Gas6 Levels Research Articles

Relationship Between Increased Expression of the Axl/Gas6 Signal Cascade and Prognosis of Patients with Upper Tract Urothelial Carcinoma.

Axl, which is in the TAM family of receptor tyrosine kinases, and its ligand, growth arrest-specific gene 6 (Gas6), have been associated with worse prognoses after the surgical treatment of some types of cancers. We herein investigated the biological significance of the protein expression of Axl and Gas6 on the outcomes of patients with upper tract urothelial carcinoma (UTUC). The protein expression of Axl and Gas6 was evaluated by immunohistochemistry, and their relationships with clinicopathological features were investigated in surgical specimens obtained from 161 patients who had been surgically treated for UTUC. Axl labeling was strong in 67 of 161 (42 %) cases, while Gas6 labeling was strong in 72 of 161 (45 %) cases. The strong expression of Axl correlated with that of Gas6. A high pathological stage (p = 0.009), strong expression of Gas6 (p = 0.038), and strong expression of Axl (p = 0.016) were independent factors for predicting worse cancer-specific survival (CSS). In a subgroup analysis of patients with pT < 2 (N = 53), no significant difference in CSS was observed between patients weakly and strongly expressing Axl/Gas6. In contrast, a subgroup analysis of patients with pT ≥ 2 (N = 108) revealed that the expression levels of Axl and Gas6 correlated with CSS. The protein expression of Axl and its ligand Gas6 may be a useful indicator for a worse clinical outcome in UTUC patients, especially patients with pT ≥ 2, who underwent radical nephroureterectomy.

Gas6/Axl in arginine-starvation therapy.

Exploiting abnormal amino acid metabolism for cancer therapy dated back to more than four decades ago by the discovery that L-asparaginase was effective in treating childhood acute lymphocytic leukemia (ALL). ALL lacks asparagine (Asn) because the key enzyme for the biosynthesis of Asn from aspartic acid, asparagine synthetase, is silenced, therefore, ALL requires Asn from the circulation for growth. Asparaginase (trade names Elspa or Concapar) catalyzes the hydrolysis of Asn in the circulation, resulting in Asn starvation and cell death. Asn starvation therapy has shown remarkable treatment efficacies [1]. Another amino acid starvation therapy strategy that has been under development is the use of pegylated arginine deiminase (ADI-PEG20) which degrades arginine (Arg) into citrulline and ammonia. Arg is an important metabolite in the urea cycle for tumor cell growth. De novo biosynthesis of Arg is regulated by the rate-limiting enzyme, argininosuccinate synthetase 1 (ASS1). Reduced levels of ASS1 expression has been found in large subpopulations of solid tumors, including malignant melanoma, hepatocellular carcinoma, prostate cancers, lung cancers and breast cancers. These ASS1low tumors require extracellular Arg to support their highly proliferative activities. ADI-PEG20 treatment causes Arg starvation resulting in tumor cell death of autophagy/apoptosis. ADI-PEG20 has been in clinical investigations for treating ASS1low tumors with promising results [2]. One major problem associated with ADI-PEG20 treatment is drug resistance due mostly to induction of ASS1 expression. Previous studies showed that ASS1 silencing is mediated by an epigenetic mechanism due to DNA methylation at the promoter region of ASS1 [3]. Tsai et al [4] demonstrated that ASS1 silencing in melanoma cells is transcriptional regulated by repressor HIF-1α which binds to the E-box of ASS1 promoter. Arg deprivation induces c-Myc expression which de-represses HIF-1α and induces ASS1 expression. The recent article by Tsai et al [5] presented a comprehensive signaling mechanism of how ASS1low cells response to Arg starvation stress leading to ASS1 activation and ADI-PEG20 resistance. These investigators reported that within minutes after Arg depletion of ASS1low cells, a reactive oxygen species-related mechanism triggers Gas6 externalization to interact with its membrane-bound receptor Axl, which then activates the downstream signal Ras/PI3K/Akt/GSK-3β growth signal, leading to accumulation of c-Myc by protein stabilization. c-Myc can also transcriptionally upregulate itself and Axl, thereby amplifying the Arg stress response signal. Upregulation of ASS1 expression by c-Myc enhances Arg production and relieves Arg-starvation stress pressure; elevated ASS1 can also feedback-downregulate c-Myc and Axl, providing a self-guarding control mechanism for Arg biosynthesis. These results demonstrated that multiple inter-regulatory pathways involving Axl, c-Myc, ASS1 regulate mammalian Arg homeostasis system and sensitivity to Arg starvation therapy using ADI-PEG20. These authors also demonstrated that expression of ASS1, c-Myc, Gas6 and Axl were elevated in primary cultures derived from two melanoma patients failed ADI-PEG20 treatments as correspondingly compared with those derived from the same patients prior to ADI-PEG20 treatment, demonstrating the clinical relevance of the observations. The mechanism that ASS1low cancer cells use Gas6/Axl growth signal in response to Arg starvation differs from the conserved general amino acid control pathway which involves rapid repressing protein synthesis due to phosphorylation of translation initiation factor 2 (eIF2) [6]. Recent studies have demonstrated that high levels of Gas6/Axl are associated with tumor aggressiveness. Moreover, elevated expression of Gas6/Axl can also affect the treatment efficacies with many cancer chemotherapeutics. Many small molecules targeting Gas6/Axl have been under development [7]. Moreover, Gas6/Axl may serve as important therapeutic markers for Arg starvation treatment. Because Gas6 secretion is the early event associated with Arg deprivation treatment, Gas6 in the liquid biopsy could become a non-invasive therapeutic marker, although a sensitive detection method may have to develop because basal level Gas6 levels in human serum is very low. Thus, the work by Tsai et al [5] provides an important rationale for further research on improving the treatment of Arg-auxotrophic tumors.

Postmortem serum protein growth arrest-specific 6 levels in sepsis-related deaths.

Growth arrest-specific 6 (Gas6) is widely expressed in leukocytes, platelets, endothelial cells, and monocytes. It regulates various processes including granulocyte adhesion to the endothelium, cell migration, thrombus stabilization, and cytokine release. In humans, increased plasma Gas6 levels have been described in patients with sepsis and septic shock. In this study, Gas6 concentrations were measured in postmortem serum from femoral blood in a series of sepsis-related fatalities and control cases. The aims were twofold: first, to determine whether Gas6 can be reliably determined in postmortem serum; and second, to assess its diagnostic potential in identifying sepsis-related deaths. Two study groups were prospectively formed, a sepsis-related fatalities group (24 cases) and a control group (24 cases) including cases of deep vein thrombosis and fatal pulmonary embolism, cases of systemic inflammatory response syndrome in severe trauma, cases of end-stage renal failure, and cases of hanging (non-septic, non-SIRS, non-end stage renal failure cases). The preliminary results of this study seem to indicate that Gas6 can be effectively measured in postmortem serum. However, Gas6 levels in sepsis-related fatalities do not appear to be clearly distinguishable from concentrations in pulmonary embolism, severe trauma, and end-stage renal failure cases. These findings tend to support previous reports that indicated that Gas6 behaves as an acute phase reactant and can be considered a general marker of inflammation rather than a specific biomarker of sepsis.

Elevated Serum Gas6 Is a Novel Prognostic Biomarker in Patients with Oral Squamous Cell Carcinoma.

ObjectiveThis study explored the level and clinical significance of serum Gas6 in patients with oral squamous cell carcinoma (OSCC).MethodsA total of 128 OSCC patients and 145 normal controls were selected. Enzyme-linked immunosorbent assay was used to detect Gas6 concentration in sera from the OSCC patients and controls. The correlations of serum Gas6 concentration and clinicopathological characteristics of OSCC patients were assessed, and the prognostic significance of serum Gas6 was evaluated with a Kaplan–Meier curve and log-rank test.ResultsThe results showed that serum Gas6 concentration was significantly higher in OSCC patients than in controls (P < 0.05). OSCC patients with late TNM stage (III, IV) had a relatively high serum Gas6 concentration compared with those with early stage (I, II) (P < 0.01) and patients with poorly differentiated tumors had a higher level of serum Gas6 than those with well-differentiated tumors (P < 0.01). Multivariate logistic regression analysis demonstrated that high serum Gas6 was an independent risk factor for lymph nodal metastases in OSCC patients (OR = 2.79, 95% CI: 1.72–4.48). For predicting OSCC development, ROC curve analysis showed a sensitivity of 0.63 with a specificity of 0.92 (AUC = 0.79, 95% CI: 0.74–0.85). Cox analysis revealed that high serum Gas6 was an independent biomarker for predicting poor overall survival in OSCC patients (HR = 2.07, 95% CI: 1.79–3.62). In addition, we found that Gas6 expression was increased in OSCC tissues and it may significantly decrease E-cadherin expression, and increase P-cadherin and N-cadherin expression, in OSCC cells. Further, Gas6 could promote the migratory and invasive ability of OSCC cells in vitro.ConclusionTaken together, these results suggest that Gas6 increases the metastatic capacity of OSCC cells and serum Gas6 could be a candidate biomarker for diagnostic and prognostic use in OSCC patients.

The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization.

BackgroundThe nuclear receptors PPAR-γ and LXRs regulate macrophage lipid metabolism and macrophage mediated inflammation. We examined the influence of these molecules on macrophage alternative activation, with particular focus on differentiation of “M2c” anti-inflammatory cells.MethodsWe cultured human monocytes in M0, M1, M2a or M2c macrophage differentiating conditions, in the presence or absence of PPAR-γ and LXR ligands. Flow cytometry was used to analyze membrane expression of phenotypic markers. Basal and LPS-stimulated production of soluble mediators was measured by ELISA. Efferocytosis assays were performed by coincubating monocytes/macrophages with apoptotic neutrophils.ResultsWe found that PPAR-γ inhibition, using the PPAR-γ antagonist GW9662, elicits differentiation of M2c-like (CD206+ CD163+ CD16+) cells and upregulation of the MerTK/Gas6 axis. Exposure of differentiating macrophages to IFN-γ, GM-CSF or LPS (M1 conditions), however, hampers GW9662 induction of MerTK and Gas6. When macrophages are differentiated with IL-4 (M2a conditions), addition of GW9662 results into an M2a (CD206+ CD209+ CD163− MerTK−) to M2c (CD206high CD209− CD163+ MerTK+) polarization shift. Conversely, in the presence of dexamethasone (M2c conditions), the PPAR-γ agonist rosiglitazone attenuates CD163 and MerTK upregulation. The LXR agonist T0901317 induces MerTK independently of M2c polarization; indeed, CD206, CD163 and CD16 are downregulated. GW9662-differentiated M2c-like cells secrete high levels of Gas6 and low amounts of TNF-α and IL-10, mimicking dexamethasone effects in vitro. However, unlike conventional M2c cells, GW9662-differentiated cells do not show enhanced efferocytic ability.ConclusionsOur results provide new insights into the role of PPAR-γ and LXR receptors in human macrophage activation and reveal the existence of different patterns regulating MerTK expression. Unexpectedly, PPAR-γ appears to negatively control the expansion of a discrete subset of M2c-like anti-inflammatory macrophages.

Association between the growth arrest-specific 6 (Gas6) gene polymorphism c.834 + 7G>A and preeclampsia

Objective: Preeclampsia (PE) is a hypertensive disease of pregnancy complicating 2–8% of all pregnancies. The exact pathophysiology still remains unknown. Growth arrest-specific 6 (Gas6) is a member of the vitamin K-dependent protein family and it has been suggested as a novel atherothrombotic risk factor with anti-angiogenic and pro-atherogenic properties. The goal of the our study was to investigate the relationships between the c.834 + 7G > A polymorphism of GAS6, plasma Gas6 levels and PE.Methods: A total of 150 women, including 82 preeclamptic pregnant women and 68 normotensive pregnant (NP) women, were recruited in the current study. Blood samples were taken from all participitants. Plasma Gas6 levels measured by an enzyme-linked immunosorbent assay. GAS6 polymorphism was determined using a PCR-RFLP method.Results: The plasma Gas6 levels of preeclamptic patients were significantly lower than those of NP women (8.65 ± 3.70 ng/ml and 10.89 ± 4.23 ng/ml respectively, p < 0.001). The GG genotype was the most prevalent, and the risk of PE was 3.5-fold higher in pregnant women with GG genotype compared to woman with AA genotype (p < 0.01). The A allele was less frequent in preeclamptic patients than in control subjects (OR = 2.118, 95% CI = 1.330–3.371, p < 0.001).Conclusions: Our results suggest that GAS6 c.834 + 7G > A polymorphism may have a pivotal role in the pathogenesis of PE suggesting that the A allele has a protective role for PE.

Gas6/Axl pathway is activated in chronic liver disease and its targeting reduces fibrosis via hepatic stellate cell inactivation

Liver fibrosis, an important health concern associated to chronic liver injury that provides a permissive environment for cancer development, is characterized by accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells (HSCs). Axl, a receptor tyrosine kinase and its ligand Gas6, are involved in cell differentiation, immune response and carcinogenesis. HSCs were obtained from WT and Axl(-/-) mice, treated with recombinant Gas6 protein (rGas6), Axl siRNAs or the Axl inhibitor BGB324, and analyzed by western blot and real-time PCR. Experimental fibrosis was studied in CCl4-treated WT and Axl(-/-) mice, and in combination with Axl inhibitor. Gas6 and Axl serum levels were measured in alcoholic liver disease (ALD) and hepatitis C virus (HCV) patients. In primary mouse HSCs, Gas6 and Axl levels paralleled HSC activation. rGas6 phosphorylated Axl and AKT prior to HSC phenotypic changes, while Axl siRNA silencing reduced HSC activation. Moreover, BGB324 blocked Axl/AKT phosphorylation and diminished HSC activation. In addition, Axl(-/-) mice displayed decreased HSC activation in vitro and liver fibrogenesis after chronic damage by CCl4 administration. Similarly, BGB324 reduced collagen deposition and CCl4-induced liver fibrosis in mice. Importantly, Gas6 and Axl serum levels increased in ALD and HCV patients, inversely correlating with liver functionality. The Gas6/Axl axis is required for full HSC activation. Gas6 and Axl serum levels increase in parallel to chronic liver disease progression. Axl targeting may be a therapeutic strategy for liver fibrosis management.

Growth Arrest-Specific 6 (Gas6) and TAM Receptors in Mouse Platelets.

Objective:Growth arrest-specific 6 (Gas6) is a newly discovered vitamin K-dependent protein, which is a ligand for TAM receptors [Tyro3 (Sky), Axl, and Mer] from the tyrosine kinase family. Gas6 knockout mice were resistant to venous and arterial thrombosis. There are contradictory reports on the presence of Gas6 and its receptors in mouse platelets. The objective of this study was to investigate whether Gas6 and its receptors were present in mouse platelets or not. Materials and Methods:Specific pathogen-free BALB/c male and female mice of 8-10 weeks old and 25-30 g in weight were anesthetized under light ether anesthesia and blood samples were taken from their hearts. RNAs were isolated from isolated platelets, and then mRNAs encoding Gas6 and TAM receptors were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA, but not those of Mer, because of the absence of any commercial ELISA kit for mouse specimens.Results:RT-PCR results indicated the presence of mRNAs encoding Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro3 (Sky). Receptor protein levels of Axl and Tyro3 were below the detection limits of the ELISA method.Conclusion:We found the presence of mRNAs encoding Gas6 and the receptor Mer in mouse platelets, but not Axl and Tyro3. Gas6, Axl, and Tyro3 protein levels were below the detection limits of the ELISA. The presence of mRNA is not obvious evidence of protein expression in platelets that have no nucleus or DNA. Further studies are required to clarify the presence of Gas6/TAM receptors in platelets using real-time PCR and more sensitive immunological methods, and future studies on mechanisms will indicate whether the Gas6/TAM pathway is a strategy for treatment of disorders.

Maternal high-fat diet and offspring expression levels of vitamin K-dependent proteins.

Studies suggest that bone growth and development and susceptibility to vascular disease in later life are influenced by maternal nutrition during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including osteocalcin, matrix Gla protein, periostin, and growth-arrest specific- protein 6, in both bone and vascular development. We have examined whether there are alterations in these VKDPs in bone and vascular tissue from offspring of mothers subjected to a nutritional challenge: a high-fat diet during pregnancy and postnatally, using 6-week-old mouse offspring. Bone site-specific and sex-specific differences across femoral and vertebral bone in male and female offspring were observed. Overall a high-fat maternal diet and offspring diet exacerbated the bone changes observed. Sex-specific differences and tissue-specific differences were observed in VKDP levels in aorta tissue from high-fat diet-fed female offspring from high-fat diet-fed mothers displaying increased levels of Gas6 and Ggcx compared with those of female controls. In contrast, differences were seen in VKDP levels in femoral bone of female offspring with lower expression levels of Mgp in offspring of mothers fed a high-fat diet compared with those of controls. We observed a significant correlation in Mgp expression levels within the femur to measures of bone structure of the femur and vertebra, particularly in the male offspring cohort. In summary, the current study has highlighted the importance of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure.

Abstract 2780: Tyro3 and Axl receptors tyrosine kinase as potential therapeutic targets in leiomyosarcoma

Abstract Leiomyosarcoma (LMS) represents 15% of adult sarcomas. Once metastatic, curative options are limited and innovative strategies are needed. TYRO3 and AXL proto-oncogenes belong to TAM family (Tyro3/Axl/Mer) of tyrosine kinase receptors, with oncogenic properties that are often overexpressed or activated in various malignancies. Using kinexus phosphoprotein microarrays (KAM-1.1) and western blot analyses, we evidenced that Tyro3 and Axl are overexpressed in LMS along with Gas6, their ligand. Interestingly LMS samples harbouring high levels of Gas6 and its receptors also presented AKT phosphorylation on S473 residue keeping with previous studies that highlighted AKT- mTOR activation in leiomyosarcomas. Based on these premises, we asked whether Tyro3-Axl axis may drive LMS proliferation and could represent potential targets in these tumors. The overexpression of Tyro3 was found to be associated with a specific miRNA signature, notably with an overexpression of miR22 in LMS tumors. Furthermore Tyro3 and Axl were found overexpressed in a panel of 6 LMS cell lines (SK-LMS-1 from ATCC and IB112, IB118, IB133, IB134 and IB136 from Institut Bergonié), compared with normal human smooth muscle. Tyro3 or Axl knockdown via shRNA retroviral vector reduced cell proliferation by up to 50 and 64% respectively and decreased soft agar colony-forming ability. Crizotinib and foretinib are two tyrosine kinase inhibitors (TKI) targeting Met and Axl. Treatment of LMS cell lines with both TKIs resulted in a significant increase in cell death, with IC50 values ranging from 3.6 to 14.6µM and 0.7 to 1.8µM, respectively. Annexin V and PI staining revealed that cell death was mainly due to apoptosis. Flow cytometric cell cycle analysis demonstrates that both TKIs altered cell cycle phase distribution. A sub-G1 phase accumulation was observed for one of the cell lines; meanwhile the other cell lines presented a G2 arrest and 4n chromosomes accumulation accompanied by an increase in cell and nucleus sizes. Only foretinib induced nuclear fragmentation consistent with late apoptosis. Pharmacological inhibition also reduced migration and colony formation in soft agar. Drug combinations with an AKT inhibitor led to significant increase in growth inhibition in all tested cell lines. Crizotinib and foretinib reduced activation of Tyro3 or Axl-mediated downstream signaling including MAPK/ERK, PI3K/AKT along with an inhibition of Axl phosphorylation in a dose dependent manner. By immunoprecipitation we observed also a reduction in Tyro3 phosphorylation, following exposure to both agents, showing that Tyro3 is a new target for those drugs. IHC analysis of Tyro3, Axl and Gas6 expression in a cohort of LMS patient is ongoing. In vivo analysis wil be performed. In conclusion these results strongly suggest that Tyro3 and Axl are relevant therapeutic targets for LMS treatment and will be investigated in a proof of concept clinical trial. Citation Format: Carmela Dantas-Barbosa, François Le Loarer, Marta Mendiola, Isabelle Treilleux, Frederic Chibon, Hyba El Sayadi, Jean Michel Coindre, Laurent Alberti, Jean-Yves Blay. Tyro3 and Axl receptors tyrosine kinase as potential therapeutic targets in leiomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2780. doi:10.1158/1538-7445.AM2014-2780

Serum growth arrest specific protein 6 and its receptor (Axl) in SLE patients

Aim of the workGrowth arrest-specific protein 6 (Gas6) and its soluble tyrosine kinase receptor sAxl are known to have antiapoptotic function which may be implicated in systemic lupus erythematosus (SLE) pathogenesis. We aimed to assess serum levels of GAS6 and its receptor sAxl in SLE patients and to correlate these levels with disease activity index and renal involvement. Patients and methodsGas6 and sAxl were measured by enzyme linked immunosorbent assay (ELISA) in sera obtained from 50 female patients with SLE and 40 healthy age-matched females serving as controls. Disease activity was calculated according to the SLE disease activity index (SLEDAI). ResultsSerum levels of Gas6 and sAxl were significantly increased in patients with SLE in comparison to controls (p<0.001). Serum Gas6 levels increased significantly in active SLE patients and lupus nephritis patients (p<0.001). Serum levels of Gas6 correlated positively with SLEDAI, double-stranded DNA titers and proteinuria. In addition, there was a significant negative correlation between Gas6 serum levels and both C3 and C4; while, serum sAxl levels did not show these correlations. ConclusionsGas6 and sAxl may have a role in the pathogenesis of SLE. Increased serum Gas6 level may be an important risk factor for the development and severity of lupus nephritis. So, Gas6 may have a potential role to serve as a disease activity marker and may have a role as a therapeutic target for SLE.

Axl Tyrosine Kinase Receptor As a Key Regulator of Proliferation and Survival in Colorectal Cancer (Crc)

ABSTRACT Background: Novel therapeutic strategies are evolving in CRC to selectively inhibit oncogenic pathways. In this scenario we identified the tyrosine kinase receptor AXL as a potential target. Methods: We evaluated the expression and activation of AXL kinase and its ligand GAS6 in a panel of CRC cell lines by receptor tyrosine kinase array (RTK), western blot and Real Time PCR. In the AXL expressing cell lines we analyzed cell survival and drug sensitivity (using the AXL inhibitor foretinib) by MTT assay. Finally we generated stable short hairpin RNA (shRNA)-sh-AXL cell line in order to evaluate the effect of AXL specific knockdown in a model of CRC in vitro and to analyze the mechanism underlying AXL activation. Moreover, TGFb expression was measured by Luminex assay. Results: We found AXL expression in LOVO, HCT116, SW480 and SW620 CRC cells whereas the HT29, SW48, SW48-CR (Cetuximab Resistant), Colo205, GEO, GEO-CR (Cetuximab Resistant) and HCT15 CRC cells were AXL-negative. AXL-expressing cell lines were sensitive to foretinib with an IC50 range from 0,5 to 2 mM. AXL inhibition caused a significant phosphorylation decrease of MAPK, AKT and S6 ribosomal protein. This data were further confirmed by stable silencing of AXL with sh-RNA in LOVO cells that resulted in a dramatic reduction of cell proliferation and activation of downstream pathway. Since no GAS6 levels were found by ELISA in the supernatant of cells tested, we tried to investigate the possible mechanism of AXL activation in our model. We found highest levels of TGFb in HCT116 and SW620 cells in medium. The stimulation of AXL expressing cells with a recombinant TGFb, for 20 and 120 minutes, resulted in increase of downstream pathway. Conclusions: This data highlighted AXL as a potential target in CRC. AXL-expressing cells are resistant to anti-EGFR therapies, thus it could be a relevant target for cancer treatment, in fact AXL inhibition by foretinib or sh-RNA resulted in a significant cell proliferation and survival reduction. Finally, we identified the TGFb as a potential activator of AXL-mediated signaling pathway. The in vivo study is currently ongoing on HCT116 ortotopic xenografts in nude mice treated with foretinib and final results will be presented. Disclosure: All authors have declared no conflicts of interest.

Assay of serum growth arrest-specific gene product 6 and stromal cell-derived factor isoform-1 in patients with diabetic retinopathy

Background Diabetic retinopathy (DR) is a progressive vision-threatening complication of diabetes mellitus (DM),but its pathogenic mechanism is still unclear.Researches showed that growth arrest-specific gene product 6 (Gas6) /TAM system participates in pathogenesis and development of DR,and stromal-derived factors (SDF) vary in 2-type DM patients.However,whether Gas6/TAM and SDF-1 are associated with DR is below understood.Objective This study was to determine the relationship between the staging of DR and the levels of serum Gas6,SDF-1α and SDF-1β in DM patients.Methods A prospective cohord study was designed in this study.Ninety 2-type DM patients were included in the 3rd Affiliated Hospital of Southern Medical University Hospital from January to August in 2013.The patients were grouped into the non-diabetic retinopathy (NDR) group,background DR group (BDR) and proliferative DR (PDR) group,with 30 for each group.Thirty normal volunteers were enrolled in the same hospital and same period.The periphery blood 2 ml was collected from all the subjects under the consent inform.The levels of serum Gas6,SDF-1α and SDF-1β were assayed by ELISA,and leukocytes,neutrophils,plasma triglycerides (TG),total cholesterol (CHOL),high density lipoprotein cholesterol (H DL-C) and low density lipoprotein cholesterol (LDL-C) levels were detected and compared among the 4 groups.The correlations of serum Gas6,SDF-1 α and SDF-1 β changes with blood inflammatory cells and blood lipid were analyzed.Results The plasma CHOL concentrations were 4.93(4.14,5.44),5.02(4.35,5.69),4.54(3.85,5.93) and 5.99(5.11,6.89)mmol/L in the normal control group,NDR group,BDR group and PDR group,respectively,and the blood CHOL concentrations were significantly higher in the PDR group than those of the normal control group,NDR group,BDR group (P =0.002,P =0.007,P =0.006).White blood cell counts in the normal control group,BDR group were higher than those of the PDR group (P =0.034,P =0.015),neutrophil counts in the BDR group were higher than those of the PDR group (P =0.024),HDL-C in the NDR group was higher than that in the PDR group (P =0.032).LDL-C in the PDR group was higher than that in the normal control group.Compared with the normal control group,serum Gas6 levels were significantly lower in the NDR group and BDR group (P =0.048,P =0.006),and the serum Gas6 level showed an insignificant increase in the PDR group in comparison with BDR group (P =0.297).Serum SDF-1α levels in the PDR group was significantly higher than that in the BDR group (P =0.033) ;serum SDF-1β levels in the PDR group,BDR group were significantly higher than that in the NDR group (P =0.011,P =0.008) and normal control group (P =0.030,P =0.002).Weaker positive correlation was observed between the serum Gas6 and CHOL,TG,LDL-C levels (r=0.285,r=0.200,r=0.241,all at P＜0.05),between SDF-1α and SDF-1β (r=0.190,P＜0.05) as well as between SDF-1β and white blood cell (r=0.183,P＜0.05).Serum Gas6 served as dependent variable,while white blood cell,neutrophil,CHOL,TG,HDL-C,LDL-C,SDF-1α,SDF-1β served as independent variables,multiple stepwise regression analysis showed Gas6 =170.791 + 5.283CHOL (F =5.021,P =0.027).Conclusions Serum Gas6,SDF-1α and SDF-1β probably participate in the development of DR in 2-type diabetic patients.Gas6,SDF-1 α,SDF-1 β may play roles by affecting blood glucose level,angiogenesis,inflammatory cells and blood lipid metabolism. Key words: Growth arrest-specific gene 6; Stromal cell-derived factor-1; Diabetes mellitus; Complication; Diabetic retinopathy

C0558: The Plasma Levels of Gas6 in Recurrent Pregnancy Losses

C0558: The Plasma Levels of Gas6 in Recurrent Pregnancy Losses

Diminished soluble levels of growth arrest specific protein 6 and tyrosine kinase receptor Axl in patients with rheumatoid arthritis.

Growth arrest specific protein 6 (Gas-6) and its tyrosine kinase receptor Axl plays an important role in apoptosis, and regulation of innate immune response, therefore, we investigated their plasma concentrations in Rheumatoid arthritis (RA) patients and correlated them to clinical, laboratory and radiological parameters of the disease. Plasma from 77 RA patients and 50 normal healthy subjects were assayed for plasma Gas6 and Axl levels. Demographic, clinical and serological data were prospectively assessed. Rheumatoid arthritis disease activity was assessed using 28-joint Disease Activity Score (DAS-28) and functional capacity by modified health assessment questionnaire (mHAQ). Standardized x-rays for hands and feet were done to all participants. The level of Gas6 and Axl were significantly decreased in the RA patients compared to those of the healthy control subjects. Levels of Gas6 correlated positively with Axl levels in both patients and healthy control. Gas6 levels were remarkably reduced in those patients with erosive RA than those without. Levels of Gas6 were found to be negatively correlated with the presence of erosive disease and positively correlated with DAS-28, ESR, Leucocytosis and IL6. The plasma concentrations of Gas6 and Axl are altered in RA patients and thus may have a role in RA pathogenesis. Further mechanistic studies on the involvement of all TAM receptors tyrosine kinases pathway in RA are needed to help in understanding the pathogenesis and possibly aid in diagnosis and future treatments of RA especially for patients with erosive disease.

P611 GAS6/AXL AXIS AS PROGNOSTIC AND THERAPEUTIC TARGET IN LIVER DISEASE

Background and Aims: AXL, a receptor tyrosin kinase, and its ligand GAS6 are related to processes leading to cell differentiation and carcinogenesis. The role of GAS6/AXL signaling in hepatic stellate cell (HSC) transformation during liver disease has been insufficiently addressed. Methods: HSCs were isolated from wild-type and AXL knockout (KO) mice. HSCs and LX2 human activated HSCs were exposed to recombinant GAS6, AXL inhibitor (BGB324, BerGenBio) or siRNA silencing. p-AXL, p-AKT and HSC transformation were evaluated by western blotting and qPCR. CCl4-induced fibrosis was analyzed in wild-type and AXL KO mice. Gas6 and soluble AXL (sAXL) levels were measured by ELISA in serum from alcoholic patients with normal liver, compensated cirrhosis and decompensated cirrhosis, and in control individuals (n = 10, each group). Results: Recombinant GAS6 stimulated AXL and AKT activation, inducing phenotypic changes in primary HSCs. HSCs from AXL KO mice displayed reduced capacity to be activated in vitro, and AXL inhibition (BGB324, Bergenbio) reduced HSC activation and proliferation. Similar results were obtained in LX2 cells and after RNA silencing of AXL. Chronic CCl4 administration increased serum levels of sAXL and GAS6, while AXL deficient mice exhibited reduced liver fibrosis. More importantly, AXL inhibition (BGB324) efficiently diminished CCl4-induced liver fibrosis in wild-type mice. In parallel, GAS6 and sAXL levels in serum were increased in alcoholic liver disease (ALD) patients, inversely correlating with liver functionality. Conclusions: AXL inhibitors have clinical interest since AXL targeting is effective to treat experimental liver fibrosis. In addition, GAS6 and sAXL levels could be proposed as early prognostic markers in liver disease.

Plasma growth arrest-specific protein 6 levels in premenopausal and postmenopausal women: the role of endogenous estrogen

Growth arrest-specific 6 (Gas6) is a vitamin K-dependent protein that interacts with receptor tyrosine kinases of the Tyro-3, AXL, Mer (TAM) family. The Gas6/TAM system contributes to the regulation of cell survival and proliferation, cell adhesion and migration, and inflammatory cytokines release. Plasma Gas6 plays an important role in the inflammatory process, and is involved in diverse human diseases. Few studies have investigated gender-specific variations in plasma Gas6 concentration. Hence, the aim of this study was to determine whether plasma Gas6 levels are associated with sex hormones in premenopausal and postmenopausal women. A total of 103 premenopausal and 135 postmenopausal women were recruited. Plasma Gas6 concentration, estradiol (E2), and sex hormone-binding globulin were assayed. The free estrogen index (FEI) was calculated. The results showed significantly lower Gas6 levels in the postmenopausal compared to the premenopausal women (P < 0.005). Plasma Gas6 levels were positively correlated with E2 levels in the pre- and postmenopausal women (r = 0.359, P < 0.001 and r = 0.261, P = 0.002, respectively). Gas6 levels were also correlated with FEI in the pre- and postmenopausal women (r = 0.234, P = 0.017 and r = 0.188, P = 0.029, respectively). After adjusting for confounders, the correlations still remained significant. In multiple stepwise regression analysis, only E2 in premenopausal and both age and E2 in postmenopausal women were independently correlated with the plasma Gas6 levels (all P < 0.001). These results suggest that plasma Gas6 is associated with sex hormones in both pre- and postmenopausal women, indicating a potential role of sex hormones in the Gas6/TAM system.

Expression of growth arrest-specific protein 6 and Axl molecules in the left internal mammary artery of patients undergoing coronary artery bypass grafting

AimsStudies have demonstrated that using a left internal mammary artery (LIMA) graft yields excellent long-term results in coronary artery bypass grafting (CABG). The growth arrest-specific 6 (Gas6) gene and its...

Association of plasma growth arrest-specific protein 6 (Gas6) concentrations with albuminuria in patients with type 2 diabetes

Aims: New biomarkers are required to detect diabetic nephropathy earlier in persons with type 2 diabetes mellitus. Recent experimental studies indicate that growth arrest-specific protein 6 (Gas6) may have a role in pathogenesis of complications associated with diabetes. The objective of the current study is to examine whether plasma Gas6 concentrations are associated with albuminuria in persons with type 2 diabetes mellitus. Methods: About 32 patients with diabetes which have micro or macroalbuminuria, 37 patients with diabetes and normoalbuminuria, and 30 healthy volunteers were recruited. Plasma Gas6 levels were measured by ELISA. Hemoglobin A1c (HbA1c), serum C reactive protein, fibrinogen and 24-h urine samples for microalbuminuria were analyzed by Primus PDQ, Beckman Coulter Immage 800, STA Compact and Roche Cobas Integra 800 analyzer, respectively. Statistical analysis was performed using SPSS (Statistical Package for Social Sciences) for Windows 11.5. Results: There was a noteworthy difference among the three groups for Gas6 according to the Kruskal–Wallis test (p < 0.01). Plasma Gas6 concentrations were higher in patients with micro or macroalbuminuria [20.9 ng/mL (16.7–27.0); median (25–75% percentile)] compared to patients with normoalbuminuria [16.5 ng/mL (13.1–22.9)], and healthy controls [15.3 ng/mL (8.3–33.6)]. Conclusions: In conclusion, this is the first study indicating that plasma Gas6 levels are associated with albuminuria in patients with type 2 diabetes. This study could be considered a starting point to focus on the association between Gas6 and diabetic nephropathy.

Different expression patterns and clinical significance of mAxl and sAxl in systemic lupus erythematosus

Axl is one of the TAM family members that downregulates activated immune responses to maintain immune homeostasis. We analyzed the expression and clinical relevance of Axl on the surface of CD14+ monocytes/macrophages (mAxl, membrane Axl) and in the plasma (sAxl, soluble Axl) from patients with systemic lupus erythematosus (SLE). Compared to healthy subjects, the concentrations of sAxl were significantly elevated in plasma from SLE patients, while the mAxl expression on CD14+ monocytes/macrophages from SLE patients was significantly downregulated. A series of severe disease clinical manifestations and laboratory features such as presence of autoantibodies, 24-hour proteinuria excretion or SLEDAI ≥10 were associated with decreased mAxl expression on monocytes/macrophages but elevated sAxl levels in plasma. The plasma level of Gas6, the main ligand of Axl, was slightly decreased in SLE patients, and was negatively correlated with anti-dsDNA antibodies and C-reactive protein. SLE patients with SLEDAI ≥10 showed significantly lower Gas6 levels. Our study suggests that abnormal mAxl and sAxl expression may be involved in the imbalance of immune regulation in SLE.