key: cord-0299929-txnp64dx
authors: Villalba, Nuria; Sackheim, Adrian M.; Lawson, Michael A.; Haines, Laurel; Chen, Yen-Lin; Sonkusare, Swapnil K.; Ma, Yong-Tao; Li, Jianing; Majumdar, Dev; Bouchard, Beth A.; Boyson, Jonathan E.; Poynter, Matthew E.; Nelson, Mark T.; Freeman, Kalev
title: The polyanionic drug suramin neutralizes histones and prevents endotheliopathy
date: 2021-12-10
journal: bioRxiv
DOI: 10.1101/2021.12.09.469611
sha: 637df4c8f668971f4fa411622a29ebcb21be239e
doc_id: 299929
cord_uid: txnp64dx

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but may cause secondary, deleterious effects such as excessive bleeding. Here, we demonstrate that suramin—a widely available polyanionic drug—completely neutralizes the toxic effects of histones. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sub-lethal doses of histones in vivo. Suramin also prevented lung edema, intra-alveolar hemorrhage and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels. Significance Statement Pathologic levels of circulating histones cause acute endotheliopathy, characterized by widespread disruption of critical endothelial functions and thromboinflammation. We discovered that suramin binds histones and prevents histone-induced endothelial dysfunction, thrombin generation, lung injury, and death. Histone binding is a novel mechanism of action for suramin, considered among the safest and most effective drugs by the World Health Organization. These results support the use of suramin for protection of blood vessels in conditions exacerbated by circulating histones including trauma and sepsis.

a surface-charge dependent fashion causing atherosclerosis (4). Elevated histone levels have 96 been linked to widespread endothelial injury and organ damage in human patients after trauma 97 (19, (32) (33) (34) (35) (36) (37) (38) and other conditions including ischemic stroke (39), sepsis (3), pancreatitis (40), and 98 acute respiratory distress syndrome (ARDS) (41, 42) .

The critical unmet need for therapeutics that protect the vascular endothelium from histone-100 mediated injury has become of immediate relevance in the context of the SARS-CoV-2 pandemic

(1, 43). We previously used native blood vessels from humans and mice to show the spatio-102 temporal distribution of endothelial cell calcium signals after histone exposure (20). Using 103 resistance-sized mesenteric arteries from mice expressing an endothelial cell-specific fluorescent 104 calcium biosensor, we found that histone-induced calcium signals were blocked by the removal of 105 extracellular calcium, but unaffected by depletion of intracellular calcium stores with cyclopiazonic 106 acid (CPA). Because TLR receptors have been implicated in histone effects in platelets and other 107 cells (6, 25, 44), and TLR agonists can trigger store-operated calcium (SOC) entry in endothelial 108 cells (45), we suspected that TLR activation might explain histone-induced calcium influx. However, 109 we and others have shown that TLR2 and TLR4 are not significantly involved in endothelial calcium 110 entry or cytotoxicity induced by histones (19, 20, 46) . We then hypothesized that histone-induced 

These unexpected observations were interesting, because suramin is not only a useful tool in 129 pharmacology, but also a safe and widely available drug. First synthesized by Bayer in 1917 as 130 part of a drug discovery program for trypanosomiasis (African sleeping sickness), suramin is a bis-131 polysulfonated naphthylurea hexaanion with activity against trypanosomes in both animal models 132 and humans (51). Suramin has been used clinically for over 100 years as an anti-parasite and anti-133 cancer agent, and, importantly, is considered among the safest and most effective drugs for health 134 care by the World Health Organization. Unlike heparan sulfate or heparin synthetic polyanions, 135 which also bind histones, suramin dosing is infrequent (usually once per week), and is inexpensive, 136 well-tolerated, and does not cause complications associated with anticoagulation.

Together, the published literature and our preliminary data provided a strong rationale for us to 138 investigate suramin as a candidate drug to prevent histone-induced endotheliopathy. The objective 139 of this study was to test the hypothesis that suramin can protect against histone-induced endothelial 140 dysfunction. We demonstrate that histones activate human endothelial cells to promote rapid 141 thrombin generation, a reaction that is blocked by suramin. In pressurized murine vascular 142 preparations, we directly tested the efficacy of suramin for preventing histone-induced aberrant 143 endothelial calcium signaling and vasodilatory dysfunction. In animal models of trauma, it is the 144 lung tissue and not kidney or liver, which is the primary target for circulating damage-associated 145 molecular pattern proteins (19) . In a histone infusion model, we found that suramin prevented 146 4 histone-induced lung injury, endothelial cell activation, adhesion molecule expression, and 147 pulmonary barrier disruption. Importantly, we also show that suramin completely protects against 148 a lethal dose of histones in vivo. Thus, histone binding is a novel mechanism of action for suramin, 149 providing a rationale for its use to protect the endothelium in conditions exacerbated by circulating 150 histones, such as trauma and sepsis. Together, these experiments provide new insights into the 151 deleterious effects of histones on endothelial function, thrombin generation, and lung damage, and 152 provide support for the use of suramin as a strategy to protect against histone-induced 153 endotheliopathy.

Suramin binds histones in solution. Based on its molecular structure ( Fig. 1 A) , we hypothesized 156 that suramin, a highly charged polysulfonated napthylurea, would bind avidly to cationic histone 157 complexes. When NETs or nucleosomes enter the bloodstream, they are exposed to endogenous 158 nucleases that rapidly digest DNA, leaving free histone proteins (15). Therefore, we focused on 159 testing the interactions between suramin and histones. First, fluorescent spectroscopy studies were injury is a significant concern in conditions characterized by high levels of histones such as ARDS 206 (19) , we also studied vascular preparations from small mouse pulmonary arteries. These blood 207 vessels were surgically opened on one side to expose the endothelial cell layer for direct 208 measurement of a fluorescent calcium indicator using confocal microscopy ( Fig. 3 C and D) . Similar humans and animal models of trauma, lung tissue is particularly vulnerable to circulating damage 221 associated molecular pattern proteins (19) . Therefore, we next tested the hypothesis that suramin Molecule-1 (ICAM-1) expression was also significantly increased by histones (Fig. 4 B) . Suramin 

Our results provide new insight into the pathophysiological outcomes of histone-induced organ 272 injury. We provide the first demonstration that in native, pulmonary artery preparations, histones 273 elicit calcium-mediated events similar to those we previously observed in mesenteric resistance 274 arteries from human and mouse. We also found that histone infusion caused endothelial barrier 275 breakdown of small blood vessels in both kidney and lung, with increased extravasation of the 70-276 kDa dextran, but not brain. This is consistent with other evidence that pulmonary and renal (6, 17, 277 19) tissue beds are highly sensitive to histone-induced injury. It was recently shown that histones 278 increased paracellular permeability in the hippocampus but not cortical brain regions (23). It is 279 possible that we missed these regional cerebrovascular effects because we quantified vascular 280 leak for the entire brain and not specific regions, or because we used a 70-kDa tracer rather than 281 a smaller sized dextran which would more specifically target blood-brain barrier permeability. Here, 

In summary, we demonstrate a new and previously unreported mechanism of action for an old 

Additional survival studies were also performed in mice that received a lethal dose of histones (75 362 mg/Kg; i.v.) (1); the survival rates were determined every five minutes, for 1 hour.

To assess for markers of activated endothelium, blood was collected via cardiac puncture in BD

Microtainer® blood collection tubes (BD Biosciences). Sera was obtained by centrifugation (1,300 365 x g, 10 min) and frozen. Thawed sera were diluted two-fold and cardiovascular markers were 

The samples were excited at 315 nm and the emission spectrum was measured between 370-480 406 nm. Histones did not show spectral overlap in that range (Supplemental Fig. 1 A) 

The Van der Waals and short-range electrostatics were cut off at 9.0 Å. Hydrogen atoms were 425 constrained using the SHAKE algorithm. Each simulation has two 700-ns replicas. Fig. 1 A) , and then measured suramin sodium salt intrinsic 670 fluorescence using increasing concentrations of suramin to determine the saturation range of the 671 detector (Supplemental Fig. 1 B) . 

Several exposed amino acid residues including arginine, asparagine, lysine, and threonine form 675 hydrogen bonds with the sulfate groups on suramin (Lys118, Thr76, Arg71, Lys77, Thr116, Arg45, 676 Asn108, Arg23, Arg63, Lys91, Arg83, Arg28). These include residues on H2A, H2B, H3, and H4, 677 which are predicted to form stable electrostatic interactions with the sulfate groups on suramin.

Thrombocytopathy and endotheliopathy: crucial contributors to COVID-19 492 thromboinflammation

Extracellular histones are major mediators of death in sepsis

Endotoxinemia Accelerates Atherosclerosis Through Electrostatic

Apoptotic release of histones from nucleosomes

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4

Blebs produced by actin-myosin contraction during apoptosis release 503 damage-associated molecular pattern proteins before secondary necrosis occurs

Histones, DNA, and Citrullination Promote Neutrophil 506

Extracellular Trap Inflammation by Regulating the Localization and Activation of TLR4

Autoantibodies stabilize neutrophil extracellular traps in COVID-19

Peptidylarginine deiminase inhibition reduces vascular damage and 511 modulates innate immune responses in murine models of atherosclerosis

Externalized histone H4 orchestrates chronic inflammation by 514 inducing lytic cell death

VWF-mediated leukocyte recruitment with chromatin decondensation 516 by PAD4 increases myocardial ischemia/reperfusion injury in mice

Neutrophil extracellular traps promote deep vein thrombosis in mice

Neutrophil extracellular traps form predominantly during the 521 organizing stage of human venous thromboembolism development

Extracellular histones, cell-free DNA, or 524 nucleosomes: differences in immunostimulation

The role of extracellular histone in organ injury

Histones and Neutrophil Extracellular Traps Enhance Tubular Necrosis 528 and Remote Organ Injury in Ischemic AKI

Circulating histones play a central role in COVID-19-associated 530 coagulopathy and mortality

Circulating Histones Are Mediators of Trauma-associated Lung Injury

Extracellular histones induce calcium signals in the endothelium of 534 resistance-sized mesenteric arteries and cause loss of endothelium-dependent dilation

Extracellular Histones Induced Eryptotic Death in Human Erythrocytes

Extracellular histones play an inflammatory role in acid aspiration-induced 587 acute respiratory distress syndrome

Treating the endotheliopathy of SARS-CoV-2 infection with plasma: Lessons 589 learned from optimized trauma resuscitation with blood products

Extracellular histones are 592 mediators of death through TLR2 and TLR4 in mouse fatal liver injury

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-595 induced vascular inflammation

Histones Induce the Procoagulant Phenotype of 597

Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-598 Regulation

Elementary Ca 2+ signals through endothelial TRPV4 channels 600 regulate vascular function

Pannexin-1 channels on endothelial cells mediate vascular inflammation 602 during lung ischemia-reperfusion injury

Systemic ATP impairs neutrophil chemotaxis and host defense in sepsis

Mitochondria regulate TRPV4-607 mediated release of ATP

Thrombin Formation

Bioactive DNA from extracellular vesicles and particles. Cell 612 Death Dis

Histone-induced damage of a mammalian 614 epithelium: the conductive effect

Histones and basic 616 polypeptides activate Ca 2+ /cation influx in various cell types

Histones stimulate von Willebrand factor 619 release in vitro and in vivo

Extracellular histones increase 621 plasma thrombin generation by impairing thrombomodulin-dependent protein C activation

TMEM16F forms a Ca 2+ -activated cation channel required for lipid scrambling 624 in platelets during blood coagulation

Thrombotic Role of Blood and Endothelial Cells in Uremia through 626

Nonanticoagulant heparin prevents histone-mediated 628 cytotoxicity in vitro and improves survival in sepsis

Heparins attenuated histone-mediated cytotoxicity in vitro and improved the 630 survival in a rat model of histone-induced organ dysfunction

Neutralization of heparin by histone and its subfractions

Therapeutic Anticoagulation with Heparin in Critically Ill 635 Patients with Covid-19

Therapeutic Anticoagulation with Heparin in Noncritically Ill 637 Patients with Covid-19

Protective effect of the long pentraxin PTX3 against histone-mediated 639 endothelial cell cytotoxicity in sepsis

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung 641 Injury

Endothelium-protective, histone-neutralizing properties of the polyanionic agent 643 defibrotide

Histone induced platelet aggregation is inhibited by normal albumin

Increased Histone-DNA Complexes and Endothelial-Dependent 647

Thrombin Generation in Severe COVID-19

The Use of Cyclodextrin or its Complexes 649 as a Potential Treatment Against the

Unfractioned heparin for treatment of sepsis: A randomized clinical trial (The 652 HETRASE Study)

Covid-19: trials of four potential treatments to generate "robust data" of what 654 works

Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-656

Endothelial pannexin 1-TRPV4 channel signaling lowers pulmonary arterial 658 pressure in mice

Melittin Aggregation in Aqueous Solutions: Insight from Molecular Dynamics 660 Simulations

A computational study of cooperative 662 binding to multiple SARS-CoV-2 proteins

Histones drive rapid thrombin generation on human endothelial cells which is blocked by 680 suramin. (A) Calibrated automated thrombogram (CAT) tracings of thrombin generation (nM) vs

time (min) by cultured human endothelial cells (Ea.hy926) in re-calcified, pooled, healthy human 682 plasma. Histones (50 µg/mL), suramin (50 µM) or a combination of both were exogenously added 683 to the cell culture and plasma samples as needed. (B) Summary data for lag time (min)

± 0.1 min; n=6) samples. (C) Summary data for peak thrombin (nM) in control

suramin (94 ± 4 nM; n=6), suramin and histones (106 ± 2 nM; n=6) and histones (127 ± 2 nM

Summary data for area under the curve (AUC; nM thrombin) in control (143732 688 ± 2498 nM; n=6), suramin (139915 ± 2355 nM; n=6)

± 3977 nM; n=6) samples. (E) Summary data for time to peak (min) in 690 control (31 ± 0.8 min; n=6), suramin (29 ± 0.8 min; n=6), suramin and histones (28 ± 0.6 min

(F) Summary data for velocity (nM thrombin/min) in control 692 (3.0 ± 0.1 nM thrombin/min; n=6), suramin (3.2 ± 0.1 nM thrombin/min; n=6), suramin and histones 693 (3.8 ± 0.04 nM thrombin/min; n=6) and histone (13 ± 0.3 nM thrombin/min; n=6) samples. Data are 694 expressed as mean ± SEM. Ordinary one-way ANOVA

Suramin prevents endothelial dysfunction and calcium overload caused by histones

Representative tracings of pressurized (80 mm Hg), third-order, mouse mesenteric arteries

µg/mL) or saline (control) was flowed through the lumen at 2 µL/min (<5 dynes

3; 1 µM) pre-flow 702 and post-flow were recorded. In one subset of experiments suramin (50 µM) was superfused 703 abluminally for 10 minutes prior to and then continuously during histones flow. Maximal dilations 704 were elicited at the end of the experiments using 0-Ca 2+ PSS. (B) Paired summary data of percent 705 dilation to 1 µM NS309 pre-flow and post-flow of saline (pre-sal 99 ± 1 vs

histones (10 µg/mL) (pre-his 97 ± 2 vs. post-his 33 ± 2 %; n=5

05; Paired Student's T-707 test), and suramin (50 µM) with histones (pre-sur+his 99 ± 1 vs. post-sur-his 98 ± 1 %

Representative images from en face mouse pulmonary arteries loaded with Fluo-4 (10 µM) on 709 a spinning disk confocal microscope. All images are from the same field of view recorded over 2 710 minutes. Arrows indicate large histone-induced calcium event F/F0 ROIs. (D) Summary data of the 711 paired total number of events per field after saline

µg/mL; 41 ± 6 events; n=4), and suramin (50 µM) and histones (Sur+His; 27 ± 6 events

Significant differences were determined using a repeated measures one-way ANOVA 714 test with a Holm-Sidak correction for multiple comparisons for all three groups

Histone-induced neutrophil recruitment and adhesion molecule expression is blocked by 719 suramin. (A) Live cells were gated and doublets excluded (FSC-A vs FSC-H). CD45 + cells were 720 selected and CD11c -cells identified. Neutrophils (CD11b + Ly6G + ) were identified, and the frequency 721 of neutrophils per live cells determined. Summary data of the frequency of neutrophil levels in lung 722 tissue 4 hours after saline (Control

± 0.4 frequency; n=4), or suramin (50 mg/Kg) and histone injection

Endothelial cells (CD31 + CD141 + , Q2) were assessed 726 for CD54 expression (geometric mean intensity). Summary data for endothelial ICAM-1 (CD54) 727 expression geometric means (GM) in lung tissue 4 hours after saline (Control

histones (Hist; 45 mg/Kg; 45314 ± 2126 GM; n=5), or suramin (50 mg/Kg) and histone 729 injection (Sur+His; 34288 ± 1957 GM; n=5). Data are expressed as mean ± SEM. Two-way ANOVA 730 with Bonferroni's correction for multiple comparisons; P<0.05. (C) Suramin decreases histone-731 induced adhesion molecule levels in serum. Adhesion molecule levels measured in serum with a 732 multiplex immunoassay (Luminex)

and histone with suramin (50 mg/Kg) injection (Sur+His)

ICAM-1 and PECAM-1. Data are expressed as fold change, mean ± SEM

Student's T-test

Figure 5. Suramin improves survival and prevents lung injury and edema caused by histones

Saline injected (control; n=6), a lethal dose of histones (His; 75 mg/Kg; n=10), and a lethal dose of 739 histones with suramin (Sur+His; 20 mg/Kg n=11 or 50 mg/Kg n=6) was injected into mice and 740 survival was recorded over the course of 35 minutes

05; *, for each group compared to saline. (B) Representative images of 742 hematoxylin and eosin stain (H&E) of a histological section of paraffin-embedded fixed lung tissue 743 from a mouse from each group. The dark blue color denotes cell nuclei

Summary data of the total, non-745 differentiated cell counts in the bronchial-alveolar lavage fluid (BALF) at 4 hours after saline 746 (control; 43333 ± 4410 cells/mL; n=3), histones (His; 45 mg/Kg; 167847 ± 22008 cells/mL; n=7), or 747 suramin (50 mg/Kg and histone injection (Sur+His; 41667 ± 5725 cells/mL; n=6) and 24 hours after 748 saline

or suramin (50 mg/Kg) and histone injection (Sur+His; 45000 ± 10247 cells/mL; n=6)

Summary data for the total protein leakage into the BALF at 4 hours after saline (control

µg/mL; n=5), histones (His; 45 mg/Kg; 1215 ± 186 µg/mL; n=9), or suramin (50 mg/Kg) and histone 752 injection (Sur+His; 507 ± 66 µg/mL; n=5) and 24 hours after saline

histones (His; 45 mg/Kg; 901 ± 249 µg/mL; n=9), or suramin (50 mg/Kg) and histone injection 754 (Sur+His; 225 ± 39 µg/mL; n=7). Data are expressed as mean ± SEM

Bonferroni's correction for multiple comparisons; P<0.05. (D) Summary data for FITC-dextran 756 extravasation using the modified Mile's Assay in lung, kidney

mg/Kg) and suramin (50 mg/Kg) and histone (Sur+His) injected mice at 4 hr. Lung 758 permeability in saline

7 ng FITC/mg tissue; n=5) 760 injected mice. Kidney permeability in saline (control; 12 ± 2.7 ng FITC/mg tissue; n=6), histones 761 (His; 94 ± 7.7 ng FITC/mg tissue; n=6), and suramin and histone (Sur+His; 36 ± 11 ng FITC/mg 762 tissue; n=6) injected mice

histones (His; 3.1 ± 0.6 ng FITC/mg tissue; n=6), and suramin and histone

FITC/mg tissue; n=6) injected mice. Data are expressed as mean ± SEM. Two-way ANOVA with 765

Bonferroni's correction for multiple comparisons

Suramin prevents histone-induced calcium overload and death, impaired vasodilation, 771 endothelial barrier breakdown, neutrophil migration, adhesion molecule expression

4). λexc = 315 nm and λem = 405 nm were used for the emission and excitation 777 scan, respectively, of suramin fluorescence, whereas λexc = 278 nm and λem = 305 nm were used 778 for the emission and excitation scan, respectively, of histones. (A) Dual plot of both histones and 779 suramin fluorescence and absorbance. (B) Suramin intrinsic fluorescence

Supplemental video 1. A molecular dynamics simulation shows electrostatic interactions between 782 suramin molecules (stick representation) and the histone octamer (cartoon representation), with 783 hidden water molecules and counter ions for clarity. Six suramin molecules were first arbitrarily 784 placed in the simulation box at the proximity of histone