Endothelial Dysfunction Criteria in Critically Ill Children: The PODIUM Consensus Conference

The PODIUM Collaborative sought to develop evidence-based criteria for organ dysfunction in critically ill children. The present article reports on the systematic review on endothelial dysfunction scoring tools performed as part of PODIUM, provides a critical evaluation of the available literature, and proposes recommendations for future research. The PODIUM Executive Summary details Population, Interventions, Comparators, and Outcomes questions; search strategies; study inclusion and exclusion criteria; and processes for risk of bias assessment, data abstraction and synthesis, and for drafting and developing agreement for criteria indicating endothelial dysfunction.⁷  A quantitative meta-analysis was not performed for several reasons:

1. relative estimates of effect could not be calculated for studies lacking control groups;

2. studies included patients with a variety of etiologies of critical illness; and

3. heterogeneity existed in the hemodynamic parameters and timing of assessments relative to disease onset or biomarker evaluation.

Therefore, all studies were qualitatively analyzed.

Of 1071 unique citations identified between 1992 and 2020, 156 articles met the criteria for full text review. Less than half (n = 62 studies) measured 36 unique biomarkers, which were included in the final analysis, as shown in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses flowchart (Fig 1). Data tables (Supplemental Tables 1 and 2) and risk of bias assessment summaries (Supplemental Fig 1) are detailed in the Supplemental Information.

Of the 62 studies included, 51% (32 of 62) were prospective cohort investigations, 8% (5 of 62) were retrospective cohort investigations, 16% (10 of 62) were case-control studies, and 18% (11 of 62) were case series. Some studies incorporated more than 1 design. Twenty-six percent were conducted in the United States (16 of 62), 24% (15 of 62) in Asia, and 21% (13 of 62) in Africa. Protein biomarkers measured from the serum or plasma were examined in the majority of studies (92%, 57 of 62) to predict outcomes including mortality (35%, 22 of 62), functional status (11%, 7 of 62), or organ-specific outcomes including morbidity (10%, 6 of 62). The quality of collected evidence for identifying an endothelial biomarker for MODS is poor. Risk of bias for the collected literature was high in 44% (27 of 62) of studies assessed, moderate in 24% (15 of 62), and low in 24% (15 of 62) (Supplemental Fig 1). The collected studies enrolled low numbers of subjects, with heterogeneous diagnoses and highly variable severity of illness. Of the identified studies, most (42% [26 of 62]) focused on sepsis, including 8 studies of children with malaria-induced MODS. Acute lung injury was the second most studied diagnosis and reflected 15% (9 of 62) of studies, followed by neurologic injury (infection, trauma, and stroke) and cardiopulmonary bypass-related vascular dysfunction (both 13% [8 of 62]) of the reviewed studies. Finally, congenital heart disease, including pulmonary hypertension, was involved in 7% (4 of 62) of studies and necrotizing enterocolitis, lupus, bone marrow transplantation, extracorporeal life support, hemolytic uremic syndrome, scorpion bite, and disseminated intravascular coagulation were each the focus of a single study.

Many studies surveyed biomarkers relative to more than one EC function, resulting in 84 assessments of different EC functions across the 62 studies. Of the measured biomarkers, 33% (28 of 84) involved leukocyte recruitment, 21% were markers of permeability (18 of 84), 14% were involved in inflammation (12 of 84), 14% described coagulation (12 of 82), and 13% assessed markers of blood flow (11 of 84). Notably, no studies investigated endothelial junctional proteins such as claudin-5 or vascular endothelial (VE)-cadherin, which are selectively expressed in ECs.

Our comprehensive literature review revealed that no single biomarker of endothelial dysfunction in critically ill pediatric patients with MODS has been identified. The endothelium supports many functions essential for homeostasis and extends to close proximity (microns) of all organ parenchymal cells. Disruption of EC functions, such as systemic inflammation (leukocyte recruitment and activation), capillary leak (permeability), coagulopathies (coagulation), and capillary shunting (heterogenous microvascular blood flow), have all been associated with MODS. It is of critical importance to identify endothelial biomarkers related to MODS diagnosis, progression, and resolution. The lack of sufficient prospective trials, use of multiple exposures and outcomes, and lack of biomarker correlation with EC function resulted in insufficient evidence for a consensus biomarker-based definition of endothelial dysfunction in pediatric MODS.

Biomarkers of leukocyte adhesion, including intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E, L and P-selectin, are the most commonly surveyed biomarkers of EC dysfunction in MODS.⁸  In intact ECs, these molecules are upregulated in response to inflammatory cytokines like tumor necrosis factor or interleukin-1. Upon stimulation, these molecules collect on the luminal surface of EC, where they facilitate leukocyte adhesion and transmigration. However, all studies reviewed measured the soluble forms of these molecules after release from the EC, which may represent fragments of dead or sloughed EC. These markers do not provide information on specific organ dysfunction and are not EC specific because many molecules are expressed on circulating immune cells. Although soluble adhesion molecules were the most commonly investigated in our survey of existing literature, these may be poor biomarkers of EC dysfunction.

In addition to alterations in leukocyte recruitment, vascular leak can be significantly deranged during endothelial dysfunction in pediatric MODS. The majority of studies focusing on EC permeability investigated the angiopoietin (Angpt)-TIE2 axis.⁹  Angpt-1 is a pleotropic glycoprotein that promotes vascular quiescence and stabilizes EC barrier function. Angpt-2 predominately functions as an antagonist of Angpt-1 at the receptor tyrosine kinase, TIE2. However, Angpt-2 has several other independent, context-specific functions in vasculogenesis.¹⁰  Angpt-2 is produced by EC and stored in Weibel Palade bodies, along with E-selectin, von Willebrand factor, and other proteins,¹¹  which are exocytosed in response to inflammation, hypoxemia, and alterations in blood flow.¹²  Recently, Angpt-2 has been shown to alter vascular permeability in a concentration-dependent fashion, which differs in vascular segments and organs.¹⁰  A direct link between Angpt-2 concentrations and the progression or resolution of capillary leak is not established. Indeed, Angpt-2 does not affect permeability of tight junction architecture in cultured cells.¹³  Specifically, there appears to be no interaction with Angpt-2 and capillary tight junction proteins such as claudin-5 or zonula occludens protein-1, or with adherens junction proteins such as VE-cadherin. Furthermore, there are currently no studies investigating the role of molecules specific for EC junctions, such as claudin-5 or VE-cadherin, in pediatric MODS.

Interleukins modulate the activity of recruited immune cells and are elevated in critical illness.¹⁴  These can be secreted by endothelial cells and may recruit adhesion proteins to areas of injury.¹⁵  However, the effects of these cytokines on EC function are poorly defined and these molecules are unlikely to represent autocrine signaling. For example, soluble Fas is thought to be antiapoptotic, whereas soluble Fas Ligand is proapoptotic,¹⁶  although these functions are not unique to ECs.

Several studies investigated damage to the endothelial glycocalyx, a glycoprotein monolayer important in vascular integrity and permeability selectivity.^17–21  It is not clear from the current literature which factors induce sloughing of the glycocalyx and what consequences ensue from glycocalyx exposure. It is also not clear which vascular segments are most affected when this occurs. More research is needed to answer these important questions.

Vascular endothelial growth factor and its receptor, soluble fms-like tyrosine kinase-1, are thought to aid in growth and repair of the vascular intima.²²  Several studies suggest the association between increased circulating levels and critical illness states, but there has not been a documented association with MODS.

Regulation of blood flow is a critical measure of EC function and of clear importance to the intensivist. Investigations of EC blood flow regulation predominately focus on reactive nitrogen species and particularly reflect the production, and therefore action, of nitric oxide (NO), a potent vasodilator that is locally-acting and extremely short-lived.²³  However, reactive nitrogen species levels may be altered by numerous other cell types and are not clearly linked to abundance or regional activity of NO. Systemic levels of NO degradation byproducts (nitrite/nitrate) do not accurately reflect regulation of local blood flow. Similarly, inhibitors of NO production have not been demonstrated to relate to EC function.

The regulation of blood fluidity and coagulation is intimately linked to inflammation and predominately regulated by ECs.²⁴  Several studies investigated levels of EC-derived coagulation factors.²⁵  Many of these proteins are fairly specific to ECs. Secreted markers, such as von Willebrand factor and Factor VIII, are released by activated EC, but are not differentiated between EC in different vascular segments or organs. Other markers, such as fibrinogen, may be secreted from EC and other parenchymal cells.²⁶  Some of the surveyed markers of dysregulated coagulation, such as the extensively studied activated protein C (also thrombomodulin, tissue factor pathway inhibitor, and plasminogen activator inhibitor) are bound to EC membranes and the significance of soluble concentrations of these proteins is not clear. Functional markers, such as international normalized ratio, prothrombin time, partial thromboplastin time, and thromboelastography could be coupled with levels of specific EC-derived molecules and accurately reflect vascular function in this domain. More investigation would be required to link functional coagulation markers to MODS.

Finally, a single study found endothelial-derived (endoglin, platelet endothelial cell adhesion module-1, vascular endothelial growth factor receptor-2, and mucosal vascular addressin cell adhesion molecule-1–positive) microvesicles (MVs) were increased in the blood of neonates with MODS requiring extracorporeal life support.²¹  MVs, also known as ectosomes or microparticles, are released from ECs in response to a variety of stimulation.²⁷  MVs contain microRNA, protein, and lipid-signaling molecules, and are coated with surface markers providing information of their cells of origin. The identified study only quantified EC-derived MVs in MODS and did not investigate differences in MVs contents. An important area of future research will be to categorize surface protein expression on MVs that reflects vascular segments or organ-specific EC function and decipher the signaling cargo within MVs. If surface markers can be correlated with cellular origin and their contents to function, MVs may provide specificity for vascular segment and organ–EC function.

The Endothelial Subgroup concludes that there are no single or combination of endothelial-derived biomarkers for pediatric MODS. Nearly all biomarkers studied lacked vascular segment and organ specificity. Most biomarkers indirectly surveyed EC functions or relied on circulating markers of unknown biologic significance. Scientific priorities for future research include further study of the endothelial glycocalyx, which may provide vascular segment and organ specificity, and MVs, which may also provide specificity with additional insights into vascular function.

Angpt

angiopoietin

EC

endothelial cell

MODS

multiple organ dysfunction syndrome

MVs

microvesicles

NO

nitric oxide

PODIUM

Pediatric Organ Dysfunction Information Update Mandate

VE

vascular endothelial