Hyaluronidase had no effect on the effective stiffness parameter for albumin at either 1% (P = 0.063) or 4% (P = 0.170; Figure 9A). Likewise, hyaluronidase had no effect on effective stiffness in the presences of 4% HES compared with untreated control cells (Figure 9B, P = 0.468).
Overview of Results
The results of this study demonstrate complex and anomalous effects of 2 different colloids (albumin versus HES) on biomechanical properties of the lung endothelial glycocalyx. These findings provide unique insight into the disparate site-dependent effects of albumin and starch on the interior of the glycocalyx (assessed by AFM) and at the glycocalyx—fluid interface (assessed by RICM). These variable, site-dependent effects of colloids may affect barrier function, mechanotransduction, and vascular permeability differently in the intact glycocalyx, leading to divergent clinical properties. We found that, during severe inflammatory states when clinicians are most likely to consider colloid use, the glycocalyx is compromised by proteases and other glycoprotein-degrading processes, and under such conditions, albumin has opposing effects versus HES on biomechanical properties. Collectively, these results suggest that HES is not comparable with albumin when considering their biomechanical effects on the glycocalyx or on the interactions of each colloid with the glycocalyx, per se, and therefore, the effects of colloids on glycocalyx-dependent processes that influence vascular physiology.
Atomic Force Microscopy
We previously established 2 complementary techniques to evaluate the soft-layer mechanics of the glycocalyx11,32: RICM and atomic force microscopy (AFM) each use an 18-μm diameter spherical probe to impart a loading force onto the glycocalyx. AFM measures mechanical properties with progressive indentation to approximately 700 nm into the glycocalyx, whereas RICM measures equilibrium mechanics at much shallower (<10 nm) depths and with very fast indentation rates. These techniques have allowed us to analyze glycocalyx mechanics and to quantify the mechanical effects of albumin and HES over a clinically relevant range. In addition, to simulate glycocalyx degradation as might occur during acute inflammation, we measured the mechanical effects of albumin and HES on the glycocalyx in the presence of hyaluronidase.11,28
In this study, we determined the elastic modulus, E, of BLMVEC monolayers. As evident in Figure 3, albumin was associated with a softer glycocalyx (lower E), whereas HES resulted in an increase in glycocalyx stiffness at indentations depths from 300 to 700 nm. This effect is, in part, attributed to a reduction in the thickness of the glycocalyx in the presence of HES (Table 3) because the AFM loading forces are more effectively transmitted to the cell cytoskeleton. By using depolymerization of actin with cytochalasin D in BMLVEC, we previously determined that, at indentations depths deeper than 200 to 300 nm, AFM measurements largely evaluate cytoskeleton mechanics. On the basis of those data, we conclude that the glycocalyx is approximately 10 times softer than the underlying cellular structures. Given limitations in vertical AFM resolution, we assigned the glycocalyx modulus at the indentation depth of 100 nm (E100) as a measure of glycocalyx stiffness (Figure 6). For each colloid, the 1% concentration resulted in the stiffest mean elastic modulus, and increasing or decreasing the albumin or HES concentration produced a softer glycocalyx.
Glycocalyx thickness (δg) was greater with albumin than with HES. In the presence of 1% albumin, δg = 532 ± 102 nm vs 317 ± 38 nm with 1% HES. These changes in the glycocalyx thickness were best revealed by analyzing ΔE(δ) curves as shown in Figures 4 and 5. A ΔE(δ) < 0 (Figure 4, A and B) indicates glycocalyx thickening, and our data suggest that reducing albumin from 1% to 0.1% doubled glycocalyx thickness (at 0.1 %, δg = 1267 ± 68 nm vs at 1 %, δg = 532 ± 102 nm; Table 4 and Figure 4A). The 2-fold increase in glycocalyx thickness is most likely caused by a reduced number of albumin-dependent cross-links between HA and other glycocalyx components, allowing the glycosaminoglycan (GAG) chains to become unrestrained. Interestingly, an increase in albumin concentration from 1% to 4% also increased the glycocalyx thickness, from 532 ± 102 nm to 910 ± 49 nm, respectively (Table 4). This effect on glycocalyx thickness likely occurs as a result of excess albumin molecules within the glycocalyx and additional water and electrolytes that osmotically accompany albumin. The functional relationship that emerges between albumin concentration and glycocalyx thickness can be summarized as follows: at 1% albumin, the glycocalyx is in a cross-linked or restrained state. When albumin concentration is reduced to 0.1%, the albumin-dependent cross-linking is lost, and the GAG chains become unrestrained, increasing glycocalyx thickness. When albumin concentration is increased to 4%, the excess albumin, water, and electrolytes induce a swelling effect that also increases glycocalyx thickness. Thus, at both high and low albumin concentrations, the glycocalyx structure thickens, but by different mechanisms.
RICM provided detailed assessment at the outermost regions of the glycocalyx, eg, at the aqueous–fiber interface. Albumin induced a concentration-dependent softening of the glycocalyx over a 0.1% to 4% range. Conversely, HES increased the effective stiffness over a 0% to 4% range. These results differ from measurements made by AFM from the interior of the glycocalyx. The easiest explanation for these findings is that outermost regions of the glycocalyx have different structural components and, therefore, interact differently with albumin and HES. However, these dichotomous findings might also have resulted from the difference in the speed with which loading forces were applied by AFM versus RICM. The AFM loading rate was quite low compared with the fast fluctuations in RICM. Because of these loading rate differences, the RICM-measured stiffness could have been influenced by viscous forces and related molecular rearrangements in a different way compared with the AFM modulus. Thus, the potential role of loading rates and viscous forces add an additional level of mechanical interactions that may influence endothelial signaling.
Are There Other Effects of Albumin and HES that May Explain These Data?
It is possible that increasing the colloid oncotic pressure altered cell physiology sufficiently to also alter cell surface biomechanics. Chiang et al33 observed that polyethylene glycol, a supposedly inert neutral polymer, had a biphasic effect on endothelial permeability and actin organization, increasing transendothelial electrical resistance over the range of 0% to 8%, but inducing barrier dysfunction above 8%. The increase in electrical resistance was associated with a reduction of actin stress fiber and an increase in peripheral actin. The effect of polyethylene glycol on trans endothelial electrical resistance (TEER) and actin reorganization were complete in approximately 1 hour. In contrast, in our study, HES had a biphasic effect of glycocalyx stiffness over the range of 0% to 4%. Along with the increase in E100, which should exclude a cytoskeletal mechanism, this finding suggests that HES has direct effects on glycocalyx mechanics. However, in Figure 4, 4% HES also affected cell mechanics at depths below 300 nm, which may indicate a cytoskeletal effect on the cytoskeleton. More work is needed to understand the effects on colloids on cytoskeletal mechanics.
Glycocalyx Degradation: The Effect on Biomechanics
During acute inflammation, glycocalyx constituents are shed by plasma proteases, neutrophil proteases, and endothelial membrane-associated metalloproteases.23,34 For example, activated neutrophils release proteases and have membrane-bound proteases that participate in neutrophil transmigration out of the vascular system. Major constituents of the glycocalyx, such as syndecan, can be cleaved by several common proteases.35 Plasma syndecan-1 has become a marker for the magnitude of vascular injury after trauma and hemorrhagic shock,17,19 and plasma GAGs can be a signature for respiratory failure.18
On the basis of our previous biophysical characterization of BLMVEC glycocalyx, we concluded that HA is a major structural constituent and, on its removal from the glycocalyx, albumin dynamics within the glycocalyx was altered dramatically.24 Likewise, others have demonstrated that HA removal significantly alters endothelial function.36,37 Therefore, we used hyaluronidase to selectively remove HA to mimic the degradation of the glycocalyx that may occur during acute inflammation. The removal of HA altered both the physical and mechanical properties of glycocalyx. The fitted thickness parameter (Table 4) indicated that HA caused a reduction in the glycocalyx thickness from 532 ± 102 nm (1% albumin) to 450 ± 4 and 322 ± 4 nm, for 1% and 4% albumin, respectively. The changes in the fitted glycocalyx modulus (Eglycocalyx, Table 4) also agreed very well with the RICM-measured effective stiffness of the outermost part of the glycocalyx layer (Figure 8B); both parameters decreased significantly with 4% albumin in the absence of HA (compared with 1% albumin before or after removal of HA).
The changes in effective stiffness for 4% HES before and after removal of HA were statistically insignificant (Figure 9B), as were changes in the fitted δg parameter at these conditions (Figure 5, B and C and Table 4). Overall, the mechanical parameters measured after HA removal from the glycocalyx support our previous observation that the HA chains are a major component of the glycocalyx and are responsible for interactions with albumin.24 These results also support the conclusion that HA chains are inert toward HES. Finally, our results support the findings of Zeng et al,25 who demonstrated that enzymatic removal of HA reduced the thickness of the cell surface albumin layer on bovine aortic endothelial cells.
The primary limitation to our study is the necessity to conduct these experiments using an in vitro model. This methodology is both a strength and weakness, because RICM and AFM make precise measurements on a scale that is otherwise unattainable in vivo. However, there is controversy as to the composition and thickness of the glycocalyx of cultured cells versus its in vivo state. Measurements of the glycocalyx in vivo range from 0.4 to 5.0 μm,38–40 depending on the vascular bed and species studied. The controversy regarding the use of cell culture models is also driven by the type of endothelial cells chosen for comparison. For example, based on electron microscopy and micro-velocimetry, human umbilical vein endothelial cells (HUVECs) have little, if any, glycocalyx.41 This finding should not be surprising, because HUVECs are derived from a conduit vessel where permeability of the umbilical vein has little physiologic significance, limiting the value of HUVECs for studies on barrier properties.
We and others6,24,25,40,42 have used a variety of methodologies, including fluorescence correlation spectroscopy (FCS), rapid freezing/freeze substitution transmission electron microscopy, and high-resolution confocal microscopy, to demonstrate that cultured endothelial cells from the bovine lung (BLMVEC), bovine aorta (bovine aortic endothelial cell, BAEC), and rat pad (rat fat pad endothelial cell, RFPEC) have a glycocalyx comparable in thickness with the in vivo state. By using rapid freezing/freeze substitution transmission electron microscopy, Ebong et al40 reported a thickness of 11 μm for BAEC glycocalyx and 5 μm for rat fad pad endothelial cells. However, they used standard fixation techniques on these cells and observed a significant decrease in thickness of the glycocalyx. Thus, some forms of processing and fixation alter the measurable thickness. We used FCS, a biophysical modality that has submicron resolution, on live BLMVEC and measured a glycocalyx thickness of 1 to 3 μm. Using confocal microscopy and immunostaining for heparan sulfates, we observed a cell surface layer that was approximately 3.0-μm thick, providing good correlation with FCS. Tarbell and coworkers6,25,42 have published a series of articles using high-resolution confocal microscopy of BAECs and RFPEC after immunostaining for syndecan, glypican, heparan sulfate, and albumin and consistently reported a glycocalyx thickness of 1.5 to 3.0 μm.
The resolution of standard confocal microscopy is approximately 0.5 to 1.0 µ, depending on the magnification and numerical aperture. In this study, AFM and our 2-layer composite model can derive changes of as little as 10 to 20 nm. The difference in glycocalyx thickness between albumin-treated and HES-treated cell was 200 nm, a difference that could not be measured with confocal imaging. This increased resolution highlights the value of biophysical techniques such as FCS (resolution, 0.1 µm), RICM (resolution subnanometer), and AFM (resolution, 10–20 nm) over other imaging modalities.
The endothelial glycocalyx plays an important role in vascular barrier regulation,4 white blood cell adhesion,43 coagulation,44,45 angiogenesis,46 and tissue repair.47 Nearly all these functions are relevant to perioperative clinicians. Moreover, understanding how resuscitation fluids like albumin and HES interact with the glycocalyx, and alter the structural and mechanical properties of the glycocalyx, has clinical implications in patient management and, potentially, in the development of novel resuscitation colloids.48,49
Safety concerns have been raised regarding the use of HES in specific clinical settings (eg, sepsis), because of increased risk of acute kidney injury. Low MW 130/0.4 HES has largely replaced the use of high MW 600/0.7 HES, but high MW HES is still available in the United States. Excluding sepsis and intensive care unit patients, the risk associated with the use of any HES solutions, especially in healthy patients, has not been adequately addressed in randomized controlled clinical trials.50 At the time we began these studies, high MW HES was in routine use and we selected it as a prototype colloid that was structurally distinct from albumin. Clearly, the shift to low MW HES (130/0.4) may alter the results of our study. However, we would not anticipate significant differences between 600 and 130 MW HES on the biomechanical measurements, because both solutions are polydispersed, eg, have a wide range of polymer sizes. Predictably, solubility and absorbability (larger macromolecules tend to be less soluble but adsorb better) may differ with HES MW, but elastically the 2 HES fractions are similar. Overall, larger MW HES could adsorb more onto the glycocalyx, but produce a larger steric hindrance effect, whereas smaller MW starch solutions might have smaller adsorption, but allow better space usage (less steric hindrance). Ultimately, these 2 effects should compensate each other, meaning that their mechanical behavior as measured by AFM and RICM should remain unchanged.
Clinical choices for resuscitation fluids include crystalloid (most commonly lactated Ringer’s solution or normal saline), human albumin solution, or HES. Although we chose to focus on the effects of colloids on the biomechanical properties of the intact and compromised glycocalyx, the effects of crystalloids that are clinically used much more often are equally important. Although there are no direct studies on the effect of crystalloids on glycocalyx biomechanics, a few points can be gleaned from our data. We assessed the effect of albumin over a concentration range from 0.1% to 4%; a plasma albumin concentration in this range would be considered hypoproteinemic by clinical standards but could easily result from hemorrhagic shock, where blood loss was replaced by a crystalloid solution. Reductions in plasma proteins on the interior mechanics of the glycocalyx are presented in Figure 6, demonstrating a biphasic effect on stiffness. In Figure 8, we observed a linear increase in glycocalyx stiffness as albumin concentration was reduced from 4% to 0.1%; these measurements were derived using RICM that measures the mechanics of the outmost structure of the glycocalyx. We can conclude that crystalloid resuscitation and the associated reduction in plasma protein would likely make the outer layer of the glycocalyx stiffer.
The ionic composition of resuscitation fluids, particularly hypertonic sodium solutions, and even 0.9% saline, will likely also affect the surface mechanics of the glycocalyx. Acute changes in extracellular sodium, >135 mEq/L, result in stiffening of endothelial cells and reduce NO production.51 In these studies, however, the increase in cell stiffness occurred within minutes, but the change in NO production occurred over days. It is not known whether the acute change in cell stiffness is associated with rapid changes in NO production or glycocalyx shedding.
In addition to the composition of resuscitation fluid, the volume of fluid administered also affects the integrity of the glycocalyx. Acute hypervolemic volume loading with 6% HES solution (130/0.4) increased plasma levels of atrial natriuretic peptide (ANP), a protein that causes glycocalyx shedding.52,53 Volume loading was associated with increased plasma syndecan-1, HA, and heparan sulfate concentrations, consistent with ANP-induced glycocalyx breakdown. The mechanism(s) by which ANP promotes widespread breakdown of the glycocalyx remain unknown. In contrast, acute normovolemic hemodilution with 6% HES did not increase plasma ANP and was not associated with changes in plasma markers of glycocalyx breakdown.
Albumin and hetastarches differ in their effects on the biomechanical properties of the intact and partially degraded glycocalyx. These changes in colloid-dependent glycocalyx stiffness may have important implications in glycocalyx-dependent mechanotransduction and barrier function. It is clear that albumin and hetastarch are not comparable when used in studies of vascular function when factors other than oncotic pressure are operational. Whether a thicker or stiffer glycocalyx is better or worse is a complex question that cannot easily be answered given our current level of understanding. In terms of the glycocalyx as a passive barrier, thicker is considered better if other parameters (eg, porosity, composition) have not changed. Glycocalyx stiffness is related to signaling sensitivity in a biphasic manner; if the glycocalyx is too soft, it will be unable to transmit forces; if the glycocalyx is too stiff, it will not be stressed by the prevailing forces. In summary, understanding the molecular and biomechanical effects of resuscitation colloids should inform the practitioner about their indicated uses and provide the best possible clinical effects to enhance patient outcomes.
Name: Kathleen M. Job, PhD.
Contribution: This author designed the studies, conducted the experiments, analyzed data, and assisted in manuscript preparation.
Name: Ryan O’Callaghan, MS.
Contribution: This author helped design the studies, conducted some of the experiments, and analyzed data.
Name: Vladimir Hlady, PhD.
Contribution: This author planned experiments, assisted in data interpretation, assisted in manuscript preparation.
Name: Alexandra Barabanova, MS.
Contribution: This author assisted in data interpretation and analysis and assisted in manuscript preparation.
Name: Randal O. Dull, MD, PhD.
Contribution: This author planned experiments, assisted in data interpretation, and assisted in manuscript preparation.
This manuscript was handled by: Avery Tung, MD.
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© 2016 International Anesthesia Research Society
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