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Retention of circulating lipoproteins by their interaction with extracellular matrix molecules has been suggested as an underlying mechanism for atherosclerosis. We investigated the role of glypican-4 (GPC4), a heparan sulfate (HS) proteoglycan, in the development of endothelial dysfunction and plaque progression; Expression of GPC4 and HS was investigated in human umbilical vein/artery endothelial cells (HUVECs/HUAECs) using flow cytometry, qPCR, and immunofluorescent staining. Leukocyte adhesion was determined in HUVECs in bifurcation chamber slides under dynamic flow. The association between the degree of inflammation and GPC4, HS, and syndecan-4 expressions was analyzed in human carotid plaques; GPC4 was expressed in HUVECs/HUAECs. In HUVECs, GPC4 protein expression was higher in laminar than in non-uniform shear stress regions after a 1-day or 10-day flow ( p < 0.01 each). The HS expression was higher under laminar flow after a 1 day ( p < 0.001). Monocytic THP-1 cell adhesion to HUVECs was facilitated by GPC4 knock-down ( p < 0.001) without affecting adhesion molecule expression. GPC4 and HS expression was lower in more-inflamed than in less-inflamed plaque shoulders ( p < 0.05, each), especially in vulnerable plaque sections; Reduced expression of GPC4 was associated with atherogenic conditions, suggesting the involvement of GPC4 in both early and advanced stages of atherosclerosis.

Atherosclerotic lesions preferentially develop at bifurcations, characterized by non-uniform shear stress (SS). The aim of this study was to investigate SS-induced endothelial activation, focusing on stress-regulated mitogen-activated protein kinases (MAPK) and downstream signaling, and its relation to gap junction proteins, Connexins (Cxs). Human umbilical vein endothelial cells were exposed to flow ("mechanical stimulation") and stimulated with TNF-α ("inflammatory stimulation"). Phosphorylated levels of MAPKs (c-Jun N-terminal kinase (JNK1/2), extracellular signal-regulated kinase (ERK), and p38 kinase (p38K)) were quantified by flow cytometry, showing the activation of JNK1/2 and ERK. THP-1 cell adhesion under non-uniform SS was suppressed by the inhibition of JNK1/2, not of ERK. Immunofluorescence staining and quantitative real-time PCR demonstrated an induction of c-Jun and c-Fos and of Cx43 in endothelial cells by non-uniform SS, and the latter was abolished by JNK1/2 inhibition. Furthermore, plaque inflammation was analyzed in human carotid plaques ( n = 40) using immunohistochemistry and quanti-gene RNA-assays, revealing elevated Cx43 + cell counts in vulnerable compared to stable plaques. Cx43 + cell burden in the plaque shoulder correlated with intraplaque neovascularization and lipid core size, while an inverse correlation was observed with fibrous cap thickness. Our results constitute the first report that JNK1/2 mediates Cx43 mechanoinduction in endothelial cells by atheroprone shear stress and that Cx43 is expressed in human carotid plaques. The correlation of Cx43 + cell counts with markers of plaque vulnerability implies its contribution to plaque progression.

To facilitate the recovery process of chronic and hard-to-heal wounds novel pro-resolving treatment options are urgently needed. We investigated the pro-regenerative properties of soluble CD83 (sCD83) on cutaneous wound healing, where sCD83 accelerated wound healing not only after systemic but also after topical application, which is of high therapeutic interest. Cytokine profile analyses revealed an initial upregulation of inflammatory mediators such as TNFα and IL-1β, followed by a switch towards pro-resolving factors, including YM-1 and IL-10, both expressed by tissue repair macrophages. These cells are known to mediate resolution of inflammation and stimulate wound healing processes by secretion of growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), which promote vascularization as well as fibroblast and keratinocyte differentiation. In conclusion, we have found strong wound healing capacities of sCD83 beyond the previously described role in transplantation and autoimmunity. This makes sCD83 a promising candidate for the treatment of chronic- and hard-to-heal wounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Royzman, Peckert-Maier, Stich, König, Wild, Tauchi, Ostalecki, Kiesewetter, Seyferth, Lee, Eming, Fuchs, Kunz, Stürmer, Peters, Berking, Zinser and Steinkasserer.)

Background: Bispectral index (BIS) monitoring is a widely used non-invasive method to monitor the depth of anesthesia. However, in the event of surgeries requiring a frontal approach, placement of the electrode may be impossible at the designated area to achieve a proper BIS measurement., Methods: We developed an investigational interface device to connect needle-electrodes to BIS sensors. The safety and clinical performance were investigated in patients who underwent surgery. Direct BIS values from a disposable BIS electrode and indirect values via the interface device were simultaneously recorded from the same areas of electrode placement in a single patient. The agreement between the direct and indirect BIS values was statistically analyzed., Results: The interface device with a silver electrode demonstrated sufficient electric conduction to transmit electroencephalogram signals. The overall BIS curves were similar to those of direct BIS monitoring. Direct and indirect BIS values from 18 patients were statistically analyzed using a linear mixed model and a significant concordance was confirmed (indirect BIS = 7.0405 + 0.8286 * direct BIS, p<0.0001). Most observed data (2582/2787 data points, 92.64%) had BIS unit differences of 10 or less., Conclusions: The interface device provides an opportunity for intraoperative BIS monitoring of patients, whose clinical situation does not permit the placement of conventional adhesive sensors at the standard location., Competing Interests: The authors have declared that no competing interests exist.

In the 1900s, researchers established animal models experimentally to induce atherosclerosis by feeding them with a cholesterol-rich diet. It is now accepted that high circulating cholesterol is one of the main causes of atherosclerosis; however, plaque localization cannot be explained solely by hyperlipidemia. A tremendous amount of studies has demonstrated that hemodynamic forces modify endothelial athero-susceptibility phenotypes. Endothelial cells possess mechanosensors on the apical surface to detect a blood stream-induced force on the vessel wall, known as "wall shear stress (WSS)", and induce cellular and molecular responses. Investigations to elucidate the mechanisms of this process are on-going: on the one hand, hemodynamics in complex vessel systems have been described in detail, owing to the recent progress in imaging and computational techniques. On the other hand, investigations using unique in vitro chamber systems with various flow applications have enhanced the understanding of WSS-induced changes in endothelial cell function and the involvement of the glycocalyx, the apical surface layer of endothelial cells, in this process. In the clinical setting, attempts have been made to measure WSS and/or glycocalyx degradation non-invasively, for the purpose of their diagnostic utilization. An increasing body of evidence shows that WSS, as well as serum glycocalyx components, can serve as a predicting factor for atherosclerosis development and, most importantly, for the rupture of plaques in patients with high risk of coronary heart disease.

Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ-strand branching points and contribute to the maintenance of epithelial barrier integrity.

Background and Aims: Oxidation of low-density lipoprotein (LDL) and oxidized LDL-mediated activation of the innate immune system have been recognized as early key events during the pathogenesis of atherosclerosis. Recent evidence identified eosinophils as a major source of enzymatic lipid oxidation and suggested a potential role of type 2 immunity in atherogenesis. However, the involvement of individual type 2 immune cell subsets involved in this process has been incompletely defined. We therefore sought to determine the role of eosinophils during LDL oxidation and the pathogenesis of this disease., Methods: Using eosinophil-deficient dblGATA1 mice, we studied the role of eosinophils in two established mouse models of atherosclerosis., Results: These experiments revealed that the presence of eosinophils did neither affect biomarkers of LDL oxidation nor atherosclerotic lesion development., Conclusions: The obtained results show that LDL oxidation and development of atherosclerosis are largely independent of eosinophils or eosinophil-mediated LDL oxidation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Targeted temperature management, or therapeutic hypothermia, is a potent neuroprotective approach after ischemic brain injury. Hypothermia should be induced as soon as possible after the onset of acute stroke to assure better outcomes. Accordingly, drugs with a fast-acting hypothermic effect sustainable through the period of emergency transportation to hospital would have clinical advantages. Activation of the transient receptor potential vanilloid-1 (TRPV1) can induce hypothermia. Our immunohistochemical investigations confirmed that TRPV1 was distributed to perivascular and periventricular regions of the rat brain, where TRPV1 can be easily detected by TRPV1 agonists. An endogenous TRPV1 selective agonist, N -oleoyldopamine (OLDA), and a synthetic antagonist, AMG 9810, were injected intraperitoneally into healthy adult male Wister rats, and brain and core temperatures and gross motor activities were monitored. Comparison with baseline temperatures showed that TRPV1 injection immediately induced mild hypothermia ( p  < 0.05 in brain and p  < 0.01 in body), and AMG 9810 induced immediate mild hyperthermia (not significant). However, the OLDA-induced hypothermia did not decrease lesion volume after middle carotid artery occlusion in rats. Relative to vehicle, OLDA yielded poorer outcomes and AMG 9810 yielded better outcomes in neurological scores and lesion size. Our study showed that, as an agonist of TRPV1, OLDA has suitable hypothermia-inducing properties, but did not decrease lesion volume. Therefore, the search for novel TRPV1 agonists and/or antagonists providing hypothermia and neuroprotection should continue. Further investigations should also target OLDA-induced transient hypothermia combined with long-term hypothermia maintenance with surface cooling, which mimics the anticipated clinical use of this class of drug.

Single nucleotide polymorphisms (SNPs) in vascular endothelial growth factor receptor 2 (VEGFR2) are associated with coronary artery disease, hypertension and myocardial infarction. However, their association with atherosclerosis remains to be fully elucidated. The purpose of the present study was to determine whether SNPs are involved in atherogenesis, by analyzing their impact on human umbilical vein endothelial cells (HUVECs) under laminar and non‑uniform shear stress in a well‑established in vitro model that simulates shear stress‑induced proatherogenic processes at vessel bifurcations. All experiments were performed using freshly isolated HUVECs. Three SNPs in the VEGFR2 gene (rs1870377 T>A, rs2071559 A>G and rs2305948 C>T) were genotyped and the expression levels of VEGFR2 were semi‑quantitatively determined using western blotting. Subsequently, the HUVECs were seeded in bifurcating flow‑through cell culture slides and flow (9.6 ml/min) was applied for 19 h, including tumor necrosis factor‑α stimulation during the final 2 h of flow. The protein expression levels of VCAM‑1, E‑selectin and VEGFR2 and the adhesion of THP‑1 cells were analyzed in laminar and non‑uniform shear stress regions. Data were analyzed for associations with the respective SNPs. The total expression of VEGFR2 was significantly lower under non‑uniform shear stress than under laminar shear stress conditions, independent of the genotype. The expression of VEGFR2 between the different shear stress patterns was not significantly altered by the different SNPs. The expression levels of VCAM‑1 and E‑selectin were lower in the A/A genotype compared with those in other genotypes in rs1870377 T>A and rs2071559 A>G. In conclusion, the results suggested that SNPs within the VEGFR2 gene have a significant impact on shear stress‑related endothelial activation.

Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) in the neonatal intensive care unit (NICU) have been reported worldwide. Some outbreaks were caused by USA300, which is a community-associated MRSA clone. In 2011, polymerase chain reaction-based open reading frame typing (POT) for the initial MRSA isolates from all inpatients was started at the Tokyo Metropolitan Children's Medical Center. From March 2014 to April 2015, a total of 131 MRSA strains were isolated, 104 of which were analyzed as healthcare-associated MRSA. Thirteen stains (12.5%) had a POT number of 106-9-93, which strongly suggested USA300; these included 6 from nasal swabs, 6 from blood cultures and 1 from subcutaneous pus. All the MRSA strains were isolated from patients in the NICU; were typed as sequence type 8, spa type t008, and staphylococcal cassette chromosome type mec IVa; and possessed the lukS-lukF and arginine catabolic mobile element-arcA gene. Pulsed-field gel electrophoresis of all the strains, with USA300-0114 as a reference, showed indistinguishable banding pattern. Based on these results, POT was useful in recognizing this first MRSA outbreak of USA300 in a Japanese NICU and was advantageous in terms of swiftness, less cost and monitoring change of the epidemic MRSA lineage., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

A type 1 immune response is involved in atherosclerosis progression, whereas the role of a type 2 polarization, especially with regard to an enhanced T helper (T h )2 cell differentiation, is still unclear. Helminths trigger type 2 immune responses, protecting the host from inflammatory disorders. We investigated whether an increased type 2 polarization by administration of Litomosoides sigmodontis adult worm extract (LsAg) affects atherosclerosis in apolipoprotein E-deficient (ApoE -/- ) mice. Injections of 50 µg LsAg, i.p. into ApoE -/- mice induced a type 2 immune response shown by increased frequencies of peritoneal eosinophils and alternatively activated macrophages. To analyze the effect of LsAg on atherosclerosis initiation, ApoE -/- mice received a high-fat diet for 12 wk and weekly injections of 50 µg LsAg from wk 5 to 12. Therapeutic effects on advanced atherosclerosis were analyzed in mice that were fed a high-fat diet for 12 wk followed by 12 wk of normal chow and weekly LsAg injections. Both preventive and therapeutic LsAg application significantly decreased plaque size. Therapeutic treatment even caused regression of plaque size and macrophage density in the aortic root and reduced T h 1-specific gene expression and intraplaque inflammation. In addition, plaque size after therapeutic treatment was inversely correlated with plaque-infiltrated alternatively activated macrophages. In vitro , LsAg treatment of HUVECs reduced intracellular levels of phosphorylated NF-κB-p65, IκB-α, and JNK1/2. In bifurcation flow-through slides, THP-1 cell adhesion to a HUVEC monolayer was decreased by LsAg in regions of nonuniform shear stress. Applying inhibitors of the respective kinases suggests JNK1/2 inhibition is involved in the suppressed cell adhesion. A switch to an enhanced type 2 immune response by LsAg exerts antiatherogenic effects on murine plaque development, indicating a protective role of a hampered type 1 polarization. In vitro , LsAg affects endothelial signaling pathways, among which JNK1/2 inhibition seems to be involved in the suppression of monocytic cell adhesion under proatherogenic shear stress.-Constanze, K., Tauchi, M., Furtmair, R., Urschel, K., Raaz-Schrauder, D., Neumann, A.-L., Frohberger, S. J., Hoerauf, A., Regus, S., Lang, W., Sagban, T. A., Stumpfe, F. M., Achenbach, S., Hübner, M. P., Dietel, B. Filarial extract of Litomosoides sigmodontis induces a type 2 immune response and attenuates plaque development in hyperlipidemic ApoE-knockout mice.

Targeted temperature management (TTM), or therapeutic hypothermia, is one of the most potent neuroprotective approaches after ischemic and traumatic brain injuries. TTM has been applied clinically with various methods, but effective achievement and maintenance of the target temperature remain challenging. Furthermore, timing of cooling and target body and brain temperature to optimize effectiveness for neuroprotection and to minimize side effects are yet to be standardized. Focal brain cooling is a potential strategy to minimize adverse effects of systemic TTM. In this study, we report on a focal brain cooling device for animals and its effectiveness of focal cooling in several animal models of ischemic cerebral stroke. A focal brain cooling device was constructed using a Peltier's element, a thermoelectric heat pump. The device was validated for its cooling ability, and optimal settings to induce an effective intracranial temperature were determined using male Sprague-Dawley rats. Transient and permanent middle cerebral artery occlusions were experimentally induced, and focal brain cooling was applied using the device varying the timing and duration of cooling. The stroke-induced infarct and edema volumes were evaluated from Nissl-stained cryosections. The focal brain cooling device was able to decrease and subsequently maintained cerebral hypothermia in free-moving rats without altering the core temperature. The device with validated intracranial temperatures produced neuroprotective effects in the acute phase of ischemic neural death, reperfusion injury, progressing damage to the penumbra, and edema formation. In conclusion, our validated focal cooling device enabled rapid and accurate cerebral TTM in rats. Using this device, we were able to test the neuroprotective effect of focal TTM in several pathological stages of cerebral ischemia, which warrants further studies to develop clinically feasible TTM procedures for patients with cerebral stroke.