Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic Machinery for Biomedical Applications.

Glycosaminoglycans (GAGs) are polysaccharides ubiquitously found on cell surfaces and in the extracellular matrix (ECM). They regulate numerous cellular signaling events involved in many developmental and pathophysiological processes. GAGs are composed of complex sequences of repeating disaccharide units, each of which can carry many different modifications. The tremendous structural variations account for their ability to bind many proteins and thus, for their numerous functions. Although the sequence of GAG biosynthetic events and the enzymes involved mostly were deduced a decade ago, the emergence of tissue or cell specific GAGs from a nontemplate driven process remains an enigma. Current knowledge favors the hypothesis that macromolecular assemblies of GAG biosynthetic enzymes termed "GAGOSOMEs" coordinate polymerization and fine structural modifications in the Golgi apparatus. Distinct GAG structures arise from the differential channeling of substrates through the Golgi apparatus to various GAGOSOMEs. As GAGs perform multiple regulatory roles, it is of great interest to develop molecular strategies to selectively interfere with GAG biosynthesis for therapeutic applications. In this Account, we assess our present knowledge on GAG biosynthesis, the manipulation of GAG biosynthesis using synthetic xylosides, and the unrealized potential of these xylosides in various biomedical applications. Synthetic xylosides are small molecules consisting of a xylose attached to an aglycone group, and they compete with endogenous proteins for precursors and biosynthetic enzymes to assemble GAGs. This competition reduces endogenous proteoglycan-bound GAGs while increasing xyloside-bound free GAGs, mostly chondroitin sulfate (CS) and less heparan sulfate (HS), resulting in a variety of biological consequences. To date, hundreds of xylosides have been published and the importance of the aglycone group in determining the structure of the primed GAG chains is well established. However, the structure-activity relationship has long been cryptic. Nonetheless, xylosides have been designed to increase HS priming, modified to inhibit endogenous GAG production without priming, and engineered to be more biologically relevant. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. They are small, orally bioavailable, easily excreted, and utilize the host cell biosynthetic machinery to assemble GAGs that are likely nonimmunogenic. Various xylosides have been shown, in different biological systems, to have anticoagulant effects, selectively kill tumor cells, abrogate angiogenic and metastatic pathways, promote angiogenesis and neuronal growth, and affect embryonic development. However, most of these studies utilized the commercially available one or two ß-D-xylosides and focused on the impact of endogenous proteoglycan-bound GAG inhibition on biological activity. Nevertheless, the manipulation of cell behavior as a result of stabilizing growth factor signaling with xyloside-primed GAGs is also reckonable but underexplored. Recent advances in the use of molecular modeling and docking simulations to understand the structure-activity relationships of xylosides have opened up the possibility of a more rational aglycone design to achieve a desirable biological outcome through selective priming and inhibitory activities. We envision these advances will encourage more researchers to explore these fascinating xylosides, harness the GAG biosynthetic machinery for a wider range of biomedical applications, and accelerate the successful transition of xyloside-based therapeutics from bench to bedside.

Methods for Assessing the Effects of Xylosides on Angiogenesis.

Xylosides are small synthetic molecules consisting of a xylose molecule attached to an aglycone group and serve as primers in the assembly of core protein free glycosaminoglycans using cellular machinery. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. Recent advances in the study of xylosides have opened up the possibility of developing xylosides as therapeutics to achieve a desirable biological outcome through their selective priming and inhibitory activities toward glycosaminoglycan biosynthesis. The approach described, herein, will serve as a general strategy to comprehensively screen xylosides and evaluate their ability to promote or inhibit angiogenesis, a critical biological process that is dysregulated in over 70 human diseases.

Manipulation of Glycosaminoglycans Using Synthetic Xylosides to Study Their Roles in Lung Branching Morphogenesis in Ex Vivo Lung Bud Culture System.

The primary left and right bronchial buds grow and sprout secondary bronchi, which in turn develop tertiary bronchi, and so on. Branching continues for a total of 6-8 generations in the mouse and for about 23 generations in humans, forming the estimated 50 million branches of the human lung. Thus, patterns of branching are incalculably complex. However, these branches are rarely random, implying that they are under genetic control. Genomic information alone cannot specify the patterning information in terms of where the branching occurs and the direction it grows as well as their size and shape. There is a complex choreography among glycosaminoglycans and growth factors/morphogens that provide a highly complex instructive cues that control lung branching and development of the functional lung. Herein, we describe the use of xylosides in the manipulation of glycosaminoglycan (GAG) biosynthesis and study the effect of xyloside-primed GAGs in the regulation of lung branching events.

Applications of Xylosides in the Manipulation of Stem Cell Niche to Regulate Human Neural Stem Cell Differentiation and Neurite Outgrowth.

The extracellular matrix (ECM) plays a pivotal role in the regulation of neural stem cell differentiation, axon guidance and growth, and neural plasticity. Glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, are significant components of brain ECM that dictates neurogenesis and neural repair. Herein, we describe a simple method to assess the effect of xylsoides, which serve as primers and inhibitors of GAG biosynthesis, on human neural stem cell differentiation and neurite outgrowth in in vitro culture conditions.

Methods to Analyze the Effect of Diet-Derived Metabolites on Endothelial Inflammation and Cell Surface Glycosaminoglycans.

The glycocalyx is a biologically active barrier that covers the luminal side of the vascular endothelium and it is comprised of proteoglycans [core proteins with glycosaminoglycans (GAG) side chains], glycoproteins, and plasma proteins. Evidence shows that the disruption in the structure and function of the endothelial glycocalyx exacerbates vascular inflammation and atherosclerosis. The GAG components of the glycocalyx undergo remodeling in the setting of diabetes and these alterations in endothelial GAGs negatively impact the vascular function. Hence, the preservation and restoration of GAGs in altered vasculature may be a novel strategy to ameliorate vascular complications in diabetes and metabolic syndrome. Human studies support the beneficial vascular effects of flavonoids which are widely found in fruits and vegetables. Flavonoids are extensively metabolized by the intestinal microbiota and digestive enzymes in humans, suggesting that their biological activities may be mediated by their circulating metabolites. Studies indicate that counteracting the damage to GAGs using dietary compounds improve vascular complications. In this article, we describe the methods to analyze the effect of diet-derived metabolites such as metabolites of flavonoids on endothelial inflammation and cell surface glycosaminoglycans.

Visualizing antithrombin-binding 3-O-sulfated heparan sulfate motifs on cell surfaces.

To map the cellular topography of the rare 3-O-sulfated structural motif of heparan sulfate (HS), we constructed quantum dot-based probes for antithrombin and FGF2, which reveal widely different distribution of the targeted HS motifs. The technology helps show that old and young aortic endothelia display widely different levels of the antithrombin-binding 3-O-sulfated HS motif.

Blueberry metabolites restore cell surface glycosaminoglycans and attenuate endothelial inflammation in diabetic human aortic endothelial cells.

BACKGROUND: Glycosaminoglycan (GAG), a major component of the endothelial glycocalyx, is severely perturbed in diabetic vasculature leading to endothelial inflammation and vascular disease in diabetes. We tested the hypothesis that blueberry metabolites (BBM) ameliorate endothelial inflammation in diabetic endothelial cells (ECs) by restoring cell surface GAGs. METHODS: ECs isolated from healthy individuals [human aortic ECs (HAECs)] and diabetic patients (diabetic HAECs) were treated with ±BBM (benzoic acid-4-sulfate, hippuric acid, hydroxyhippuric acid, isovanillic acid-3-sulfate, and vanillic acid-4-sulfate at concentrations known to circulate in human plasma following blueberry consumption) for 3 days, and indices for endothelial inflammation were measured. To analyze GAGs, ECs were incubated with sulfate-free medium supplemented with [35S] Na2SO4 ±â€¯BBM. Total GAGs in ECs and medium were purified using DEAE-Sepharose column and were analyzed with high-pressure liquid chromatography coupled to an inline flow scintillation analyzer. Heparan sulfate/chondroitin sulfate ratio and disaccharide composition of GAGs from the medium were analyzed using DEAE-3SW column and Dionex CarboPac PA1 column, respectively. RESULTS: BBM suppressed diabetes-induced monocyte binding to ECs, and reduced the expression of inflammatory markers in diabetic HAECs. Diabetic HAECs displayed a decrease in [35S] sulfate incorporation into the cell surface GAGs indicating the dysregulation of sulfated GAGs. However, treatment with BBM restored the levels of GAGs in diabetic HAECs. The composition, heparan sulfate/chondroitin sulfate ratio, and disaccharide composition of GAGs from medium were similar among groups. CONCLUSIONS: BBM restored cell surface GAGs and attenuated endothelial inflammation in diabetic HAECs. Blueberry might complement conventional therapies to improve vascular complications in diabetes.

A glycan-based approach to therapeutic angiogenesis.

Angiogenesis, the sprouting of new blood vessels from existing vasculature, involves multiple complex biological processes, and it is an essential step for hemostasis, tissue healing and regeneration. Angiogenesis stimulants can ameliorate human disease conditions including limb ischemia, chronic wounds, heart disease, and stroke. The current strategies to improve the bioavailability of pro-angiogenic growth factors, including VEGF and FGF2, have remained largely unsuccessful. This study demonstrates that small molecules, termed click-xylosides, can promote angiogenesis in the in vitro matrigel tube formation assay and the ex ovo chick chorioallantoic membrane assay, depending on their aglycone moieties. Xyloside treatment enhances network connectivity and cell survivability, thereby, maintaining the network structures on matrigel culture for an extended period of time. These effects were achieved via the secreted xyloside-primed glycosaminoglycans (GAG) chains that in part, act through an ERK1/2 mediated signaling pathway. Through the remodeling of GAGs in the extracellular matrix of endothelial cells, the glycan approach, involving xylosides, offers great potential to effectively promote therapeutic angiogenesis.

Extending neurites sense the depth of the underlying topography during neuronal differentiation and contact guidance.

The topography of the extracellular microenvironment influences cell morphology, provides conduct guidance and directs cell differentiation. Aspect ratio and dimension of topography have been shown to affect cell behaviours, but the ability and mechanism of depth-sensing is not clearly understood. We showed that murine neural progenitor cells (mNPCs) can sense the depth of the micro-gratings. Neurite elongation, alignment and neuronal differentiation were observed to increase with grating depth. We proposed a mechanism for depth-sensing by growing neurites: filopodial adhesion in the growth cones favour elongation but the bending rigidity of the neurite cytoskeleton resists it. Thus, perpendicular extension on deeper grooves is unfavourable as neurites need to bend over a larger angle. A quantitative model was developed and its prediction of neurite growth on gratings fit well with the experimental data. The results indicated that mNPC fate can be directed by appropriately designed patterned surfaces.

Cultivation of human microvascular endothelial cells on topographical substrates to mimic the human corneal endothelium.

Human corneal endothelial cells have a limited ability to replicate in vivo and in vitro. Allograft transplantation becomes necessary when an accident or trauma results in excessive cell loss. The reconstruction of the cornea endothelium using autologous cell sources is a promising alternative option for therapeutic or in vitro drug testing applications. The native corneal endothelium rests on the Descemet's membrane, which has nanotopographies of fibers and pores. The use of synthetic topographies mimics the native environment, and it is hypothesized that this can direct the behavior and growth of human microvascular endothelial cells (HMVECs) to resemble the corneal endothelium. In this study, HMVECs are cultivated on substrates with micron and nano-scaled pillar and well topographies. Closely packed HMVEC monolayers with polygonal cells and well-developed tight junctions were formed on the topographical substrates. Sodium/potassium (Na+/K+) adenine triphosphatase (ATPase) expression was enhanced on the microwells substrate, which also promotes microvilli formation, while more hexagonal-like cells are found on the micropillars samples. The data obtained suggests that the use of optimized surface patterning, in particular, the microtopographies, can induce HMVECs to adopt a more corneal endothelium-like morphology with similar barrier and pump functions. The mechanism involved in cell contact guidance by the specific topographical features will be of interest for future studies.