The Biomechanical Effects of Resuscitation Colloids on the Compromised Lung Endothelial Glycocalyx.

BACKGROUND: The endothelial glycocalyx is an important component of the vascular permeability barrier, forming a scaffold that allows serum proteins to create a gel-like layer on the endothelial surface and transmitting mechanosensing and mechanotransduction information that influences permeability. During acute inflammation, the glycocalyx is degraded, changing how it interacts with serum proteins and colloids used during resuscitation and altering its barrier properties and biomechanical characteristics. We quantified changes in the biomechanical properties of lung endothelial glycocalyx during control conditions and after degradation by hyaluronidase using biophysical techniques that can probe mechanics at (1) the aqueous/glycocalyx interface and (2) inside the glycocalyx. Our goal was to discern the location-specific effects of albumin and hydroxyethyl starch (HES) on glycocalyx function. METHODS: The effects of albumin and HES on the mechanical properties of bovine lung endothelial glycocalyx were studied using a combination of atomic force microscopy and reflectance interference contrast microscopy. Logistic regression was used to determine the odds ratios for comparing the effects of varying concentrations of albumin and HES on the glycocalyx with and without hyaluronidase. RESULTS: Atomic force microscopy measurements demonstrated that both 0.1% and 4% albumin increased the thickness and reduced the stiffness of glycocalyx when compared with 1% albumin. The effect of HES on glycocalyx thickness was similar to albumin, with thickness increasing significantly between 0.1% and 1% HES and a trend toward a softer glycocalyx at 4% HES. Reflectance interference contrast microscopy revealed a concentration-dependent softening of the glycocalyx in the presence of albumin, but a concentration-dependent increase in stiffness with HES. After glycocalyx degradation with hyaluronidase, stiffness was increased only at 4% albumin and 1% HES. CONCLUSIONS: Albumin and HES induced markedly different effects on glycocalyx mechanics and had notably different effects after glycocalyx degradation by hyaluronidase. We conclude that HES is not comparable with albumin for studies of vascular permeability and glycocalyx-dependent signaling. Characterizing the molecular and biomechanical effects of resuscitation colloids on the glycocalyx should clarify their indicated uses and permit a better understanding of how HES and albumin affect vascular function.

Exploiting differential surface display of chondroitin sulfate variants for directing neuronal outgrowth.

Chondroitin sulfate (CS) proteoglycans (CSPGs) are known to be primary inhibitors of neuronal regeneration at scar sites. However, a variety of CSPGs are also involved in neuronal growth and guidance during other physiological stages. Sulfation patterns of CS chains influence their interactions with various growth factors in the central nervous system (CNS), thus influencing neuronal growth, inhibition, and pathfinding. This report demonstrates the use of differentially sulfated CS chains for neuronal navigation. Surface-immobilized patterns of CS glycosaminoglycan chains were used to determine neuronal preference toward specific sulfations of five CS variants: CS-A, CS-B (dermatan sulfate), CS-C, CS-D, and CS-E. Neurons preferred CS-A, CS-B, and CS-E and avoided CS-C containing lanes. In addition, significant alignment of neurites was observed using underlying lanes containing CS-A, CS-B, and CS-E chains. To utilize differential preference of neurons toward the CS variants, a binary combinations of CS chains were created by backfilling a neuro-preferred CS variant between the microcontact printed lanes of CS-C stripes, which are avoided by neurons. The neuronal outgrowth results demonstrate for the first time that a combination of sulfation variants of CS chains without any protein component of CSPG is sufficient for directing neuronal outgrowth. Biomaterials with surface immobilized GAG chains could find numerous applications as bridging devices for tackling CNS injuries where directional growth of neurons is critical for recovery.

Use of reflectance interference contrast microscopy to characterize the endothelial glycocalyx stiffness.

Reflectance interference contrast microscopy (RICM) was used to study the mechanics of the endothelial glycocalyx. This technique tracks the vertical position of a glass microsphere probe that applies very light fluctuating loads to the outermost layer of the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx. Fluctuations in probe vertical position are used to estimate the effective stiffness of the underlying layer. Stiffness was measured before and after removal of specific glycocalyx components. The mean stiffness of BLMVEC glycocalyx was found to be ~7.5 kT/nm(2) (or ~31 pN/nm). Enzymatic digestion of the glycocalyx with pronase or hyaluronan with hyaluronidase increased the mean effective stiffness of the glycocalyx; however, the increase of the mean stiffness on digestion of heparan sulfate with heparinase III was not significant. The results imply that hyaluronan chains act as a cushioning layer to distribute applied forces to the glycocalyx structure. Effective stiffness was also measured for the glycocalyx exposed to 0.1%, 1.0%, and 4.0% BSA; glycocalyx compliance increased at two extreme BSA concentrations. The RICM images indicated that glycocalyx thickness increases with BSA concentrations. Results demonstrate that RICM is sensitive to detect the subtle changes of glycocalyx compliance at the fluid-fiber interface.

Multiprotein microcontact printing with micrometer resolution.

Depositing multiple proteins on the same substrate in positions similar to the natural cellular environment is essential to tissue engineering and regenerative medicine. In this study, the development and verification of a multiprotein microcontact printing (µCP) technique is described. It is shown that patterns of multiple proteins can be created by the sequential printing of proteins with micrometer precision in registration using an inverted microscope. Soft polymeric stamps were fabricated and mounted on a microscope stage while the substrate to be stamped was placed on a microscope objective and kept at its focal distance. This geometry allowed for visualization of patterns during the multiple stamping events and facilitated the alignment of multiple stamped patterns. Astrocytes were cultured over stamped lane patterns and were seen to interact and align with the underlying protein patterns.

Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy.

The mechanical properties of endothelial glycocalyx were studied using atomic force microscopy with a silica bead (diameter ∼18 µm) serving as an indenter. Even at indentations of several hundred nanometers, the bead exerted very low compressive pressures on the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx and allowed for an averaging of stiffness in the bead-cell contact area. The elastic modulus of BLMVEC glycocalyx was determined as a pointwise function of the indentation depth before and after enzymatic degradation of specific glycocalyx components. The modulus-indentation depth profiles showed the cells becoming progressively stiffer with increased indentation. Three different enzymes were used: heparinases III and I and hyaluronidase. The main effects of heparinase III and hyaluronidase enzymes were that the elastic modulus in the cell junction regions increased more rapidly with the indentation than in BLMVEC controls, and that the effective thickness of glycocalyx was reduced. Cytochalasin D abolished the modulus increase with the indentation. The confocal profiling of heparan sulfate and hyaluronan with atomic force microscopy indentation data demonstrated marked heterogeneity of the glycocalyx composition between cell junctions and nuclear regions.

Using microcontact printing of fibrinogen to control surface-induced platelet adhesion and activation.

The ability to promote or inhibit specific platelet-surface interactions in well-controlled environments is crucial to studying fundamental adhesion and activation mechanisms. Here, microcontact printing was used to immobilize human fibrinogen covalently in the form of randomly placed, micrometer-sized islands at an overall surface coverage of 20, 50, or 85%. The nonprinted background region was blocked with covalently immobilized human albumin. Platelet adhesion and morphology on each substrate were assessed using combined differential interference and fluorescence microscopy. At 20% coverage, most of the fibrinogen surface features were small round islands, and platelet adhesion and spreading areas were limited by the position and the size of the islands. Platelet circularity, indicated the morphology was mostly rounded. At 50% coverage, some fibrinogen islands coalesced and platelet adhesion and spreading areas increased. Platelet morphology was controlled by the shape of underlying fibrinogen islands, leading to more irregular spreading. At 85% coverage, the fibrinogen pattern was completely interconnected and both platelet adhesion and the spreading area were significantly higher than at lower coverage. In addition, platelets also spread over the albumin regions, suggesting that after a critical surface density of fibrinogen ligands is reached, platelet spreading is no longer inhibited by albumin. Increasing the overall fibrinogen coverage resulted in higher activation levels defined by key morphological characteristics of the spreading platelet.

Microfluidic assay of antiplatelet agents for inhibition of shear-induced platelet adhesion and activation.

We have developed a microfluidic system to perfuse whole blood through a flow channel with an upstream stenotic region and a downstream protein capture region. This flow-based system was used to assay how effectively antiplatelet agents suppress shear-induced platelet adhesion and activation downstream of the stenotic region. Microcontact printing was used to covalently attach one of three platelet binding proteins [fibrinogen, collagen, or von Willebrand factor (vWf)] to the surface of the downstream capture region. Whole blood with an antiplatelet agent was transiently exposed to an upstream high wall shear rate (either 4860 s-1 or 11 560 s-1), and subsequently flowed over the downstream capture region where the platelet adhesion was measured. Several antiplatelet agents (acetylsalicylic acid, tirofiban, eptifibatide, anti-vWf, and anti-GPIbα) were evaluated for their efficacy in attenuating downstream adhesion. Following antibody blocking of vWf or GPIbα, downstream platelet activation was also assessed in perfused blood by flow cytometry using two activation markers (active GPIIb/IIIa and P-selectin). Acetylsalicylic acid demonstrated its inability to diminish shear-induced platelet adhesion to all three binding proteins. GPIIb/IIIa inhibitors (tirofiban and eptifibatide) significantly reduced platelet adhesion to fibrinogen. Antibody blocking of vWf or GPIbα effectively diminished platelet adhesion to all three capture proteins as well as platelet activation in perfused blood, indicating an essential role of vWf-GPIbα interaction in mediating shear-induced platelet aggregation.

Effect of upstream priming on transient downstream platelet-substrate interactions.

Upstream exposure of platelets to activating proteins 'primes' platelets for increased downstream adhesion, though the mechanics of platelet translocation before permanently arresting are not well understood. To investigate platelet translocation on platelet-binding proteins, primed platelets' transient contacts with immobilized proteins were recorded and analyzed. Using a microfluidic channel, representative of a vascular graft, platelet-activating proteins were covalently attached to the upstream priming, center, and downstream capture positions. Image sequences of platelet interactions with the center protein were captured as platelet-rich plasma (PRP) was perfused through the channel. There was an increase in both platelet pause events and net platelet adhesion on von Willebrand factor, collagen, or fibrinogen following upstream exposure to the same protein. Upstream priming also caused a decrease in average platelet velocity. The duration of transient platelet arrests on the protein-coated surface and the distance that platelets travel between pause events depended on the protein with which they were interacting. The most significant increase in platelet pause events frequency and decrease in average velocity occurred on immobilized von Willebrand factor, compared to the control with no upstream priming. These results demonstrate that platelet priming increases downstream platelet-protein interactions prior to permanent adhesion.

Spatial variation of the charge and sulfur oxidation state in a surface gradient affects plasma protein adsorption.

A gradient of negative surface charge based on the 1D spatial variation from surface sulfhydryl to mixed sulfhydryl-sulfonate moieties was prepared by the controlled UV oxidation of a 3-mercaptopropylsilane monolayer on fused silica. The adsorption of three human plasma proteins--albumin (HSA), immunoglobulin G (IgG), and fibrinogen (Fgn)--onto such a surface gradient was studied using spatially resolved total internal reflection fluorescence (TIRF) and autoradiography. Adsorption was measured from dilute solutions equivalent to 1/100 (TIRF, autoradiography), 1/500, and 1/1000 (autoradiography) of protein physiological concentrations in plasma. All three proteins adsorbed more to the nonoxidized sulfhydryl region than to the oxidized, mixed sulfhydryl-sulfonate region of the gradient. In the case of HSA, the adsorption contrast along the gradient was largest when the adsorption took place from more dilute protein solutions. Increasing the concentration to 1/100 of the protein plasma concentration eliminated the effect of the gradient on HSA adsorption and, to the lesser extent, on IgG adsorption. In the case of Fgn, the greatest adsorption contrast was observed at the highest concentration used. On the basis of adsorption kinetics, the estimated binding affinity of HSA for the sulfhydryl region was twice the affinity for the mixed sulfhydryl-sulfonate region of the gradient. For IgG and Fgn, the initial adsorption was transport-limited and the initial adsorption rates approached the computed flux of the protein to the surface.

Effects of elapsed time on downstream platelet adhesion following transient exposure to elevated upstream shear forces.

Transient exposure to elevated shear forces is known to prime platelets for enhanced downstream adhesion, but how far downstream these priming effects persist is not known. In the present study, the platelet capture regions, prepared by immobilizing fibrinogen, collagen, or von Willebrand factor, were placed at three different distances from the upstream stenotic region to vary the elapsed time of circulating platelets downstream. Platelet adhesion increased with the increase of upstream wall shear rates from 1620 s-1 to 11,560 s-1 for all three downstream proteins, but only the adhesion to fibrinogen increased significantly with the distance between the upstream stenotic region and the downstream capture region. In contrast, platelet adhesion to downstream collagen remained essentially independent on the distance and the adhesion to von Willebrand factor marginally increased with the distance after transient platelet exposure to upstream wall shear rates of 2145 s-1 and 11,560 s-1. The results implied that the activation of fibrinogen receptor GPIIb/IIIa by transient exposure to high upstream wall shear rates progresses in a time-dependent manner during the downstream flow of platelets. The highly elevated upstream wall shear rate of 11,560 s-1 altered the morphology of many platelets adhered to downstream fibrinogen from their native ellipsoidal to spread circular form. The platelet shape analysis showed that longer periods of post-stenotic flow increased the surface coverage fraction of ellipsoidal platelet population and decreased the surface coverage fraction of fully spread platelets on fibrinogen for both transiently elevated upstream wall shear rates.