P. García de Frutos

Genetic loss of Gas6 induces plaque stability in experimental atherosclerosis

The growth arrest‐specific gene 6 (Gas6) plays a role in pro‐atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that Gas6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. Gas6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of Gas6 does not affect the number and size of initial and advanced plaques in ApoE−/− mice, but alters its plaque composition. Compared to Gas6+/+: ApoE−/− mice, initial and advanced plaques of Gas6−/−: ApoE−/− mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFβ was increased. In addition, fewer macrophages were found in advanced plaques of Gas6−/−: ApoE−/− mice. Hence, loss of Gas6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for Gas6 in plaque composition and stability. Copyright

Association study between polymorphims in GAS6-TAM genes and carotid atherosclerosis

Carotid atherosclerosis (CA) is one of the most common causes of stroke, and recent studies suggest that pathways initiated by the interaction of the plasma vitamin K-dependent protein GAS6 with the tyrosine kinase receptors TYRO3, AXL and MERTK (TAM) may have a relevant role in atherogenesis. Furthermore, our previous studies indicated an association between GAS6 and stroke. The aim of this study was to analyse the genetic association between SNPs and haplotypes in GAS6-TAM genes and CA. We performed a case-control study with 233 CA patients confirmed by nuclear magnetic resonance angiography and 202 patients who suffered from cardioembolic (non atherogenic) stroke. For all included subjects information on established risk factors was available. Genotyping of 16 selected tagSNPs was performed by real-time PCR, using either FRET or TaqMan probes. Adjusted logistic regression (LR) analyses indicated that rs2289743 in TYRO3 and rs869016 in MERTK were associated to CA, decreasing its risk (OR [95%CI]=0.39 [0.16-0.94] and OR [95%CI]=0.31 [0.14-0.69], respectively). Linkage disequilibrium results were consistent with the haplotype blocks described in HapMap and adjusted LR analyses revealed that the haplotype ACAA in MERTK , containing the minor allele of the associated SNP, was also associated to CA. No association was observed with GAS6 and AXL variants, which suggests that CA is not the mechanism underlying the reported association between GAS6 and stroke. The association between TYRO3 and MERTK variants and carotid atherosclerosis found in this study reinforces a physiological role of the GAS6-TAM pathway in atherogenesis.

AXL receptor tyrosine kinase is increased in patients with heart failure

BACKGROUND AXL is a membrane receptor tyrosine kinase highly expressed in the heart and has a conspicuous role in cardiovascular physiology. The role of AXL in heart failure (HF) has not been previously addressed. METHODS AND RESULTS AXL protein was enhanced 6-fold in myocardial biopsies of end-stage HF patients undergoing heart transplantation compared to controls from heart donors (P<0.0001). Next, we performed a transversal study of patients with chronic HF (n=192) and a group of controls with no HF (n=67). sAXL and BNP circulating levels were quantified and clinical and demographic data were collected. sAXL levels in serum were higher in HF (86.3 ± 2.0 ng/mL) than in controls (67.8 ± 2.0 ng/mL; P<0.0001). Also, sAXL correlated with several parameters associated with worse prognosis in HF. Linear regression analysis indicated that serum creatinine, systolic blood pressure and atrial fibrillation, but not BNP levels, were predictive of sAXL levels. Cox regression analysis indicated that high sAXL values at enrollment time were related to the major HF events (all-cause mortality, heart transplantation and HF hospitalizations) at one year follow-up (P<0.001), adding predictive value to high BNP levels. CONCLUSIONS Myocardial expression and serum concentration of AXL is elevated in HF patients compared to controls. Furthermore, peripheral sAXL correlates with parameters associated with the progression of HF and with HF events at short term follow-up. All together these results suggest that sAXL could belong to a new molecular pathway involved in myocardial damage in HF, independent from BNP.

Coagulation factor and protease pathways in thrombosis and cardiovascular disease

The biochemical characterisation of the proteolytic pathways that constitute blood coagulation was one of the most relevant achievements in biomedical research during the second half of the 20th century. Understanding these pathways was of crucial importance for improving global health through application in haemostasis and thrombosis pathologies. Immediately after the cloning of the genes corresponding to these proteins, mutations were discovered in them that were associated with imbalances in haemostasis. Later, the importance of coagulation pathways in other pathological processes was demonstrated, such as in atherosclerosis and inflammation, both essential processes involved in vascular disease. In the present review we evaluate the concepts that have allowed us to reach the integrated vision on coagulation that we have today. The thrombo-inflammation model encompassing these aspects includes a pivotal role for the proteases of the coagulation pathway as well as the regulatory proteins thereof. These concepts illustrate the importance of the coagulation cascade in cardiovascular pathology, not only in thrombotic processes, but also in atherosclerotic processes and in the response to ischaemia-reperfusion injury, making it a central mechanism in cardiovascular disease.

Use of serum levels of high sensitivity troponin T, galectin-3 and C-terminal propeptide of type I procollagen at long term follow-up in heart failure patients with reduced ejection fraction: Comparison with soluble AXL and BNP

BACKGROUND Prognostic biomarkers are needed to improve the management of the heart failure (HF) epidemic, being the brain natriuretic peptides the most valuable. Here we evaluate 3 biomarkers, high sensitivity troponin T (hs-TnT), galectin-3 (Gal-3) and C-terminal propeptide of type I procollagen (CICP), compare them with a recently described new candidate (sAXL), and analyze their relationship with BNP. METHODS HF patients with reduced ejection fraction (n=192) were included in this prospective observational study, with measurements of candidate biomarkers, functional, clinical and echocardiographic variables. A Cox regression model was used to determine predictors for clinical events, i.e. all-cause mortality and heart transplantation. RESULTS Hs-TnT circulating values were correlated to clinical characteristics indicative of more advanced HF. When analyzing the event-free survival at a mean follow-up of 3.6years, patients in the higher quartile of either BNP, hs-TnT, CICP and sAXL had increased risk of suffering a clinical event, but not Gal-3. Combination of high sAXL and BNP values had greater predictive value (HR 6.8) than high BNP alone (HR 4.9). In a multivariate Cox regression analysis, BNP, sAXL and NYHA class were independent risk factors for clinical events. CONCLUSIONS In this HF cohort, hs-TnT is a good HF marker and has a very significant prognostic value. The prognostic value of CICP and sAXL was of less significance. However, hs-TnT did not add predictive value to BNP, while sAXL did. This suggests that elevated troponin has a common origin with BNP, while sAXL could represent an independent pathological mechanism.

An electrochemical immunosensor for brain natriuretic peptide prepared with screen-printed carbon electrodes nanostructured with gold nanoparticles grafted through aryl diazonium salt chemistry

A sensitive amperometric immunosensor has been prepared by immobilization of capture antibodies onto gold nanoparticles (AuNPs) grafted on a screen-printed carbon electrode (SPCE) through aryl diazonium salt chemistry using 4-aminothiophenol (AuNPs-S-Phe-SPCE). The immunosensor was designed for the accurate determination of clinically relevant levels of B-type natriuretic peptide (BNP) in human serum samples. The nanostructured electrochemical platform resulted in an ordered layer of AuNPs onto SPCEs which combined the advantages of high conductivity and improved stability of immobilized biomolecules. The resulting disposable immunosensor used a sandwich type immunoassay involving a peroxidase-labeled detector antibody. The amperometric transduction was carried out at -0.20V (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. The nanostructured immunosensors show a storage stability of at least 25 days, a linear range between 0.014 and 15ngmL-1, and a LOD of 4pgmL-1, which is 100 times lower than the established cut-off value for heart failure (HF) diagnosis. The performance of the immunosensor is advantageously compared with that provided with immunosensors prepared by grafting SPCE with p-phenylendiamine (H2N-Phe-SPCE) and attaching AuNPs by immersion into an AuNPs suspension or by electrochemical deposition, as well as with immunosensors constructed using commercial AuNPs-modified SPCEs. The developed immunosensor was applied to the successful analysis of human serum from heart failure (HF) patients upon just a 10-times dilution as sample treatment.

A novel method to detect causative mutations in fibrinogen, using a small amount of blood processed through HPLC and time of flight (TOF) mass spectrometry

A novel method to detect causative mutations in fibrinogen, using a small amount of blood processed through HPLC and time of flight (TOF) mass spectrometry -

New genetic association for the kinin system: the L-arginine connection

New genetic association for the kinin system: the L-arginine connection - Insight: association of L-arginine levels and two loci in genes of the kallikrein-kinin system

Protein S: an anticoagulant in its own right.

Editorial on Heeb et al. Thromb Haemost 2012; 107: 690-698. Since the isolation of protein S in 1979, initial studies demonstrated that protein S acted as a natural anticoagulant by promoting the serine protease activity of activated protein C (APC) on its substrates, the coagulation factors Va and VIIIa. This interaction required binding to phospholipid membranes through the vitamin K-dependent Gla residues of protein S. Later work showed that protein S interacts with other blood components, including a regulator of complement (C4b-binding protein), other components of the coagulation cascade (tissue factor pathway inhibitor and factor Xa), plasma lipoproteins and cells. The later interaction is mediated through specific membrane receptor tyrosine kinases belonging to the TAM family. This choreography of interactions predicted new biological functions, and protein S is now recognised as a regulator of essential processes in the response to damage, including regulation of phagocytosis of apoptotic cells (efferocytosis), cell survival and activation of innate immunity. Still, the anticoagulant function of protein S is central to its biological role, as it was first proposed by the association of protein S deficiency and thrombosis in human cohorts. This has been experimentally corroborated by the recent characterisation of the protein S knockout mouse phenotype (1). In the present issue of Thrombosis and Haemostasis, Heeb et al. (2) provide further evidence of the use of protein S in therapeutic anticoagulation independently of the infusion of APC. One of the reasons behind the late recognition of the APC-independent anticoagulant function of protein S derives from basic biochemical reasons. This functional property depends on the presence of Zn2+ ions, which were often removed during protein S purification procedures (3). The present study shows promises of using protein S as an anticoagulant drug on its own, independently of APC. This would add a new therapeutic substance that could be especially interesting for situations where control of both anticoagulation and inflammation has to be provided. This includes treatments for sepsis and atherosclerotic disease, were the role of protein S has been recently evaluated (4). Protein S is a complex molecule with several posttranslational modifications, and needs suitable animal models where its functional requirements are validated. The present work is an important step forward in this direction.Editorial on Heeb et al. Thromb Haemost 2012; 107: 690-698. Since the isolation of protein S in 1979, initial studies demonstrated that protein S acted as a natural anticoagulant by promoting the serine protease activity of activated protein C (APC) on its substrates, the coagulation factors Va and VIIIa. This interaction required binding to phospholipid membranes through the vitamin K-dependent Gla residues of protein S. Later work showed that protein S interacts with other blood components, including a regulator of complement (C4b-binding protein), other components of the coagulation cascade (tissue factor pathway inhibitor and factor Xa), plasma lipoproteins and cells. The later interaction is mediated through specific membrane receptor tyrosine kinases belonging to the TAM family. This choreography of interactions predicted new biological functions, and protein S is now recognised as a regulator of essential processes in the response to damage, including regulation of phagocytosis of apoptotic cells (efferocytosis), cell survival and activation of innate immunity. Still, the anticoagulant function of protein S is central to its biological role, as it was first proposed by the association of protein S deficiency and thrombosis in human cohorts. This has been experimentally corroborated by the recent characterisation of the protein S knockout mouse phenotype (1). In the present issue of Thrombosis and Haemostasis, Heeb et al. (2) provide further evidence of the use of protein S in therapeutic anticoagulation independently of the infusion of APC. One of the reasons behind the late recognition of the APC-independent anticoagulant function of protein S derives from basic biochemical reasons. This functional property depends on the presence of Zn2+ ions, which were often removed during protein S purification procedures (3). The present study shows promises of using protein S as an anticoagulant drug on its own, independently of APC. This would add a new therapeutic substance that could be especially interesting for situations where control of both anticoagulation and inflammation has to be provided. This includes treatments for sepsis and atherosclerotic disease, were the role of protein S has been recently evaluated (4). Protein S is a complex molecule with several posttranslational modifications, and needs suitable animal models where its functional requirements are validated. The present work is an important step forward in this direction.