Rationale: Atherosclerosis preferentially occurs at specific sites of the vasculature where endothelial cells (ECs) are exposed to disturbed blood flow. Translocation of integrin α5 to lipid rafts promotes integrin activation and ligation, which is critical for oscillatory shear stress (OSS)-induced EC activation. However, the underlying mechanism of OSS promoted integrin α5 lipid raft translocation has remained largely unknown. Objective: The objective of this study was to specify the mechanotransduction mechanism of OSS-induced integrin α5 translocation and subsequent EC activation. Methods and Results: Mass spectrometry studies identified endothelial ANXA2 (annexin A2) as a potential carrier allowing integrin α5β1 to traffic in response to OSS. Interference by siRNA of AnxA2 in ECs greatly decreased OSS-induced integrin α5β1 translocation to lipid rafts, EC activation, and monocyte adhesion. Pharmacological and genetic inhibition of PTP1B (protein tyrosine phosphatase 1B) blunted OSS-induced integrin α5β1 activation, which is dependent on Piezo1-mediated calcium influx in ECs. Furthermore, ANXA2 was identified as a direct substrate of activated PTP1B by mass spectrometry. Using bioluminescence resonance energy transfer assay, PTP1B-dephosphorylated ANXA2 at Y24 was found to lead to conformational freedom of the C-terminal core domain from the N-terminal domain of ANXA2. Immunoprecipitation assays showed that this unmasked ANXA2-C-terminal core domain specifically binds to an integrin α5 nonconserved cytoplasmic domain but not β1. Importantly, ectopic lentiviral overexpression of an ANXA2 ^Y24F mutant increased and shRNA against Ptp1B decreased integrin α5β1 ligation, inflammatory signaling, and progression of plaques at atheroprone sites in apolipoprotein E ( ApoE ) ^−/− mice. However, the antiatherosclerotic effect of Ptp1B shRNA was abolished in AnxA2 ^−/− ApoE ^−/− mice. Conclusions: Our data elucidate a novel endothelial mechanotransduction molecular mechanism linking atheroprone flow and activation of integrin α5β1, thereby identifying a class of potential therapeutic targets for atherosclerosis. Graphic An graphic abstract is available for this article.

The Hippo/Yes-associated protein (YAP) pathway has pivotal roles in innate immune responses against pathogens in macrophages. However, the role of YAP in macrophages during atherosclerosis and its mechanism of YAP activation remain unknown. Here, we find that YAP overexpression in myeloid cells aggravates atherosclerotic lesion size and infiltration of macrophages, whereas YAP deficiency reduces atherosclerotic plaque. Tumor necrosis factor receptor-associated factor 6 (TRAF6), a downstream effector of interleukin-1β (IL-1β), triggers YAP ubiquitination at K252, which interrupts the interaction between YAP and angiomotin and results in enhanced YAP nuclear translocation. The recombinant IL-1 receptor antagonist anakinra reduces atherosclerotic lesion formation, which is abrogated by YAP overexpression. YAP level is increased in human and mouse atherosclerotic vessels, and plasma IL-1β level in patients with STEMI is correlated with YAP protein level in peripheral blood mononuclear cells. These findings elucidate a mechanism of YAP activation, which might be a therapeutic target for atherosclerosis.

Rationale: Timely inhibition of inflammation and initiation of resolution are important to repair injured tissues. MST1/2 (mammalian STE20-like protein kinase 1/2) acts as a regulator of macrophage-associated immune responses to bacterial infections. However, the role of MST1/2 in regulating macrophage phenotype and function in myocardial infarction (MI) remains unclear. Objective: To determine the function and underlying mechanism of macrophage MST1/2 in cardiac repair post-MI. Methods and Results: Using LysMCre -mediated Mst1/2 -deficient mice, we found that MST1 deficiency exacerbated cardiac dysfunction after MI. Single-cell RNA sequencing assay indicated that the effect was attributed to a shift of macrophage subtypes from those expressing Cxcl2 and Cd163 toward Ccl2 and Ccl4 expression. Mass spectrometry identified LTB4 (leukotriene B4) as the lipid mediator that was upregulated in the absence of MST1. We found that MST1 phosphorylated 5-LOX (5-lipoxygenase) at its T218 residue, disrupting the interaction between 5-LOX and 5-LOX-activating protein, resulting in a reduction of LTB4 production. In contrast, a 5-LOX ^T218A variant showed no response to MST1. Moreover, treatment of peritoneal macrophages with LTB4 or medium conditioned by Mst1 -deficient macrophages resulted in high Ccl2 and Ccl4 expression and low Cxcl2 and Cd163 expression, except when the cells were co-treated with the BLT1 (LTB4 receptor 1) antagonist CP105696. Furthermore, CP105696 ameliorated cardiac dysfunction in LysMCre -mediated Mst1/2 -deficient mice and enhanced cardiac repair in wild-type mice treated with XMU-MP-1 (4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide) after MI. Conclusions: Taken together, our results demonstrate that inhibition of MST1/2 impaired post-MI repair through activating macrophage 5-LOX–LTB4–BLT1 axis.

Background: Ischemia-reperfusion (I/R)-induced vascular dysfunction is the main factor to acute ischemic stroke. Sirt3 is one of the sirtuin family members, which plays an important role in the development of neurological diseases. Methods: In this study, we constructed I/R injury model on HBMEC cells and induced the overexpression of Sirt3 in model cells. Meanwhile, the p38 activator U-46619 was used to examine the connection between Sirt3 and p38. We also examined the level of endothelial associated proteins, including occluding, ZO-1 and claudin-4 by using qRT-PCR and western blot. Results: Our findings indicated that overexpression of Sirt3 decreased the permeability of model cells and promoted in the growth of endothelial cells. However, the activation of p38 could antagonize the function of Sirt3 in HBMEC cells. Moreover, Our results indicated a positive correlation between Sirt3 and inter-endothelial junction proteins. Importantly, PPAR-γ agonist and inhibitor were utilized to investigate the role of PPAR-γ in Sirt3 mediated cell function. Sirt3 was targeted by PPAR-γ in model cells. Conclusions: Taken together, this research not only demonstrated PPAR-γ might benefit to the growth of endothelial cell though activating Sirt3 but also indicated its potential value in the treatment for ischemic stroke.

BACKGROUND: Metabolic syndrome heightens cardiovascular disease risk primarily through increased arterial stiffness. We previously demonstrated the involvement of YAP (Yes-associated protein) in high-fat/high-sucrose diet (HFHSD)–induced arterial stiffness via modulation of PPM1B (protein phosphatase Mg ²⁺ /Mn ²⁺ –dependent 1B)-lysine 63(K63) deubiquitination. In this study, we aimed to elucidate the role and mechanisms underlying PPM1B deubiquitination in HFHSD-induced arterial stiffness. METHODS: Enzymes governing PPM1B deubiquitination were identified through small interfering RNA (siRNA) screening and mass spectrometry. Glutathione S-transferase pull-down, coimmunoprecipitation, protein purification, and immunofluorescence were used to explore the mechanism underlying PPM1B deubiquitination. Doppler ultrasound was used to evaluate HFHSD-induced arterial stiffness in mice, and telemetry was used to record pulsatile (systolic and diastolic) blood pressure. RESULTS: Smooth muscle cell–specific PPM1B overexpression attenuated HFHSD-induced arterial stiffness in mice in a PPM1B-K326-K63–linked polyubiquitination-dependent manner. Mechanistically, ABRO1 (Abraxas brother 1; a core BRCC36 [BRCA1/BRCA2 (breast cancer type 1/2)–containing complex subunit 36] isopeptidase complex component) directly bound YAP and underwent liquid-liquid phase separation with YAP and PPM1B in a YAP-dependent manner, which in turn promoted PPM1B deubiquitination. Furthermore, smooth muscle cell–specific Abro1 -knockout mice and Brcc3 -knockout mice showed attenuated HFHSD-induced arterial stiffness and activation of transforming growth factor-β–Smad (mothers against decapentaplegic homolog) signaling. CONCLUSIONS: We elucidated the PPM1B deubiquitination mechanisms and highlighted a potential therapeutic target for metabolic syndrome–related arterial stiffness.

Prolonged length of stay (LOS) of sepsis can drain a hospital's material and human resources. This study investigated the correlations between T helper type 17 (Th17) and regulatory T (Treg) balance with LOS in sepsis. A prospective clinical observational study was designed in Changhai Hospital affiliated to Naval Medical University in Shanghai, China, from January to October 2020. The patients diagnosed with sepsis and who met the inclusion and exclusion criteria were recruited and whether the levels of cytokines, procalcitonin, subtypes, and biomarkers of T cells in the peripheral blood were detected. We analyzed the correlation between these and LOS. Sixty septic patients were classified into two groups according to whether their intensive care unit (ICU) stay exceeded 14 days. The patients with LOS ≥14 days were older ([72.6±7.5] years vs. [63.3±10.4] years, P=0.015) and had higher Sequential Organ Failure Assessment (SOFA) (median [interquartile range]: 6.5 [5.0–11.0] vs. 4.0 [3.0–6.0], P=0.001) and higher Acute Physiology and Chronic Health Evaluation (APACHE) II scores (16.0 [13.0–21.0] vs. 8.5 [7.0–14.0], P=0.001). There was no difference in other demographic characteristics and cytokines, interleukin-6, tumor necrosis factor-α, and interleukin-10 between the two groups. The Th17/Treg ratio of sepsis with LOS <14 days was considerably lower (0.48 [0.38–0.56] vs. 0.69 [0.51–0.98], P=0.001). For patients with LOS ≥14 days, the area under the receiver operating characteristic curve for the Th17/Treg ratio was 0.766. It improved to 0.840 and 0.850 when combined with the SOFA and APACHE II scores, respectively. The Th17/Treg ratio was proportional to septic severity and can be used as a potential predictor of ICU stay in sepsis, presenting a new option for ICU practitioners to better care for patients with sepsis.