Amphiphilic Heparinoids as Potent Antiviral Agents against SARS-CoV-2.

Herein, we report the synthesis and biological evaluation of a novel series of heparinoid amphiphiles as inhibitors of heparanase and SARS-CoV-2. By employing a tailor-made synthetic strategy, a library of highly sulfated homo-oligosaccharides bearing d-glucose or a C5-epimer (i.e., l-idose or l-iduronic acid) conjugated with various lipophilic groups was synthesized and investigated for antiviral activity. Sulfated higher oligosaccharides of d-glucose or l-idose with lipophilic aglycones displayed potent anti-SARS-CoV-2 and antiheparanse activity, similar to or better than pixatimod (PG545), and were more potent than their isosteric l-iduronic acid congeners. Lipophilic groups such as cholestanol and C18-aliphatic substitution are more advantageous than functional group appended lipophilic moieties. These findings confirm that fine-tuning of higher oligosaccharides, degree of sulfation, and lipophilic groups can yield compounds with potent anti-SARS-CoV-2 activity.

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.

Quantitative vestibular function tests in posterior circulation stroke patients: A review.

While specific bedside examinations are known to be sensitive in identifying stroke among acute vestibular syndrome patients, complementary quantitative vestibular function testing can be helpful to quantify vestibular loss due to stroke. In contrast to peripheral vestibular dysfunction, diagnosis of central vestibular dysfunction can be challenging for unskilful clinicians. This article presents a comprehensive overview of quantitative vestibular function test findings such as the video head impulse test (vHIT), cervical vestibular evoked myogenic potentials (cVEMPs), ocular vestibular evoked myogenic potentials (oVEMPs), videonystagmography (VNG) and caloric test among stroke patients. Vestibulo-ocular reflex (VOR) gain is usually found normal among posterior inferior cerebellar artery (PICA) stroke patients but varies among anterior inferior cerebellar artery (AICA) stroke patients. Abnormal contralesional posterior semicircular canal VOR gain can be observed due to lesions in the medial longitudinal fasciculus (MLF). AICA and PICA stroke can impair cVEMPs, oVEMPs, and VNG (i.e., smooth pursuit and saccade functions). Strokes, particularly those involving the vestibular nucleus, including both upper, lower brainstem and cerebellum, can result in various abnormalities of smooth pursuit, saccade or calorics testing. The combined evaluations of VNG, vHIT, and VEMPs can be accurately used to complement and quantify bedside vestibular evaluation in diagnosing central vestibular dysfunction. In addition, as most studies were conducted amongst acute vestibular syndrome (AVS) patients, future studies that investigate the prevalence of vestibular dysfunction in recovering stroke patients are required.

A review of idiopathic inflammatory myopathy cases in Terengganu, Malaysia: A single centre experience.

INTRODUCTION: The purpose of this study was to analyse the clinical characteristics of patients with idiopathic inflammatory myopathy (IIM) in Hospital Sultanah Nurzahirah (HSNZ), Terengganu, Malaysia. It also aimed to describe the disease manifestations in association with malignancy and other CTD. METHODS: This was a retrospective descriptive study involving all IIM patients who were managed by the Rheumatology Unit HSNZ from January 2010 to December 2019. RESULTS: In this review we described 15 cases wherein malignancy was detected in 4 patients after the diagnosis of IIM was made and 4 patients with overlap syndrome. One third of patients with malignancy and overlap syndrome had poor treatment response and succumbed to complications of the disease. Almost all of patients received corticosteroid as the first line therapy and nearly two thirds of them responded well to either corticosteroid alone or with combination therapy. CONCLUSION: Although this study did not represent the whole population in Malaysia, it does provide a better understanding of the disease manifestation, treatment and disease complications in our cohort of patients.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Risk factors and predictors of levodopa-induced dyskinesia among multiethnic Malaysians with Parkinson's disease.

UNLABELLED: Chronic pulsatile levodopa therapy for Parkinson's disease (PD) leads to the development of motor fluctuations and dyskinesia. We studied the prevalence and predictors of levodopa-induced dyskinesia among multiethnic Malaysian patients with PD. METHODS: This is a cross-sectional study involving 95 patients with PD on uninterrupted levodopa therapy for at least 6 months. The instrument used was the UPDRS questionnaires. The predictors of dyskinesia were determined using multivariate logistic regression analysis. RESULTS: The mean age was 65.6 ± 8.5 years. The mean onset age was 58.5 ± 9.8 years. The median disease duration was 6 (7) years. Dyskinesia was present in 44% (n = 42) with median levodopa therapy of 3 years. There were 64.3% Chinese, 31% Malays, and 3.7% Indians and other ethnic groups. Eighty-one percent of patients with dyskinesia had clinical fluctuations. Patients with dyskinesia had lower onset age ( p < 0.001), longer duration of levodopa therapy ( p < 0.001), longer disease duration ( p < 0.001), higher total daily levodopa dose ( p < 0.001), and higher total UPDRS scores ( p = 0.005) than patients without dyskinesia. The three significant predictors of dyskinesia were duration of levodopa therapy, onset age, and total daily levodopa dose. CONCLUSIONS: The prevalence of levodopa-induced dyskinesia in our patients was 44%. The most significant predictors were duration of levodopa therapy, total daily levodopa dose, and onset age.

Prevalence of depression in stroke patients with vascular dementia in universiti kebangsaan malaysia medical center.

OBJECTIVE: Depression among patients with vascular dementia is frequently overlooked and potentially causes significant morbidity. There is limited data in Malaysia on the subject and this study was conducted to determine the prevalence of depression in vascular dementia (VaD) in UKMMC. METHODS: This was a cross-sectional study involving diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM IV) criteria and who had a mini mental state examination (MMSE) score of less than 26. All patients were interviewed, examined clinically and their previous brain computer tomography (CT) were reviewed. The prevalence of depression was determined using the Cornell scale of depression. RESULTS: A total of 76 patients were recruited with a mean age of 70.5 ± 9.5 years. The median duration of illness was 2.0 (1.0-4.8) years. The prevalence of depression in the study population was 31.6%. The patients with depression had a significant older mean age (74.5±8.7 years old) compared to those without depression (68.6±9.4 years old). Patients with large artery stroke of less than 3 years had significant higher frequency of depression (53.6%) compared to patients with small artery stroke (23.8%) and patients with right sided large artery stroke had significantly higher frequency of depression compared to left (70% vs. 44.4%). Median MMSE score (17.0) for depressed patients was significantly lower compared with median MMSE score (22.5) for non depressed patients. Median Barthel Index (30.0) for depressed patients was significantly lower compared with median Barthel score for non depressed patients. CONCLUSIONS: Depression was prevalent among post stroke patients with VaD in UKMMC particularly for patients with older age, large artery stroke, right sided large artery stroke, low MMSE score and low Barthel Index. Early recognition of high risk patients is important in the holistic management of patients to prevent significant morbidity arising from depression.