict that this will allow us to control ES cell pluripotency and differentiation in a three-dimensional setting, allowing the generation of differentiated cell types for use in drug discovery/testing or in therapeutics.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

Human mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

Adding the female condom to the public health agenda on prevention of HIV and other sexually transmitted infections among men and women during anal intercourse.

Legal barriers to conducting public health research on methods of protection for anal intercourse were lifted in the United States in 2003 when the US Supreme Court invalidated all state antisodomy laws. Although research funding has been available for the development of rectal microbicides, the female condom, which has already been approved for vaginal use, has not been evaluated for anal use. Although there is no evidence that the female condom is safe for anal intercourse, it has already been taken up for off-label use by some men who have sex with men. This demonstrates the urgent need for more protection options for anal intercourse and, more immediately, the need to evaluate the safety and efficacy of the female condom for anal intercourse.

A Heparan Sulfate Device for the Regeneration of Osteochondral Defects.

IMPACT STATEMENT: Repairing damaged joint cartilage remains a significant challenge. Treatment involving microfracture, tissue grafting, or cell therapy provides some benefit, but seldom regenerates lost articular cartilage. Providing a point-of-care solution that is cell and tissue free has the potential to transform orthopedic treatment for such cases. Glycosaminoglycans such as heparan sulfate (HS) are well suited for this purpose because they provide a matrix that enhances the prochondrogenic activities of growth factors normally found at sites of articular damage. In this study, we show the potential of a novel HS device, which is free of exogenous cells or growth factors, in regenerating osteochondral defects.

Minimum structural requirements for BMP-2-binding of heparin oligosaccharides.

Bone morphogenetic proteins (BMPs) are essential during tissue repair and remodeling after injury. Glycosaminoglycan (GAG) sugars are known to enhance BMP activity in vitro and in vivo; here the interactions of BMP-2 with various glycosaminoglycan classes were compared and shown to be selective for heparin over other comparable saccharides. The minimal chain lengths and specific sulfate moieties required for heparin-derived oligosaccharide binding to BMP-2, and the ability of such oligosaccharides to promote BMP-2-induced osteogenic differentiation in vitro were then determined. BMP-2 could bind to heparin hexasaccharides (dp6) and octasaccharides (dp8), but decasaccharides (dp10) were the minimum chain length required for both efficient binding of BMP-2 and consequent heparin-dependent cell responses. N-sulfation is the most important, and 6-O-sulfation moderately important for BMP-2 binding and activity, whereas 2-O-sulfation was much less critical. Bone formation assays in vivo further confirmed that dp10, N-sulfated heparin oligosaccharides were the minimal requirement for effective enhancement of BMP-2-induced bone formation. Such information is necessary for the rational design of the next generations of heparan-based devices for bone tissue repair.

Retention of the Structure and Function of Heparan Sulfate Biomaterials After Gamma Irradiation.

Heparan sulfate (HS) is a highly heterogeneous polysaccharide implicated in many important biological processes. Our previous work has demonstrated that a particular affinity-selected HS (referred to henceforth as "HS3") is capable of enhancing the osteogenic effects of bone morphogenetic protein 2 (BMP2). Here, we gamma-irradiated HS with 26 kGy of ionizing radiation to determine how this affected the structure, composition, and function. Initial structural studies were performed on a commercial preparation of HS as a proof-of-concept. Gamma irradiation of this HS preparation did not significantly alter its structure or composition compared to nonirradiated material, as demonstrated by proton nuclear magnetic resonance spectroscopy, molecular weight analysis using size exclusion chromatography, and disaccharide compositional analysis. When HS3 was gamma irradiated, no significant effect on binding affinity toward BMP2 was observed, based on competitive surface plasmon resonance and differential scanning fluorimetry assays. Furthermore, irradiation did not significantly affect HS3's ability to synergistically enhance the osteogenic effects of BMP2 in vitro; as measured by the relative abundance of osteogenic transcripts in transdifferentiating C2C12 murine myoblasts. Additionally, no significant differences were observed in the levels of alkaline phosphatase (ALP) or calcium deposition in C2C12s treated with BMP2, together with the irradiated, or nonirradiated HS3. Irradiation of HS3 incorporated into collagen type I sponges did not affect its ability to enhance BMP2-mediated ALP expression in C2C12 cells. Our data confirm that gamma irradiation is a cost-effective and viable solution for the sterilization of HS species that allows the retention of its structure and biological function. The work suggests an effective way to incorporate clinically compatible HS species into orthotic implants, scaffolds, and other medical devices for use in the treatment of a range of diseases and disorders.

Growth Differentiation Factor 5-Mediated Enhancement of Chondrocyte Phenotype Is Inhibited by Heparin: Implications for the Use of Heparin in the Clinic and in Tissue Engineering Applications.

The highly sulfated glycosaminoglycan (GAG) heparin is widely used in the clinic as an anticoagulant, and researchers are now using it to enhance stem cell expansion/differentiation protocols, as well as to improve the delivery of growth factors for tissue engineering (TE) strategies. Growth differentiation factor 5 (GDF5) belongs to the bone morphogenetic protein family of proteins and is vital for skeletal formation; however, its interaction with heparin and heparan sulfate (HS) has not been studied. We identify GDF5 as a novel heparin/HS binding protein and show that HS proteoglycans are vital in localizing GDF5 to the cell surface. Clinically relevant doses of heparin (≥10 nM), but not equivalent concentrations of HS, were found to inhibit GDF5's biological activity in both human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures and the skeletal cell line ATDC5. We also found that heparin inhibited both GDF5 binding to cell surface HS and GDF5-induced induction of Smad 1/5/8 signaling. Furthermore, GDF5 significantly increased aggrecan gene expression in chondrocyte pellet cultures, without affecting collagen type X expression, making it a promising target for the TE of articular cartilage. Importantly, this study may explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE and the adverse skeletal effects reported in the clinic following long-term heparin treatment. Our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g., HS derivatives), with better-defined structures and fewer off-target effects.

Glycocalyx Remodeling with Glycopolymer-Based Proteoglycan Mimetics.

The cellular glycocalyx controls many of the crucial signaling pathways involved in cellular development. Synthetic materials that can mimic the multivalency and three-dimensional architecture of native glycans serve as important tools for deciphering and exploiting the roles of these glycans. Here we describe a chemical approach for the engineering of growth-factor interactions at the surfaces of stem cells using synthetic glycomimetic materials, with an eye towards promoting their commitment towards specific cell lineages with therapeutic potential.

Determining the extent of heparan sulfate depolymerisation following heparin lyase treatment.

The depolymerisation of porcine mucosal heparan sulfate under the action of heparin lyases and analysis by size-exclusion chromatography (SEC) is described. Heparan sulfate treated to enzymic bond scission producing a Δ4,5 double-bond and quantified by SEC with ultraviolet-visible (UV) spectroscopic detection (230nm) indicated that the majority of the biopolymer (>85%) was reduced to disaccharides (degree of polymerisation (DP)=2). However, analysis of the SEC eluant using refractive index (RI), which reflects the mass contribution of the oligosaccharides rather than the molar response of a UV chromophore, indicated that a considerable proportion of the digested HS, up to 43%, was present with DP >2. This was supported by a mass balance analysis. These results contradict the accepted literature where "complete digestion" is routinely reported. Herein we report on the composition and methodology utilised to ascertain the extent of depolymerization and disaccharide composition of this important biopolymer.

Use of flow cytometry for characterization and fractionation of cell populations based on their expression of heparan sulfate epitopes.

The ability to characterize alterations in heparan sulfate (HS) structure during development or as a result of loss or mutation of one or more components of the HS biosynthetic pathway is essential for broad understanding of the effects these changes may have on cell/tissue function. The use of anti-HS antibodies provides an opportunity to study HS chain composition in situ, with a multitude of different antibodies having been generated that recognize subtle differences in HS patterning, with the number and positioning of sulfate groups influencing antibody binding affinity. Flow cytometry is a valuable technique to enable the rapid characterization of the changes in HS-specific antibody binding in situ, allowing multiple cell types to be directly compared. Additionally fluorescent-activated cell sorting (FACS) allows fractionation of cells based on their HS-epitope expression.