Shedding of the Vascular Endothelial Glycocalyx: A Common Pathway to Severe Malaria?

(See the Major article by Yeo et al on pages 1712–20.) The glycocalyx (literally meaning “sweet husk”) is a surface layer separating vascular endothelial cells from the circulating components of the blood. The glycocalyx is actually more gel-like than husk, composed of complex proteoglycans, glycoproteins, and soluble proteins, constantly shedding and regenerating with variation in composition, depth, and surface structure to enable it to fulfil a myriad of biological functions [1–3]. The glycocalyx is often omitted from illustrations of vascular endothelial physiology, but it is increasingly recognized to be crucial for proper function of the vascular endothelium and maintenance of homeostasis. In addition to forming a protective coating over the vascular endothelium, the glycocalyx prevents clotting activation, limits cellular interactions by physically separating circulating cells from the endothelium, transduces shear stress due to alterations in blood flow, and regulates vascular permeability [1, 2, 4]. Shedding of the glycocalyx is greatly increased in a variety of pathological conditions, including severe sepsis and dengue [4–6], where loss of the glycocalyx is thought to contribute to vascular leak, impaired perfusion, thrombosis, and leukocyte adhesion. Mechanistically, glycocalyx shedding can occur through convergence of a variety of stimuli in severe infections, including tumor necrosis factor α (TNF-α), lipopolysaccharide, endothelial activation, ischemia-reperfusion, hyperglycemia, and reactive oxygen species (ROS) [1, 4]. In this issue, Yeo and colleagues provide the first compelling evidence that glycocalyx shedding is associated with severity of human malaria [7]. This association is perhaps not surprising, because many of the stimuli for glycocalyx shedding (TNF-α, ROS, endothelial activation) are also associated with severe malaria [8]. What is remarkable is that the detailed analysis of associations between glycocalyx shedding and other markers of pathophysiology indicate that the glycocalyx may play a central role in severe disease and be a tractable target for therapeutic intervention. Severe malaria can manifest in different ways (coma, respiratory distress, acute kidney injury, acidosis), which is likely explained by some differences in underlying mechanisms [8]. Broadly speaking, severe malaria is driven by high parasite load, inflammation, vascular endothelial activation and dysfunction (impaired barrier, blood flow regulation, and antithrombotic function), oxidative stress, and in the case of Plasmodium falciparum, microvascular obstruction by sequestered parasites [8–10]. Each of these mechanisms has the potential to potentiate the others in an amplifying cycle, which creates challenges for unraveling their respective contributions and for developing adjunctive therapies. Studying hospitalized Indonesian adults with a variety of manifestations of severe P. falciparum malaria, moderately severe malaria, and healthy controls, Yeo and colleagues found strong evidence to support the association of glycocalyx shedding with each of the key pathological processes of severe malaria. Urinary levels of the glycosaminoglycan (GAG) components of the glycocalyx, and plasma levels of the syndecan-1 protein component, were substantially increased in severe disease. They were also strongly associated with parasite load, cell free hemoglobin (a driver of oxidative stress), endothelial activation markers such as angiopoietin-2 and intercellular adhesion molecule 1, and reactive hyperemia-peripheral arterial tonometry measurements (a functional assessment of nitric-oxide dependent regulation of blood flow by the vascular endothelium). In P. falciparum malaria, a substantial proportion of parasites can be sequestered in the microvasculature rather than circulating in the blood, and levels of parasite histidine rich protein 2 in plasma can be used as a biomarker of the total parasite load [11]. Yeo and colleagues found that the association between glycocalyx shedding and total body parasite load was generally stronger than the association of glycocalyx shedding with circulating parasitemia. This may hint that sequestered parasites play a more important role in glycocalyx shedding than those in the circulation and would be consistent with a previously proposed mechanism, that parasitized red cells initially bind to glycosaminoglycans on intact glycocalyx before triggering glycocalyx degradation [3]. As degradation proceeds, infected erythrocytes would become close enough for parasite molecules on their surfaces to bind to endothelial cell surface receptors. These specific cytoadhesive interactions might then amplify organ-specific sequestration and further vascular endothelial and end organ dysfunction. As with any observational study, Yeo and colleagues could not definitively establish whether glycocalyx shedding is cause or consequence of other pathological changes in severe malaria. Sequential analyses in a mouse model suggest that glycocalyx shedding starts before the terminal phase of experimental cerebral malaria [12], but the coincidence with other pathological processes was not studied in detail. The order of events is important if, for example, the measurement of shed glycocalyx components might be used as a predictor of adverse outcome. Yeo and colleagues found that urinary GAGs predicted the small number of deaths in their cohort with good accuracy, and suggest that a simple colorimetric assay could be a useful prognostic indicator in resource limited settings. A larger independent prospective study will be needed to confirm this. The shedding of endothelial glycocalyx described in the mouse experimental cerebral malaria model and now in humans is potentially an important unifying aspect of pathophysiology [3, 12, 13]. There has been extensive debate in the past about the relevance of the mouse model for human disease [14]. Despite the fact that both ultimately result in brain swelling, seizures, coma, and death, they appear to do so through different mechanisms. The pathognomonic feature of human cerebral malaria, cytoadhesive sequestration of parasitized red cells to brain microvascular endothelial cells, is rarely seen in the mouse model [15]. In contrast, the mouse model is highly dependent on the host immune response and can be rescued by anti-inflammatory treatments, which have failed in clinical trials in humans [16]. Convergence of different pathogenic mechanisms on the endothelial glycocalyx offers an explanation for how these different upstream processes may lead to similar manifestations of disease. Shedding of the glycocalyx in the brain vasculature could contribute to almost all of the common pathological features observed in both human and experimental cerebral malaria, such as activation of coagulation pathways, endothelial activation, impaired barrier function, and cerebral edema [9, 17]. Further studies investigating the endothelial glycocalyx in human post-mortem brain specimens would be particularly helpful to confirm localization of glycocalyx shedding with other pathological processes. Identification of a common mechanism in human and experimental cerebral malaria opens the way for novel therapeutic interventions to be tested. A variety of pharmacological agents that help to preserve or restore the endothelial glycocalyx are available [1, 2, 4] and might be attractive adjunctive therapies. To identify those that may be useful in humans it is particularly important to test them as true adjunctive therapies in the mouse model, in appropriately powered, randomized-controlled experiments in mice with established severe disease and alongside curative antimalarial treatment [17]. We suspect that greatest benefit could come from attempting to restore the glycocalyx, for example, using infusions of natural or bioengineered GAGs or sphingosine-1-phosphate, which may also be beneficial in sepsis and other pathological causes of glycocalyx shedding [4]. Although the discovery of glycocalyx shedding in human malaria by Yeo and colleagues is an exciting advance, it also raises several questions that require further investigation. They used a variety of techniques to measure shedding of different glycocalyx components, which showed different associations with disease manifestations and pathophysiological biomarkers. Is this just a consequence of multiple statistical tests on relatively small numbers of subjects, or does it indicate more nuanced underlying biology of the different glycocalyx components? There was no correlation between any markers of glycocalyx shedding and blood lactate concentrations after adjusting for severity. Hyperlactatemia is believed to reflect impaired microvascular perfusion in malaria [8], so is the regulation of blood flow by the glycocalyx less impaired than other aspects of its function? More detailed exploration in both humans and animal models will be needed to understand exactly how glycocalyx shedding is contributing to severe malaria and perhaps to develop effective therapeutics. Financial support.