Role of albumin in the preservation of endothelial glycocalyx integrity and the microcirculation: a review

The endothelial glycocalyx is a complex carbohydrate-rich gel-like layer lining the luminal surface of blood vessels [1]; it functions as a barrier between the blood and vessel wall [2, 3]. The glycocalyx layer is composed of membrane-bound proteoglycans, secreted glycosaminoglycans (GAGs), sialic acid-containing glycoproteins, and glycolipids associated with the endothelial surface (Fig. 1) [4]. The main proteoglycans of the endothelial glycocalyx are membrane-spanning syndecans and glycosylphosphatidylinositol-linked glypicans which carry the two main GAGs, heparan sulfate and chondroitin sulfate, through covalent attachment to the protein core. Syndecans carry both GAGs, while glypicans carry only heparan sulfate. Table 1 summarizes the characteristics of core proteoglycans in the glycocalyx [5]. A third major GAG, hyaluronan, is secreted by endothelial cells but is not covalently linked to a core protein; it binds to cell surface adhesion receptors such as CD44. Plasma proteins such as albumin and antithrombin are also bound within the glycocalyx [4, 6‐9].

The term endothelial surface layer is sometimes used to describe the intimal surface of blood vessels comprising the endothelial glycocalyx and associated components derived from endothelial cells and plasma [3, 10]. The thickness of the glycocalyx/endothelial surface layer (Fig. 2) [11] varies depending on the method used for measurement [9]. Intravital microscopy and orthogonal polarization spectral imaging are techniques which indirectly measure the endothelial glycocalyx in vivo, and the glycocalyx can be measured indirectly ex vivo using microparticle image velocimetry. The glycocalyx can be measured directly in vitro using transmission electron microscopy, confocal laser scanning microscopy and atomic force microscopy, and ex vivo using two-photon laser scanning microscopy [12, 13]. In humans, the mean microvascular glycocalyx thickness estimated using orthogonal polarization spectral imaging was about 0.5 µm (range 0.3 to 0.75 µm) [14].

The outermost layer of the microvasculature serves as a regulatory barrier of vascular permeability. It participates in mechanotransduction by sensing fluid shear forces and regulating the vascular tone. The endothelial glycocalyx, which has an important role in maintaining vascular homeostasis [9], also has several anti-adhesive and antithrombotic effects on the surface of endothelial cells and can protect endothelial cells from oxidative stress [3, 6‐9, 15, 16].

Although albumin has a net negative charge, its amphoteric nature promotes tight binding to the glycocalyx with the net effect of reducing hydraulic conductivity across the vascular barrier, resisting glycocalyx degradation (i.e., protecting against shedding) and thereby contributing to maintenance of vascular integrity and normal capillary permeability, and facilitating transmission of shear stress [2, 15, 23, 25].

Under physiological conditions, the concentration of intravascular albumin is the major determinant of plasma colloid osmotic pressure [18].

Exposed thiol groups on the albumin molecule act as a scavenger for ROS such as superoxide (O₂⁻) and hydroxyl (^•OH) radicals and reactive nitrogen species, e.g., peroxynitrite radicals. Albumin has an additional anti-oxidant effect through binding to free copper ions (Cu²⁺) which are known to accelerate the production of free radicals [26, 27].

Albumin also has immunomodulatory and anti-inflammatory effects through binding of bacterial products, modulation of antigen-presenting cell function, modulation of cytokine production, and reducing hypoxia-inducible factor-1α gene expression which is upregulated in response to low oxygen concentrations [25].

Along with lipoproteins, albumin has an important role in delivering sphingosine-1-phosphate (S1P) to the endothelial cell surface where it functions in maintaining normal vascular permeability [28]. S1P protects endothelial cells by suppressing the activity of metalloproteinases, stabilizes the glycocalyx by reducing GAG degradation and shedding [21, 22], and regulates barrier function by modulating the expression of vascular endothelial-cadherin and β-catenin at endothelial cell–cell contact regions [29].

Post-translational modifications of human serum albumin include glycation, cysteinylation, S-nitrosylation, S-guanylation and S-transnitrosation which can affect the binding of some exogenous drugs [30]. In addition, advanced glycation end (AGE)-modified albumin can induce proinflammatory signaling through activation of AGE receptors [31]. This was illustrated in a murine model of peritonitis and sepsis where administration of therapeutic infusion solutions containing high concentrations of AGE-modified albumin reduced survival [32].

Glycocalyx and endothelial cell damage, or endotheliopathy as it is known [33], occur in several clinical situations including ischemia–reperfusion injury, hypoxia/reoxygenation, inflammation, sepsis, hemorrhagic shock, hypervolemia, hyperglycemia, excessive shear stress and coronary artery bypass surgery [23, 34]. These injuries determine pathological changes in the endothelial glycocalyx such as impaired mechanotransduction, increased egress of leukocytes, loss of coagulation control, loss of anti-oxidant defense, loss of deposited growth factors, and increased vascular permeability (Fig. 4) [10, 35]. In a clinical context, disruption of the endothelial glycocalyx layer can lead to development of interstitial edema in some patients, notably those with inflammatory conditions such as sepsis [36].

Biomarkers of endothelial damage have been developed with most applied prognostically for conditions of systemic inflammation and sepsis [43, 62‐71] (Box 1). In patients with septic shock, increased plasma angiopoietin-2 levels were associated with higher fluid overload, hepatic and coagulation dysfunction, acute kidney injury, mortality, and plasma cytokines, likely as the result of increased vascular leakage [72].

Assessment of glycocalyx damage using a variety of biomarkers has provided evidence of glycocalyx degradation in a range of clinical conditions including trauma, CKD, myeloid leukemia, acute decompensated heart failure, and Crohn’s disease [5].

Pre-clinical studies which illustrate the mechanism of action of albumin, and its effects in models of hemorrhagic shock, endotoxemia, vascular permeability and ischemia are summarized in Table 2 [13, 14, 60, 73‐87]. Results from in vitro, in vivo, and ex vivo experiments illustrate the multifunctional nature of albumin including maintaining glycocalyx integrity and partially restoring impaired vascular permeability via release of S1P from RBCs; anti-inflammatory and anti-oxidative effects; improvement of the microcirculation and hemodynamics following hemorrhagic shock or endotoxemia; and acting as an effective plasma volume expander.

A recent study of patients with septic shock (n = 30) reported that, compared with saline, albumin improved skin endothelial cell function, improving microcirculatory blood flow. These beneficial effects may be independent of the oncotic properties of albumin as neither cardiac output nor skin blood flow differed between albumin- and saline-treated patients [88].

Several techniques are available to monitor the microcirculation and endothelial damage. These include: intravital microscopy in in vivo animal models for visualization of vascular dynamic events such as microvascular permeability, vasotone and blood flow [89] or the glycocalyx [90, 91]; assessment of the microcirculation with potential to measure the glycocalyx in critically ill patients [92]; application of near-infrared spectroscopy to measure tissue oxygenation [93, 94]; measuring skin mottling over the anterior surface of the knee [94]; measuring microalbuminuria [95]; biomarkers of acute kidney injury [96]; measuring protein concentrations in alveolar fluid lavage; and hemostasis-related biomarkers, e.g., factor VIII, von Willebrand factor, International Normalized Ratio, partial thromboplastin time and platelet count.

Intravital microscopy using sidestream darkfield (SDF) imaging is a non-invasive method increasingly used to analyze the sublingual microcirculation. The technique visualizes erythrocytes within the microvasculature due to light emitted by a light emitting diode probe which is reflected by hemoglobin and detected by a SDF camera [97]. Total vessel density, perfused vessel density, proportion of perfused vessels and microvascular flow index are traditionally estimated by offline computer analysis although, more recently, point-of-care approaches using validated automatic software platforms have been described [98, 99]. SDF imaging detection of RBCs is used as a marker of microvascular perfusion, and measurement of the perfused boundary region (PBR) as an indirect marker for endothelial glycocalyx barrier dimensions. In a large study of overweight and obese individuals, the PBR and presence of RBCs in the microvascular circulation were markedly associated [100]. Hand-held intravital microscopy showed that sublingual microvascular blood flow alterations are common in patients with sepsis, with blood flow abnormality related to disease severity [92, 101]. Furthermore, sublingual microvascular glycocalyx is damaged in critically ill patients, especially those with sepsis [102, 103], but also after cardiac surgery with cardiopulmonary bypass [104, 105] and in emergency room and intensive care unit patients [106]. However, in patients with sepsis, there was no association of PBR and syndecan-1 values with established microcirculatory parameters [102], likely indicating that both alterations occur independently. These data should be treated with caution as the reproducibility of three sublingual microcirculation parameters (vascular density, RBC filling and PBR) estimated by SDF imaging is controversial and large studies are required to achieve statistically significant effects [107]. However, some studies have shown good reproducibility with the method if consecutive measurements are averaged [106, 108, 109]. The accuracy of in vivo glycocalyx measurement has been analyzed further with an in vitro approach using atomic force microscopy [102, 110]. Consensus European Society of Intensive Care Medicine guidelines provide 15 recommendations for acquisition and interpretation of microcirculatory images obtained with hand-held vital microscopes for assessment of the microcirculation in critically ill patients [92].

According to our clinical and scientific judgement, biomarkers of endothelial damage and/or evaluation of the sublingual microcirculation may have a role in identifying subgroups of patients at risk of morbidity and mortality.

We consider that albumin should be used in accepted indications in which it has a proven positive risk:benefit balance, mainly in septic patients (for initial resuscitation after adequate crystalloid infusion and hypoalbuminemic septic shock), for burn shock resuscitation and fluid maintenance, and in some cases of liver insufficiency. The benefits of albumin may relate to its ability to restore function to damaged glycocalyx, although further studies are required to confirm this relationship and identify thresholds for optimal benefit. The mechanism of action of these restorative effects also needs to be elucidated.

As the lung and kidney are the organs most affected by septic shock, pre-clinical investigation of the effects of albumin on permeability disorders of these organs should be conducted. The development of non-invasive imaging-based analysis tools to assess changes in permeability, diameter, and blood flow of vessels in response to specific stimuli is beginning to benefit research in both pre-clinical and clinical areas. It is likely that patients’ response to albumin may differ depending on localization of the blood vessel under investigation.

Detailed studies of the effect of albumin on oxidative stress are required and could be assessed using in vitro models (e.g., cultures of human endothelial cells and glycocalyx components), as these are more reproducible than pre-clinical animal studies.

The main objective of clinical studies of albumin should not be to evaluate its effect on overall mortality, but rather on more specific endpoints such as organ dysfunction. Investigating the economic impact of albumin and its long-term consequences is also advised. Studies should accrue biological information, i.e., justification for the selected objectives. Smaller studies with homogeneous populations are preferred to large multicenter studies of patients with clinical heterogeneity.

In summary, additional research needs to be conducted to clarify the role of albumin as a protector or restorer of damaged glycocalyx, with the aim of identifying clinical applications.