Neutralization of Inflammasome-Processed Cytokines Reduces Inflammatory Mechanisms and Leukocyte Recruitment in the Vasculature of TNF-α-Stimulated Sickle Cell Disease Mice

The chronic inflammatory state associated with sickle cell disease (SCD) incurs pan-cellular activation and the recruitment of leukocytes to the activated endothelium of blood vessels. The resultant rheological alterations and red cell sickling culminate in acute vaso-occlusive processes. Cytokines IL-1β and IL-18, the bio-active products of the activated inflammasome, are elevated in the plasma of SCD patients (ASH Abstract (2016) 128 (22): 854), and a previous study reported that anti-IL1β therapy alleviated reperfusion injury and flow stasis in NY1DD transgenic sickle mice exposed to hypoxia/reoxygenation (ASH Abstract (2011) 118(21): 848). The aim of this study was to determine whether antibodies that neutralize IL-1β and IL-18 could individually, or synergistically, diminish inflammatory processes and leukocyte recruitment in mice with SCD. Townes mice (5-months old; N=4-7 per group) received an i.p. administration of either saline, 200 µg/mouse anti-murine IL-1β (01BSUR), and/or 250 or 500 µg/mouse anti-murine IL-18 (SK113AE-4), or an IgG1 control antibody (iProt105125; 200 or 500 µg/mouse). At 21h after treatments, vaso-occlusive-like processes were induced in mice by the injection of tumor necrosis factor-α (TNF; 0.5μg, i.p.). At 3h after TNF, the cremaster muscles of anesthetized mice were surgically exposed, and leukocyte TNF recruitment and extravasation in venules of the microcirculation were observed using intravital microscopy. Another set of Townes mice (N=4-12 per group) was submitted to the same procedures, with blood sampling for ELISA at 3h post TNF. Optimal concentrations of antibodies were determined by observing leukocyte recruitment by intravital microscopy (doses; 100, 200 µg/mouse anti-IL-1β [N=2; 3, respectively] and 250, 500 µg/mouse anti-IL-18, [N=2 each]) in TNF-stimulated C57BL/6J mice (data not shown). Figure 1 (A-C) demonstrates the extensive recruitment and extravasation of leukocytes that occurs in the microvasculature of Townes mice at 3h post-TNF (saline group). Pre-treatment of mice with either anti-IL-1β or anti-IL-18 significantly abrogated (P<0.01) the TNF-induced adhesion (Fig. 1B) and extravasation (Fig. 1C) of leukocytes in venules, while only anti-IL-18 significantly reduced leukocyte rolling along the venule endothelium (Fig. 1A; P<0.05). In contrast, the administration of 500 µg/mouse (Fig. 1) or 200 µg/mouse (data not shown) of a non-specific IgG1 did not significantly affect TNF-induced leukocyte recruitment/extravasation (P>0.05). The combined use of the anti-IL-1β (200 µg/mouse) together with an intermediate dose of anti-IL-18 (250 µg/mouse) did not further reduce leukocyte recruitment and extravasation in this model, when compared with the effects of anti-IL-1β alone (Fig. 1A-C; P>0.05). Investigating the effects of these biological agents on inflammatory molecules production in TNF-stimulated Townes mice, we found that the administration of anti-IL-1β (200 µg) reduced IL-6 production, a pleiotropic inflammatory molecule that is upregulated by IL-1β (Fig. 1D), as did the combination of anti-IL-1β plus anti-IL-18 (200 µg and 250 µg/mouse, respectively). Pre-treatment of TNF-stimulated SCD mice with anti-IL-1β plus anti-IL-18 decreased Interferon (IFN)-γ production, a molecule that is upregulated synergistically by IL-18 and IL-12 and that mediates early host immune defenses (Fig. 1E). In contrast, anti-inflammatory IL-10 production was not significantly modulated by the pre-treatment of TNF-stimulated mice with these biological agents (Fig. 1F). As such, IL-1β and IL-18 neutralization significantly reduced inflammatory processes and leukocyte recruitment in the microvasculature of mice with SCD, indicating that biological agents that inhibit the effects of inflammasome-processed cytokines may hold potential for reducing vaso-occlusive processes in patients with this disease. Figure 1 Figure 1. Disclosures Kovarik: Novartis Institutes for Biomedical Research: Current Employment. Costa: Novartis: Consultancy. Conran: Novartis Pharma AG: Research Funding.

Abstract 1122‐000190: Persistent DWI Signal for 18 Months in Ischemic Stroke Patient with Carotid Web

Introduction : Diagnostic tools for acute ischemic infarcts include the use of DWI sequence on MRI to identify acute infarcts is especially useful since lesions can become hyperintense on this sequence very rapidly (Albers 1998). Over the next 15 days, DWI hyperintensity slowly decreases back to isointense. In some patients, however, there is persistent DWI hyperintensity past 1 month. There are theories that these persistent areas exhibit delayed onset infarct, prolonged ischemia, or perhaps different repair processes (Rivers, et al 2006). To this day, all DWI signals have been known to resolve within a few months even for persistent hyperintensities (Rivers, et al 2006). Carotid webs are a rare form of fibromuscular dysplasia that protrudes from the intimal tissues of carotid arteries. They are shelf‐like projections that grow into the lumen and disrupt normal blood flow (Zhang, et al 2018). These outgrowths are theorized to lead to ischemic strokes due to flow stasis and subsequent embolization of clots that form (Zhang, et al 2018). There is no consensus on the best management of carotid webs, and secondary prevention of recurrent strokes range from medical management to carotid stenting. Methods : This is a case report, and information for the patient was gathered through review of medical records on the EMR. Results : We present a case of ischemic stroke in the right basal ganglia/corona radiata, who presented with left sided weakness. The patient was found to have prediabetes, HTN, and HLD. However, she had recurrence of her symptoms over the next 18 months (figure 1). Repeat MRIs showed persistent DWI hyperintensity that slowly decreased in size and signal intensity over this period but in the same area as the initial infarct. The rest of the work up was only significant for a carotid web in the right internal carotid artery identified on conventional angiography. Ultimately she was managed with medical therapy including aspirin, statin, and antihypertensives. Conclusions : It is unclear whether the carotid web is associated with persistent DWI for such an extended time frame. There is very little research that explores the pathophysiology of ischemic strokes from carotid webs. In addition, there is even less information about the physiology of an evolving infarct that shows persistent DWI signals for such an extended time frame. Further studies that look into carotid webs may help us understand the best long term management in such patients. Future studies that explore the physiology of ischemic strokes that show such persistent DWI signals may elucidate and perhaps expand upon current management options and possibly identify new areas for intervention.

Methods for making animal models of retinal vein occlusion

Retinal vein occlusion (RVO) is divided into branch retinal vein occlusion and central retinal vein occlusion. It is characterized by retinal vein dilatation and tortuosity, blood flow stasis, bleeding and edema. It is often accompanied by macular edema (ME) and neovascularization. Neovascular glaucoma is the most serious complications. RVO is the second most common cause of visual loss classified under retinal vascular disorders after diabetic retinopathy. So far, the number of patients suffering from retinal vein occlusion has increased, but the pathogenesis of retinal vein occlusion has not been fully understood and there are no treatments that are very long-lasting. The research of animal models on the pathogenesis and treatment of the RVO is very important. Therefore, this article gives a briefly review to the animals and model making methods used in retinal vein occlusion experiments, and discusses the advantages and disadvantages of various RVO animal models.

Carotid webs produce greater hemodynamic disturbances than atherosclerotic disease: a DSA time–density curve study

BackgroundCarotid webs (CaWs) are associated with ischemic strokes in younger patients without degrees of stenosis that are traditionally considered clinically significant.ObjectiveTo compare the hemodynamic parameters in the internal carotid artery (ICA) bulbar segment in patients with CaW with those in patients with atherosclerotic lesions using time–density curve (TDC) analysis of digital subtraction angiography (DSA) images.MethodsWe retrospectively assessed DSA images of 47 carotid arteries in 41 adult patients who underwent ICA catheter angiography for evaluation after ischemic stroke. Hemodynamic parameters, including full width at half maximum (FWHM) and area under the time–density curve (AUC) as proxies for increased flow stasis, were calculated using TDC analyses of a region of interest (ROI) in the ICA bulb immediately rostral to the web/atherosclerotic plaque, relative to a standardized ROI in the ipsilateral distal common carotid artery (eg, relative FWHM (rFWHM)). Hemodynamic parameters were compared using non-parametric Kruskal-Wallis tests. Logistic regression was used to predict CaW versus mild/moderate atherosclerosis for each hemodynamic parameter, adjusting for degree of stenosis.ResultsMean age of patients was 56.0±13 years, with 22 (53.7%) women. 17 CaWs, 22 atherosclerotic plaques (15 mild/moderate and 7 severe), and eight normal carotid arteries were assessed. Significant between-group differences were present in the relative total AUC (p<0.001), relative AUC at wash out (p=0.031), and relative FWHM (p=0.001). Logistic regression to predict CaW versus mild/moderate atherosclerosis showed that rAUC total had the highest predictive value (pAUC=0.96, 95% CI 0.90 to 1.00), followed by rFWHM (0.87, 95% CI 0.74 to 1.00), and rAUC WO (0.74, 95% CI (0.57 to 0.91).ConclusionCaW results in larger local hemodynamic disruption, characterized by flow stasis, than mild/moderate carotid atherosclerotic lesions, suggesting that CaWs may produce larger regions of thrombogenic flow stasis.

Angiographic Predictors of Spontaneous Obliteration of Transarterial Partially Embolized Brain Arteriovenous Malformations.

Background: spontaneous obliteration of cerebral arteriovenous malformations is uncommon but could occur after partial embolization. Materials and methods:A retrospective study of 140 patients that underwent embolization for cerebral AVMs from 2005 to August 2019 using liquid embolic agents. The angiographic outcome of patients was classified as regard complete embolization, partial embolization, and complete obliteration after partial embolization. The parameters studied included size, location, number of arterial feeders, number of draining veins, rupture status, embolic agent, and patient factors as well.Results: The study patients included 74 (53%) females and 66 (47%) males. Their age ranged from 7 to 43 years old. One hundred and eight patients (77%) presented with hemorrhage. The AVM grades were grade II in 57 (40.7%) patients and grade III in 56 (39.3%) patients. Sixty-one (43.57%) patients were treated by n-Butyl Cyanoacrylate and 71(50.71%) patients were treated with Onyx, and both materials were used together in 8 cases. Follow-up angiography was done from 6 to 36 months after embolization. The rate of complete occlusion in all patients was 61.43% (86 patients). There were three groups of patients, the first group had complete occlusion of the nidus at the time of embolization and included 68 (48.57%) patients. The second group had partial embolization with partial occlusion of the nidus 54 patients (38.57%). The 3rd group included 18 patients (12.85%) with complete nidal occlusion on follow up after partial embolization. The delay in the venous drainage of the AVM to the late arterial phase or early venous phase with flow stasis was a significant predictor of future obliteration on follow up after partial embolization. Other significant parameters that were associated with the progressive disappearance of the AVM nidus on follow up after partial embolization are presentation with hemorrhage, AVMs size less than 3 cm, the presence of single draining or double draining veins, superficial venous drainage, and one or 2 arterial feeders.Conclusion: spontaneous closure of intracranial arteriovenous malformations after partial embolization may be encountered in cases of stasis of flow during embolization procedure with a delay of the venous drainage. A long-term follow-up of more cases over many years is required to confirm the validity of this conclusion.

Left Atrial Flow Stasis in Patients Undergoing Pulmonary Vein Isolation for Paroxysmal Atrial Fibrillation Using 4D-Flow Magnetic Resonance Imaging

Atrial fibrillation (AF) is associated with systemic thrombo-embolism and stroke events, which do not appear significantly reduced following successful pulmonary vein (PV) ablation. Prior studies supported that thrombus formation is associated with left atrial (LA) flow alterations, particularly flow stasis. Recently, time-resolved three-dimensional phase-contrast (4D-flow) showed the ability to quantify LA stasis. This study aims to demonstrate that LA stasis, derived from 4D-flow, is a useful biomarker of LA recovery in patients with AF. Our hypothesis is that LA recovery will be associated with a reduction in LA stasis. We recruited 148 subjects with paroxysmal AF (40 following 3–4 months PV ablation and 108 pre-PV ablation) and 24 controls (CTL). All subjects underwent a cardiac magnetic resonance imaging (MRI) exam, inclusive of 4D-flow. LA was isolated within the 4D-flow dataset to constrain stasis maps. Control mean LA stasis was lower than in the pre-ablation cohort (30 ± 12% vs. 47 ± 18%, p < 0.001). In addition, mean LA stasis was reduced in the post-ablation cohort compared with pre-ablation (36 ± 15% vs. 47 ± 18%, p = 0.002). This study demonstrated that 4D flow-derived LA stasis mapping is clinically relevant and revealed stasis changes in the LA body pre- and post-pulmonary vein ablation.

Vascular targeted photodynamic therapy: A review of the efforts towards molecular targeting of tumor vasculature

The therapeutic value of vascular targeted photodynamic therapy (VTP) for cancer has already been recognized in the clinic: TOOKAD® has been clinically approved in Europe and Israel for treatment of men with low-risk prostate cancer. When light is applied shortly after intravenous administration of the photosensitizer, the damage is primarily done to the vasculature. This results in vessel constriction, blood flow stasis, and thrombus formation. Subsequently, the tumor is killed due to oxygen and nutrient deprivation. To further increase treatment specificity and to reduce undesired side effects such as damaging to the surrounding healthy tissues, efforts have been made to selectively target the PS to the tumor vasculature, an approach named molecular targeted VTP (molVTP). Several receptors have already been explored for this approach, namely CD13, CD276, Extra domains of fibronectin (A, B), Integrin [Formula: see text]v[Formula: see text]3, Neuropilin-1, Nucleolin, PDGFR[Formula: see text], tissue factor, and VEGFR-2, which are overexpressed on tumor vasculature. Preclinical studies have shown promising results, further encouraging the investigation and future application of molVTP, to improve selectivity and efficacy of cancer treatment. This strategy will hopefully lead to even more selective treatments for many cancer patients.