Vascular Leakage Research Articles

Toxicities of CAR T-cell therapy: a review of current literature

The chimeric antigen receptor (CAR) design, first invented by Zelig Eshhar, paved the way for the use of genetically modified T-cells in targeted therapy against cancer cells. Since then, it has gone through many generations, especially with the integration of co-stimulation in the second and third-generation CARs. However, it also mounts a hyperactive immune response named as cytokine release syndrome with the release of several cytokines eventually resulting in multiple end-organ toxicities. The severity of cytokine release syndrome depends upon certain factors such as the tumor burden, choice of co-stimulation, and degree of lymphodepletion, and can manifest as pulmonary edema, vascular leak, renal dysfunction, cardiac problems, hepatic failure, and coagulopathy. Many grading criteria have been used to define these clinical manifestations but they lack harmonization. Neurotoxicity has also been significantly associated with CAR T-cell therapy but it has not been studied much in previous literature. This review aims to provide a comprehensive account of the clinical manifestations, diagnosis, management, and treatment of CAR T-cell associated neurotoxicity.

Fingolimod (FTY720), an FDA-approved sphingosine 1-phosphate (S1P) receptor agonist, restores endothelial hyperpermeability in cellular and animal models of dengue virus serotype 2 infection.

Extensive vascular leakage and shock is a major cause of dengue-associated mortality. At present, there are no specific treatments available. Sphingolipid pathway is a key player in the endothelial barrier integrity; and is mediated through the five sphingosine-1-phosphate receptors (S1PR1-S1PR5). Signaling through S1PR2 promotes barrier disruption; and in Dengue virus (DENV)-infection, there is overexpression of this receptor. Fingolimod (FTY720) is a specific agonist that targets the remaining barrier-protective S1P receptors, without targeting S1PR2. In the present study, we explored whether FTY720 treatment can alleviate DENV-induced endothelial hyperpermeability. In functional assays, in both in vitro systems and in AG129 animal models, FTY720 treatment was found effective. Upon treatment, there was complete restoration of the monolayer integrity in DENV serotype 2-infected human microvascular endothelial cells (HMEC-1). At the molecular level, the treatment reversed activation of the S1P pathway. It significantly reduced the phosphorylation of the key molecules such as PTEN, RhoA, and VE-Cadherin; and also, the expression levels of S1PR2. In DENV2-infected AG129 mice treated with FTY720, there was significant improvement in weight gain, in overall clinical symptoms, and in survival. Whereas 100% of the DENV2-infected, untreated animals died by day-10 post-infection, 70% of the FTY720-treated animals were alive; and at the end of the 15-day post-infection observation period, 30% of them were still surviving. There was a significant reduction in the Evan's-blue dye permeability in the organs of FTY720-treated, DENV-2 infected animals; and also improvement in the hemogram, with complete restoration of thrombocytopenia and hepatic function. Our results show that the FDA-approved molecule Fingolimod (FTY720) is a promising therapeutic intervention in severe dengue.

First magnetic particle imaging to assess pulmonary vascular leakage in vivo in the acutely injured and fibrotic lung.

Increased pulmonary vascular permeability is a characteristic feature of lung injury. However, there are no established methods that allow the three-dimensional visualization and quantification of pulmonary vascular permeability in vivo. Evans blue extravasation test and total protein test of bronchoalveolar lavage fluid (BALF) are permeability assays commonly used in research settings. However, they lack the ability to identify the spatial and temporal heterogeneity of endothelial barrier disruption, which is typical in lung injuries. Magnetic resonance (MR) and near-infrared (NIR) imaging have been proposed to image pulmonary permeability, but suffer from limited sensitivity and penetration depth, respectively. In this study, we report the first use of magnetic particle imaging (MPI) to assess pulmonary vascular leakage noninvasively in vivo in mice. A dextran-coated superparamagnetic iron oxide (SPIO), synomag®, was employed as the imaging tracer, and pulmonary SPIO extravasation was imaged and quantified to evaluate the vascular leakage. Animal models of acute lung injury and pulmonary fibrosis (PF) were used to validate the proposed method. MPI sensitively detected the SPIO extravasation in both acutely injured and fibrotic lungs in vivo, which was confirmed by ex vivo imaging and Prussian blue staining. Moreover, 3D MPI illustrated the spatial heterogeneity of vascular leakage, which correlated well with CT findings. Based on the in vivo 3D MPI images, we defined the SPIO extravasation index (SEI) to quantify the vascular leakage. A significant increase in SEI was observed in the injured lungs, in consistent with the results obtained via ex vivo permeability assays. Overall, our results demonstrate that 3D quantitative MPI serves as a useful tool to examine pulmonary vascular integrity in vivo, which shows promise for future clinical translation.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure

BackgroundAtrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. ObjectivesIn this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. MethodsThe authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. ResultsAF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. ConclusionsThese results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

Targeting Endothelial PERK-DLL4 Axis to Enhance Hematopoietic Stem Cell and Lymphoid Progenitor Regeneration

Despite our current information on hundreds of genes implicated in hematopoietic stem cells (HSC) biology, major gaps still exist in our understanding of HSC physiology and their regeneration mechanism under disease and stress conditions, hindering the development of strategies to enhance HSC regeneration. HSCs reside in a tightly controlled bone marrow HSC niche that regulates and maintains hematopoietic homeostasis. HSC niche is composed of a heterogeneous population of specialized stroma cells. Among these, the endothelial cells and the perivascular stroma cells provide critical pro-hematopoietic factors to support HSC pool and committed lineage progenitors particularly lymphoid progenitors. In addition, evidence suggests that endothelium-expressing Notch ligand may promote Notch-dependent long-term HSC proliferation. Recent single cell transcriptome profiling revealed that Delta-like 4 (DLL4) is expressed by the vascular endothelial cells at a higher level than other Notch ligands while its expression down-regulates in response to myeloablation. However, the molecular mechanism regulating the dynamic DLL4 expression during hematopoietic regeneration in response to myeloablative stimuli is unknown. Unfolded protein response (UPR) (also known as endoplasmic reticulum stress, ER stress) has been increasingly recognized as crucial for HSC maintenance. During hematopoietic regeneration, UPR is critical for meeting the increased demand for protein synthesis required for rapid cellular proliferation and for adapting to pathological conditions such as increased levels of reactive oxygen species following irradiation or chemotherapy. Unlike HSCs, how HSC niche endothelial cells deal with redox imbalance and higher requirements of vessel regeneration to regulate stress hematopoietic regeneration is not clear. In this study, using genetically engineered mouse models, we examined the dynamic expression and function of PERK, one arm of UPR, and its potential downstream target, Dll4, during the stress hematopoiesis triggered by transplantation. We found that PERK is activated in the mouse bone marrow endothelial cells following irradiation. Ablation of endothelial Perk in mice (referred as Perk iΔEC mice) promoted post-transplantation immediate recovery of blood cells derived from wild type donor mice and enhanced HSC and lymphoid progenitor regeneration with the expansion of B lineage progenitors including pre-proB, pro-B, and pre-B cells. In addition, by whole-mount immunostaining and intravital microscopy, we found that endothelial Perk deletion restrained disorganized angiogenesis and vascular leakage provoked by irradiation. Although irradiation itself had no effect on endothelial Dll4, Perk ablation markedly increased endothelial Dll4 expression. We found that Dll4 is essential for the post-irradiation HSC and lymphoid progenitor rejuvenation as ablation of endothelial Dll4 in mice (referred as Dll4 iΔEC mice) led to impaired short-term and long-term hematopoietic regeneration and markedly decreased wild type donor derived peripheral B cell and lymphoid progenitor recovery. We found that endothelial Dll4 is required for HSC quiescence maintenance, HSC niche retention and HSC self-renewal. Further, ablation of endothelial Dll4 led to disorganized bone marrow angiogenesis and increased marrow vascular leakage. Finally, we showed that deletion of Dll4 in Perk iΔEC mice completely abrogated the increased numbers of HSC and lymphoid progenitors mediated by Perk deletion, suggesting that up-regulation of endothelial Dll4 is likely responsible for Perk ablation-mediated enhanced HSC and lymphoid progenitor regeneration. Taken together, our results revealed a novel regulatory mechanism by which endothelial Dll4 expression is suppressed by PERK of UPR during transplantation while ablating Perk increases Dll4 expression. Endothelial DLL4 in the bone marrow endothelial cells is critical for supporting vascular integrity and myeloablation-induced HSC and lymphoid progenitor regeneration. Our findings suggest that targeting endothelial PERK-DLL4 axis could be a viable option to improve post-irradiation regeneration of HSC and lymphoid progenitors.

Survival Advantage Associated with Heterozygous Factor V Leiden Mutation in Sars-Cov-2-Infected K18-hACE2 Mice

The pathogenesis of coronavirus disease 2019 (COVID-19) complications in critically ill patients involves virus-mediated cellular damage in infected tissue and secondary organ damage associated with a hyperinflammatory response. The proinflammatory milieu can result in endothelial dysfunction, predisposing patients to thrombosis leading to a life-threatening condition known as COVID-19-associated coagulopathy (CAC) which is driven by cellular and molecular mechanisms that are still unclear. We developed a mouse model based on the K18-hACE2 transgenic mouse recapitulating many of the CAC-associated pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Plasma analysis revealed virus-induced upregulation of endothelial activation markers and alterations of all the measured coagulation factors' activities, resulting first in increased vascular leakage in the lung on day 5 post-infection, followed by the appearance of focal hemorrhagic lesions potentially associated with local thrombotic events on day 7. The infection-associated procoagulant phenotype appeared as early as day 3 post-infection (p.i.). Prothrombin time showed a 1/3 reduction in the normal clotting time in males. The overall procoagulant state was also reflected in the alteration of Factor(F) VIII and FX activity increasing to ~150% compared to uninfected controls. FIX showed the most significant alteration and was ~200% of the normal activity on day 3 p.i. and gradually increased over time, reaching ~350%. We also observed an increase in the activity of FV starting 3 days p.i. at ~210% of the normal level and persisting unchanged up to day 7 p.i. As reported during human infections, FV activity increased without significant changes in protein concentration or FVa levels. Natural anticoagulant and fibrinolytic mechanisms were dysregulated during virus infection resulting in a profound imbalance in thrombus formation and resolution. Infected K18-hACE2 mice showed downregulation of plasma protein C concentration which was not associated with any alteration of liver function, while plasma protein S concentration remained unaltered. Given the procoagulant environment generated after SARS-CoV-2 infection, reducing activated protein C (APC) anticoagulant and cytoprotective activities by blocking APC's active site with the SPC-54 monoclonal antibody worsened disease outcome and brought the mortality rate from 50% to 90%. In this observed procoagulant pathological context, where we observe increased activity of FV and where a deficiency of APC functions resulted in increased mortality, it wouldn't be surprising to observe an even more severe phenotype with the addition of APC resistance. To test this idea, we generated K18-hACE2 mice harboring the homologous human FV Leiden (FV L) mutation (R504Q). The Leiden mutation in FV eliminates one of several sites in FVa that are substrates for proteolysis by APC, resulting in diminished inactivation of FVa by APC and preventing the formation of the anticoagulant form of FV that serves as a cofactor for protein S-dependent inactivation of FVIIIa by APC. Upon SARS-CoV-2 infection, the presence of the homozygous FV L mutation in K18-hACE2 mice didn't affect the severity of the disease as measured by body weight loss during the course of the infection, and homozygous mutated mice had the same mortality rate as FV WT mice. Surprisingly, K18-hACE2 male mice carrying a heterozygous FV L mutation were extremely protected from SARS-CoV-2 infection, as their mortality dropped from 50% to only 15%. Histological analysis reveals how heterozygous FV L mice displayed a great reduction in immune infiltrate-associated fibrin deposition, suggesting an unexpected protective role of this prothrombotic mutation in the development of CAC. The protective effect of FV L heterozygosity may be attributable to enhanced thrombin formation which boosts inflammation and enhanced APC generation controlling proinflammatory and prothrombotic signaling, thus showing a delicate balance achieved by FV L heterozygosity that was lost for FV L homozygosity. As seen also in mouse models of bacterial sepsis, such favorable effects associated with FV L support the somewhat counterintuitive speculation that a partial increase in thrombin production in the early phase of the infection may support the resolution of COVID-19 complications.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

1762. Clinical application of handheld ultrasound devices during the 2022 Sudan Virus disease Ebola outbreak in Uganda

Abstract Background The fluid status and causes of respiratory failure in patients with Ebola disease can be challenging to determine due to concomitant severe diarrhea, vascular leakage, and multiorgan failure. Point-of-care ultrasound (POCUS) could guide fluid resuscitation for patients in resource-limited settings. We present the clinical application of POCUS in the 2022 Sudan Virus disease Ebola outbreak in Uganda. Figure. Ultrasound findings from patient 2. 2A: Discrete B lines observed with an irregular pleural line; 2B: Inferior vena cava collapsibility <50% suggestive of adequate fluid resuscitation; 2C: parasternal short cardiac view with small pericardial effusion identified; 2D: apical cardiac view with small pericardial effusion redemonstrated. Methods A clinical research team in Fort Portal, Uganda was trained in advanced Infection Prevention and Control (IPC) to care for patients with Ebola disease and in POCUS for septic shock. The clinical team learned a standardized lung and cardiac critical care ultrasound approach to evaluate patients in septic shock. The scanning procedure was used during the care of 6 patients with confirmed Sudan virus disease within 2 weeks of outbreak declaration to make critical patient care decisions like fluid management. Chest X-ray services and mechanical ventilation were not accessible. A handheld Philips Lumify device was kept in the biocontainment area and was used in full personal protective equipment. Scanning procedures included 12-zone lung ultrasound and 4-view cardiac ultrasound scans to determine causes of respiratory disease and evaluate fluid status. Results Among 6 patients (age range 27 to 58 years; 1/6 female), 5/6 were in their third week of illness, and 1/6 was in his second week of illness. Lung ultrasound abnormalities included discrete B-lines (5/6 patients), confluent B-lines (1/6), irregular pleural lines (3/6 patients), and subpleural consolidations (1/6 patients). Two participants in respiratory distress had cardiac POCUS. Both had hyperdynamic left ventricular function and one had a small pericardial effusion (Figure). One patient had inferior vena cava (IVC) collapsibility < 50% (1.5 cm diameter), and another with a >50% IVC collapsibility (1.3 cm diameter). These findings contributed to a decision to continue fluid resuscitation. Conclusion Portable handheld ultrasound devices identified previously undescribed ultrasound features of Sudan virus disease. POCUS helped determine causes of respiratory failure and guided fluid resuscitation in a biocontainment ward without access to mechanical ventilation. Disclosures All Authors: No reported disclosures

Leakage beyond the primary lesion: A temporal analysis of cerebrovascular dysregulation at sites of hippocampal secondary neurodegeneration following cortical photothrombotic stroke.

We have previously demonstrated that a cortical stroke causes persistent impairment of hippocampal-dependent cognitive tasks concomitant with secondary neurodegenerative processes such as amyloid-β accumulation in the hippocampus, a region remote from the primary infarct. Interestingly, there is emerging evidence suggesting that deposition of amyloid-β around cerebral vessels may lead to cerebrovascular structural changes, neurovascular dysfunction, and disruption of blood-brain barrier integrity. However, there is limited knowledge about the temporal changes of hippocampal cerebrovasculature after cortical stroke. In the current study, we aimed to characterise the spatiotemporal cerebrovascular changes after cortical stroke. This was done using the photothrombotic stroke model targeting the motor and somatosensory cortices of mice. Cerebrovascular morphology as well as the co-localisation of amyloid-β with vasculature and blood-brain barrier integrity were assessed in the cortex and hippocampal regions at 7, 28 and 84 days post-stroke. Our findings showed transient cerebrovascular remodelling in the peri-infarct area up to 28 days post-stroke. Importantly, the cerebrovascular changes were extended beyond the peri-infarct region to the ipsilateral hippocampus and were sustained out to 84 days post-stroke. When investigating vessel diameter, we showed a decrease at 84 days in the peri-infarct and CA1 regions that were exacerbated in vessels with amyloid-β deposition. Lastly, we showed sustained vascular leakage in the peri-infarct and ipsilateral hippocampus, indicative of a compromised blood-brain-barrier. Our findings indicate that hippocampal vasculature may represent an important therapeutic target to mitigate the progression of post-stroke cognitive impairment.

A ligand-independent Tie2-activating antibody reduces vascular leakage in models of Clarkson disease.

Vascular dysfunction resulting from endothelial hyperpermeability is a common and important feature of critical illness due to sepsis, trauma, and other conditions associated with acute systemic inflammation. Clarkson disease [monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS)] is a rare, orphan disorder marked by spontaneous and recurrent episodes of hypotensive shock and peripheral edema due to widespread vascular leakage in peripheral tissues. Mortality from acute flares approaches 30% due to lack of effective therapies. We evaluated a monoclonal antibody (4E2) specific for the endothelial receptor tyrosine kinase Tie2 in ISCLS models. 4E2 activated Tie2 in ISCLS patient-derived endothelial cells and reduced baseline and proinflammatory mediator-induced barrier dysfunction. 4E2 also reduced mortality and/or vascular leakage associated with systemic histamine challenge or influenza infection in the SJL/J mouse model of ISCLS. These findings support a critical role for Tie2 dysregulation in ISCLS and highlight a viable therapeutic approach to this catastrophic disorder.

Deficiency of S100A8/A9 attenuates pulmonary microvascular leakage in septic mice

BackgroundWe have reported a positive correlation between S100 calcium-binding protein (S100) A8/S100A9 and sepsis-induced lung damage before. However, limited knowledge exists concerning the biological role of S100A8/A9 in pulmonary vascular endothelial barrier dysfunction, as well as the diagnostic value of S100A8/A9 in sepsis.MethodsSepsis was induced in C57BL/6J mice and S100A9-knockout (KO) mice through the cecal ligation and puncture (CLP). Pulmonary vascular leakage was determined by measuring extravasated Evans blue (EB). Reverse transcription polymerase chain reaction and the histological score were used to evaluate inflammation and lung injury, respectively. Recombinant S100A8/A9 (rhS100A8/A9) was used to identify the effects of S100A8/A9 on endothelial barrier dysfunction in human umbilical vein endothelial cells (HUVECs). Additionally, the diagnostic value of S100A8/A9 in sepsis was assessed using receiver operating characteristic.ResultsS100A8/A9 expression was up-regulated in the lungs of CLP-operated mice. S100A9 KO significantly reversed CLP-induced hypothermia and hypotension, resulting in an improved survival rate. S100A9 KO also decreased the inflammatory response, EB leakage, and histological scores in the lungs of CLP-operated mice. Occludin and VE-cadherin expressions were decreased in the lungs of CLP-operated mice; However, S100A9 KO attenuated this decrease. Moreover, CLP-induced signal transducer and activator of transcription 3 (STAT3) and p38/extracellular signal-regulated kinase (ERK) signalling activation and apoptosis were mitigated by S100A9 KO in lungs. In addition, rhS100A8/A9 administration significantly decreased occludin and VE-cadherin expressions, increased the phosphorylated (p)-ERK/ERK, p-p38/p38, and B-cell leukaemia/lymphoma 2 protein (Bcl-2)-associated X protein/Bcl-2 ratios in HUVECs.ConclusionThe present study demonstrated S100A8/A9 aggravated sepsis-induced pulmonary inflammation, vascular permeability, and lung injury. This was achieved, at least partially, by activating the P38/STAT3/ERK signalling pathways. Moreover, S100A8/A9 showed the potential as a biomarker for sepsis diagnosis.

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

TMIC-23. UNRAVELLING THE HETEROGENEITY OF GLIOBLASTOMA'S VASCULAR LANDSCAPE

Abstract BACKGROUND Glioblastoma tumours are highly vascular and angiogenic. However, they are also heterogeneous, comprising many different macro- and microenvironments. Reflecting this, tumour-associated blood vessels are variable in their distribution, structure, and function. Despite the significance of vasculature in the development and progression of glioblastoma, its complexity remains poorly defined. Therefore, characterising vascular diversity across the glioblastoma landscape is a crucial step in the development of targeted therapies. METHODS 40 glioblastoma and 5 epilepsy cases were immunohistochemically labelled with a vascular antibody panel (PDGFRβ, ColIV, αSMA, UEA-1 and GFAP) to identify and subtype vessels. Single-vessel image analysis was performed to analyse vascular coverage and to classify vascular subtypes based on marker co-expression. Labelling with haemoglobin, ZO-1, and claudin-5 allowed for the evaluation of vascular leakage and blood-brain barrier integrity. Tissue segmentation was performed to analyse vascular coverage, vascular subtypes, and vascular integrity across the tumour macro-environment. RESULTS No significant difference in gross vascular coverage was found between tumour and epilepsy tissue. However, vascular coverage within glioblastoma varied significantly depending on the tumour macro-environment. Whilst vascular coverage in the tumour periphery was comparable to epilepsy tissue, increased vessel density was observed in the intermediate tumour zone, followed by decreased vessel density towards the tumour core. Reflecting this variation, multiple vessel subtypes, including previously described glomerular microvascular proliferation, mother vessels, vascular malformations and hyperplastic vessels, were identified. These tumour vessels display highly irregular shapes, sizes, and cellular compositions. Finally, glioblastoma tissue displayed a significant increase in haemoglobin leakage and a decrease in vascular integrity compared to epilepsy tissue. CONCLUSION Characterising this vascular landscape provides valuable insights for treatment strategies related to hypoxia and angiogenesis, two key factors driving tumour growth. Indeed, targeting specific vessel subtypes to address compromised vascular function and aberrant vascularisation may improve outcomes for glioblastoma patients.

RNA surveillance by ADAR1 is required for vascular endothelial resilience

Abstract Introduction Long dsRNAs are recognised as danger-associated molecular patterns by the cytosolic innate immune sensors inducing thus innate immune responses. RNA editing is a post-transcriptional modification induced by the binding of Adenosine Deaminases Acting on RNA (ADARs) to double-stranded RNA structures (dsRNAs). We have recently reported that ADAR1 is the main RNA editor in endothelial cells (ECs) but the role of ADAR1 in vascular homeostatic processes remains yet elusive. Methods Genetic deletion strategies were employed by crossing mice carrying a conditional (floxed) Adar1 , Ifh1 or Tlr3 allele with either a Tie2-Cre or with a tamoxifen-inducible VE-Cadherin-CreERT2 mouse line. Primary human and murine vascular EC culture assays, dsRNA metabolism studies, gene-silencing and expression techniques, immunohistochemistry and confocal microscopy were used to assess the EC-specific ADAR1 effects. Results EC-specific ADAR1 deletion resulted in prenatal embryonic lethality evidencing an absolute requirement of EC-ADAR1 in life. Inducible EC-ADAR1 ablation in adult mice elicited premature sudden death due to a widespread lung vascular leakage and pleural effusion, reflecting a compromised EC barrier function. Mechanistically, silencing of endothelial ADAR1 disrupted long-to-short dsRNA metabolism witnessed by the striking accumulation of cytoplasmic long dsRNAs. Subsequently, the cytoplasmic accumulation of endogenous long dsRNAs induced aberrant levels of interferon-β and activation of innate immune system. Finally, activation of the cytosolic dsRNA sensors inflicted the dissociation of β-catenin from VE-cadherin in EC junctions, while, co-silencing of the dsRNA sensor, MDA5, was sufficient to restore the integrity of the ADAR1-deficient endothelium. While double EC-specific ADAR1 and TLR3 did not fully rescue the lethality of the EC-ADAR1 animals, in vivo experiments using double EC-specific ADAR1 and MDA5 KO mice fully rescued the phenotype. Validation experiments using murine vascular ECs isolated from the mouse strains of our studies confirmed our mechanistic and phenotypic findings. Conclusion Endogenous suppression of double-stranded RNA-induced MDA5 autoinflammatory signaling by the RNA editor ADAR1 is essential for the maintenance of vascular integrity in adult mice.

Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice.

Dengue is a major health problem in tropical and subtropical regions. Some patients develop a severe form of dengue, called dengue hemorrhagic fever, which can be fatal. Severe dengue is associated with a transient increase in vascular permeability. A cytokine storm is thought to be the cause of the vascular leakage. Although there are various research reports on the pathogenic mechanism, the complete pathological process remains poorly understood. We previously reported that dengue virus (DENV) type 3 P12/08 strain caused a lethal systemic infection and severe vascular leakage in interferon (IFN)-α/β and γ receptor knockout mice (IFN-α/β/γRKO mice), and that blockade of TNF-α signaling protected mice. Here, we performed transcriptome analysis of liver and small intestine samples collected chronologically from P12/08-infected IFN-α/β/γRKO mice in the presence/absence of blockade of TNF-α signaling and evaluated the cytokine and effector-level events. Blockade of TNF-α signaling mainly protected the small intestine but not the liver. Infection induced the selective expansion of IL-17A-producing Vγ4 and Vγ6 T cell receptor (TCR) γδ T cells in the small intestine, and IL-17A, together with TNF-α, played a critical role in the transition to severe disease via the induction of inflammatory cytokines such as TNF-α, IL-1β, and particularly the excess production of IL-6. Infection also induced the infiltration of neutrophils, as well as neutrophil collagenase/matrix metalloprotease 8 production. Blockade of IL-17A signaling reduced mortality and suppressed the expression of most of these cytokines, including TNF-α, indicating that IL-17A and TNF-α synergistically enhance cytokine expression. Blockade of IL-17A prevented nuclear translocation of NF-κB p65 in stroma-like cells and epithelial cells in the small intestine but only partially prevented recruitment of immune cells to the small intestine. This study provides an overall picture of the pathogenesis of infection in individual mice at the cytokine and effector levels.

Blocked-flow vs. free-flow cyanoacrylate glue embolization: Histological differences in an in vivo rabbit renal artery model

PurposeThe purpose of this in vivo animal study was to compare the acute histological effects on the arterial vessel wall of free-flow vs. blocked-flow embolization with metacryloxysulfolane-n‑butyl cyanoacrylate (MS-NBCA) in several concentrations. Materials and methodsA total of 42 rabbit renal arteries were embolized using MS-NBCA mixed with ethiodized oil. The MS-NBCA concentration was 12.5%, 25%, or 50%. All mixtures were injected under both free-flow and blocked-flow conditions. The rabbits were euthanised 30 min after arterial embolization. Arterial-lumen distension, intimal inflammation and necrosis, peri‑arterial edema, and distality of MS-NBCA penetration were assessed histologically. Multivariable regression analyses were performed using a manual backward procedure, with linear, ordinal and logistic regression to search for factors associated with these outcomes ResultsMarked or severe dilatation was observed in 36 out of 42 arteries (86%) and marked or transmural intimal arteritis in all 42 arteries (42/42; 100%). Lumen dilatation caused focal vessel-wall flattening, which resulted in intimal necrosis. Multifocal necrosis extending from the intima to the media occurred in 23 out of 42 kidneys (55%) and peri‑arterial edema with multifocal vascular leakage in 19 out of 42 kidneys (45%). At multivariable analysis, blocked-flow MS-NBCA injection was associated with greater severity of vessel-wall lesions, including intimal arteritis (P = 0.003) and intimal necrosis (P = 0.014), compared to free-flow injection. Blocked-flow injection was also associated with peri‑arterial edema (P = 0.008) and greater distality of MS-NBCA penetration (P = 0.001). ConclusionBlocked-flow MS-NBCA injection during renal artery embolization is significantly associated with more acute arterial-wall damage and greater distality of glue penetration compared to free-flow injection in a rabbit model. These preliminary findings may have clinical implications, as blocked-flow injection is routinely used to treat specific vascular diseases or malformations in human.

Abstract 17142: Deficiency of Smooth Muscle ADAR1 Exacerbates Vascular Remodeling and Pulmonary Hypertension

Background: Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine (A) to inosine (I) in double-stranded RNA, which is critical to prevent auto-inflammatory responses mediated by activation of the type I interferon (IFN) signaling. Here, we define the role of ADAR1-dependent RNA editing in IFNβ activation and pulmonary artery smooth muscle cell (PASMC) remodeling in pulmonary arterial hypertension (PAH), a devastating disease leading to right heart failure and premature death. Methods: Total RNAs from IPAH PASMCs (N=3) and healthy control (N=3) were deep sequenced by RNA-seq and determined the fraction of reads with a ‘G’ nucleotide editing sites using RADAR database. A transgenic line ADAR1 SMC-KO was generated by knocking down ADAR1 selectively in αSMA+ cells, followed by 3 weeks of hypoxia. PKR inhibitor Imoxin was used to treat ADAR1 SMC-KO . Results: SMC layers of human IPAH tissues expressed reduced levels of ADAR1. Moreover, IPAH PASMCs displayed decreased mRNA and protein levels of ADAR1, along with reduced editing sites compared to healthy PASMCs (Fig.1A). The knockdown of ADAR1 in PASMCs resulted in the activation of the MDA5-PKR-IFNβ signaling pathway. Compared with controls in vivo , hypoxic ADAR1 SMC-KO mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles (Fig.1B) and increased vascular leakage resulting in elevated right ventricular systolic pressure (37 vs 23 mmHg) and right ventricular hypertrophy by Fulton Index (46 vs. control 20%) (Fig.1C). The immunofluorescence staining showed higher expression of IFNβ and increased recruitment of inflammatory immune cells. Imoxin treatment prevented PH & RVH. Conclusions: The deficiency of ADAR1 leads to the activation of IFNβ thus contributing to the pathological processes observed in PAH. Inhibiting PKR activity could attenuate the aberrant IFNβ activation and potentially serve as a new therapeutic strategy to treat PAH.

MicroRNA-10a enrichment in factor VIIa–released endothelial extracellular vesicles: potential mechanisms

BackgroundFactor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa–released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. ObjectivesTo investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. MethodsActivation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. ResultsFVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa–driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa–EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM−/− mice against lipopolysaccharide-induced inflammation and vascular leakage. ConclusionOur data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.

Effects of tumor necrosis factor-α and interleukin-1β on human retinal endothelial cells

Uveitis, or intraocular inflammation, is a potentially blinding condition that mostly affects the working-age population. The cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play a role in the pathogenesis of non-infectious uveitis and have been linked to the breakdown of the inner blood-retinal barrier, composed mainly of retinal endothelial cells, leading to macular oedema and vascular leakage. However, the effects of TNF-α and IL-1β on human retinal endothelial function are not fully understood. In this work, we investigated the impact of TNF-α and IL-1β on several aspects of human retinal endothelial cell biology. Through a real-time biosensor, the impact of TNF-α and IL-1β on formation of a retinal endothelial cell barrier was analyzed. Changes in junctional components were assessed via RT-qPCR and immunolabelling. Cell survival, necrosis and apoptosis were appraised via cell proliferation and flow cytometric studies. Tumor necrosis factor-α and IL-1β impaired the electrical resistance of the retinal endothelial cell barrier, while the addition of a potentially barrier-impairing cytokine, IL-6, did not enhance the effect of TNF-α and IL-1β. Level of the gene transcript encoding zonula occludens (ZO)-1 was diminished, while ZO-1 protein configuration was changed by TNF-α and IL-1β. Both cytokines affected human retinal endothelial cell proliferation and viability, while only TNF-α increased rates of necrosis. These results indicate that TNF-α and IL-1β are important drivers of retinal endothelial dysfunction in non-infectious uveitis, suggesting that targeting these cytokines is critical when treating complications of uveitis, such as macular oedema and vascular leakage.

Flu Induced Acute Chest Syndrome in Sickle Cell Disease Mice

Background: Sickle cell disease (SCD) affects over ~8 million people world-wide. Acute chest syndrome, a type of acute lung injury (ALI) is a major cause of mortality among SCD patients. Flu is an acute respiratory disease primarily caused by the influenza A virus (IAV), which is typically a self-resolving upper airway inflammation. However, epidemiological evidence suggests that flu can result in severe ACS in SCD patients. Although flu associates with a 50-fold higher risk of ICU admission among SCD than non-SCD humans, the etiological mechanism underlying the flu severity in SCD remains unknown. Aim: To determine whether a mild dose of IAV promotes severe lung injury in SCD mice. Methods: We have developed a model of A/PR/8/34 (H1N1) IAV induced respiratory infection in knock-in, humanized SCD mice. Mice were intranasally inoculated with a mild dose of IAV. The body weight was measured every day for three weeks after intranasal IAV administration followed by assessment of lung injury at day 10 and 14 post infection. Severity of lung injury was evaluated using histological scoring and oxygen saturation measurements. Real-time intravital ( in vivo) multi-photon-excitation fluorescence microscopy (MPE) was used to assess thrombo-inflammation and vascular leakage in the lung of mice at 10 and 14 days post IAV-infection. Results: Over the first 8 days post-IAV infection, both SCD and control mice manifested comparable levels of mild lung injury and a mild drop in body weight (15%). Unlike the control mice that started to recover, the lung injury and drop in body weight continued to worsen in SCD mice at 8-10 days post IAV infection. By day 14 post IAV infection, the lung injury was resolved and body weight was recovered in control mice, while SCD mice still manifested severe lung injury and ~20% drop in body weight. Intravital lung microscopy revealed large areas with vascular occlusion, ischemia, neutrophil infiltration and vascular leakage in the lung of SCD mice 10 days post infection, however, such pathophysiology was rarely present in the lung of control mice at 10 days post infection. Conclusions: Our current findings are the first to suggest that the inflammatory milieu in SCD mice promotes a severe non-resolving ALI following an IAV infection, which otherwise leads to a mild self-resolving lung inflammation in control mice.