Anexelekto (AXL) no more: microRNA-155 (miR-155) controls the "Uncontrolled" in SARS-CoV-2.

AXL is the gene that encodes the Anexelekto (AXL) receptor tyrosine kinase that demonstrates significant roles in various cellular processes, including cell growth, survival, and migration. Anexelekto is a Greek word meaning excessive and uncontrolled, semantically implying the crucial involvement of AXL in cancer and immune biology, and in promoting cancer metastasis. AXL overexpression appears to drive epithelial to mesenchymal transition, tumor angiogenesis, decreased antitumor immune response, and resistance to therapeutic agents. Recently, AXL has been reported to play important roles in several viral infections, including SARS-CoV-2. We have previously outlined the importance of microRNAs (miRNAs, miRs) and especially miR-155 in SARS-CoV-2 pathophysiology through regulation of the Renin-Angiotensin Aldosterone System (RAAS) and influence on several aspects of host innate immunity. MiRNAs are negative regulators of gene expression, decreasing the stability of target RNAs or limiting their translation and, enthrallingly, miR-155 is also involved in AXL homeostasis-both endogenously and pharmaceutically using repurposed drugs (e.g., metformin)-highlighting thrifty evolutionary host innate immunity mechanisms that successfully can thwart viral entry and replication. Cancer, infections, and immune system disturbances will increasingly involve miRNA diagnostics and therapeutics in the future.

Utility of Serum Growth Arrest-Specific Protein 6 as a Biomarker of Severity and Prognosis After Severe Traumatic Brain Injury: A Prospective Observational Study.

Objective: Growth arrest-specific protein 6 (Gas6) may harbor protective effects in acute brain injury. This study was designed to determine the relation of serum Gas6 levels to severity and prognosis after traumatic brain injury (TBI). Methods: In this prospective cohort study of 114 controls and 114 patients with severe TBI, multivariate analysis was used to assess relationships between serum Gas6 levels, Glasgow coma scale (GCS) score, Rotterdam computed tomography (CT) score, postinjury 180-day mortality, overall survival and poor prognosis (Extended Glasgow outcome scale score 1-4). Results: Significantly increased serum Gas6 levels of patients (median, 10.3 ng/mL versus 32.5 ng/mL; P < 0.001), as compared with controls, were independently correlated with Rotterdam CT score (t = 3.629, P < 0.001) and GCS score (t=-3.393, P = 0.001), and independently predicted 180-day mortality (odds ratio, 1.078; 95% confidence interval (CI), 1.007-1.154), overall survival (hazard ratio, 1.074; 95% CI, 1.012-1.139) and poor prognosis (odds ratio, 1.129; 95% CI, 1.059-1.205). Areas under receiver operating characteristic curve (AUCs) of serum Gas6 levels for discriminating risks of 180-day mortality and poor prognosis were 0.785 (95% CI, 0.699-0.857) and 0.793 (95% CI, 0.707-0.863), respectively; and serum Gas6 levels above 30.9 ng/mL and 28.3 ng/mL predicted 180-day mortality and poor prognosis with maximum Youden indices of 0.451 and 0.468, respectively. The predictive ability of serum Gas6 levels for mortality was similar to those of GCS score (AUC, 0.833; 95% CI, 0.751-0.896; P = 0.286) and Rotterdam CT score (AUC, 0.823; 95% CI, 0.740-0.888; P = 0.432). The discriminatory capability of serum Gas6 levels for the risk of poor prognosis was in the range of GCS score (AUC, 0.846; 95% CI, 0.766-0.906; P = 0.178) and Rotterdam CT score (AUC, 0.831; 95% CI, 0.750-0.895; P = 0.368). Conclusion: Serum Gas6 may appear as a promising biochemical parameter for aiding in the assessment of trauma severity and prediction of prognosis among patients with severe TBI.

Growth Arrest-Specific 6 in Chromophobe Renal Cell Carcinoma.

BACKGROUND: Overexpression of tumor-associated growth arrest-specific protein 6 (Gas6) is found in many tumor entities. The prognostic value of Gas6 in renal cell carcinoma (RCC), especially in non-clear cell RCC, is still unclear. AIM: The aim of the study was to evaluate the prognostic impact of Gas6 expression in a large cohort of patients with chromophobe RCC (chRCC). MATERIAL AND METHODS: Patients who underwent renal surgery due to chRCC were retrospectively evaluated. Tumor specimens were analyzed for Gas6 expression by immunohistochemistry. RESULTS: Eighty-one chRCC patients were eligible for analysis; of these, 24 (29.6%) patients were positive for Gas6. No significant associations were found for Gas6 expression and clinical attributes in patients with chRCC. The Kaplan-Meier analysis revealed no differences in 5-year overall survival for Gas6- compared to Gas6+ (89.6% vs. 100.0%; p = 0.288) tumors. CONCLUSION: In chRCC, Gas6 expression is not associated with survival and other parameters of aggressiveness. Due to the rare incidence of chRCC, further studies with larger cohorts are warranted.

Serum Gas6 contributes to clinical outcome after aneurysmal subarachnoid hemorrhage: A prospective cohort study.

BACKGROUND: Growth-arrest-specific protein 6 (Gas6) exerts nervous protective effects on acute brain injury. We endeavored to ascertain whether serum Gas6 concentrations are associated with severity, delayed cerebral ischemia (DCI) and prognosis following aneurysmal subarachnoid hemorrhage (aSAH). METHODS: We measured serum Gas6 concentrations of 124 aSAH patients. The Hunt-Hess scale and modified Fisher grading scale were used to evaluate illness severity. Multivariate analysis was utilized to determine relationships between serum Gas6 concentrations and severity, DCI plus 90-day unfavorable outcome (Glasgow outcome scale score of 1-3). RESULTS: Patients with unfavorable outcome or DCI had significantly higher serum Gas6 concentrations than other remainders (median, 35.0 vs 23.3 ng/ml; 36.1 vs 25.3 ng/ml; both P < 0.001). Serum Gas6 concentrations displayed independent correlations with Hunt-Hess scores (t = 5.518, P < 0.001) and modified Fisher scores (t = 3.531, P = 0.001). Serum Gas6 concentrations were independently associated with unfavorable outcome (OR: 1.125; 95% CI, 1.063-1.190; P = 0.014) and DCI (OR: 1.104; 95% CI, 1.041-1.170; P = 0.010) as well as exhibited AUCs of 0.786 (95% CI, 0.703-0.854) and 0.753 (95% CI, 0.668-0.826) for predicting unfavorable outcome and DCI respectively. Its discriminatory ability for risk of unfavorable outcome or DCI was similar to those of Hunt-Hess scores and modified Fisher scores (all P > 0.05). CONCLUSIONS: Serum Gas6 concentrations are independently associated with stroke severity and worse clinical outcome after aSAH, indicating serum Gas6 may be a potential prognostic biomarker for aSAH.

Inhibition of Gas6 promotes crystalline silica-induced inflammatory response of macrophages via blocking autophagy flux.

Inhalation of crystalline silica (CS) can cause silicosis, which is one of the most serious interstitial lung diseases worldwide. Autophagy dysfunction is an essential step in silicosis progression. In this study, we aim to identify the effect of growth arrest-specific protein 6 (Gas6) during autophagy induction and macrophage inflammatory response caused by CS. After RAW 264.7 macrophages exposed to CS, the levels of Gas6 and autophagy markers (p62, Beclin1, and LC3-II/LC3-I) were increased, accompanied with enhanced inflammatory cytokines secretion. Using autophagy activator (rapamycin) repressed, whereas autophagy inhibitor (3-methyladenine) promoted inflammatory cytokines release. Besides, inhibition of Gas6 aggravated CS-induced inflammatory response, and autophagy inhibition facilitated the promoted effect of Gas6 silencing, resulting in elevated expression of inflammatory cytokines. These findings reveal the protective effects of Gas6 and autophagy in macrophages in response to CS exposure, and highlight the autophagy regulated by Gas6 may be a potential prevention target for CS-induced lung inflammatory response.

Ginsenoside Rb1 attenuates age-associated vascular impairment by modulating the Gas6 pathway.

CONTEXT: Ginsenoside Rb1 (Rb1) exerts many beneficial effects and protects against cardiovascular disease. OBJECTIVE: To investigate whether Rb1 could attenuate age-related vascular impairment and identify the mechanism. MATERIALS AND METHODS: Female C57BL/6J mice aged 2 and 18 months, randomly assigned to Young, Young + 20 mg/kg Rb1, Old + vehicle, Old + 10 mg/kg Rb1 and Old + 20 mg/kg Rb1 groups, were daily intraperitoneal injected with vehicle or Rb1 for 3 months. The thoracic aorta segments were used to inspect the endothelium-dependent vasorelaxation. Left thoracic aorta tissues were collected for histological or molecular expression analyses, including ageing-related proteins, markers relevant to calcification and fibrosis, and expression of Gas6/Axl. RESULTS: We found that in Old + vehicle group, the expression of senescence proteins and cellular adhesion molecules were significantly increased, with worse endothelium-dependent thoracic aorta relaxation (58.35% ± 2.50%) than in Young group (88.84% ± 1.20%). However, Rb1 treatment significantly decreased the expression levels of these proteins and preserved endothelium-dependent relaxation in aged mice. Moreover, Rb1 treatment also reduced calcium deposition, collagen deposition, and the protein expression levels of collagen I and collagen III in aged mice. Furthermore, we found that the downregulation of Gas6 protein expression by 41.72% and mRNA expression by 52.73% in aged mice compared with young mice was abrogated by Rb1 treatment. But there was no significant difference on Axl expression among the groups. CONCLUSIONS: Our study confirms that Rb1 could ameliorate vascular injury, suggesting that Rb1 might be a potential anti-ageing related vascular impairment agent.

Gas6/Axl signaling pathway promotes proliferation, migration and invasion and inhibits apoptosis in A549 cells.

Several studies have demonstrated that growth arrest-specific protein 6 (Gas6) and Axl are highly expressed in various tumor tissues, such as renal cell and esophageal carcinoma. However, the effect of the Gas6/Axl signaling pathway on lung adenocarcinoma is still unclear. The aim of the present study was to investigate the effect of the Gas6/Axl signaling pathway on lung adenocarcinoma cells and its mechanism of action, which may provide a novel target for the clinical treatment of lung adenocarcinoma. Human lung adenocarcinoma tissues were used to examine the activation of the Gas6/Axl signaling pathway. In addition, the human lung adenocarcinoma cell line A549 was employed to study the effects of the Gas6/Axl signaling pathway on the proliferation, migration, invasion and apoptosis of lung adenocarcinoma cells. Recombinant human Gas6 protein and inhibitor TP-0903 were used to activate and inhibit the Gas6/Axl signaling pathway, respectively. The results revealed that Gas6 and Axl expression level was increased in human lung adenocarcinoma tissues compared with adjacent healthy tissues. After inhibition of the Gas6/Axl signaling pathway with TP-0903, p21, p53, caspase 3, caspase 8 and caspase 9 exhibited higher expression level in A549 cells. The opposite effect was observed when the Gas6/Axl signaling pathway was activated. In addition, the migratory and invasive ability of A549 cells was determined via wound-healing and Transwell invasion assays. The results indicated that the migratory and invasive ability of A549 cells was significantly increased when the Gas6/Axl signaling pathway was activated and inhibition of Gas6/Axl signaling pathway caused the opposite results. Activity of Gas6/Axl signaling pathway was shown to be positively associated with cell proliferation by Cell Counting Kit 8 and clone formation assays. In conclusion, the Gas6/Axl signaling pathway was revealed to promote the proliferation, migration and invasion and inhibit the apoptosis of lung adenocarcinoma cells, which serve important roles in the progression of lung adenocarcinoma.

Gamma-glutamyl carboxylated Gas6 facilitates the prophylactic effect of vitamin K in inhibiting hyperlipidemia-associated inflammatory pathophysiology via arresting MCP-1/ICAM-1 mediated monocyte-hepatocyte adhesion.

Role of growth arrest-specific 6 (Gas6), member of vitamin K (VK)-dependent protein family in hyperlipidemia-associated inflammation remains unresolved. To address this, blood samples were collected from hyperlipidemic subjects and age-matched healthy controls and observed that gamma-glutamyl carboxylated Gas6 (Gla-Gas6) but not total Gas6 were significantly lower while pro-inflammatory markers, MCP-1 and ICAM-1 were remarkably higher in hyperlipidemic subjects compared to control. Correlation analyses demonstrated that Gla-Gas6 levels were inversely correlated with MCP-1 and ICAM-1 but positively with plasma VK in hyperlipidemic subjects but not in control. This suggests that boosting VK level might ameliorate the hyperlipidemia-associated inflammatory pathophysiology via augmenting Gla-Gas6. Further studies with high fat diet (HFD)-fed mice demonstrated that VK supplementation (1, 3, and 5 µg/kg BW, 8 weeks) dose-dependently reduced both hepatic and plasma levels of MCP-1 and ICAM-1 while elevating that of Gla-Gas6 but not total Gas6 in HFD-fed mice. Cell culture studies with gamma-glutamyl carboxylase (enzyme causes VK-dependent carboxylation of Gas6) knockdown hepatocytes and monocytes dissected the direct role of Gla-Gas6 in inhibiting high palmitic acid (0.75 mM)-induced inflammation via arresting MCP-1/ICAM-1 mediated hepatocyte-monocyte adhesion. The present study demonstrated an important role of Gla-Gas6 in facilitating the prophylactic effect of VK against hyperlipidemia associated inflammation.

Screening of Differential Genes for Endometriosis and Expression Validation of the Potential Target GAS6 / 中国生物化学与分子生物学报

Growth arrest specific protein 6 (GAS6) plays an important role in the occurrence and development of tumors, and its signal transduction is involved in cell proliferation, adhesion and migration, but its related functions and molecular mechanisms in endometriosis (EMs) are still unclear. In this study, we searched and downloaded the transcriptome datasets of EMs from GEO database and performed GEO online analysis, and then screened out the differentially expressed genes and performed cluster analysis based on GO and KEGG pathway. The mRNA levels of the differentially expressed genes shared by more than three datasets were verified by qRT-PCR in the endometrium of ten women with no endometriosis and no clear disease and the ectopic endometrium of 11 patients with ovarian chocolate cysts. Immunohistochemistry and qRT-PCR were used to verify the expression of GAS6 and epithelial mesenchymal transition (EMT) marker genes, and immunofluorescence was used to co-label GAS6 and E-cadherin in endometriosis clinical samples. In this study, a total of 47 differentially expressed genes were screened out of the four transcriptome datasets, which were mainly enriched in processes such as cell migration and related signal pathways such as MAPK, PI3K-AKT, and tight junction. The mRNA levels of the nine differentially expressed genes shared by more than three datasets in endometriosis patients were consistent with the results of bioinformatics analysis. GAS6 expression levels in ectopic endometrium of EMs patients are higher than the control group (P < 0. 05), and EMs patients have the characteristics of EMT in the ectopic endometrial tissue, that is, the expression of E-cadherin is down-regulated (P < 0. 05) and the expression of vimentin is up-regulated (P < 0. 01). The expression of E-cadherin in the ectopic endometrial glandular epithelial cells of EMs patients is low while the expression of GAS6 is up-regulated, suggesting that GAS6 may mediate the EMT process in endometriosis. In conclusion, this study reveals that GAS6 is highly expressed in endometriosis patients and may mediate the EMT process to participate in the occurrence and development of endometriosis, providing a potential target for clinical treatment of endometriosis.

TAM receptors: A phosphatidylserine receptor family and its implications in viral infections.

Phosphatidylserine (PS) is an anionic phospholipid that is usually localized in the inner leaflets of the plasma membrane. However, the enzyme scramblase catalyzes the externalization of PS on the outer leaflet of the plasma membrane during apoptosis or cellular stress. This event prompts the recognition of PS displaying cells by phagocytes leading to "apoptotic clearance." Multiple PS receptors (PSRs) mediate this process including members from the TAM (Tyro3, Axl, Mertk) receptor Tyrosine kinases (RTKs) by interacting with PS via bridging proteins like Gas6 and ProS1. Ironically, this network (PS/TAM) that evolved for boosting cellular health through clearance of apoptotic and necrotic cells, has been manoeuvred by pathogens and tumor cells using "apoptotic mimicry." Enveloped viruses, responsible for most of the lethal epidemics and pandemics including the current SARS-CoV2 outbreak, have employed apoptotic mimicry to their advantage. In the current chapter, we summarize the existing knowledge regarding the involvement of PS/Gas6, ProS1/TAM in facilitating infectivity in a diverse set of cell lines, animals as well as organoids. This network executes a largely proviral role in facilitating infection as seen with Zika, Ebola, Influenza and Dengue viruses. However, this response varies with strains and the cells infected, and in some cases, this same signaling displays an antiviral function. We also report multiple studies that have used neutralizing antibodies and small molecule inhibitors in successfully reducing viral replication and ameliorating pathogenicity. Knowledge about this unique signaling pathway and measures that can be taken to inhibit it is most valuable now given how enveloped viruses lead to plagues on the entire globe.

[Role of growth arrest-specific protein 6 in migration and osteogenic differentiation of human periodontal ligament cells].

OBJECTIVE: To investigate the role of growth arrest-specific protein 6 (Gas6) in the process of the migration and osteogenic differentiation of human periodontal ligament cells (hPDLCs). METHODS: After different concentrations of recombinant human Gas6 (rhGas6) were added to hPDLCs, cell prolife-ration experiment (CCK-8) was taken to observe the effect of rhGas6 on hPDLCs cell proliferation. Scratch test and cell migration test (Transwell) were taken to analyze the migratory ability of hPDLCs in different concentrations of rhGas6 groups. After osteogenic induction, real-time quantitative polymerase chain reaction (real-time PCR) was taken to detect the expression of the Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP). ALP staining was used to detect the amount of mineralized nodules. RESULTS: After adding different concentrations of rhGas6, there were no statistically significant differences in hPDLCs cell proliferation among the experimental groups and the control group at 24, 48 and 72 hours (P>0.05). After 24 h of scratch, the healing area in the 800 µg/L of the rhGas6 group was greater than that in the control group, but without statistically significant difference (31.06%±13.70% vs. 21.79%±9.51%, P>0.05). In the migration test, after 24 h, the number of hPDLCs cells which penetrated through the membrane in the 800 µg/L rhGas6 group was significantly higher than that in the control group (P < 0.01). After rhGas6 was added and osteogenic induction, Runx2 and ALP gene expressions of hPDLCs in the 800 µg/L group were significantly higher than those in the control group (1.60±0.30 vs. 0.91±0.10, 2.81±0.61 vs. 0.86±0.12, P < 0.01). After Gas6 was knocked down, the ALP expression of hPDLCs was significantly lower than that of the control group (0.39±0.07 vs. 0.92±0.14, P < 0.01). There was no significant change in Runx2 expression (P>0.05). After 7 days of osteogenic induction, the mineralized nodules formed in the Gas6 knockdown group were significantly less than those in control group (0.25±0.04 vs. 1.00±0.11, P < 0.001). After 14 days of induction, the staining degree of the Gas6 knockdown group was lower than that of the control group, but there was no significant difference (0.86±0.04 vs. 1.00±0.16, P>0.05). CONCLUSION: After downregulation of Gas6 gene, mineralized nodule formation was reduced and ALP gene expressions were decreased in the early stage of osteogenic induction (7 days). After addition of rhGas6, Runx2 and ALP gene expressions were increased and the number of cell migration was increased, suggesting that Gas6 might play a promoting role in the migration and osteogenic differentiation of human periodontal ligament cells.

Role of growth arrest-specific protein 6 in migration and osteogenic differentiation of human periodontal ligament cells / 北京大学学报(医学版)

OBJECTIVE@#To investigate the role of growth arrest-specific protein 6 (Gas6) in the process of the migration and osteogenic differentiation of human periodontal ligament cells (hPDLCs).@*METHODS@#After different concentrations of recombinant human Gas6 (rhGas6) were added to hPDLCs, cell prolife-ration experiment (CCK-8) was taken to observe the effect of rhGas6 on hPDLCs cell proliferation. Scratch test and cell migration test (Transwell) were taken to analyze the migratory ability of hPDLCs in different concentrations of rhGas6 groups. After osteogenic induction, real-time quantitative polymerase chain reaction (real-time PCR) was taken to detect the expression of the Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP). ALP staining was used to detect the amount of mineralized nodules.@*RESULTS@#After adding different concentrations of rhGas6, there were no statistically significant differences in hPDLCs cell proliferation among the experimental groups and the control group at 24, 48 and 72 hours (P>0.05). After 24 h of scratch, the healing area in the 800 μg/L of the rhGas6 group was greater than that in the control group, but without statistically significant difference (31.06%±13.70% vs. 21.79%±9.51%, P>0.05). In the migration test, after 24 h, the number of hPDLCs cells which penetrated through the membrane in the 800 μg/L rhGas6 group was significantly higher than that in the control group (P < 0.01). After rhGas6 was added and osteogenic induction, Runx2 and ALP gene expressions of hPDLCs in the 800 μg/L group were significantly higher than those in the control group (1.60±0.30 vs. 0.91±0.10, 2.81±0.61 vs. 0.86±0.12, P < 0.01). After Gas6 was knocked down, the ALP expression of hPDLCs was significantly lower than that of the control group (0.39±0.07 vs. 0.92±0.14, P < 0.01). There was no significant change in Runx2 expression (P>0.05). After 7 days of osteogenic induction, the mineralized nodules formed in the Gas6 knockdown group were significantly less than those in control group (0.25±0.04 vs. 1.00±0.11, P < 0.001). After 14 days of induction, the staining degree of the Gas6 knockdown group was lower than that of the control group, but there was no significant difference (0.86±0.04 vs. 1.00±0.16, P>0.05).@*CONCLUSION@#After downregulation of Gas6 gene, mineralized nodule formation was reduced and ALP gene expressions were decreased in the early stage of osteogenic induction (7 days). After addition of rhGas6, Runx2 and ALP gene expressions were increased and the number of cell migration was increased, suggesting that Gas6 might play a promoting role in the migration and osteogenic differentiation of human periodontal ligament cells.

RhoA-Dependent HGF and c-Met Mediate Gas6-Induced Inhibition of Epithelial-Mesenchymal Transition, Migration, and Invasion of Lung Alveolar Epithelial Cells.

Previously, we demonstrated that growth arrest-specific protein 6 (Gas6)/Axl or Mer signaling inhibited the transforming growth factor (TGF)-ß1-induced epithelial-mesenchymal transition (EMT) in lung epithelial cells. Hepatocyte growth factor (HGF) has also been shown to inhibit TGF-ß1-induced changes in EMT markers. Here, we examined whether Gas6 signaling can induce the production of HGF and c-Met in lung alveolar epithelial cells to mediate the inhibition of EMT and to inhibit the migration and invasion of epithelial cells. The inhibition of the RhoA/Rho kinase pathway, using either a RhoA-targeted small interfering RNA (siRNA) or the Rho kinase pharmacologic inhibitor Y27362, prevented the inhibition of TGF-ß1-induced EMT in LA-4 cells and primary alveolar type II (AT II) epithelial cells. The c-Met antagonist PHA-665752 also blocked the anti-EMT effects associated with Gas6. Moreover, treatment with Y27362 or PHA-665752 prevented the Gas6-mediated inhibition of TGF-ß1-induced migration and invasion. Our data provided evidence that the RhoA-dependent production of HGF and c-Met mediated the Gas6-induced inhibition of EMT, migration and invasion in lung alveolar epithelial cells. Thus, Gas6/Axl and Mer/RhoA signaling may be necessary for the maintenance of homeostasis in the alveolar epithelium, via HGF and c-Met.

Gamma-glutamyl carboxylated Gas6 mediates the beneficial effect of vitamin K on lowering hyperlipidemia via regulating the AMPK/SREBP1/PPARα signaling cascade of lipid metabolism.

The present study for the first time aims to examine the hypothesis that circulating gamma-glutamyl carboxylated growth arrest specific protein 6 (Gla-Gas6) deficiency may be associated with hyperlipidemia and vitamin K (VK) supplementation may ameliorate the impaired lipid homeostasis via activating Gas6 protein. Subjects with hyperlipidemia (n=22) and age-matched healthy controls (n=19) were included in this study. Results showed that plasma levels of Gla-Gas6 protein and VK were significantly lower in hyperlipidemic subjects compared to control. Moreover, Gla-Gas6 levels were significantly and positively correlated with VK (P=.034, r=0.452) and negatively with triglyceride (P=.022, r=-0.485) and total cholesterol (P=.043, r=-0.435) in hyperlipidemic subjects, which suggest that VK supplementation may have a positive effect in activating Gas6 protein and thereby reducing the aberrant plasma lipid levels. Further studies with high-fat diet (HFD)-fed animal model of hyperlipidemia demonstrated that VK supplementation (5 µg/kg body weight, 8 weeks) reduced the plasma lipid levels, stimulated both the plasma levels and the hepatic protein expression of Gla-Gas6 protein, and regulated the AMPK/SREBP1/PPARα signaling pathways of hepatic lipid metabolism in HFD-fed mice. Moreover, by using palmitic acid (PA, 0.75 mM)-treated both control and GGCX knockdown hepatocytes, this study dissected the direct role of Gla-Gas6 in mediating the positive effect of VK on preventing the PA-induced impaired hepatic lipid metabolism via regulating AMPK/SREBP1/PPARα pathways. Combining all, the present study demonstrated the beneficial effect of VK supplementation in preventing the impaired lipid homeostasis via activating VK-dependent Gas6 protein.

Growth arrest-specific protein 6 (Gas6) attenuates inflammatory injury and apoptosis in iodine-induced NOD.H-2h4 mice.

PURPOSE: Growth arrest-specific protein 6 (Gas6) is a vitamin K-dependent protein that plays an important role in the pathogenesis of autoimmune diseases. The purpose of this study was to explore the expression of Gas6 and its effects on autoimmune thyroiditis (AIT). METHOD: A total of 24 male NOD.H-2h4 mice were randomly assigned to three groups: (1) a control group supplied with regular water; (2) a sodium iodide (NaI) group supplied with 0.005% sodium iodide water; and (3) a group treated with recombinant mouse Gas6 (rmGas6) after iodine supplementation (NaI + Gas6 group). The severity of lymphocytic infiltration in the thyroid was measured through histopathology. Serum levels of tumor necrosis factor α (TNF-α), interleukin (IL) 6 and IL-1ß, as well as anti-thyroglobulin antibody (TgAb) titers were measured using an enzyme-linked immunosorbent assay. In addition, the expression of Gas6, Caspase 3, TAM receptors (Axl and MerTK), nuclear factor κB (NF-κB) and I-kappa-B α (IκB-α) were measured by Western blotting. Finally, the proportions of T cells were determined in the splenocytes of NOD.H-2h4 mice by flow cytometry. RESULTS: The mRNA and protein expression of Gas6 was significantly lower in the NaI group compared to the control group. Serum levels of TgAb, TNF-α, IL-6 and IL-1ß were also significantly higher in the NaI group but were dramatically reduced after rmGas6 injection. The prevalence of thyroiditis and the infiltration of lymphocytes were significantly lower in the NaI + Gas6 group compared to the NaI group. The protein expression of cleaved-Caspase 3, phosphorylation of MerTK, and NF-κB and IκB-α in the thyroid gland were significantly reduced after rmGas6 administration. The proportion of Th1, Th2 and Th17 cells in splenocytes were also significantly reduced after rmGas6 treatment, whereas there was a dramatic increase in the proportion of Treg cells. CONCLUSION: Gas6 exerts an anti-inflammatory effect in a mouse model of AIT and may therefore be a potential therapeutic target.

Vascular calcification and growth arrest specific protein 6 levels in chronic renal failure / Calcificación vascular y detención de crecimiento de proteína específica 6 niveles en insuficiencia renal crónica

INTRODUCTION: Cardiovascular disease is the main cause of mortality and morbidity in chronic renal failure. It's known that vascular calcification (VC) and carotid intima media thickness (CIMT) are strongly associated with cardiovascular diseases. Growth arrest specific protein 6 (Gas6) is a vitamin K-dependent protein and regulates various processes such as proliferation, cell survival, migration and inflammation. Gas6 is known to protect endothelial cells and vascular smooth muscle cells against apoptosis by inhibiting Bcl-2 induced Caspase 3 activation. The relationship between Gas6 and cardiovascular diseases has been demonstrated in many mouse models and cell cultures. However, there are conflicting reports whether Gas6 levels are increasing or decreasing in human studies of diabetic and/or chronic renal failure. In present study the aim was to examine plasma Gas6 levels and its relation with CIMT and coronary artery calcification score (CACS) in chronic kidney disease (CKD) patients. METHODS: Total of 137 patients of which 32 chronic hemodialysis and 105 predialysis patients as well as 73 healthy controls were enrolled in the study. Human Gas6 levels in serum samples were studied by ELISA method. CIMT was measured by ultrasonography. CACS was measured by multislice computed tomography. RESULTS: The mean age was 54.37±16.61 years in dialysis group, 55.20±14.80 years in predialysis group and 53.26±9.04 years in control group. Serum creatinine was 0.78±0.16 mg/dl in the control group and 1.96±1.64 mg/dl in the predialysis group and 5.94±1.55 mg/dl in the dialysis group. 24 hours urine protein levels were significally higher in the dialysis group than the predialysis and the control group. CIMT values were similar in predialysis and dialysis groups. These values were significantly higher than the control group. Although CACS was higher in dialysis group than predialysis and control group, the results were not statistically significant since the distribution range was very wide. Gas6 was 98.84±53.32 ng/mL in the control group and statistically higher than the dialysis (63.85±38.92 ng/mL) and the predialysis groups (54.96±38.49 ng/mL) (p=0.001). Gas6 levels were lower in diabetic patients than non-diabetics (53.69±35.26 ng/mL, 69.26±47.50 ng/mL, p=0.023, respectively). Negative correlation was detected between Gas6 and age, BMI, CACS, carotid IMT and proteinuria. In the logistic regression analysis, Gas6 remained significantly associated with BMI, CIMT and proteinuria. CONCLUSION: In our study, a negative correlation of Gas6 with BMI, CACS, CIMT and proteinuria and lower Gas6 levels in diabetic patients support that decreased Gas6 levels in chronic renal failure may have a role in vascular calcification through altered glucose tolerance, chronic inflammation, endothelial dysfunction and increased apoptosis. Our study has an importance because it is the first study showing a relation between Gas6 and proteinuria, CACS and carotid IMT in patients with chronic renal failure

INTRODUCCIÓN: La enfermedad cardiovascular es la principal causa de mortalidad y morbilidad en la insuficiencia renal crónica. Se sabe que la calcificación vascular (CV) y el grosor de la íntima-media de la carótida (CIMT, por sus siglas en inglés) están vinculados de forma muy estrecha con enfermedades cardiovasculares. La proteína específica del gen 6 de la detención de crecimiento (Gas6) es una proteína dependiente de la vitamina K y regula diversos procesos, como la proliferación, la supervivencia celular, la migración y la inflamación. La proteína Gas6 es conocida por proteger las células endoteliales y las células musculares lisas vasculares contra la apoptosis mediante la inhibición de la activación de la caspasa-3 inducida por la proteína Bcl-2. Se ha demostrado la relación entre la Gas6 y las enfermedades cardiovasculares en muchos modelos de ratones y cultivos celulares. Sin embargo, existen informes contradictorios acerca de si los niveles de Gas6 aumentan o disminuyen en estudios de humanos con insuficiencia renal crónica y/o diabética. En este estudio, el objetivo fue examinar los niveles plasmáticos de Gas6 y su relación con el CIMT y la puntuación de calcificación de las arterias coronarias (CACS, por sus siglas en inglés) en pacientes con enfermedad renal crónica (ERC). MATERIAL Y MÉTODOS: Un total de 137 pacientes fueron incluidos en el estudio, de los cuales 32 estaban en hemodiálisis crónica, 105 en prediálisis, y 73 pacientes representaban controles sanos. Se esudiaron los niveles de Gas6 en muestras de suero mediante el método ELISA. El CIMT se midió por medio de ecografía. La CACS se midió mediante tomografía computarizada multicorte. RESULTADOS: La edad media fue de 54,37 ± 16,61 años en el grupo de diálisis; 55,20 ± 14,80 años en el grupo de prediálisis, y 53,26 ± 9,04 años en el grupo de control. La creatinina sérica fue de 0,78 ± 0,16 mg/dl en el grupo de control; 1,96 ± 1,64 mg/dl en el de prediálisis, y 5,94 ± 1,55 mg/dl en el de diálisis. Las concentraciones de proteína en orina de 24 horas fueron significativamente más altas en el grupo de diálisis que en los de prediálisis y control. Los valores del CIMT fueron similares en los grupos de prediálisis y de diálisis. Estos valores fueron considerablemnete más altos que en el grupo de control. Aunque la CACS fue más alta en el grupo de diálisis que en los otros dos, los resultados no fueron estadísticamente significativos, ya que el rango de distribución fue muy amplio. La proteína Gas6 fue de 98,84 ± 53,32 ng/ml en el grupo de control y estadísticamente más alta que en los grupos de diálisis (63,85 ± 38,92 ng/ml) y de prediálisis (54,96 ± 38,49 ng/ml) (p = 0,001). Los niveles de Gas6 fueron más bajos en los pacientes diabéticos que en los no diabéticos (53,69 ± 35,26 ng/ml; 69,26 ± 47,50 ng/ml, [p = 0,023], respectivamente). Se detectó una correlación negativa entre la proteína Gas6 y la edad, el IMC, la CACS, el CIMT y la proteinuria. En el análisis de regresión logística, la Gas6 se mantuvo estrechamente relacionada con el IMC, el CIMT y la proteinuria. CONCLUSIÓN: En nuestro estudio, la correlación negativa de Gas6 con IMC, CACS, CIMT y proteinuria, y los niveles más bajos de Gas6 en pacientes diabéticos sustentan la idea de que la disminución de los niveles de Gas6 en la insuficiencia renal crónica puede jugar un papel en la calcificación vascular a través de la tolerancia alterada a la glucosa, la inflamación crónica, la disfunción endotelial y el aumento de la apoptosis. La importancia de nuestro estudio radica en que es el primero que muestra una relación entre la Gas6 y la proteinuria, la CACS y el CIMT en pacientes con insuficiencia renal crónica

Plasma sMer, sAxl and GAS6 levels correlate with disease activity and severity in lupus nephritis.

OBJECTIVE: The purpose of this study is to determine whether TAM receptors and ligands associated with the activity and severity of lupus nephritis. METHODS: Clinical data were statistically analysed and studied in 122 SLE patients, diagnosed from 2013 to 2016 in First Hospital Affiliated to Harbin Medical University. Levels of TAM receptors and ligands in the plasma of 122 SLE patients were measured by ELISA. Renal biopsies were performed to confirm lupus nephritis (LN) by histopathology in 68 patients. The associations of TAM receptors and ligands with clinical and serological parameters were analysed in 68 LN patients. RESULTS: Amongst patients with SLE, those with LN had significantly higher plasma sMer, sAxl and GAS6 levels than those without renal involvement (P < 0.01 for all comparisons). Additional comparisons on the renal function-associated clinical parameters confirmed an indicative role of the sMer, sAXL and GAS6 levels in the cohort of patients with more severe nephritis. Patients with higher sMer, sAXL and GAS6 levels of LN patients tended to suffer from proliferative glomerulonephritis. The sAXL and GAS6 levels had a strong positive correlation with activity index (AI) in LN patients. Furthermore, there was a significant drop of the sMer, sAXL and GAS6 concentrations from the time of the biopsy to month t6, but no further decrease from months t6 to t12. CONCLUSIONS: These results suggest that plasma sMer, sAxl and GAS6 can be an additional clinical marker related to the disease activity and severity in LN.

Plasma Growth Arrest-Specific 6 Protein and Genetic Variations in the GAS6 Gene in Patients with Metabolic Syndrome.

BACKGROUND: Growth arrest-specific 6 (Gas6) is a vitamin K-dependent protein secreted by immune cells, endothelial cells, vascular smooth muscle cells, and adipocytes. Recent studies indicate that Gas6 and receptors of the TAM (Tyro3, Axl, and Mer) family may be involved in the pathogenesis of obesity, systemic inflammation, and insulin resistance. The aim of this study was to investigate the association between plasma Gas6 protein and the c.843 + 7G>A Gas6 polymorphism in metabolic syndrome (MetS). METHODS: Two hundred five adults (88 men and 117 women) were recruited in this study. Plasma Gas6 concentration, general, and biochemical data were measured. All subjects were genotyped for the c.843 + 7G>A Gas6 polymorphism. RESULTS: Plasma Gas6 concentrations decreased in parallel with various MetS components in all groups (P = 0.017 for trend). Patients in the second and third tertiles of Gas6 level had higher high-density lipoprotein cholesterol (HDL-C) levels than those in the first tertile overall and in the female group. Plasma Gas6 levels were significantly positively correlated with HDL-C level and negatively with fasting glucose level in the female patients. The A allele and genotype AA in single nucleotide polymorphism c.843 + 7G>A were less frequent in the subjects with MetS compared to those without MetS. CONCLUSIONS: Our results demonstrated a positive correlation between Gas6 protein values and HDL-C and reinforce the association with fasting glucose. In addition, the presence of c.843 + 7G>A Gas6 polymorphisms, especially the AA genotype, had an association with MetS. The potential role of the Gas6/TAM system in MetS deserves further investigation.

The small-molecule MERTK inhibitor UNC2025 decreases platelet activation and prevents thrombosis.

Essentials Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation. UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase. UNC2025 decreases platelet activation in vitro and thrombus formation in vivo. UNC2025's anti-platelet effect is synergistic with inhibition of the ADP receptor, P2Y12 . SUMMARY: Background Growth arrest-specific protein 6 signals through the TAM (TYRO-3-AXL-MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing αIIb ß3 integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP-P2Y1&12 pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP-P2Y1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP-P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.

Axl activation attenuates neuroinflammation by inhibiting the TLR/TRAF/NF-κB pathway after MCAO in rats.

BACKGROUND AND PURPOSE: Ischemic stroke activates Toll-like receptors (TLRs), triggering rapid inflammatory cytokine production. Axl signaling has multiple roles, including regulating cytokine secretion, clearing apoptotic cells, and maintaining cell survival, however, its role in inflammation after ischemic stroke has not been examined. We hypothesized that activation of Axl by recombinant Growth-arrest-specific protein 6 (rGas6) attenuates neuroinflammation by inhibiting the TLR/TRAF/NF-κB pathway after middle cerebral artery occlusion (MCAO) in rats. METH: Sprague-Dawley rats were subjected to 2h of MCAO. One hour after reperfusion, the rats were given an intranasal injection of rGas6, vehicle, or R428 (Axl receptor inhibitor). Neurological scores, infarct volumes, immunofluorescence staining, Morris Water Maze, rotarod test and histology alterations were analyzed. The expressions of proinflammatory cytokines, including IL-1ß, IL-6, TNF-α, and Gas6, Axl, STAT1, SOCS1, SOCS3 and the TLR/TRAF/NF-κB pathway were quantified using Western blot. RESULTS: Endogenous expressions of Gas6 and Axl decreased significantly by 24h after MCAO. rGas6 reduced brain infarction and improved neurologic deficits scores, and increased expression of Axl and decreased the expressions of TRAF3, TRAF6 and inflammatory factors IL-1ß, IL-6, and TNF-α. Four weeks after MCAO, rGas6 improved long-term neurological behavior and memory. Inhibition of the Axl/TLR/TRAF/NF-κB pathway reversed the brain protection by rGas6. CONCLUSION: rGas6 reduced the neurological deficits by inhibiting neuroinflammation via the TLR/TRAF/NF-κB signaling pathway. rGas6 can be used as potential treatment to ischemic stroke.