Piezo1 activation attenuates thrombin-induced blebbing in breast cancer cells.

Cancer cells exploit a variety of migration modes to leave primary tumors and establish metastases, including amoeboid cell migration, which is typically reliant on bleb formation. Here we demonstrate that thrombin induces dynamic blebbing in the MDA-MB-231 breast cancer cell line and confirm that protease-activated receptor 1 (PAR1) activation is sufficient to induce this effect. Cell confinement has been implicated as a driving force in bleb-based migration. Unexpectedly, we found that gentle contact compression, exerted using a custom built 'cell press' to mechanically stimulate cells, reduced thrombin-induced blebbing. Thrombin-induced blebbing was similarly attenuated using the small molecule Yoda1, an agonist of the mechanosensitive Ca2+ channel Piezo1, and this attenuation was impaired in Piezo1-depleted cells. Additionally, Piezo1 activation suppressed thrombin-induced phosphorylation of ezrin, radixin and moesin (ERM) proteins, which are implicated in the blebbing process. Our results provide mechanistic insights into Piezo1 activation as a suppressor of dynamic blebbing, specifically that which is induced by thrombin.

Concurrent measurement of serum and radiomic biomarkers in the clinical investigation of equine musculoskeletal injuries: A prospective pilot study.

The prevention of musculoskeletal injuries and their related welfare and economic impacts represent an immediate priority for the horse racing industry. This prospective pilot study aimed to evaluate a method to quantitatively analyze scintigraphic features of specific anatomical regions of the horse's appendicular skeleton in combination with secondary measures of musculoskeletal metabolism in blood. Twelve horses referred for scintigraphic assessment of lameness were enrolled. Blood samples were collected immediately prior to the administration of radiotracer. Serum concentrations associated with bone turnover were determined for the following biomarkers: C-terminal telopeptides of type I collagen, proteoglycans and sulfated glycosaminoglycans, collagen type II, osteocalcin, and procollagen II C-terminal propeptide. Scintigraphic images underwent radiomic analysis of discrete regions of the distal limbs and these data were correlated to bone turnover markers. Three lame horses demonstrated asymmetrical radiomic abnormalities. The concentration of osteocalcin in the lame horses was significantly higher when compared to the control group, while no significant changes were observed for the other screened serum biomarkers. Findings from the current study provided evidence that radiomic analysis of equine scintigraphy is feasible. This method has the potential to interrogate which serum markers are associated with musculoskeletal injuries.

Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-ß aggregates.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aß) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aß/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear. METHODS: The aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aß pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aß and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized "close-culture" chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space. RESULTS: Our data show that intracellular deposits of αSYN and Aß are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the "close-culture" chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte. CONCLUSIONS: Taken together, our data demonstrate the importance of astrocyte and microglia interactions in Aß/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.

Mediators of focused psychosocial support interventions for children in low-resource humanitarian settings: analysis from an Individual Participant Dataset with 3,143 participants.

BACKGROUND: Research on psychosocial interventions has been focused on the effectiveness of psychosocial interventions on mental health outcomes, without exploring how interventions achieve beneficial effects. Identifying the potential pathways through which interventions work would potentially allow further strengthening of interventions by emphasizing specific components connected with such pathways. METHODS: We conducted a preplanned mediation analysis using individual participant data from a dataset of 11 randomized controlled trials (RCTs) which compared focused psychosocial support interventions versus control conditions for children living in low- and middle-income countries (LMICs) affected by humanitarian crises. Based on an ecological resilience framework, we hypothesized that (a) coping, (b) hope, (c) social support, and (d) functional impairment mediate the relationship between intervention and outcome PTSD symptoms. A systematic search on the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, PubMed, PyscARTICLES, Web of Science, and the main local LMICs databases was conducted up to August 2018. The hypotheses were tested by using individual participant data obtained from study authors of all the studies included in the systematic review. RESULTS: We included 3,143 children from 11 studies (100% of data from included studies), of which 1,877 from six studies contributed to the mediation analysis. Functional impairment was the strongest mediator for focused psychosocial interventions on PTSD (mediation coefficient -0.087, standard error 0.040). The estimated proportion of effect mediated by functional impairment, and adjusted for confounders, was 31%. CONCLUSIONS: Findings did not support the proposed mediation hypotheses for coping, hope, and social support. The mediation through functional impairment may represent unmeasured proxy measures or point to a broader mechanism that impacts self-efficacy and agency.

Analysis of adoption rates for Needs Driven versus Value Driven innovation water technologies.

This paper analyzes six case studies of new water technology innovations in the last three decades and investigates the differences in timelines for moving through the various stages of water technology commercialization. The concept of two different types of innovation was explored: Crisis/Needs Driven and Value Driven. It was found that the case studies that mapped to the Crisis/Needs Driven innovation moved relatively quickly compared to Value Driven innovations and in most cases involved new entrants. New entrants refer to new companies or start-ups that have recently entered the water technology market. The case studies, which could be mapped to Value Driven innovation, had a slower rate of technology diffusion, and they involved a combination of existing companies as well as new entrants. PRACTITIONER POINTS: The paper identifies two key types of innovation: Crisis/Needs Driven and Value Driven. Legislation was observed to be a key driver for the adoption of new technology innovation in the water sector. The Crisis/Needs driven innovations studied were observed to diffuse through the Water Technology Diffusion model at up to twice the pace of Value driven innovation. Crisis/Needs driven innovation typically involves disruptive innovation offered by new entrants, whereas with Value driven innovation, the solutions are provided by both existing companies as well as new entrants. It is also observed that in most cases a technology that is adopted in order to meet a crisis or need in the market is more expensive at the outset compared with incumbent solutions. While value driven adoption has a slower cycle for adoption, it presents a lower risk as it is less dependent on external factors and timing of implementation of regulations or the occurrence of some public health related or environmental crisis.

Quality assurance issues in the teacher-based assessment of students with literacy difficulties for examination access arrangements.

This paper considers two major concerns centring on the Joint Council for Qualifications (JCQ) regulations for access arrangement and reasonable adjustments and qualifications for teachers who take on the role of Level 7 access arrangements assessor. Thus, the paper is divided into two parts. First, the JCQ 2017-2018 regulations are critically evaluated highlighting the areas of need for which greater clarity and more extensive detail is required in these regulations. The second part of the paper discusses the findings of research on teacher competence in test administration, scoring, and reporting. Drawing on evidence from the first stage of this research, McMurray, O'Callaghan, and McVeigh highlight the extensive formative process required to build a high-level skill set required for competent assessment involving the use of high level tests and also the specialist knowledge required to analyse and accurately report assessment findings to make recommendations for students with specific literacy difficulties. Recommendations are provided both at policy and practice levels regarding the content of courses and the assessment involving the use of high level tests and also the specialist knowledge required to analyse and accurately report assessment findings to make recommendations for students with specific literacy difficulties. The authors provide recommendations for test publishers, course providers, and assessors.

Development and Application of a Model to Study Water Technology Adoption.

This paper develops a set of criteria that can be used to study industry adoption and dissemination of water technologies through various stages of a market adoption model. It tests the applicability of these criteria on a diverse array of over 488 water technologies. Based on case studies, it seeks to define the typical and reasonable time frames in which a water technology moves through these defined stages of industry adoption and dissemination. The development of these criteria, and the definition of reasonable industry average timelines to move through these stages, is an important contribution to the understanding of the process of water technology development. The criteria and defined timelines described in this paper are foundational, and will be used as the basis for subsequent research and analysis to examine the success rates for different water technologies, and common factors linked to why some technologies succeed and others fail.

Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response.

Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1ß (IL1ß) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1ß. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism.

MicroRNA-24 suppression of N-deacetylase/N-sulfotransferase-1 (NDST1) reduces endothelial cell responsiveness to vascular endothelial growth factor A (VEGFA).

Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.

Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein.

Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.

Quantitative imaging of doxorubicin diffusion and cellular uptake in biomimetic gels with human liver tumor cells.

Novel tumor-on-a-chip approaches are increasingly used to investigate tumor progression and potential treatment options. To improve the effect of any cancer treatment it is important to have an in depth understanding of drug diffusion, penetration through the tumor extracellular matrix and cellular uptake. In this study, we have developed a miniaturized chip where drug diffusion and cellular uptake in different hydrogel environments can be quantified at high resolution using live imaging. Diffusion of doxorubicin was reduced in a biomimetic hydrogel mimicking tissue properties of cirrhotic liver and early stage hepatocellular carcinoma (373 ± 108 µm2/s) as compared to an agarose gel (501 ± 77 µm2/s, p = 0.019). The diffusion was further lowered to 256 ± 30 µm2/s (p = 0.028) by preparing the biomimetic gel in cell media instead of phosphate buffered saline. The addition of liver tumor cells (Huh7 or HepG2) to the gel, at two different densities, did not significantly influence drug diffusion. Clinically relevant and quantifiable doxorubicin concentration gradients (1-20 µM) were established in the chip within one hour. Intracellular increases in doxorubicin fluorescence correlated with decreasing fluorescence of the DNA-binding stain Hoechst 33342 and based on the quantified intracellular uptake of doxorubicin an apparent cell permeability (9.00 ± 0.74 × 10-4 µm/s for HepG2) was determined. Finally, the data derived from the in vitro model were applied to a spatio-temporal tissue concentration model to evaluate the potential clinical impact of a cirrhotic extracellular matrix on doxorubicin diffusion and tumor cell uptake.

Group trauma-focused cognitive-behavioural therapy with former child soldiers and other war-affected boys in the DR Congo: a randomised controlled trial.

BACKGROUND: The Democratic Republic of Congo (DRC) has been home to the world's deadliest conflict since World War II and is reported to have the largest number of child soldiers in the world. Despite evidence of the debilitating impact of war, no group-based mental health or psychosocial intervention has been evaluated in a randomised controlled trial for psychologically distressed former child soldiers. METHOD: A randomised controlled trial involving 50 boys, aged 13-17, including former child soldiers (n = 39) and other war-affected boys (n = 11). They were randomly assigned to an intervention group, or wait-list control group. The intervention group received a 15-session, group-based, culturally adapted Trauma-Focused Cognitive-Behavioural Therapy (TF-CBT) intervention. Assessment interviews were completed at baseline, postintervention and 3-month follow-up (intervention group). RESULTS: Analysis of Covariance (ANCOVA) demonstrated that, in comparison to the wait-list control group, the TF-CBT intervention group had highly significant reductions in posttraumatic stress symptoms, overall psychosocial distress, depression or anxiety-like symptoms, conduct problems and a significant increase in prosocial behaviour (p < .001 for all). Effect sizes were higher when former child soldier scores were separated for sub-analysis. Three-month follow-up of the intervention group found that treatment gains were maintained. CONCLUSIONS: A culturally modified, group-based TF-CBT intervention was effective in reducing posttraumatic stress and psychosocial distress in former child soldiers and other war-affected boys.

Fe and C additions decrease the dissolution rate of silicon nitride coatings and are compatible with microglial viability in 3D collagen hydrogels.

Silicon nitride (SiN) coatings may reduce unwanted release of metal ions from metallic implants. However, as SiN slowly dissolves in aqueous solutions, additives that reduce this dissolution rate would likely increase the lifetime and functionality of implants. Adding iron (Fe) and carbon (C) permits tuning of the SiN coatings' mechanical properties, but their effect on SiN dissolution rates, and their capacity to reduce metal ion release from metallic implant substrates, have yet to be investigated. Such coatings have recently been proposed for use in spinal implants; therefore, it is relevant to assess their impact on the viability of cells expected at the implant site, such as microglia, the resident macrophages of the central nervous system (CNS). To study the effects of Fe and C on the dissolution rate of SiN coatings, compositional gradients of Si, Fe and C in combination with N were generated by physical vapor deposition onto CoCrMo discs. Differences in composition did not affect the surface roughness or the release of Si, Fe or Co ions (the latter from the CoCrMo substrate). Adding Fe and C reduced ion release compared to a SiN reference coating, which was attributed to altered reactivity due to an increase in the fraction of stabilizing Si-C or Fe-C bonds. Extracts from the SiN coatings containing Fe and C were compatible with microglial viability in 2D cultures and 3D collagen hydrogels, to a similar degree as CoCrMo and SiN coated CoCrMo reference extracts. As Fe and C reduced the dissolution rate of SiN-coatings and did not compromise microglial viability, the capacity of these additives to extend the lifetime and functionality of SiN-coated metallic implants warrants further investigation.

The ß-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signaling.

The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting ß-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet ß-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.

Heparanase overexpression impairs inflammatory response and macrophage-mediated clearance of amyloid-ß in murine brain.

Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimer's disease, as well as after traumatic injury and pathogen infection. Resident immune cells, microglia and astrocytes, are activated and joined by blood-borne monocytes that traverse the blood-brain barrier and convert into activated macrophages. The activated cells express various cytokines, chemokines and proteolytic enzymes. To study the role of heparan sulfate proteoglycans in neuroinflammation, we employed a transgenic mouse overexpressing heparanase, an endoglucuronidase that specifically degrades heparan sulfate side chains. Neuroinflammation was induced by systemic challenge with lipopolysaccharide, or by localized cerebral microinjection of aggregated amyloid-ß peptide, implicated in Alzheimer's disease. Lipopolysaccharide-treated control mice showed massive activation of resident microglia as well as recruitment of monocyte-derived macrophages into the brain parenchyma. Microinjection of aggregated amyloid-ß elicited a similar inflammatory response, albeit restricted to the injection site, which led to dispersion and clearance of the amyloid. In the heparanase-overexpressing mice, all aspects of immune cell recruitment and activation were significantly attenuated in both inflammation models, as was amyloid dispersion. Accordingly, an in vitro blood-brain barrier model constructed from heparanase-overexpressing cerebral vascular cells showed impaired transmigration of monocytes compared to a corresponding assembly of control cells. Our data indicate that intact heparan sulfate chains are required at multiple sites to mediate neuroinflammatory responses, and further point to heparanase as a modulator of this process, with potential implications for Alzheimer's disease.

Screening for traumatic exposure and psychological distress among war-affected adolescents in post-conflict northern Uganda.

BACKGROUND: The war in northern Uganda has had a debilitating effect on the mental health of children and adolescents in the population. This study measures the prevalence and considers the aetiology of psychological distress in war-affected adolescents 4 years after the end of the conflict. METHODS: This is a cross-sectional study of 205 adolescents, aged 12-19, from a boarding primary school in Gulu, northern Uganda. A war experiences checklist was developed with the assistance of local professionals. The Impact of Event Scale-Revised (IES-R) measured post-traumatic stress symptoms. Finally, the Acholi Psychosocial Assessment Instrument (APAI) was used to measure locally described mental health constructs similar to the Western concepts of depression and anxiety. RESULTS: Four years after the end of the war, 57% of the students were still found to have clinically significant levels of post-traumatic stress symptoms using a similar cut-off score to previous studies among the same population. Both components of traumatic exposure: (i) the number of types of traumatic event experienced; and (ii) whether the adolescent was abducted were significantly associated with psychological distress. There was a strong correlation between post-traumatic stress symptoms and internalising symptoms. CONCLUSION: War-affected adolescents may continue to suffer from significant psychological stress in the years following the cessation of conflict. Multiple exposure to a number of different types of traumatic event may directly increase the likelihood of psychological distress especially for those exposed to the most extreme violence. The feasibility of employing a locally developed and validated screening instrument is demonstrated. Implications for future research and intervention in post-conflict areas are considered.

Introducing or removing heparan sulfate binding sites does not alter brain uptake of the blood-brain barrier shuttle scFv8D3.

The blood-brain barrier (BBB) greatly limits the delivery of protein-based drugs into the brain and is a major obstacle for the treatment of brain disorders. Targeting the transferrin receptor (TfR) is a strategy for transporting protein-based drugs into the brain, which can be utilized by using TfR-binding BBB transporters, such as the TfR-binding antibody 8D3. In this current study, we investigated if binding to heparan sulfate (HS) contributes to the brain uptake of a single chain fragment variable of 8D3 (scFv8D3). We designed and produced a scFv8D3 mutant, engineered with additional HS binding sites, HS(+)scFv8D3, to assess whether increased HS binding would improve brain uptake. Additionally, a mutant with a reduced number of HS binding sites, HS(-)scFv8D3, was also engineered to see if reducing the HS binding sites could also affect brain uptake. Heparin column chromatography showed that only the HS(+)scFv8D3 mutant bound HS in the experimental conditions. Ex vivo results showed that the brain uptake was unaffected by the introduction or removal of HS binding sites, which indicates that scFv8D3 is not dependent on the HS binding sites for brain uptake. Conversely, introducing HS binding sites to scFv8D3 decreased its renal excretion while removing them had the opposite effect.

An open source extrusion bioprinter based on the E3D motion system and tool changer to enable FRESH and multimaterial bioprinting.

Bioprinting is increasingly used to create complex tissue constructs for an array of research applications, and there are also increasing efforts to print tissues for transplantation. Bioprinting may also prove valuable in the context of drug screening for personalized medicine for treatment of diseases such as cancer. However, the rapidly expanding bioprinting research field is currently limited by access to bioprinters. To increase the availability of bioprinting technologies we present here an open source extrusion bioprinter based on the E3D motion system and tool changer to enable high-resolution multimaterial bioprinting. As proof of concept, the bioprinter is used to create collagen constructs using freeform reversible embedding of suspended hydrogels (FRESH) methodology, as well as multimaterial constructs composed of distinct sections of laminin and collagen. Data is presented demonstrating that the bioprinted constructs support growth of cells either seeded onto printed constructs or included in the bioink prior to bioprinting. This open source bioprinter is easily adapted for different bioprinting applications, and additional tools can be incorporated to increase the capabilities of the system.

Heparanase overexpression impedes perivascular clearance of amyloid-ß from murine brain: relevance to Alzheimer's disease.

Defective amyloid-ß (Aß) clearance from the brain is a major contributing factor to the pathophysiology of Alzheimer's disease (AD). Aß clearance is mediated by macrophages, enzymatic degradation, perivascular drainage along the vascular basement membrane (VBM) and transcytosis across the blood-brain barrier (BBB). AD pathology is typically associated with cerebral amyloid angiopathy due to perivascular accumulation of Aß. Heparan sulfate (HS) is an important component of the VBM, thought to fulfill multiple roles in AD pathology. We previously showed that macrophage-mediated clearance of intracortically injected Aß was impaired in the brains of transgenic mice overexpressing heparanase (Hpa-tg). This study revealed that perivascular drainage was impeded in the Hpa-tg brain, evidenced by perivascular accumulation of the injected Aß in the thalamus of Hpa-tg mice. Furthermore, endogenous Aß accumulated at the perivasculature of Hpa-tg thalamus, but not in control thalamus. This perivascular clearance defect was confirmed following intracortical injection of dextran that was largely retained in the perivasculature of Hpa-tg brains, compared to control brains. Hpa-tg brains presented with thicker VBMs and swollen perivascular astrocyte endfeet, as well as elevated expression of the BBB-associated water-pump protein aquaporin 4 (AQP4). Elevated levels of both heparanase and AQP4 were also detected in human AD brain. These findings indicate that elevated heparanase levels alter the organization and composition of the BBB, likely through increased fragmentation of BBB-associated HS, resulting in defective perivascular drainage. This defect contributes to perivascular accumulation of Aß in the Hpa-tg brain, highlighting a potential role for heparanase in the pathogenesis of AD.

Heparan sulfate mediates amyloid-beta internalization and cytotoxicity.

Heparan sulfate (HS) has been found associated with amyloid deposits, including the toxic amyloid-beta (Abeta) peptide aggregates in cerebral vasculature and neuronal tissues in patients with Alzheimer's disease. However, the pathophysiological significance of the HS-Abeta interaction has remained unclear. In the present study, we applied cell models to gain insight into the roles of HS in relation to Abeta toxicity. Wild-type Chinese hamster ovary (CHO-WT) cells showed loss of viability following exposure to Abeta40, whereas the HS-deficient cell line, pgsD-677, was essentially resistant. Immunocytochemical analysis showed Abeta internalization by CHO-WT, but not pgsD-677 cells. Abeta40 toxicity was also attenuated in human embryonic kidney cells overexpressing heparanase. Finally, addition of heparin to human umbilical vein endothelial cells prevented internalization of added Abeta40 and protected against Abeta toxicity. Taken together, these findings suggest that cell-surface HS mediates Abeta internalization and toxicity.