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Metabolic dysfunction-associated fatty liver disease (MASLD) is a common liver and health issue associated with heightened cardiovascular disease (CVD) risk, with Cytokeratin 18 (CK-18) as a marker of liver injury across the MASLD to cirrhosis spectrum. Autoantibodies against apolipoprotein A-1 (AAA-1s) predict increased CVD risk, promoting atherosclerosis and liver steatosis in apoE-/- mice, though their impact on liver inflammation and fibrosis remains unclear. This study examined AAA-1s' impact on low-grade inflammation, liver steatosis, and fibrosis using a MASLD mouse model exposed to AAA-1s passive immunization (PI). Ten-week-old male C57BL/6J mice under a high-fat diet underwent PI with AAA-1s or control antibodies for ten days. Compared to controls, AAA-1-immunized mice showed higher plasma CK-18 (5.3 vs. 2.1 pg/mL, p = 0.031), IL-6 (13 vs. 6.9 pg/mL, p = 0.035), IL-10 (27.3 vs. 9.8 pg/mL, p = 0.007), TNF-α (32.1 vs. 24.2 pg/mL, p = 0.032), and liver steatosis (93.4% vs. 73.8%, p = 0.007). Transcriptomic analyses revealed hepatic upregulation of pro-fibrotic mRNAs in AAA-1-recipient mice, though histological changes were absent. In conclusion, short-term AAA-1 PI exacerbated liver steatosis, inflammation, and pro-fibrotic gene expression, suggesting that AAA-1s may play a role in MASLD progression. Further research with prolonged AAA-1 exposure is warranted to clarify their potential role in liver fibrosis and associated complications.

Background: Chondrocyte-based cell therapy to repair cartilage has been used for >25 years despite current limitations. This work presents a new treatment option for cartilage lesions., Hypothesis: High-quality hyaline cartilage microtissues called Cartibeads are capable of treating focal chondral lesions once implanted in the defect, by complete fusion of Cartibeads among themselves and their integration with the surrounding native cartilage and subchondral bone., Study Design: Controlled laboratory study., Methods: Cartibeads were first produced from human donors and characterized using histology (safranin O staining of glycosaminoglycan [GAG] and immunohistochemistry of collagen I and II) and GAG dosage. Cartibeads from 6 Göttingen minipigs were engineered and implanted in an autologous condition in the knee (4 or 5 lesions per knee). One group was followed up for 3 months and the other for 6 months. Feasibility and efficacy were measured using histological analysis and macroscopic and microscopic scores., Results: Cartibeads revealed hyaline features with strong staining of GAG and collagen II. High GAG content was obtained: 24.6-µg/mg tissue (wet weight), 15.52-µg/mg tissue (dry weight), and 35 ± 3-µg GAG/bead (mean ± SD). Histological analysis of Göttingen minipigs showed good integration of Cartibeads grafts at 3 and 6 months after implantation. The Bern Score of the histological assay comparing grafted versus empty lesions was significant at 3 months (grafted, n = 10; nongrafted, n = 4; score, 3.3 and 5.3, respectively) and 6 months (grafted, n = 11; nongrafted, n = 3; score, 1.6 and 5.1)., Conclusion: We developed an innovative 3-step method allowing, for the first time, the use of fully dedifferentiated adult chondrocytes with a high number of cell passage (owing to the extensive amplification in culture). Cartibeads engineered from chondrocytes hold potential as an advanced therapy medicinal product for treating cartilage lesions with established efficacy., Clinical Relevance: This successful preclinical study, combined with standardized manufacturing of Cartibeads according to good manufacturing practice guidelines, led to the approval of first-in-human clinical trial by the ethics committee and local medical authority. The generated data highlighted a promising therapy to treat cartilage lesions from a small amount of starting biopsy specimen. With our innovative cell amplification technology, very large lesions can be treated, and older active patients can benefit from it.

The repair of damaged articular cartilage is an unmet medical need. Chondrocyte-based cell therapy has been used to repair cartilage for over 20 years despite current limitations. Chondrocyte dedifferentiation upon expansion in monolayer is well known and is the main obstacle to their use as cell source for cartilage repair. Consequently, current approaches often lead to fibrocartilage, which is biomechanically different from hyaline cartilage and not effective as a long-lasting treatment. Here, we describe an innovative 3-step method to engineer hyaline-like cartilage microtissues, named Cartibeads, from high passage dedifferentiated chondrocytes. We show that WNT5A/5B/7B genes were highly expressed in dedifferentiated chondrocytes and that a decrease of the WNT signaling pathway was instrumental for full re-differentiation of chondrocytes, enabling production of hyaline matrix instead of fibrocartilage matrix. Cartibeads showed hyaline-like characteristics based on GAG quantity and type II collagen expression independently of donor age and cartilage quality. In vivo, Cartibeads were not tumorigenic when transplanted into SCID mice. This simple 3-step method allowed a standardized production of hyaline-like cartilage microtissues from a small cartilage sample, making Cartibeads a promising candidate for the treatment of cartilage lesions., (© The Author(s) 2022. Published by Oxford University Press.)

Background: Characterization of the clot occluding the arteries in acute ischemic stroke received ample attention, in terms of elucidating the relationship between the clot composition, its etiology and its amenability for pharmacological treatment and mechanical thrombectomy approaches. Traditional analytical techniques such as conventional 2D histopathology or electron microscopy sample only small parts of the clot. Visualization and analysis in 3D are necessary to depict and comprehend the overall organization of the clot. The aim of this study is to investigate the potential of microCT for characterizing the clot composition, structure, and organization., Methods: In a pilot study, we analyzed with microCT clots retrieved from 14 patients with acute ischemic stroke. The following parameters were analyzed: overall clot density, clot segmentation with various density thresholds, clot volume., Results: Our findings show that human clots are heterogeneous in terms of CT intra-clot density distribution. After fixation in formalin, the clots display a shift toward negative values. On average, we found the mean HU values of red clots retrieved from patients to be -153 HU, with SD = 23.8 HU, for the intermediate clots retrieved from patients -193 HU, SD = 23.7 HU, and for the white clots retrieved from patients -229 HU, SD = 64.8 HU., Conclusion: Our study shows that volumetric and density analysis of the clot opens new perspectives for clot characterization and for a better understanding of thrombus structure and composition., 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 Dumitriu LaGrange, Braunersreuther, Wanke, Berberat, Luthman, Fitzgerald, Doyle, Brina, Reymond, Platon, Muster, Machi, Poletti, Vargas and Lövblad.)

Objective: Intracranial aneurysms (IAs) are more often diagnosed in women. Hormones and vessel geometry, which influences wall shear stress, may affect pathophysiological processes of the arterial wall. Here, the authors investigated sex-related differences in the remodeling of the aneurysm wall and in intraluminal thrombus resolution., Methods: A well-characterized surgical side-wall aneurysm model was used in female, male, and ovariectomized rats. Decellularized grafts were used to model highly degenerated and decellularized IA walls and native grafts to model healthy IA walls. Aneurysm growth and thrombus composition were analyzed at 1, 7, 14, and 28 days. Sex-related differences in vessel wall remodeling were compared with human IA dome samples of men and pre- and postmenopausal women., Results: At 28 days, more aneurysm growth was observed in ovariectomized rats than in males or non-ovariectomized female rats. The parent artery size was larger in male rats than in female or ovariectomized rats, as expected. Wall inflammation increased over time in all groups and was most severe in the decellularized female and ovariectomized groups at 28 days compared with the male group. Likewise, in these groups the most elastin fragmentation was seen at 28 days. In female rats, on days 1, 7, and 14, the intraluminal thrombus was mainly composed of red blood cells and fibrin. On days 14 and 28, macrophage and smooth muscle cell invasion inside the thrombus was shown, leading to the removal of red blood cells and deposition of collagen and elastin. On days 14 and 28, similar profiles of thrombus reorganization were observed in male and ovariectomized female rats. However, collagen content in thrombi and vessel wall macrophage content were higher in aneurysms of male rats at 28 days than in those of female rats. On day 28, thrombus coverage by endothelial cells was lower in ovariectomized than in female or male rats. Finally, analysis of human IA domes showed that endothelial cell coverage was lower in men and postmenopausal women than in younger women., Conclusions: Aneurysm growth and intraluminal thrombus resolution show sex-dependent differences. While certain processes (endothelial cell coverage and collagen deposition) point to a strong hormonal dependence, others (wall inflammation and aneurysm growth) seem to be influenced by both hormones and parent artery size.

Pathogenesis of intracranial aneurysm is complex and the precise biomechanical processes leading to their rupture are uncertain. The goal of our study was to characterize the aneurysmal wall histologically and to correlate histological characteristics with clinical and radiological factors used to estimate the risk of rupture. A new biobank of aneurysm domes resected at the Geneva University Hospitals (Switzerland) was used. Histological analysis revealed that unruptured aneurysms have a higher smooth muscle cell (SMC) content and a lower macrophage content than ruptured domes. These differences were associated with more collagen in unruptured samples, whereas the elastin content was not affected. Collagen content and type distribution were different between thick and thin walls of unruptured aneurysms. Classification of aneurysm domes based on histological characteristics showed that unruptured samples present organized wall rich in endothelial and SMCs compared with ruptured samples. Finally, aneurysm wall composition was altered in unruptured domes of patients presenting specific clinical factors used to predict rupture such as large dome diameter, dome irregularities, and smoking. Our study shows that the wall of aneurysm suspected to be at risk for rupture undergoes structural alterations relatively well associated with clinical and radiological factors currently used to predict this risk.

Objectives: We validate a 4D strategy tailored for 3T clinical systems to simultaneously quantify function and infarct size in wild type mice after ischemia/reperfusion, with improved spatial and temporal resolution by comparison to previous published protocols using clinical field MRI systems., Methods: C57BL/6J mice underwent 60min ischemia/reperfusion (n=14) or were controls without surgery (n=6). Twenty-four hours after surgery mice were imaged with gadolinium injection and sacrificed for post-mortem MRI and histology with serum also taken for Troponin I levels. The double ECG- and respiratory-triggered 3D FLASH (Fast Low Angle Shot) gradient echo (GRE) cine sequence had an acquired isotropic resolution of 344μm, TR/TE of 7.8/2.9ms and acquisition time 25-35min. The conventional 2D FLASH cine sequence had the same in-plane resolution of 344μm, 1mm slice thickness and TR/TE 11/5.4ms for an acquisition time of 20-25min plus 5min for planning. Left ventricle (LV) and right ventricle (RV) volumes were measured and functional parameters compared 2D to 3D, left to right and for inter and intra observer reproducibility. MRI infarct volume was compared to histology., Results: For the function evaluation, the 3D cine outperformed 2D cine for spatial and temporal resolution. Protocol time for the two methods was equivalent (25-35min). Flow artifacts were reduced (p=0.008) and epi/endo-cardial delineation showed good intra and interobserver reproducibility. Paired t-test comparing ejection volume left to right showed no significant difference for 3D (p=0.37), nor 2D (p=0.30) and correlation slopes of left to right EV were 1.17 (R 2 =0.75) for 2D and 1.05 (R 2 =0.50) for 3D. Quantifiable 'late gadolinium enhancement' infarct volume was seen only with the 3D cine and correlated to histology (R 2 =0.89). Left ejection fraction and MRI-measured infarct volume correlated (R 2 >0.3)., Conclusions: The 4D strategy, with contrast injection, was validated in mice for function and infarct quantification from a single scan with minimal slice planning., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Extracellular ATP is a central signaling molecule in inflammatory responses. Pannexin1 (Panx1) channels release ATP in a controlled manner and have been implicated in various inflammatory pathologies, but their role in atherogenesis remains elusive. Using atherosclerosis-susceptible mouse models with ubiquitous deletion of Panx1 (Panx1 -/- Apoe -/- ) or with Cre recombinase-mediated deletion of Panx1 in endothelial cells and monocytes (Tie2-Cre Tg Panx1 fl/fl Apoe -/- ; Panx1 del Apoe -/- ), we identified a novel role for Panx1 in the lymphatic vasculature. Atherosclerotic lesion development in response to high-cholesterol diet was enhanced in Panx1 del Apoe -/- mice, pointing to an atheroprotective role for Panx1 in endothelial and/or monocytic cells. Unexpectedly, atherogenesis was not changed in mice with ubiquitous Panx1 deletion, but Panx1 -/- Apoe -/- mice displayed reduced body weight, serum cholesterol, triglycerides and free fatty acids, suggesting altered lipid metabolism in these Panx1-deficient mice. Mechanistically, Panx1 -/- Apoe -/- mice showed impairment of lymphatic vessel function with decreased drainage of interstitial fluids and reduced dietary fat absorption. Thus, the detrimental effect of Panx1 deletion in endothelial and/or monocytic cells during atherogenesis is counterbalanced by an opposite effect resulting from impaired lymphatic function in ubiquitous Panx1-deficient mice. Collectively, our findings unveil a pivotal role of Panx1 in linking lymphatic function to lipid metabolism and atherosclerotic plaque development.

Selective pharmacological treatments targeting reperfusion injury produced modest protective effects and might be associated with immunosuppression. In order to identify novel and better-tolerated approaches, we focused on the neutralization of receptor activator of nuclear factor kappa-B ligand [RANKL], a cytokine recently shown to activate inflammatory cells (i.e. neutrophils) orchestrating post-infarction injury and repair. Myocardial ischemia (60min) and reperfusion injury was surgically induced in C57Bl/6 mice. In hearts and serum, RANKL was early upregulated during reperfusion. A "one-shot" injection with neutralizing anti-RANKL IgG during ischemia ameliorated myocardial infarct size and function, but not adverse remodeling (determined by Magnetic Resonance Imaging [MRI]) as compared to Vehicle or control IgG. These beneficial effects were accompanied in vivo by reduction in cardiac neutrophil infiltration, reactive oxygen species (ROS) and MMP-9 release. Anti-RANKL IgG treatment suppressed sudden peak of neutrophil granule products in mouse serum early after reperfusion onset. In vitro, RANK mRNA expression was detected in isolated mouse neutrophils. Co-incubation with neutralizing anti-RANKL IgG abrogated RANKL-induced mouse neutrophil degranulation and migration, suggesting a critical role of RANKL in neutrophil-mediated injury. Conversely, anti-RANKL IgG did not affect salvage pathways in cardiac cells (i.e. ERK p42/p44, Akt and STAT-3) or macrophage cardiac infiltration. Finally, treatment with anti-RANKL IgG showed no effect on B and T lymphocyte polarization (in serum, spleen and infarcted myocardium) and circulating chemokines as compared with Vehicle or control IgG. In conclusion, acute treatment with anti-RANKL IgG improved cardiac infarct size and function by potentially impacting on neutrophil-mediated injury and repair., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Significance: Phagocytes play a key role in promoting the oxidative stress after ischemic stroke occurrence. The phagocytic NADPH oxidase (NOX) 2 is a membrane-bound enzyme complex involved in the antimicrobial respiratory burst and free radical production in these cells., Recent Advances: Different oxidants have been shown to induce opposite effects on neuronal homeostasis after a stroke. However, several experimental models support the detrimental effects of NOX activity (especially the phagocytic isoform) on brain recovery after stroke. Therapeutic strategies selectively targeting the neurotoxic ROS and increasing neuroprotective oxidants have recently produced promising results., Critical Issues: NOX2 might promote carotid plaque rupture and stroke occurrence. In addition, NOX2-derived reactive oxygen species (ROS) released by resident and recruited phagocytes enhance cerebral ischemic injury, activating the inflammatory apoptotic pathways. The aim of this review is to update evidence on phagocyte-related oxidative stress, focusing on the role of NOX2 as a potential therapeutic target to reduce ROS-related cerebral injury after stroke., Future Directions: Radical scavenger compounds (such as Ebselen and Edaravone) are under clinical investigation as a therapeutic approach against stroke. On the other hand, NOX inhibition might represent a promising strategy to prevent the stroke-related injury. Although selective NOX inhibitors are not yet available, nonselective compounds (such as apocynin and fasudil) provided encouraging results in preclinical studies. Whereas additional studies are needed to better evaluate this therapeutic potential in human beings, the development of specific NOX inhibitors (such as monoclonal antibodies, small-molecule inhibitors, or aptamers) might further improve brain recovery after stroke.

Auto-antibodies to apolipoprotein A-1 (anti-apoA-1 IgG) were shown to promote inflammation and atherogenesis, possibly through innate immune receptors signalling. Here, we aimed at investigating the role of Toll-like receptors (TLR) 2 and 4 on anti-apoA-1 IgG-induced atherosclerotic plaque vulnerability, myocardial necrosis and mortality in mice. Adult male apolipoprotein E knockout (ApoE)-/- (n=72), TLR2-/-ApoE-/- (n=36) and TLR4-/-Apo-/- (n=28) mice were intravenously injected with 50 µg/mouse of endotoxin-free polyclonal anti-apoA-1 IgG or control isotype IgG (CTL IgG) every two weeks for 16 weeks. Atherosclerotic plaque size and vulnerability were assessed by histology. Myocardial ischaemia and necrosis, respectively, were determined by electrocardiographic (ECG) changes assessed by telemetry and serum troponin I (cTnI) measurements. Impact on survival was assessed by Kaplan-Meier analyses. In ApoE-/- mice, anti-apoA-1 IgG passive immunisation enhanced histological features of atherosclerotic plaque vulnerability (increase in neutrophil and MMP-9 and reduction in collagen content), induced a substantial cTnI elevation (p=0.001), and increased mortality rate by 23 % (LogRank, p=0.04) when compared to CTL IgG. On a subgroup of ApoE-/- mice equipped with telemetry (n=4), a significant ST-segment depression was noted in anti-apoA-1 IgG-treated mice when compared to CTL IgG recipients (p< 0.001), and an acute ST-segment elevation myocardial infarction preceding mouse death was observed in one case. The deleterious effects of anti-apoA-1 IgG on atherosclerotic plaque vulnerability, myocardial necrosis and death were partially reversed in TLR2-/-ApoE-/- and TLR4-/-ApoE-/- backgrounds. In conclusion, anti-apoA-1 auto-antibodies seem to be active mediators of atherosclerotic plaque vulnerability, myocardial necrosis, and mortality in mice through TLR2- and TLR4-mediated pathways.

Background: New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects., Objective: The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations., Methods and Results: The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p<0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p<0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo., Conclusion: HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte.

Aims: Several intracellular mediators have been implicated as new therapeutic targets against myocardial ischaemia and reperfusion injury. However, clinically effective salvage pathways remain undiscovered. Here, we focused on the potential role of the adaptor protein p66(Shc) as a regulator of myocardial injury in a mouse model of cardiac ischaemia and reperfusion., Methods and Results: Adult male p66(Shc) deficient (p66(Shc) (-/-)) and C57Bl/6 wild-type (WT) mice were exposed to 30, 45, or 60 min of ischaemia and reperfusion (5, 15 min, or 24 h). Infarct size, systemic and intracardiac inflammation and oxidants, as well as cytosolic and mitochondrial apoptotic pathways were investigated. Following 30, but not 45 or 60 min of ischaemia, genetic p66(Shc) deficiency was associated with larger infarcts. In WT mice, in vivo p66(Shc) knock down by siRNA with transient protein deficiency confirmed these findings. P66(Shc) inhibition was not associated with any modification in post-infarction inflammation, oxidative burst nor cardiac vessel density or structure. However, in p66(Shc) (-/-) mice activation of the protective and anti-apoptotic Reperfusion Injury Salvage Kinases and Survivor Activating Factor Enhancement pathways were blunted and mitochondrial swelling and cellular apoptosis via the caspase-3 pathway increased compared with WT., Conclusions: Genetic deletion of p66(Shc) increased susceptibility to myocardial injury in response to short-term ischaemia and reperfusion in mice. Still, additional studies are needed for assessing the role of this pathway in acute coronary syndrome patients., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS-producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase-induced emphysema using NOX2- and NOX1-deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2-positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase-induced alveolar airspace enlargement and elastin degradation were prevented in NOX2-deficient mice, but not in NOX1-deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase-9 (MMP-9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage-restricted functional NOX2 in elastase-induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68-positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase-induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage-specific NOX2 participates in elastase-induced emphysema through the involvement of SIRT1/MMP-9 pathways in mice., (© 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)