F. E. Curry

Oncotic pressures opposing filtration across non-fenestrated rat microvessels

We hypothesized that ultrafiltrate crossing the luminal endothelial glycocalyx through infrequent discontinuities (gaps) in the tight junction (TJ) strand of endothelial clefts reduces albumin diffusive flux from tissue into the ‘protected region’ of the cleft on the luminal side of the TJ. Thus, the effective oncotic pressure difference (σ□π) opposing filtration is greater than that measured between lumen and interstitial fluid. To test this we measured σ□π across rat mesenteric microvessels perfused with albumin (50 mg ml−1) with and without interstitial albumin at the same concentration within a few micrometres of the endothelium as demonstrated by confocal microscopy. We found σ□π was near 70% of luminal oncotic pressure when the tissue concentration equalled that in the lumen. We determined size and frequency of TJ strand gaps in endothelial clefts using serial section electron microscopy. We found nine gaps in the reconstructed clefts having mean spacing of 3.59 μm and mean length of 315 nm. The mean depth of the TJ strand near gaps was 67 nm and the mean cleft path length from lumen to interstitium was 411 nm. With these parameters our three‐dimensional hydrodynamic model confirmed that fluid velocity was high at gaps in the TJ strand so that even at relatively low hydraulic pressures the albumin concentration on the tissue side of the glycocalyx was significantly lower than in the interstitium. The results conform to the hypothesis that colloid osmotic forces opposing filtration across non‐fenestrated continuous capillaries are developed across the endothelial glycocalyx and that the oncotic pressure of interstitial fluid does not directly determine fluid balance across microvascular endothelium.

Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice

Previous experiments using cultured endothelial monolayers indicate that Rho‐family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin‐based contractility and junctional distribution of the Ca2+‐dependent endothelial cell adhesion molecule, VE‐cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng ml−1) inactivated 50–90 % of all endothelial Rho proteins within 60–90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F‐actin in cultured endothelial monolayers and in mouse aortic endothelium in situ. Also, VE‐cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y‐27632 (30 μm) for 90–120 min induced F‐actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE‐cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability (Lp) but did not attenuate the transient increase in Lp of microvessels exposed to bradykinin. Perfusion of microvessels with Y‐27632 (30 μm) for up to 100 min reduced basal Lp but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B‐mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA‐mediated contractile mechanisms are not involved in bradykinin‐ or PAF‐induced hyperpermeability of intact microvessels.

Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge

Multiple processes modulate net blood-to-tissue exchange in a microvascular unit in normal and pathophysiological conditions. These include mechanisms that control the number and type of microvessels perfused, the balance of adhesion and contractile forces that determine the conductance of the spaces between endothelial cells to water and solutes, the pressure and chemical potential gradients determining the driving forces through these conductive pathways, and the organization of barriers to macromolecules in the endothelial glycocalyx. Powerful methods are available to investigate these mechanisms at the levels of cultured endothelial monolayers, isolated microvessels, and the microvascular units within intact organs. Here we focus on current problems that limit the integration of our knowledge of mechanisms investigated in detail at the cellular level into a more complete understanding of modulation of blood-to-tissue exchange in whole organs when the endothelial barrier is exposed to acute and more long-term inflammatory conditions. First, we review updated methods, applicable in mouse models of vascular permeability regulation, to investigate both acute and long-term changes in permeability. Methods to distinguish tracer accumulation due to change in perfusion from real increases in extravascular accumulation are emphasized. The second part of the review compares normal and increased permeability in individually perfused venular microvessels and endothelial cell monolayers. The heterogeneity of endothelial cell phenotypes in the baseline state and after exposure to injury and inflammatory conditions is emphasized. Lastly, we review new approaches to investigation of the glycocalyx barrier properties in cultured endothelial monolayers and in whole-body investigations.

Modulation of venular microvessel permeability by calcium influx into endothelial cells.

It has been proposed that calcium ion influx into endothelial cells modulates the permeability of venular microvessels via a calcium‐dependent contractile process. The results of recent investigations using permeabilized endothelial cell monolayers conform to this hypothesis by demonstrating a calcium‐dependent interaction of endothelial actin and myosin during the retraction of adjacent endothelial cells exposed to inflammatory agents. Little is known about the pathway for calcium influx into endothelial cells after exposure to mediators of inflammation, but evidence suggests that the properties of the calcium entry pathways are similar to the calcium entry pathways that regulate the release of endothelium‐derived relaxing factor (EDRF). Substances that stimulate EDRF release from arterial endothelium also increase venular microvessel permeability. Recently developed methods to measure cytoplasmic calcium concentration in the endothelial cells forming the walls of individually perfused microvessels enable a direct investigation of the modulation of the permeability of venular microvessels by calcium influx. These experiments demonstrate that the magnitude of the initial increase in the permeability of microvessels after exposure to an agent that increases permeability, such as a calcium ionophore, is determined by the magnitude of calcium ion influx into the endothelial cells. Furthermore, the magnitude of the calcium influx into endothelial cells is modulated by the membrane potential of the endothelial cells. Depolarization of the endothelial cell membrane reduces calcium influx and attenuates increases in permeability whereas hyperpolarization of the endothelial membrane increases calcium influx and potentiates increases in permeability. These data conform to the hypothesis that a passive conductance channel for calcium is a major pathway for calcium ion flux responsible to eliciting an increase in the permeability of the endothelial barrier in microvessels.—Curry, F. E. Modulation of venular microvessel permeability by calcium influx into endothelial cells. FASEB J. 6: 2456‐2466; 1992.

Dynamics of Neutrophil Infiltration during Cutaneous Wound Healing and Infection Using Fluorescence Imaging

Neutrophil influx is an early inflammatory response that is essential for the clearance of bacteria and cellular debris during cutaneous wounding. A non-invasive real-time fluorescence imaging technique was developed to examine the kinetics of enhanced green fluorescence protein-polymorphonuclear leukocyte (EGFP-PMN) influx within a wound. We hypothesized that infection or systemic availability would directly regulate the dynamics of EGFP-PMN recruitment and the efficiency of wound closure. Neutrophil recruitment increased dramatically over the first 24 hours from 10(6) at 4 hours up to a maximum of 5 x 10(6) EGFP-PMNs at 18 hours. A high rate of EGFP-PMN turnover was evidenced by approximately 80% decrease in EGFP signal within 6 hours. In response to wound colonization by Staphylococcus aureus or injection of GM-CSF, systemic PMNs increased twofold above saline control. This correlated with an increase in EGFP-PMN recruitment up to approximately 10(7) within the wound. Despite this effect by these distinct inflammatory drivers, wound closure occurred at a rate similar to the saline-treated control group. In summary, a non-invasive fluorescence-based imaging approach combined with genetic labeling of neutrophils provides a dynamic inner view of inflammation and the kinetics of neutrophil infiltration into the wounded skin over extended durations.

Determinants of capillary permeability: a review of mechanisms based on single capillary studies in the frog.

THE objective of this article is to examine two complementary hypotheses that, together, describe the role of the intercellular junction in the regulation of capillary permeability. One hypothesis states that the permeability of capillaries with continuous endothelium is modulated by the area available for the diffusion of small solutes in the spaces between adjacent endothelial cells. The second hypothesis states that the entry of solute into the junctional pathway and the diffusion of solute within the junctional pathway are regulated by the size and distribution of a network of fibrous molecules within the wide part of the junction. Transport through the junction may also be modulated by charge and specific chemical interactions between the solute and side chains on the fibrous network. When evaluating recent research designed to test the two hypotheses, I will draw mainly on experimental data obtained on individually perfused capillaries in frog mesentery and frog muscle. For these capillaries, methods have been developed to measure the permeability properties of the capillary wall under conditions where the area for transcapillary exchange and the forces determining exchange across the capillary wall are directly measured. Many of the problems to be discussed in this review were identified first from experiments on the microvascular bed in whole organs. Studies based on single capillaries enable the mechanisms of exchange across the capillary wall to be studied directly, removing some of the uncertainties that arise when 1) microvessels, having a range of permeability properties, contribute to exchange in a whole microvascular bed, 2) it is not possible to obtain a precise measurement of exchange area, and 3) the forces determining exchange at the capillary wall (hydrostatic pressure difference, solute concentration difference) are estimated indirectly from measurements in large arteries and veins. For a review of the advantages and limitations of single capillary studies see

Microvascular solute and water transport.

Objective: This review evaluate [1] the regulation of water and solute transport across the endothelial barrier in terms of pore theory and the glycocalyx‐junction‐break model of capillary permeability; and [2] the mechanisms regulating permeability based on experiments using cultured endothelial cells and intact microvessels.

A Junction-Orifice-Fiber Entrance Layer Model for Capillary Permeability: Application to Frog Mesenteric Capillaries

The recent serial section electron microscopic studies by Adamson and Michel (1993) on microves gels of frog mesentery have revealed that the large pores in the junction strand of the interendothelial cleft are widely separated 150 nm wide orifice-like breaks whose gap height 20 nm is the same as the wide part of the cleft. In this paper a modified version of the model in Weinbaum et al. (1992) is first developed in which this orifice structure is explored in combination with a random or ordered fiber matrix layer that is at the luminal surface and/or occupies a fraction of the wide part of the cleft. This basic orifice model predicts that for the measured Lp to be achieved the fiber layer must be confined to a relatively narrow region at the entrance to the cleft where it serves as the primary molecular filter. The model provides a much better fit of the permeability P for intermediate size solutes between 1 and 2 nm radius than the previous model in Weinbaum et al., where the junction strand breaks were treated as finite depth circular or rectangular pores, but like the previous model significantly underestimates P for small ions. However, it is shown that if a small frequent pore of 1.5 nm radius with characteristic spacing comparable to the diameter of the junction proteins or a continuous narrow slit of approximately 1.5 to 2.3 nm gap height is also present in the continuous part of the junction strand, small ion permeability can also be satisfied. The 1.5 nm radius pore does not significantly change Lp, whereas the continuous narrow slit provides a contribution to Lp that is comparable to, or in the case of the 2.3 nm slit greater than, the widely spaced 150 nm orifices. Thus, for the narrow slit the contribution to Lp from the orifices can be as low as 1.0 x 10(-7) cm/s/cm H2O and it is also possible to satisfy the 2.5 fold increase in permeability that occurs when the matrix is enzymatically removed from the luminal side of the cleft, Adamson (1990). The likelihood of each of these cleft structures is discussed.

Sphingosine-1-phosphate protects endothelial glycocalyx by inhibiting syndecan-1 shedding.

Endothelial cells (ECs) are covered by a surface glycocalyx layer that forms part of the barrier and mechanosensing functions of the blood-tissue interface. Removal of albumin in bathing media induces collapse or shedding of the glycocalyx. The electrostatic interaction between arginine residues on albumin, and negatively charged glycosaminoglycans (GAGs) in the glycocalyx have been hypothesized to stabilize the glycocalyx structure. Because albumin is one of the primary carriers of the phospholipid sphingosine-1-phosphate (S1P), we evaluated the alternate hypothesis that S1P, acting via S1P1 receptors, plays the primary role in stabilizing the endothelial glycocalyx. Using confocal microscopy on rat fat-pad ECs, we demonstrated that heparan sulfate (HS), chondroitin sulfate (CS), and ectodomain of syndecan-1 were shed from the endothelial cell surface after removal of plasma protein but were retained in the presence of S1P at concentrations of >100 nM. S1P1 receptor antagonism abolished the protection of the glycocalyx by S1P and plasma proteins. S1P reduced GAGs released after removal of plasma protein. The mechanism of protection from loss of glycocalyx components by S1P-dependent pathways was shown to be suppression of metalloproteinase (MMP) activity. General inhibition of MMPs protected against loss of CS and syndecan-1. Specific inhibition of MMP-9 and MMP-13 protected against CS loss. We conclude that S1P plays a critical role in protecting the glycocalyx via S1P1 and inhibits the protease activity-dependent shedding of CS, HS, and the syndecan-1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.

Dynamics of neutrophil extravasation and vascular permeability are uncoupled during aseptic cutaneous wounding

Transport of macromolecules and transmigration of leukocytes across vascular endothelium are regulated by a tight molecular junction, but the mechanisms by which these two inflammatory events are differentially controlled in time and magnitude during aseptic cutaneous wounding remain elusive. A real-time fluorescence imaging technique was developed to simultaneously track influx of Alexa 680-labeled albumin and genetically tagged enhanced green fluorescent protein-neutrophils [polymorphonuclear neutrophils (PMN)] within the wound bed. Vascular permeability increased approximately threefold more rapidly than the rate of PMN influx, reaching a maximum at 12 h, on the order of approximately 0.15% per minute versus approximately 0.05% per minute for PMN influx, which peaked at 18 h. Systemic depletion of PMN with antibody blocked their extravasation to the wound but did not alter the increase in vascular permeability. In contrast, pretreatment with antiplatelet GPIb decreased permeability by 25% and PMN influx by 50%. Hyperpermeability stimulated by the endothelium-specific agonists VEGF or thrombin at 24 h postwounding was completely inhibited by blocking Rho-kinase-dependent signaling, whereas less inhibition was observed at 1 h and neutrophil influx was not perturbed. These data suggest that in aseptic wounds, the endothelium maintains a tight junctional barrier to protein leakage that is independent of neutrophil transmigration, partially dependent on circulating platelets, and associated with Rho-kinase-dependent signaling.