C5a and C5aR1 are key drivers of microvascular platelet aggregation in clinical entities spanning from aHUS to COVID-19

Unrestrained activation of the complement system till the terminal products, C5a and C5b-9, plays a pathogenetic role in acute and chronic inflammatory diseases. In endothelial cells, complement hyperactivation may translate into cell dysfunction, favoring thrombus formation. The aim of this study was to investigate the role of the C5a/C5aR1 axis as opposite to C5b-9 in inducing endothelial dysfunction and loss of anti-thrombogenic properties. In vitro and ex vivo assays with serum from patients with atypical hemolytic uremic syndrome (aHUS) -a prototype rare disease of complement-mediated microvascular thrombosis due to genetically determined alternative pathway dysregulation- and cultured microvascular endothelial cells, demonstrated that the C5a/C5aR1 axis is a key player of endothelial thromboresistance loss. C5a added to normal human serum, fully recapitulated the pro-thrombotic effects of aHUS serum. Mechanistic studies showed that C5a caused RalA-mediated exocytosis of vWF and P-selectin from Weibel-Palade bodies, which favored further vWF binding on the endothelium and platelet adhesion and aggregation. In patients with severe COVID-19 -who suffered from acute activation of complement triggered by SARS-CoV-2 infection- we found the same C5a-dependent pathogenic mechanisms. These results highlight C5a/C5aR1 as a common pro-thrombogenic effector spanning from genetic rare diseases to viral infections, and may have clinical implications. Selective C5a/C5aR1 blockade could have advantages over C5 inhibition, since the former preserves the formation of C5b-9 that is critical to control bacterial infections that often develop as comorbidities in severely ill patients. (Clinicaltrials.gov identifier NCT02464891)

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Complement and the prothrombotic state

Blood ◽

10.1182/blood.2020007206 ◽

2021 ◽

Author(s):

Christoph Q Schmidt ◽

Hubert Schrezenmeier ◽

David Kavanagh

Keyword(s):

Complement Activation ◽

Ex Vivo ◽

Atypical Hemolytic Uremic Syndrome ◽

Protein Losing Enteropathy ◽

Mortality And Morbidity ◽

Life Threatening ◽

Uremic Syndrome ◽

Clinical Conditions ◽

Distinct Features

In 2007 and 2009 the regulatory approval of the first-in-class complement inhibitor Eculizumab has revolutionized the clinical management of two rare, life-threatening clinical conditions: paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). While being completely distinct diseases affecting blood cells and the glomerulus, PNH and aHUS remarkably share several features in their etiology and clinical presentation. An imbalance between complement activation and regulation at host surfaces underlies both diseases precipitating in severe thrombotic events that are largely resistant to anti-coagulant and/or anti-platelet therapies. Inhibition of the common terminal complement pathway by Eculizumab prevents the frequently occurring thrombotic events responsible for the high mortality and morbidity observed in patients not treated with anti-complement therapy. While many in vitro and ex vivo studies elaborate numerous different molecular interactions between complement activation products and hemostasis, this review focuses on the clinical evidence that links these two fields in humans. Several non-infectious conditions with known complement involvement are scrutinized for common patterns concerning a prothrombotic statues and the occurrence of certain complement activation levels. Next to PNH and aHUS, germline encoded CD59 or CD55 deficiency (the latter causing the disease Complement Hyperactivation, Angiopathic thrombosis, and Protein-Losing Enteropathy; CHAPLE), autoimmune hemolytic anemia (AIHA), (catastrophic) anti-phospholipid syndrome (APS, CAPS) and C3 glomerulopathy are considered. Parallels and distinct features among these conditions are discussed against the background of thrombosis, complement activation, and potential complement diagnostic and therapeutic avenues.

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Hyperfunctional C3 convertase leads to complement deposition on endothelial cells and contributes to atypical hemolytic uremic syndrome

Blood ◽

10.1182/blood-2009-01-197640 ◽

2009 ◽

Vol 114 (13) ◽

pp. 2837-2845 ◽

Cited By ~ 98

Author(s):

Lubka T. Roumenina ◽

Mathieu Jablonski ◽

Christophe Hue ◽

Jacques Blouin ◽

Jordan D. Dimitrov ◽

...

Keyword(s):

Endothelial Cells ◽

Hemolytic Uremic Syndrome ◽

Umbilical Vein ◽

Alternative Pathway ◽

Atypical Hemolytic Uremic Syndrome ◽

Factor H ◽

Immune Defense ◽

Factor B ◽

Uremic Syndrome ◽

Complement Deposition

Abstract Complement is a major innate immune defense against pathogens, tightly regulated to prevent host tissue damage. Atypical hemolytic uremic syndrome (aHUS) is characterized by endothelial damage leading to renal failure and is highly associated with abnormal alternative pathway regulation. We characterized the functional consequences of 2 aHUS-associated mutations (D254G and K325N) in factor B, a key participant in the alternative C3 convertase. Mutant proteins formed high-affinity C3-binding site, leading to a hyperfunctional C3 convertase, resistant to decay by factor H. This led to enhanced complement deposition on the surface of alternative pathway activator cells. In contrast to native factor B, the 2 mutants bound to inactivated C3 and induced formation of functional C3-convertase on iC3b-coated surface. We demonstrated for the first time that factor B mutations lead to enhanced C3-fragment deposition on quiescent and adherent human glomerular cells (GEnCs) and human umbilical vein endothelial cells (HUVECs), together with the formation of sC5b-9 complexes. These results could explain the occurrence of the disease, since excessive complement deposition on endothelial cells is a central event in the pathogenesis of aHUS. Therefore, risk factors for aHUS are not only mutations leading to loss of regulation, but also mutations, resulting in hyperactive C3 convertase.

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Antiangiogenic effects of phospholipase A2 Lys49 BnSP-7 from Bothrops pauloensis snake venom on endothelial cells: An in vitro and ex vivo approach

Toxicology in Vitro ◽

10.1016/j.tiv.2021.105099 ◽

2021 ◽

Vol 72 ◽

pp. 105099

Author(s):

Lorena Polloni ◽

Fernanda Van Petten Vasconcelos Azevedo ◽

Samuel Cota Teixeira ◽

Eloá Moura ◽

Tassia Rafaela Costa ◽

...

Keyword(s):

Endothelial Cells ◽

Phospholipase A2 ◽

Snake Venom ◽

Ex Vivo ◽

Ex Vivo Approach

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Neutrophil extracellular traps induce MCP-1 release from endothelial cells at the plaque rupture site in acute myocardial infarction

European Heart Journal ◽

10.1093/ehjci/ehaa946.3834 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

A Ondracek ◽

T.M Hofbauer ◽

A Mangold ◽

T Scherz ◽

V Seidl ◽

...

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Endothelial Cells ◽

Plaque Rupture ◽

Ex Vivo ◽

Coronary Intervention ◽

Neutrophil Extracellular Traps ◽

Funding Source ◽

Extracellular Traps

Abstract Introduction Leukocyte-mediated inflammation is crucial in acute myocardial infarction (AMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in AMI. Methods Culprit site and femoral blood of AMI patients was drawn during percutaneous coronary intervention. We characterized CCR2 expression of fibrocytes by flow cytometry. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA. Fibrocytes were treated in vitro with MCP-1. Human coronary arterial endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 was measured by ELISA and qPCR. The influence of MCP-1 on NET formation in vitro was assessed using isolated neutrophils. Results We have included 50 consecutive AMI patients into the study. NETs and concentrations of MCP-1 were increased at the CLS. NET stimulation of hCAECs induced MCP-1 on mRNA and protein level. Increasing MCP-1 gradient was associated with fibrocyte accumulation at the site of occlusion. In the presence of higher MCP-1 these fibrocytes expressed proportionally less CCR2 than peripheral fibrocytes. In vitro, MCP-1 dose-dependently decreased fibrocyte CCR2 and reduced ex vivo NET release of healthy donor neutrophils. Conclusions NETs induce endothelial MCP-1 release, presumably promoting a chemotactic gradient for leukocyte and fibrocyte migration. MCP-1 mediated inhibition of NET formation could point to a negative feedback loop. These data will shed light on vascular healing. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund

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Antimalarial drugs—are they beneficial in rheumatic and viral diseases?—considerations in COVID-19 pandemic

Clinical Rheumatology ◽

10.1007/s10067-021-05805-5 ◽

2021 ◽

Author(s):

Bogna Grygiel-Górniak

Keyword(s):

Connective Tissue ◽

Viral Infections ◽

Current Knowledge ◽

Inflammatory Diseases ◽

Antiviral Drugs ◽

In Vivo Studies ◽

Viral Diseases ◽

Cardiac Complications

AbstractThe majority of the medical fraternity is continuously involved in finding new therapeutic schemes, including antimalarial medications (AMDs), which can be useful in combating the 2019-nCoV: coronavirus disease (COVID-19). For many decades, AMDs have been widely used in the treatment of malaria and various other anti-inflammatory diseases, particularly to treat autoimmune disorders of the connective tissue. The review comprises in vitro and in vivo studies, original studies, clinical trials, and consensus reports for the analysis, which were available in medical databases (e.g., PubMed). This manuscript summarizes the current knowledge about chloroquine (CQ)/hydroxychloroquine (HCQ) and shows the difference between their use, activity, recommendation, doses, and adverse effects on two groups of patients: those with rheumatic and viral diseases (including COVID-19). In the case of connective tissue disorders, AMDs are prescribed for a prolonged duration in small doses, and their effect is observed after few weeks, whereas in the case of viral infections, they are prescribed in larger doses for a short duration to achieve a quick saturation effect. In rheumatic diseases, AMDs are well tolerated, and their side effects are rare. However, in some viral diseases, the effect of AMDs is questionable or not so noticeable as suggested during the initial prognosis. They are mainly used as an additive therapy to antiviral drugs, but recent studies have shown that AMDs can diminish the efficacy of some antiviral drugs and may cause respiratory, kidney, liver, and cardiac complications.

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Complement-Mediated Thrombotic Microangiopathy Associated with Lupus Nephritis Treated with Eculizumab: A Case Report

Case Reports in Nephrology and Dialysis ◽

10.1159/000512227 ◽

2021 ◽

pp. 95-102

Author(s):

Everardo Arias Torres ◽

Yongen Chang ◽

Sheetal Desai ◽

Ian Chang ◽

Jonathan E. Zuckerman ◽

...

Keyword(s):

Lupus Erythematosus ◽

Alternative Pathway ◽

Atypical Hemolytic Uremic Syndrome ◽

Homozygous Deletion ◽

Multiple Organ ◽

Thrombotic Microangiopathies ◽

Clinical Criteria ◽

Organ Systems ◽

Oral Steroids ◽

Uremic Syndrome

Thrombotic microangiopathies (TMAs) involve multiple organ systems due to the presence of microangiopathic hemolysis. One such condition, atypical hemolytic uremic syndrome (aHUS), is a complement-mediated process that is part of a spectrum of disorders that have underlying complement dysfunction of the alternative pathway due to overactivity or decreased self-nonself discrimination by innate immunity. Complement-amplifying conditions such as pregnancy may unmask a diagnosis of aHUS. We present an important case of a pregnant 23-year-old Hispanic female who presented in mid-gestation (21 weeks) with an initial diagnosis of systemic lupus erythematosus (SLE) complicated by aHUS. She met clinical criteria for aHUS on presentation and was found to have a pathogenic CFHR1–3 homozygous deletion. She has been treated with intravenous and oral steroids, cyclophosphamide, subsequently also with plasma exchange, and finally with eculizumab with partial improvement in renal function. This case adds to the emerging literature showing that SLE and aHUS (or complement-mediated TMA) can be successfully treated with C5 blockade.

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Biochemical and Pharmacological Properties of SANORG 34006, a Potent and Long-Acting Synthetic Pentasaccharide

Blood ◽

10.1182/blood.v91.11.4197 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4197-4205 ◽

Cited By ~ 151

Author(s):

J.M. Herbert ◽

J.P. Hérault ◽

A. Bernat ◽

R.G.M. van Amsterdam ◽

J.C. Lormeau ◽

...

Keyword(s):

Thrombin Generation ◽

Ex Vivo ◽

Thrombus Formation ◽

Factor Xa ◽

Therapeutic Index ◽

Inhibitory Effect ◽

Experimental Models ◽

Treatment And Prevention ◽

Long Acting

Abstract SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the “synthetic pentasaccharide” (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 ± 15 anti–factor Xa U/mg v850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti–factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti–factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti–factor Xa activity. SANORG 34006 enhanced rt-PA–induced thrombolysis and inhibited accretion of125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.

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Patient-specific iPSC-derived endothelial cells reveal aberrant p38 MAPK signaling in atypical hemolytic uremic syndrome

Stem Cell Reports ◽

10.1016/j.stemcr.2021.07.011 ◽

2021 ◽

Author(s):

Danni Zhou ◽

Ying Tan ◽

Xiaoling Liu ◽

Ling Tang ◽

Hao Wang ◽

...

Keyword(s):

Endothelial Cells ◽

Hemolytic Uremic Syndrome ◽

P38 Mapk ◽

Atypical Hemolytic Uremic Syndrome ◽

Mapk Signaling ◽

Patient Specific ◽

Uremic Syndrome

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Can Be miR-126-3p a Biomarker of Premature Aging? An Ex Vivo and In Vitro Study in Fabry Disease

Cells ◽

10.3390/cells10020356 ◽

2021 ◽

Vol 10 (2) ◽

pp. 356 ◽

Cited By ~ 1

Author(s):

Alessia Lo Curto ◽

Simona Taverna ◽

Maria Assunta Costa ◽

Rosa Passantino ◽

Giuseppa Augello ◽

...

Keyword(s):

Endothelial Cells ◽

Fabry Disease ◽

Healthy Subjects ◽

Aging Process ◽

Replicative Senescence ◽

Ex Vivo ◽

Umbilical Vein ◽

Quantitative Polymerase Chain Reaction ◽

Premature Aging

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by lysosomal accumulation of glycosphingolipids in a wide variety of cytotypes, including endothelial cells (ECs). FD patients experience a significantly reduced life expectancy compared to the general population; therefore, the association with a premature aging process would be plausible. To assess this hypothesis, miR-126-3p, a senescence-associated microRNA (SA-miRNAs), was considered as an aging biomarker. The levels of miR-126-3p contained in small extracellular vesicles (sEVs), with about 130 nm of diameter, were measured in FD patients and healthy subjects divided into age classes, in vitro, in human umbilical vein endothelial cells (HUVECs) “young” and undergoing replicative senescence, through a quantitative polymerase chain reaction (qPCR) approach. We confirmed that, in vivo, circulating miR-126 levels physiologically increase with age. In vitro, miR-126 augments in HUVECs underwent replicative senescence. We observed that FD patients are characterized by higher miR-126-3p levels in sEVs, compared to age-matched healthy subjects. We also explored, in vitro, the effect on ECs of glycosphingolipids that are typically accumulated in FD patients. We observed that FD storage substances induced in HUVECs premature senescence and increased of miR-126-3p levels. This study reinforces the hypothesis that FD may aggravate the normal aging process.

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