MINI REVIEW published: 25 April 2022 doi: 10.3389/fmed.2022.811504 Complement Mediated Endothelial Damage in Thrombotic Microangiopathies Miquel Blasco 1,2† , Elena Guillén-Olmos 1† , Maribel Diaz-Ricart 3,4 and Marta Palomo 3,4,5* 1 Department of Nephrology and Kidney Transplantation, Hospital Clínic, Centro de Referencia en Enfermedad Glomerular Compleja del Sistema Nacional de Salud (CSUR), University of Barcelona, Barcelona, Spain, 2 Institute of Biomedical Research August Pi i Sunyer (IDIPABS), Malalties Nefro-Urològiques i Trasplantament Renal, Barcelona, Spain, 3 Hematopathology Unit, Department of Pathology, Hospital Clínic of Barcelona, Biomedical Diagnosis Centre (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain, 4 Barcelona Endothelium Team, Barcelona, Spain, 5 Josep Carreras Leukaemia Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain Thrombotic microangiopathies (TMA) constitute a group of different disorders that have a common underlying mechanism: the endothelial damage. These disorders may exhibit different mechanisms of endothelial injury depending on the pathological trigger. However, over the last decades, the potential role of the complement system (CS) has gained prominence in their pathogenesis. This is partly due to the great efficacy of complement-inhibitors in atypical hemolytic syndrome (aHUS), a TMA form where Edited by: Eleni Gavriilaki, the primary defect is an alternative complement pathway dysregulation over endothelial G. Papanikolaou General Hospital, cells (genetic and/or adquired). Complement involvement has also been demonstrated Greece in other forms of TMA, such as thrombotic thrombocytopenic purpura (TTP) and in Reviewed by: Shiga toxin-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS), as Peter Hughes, Royal Melbourne Hospital, Australia well as in secondary TMAs, in which complement activation occurs in the context of Jeffrey Laurence, other diseases. However, at present, there is scarce evidence about the efficacy of Cornell University, United States complement-targeted therapies in these entities. The relationship between complement *Correspondence: Marta Palomo dysregulation and endothelial damage as the main causes of TMA will be reviewed mpalomo@carrerasresearch.org here. Moreover, the different clinical trials evaluating the use of complement-inhibitors † These authors have contributed for the treatment of patients suffering from different TMA-associated disorders are equally to this work summarized, as a clear example of the entry into a new era of personalized medicine in Specialty section: its management. This article was submitted to Keywords: C5b-9 deposition, complement system activation, complement blockade, endothelia cells, membrane Hematology, attack complex, thrombotic microangiopathies a section of the journal Frontiers in Medicine Received: 08 November 2021 INTRODUCTION Accepted: 14 March 2022 Published: 25 April 2022 Thrombotic microangiopathies (TMA) constitute a group of disorders characterized by Citation: micoangiopathic hemolysis, platelet consumption and systemic organ damage. The identification of Blasco M, Guillén-Olmos E, the underlying etiology among TMA-associated disorders is a major challenge due to the variability Diaz-Ricart M and Palomo M (2022) of clinical presentation and the absence of pathognomonic histological findings. Despite these Complement Mediated Endothelial Damage in Thrombotic factors, its assessment is crucial for directed therapy (1). Microangiopathies. The vascular endothelium plays a pivotal role in the regulation of the hemostatic balance. In this Front. Med. 9:811504. regard, endothelial cells (EC) provide a non-thrombogenic inner layer of the vascular wall which doi: 10.3389/fmed.2022.811504 maintains the blood fluidity, prevent thrombosis through different anticoagulant, and antiplatelet Frontiers in Medicine | www.frontiersin.org 1 April 2022 | Volume 9 | Article 811504 Blasco et al. Endothelium and Complement in TMA mechanisms, and regulate clot formation, limiting it to those complement factor H) have been identified in about 40–60% of areas without vascular integrity (2). Endothelial injury is the patients (10–12). Coexistence of these predisposing factors with common underlying mechanism among different TMA, leading environmental triggers or complement amplifying conditions to the microvasculature thrombosis observed in these conditions (i.e., infections, drugs, surgery, pregnancy) may lead to disease (3) (Figure 1). onset (13, 14). aHUS is an ultra-rare disease, that affects The complement system (CS) is a central component both pediatric and adult populations, of a systemic nature of innate immunity and bridges the innate to the adaptive and with highly morbidity and mortality rates (14–16). These immune response. Activation of complement pathways characteristics, together with the need for a clinical diagnosis under physiological conditions facilitates the clearance of (exclusion of all potential TMA causes), could delay an early microbes, damaged cells and immune complexes from an etiological treatment with terminal complement blockers (17– organism, promotes inflammation, and attacks pathogen’s cell 22), decreasing organ function recovery outcomes (especially membrane. To prevent undesirable activation and tissue damage, kidney function). complement activation is strictly controlled by numerous Endothelial activation and injury in aHUS occur mainly regulators under normal circumstances (4). due to immune innate system dysregulation and the loss of During the last decades, the contribution of dysregulated protection at cell surfaces. Disease phenotype and severity will complement activation to endothelial damage has been widely depend on pro- and anti-inflammatory cytokines balance and demonstrated in some TMA forms. Among them, the prototype regulation of individual complement components (23, 24). Once is atypical hemolytic syndrome (aHUS), in which complement the complement cascade is amplified, the terminal phase will dysregulation occurs as the primary event (genetic and/or play a key role, especially, the cleavage of C5 molecule into adquired alternative complement pathway dysregulation). C5a and the subsequent generation of membrane attack complex Complement involvement has also been demonstrated in (MAC), also called C5b-9 (16). C5a is a potent anaphylatoxin, thrombotic thrombocytopenic purpura (TTP) and in Shiga promoting the recruitment of platelets and leukocytes to the toxin-producing Escherichia coli hemolytic uremic syndrome endothelial surface. In addition, it causes endothelial retraction (STEC-HUS), but, in these cases, it occurs as a secondary event, with the consequent exposure of the underlying basement triggered either by ADAMTS13-deficiency or toxin-mediated membrane and overexpression of procoagulant tissue factor (25). injury, respectively (5). Finally, the role of complement has MAC forms pores in pathogens or targeted cell membranes also been suggested to be involved in other TMA-associated leading to osmolysis (cytolytic effector of innate and adaptative disorders, although it has not been completely elucidated (6). immunity). It can also induce cell activation and intracellular Distinguishing among the different causes of TMA may signaling (26), promoting a switch from an anti-coagulant and be challenging, leading to a delay in etiologic diagnosis and, anti-inflammatory endothelial phenotype to a highly active, therefore, in the initiation of the most appropriate treatment. pro-coagulant phenotype. Although it may not be identified The development of diagnostic tools based on functional and in all cases, aHUS patients present a defect of AP regulation genetic studies to assess the involvement of complement in over EC surfaces, allowing an abnormal formation of terminal the pathogenesis of the different clinical entities is crucial complement phase (C5a and C5b-9) on the endothelium, which (1). In this regard, new management approaches of TMA lead to cell apoptosis and TMA development, with a special are being evaluated, and complement-targeted therapies are predilection for the kidney vasculature (27). gaining importance since they selectively block this inflammation pathway, thus avoiding the side effects of some traditional therapies (7). Thrombotic Thrombocytopenic Purpura This review addresses the relationship between endothelial ADAMTS-13 (a disintegrin and metalloproteinase with a damage and complement dysregulation among the main causes thrombospondin type 1 motif, member 13) deficiency is of TMA. We also summarize the clinical trials carried out in the key event in the pathophysiology of TTP (28). When TMA-associated disorders treated with complement inhibitors functional, ADAMTS-13 cleaves the ultra-Large von Willebrand (Table 1) and the need to consolidate and develop different factor (ULVWF) multimers, which are highly reactive to biomarkers that may allow an individualized treatment in platelets, before VWF is released to plasma. ADAMTS-13 the near future. deficiency, which can be congenital or acquired, leads to the accumulation of ULVWF secreted by the endothelium, causing platelet aggregation, formation of microthrombi and ultimately COMPLEMENT ENDOTHELIAL DAMAGE endothelial damage (29). However, an important factor in the IN THROMBOTIC MICROANGIOPATHIES pathogenesis of this disease is the different susceptibility of the vascular beds. The selective injury to dermal, renal, and cerebral Atypical Hemolytic Uremic Syndrome microvasculature does not occur in EC from lung and liver Dysregulation of the alternative complement pathway over (30). Notably, this different susceptibility is not appreciated in EC surfaces is the main cause of TMA development in cells with a macrovascular origin. This phenomenon has not atypical hemolytic uremic syndrome (aHUS) (8, 9). Genetic been described only in vitro experiments (31) but also in animal variants in complement components (gain or loss function models (32), and offers the potential explanation for tissue mutations) and/or acquired conditions (antibodies against distribution damage in TTP. Frontiers in Medicine | www.frontiersin.org 2 April 2022 | Volume 9 | Article 811504 Blasco et al. Endothelium and Complement in TMA FIGURE 1 | Pathophysiology of complement mediated endothelial damage in TMA. ICAM-1, Intercellular Adhesion Molecule 1; VCAM-1, Vascular cell adhesion protein 1; TF, tissue factor; TM, thrombomodulin; vWF, von Willebrand factor; MAC, membrane attack complex. Although the role of complement dysregulation in TTP is Therefore, antigen-antibody complexes formed by ADAMTS- not as well-defined as it is in aHUS etiology, it has been 13 and anti-ADAMTS-13 IgG directly activate the classical demonstrated through several approaches. Increased levels of complement pathway, leading to downstream activation of C3 complement anaphylatoxins and soluble C5b-9 (sC5b-9), have and formation of C3a (34). Moreover, the increased thrombi been detected in patients with acute TTP when compared with formation in acute TTP could also accelerate complement remission and also with healthy controls (33, 34). Furthermore, activation (36) through its activity as C5 convertase by forming significant decrease of C3a and sC5b9 has been observed during positive feedback loops (37). In keeping with this, it has plasma exchange in TTP patients (34). Higher complement been documented that TTP could trigger aHUS in susceptible activity has been also reported in patients dying during an individuals presenting mutations in CFH or other complement- acute TTP episode compared to patients responding to treatment related proteins (38). Finally, there is evidence showing that (35). From the three different pathways through which the anti-C5 therapy is effective in refractory TTP unresponsive to CS can be activated, the classical is the one triggered by plasma (39). immunocomplexes. The majority of TTP cases are idiopathic and In addition, the role of the lectin complement pathway mediated by immunoglobulin G antibodies to ADAMTS-13 (29). (LP) has recently been postuated in TTP through the finding Frontiers in Medicine | www.frontiersin.org 3 April 2022 | Volume 9 | Article 811504 Blasco et al. Endothelium and Complement in TMA of mannose-binding lectin associated serine protease (MASP2) Transplantation Associated Thrombotic elevated levels in the sera from acute phase TTP patients. Microangiopathy Moreover, in vitro experiments using plasma from PTT patients Transplantation associated thrombotic microangiopathy (TA- suggest a role of this pathway in microvascular endothelial TMA) is a severe complication associated with allogeneic cell injury through an specific caspase 8 activation that can hematopoietic cell transplantation (HCT) that occurs when be blocked by the anti-MASP2 human monoclonal antibody endothelial dysfunction triggers the CS inducing the formation narsoplimab (40). of platelet enriched thrombi in the microvasculature and a consequent hemolytic anemia (51). This complication can Shiga Toxin-Producing Escherichia coli constitute a mild self-limiting disease or be associated with multiorgan disorder leading to death (52). The incidence Hemolytic Uremic Syndrome and mortality of TA-TMA varies widely among studies and Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 hospitals due to heterogeneous diagnostic criteria and under- is the main cause of hemorrhagic colitis, occurring mainly in recognition (53). childhood. In 5–15% of cases it progresses to hemolytic uremic The endothelial damage in HCT is multifactorial and syndrome (HUS), and constitutes the leading cause of HUS cumulative (54, 55) due to the effect of the conditioning regimen, worldwide (41). calcineurin inhibitors, infections, graft vs. host disease and Endothelial damage plays a central role in the underlying processes inherent to HCT such as the engraftment syndrome, pathogenesis of STEC-HUS, and Stx (principally Stx subtype among others (56–59). This endothelial damage leads to a 2) is thought to be the key in this microangiopathic process release of proinflammatory cytokines and procoagulant factors through several mechanisms. In vitro studies have demonstrated together with nitric oxide depletion and an increase in the that Stx upregulates the generation of adhesive molecules expression of adhesion molecules at cell surface. All these (E-selectin, ICAM-1, and VCAM-1) and chemokines (MCP-1, phenomena promote further endothelial injury and leads to IL-8, fractalkine), promoting the adhesion of leukocytes to platelet aggregation and the initiation and propagation of cultured human EC under flow conditions (42, 43). Stx also the complement cascade (60, 61). There is evidence showing induces rapid release of ULVWF multimers from EC and inhibits that neutrophil extracellular traps (NETs) could constitute the the multimer cleavage by ADAMTS13, thus favoring platelet mechanistical link between endothelial damage and complement adhesion and clot formation in the microvasculature (41, 44). activation (62). Several approaches have identified increased Moreover, Stx promote changes in gene expression, stimulating levels of C3b and sC5b-9 (63, 64), being the last included mRNA and protein production of chemokines and cytokines in TMA diagnostic algorithm proposed by Jodele et al. (63). which may exacerbate endothelial damage (45). All these events In this algorithm the authors assessed that TA-TMA should generate changes in the endothelial phenotype, resulting in a be suspected in HCT recipients with an acute elevation of prothrombogenic intravascular environment. LDH, proteinuria >30 mg/dL, and hypertension more severe Another key element in STEC-HUS pathogenesis is than expected with calcineurin or steroid therapy, and that dysregulation of the CS, and, likewise, it is mainly driven clinical interventions should be considered for patients with by Stx. Stx activates complement in the fluid phase, with reduced both proteinuria >30 mg/dL and elevated sC5b-9. All these plasma levels of C3 and augmented levels of C3a, Bb and sC5b-9 phenomena have been included in the “Three-Hit Hypothesis” during the active phase of the disease (46, 47). This evidence, postulated by Dvorak et al. (65). This hypothesis includes along with the demonstration in murine models of disease the different risk factors for the development of TA-TMA in that factor B knockout mices were protected from Stx induced three potential consecutive events that culminate in a cycle of renal damage (48), indicates that complement activation is activation of endothelial cells: Hit 1, an underlying predisposition produced via alternative pathway (AP) in STEC-HUS patients. to complement activation (genetical susceptibility due to Moreover, deposits of C3 and C9 have been detected on complement gene variants) or pre-existing endothelial injury; Hit platelet-leukocyte heterotypic aggregates and microvesicles 2, the direct effect on endothelium of toxic conditioning regimen; obtained from these patients (49, 50), thus indicating that and Hit 3, the additional insults triggered by medications, complement AP might contribute to endothelial damage and alloreactivity, infections, and/or antibodies that occur post- thrombosis in STEC-HUS. transplant. Furthermore, the anti-C5 monoclonal antibody Two double-blinded placebo controlled trials with eculizumab eculizumab has proven to be an effective therapeutic strategy (C5 inhibitor) has been performed to evaluate its efficacy for TA-TMA, both in pediatric (66) and adult (67) patients, for STEC-HUS treatment: ECULISHU (NCT02205541) in supporting the involvement of CS in the pathogenesis of TA- France looking at renal outcome in pediatric population, TMA. completed in 2019, and ECUSTEC (NCT01410916) in the United Kingdom looking at overall disease severity, completed in 2015 (Table 1). Until results become available from Kidney Transplantation Associated these prospective, randomized control trials, practitioners will Thrombotic Microangiopathies continue to rely on small case series, single-center studies, and TMA associated with kidney transplantation may occur as a case reports to guide the eculizumab off-label indication for recurrence of aHUS or without previous history of thrombotic STEC-HUS treatment. microangiopathy (de novo TMA). Kidney transplantation Frontiers in Medicine | www.frontiersin.org 4 April 2022 | Volume 9 | Article 811504 Blasco et al. Endothelium and Complement in TMA TABLE 1 | Complement inhibitors clinical trials. Drug Function Status Phase Description Population No. of Identifier participants SHUa Crovalimab C5 inhibitor Recruiting 3 Multicenter, single-arm Adults and adolescents 90 NCT04861259 study Crovalimab C5 inhibitor Not yet 3 Multicenter, single-arm Pediatric patients 35 NCT04958265 recruiting study Eculizumab C5 inhibitor Completed 2 Open-label, Adult, older adult 44 NCT01194973 (May 30, 2017) Multi-center clinical trial Eculizumab C5 inhibitor Completed 2 Open-label, Pediatric 22 NCT01193348 (April 29, 2015) multi-center clinical trial Eculizumab C5 inhibitor Completed 2 Open-label, Adults 16 NCT00844545 (July 23, 2015) multi-center controlled clinical trial in patients with plasma therapy-resistance Iptacopan Factor B inhibitor Recruiting 3 Multicenter, single-arm, Adult, older adult 50 NCT02604420 open label trial Ravulizumab C5 inhibitor Active, not 3 Single-arm study Adults and adolescents 58 NCT02949128 recruiting Ravulizumab C5 inhibitor Active, not 3 Open-label, multicenter Children and adolescents 31 NCT03131219 recruiting study MAT related with autoimmune disorders Ravulizumab C5 inhibitor Recruiting 2 Double-blind, Adults 120 NCT04564339 randomized, placebo-controlled study in participants with proliferative lupus nephritis (LN) or immunoglobulin A nephropathy (IgAN) Shiga-toxin Related SHUa Eculizumab C5 inhibitor Completed 3 Prospective Pediatric 100 NCT02205541 (July 12, 2019) randomized controlled therapeutic trial versus placebo Eculizumab C5 inhibitor Completed 3 Open-label, Child and adult 198 NCT01410916 (April 5, 2013) multi-center trial TMA after hematopoietic stem cell transplantation Eculizumab C5 inhibitor Recruiting 2 Early intervention to Children and young adults 21 NCT03518203 treat TMA/aHUS-associated MODS LFG316 C5 inhibitor Terminated 2 Randomized, open Adult, older adult 7 NCT02763644 (January 5, label, controlled, 2021) multiple dose study Ravulizumab C5 inhibitor Recruiting 3 Randomized, Adults and adolescents 184 NCT04543591 double-blind, placebo-controlled, multicenter study Ravulizumab C5 inhibitor Recruiting 3 Open-label, single arm, Pediatric 40 NCT04557735 multicenter study Infections: COVID AMY-101 C3 inhibitor Not yet 2 Randomized, parallel Adult, older adult 144 NCT04395456 recruiting assignment study Bradykinin C1 inhibitor Completed 2 Prospective, Adult, older adult 44 NCT05010876 (August 18, randomized, 2021) double-blind, multicenter, prospective study (Continued) Frontiers in Medicine | www.frontiersin.org 5 April 2022 | Volume 9 | Article 811504 Blasco et al. Endothelium and Complement in TMA TABLE 1 | (Continued) Drug Function Status Phase Description Population No. of Identifier participants Conestat Alfa rhC1-INH Active, not 2 Randomized, Adult, older adult 129 NCT04414631 recruiting parallel-group, open-label, multi-center pilot trial Icatibant C1 inhibitor Recruiting 2 Randomized, open, Adult, older adult 120 NCT04978051 multicenter, proof of concept Eculizumab C5 inhibitor Recruiting 2 Bayesian open labeled Adult, older adult 120 NCT04346797 randomized clinical trial (nested in the CORIMUNO-19 cohort) Ravulizumab C5 inhibitor Recruiting 3 Open labe, randomized Adult, older adult 32 NCT04570397 study Ravulizumab C5 inhibitor Terminated 3 Open-label, Adult, older adult 202 NCT04369469 randomized, controlled study Ruconest rhC1-INH Recruiting 2 Randomized, Adult, older adult 120 NCT04530136 parallel-group, open-label, multi-center pilot trial Zilucoplan C5 inhibitor Completed 2 Prospective, Adult, older adult 81 NCT04382755 (July 2, 2021) randomized, open-label study Infections: Sepsis CaCP29 C5 inhibitor Completed 2 Randomized, Adult, older adult 72 NCT02246595 (April 25, 2016) placebo-controlled, double-blind, dose controlled trial C1-esterase inhibitor C1-esterase inhibitor Completed 3 A randomized Adults 20 NCT01766414 (December 2, controlled pilot study 2014) TMA associated with a trigger Ravulizumab C5 inhibitor Recruiting 3 Randomized, Adult, older adult 100 NCT04743804 double-blind, placebo-controlled, multicenter study in participants who have thrombotic microangiopathy associated with a trigger For the resulting table several searches in clinicaltrial.gov were perform including all countries and interventional clinical studies that were completed, terminated, recruiting and not recruiting. Studies with less than 5 participants have been excluded. In those that are already completed the last update is included between parenthesis. The searches performed were: (1) combination of the concepts “thrombotic microangiopathy” and “complement inhibitors”, (2) the pathologies included in the present review and “complement inhibitors”, and (3) the search of several individual drugs. recipients are exposed to many triggers that can damage be injured by important number of factors in transplanted the endothelium and produce TMA, which can have a patients, triggering de novo TMA. Calcineurin inhibitors (CNI) significant impact on allograft survival (68). The incidence can induce TMA through arteriolar vasoconstriction (increased is concentrated in the first months after transplantation synthesis of endothelin and thromboxane A2, and reduced and the main causes are ischemia-reperfusion damage, acute expression of prostacyclin and prostaglandin E2) (72–74), humoral rejection, medications, and opportunistic infections platelet activation, anti-fibrinolytic and pro-coagulant effects (75) (69). Unlike TMA that occurs in native kidneys, graft biopsy and, by activation of AC pathway (microparticles released by is very useful since it can offer an etiological diagnosis injured EC) (76). m-TOR inhibitors block cell cycle progression in most cases. and proliferation, induce endothelial progenitor cells death, aHUS recurrence after transplantation depends on genetic increase procoagulant status, reduce fibrinolytic state and predisposition and endothelial damage is mediated by a decrease renal expression of endothelial growth factor (77–80). dysregulation of AC pathway (70, 71). Endothelium could also All these processes can contribute to the endothelial damage Frontiers in Medicine | www.frontiersin.org 6 April 2022 | Volume 9 | Article 811504 Blasco et al. Endothelium and Complement in TMA that triggers de novo TMA onset, described with the use of with an increased risk for venous thrombosis in SLE CNI, m-TOR, or a combination of both (possibly related to patients (92). elevated serum levels) (81, 82). Acute humoral rejection is an important cause of endothelial damage in kidney grafts, frequently associated with TMA development. Donor specific Thrombotic Microangiopathies antibodies bind to major histocompatibility complexes on EC, Associated With Infections fixing the CS and activating them through the over-expression Besides the above-mentioned STEC-HUS, TMA has been of pro-inflammatory genes. Terminal complement phase (C5b-9) associated with a large number of infectious diseases, has been also related to allograft vasculopathy development specially viral (6). Mechanisms of TMA-associated with in humoral response, since it can activate EC, favoring T infectious diseases are complex, and differ depending on the cell recruitment and secretion of cytokines and interferon pathogens causing them. γ. Finally, ischemia-reperfusion injury is also associated with Cytomegalovirus (CMV) can directly damage EC and cause endothelial damage and TMA onset. The changes induced on platelet adhesion by inducing the expression of adhesion EC produce complement activation that in turn accentuates molecules and release of vWF (93). Moreover, CMV activates and perpetuates endothelial damage at the expense of MAC the complement classical pathway by binding of C1q to CMV (83). Animal models have shown a clear benefit of blocking the infected cells (94). terminal phase of complement by different therapeutic strategies TMA is also a known complication of HIV infection, with (84–86). different forms of presentation. Cases of both classic TTP and aHUS have been described in HIV-infected patients (95, 96), although the exact mechanisms involved remain unclear. Thrombotic Microangiopathies Related Most cases present without a severe reduction in ADAMTS-13 to Autoimmune Disorders activity levels, which support the hypothesis that the underlying Antiphospholipid syndrome (APS) and systemic erythematous mechanism may be different from classic TTP. It has been lupus (SLE) are the autoimmune disorders most frequently suggested that endothelial cells can be infected by HIV (96). associated with TMA, which can result in a life-threating Moreover, TMA induced by influenza A virus (H1N1) complication in these contexts. infection was reported during the 2009 pandemic. The APS is a prothrombotic disorder characterized by thrombosis neuraminidase produced by this virus, as well as by the and/or pregnancy morbidity, which occurs in the presence bacteria Streptococcus pneumoniae, causes erythrocyte fusion of antiphospholipid antibodies (aPL). Among them, anti- and hemolysis, activation of platelets and generation of β2-glycoprotein I (B2GPI) antibodies are one of the main thrombin, leading to TMA in both cases. Furthermore, low drivers of the endothelial damage, since they upregulate levels of ADAMTS-13 and elevated sC5b-9 have been detected in the endothelial expression of prothrombotic factors and patients with H1N1 infection (97). adhesion molecules. B2GPI also interacts with von Willebrand More recently, the severe acute respiratory syndrome factor (vWF), leading to platelet activation (87). Moreover, coronavirus 2 (SARS-COV-2) has emerged as another clear murine models have demonstrated that increased complement example of viral infection in which endothelial damage occurs activation plays a role in APS pathogenesis, and the interaction in parallel to an overactivation of complement cascade. Patients of C5a with its receptor C5aR leads to inflammation, with moderate and severe COVID-19 disease exhibit elevated C5a placental insufficiency, and thrombosis (88, 89). APS can and sC5b-9 plasma levels compared with healthy controls, the occur in isolation or in association with other autoimmune later correlating with vWF plasma levels and disease severity (98). diseases, such as SLE. Moreover, COVID-19 patients were also found to have C5b-9 SLE is a multisystem autoimmune disorder associated with and C4d skin deposits, as well as mannose-binding lectin (MBL) the presence of autoantibodies against double-stranded DNA deposits in lungs, suggesting the overactivation of alternative (ds-DNA), among others, which can produce endothelial and lectin-complement pathways (99–101). These observations damage, either directly binding to EC or forming circulating may reflect the close relationship between endothelial stress immunocomplexes that deposit on vessels. In this regard, it and complement dysregulation in this condition. In this regard, has been demonstrated that several indicators of endothelial isolated experiences using complement inhibitors under a dysfunction (Pentraxin 3, E-selectin, VCAM-1) are higher compassionate-use program, such as narsoplimab (102), or AMY- in patients with SLE compared to healthy controls (90). 101, a compstatin-based C3 inhibitor (103) has been publicated. CS is also involved in SLE pathogenesis, since these The former (narsoplimab) demonstrated rapid and sustained immunocomplexes activate the classical complement pathway reduction of circulating endothelial cells, as well as with decreased and cause tissue damage, as can be observed in kidney circulating inflammatory cytokines, while the second (AMY-101) biopsies of patients with lupus nephritis, in which C1q, C3 was associated with a favorable clinical evolution in a patient and C4d deposition is common (90, 91). Moreover, there with COVID-19 severe pneumonia. All together, these results is increasing evidence that CS is linked with thrombotic suggest that CS plays a central role in the pathophysiology of events occurring is SLE. An example is the relationship that COVID-19-related lung injury. has been observed between the deposition of complement Moreover, several clinical trials targeting the complement activation products (C1q, C3d, C4d) on platelets surfaces system are currently ongoing in patients with COVID-19 Frontiers in Medicine | www.frontiersin.org 7 April 2022 | Volume 9 | Article 811504 Blasco et al. Endothelium and Complement in TMA (Table 1). Among them, two have been completed: AntagoBrad- management, especially with early therapy introduction. The Cov (NCT05010876), a prospective, randomized, double-blind, excellent response to these treatments has led to the research of multicenter study of three parallel groups of patients comparing the potential pathological role of the CS in other TMA forms the efficacy of human C1 inhibitor, administered alone with higher prevalence and without known etiological treatment. or in combination with icatibant (a specific bradykinin Figure 1 summarizes the pathophysiological process through B2 receptor antagonist) on the pulmonary manifestations which complement could damage the endothelium in different of COVID-19 infections; and ZILU-COV (NCT04382755), TMA forms, as well as the resulting functional and molecular a prospective, randomized, open-label, study to investigate endothelial changes. One of the difficulties faced in making the efficacy of a complement C5 inhibitor (Zilucoplan ) in R a diagnosis of complement mediated TMA is the lack of improving oxygenation and short-and long-term outcome of reliable markers of CS hyperactivation. For instance, in the COVID-19 patients with acute hypoxic respiratory failure. The case of the paradigmatic complement mediated TMA, aHUS, results of the latter haven’t been published yet. relatively few patients have consumption of complement factors, Interestingly, samples from septic shock (SS) patients induced complement mutations are heterozygous-and the corresponding higher C5b-9 deposition on EC than those from COVID-19 protein concentrations in blood are not consistently abnormal- patients, whereas there were no differences regarding sC5b-9 and genetic screenings are slow and even uninformative in levels between both groups. Thus, COVID-19 endotheliopathy up to 50% of cases (106). In general, the quantification of may differs from SS, in which endothelial damage and levels of individual complement components in serum is not complement may also play an important pathogenic role a straightforward approach in TMA diagnosis, as soluble levels (98). In this regard, two clinical trials have been performed are not reliable biomarkers of complement activation in any (Table 1). The first one, completed in 2014, is VECTORII form of TMA (107), although the quantification of sC5b-9 has Study (NCT01766414), a randomized, controlled, pilot study to shown some usefulness in the management of patients with TA- evaluate the role of a C1-esterasa inhibitor in the modulation TMA (64) and is available clinically (commercial kits). For this of innate immune response in a human endotoxemia model. reason, new diagnostic tools are needed for TMAs management. The inhibition of C1-esterase exerted anti-inflammatory effects in In this regard, functional studies may help to demonstrate the the absence of classic complement activation (104). The second role of complement activation not only at diagnosis, but also to one, completed in 2016, is SCIENS Study (NCT02246595), a monitor treatment response. Among them, the analysis of C5b- phase II clinical trial conducted to study safety, tolerability, 9 deposition on endothelial cells culture could be an attractive pharmacokinetics, and pharmacodynamics of Vilobelimab (IFX- option, since it has shown a great correlation in different clinical 1; CaCP 29), a recombinant monoclonal antibody against C5a, stages of aHUS patients, as well as in certain secondary TMA in patients with severe sepsis or septic shock. Vilobelimab forms by different research groups (108–112). However, there is demonstrated to neutralize selectively C5a in a dose-dependent still an open discussion about their utility, especially because it is manner without blocking formation of the membrane attack not a quick procedure, requires specialized and trained personnel, complex, and without resulting in detected safety issues (105). and is not commercially available (1). In section 2, we have reviewed the evidence generated regarding the participation of the CS in various TMA forms. DISCUSSION Since the approval of eculizumab for the treatment of aHUS in 2011, the development of therapies that directly target CS Clinical TMA management (diagnostic-therapeutic process) at different levels has enabled a large number of clinical trials is a great challenge due to its systemic nature (variable focused on complement blockers in patients with different signs and symptoms) and high associated morbidity and TMA-associated disorders (Table 1). Most of them have mortality. Enhancing the etiological knowledge of the evaluated the role of C5-inhibitors, mainly eculizumab and different clinical entities involved in TMA will allow the ravulizumab (a long-acting C5-inhibitor), in the treatment development of targeted therapies that may improve their of various conditions. Among them, those performed with poor prognosis. The cornerstone for thrombi development in eculizumab in patients with aHUS and STEC-HUS have the microvasculature is the endothelial cell damage, common been already completed. In recent decades, a large number to all TMA-associated disorders. However, these pathological of potential complement-related targets have progressively processes exhibit differential patterns and mechanisms of emerged, such as C3, factor B and C1q. Probably, this fact endothelial injury, although occasionally there may be also a may be related with the large amount of scientific evidence pathophysiological overlap. generated regarding the role of complement in the pathogenesis CS, a key element of the innate immune system, requires of the different conditions reviewed in this work. As shown precise regulation. Defects in the elements involved result in Table 1, several clinical trials evaluating these factors in dysregulation and over-activation. In the case of TMA, are ongoing, and their results may probably help to extend an acquired or congenital dysregulation of the alternative the therapeutic options for TMA-associated conditions in complement pathway is primarily responsible for the the near future. endothelial damage that occurs in aHUS patients. Therefore, the In conclusion, endothelial damage in TMA can have different development of monoclonal antibodies that block the terminal origins depending on the responsible pathological process. complement phase (C5) has led to a great advance in aHUS However, the potential role of complement system in many Frontiers in Medicine | www.frontiersin.org 8 April 2022 | Volume 9 | Article 811504 Blasco et al. Endothelium and Complement in TMA of them represents a real and current treatment opportunity. autoimmune disorders, and TMA associated with infections Identifying the exact factors involved in each disease will allow sections. MD-R contributed to the manuscript with her critical an individualized patient management. This is a challenge that review and comments. All authors contributed to the article and will require a great effort involving clinicians, immunologists, approved the submitted version. geneticists, and basic researchers. AUTHOR CONTRIBUTIONS FUNDING MB reviewed the bibliography and wrote aHUS, kidney This work was supported by Fundació Miarnau (Spain), transplantation associated TMA and discussion sections. MP Fundació La Marató de TV3 (202026-10), Instituto de Salud reviewed and wrote the TTP and TA-TMA sections, and designed Carlos III from Spanish Government (PI19/00888), Shook Studio Table 1 and Figure 1 with the support of MB and EG-O. EG-O for figure design, and Ayuda a la Investigación de la Fundación reviewed and wrote introduction, STEC-HUS, TMA related to Senefro 2020. REFERENCES 15. Constantinescu AR, Bitzan M, Weiss LS, Christen E, Kaplan BS, Cnaan A, et al. Non-enteropathic hemolytic uremic syndrome: causes and short-term 1. Blasco M, Guillén E, Quintana LF, Garcia-Herrera A, Piñeiro G, Poch E, course. Am J Kidney Dis. (2004) 43:976–82. doi: 10.1053/j.ajkd.2004.02.010 et al. Thrombotic microangiopathies assessment: mind the complement. Clin 16. Loirat C, Frémeaux-Bacchi V. 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Efficacy and safety of vilobelimab (IFX-1), a novel monoclonal anti-C5a Conflict of Interest: MB reports advisory boards and symposium speaker antibody, in patients with early severe sepsis or septic shock-A randomized, honoraria from Alexion Pharmaceuticals. MD-R has received research funding placebo-controlled, double-blind, multicenter, phase IIa trial (SCIENS and speaker fees from Jazz Pharmaceuticals. study). Crit Care Explor. (2021) 3:e0577. doi: 10.1097/CCE.00000000000 00577 The remaining authors declare that the research was conducted in the absence of 106. Gavriilaki E, Yuan X, Ye Z, Ambinder AJ, Shanbhag SP, Streiff MB, et al. any commercial or financial relationships that could be construed as a potential Modified ham test for atypical hemolytic uremic syndrome. Blood. (2015) conflict of interest. 125:3637–46. doi: 10.1182/blood-2015-02-629683 107. Bu F, Meyer NC, Zhang Y, Borsa NG, Thomas C, Nester C, et al. Soluble C5b- Publisher’s Note: All claims expressed in this article are solely those of the authors 9 as a biomarker for complement activation in atypical hemolytic uremic and do not necessarily represent those of their affiliated organizations, or those of syndrome. Am J Kidney Dis. (2015) 65:968–9. doi: 10.1053/j.ajkd.2015.02. the publisher, the editors and the reviewers. Any product that may be evaluated in 326 this article, or claim that may be made by its manufacturer, is not guaranteed or 108. Noris M, Galbusera M, Gastoldi S, Macor P, Banterla F, Bresin E, endorsed by the publisher. et al. Dynamics of complement activation in aHUS and how to monitor eculizumab therapy. Blood. (2014) 124:1715–26. doi: 10.1182/blood-2014- Copyright © 2022 Blasco, Guillén-Olmos, Diaz-Ricart and Palomo. This is an open- 02-558296 access article distributed under the terms of the Creative Commons Attribution 109. Timmermans SAMEG, Abdul-Hamid MA, Potjewijd J, Theunissen License (CC BY). The use, distribution or reproduction in other forums is permitted, ROMFIH, Damoiseaux JGMC, Reutelingsperger CP, et al. C5b9 formation provided the original author(s) and the copyright owner(s) are credited and that the on endothelial cells reflects complement defects among patients with renal original publication in this journal is cited, in accordance with accepted academic thrombotic microangiopathy and severe hypertension. J Am Soc Nephrol. practice. No use, distribution or reproduction is permitted which does not comply (2018) 29:2234–43. doi: 10.1681/ASN.2018020184 with these terms. Frontiers in Medicine | www.frontiersin.org 12 April 2022 | Volume 9 | Article 811504