MICROVASCULAR ENDOTHELIAL ACTIVATION/DYSFUNCTION AND DYSREGULATION OF THE ANGIOPOIETIN-TIE2 SYSTEM IN THE PATHOGENESIS OF LIFE-THREATENING INFECTIONS

ABSTRACT

Microvascular endothelial activation/dysfunction has emerged as an important mechanistic pathophysiological process in the development of morbidity and mortality in life-threatening infections. The angiopoietin-Tie2 system plays an integral role in the regulation of microvascular endothelial integrity. Angiopoietin-1 (Ang-1), produced by platelets and pericytes, is the cognate agonistic ligand for Tie2, promoting endothelial quiescence and inhibiting microvascular leak. Angiopoietin-2 (Ang-2), released from activated endothelial cells in Weibel-Palade bodies, competes with Ang-1 for binding to Tie-2, thereby promoting endothelial activation/dysfunction and microvascular leak. In healthy homeostasis, levels of Ang-1 far exceed Ang-2 in circulating serum/plasma. In diseases associated with systemic inflammation, Ang-1 falls and Ang-2 rises (i.e., Ang-1/2 dysregulation). Our research has shown that Ang-1/2 dysregulation is a prominent feature in a number of life-threatening infections and critical illnesses, including sepsis, cerebral malaria, COVID-19, streptococcal toxic shock syndrome (STSS), hemolytic-uremic syndrome (HUS), dengue, and CAR T-cell-associated neurotoxicity. Further work has implicated Ang-1/2 dysregulation in the development of end-organ injury, including acute lung injury/ARDS, acute kidney injury (AKI), and blood-brain-barrier (BBB) breakdown. Current studies are focused in three areas: (a) translation of Ang-1 and -2 as clinically informative prognostic and “theranostic” biomarkers in critically ill individuals; (b) incorporation of Ang-1/2 assays in a point of care device for clinical triage decision making; and (c) development of an engineered Ang-1 super agonist nanoparticle as a novel pathogen-agnostic therapeutic to prevent and/or mitigate end-organ dysfunction in individuals with life-threatening infections and critical illnesses associated with systemic inflammation.

INTRODUCTION

Antimicrobials are not enough.

“It appears that patients are dying not from their infections, but rather their reaction to them.”

Sir William Osler, 1904

Sepsis, the clinical syndrome of infection-related systemic inflammatory response syndrome (SIRS), is recognized as one of the oldest and most confounding enigmatic problems in medicine. It is a common and devastating complication in critically ill patients and remains a major cause of morbidity and mortality in the intensive care unit (ICU), especially when patients progress to septic shock. In large part, the development of major end-organ (i.e., lung, kidney, liver, heart) injury/dysfunction is responsible for the high morbidity and mortality of clinical sepsis.

Despite decades of research, an effective treatment for sepsis and sepsis-related multiple organ dysfunction syndrome (MODS) has remained elusive. No drug is currently approved by the Food and Drug Administration (FDA) for the specific treatment of sepsis and sepsis-related complications. This lack of an effective therapeutic approach for sepsis represents a major unmet need in clinical medicine.

Infection by a microbial pathogen is necessary but not sufficient to cause sepsis. The pathophysiology induced by the host response to a microbial pathogen defines sepsis and the clinical course of an infected individual. Endothelial activation/dysfunction and associated microvascular leak have emerged as critical pathogenic mechanisms in sepsis and MODs. In June 2010, the Division of Blood Diseases and Resources of the National Heart, Lung, and Blood Institute (NHLBI) convened a workshop involving a multidisciplinary group of experts, entitled “Blood and Vascular Systems Response to Sepsis,” to address the challenges posed by sepsis and sepsis-induced MODS. This group concluded that sepsis represents a syndrome of severe endothelial dysfunction that causes MODS in response to intra- or extravascular infection. The panel of experts recommended “development and application of novel methods for studies of vascular integrity and vascular injury in animal models of sepsis, and innovative approaches to cytoprotection of the vasculature.” (https://www.nhlbi.nih.gov/events/2010/sepsis-meeting) The recognition of the critical mechanistic role of endothelial dysfunction in the pathogenesis of sepsis has generated a new direction for novel, innovative treatment strategies to improve clinical outcomes in critically ill patients—namely, the development of agents to prevent or decrease endothelial dysfunction and consequently preserve or enhance endothelial barrier integrity in sepsis.

The angiopoietin-1/2-Tie-2 axis has emerged as a critical system regulating endothelial activation/dysfunction in a number of pathologic conditions associated with systemic inflammation (13-26). Angiopoeitin-1 (Ang-1; produced primarily by pericytes) and -2 (Ang-2; produced primarily by endothelial cells and stored in Weibel-Palade bodies), initially implicated in embryonic vasculogenesis and angiogenesis, play divergent roles in mediating inflammation and vascular quiescence. Angiopoietin-1 and -2 are antagonistic ligands that bind to the extracellular domain of the Tie-2 receptor, expressed almost exclusively in endothelial cells. Ang-1 stabilizes the endothelium, inhibits vascular leakage, and suppresses inflammatory and coagulation gene expression through constitutive activation (phosphorylation) of Tie-2, while Ang-2 generally promotes loss of barrier integrity by sensitizing endothelium to stimuli that disrupt microvascular integrity, resulting in vascular leak and end-organ dysfunction (1-7).

ANG-1/2: CLINICALLY INFORMATIVE PROGNOSTIC BIOMARKERS FOR SYSTEMIC INFECTIOUS DISEASES OF PUBLIC HEALTH IMPORTANCE

Over the past 15 years, we have investigated the role of serum/plasma Ang-1 and -2 as clinically informative biomarkers in a number of sepsis-related conditions. A number of these studies are reviewed below and outlined in Table 1.

TABLE 1.

Selected Studies of Ang-1 and -2 as Clinically Informative Biomarkers in Life-Threatening Infectious Diseases

Disease	Results	Reference(s)
Sepsis—ICU	Ang-1/2 dysregulation is associated with severity of illness and subsequent adverse clinical outcomes (e.g., AKI ARDS, shock, death) in critically ill ICU patients. Circulating levels of Ang-2 remain elevated in patients with adverse clinical outcomes. Circulating levels of Ang-1 are persistently decreased during the course of illness in patients with adverse clinical outcomes.	8-18
Sepsis—Developing World	Circulating Ang-2 levels contributed to identification of three biological sub-phenotypes of sepsis with differing clinical prognoses.	19
Malaria	Magnitude of Ang-1/2 dysregulation correlated with disease severity. High Ang-2: Ang-1 ratio was associated with progression to cerebral malaria and mortality.	20-23
Toxic Shock Syndrome (TSS)	Systemic Ang-1/2 dysregulation at clinical presentation was associated with progression to TSS in patients with invasive group A streptococcal infection.	24
Hemolytic-Uremic Syndrome (HUS)	Systemic Ang-1/2 dysregulation at clinical presentation was associated with progression to HUS in children with E. coli O157:H7-associated bloody diarrhea.	25
COVID-19	Systemic Ang-1/2 dysregulation was observed in severe COVID-19, at levels similar to that observed in severe non-COVID-19 sepsis.	18

SEPSIS IN THE INTENSIVE CARE UNIT (DEVELOPED WORLD)

In healthy individuals, circulating levels of plasma Ang-1 are high, and circulating plasma Ang-2 is barely detectable by immunoassay. During systemic inflammation, Ang-1 levels decline, and Ang-2 levels rise. In a cohort of 70 adult patients with severe sepsis, low plasma levels of Ang-1 on ICU admission correlated with 28-day mortality. Both low Ang-1 and high Ang-2 levels measured serially were associated with 28-day mortality. Ang-2 levels correlated with end-organ dysfunction/injury [i.e., acute kidney injury (AKI), acute respiratory distress syndrome (ARDS), and septic shock]. These results suggested the potential for Ang-1/2 to function as clinically informative biomarkers of disease severity and clinical outcome in sepsis (8).

A subsequent study that enrolled 943 critically ill adult patients with SIRS who were admitted to an ICU of an academic medical center confirmed the findings discussed above. High levels of plasma Ang-2 and low levels of plasma Ang-1 were associated with increased mortality. This study concluded that early measurements of Ang-1 and -2 in critically ill patients with SIRS were associated with death and organ dysfunction, especially AKI, independently of biomarkers of inflammation measured simultaneously (9-15). Similar findings were also observed in a study of pediatric sepsis (16). Importantly, Ang-1/2 dysregulation appears to be a general pathophysiological process in sepsis, regardless of the microbial pathogen responsible for the underlying infection (17,18).

SEPSIS IN THE DEVELOPING WORLD (SUB-SAHARAN AFRICA)

Sepsis is not only a major clinical problem in the developed world, but it also represents an underappreciated cause of morbidity and mortality in the developing world. In a study of 426 consecutively enrolled adult patients with sepsis in Uganda, latent class analysis revealed that circulating levels of both Ang-1 and -2 contributed to the identification of distinct subgroups of patients with differing risks for sepsis-related mortality (19). Thus, Ang-1/2 dysregulation appears to be a generalized phenomenon in sepsis beyond the ICU.

MALARIA (SEVERE AND CEREBRAL MALARIA)

Clinical malaria can range in severity from a mild febrile syndrome to cerebral malaria, a life-threatening disease. Individuals infected with Plasmodium falciparum are at risk of developing cerebral malaria, but tools to identify those at risk of progression from uncomplicated or severe malaria to potentially fatal cerebral malaria are currently lacking. Cerebral malaria remains a major source of significant morbidity and mortality in children in malaria-endemic regions of the world, especially in sub-Saharan Africa. Ang-1 and -2 were investigated for their potential use as predictive biomarkers in malaria in both children in Uganda and adults in Thailand in separate studies. Ang-1 (low plasma levels) and the Ang-2/1 ratio (elevated levels) were strongly associated with disease severity and progression to severe and cerebral malaria in both adults and children. These results suggested the utility of measurement of Ang-1 and -2 as prognostic biomarkers for clinical decision making and resource utilization in the management of malaria. Moreover, these studies identified the Ang-Tie2 axis as a potential target for therapeutic intervention in severe and cerebral malaria (20-23).

STREPTOCOCCAL TOXIC SHOCK SYNDROME

Streptococcal toxic shock syndrome (STSS) is a super-antigen disease mediated by invasive group A streptococcal (Streptococcus pyogenes) infection. Exotoxins released by invasive group A streptococcal bacteria can release exotoxins that activate a large number of T cells in a major histocompatibility complex class II (MHC class II)-unrestricted activation, to initiate an overwhelming inflammatory response that results in diffuse endothelial cell activation, vascular leak, hypotension, end-organ dysfunction, and coagulopathy. Prediction of infected individuals who will progress to STSS can be clinically challenging. Plasma Ang-1 and -2 concentrations were measured in 37 patients with confirmed invasive group streptococcal infection with and without concurrent STSS. Ang-1/2 dysregulation (low Ang-1, high Ang-2) was greater in STSS than in invasive infection without shock, and this dysregulation improved during convalescence. These results suggest that Ang-1 and -2 may provide useful clinical information as biomarkers in STSS and be used to monitor therapeutic response in STSS (24).

ESCHERICHIA. COLI O157:H7-ASSOCIATED HEMOLYTIC-UREMIC SYNDROME (HUS)

E. coli O157:H7-associated hemolytic-uremic syndrome (HUS) is a type of thrombotic microangiopathy characterized by endothelial cell activation/dysfunction and prothrombotic abnormalities. Based on the hypothesis that Ang-1/2 dysregulation could underlie the pathophysiology of HUS, plasma concentrations of Ang-1 and -2 were measured in 77 children with documented E. coli O157:H7 bloody diarrhea. Ang-1, Ang-2, and the Ang-2/1 ratio were significantly different in the pre-HUS phase of illness or uncomplicated infection. Moreover, Ang-1/2 dysregulation preceded HUS and worsened as HUS developed. These results demonstrated that Ang-1/2 dysregulation preceded and predicted the development of HUS in E. coli O157:H7 infection, indicating that subclinical endothelial activation/dysfunction precedes overt microangiopathy in HUS. Furthermore, these findings indicate that plasma Ang-1/2 could provide clinically useful disease information regarding prognosis and therapeutic response when employed for evaluation of individuals with E. coli O157:H7 infection (25).

COVID-19

Ang-1/2 dysregulation was investigated in severe COVID-19 (n=78 ICU patients) and compared to the level of dysfunction observed in non-COVID-19 sepsis (n=93 ICU patients). The plasma levels of Ang-1 and -2 were similar (not statistically different; p > 0.05) in both groups of patients. Notably, Ang-1/2 dysregulation, indicative of endothelial activation/dysfunction was not greater in severe COVID-19, despite previous reports of “endotheliopathy” as a unique pathogenic feature of severe COVID-19. Instead, the degree of Ang-1/2 dysregulation in severe COVID-19 sepsis was similar to that observed in non-COVID-19 severe sepsis (18).

ANG-1/2: UTILITY FOR THERAPEUTIC DECISION MAKING IN CRITICALLY ILL PATIENTS— POTENTIAL “THERANOSTIC” PRECISION MEDICINE IN THE ICU

Sepsis-Related Acute Kidney Injury (AKI) in the ICU

As noted previously, dysregulation of the Ang-1/2-Tie2 axis has been implicated mechanistically in the development of AKI related to septic shock (10,12,13). The potential utility of circulating Ang levels to serve as “theranostic” biomarkers (biomarkers used in a “precision medicine” approach to guide therapeutic decision making) was recently demonstrated in a retrospective post-hoc analysis of the Vasopressin in Septic Shock Trial (VASST). Latent class analysis identified two biological sub-phenotypes of septic shock related AKI, based in part on circulating Ang-2 levels, with differential responses in terms of mortality when vasopressin was added to standard norepinephrine vasopressor therapy (26). If these findings can be confirmed in a subsequent, planned prospective clinical trial, then Ang-2 could be employed as a “theranostic” biomarker to decide which patients should receive vasopressin support to improve clinical outcomes and survival in septic shock.

ANG-1/2-TIE2 AXIS SYSTEM: POTENTIAL THERAPEUTIC TARGET TO IMPROVE CLINICAL OUTCOMES IN LIFE-THREATENING DISEASES

Cerebral Malaria

As noted previously, cerebral malaria persists as a leading cause of global morbidity and mortality, especially in children in sub-Saharan Africa. Amg-1/dysregulation was linked mechanistically in the pathogenesis of cerebral malaria in a manuscript published by Higgins et al (23). First, Ang-1 expression and soluble Tie-2 expression were associated with disease severity and clinical outcome in a prospective study of Ugandan children. Second, a preclinical murine model of cerebral malaria (caused by experimental infection with Plasmodium berghei ANKA) was utilized to demonstrate that Ang-1 was necessary for maintenance of vascular integrity, preservation of the blood-brain-barrier, and survival following infection. Therapeutic administration of an Ang-1 derivative, BowAng1, preserved blood-brain-barrier integrity and prolonged survival in combination with artesunate (the anti-malarial antimicrobial of choice for treatment of P. falciparum malaria), beyond that conferred by treatment with artesunate alone. These results indicate a potential role for therapeutic agents that target the Ang-Tie2 axis system as adjunctive treatment strategies to improve clinical outcomes in malaria.

CAR T CELL MEDIATED CYTOKINE RELEASE SYNDROME AND NEUROTOXICITY

CD19-targeted chimeric antigen receptor modified T (CAR T) cell therapy has emerged as a promising therapeutic modality for the treatment of patients with refractory B cell malignancies. However, infusion of CAR T cells can be associated with cytokine release syndrome (CRS) that can be associated with end-organ dysfunction and injury, including potentially fatal CNS neurotoxicity [immune effector cell-associated neurotoxicity syndrome (ICANS)]. Blood-brain-barrier breakdown may occur in fatal CAR T cell related neurotoxicity, and Ang-1/2 dysregulation has been implicated in this pathogenic process. Patients with evidence of Ang-1/2 dysregulation prior to the lymphodepletion procedure involved in CAR T cell therapy may increase their risk for subsequent development of ICANS. These findings indicate that the Ang-Tie2 axis represents a target for development of novel therapeutic strategies to reduce CRS-induced morbidity and prevent fatal neurotoxicity in CAR T cell therapy for malignancies (27-29).

DEVELOPMENT OF THERAPEUTIC TIE2 SUPERAGONIST NANOPARTICLES FOR PATHOGEN-AGNOSTIC TREATMENT OF LIFE-THREATENING INFECTIONS

We are currently collaborating with the Institute for Protein Design (https://www.ipd.uw.edu/) at the University of Washington to develop an engineered therapeutic Angiopoietin-1 superagonist nanoparticle for treatment of microvascular leak in diseases associated with systemic inflammation. This nanoparticle, designed to cluster and promote Tie2 signaling to preserve endothelial integrity, could be administered in a pathogen-agnostic manner to prevent or ameliorate end-organ injury and maintain functional end-organ integrity in patients at risk for AKI, ARDS, septic shock, and neurotoxicity associated with BBB breakdown.

ANG-TIE2 AXIS: SUMMARY OF POTENTIAL APPLICATIONS TO PATIENT CARE

1. Biomarker for life-threatening infections

2. Identification of patients at risk for deleterious clinical outcomes at clinical presentation

3. Evidence-based decision making

4. Prognostic biomarker for risk stratification and resource utilization

5. Monitor response to therapy

6. Potential real-time precision medicine in critically ill patient populations

SUMMARY: DYSREGULATION OF THE ANG-TIE2 SYSTEM IN LIFE-THREATENING INFECTIOUS DISEASES

1. Biological plausibility—preclinical models support causal/mechanistic association with endothelial cell dysfunction, vascular leak, and end-organ injury

2. Consistency—strong and consistent associations reported in multiple studies

3. Proportional (“dose-dependent”) associations—high plasma/serum Ang-2 values are associated with increased disease severity and adverse clinical outcomes

4. Broad applicability—circulating Ang-2 is elevated in a wide variety of diseases characterized by vascular injury and microvascular leak

5. Temporal relationship—increased circulating Ang-2 precedes development of adverse clinical outcomes

CURRENT RESEARCH PRIORITIES

1. Establish use cases for employment of Ang-2 in predictive, prognostic, and “theranostic” clinical decision making.

2. Seek FDA approval of the Amg-2 prototype assay as a clinical assay.

3. Combine assays for Ang-2 and soluble triggering receptor expressed on myeloid cells 1 (TREM-1) (30,31), a novel biomarker of innate immune activation, in a rapid point-of-care multiplex assay platform of critically ill inpatients and outpatients.

4. Develop an effective novel Ang-1 therapeutic drug to prevent end-organ injury in a pathogen-agnostic manner.

DISCUSSION

Tweardy, Houston: Really exciting work—you showed the benefit of your stabilized angiopoietin-1 in cerebral malaria models. Given the broad possibilities for the use of such an agent, did you use some of the more conventional models of sepsis such as the cecal ligation model?

Liles, Seattle: We've done a lot of cecal ligation work but to investigate stabilization of the blood-brain-barrier we chose the experimental cerebral malaria model in order to prove the concept per se. We have worked a lot in cecal ligation and puncture (CLP) investigating a number of therapeutic strategies for sepsis. One such approach in which we have been involved is mesenchymal stromal cell therapy, now in phase III clinical trials both in the United States and Canada. All the preclinical work supporting the mesenchymal stroma and cell therapy clinical trials were performed in collaboration with my Canadian colleagues using the CLP model of polymicrobial sepsis, which is a great clinical model.

Sharma, San Antonio: Great work. I really like the biomarker approach in acute kidney injury (AKI). Could you comment on the role of angiopoietin-1 and -2 in chronic diseases such as chronic kidney disease which is also characterized by endothelial dysfunction?

Liles, Seattle: Yes, I've done a bit of this work with my wife, who's also an academic, and we've looked at human immunodeficiency virus (HIV). What we found in HIV is that biomarkers of endothelial activation/dysfunction are elevated in virally suppressed individuals living with HIV who subsequently develop adverse cardiovascular outcomes including stroke and myocardial infarction (MI). I didn't talk about those studies today. Biomarkers of endothelial activation/dysfunction have clinical utility in some chronic diseases, such as progression of chronic kidney disease. I also did not have time today to discuss recent work in which urinary angiopoietin-2 functions as a predictive biomarker for sepsis-induced AKI. Conceivably, urinary angiopoietin-2 could serve as a biomarker to guide novel therapeutic interventions to prevent or reduce adverse kidney outcomes in sepsis.

Ross, St. Louis: I found out that the ACCA is populated by a lot of nephrologists and that's a good thing! I’m looking at your data and realizing that a lot of those effects could be from vascular endothelial growth factor (VEGF) and so I’m just curious if you looked at VEGF.

Liles, Seattle: Yes, we have looked at VEGF in comparison with these other biomarkers of endothelial/activation dysfunction. Of these, the most telling, most positive, and most clinically informative was the Ang-1/2-Tie2 system over the VEGF system. However, there is interplay between the VEGF system and the Ang-1/2-Tie2 system that has not been fully elucidated and is not completely understood.