Cerebral microvascular endothelial glycocalyx damage, its implications on the blood–brain barrier and a possible contributor to cognitive impairment

The socio-economic impact of diseases associated with cognitive impairment is increasing. According to the Alzheimer’s Society there are over 850,000 people with dementia in the UK, costing the UK £26 billion in 2013. Therefore, research into treatment of those conditions is vital. Research into the cerebral endothelial glycocalyx (CeGC) could offer effective treatments.The CeGC, consisting of proteoglycans, glycoproteins and glycolipids, is a dynamic structure covering the luminal side of the endothelial cells of capillaries throughout the body. The CeGC is thicker in cerebral micro vessels, suggesting specialisation for its function as part of the blood–brain barrier (BBB). Recent research evidences that the CeGC is vital in protecting fragile parenchymal tissue and effective functioning of the BBB, as one particularly important CeGC function is to act as a protective barrier and permeability regulator.CeGC degradation is one of the factors which can lead to an increase in BBB permeability. It occurs naturally in aging, nevertheless, premature degradation has been evidenced in multiple conditions linked to cognitive impairment, such as inflammation, brain edema, cerebral malaria, Alzheimer’s and recently Covid-19. Increasing knowledge of the mechanisms of CeGC damage has led to research into preventative techniques showing that CeGC is a possible diagnostic marker and a therapeutic target. However, the evidence is relatively new, inconsistent and demonstrated mainly in experimental models.This review evaluates the current knowledge of the CeGC, its structure, functions, damage and repair mechanisms and the impact of its degeneration on cognitive impairment in multiple conditions, highlighting the CeGC as a possible diagnostic marker and a potential target for therapeutic treatment

Poly(ethylene glycol)-crosslinked gelatin hydrogel substrates with conjugated bioactive peptides influence endothelial cell behavior

The basement membrane is a specialized extracellular matrix substrate responsible for support and maintenance of epithelial and endothelial structures. Engineered basement membrane-like hydrogel systems have the potential to advance understanding of cell-cell and cell-matrix interactions by allowing precise tuning of the substrate or matrix biochemical and biophysical properties. In this investigation, we developed tunable hydrogel substrates with conjugated bioactive peptides to modulate cell binding and growth factor signaling by endothelial cells. Hydrogels were formed by employing a poly(ethylene glycol) crosslinker to covalently crosslink gelatin polymers and simultaneously conjugate laminin-derived YIGSR peptides or vascular endothelial growth factor (VEGF)-mimetic QK peptides to the gelatin. Rheological characterization revealed rapid formation of hydrogels with similar stiffnesses across tested formulations, and swelling analysis demonstrated dependency on peptide and crosslinker concentrations in hydrogels. Levels of phosphorylated VEGF Receptor 2 in cells cultured on hydrogel substrates revealed that while human umbilical vein endothelial cells (HUVECs) responded to both soluble and conjugated forms of the QK peptide, conditionally-immortalized human glomerular endothelial cells (GEnCs) only responded to the conjugated presentation of the peptide. Furthermore, whereas HUVECs exhibited greatest upregulation in gene expression when cultured on YIGSR- and QK-conjugated hydrogel substrates after 5 days, GEnCs exhibited greatest upregulation when cultured on Matrigel control substrates at the same time point. These results indicate that conjugation of bioactive peptides to these hydrogel substrates significantly influenced endothelial cell behavior in cultures but with differential responses between HUVECs and GEnCs.</p

Response to There is little evidence that the endothelial glycocalyx has a specific role in glomerular permeability of albumin

We write in response to the letter entitled “There is little evidence that the endothelial glycocalyx has a specific role in glomerular permeability of albumin,”1 in reference to our original publication.2 The letter title is very misleading. It represents only the opinion of this author and is diametrically opposed to the data presented in our paper and an accumulating body of evidence including in vivo multiphoton measurements.3 This author’s claims are not supported by experimental observations and we and others have addressed them extensively including in a response4 to a previous similar letter. It is curious that the major focus of the letter seems to be on charge selectivity when charge is not mentioned in our paper

Transport of Gold Nanoparticles by Vascular Endothelium from Different Human Tissues

The selective entry of nanoparticles into target tissues is the key factor which determines their tissue distribution. Entry is primarily controlled by microvascular endothelial cells, which have tissue-specific properties. This study investigated the cellular properties involved in selective transport of gold nanoparticles (<5 nm) coated with PEG-amine/galactose in two different human vascular endothelia. Kidney endothelium (ciGENC) showed higher uptake of these nanoparticles than brain endothelium (hCMEC/D3), reflecting their biodistribution in vivo. Nanoparticle uptake and subcellular localisation was quantified by transmission electron microscopy. The rate of internalisation was approximately 4x higher in kidney endothelium than brain endothelium. Vesicular endocytosis was approximately 4x greater than cytosolic uptake in both cell types, and endocytosis was blocked by metabolic inhibition, whereas cytosolic uptake was energy-independent. The cellular basis for the different rates of internalisation was investigated. Morphologically, both endothelia had similar profiles of vesicles and cell volumes. However, the rate of endocytosis was higher in kidney endothelium. Moreover, the glycocalyces of the endothelia differed, as determined by lectin-binding, and partial removal of the glycocalyx reduced nanoparticle uptake by kidney endothelium, but not brain endothelium. This study identifies tissue-specific properties of vascular endothelium that affects their interaction with nanoparticles and rate of transpor

Direct detection and measurement of wall shear stress using a filamentous bio-nanoparticle

The wall shear stress (WSS) that a moving fluid exerts on a surface affects many processes including those relating to vascular function. WSS plays an important role in normal physiology (e.g. angiogenesis) and affects the microvasculature's primary function of molecular transport. Points of fluctuating WSS show abnormalities in a number of diseases; however, there is no established technique for measuring WSS directly in physiological systems. All current methods rely on estimates obtained from measured velocity gradients in bulk flow data. In this work, we report a nanosensor that can directly measure WSS in microfluidic chambers with sub-micron spatial resolution by using a specific type of virus, the bacteriophage M13, which has been fluorescently labeled and anchored to a surface. It is demonstrated that the nanosensor can be calibrated and adapted for biological tissue, revealing WSS in micro-domains of cells that cannot be calculated accurately from bulk flow measurements. This method lends itself to a platform applicable to many applications in biology and microfluidics

Effect of endothelial cell heterogeneity on nanoparticle uptake

Endothelial cells exhibit distinct properties in morphology and functions in different organs that can be exploited for nanomedicine targeting. In this work, endothelial cells from different organs, i.e. brain, lung, liver, and kidney, were exposed to plain, carboxylated, and amino-modified silica. As expected, different protein coronas were formed on the different nanoparticle types and these changed when foetal bovine serum (FBS) or human serum were used. Uptake efficiencies differed strongly in the different endothelia, confirming that the cells retained some of their organ-specific differences. However, all endothelia showed higher uptake for the amino modified silica in FBS, but, interestingly, this changed to the carboxylated silica when human serum was used, confirming that differences in the protein corona affect uptake preferences by cells. Thus, uptake rates of fluid phase markers and transferrin were determined in liver and brain endothelium to compare their endocytic activity. Overall, our results showed that endothelial cells of different organs have very different nanoparticle uptake efficiency, likely due to differences in receptor expression, affinity, and activity. A thorough characterization of phenotypic differences in the endothelia lining different organs is key to the development of targeted nanomedicine

GlomSpheres as a 3D co-culture spheroid model of the kidney glomerulus for rapid drug-screening

The glomerulus is the filtration unit of the kidney. Injury to any component of this specialised structure leads to impaired filtration and eventually fibrosis and chronic kidney disease. Current two and three dimensional (2D and 3D) models that attempt to recreate structure and interplay between glomerular cells are imperfect. Most 2D models are simplistic and unrepresentative, and 3D organoid approaches are currently difficult to reproduce at scale and do not fit well with current industrial drug-screening approaches. Here we report a rapidly generated and highly reproducible 3D co-culture spheroid model (GlomSpheres), better demonstrating the specialised physical and molecular structure of a glomerulus. Co-cultured using a magnetic spheroid formation approach, conditionally immortalised (CI) human podocytes and glomerular endothelial cells (GEnCs) deposited mature, organized isoforms of collagen IV and Laminin. We demonstrate a dramatic upregulation of key podocyte (podocin, nephrin and podocalyxin) and GEnC (pecam-1) markers. Electron microscopy revealed podocyte foot process interdigitation and endothelial vessel formation. Incubation with pro-fibrotic agents (TGF-β1, Adriamycin) induced extracellular matrix (ECM) dysregulation and podocyte loss, which were attenuated by the anti-fibrotic agent Nintedanib. Incubation with plasma from patients with kidney disease induced acute podocyte loss and ECM dysregulation relative to patient matched remission plasma, and Nintedanib reduced podocyte loss. Finally, we developed a rapid imaging approach to demonstrate the model’s usefulness in higher throughput pharmaceutical screening. GlomSpheres therefore represent a robust, scalable, replacement for 2D in vitro glomerular disease models

Matrix metalloproteinase-9 mediated shedding of syndecan-4 in glomerular endothelial cells

Background - Diabetic nephropathy is the most common cause of end‐stage renal failure in the western world and Asia. The mechanisms are not fully elucidated, but disruption of glomerular endothelial glycocalyx and shedding of its components including syndecans has been implicated. Aims - We hypothesize that reduced glomerular filtration in diabetes is caused by disruption of endothelial glycocalyx in glomeruli, including increased shedding of syndecan‐4. The aim of this study was to determine the effects of experimental diabetic conditions by means of hyperglycemia and IL‐1β exposure on syndecan‐4 shedding in GEnC, and to investigate regulation of shedding by sheddases. Results - We found that in GEnC the expression of syndecan‐4 is higher than that of the other syndecans. In polarized GEnC, apical shedding of syndecan‐4 and syndecan‐4 gene expression was increased by 60% after IL‐1β‐stimulation, but not affected by hyperglycemic conditions. This was accompanied by a 50% increase in MMP9 gene expression in IL‐1β‐stimulated cells but not hyperglycemia. MMP9 knockdown reduced syndecan‐4 shedding by 50%. Conclusion - IL‐1β but not hyperglycemia increases the shedding of syndecan‐4 from GEnC in an MMP9‐dependent manner. This provides a potential mechanism of GEnC damage in diabetes and other inflammatory conditions

A Composite Hydrogel Scaffold Permits Self‐Organization and Matrix Deposition by Cocultured Human Glomerular Cells

Three-dimensional scaffolds provide cells with a spatial environment that more closely resembles that of in vivo tissue, when compared to 2D culture on a plastic substrate. However, many scaffolding materials commonly used in tissue engineering tend to exhibit anisotropic morphologies that exhibit a narrow range of fibre diameters and pore-sizes, which do not recapitulate extracellular matrices. In this study, a fibrin hydrogel is formed within the interstitial spaces of an electrospun poly(glycolic) acid (PGA) monolith to generate a composite, bimodal scaffold for the co-culture of kidney glomerular cell lines. This new scaffold exhibits multiple fibre morphologies, containing both PGA microfibres (14.5 ± 2 µm) and fibrin gel nanofibres (0.14 ± 0.09 µm), which increase the compressive Young’s modulus beyond that of either of the constituents. The composite structure provides an enhanced 3D environment that increases proliferation and adhesion of immortalised human podocytes and glomerular endothelial cells. Moreover, the micro/nanoscale fibrous morphology promotes motility and reorganisation of the glomerular cells into glomerulus-like structures, resulting in the deposition of organised collagen IV; the primary component of the glomerular basement membrane (GBM)