PBMC-derived extracellular vesicles in a smoking-related inflammatory disease model.

Extracellular vesicles (EVs) function as mediators of intercellular communication and as such influence the recipient cell function. EVs derived from immune cells can carry out many of the same functions as their parental cells, as they carry costimulatory molecules, antigens, and antigen-MHC complexes. As a result, there is a strong interest in understanding the composition and origin of immune cell-derived EVs in order to understand their role in the pathogenesis of diseases. This study aimed to optimize methodologies to study immune cell-derived EVs. Peripheral blood mononuclear cell-derived small EVs were isolated and observed using conventional transmission electron microscopy and sized by nanoparticle tracking analysis. They were then enumerated and profiled using imaging flow cytometry and were further characterized using a flow cytometric multiplex bead assay. These techniques were then applied to our current research, namely smoking-related inflammatory disease. We present here a comprehensive approach to analyze PBMC-derived small EVs in smoking-related inflammatory disease following the Minimal Information for Studies of Extracellular Vesicle 2018 guidelines.

Heparanase inhibition as a systemic approach to protect the endothelial glycocalyx and prevent microvascular complications in diabetes.

BACKGROUND: Diabetes mellitus is a chronic disease which is detrimental to cardiovascular health, often leading to secondary microvascular complications, with huge global health implications. Therapeutic interventions that can be applied to multiple vascular beds are urgently needed. Diabetic retinopathy (DR) and diabetic kidney disease (DKD) are characterised by early microvascular permeability changes which, if left untreated, lead to visual impairment and renal failure, respectively. The heparan sulphate cleaving enzyme, heparanase, has previously been shown to contribute to diabetic microvascular complications, but the common underlying mechanism which results in microvascular dysfunction in conditions such as DR and DKD has not been determined. METHODS: In this study, two mouse models of heparan sulphate depletion (enzymatic removal and genetic ablation by endothelial specific Exotosin-1 knock down) were utilized to investigate the impact of endothelial cell surface (i.e., endothelial glycocalyx) heparan sulphate loss on microvascular barrier function. Endothelial glycocalyx changes were measured using fluorescence microscopy or transmission electron microscopy. To measure the impact on barrier function, we used sodium fluorescein angiography in the eye and a glomerular albumin permeability assay in the kidney. A type 2 diabetic (T2D, db/db) mouse model was used to determine the therapeutic potential of preventing heparan sulphate damage using treatment with a novel heparanase inhibitor, OVZ/HS-1638. Endothelial glycocalyx changes were measured as above, and microvascular barrier function assessed by albumin extravasation in the eye and a glomerular permeability assay in the kidney. RESULTS: In both models of heparan sulphate depletion, endothelial glycocalyx depth was reduced and retinal solute flux and glomerular albumin permeability was increased. T2D mice treated with OVZ/HS-1638 had improved endothelial glycocalyx measurements compared to vehicle treated T2D mice and were simultaneously protected from microvascular permeability changes associated with DR and DKD. CONCLUSION: We demonstrate that endothelial glycocalyx heparan sulphate plays a common mechanistic role in microvascular barrier function in the eye and kidney. Protecting the endothelial glycocalyx damage in diabetes, using the novel heparanase inhibitor OVZ/HS-1638, effectively prevents microvascular permeability changes associated with DR and DKD, demonstrating a novel systemic approach to address diabetic microvascular complications.

A Systematic Review and Meta-Analysis of the Pathology Underlying Aneurysm Enhancement on Vessel Wall Imaging.

Intracranial aneurysms are common, but only a minority rupture and cause subarachnoid haemorrhage, presenting a dilemma regarding which to treat. Vessel wall imaging (VWI) is a contrast-enhanced magnetic resonance imaging (MRI) technique used to identify unstable aneurysms. The pathological basis of MR enhancement of aneurysms is the subject of debate. This review synthesises the literature to determine the pathological basis of VWI enhancement. PubMed and Embase searches were performed for studies reporting VWI of intracranial aneurysms and their correlated histological analysis. The risk of bias was assessed. Calculations of interdependence, univariate and multivariate analysis were performed. Of 228 publications identified, 7 met the eligibility criteria. Individual aneurysm data were extracted for 72 out of a total of 81 aneurysms. Univariate analysis showed macrophage markers (CD68 and MPO, p = 0.001 and p = 0.002), endothelial cell markers (CD34 and CD31, p = 0.007 and p = 0.003), glycans (Alcian blue, p = 0.003) and wall thickness (p = 0.030) were positively associated with enhancement. Aneurysm enhancement therefore appears to be associated with inflammatory infiltrate and neovascularisation. However, all these markers are correlated with each other, and the literature is limited in terms of the numbers of aneurysms analysed and the parameters considered. The data are therefore insufficient to determine if these associations are independent of each other or of aneurysm size, wall thickness and rupture status. Thus, the cause of aneurysm-wall enhancement currently remains unknown.

John squire and endothelial glycocalyx structure: an unfinished story.

John Squire did not only produce leading works in the muscle field, he also significantly contributed to the vascular permeability field by ultrastructural analysis of the endothelial glycocalyx. Presented here is a review of his involvement in the field by his main collaborator C.C. Michel and his last postdoctoral researcher KP Arkill. We end on a reinterpretation of his work that arguably links to our current understanding of endothelial glycocalyx structure and composition predicting 6 glycosaminoglycans fibres per syndecan core protein, only achieved in the endothelium by dimerization.

Hydrogel-Based Pre-Clinical Evaluation of Repurposed FDA-Approved Drugs for AML.

In vivo models of acute myeloid leukemia (AML) are low throughput, and standard liquid culture models fail to recapitulate the mechanical and biochemical properties of the extracellular matrix-rich protective bone marrow niche that contributes to drug resistance. Candidate drug discovery in AML requires advanced synthetic platforms to improve our understanding of the impact of mechanical cues on drug sensitivity in AML. By use of a synthetic, self-assembling peptide hydrogel (SAPH) of modifiable stiffness and composition, a 3D model of the bone marrow niche to screen repurposed FDA-approved drugs has been developed and utilized. AML cell proliferation was dependent on SAPH stiffness, which was optimized to facilitate colony growth. Three candidate FDA-approved drugs were initially screened against the THP-1 cell line and mAF9 primary cells in liquid culture, and EC50 values were used to inform drug sensitivity assays in the peptide hydrogel models. Salinomycin demonstrated efficacy in both an 'early-stage' model in which treatment was added shortly after initiation of AML cell encapsulation, and an 'established' model in which time-encapsulated cells had started to form colonies. Sensitivity to Vidofludimus treatment was not observed in the hydrogel models, and Atorvastatin demonstrated increased sensitivity in the 'established' compared to the 'early-stage' model. AML patient samples were equally sensitive to Salinomycin in the 3D hydrogels and partially sensitive to Atorvastatin. Together, this confirms that AML cell sensitivity is drug- and context-specific and that advanced synthetic platforms for higher throughput are valuable tools for pre-clinical evaluation of candidate anti-AML drugs.

Endothelial glycocalyx is damaged in diabetic cardiomyopathy: angiopoietin 1 restores glycocalyx and improves diastolic function in mice.

AIMS/HYPOTHESIS: Diabetic cardiomyopathy (DCM) is a serious and under-recognised complication of diabetes. The first sign is diastolic dysfunction, which progresses to heart failure. The pathophysiology of DCM is incompletely understood but microcirculatory changes are important. Endothelial glycocalyx (eGlx) plays multiple vital roles in the microcirculation, including in the regulation of vascular permeability, and is compromised in diabetes but has not previously been studied in the coronary microcirculation in diabetes. We hypothesised that eGlx damage in the coronary microcirculation contributes to increased microvascular permeability and hence to cardiac dysfunction. METHODS: We investigated eGlx damage and cardiomyopathy in mouse models of type 1 (streptozotocin-induced) and type 2 (db/db) diabetes. Cardiac dysfunction was determined by echocardiography. We obtained eGlx depth and coverage by transmission electron microscopy (TEM) on mouse hearts perfusion-fixed with glutaraldehyde and Alcian Blue. Perivascular oedema was assessed from TEM images by measuring the perivascular space area. Lectin-based fluorescence was developed to study eGlx in paraformaldehyde-fixed mouse and human tissues. The eGlx of human conditionally immortalised coronary microvascular endothelial cells (CMVECs) in culture was removed with eGlx-degrading enzymes before measurement of protein passage across the cell monolayer. The mechanism of eGlx damage in the diabetic heart was investigated by quantitative reverse transcription-PCR array and matrix metalloproteinase (MMP) activity assay. To directly demonstrate that eGlx damage disturbs cardiac function, isolated rat hearts were treated with enzymes in a Langendorff preparation. Angiopoietin 1 (Ang1) is known to restore eGlx and so was used to investigate whether eGlx restoration reverses diastolic dysfunction in mice with type 1 diabetes. RESULTS: In a mouse model of type 1 diabetes, diastolic dysfunction (confirmed by echocardiography) was associated with loss of eGlx from CMVECs and the development of perivascular oedema, suggesting increased microvascular permeability. We confirmed in vitro that eGlx removal increases CMVEC monolayer permeability. We identified increased MMP activity as a potential mechanism of eGlx damage and we observed loss of syndecan 4 consistent with MMP activity. In a mouse model of type 2 diabetes we found a similar loss of eGlx preceding the development of diastolic dysfunction. We used isolated rat hearts to demonstrate that eGlx damage (induced by enzymes) is sufficient to disturb cardiac function. Ang1 restored eGlx and this was associated with reduced perivascular oedema and amelioration of the diastolic dysfunction seen in mice with type 1 diabetes. CONCLUSIONS/INTERPRETATION: The association of CMVEC glycocalyx damage with diastolic dysfunction in two diabetes models suggests that it may play a pathophysiological role and the enzyme studies confirm that eGlx damage is sufficient to impair cardiac function. Ang1 rapidly restores the CMVEC glycocalyx and improves diastolic function. Our work identifies CMVEC glycocalyx damage as a potential contributor to the development of DCM and therefore as a therapeutic target.

Serine-arginine-rich protein kinase-1 inhibition for the treatment of diabetic retinopathy.

Angiogenic VEGF isoforms are upregulated in diabetic retinopathy (DR), driving pathological growth and fluid leakage. Serine-arginine-rich protein kinase-1 (SRPK1) regulates VEGF splicing, and its inhibition blocks angiogenesis. We tested the hypothesis that SRPK1 is activated in diabetes, and an SRPK1 inhibitor (SPHINX31) switches VEGF splicing in DR and prevents increased vascular permeability into the retina. SRPK1 was activated by high glucose (HG), in a PKC-dependent manner, and was blocked by SPHINX31. HG induced release of SRSF1 from the nuclear speckles, which was also SRPK1 dependent, and increased retinal pigment epithelial (RPE) monolayer admittance, which was reversed by SRPK1 inhibition (P < 0.05). Diabetes increased retinal permeability and thickness after 14 days which was blocked by treatment with SPHINX31 eye drops (P < 0.0001). These results show that SRPK1 inhibition, administered as an eye drop, protected the retinal barrier from hyperglycemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. A clinical trial of another SRPK1 inhibitor has now been initiated in patients with diabetic macular edema.NEW & NOTEWORTHY VEGF-A165b splicing is induced by hyperglycemia through PKC-mediated activation of SRPK1 in RPE cells, increasing their permeability and angiogenic capability. SRPK1 inhibitors can be given as eye drops to reduce retinal permeability and edema in diabetic retinopathy.

A new view of macula densa cell microanatomy.

Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation, and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells, were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy of the rat, rabbit, and human kidney. An elaborate network of major and minor cell processes, here named maculapodia, were found at the cell base, projecting toward other MD cells and the glomerular vascular pole. The extent of maculapodia showed upregulation by low dietary salt intake and the female sex. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. Electron microscopy of rat, rabbit, and human kidneys and three-dimensional volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD cells and between MD and other target cells.NEW & NOTEWORTHY This study illuminated a physiologically regulated dense network of basal cell major and minor processes (maculapodia) in macula densa (MD) cells. The newly identified dynamic and secretory features of these microanatomical structures suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD and other target cells. Detailed characterization of the function and molecular details of MD cell intercellular communications and their role in physiology and disease warrant further studies.

Non-invasive measurement of retinal permeability in a diabetic rat model.

OBJECTIVE: The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. METHODS: Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. RESULTS: In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P < .01, P < .001, P < .0001). CONCLUSIONS: This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.

Novel hemodynamic structures in the human glomerulus.

To investigate human glomerular structure under conditions of physiological perfusion, we have analyzed fresh and perfusion-fixed normal human glomeruli at physiological hydrostatic and oncotic pressures using serial resin section reconstruction, confocal, multiphoton, and electron microscope imaging. Afferent and efferent arterioles (21.5 ± 1.2 µm and 15.9 ± 1.2 µm diameter), recognized from vascular origins, lead into previously undescribed wider regions (43.2 ± 2.8 µm and 38.4 ± 4.9 µm diameter) we have termed vascular chambers (VCs) embedded in the mesangium of the vascular pole. Afferent VC (AVC) volume was 1.6-fold greater than efferent VC (EVC) volume. From the AVC, long nonbranching high-capacity conduit vessels ( n = 7) (Con; 15.9 ± 0.7 µm diameter) led to the glomerular edge, where branching was more frequent. Conduit vessels have fewer podocytes than filtration capillaries. VCs were confirmed in fixed and unfixed specimens with a layer of banded collagen identified in AVC walls by multiphoton and electron microscopy. Thirteen highly branched efferent first-order vessels (E1; 9.9 ± 0.4 µm diameter) converge on the EVC, draining into the efferent arteriole (15.9 ± 1.2 µm diameter). Banded collagen was scarce around EVCs. This previously undescribed branching topology does not conform to the branching of minimum energy expenditure (Murray's law), suggesting that even distribution of pressure/flow to the filtration capillaries is more important than maintaining the minimum work required for blood flow. We propose that AVCs act as plenum manifolds possibly aided by vortical flow in distributing and balancing blood flow/pressure to conduit vessels supplying glomerular lobules. These major adaptations to glomerular capillary structure could regulate hemodynamic pressure and flow in human glomerular capillaries.

Dye Aggregate-Mediated Self-Assembly of Bacteriophage Bioconjugates.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function.

KEY POINTS: We have developed novel techniques for paired, direct, real-time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability. Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel. The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth. Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin. ABSTRACT: The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real-time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17-3.02 µm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 µm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a time-dependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin.

A High Resolution Study of Dynamic Changes of Ce2O3 and CeO2 Nanoparticles in Complex Environmental Media.

Ceria nanoparticles (NPs) rapidly and easily cycle between Ce(III) and Ce(IV) oxidation states, making them prime candidates for commercial and other applications. Increased commercial use has resulted in increased discharge to the environment and increased associated risk. Once in complex media such as environmental waters or toxicology exposure media, the same redox transformations can occur, causing altered behavior and effects compared to the pristine NPs. This study used high resolution scanning transmission electron microscopy and electron energy loss spectroscopy to investigate changes in structure and oxidation state of small, polymer-coated ceria suspensions in complex media. NPs initially in either the III or IV oxidation states, but otherwise identical, were used. Ce(IV) NPs were changed to mixed (III, IV) NPs at high ionic strengths, while the presence of natural organic macromolecules (NOM) stabilized the oxidation state and increased crystallinity. The Ce(III) NPs remained as Ce(III) at high ionic strengths, but were modified by the presence of NOM, causing reduced crystallinity and degradation of the NPs. Subtle changes to NP properties upon addition to environmental or ecotoxicology media suggest that there may be small but important effects on fate and effects of NPs compared to their pristine form.

Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.

Optical micro-spectroscopy of single metallic nanoparticles: quantitative extinction and transient resonant four-wave mixing.

We report a wide-field imaging method to rapidly and quantitatively measure the optical extinction cross-section σ(ext) (also polarisation resolved) of a large number of individual gold nanoparticles, for statistically-relevant single particle analysis. We demonstrate a sensitivity of 5 nm(2) in σ(ext), enabling detection of single 5 nm gold nanoparticles with total acquisition times in the 1 min range. Moreover, we have developed an analytical model of the polarisation resolved σ(ext), which enabled us to extract geometrical particle aspect ratios from the measured σ(ext). Using this method, we have characterized a large number of nominally-spherical gold nanoparticles in the 10-100 nm size range. Furthermore, the method provided measurements of in-house fabricated nanoparticle conjugates, allowing distinction of individual dimers from single particles and larger aggregates. The same particle conjugates were investigated correlatively by phase-resolved transient resonant four-wave mixing micro-spectroscopy. A direct comparison of the phase-resolved response between single gold nanoparticles and dimers highlighted the promise of the four-wave mixing technique for sensing applications with dimers as plasmon rulers.

Origin of parietal podocytes in atubular glomeruli mapped by lineage tracing.

Parietal podocytes are fully differentiated podocytes lining Bowman's capsule where normally only parietal epithelial cells (PECs) are found. Parietal podocytes form throughout life and are regularly observed in human biopsies, particularly in atubular glomeruli of diseased kidneys; however, the origin of parietal podocytes is unresolved. To assess the capacity of PECs to transdifferentiate into parietal podocytes, we developed and characterized a novel method for creating atubular glomeruli by electrocoagulation of the renal cortex in mice. Electrocoagulation produced multiple atubular glomeruli containing PECs as well as parietal podocytes that projected from the vascular pole and lined Bowman's capsule. Notably, induction of cell death was evident in some PECs. In contrast, Bowman's capsules of control animals and normal glomeruli of electrocoagulated kidneys rarely contained podocytes. PECs and podocytes were traced by inducible and irreversible genetic tagging using triple transgenic mice (PEC- or Pod-rtTA/LC1/R26R). Examination of serial cryosections indicated that visceral podocytes migrated onto Bowman's capsule via the vascular stalk; direct transdifferentiation from PECs to podocytes was not observed. Similar results were obtained in a unilateral ureter obstruction model and in human diseased kidney biopsies, in which overlap of PEC- or podocyte-specific antibody staining indicative of gradual differentiation did not occur. These results suggest that induction of atubular glomeruli leads to ablation of PECs and subsequent migration of visceral podocytes onto Bowman's capsule, rather than transdifferentiation from PECs to parietal podocytes.

Resolution of the three dimensional structure of components of the glomerular filtration barrier.

BACKGROUND: The human glomerulus is the primary filtration unit of the kidney, and contains the Glomerular Filtration Barrier (GFB). The GFB had been thought to comprise 3 layers - the endothelium, the basement membrane and the podocyte foot processes. However, recent studies have suggested that at least two additional layers contribute to the function of the GFB, the endothelial glycocalyx on the vascular side, and the sub-podocyte space on the urinary side. To investigate the structure of these additional layers is difficult as it requires three-dimensional reconstruction of delicate sub-microscopic (<1 µm) cellular and extracellular elements. METHODS: Here we have combined three different advanced electron microscopic techniques that cover multiple orders of magnitude of volume sampled, with a novel staining methodology (Lanthanum Dysprosium Glycosaminoglycan adhesion, or LaDy GAGa), to determine the structural basis of these two additional layers. Serial Block Face Scanning Electron Microscopy (SBF-SEM) was used to generate a 3-D image stack with a volume of a 5.3 x 105 µm3 volume of a whole kidney glomerulus (13% of glomerular volume). Secondly, Focused Ion Beam milling Scanning Electron Microscopy (FIB-SEM) was used to image a filtration region (48 µm3 volume). Lastly Transmission Electron Tomography (Tom-TEM) was performed on a 0.3 µm3 volume to identify the fine structure of the glycocalyx. RESULTS: Tom-TEM clearly showed 20 nm fibre spacing in the glycocalyx, within a limited field of view. FIB-SEM demonstrated, in a far greater field of view, how the glycocalyx structure related to fenestrations and the filtration slits, though without the resolution of TomTEM. SBF-SEM was able to determine the extent of the sub-podocyte space and glycocalyx coverage, without additional heavy metal staining. Neither SBF- nor FIB-SEM suffered the anisotropic shrinkage under the electron beam that is seen with Tom-TEM. CONCLUSIONS: These images demonstrate that the three dimensional structure of the GFB can be imaged, and investigated from the whole glomerulus to the fine structure of the glycocalyx using three dimensional electron microscopy techniques. This should allow the identification of structural features regulating physiology, and their disruption in pathological states, aiding the understanding of kidney disease.

Modulation of the pre-metastatic bone niche: molecular changes mediated by bone-homing prostate cancer extracellular vesicles.

Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have emerged as key players in cancer communication and metastasis, promoting the formation of supportive microenvironments in distant sites. Our previous studies have highlighted the role of PCa EVs in modulating osteoblasts and facilitating tumor progression. However, the early pre-metastatic changes induced by PCa EVs within the bone microenvironment remain poorly understood. To investigate the early effects of repeated exposure to PCa EVs in vivo, mimicking EVs being shed from the primary tumor, PCa EVs isolated from cell line PC3MLuc2a were fluorescently labelled and repeatedly administered via tail vein injection to adult CD1 NuNu male mice for a period of 4 weeks. In vivo imagining, histological analysis and gene expression profiling were performed to assess the impact of PCa EVs on the bone microenvironment. We demonstrate for the first time that PCa EVs home to both bone and lymph nodes following repeated exposures. Furthermore, the accumulation of EVs within the bone leads to distinct molecular changes indicative of disrupted bone homeostasis (e.g., changes to signaling pathways such as Paxillin p = 0.0163, Estrogen Receptor p = 0.0271, RHOA p = 0.0287, Ribonucleotide reductase p = 0.0307 and ERK/MAPK p = 0.0299). Changes in key regulators of these pathways were confirmed in vitro on human osteoblasts. In addition, our data compares the known gene signature of osteocytes and demonstrates a high proportion of overlap (52.2%), suggesting a potential role for this cell type in response to PCa EV exposure. No changes in bone histology or immunohistochemistry were detected, indicating that PCa EV mediated changes were induced at the molecular level. This study provides novel insights into the alterations induced by PCa EVs on the bone microenvironment. The observed molecular changes indicate changes in key pathways and suggest a role for osteocytes in these EV mediated early changes to bone. Further research to understand these early events may aid in the development of targeted interventions to disrupt the metastatic cascade in PCa.

Measurement of solute permeability in the mouse spinal cord.

BACKGROUND: Sensory perception and motor dexterity is coordinated by the spinal cord, which remains effective due to maintenance of neuronal homeostasis. This is stringently controlled by the blood spinal cord barrier. Therefore, the function of the spinal cord is susceptible to alterations in the microvessel integrity (e.g. vascular leakage) and/or perfusion (e.g. changes in blood flow). NEW METHOD: Spinal cord solute permeability was measured in anaesthetised mice. The lumbar spinal cord vertebra were stabilised and a coverslip secured to allow fluorescent tracers of vascular function and anatomy to be visualised in the vascular network. Fluorescence microscopy allowed real time measurements of vascular leakage and capillary perfusion within the spinal cord. RESULTS: Capillaries were identified through fluorescent labelling of the endothelial luminal glycocalyx (wheat germ agglutin 555). Real time estimation of vascular permeability through visualisation of sodium fluorescein transport was recorded from identified microvessels in the lumbar dorsal horn of the spinal cord. COMPARISON WITH EXISTING METHOD(S): Current approaches have used histological and/or tracer based in-vivo assays alongside cell culture to determine endothelium integrity and/or function. These only provide a snapshot of the developing vasculopathy, restricting the understanding of physiological function or disease progression over time. CONCLUSIONS: These techniques allow for direct visualisation of cellular and/or mechanistic influences upon vascular function and integrity, which can be applied to rodent models including disease, transgenic and/or viral approaches. This combination of attributes allows for real time understanding of the function of the vascular network within the spinal cord.

3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography.

OBJECTIVE: Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the lumen and directly applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed. METHODS: Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained glycocalyx preparations. A tannic acid staining method and a novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method). RESULTS: 3D electron tomography reveals details of the architecture of the glycocalyx just above the endothelial cell layer. The LaDy GAGa method visually appears to show more complete coverage and more depth than the Tannic Acid staining method. CONCLUSION: The tomographic reconstructions show a potentially significant improvement in determining glycocalyx structure over standard transmission electron microscopy.