Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter's syndrome.

Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

Ultrastructural Characterization of Human Bronchial Epithelial Cells during SARS-CoV-2 Infection: Morphological Comparison of Wild-Type and CFTR-Modified Cells.

SARS-CoV-2 replicates in host cell cytoplasm. People with cystic fibrosis, considered at risk of developing severe symptoms of COVID-19, instead, tend to show mild symptoms. We, thus, analyzed at the ultrastructural level the morphological effects of SARS-CoV-2 infection on wild-type (WT) and F508del (ΔF) CFTR-expressing CFBE41o- cells at early and late time points post infection. We also investigated ACE2 expression through immune-electron microscopy. At early times of infection, WT cells exhibited double-membrane vesicles, representing typical replicative structures, with granular and vesicular content, while at late time points, they contained vesicles with viral particles. ∆F cells exhibited double-membrane vesicles with an irregular shape and degenerative changes and at late time of infection, showed vesicles containing viruses lacking a regular structure and a well-organized distribution. ACE2 was expressed at the plasma membrane and present in the cytoplasm only at early times in WT, while it persisted even at late times of infection in ΔF cells. The autophagosome content also differed between the cells: in WT cells, it comprised vesicles associated with virus-containing structures, while in ΔF cells, it comprised ingested material for lysosomal digestion. Our data suggest that CFTR-modified cells infected with SARS-CoV-2 have impaired organization of normo-conformed replicative structures.

CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells.

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19.

The transcriptional profile of adipose-derived stromal cells (ASC) mirrors the whitening of adipose tissue with age.

Multipotent stem cells persist within the stromal vascular fraction (SVF) of adipose tissue during adulthood. These cells, commonly referred to as adipose-derived stromal cells (ASC), have been extensively investigated over the past years as a promising therapeutic tool based on their regenerative and immunomodulatory properties. However, how ASC might mirror the age-related alteration of the fat they reside in remains unclear. Herein, we show that inguinal adipose tissue in mice turns from brown/beige- to white-like with age and resident ASC readily mirror these changes both at mRNA and microRNA transcriptional level. Mechanistically, our data suggest that these brown/age-related changes in ASC transcription rely on changes in the activity of E2F1 and NFkB transcription factors.

MRI characterization of rat brain aging at structural and functional level: Clues for translational applications.

Magnetic resonance imaging (MRI) paradigms, using non-invasive approaches, can provide relevant findings about brain aging. The attention has been primarily focused on neurodegenerative diseases, while little or nothing has been done to differentiate physiology from pathology. The present study aimed to test diffusion tensor imaging (DTI) and functional MRI (fMRI) metrics to analyze physiological age-related changes in rats at myelin structure and activation level; findings were validated by ex vivo histology. The purpose is to find comparable biomarkers in rodents and humans to allow a reliable translation from pre-clinical to clinical settings. Data evidenced: i) a significantly higher cerebrospinal fluid volume in middle-aged and aged vs. young rats; ii) a progressive alteration of white matter; iii) a significant reduction of evoked activity in aged animals. These results partially mirror the age-related changes in humans and may represent a preliminary step to find reliable tools for a lifelong monitoring with a value for the clinical practice (e.g., to provide support to the early diagnosis of dementia in asymptomatic subjects).

Ultrastructural Analysis of Rehydrated Human Donor Corneas After Air-Drying and Dissection by Femtosecond Laser.

Purpose: To evaluate the efficiency of femtosecond laser (FSL) incision of rehydrated human donor corneas after air-drying and its effects on corneal structure. Methods: We compared the rehydrated and fresh-preserved corneas by microscopy following Victus-Tecnolas FSL treatment for straight-edge anterior lamellar keratoplasty (ALK). The corneas were dehydrated at room temperature under a laminar-flow hood. Results: To obtain the horizontal cut in rehydrated corneas, we increased the FSL pulse energy to 1.2 µJ from 0.80 µJ applied for the fresh corneas and obtained a clear-cut separation of the lamellar lenticule cap from the corneal bed. Light microscopy showed regular arrangement of stromal collagen lamellae, with spaces in between the fibers in the corneal stroma in the fresh and the rehydrated corneas, but the uppermost epithelial layers in the rehydrated corneas were lost. Transmission electron microscopy (TEM) revealed no signs of thermal or mechanical damage to the corneal structure. The epithelial basal membrane and Bowman's layer maintained their integrity. The epithelial basal layer and cells were separated by large spaces due to junction alteration in the rehydrated corneas. There were gaps between the lamellar layers in the stroma, especially in the rehydrated corneas. Keratocytes displayed normal structure in the fresh corneas but were devoid of microorganules in the rehydrated corneas. Minor irregularities were observed in the vertical incision and the horizontal stroma appeared smooth on scanning electron microscopy. Conclusion: The corneal stroma of rehydrated corneas maintained morphology and integrity, while corneal cellular components were generally altered. When corneas are intended for FSL-assisted ALK, effective stromal bed incision is best achieved at a laser power higher than that currently adopted for fresh corneas.

Immunolocalization of leptin and leptin receptor in colorectal mucosa of ulcerative colitis, Crohn's disease and control subjects with no inflammatory bowel disease.

The expression of leptin and leptin receptor (Ob-R) has been partially elucidated in colon of patients with inflammatory bowel diseases (IBDs), even though leptin is involved in angiogenesis and inflammation. We previously reported overexpression of GLUT5 fructose transporter, in aberrant clusters of lymphatic vessels in lamina propria of IBD and controls. Here, we examine leptin and Ob-R expression in the same biopsies. Specimens were obtained from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls who underwent screening for colorectal cancer, follow-up after polypectomy or with a history of lower gastrointestinal symptoms. Immunohistochemistry revealed leptin in apical and basolateral membranes of short epithelial portions, Ob-R on the apical pole of epithelial cells. Leptin and Ob-R were also identified in structures and cells scattered in the lamina propria. In UC, a significant correlation between leptin and Ob-R in the lamina propria was found in all inflamed samples, beyond non-inflamed samples of the proximal tract, while in CD, it was found in inflamed distal samples. Most of the leptin and Ob-R positive areas in the lamina propria were also GLUT5 immunoreactive in inflamed and non-inflamed mucosa. A significant correlation of leptin or Ob-R expression with GLUT5 was observed in the inflamed distal samples from UC. Our findings suggest that there are different sites of leptin and Ob-R expression in large intestine and those in lamina propria do not reflect the status of mucosal inflammation. The co-localization of leptin and/or Ob-R with GLUT5 may indicate concomitance effects in colorectal lamina propria areas.

Artificial dermal substitutes for tissue regeneration: comparison of the clinical outcomes and histological findings of two templates.

OBJECTIVE: Artificial dermal substitutes (DSs) are fundamental in physiological wound healing to ensure consistent and enduring wound closure and provide a suitable scaffold to repair tissue. We compared the clinical and histological features of two DSs, Pelnac and Integra, in the treatment of traumatic and iatrogenic skin defects. METHODS: This prospective observational study involved 71 randomly selected patients from our hospital. Wound healing was analyzed using the Wound Surface Area Assessment, the Vancouver Scar Scale, and a visual analog scale. Histological and immunohistochemical evaluations were also performed. RESULTS: At 2 weeks, greater regeneration with respect to proliferation of the epidermis and renewal of the dermis was observed with Pelnac than with Integra. At 4 weeks, the dermis had regenerated with both DSs. Both templates induced renewed collagen and revascularization. Differences in the Vancouver Scar Scale score were statistically significant at 4 weeks and 1 year. Pelnac produced a significant increase in contraction at 2 weeks with increasing effectiveness at 4 weeks. Integra produced a higher percentage reduction in the wound surface area and a shorter healing time than Pelnac for wounds >1.5 cm deep. CONCLUSION: Our observational data indicate that both DSs are effective and applicable in different clinical contexts.

Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.

OBJECTIVES: Among the factors that could determine neurological outcome after hypothermic circulatory arrest (HCA) rewarming is rarely considered. The optimal rewarming rate is still unknown. The goal of this study was to investigate the effects of 2 different protocols for rewarming after HCA on neurological outcome in an experimental animal model. METHODS: Forty-four Sprague Dawley rats were cooled to 19 ± 1°C body core temperature by cardiopulmonary bypass (CPB). HCA was maintained for 60 min. Animals were randomized to receive slow (90 min) or fast (45 min) assisted rewarming with CPB to a target temperature of 35°C. After a total of 90 min of reperfusion in both groups, brain samples were collected and analysed immunohistochemically and with immunofluorescence. In 10 rats, magnetic resonance imaging was performed after 2 and after 24 h to investigate cerebral perfusion and cerebral oedema. RESULTS: Interleukin 6, chemokine (C-C motif) ligand 5, intercellular adhesion molecule 1 and tumour necrosis factor α in the hippocampus are significantly less expressed in the slow rewarming group, and microglia cells are significantly less activated in the slow rewarming group. Magnetic resonance imaging analysis demonstrated better cerebral perfusion and less water content in brains that underwent slow rewarming at 2 and 24 h. CONCLUSIONS: Slow rewarming after HCA might be superior to fast rewarming in neurological outcome. The present experimental study demonstrated reduction in the inflammatory response, reduction of inflammatory cell activation in the brain, enhancement of cerebral blood flow and reduction of cerebral oedema when slow rewarming was applied.

Cocoa Flavonoids Reduce Inflammation and Oxidative Stress in a Myocardial Ischemia-Reperfusion Experimental Model.

: Consumption of flavonoid-rich nutraceuticals has been associated with a reduction in coronary events. The present study analyzed the effects of cocoa flavonols on myocardial injury following acute coronary ischemia-reperfusion (I/R). A commercially available cocoa extract was identified by chromatographic mass spectrometry. Nineteen different phenolic compounds were identified and 250 mg of flavan-3-ols (procyanidin) were isolated in 1 g of extract. Oral administration of cocoa extract in incremental doses from 5 mg/kg up to 25 mg/kg daily for 15 days in Sprague Dawley rats (n = 30) produced a corresponding increase of blood serum polyphenols and become constant after 15 mg/kg. Consequently, the selected dose (15 mg/kg) of cocoa extract was administered orally daily for 15 days in a treated group (n = 10) and an untreated group served as control (n = 10). Both groups underwent surgical occlusion of the left anterior descending coronary artery and reperfusion. Cocoa extract treatment significantly reversed membrane peroxidation, nitro-oxidative stress, and decreased inflammatory markers (IL-6 and NF-kB) caused by myocardial I/R injury and enhanced activation of both p-Akt and p-Erk1/2. Daily administration of cocoa extract in rats is protective against myocardial I/R injury and attenuate nitro-oxidative stress, inflammation, and mitigates myocardial apoptosis.

The Anti-Apoptotic Effect of ASC-Exosomes in an In Vitro ALS Model and Their Proteomic Analysis.

Stem cell therapy represents a promising approach in the treatment of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). The beneficial effect of stem cells is exerted by paracrine mediators, as exosomes, suggesting a possible potential use of these extracellular vesicles as non-cell based therapy. We demonstrated that exosomes isolated from adipose stem cells (ASC) display a neuroprotective role in an in vitro model of ALS. Moreover, the internalization of ASC-exosomes by the cells was shown and the molecules and the mechanisms by which exosomes could exert their beneficial effect were addressed. We performed for the first time a comprehensive proteomic analysis of exosomes derived from murine ASC. We identified a total of 189 proteins and the shotgun proteomics analysis revealed that the exosomal proteins are mainly involved in cell adhesion and negative regulation of the apoptotic process. We correlated the protein content to the anti-apoptotic effect of exosomes observing a downregulation of pro-apoptotic proteins Bax and cleaved caspase-3 and upregulation of anti-apoptotic protein Bcl-2 α, in an in vitro model of ALS after cell treatment with exosomes. Overall, this study shows the neuroprotective effect of ASC-exosomes after their internalization and their global protein profile, that could be useful to understand how exosomes act, demonstrating that they can be employed as therapy in neurodegenerative diseases.

Bitter tastants and artificial sweeteners activate a subset of epithelial cells in acute tissue slices of the rat trachea.

Bitter and sweet receptors (T2Rs and T1Rs) are expressed in many extra-oral tissues including upper and lower airways. To investigate if bitter tastants and artificial sweeteners could activate physiological responses in tracheal epithelial cells we performed confocal Ca2+ imaging recordings on acute tracheal slices. We stimulated the cells with denatonium benzoate, a T2R agonist, and with the artificial sweeteners sucralose, saccharin and acesulfame-K. To test cell viability we measured responses to ATP. We found that 39% of the epithelial cells responding to ATP also responded to bitter stimulation with denatonium benzoate. Moreover, artificial sweeteners activated different percentages of the cells, ranging from 5% for sucralose to 26% for saccharin, and 27% for acesulfame-K. By using carbenoxolone, a gap junction blocker, we excluded that responses were mainly mediated by Ca2+ waves through cell-to-cell junctions. Pharmacological experiments showed that both denatonium and artificial sweeteners induced a PLC-mediated release of Ca2+ from internal stores. In addition, bitter tastants and artificial sweeteners activated a partially overlapping subpopulation of tracheal epithelial cells. Our results provide new evidence that a subset of ATP-responsive tracheal epithelial cells from rat are activated by both bitter tastants and artificial sweeteners.

Glucose transporter expression in the human colon.

AIM: To investigate by immunostaining glucose transporter expression in human colorectal mucosa in controls and patients with inflammatory bowel disease (IBD). METHODS: Colorectal samples were obtained from patients undergoing lower endoscopic colonoscopy or recto-sigmoidoscopy. Patients diagnosed with ulcerative colitis (n = 18) or Crohn's disease (n = 10) and scheduled for diagnostic colonoscopy were enrolled. Patients who underwent colonoscopy for prevention screening of colorectal cancer or were followed-up after polypectomy or had a history of lower gastrointestinal symptoms were designated as the control group (CTRL, n = 16). Inflammatory status of the mucosa at the sampling site was evaluated histologically and/or endoscopically. A total of 147 biopsies of colorectal mucosa were collected and processed for immunohistochemistry analysis. The expression of GLUT2, SGLT1, and GLUT5 glucose transporters was investigated using immunoperoxidase labeling. To compare immunoreactivity of GLUT5 and LYVE-1, which is a marker for lymphatic vessel endothelium, double-labeled confocal microscopy was used. RESULTS: Immunohistochemical analysis revealed that GLUT2, SGLT1, and GLUT5 were expressed only in short epithelial portions of the large intestinal mucosa. No important differences were observed in glucose transporter expression between the samples obtained from the different portions of the colorectal tract and between the different patient groups. Unexpectedly, GLUT5 expression was also identified in vessels, mainly concentrated in specific areas where the vessels were clustered. Immunostaining with LYVE-1 and GLUT5 antibodies revealed that GLUT5-immunoreactive (-IR) clusters of vessels were concentrated in areas internal to those that were LYVE-1 positive. GLUT5 and LYVE-1 did not appear to be colocalized but rather showed a close topographical relationship on the endothelium. Based on their LYVE-1 expression, GLUT5-IR vessels were identified as lymphatic. Both inflamed and non-inflamed mucosal colorectal tissue biopsies from the IBD and CTRL patients showed GLUT5-IR clusters of lymphatic vessels. CONCLUSION: Glucose transporter immunoreactivity is present in colorectal mucosa in controls and IBD patients. GLUT5 expression is also associated with lymphatic vessels. This novel finding aids in the characterization of lymphatic vasculature in IBD patients.

Theranostic Role of 32P-ATP as Radiopharmaceutical for the Induction of Massive Cell Death within Avascular Tumor Core.

Drug inaccessibility to vast areas of the tumor parenchyma is amongst the major hurdles for conventional therapies. Treatment efficacy rapidly decreases with distance from vessels and most of the tumor cells survive therapy. Also, between subsequent cycles of treatment, spared cancer cells replace those killed near the vessels, improving their access to nutrients, boosting their proliferation rate, and thus enabling tumor repopulation. Because of their property of "acting at a distance," radioisotopes are believed to overcome the physical barrier of vascular inaccessibility. Methods A novel molecular imaging tool called Cerenkov Luminescence Imaging (CLI) was employed for the detection of Cerenkov radiation emitted by beta particles, allowing in vivo tracking of beta-emitters. More precisely we investigated using a xenograft model of colon carcinoma the potential use of 32P-ATP as a novel theranostic radiopharmaceutical for tracing tumor lesions while simultaneously hampering their growth. Results Our analyses demonstrated that 32P-ATP injected into tumor-bearing mice reaches tumor lesions and persists for days and weeks within the tumor parenchyma. Also, the high-penetrating beta particles of 32P-ATP exert a "cross-fire" effect that induces massive cell death throughout the entire tumor parenchyma including core regions. Conclusion Our findings suggest 32P-ATP treatment as a potential approach to complement conventional therapies that fail to reach the tumor core and to prevent tumor repopulation.

All muscarinic acetylcholine receptors (M1-M5) are expressed in murine brain microvascular endothelium.

Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors.

Are they in or out? The elusive interaction between Qtracker ® 800 vascular labels and brain endothelial cells.

AIM: Qtracker(®)800 Vascular labels (Qtracker(®)800) are promising biomedical tools for high-resolution vasculature imaging; their effects on mouse and human endothelia, however, are still unknown. MATERIALS & METHODS: Qtracker(®)800 were injected in Balb/c mice, and brain endothelium uptake was investigated by transmission electron microscopy 3-h post injection. We then investigated, in vitro, the effects of Qtracker(®)800 exposure on mouse and human endothelial cells by calcium imaging. RESULTS: Transmission electron microscopy images showed nanoparticle accumulation in mouse brain endothelia. A subset of mouse and human endothelial cells generated intracellular calcium transients in response to Qtracker(®)800. CONCLUSION: Qtracker(®)800 nanoparticles elicit endothelial functional responses, which prompts biomedical safety evaluations and may bias the interpretation of experimental studies involving vascular imaging.

Oxygenator Is the Main Responsible for Leukocyte Activation in Experimental Model of Extracorporeal Circulation: A Cautionary Tale.

In order to assess mechanisms underlying inflammatory activation during extracorporeal circulation (ECC), several small animal models of ECC have been proposed recently. The majority of them are based on home-made, nonstandardized, and hardly reproducible oxygenators. The present study has generated fundamental information on the role of oxygenator of ECC in activating inflammatory signaling pathways on leukocytes, leading to systemic inflammatory response, and organ dysfunction. The present results suggest that experimental animal models of ECC used in translational research on inflammatory response should be based on standardized, reproducible oxygenators with clinical characteristics.

Chronic overcirculation-induced pulmonary arterial hypertension in aorto-caval shunt.

Pulmonary arterial hypertension is a common complication of congenital heart defects with left-to-right shunts. Current preclinical models do not reproduce clinical characteristics of shunt-related pulmonary hypertension. Aorto-caval shunt was firstly described as a model of right ventricle volume overload. The pathophysiology and the possible determination of pulmonary arterial hypertension of different periods of shunt exposure are still undefined. A method to create standardized, reproducible aorto-caval shunt was developed in growing rats (260±40 g). Three groups of animals were considered: shunt exposure for 10 weeks, shunt exposure for 20 weeks and control (sham laparotomy). Echocardiography and magnetic resonance revealed increased right ventricular end diastolic area in shunt at 10 weeks compared to control. Hemodynamic analysis demonstrated increased right ventricular afterload and increased effective pulmonary arterial elastance (Ea) in shunt at 20 weeks compared to control (1.29±0.20 vs. 0.14±0.06 mmHg/µl, p=0.004). At the same time point, the maximal slope of end-systolic pressure-volume relationship (Ees) decreased (0.5±0.2 mmHg/ml vs. 1.2±0.3, p<0.001). Consequently, right ventricular-arterial coupling was markedly deteriorated with a ≈50% decrease in the ratio of end-systolic to pulmonary artery elastance (Ees/Ea). Finally, left ventricular preload diminished (≈30% decrease in left ventricular end-diastolic volume). Histology demonstrated medial hypertrophy and small artery luminal narrowing. Chronic exposure to aorto-caval shunt is a reliable model to produce right ventricular volume overload and secondary pulmonary arterial hypertension. This model could be an alternative with low mortality and high reproducibility for investigators on the underlying mechanisms of shunt-related pulmonary hypertension.

Expansion of necrotic core and shedding of Mertk receptor in human carotid plaques: a role for oxidized polyunsaturated fatty acids?

AIMS: Expansion of necrotic core (NC), a major feature responsible for plaque disruption, is likely the consequence of accelerated macrophage apoptosis coupled with defective phagocytic clearance (efferocytosis). The cleavage of the extracellular domain of Mer tyrosine kinase (Mertk) by metallopeptidase domain17 (Adam17) has been shown to produce a soluble Mertk protein (sMer), which can inhibit efferocytosis. Herein, we analysed the expression and localization of Mertk and Adam17 in the tissue around the necrotic core (TANC) and in the periphery (P) of human carotid plaques. Then we studied the mechanisms of NC expansion by evaluating which components of TANC induce Adam17 and the related cleavage of the extracellular domain of Mertk. METHODS AND RESULTS: We studied 97 human carotid plaques. The expression of Mertk and Adam17 was found to be higher in TANC than in P (P < 0.001). By immunohistochemistry, Mertk was higher than Adam17 in the area of TANC near to the lumen (P < 0.01) but much lower in the area close to NC (P < 0.01). The extract of this portion of TANC increased the expression (mRNA) of Adam17 and Mertk (P < 0.01) in macrophage-like THP-1 cells but it also induced the cleavage of the extracellular domain of Mertk, generating sMer in the medium (P < 0.01). This effect of TANC extract was most evoked by its content in F(2)-isoprostanes, hydroxyoctadecadienoic acids, and hydroxytetraenoic acids. CONCLUSION: Some oxidized derivatives of polyunsaturated fatty acids contained in TANC of human carotid plaques are strong inducers of Adam17, which in turn leads to the generation of sMer, which can inhibit efferocytosis.