Hallmarks of pancreatic cancer: spotlight on TAM receptors.

Pancreatic ductal adenocarcinoma (PDAC) represents the most prevalent type of pancreatic cancer and ranks among the most aggressive tumours, with a 5-year survival rate of less than 11%. Projections indicate that by 2030, it will become the second leading cause of cancer-related deaths. PDAC presents distinctive hallmarks contributing to its dismal prognosis: (i) late diagnosis, (ii) heterogenous and complex mutational landscape, (iii) high metastatic potential, (iv) dense fibrotic stroma, (v) immunosuppressive microenvironment, and (vi) high resistance to therapy. Mounting evidence has shown a role for TAM (Tyro3, AXL, MerTK) family of tyrosine kinase receptors in PDAC initiation and progression. This review aims to describe the impact of TAM receptors on the defining hallmarks of PDAC and discuss potential future directions using these proteins as novel biomarkers for early diagnosis and targets for precision therapy in PDAC, an urgent unmet clinical need.

Dynamic changes in immune cell populations by AXL kinase targeting diminish liver inflammation and fibrosis in experimental MASH.

Background and aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Methods: Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. Results: In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB- NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Conclusion: Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

Induction of the Inflammasome Pathway by Tyrosine Kinase Inhibitors Provides an Actionable Therapeutic Target for Hepatocellular Carcinoma.

During the last decade, tyrosine kinase inhibitors (TKIs) sorafenib and regorafenib have been standard systemic treatments for advanced hepatocellular carcinoma (HCC). Previous data associated sorafenib with inflammasome activation. However, the role of the inflammasome in sorafenib and regorafenib signaling has not been described in liver cancer patients. For this purpose, we analyzed inflammasome-related transcriptomic changes in a murine HCC model. Our data confirmed inflammasome activation after both TKI treatments, sharing a similar pattern of increased gene expression. According to human database results, transcriptional increase of inflammasome genes is associated with poorer prognosis for male liver cancer patients, suggesting a sex-dependent role for inflammasome activation in HCC therapy. In biopsies of HCC and its surrounding tissue, we detected durable increases in the inflammasome activation pattern after sorafenib or regorafenib treatment in male patients. Further supporting its involvement in sorafenib action, inflammasome inhibition (MCC950) enhanced sorafenib anticancer activity in experimental HCC models, while no direct in vitro effect was observed in HCC cell lines. Moreover, activated human THP-1 macrophages released IL-1ß after sorafenib administration, while 3D Hep3B spheres displayed increased tumor growth after IL-1ß addition, pointing to the liver microenvironment as a key player in inflammasome action. In summary, our results unveil the inflammasome pathway as an actionable target in sorafenib or regorafenib therapy and associate an inflammasome signature in HCC and surrounding tissue with TKI administration. Therefore, targeting inflammasome activation, principally in male patients, could help to overcome sorafenib or regorafenib resistance and enhance the efficacy of TKI treatments in HCC.

GAS6/TAM Axis as Therapeutic Target in Liver Diseases.

TAM (TYRO3, AXL, and MERTK) protein tyrosine kinase membrane receptors and their vitamin K-dependent ligands GAS6 and protein S (PROS) are well-known players in tumor biology and autoimmune diseases. In contrast, TAM regulation of fibrogenesis and the inflammation mechanisms underlying metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and, ultimately, liver cancer has recently been revealed. GAS6 and PROS binding to phosphatidylserine exposed in outer membranes of apoptotic cells links TAMs, particularly MERTK, with hepatocellular damage. In addition, AXL and MERTK regulate the development of liver fibrosis and inflammation in chronic liver diseases. Acute hepatic injury is also mediated by the TAM system, as recent data regarding acetaminophen toxicity and acute-on-chronic liver failure have uncovered. Soluble TAM-related proteins, mainly released from activated macrophages and hepatic stellate cells after hepatic deterioration, are proposed as early serum markers for disease progression. In conclusion, the TAM system is becoming an interesting pharmacological target in liver pathology and a focus of future biomedical research in this field.

Direct actions of dapagliflozin and interactions with LCZ696 and spironolactone on cardiac fibroblasts of patients with heart failure and reduced ejection fraction.

AIMS: Inhibitors of SGLT2 (SGLT2i) have shown a positive impact in patients with chronic heart failure and reduced ejection fraction (HFrEF). Nonetheless, the direct effects of SGLT2i on cardiac cells and how their association with main drugs used for HFrEF affect the behaviour and signalling pathways of myocardial fibroblasts are still unknown. We aimed to determine the effects of dapagliflozin alone and in combination with sacubitril/valsartan (LCZ696) or spironolactone on the function of myocardial fibroblasts of patients with heart failure and reduced ejection fraction (HFrEF). METHODS AND RESULTS: Myocardial fibroblasts isolated from HFrEF patients (n = 5) were treated with dapagliflozin alone (1 nM-1 µM) or combined with LCZ696 (100 nM) or spironolactone (100 nM). The migratory rate was determined by wound-healing scratch assay. Expression of heart failure (HF) markers and signalling pathways activation were analysed with multiplexed protein array. Commercially available cardiac fibroblasts from healthy donors were used as Control (n = 4). Fibroblasts from HFrEF show higher migratory rate compared with control (P = 0.0036), and increased expression of HF markers [fold-change (Log2): COL1A1-1.3; IL-1b-1.9; IL-6-1.7; FN1-2.9 (P < 0.05)]. Dapagliflozin slowed the migration rate of HFrEF fibroblasts in a dose-dependent manner and markedly decreased the expression of IL-1ß, IL-6, MMP3, MMP9, GAL3, and FN1. SGLT2i had no effect on control fibroblasts. These effects were associated with decreased phosphorylation of AKT/GSK3 and PYK2 kinases and the signal transducer and activator of transcription (STAT). A combination of dapagliflozin + LCZ696 further decreased fibroblast migration, although it did not have a significant effect on the regulation of signalling pathways and the expression of biomarkers induced by SGLT2 inhibition alone. In contrast, the combination of dapagliflozin + spironolactone did not change the migration rate of fibroblast but significantly altered SGLT2i responses on MMP9, GAL3, and IL-1b expression, in association with increased phosphorylation of the kinases AKT/GSK3 and ERK1/2. CONCLUSIONS: SGLT2i, LCZ696, and spironolactone modulate the function of isolated myocardial fibroblasts from HFrEF patients through the activation of different signalling pathways. The combination of SGLT2i + LCZ696 shows an additive effect on migration, while spironolactone modifies the signalling pathways activated by SGLT2i and its beneficial effects of biomarkers of heart failure.

Hepatocellular Carcinoma: Molecular Pathogenesis and Therapeutic Advances.

Hepatocellular carcinoma (HCC), the most common form of liver cancer, continues to be a serious medical problem with poor prognosis, without major therapeutic improvement for years and increasing incidence. Fortunately, advances in systemic treatment options are finally arriving for HCC patients. After a decade of sorafenib as a standard therapy for advanced HCC, several tyrosine kinase inhibitors (TKIs), antiangiogenic antibodies, and immune checkpoint inhibitors have reached the clinic. Although infections by hepatitis B virus and hepatitis C virus remain principal factors for HCC development, the rise of non- alcoholic steatohepatitis from diabetes mellitus or metabolic syndrome is impeding HCC decline. Knowledge of specific molecular mechanisms, based on the etiology and the HCC microenvironment that influence tumor growth and immune control, will be crucial for physician decision-making among a variety of drugs to prescribe. In addition, markers of treatment efficacy are needed to speed the movement of patients towards other potentially effective treatments. Consequently, research to provide scientific data for the evidence-based management of liver cancer is guaranteed in the coming years and discussed here.

Soluble AXL is a novel blood marker for early detection of pancreatic ductal adenocarcinoma and differential diagnosis from chronic pancreatitis.

BACKGROUND: Early diagnosis is crucial for patients with pancreatic ductal adenocarcinoma (PDAC). The AXL receptor tyrosine kinase is proteolytically processed releasing a soluble form (sAXL) into the blood stream. Here we explore the use of sAXL as a biomarker for PDAC. METHODS: AXL was analysed by immunohistochemistry in human pancreatic tissue samples. RNA expression analysis was performed using TCGA/GTEx databases. The plasma concentrations of sAXL, its ligand GAS6, and CA19-9 were studied in two independent cohorts, the HMar cohort (n = 59) and the HClinic cohort (n = 142), including healthy controls, chronic pancreatitis (CP) or PDAC patients, and in a familial PDAC cohort (n = 68). AXL expression and sAXL release were studied in PDAC cell lines and murine models. FINDINGS: AXL is increased in PDAC and precursor lesions as compared to CP or controls. sAXL determined in plasma from two independent cohorts was significantly increased in the PDAC group as compared to healthy controls or CP patients. Patients with high levels of AXL have a lower overall survival. ROC analysis of the plasma levels of sAXL, GAS6, or CA19-9 in our cohorts revealed that sAXL outperformed CA19-9 for discriminating between CP and PDAC. Using both sAXL and CA19-9 increased the diagnostic value. These results were validated in murine models, showing increased sAXL specifically in animals developing PDAC but not those with precursor lesions or acinar tumours. INTERPRETATION: sAXL appears as a biomarker for early detection of PDAC and PDAC-CP discrimination that could accelerate treatment and improve its dismal prognosis. FUNDING: This work was supported by grants PI20/00625 (PN), RTI2018-095672-B-I00 (AM and PGF), PI20/01696 (MG) and PI18/01034 (AC) from MICINN-FEDER and grant 2017/SGR/225 (PN) from Generalitat de Catalunya.

Antioxidants Threaten Multikinase Inhibitor Efficacy against Liver Cancer by Blocking Mitochondrial Reactive Oxygen Species.

Sorafenib and regorafenib, multikinase inhibitors (MKIs) used as standard chemotherapeutic agents for hepatocellular carcinoma (HCC), generate reactive oxygen species (ROS) during cancer treatment. Antioxidant supplements are becoming popular additions to our diet, particularly glutathione derivatives and mitochondrial-directed compounds. To address their possible interference during HCC chemotherapy, we analyzed the effect of common antioxidants using hepatoma cell lines and tumor spheroids. In liver cancer cell lines, sorafenib and regorafenib induced mitochondrial ROS production and potent cell death after glutathione depletion. In contrast, cabozantinib only exhibited oxidative cell death in specific HCC cell lines. After sorafenib and regorafenib administration, antioxidants such as glutathione methyl ester and the superoxide scavenger MnTBAP decreased cell death and ROS production, precluding the MKI activity against hepatoma cells. Interestingly, sorafenib-induced mitochondrial damage caused PINK/Parkin-dependent mitophagy stimulation, altered by increased ROS production. Finally, in sorafenib-treated tumor spheroids, while ROS induction reduced tumor growth, antioxidant treatments favored tumor development. In conclusion, the anti-tumor activity of specific MKIs, such as regorafenib and sorafenib, is altered by the cellular redox status, suggesting that uncontrolled antioxidant intake during HCC treatment should be avoided or only endorsed to diminish chemotherapy-induced side effects, always under medical scrutiny.

Electrochemical Immunosensing of ST2: A Checkpoint Target in Cancer Diseases.

A magnetic beads (MB)-involved amperometric immunosensor for the determination of ST2, a member of the IL1 receptor family, is reported in this work. The method utilizes a sandwich immunoassay and disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates built on the surface of carboxylic acid-microsized magnetic particles (HOOC-MBs) were used to selectively capture ST2. A biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP) was used to accomplish the sandwiching of the target protein. The immune platform exhibits great selectivity and a low limit of detection (39.6 pg mL-1) for ST2, allowing the determination of soluble ST2 (sST2) in plasma samples from healthy individuals and patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in only 45 min once the immunoconjugates have been prepared. The good correlation of the obtained results with those provided by an ELISA kit performed using the same immunoreagents demonstrates the potential of the developed strategy for early diagnosis and/or prognosis of the fatal PDAC disease.

Growth Arrest-Specific Factor 6 (GAS6) Is Increased in COVID-19 Patients and Predicts Clinical Outcome.

BACKGROUND: Growth arrest-specific factor 6 (GAS6) and the Tyro3, AXL, and MERTK (TAM) receptors counterbalance pro-inflammatory responses. AXL is a candidate receptor for SARS-CoV-2, particularly in the respiratory system, and the GAS6/AXL axis is targeted in current clinical trials against COVID-19. However, GAS6 and TAMs have not been evaluated in COVID-19 patients at emergency admission. METHODS: Plasma GAS6, AXL, and MERTK were analyzed in 132 patients consecutively admitted to the emergency ward during the first peak of COVID-19. RESULTS: GAS6 levels were higher in the SARS-CoV-2-positive patients, increasing progressively with the severity of the disease. Patients with initial GAS6 at the highest quartile had the worst outcome, with a 3-month survival of 65%, compared to a 90% survival for the rest. Soluble AXL exhibited higher plasma concentration in deceased patients, without significant differences in MERTK among SARS-CoV-2-positive groups. GAS6 mRNA was mainly expressed in alveolar cells and AXL in airway macrophages. Remarkably, THP-1 human macrophage differentiation neatly induces AXL, and its inhibition (bemcentinib) reduced cytokine production in human macrophages after LPS challenge. CONCLUSIONS: Plasma GAS6 and AXL levels reflect COVID-19 severity and could be early markers of disease prognosis, supporting a relevant role of the GAS6/AXL system in the immune response in COVID-19.

Role of Vitamin K-Dependent Factors Protein S and GAS6 and TAM Receptors in SARS-CoV-2 Infection and COVID-19-Associated Immunothrombosis.

The vitamin K-dependent factors protein S (PROS1) and growth-arrest-specific gene 6 (GAS6) and their tyrosine kinase receptors TYRO3, AXL, and MERTK, the TAM subfamily of receptor tyrosine kinases (RTK), are key regulators of inflammation and vascular response to damage. TAM signaling, which has largely studied in the immune system and in cancer, has been involved in coagulation-related pathologies. Because of these established biological functions, the GAS6-PROS1/TAM system is postulated to play an important role in SARS-CoV-2 infection and progression complications. The participation of the TAM system in vascular function and pathology has been previously reported. However, in the context of COVID-19, the role of TAMs could provide new clues in virus-host interplay with important consequences in the way that we understand this pathology. From the viral mimicry used by SARS-CoV-2 to infect cells, to the immunothrombosis that is associated with respiratory failure in COVID-19 patients, TAM signaling seems to be involved at different stages of the disease. TAM targeting is becoming an interesting biomedical strategy, which is useful for COVID-19 treatment now, but also for other viral and inflammatory diseases in the future.

Regorafenib Alteration of the BCL-xL/MCL-1 Ratio Provides a Therapeutic Opportunity for BH3-Mimetics in Hepatocellular Carcinoma Models.

BACKGROUND: The multikinase inhibitor regorafenib, approved as second-line treatment for hepatocellular carcinoma (HCC) after sorafenib failure, may induce mitochondrial damage. BH3-mimetics, inhibitors of specific BCL-2 proteins, are valuable drugs in cancer therapy to amplify mitochondrial-dependent cell death. METHODS: In in vitro and in vivo HCC models, we tested regorafenib's effect on the BCL-2 network and the efficacy of BH3-mimetics on HCC treatment. RESULTS: In hepatoma cell lines and Hep3B liver spheroids, regorafenib cytotoxicity was potentiated by BCL-xL siRNA transfection or pharmacological inhibition (A-1331852), while BCL-2 antagonism had no effect. Mitochondrial outer membrane permeabilization, cytochrome c release, and caspase-3 activation mediated A-1331852/regorafenib-induced cell death. In a patient-derived xenograft (PDX) HCC model, BCL-xL inhibition stimulated regorafenib activity, drastically decreasing tumor growth. Moreover, regorafenib-resistant HepG2 cells displayed increased BCL-xL and reduced MCL-1 expression, while A-1331852 reinstated regorafenib efficacy in vitro and in a xenograft mouse model. Interestingly, BCL-xL levels, associated with poor prognosis in liver and colorectal cancer, and the BCL-xL/MCL-1 ratio were detected as being increased in HCC patients. CONCLUSION: Regorafenib primes tumor cells to BH3-mimetic-induced cell death, allowing BCL-xL inhibition with A-1331852 or other strategies based on BCL-xL degradation to enhance regorafenib efficacy, offering a novel approach for HCC treatment, particularly for tumors with an elevated BCL-xL/MCL-1 ratio.

A Functional Role of GAS6/TAM in Nonalcoholic Steatohepatitis Progression Implicates AXL as Therapeutic Target.

BACKGROUND AND AIMS: GAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development. METHODS: GAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl-/-, Mertk-/- and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients. RESULTS: In primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD- and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibrotic- and inflammatory-related genes in HFD-fed mice after bemcentinib administration. HFD-fed Mertk-/- mice exhibited enhanced NASH, while Axl-/- mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH. CONCLUSION: AXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, reduces experimental NASH, revealing AXL as an effective therapeutic target for clinical practice.

Axl expression is increased in early stages of left ventricular remodeling in an animal model with pressure-overload.

BACKGROUND: AXL is a receptor tyrosine kinase that has been related to kidney and vascular disorders. Heart failure patients with reduced ejection fraction have higher AXL in serum than controls. No information about Axl expression with HF progression is available. METHODS: Thoracic transverse aortic constriction (TAC) was successfully performed on male Wistar rats (n = 25) with different constriction levels. Controls underwent sham surgery (n = 12). Echocardiography measurements were performed 4-8 weeks after surgery. Collagen deposition was measured with picrosirius red staining. Axl mRNA levels in left ventricle (LV), left kidney (LK) and ascending aorta (aAo) and the LV expression of cardiac remodeling and fibrogenic factors were quantified with real-time PCR. AXL LV protein levels were quantified with western blot and localization was analyzed by immunohistochemistry. Soluble AXL levels in plasma were assayed with ELISA. RESULTS: Successful TAC rats were classified into LV hypertrophy (LVH) or heart failure (HF), modeling the progressive cardiac changes after pressure overload. Collagen deposition was increased only in the HF group. LV Axl mRNA levels were higher in LVH and HF than in Sham rats, and correlated with LVHi, and hypertrophic and fibrogenic mediators. However, no association was found with LV systolic function. AXL was expressed in LV myocytes and other cell types. Concentration of circulating sAXL in plasma was increased in the LVH group compared to Sham and HF rats. Axl mRNA levels were similar in all groups in the LK and aAo. CONCLUSIONS: Axl expression pattern suggests a role in the early progression of LV remodeling in HF but not in the later systolic dysfunction. The higher levels of circulating AXL found in HF patients most probably shed from the heart.

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

Serum levels of Growth Arrest-Specific 6 protein and soluble AXL in patients with ST-segment elevation myocardial infarction.

BACKGROUND: Serum soluble AXL (sAXL) and its ligand, Growth Arrest-Specific 6 protein (GAS6), intervene in tissue repair processes. AXL is increased in end-stage heart failure, but the role of GAS6 and sAXL in ST-segment elevation myocardial infarction (STEMI) is unknown. OBJECTIVES: To study the association of sAXL and GAS6 acutely and six months following STEMI with heart failure and left ventricular remodelling. METHODS: GAS6 and sAXL were measured by enzyme-linked immunosorbent assay at one day, seven days and six months in 227 STEMI patients and 20 controls. Contrast-enhanced magnetic resonance was performed during admission and at six months to measure infarct size and left ventricular function. RESULTS: GAS6, but not sAXL, levels during admission were significantly lower in STEMI than in controls. AXL increased progressively over time (p<0.01), while GAS6 increased only from day 7. GAS6 or sAXL did not correlate with brain natriuretic peptide or infarct size. However, patients with heart failure (Killip >1) had higher values of sAXL at day 1 (48.9±11.9 vs. 44.0±10.7 ng/ml; p<0.05) and at six months (63.3±15.4 vs. 55.9±13.7 ng/ml; p<0.05). GAS6 levels were not different among subjects with heart failure or left ventricular remodelling. By multivariate analysis including infarct size, Killip class and sAXL at seven days, only the last two were independent predictors of left ventricular remodelling (odds ratio 2.24 (95% confidence interval: 1.08-4.63) and odds ratio 1.04 (95% confidence interval: 1.00-1.08) respectively). CONCLUSION: sAXL levels increased following STEMI. Patients with heart failure and left ventricular remodelling have higher sAXL levels acutely and at six month follow-up. These findings suggest a potential role of the GAS6-AXL system in the pathophysiology of left ventricular remodelling following STEMI.

High-Sensitivity Troponin T and Soluble Form of AXL as Long-Term Prognostic Biomarkers after Heart Transplantation.

ANTECEDENTS: Cardiac allograft vasculopathy (CAV) is a frequent complication limiting the long-term (>1 year) survival after heart transplantation (HTx). CAV is initiated by endothelial dysfunction and can lead to severe cardiovascular (CV) complications. Since CAV is often clinically silent, biomarkers could help identifying HTx patients at risk of CAV and their severe complications. AIM: Evaluate the clinical yield of high-sensitivity cardiac troponin T (hs-cTnT), marker of cardiomyocyte damage, and the soluble form of AXL (sAXL), biomarker of endothelial dysfunction, to assess the prognosis of long-term cardiovascular (CV) events occurring after HTx. METHODS: 96 patients were evaluated at least > 1 year after HTx. CAV was evaluated by coronary angiography or multisliced tomography, and hs-cTnT and sAXL measured 6 months before or after CAV evaluation. Patients were followed during 42 ± 15 months for a combined end point including cardiac death, angina or acute myocardial infarction, left ventricular ejection fraction < 50%, or heart failure not due to an acute rejection. RESULTS: 51 patients (53%) presented CAV at evaluation; 21 of them had CV events. Hs-cTnT (56 ± 45 versus 20 ± 18 ng/L; p = 0.04) and sAXL concentrations (98 ± 51 versus 26 ± 26 ng/L; p = 0.01) were significantly higher in patients with CV events. Hs-cTnT (HR 1.03; 95% CI 1.015-1.042, p = 0.0001) and sAXL (HR 1.01; 95% CI 1.001-1.019, p = 0.02) were independent predictors of CV events. A hs-cTnT concentration < 21 ng/L, detected by AUC ROC, predicted the absence of CV events with a predictive value of 91%; sAXL did not add more predictive value to hs-cTnT. Survival free of CV events was 92% in patients with hs-cTnT < 21 ng/L and 57% in those with hs-cTnT > 21 ng/L (p < 0.001). CONCLUSION: Hs-cTnT, but not sAXL, measured during the long-term follow-up of HTx patients appears as a helpful biomarker to identify patients at low risk of adverse CV outcomes.

Melatonin induces mechanisms of brain resilience against neurodegeneration.

Melatonin is an endogenous pleiotropic molecule which orchestrates regulatory functions and protective capacity against age-related ailments. The increase in circulating levels of melatonin through dietary supplements intensifies its health benefits. Investigations in animal models have shown that melatonin protects against Alzheimer's disease (AD)-like pathology, although clinical studies have not been conclusive. We hypothesized that melatonin induces changes in the brain that prevent or attenuate AD by increasing resilience. Therefore, we treated healthy nontransgenic (NoTg) and AD transgenic (3xTg-AD) 6-month-old mice with a daily dose of 10 mg/kg of melatonin until 12 months of age. As expected, melatonin reversed cognitive impairment and dementia-associated behaviors of anxiety and apathy and reduced amyloid and tau burden in 3xTg-AD mice. Remarkably, melatonin induced cognitive enhancement and higher wellness level-related behavior in NoTg mice. At the mechanism level, NF-κB and proinflammatory cytokine expressions were decreased in both NoTg and 3xTg-AD mice. The SIRT1 pathway of longevity and neuroprotection was also activated in both mouse strains after melatonin dosing. Furthermore, we explored new mechanisms and pathways not previously associated with melatonin treatment such as the ubiquitin-proteasome proteolytic system and the recently proposed neuroprotective Gas6/TAM pathway. The upregulation of proteasome activity and the modulation of Gas6 and its receptors by melatonin were similarly displayed by both NoTg and 3xTg-AD mice. Therefore, these results confirm the potential of melatonin treatment against AD pathology, by way of opening new pathways in its mechanisms of action, and demonstrating that melatonin induces cognitive enhancement and brain resilience against neurodegenerative processes.

The α7ß0 isoform of the complement regulator C4b-binding protein induces a semimature, anti-inflammatory state in dendritic cells.

The classical pathway complement regulator C4b-binding protein (C4BP) is composed of two polypeptides (α- and ß-chains), which form three plasma oligomers with different subunit compositions (α7ß1, α7ß0, and α6ß1). We show in this article that the C4BP α7ß0 isoform (hereafter called C4BP[ß(-)] [C4BP lacking the ß-chain]), overexpressed under acute-phase conditions, induces a semimature, tolerogenic state on human monocyte-derived dendritic cells (DCs) activated by a proinflammatory stimulus. C4BP isoforms containing ß-chain (α7ß1 and α6ß1; C4BP[ß(+)]) neither interfered with the normal maturation of DCs nor competed with C4BP(ß(-)) activity on these cells. Immature DCs (iDCs) treated with C4BP(ß(-)) retained high endocytic activity, but, upon LPS treatment, they did not upregulate surface expression of CD83, CD80, and CD86. Transcriptional profiling of these semimature DCs revealed that treatment with C4BP(ß(-)) prevented the induction of IDO and BIC-1, whereas TGF-ß1 expression was maintained to the level of iDCs. C4BP(ß(-))-treated DCs were also unable to release proinflammatory Th1 cytokines (IL-12, TNF-α, IFN-γ, IL-6, IL-8) and, conversely, increased IL-10 secretion. They prevented surface CCR7 overexpression and, accordingly, displayed reduced chemotaxis, being morphologically indistinguishable from iDCs. Moreover, C4BP(ß(-))-treated DCs failed to enhance allogeneic T cell proliferation, impairing IFN-γ production in these cells and, conversely, promoting CD4(+)CD127(low/neg)CD25(high)Foxp3(+) T cells. Deletion mutant analysis revealed that the complement control protein-6 domain of the α-chain is necessary for the tolerogenic activity of C4BP(ß(-)). Our data demonstrate a novel anti-inflammatory and immunomodulatory function of the complement regulator C4BP, suggesting a relevant role of the acute-phase C4BP(ß(-)) isoform in a number of pathophysiological conditions and potential applications in autoimmunity and transplantation.