Kenton P. Arkill

Similar Endothelial Glycocalyx Structures in Microvessels from a Range of Mammalian Tissues: Evidence for a Common Filtering Mechanism?

The glycocalyx or endocapillary layer on the luminal surface of microvessels has a major role in the exclusion of macromolecules from the underlying endothelial cells. Current structural evidence in the capillaries of frog mesentery indicates a regularity in the structure of the glycocalyx, with a center-to-center fiber spacing of 20 nm and a fiber width of 12 nm, which might explain the observed macromolecular filtering properties. In this study, we used electron micrographs of tissues prepared using perfusion fixation and tannic acid treatment. The digitized images were analyzed using autocorrelation to find common spacings and to establish whether similar structures, hence mechanisms, are present in the microvessel glycocalyces of a variety of mammalian tissues. Continuous glycocalyx layers in mammalian microvessels of choroid, renal tubules, glomerulus, and psoas muscle all showed similar lateral spacings at ∼19.5 nm (possibly in a quasitetragonal lattice) and longer spacings above 100 nm. Individual glycocalyx tufts above fenestrations in the first three of these tissues and also in stomach fundus and jejunum showed evidence for similar short-range structural regularity, but with more disorder. The fiber diameter was estimated as 18.8 (± 0.2) nm, but we believe this is an overestimate because of the staining method used. The implications of these findings are discussed.

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

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

Transformations of citrate and Tween coated silver nanoparticles reacted with Na2S

Silver nanoparticles (Ag NPs) are susceptible to transformations in environmental and biological media such as aggregation, oxidation, dissolution, chlorination, sulfidation, formation/replacement of surface coatings following interaction with natural organic matter (NOM). This paper investigates the impact of surface coating and Suwannee River fulvic acid (SRFA) on the transformations and behavior of Ag NPs (citrate coated and Tween coated; cit-Ag NPs and Tween-Ag NPs, respectively), following reaction with different concentrations of Na2S solution (as a source of sulfide species, H2S and HS(-)). These transformations and the dominant mechanisms of transformations were investigated using UV-vis and scanning transmission electron microscopy coupled with electron energy loss spectroscopy. Here, we have shown that Ag NP surface coating impacts their dissolution following dilution in ultrahigh purity water, with higher extent of dissolution of Tween-Ag NPs compared with cit-Ag NPs. Tween-Ag NPs are susceptible to dissolution following their sulfidation at low S/Ag molar ratio. Suwannee River fulvic acid (SRFA) slows down the dissolution of Tween-Ag NPs at low sulfide concentrations and reduces the aggregation of cit-Ag NP in the presence of sodium sulfide. Sulfidation appears to occur by direct interaction of sulfide species with Ag NPs rather than by indirect reaction of sulfide with dissolved Ag species subsequent to dissolution. Furthermore, the sulfidation process results in the formation of partially sulfidized Ag NPs containing unreacted (metallic) subgrains at the edge of the NPs for Tween-Ag NPs in the presence of high sulfide concentration (2000nM Na2S), which occurred to less extent at lower Na2S concentration for Tween-Ag NPs and at all concentrations of Na2S for cit-Ag NPs. Thus, sulfidized Ag NPs may preserve some of the properties of the Ag NPs such as their potential to shed Ag(+) ions and their toxic potential of Ag NPs.

Origin of Parietal Podocytes in Atubular Glomeruli Mapped by Lineage Tracing

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

Modeling flow in collecting lymphatic vessels: one-dimensional flow through a series of contractile elements

The lymphatic system comprises a series of elements, lymphangions, separated by valves and possessed of active, contractile walls to pump interstitial fluid from its collection in the terminal lymphatics back to the main circulation. Despite its importance, there is a dearth of information on the fluid dynamics of the lymphatic system. In this article, we describe linked experimental and computational work aimed at elucidating the biomechanical properties of the individual lymphangions. We measure the static and dynamic mechanical properties of excised bovine collecting lymphatics and develop a one-dimensional computational model of the coupled fluid flow/wall motion. The computational model is able to reproduce the pumping behavior of the real vessel using a simple contraction function producing fast contraction pulses traveling in the retrograde direction to the flow.

The structure and mechanical properties of collecting lymphatic vessels: an investigation using multimodal nonlinear microscopy

This study employed nonlinear microscopy on fresh, unstained and unfixed collecting lymphatic vessels to determine the wall structure and its relationships to the mechanical properties of the tissue. Fresh bovine mesenteric collecting lymphatic vessels were mounted in a vessel bath and imaged under different luminal pressures (0–30 cmH2O pressure head), and longitudinal tensions. The entire wall thickness was imaged, using two‐photon fluorescence to visualize elastin, second harmonic generation to image the collagen, and coherent anti‐Stokes Raman scattering to image the cell membrane. The adventitial fat cells were coupled to the wall within the elastin‐rich network of fibres. The medial smooth muscle cells were too densely packed to resolve the boundaries of individual cells in en face images, but in tissue sections their appearance was consistent with electron microscopic data. Two distinct populations of collagen fibre were revealed. Large fibre (15–25 μm diameter) bundles were present in the inner media and small fibres (2–5 μm diameter) were distributed throughout the wall. The responses to longitudinal tension and luminal pressure indicated that the larger fibres resist the longitudinal strain and the smaller oppose pressure forces. Individual elastin fibres were of uniform thickness (1–3 μm) and interwove amongst themselves and between the collagen fibres. The network was probably too sparse directly to support mechanical loads and we speculate that its main function is to maintain the organization of collagen bundles during recovery from contraction.

3D Reconstruction of the Glycocalyx Structure in Mammalian Capillaries using Electron Tomography

Please cite this paper as: Arkill KP, Neal CR, Mantell JM, Michel CC, Qvortrup K, Rostgaard J, Bates DO, Knupp C, Squire JM. 3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography. Microcirculation 19: 343–351, 2012.

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

We have developed novel techniques for paired, direct, real‐time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability. Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel. The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth. Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin.

Cerium oxide nanoparticles induce oxidative stress in the sediment-dwelling amphipod Corophium volutator

Abstract Cerium oxide nanoparticles (CeO2 NPs) exhibit fast valence exchange between Ce(IV) and Ce(III) associated with oxygen storage and both pro and antioxidant activities have been reported in laboratory models. The reactivity of CeO2 NPs once they are released into the aquatic environment is virtually unknown, but this is important to determine for assessing their environmental risk. Here, we show that amphipods (Corophium volutator) grown in marine sediments containing CeO2 NPs showed a significant increase in oxidative damage compared to those grown in sediments without NPs and those containing large-sized (bulk) CeO2 particles. There was no exposure effect on survival, but significant increases in single-strand DNA breaks, lipid peroxidation and superoxide dismutase activity were observed after a 10-day exposure to 12.5 mg L−1 CeO2. Characterisation of the CeO2 NPs dispersed in deionised or saline exposure waters revealed that more radicals were produced by CeO2 NPs compared with bulk CeO2. Electron energy loss spectroscopy (EELS) analysis revealed that both CeO2 NPs were predominantly Ce(III) in saline waters compared to deionised waters where they were predominantly Ce(IV). In both types of medium, the bulk CeO2 consisted mainly of Ce(IV). These results support a model whereby redox cycling of CeO2 NPs between Ce(III) and Ce(IV) is enhanced in saline waters, leading to sublethal oxidative damage to tissues in our test organism.

Using size-selected gold clusters on graphene oxide films to aid cryo-transmission electron tomography alignment.

A three-dimensional reconstruction of a nano-scale aqueous object can be achieved by taking a series of transmission electron micrographs tilted at different angles in vitreous ice: cryo-Transmission Electron Tomography. Presented here is a novel method of fine alignment for the tilt series. Size-selected gold clusters of ~2.7 nm (Au561 ± 14), ~3.2 nm (Au923 ± 22), and ~4.3 nm (Au2057 ± 45) in diameter were deposited onto separate graphene oxide films overlaying holes on amorphous carbon grids. After plunge freezing and subsequent transfer to cryo-Transmission Electron Tomography, the resulting tomograms have excellent (de-)focus and alignment properties during automatic acquisition. Fine alignment is accurate when the evenly distributed 3.2 nm gold particles are used as fiducial markers, demonstrated with a reconstruction of a tobacco mosaic virus. Using a graphene oxide film means the fiducial markers are not interfering with the ice bound sample and that automated collection is consistent. The use of pre-deposited size-selected clusters means there is no aggregation and a user defined concentration. The size-selected clusters are mono-dispersed and can be produced in a wide size range including 2–5 nm in diameter. The use of size-selected clusters on a graphene oxide films represents a significant technical advance for 3D cryo-electron microscopy.