Injury Induced Endotheliopathy: Overview, Diagnosis, and Management

Abstract

Purpose of Review:

This review aims to examine recent advances in the understanding of injury-induced endotheliopathy and therapeutics to mitigate its development in critically injured patients.

Recent Findings:

Clinical studies have clearly demonstrated that syndecan-1 ectodomains can be found in circulation after various types of trauma and injury and correlates with worse outcomes. As the mechanisms of endotheliopathy are better understood, pathologic hyperadhesive forms of von Willebrand factor, along with a relative deficiency of its cleaving enzyme, a disintegrin and metalloprotease with thrombospondin type I motifs, member 13 (ADAMTS13), have emerged as additional biomarkers. Therapeutics to date have focused primarily on the protective effects of fresh frozen plasma and its constituents to restore the glycocalyx. Human recombinant ADAMTS13 holds promise, as do synthetic variants of heparan sulfate and activated protein C, although all data to date are preclinical.

Summary:

Injury-induced endotheliopathy represents an important pathologic response to trauma. Key biomarkers, such as syndecan-1, can aid in the diagnosis, but testing is not yet available clinically. As the mechanisms of endotheliopathy are better understood, therapeutics are being identified and show promise. To date, plasma has been the most widely studied; however, like all therapeutics for injury-induced endotheliopathy, it has primarily been studied in the preclinical setting.

INTRODUCTION

Trauma is the leading cause of disability and death worldwide, with uncontrolled hemorrhage accounting for approximately 40% of trauma fatalities (1). While hemorrhagic shock is the major cause of early deaths, it is not the sole driver of mortality, as many patients die from bleeding despite hemorrhage control and adequate resuscitation. A more insidious cause of morbidity and mortality after severe trauma and hemorrhage is direct injury to vascular endothelial cells (ECs), which leads to endothelial dysfunction, impaired vascular barrier integrity, and activation of interconnected inflammation and coagulopathy pathways (2, 3), referred to as the endotheliopathy of trauma (EOT). EOT is strongly linked with poor outcomes (4-6). Therefore, prevention, diagnosis, and treatment has been a focus of modern trauma resuscitation strategies (7). This review explores recent advances in the understanding of injury-induced endotheliopathy and therapies to mitigate its development in critically injured patients.

STRUCTURE AND FUNCTION OF THE ENDOTHELIUM

The vascular endothelium plays a central role in homeostasis by regulating coagulation, inflammation, and the vascular barrier. Under physiologic conditions, ECs express membrane-bound sulfated proteoglycans (e.g., syndecan-1, glypican), glycoproteins (e.g., thrombomodulin), and side-chains of glycosaminoglycans (e.g. heparan sulfate), which form a gel-like glycocalyx coating over the luminal surface. This barrier not only physically separates blood from the underlying endothelium, but also interacts with circulating platelets, leukocytes, and soluble factors to inhibit coagulation and maintain endothelial structure (2). Glycocalyx components, such as heparan sulfate and thrombomodulin, regulate the activity of thrombin through the modulation of antithrombin and activated protein C, respectively (8). ECs also modulate platelet activity through the expression of von Willebrand factor (vWF) which promotes platelet plug formation upon injury. Thrombosis and inflammation are co-regulated through the production of nitric oxide and prostacyclin, which suppress platelet activity and inhibit inflammatory processes by reducing leukocyte recruitment, activation, and adhesion to the vessel wall (3). Lastly, the EC cytoskeleton and cell junctions maintain the semipermeable barrier between the intravascular space and other tissues. The expression and dissociation of these proteins are modulated by inflammatory mediators and by the transduction of mechanical shear stress signals by transmembrane glycoproteins.

Endothelium After Injury

The endothelium is a complex and heterogeneous system. Dynamic changes that occur post-injury are interconnected and act synergistically. In cases of mild injury, these changes restore the system to homeostasis. However, in patients with severe trauma or hemorrhage, systemic responses can become exaggerated, leading to a cascade of worsening shock and even death.

Glycocalyx

Following major injury and hemorrhagic shock, proinflammatory cytokines such as tumor necrosis factor-alpha are released from damaged tissues into plasma. This triggers the release of proteases (e.g., matrix metalloproteinases, MMPs) from activated leukocytes, platelets, and ECs. These proteases, or sheddases, cleave syndecan-1 and GAGs, and the fragments are released into circulation (9). The loss of syndecan-1 hinders the mechanical transduction signaling pathways that maintain the cytoskeletal structure of ECs, while cleavage of heparan sulfate results in decreased antithrombin activity and disinhibition of factor Xa and thrombin (factor IIa). Unlike cleaved syndecan-1, which does not elicit an inflammatory response, the cleaved heparan sulfate fragments can downregulate the expression of intercellular junction proteins, thereby disrupting the integrity of the vascular barrier (10). They also function as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response through toll-like receptor signaling (11, 12). Nonenzymatic changes compound glycocalyx damage through processes such as depolarization of GAGs by reactive oxygen species and membrane reorganization driven by succinate accumulation within ECs (13, 14). Systemic shedding and depletion of the glycocalyx layer is an integral mechanism by which endotheliopathy contributes to microvascular inflammation, tissue edema, coagulopathy, and end-organ injury.

Von Willebrand Factor and ADAMTS13

vWF is an important hemostatic factor. It is synthesized in endothelial cells and megakaryocytes and can be secreted constitutively or stored in Weibel-Palade bodies of endothelial cells or in alpha granules of platelets, where multimerization occurs. With endothelial cell activation or injury, such as that occurring after trauma, vWF is released as ultralarge multimers (ULVWF) onto the surface of the EC where it is cleaved by circulating metalloproteinase a disintegrin and metalloprotease with thrombospondin type I motifs, member 13 (ADAMTS13) into its smaller multimers. In this smaller form, vWF does not easily bind platelets unless it is first bound to and immobilized by the subendothelium at the site of localized injury. However, after severe injury, there is a relative reduction in ADAMTS13 compared to vWF, resulting in ultralarge multimers that are intrinsically hyperadhesive (15). As a constitutively expressed protein, its concentration does not rise as rapidly as that of vWF, and its activity is further inhibited by proinflammatory cytokines and reactive oxygen species.

DIAGNOSING INJURY-INDUCED ENDOTHELIOPATHY

The multifactorial pathophysiology of endotheliopathy presents a challenge for diagnostic interpretation, as it can manifest with various phenotypes and implicates many pathways (16). Nevertheless, several biomarkers of endotheliopathy have been identified, including syndecan-1, soluble thrombomodulin, and VWF:ADAMTS13 ratio.

Syndecan-1

Soluble syndecan-1 is a well-recognized biomarker of glycocalyx shedding and endothelial damage following traumatic injury. A study of 410 trauma patients found that a syndecan-1 level >40 ng/dL on admission was associated with increased transfusion needs, resuscitation requirements, and 2.23-fold higher odds of 30-day in-hospital mortality compared to trauma patients with lower levels (17). Multiple studies have shown a strong correlation between elevated syndecan-1 levels and morbidity and mortality in severely injured trauma patients, regardless of age or injury type, including blunt and penetrating trauma, hemorrhagic shock, septic shock, and burns (5, 16, 18-23). Syndecan-1 is also as a marker of endotheliopathy in traumatic brain injury (TBI), reflecting its role in maintaining the structure of the blood-brain barrier (24). As such, syndecan-1 is a promising target for both diagnosis and mitigation of endotheliopathy.

Thrombomodulin

Thrombomodulin is an EC glycoprotein that has both anticoagulant and anti-inflammatory properties. It binds to thrombin on the EC surface to activate protein C, which inhibits factors Va and VIIIa to limit thrombin formation. Thrombomodulin also has domains that scavenge and bind pro-inflammatory cytokines before they can reach their target (25). In a stress response, such as after injury, proteases and reactive oxygen species lead to the release of membrane-bound thrombomodulin into circulation. Like syndecan-1, soluble thrombomodulin is a recognized biomarker of endotheliopathy, though correlations with outcomes are not as strong, and efforts to target it as a therapeutic have not yet been successful (5, 16, 26).

vWF and ADAMTS13

There have been several studies in trauma demonstrating a decrease in ADAMTS13 activity and an increase in hyperadhesive vWF. Matsumoto et al. showed that early after trauma, ADAMTS13 activity is reduced and significantly correlates with changes in prothrombin time and fibrin/fibrinogen degradation products. ADAMTS13 activity at less than 50% of normal levels also correlates with the development of disseminated intravascular coagulation and an increased need for transfusion (27). Dyer et al. found similar associations between ADAMTS13 and coagulopathy (15). Since plasma levels of vWF and ADAMTS13 vary widely among healthy individuals and can even vary by blood type, the ADAMTS13:vWF ratio may prove to be a more useful biomarker than either level alone (28, 29).

Extracellular Vesicles

Extracellular vesicles (EVs) are small membrane-bound structures released from multiple cell types in response to external stimuli during cell activation or injury. Bioactive molecules released from EVs can cause secondary injury both locally and systemically. EVs isolated from blood can potentially be used as biomarkers of endotheliopathy. Their membranes often express anionic phospholipids that initiate and propagate coagulation (30). They can also have surface-anchored proteins, such as vWF and tissue factor (TF), that aberrantly promote binding of the EV to ECs and recruitment of leukocytes at locations far from the initial site of injury. Through adoptive transfer experiments, EVs collected from patients with traumatic brain injury or from patients with hemorrhagic shock have been shown to induce endothelial dysfunction and coagulopathy when injected into non-injured mice (31-33). The precise mechanisms by which this occurs remain unclear.

Thromboelastography

One of the key challenges in diagnosing endotheliopathy is that it is driven by many processes. Several distinct phenotypes of endotheliopathy have been identified, with varying degrees of pro- and anti-thrombotic as well as pro- and anti-fibrinolytic properties (16). Given the complexity of changes that occur after injury, whole blood tests of coagulative kinetics and clot strength, such as thromboelastography (TEG), have gained popularity as possible tools for phenotyping. Some studies have demonstrated that TEG results can diagnose endotheliopathy and coagulopathy, correlating with biomarkers such as syndecan-1 or ADAMTS13 (34-36). However, no studies to date have outlined standardized value ranges or guidelines for definitive phenotyping. Routine blood tests such as PT, PTT, and INR indicate coagulopathy but correlate poorly with clinical bleeding and the need for transfusion, especially in patients without active hemorrhage (37). Nevertheless, a recent study of over 20,000 trauma patients by Teeter et al. found that abnormal PT and PTT values are independent predictors of mortality in trauma patients, even in those with low injury severity (6), underscoring our current lack of mechanistic insight into coagulopathy and highlighting the limited use of routine coagulation studies in diagnosing endotheliopathy.

POTENTIAL THERAPEUTICS TO MITIGATE INJURY-INDUCED ENDOTHELIOPATHY

Clinical evidence of glycocalyx degradation, increased fibrinolysis, platelet dysfunction, and increased vascular permeability can be seen within minutes after traumatic insult (4). These changes persist well into the resuscitative period and have been strongly linked to significant morbidity and mortality (38).

Plasma

Timely treatment of endotheliopathy is vital for optimizing patient outcomes. The landmark PROMMTT, PROPPR, and PAMPer trials all demonstrated that early resuscitation with plasma improved survival in patients with hemorrhagic shock, with clinical and preclinical evidence suggesting its protective role is due in part to maintaining the glycocalyx (39-42). In a secondary analysis of PAMPer, Gruen et al. found that plasma administration was associated with a decrease in endothelial and glycocalyx biomarkers (syndecan-1, thrombomodulin, and vascular endothelial growth factor) in a subgroup of patients with higher injury severity scores and a higher incidence of blunt trauma (43). Plasma contains several bioactive agents which may contribute to its protective effects against glycocalyx degradation including fibrinogen, sphingosine-1 phosphate, antithrombin, and adiponectin (44).

Fibrinogen

Fibrinogen has been identified as an important mediator of endothelial protection by binding glycosaminoglycans and stabilizing syndecan-1 (45, 46). It has also been found to be an independent predictor of mortality in patients with severe trauma (47). Preclinical studies have demonstrated that fibrinogen alone has endothelial protective effects (48). Fibrinogen is a key constituent of cryoprecipitate, which can also attenuate endotheliopathy (49, 50). A recent clinical study, however, failed to demonstrate benefit from the early use of empiric cryoprecipitate in patients at risk for bleeding, though the study had limitations and did not assess endotheliopathy (51).

Activated Protein C

Activated Protein C has both pro-coagulant and anti-inflammatory properties. It depresses fibrinolysis by inactivating factors Va and VIIIa, while also exerting anti-apoptotic effects through inhibition of protease-activated receptor-1 (PAR-1) signaling. A synthetic variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity. In vitro studies show that treatment with 3K3A-aPC can mitigate endotheliopathy by reducing endothelial cell permeability (52, 53). The early results are promising, but further research is warranted.

Sheddase Inhibitors

Therapeutics that target the release and activity of sheddases, including etanercept, angiopoietin-1, and hydrocortisone, have been studied in preclinical models. In a recent randomized controlled trial by Gao et al., patients who received doxycycline, an MMP inhibitor, prior to undergoing cardiopulmonary bypass exhibited reduced glycocalyx shedding (54). Synthetic heparan sulfate (dekaparin) has also been studied as a possible therapeutic. Vidaurre et al. demonstrated that dekaparin had similar anti-inflammatory and organ-protective properties to plasma in a mouse model of trauma and hemorrhagic shock (55). Inhibition of sheddases and restoration of shed glycocalyx components could be a novel strategy for mitigating endotheliopathy.

vWF and ADAMTS13

Preclinical studies have examined the role of recombinant human ADAMTS13 (rhADAMTS13) in regulating vWF activity. rhADAMTS13 has been shown to reduce inflammation and EC function in mice with renal ischemia and decrease lung permeability, lung and kidney injury, and syndecan-1 shedding in rats with hemorrhagic shock (56, 57). An FDA-approved human recombinant ADAMTS13 is now available for clinical use in patients with thrombotic thrombocytopenic purpura with promising results, but rhADAMTS13 has not yet been studied in trauma patients (58).

Removal of Extracellular Vesicles

By transporting pro-inflammatory and pro-coagulant molecules throughout the bloodstream, injury-induced extracellular vesicles contribute to the systemic response after trauma. Lactadherin is an apoptotic cell-scavenging molecule that can remove pathologic EVs by coupling them to macrophages and facilitating phagocytosis. It has been shown to promote the clearance of EVs, decrease coagulopathy and EC permeability, and improve neurologic outcomes and survival in mouse TBI models (59). Lactadherin has not yet been studied in trauma models, and its use may be somewhat limited as it binds and clears all phosphatidylserine expressing cells as well as EVs.

CONCLUSION

Injury-induced endotheliopathy represents an important pathologic response to trauma. Key biomarkers such as syndecan-1 can aid in the diagnosis, but testing is not yet available clinically. As the mechanisms of endotheliopathy are better understood, therapeutics are being identified (Figure 1) and show promise although data is primarily preclinical.

Figure 1.

Several potential therapeutic targets to preserve the endothelial glycoclayx to prevent injury-induced endotheliopathy have been identified. Plasma resuscitation has a protective effect on the endothelial glycocalyx by preventing glycocalyx degradation. Heparan sulfates which are bound to a syndecan core show anticoagulant mechanisms. Heparan sulfates bind antithrombin 3 to inhibit thrombin (IIa). Dekaparin, a synthetic variant of heparan sulfate, interacts with antithrombin 3 and inhibits nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), Factor Xa, Factor IIa and upregulate prostacyclin. Doxycycline inhibits matrix metaloproteases (MMPs) and prevents endothelial glycocalyx degradation. Fibrinogen, a component of cryoprecipitate used in resuscitation, binds with glycosaminoglycans (GAGs) and stabilizes syndecan-1 hence preventing syndecan-1 shedding and protecting the endothelial glycocalyx. Endothelial cells can potently activate the coagulation system through secretion of von Willebrand Factor (vWF) from Weibel-Palade Bodies, which bind platelets to the sub endothelium. Recombinant a disintegrin and metalloprotease with thrombospondin type I motifs, member 13 (ADAMTS-13) regulates VWF activity and reduces inflammation and syndecan-1 shedding. Endothelial barrier function is regulated by the transcellular pathway through caveolae. The paracellular pathway is regulated through expression of junctional proteins such as junctional adhesion molecules (JAM), gap, and adherens, and junctional proteins. 3K3A-aPC, which is a synthetic variant of activated protein C, decreases endothelial membrane permeability and prevents injury induced endotheliopathy. Lactadherin is a cell scavenging molecule which removes pathologic extracellular vesicles (EVs) by integrating with macrophages and promoting phagocytosis. Created in BioRender. Abdullah, S. (2024) https://BioRender.com/z80s194

KEY POINTS:

• Injury-induced endotheliopathy describes the constellation of endothelial dysfunction, inflammation and coagulopathy that develops soon after injury.

• Several biomarkers of endotheliopathy have been identified with syndecan-1 being the most widely studied including its correlation with adverse outcomes after injury.

• Preclinical therapeutics to mitigate endotheliopathy are being investigated, but to date, only fresh frozen plasma has any supportive clinical data.