In the developing embryo, blood vessel formation and growth are necessary at an early stage to guarantee cellular supply with oxygen and nutrients. In general, two distinct processes can be distinguished in the development of vasculature. Vasculogenesis describes the de novo formation of vessels by differentiation, proliferation and migration of endothelial progenitor cells. In contrast, angiogenesis characterizes the generation of new vessels from existing ones by sprouting and intussusception (i.e., splitting of an existing vessel) (Conway et al., 2001; Naito et al., 2020). Components of the glycocalyx have been shown to be critically involved in both processes (Iozzo and San Antonio, 2001; Piecewicz and Sengupta, 2011). It is long known that pro-angiogenic factors critical for vasculo- and angiogenesis, including vascular endothelial growth factor (VEGF) and fibroblast growth fact-2 (FGF-2), bind to heparan sulfate proteoglycans (HSPG), the most abundant component of the EG (Yayon et al., 1991; Gitay-Goren et al., 1992; Lundin et al., 2000). As demonstrated by Harfouche et al. (2009), differentiation of embryonic stem cells into endothelial cells is paralleled by an increase in the synthesis of di- and trisulfated heparan sulfate glycosaminoglycans (HSGAG). Vice versa, inhibition of HSGAG sulfation by treatment with sodium chloride or digestion of HSGAGs by heparinase led to a significantly lower expression of endothelial markers such as von Willebrand factor and angiopoetin-2 (Harfouche et al., 2009). These in vitro findings were validated in zebrafish embryos showing that knock-down of the enzyme N-deacetylase/N-sulfotransferase 1 (NDST1), which is critical for posttranslational sulfation of glycosaminoglycans, led to impaired vessel formation (Harfouche et al., 2009).

Syndecan-2, a plasma membrane-bound HSPG expressed on human microvascular endothelial cells (EC), is upregulated under stimulation with FGF or VEGF. Inhibition of Syndecan-2 gene transcription using antisense oligonucleotides led to impaired EC adhesion (i.e., attachment of EC to fibronectin coated culture dishes), spreading (i.e., number of attached ECs showing extended cytoplasm) and capillary tube formation in vitro (Noguer et al., 2009). In vivo, knock-down of Syndecan-2 by injection of morpholino designed against the 5′ UTR region of Syndecan-2 mRNA, led to impaired VEGF-dependent angiogenic sprouting in the zebrafish (Chen et al., 2004). These studies point at the importance of the EG, and HSPGs in particular, during vasculogenesis and angiogenesis. As reviewed by Iozzo and San Antonio, HSPGs act in concert with pro-angiogenic factors to control vascular development by providing a depot for these factors, limiting their diffusion and promoting receptor-ligand interaction and intracellular signaling (Iozzo and San Antonio, 2001).

More recent studies by the group of D’amore investigated the function of endomucin (EMCN), an integral sialoglycoprotein present in the EG of capillaries and veins, during angiogenesis (Park-Windhol et al., 2017; LeBlanc et al., 2019). Using a model of mouse retinal vascularization, it was demonstrated that silencing of EMCN resulted in a significant reduction of retinal vessel density and branching (Park-Windhol et al., 2017). Further analyses with human retinal endothelial cells lacking or overexpressing EMCN corroborated the role of EMCN in VEGF-induced signaling pathways by modulating internalization of the VEGF receptor 2 (VEGFR2), thereby regulating EC proliferation and migration (LeBlanc et al., 2019). It was recently shown that this effect of EMCN was dependent on N-glycosylation of its extracellular domain (Hu et al., 2020). Taken together, these studies highlight the essential involvement and contribution of the EG in vessel formation.

One of the challenges in interpreting the results of studies on the EG in pediatric diseases is the lack of reference values. Recent investigations in animals and humans have provided evidence that aging is accompanied by a reduction in the EG size possibly due to increased EG shedding in combination with decreased synthesis of glycocalyx components (Machin et al., 2018; Majerczak et al., 2019). The glycocalyx thickness in sublingual capillaries of old study participants (mean age 60 ± 2 years) compared to young (mean age 29 ± 1 years) decreased by around 30%. Interestingly, a significant reduction in glycocalyx thickness was also demonstrated in the aging lymphatic vasculature of the rat mesentery with a decrease by more than 50% in 24-month old animals versus 9 month old animals (note: 1 month of life in a rat equals about 3 years in a human) (Zolla et al., 2015). Considering the profound physiological changes occurring in the growing fetus, neonate and child, it seems very likely that the EG is also subject to age-dependent variations. At present, very limited data is available on the ontogeny of the EG. As reported by Henderson-Toth et al. (2012), the EG can be detected in the dorsal aorta of quail embryos at an early developmental stage (14 somites) as soon as blood flow commences. Immunohistochemistry confirmed the presence of functionally important EG components, including hyaluronic acid, heparan sulfate and chondroitin sulfate. Selective enzymatic digestion of these components demonstrated a role of hyaluronan (and chondroitin-/dermatan-sulfate) in maintaining blood flow as well as vascular barrier function, thereby emphasizing the functional importance of the EG at this early developmental stage. Using SDF-imaging for PBR measurements in the cutaneous microcirculation, we have recently shown that the endothelial glycocalyx in preterm and term neonates depends on gestational age at birth (Puchwein-Schwepcke et al., 2021). Intriguingly, we observed an inverse correlation of the EG dimension with gestational age, i.e., the most immature neonates exhibited the thickest EG (represented by low PBR values). Whether this finding reflects the functionally importance of the EG in vascular development remains speculative due to the observational nature of the study. Longitudinal follow-up in the group of preterm infants further demonstrated an effect of postnatal age on the EG with a gradual decrease of EG thickness (increase in PBR). This effect was most pronounced in the group of extremely preterm neonates resulting in significantly higher PBR values (smaller EG) when reaching term age compared to term born neonates. This acceleration of PBR changes over time might be due to the frequent presence of multiple EG stressors (e.g., hyperglycemia, sepsis, reactive oxygen species) and could possibly contribute to a higher vascular vulnerability in this patient group.

Interestingly, PBR values reported for neonates and infants are consistently higher than in adults. In healthy mature newborns (mean age 3 days) the PBR was 2.14 ± 0.25 μm (Puchwein-Schwepcke et al., 2021) versus a PBR of 1.88 ± 0.2 μm measured in healthy adults (mean age 20.7 years) (Astapenko et al., 2019). Likewise, infants with cardiac defects (mean age 8.9 month) had a higher baseline PBR than adult cardiac patients (median age 64–69 years) before undergoing surgery on cardiopulmonary bypass (2.5 μm [2.44–2.7 IQR] vs. 2.0 ± 0.2 μm, respectively) (Nussbaum et al., 2015; Dekker et al., 2019; Dekker et al., 2020). At present it remains unclear whether these differences in PBR magnitude are due to methodological differences (e.g., measurements obtained sublingually versus the fossa auricularis of the ear conch) or truly reflect an age-dependence in PBR values.

Further studies in various age groups and in larger cohorts are needed to better understand the natural course of EG development and establish normal values necessary for the implementation of EG measurements in the clinical routine.

Surgery on cardiopulmonary bypass has been shown to acutely and severely affect the integrity of the EG in adults (Rehm et al., 2007). In children undergoing cardiac surgery on cardiopulmonary bypass (CPB), an increase of circulating hyaluronan and syndecan-1 was witnessed in dependence of the ischemic impact indicating acute shedding of the EG (Bruegger et al., 2015). This was further confirmed in a longitudinal cohort study investigating 40 children that underwent cardiac surgery (36 with and four without CPB) using SDF-imaging to visualize the microcirculation at the ear conch (Nussbaum et al., 2015). A significant reduction in glycocalyx thickness (indicated by an increased PBR) was observed after cardiac surgery with cardiopulmonary bypass compared to preoperative values. In contrast, no significant change in PBR was observed in control patients subjected to a different procedure requiring general anesthesia (cleft palate surgery, cardiac catheterization), indicating a direct effect of the cardiopulmonary bypass in perturbation of the microvascular glycocalyx in pediatric heart surgery (Nussbaum et al., 2015). Similar results have been obtained in adult patients undergoing coronary bypass operation on CPB, demonstrating a significant increase in PBR during surgery. However, the time course of PBR changes described in adults differs from that in infants. While PBR values were shown to further increase during the first three postoperative days in adults following surgery on cardiopulmonary bypass (Dekker et al., 2019, 2020), in infants PBR values were already decreasing 24 h after surgery (Nussbaum et al., 2015). As the studies vary largely with respect to the underlying cardiac disease (congenital heart defect vs. coronary artery disease), surgical procedures applied and presence of cardiac risk factors, it is impossible to draw a conclusion from these studies regarding possible age-dependent differences in shedding and recovery of the glycocalyx.

In two double-blinded, randomized, placebo-controlled trials, syndecan-1 plasma levels were evaluated in neonates subjected to open heart surgery (neonatal trial) and in infants undergoing correction of a ventricular septal defect (VSD trial) to determine whether high-dose steroid treatment might have a protective effect on the glycocalyx. The authors could prove that in complex heart surgery in neonates, high-dose steroid treatment resulted in lower syndecan-1 levels compared to a placebo group. However, there were no differences in syndecan-1 levels between treatment and placebo groups in older children after VSD repair (Pesonen et al., 2016).

In a prospective cohort study on 289 children undergoing cardiac surgery, higher syndecan-1 levels were associated with poor outcomes and postoperative acute kidney disease (de Melo Bezerra Cavalcante et al., 2016). Similar results were found in a prospective cohort study on 86 pediatric patients recovering from heart surgery. Postoperative urinary syndecan-1 was collected within 2 h after surgery and was higher in patients suffering from acute kidney injury in the follow-up. In addition, the prediction of acute kidney injury in a risk-stratified statistical model of clinical outcome was improved after adding urinary syndecan-1 (Ferrer et al., 2018). These data were recently confirmed and expanded by Bangalore et al. (2021), demonstrating an association of the amount of circulating heparan sulfate with metabolic acidosis, renal dysfunction and capillary leak in 27 neonates and infants following cardiopulmonary bypass surgery.

Collectively, these studies provide univocal evidence for EG alterations in pediatric cardiac surgery contributing to adverse outcomes. Thus, assessment of the EG might offer the potential to identify patients at risk for postoperative complications and serve as a monitoring parameter to evaluate treatment strategies aiming at EG restoration.

Multiorgan failure after pediatric trauma has been discussed to be associated with an imbalanced inflammatory reaction that may lead to endothelial disruption and impairment of the glycocalyx. An increase of endothelial-derived angiopoietins (angiopoietin-1 and angiopoietin-2) indicates a developing endotheliopathy, whereas circulating syndecan-1 can be interpreted as a sign of glycocalyx injury. In a prospective cohort study, 52 pediatric trauma patients were compared to 12 pediatric controls with respect to angiopoietin levels, syndecan-1 levels and clinical outcome. The authors could show that higher angiopoietin-2 levels were associated with worse clinical outcomes and were positively correlated to syndecan-1 levels. This may indicate that glycocalyx injury results in adverse outcome (Richter et al., 2019).

Similar findings were observed in another prospective cohort study on 149 pediatric trauma patients and 62 pediatric controls studying the role of histonic DNA (hcDNA) as a marker of damage-associated molecular patterns (DAMPs) and circulating syndecan-1 levels as a marker of EG shedding (Russell et al., 2018). Syndecan-1 levels were evaluated in relation to hcDNA levels at admission and after 24 h. Control patients had low levels of both syndecan-1 and hcDNA, whereas these parameters were significantly higher in the pediatric trauma group, with the highest hcDNA levels corresponding to the highest levels of syndecan-1 and poor outcome. This indicates a link between trauma-induced extracellular hcDNA release and endothelial glycocalyx degradation. However, the causality of the association and the underlying mechanisms still need to be established.

Similarly, infectious diseases may result in acute effects on the microvasculature and the glycocalyx. During sepsis, shedding of the endothelial glycocalyx has been well established in the adult population and linked to mortality (Puskarich et al., 2016; Rovas et al., 2019; Beurskens et al., 2020; Saoraya et al., 2021). In the pediatric population, primarily Kawasaki disease and Malaria were studied for their association with glycocalyx damage.