Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by the Helicobacter pylori type IV secretion system.

The bacterial human pathogen Helicobacter pylori produces a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. The cagT4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shape N-terminal domains (CagIN) prolonged by globular C-terminal domains (CagIC). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagIC with subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagIC were found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagIC plays a key role in cagT4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of the cagT4SS, a crucial risk factor for gastric cancer development.

Extracellular vesicle species differentially affect endothelial cell functions and differentially respond to exercise training in patients with chronic coronary syndromes.

BACKGROUND: Extracellular vesicles are released upon cellular activation and mediate inter-cellular communication. Individual species of extracellular vesicles might have divergent roles in vascular homeostasis and may show different responses to therapies such as exercise training. AIMS: We examine endothelial effects of medium-size and small extracellular vesicles from the same individual with or without chronic coronary syndrome, and in chronic coronary syndrome patients participating in a four-week high-intensity interval training intervention. METHODS: Human aortic endothelial cells were exposed to medium-size extracellular vesicles and small extracellular vesicles isolated from plasma samples of study participants. Endothelial cell survival, activation and re-endothelialisation capacity were assessed by respective staining protocols. Extracellular vesicles were quantified by nanoparticle tracking analysis and flow cytometry. Extracellular vesicle microRNA expression was quantified by realtime-quantitative polymerase chain reaction. RESULTS: In patients with chronic coronary syndrome (n = 25), plasma counts of leukocyte-derived medium-size extracellular vesicles were higher than in age-matched healthy controls (n = 25; p = 0.04) and were reduced by high-intensity interval training (n = 15; p = 0.01 vs baseline). Re-endothelialisation capacity was promoted by medium-size extracellular vesicles from controls, but not by medium-size extracellular vesicles from chronic coronary syndrome patients. High-intensity interval training for 4 weeks enhanced medium-size extracellular vesicle-mediated support of in vitro re-endothelialisation. Small extracellular vesicles from controls or chronic coronary syndrome patients increased endothelial cell death and reduced repair functions and were not affected by high-intensity interval training. CONCLUSION: The present study demonstrates that medium-size extracellular vesicles and small extracellular vesicles differentially affect endothelial cell survival and repair responses. This equilibrium is unbalanced in patients with chronic coronary syndrome where leukocyte-derived medium-size extracellular vesicles are increased leading to a loss of medium-size extracellular vesicle-mediated endothelial repair. High-intensity interval training partially restored medium-size extracellular vesicle-mediated endothelial repair, underlining its use in cardiovascular prevention and therapy to improve endothelial function.

AP-1 (Activated Protein-1) Transcription Factor JunD Regulates Ischemia/Reperfusion Brain Damage via IL-1ß (Interleukin-1ß).

Background and Purpose- Inflammation is a major pathogenic component of ischemia/reperfusion brain injury, and as such, interventions aimed at inhibiting inflammatory mediators promise to be effective strategies in stroke therapy. JunD-a member of the AP-1 (activated protein-1) family of transcription factors-was recently shown to regulate inflammation by targeting IL (interleukin)-1ß synthesis and macrophage activation. The purpose of the present study was to assess the role of JunD in ischemia/reperfusion-induced brain injury. Methods- WT (wild type) mice randomly treated with either JunD or scramble (control) siRNA were subjected to 45 minutes of transient middle cerebral artery occlusion followed by 24 hours of reperfusion. Stroke size, neurological deficit, plasma/brain cytokines, and oxidative stress determined by 4-hydroxynonenal immunofluorescence staining were evaluated 24 hours after reperfusion. Additionally, the role of IL-1ß was investigated by treating JunD siRNA mice with an anti-IL-1ß monoclonal antibody on reperfusion. Finally, JunD expression was assessed in peripheral blood monocytes isolated from patients with acute ischemic stroke. Results- In vivo JunD knockdown resulted in increased stroke size, reduced neurological function, and increased systemic inflammation, as confirmed by higher neutrophil count and lymphopenia. Brain tissue IL-1ß levels were augmented in JunD siRNA mice as compared with scramble siRNA, whereas no difference was detected in IL-6, TNF-α (tumor necrosis factor-α), and 4-hydroxynonenal levels. The deleterious effects of silencing of JunD were rescued by treating mice with an anti-IL-1ß antibody. In addition, JunD expression was decreased in peripheral blood monocytes of patients with acute ischemic stroke at 6 and 24 hours after onset of stroke symptoms compared with sex- and age-matched healthy controls. Conclusions- JunD blunts ischemia/reperfusion-induced brain injury via suppression of IL-1ß.

Deleterious role of endothelial lectin-like oxidized low-density lipoprotein receptor-1 in ischaemia/reperfusion cerebral injury.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is implicated in cardiovascular disease by modulating apoptosis and oxidative stress. We hypothesized that LOX-1 may be involved in pathophysiology of stroke by mediating ischaemia/reperfusion (I/R)-dependent cell death. Transient middle cerebral artery occlusion (tMCAO) was performed in wild-type (WT) mice, endothelial-specific LOX-1 transgenic mice (eLOX-1TG) and WT animals treated with LOX-1 silencing RNA (siRNA). In WT mice exposed to tMCAO, LOX-1 expression and function were increased in the MCA. Compared to WT animals, eLOX-1TG mice displayed increased stroke volumes and worsened outcome after I/R. Conversely, LOX-1-silencing decreased both stroke volume and neurological impairment. Similarly, in HBMVECs, hypoxia/reoxygenation increased LOX-1 expression, while LOX-1 overexpressing cells showed increased death following hypoxia reoxygenation. Increased caspase-3 activation was observed following LOX-1 overexpression both in vivo and in vitro, thus representing a likely mediator. Finally, monocytes from ischaemic stroke patients exhibited increased LOX-1 expression which also correlated with disease severity. Our data unequivocally demonstrate a key role for LOX-1 in determining outcome following I/R brain damage. Our findings could be corroborated in human brain endothelial cells and monocytes from patients, underscoring their translational relevance and suggesting siRNA-mediated LOX-1 knockdown as a novel therapeutic strategy for stroke patients.

Sirtuin 5 as a novel target to blunt blood-brain barrier damage induced by cerebral ischemia/reperfusion injury.

BACKGROUND: In acute ischemic stroke (AIS) patients, impaired blood-brain barrier (BBB) integrity is associated with hemorrhagic transformation and worsened outcome. Yet, the mechanisms underlying these relationships are poorly understood and consequently therapeutic strategies are lacking. This study sought to determine whether SIRT5 contributes to BBB damage following I/R brain injury. METHODS AND RESULTS: SIRT5 knockout (SIRT5-/-) and wild type (WT) mice underwent transient middle cerebral artery (MCA) occlusion (tMCAO) followed by 48h of reperfusion. Genetic deletion of SIRT5 decreased infarct size, improved neurological function and blunted systemic inflammation following stroke. Similar effects were also achieved by in vivo SIRT5 silencing. Immunohistochemical analysis revealed decreased BBB leakage and degradation of the tight junction protein occludin in SIRT5-/- mice exposed to tMCAO as compared to WT. In primary human brain microvascular endothelial cells (HBMVECs) exposed to hypoxia/reoxygenation (H/R), SIRT5 silencing decreased endothelial permeability and upregulated occludin and claudin-5; this effect was prevented by the PI3K inhibitor wortmannin. Lastly, SIRT5 gene expression was increased in peripheral blood monocytes (PBMCs) of AIS patients at 6h after onset of stroke compared to sex- and age-matched healthy controls. CONCLUSION: SIRT5 is upregulated in PBMCs of AIS patients and in the MCA of WT mice exposed to tMCAO; SIRT5 mediates I/R-induced brain damage by increasing BBB permeability through degradation of occludin. This effect was reproduced in HBMVECs exposed to H/R, mediated by the PI3K/Akt pathway. Our findings shed new light on the mechanisms of I/R-dependent brain damage and suggest SIRT5 as a novel therapeutic target.

Profiling and validation of circulating microRNAs for cardiovascular events in patients presenting with ST-segment elevation myocardial infarction.

AIMS: MicroRNAs (miRNA) are important non-coding modulators controlling patterns of gene expression. However, profiling and validation of circulating miRNA levels related to adverse cardiovascular outcome has not been performed in patients with an acute coronary syndrome (ACS). METHODS AND RESULTS: In a multicentre, prospective ACS cohort, 1002 out of 2168 patients presented with ST-segment elevation myocardial infarction (STEMI). Sixty-three STEMI patients experienced an adjudicated major cardiovascular event (MACE, defined as cardiac death or recurrent myocardial infarction) within 1 year of follow-up. From a miRNA profiling in a matched derivation case-control cohort, 14 miRNAs were selected for validation. Comparing 63 cases vs. 126 controls, 3 miRNAs were significantly differentially abundant. In patients with MACE, miR-26b-5p levels (P = 0.038) were decreased, whereas miR-320a (P = 0.047) and miR-660-5p (P = 0.01) levels were increased. MiR-26b-5p has been suggested to prevent adverse cardiomyocyte hypertrophy, whereas miR-320a promotes cardiomyocyte death and apoptosis, and miR-660-5p has been related to active platelet production. This suggests that miR-26b-5p, miR-320a, and miR-660-5p may reflect alterations of different pathophysiological pathways involved in clinical outcome after ACS. Consistently, these three miRNAs reliably discriminated cases from controls [area under the receiver-operating characteristic curve (AUC) in age- and sex-adjusted Cox regression for miR-26b-5p = 0.707, miR-660-5p = 0.683, and miR-320a =0.672]. Combination of the three miRNAs further increased AUC to 0.718. Importantly, addition of the three miRNAs to both, the Global Registry of Acute Coronary Events (GRACE) score and a clinical model increased AUC from 0.679 to 0.720 and 0.722 to 0.732, respectively, with a net reclassification improvement of 0.20 in both cases. CONCLUSION: This is the first study performing profiling and validation of miRNAs that are associated with adverse cardiovascular outcome in patients with STEMI. MiR-26b-5p, miR-320a, and miR-660-5p discriminated for MACE and increased risk prediction when added to the GRACE score and a clinical model. These findings suggest that the release of specific miRNAs into circulation may reflect the activation of molecular pathways that impact on clinical outcome after STEMI.

Epigenetic regulation of tissue factor inducibility in endothelial cell senescence.

Cellular senescence, a programmed state induced by multiple deleterious triggers, is characterised by permanent cell-cycle exit and altered gene expression and cell morphology. In humans it is considered a tumor suppressor mechanism, mediating removal of damaged or mutated cells from the cell-cycle pool, and may also contribute to the ageing process. In this study, we show that senescent human umbilical vein endothelial cells lose their ability to induce tissue factor (TF), a transmembrane protein with important roles in hemostasis and cancer progression, in response to thrombin or - independently of cell-surface receptors - phorbol-12-myristate-13-acetate. This phenomenon could not be explained by senescence-related alterations in the downstream signal transduction cascade or by accelerated TF mRNA degradation. Rather, using chromatin immuno-precipitation we could show that loss of TF gene inducibility during senescence occurs following chromatin remodelling of the TF promoter resulting from hypo-acetylation of histone H3. These findings were reversible after transduction of presenescent cultures with telomerase reverse transcriptase, enabling late-passage cultures to escape senescence. These results extend the involvement of heterochromatic gene silencing in senescence beyond cell cycle-related genes and suggest a novel anti-cancer mechanism of senescence through inhibition of TF inducibility.