The endothelial glycocalyx is a complex fibrous network of molecules that extends from the endothelial cell membrane for distances that range from less than 0.3 micron to several microns. The mechanisms determining the overall organization of the glycocalyx are still poorly understood, but our understanding is informed by high-resolution methods to analyze periodicity in electron micrographs of the glycocalyx revealed by chemical stains, and super-resolution optical imaging of specific moieties such as heparan sulfate and hyaluronic acid. The interpretation of these images in terms of the permeability properties of the vascular wall requires additional modeling of water and macromolecule flows through ordered molecular structures. While hydrodynamic approaches have been the primary focus of these efforts, extension of the fiber matrix theories that account for the space available to water and solutes within the matrix provides new ways to interpret images where detailed hydrodynamic models are not yet available. A combination of both approaches suggests ways to evaluate the primary molecular filter formed by the spaces between fibers in the glycocalyx in two possible arrangements: Thicker 10-12 nm diameter fibers formed by the core proteins of syndecans and glypicans and their side chains with adsorbed plasma components or thinner (<2 nm diameter) fibers of glycoprotein sidechains that extend above the core protein. This chapter is dedicated to Charles Michel, whose novel experimental methods to investigate the glycocalyx in microvessels, where permeability was measured, and associated theoretical analyses are starting points for the material described in this chapter.

INTRODUCTION:Investigations of responses of animals and humans to changes of plasma volume are usually reported as average responses of groups of individuals. This ignores considerable quantitative variation between individuals. We examined the hypothesis that individual responses follow a common temporal pattern with variations reflecting different parameters describing that pattern. METHODS:We illustrate this approach using data of Hahn, Lindahl and Drobin (Acta Anaesthesiol Scand.2011, 55:987-94) who measured urine volume and haemoglobin dilution of 10 female subjects during intravenous Ringer infusions for 30 min and subsequent 3.5 h. The published time courses were digitised and analysed to determine if a family of mathematical functions accounted for the variation in individual responses. RESULTS:Urine excretion was characterised by a time delay (Td) before urine flow increased and a time course of cumulative urine excretion described by a logarithmic function. This logarithmic relation forms the theoretical basis of a family of linear relations describing urine excretion as a function of Td. Measurement of Td enables estimation of subsequent values of urine excretion and thereby the fraction of infused fluid retained in the body. CONCLUSION:The approach might be useful for physiologists and clinical investigators to compare the response to infusion protocols when both test and control responses can be described by linear relations between cumulative urine volume at specific times and Td. The approach may also be useful for clinicians by complementing strategies to guide fluid therapy by enabling the later responses of an individual to be predicted from their earlier response.

<p>Supplementary Figures S1-S2.</p>

PDF file - 137K, Detailed relationship between the PAP parameter derived through the image-guided model with the classical parameters of solute transport coefficient, clearance, and 30 minute albumin clearance (Cl30).

PDF file - 51K, Representative biodistribution of tissues 48 hours after nanoparticle injection (N=8). Accumulation was significantly greater for insonified tumors (p<0.001).

The primary purpose of these investigations is to integrate our growing knowledge about the endothelial glycocalyx as a permeability and osmotic barrier into models of trans-vascular fluid exchange in whole organs. We describe changes in the colloid osmotic pressure (COP) difference for plasma proteins across the glycocalyx after an increase or decrease in capillary pressure. The composition of the fluid under the glycocalyx changes in step with capillary pressure whereas the composition of the interstitial fluid takes many hours to adjust to a change in vascular pressure. We use models where the fluid under the glycocalyx mixes with sub-compartments of the interstitial fluid (ISF) whose volumes are defined from the ultrastructure of the inter-endothelial cleft and the histology of the tissue surrounding the capillaries. The initial protein composition in the sub-compartments is that during steady state filtration in the presence of a large pore pathway in parallel with the “small pore” glycocalyx pathway. Changes in the composition depend on the volume of the sub-compartment and the balance of convective and diffusive transport into and out of each sub-compartment. In skeletal muscle the simplest model assumes that the fluid under the glycocalyx mixes directly with a tissue sub-compartment with a volume less than 20% of the total skeletal muscle interstitial fluid volume. The model places limits on trans-vascular flows during transient filtration and reabsorption over periods of 30–60 min. The key assumption in this model is compromised when the resistance to diffusion between the base of the glycocalyx and the tissue sub-compartment accounts for more than 1% of the total resistance to diffusion across the endothelial barrier. It is well established that, in the steady state, there can be no reabsorption in tissue such as skeletal muscle. Our approach extends this idea to demonstrate that transient changes in vascular pressure favoring initial reabsorption from the interstitial fluid of skeletal muscle result in much less fluid exchange than is commonly assumed. Our approach should enable critical evaluations of the empirical models of trans-vascular fluid exchange being used in the clinic that do not account for the hydrostatic and COPs across the glycocalyx.

AIM:The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium.METHODS:In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin.RESULTS:Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide.CONCLUSION:Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.

The Starling Principle states that fluid movements between blood and tissues are determined by differences in hydrostatic and colloid osmotic (oncotic) pressures between plasma inside microvessels and fluid outside them. The Revised Starling Principle recognizes that, because microvessels are permeable to macromolecules, a balance of pressures cannot halt fluid exchange. In most tissues, steady oncotic pressure differences between plasma and interstitial fluid depend on low levels of steady filtration from plasma to tissues for which the Revised Principle provides the theory. Plasma volume is normally maintained by fluid losses from filtration being matched by fluid gains from lymph. Steady state fluid uptake into plasma only occurs in tissues such as intestinal mucosa and renal peri-tubular capillaries where a protein-free secretion of adjacent epithelia contributes significantly to interstitial fluid volume and keeps interstitial oncotic pressure low. Steady filtration rates in different tissues are disturbed locally by reflex changes in capillary pressure and perfusion. The rapid overall decline in capillary pressure after acute blood loss initiates rapid fluid uptake from tissue to plasma, that is, autotransfusion. Fluid uptake is transient, being rapid at first then attenuating but low levels may continue for more than an hour. The Revised Principle highlights the role of oncotic pressure of small volumes of interstitial fluid within a sub-compartment surrounding the microvessels rather than the tissue's mean interstitial fluid oncotic pressure. This maximizes oncotic pressure differences when capillary pressure are high and enhances initial absorption rates when pressures are low, accelerating short-term regulation of plasma volume.

Regulation of intercellular junctional complexes is critical for the control of endothelial barrier function and dependent on cAMP‐mediated Rac1 activation. Recently, the exchange protein activated by cAMP (Epac1) has been shown to serve as a tonic stabilizer of the endothelial barrier. Here, we further elucidate the role of Epac1 in cAMP and Rac1‐mediated endothelial barrier regulation.Transendothelial‐electrical‐resistance (TER) measurements in newly generated immortalized myocardial endothelial cells derived from Epac1‐knockout (KO) mice revealed significant reduction in the baseline TER, when compared to wild‐type (WT) cells, indicating that Epac1 is required for maintenance of endothelial barrier function. This effect was associated with fragmented VE‐cadherin staining and reduced localization of tight junctional proteins ZO‐1 and occludin along cell borders. In WT‐cells, application of forskolin (F) and rolipram (R) resulted in increased TER associated with linearization and augmentation of VE‐cadherin immunostaining at adherens junctions. In contrast, Epac1‐KO‐cells did not respond to F/R treatment indicating that Epac1 is crucial for cAMP‐mediated endothelial barrier stabilization. In comparison to WT‐cells, Epac1‐KO‐cells revealed significantly increased protein levels of Rac1, whereas Rac1 basal activity was not enhanced, indicating a defect in Rac1 activation. However, in Epac1‐KOcells, cAMP‐mediated Rac1 activation was not abolished and the basal cAMP‐concentration was significantly increased. Additionally, in Epac1‐deficient cells F/R‐mediated increase in cAMP levels was less compared to WT cells. Taken together, our data show that Epac1 is crucial for cAMP‐mediated barrier stabilization by mechanisms which at least in part are independent of Rac1 regulation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ultrastructural investigations of the endothelial glycocalyx reveal a layer adjacent to the cell surface with a structure consistent with the primary ultrafilter of vascular walls. Theory predicts this inner layer can be no greater than 200–300 nm thick, a result to be reconciled with observations that red cells and large macromolecules are excluded from a region up to 1 micron or more from the cell membrane. We tested whether this apparent inconsistency might be accounted for by a model of steady state water and protein transport through a glycocalyx bi‐layer. Expressions for coupled water and albumin fluxes through the two layers were used to describe steady state ultrafiltration. The model demonstrated that albumin may accumulate at the interface between the porous layer and the selective inner layer. The osmotic pressure of accumulated albumin would significantly modify the observed permeability properties of the microvessel wall by an effective unstirred layer effect. Our results place constraints on the outer layer permeability properties. For example, with an perfusate albumin concentration of 50 mg/ml, Adamson et al ( Am. J. Physiol. 557: 889–907,2004) reported that the hydraulic conductivity measured in rat mesenteric microvessels under conditions of steady state ultrafiltration were similar to that measured before steady ultrafiltration was established. The only properties of the outer layer consistent with these observations are albumin permeability coefficients and hydraulic conductivities close to an order of magnitude larger than that of the inner layer. A high filtration coefficient for the outer layer is also consistent with estimates of the hydraulic conductance of the outer layer needed to account for red cell movement over the endothelial surface up to 1 micron thick.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AIM:Epac1-/- mice, but not Epac2-/- mice have elevated baseline permeability to albumin. This study extends the investigations of how Epac-dependent pathways modulate transvascular exchange in response to the classical inflammatory agent histamine. It also evaluates the limitations of models of blood-to-tissue exchange in transgenic mice in DCE-MRI measurements.METHODS:We measured DCE-MRI signal intensity in masseter muscle of wt and Epac1-/- mice with established approaches from capillary physiology to determine how changes in blood flow and vascular permeability contribute to overall changes of microvascular flux. We used two tracers, the high molecular weight tracer (Gadomer-17, MW 17 kDa, apparent MW 30-35 kDa) is expected to be primarily limited by diffusion and therefore less dependent on changes in blood flow and the low molecular weight tracer (Dotarem (MW 0.56 kDa) whose transvascular exchange is determined by both blood flow and permeability. Paired experiments in each animal combined with analytical methods provided an internally consistent description of microvascular transport.RESULTS:Epac1-/- mice had elevated baseline permeability relative to wt control mice for Dotarem and Gadomer-17. In contrast to wt mice, Epac1-/- mice failed to increase transvascular permeability in response to histamine. Dotarem underestimated blood flow and vascular volume and Gadomer-17 has limited sensitivity in extravascular accumulation.CONCLUSION:The study suggests that the normal barrier loosening effect of histamine in venular microvessels do not function when the normal barrier tightening effect of Epac1 is already compromised. The study also demonstrated that the numerical analysis of DCE-MRI data with tracers of different molecular weight has significant limitations.

There has been rapid progress over the past decade to extend the concept that a quasiperiodic inner endothelial glycocalyx layer (EGL, < 300 nm thick, with key components associated with the endothelial cell membrane) forms the primary molecular filter between circulating blood and the body tissues. The EGL is common to both continuous and fenestrated microvessels. The revised Starling Principle describing steady-state fluid exchange across the EGL describes new ways to understand transvascular exchange of water and plasma proteins in microvessels in both normal and disturbed states such as hemorrhage and fluid replacement during surgery. At the same time, direct optical observations describe endothelial surface layers (ESLs) with porous outer layers that extend 1-2 mu m beyond the EGL. Preliminary analyses of water and plasma protein transport through barriers formed by a thick ESL in series with the EGL indicate that such two-layer structures can have permeability properties that are not consistent with measured water and plasma exchange in microvessels. Such multilayer models provide a basis for future detailed evaluations of both transports across endothelial surface layers and the methods to image components of both the EGL and the ESL. Furthermore changes in the thickness and distribution of thick ESLs in vessels with diameters larger than 50 mu m may not reflect functional changes in the inner glycocalyx layer.

The paper by Lukasz et al . 1 in this issue contributes to the growing understanding of the role of the glycocalyx in microvascular function, not only because it describes a novel role for angiopoietin-2 (Angpt-2) in the enzymatic degradation of the heparan sulfate side chains of endothelial glycocalyx glycoproteins, but also because it demonstrates both the strengths and limitations of translating observations of the glycocalyx made on cultured endothelial cell monolayers to intact microvasculature function. Angpt-2 is a natural Tie2 endothelial receptor antagonist, which, by opposing the stabilizing functions of the growth factor angiopoietin-1 (Angtp-1), has been implicated in various forms of microvascular dysfunction and disease states such as sepsis where it can be present at concentrations up to 50 ng/mL. The current model of the endothelial glycocalyx describes an inner dense matrix layer, associated with membrane-attached glycoproteins (up to 200–300nm thick) that forms a primary selective barrier to plasma macromolecules, and an outer less dense layer that may extend one or more microns into the vessel lumen,

We tested the hypothesis that the resistance to fluid movement within the endothelial glycocalyx is better described by models with an inner semi‐periodic structure and an outer less porous layer, likely stabilized by hyaluronic acid. Previous detailed hydrodynamic modeling has demonstrated that periodic structures close to the membrane, characteristic of membrane‐bound glycoproteins in a quasi‐periodic array extending 200–300nm from the endothelial membrane surface and having a significant resistance to water flow (Darcy coefficient (Kw) in the range 10 −13 to 10 −14 cm 2 s) are sufficient to describe both the selectivity and permeability of the glycocalyx to macromolecules and an effective lubrication layer mechanism for red cells. However, a thicker glycocalyx of similar uniform composition does not provide the same consistent description of both permeability and red cell mechanics (Annals of Biomedical Eng. 40: 828,2011). The problem is resolved if the outer layers of the glycocalyx (thickness L) have less resistance to water flows. For example, the criterion for an effective lubrication layer (L/Kw 0.5 >100) is met with a outer glycocalyx thickness up to 1 micron but with a hydraulic resistance more that an order of magnitude less than the inner core. These observations are consistent with the conclusion that the fraction of the circulating plasma volume that partitions within the glycocalyx is much smaller than estimates based on the characteristics of red cell flows, total glycocalyx thickness, and glycocalyx volume (for review and clinical significance see Chapters 2 and 3 of Perioperative Fluid Management (E. Farag and A. Kurz Eds) Springer 2016)

Temperature-sensitive liposomal formulations of chemotherapeutics, such as doxorubicin, can achieve locally high drug concentrations within a tumor and tumor vasculature while maintaining low systemic toxicity. Further, doxorubicin delivery by temperature-sensitive liposomes can reliably cure local cancer in mouse models. Histological sections of treated tumors have detected red blood cell extravasation within tumors treated with temperature-sensitive doxorubicin and ultrasound hyperthermia. We hypothesize that the local release of drug into the tumor vasculature and resulting high drug concentration can alter vascular transport rate constants along with having direct tumoricidal effects. Dynamic contrast enhanced MRI (DCE-MRI) coupled with a pharmacokinetic model can detect and quantify changes in such vascular transport rate constants. Here, we set out to determine whether changes in rate constants resulting from intravascular drug release were detectable by MRI. We found that the accumulation of gadoteridol was enhanced in tumors treated with temperature-sensitive liposomal doxorubicin and ultrasound hyperthermia. While the initial uptake rate of the small molecule tracer was slower (k1=0.0478±0.011s-1 versus 0.116±0.047s-1) in treated compared to untreated tumors, the tracer was retained after treatment due to a larger reduction in the rate of clearance (k2=0.291±0.030s-1 versus 0.747±0.24s-1). While DCE-MRI assesses a combination of blood flow and permeability, ultrasound imaging of microvascular flow rate is sensitive only to changes in vascular flow rate; based on this technique, blood flow was not significantly altered 30min after treatment. In summary, DCE-MRI provides a means to detect changes that are associated with treatment by thermally-activated particles and such changes can be exploited to enhance local delivery.

Spontaneous formation of defects in the walls of leaky tumour blood vessels may explain the increased accumulation of large nanoparticles in certain tumours.