The blood-nerve barrier (BNB) is a dynamic and competent interface between the endoneurial microenvironment and the surrounding extracellular space or blood. It is localised at the innermost layer of ...the multilayered ensheathing perineurium and endoneurial microvessels, and is the key structure that controls the internal milieu of the peripheral nerve parenchyma. Since the endoneurial BNB is the point of entry for pathogenic T cells and various soluble factors, including cytokines, chemokines and immunoglobulins, understanding this structure is important to prevent and treat human immune mediated neuropathies such as Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein and skin changes) syndrome and a subset of diabetic neuropathy. However, compared with the blood-brain barrier, only limited knowledge has been accumulated regarding the function, cell biology and clinical significance of the BNB. This review describes the basic structure and functions of the endoneurial BNB, provides an update of the biology of the cells comprising the BNB, and highlights the pathology and pathomechanisms of BNB breakdown in immune mediated neuropathies. The human immortalised cell lines of BNB origin established in our laboratory will facilitate the future development of BNB research. Potential therapeutic strategies for immune mediated neuropathies manipulating the BNB are also discussed.
In diabetic peripheral neuropathy (DPN), metabolic disorder by hyperglycemia progresses in peripheral nerves. In addition to the direct damage to peripheral neural axons, the homeostatic mechanism of ...peripheral nerves is disrupted by dysfunction of the blood-nerve barrier (BNB) and Schwann cells. The disruption of the BNB, which is a crucial factor in DPN development and exacerbation, causes axonal degeneration via various pathways. Although many reports revealed that hyperglycemia and other important factors, such as dyslipidemia-induced dysfunction of Schwann cells, contributed to DPN, the molecular mechanisms underlying BNB disruption have not been sufficiently elucidated, mainly because of the lack of in vitro studies owing to difficulties in establishing human cell lines from vascular endothelial cells and pericytes that form the BNB. We have developed, for the first time, temperature-sensitive immortalized cell lines of vascular endothelial cells and pericytes originating from the BNB of human sciatic nerves, and we have elucidated the disruption to the BNB mainly in response to advanced glycation end products in DPN. Recently, we succeeded in developing an in vitro BNB model to reflect the anatomical characteristics of the BNB using cell sheet engineering, and we established immortalized cell lines originating from the human BNB. In this article, we review the pathologic evidence of the pathology of DPN in terms of BNB disruption, and we introduce the current in vitro BNB models.
Background:The randomized controlled VOLCANO trial demonstrated comparable 1-year rhythm outcomes between patients with and without ablation targeting low-voltage areas (LVAs) in addition to ...pulmonary vein isolation among paroxysmal atrial fibrillation (PAF) patients with LVAsMethods and Results:An extended-follow-up study of 402 patients enrolled in the VOLCANO trial with PAF, divided into 4 groups based on the results of voltage mapping: group A, no LVA (n=336); group B, LVA ablation (n=30); group C, LVA without ablation (n=32); and group D, incomplete voltage map (n=4). At 25 (23, 31) months after the initial ablation, AF/atrial tachycardia (AT) recurrence rates were 19% in group A, 57% in group B, 59% in group C, and 100% in group D. Recurrence rates were higher in patients with LVAs than in those without (group A vs. B+C, P<0.0001), and were comparable between those with and without LVA ablation (group B vs. C, P=0.83). Among patients who underwent repeat ablation, ATs were more frequently observed in patients with LVAs (Group B+C, 50% vs. A, 14%, P<0.0001). In addition, LVA ablation increased the incidence of AT development (group B, 71% vs. C, 32%, P<0.0001).Conclusions:Patients with LVAs demonstrated poor long-term rhythm outcomes irrespective of LVA ablation. ATs were frequently observed in patients with LVAs, and LVA ablation might exacerbate the occurrence of iatrogenic ATs.
Background:The efficacy of ablation targeting low-voltage areas (LVAs) is controversial, although LVA presence is well known to be associated with atrial fibrillation (AF) recurrence after ablation. ...AF substrate may not localize within LVAs.Methods and Results:This observational study enrolled 405 consecutive patients who underwent an initial AF ablation procedure. The left atrial (LA) voltage map was obtained after pulmonary vein isolation. LVAs were defined as areas with voltage <0.5 mV. To estimate whole LA electrophysiological degeneration, mean regional voltage at each of the 6 regions and LA total conduction velocity were measured. LVAs existed in 143 of 405 (35.3%) patients. Patients with LVAs demonstrated lower mean regional voltages throughout all 6 regions compared to those without LVAs (1.3 1.8, 0.8 vs. 0.6 1.0, 0.2 mV for the anterior wall, P<0.001). In contrast, LA conduction velocity was lower in patients with LVAs than in those without (0.89 1.01, 0.74 vs. 0.93 1.03, 0.87 m/s, P<0.001). Multivariate analysis revealed that low LA total conduction velocity and a higher number of regions with mean voltage reduction were independently associated with AF recurrence, although LVA presence was not.Conclusions:Patients with localized LA LVAs were characterized by whole LA electrophysiological degeneration as assessed by mean regional voltage and conduction velocity. In addition, whole LA electrophysiological degeneration parameters were well associated with AF recurrence.
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Sphingosine 1 phosphate (S1P) is a bioactive sphingolipid that exerts several functions in physiological and pathological conditions. The modulation of one of its receptors, S1P1, ...plays an important role in the egress of lymphocytes from lymph nodes and is a useful target in multiple sclerosis (MS) treatment. A new drug, siponimod (BAF-312) has been recently approved for the treatment of secondary progressive MS and has affinity for two S1P receptors, S1P1 and S1P5. The two receptors are expressed by endothelial cells that, as components of the blood–brain barrier (BBB), prevent the access of solutes and lymphocytes into the central nervous system, function often compromised in MS. Using an in vitro BBB model exposed to inflammatory cytokines (TNFα and IFNγ, 5 UI and 10 UI respectively), we evaluated the effects of BAF-312 (100 nM) on expression and function of endothelial tight junctional proteins (Zo-1 and claudin-5), regulation of transendothelial electrical resistance (TEER) and permeability to FITC-conjugated dextran. Zo-1 expression, as well as TEER values, were promptly recovered (24 h) when both S1P1 and S1P5 were activated by BAF-312. In contrast, at this time point, activation of S1P5 with the selective agonist UC-42-WP04 (300 nM) or with BAF-312, under blockade of S1P1 with the selective antagonist NIBR-0213 (1 μM), resulted in recovery of expression and localization of claudin-5 and reduction of TNFα/INFγ-induced expression of metalloproteinase 9. Only after a prolonged BAF-312 exposure (48 h), S1P1 was involved through activation of the PI3K/Akt pathway. The PI3K inhibitor LY294002 (10 µM) prevented in fact the effects of BAF-312 on all the parameters examined. In conclusion, BAF-312, by modulating both S1P1 and S1P5, may strengthen BBB properties, thus providing additional effects in the treatment of MS.
Background:Although patients with poor ability to perform activities of daily living, such as those with high Clinical Frailty Score (CFS), will often receive a cardiac implantable electric device ...(CIED), the indications for implantation in these patients have not been clearly defined. We investigated the association between CFS and prognosis in patients with a CIED.Methods and Results:We retrospectively enrolled 323 consecutive patients who underwent initial device implantation (age, 77 (70–83) years; male, 181 56% patients; high-voltage device, 49 15% patients), and the CFS was retrospectively estimated. Primary outcome was all-cause death, and the secondary outcome was hospitalization due to heart failure (HF). Median CFS was 4 (3–5) points. During 2 years’ follow-up, all-cause death occurred in 32 patients (10%). Freedom from all-cause death was significantly lower in patients with a high CFS than in those with a low score (1–2 points: 100%, 3–4 points: 92.9%, 5–9 points: 77.3%, P<0.01). After adjustment for age and sex, the CFS was an independent predictor of the primary outcome (hazard ratio HR 2.0, 95% confidence interval CI 1.6–2.5, P<0.01), and of the secondary outcome (HR 1.6 95% CI 1.2–2.0, P<0.01).Conclusions:The CFS is an independent predictor of both death and hospitalization due to HF in patients with a CIED.
Background:The purpose of this study was to elucidate the effect of the temporal relationship between atrial fibrillation (AF) and heart failure (HF) on clinical outcomes after catheter ...ablation.Methods and Results:We included 129 consecutive patients with AF and HF who underwent catheter ablation in hospital from December 2014 to September 2017. The patients were divided into 2 groups based on the temporal relationship between AF and HF. Group 1 consisted of 42 patients with AF following HF while Group 2 consisted of 87 patients with AF preceding HF or those who developed both of them simultaneously at the timing of first visit to a doctor. The primary endpoint was a composite of death and hospitalization due to HF during a 2-year follow-up. AF recurrence was more common in Group 1 (45% vs. 23%; hazard ratio HR, 2.49; 95% confidence interval CI, 1.25–4.94; P=0.009). Death and HF hospitalization were more frequent in Group 1 (19 45%, 6 7% patients, respectively, P<0.0001). After adjustment for several covariates, patients in Group 1 were independently associated with poorer outcomes after AF ablation (HR, 8.66; 95% CI, 2.942–5.5; P<0.0001).Conclusions:Adverse clinical outcomes of death, HF hospitalization and AF recurrence were more frequent in patients with AF following HF than in those with AF preceding HF.
Receptor-mediated transcytosis is one of the major routes for drug delivery of large molecules into the brain. The aim of this study was to develop a novel model of the human blood-brain barrier ...(BBB) in a high-throughput microfluidic device. This model can be used to assess passage of large biopharmaceuticals, such as therapeutic antibodies, across the BBB.
The model comprises human cell lines of brain endothelial cells, astrocytes, and pericytes in a two-lane or three-lane microfluidic platform that harbors 96 or 40 chips, respectively, in a 384-well plate format. In each chip, a perfused vessel of brain endothelial cells was grown against an extracellular matrix gel, which was patterned by means of surface tension techniques. Astrocytes and pericytes were added on the other side of the gel to complete the BBB on-a-chip model. Barrier function of the model was studied using fluorescent barrier integrity assays. To test antibody transcytosis, the lumen of the model's endothelial vessel was perfused with an anti-transferrin receptor antibody or with a control antibody. The levels of antibody that penetrated to the basal compartment were quantified using a mesoscale discovery assay.
The perfused BBB on-a-chip model shows presence of adherens and tight junctions and severely limits the passage of a 20 kDa FITC-dextran dye. Penetration of the antibody targeting the human transferrin receptor (MEM-189) was markedly higher than penetration of the control antibody (apparent permeability of 2.9 × 10
versus 1.6 × 10
cm/min, respectively).
We demonstrate successful integration of a human BBB microfluidic model in a high-throughput plate-based format that can be used for drug screening purposes. This in vitro model shows sufficient barrier function to study the passage of large molecules and is sensitive to differences in antibody penetration, which could support discovery and engineering of BBB-shuttle technologies.
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