Methods for predicting characteristics of endothelial cell glycocalyx

Abstract

The present invention provides a method of predicting one or more characteristics of endothelial cell glycocalyx in a subject wherein the method comprises measuring the corresponding one or more characteristics of red blood cell glycocalyx from the subject. This is useful in the diagnosis and monitoring of diseases associated with endothelial cell damage.

Description

[0001] Methods for predicting characteristics of endothelial cell glycocalyx

[0002] This application claims priority from GB2410240.2 filed 12 July 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Field of the Invention

[0004] The present invention relates to methods of predicting characteristics of endothelial cell glycocalyx, and particularly, although not exclusively, to using these predictions to screen for, diagnose, and monitor disease progression or response to clinical intervention, complete clinical risk assessment or aid the management of human or animal disease.

[0005] Background

[0006] The endothelial glycocalyx is an interwoven network of proteoglycans and glycoproteins, as well as other endothelium-derived or plasma-derived molecules. It covers the apical surface of the vascular endothelium and is present on all blood vessels (Yilmaz et al., 2019). Functions of the endothelial cell glycocalyx (EnC Glx) include modulation of inflammatory cell and platelet adhesion to the endothelial surface, sensing fluid shear stress and acting as a mechanotransducer of these stresses, acting as a fluid and protein permeability barrier, and as a circulating cell “bumper” (Yilmaz et al., 2019).

[0007] Endothelial cell (EnC) damage contributes to multiple human diseases (Prober et al., 2007; Butler et al., 2020). One of the earliest manifestations of EnC damage is loss of the delicate sugar-dense glycocalyx (Glx) layer (Butler et al., 2020; Nieuwdorp et al., 2006; Satchell, 2012; Salmon and Satchell, 2012). In the kidney glomerulus, EnC Glx damage causes increased albuminuria - a hallmark of kidney disease and a strong predictor of cardiovascular mortality (Satchell., 2013). Glx damage has also been implicated in the pathogenesis of sepsis (Sullivan et al., 2021), preeclampsia (Carlberg et al., 2022; Weissgerber et al., 2019), thrombotic microangiopathy (Teoh et al., 2023; Bowen et al., 2023) and vascular disease (Butler et al., 2020) amongst other conditions.

[0008] Key building blocks are frequently found in all Glx including core proteoglycans including syndecans and glypicans, which carry glycosaminoglycan (GAG) side-chains such as heparan sulphate, and hyaluronan which is synthesised as long chains on the EnC surface (Weinbaum et al., 2007). Terminal sialic acid residues decorate multiple components of the Glx contributing to the negative charge of the Glx structure (Weinbaum et al., 2007).

[0009] To date, the detection of Glx damage for in vivo research in this field has relied upon perfusion fixation and electron microscopy techniques. However, Glx depth is influenced by perfusion and fixation artifacts, and this method cannot be used in human patients. Measurement of EnC in patients has therefore relied on the measurement of circulating shed Glx fragments or side stream dark field imaging techniques such as Glycocheck™ - a sublingual imaging system (Liew et al., 2021 ; Reitsma et al., 2007). Whilst these techniques have helped confirm that EnC Glx damage is frequently systemic, they lack practicality for widespread Glx surveillance in clinical practice (Eickhoff et al., 2020; Bol et al., 2022). Furthermore, Glycocheck™ requires the patient to avoid eating for 4 hours prior to testing, avoid exercise for 12 hours prior to testing, not consume alcohol within 24 hours prior testing, avoid caffeine on the day of their test and rest for 15 to 20 minutes prior to testing to reduce their stress level, which can be inconvenient for the patient. Glycocheck™ also requires a skilled and fully trained operator, bespoke equipment and takes a minimum of 10 minutes per patient.

[0010] Recently published papers demonstrate that confocal microscopy can be used to measure EnC Glx damage on ex vivo tissue (Gamez et al., 2024; Crompton et al., 2023). These novel techniques provided reproducible, accurate measures of EnC Glx and were able to predict EnC Glx function better than electron microscopy-based measures (Crompton et al., 2023). However, these techniques also lack practicality for widespread Glx surveillance in clinical practice and require invasive procedures to obtain the tissue samples.

[0011] The present invention has been devised in light of the above considerations.

[0012] Summary of the Invention

[0013] The present inventors have surprisingly found that the red blood cell glycocalyx (RBC Glx) is able to “mirror” the EnC Glx. Measuring characteristics of RBC Glx therefore provides an indirect measurement of the corresponding characteristics of EnC Glx. Using a series of experiments, the inventors have discovered that Glx components can be transferred between the two cell types during contact. Without being bound by any theory, it is believed that the observed “mirroring” effect is as a result of this transfer. This exchange of Glx components between EnCs and RBCs can be harnessed to monitor endothelial injury. The inventors have also developed a unique method for measuring RBC Glx - allowing a quick and a practical method of assessing EnC Glx health. This can aid in diagnosis and monitoring of conditions linked with changes in the EnC Glx, which is an early indicator of endothelial damage.

[0014] Accordingly, in a first aspect the present invention provides a method of predicting one or more characteristics of EnC Glx in a subject wherein the method comprises measuring the corresponding one or more characteristics of RBC Glx from the subject.

[0015] In a second aspect, the present invention provides a method of diagnosing, monitoring, prognosticating, or stratifying a disease linked with changes in one or more characteristics of EnC Glx in a subject by carrying out the method of the first aspect of the invention. The disease may be an acute disease or a chronic disease.

[0016] In some embodiments, monitoring a disease linked with changes in one or more characteristics of EnC Glx in a subject comprises measuring the corresponding one or more characteristics of RBC Glx from the subject before and after a therapeutic is administered to the subject. In some embodiments, the one or more characteristics of RBC Glx may also be measured during administration of the therapeutic to the subject. In some embodiments, measuring the one or more characteristics of RBC Glx comprises measuring RBC Glx depth, and an increase in RBC Glx depth after the therapeutic is administered compared to before the therapeutic was administered is indicative of improved disease status in the subject.

[0017] In some embodiments, the one or more characteristics of RBC Glx are measured from a blood sample obtained from the subject. The measurement may be carried out in vitro on the sample.

[0018] In some embodiments, the one or more characteristics comprises glycocalyx depth, charge, density, elasticity, and molecular and chemical composition.

[0019] In one embodiment, the molecular and chemical composition comprises at least one glycocalyx component selected from proteoglycans, glycosaminoglycans, glycoproteins, selectins, integrins, syndecans, intercellular adhesion molecules, glycolipids, glycophorins, glypicans glycans and sialic acids.

[0020] In one embodiment, the molecular and chemical composition comprises at least one glycocalyx component selected from CD44, perlecan (also known as HSPG2), syndecan 3, versican, decorin, biglycan, mimecan, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, hyaluronic acid, ITGA5, ITGB3, ITGA3, ICAM1 , platelet / endothelial cell adhesion molecules (PECAM-1), vascular cell adhesion molecule (VCAM-1), Tamm-Horsfall glycoprotein, basigin, glucose, galactose, mannose, N- acetylneuraminic acid, N-acetylglucosamine, Gal-P(1-3)-GalNAc, N-acetylgalactosamine, glucuronic acid, xylose and fucose.

[0021] In one embodiment, a reduction in RBC Glx depth (e.g. relative to the glx depth of a control red blood cell or relative to an earlier RBC Glx depth measurement from the same subject) is indicative of a reduction in EnC Glx depth of the subject, e.g. relative to the control subject or relative to the EnC Glx depth of the subject at the earlier time point.

[0022] In one embodiment, a reduction in RBC Glx depth is indicative of a disease linked with changes in one or more characteristics of EnC Glx.

[0023] In some embodiments, the EnC Glx is cardiac, renal, cerebral, pulmonary, cutaneous, hepatic, gut, arterial, systemic or sublingual EnC Glx.

[0024] In some embodiments, the one or more characteristics of RBC Glx is measured using a glycocalyx binding molecule, wherein the glycocalyx-binding molecule selectively binds to a RBC Glx component.

[0025] In some embodiments, the glycocalyx-binding molecule comprises a lectin, antibody, glycosaminoglycan- binding protein, aptamer, adsorbent protein, or small molecule.

[0026] The lectin may be any lectin that can bind to a RBC Glx component. For example, the lectin may be selected from wheat germ agglutinin, peanut agglutinin, Lycopersicon esculentum lectin, Ulex europaeus agglutinin I, Sambucus Nigra lectin, Wisteria floribunda lectin, Ricinus Communis Agglutinin I, Dolichos Biflorus Agglutinin, Dioclea bicolor lectin and / or Marasmius oreades agglutinin.

[0027] The antibody may be any that can bind to a RBC Glx component. For example, the glycocalyx component may be (but is not limited to) CD44, perlecan (also known as HSPG2), syndecan 3, versican, decorin, biglycan, mimecan, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, hyaluronic acid, ITGA5, ITGB3, ITGA3, ICAM1 , platelet / endothelial cell adhesion molecules (PECAM-1), vascular cell adhesion molecule (VCAM-1), Tamm-Horsfall glycoprotein, basigin, glucose, galactose, mannose, N- acetylneuraminic acid, N-acetylglucosamine, Gal-P(1-3)-GalNAc, N-acetylgalactosamine, glucuronic acid, xylose or fucose.

[0028] The glycosaminoglycan-binding protein may be any that can bind to a RBC Glx component. For example, hyaluronic acid-binding protein.

[0029] The small molecule may be any that can bind to a RBC Glx component. For example, phenyl boronic acids or alcian blue.

[0030] The aptamer may be any that can bind to a RBC Glx component. For example, the glycocalyx component may be, but is not limited toCD44, perlecan (also known as HSPG2), syndecan 3, versican, decorin, biglycan, mimecan, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, hyaluronic acid, ITGA5, ITGB3, ITGA3, ICAM1 , platelet / endothelial cell adhesion molecules (PECAM-1), vascular cell adhesion molecule (VCAM-1), Tamm-Horsfall glycoprotein, basigin, glucose, galactose, mannose, N- acetylneuraminic acid, N-acetylglucosamine, Gal-0(1-3)-GalNAc, N-acetylgalactosamine, glucuronic acid, xylose or fucose.

[0031] The glycocalyx-binding molecule may be conjugated to a detection label, optionally fluorescent label, bioluminescent label, colourimetric label, absorbent label, electrochemical label, quantum dot, nanoparticle, peptide tag, electrochemical label, enzyme, enzyme substrate or biotin. The glycocalyxbinding molecule may be immobilised on a solid substrate or hydrogel.

[0032] In some embodiments, measurement of the one or more characteristics of red blood cell glycocalyx comprises labelling the red blood cell glycocalyx with quantum dots or other electron dense probe and imaging using electron microscopy, labelling the red blood cell glycocalyx and measuring the labels using flow cytometry, labelling the red blood cell glycocalyx and imaging using confocal microscopy, mass spectrometry of the red blood cell glycocalyx composition, proteomic measurement of the red blood cell glycocalyx, or glycomic measurement of the red blood cell glycocalyx.

[0033] In some embodiments, measurement of the one or more characteristics of RBC Glx comprises assaying RBC aggregation, RBC mobility, and / or RBC morphology.

[0034] In some embodiments, the one or more characteristics of RBC Glx is measured by proteomics.

[0035] In some embodiments, measurement of the one or more characteristics of RBC Glx comprises labelling the RBC Glx with quantum dots and imaging using electron microscopy, labelling the red blood cell glycocalyx and measuring the labels using flow cytometry, or labelling the red blood cell glycocalyx and imaging using confocal microscopy.

[0036] In some embodiments, measuring RBC Glx depth comprises:

[0037] (i) labelling a red blood cell obtained from a subject with a labelled marker of RBC Glx and a labelled marker of red blood cell membrane; (ii) measuring signals generated by the labelled marker of RBC Glx and labelled marker of red blood cell membrane; and

[0038] (iii) calculating the distance between peak signals of the labelled marker of red blood cell glycocalyx and labelled marker of red blood cell membrane, wherein the distance is indicative of RBC Glx depth.

[0039] The distance between peak signals may be calculated at a plurality of points along the circumference of a red blood cell and a mean and / or median RBC Glx depth may be calculated for the red blood cell.

[0040] In some embodiments, the labelled marker of RBC Glx is a labelled lectin or labelled antibody.

[0041] In some embodiments, a red blood cell in which at least 50% of the cell membrane is not in contact with another cell contributes to the indication of RBC Glx depth.

[0042] In some embodiments, steps (ii) and / or (iii) are implemented by a computer.

[0043] The negative binding assay can be used to measure the one or more RBC characteristics. In this embodiment, measurement of the one or more characteristics of RBC Glx comprises:

[0044] (i) incubating a blood sample obtained from a subject with one or more labelled markers of RBC Glx;

[0045] (ii) separating the red blood cells to produce a substantially cell free portion; and

[0046] (iii) measuring the amount of residual labelled marker present in the substantially cell free portion, wherein the amount of residual labelled marker present in the substantially cell free portion is inversely proportional to the amount of labelled marker bound to the RBC Glx. By extension this is inversely proportional to the integrity of the EnC Glx.

[0047] In some embodiments, the labelled marker of RBC Glx may be specific for intact RBC Glx. For example, the marker may be wheat germ agglutinin.

[0048] In some embodiments, the labelled marker of RBC Glx is specific for damaged RBC Glx. For example, the marker may be peanut agglutinin or Wisteria floribunda lectin.

[0049] In some embodiments, the disease linked with changes in one or more characteristics of EnC Glx comprises a vascular disease. In some embodiments, the disease linked with changes in one or more characteristics of EnC Glx comprises a microvascular disease.

[0050] In some embodiments, the disease (in early, acute or later stages) linked with changes in in one or more characteristics of EnC Glx comprises a disease such as diabetes, early diabetes, heart failure, mitral valve disease, preeclampsia, ischaemic reperfusion injury, sepsis, delirium, dementia, cerebral oedema (post head injury or surgery), cancer, cancer metastasis, haemolytic uremic syndrome, compliment disorders, atherosclerosis, venous thrombosis, thin basement disease, acute kidney injury minimal change nephrotic syndrome, membranous nephropathy, focal segmental glomerulosclerosis, chronic kidney disease, acute respiratory distress syndrome (ARDS), IgA nephropathy, transplant rejection, radiation injury and COVID-19.

[0051] The diabetes may be diabetic retinopathy, diabetic nephropathy or diabetic vascular disease. In a further aspect, the present invention provides a device for carrying out the negative binding assay.

[0052] In a further aspect, the present invention provides a kit for carrying out the negative binding assay, wherein the kit comprises: (i) a receptacle for holding blood, wherein the receptacle comprises a labelled marker; and (ii) a device for measuring the labelled marker. In some embodiments, the labelled marker is a labelled glycocalyx binding molecule. For example, a labelled lectin. In some embodiments the label is a fluorescent label. In some embodiments, the device measures fluorescence. In some embodiments, the device is portable.

[0053] In a further aspect, the present invention provides use of a red blood cell sample from a subject as a biomarker for determining the status, e.g. dysregulation, of the EnC Glx of the subject.

[0054] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0055] Summary of the Figures

[0056] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0057] Figure 1. A. Alcian blue perfusion fixed rat glomerular EnC Glx and RBC Glx imaged by electron microscopy. Scale bar represents 200 nm. B. Biotinylated lectins were perfused into donated human placentae before perfusion fixing the specimen with glutaraldehyde. The lectin-biotin tag was then conjugated to quantum dots (Qdots) before imaging their distribution by transmission electron microscopy. Image shows maternal RBC within the placental circulation following perfusion with biotinylated Lycopersicon esculentum (LEL) lectin. Scale bar represents 1000 nm.

[0058] Figure 2. Lectin based measurement of Glx depth in human kidney biopsies from patients with thin basement membrane disease (TMD) and minimal change nephrotic syndrome (MCNS). All samples were stained with FITC-UEA1 lectin, R18 and DAPI for staining Glx, the cell membranes and nuclei, respectively. All scale bars represent 10 pm. A. A confocal image of a glomeruli taken from a patient with TMD illustrating dense lectin labelling within the glomerular capillary loops consistent with an intact EnC Glx, inset illustrates RBC surface lectin, again consistent with intact RBC Glx. B. A representative confocal image of a glomeruli from a patient with MCNS with minimal lectin staining, inset highlights the additional lack of RBC surface lectin. C. Analysis of glomerular EnC Glx thickness by patient and disease. Patients with MCNS had a significantly thinner EnC Glx compared to patients with TMD (p<0.0001). D. Linear regression of the RBC Glx thickness (measured on isolated RBC within capillary loops) and EnC Glx measurement taken from the same patient confirmed a significant relationship between RBC Glx thickness and glomerular EnC Glx (p=0.02), peri-tubular EnC Glx (p=0.03) and combined glomerular and peritubular EnC Glx (illustrated, p=<0.0001 , r2=0.9226). Figure 3. A. Confocal image of a FITC-LEL (to stain Glx) and R18 (to stain cell membrane) labelled RBC aligned parallel to a slide on a methanol fixed human blood smear. The illustrated line profile at 90 degrees to the membrane was used to generate an illustrative “peak to peak” measure of the RBC Glx shown in B. C. Artificial intelligence was trained to identify RBCs on blood smears and differentiate them from other cells such as white blood cells and platelets. D. Identified cells were then examined automatically by placing line profiles every 15 degrees around the circumference of each identified RBC. Provided the line profile generated a signal which passed the quality control step, the resulting “peak to peak” analysis was included in the calculations for that cell. If the profile did not pass the quality control steps, these profiles were excluded. The artificial intelligence model was able to mark which profiles are included and which were not on the cell. E. A representative population of RBC from a single blood sample. The “peak to peak” distribution is normally distributed. All scale bars represent 5 pm unless otherwise indicated. F. The artificial intelligence software can also provide secondary measures for every line profile generated.

[0059] Figure 4. A, B RBCs were labelled with FITC-LEL lectin and R18 and cells were exposed to 1 , 4, 16 or 64 Sigma milliunit / ml neuraminidase or no neuraminidase. “Peak to peak” measurements (A) or LEL lectin intensity measurements (B) were then carried out. Each dot represents a single RBC. RBC Glx structure appeared to collapse after 30m exposure to 16mu / ml neuraminidase. C, D. RBC exposed to neuraminidase for 10, 20, 30 or 60 minutes. Data shown are median area under the curve measures for LEL lectin (C) and WGA lectin (D).

[0060] Figure 5. A. Individual patient’s data generated by GlycoCheck ™ across various capillary diameters (bottom line 5-9 pm, middle line 10-19 pm, top line 20-25 pm) correlated with their measured RBC peak to peak (p2p). B. Combined PBR assessment across all vessel sizes corelated against RBC “peak to peak” measurements.

[0061] Figure 6. A. Rats were treated with vehicle or STZ at week 0. At week 4, they were treated with vehicle or spironolactone daily. B. RBCs were labelled with FITC-WGA lectin and R18 and “peak to peak” measurements to assess RBC Glx depth were carried out. Heart (left ventricle) capillary Glx was assessed using alcian blue perfusion fixation and electron microscopy. RBC Glx correlated with EnC Glx depth. Each point represents an individual rat. Square represents diabetes rats, triangles represent diabetes plus spironolactone rats, circles represent control rats. C. RBCs and kidney (glomerulus) capillary were labelled with FITC-WGA lectin and R18 and “peak to peak” measurements were carried out to assess RBC Glx depth and EnC Glx depth, respectively. Each point represents an individual rat. Square represents diabetes rats, triangles represent diabetes plus spironolactone rats, circles represent control rats.

[0062] Figure 7. A. Rats were treated with vehicle or STZ at week 0 to render rats diabetic. At week 4, they were treated with vehicle or spironolactone daily B. RBCs from samples taken at various time points were labelled with FITC-WGA lectin and R18 and “peak to peak” measurements to assess RBC Glx depth were carried out. RBC Glx was shown to be impaired 3 weeks post STZ (diabetes) (T-test comparison of two groups, n=10 control, 12 diabetic). However, Glx repair following the initiation of spironolactone could be detected after 2 weeks treatment and remained significant following 4 weeks of treatment (ANOVA, n=6 per group). P<0.05*, p<0.01**, p<0.005***, p<0.0001****. Square represents diabetes rats, triangles represent diabetes plus spironolactone rats, circles represent control rats.

[0063] Figure 8. Rat RBCs were labelled with FITC-WGA or FITC-MOA lectin and R18 to stain the Glx and cell membrane, respectively. “Peak to peak” measurements were carried out to assess RBC Glx depth. RBC Glx depth correlated with glomerular leakage of albumin. Square represents diabetes rats, triangles represent diabetes plus spironolactone rats, circles represent control rats.

[0064] Figure 9. A. Mice were treated with vehicle or STZ at week 0. At week 4, they were treated with vehicle or SB-3CT (an MMP inhibitor) daily. B. Mouse RBCs were labelled with FITC-LEL lectin and R18 to stain the Glx and cell membrane, respectively. Mouse cardiac capillary EnC Glx was labelled with SNA lectin and R18 to stain the Glx and cell membrane, respectively. “Peak to peak” measurements were carried out to assess Glx depth. C. Echo cardiography measures of cardiac function. E / A ratios of mice from the control, diabetes and diabetes plus SB-3CT group are plotted. The E / A ratios are a marker of the function of the left ventricle of the heart.

[0065] Figure 10. A. Dog RBCs were obtained from dogs in either the A, B1 or B2 stage of mitral valve disease. The RBCs were labelled with FITC-LEL lectin and R18 to stain the RBC Glx and cell membrane, respectively. “Peak to peak” measurements were carried out to assess Glx depth. B. Left ventricular internal dimension in diastole (LVIDd-N) was used to assess severity of dog mitral valve disease and plotted against the “peak to peak” measurements.

[0066] Figure 11. Mice were treated with control or LPS to induce sepsis. RBC Glx and cell membrane were labelled and “peak to peak” measurements carried out.

[0067] Figure 12. A. RBCs from patients enrolled in the GlycoSAVe trial were labelled with FITC-LEL lectin and R18 to stain the RBC Glx and cell membrane, respectively. “Peak to peak” measurements were carried out to assess Glx depth. Patients admitted to hospital with bacterial sepsis were compared to age matched controls. B. RBCs from patients enrolled in the GlycoSAVe trial were labelled with FITC-LEL lectin. Peak lectin intensity was measured. Patients admitted to hospital with bacterial sepsis were compared to age matched controls. C. RBCs from patients enrolled in the GlycoSAVe trial were labelled with FITC-LEL lectin. Each dot represents an RBC (median area under the Gaussian modelled lectin light ‘peak’). Patients admitted to hospital with bacterial sepsis were compared to age matched controls. D. RBCs from patients enrolled in the GlycoSAVe trial were labelled with FITC-LEL lectin and R18 to stain the RBC Glx and cell membrane, respectively. “Peak to peak” measurements were carried out to assess Glx depth and plotted against acute kidney injury stage.

[0068] Figure 13. RBCs from patients admitted to hospital with COVID-19 were labelled with FITC-WGA or FITC-LEL lectin and R18 to stain the RBC Glx and cell membrane. “Peak to peak” measurements were carried out to assess Glx depth.

[0069] Figure 14. RBCs were incubated with fluorescently labelled WGA and PNA lectin in solution. Various concentrations of heparanase were added to the RBCs to induce Glx damage. The RBCs were separated from the solution. Residual lectin present in the solution was measured. Squares represent residual WGA in solution and circles represent residual PNA in solution. Figure 15. RBCs were incubated with A. biotinylated LEL or B. biotinylated UEA. R18 was used to stain the RBC membrane The biotinylated lectins bound to the RBC Glx (green) external to the cell membrane

[0070] (red). “Peak to peak” measurements were carried out to assess Glx depth.

[0071] Figure 16. RBCs from A. healthy control or B. sepsis patients were incubated with FITC-peanut lectin and imaged using confocal microscopy. C. 3D image generated from a z-stack of confocal images of RBC taken from a patient with sepsis.

[0072] Figure 17. A. Antibody staining of ICAM-1 and PECAM-1 within the RBC Glx. B. RBCs from littermate control mice stained with 3G10 antibody to detect heparan sulphate. C. RBCs from endothelial-specific EXT1 knockdown mice stained with 3G10 antibody to detect heparan sulphate. These cells lacked heparan sulphate within their glycocalyx. D. RBCs from littermate control mice stained with an antibody which binds HSPG2. E. RBCs from endothelial-specific EXT1 knockdown mice stained with an antibody which binds HSPG2. These cells lacked HSPG2 within their Glx.

[0073] Figure 18 A. Electron micrographs of endothelial and RBC Glx stained with alcian blue. B. Alcian blue was added to fresh blood or stored blood in solution where it was allowed to bind to the cell surface. Following removal of the RBCs, the concentration of alcian blue left in the samples was measured and used as an indirect measure of alcian blue Glx binding. C. 3-Dansylaminophenylboronic acid (blue) bound to sialic acids within the RBCGIx. D. The 3-Dansylaminophenylboronic acid bound cells were also stained with R18 to detect the cell membrane (red). This confirmed that 3-Dansylaminophenylboronic acid was binding the RBC Glx. E. Measurement of peak signal locations on confocal images were used to detect changes in RBC Glx.

[0074] Figure 19. RBCs were incubated in media containing A. AlexaFluor488 (AF488)-tagged albumin, or B. AF488-tagged fibrinogen. Confocal imaging shows binding of these tagged proteins to the RBC Glx.

[0075] Figure 20 A. Confocal imaging of endothelial monolayers highlights universal ManNAz integration. B. High resolution confocal imaging of cells stained to detect ManNAz (green), cell membrane (red) and DNA (blue) confirm ManNAz labeled sialic acids are expressed on the endothelial cell surface external to the cell membrane. C. RBCs exposed to control endothelial cells for 24 hours show minimal background florescence. D. RBCs exposed to ManNAz labelled endothelial cells display the Click™ binding site on their surface following interaction suggesting transfer E. Quantification of individual RBC Click™ signal intensity at the membrane confirms eGlx - RBC sialic acid transfer (t-test p<0.0001 ) F. Endothelial cells exposed (4 hours) to RBCs previously interacted with control endothelial cells for 24 hours demonstrate no significant ‘click’ binding, as shown by lack of green fluorescence. G. Endothelial cells exposed (4 hours) to RBCs ‘loaded’ with ‘click’ labelled EnC Glx exhibit dense patches of click labelled glycocalyx following RBC removal, inset highlights transferred material (green) remains on the cell surface. H. Quantification of green ‘click’ label intensity on cultured endothelial monolayers after RBC contact confirmed significant transfer from ‘loaded’ RBC onto the endothelial surface (one sample t and Wilcoxon test (p<0.01 )) (scale bar 5pm (p<0.01 **, p<0.005 ***, p<0.0001 ****).

[0076] Figure 21. Endothelial cells were fed ManNAz to integrate the 'tag' into sialic acids which are bound to multiple surface proteins. RBCs were interacted with these endothelial cell monolayers, resulting in transfer of the tagged proteins into the RBC Glx. Following isolation of the RBC, Click chemistry was used to refine the proteins present within the RBCGIx that were originally generated by EnC. Displayed are selected proteins shown to transfer into the RBC Glx from EnCs. All proteins within the grey area were statistically significantly refined on the RBC compared to cells media and control cells.

[0077] Figure 22. A. RBCs were treated with neuraminidase (30mU / ml; 1 hour) and labelled with LEL lectin and R18. ‘Peak to peak’ measurements were carried out to assess Glx depth. Following neuraminidase treatment, LEL lectin ‘peak to peak’ thickness was significantly reduced on human RBCs. One hour interaction between depleted RBC and control endothelial cells had no measurable effect. In contrast, one hour interaction between depleted RBC and EXT1 over-expressing endothelial cells (EXT1 +) significantly restored the RBC Glx (ANOVA p<0.005). B. After 14 hours interaction significant recovery of the RBCGIx was seen but no significant residual effect from EXT1 over expression was detected. Circulating RBC in endothelial cell-conditioned media for 14 hours had no effect on the RBCGIx thickness. C. Representative 3D image of RBC labelled with LEL lectin following neuraminidase and one hour interaction with control endothelial cells. The inset displays a single z stack image of the same cells , and a 5pm scale bar. D. Representative 3D image of RBC labelled with LEL lectin (green) following neuraminidase and one hour interaction with endothelial cells over expressing EXT 1 . The inset displays single z stack image of the same cells and a 5pm scale bar. Both images C. and D. were taken whilst blinded using fixed settings. (n= RBC, 3 experimental repeats, Kruskal-Wallis analysis) (p<0.05 *, p<0.01 **, p<0.005 ***, p<0.0001 ****).

[0078] Figure 23. Blood smears were prepared from A. litter mate control mice, and B. mice with conditional endothelial specific deletion of EXT 1 . The cells were labelled identically with R18 and LEL lectin and were imaged blind. The left panels of A. and B. show a combined image of R18 and LEL. The right panels show an identical image but where only the green channel (to visualise LEL lectin) is displayed. The Images highlight visible differences in RBC Glx, with visible RBC Glx loss seen in EXT1 - mice. C. RBC surface LEL lectin intensity was significantly reduced in EXT1 knock down mice, (t-test p<0.005) D. Following conditional knock down of endothelial EXT 1 , mice had a significantly thinner RBC Glx compared to littermate controls (Kruskal-Wallis). n=individual mice, (p<0.05 *, p<0.01 **, p<0.005 ***, p<0.0001 ““).

[0079] Detailed Description of the Invention

[0080] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0081] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0082] RBC Glx as an indicator of EnC Glx

[0083] The present inventors surprisingly found that RBC Glx “mirrors” EnC Glx from the same subject and as such can be used as an indirect measurement of EnC Glx characteristics. In humans EnC Glx measurements can be currently estimated using GlycoCheck™. The inventors approach, using RBC Glx to monitor EnC Glx changes holds significant advantages over GlycoCheck™. GlycoCheck™ is reliant on the localised motion of RBC in the sublingual circulation, thus subtle changes in blood pressure and microvascular flow (after caffeine or food for example) may affect the readings. Trained operators are also needed and for the patients GlycoCheck™ is time consuming (Eickhoff et al., 2017). Another method currently used is measurement of shed Glx components. The use of shed Glx components in the serum is also indirect and likely to be relatively unreliable in chronic disease because they provide a composite measure influenced dynamically by the rate of production, shedding and clearance of individual Glx components.

[0084] Accordingly, the invention provides a method of predicting one or more characteristics of EnC Glx in a subject wherein the method comprises measuring the corresponding one or more characteristics of RBC Glx from the subject.

[0085] The importance of the EnC Glx in maintaining endothelial function is well established (Butler et al., 2020; Nieuwdorp et al., 2006; Teoh et al., 2023; Gamez et al., 2024; Padberg et al., 2014). However, the EnC Glx is frequently damaged in human disease. Assessing damage to EnC Glx via measurements of RBC Glx could therefore aid in diagnosing diseases linked with changes in EnC Glx characteristics. Damage to EnC Glx frequently occurs early in the disease course (Butler et al., 2020; Crompton et al., 2023). For example, in the case of diabetic animal models, EnC Glx damage is detectable before albuminuria develops suggesting that Glx damage pre-dates irreversible microvascular injury (Crompton et al., 2023). Targeted interventions to preserve the EnC Glx at this early stage have been shown to delay the onset of microvascular damage (Gamez et al., 2024; Crompton et al., 2023). As the move towards individualised medicine occurs, detecting early microvascular injury in the form of Glx loss or alteration could therefore identify the subset of patients that will benefit most from targeted intervention.

[0086] Measuring EnC Glx following a treatment is useful in assessing response to that treatment. For example, if the Glx damage appears to have improved compared to a previous measurement, this may indicate that the subject has responded well to treatment. If the Glx does not appear to have improved compared to a previous measurement, this may indicate that the subject has not responded well to treatment, and this may shape the choice of treatment going forward. Accordingly, provided herein is a method of diagnosing a disease or monitoring progression of a disease linked with changes in one or more characteristics of EnC Glx in a subject, wherein the method comprises measuring the corresponding one or more characteristics of RBC Glx from the subject.

[0087] Also provided is a method of diagnosing, monitoring, prognosticating, or stratifying a disease linked with changes in one or more characteristics of endothelial cell glycocalyx in a subject, wherein the method comprises measuring the corresponding one or more characteristics of RBC Glx from the subject.

[0088] Monitoring (progression of) a disease linked with changes in one or more characteristics of EnC Glx in a subject may comprise measuring the corresponding one or more characteristics of RBC Glx from the subject at several timepoints. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more time points. Changes in the RBC Glx, and therefore EnC Glx, may be indicative of presence, worsening or improvement of a disease.

[0089] Monitoring (progression of) a disease linked with changes in one or more characteristics of EnC Glx in a subject may comprise measuring the corresponding one or more characteristics of RBC Glx from the subject before and after a therapeutic is administered to the subject.

[0090] Changes in the RBC Glx, and therefore EnC Glx, after a therapeutic is administered can indicate whether the subject has responded to the therapeutic. For example, whether disease has progressed, regressed or stayed the same.

[0091] In one example, measuring the one or more characteristics of RBC Glx comprises measuring RBC Glx depth, and an increase in RBC Glx depth after the therapeutic is administered compared to before the therapeutic was administered is indicative of improved disease status in the subject.

[0092] In another example, measuring the one or more characteristics of RBC Glx comprises measuring RBC Glx depth, and a decrease in RBC Glx depth after the therapeutic is administered compared to before the therapeutic was administered is indicative of worsening disease status in the subject. This may indicate that the subject is not responsive to the therapeutic.

[0093] In another example, measuring the one or more characteristics of RBC Glx comprises measuring RBC Glx depth, and no change in RBC Glx depth after the therapeutic is administered compared to before the therapeutic was administered is indicative of unchanged disease status in the subject.

[0094] The one or more characteristics of RBC Glx may be measured from a blood sample obtained from the subject. The blood sample may be taken on the same day as the RBC Glx measurement is carried out. The blood sample may be taken on the previous day before RBC Glx measurement is carried out. The RBC Glx measurement may be carried out as soon as the blood is taken. For example, less than 10 minutes, less than 20 minutes, less than 30 minutes or less than an hour after the blood is taken. The blood may be taken about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours before the RBC Glx measurement is carried out. The blood may be taken about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 2 weeks before the RBC Glx measurement is carried out. For example, blood collected in Transfix™ filled vacutainers may be taken up to 2 weeks before the RBC Glx measurement is carried out. The term “subject” can mean any subject with endothelial cells and red blood cells lined with glycocalyx. Preferably, the subject is a mammal. For example, a rabbit, a mouse, a rat, a Guinea pig, a hamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey, an ape or a human. Thus, the methods, uses and compositions described in this document are applicable to both human and veterinary disease. In preferred embodiments, the subject is a human. The subject may be male or female and may have any blood type.

[0095] Any characteristic of Glx may be measured. For example, Glx depth (i.e thickness) may be measured. The terms “depth” and “thickness” are used interchangeably throughout this disclosure. Depth is the distance from the cell membrane to which the Glx is attached to the end of the Glx, which protrudes into the lumen of the blood vessel. Other characteristics of Glx include but are not limited to composition, density, elasticity, charge and qualitive changes in the biochemical repertoire.

[0096] In one embodiment, the one or more characteristics comprises Glx depth.

[0097] In one embodiment, a reduction in RBC Glx depth is indicative of a reduction in EnC Glx depth.

[0098] In one embodiment, a reduction in RBC Glx depth is indicative of a disease linked with changes in one or more characteristics of EnC Glx.

[0099] In one embodiment, the one or more characteristics comprises Glx composition. Alteration in RBC Glx composition i.e. the alteration in the component parts of the RBC Glx or their relative expression, is indicative of alteration of the composition of the EnC Glx. Alteration may be an increase or decrease in the component part, or an alteration in the ratio between different component parts. The alteration may be relative to the Glx composition of a control RBC or relative to an earlier RBC Glx composition measurement from the same subject.

[0100] Examples of EnC Glx component parts that may be determined by measuring the corresponding component part on RBC Glx include: Proteoglycans, examples including CD44, perlecan, syndecan 3, versican, decorin, biglycan, mimecan and glypicans, which carry glycosaminoglycan (GAG) side-chains such as heparan sulphate, and hyaluronan; glycosaminoglycans including heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid; glycoproteins including selectins, integrins (e.g. ITGA5, ITGB3 and ITGA3), intercellular adhesion molecules (e.g. ICAM-1), vascular cell adhesion molecules (VCAM-1 ), platelet / endothelial cell adhesion molecule (PECAM-1), Tamm-Horsfall glycoprotein and basigin; glycolipids (e.g. those containing a-linked fucose residues); glycophorins; and specific glycans including glucose (Glc), galactose (Gal), mannose (Man), N-acetylneuraminic acid (NeuAc), N-acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), Gal-0(1-3)-GalNAc, glucuronic acid (GlaA), xylose (Xyl) and fucose (Fuc) and sialic acids.

[0101] In one embodiment, the one or more characteristics comprises Glx density. Alteration in RBC Glx density i.e. the alteration in the number or expression of component parts within a volume measure, is indicative of an alteration in the density of the EnC Glx. Alteration may be an increase or decrease. In some embodiments a reduction in RBC Glx density is indicative of a reduction in EnC Glx density. In one embodiment, a reduction in RBC Glx density is indicative of a disease linked with changes in one or more characteristics of EnC Glx. The alteration may be relative to the Glx density of a control RBC or relative to an earlier RBC Glx density measurement from the same subject.

[0102] In one embodiment, the one or more characteristics comprises Glx elasticity. Alteration in RBC modulus of elasticity i.e. the alteration in the physical characteristics of the RBC Glx to resist deformation, is indicative of an alteration in the elasticity of EnC Glx. Alteration may be an increase or decrease. In some embodiments a reduction in RBC Glx elasticity is indicative of a reduction in EnC Glx elasticity. In one embodiment, a reduction in RBC Glx elasticity is indicative of a disease linked with changes in one or more characteristics of EnC Glx. In some embodiments an increase in RBC Glx elasticity is indicative of an increase in EnC Glx elasticity. In one embodiment, an increase in RBC Glx elasticity is indicative of a disease linked with changes in one or more characteristics of EnC Glx. The alteration may be relative to the Glx elasticity of a control red blood cell or relative to an earlier RBC Glx elasticity measurement from the same subject.

[0103] In one embodiment, the one or more characteristics comprises Glx charge. Alteration in the charge of the RBC Glx is indicative of an alteration in charge of the EnC Glx. Alteration may mean that the Glx becomes more negatively charged or more positively charged. In some embodiments a reduction in RBC Glx charge is indicative of a reduction in EnC Glx charge. In one embodiment, a reduction in RBC Glx charge is indicative of a disease linked with changes in one or more characteristics of EnC Glx. In some embodiments an increase in RBC Glx charge is indicative of an increase in EnC Glx charge. In one embodiment, an increase in RBC Glx charge is indicative of a disease linked with changes in one or more characteristics of EnC Glx. The alteration may be relative to the Glx charge of a control red blood cell or relative to an earlier RBC Glx charge measurement from the same subject.

[0104] The characteristic of the sample red blood cell may be compared to the same characteristic of a control red blood cell (e.g. the glycocalyx depth of a control red blood cell) in order to diagnose a subject as having a disease or to monitor disease progression. The characteristic (e.g. depth) may be compared to a threshold value in order to diagnose a subject as having a disease or monitor disease progression. The threshold value may be based on the characteristic (e.g. glycocalyx depth) of control red blood cells. The characteristic measurement (e.g. depth measurement) may be compared to a measurement^. g. depth measurement) taken at an earlier time point from the same subject, in order to diagnose a subject as having a disease or to monitor disease progression.

[0105] In one embodiment the characteristic may comprise the molecular composition of the RBC Glx.

[0106] A qualitative change in the Glx molecular composition may reflect changes in specific Glx moieties or components detected by moiety or component specific lectins or similar identifiers. A qualitative change may be absolute or relative to other components.

[0107] Blood vessels lined with EnC Glx supply all parts of the body with blood. As such the EnC Glx may be EnC Glx of any part of the body, for example any tissue or organ. The examples in this disclosure show that cardiac, renal and sublingual EnC Glx correlate with RBC Glx. Without wishing to be bound by any particular theory, the present inventors believe that the RBC Glx reflects changes at all sites. In some embodiments, the EnC Glx is cardiac, renal, cerebral, pulmonary, cutaneous, hepatic, gut, arterial lung, systemic or sublingual EnC Glx. In one embodiment, the EnC Glx is cardiac, renal or sublingual EnC Glx.

[0108] The RBC Glx is measured using one or more agents which bind to one or more RBC Glx components.

[0109] In some embodiments, the one or more characteristics of RBC Glx is measured using a glycocalyx binding molecule, wherein the glycocalyx-binding molecule selectively binds to a RBC Glx component.

[0110] In some embodiments, the glycocalyx-binding molecule comprises a lectin, antibody, glycosaminoglycan- binding protein, aptamer, adsorbent protein, or small molecule.

[0111] Components of the Glx are known in the art. They include proteoglycans including syndecans, perlecan (also known as HSPG2), versican, decorin, biglycan and mimecan and glypicans, which carry glycosaminoglycan (GAG) side-chains such as heparan sulphate, and hyaluronan (Weinbaum et al., 2007). Various sialic acid residues decorate multiple components of the glycocalyx. These are known in the art. Glx components also include glycosaminoglycans, glycoproteins, selectins, integrins, intercellular adhesion molecules, glycolipids, glycophorins, glypicans glycans and sialic acids.

[0112] Examples of RBC Glx components include CD44, perlecan, syndecan 3, versican, decorin, biglycan, mimecan, keratan sulfate, dermatan sulfate, hyaluronic acid, ITGA5, ITGB3, ITGA3, ICAM1 , platelet / endothelial cell adhesion molecules (PECAM-1 ), vascular cell adhesion molecule (VCAM-1 ), glucose, mannose, N-acetylneuraminic acid, N-acetylgalactosamine, glucuronic acid, xylose, fucose, heparan sulphate, basigin, chondroitin sulphate, N-acetyl-d-glucosamine, Gal-0(1-3)-GalNAc, 1 ,3-N- acetylglucosamine, glycophorin, Tamm-Horsfall glycoprotein, glycoproteins and glycolipids containing a- linked fucose residues, sialic acid attached to terminal galactose in a-2,6 or a-2,3 linkage, sialic acids linked to N-acetylgalactosamine or galactose and Gala1-3Gal containing carbohydrates.

[0113] In some embodiments, the one or more characteristics of RBC Glx are measured using a lectin which selectively binds to a RBC Glx component. The lectin may bind a proteoglycan, GAG, glycoprotein proteoglycan, selectin, integrin, intercellular adhesion molecule, glycolipid, glycophorin, glypican, glycan or sialic acid. The lectin may bind to any RBC Glx component known in the art. Examples are described herein, and include CD44, perlecan, syndecan 3, versican, decorin, biglycan, mimecan, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, hyaluronic acid, ITGA5, ITGB3, ITGA3, ICAM1 , platelet / endothelial cell adhesion molecules (PECAM-1 ), vascular cell adhesion molecule (VCAM-1 ), Tamm-Horsfall glycoprotein, basigin, glucose, galactose, mannose, N-acetylneuraminic acid, N- acetylglucosamine, Gal-0(1-3)-GalNAc, N-acetylgalactosamine, glucuronic acid, xylose and fucose.

[0114] Lectins are carbohydrate binding proteins. They come from plants, and in particular legumes such as beans, soybeans, and peanuts. Lectins and their carbohydrate specificities are known in the art (see for example Kobayashi et al., 2014).

[0115] The lectins may be from any family. For example, the lectin may be an R-type lectin, L-type lectin, P-type lectin, C-type lectin, l-type lectin, Galectin or microbial lectin. Examples of lectins include but are not limited to wheat germ agglutinin, peanut agglutinin, Lycopersicon esculentum lectin, Ulex europaeus agglutinin I, Sambucus Nigra lectin, Marasmius oreades agglutinin,

[0116] Wisteria floribunda lectin, Aleuria aurantia lectin, Bauhinia purpurea lectin, Concanavalin A, Datura Stramonium lectin, Dolichos biflorus lectin, Dioclea bicolor lectin, Erythrina cristagalli agglutinin, Euonymus-related lectin, Galanthus Nivalis lectin, Griffonia (Bandeiraea) simplicifolia I, Griffonia (Bandeiraea) simplicifolia I Isolectin B4, Griffonia (Bandeiraea) simplicifolia II, Hemagglutinins, Jacalins, Lens culinaris, Lotus tetragonolobus lectin, Maackia amurensis I, Maackia amurensis II, Maclura Pomifera, Narcissus pseudonarcissus, Phaseolus vulgaris erythroagglutinin, Phaseolus vulgaris leucoagglutinin, Pisum sativum agglutinin, Ricinus communis agglutinin I, Ricinus communis agglutinin II, Solanum tuberosum lectin, soybean lectin, Vicia villosa lectin and Succinylated Wheat Germ agglutinin

[0117] Further examples of lectins include, but are not limited to, wheat germ agglutinin, peanut agglutinin, Lycopersicon esculentum lectin, Ulex europaeus agglutinin I, Sambucus Nigra lectin, Marasmius oreades agglutinin, Wisteria floribunda lectin, Helix pomatia agglutinin, lotus seed lectin, Morus nigra agglutinin, Glechoma hederacea lectin, potato lectin, lentil lectin, galectin, cygrec, selectin, type C lectin, CD301 , Clerodendrum trichotomum lectin, Phytololacca American lectin, Helix pomatia agglutinin, CLEC17A, Sclerotium rolfsii lectin, Eucheuma serra agglutinin, Sambucus sieboldiana lectin, Glechoma hederacea lectin , Morus nigra agglutinin, Salvia sclarea lectin, Salvia bogotensis lectin, Salvia horminum lectin, Clerodendrum trichotomum lectin, Moluccella laevis lectin, Psophocarpus tetragonolobus, Abrus precatorius lectin, and Amar Anthus caudatus lectin.

[0118] In the disclosed methods to measure the RBC Glx characteristics, preferably lectins that bind uniformly within the glycocalyx and / or are not affected by blood group are used (Padberg et al., 2014; Bojar et al., 2022).

[0119] In preferred embodiments, the lectin is selected from wheat germ agglutinin (WGA), peanut agglutinin (PNA), Lycopersicon esculentum lectin (LEL), Ulex europaeus agglutinin I (UEA I), Sambucus Nigra lectin (SNA), Wisteria floribunda lectin (WFL), Ricinus Communis Agglutinin I (RCA), Dolichos Biflorus Agglutinin (DBA), Dioclea bicolor lectin (DBL) and / or Marasmius oreades agglutinin (MOA).

[0120] Wheat germ agglutinin binds to N-acetyl-d-glucosamine, peanut agglutinin binds to Gal-0(1-3)-GalNAc, Lycopersicon esculentum lectin binds to 1 ,3-N-acetylglucosamine, glycophorin, and Tamm-Horsfall glycoprotein, Ulex europaeus agglutinin I binds to many glycoproteins and glycolipids containing a-linked fucose residues, Sambucus Nigra lectin binds to sialic acid attached to terminal galactose in a-2,6 or a- 2,3 linkage as well as sialic acid linked to N-acetylgalactosamine or galactose, Wisteria floribunda lectin binds to carbohydrate structures terminating in N-acetylgalactosamine linked a or 0 to the 3 or 6 position of galactose, Ricinus Communis Agglutinin I binds galactose or N-acetylgalactosamine residues of membrane glycoconjugates, Dolichos Biflorus Agglutinin binds a-linked N-acetylgalactosamine and Marasmius oreades agglutinin binds to Gala1-3Gal containing carbohydrates.

[0121] The lectin used can also be based on the type of subject. For example, MOA, LEL and Maackia amurensis lectin (MAL) bind strongly to rodent Glx. WGA, UEA1 and LEL bind strongly to human Glx. LEL also binds strongly to dog and pig Glx. Conclusions regarding the health of the EnC Glx can also be made by choosing specific detection methods. For example, wheat germ agglutinin binds to terminal sialic acids exposed on intact RBC Glx. On the other hand, peanut lectin does not bind to intact residues within the RBC Glx. Peanut lectin’s binding sites are exposed by cleaving of the terminal sialic acid. These specificities can be exploited in the methods of the current invention to determine EnC Glx health.

[0122] In some embodiments, the one or more characteristics of RBC Glx are measured using an antibody which selectively binds to a RBC Glx component.

[0123] The antibody may bind any component of the RBC Glx. For example, the antibody may target a proteoglycan, GAG, glycoprotein, proteoglycan, selectin, integrin, intercellular adhesion molecule, glycolipid, glycophorin, glypican, glycan or sialic acids. The antibody may bind to any RBC Glx component known in the art. Non limiting examples of proteoglycans include, syndecan 3, CD44, perlecan, versican, decorin, biglycan, mimecan and glypicans. Non limiting examples of GAGs include heparan sulfate, dermatan sulfate, chondroitin sulfate, keratan sulfate and hyaluronic acid. Non limiting examples of glycoproteins include selectins, integrins (e.g. ITGA5, ITGB3 and ITGA3), intercellular adhesion molecules (ICAMs), vascular cell adhesion molecules (VCAM-1), platelet / endothelial cell adhesion molecule (PECAM-1), Tamm-Horsfall glycoprotein and basigin. Non-limiting examples of glycolipids include those containing a-linked fucose residues. Non-limiting examples of glycans include glucose (Glc), galactose (Gal), mannose (Man), N-acetylneuraminic acid (NeuAc), N-acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), Gal-0(1-3)-GalNAc, glucuronic acid (GlaA), xylose (Xyl) and fucose (Fuc).

[0124] As described in Example 10, the glycocalyx binding molecule may be an antibody which binds to ICAM-1 , PECAM-1 , heparan sulphate or perlecan (HSPG2). The antibody targeting heparan sulphate may bind one or more isoforms of heparan sulphate. The antibody targeting heparan sulphate may bind an epitope in the common “stump” region. An example of such an antibody is the 3G10 antibody (Amsbio 370260).

[0125] A labelled secondary antibody may be used to bind the primary antibody. The label may be any known to the skilled person, including those described herein.

[0126] In some embodiments, the one or more characteristics of RBC Glx are measured using a glycosaminoglycan-binding protein which selectively binds to a RBC Glx component (including but not limited to glycosaminoglycans such as heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid). Non limiting examples of glycosaminoglycan-binding proteins include hyaluronic acid (HA)-binding protein, heparan sulfate-binding proteins (antithrombin, FGF etc etc), albumin, supercharged fluorescent proteins and de novo designed peptides and proteins.

[0127] In some embodiments, the one or more characteristics of RBC Glx are measured using an aptamer which selectively binds to a RBC Glx component. For example, the aptamer may target a proteoglycan, GAG, glycoprotein, proteoglycan, selectin, integrin, intercellular adhesion molecule, glycolipid, glycophorin, glypican, glycan or sialic acids. The antibody may bind to any RBC Glx component known in the art. Non limiting examples of proteoglycans include, syndecan 3, CD44, perlecan, versican, decorin, biglycan, mimecan and glypicans. Non limiting examples of GAGs include heparan sulfate, dermatan sulfate, chondroitin sulfate, keratan sulfate and hyaluronic acid. Non limiting examples of glycoproteins include selectins, integrins (e.g. ITGA5, ITGB3 and ITGA3), intercellular adhesion molecules (ICAMs), vascular cell adhesion molecules (VCAM-1), platelet / endothelial cell adhesion molecule (PECAM-1 ), Tamm- Horsfall glycoprotein and basigin. Non-limiting examples of glycolipids include those containing a-linked fucose residues. Non-limiting examples of glycans include glucose (Glc), galactose (Gal), mannose (Man), N-acetylneuraminic acid (NeuAc), N-acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), Gal-P(1-3)-GalNAc, glucuronic acid (GlaA), xylose (Xyl) and fucose (Fuc).

[0128] In some embodiments, the one or more characteristics of RBC Glx are measured using an adsorbent protein. As described in example 10, the adsorbent protein may be albumin or fibrinogen. The protein may be labelled with any label known in the art, for example with any fluorescent label such as Alexa Fluor 488.

[0129] In some embodiments, the one or more characteristics of RBC Glx are measured using a small molecule which selectively binds to a RBC Glx component (e.g. including but not limited to those described herein). Non limiting examples of small molecules include phenylboronic acids (e.g. 3- Dansylaminophenylboronic acid), alcian blue, azure A and macrocycles.

[0130] The glycocalyx binding molecule (e.g. lectin or antibody) according to the present disclosure may be detectably labelled or, at least, capable of detection. For example, glycocalyx binding molecule (e.g. the lectin or antibody) may be labelled with a radioactive atom or a coloured molecule or a fluorescent molecule or a molecule which can be readily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferase, enzymes, enzyme substrates, and radiolabels. The glycocalyx binding molecule may be directly labelled with a detectable label or it may be indirectly labelled. For example, an unlabelled antibody may be detected by another antibody which is itself labelled.

[0131] Alternatively, the second antibody may have bound to it biotin and binding of labelled streptavidin to the biotin is used to indirectly label the first antibody. Non limiting examples include fluorescent labels (e.g. small molecule dyes such as fluorescein, fluorescein isothiocyanate (FITC) or rhodamine, or fluorescent proteins such as green fluorescent protein (GFP), mCHERRY, yellow fluorescent protein (YFP) or mRUBY), luminescent labels (e.g. luciferin), colorimetric labels, biotin, quantum dots, nanoparticles (e.g. Au), peptide tags (e.g. his-tags, SpyTags, flag tags), electrochemical probes (e.g. ferrocene), enzymes (e.g., horse radish peroxidase), and enzyme substrates (e.g. peroxidase substrates). In some embodiments the glycocalyx binding molecule (e.g. lectin or antibody) is conjugated to a detection label.

[0132] In some embodiments, the glycocalyx binding molecule is directly labelled. For example, the lectin may be directly fluorescently labelled. Examples include fluorescently labelled peanut lectin or fluorescently labelled LEL lectin. The fluorescent label may be any fluorescent label known to the skilled person. For example, FITC.

[0133] In some embodiments, the glycocalyx binding molecule comprises a biotinylated lectin. Any of the lectins described herein may be biotinylated. Examples include biotinylated LEL lectin or biotinylated UEA. The biotinylated lectin may be used in combination with fluorescently labelled streptavidin, for example streptavidin-Alexa Flour 488. Methods of measuring RBC Glx

[0134] Any suitable method can be used to measure the one or more characteristics of RBC Glx, and therefore EnC Glx. Conclusions regarding the health of the Glx can also be made by choosing specific detection methods. For example, some lectins are specific for intact RBC Glx. Damage to the Glx can result in cleavage of terminal sialic acid residues, exposing previously hidden binding sites for other lectins.

[0135] Lectins specific for intact RBC Glx include wheat germ agglutinin, Lycopersicon esculentum lectin, Ulex europaeus agglutinin I, Sambucus Nigra lectin, Marasmius oreades agglutinin, Aleuria aurantia lectin, Bauhinia purpurea lectin, Concanavalin A, Datura Stramonium lectin, Dolichos biflorus lectin, Erythrina cristagalli agglutinin, Euonymus-related lectin, Galanthus Nivalis lectin, Griffonia (Bandeiraea) simplicifolia I, Griffonia (Bandeiraea) simplicifolia I Isolectin B4, Griffonia (Bandeiraea) simplicifolia II, Hemagglutinins, Jacalins, Lens culinaris, Lotus tetragonolobus lectin, Maackia amurensis I, Maackia amurensis II, Maclura Pomifera, Narcissus pseudonarcissus, Phaseolus vulgaris erythroagglutinin, Phaseolus vulgaris leucoagglutinin, Pisum sativum agglutinin, Ricinus communis agglutinin I, Ricinus communis agglutinin II, Solanum tuberosum lectin, soybean lectin, Vicia villosa lectin and Succinylated Wheat Germ agglutinin.

[0136] Lectins specific for damaged RBC Glx include peanut agglutinin and Wisteria floribunda lectin..

[0137] Characteristics of RBC Glx can also be measured using label free techniques. For example, by measuring red blood cell agglutination, red blood cell morphology or red blood cell mobility. Red blood cell mobility may be measured through a charged substrate under flow, through a microcapillary or through a charged substrate by cell electrophoresis. Further techniques include proteomics, glycomics electron microscopy, atomic force microscopy, mass spectrometry, high performance liquid chromatography (HPLC) or tracer exclusion using dextran or other tracers.

[0138] In some embodiments, measurement of the one or more characteristics of RBC Glx may comprise using electron microscopy, confocal microscopy or flow cytometry.

[0139] In some embodiments, measurement of the one or more characteristics of RBC Glx may comprise labelling the RBC Glx with quantum dots, or other electron-dense labels, and imaging using electron microscopy, labelling the RBC Glx and measuring the labels using flow cytometry, or RBC Glx and imaging using confocal microscopy.

[0140] In some embodiments, measurement of the one or more characteristics of red blood cell glycocalyx comprises mass spectrometry of the red blood cell glycocalyx composition, proteomic measurement of the red blood cell glycocalyx, or glycomic measurement of the red blood cell glycocalyx.

[0141] The inventors have developed several assays for accurate measurement of RBC Glx characteristics.

[0142] In one embodiment, measuring one or more characteristics of RBC Glx comprises:

[0143] (i) labelling a red blood cell obtained from a subject with a labelled marker of RBC Glx; and

[0144] (ii) measuring a signal generated by the labelled marker of RBC Glx. The red blood cell may have been obtained from the subject by standard methods. The red blood cells may be “smeared” on a slide to form a monolayer for subsequent fixing and labelling. The labelled marker may be any glycocalyx binding molecule (e.g. a lectin or antibody). The term “labelled” is used to refer to a “detection label” as described earlier in this disclosure. In some embodiments, the labelled marker of RBC Glx is specific for intact RBC Glx. In some embodiments, the labelled marker of RBC Glx is specific for damaged red blood cell glycocalyx.

[0145] The labelled cells may be detected using methods known in the art e.g. confocal microscopy. Signals generated from the labelled markers may then be measured. To enable quick, accurate and reproducible measurement, this measurement may be implemented by a computer. The signal may represent the total labelled marker binding e.g. total lectin binding.

[0146] The inventors have developed a programme to automate measurement of signals from labelled RBC Glx. Here, the software is able to detect the labelled cell and measures the signal intensity along a line 90 degrees to the cell membrane, a so-called region of interest (“ROI”). Several measurements may be made in the same way by measuring signal intensity of several regions of interest along the membrane and a mean and / or median RBC Glx can be calculated for the red blood cell. In some embodiments, signals are measured every 15 degrees along the membrane.

[0147] The software is able to exclude non-red blood cells such as white blood cells or platelets which would also be present in the blood sample. The software has also been programmed with inclusion criteria. For example, only red blood cell in which at least 50% of the membrane is not in contact with another cell may contribute to final measured signal.

[0148] In particular, the inventors developed a “peak to peak” method for measuring RBC Glx depth.

[0149] In some embodiments, measuring RBC Glx depth comprises:

[0150] (i) labelling a red blood cell obtained from a subject with a labelled marker of RBC Glx and a labelled marker of red blood cell membrane;

[0151] (ii) measuring signals generated by the labelled marker of RBC Glx and labelled marker of red blood cell membrane; and

[0152] (iii) calculating the distance between peak signals of the labelled marker of RBC Glx and labelled marker of red blood cell membrane, wherein the distance is indicative of red blood cell glycocalyx depth.

[0153] The red blood cell may have been obtained from the subject by standard methods. The red blood cells may be “smeared” on a slide to form a monolayer for subsequent fixing and labelling. In this method, both the RBC Glx and the RBC membrane are labelled. The labelled marker of RBC Glx may be a labelled antibody or lectin. The labelled marker of RBC membrane may be any marker of RBC membrane known in the art, for example Octadecyl rhodamine B chloride (R18). In this way, the distance between the peak signals can be measured and used to calculate the depth of the RBC Glx.

[0154] In some embodiments, the distance between peak signals is calculated at a plurality of points along the circumference of a red blood cell and a mean and / or median RBC Glx is calculated for the red blood cell. In some embodiments, the labelled marker of RBC Glx is a labelled glycocalyx binding molecule (e.g. a lectin or antibody).

[0155] In some embodiments, a red blood cell in which at least 50% of the cell membrane is not in contact with another cell contributes to the indication of RBC Glx depth.

[0156] In some embodiments, the labelled marker of RBC Glx is specific for intact RBC Glx.

[0157] Following step (i) the labelled markers may be detected using methods known in the art e.g. confocal microscopy. Signals generated from the labelled markers may then be measured.

[0158] In some embodiments, step (ii) and / or (iii) may be implemented by a computer.

[0159] As described above, the inventors have developed a programme to automate measuring of signals from labelled RBC Glx. Here, the software is able to detect the labelled cell and measures the signal intensity of both the labelled Glx and labelled cell membrane along a line 90 degrees to the cell membrane, a so called region of interest. Several measurements may be made in the same way by measuring signal intensity of several regions of interest along the membrane. In some embodiments, signals are measured every 15 degrees along the membrane. The software measures both the Glx and membrane light signals and applies Gaussian curves to the detection profiles. The distance between the two peaks of the two curves is then calculated and used as an indication of RBC Glx depth, and therefore EnC Glx depth.

[0160] The software is able to exclude non-red blood cells such as white blood cells or platelets which would also be present in the blood sample. The software has also been programmed with inclusion criteria. For example, only red blood cell in which at least 50% of the membrane is not in contact with another cell may contribute to the indication of RBC Glx depth.

[0161] Another assay developed by the inventors, termed the “negative binding assay” is described. The terms “negative binding assay” and “reverse binding assay” are used interchangeably throughout this disclosure.

[0162] Here, measuring one or more characteristics of RBC Glx comprises:

[0163] (i) incubating a blood sample obtained from a subject with one or more labelled markers of RBC Glx;

[0164] (ii) separating the red blood cells to produce a substantially cell free portion; and

[0165] (iii) measuring the amount of residual labelled marker present in the substantially cell free portion, wherein the amount of residual labelled marker present in the substantially cell free portion is inversely proportional to the amount of labelled marker bound to the red blood cell glycocalyx.

[0166] The blood sample may be mixed with a solution containing labelled marker(s) e.g. labelled glycocalyx binding molecules such as lectins, antibodies, small molecules, aptamers, and GAG-binding proteins. The labelled markers are allowed to bind to the RBC Glx. Following incubation for a period of time, for example 20 minutes, the red blood cells are separated to produce a substantially cell free portion. This can be done for example, by spinning the cells down to form a cell pellet. In some cases, no cells will remain in the cell free portion. In some cases, trace cells may still be present in the cell free portion. As such, the term “substantially cell free” refers to the supernatant portion which is obtained following standard methods of separation.

[0167] The residual labelled marker in the substantially cell free portion can be measured using standard techniques. For example, the fluorescent signal(s) could be detected using a plate reader. The amount of marker present in the supernatant is inversely proportional to the amount bound to the RBC Glx. The amount of binding of the marker to RBC Glx indicates the status of RBC Glx, and therefore the EnC Glx.

[0168] In some embodiments, the labelled marker of RBC Glx is specific for intact RBC Glx.

[0169] In some embodiments, the labelled marker of RBC Glx is specific for damaged RBC Glx.

[0170] The one or more characteristics of RBC Glx may be any characteristic, including those specified in this disclosure (e.g. depth, density, charge elasticity and composition).

[0171] In some embodiments the negative binding assay may provide results in less than 2 hours, in less than one hour or more preferably less than 30 minutes.

[0172] In a further aspect, the present invention provides a device for carrying out the negative binding assay.

[0173] The negative binding assay comprises measuring one or more characteristics of RBC Glx comprising:

[0174] (i) incubating a blood sample obtained from a subject with one or more labelled markers of RBC Glx;

[0175] (ii) separating the red blood cells to produce a substantially cell free portion; and

[0176] (iii) measuring the amount of residual labelled marker present in the substantially cell free portion, wherein the amount of residual labelled marker present in the substantially cell free portion is inversely proportional to the amount of labelled marker bound to the red blood cell glycocalyx.

[0177] The blood sample may be collected into a custom tube with labelled glycocalyx binding molecule present. The sample may then be left to stand allowing the glycocalyx binding molecule to bind to saturation on the RBC surface. RBCs are allowed to sediment to the base of the tube and the concentration of residual labelled glycocalyx binding molecule in solution (i.e. the substantially cell free portion” may be analysed.

[0178] In preferred embodiments, the glycocalyx binding molecule is a lectin. In preferred embodiments, the label is a fluorescent label. In preferred embodiments, the apparatus measures fluorescence. In some embodiments, the apparatus is portable.

[0179] In a further aspect, the present invention provides a kit for carrying out the negative binding assay, wherein the kit comprises: (i) a receptacle for holding blood, wherein the receptacle comprises a labelled marker; and (ii) a device for measuring the labelled marker. In some embodiments, the labelled marker is a labelled glycocalyx binding molecule. The glycocalyx binding molecule may be any of the glycocalyx binding molecules that are disclosed herein in relation to any of the aspects or embodiments described. For example, the glycocalyx binding molecule may be a labelled lectin. In some embodiments the label is a fluorescent label. In some embodiments, the device measures fluorescence. In some embodiments, the kit is a portable kit. In some embodiments, the device is a portable device. In some embodiments, the device is a bench-top device. In some embodiments the apparatus may provide results in less than 2 hours, in less than one hour or more preferably less than 30 minutes. “Results” refers to signal measurements e.g. fluorescence.

[0180] Medical conditions

[0181] Diseases linked to endothelial cell damage are known (Prober et al., 2007; Butler et al., 2020).

[0182] The disease referred to in the methods disclosed herein may be any disease linked with damage to the vasculature. Since damage to the vascular system begins with damage to the EnC Glx, the disease may be any disease linked with changes in EnC Glx characteristics.

[0183] In some embodiments, the disease linked with changes in one or more characteristics of endothelial cell glycocalyx comprises a vascular disease. In some embodiments, the disease linked with changes in one or more characteristics of endothelial cell glycocalyx comprises a microvascular disease.

[0184] In some embodiments, the disease linked with changes in in one or more characteristics of endothelial cell glycocalyx comprises a disease selected from diabetes, early diabetes, heart failure, mitral valve disease, preeclampsia, ischaemic reperfusion injury, sepsis, delirium, dementia, cerebral oedema (post head injury or surgery), cancer, cancer metastasis, haemolytic uremic syndrome, compliment disorders, atherosclerosis, venous thrombosis, thin basement disease, acute kidney injury, minimal change nephrotic syndrome, membranous nephropathy, focal segmental glomerulosclerosis, chronic kidney disease, acute respiratory distress syndrome (ARDS), IgA nephropathy, transplant rejection, radiation injury and COVID-19.

[0185] The diabetes may be diabetic retinopathy, diabetic nephropathy or diabetic vascular disease.

[0186] The disease may be in its early, acute or later stages.

[0187] Any of the glycocalyx binding molecules may be used to measure RBC EnC characteristics for detection of changes linked to the above-mentioned diseases. For example, peanut lectin may be used to detect sepsis. As described in example 10, the peanut lectin may be labelled directly with a fluorophore.

[0188] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0189] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0190] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0191] Examples

[0192] EXAMPLE 1

[0193] RBC have a Glx

[0194] Endothelial cell (EnC) damage contributes to multiple human diseases (Pober et al., 2007; Butler et al., 2020). The ability to monitor EnC health is therefore pivotal in allowing diagnosis and early interventions to treat conditions associated with EnC damage.

[0195] Previously the only way to directly measure EnC Glx depth was by perfusion-fixing tissue and electron microscopy. As a result, human data has been limited to derived measurements of Glx health. For example, shed fragment concentrations and Glycocheck™. Glycocheck™ analyses the sublingual microvasculature, deriving a measure of EnC Glx depth from oscillations in the red blood cell (RBC) column width. However, this procedure lacks sensitivity, and is time consuming, requires a skilled operator limiting its utility to the academic, research or speculative domains and hindering research development in this area.

[0196] The inventors set out to find improved methods of measuring EnC Glx health.

[0197] Figure 1 shows a transmission electron microscopy (TEM) image of perfusion-fixed Alcian blue (an electron dense dye which binds to the glycocalyx) labelled rat glomeruli. Using this method, it was observed that RBCs had a negatively charged Glx that looked comparable to the adjacent EnC Glx (Figure 1a). However, the frequency of finding intact, perfusion fixed RBC was very low and thus novel approaches were sought to explore this relationship further.

[0198] In this regard, biotinylated lectins were perfused into donated human placentae before perfusion fixing the specimen with glutaraldehyde. The lectin-biotin tag was then conjugated to quantum dots (Qdots) before imaging their distribution by TEM. Figure 1 B illustrates maternal RBC within the placental circulation following perfusion with biotinylated Lycopersicon esculentum (LEL) lectin. Qdots confirmed that lectins known to bind to the EnCGIx bound to the human RBC surface and distributed uniformly within the RBCGIx structure.

[0199] Further techniques were then developed to image Glx using light microscopy and lectins. These techniques were validated using Glx measurements made using electron microscopy.

[0200] Using confocal microscopy and fluorescently labelled lectins, reliable measurements of the EnC Glx thickness on renal biopsy specimens from patients biopsied due to haematuria and subsequently found to have thin basement membrane disease (TMD) (Figure 2A) and from patients with minimal change nephrotic syndrome (MCNS) (Figure 2B) were carried out. These experiments confirmed that EnC Glx damage occurs in human renal disease (Figure 20).

[0201] Whilst carrying out this work, it was surprisingly observed that entrapped RBC within the tissue sections had a visible Glx which appeared to mirror the thickness of the EnC Glx (Figure 2A and B insets and Figure 2D).

[0202] EXAMPLE 2

[0203] Development of blood smear imaging protocol

[0204] To investigate the changes occurring on the RBC surface further a blood smear-based imaging protocol was developed. This novel technique allowed measurement of the relative positions of the RBC Glx and cell membrane around RBCs circumference using high resolution confocal microscopy.

[0205] A blood sample was smeared onto a glass slide to make a monolayer of cells and fixed for imaging. Figure 3A is a representative confocal image of a single RBC optically sectioned using through its axis. The FITC conjugated LEL lectin (green) can be seen to bind to the exterior surface of the Octadecyl rhodamine B chloride (R18) (red)-labelled cell membrane. A single illustrative line profile is shown placed at 90 degrees to the cell membrane. The relative intensities of the light detected along this region of interest (ROI) are displayed (Figure 3B). The presence of the lectin peak exterior to the R18 peak confirmed RBC Glx labelling. The distance between the peak lectin and R18 signals is referred to as the ‘peak to peak’ measurement. On EnC this measure has been extensively validated and shown to provide a highly reliable index of Glx depth that is superior to TEM imaging in predicting deficits in EnC Glx function (Crompton et al., 2020).

[0206] To facilitate a high throughput analysis workflow, an automated, artificial intelligence assisted software package was developed to analyse RBC Glx changes on blood smears. This software identifies all RBC (Figure 3C) that meet preselected inclusion criteria whilst ignoring platelets and white blood cells, places ROIs (every 15 degrees) around their circumference (figure 3D), measures the light signals and applies Gaussian curves to the detected profiles. A median ‘peak to peak’ measure is then produced from the Gaussian curves. These curves ‘smooth’ the light signal to reduce the influence of single pixel noise, for each profile included. Natural variation in the RBC Glx thickness was seen across the RBC population resulting in a normal distribution (Figure 3E). However, no significant variations were seen between a sample’s mean and median RBC Glx ‘peak to peak’ values provided >50 RBC were included in the analysis.

[0207] Where an adjacent RBC was too close (or non-specific signal is detected) the profiles were excluded. However, RBC can be identified and analysed even on a suboptimal blood smear (i.e. when RBCs remain in contact). The software can identify and analyze only the appropriate segments of RBC membrane where there is no contact with adjacent cells. By default, >50% of the membrane must be analysed for the cell to be included in the final value. Each cell and line profile is numbered so that raw data can be traced.

[0208] As well as “peak to peak” measurements, the analysis software was also able to measure total lectin binding (area under the curve), peak signal, half width at half maximum (interior and exterior signals) and full width at half maximum (Figure 3F).

[0209] To confirm sensitivity of the technology, experiments were carried out in the presence of neuraminidase, which cleaves sialic acids from RBC membranes. The RBC Glx ‘peak to peak’ measure, using LEL lectin and R18, was able to detect RBC Glx changes induced by 30 minutes exposure to very low-level enzyme concentrations (1 Sigma milliunit / ml neuraminidase) (Figure 4A). Data suggest collapse of the RBC Glx structure after 30m exposure to 16mu / ml.

[0210] The custom analysis software was also able to provide a measure of the peak LEL lectin binding density within the RBC Glx. Neuraminidase rapidly reduced the number of LEL binding sites even at the lowest dose (Figure 4B).

[0211] When RBCs were exposed to neuraminidase for varying durations, data confirmed damage induced by neuraminidase can be detected and the reduction in sialic acids (WGA binding site) seen after 60 minutes sample storage (room temperature, lithium heparin vacutainer) was highlighted (Figure 4C and D).

[0212] EXAMPLE 3

[0213] To confirm and further investigate the initial surprising observation that RBC Glx “mirrors” EnC Glx, several experiments were carried out.

[0214] Changes in RBC Glx mirror changes in EnC Glx in pregnant women.

[0215] GlycoCheck™ studies the movement of RBC as they ‘bounce’ into the EnC Glx to produce a measure of EnC Glx depth (PBR). The process takes 15 minutes in a fasted patient that has avoided caffeine for >6h. The PBR increases as the glycocalyx depth decreases. The PBR measurement is currently one of the most widely used research tools for assessing EnC Glx integrity currently.

[0216] RBC Glx from healthy pregnant women with and without diabetes were measured using the newly developed “peak to peak” method and compared to a measure of sublingual EnC Glx obtained using GlycoCheck™. Figure 5A shows that individual patient’s data generated by GlycoCheck ™ across various capillary diameters corelated with their measured RBC peak to peak (p2p). Significant linear correlations were confirmed across all vessel size ranges. Figure 5B shows combined PBR assessment across all vessel sizes corelated against RBC peak to peak measurements.

[0217] During this work, it was found that very careful sample handling, storage and processing was needed to ensure the RBC Glx was optimally preserved. Samples collected into lithium heparin (LiH) vacutainers saw significant RBC Glx degradation beyond 4 hours storage at 4 degrees centigrade. To facilitate subsequent clinical studies (where delays in processing blood samples were unavoidable) the use of Transfix™ filled vacutainers for sample collection and storage was developed. These tubes contain a fixative which stabilises the RBCGIx at the point of collection, facilitating stable storage at 4 degrees centigrade for up to 2 weeks.

[0218] EXAMPLE 4

[0219] Changes in RBC Glx mirror changes in EnC Glx in a diabetic rat model

[0220] A rat model of diabetes was used to test if the RBC Glx continues to mirror EnC Glx in disease. Blood was sampled from vehicle treated control Wistar rats, diabetic rats (type 1 diabetes induced 8 weeks previously with STZ) and diabetic rats treated with daily intraperitoneal spironolactone for 4 weeks (Figure 6A). STZ damages the pancreas. Spironolactone is a drug used clinically in kidney and heart disease that has been shown to protect the EnC Glx (Crompton et al., 2023).

[0221] Peripherally sampled RBC Glx depth measured using WGA lectin and R18 ‘peak to peak’ analysis corelated significantly (linear regression) with heart (left ventricle) capillary glycocalyx depth assessed using alcian blue perfusion fixation and electron microscopy (Figure 6B).

[0222] Peripherally sampled RBC Glx depth also corelated significantly with kidney (glomerulus) capillary glycocalyx depth (Figure 6C). Both Glx were measured using WGA lectin and R18 ‘peak to peak’ analysis.

[0223] The RBC Glx alters in response to therapeutics targeting EnC Glx repair in rats.

[0224] 4 blood samples were taken from rats’ peripheral veins during the 8-week study. Samples were taken at baseline (before diabetes was induced) and before and after the initiation of spironolactone or vehicle (Figure 7A).

[0225] Serial blood sampling confirmed that diabetic rats had significant RBC Glx damage by week 4 of the study, measured using the peak to peak method (with WGA lectin and R18). After 2 weeks spironolactone treated rats’ RBC Glx significantly increased in depth. Protection of the RBC Glx in diabetes persisted to the experimental end point at 8 weeks (Figure 7B).

[0226] This new data suggests that the RBC Glx measures could be used to monitor the effectiveness of therapies targeting EnC Glx restoration.

[0227] The RBC Glx depth predicts glomerular function Work from the inventor’s laboratory (Crompton et al., 2023) showed that the depth of the EnC Glx measured “peak to peak” using MOA or WGA lectin was able to predict kidney function, determined by albumin leakage across the filtration barrier of glomeruli in an ex vivo assay. This assay provides a highly sensitive measure of the functional integrity of the glomerular filtration barrier, including the Glx on the surface of the fenestrated glomerular EnC, free from confounding by haemodynamic changes or alterations in renal tubular albumin handling (Desideri et al., 2018). RBC collected during the same study 3 days before the experimental end points were analysed to produce “peak to peak” measures of RBC Glx thickness. The thickness of the RBC Glx predicted the glomerular leakage of albumin with significant liner correlations seen between Ps’alb (cm / s) and RBC Glx (peak to peak) in two experimental series using two different RBC lectins (WGA and MOA) (Figure 8).

[0228] EXAMPLE 5

[0229] RBC Glx depth corelates with mouse heart capillary endothelial qlycocalyx depth in mice

[0230] A mouse model of diabetes was used to further test if the RBC Glx continues to mirror EnC Glx in disease. Blood was sampled from vehicle treated control mice, diabetic mice (type 1 diabetes induced 8 weeks previously with STZ) and diabetic mice treated with daily MMP inhibitor, SB-3CT, for 4 weeks (Figure 9A).

[0231] Results showed that RBC Glx was significantly depleted in diabetic mice compared to controls at the study endpoint (p=0.0006). Significant recovery of the RBC Glx was seen in mice treated with the MMP inhibitor, SB-3CT (p=0.0009). The RBC Glx again corelated with cardiac capillary EnGIx depth, confirming the RBC provides a reliable measure of EnGIx changes in mouse models (Figure 9B). Furthermore, the EnC Glx depth predicted echo cardiography measures of cardiac function, suggesting that Glx damage was contributing to cardiac dysfunction (Figure 9C).

[0232] Therefore, measurement of RBC Glx depth is a reliable measure of EnC Glx depth, which in turn can predict cardiac function.

[0233] EXAMPLE 6

[0234] RBC Glx can predict mitral valve disease stage in dogs

[0235] Some dog breeds develop mitral valve disease spontaneously. Dogs are currently categorized according to stages A-D. Dogs characterised as stage A are predisposed to mitral valve disease. Dogs in stage B1 do not have evidence of cardiomegaly (heart enlargement) but do have evidence of valve disease. Current vet practice is to screen at risk dogs with ultrasound. Cardiomegaly resulting from the leaking mitral valve moves dogs into stage B2. At this stage, treatment with pimobendane in indicated. It was discovered that dog RBC Glx is significantly altered between dogs in the B1 and B2 stages (Figure 10A). These data suggest the RBC Glx could be applied in practice to help decide when to initiate therapy in the absence of echocardiography. Furthermore, it was shown that RBC Glx depth correlates significantly with continuous echocardiographic measures of left ventricular function e.g., LVIDd-N (left ventricular internal dimension in diastole), a marker commonly used to assess dog mitral valve disease severity (Figure 10B).

[0236] EXAMPLE 7

[0237] The RBC Glx reduces in depth in sepsis

[0238] It was next assessed whether RBC Glx could be used as a marker for sepsis.

[0239] It was shown that LPS (lipopolysaccharide) a serologically reactive bacterial toxin that induces a sepsis response, rapidly (<18h) caused detectable damage to the RBC Glx in adult mice (Figure 11 ).

[0240] Furthermore, human patients admitted to NBT (North Bristol) NHS trust and enrolled in the GlycoSAVe trial had significantly thinner RBC Glx when measured using the “peak to peak” method (LEL-R18) compared to age matched controls (Figure 12A).

[0241] The peak lectin intensity at the RBC membrane in patients admitted to NBT NHS trust and enrolled in the GlycoSAVe trial were significantly lower than age matched control samples, and correlated significantly with the ‘peak to peak’ measure (p=0.0006 (Figure 12B)). RBC LEL lectin binding capacity was also reduced in patients with sepsis compared to healthy age-matched controls (Figure 12C). These data suggest peak lectin binding capacity of RBC reduces in sepsis.

[0242] Furthermore, data from the same study indicated that patients with the thinnest RBC Glx were most likely to develop sepsis-related acute kidney injury (sAKI) (Figure 12D).

[0243] EXAMPLE 8

[0244] The RBC Glx rapidly reduces in depth in COVID-19 patients

[0245] Next, it was tested whether pathological changes to the RBC Glx in human RBC samples collected as part of the Diagnostic and Severity markers of COVID-19 to Enable Rapid triage (DISCOVER) trial could be detected (Arnold et al., 2021a; Arnold et al., 2021 b). This study confirmed that hospitalised patients with COVID-19 had significant RBC Glx damage compared to healthy control samples. Labelling using both WGA and LEL lectins and R18 confirmed significant thinning of the RBC Glx in patients with COVID- 19 using the “peak to peak” method (Kruskal-Wallis p<0.05 and <0.005 respectively) (Figure 13), as predicted from the published literature suggesting EnC Glx damage in this condition (Fels et al., 2022; Rovas et al., 2021 ). EXAMPLE 9

[0246] Negative binding assay

[0247] The negative binding assay was also developed as a way to measure characteristics of RBC Glx. Here, blood from a patient is transferred to a dark tube pre-filled with lectins conjugated to fluorophores, for example WGA-633 and PNA-488. This is then mixed and incubated for 20 minutes. The cells are then separated by e.g. spinning or agglutination. The cell free supernatant can then be transferred to a plate and a plate reader used to measure the 633 and 488 signals. The lectin remaining in solution is inversely proportional to the lectin bound to RBCs.

[0248] To validate this assay, increasing concentrations of heparinase were added to a mixture of blood cells with WGA-633 and PNA-488 to induce RBC Glx damage. As shown in Figure 14, the negative binding assay is highly sensitive to enzyme induced RBC Glx damage. In this assay, PNA RBC binding increases with Glx damage and WGA RBC binding decreases with Glx damage.

[0249] EXAMPLE 10

[0250] A range of glycocalyx binding molecules and detection methods can be used to label and measure RBC Glx.

[0251] While the above examples focus on using fluorescently labelled lectins for measuring RBC Glx, the invention is not limited to this method of detection. A range of glycocalyx binding molecules and detection methods can be used. Further non-limiting examples are described below.

[0252] (i) Biotinylated lectins

[0253] In this example, blood smears were incubated with either biotinylated LEL lectin or biotinylated UEA, and then further incubated with fluorescently labelled streptavidin. RBC Glx depth was measured using lectin and R18 ‘peak to peak’ analysis, as described above (Figures 15A and B). As with the examples above, the location of the signal peaks generated using this method can be used to provide measures of RBC Glx.

[0254] (ii) Fluorescently labelled lectins

[0255] Lectins can also be directly fluorescently labelled, as already described in the above examples. In this example, RBCs taken from a healthy individual and sepsis patient were stained with peanut lectin, which had been directly conjugated to FITC. No binding to the RBC Glx was seen in cells from healthy individuals (Figure 16A). On the other hand, peanut lectin avidly bound to the RBC glycocalyx following loss of terminal sialic acid residues as seen in sepsis patients (Figure 16B). The same binding was demonstrated following exposure of RBCs to neuraminidase (data not shown). 3D images were generated from a z-stack of confocal images of RBC taken from the patient with sepsis. This imaging confirmed the RBC cell membranes were intact but that the residual RBC Glx composition had altered to permit peanut lectin binding (Figure 16C). Similar results were also obtained using identical protocols involving different lectins such as fluorescently labelled LEL lectin (data not shown).

[0256] (Hi) Antibodies

[0257] Antibodies were used to label specific RBC Glx components.

[0258] Figure 17A shows binding of antibodies to ICAM-1 and PECAM-1 on the RBC surface. Measurements of ICAM-1 and PECAM-1 within the RBC Glx can be used to derive measurements of their expression within the EnC Glx.

[0259] Heparan sulphate is another component of the EnC and RBC Glx. Knocking down EXT 1 (an enzyme involved in the biosynthesis of heparan sulphate) in EnC results in heparan sulphate loss from the EnC Glx (Gamez et al., 2024). To investigate whether endothelial specific knockdown of EXT1 also affects the composition of RBC Glx, RBCs were taken from littermate control mice and endothelial-specific EXT1 knock down mice, and stained with anti-heparan sulphate antibodies. Heparan sulphate expression was seen on the surface of the RBCs taken from the control mice, whereas RBCs taken from endothelial- specific knock down animals lacked heparan sulphate within their Glx (Figures 17B and C). RBCs from the control mice also express the heparan sulphate carrier protein HSPG2, as demonstrated using antibody staining of the RBCs (Figure 17D). On the other hand, endothelial-specific knock down of EXT 1 resulted in reduced RBC expression of HSPG2 (Figure 17E).

[0260] Altogether this demonstrates that the composition of the RBC Glx reflects to the composition of the EnC Glx, and that components of the Glx can be detected using antibodies.

[0261] (iv) Small molecules

[0262] Glx can also be detected using small molecules.

[0263] For example, Figure 18A shows the detection of endothelial and RBC Glx by staining of cells with alcian blue and imaging using electron microscopy. Binding of alcian blue can also be measured using a “negative binding assay”, also known as a “reverse binding assay”. Here, alcian blue is added to RBCs in solution, where it binds to the cell surface. When using a standard concentration of alcian blue and a standard quantity of RBC, the amount of alcian blue left in solution after incubation for 30 minutes is proportional to 1 / RBCGIx alcian blue binding. The concentration of alcian blue in solution can be measured using the light absorbance properties of the small molecule. Using this method, RBC Glx damage associated with prolonged blood sample storage was detected (Figure 18B).

[0264] 3-Dansylaminophenylboronic acid (a phenylboronic acid (PBA)) (blue) was shown to bind to sialic acids within the RBC Glx (Figure 18C). This was confirmed using R18 to label the cell membrane (Figure 18D). Changes in the RBC Glx could be quantified by measuring peak signal locations on confocal images (Figure 18E).

[0265] (v) Adsorbent protein

[0266] RBCs were incubated in media containing AlexaFluor488 (AF488)-tagged albumin or AF488-tagged fibrinogen. The RBCs became labelled with the tagged Glx component (Figures 19A and B). The degree of binding of these components to the RBC Glx can be used to derive a measure of Glx integrity on the RBC.

[0267] EXAMPLE 11

[0268] Enc Glx composition can be evaluated by measuring RBC Glx composition

[0269] EnC surface proteins can be traced by introducing synthetic bonds into their binding sugars during culture. This is done using the azide-labelled sugar N-azidoacetylmannosamine (ManNAz). ManNAz compounds are incorporated within the EnC Glx by glycosylation events to effectively 'tag' sialic acids which are bound to glycoproteins and proteoglycans with the azide group.

[0270] Universal ManNAz integration in endothelial monolayers can be observed using confocal imaging (Figure 20A). High resolution confocal imaging of cells labelled with Alexa Fluor™ 488, R18 and DAPI (for ManNAz, cell membrane and DNA labelling, respectively) confirmed that ManNAz labelled sialic acids were expressed on the endothelial cell surface external to the cell membrane (Figure 20B).

[0271] RBCs exposed to control endothelial cells for 24 hours (i.e. endothelial cells which were not fed ManNAz but processed with Click™ fluorophore) showed minimal background florescence, while RBCs exposed to ManNAz labelled endothelial cells display the Click™ binding site on their surface following interaction suggesting transfer of sialic acids (Figure 20C and D). This was confirmed by quantification of individual RBC Click™ signal intensity at the membrane (Figure 20E). These data indicate that changes in the glycan composition of EnC Glx can be evaluated by detecting these molecules on RBC Glx.

[0272] Endothelial cells exposed for 4 hours to RBCs previously interacted with control endothelial cells for 24 hours demonstrated no significant ‘click’ binding (Figure 20F). On the other hand, endothelial cells exposed for 4 hours to RBCs ‘loaded’ with ‘click’ labelled EnC Glx exhibited dense patches of click labelled glycocalyx following RBC removal (Figure 20G). This suggests that RBC can redistribute glycocalyx components from one endothelial population to another. The inset in Figure 20G highlights that transferred material remains on the cell surface. Quantification of green ‘click’ label intensity on cultured endothelial monolayers after RBC contact confirmed significant transfer from ‘loaded’ RBC onto the endothelial surface (Figure 20H).

[0273] Following isolation of the RBCs which had been interacted with “tagged” EnC derived surface proteins, Click chemistry was used to refine the proteins present within the RBC Glx that were originally generated in the EnC. Figure 21 shows proteins observed to transfer into the RBC Glx from the EnC Glx. All proteins within the grey area were statistically significantly refined on the RBC compared to cells media and control cells. These data indicate that the glycoprotein and proteoglycan composition of EnC Glx can be evaluated by detecting these molecules on RBC Glx. Named examples of transferred proteins include HSPG2 (perlecan), PECAM1 , CD44, ICAM1 , Basigin (BSG), and Integrins, ITGB3, ITGA3 and ITGA5.

[0274] Further experiments were carried out to investigate the relationship between EnC and RBC Glx, RBCs were treated with neuraminidase to cleave sialic acids from RBC membranes. The cells were labelled with LEL lectin and R18 and ‘peak to peak’ measurements carried out. Following neuraminidase treatment, LEL lectin ‘peak to peak’ thickness was significantly reduced on human RBC compared to cells which had not been treated with neuraminidase. Interacting these depleted RBCs with control endothelial cells for one hour had no measurable effect. In contrast, a one hour interaction between depleted RBC and EXT1 over-expressing endothelial cells (EXT1 +) significantly restored the RBC Glx (Figure 22A). This shows that modulation of endothelial heparan sulphate (HS) alters human RBC Glx recovery in vitro.

[0275] After 14 hours interaction, significant recovery of the RBC Glx was seen but no significant residual effect from EXT1 over expression was detected (Figure 22B). Circulating RBCs in endothelial cell-conditioned media for 14 hours had no effect on the RBC Glx thickness. Representative 3D images of RBCs labelled with LEL lectin following neuraminidase treatment and following one hour interaction with control endothelial cells are shown in Figure 22C and D.

[0276] Similar recovery results were seen in RBCs treated with heparinase III (an enzyme that degrades heparan sulphate).

[0277] To investigate the relationship between EnC Glx and RBC Glx in vivo, blood smears were isolated from littermate control mice and mice with conditional endothelial specific deletion of EXT 1 . Cells were labelled with LEL lectin and R18. Visible Glx loss was seen in the RBCs isolated from mice with conditional endothelial specific deletion of EXT1 (Figure 23A and B). This effect was not seen on platelets or leukocytes. LEL lectin intensity and Glx thickness were significantly reduced in RBCs obtained from EXT1 knockdown mice compared to controls, confirming the visual results (Figure 23C and D). Altogether, this shows that RBC from mice with endothelial specific deletion of heparan sulphate have reduced RBC Glx.

[0278] DISCUSSION

[0279] The importance of the EnC Glx in maintaining endothelial function is well established (Butler et al., 2020; Nieuwdorp et al., 2006; Teoh et al., 2023; Gamez et al., 2024; Padberg et al., 2014).

[0280] However, methods to accurately and efficiently measure EnC Glx health have been lacking. Here, it has been shown that the RBC Glx mirrors the EnC Glx and therefore RBC Glx can be used as a simple way to indirectly measure EnC Glx. RBC Glx can be used to measure various characteristics of EnC Glx, including glycocalyx depth, charge, density, elasticity and molecular and chemical composition. The inventors have shown that direct contact between the two cell types can lead to transfer of components from the Glx of one cell type to the other. This phenomenon likely contributes to the “mirroring” effect, and can be exploited to determine EnC health.

[0281] Determination of EnC Glx is particularly important early in disease as it permits earlier intervention to prevent the diseases from developing. The methods described herein are therefore useful in diagnosing and monitoring progression of diseases in which dysfunctional vascular function is a symptom.

[0282] The inventors have developed several methods to measure characteristics of RBC Glx including the “peak to peak” method and negative binding assay. Key to the “peak to peak” method is the use of the cell membrane as a reference point and the application of Gaussian models to reduce noise in the generated light signals (Crompton et al., 2023). Other validated measures of RBC Glx include measuring the total fluorescent signal or total lectin binding. The inventors have demonstrated that RBC Glx can be detected and measured using various techniques including fluorescently labelled or biotinylated lectins, antibodies, small molecules and adsorbent proteins. Thus the invention is not limited to any particular method of detection. The application of Al and automated computer analysis of images has allowed measurement of RBC Glx characteristics with great reproducibility despite the limited absolute resolving power associated with light microscopy. These elements combined with the large quantity of measurement made on each sample ensure that even the subtle changes associated with very low-level enzyme exposure can be seen. It seems likely therefore that even subtle damage in early disease can be reliably detected using these techniques.

[0283] METHODS

[0284] Rats

[0285] Male Wistar rats (150-200g, Charles River) were maintained by the Animal Services Unit, University of Bristol. Animals were housed in a conventional facility with a controlled environment (21-24 °C and 12:12 hour light-dark cycle).

[0286] Blood was sampled during brief isoflurane induced anaesthesia from the tail vein using a 1 ml syringe and 27gauge sterile needle and standard aseptic techniques. Approximately 50 microlitres was collected from each animal on each sampling occasion.

[0287] Blood sampling from humans

[0288] Whole blood samples were either collected into LiH blood tubes, stored at 4 degrees and processed within 4hours or collected into transfix™ EDTA vacutainers. Blood samples were collected as part of ongoing studies with full consent and ethics in place.

[0289] Electron microscopy

[0290] The method used to image EnC Glx using Alcian blue have been extensively published (Lawrence et al., 2022).

[0291] Quantum dot imaging Quantum dots (qdots) are semiconductor nanocrystals. They are electron dense and can be observed using electron microscopy. Qdot™ 655 streptavidin conjugate (Thermo Fisher Scientific, Waltham, USA, Q10123MP) was used at a working concentration of 1 :100. Qdot™ Streptavidin conjugate was freshly prepared in 1% BSA in PBS - 0.1% Tween, pH 6.8 and the tissue incubated at room temperature for one hour on placental tissue sections were prepared with biotinylated-LEL. The sections were then post-fixed with 1% glutaraldehyde in 0.1 M phosphate buffer for 15 minutes and processed as previously for electron microscopy and imaged using standard techniques on Tecnai 12 - FEI BioTwin Spirit (Field Electron and Ion Company, Hillsboro, USA) transmission electron microscope (TEM).

[0292] Blood smear

[0293] To make blood smears 3-5 microlitres of blood was pipetted onto clean plain glass microscope slides approximately 5mm from the frosted area. A second bevel edged slide was then advanced to the blood drop meeting the sample at 30 degrees from the horizontal first slide. The blood rapidly advances towards the slide’s margins due to capillary action. Once 2 / 3 of the slide were covered the second slide was rapidly moved away from the frosted label whilst maintaining steady downward pressure. Typically, a ‘thumb print’ shaped smear of blood was then visible on the slide. This was left to air dry before the sample was fixed by immersing it in ice cold 100% methanol for 30 seconds. The slide was then left to dry before being used in the lectin or antibody staining protocol.

[0294] Lectin staining

[0295] Following fixation, red cell smears were washed (3*5 minutes sterile PBS (ph7.4)) in slide baths. A 1% bovine serum albumin (BSA) (catalogue no) PBS solution was then used to cover the blood smears for 30 minutes at room temperature to block non-specific lectin / FITC binding. Following a further 3 wash cycles FITC conjugated lectins were suspended in PBS and placed on the slides in a dark moist slide box before incubating overnight at 4 degrees Celsius. Slides were then washed in dark slide baths (3*5 minutes sterile PBS (ph7.4)) before 36.5 pg / ml Octadecyl rhodamine B chloride (R18) (0246; Thermo Fisher Scientific) (1 :1000 dilution in PBS) was added for 10 minutes. Slides were then washed a further 3*5 minutes sterile PBS (ph7.4) before Vectashield mounting medium (H-1000; Vector Laboratories) and a coverslip were applied.

[0296] Biotinylated lectins

[0297] Blood smears were prepared and fixed as described above. Blocking buffer 1% BSA in PBS for 30 minutes was then added to the slides followed by endogenous biotin blocking using a streptavidin / biotin blocking kit (SP-2002; Vector Laboratories). After 2 washes, the sections were incubated with the biotinylated lectin (pH 6.8) overnight at 4°C. Buffer only was used as a negative control. After 3 washes, the sections were incubated with streptavidin-Alexa Fluor 488 (1 :500, S32354; Thermo Fisher Scientific) (pH 6.8) for 1 hour at room temperature.

[0298] Antibody staining

[0299] Blood smears were prepared and fixed as described above. Non-specific binding was reduced by blocking in 1%BSA PBS for 1 hour at room temperature. Primary antibodies were then applied at optimized concentrations ranging between 1 :50 and 1 :200 overnight. After washing, secondary antibodies (conjugated to fluorophores and species appropriate) were added at optimized concentrations (1 :200- 1 :500) for 1-2 hours at room temperature. Following washing, slides were processed as described herein, mounted and imaged using confocal microscopy. Antibody staining can also be performed in solution rather than on blood smears. For processing in solution, cells are spun at 200 RCF for 2-5 minutes to isolate RBC for each wash I incubation step. RBCs can be imaged in solution immediately or smeared onto slides and fixed once dry.

[0300] To detect heparan sulphate, using antibodies to label the many isoforms of heparan sulphate requires the generation of a common epitope. To achieve this, RBCs were exposed to Heparatinase III (Sigma H8891 , 1 u / ml) at 37 degrees for 1 hour. This exposed the common ‘stump’ of heparan suphate which was then labelled with 3G10 antibody (Amsbio 370260). Heat inactivated heparatinase III acted as a negative control. Enzymes were used in solution before RBCs were smeared and fixed. 3G10 was diluted 1 :200 and incubated with the RBCs for 2 hours before washing and secondary antibodies were applied for 1 hour. On mouse blood samples ‘mouse on mouse’ blocking kits (Vector laboratories MKB-2213-1) were used to reduce non-specific secondary antibody binding.

[0301] Alcian blue reverse binding assay

[0302] The alcian blue binding assay was performed on washed blood diluted 1 :10 in PBS. The Alcian Blue solution was prepared by diluting 5mg of alcian blue in 100 microliters of 100% ethanol before adding 10ml of PBS (Ph 7.4) containing 25mmol MgCh. 5ml of diluted blood was added to 500 pl of Alcian Blue solution, mixing by inversion and incubating for 30 minutes at room temperature. The mixture was then centrifuged to remove RBCs (and all the associated bound Alcian Blue) for 3 minutes before the supernatant was collected and analyzed on a plate reader using absorbance (wavelength selected between 490-600).

[0303] 3-Dansylaminophenylboronic acid (DBA) binding

[0304] DBA was initially dissolved in DMSO and stored at 4°C. To label RBCs DBA solution was further diluted in 100% ice cold methanol (5pl / ml) immediately prior to use. RBCs were smeared onto glass slides as previously described before the DBA-methanol solution was added for 60 seconds to simultaneously fix and label RBC Glx. Smears were subsequently washed and processed with R18 as described above.

[0305] Adsorbent proteins

[0306] To stain isolated washed RBCs in solution, PBS BSA, with glucose or cell media was used to maintain RBCs ex vivo. Compounds of interest were added to the RBC suspension at a final concentration of 0.001 mM (Albumin AF488 (A13100) and fibrinogen AF488 (Thermo F13191) overnight whilst RBCs were incubated at 37°C in cell incubators (5% CO2). RBCs were subsequently washed in PBS x3 before processing and imaging using confocal mcroscopy.

[0307] ManNAz labelling

[0308] To integrate ManNAz into the EnC Glx, terminally differentiated confluent endothelial monolayers in 10cm round culture dishes (grown in EBM2 (Lonza, cc1356) were exposed to EBM2 media supplemented with ManNAz (25 pg / ml or DMSO (solvent control)) for 5 days (Thermo Scientific, C33366). For proteomics experiments, endothelial cells were washed 3 times and swapped to control media before washed and isolated RBCs were added. Interaction was induced by oscilating the culture dishes using an orbital shaker to induce flow equivalent to 5dyn shear stress. RBCs flowed over the endothelial monolayers overnight. Controls included RBCs flowed over unlabelled endothelial layers and cell media flowed over labelled endothelial cells without RBCs. Sialated proteins transferred from the EnC Glx onto the RBC Glx were subsequently isolated using the ‘Click’ label (Thermo, Click-iT™ protein enrichment kit C10414) and processed for TMT proteomics per standard protocols.

[0309] For other transfer experiments, cells were labelled using the Click-iT™ cell reaction buffer kit (Thermo Scientific, C10269) to conjugate Alexa Fluor™ 488 Azide to the integrated sugar. The standard recommended protocol was followed to label RBC in suspension / on slides and endothelial cells. Endothelial cells were washed 3 times with PBS before standard EBM2 was added containing a suspension of RBCs for interaction. Retrieved RBCs were washed 3 times (with centrifuge isolation cycles (200rcf, 5min) before resuspension 3 times) before transfer to new unlabeled monolayers at a concentration of 100 000 RBC per ml of media. At the experimental end, RBCs were removed from the monolayers before washing the endothelial cells 3 times and fixing with PFA.

[0310] Interaction assays

[0311] To deplete the RBC Glx, isolated and washed RBCs were incubated in the presence of neuraminidase (Roche, 11 585 886 001 ). 100 000 RBC per ml of media were subsequently added to 10 cm round culture dishes containing endothelial monolayers. To induce interaction, movement of the RBC in suspension over the static monolayers was induced with an orbital shaker resulting in a modelled peak shear stress of 5 dyn / cm2 (SSM1 Stuart UK). RBCs were subsequently labelled using lectins as described above.

[0312] Generation of EXT1 conditional knockdown mice.

[0313] Endothelial specific EXT1 conditional knockdown mice (EXT:ECK0>') and litter mate control mice were generated on a C57BL / 6 background by crossing Cdh5(PAC)-CreERT2mice (Taconic) with EXTI-floxed (EXT1fl / fl) mice. In male Cdh5(PAC)-CreERT2;EXT1flox / floxmice (aged 6-10 weeks) gene excision was induced by tamoxifen IP injection (75mg / kg) for 5 consecutive days. Blood samples from Cdh5(PAC)- CreERT2;EXT1fl0X / fl0Xmice and their littermate controls were taken under terminal anaesthesia.

[0314] Imaging

[0315] RBC smears were imaged using either an AF600 LX wide-field fluorescence microscope (Leica Microsystems) (100x Oil immersion lens), pixel size set to 60nm) or a Leica SP8 confocal microscope (60x oil immersion lese) with the pixel size set to 52nm. For high resolution example images the ‘Lightning’ setting was used on the Leica SP8 microscope. Where smears were used for ‘peak to peak’ analysis the operator found suitably spaced cells ‘blind’ by using the R18 signal before imaging 3 discrete areas of the blood smear at random using 2 line sequential scanning to minimise channel overlap. The image ‘z’ focal position was chosen for each image to ensure RBC were consistently imaged through their axis at their widest point. RBC isolation

[0316] To isolate RBC from whole blood samples collected into lithium heparin vacutainers, 5ml of blood was drawn into sterile syringes before passing it through a leukocyte absorbing acrodisc syringe filter (Pall, AP 4851).

[0317] RBC Peak to Peak assessment

[0318] RBC labelled with lectins and R18 were analysed to derive a ‘peak to peak’ measure of the Glx thickness using Image J (FIJI).

[0319] Intensity profiles perpendicular to object surfaces were measured using a custom workflow created using the ModularlmageAnalysis (MIA) plugin for Imaged (Cross et al., 2023; Schindelin et al., 2012; Schneider et al., 2012). For detection of red blood cells, images were then subject to rolling ball background subtraction (radius = 100 px) and objects were detected from this image using a custom StarDist model (Schmidt et al., 2018). In the case of glomeruli, detection was achieved using a custom UNet model, run via DeepImageJ (G6mez-de-Mariscal et al., 2021). The output probability image from this process was binarised according to a fixed probability threshold and objects detected from this binary image using connected components labelling (Legland et al., 2016). Optionally, objects detected via either route could be manually refined using ImageJ’s paintbrush tools. At regular intervals along the surface of each detected object, intensity profiles were extracted perpendicular to the surface at that point. An asymmetric Gaussian profile of the form shown below was fit to each profile, where a is the peak amplitude, b is the peak maxima location, c and f are the peak standard deviations (widths) to the left and right of the maxima, d and e are the baselines (background) to the left and right of the maxima and k controls the transition between left and right sides (set to 100 to effectively give an immediate transition).

[0320] This equation utilised the Heaviside step function to yield a profile with different fitting parameters to the left and right of the peak maxima, but retain a common peak maxima location. Each parameter was subject to user-defined constraints and likewise, only fits with acceptable R2 values were retained for analysis. By fitting such profiles to two different signals, it was possible to measure the relative difference in peak maxima location.

[0321] Cell culture

[0322] Human conditionally immortalised EnC were selected for all work in vitro due to their extensively studied Glx. Cells were seeded at 20 000 cells / ml and cultured in EBM™ basal medium supplemented with EGM™-2 MV (Lonza, CC-3202) SingleQuots™ except gentamicin. Monolayers were generated using cells at passage 22-32. Cells were allowed to form monolayers up to 80% confluence at the permissive temperature of 33 degrees centigrade, before swapping to 37 degrees for a minimum of 5 days to allow terminal differentiation. Where EnC were to be imaged monolayers were cultured on fibronectin coated coverslips.

[0323] References

[0324] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

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Claims

Claims:1 . A method of predicting one or more characteristics of endothelial cell glycocalyx in a subject wherein the method comprises measuring the corresponding one or more characteristics of red blood cell glycocalyx from the subject.

2. A method of diagnosing, monitoring, prognosticating, or stratifying a disease linked with changes in one or more characteristics of endothelial cell glycocalyx in a subject by carrying out the method of claim 1 .

3. The method of claim 2 wherein monitoring a disease linked with changes in one or more characteristics of endothelial cell glycocalyx in a subject comprises measuring the corresponding one or more characteristics of red blood cell glycocalyx from the subject before and after a therapeutic is administered to the subject.

4. The method of claim 3 wherein measuring the one or more characteristics of red blood cell glycocalyx comprises measuring red blood cell glycocalyx depth, and an increase in red blood cell glycocalyx depth after the therapeutic is administered compared to before the therapeutic was administered is indicative of improved disease status in the subject.

5. The method of any one of the preceding claims wherein the one or more characteristics of red blood cell glycocalyx are measured from a blood sample obtained from the subject.

6. The method of any one of the preceding claims wherein the one or more characteristics comprises glycocalyx depth, charge, density, elasticity and molecular and chemical composition.

7. The method of claim 6 wherein the molecular and chemical composition comprises at least one glycocalyx component selected from proteoglycans, glycosaminoglycans, glycoproteins, selectins, integrins, syndecans, intercellular adhesion molecules, glycolipids, glycophorins, glypicans, glycans and sialic acids.

8. The method of claim 6 or claim 7 wherein the molecular and chemical composition comprises at least one glycocalyx component selected from CD44, perlecan (also known as HSPG2), syndecan 3, versican, decorin, biglycan, mimecan, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, hyaluronic acid, ITGA5, ITGB3, ITGA3, ICAM1 , platelet / endothelial cell adhesion molecules (PECAM-1), vascular cell adhesion molecule (VCAM-1), Tamm-Horsfall glycoprotein, basigin, glucose, galactose, mannose, N-acetylneuraminic acid, N-acetylglucosamine, Gal-0(1-3)-GalNAc, N- acetylgalactosamine, glucuronic acid, xylose and fucose.

9. The method of any one of the preceding claims wherein the endothelial cell glycocalyx is cardiac, renal, cerebral, pulmonary, cutaneous, hepatic, gut, arterial, systemic or sublingual endothelial cell glycocalyx.

10. The method of any one of the preceding claims wherein the one or more characteristics of red blood cell glycocalyx is measured using a glycocalyx-binding molecule.11 . The method of claim 10 wherein the glycocalyx-binding molecule comprises a lectin, antibody, glycosaminoglycan-binding protein, aptamer, adsorbent protein, or small molecule.

12. The method of claim 11 wherein the lectin is selected from wheat germ agglutinin, peanut agglutinin, Lycopersicon esculentum lectin, Ulex europaeus agglutinin I, Sambucus Nigra lectin, Wisteria floribunda lectin, Ricinus Communis Agglutinin I, Dolichos Biflorus Agglutinin, Dioclea bicolor lectin and / or Marasmius oreades agglutinin.

13. The method of claim 10-12 wherein the glycocalyx-binding molecule is (a) conjugated to a detection label, optionally wherein the detection label is a fluorescent label, bioluminescent label, colourimetric label, absorbent label, electrochemical label, quantum dot, nanoparticle, peptide tag, electrochemical label, enzyme, enzyme substrate or biotin, or (b) immobilised on a solid substrate or hydrogel.

14. The method of any one of the preceding claims wherein measurement of the one or more characteristics of red blood cell glycocalyx comprises labelling the red blood cell glycocalyx with quantum dots or other electron dense probe and imaging using electron microscopy, labelling the red blood cell glycocalyx and measuring the labels using flow cytometry, labelling the red blood cell glycocalyx and imaging using confocal microscopy, mass spectrometry of the red blood cell glycocalyx composition, proteomic measurement of the red blood cell glycocalyx, or glycomic measurement of the red blood cell glycocalyx.

15. The method of any one of the preceding claims wherein measurement of the one or more characteristics of red blood cell glycocalyx comprises measuring red blood cell mobility or red blood cell aggregation.

16. The method of claims 6 wherein measuring red blood cell glycocalyx depth comprises:(i) labelling a red blood cell obtained from a subject with a labelled marker of red blood cell glycocalyx and a labelled marker of red blood cell membrane;(ii) measuring signals generated by the labelled marker of red blood cell glycocalyx and labelled marker of red blood cell membrane; and(iii) calculating the distance between peak signals of the labelled marker of red blood cell glycocalyx and labelled marker of red blood cell membrane, wherein the distance is indicative of red blood cell glycocalyx depth.

17. The method of claim 16 wherein the distance between peak signals is calculated at a plurality of points along the circumference of a red blood cell and a mean and / or median red blood cell glycocalyx depth is calculated for the red blood cell.

18. The method of claim 16 or claim 17 wherein the labelled marker of red blood cell glycocalyx is a labelled glycocalyx-binding molecule, optionally a labelled lectin or labelled antibody.

19. The method of any one of claims 16-18 wherein a red blood cell in which at least 50% of the cell membrane is not in contact with another cell contributes to the indication of red blood cell glycocalyx depth.

20. The method of claims 1-11 wherein measurement of the one or more characteristics of red blood cell glycocalyx comprises:(i) incubating a blood sample obtained from a subject with one or more labelled markers of red blood cell glycocalyx;(ii) separating the red blood cells to produce a substantially cell free portion; and(iii) measuring the amount of residual labelled marker present in the substantially cell free portion, wherein the amount of residual labelled marker present in the substantially cell free portion is inversely proportional to the amount of labelled marker bound to the red blood cell glycocalyx.21 . The method of any one of claim 20 wherein the labelled marker of red blood cell glycocalyx is specific for intact red blood cell glycocalyx.

22. The method of claim 20 wherein the labelled marker of red blood cell glycocalyx is specific for damaged red blood cell glycocalyx.

23. The method of any one of claims 2-22 wherein the disease linked with changes in one or more characteristics of endothelial cell glycocalyx comprises a vascular disease.

24. The method of any one of claims 2-23 wherein the disease linked with changes in in one or more characteristics of endothelial cell glycocalyx comprises a disease selected from diabetes, early diabetes, heart failure, mitral valve disease, preeclampsia, ischaemic reperfusion injury, sepsis, delirium, dementia, cancer, cancer metastasis, hemolytic uremic syndrome, compliment disorders, atherosclerosis, venous thrombosis, thin basement disease, minimal change nephrotic syndrome, acute kidney injury, membranous nephrotic syndrome, focal segmental glomerulosclerosis, IgA nephropathy, chronic kidney disease, transplant rejection, radiation injury and COVID-19.

25. The method of claim 24 wherein the diabetes is diabetic retinopathy, diabetic nephropathy or diabetic vascular disease.

26. Use of a red blood cell sample from a subject as a biomarker for determining the status of the endothelial glycocalyx of the subject. l . A kit for carrying out the method of claim 20 wherein the kit comprises: (i) a receptacle for holding blood, wherein the receptacle comprises a labelled marker, optionally wherein labelled marker comprises a glycocalyx binding molecule; and (ii) a device for measuring the labelled marker.

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