Evolution of NETosis markers and DAMPs have prognostic value in critically ill COVID-19 patients.

Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days.

The nuclear receptor NOR-1 modulates redox homeostasis in human vascular smooth muscle cells.

The nuclear receptor NOR-1 (Neuron-derived Orphan Receptor-1) has recently been involved in vascular remodeling and coronary artery disease, however, to date, only a few NOR-1 target genes have been described. We aimed to identify genes regulated by NOR-1 in human vascular smooth muscle cells (VSMC). Lentiviral overexpression of NOR-1 increases reactive oxygen species (ROS) in human VSMC. In accordance, NOR-1 strongly increased NADPH oxidase NOX1 mRNA and protein levels, while NOR-1 silencing significantly reduced NOX1 expression. Luciferase reporter, site-directed mutagenesis and EMSA studies identified two nerve growth factor-induced clone B (NGFI-B)-response elements (NBREs) in NOX1 promoter as essential elements for NOR-1 responsiveness. NOR-1 and NOX1 were co-expressed by VSMC in human atherosclerotic lesions, and NOX1 knockdown counteracted the increased ROS production and cell migration induced by NOR-1 overexpression. NOR-1 also modulated the expression of other enzymes involved in cellular redox status, in particular, upregulated superoxide dismutase-1 (SOD1) and SOD3 while downregulated SOD2 and NOX4. NOR-1 induced SOD1 and SOD3 transcriptional activity and participated in the modulation of SOD3 by inflammatory stimuli. By contrast, NOR-1 impaired SOD2 transcription antagonizing NFκB signaling. These results indicate that NOR-1 induces NOX1 in human VSMC and participates in the complex gene networks regulating oxidative stress and redox homeostasis in the vasculature.

Antiapoptotic BCL-2 proteins determine sorafenib/regorafenib resistance and BH3-mimetic efficacy in hepatocellular carcinoma.

Sorafenib, systemic treatment for advanced hepatocellular carcinoma (HCC), and regorafenib, novel second line treatment after sorafenib failure, have efficacy limited by evasive mechanisms of acquired-drug resistance. BCL-2 proteins participate in the response to tyrosine kinase inhibitors; however, their role in HCC therapy with sorafenib/regorafenib remains uncertain. BH3-mimetic ABT-263 (navitoclax) enhanced sorafenib activity, inducing cell death via a mitochondrial caspase-dependent mechanism, after BCL-xL/BCL-2 inhibition. Sorafenib-resistant hepatoma cells (HepG2R and Hep3BR) exhibited altered mRNA expression of BCL-2 and other anti-apoptotic family members, such as MCL-1, priming drug-resistant cancer cells to death by BH3-mimetics. ABT-263 restored sorafenib efficacy in sorafenib-resistant cell lines and HCC mouse models. Moreover, in mice xenografts from patient-derived BCLC9 cells, better tumor response to sorafenib was associated to higher changes in the BCL-2 mRNA pattern. HCC non-treated patients displayed altered BCL-2, MCL-1 and BCL-xL mRNA levels respect to adjacent non-tumoral biopsies and an increased BCL-2/MCL-1 ratio, predictive of navitoclax efficacy. Moreover, regorafenib administration also modified the BCL-2/MCL-1 ratio and navitoclax sensitized hepatoma cells to regorafenib by a mitochondrial caspase-dependent mechanism. In conclusion, sorafenib/regorafenib response is determined by BCL-2 proteins, while increased BCL-2/MCL-1 ratio in HCC sensitizes drug resistant-tumors against ABT-263 co-administration. Thus, changes in the BCL-2 profile, altered in HCC patients, could help to follow-up sorafenib efficacy, allowing patient selection for combined therapy with BH3-mimetics or early switch them to second line therapy.

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

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Targeted GAS6 delivery to the CNS protects axons from damage during experimental autoimmune encephalomyelitis.

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNß injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.

Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity.

INTRODUCTION: Growth arrest-specific gene 6 protein (GAS6) and protein S (ProS) are vitamin K-dependent proteins present in plasma with important regulatory functions in systems of response and repair to damage. They interact with receptor tyrosine kinases of the Tyro3, Axl and MerTK receptor tyrosine kinase (TAM) family, involved in apoptotic cell clearance (efferocytosis) and regulation of the innate immunity. TAM-deficient mice show spontaneous lupus-like symptoms. Here we tested the genetic profile and plasma levels of components of the system in patients with systemic lupus erythematosus (SLE), and compare them with a control healthy population. METHODS: Fifty SLE patients and 50 healthy controls with matched age, gender and from the same geographic area were compared. Genetic analysis was performed in GAS6 and the TAM receptor genes on SNPs previously identified. The concentrations of GAS6, total and free ProS, and the soluble forms of the three TAM receptors (sAxl, sMerTK and sTyro3) were measured in plasma from these samples. RESULTS: Plasma concentrations of GAS6 were higher and, total and free ProS were lower in the SLE patients compared to controls, even when patients on oral anticoagulant treatment were discarded. Those parameters correlated with SLE disease activity index (SLEDAI) score, GAS6 being higher in the most severe cases, while free and total ProS were lower. All 3 soluble receptors increased its concentration in plasma of lupus patients. CONCLUSIONS: The present study highlights that the GAS6/ProS-TAM system correlates in several ways with disease activity in SLE. We show here that this correlation is affected by common polymorphisms in the genes of the system. These findings underscore the importance of mechanism of regulatory control of innate immunity in the pathology of SLE.

Effect of the Gas6 c.834+7G>A polymorphism and the interaction of known risk factors on AMD pathogenesis in Hungarian patients.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Numerous genetic factors contribute to the development of the multifactorial disease. We performed a case-control study to assess the risk conferred by known and candidate genetic polymorphisms on the development of AMD. We searched for genetic interactions and for differences in dry and wet AMD etiology. We enrolled 213 patients with exudative, 67 patients with dry AMD and 106 age and ethnically matched controls. Altogether 12 polymorphisms in Apolipoprotein E, complement factor H, complement factor I, complement component 3, blood coagulation factor XIII, HTRA1, LOC387715, Gas6 and MerTK genes were tested. No association was found between either the exudative or the dry form and the polymorphisms in the Apolipoprotein E, complement factor I, FXIII and MerTK genes. Gas6 c.834+7G>A polymorphism was found to be significantly protective irrespective of other genotypes, reducing the odds of wet type AMD by a half (OR = 0.50, 95%CI: 0.26-0.97, p = 0.04). Multiple regression models revealed an interesting genetic interaction in the dry AMD subgroup. In the absence of C3 risk allele, mutant genotypes of both CFH and HTRA1 behaved as strongly significant risk factors (OR = 7.96, 95%CI: 2.39 = 26.50, p = 0.0007, and OR = 36.02, 95%CI: 3.30-393.02, p = 0.0033, respectively), but reduced to neutrality otherwise. The risk allele of C3 was observed to carry a significant risk in the simultaneous absence of homozygous CFH and HTRA1 polymorphisms only, in which case it was associated with a near-five-fold relative increase in the odds of dry type AMD (OR = 4.93, 95%CI: 1.98-12.25, p = 0.0006). Our results suggest a protective role of Gas6 c.834+7G>A polymorphism in exudative AMD development. In addition, novel genetic interactions were revealed between CFH, HTRA1 and C3 polymorphisms that might contribute to the pathogenesis of dry AMD.

An endogenous vitamin K-dependent mechanism regulates cell proliferation in the brain subventricular stem cell niche.

Neural stem cells (NSC) persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ stem and progenitor cell proliferation are not fully elucidated. Vitamin K-dependent proteins (VKDPs) are mainly secreted factors that were initially discovered as major regulators of blood coagulation. Warfarin ((S(-)-3-acetonylbenzyl)-4-hydroxycoumarin)), a widespread anticoagulant, is a vitamin K antagonist that inhibits the production of functional VKDP. We demonstrate that the suppression of functional VKDPs production, in vitro, by exposure of SVZ cell cultures to warfarin or, in vivo, by its intracerebroventricular injection to mice, leads to a substantial increase in SVZ cell proliferation. We identify the anticoagulant factors, protein S and its structural homolog Gas6, as the two only VKDPs produced by SVZ cells and describe the expression and activation pattern of their Tyro3, Axl, and Mer tyrosine kinase receptors. Both in vitro and in vivo loss of function studies consisting in either Gas6 gene invalidation or in endogenous protein S neutralization, provided evidence for an important novel regulatory role of these two VKDPs in the SVZ neurogenic niche. Specifically, we show that while a loss of Gas6 leads to a reduction in the numbers of stem-like cells and in olfactory bulb neurogenesis, endogenous protein S inhibits SVZ cell proliferation. Our study opens up new perspectives for investigating further the role of vitamin K, VKDPs, and anticoagulants in NSC biology in health and disease.

GAS6 in systemic inflammatory diseases: with and without infection.

Vitamin K-dependent proteins are not only essential regulators of blood coagulation. A recent paper in Critical Care describes the levels of the vitamin K-dependent GAS6 and the soluble form of its receptor Axl in plasma from patients with sepsis of systemic inflammation. The results confirm that GAS6 is elevated during septicemia, but the fact that inflammatory conditions without infection produce a similar effect suggests it is inflammation that induces the synthesis of GAS6, rather than the interactions with bacteria or other infectious agents. The soluble form of the GAS6 receptor Axl was induced less compared with the effect observed in GAS6. This is important as the two proteins form an inactive complex in plasma, suggesting that a functional GAS6 form could be synthesized under these conditions. GAS6 has been proposed as a broad regulator of the innate immune response. GAS6 synthesis is therefore likely to be a regulatory mechanism during systemic inflammation. Recent advances provide the necessary tools for further research, including genetic screenings of the components of this system.

Genotype of the CYBA promoter -930A/G, polymorphism C677T of the MTHFR and APOE genotype in patients with hypertensive disorders of pregnancy: an observational study.

BACKGROUND AND OBJECTIVE: Hypertensive disorders of pregnancy could be favoured by polymorphisms in genes affecting vascular physiology. The aim of our work was to study several variants in the genes regulating oxidative stress, plasma lipids metabolism and endothelial function (observational study). MATERIAL AND METHODS: We studied the -930A/G polymorphism of the CYBA gene promoter, the apolipoprotein E (APOE) genotype and the methylene-tetrahydrofolate reductase (MTHFR) gene C677T polymorphism in 134 healthy pregnant women, 266 pregnant with non-proteinuric hypertension (NPH) and 184 patients with preeclampsia (PE). RESULTS: The GG genotype of the CYBA gene promoter was present in 32.1% of the control population, 38.7% of patients with NPH (P=0.19) and 21.2% of the women with PE (P=0.03). A higher frequency of epsilon 3/epsilon 4 and epsilon 4/epsilon 4 genotypes of APOE was observed in patients with PE or NPH compared with controls (P<0.01). There were no significant differences detected in genotype or allele distribution of the MTHFR, C677T polymorphism. APOE epsilon 3/epsilon 4 and epsilon 4/epsilon 4 genotypes had a worse lipoprotein profile characterized by higher plasma values of total cholesterol (P<0.05) and triglycerides (P<0.005). Despite no differences in MTHFR C677T polymorphism distribution, higher levels of plasma homocysteine were observed in patients with PE than in patients with NPH or controls. CONCLUSIONS: CYBA and APOE polymorphism showed a different distribution in the groups studied, while no differences were observed in MTHFR C677T polymorphism. APOE genotype was associated with changes in lipid and lipoprotein profiles in pregnant women.

Vitamin K-dependent actions of Gas6.

Gas6 (growth arrest-specific gene 6) is the last addition to the family of plasma vitamin K-dependent proteins. Gas6 was cloned and characterized in 1993 and found to be similar to the plasma anticoagulant protein S. Soon after it was recognized as a growth factor-like molecule, as it interacted with receptor tyrosine kinases (RTKs) of the TAM family; Tyro3, Axl, and MerTK. Since then, the role of Gas6, protein S, and the TAM receptors has been found to be important in inflammation, hemostasis, and cancer, making this system an interesting target in biomedicine. Gas6 employs a unique mechanism of action, interacting through its vitamin K-dependent Gla module with phosphatidylserine-containing membranes and through its carboxy-terminal LG domains with the TAM membrane receptors. The fact that these proteins are affected by anti-vitamin K therapy is discussed in detail.

Association of specific haplotypes of GAS6 gene with stroke.

The product of the growth arrest-specific gene 6 (GAS6), a ligand for tyrosine kinase receptors, is a vitamin K-dependent protein, structurally related to anticoagulant protein S. Gas6-deficient mice are protected against thrombosis, demonstrating the importance of this protein in the cardiovascular system. In a preliminary study on GAS6 polymorphisms and atherothrombotic disease we found an association between the AA genotype of the c.834 + 7G > A GAS6 polymorphism and stroke. In order to further explore this association by considering GAS6 haplotypes and the main stroke subtypes, 457 patients with ischemic stroke, 199 with hemorrhagic stroke and 150 asymptomatic controls were genotyped for eight GAS6 polymorphisms and other genetic markers in the same genome region. Association was measured by logistic regression analysis. The THESIAS program was used to measure linkage disequilibrium and haplotype frequencies. In univariate analysis, the GAS6 c.834 + 7AA genotype was found associated with decreased risk for stroke (OR: 0.59; 95%CI: 0.37-0.93). After adjustment for vascular risk factors, association was maintained when stroke subtypes affecting the microvasculature such as lacunar stroke and deep haemorrhage, were grouped together (OR: 0.44; 95%CI: 0.21-0.90). Furthermore, haplotype analysis revealed that association was even stronger when the c.834 + 7A allele was present in a specific haplotype (CACA) of four GAS6 polymorphisms. From these results we conclude that the A allele of the GAS6 c.834 + 7G > A polymorphism and more specifically, the CACA haplotype, is less prevalent in patients with stroke, suggesting a protective role for stroke of this haplotype.

Human vitamin K-dependent GAS6: gene structure, allelic variation, and association with stroke.

The product of the growth arrest-specific gene 6 (GAS6), a ligand for the Axl, Sky, and Mer tyrosine kinase receptors, is a vitamin K-dependent protein, structurally related to anticoagulant protein S. Gas6-deficient mice are protected against thrombosis, demonstrating the importance of this protein in the cardiovascular system. The present study was aimed at determining the human GAS6 intron-exon structure and analyzing the gene for the presence of allelic variants that could be associated with atherothrombotic disease. Online analyses allowed us to localize 15 GAS6 exons and to determine the sequence of their intron-flanking regions, in a chromosome 13 region spanning 43.8 kb of DNA. SSCP analysis of PCR-amplified GAS6 exons with their intron-flanking regions from a minimum of 12 control DNA samples, revealed the presence of eight different variants, which were confirmed to be single nucleotide polymorphisms (SNPs). Three of them (c.1263G>C, c.1332C>T, and c.1869T>C) are localized in exons 11, 12, and 14, and appear to be neutral since they do not modify the encoded amino acid. The other SNPs (c.280+170C>G, c.712+26G>A, c.713-155C>T, c.834+7G>A, and c.1478-94C>G) are in introns 3, 7, 8, and 12. A preliminary analysis of five of these SNPs in a group of 110 healthy controls and 188 patients with atherothrombotic disease has revealed statistically significant differences between controls and stroke patients in the allelic distributions of one of these variants (c.834+7G>A in intron 8). The SNP identification in GAS6 reported here would be very useful in future association studies aimed at determining the physiologic role of GAS6 in stroke and other human diseases.