Glycocalyx in vivo measurement

1 Introduction

Endothelial glycocalyx (EG) is a delicate, sugar-based structure lining the endoluminal surface of endothelial cells, considered as a major regulator of endothelial function (Fig. 1) [1–5]. Current studies suggest that virtually all conditions underlying critical states – infection, trauma (including surgical), hypoxia and hypovolemia – may disrupt EG structure and function (Fig. 2), and participate in the development of organ dysfunction and failure, resulting in death [6–8]. Rapid, “point of care-like” assessments of the EG in clinical medicine would be of high relevance for various medical specialties, particularly assessing EG changes during acute and chronic clinical conditions to determine the outcome of different therapeutic interventions. Moreover, EG assessment in clinical and research setting could potentially be used as a biomarker predicting or detecting early EG changes in cardiovascular diseases or assessing the risk of seizure in patients with epilepsy [9, 10]. Visualization and/or reliable quantification of EG would also bring the possibility of investigating the effect of life style intervention on vascular health, or to search for new strategies to increase endurance in sport medicine [11, 12].

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Fig.1

Healthy capillary; Intact endothelial barrier, solid layer of endothelial glycocalyx; EC – endothelial cells, GCX – glycocalyx, RBC – red blood cells, PLT – platelets, LEU – leukocytes, HSA – human serum albumin.

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Fig.2

Damaged capillary; Damaged endothelial glycocalyx, disrupted endothelial barrier with diapedesis of leukocytes, leakage of plasm, interstitial oedema and thrombosis; EC – endothelial cells, GCX – glycocalyx, RBC – red blood cells, PLT – platelets, LEU – leukocytes, HSA – human serum albumin.

The EG is a highly dynamic structure comprised of endothelium-bound molecules (proteoglycans and glycoproteins), soluble plasma components (proteoglycans, glycosaminoglycans, sialoproteins, albumin), and a large volume of non-circulating plasma (∼1 liter) as an aggregate of the hydration shells [13]. The gel-like structure of the glycocalyx makes the “circulating” concentration of its components quite unstable and scattered. The EG is not the sole product of endothelial cells, but rather a product of endothelium-plasma interaction. Due to dynamic equilibrium with plasma components, it is tempting to use humoral markers to estimate glycocalyx shedding in vivo. These markers include syndecan-1 (also known as CD138), hyaluronan, heparan sulphate, chondroitin sulphate, glypicans, and perlecans [14]. Unfortunately, none of these markers are endothelial specific, thus hampering their use in clinical medicine. The comparability of results is further complicated by the lack of reliable enzyme-linked immunosorbent assay kits. Moreover, interpreting the results is extremely difficult due to the fast, or unknown, turnover rate of these markers and the central role of hepatic uptake in their metabolism. For example, the human body contains approximately 15 grams of hyaluronan, of which about a third is replaced every day and minute amounts are excreted through urine [15]. Due to this high turnover rate (t_1/2 in circulation is ∼2– 5 min), the contribution of endothelial hyaluronan to its total plasma concentration is disputable.

3 Imaging methods

Experimentally, intravital microscopy and electron microscopy have been employed for direct EG visualization. Recently, we were able to obtain images of EG of zebrafish embryo (own data, Fig. 3). Unfortunately, elecron and invtravital microscopy in a clinical setting is of little use. In vivo, the visualization of the glycocalyx in humans is extremely difficult, mainly because of its fragility. The glycocalyx is partially accessible to flowing red blood cells (RBC) at its luminal side, called the Perfused Boundary Region (PBR). A detailed description of PBR calculation is available elsewhere [16]. In short, PBR describes the penetration extent of flowing RBC (measured in μm) into the EG, as well as RBC deviation from the central flow towards the endothelial cells. EG damage results in deeper RBC penetration and is reflected by increased PBR. The software automatically records PBR in vessels of diameters ranging from 5– 25μm, presenting an average result.

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Fig.3

Electron microscopy image (TEM, HV = 80 kV, 15000x, FAC) of a cross-sectional mount of 5d post-fertilization zebrafish embryo. Arrows indicate glycocalyx presence in the lumen of a blood vessel.

Glycocalyx damage results in increased PBR, which can be measured in human sublingual microvasculature recordings obtained by orthogonal polarization spectral (OPS), sidestream dark field (SDF), or Incident Dark Field imaging technologies. The first study utilizing SDF imaging of the human sublingual microcirculation, to non-invasively measure glycocalyx thickness, was performed by Donati et al. [7], describing a significant increment of PBR in critically ill patients with sepsis, showing a PBR of 2.76μm as the best discriminative measurement indicating the presence of sepsis. Further, PBR was correlated with the number of rolling leukocytes in post-capillary venules, confirming that glycocalyx shedding enhances leukocyte– endothelium interaction. Damiani et al. also performed a clinical study on the microcirculatory response to fresh or old blood transfusions during sepsis, including PBR as an index of glycocalyx damage [17]; as a result, the transfusion of old RBCs was associated with increased free hemoglobin (fHb) levels in the plasma of septic patients, although the change in plasma fHb after transfusion was negatively correlated with modified sublingual microvascular density and peripheral tissue hemoglobin content; regardless, the PBR was unaffected. Several authors suggest using PBR as an indicator reflecting glycocalyx thickness under various clinical scenarios [17, 18].

Improvements made in microcirculatory imaging systems, such as Incident Dark Field imaging, and the availability of automated software for the analysis of glycocalyx thickness will further facilitate glycocalyx studies in humans. Except for in vivo indirect glycocalyx visualization, using currently available video imaging methods, the only accessible cells containing glycocalyx in clinical conditions are RBC. After the tight link between the endothelial and RBC glycocalyx was demonstrated [19], a method, suitable for clinical practice, was developed for the evaluation of erythrocyte glycocalyx [20].