Endothelial Toxicity Research Articles

Comparative assessment of the endothelial toxicity of intracameral cefuroxime after phacoemulsification

Comparative assessment of the endothelial toxicity of intracameral cefuroxime after phacoemulsification

Potential mechanisms of resistance to current anti-thrombotic strategies in Multiple Myeloma.

Multiple Myeloma (MM) is a common haematological malignancy that is associated with a high rate of venous thromboembolism (VTE) with almost 10% of patients suffering thrombosis during their disease course. Recent studies have shown that, despite current thromboprophylaxis strategies, VTE rates in MM remain disappointingly high. The pathophysiology behind this consistently high rate of VTE is likely multifactorial. A number of factors such as anti-thrombin deficiency or raised coagulation Factor VIII levels may confer resistance to heparin in these patients, however, the optimal method of clinically evaluating this is unclear at present, though some groups have attempted its characterisation with thrombin generation testing (TGT). In addition to testing for heparin resistance, TGT in patients with MM has shown markedly varied abnormalities in both endogenous thrombin potential and serum thrombomodulin levels. Apart from these thrombin-mediated processes, other mechanisms potentially contributing to thromboprophylaxis failure include activated protein C resistance, endothelial toxicity secondary to chemotherapy agents, tissue factor abnormalities and the effect of immunoglobulins/“M-proteins” on both the endothelium and on fibrin fibre polymerisation. It thus appears clear that there are a multitude of factors contributing to the prothrombotic milieu seen in MM and further work is necessitated to elucidate which factors may directly affect and inhibit response to anticoagulation and which factors are contributing in a broader fashion to the hypercoagulability phenotype observed in these patients so that effective thromboprophylaxis strategies can be employed.

Disturbance of Vancomycin Infusion Flow during Multidrug Infusion: Influence on Endothelial Cell Toxicity.

Background: Phlebitis is a common side effect of vancomycin peripheral intravenous (PIV) infusion. As only one PIV catheter is frequently used to deliver several drugs to hospitalized patients through the same Y-site, perturbation of the infusion flow by hydration or other IV medication may influence vancomycin exposure to endothelial cells and modulate toxicity. Methods: We assessed the toxicity of variations in vancomycin concentration induced by drug mass flow variations in human umbilical vein endothelial cells (HUVECs), simulating a 24 h multi-infusion therapy on the same line. Results were expressed as the percentage of viable cells compared with a 100% control, and the Kruskal–Wallis test was used to assess the toxicity of vancomycin. Results: Our results showed that variations in vancomycin concentration did not significantly influence local toxicity compared to a fixed concentration of vancomycin. Nevertheless, the loss of cell viability induced by mechanical trauma mimicking multidrug infusion could increase the risk of phlebitis. Conclusion: To ensure that vancomycin-induced phlebitis must have other causes than variation in drug mass flow, further in vitro experiments should be performed to limit mechanical stress to frequent culture medium change.

Pharmacological inhibition of IRAK1 and IRAK4 prevents endothelial inflammation and atherosclerosis in ApoE-/- mice

Inflammation associated endothelial dysfunction represents a pivotal contributor to atherosclerosis. Increasingly, evidence has demonstrated that interleukin 1 receptor (IL1-R) / toll-like receptor (TLR) signaling participates in the development of atherosclerosis. Recent large-scale clinical trials have supported the therapeutic potential of anti-inflammatory therapies targeting IL-1β and IL-6 in reducing atherosclerosis. The present study examined the pharmacological effects of IL-1R-associated kinase 1 and 4 inhibitors (IRAK1/4i) in regulating inflammation of the endothelium and atherosclerosis. We demonstrate that dual pharmacological inhibition of IRAK1 and IRAK4 by an IRAK1/4i is more effective against LPS induced endothelial inflammation, compared with IRAK1 inhibitor or IRAK4 inhibitor monotherapy. IRAK1/4i showed little endothelial cell toxicity at concentrations from 1 μM up to 10 μM. Inhibition of IRAK1/4 reduced endothelial activation induced by LPS in vitro as evidenced by attenuated monocyte adhesion to the endothelium. Mechanistically, blockade of IRAK1/4 ameliorated the transcriptional activity of NF-κB. To assess the pharmacological effects of IRAK1/4i on atherosclerosis in vivo, ApoE-/- mice were orally administered IRAK1/4i (20 mg/kg/d) for 8 weeks. We show that IRAK1/4i reduced atherosclerotic lesion size in the aortic sinus and increased hepatic LDLR protein levels as well as lowered LDL-C level, without affecting other lipid parameters or glucose tolerance. Taken together, our findings demonstrate that dual pharmacological inhibition of IRAK1 and IRAK4 attenuates endothelial inflammation, lowers LDL-C levels and reduces atherosclerosis. Our study reinforces the evolving standing of anti-inflammatory approaches in cardiovascular therapeutics.

Sodium-glucose cotransporter type 2 inhibitors prevent ponatinib-induced endothelial senescence and disfunction: A potential rescue strategy

BackgroundPonatinib (PON), a third-generation tyrosine kinase inhibitor (TKI), has proven cardiovascular toxicity, with no known preventing agents usable to limit such side effect. Sodium-glucose cotransporter type 2 (SGLT2) inhibitors are a new class of glucose-lowering agents, featuring favorable cardiac and vascular effects. AimsWe assessed the effects of the SGLT2 inhibitors empagliflozin (EMPA) and dapagliflozin (DAPA) on human aortic endothelial cells (HAECs) and underlying vasculo-protective mechanisms in an in vitro model of PON-induced endothelial toxicity. Methods and resultsWe exposed HAECs to PON or vehicle (DMSO) in the presence or absence of EMPA (100 and 500 nmol/L) or dapagliflozin (DAPA) for 0–48 h exposure times. Compared with vehicle, incubations of HAECs with PON significantly reduced cell viability (0.56 ± 0.11 vs 0.23 ± 0.05 absorbance units, p < 0.01), increased the number of senescent cells at β-gal-assay (PON 9 ± 4 vs basal DMSO 3 ± 1 β-Gal+ cells/field, p < 0.01), decreased tubulization in Matrigel (PON PON: 6 ± 1 vs basal DMSO 12 ± 1 tubuli number/field, p < 0.05) with a non-statistically significant trend of PON to decrease the number of autophagic cells at immunofluorescence assay and flow cytometry. EMPA reverted the effects of PON on cell viability (E 500 + PON 0.24 ± 0.05 vs PON 0.56 ± 0.11 absorbance units, p < 0.01) and induced autophagy (E 500 7 ± 4.3 vs basal DMSO 2.6 ± 2.3 mean fluorescence vs PON 2.6 ± 2.4 mean fluorescence, p < 0.05). EMPA and DAPA also reversed the effects of PON on cell senescence (E 500 + PON 4 ± 1 and DAPA 100 4 ± 2 vs PON 9 ± 4 β-Gal+ cells/field, p < 0.01) and improved cell tubulization (E 500 + PON 21 ± 3 vs PON 6 ± 1 tubuli number/field, p < 0.05; DAPA 100 + PON 16 ± 2 vs PON 6 ± 1 tubuli number/field, p < 0.05). ConclusionEMPA and DAPA attenuate the vasculo-toxic effect exerted by PON by reverting endothelial cell senescence and dysfunction. These findings support the design of clinical studies exploring the vasculo-protective effects of EMPA or DAPA on PON-induced vascular toxicity.

Abstract 10999: Pod-Based E-liquids Induce Vascular Endothelial Cell Dysfunction

Introduction: Pod-based e-cigarettes including JUUL represent the most commonly used product type and more efficiently deliver nicotine; however, their cardiovascular effects are poorly understood. Methods: We evaluated endothelial function (cell viability by TUNEL and nitric oxide bioavailability using DAF-2DA) in human aortic endothelial cells (HAECs) exposed for 90 minutes to one of four JUUL pod flavors (Menthol, Mint, Mango and Virginia Tobacco) or constituents propylene glycol (PG)/vegetable glycerol (VG) at 30:70 ratio with and without nicotine salt, as well as their heated particle fractions separated with a mini-MOUDI impactor. In healthy young adults, we assessed endothelial cell function in nonusers of tobacco products, pod users and combustible cigarette users for eNOS activation. Results: We observed significant increases in cell death with JUUL e-liquid flavors across 0.0001-1% dilutions. PG/VG vehicle alone and with nicotine salt induced cell death to similar levels as the JUUL e-liquids. All JUUL e-liquids at 0.0001% dilution impaired A23187-stimulated nitric oxide production. Exposure to particle fractions of JUUL e-liquids at 0.001% dilution also reduced A23187-stimulated nitric oxide production. Endothelial cells from pod users showed reduced eNOS activation compared with nonusers and similar to reduced levels in combustible cigarette users (figure). Conclusions: Our data suggest endothelial toxicity of pod-based e-liquids and their constituents, including in young adult users.

N-Acetylcysteine Amide Alleviates Cyclophosphamide-Induced Endothelial Damage

Rui He 1,2, Wenyi Zheng 1,2, Terra Slof 3, Eva Wärdell 4, Agneta Månsson-Broberg 4, Svante Norgren 5, Per Ljungman 2, Ying Zhao 1, 2 , Moustapha Hassan 1, 21.Experimental Cancer Medicine, Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, 141 86 Huddinge, Sweden2.Center of Allogeneic Stem Cell Transplantation (CAST) and Clinical Research Center, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden.3.Institute for Life Sciences and Chemistry, HU University of Applied Sciences Utrecht, Oudenoord 330, 3513 EX Utrecht, Netherlands4.Department of Medicine, Division of Cardiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.5.Children´s and Women´s Health Theme, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenAimThe current study aimed to investigate the protective role of the novel antioxidant N-acetylcysteine amide (NACA) in cyclophosphamide (Cy)-induced endothelial injury and to explore the underlying mechanism.BackgroundThe alkylating agent Cy is still one of the important corner stones in cancer treatment. Cy is used also as a part of the conditioning regimens prior to hematopoietic cell transplantation (HCT) and as a prophylactic treatment post transplantation in graft-versus-host disease (GVHD). Several studies have shown that high doses of Cy are associated with a number of adverse effects including alopecia, gonadal toxicity, hemorrhagic cystitis, carcinogenesis and late cardiovascular toxicity. Moreover, we have shown that treatment with high-dose Cy caused damage to the arterial endothelium, which might contribute to late cardiovascular disorders. Hitherto, there is no prophylactic strategy for Cy-induced endothelial toxicity. Oxidative stress is one of the main players in the pathogenesis of endothelial disorders and hence is an exploitable trait for targeted treatment.MethodsThe prophylactic efficacy of NACA was evaluated using human aortic endothelial cells (HAECs) and human umbilical endothelial cells (HUVECs). The efficacy of NACA was compared with the clinically approved derivative N-acetylcysteine (NAC). Cy is a prodrug that cannot be activated in the endothelial cells. Therefore, cells were incubated with Cy active metabolite, 4-hydroxycyclophosphamide (4-OH-Cy). In order to reveal the underling mechanism of action, we have investigated and evaluated the following parameters: alteration in cell morphology, cell death pathway, inhibition of DNA damage, variation of redox hemostasis, and neovascularization.ResultsWe found that pre-incubation with 5 mM NACA preserved the normal endothelial cell morphology. However, for the quantification of the obtained results, cell colonies were visualized using crystal violet staining while cell viability was determined using WST-1 assay. Moreover, NACA was found to decrease 4-OH-Cy-induced DNA damage and cell death via caspase-dependent apoptosis in both HAECs and HUVECs. In addition, NACA significantly inhibited 4-OH-Cy-induced oxidative stress by reducing the intracellular levels of reactive oxygen species (ROS) and by improving the cellular antioxidative capacity. Increased activities of catalase and superoxide dismutase (SOD) together with increased levels of reduced glutathione (GSH) were observed. Meanwhile, NACA pre-treatment rebalanced endothelial nitric oxide synthase (eNOS) and arginase I in HAECs, suggesting that NACA may be used as prophylactic strategy to avoid the impaired endothelium-dependent vasodilation caused by 4-OH-Cy. Moreover, data from tube formation assay indicated that NACA preserved the angiogenic capability of endothelial cells that was compromised by 4-OH-Cy through blockage of Notch signaling pathway. Our results showed that in contrast to NAC, its amide derivative NACA showed remarkably superior ability in alleviating the endothelial damage caused by 4-OH-Cy.ConclusionsIn summary, the present investigation showed that the novel antioxidant NACA could be applied to protect against 4-OH-Cy-induced endothelial cell dysfunction and death. Higher bioavailability and better cellular uptake make NACA to a better prophylactic agent compared to NAC. To translate these findings into the clinic, additional in vivo studies are warranted to confirm the protective potential of NACA. DisclosuresLjungman: Takeda: Consultancy, Other: Endpoint committee, speaker; Janssen: Other: Investigator; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy.

Anti-Apoptotic Effect of Flavokawain A on Ochratoxin-A-Induced Endothelial Cell Injury by Attenuation of Oxidative Stress via PI3K/AKT-Mediated Nrf2 Signaling Cascade.

This study investigates the endothelial protective activity of flavokawain A (FKA) against oxidative stress induced by ochratoxin A (OTA), which acts as a mycotoxin, and its primary mechanisms in in vitro models. Reactive oxygen species, in general, regulate oxidative stress that significantly contributes to the pathophysiology of endothelial dysfunctions. OTA exerts toxicity through inflammation and the accumulation of ROS. This research is aimed at exploring the defensive function of FKA against the endothelial injury triggered by OTA through the Nrf2 pathway regulated by PI3K/AKT. OTA exposure significantly increased the nuclear translocation of NFκB, whereas we found a reduction in inflammation via NFκB inhibition with FKA treatment. FKA increased the PI3K and AKT phosphorylation, which may lead to the stimulation of antioxidative and antiapoptotic signaling in HUVECs. It also upregulated the phosphorylation of Nrf2 and a concomitant expression of antioxidant genes, such as HO-1, NQO-1, and γGCLC, depending on the dose under the oxidative stress triggered by OTA. Knockdown of Nrf2 through small interfering RNA (siRNA) impedes the protective role of FKA against the endothelial toxicity induced by OTA. In addition, FKA enhanced Bcl2 activation while suppressing apoptosis marker proteins. Therefore, FKA is regarded as a potential agent against endothelial oxidative stress caused by the deterioration of the endothelium. The research findings showed that FKA plays a key role in activating the p-PI3K/p-AKT and Nrf2 signaling pathways, while suppressing caspase-dependent apoptosis.

Breast cancer chemotherapy vascular toxicity: a review of mediating mechanisms and exercise as a potential therapeutic.

Breast cancer chemotherapy, although very potent against tumour tissue, results in significant cardiovascular toxicity. The focus of research in this area has been predominantly towards cardiotoxicity. There is limited evidence detailing the impact of such treatment on the vasculature despite its central importance within the cardiovascular system and resultant detrimental effects of damage and dysfunction. This review highlights the impact of chemotherapy for breast cancer on the vascular endothelium. We consider the most likely mechanisms of endothelial toxicity to be through direct damage and dysfunction of the endothelium. There are sharp consequences of these detrimental effects as they can lead to cardiovascular disease. However, there is potential for exercise to alleviate some of the vascular toxicity of chemotherapy, and the evidence for this is provided. The potential role of exercise in protecting against vascular toxicity is explained, highlighting the recent in-human and animal model exercise interventions. Lastly, the mediating mechanisms of exercise protection of endothelial health is discussed, focusing on the importance of exercise for endothelial health, function, repair, inflammation and hyperlipidaemia, angiogenesis, and vascular remodelling. These are all important counteracting measures against chemotherapy-induced toxicity and are discussed in detail.

Oxidized carbon black nanoparticles induce endothelial damage through C-X-C chemokine receptor 3-mediated pathway

Oxidation of engineered nanomaterials during application in various industrial sectors can alter their toxicity. Oxidized nanomaterials also have widespread industrial and biomedical applications. In this study, we evaluated the cardiopulmonary hazard posed by these nanomaterials using oxidized carbon black (CB) nanoparticles (CBox) as a model particle.Particle surface chemistry was characterized by X-ray photo electron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). Colloidal characterization and in vitro dosimetry modeling (particle kinetics, fate and transport modeling) were performed. Lung inflammation was assessed following oropharyngeal aspiration of CB or oxidized CBox particles (20 μg per mouse) in C57BL/6J mice. Toxicity and functional assays were also performed on murine macrophage (RAW 264.7) and endothelial cell lines (C166) with and without pharmacological inhibitors. Oxidant generation was assessed by electron paramagnetic resonance spectroscopy (EPR) and via flow cytometry. Endothelial toxicity was evaluated by quantifying pro-inflammatory mRNA expression, monolayer permeability, and wound closure.XPS and FTIR spectra indicated surface modifications, the appearance of new functionalities, and greater oxidative potential (both acellular and in vitro) of CBox particles. Treatment with CBox demonstrated greater in vivo inflammatory potentials (lavage neutrophil counts, secreted cytokine, and lung tissue mRNA expression) and air-blood barrier disruption (lavage proteins). Oxidant-dependent pro-inflammatory signaling in macrophages led to the production of CXCR3 ligands (CXCL9,10,11). Conditioned medium from CBox-treated macrophages induced significant elevation in endothelial cell pro-inflammatory mRNA expression, enhanced monolayer permeability and impairment of scratch healing in CXCR3 dependent manner.In summary, this study mechanistically demonstrated an increased biological potency of CBox particles and established the role of macrophage-released chemical mediators in endothelial damage.

NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity.

CD93 CAR T cells eliminate AML and spare HSPCs but exert on-target, off-tumor toxicity to endothelial cells. We show coexpression of other AML targets on endothelial cells, introduce a novel NOT-gated strategy to mitigate endothelial toxicity, and demonstrate use of high-dimensional transcriptomic profiling for rational design of combinatorial immunotherapies.See related commentary by Velasquez and Gottschalk, p. 559. This article is highlighted in the In This Issue feature, p. 549.

Polyethylenimine-Modified Mesoporous Silica Nanoparticles Induce a Survival Mechanism in Vascular Endothelial Cells via Microvesicle-Mediated Autophagosome Release.

Surface-modified mesoporous silica nanoparticles (MSNs) have attracted more and more attention as promising materials for biomolecule delivery. However, the lack of detailed evaluation relevant to the potential cytotoxicity of these MSNs is still a major obstacle for their applications. Unlike the bare MSNs and amino- or liposome-modified MSNs, we found that polyethylenimine-modified MSNs (MSNs-PEI) had no obvious toxicity to human umbilical vein endothelial cells (HUVECs) at the concentrations up to 100 μg/mL. However, MSNs-PEI induced autophagosomes accumulation by blocking their fusion with lysosomes, an essential mechanism for the cytotoxicity of many nanoparticles (NPs). Thus, we predicted that an alternative pathway for autophagosome clearance exists in HUVECs to relieve autophagic stress induced by MSNs-PEI. We found that MSNs-PEI prevented STX17 loading onto autophagosomes instead of influencing lysosomal pH or proteolytic activity. MSNs-PEI induced the structural alternation of the cytoskeleton but did not cause endoplasmic reticulum stress. The accumulated autophagosomes were released to the extracellular space via microvesicles (MVs) when the autophagic degradation was blocked by MSNs-PEI. More importantly, blockade of either autophagosome formation or release caused the accumulation of damaged mitochondria and excessive ROS production in the MSNs-PEI-treated HUVECs, which in turn led to cell death. Thus, we propose here that the MV-mediated autophagosome release, a compensation mechanism, allows the vascular endothelial cell survival when the degradation of autophagosomes is blocked by MSNs-PEI. Accordingly, promoting the release of accumulated autophagosomes may be a protective strategy against the endothelial toxicity of NPs.

The effects of pod‐based e‐liquids on vascular endothelial cell function

The use of electronic (e-) cigarettes has rapidly risen in youth and young adults. Pod-based e-cigarettes represent a more evolved generation of these devices that are designed to deliver higher levels of nicotine. Despite their popularity, little is known about the cardiovascular toxicity imposed by the use of e-cigarettes. The JUUL brand of pod-based e-cigarettes holds the majority of market shares in the US. The purpose of this study was to evaluate the effects of four common JUUL pod flavors (Menthol, Mint, Mango and Virginia Tobacco), the product constituents propylene glycol (PG)/vegetable glycerol (VG) vehicle at 30:70 ratio and nicotine salt in 30:70 PG/VG on vascular endothelial cell function. We evaluated endothelial function (cell viability by TUNEL and nitric oxide bioavailability using DAF-2DA) in human aortic endothelial cells (HAECs) following a 90-minute exposure to JUUL pods at dilutions of 0.00001% to 1%. Flavored JUUL pod exposure induced at least 15% endothelial cell death at dilutions of 0.0001% for Menthol, 0.1% for Mint, 0.1-1% for Mango, and 0.0001-1% for Virginia Tobacco. Cells exposed to the PG/VG vehicle alone and in the presence of nicotine salt increased cell death to similar levels as the flavored JUUL pod exposures, suggesting that the vehicle may be an important source of toxicity. All flavored JUUL pods at a dilution of 0.0001% impaired A23187-stimulated nitric oxide in HAECs, suggestive of endothelial dysfunction. Our data suggests that exposure of endothelial cells to flavored JUUL pods results in cytotoxicity and impairs nitric oxide bioavailability, which may be relevant to cardiovascular toxicity. Ongoing experiments focus on the evaluation of oxidative stress and interleukin-6 expression in vascular endothelial cells. Future work will include evaluation of the effects of JUUL e-liquid heated fractions to identify fractions and specific chemical constituents that induce endothelial cell toxicity.

Antifungal Efficacy and Safety of Cycloheximide as a Supplement in Optisol-GS.

PurposeThe incidence of fungal infection after corneal transplant has increased significantly in recent years, especially Candida spp. This study aimed to evaluate the efficacy and safety of the addition of cycloheximide in Optisol-GS media in decreasing the growth of Candida spp. strains.MethodsThis in vitro laboratory efficacy study measured fungal colony growth in 24 vials of Optisol-GS that were divided into 6 groups of 4 vials each, as follows: (1) MIC/2 cycloheximide, (2) MIC cycloheximide, (3) MICx5 cycloheximide, (4) MICx10 cycloheximide, from MIC values obtained for each strain, (5) unsupplemented optisol-GS as a positive control (added inoculum), and (6) unsupplemented optisol-GS as a negative control (no inoculum). In each group was added Candida albicans, C. glabrata and C. parapsilosis, except in the negative control. The evaluated variables were fungal colony growth from the Optisol-GS vials, corneal endothelial cell density and endothelial cell viability at different concentrations of cycloheximide.ResultsIn the efficacy study, all strains showed a reduction in fungal cell growth from the second day at all evaluated concentrations of optisol-GS supplemented with cycloheximide, even at subinhibitory concentrations (MIC/2). For C. glabrata, the colony count was reduced to 99%. No evidence of corneal endothelial toxicity was found at any concentration, in the safety study, compared with the paired control.ConclusionThe addition of cycloheximide to optisol-GS decreased the fungal growth, demonstrating fungicide action against C. glabrata and fungistatic action against C. albicans and C.parapsilosis. This drug did not demonstrate toxicity to the corneal endothelium at different concentrations.

GVHD Prophylaxis 2020.

Graft-vs. host disease (GVHD), both acute and chronic are among the chief non-relapse complications of allogeneic transplantation which still cause substantial morbidity and mortality despite significant advances in supportive care over the last few decades. The prevention of GVHD therefore remains critical to the success of allogeneic transplantation. In this review we briefly discuss the pathophysiology and immunobiology of GVHD and the current standards in the field which remain centered around calcineurin inhibitors. We then discuss important translational advances in GVHD prophylaxis, approaching these various platforms from a mechanistic standpoint based on the pathophysiology of GVHD including in-vivo and ex-vivo T-cell depletion alongwith methods of selective T-cell depletion, modulation of T-cell co-stimulatory pathways (checkpoints), enhancing regulatory T-cells (Tregs), targeting T-cell trafficking as well as cytokine pathways. Finally we highlight exciting novel pre-clinical research that has the potential to translate to the clinic successfully. We approach these methods from a pathophysiology based perspective as well and touch upon strategies targeting the interaction between tissue damage induced antigens and T-cells, regimen related endothelial toxicity, T-cell co-stimulatory pathways and other T-cell modulatory approaches, T-cell trafficking, and cytokine pathways. We end this review with a critical discussion of existing data and novel therapies that may be transformative in the field in the near future as a comprehensive picture of GVHD prophylaxis in 2020. While calcineurin inhibitors remain the standard, post-transplant eparinsphamide originally developed to facilitate haploidentical transplantation is becoming an attractive alternative to traditional calcinuerin inhibitor based prophylaxis due to its ability to reduce severe forms of acute and chronic GVHD without compromising other outcomes, even in the HLA-matched setting. In addition T-cell modulation, particularly targeting some important T-cell co-stimulatory pathways have resulted in promising outcomes and may be a part of GVHD prophylaxis in the future. Novel approaches including targeting early events in GVHD pathogenesis such as interactions bvetween tissue damage associated antigens and T-cells, endothelial toxicity, and T-cell trafficking are also promising and discussed in this review. GVHD prophylaxis in 2020 continues to evolve with novel exicitng therapies on the horizon based on a more sophisticated understanding of the immunobiology of GVHD.

Dose-dependent emergence of acute and recurrent corneal lesions in sulfur mustard-exposed rabbit eyes

Sulfur mustard (SM) is a lipid soluble alkylating agent that causes genotoxic injury. The eye is highly sensitive to SM toxicity and exposures exceeding 400 mg min/m3 can elicit irreversible corneal pathophysiologies. Development of medical countermeasures for ocular SM exposure has been hindered by a limited understanding of dose-dependent effects of SM on corneal injury. Here, clinical, histological and ultrastructural analyses were used to characterize the effects of SM dose on corneal injury progression. Corneas were evaluated for up to 20 wk following exposure to saturated SM vapor for 30−150 s, which corresponds to 300−1,500 mg min/m3. In acute studies, a ceiling effect on corneal edema developed at doses associated with full-thickness corneal lesions, implicating endothelial toxicity in corneal swelling. Recurrent edematous lesions (RELs) transiently emerged after 2 wk in a dose-dependent fashion, followed by the development of secondary corneal pathophysiologies such as neovascularization, stromal scarring and endothelial abnormalities. RELs appeared in 96 % of corneas exposed for ≥ 90 s, 52 % of corneas exposed for 60 s and 0 % of corneas exposed for 30 s. While REL latency was variable in corneas exposed for 60 s, REL emergence was synchronized at exposures ≥ 90 s. Corneas did not exhibit more than one REL, suggesting RELs are part of a programmed pathophysiological response to severe alkylating lesions. In post-mortem studies at 12 wk, corneal edema was positively correlated to severity of endothelial pathologies, consistent with previous findings that endothelial toxicity influences long-term outcomes. These results provide novel insight into long-term corneal pathophysiological responses to acute toxicity and identify exposure conditions suitable for therapeutic testing.

Deswelling kinetics in CorneaJet of organ cultured human corneas

AbstractPurposeThe deswelling of a corneal graft at the end of storage in the eye bank is essential before perforating or deep anterior lamellar keratoplasty to allow good donor/recipient congruence and to accelerate visual recovery. In France, CorneaJet (Eurobio, France) containing 5% Dextran T500 is the most commonly used medium, but its deswelling kinetics is not referenced and the duration of 48 or 72 hr is chosen purely by habit while the increase in endothelial toxicity over time is documented.AimTo determine the corneal deswelling kinetics in CorneaJet.Methods12 corneas discarded by our eye bank were used. They had an endothelial cell density at reception (day 2 to day 4) of 1713 ± 389 cell/mm2 (mean ± SD). All had been stored in organoculture in CorneaMax medium at 31°C for at least 1 week. The corneas were immersed in 50 ml vials of CorneaJet (same batch) and were stored at 31°C. Central corneal thickness (CCT in µm) and central corneal volume (on an 8mm disc, CCV8 in mm3) were measured by optical coherence tomography (OCT, Casia I, Tomey, Japan). Transparency was evaluated on a prototype measuring contrast transmission (relative scale in %). Measurements were performed before immersion in CorneaJet and repeated at hour (H)3, H6, H9, H12, H18, H24, H30, H36 and H48.ResultsAll data were expressed as mean ± SD respectively at the different times. CCT: 1284 ± 249; 1031 ± 233; 897 ± 22; 773 ± 160; 741 ± 123; 673 ± 75; 626 ± 27; 638 ± 41; 645 ± 49; 652 ± 55. CCV8: 73 ± 9; 61 ± 9; 53 ± 9; 47 ± 8; 44 ± 6; 40 ± 4; 38 ± 3; 37 ± 3; 37 ± 3 and 37 ± 3. Transparency: 13 ± 4; 19 ± 5; 24 ± 6; 29 ± 6; 33 ± 7; 40 ± 7 41 ± 7; 44 ± 9; 45 ± 8 and 44 ± 11.ConclusionsCorneal deswelling in CorneaJet is rapid during the first 12 hr and reaches a plateau at 20 hr. After 20 hr, transparency and thickness are ideal for surgery.

Shared Expression of CD93 and Other Antigens By AML and Endothelial Cells Highlights a Need for Rational Combinatorial Targeting

Shared Expression of CD93 and Other Antigens By AML and Endothelial Cells Highlights a Need for Rational Combinatorial Targeting

TLR2 signalling orchestrates neutrophil activation in acute coronary syndrome with intact fibrous cap – results from the OPTICO-ACS study

Abstract Background Neutrophil granulocytes are key players of the innate immunity, participating in the initiation and progression of atherosclerosis. However, the exact mechanisms of neutrophil activation after acute coronary syndrome (ACS) are poorly understood, especially in the context of the two predominant ACS-causing pathophysiologies - ACS with intact fibrous cap (IFC-ACS) and ACS with ruptured fibrous cap (RFC-ACS). Therefore, the current study focuses on immunophenotyping and ex-vivo functional characterization of neutrophils with regard to the molecular differences between IFC-ACS and RFC-ACS. Methods Using high-resolution optical coherence tomography (OCT) of the ACS-causing culprit lesion and re-evaluation by a second OCT-core lab, thirty-two IFC-ACS-patients were matched to thirty-two RFC-ACS-patients by gender, age and diabetes. Local and systemic blood samples were obtained from the site of the ACS-causing culprit lesion (LOC) and from the arterial sheath (SYS), respectively. Neutrophil abundance and surface marker expression were quantified by flow cytometry. Fresh neutrophils were isolated for functional analysis and ex-vivo assessment of cell-toxicity in a co-culture with human aortic endothelial cells (HAECs). Neutrophil secretion of active MMP9 was evaluated by fluorescence-based zymography in supernatants of isolated neutrophils and in patients' plasma samples. Results Neutrophils of patients with IFC-ACS show significantly higher expression of the toll-like receptor 2 (TLR2) in comparison to RFC-ACS-derived neutrophils (LOC: 1991±492.8 vs. 1615±440.2; p=0.01; SYS: 2062±464.4 vs. 1670±525.1; p=0.0056). Ex-vivo TLR2-stimulation of local neutrophils in patients with IFC-ACS led to increased toxicity of their secretome and aggravated endothelial cell death in co-culture, as compared to neutrophils from RFC-ACS patients (+59% dead HAECs, IFC-LOC vs. RFC-LOC; p=0.0078). Furthermore, TLR2-stimulation using Pam3CSK4 triggered higher activity rates of MMP9 exclusively in local neutrophils of IFC-ACS-patients (+38.9%±6.1% in IFC-LOC vs. RFC-LOC; p=0.0154). This effect was reversed in IFC-ACS-derived neutrophils being pre-treated with an anti-TLR2 neutralizing antibody (−58.4% ±5.2%, IFC-LOC-anti-TLR2 vs. IFC-LOC-vehicle; p=0.0069). Additionally, MMP9 activity was higher in plasma obtained from the culprit site of IFC-ACS patients (74.1 U/ml ±4.1 vs. 70.0 U/ml ±5.1, IFC-LOC vs. RFC-LOC; p=0.01). Conclusion The current study demonstrates novel TLR2-dependant neutrophil activation patterns at the coronary culprit lesion of IFC-ACS, leading to higher endothelial cell toxicity and MMP9 activity. Further studies need to assess whether a temporary blockade of TLR2 activation could be a possible therapeutic target in the era of personalized medicine. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Berlin Institute of Health (BIH), German Center for Cardiovascular Research

Ponatinib induces vascular toxicity through the Notch-1 signaling pathway

Abstract Background Ponatinib, a third-generation tyrosine kinase inhibitor (TKI), is the only approved TKI that is effective against T315I mutations in patients with chronic myeloid leukemia (CML). Specific activation of Notch signaling in CML cells by ponatinib can be considered as the “on-target effect” on the tumor and represents a therapeutic approach for CML. Nevertheless, ponatinib-induced vascular toxicity remains a serious concern, with underlying mechanisms poorly understood. Aims We aimed at determining mechanisms of ponatinib-induced vascular toxicity, defining associated signaling pathways and identifying potential rescue strategies. Methods and results We exposed human umbilical endothelial cells (HUVECs) to ponatinib or vehicle in the presence or absence of the neutralizing factor anti-Notch-1 antibody for exposure times of 0–72 hours. Label-free proteomics and network analysis showed that protein cargo of HUVECs treated with ponatinib triggered apoptosis, and inhibited vasculature development (Fig. 1). We validated the proteomic data showing the inhibition of matrigel tube formation, an upregulation of cleaved caspase-3 and a downregulation of phosphorylated AKT and phosphorylated eNOS. We delineated the signaling of ponatinib-induced vascular toxicity demonstrating that ponatinib inhibits endothelial survival, reduces angiogenesis and induces endothelial senescence and apoptosis via Notch-1 pathway. Conclusion Ponatinib induced endothelial toxicity in vitro. Hyperactivation of Notch-1 in the vessels can lead to abnormal vascular development and vascular dysfunction. By hyperactivating Notch-1 in the vessels, ponatinib exerts an “on-target off turmor effect”, which leads to deleterious effects and may explain the drug's vasculotoxicity. Selective blockade of Notch-1 prevented ponatinib-induced vascular toxicity. Figure 1 Funding Acknowledgement Type of funding source: None