Christine Masson

Effect of HMGCoA reductase inhibitors on cytochrome P450 expression in endothelial cell line.

Endothelial cells and smooth muscle cells are the major cells that constitute blood vessels, and endothelial cells line the lumen of blood vessels. These 2 types of cells also play an integral role in the regional specialization of vascular structure. On the basis of these observations, we designed our study to investigate the effect of various statins on CYP expression in endothelial cells. 3-hydroxymethyl coenzyme A reductase inhibitors play an important role in vascular function. The majority of the statins available on the market show extensive metabolism by cytochrome P450 (CYP) enzymes. Both cell types are involved in the bioconversion of arachidonic acid into vasoactive compounds. The aim of this study was to demonstrate the effect of statins on cytochrome P450 expression in endothelial cells. Our results show that endothelial cells expressed both CYPs involved in epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) production and the nuclear receptor implicated in cytochrome P450 regulation. Treatment of endothelial cells with lovastatin increased CYP2C9 expression. After 96 hours of treatment, fluvastatin and lovastatin clearly increased CYP2C9 protein level. CAR but not PXR was expressed in endothelial cells, indicating that the upregulating effect of statins on CYP2C9 in endothelial cells could be mediated through CAR only due to the lack of expression of PXR in these cells.

Influence of Pre-analytical Variables on VEGF Gene Expression and Circulating Protein Concentrations

BACKGROUND The extended role of vascular endothelial growth factor (VEGF) in human pathophysiology led us to evaluate pre-analytical parameters possibly influencing its levels in peripheral blood and tissues. The effects on VEGF protein levels and mRNA expression were measured after storage delay (blood and tissue), use of different types of anticoagulants (blood), and after different numbers of freeze-thaw cycles (blood). METHODS Blood from healthy donors was sampled simultaneously in ethylene diamine tetraacetic acid (EDTA), acid citrate dextrose (ACD-A), hirudin, and serum separation tubes. For each anticoagulant, VEGF was measured by enzyme-linked immunosorbent assay (ELISA) with different conditions of delay at 4°C before centrifugation (2 h, 4 h, or 48 h) and of different numbers of freeze-thaw cycles (1, 2, and 10). The transcripts coding for the VEGF165 isoform were quantified in peripheral blood mononuclear cells by RT-PCR. Muscle biopsy samples were frozen with delays of 15, 30, or 60 min after surgery. VEGF expression was quantified on immunofluorescence stained slides. RESULTS The period of storage and the number of freeze-thaw cycles correlated with an increase in the levels of circulating VEGF (for each anticoagulant but not for serum) and its expression in PBMCs. VEGF expression measured from muscle biopsy sections was higher with freezing delays, with a peak at 30 and 60 min as compared to 15 min. CONCLUSIONS The most reliable conditions for measuring both circulating VEGF and its gene expression are to reduce time between blood collection and centrifugation, and to avoid multiple freeze-thaw cycles. Serum collection tubes with no additive and no separator were less sensitive to the pre-analytical variations analyzed in this study. Freezing delay had a significant influence on VEGF protein expression in tissue samples.

Klotho KL-VS genotype is involved in blood pressure regulation.

BACKGROUND AND AIMS Genome-wide linkage analysis studies reported the importance of the long arm of chromosome 13 in systolic blood pressure regulation. Therefore, isolating a genetic variant related to this chromosomal region could be challenging. Klotho KL-VS allele is located on this chromosomal region and its relationships with cardio-vascular risk factors need extensive investigations. The aim of the present study is to examine whether the klotho KL-VS genotype is associated with cardio-vascular risk factors, more particularly hypertension, in two independent cohorts. A secondary objective was to investigate relationships with antihypertensive treatment, arterial stiffness and carotid artery parameters. METHODS AND RESULTS A total of 1023 French individuals were genotyped for klotho KL-VS. Participants were part of the French ERA and STANISLAS cohorts. In both cohorts, klotho KL-VS/KL-VS genotype was significantly associated with lower systolic blood pressure and pulse pressure when compared to homozygous and heterozygous more frequent (WT) allele carriers (p=0.003 and p<0.001 respectively). Antihypertensive treatment stratification confirmed the previous significant associations, while a significant interaction between klotho KL-VS genotype and antihypertensive treatment was also interestingly found (0.019 for p interaction). CONCLUSION Klotho KL-VS/KL-VS genotype may be associated with decreased cardio-vascular risk and may interact with antihypertensive treatment in order to reduce blood pressure. This finding could lead to identify subgroups of hypertensive adults who might benefit antihypertensive drug therapies.

Association between EGF and lipid concentrations: A benefit role in the atherosclerotic process?

CONTEXT AND OBJECTIVE Previous in vitro studies have shown a relationship between epidermal growth factor (EGF) and lipid metabolism. Indeed, EGF is able to modulate lipoprotein fractions in human fetal intestine and hepatic-derived cell lines. The aim of this study was to search for potential associations between EGF concentrations and lipid parameters in both plasma and peripheral blood mononuclear cells (PBMCs) among healthy individuals. DESIGN EGF concentrations were quantified in plasma of 273 men and 279 women and in PBMCs of 57 men and 62 women from the STANISLAS cohort. In addition, basic blood constituents including lipid parameters (apolipoproteins A1, and B, total and high-density lipid (HDL)-cholesterols and triglycerides) were measured. RESULTS Plasma EGF concentrations were significantly and positively correlated with apolipoprotein A1 and HDL-cholesterol concentrations after adjustment for age, sex, and body mass index (r=0.155, P=0.0003; and r=0.112, P=0.0086, respectively). They were also negatively correlated with the apolipoprotein B/A1 ratio (r=-0.107, P=0.012). In addition, EGF concentrations in PBMCs were negatively correlated with apolipoprotein B, total and low-density lipid-cholesterols, and triglyceride concentrations, and apolipoprotein B/A1 ratio (P=0.0018, P=0.0137, P=0.0142, P=0.0162, and P=0.0077, respectively, with r ranging between -0.287 and -0.222). No interactions were found with gender. CONCLUSION New associations between EGF and lipid concentrations have been reported, providing new perspectives with regard to the role of EGF in atherosclerosis.

Functional epistatic interaction between rs6046G>A in F7 and rs5355C>T in SELE modifies systolic blood pressure levels.

Background Although numerous genetic studies have been performed, only 0.9% of blood pressure phenotypic variance has been elucidated. This phenomenon could be partially due to epistatic interactions. Our aim was to identify epistatic interaction(s) associated with blood pressure levels in a pre-planned two-phase approach. Methods and Results In a discovery cohort composed of 3,600 French individuals, we found rs6046A allele in F7 associated with decreased blood pressure levels (P≤3.7×10−3) and rs5355T allele in SELE associated with decreased diastolic blood pressure levels (P = 5×10−3). Both variants interacted in order to influence blood pressure levels (P≤0.048). This interaction was replicated with systolic blood pressure in 4,620 additional European individuals (P = 0.03). Similarly, in this replication cohort, rs6046A was associated with decreased blood pressure levels (P≤8.5×10−4). Furthermore, in peripheral blood mononuclear cells of a subsample of 90 supposed healthy individuals, we found rs6046A positively associated with NAMPT mRNA levels (P≤9.1×10−5), suggesting an eventual involvement of NAMPT expression in blood pressure regulation. Confirming this hypothesis, further transcriptomic analyses showed that increased NAMPT mRNA levels were positively correlated with ICAM1, SELL, FPR1, DEFA1-3, and LL-37 genes expression (P≤5×10−3). The last two mRNA levels were positively associated with systolic blood pressure levels (P≤0.01) and explained 4% of its phenotypic variation. Conclusion These findings reveal the importance of epistatic interactions in blood pressure genetics and give new insights for the role of inflammation in its complex regulation.

Angiogenesis related genes NOS3, CD14, MMP3 and IL4R are associated to VEGF gene expression and circulating levels in healthy adults.

BackgroundVascular endothelial growth factor (VEGF) plays a key role in angiogenesis. The aim was to assess the genetic connections between the angiogenesis-related NOS3, CD14, MMP3, IL4R, IL4 genes and VEGF expression and plasma levels.MethodsThe associations between VEGF plasma levels with the polymorphisms of NOS3, CD14, MMP3, IL4R, and IL4 were assessed in 403 healthy unrelated adults. The epistatic and environmental interactions were explored, including four VEGF-related polymorphisms previously identified. The VEGF expression in peripheral blood mononuclear cells was quantified (n = 65) for the VEGF121, VEGF145, VEGF165, and VEGF189 isoforms.ResultsThe polymorphism rs1799983 of NOS3 was associated with the sum of all VEGF isoforms mRNA levels (P = 0.032) and VEGF145 (P = 0.033). Rs1800779 of NOS3 interacted with rs3918226 of the same gene and with the rs2569190 of CD14 (P = 0.022, P = 0.042, respectively) for VEGF plasma levels. Other epistatic interactions included the rs1801275 of IL4R with the rs6921438 (VEGF-related variant) and rs3025058 of MMP3 (P = 0.042, P = 0.010 respectively) and the rs2569190 of CD14 with the rs3025058 of MMP3 (P = 0.0119). We also identified an interaction of rs1800779 with obesity, high-density lipoprotein cholesterol and triglycerides (P = 0.018, P = 0.005, P = 0.043, respectively) as well as the interaction of rs6921438 with hypertension (P = 0.028).ConclusionsOur findings indicated that genetic variants of NOS3, CD14, MMP3 and IL4R are implicated in the determination of VEGF expression and plasma levels. Thus, they support the hypothesis that in physiological conditions there are complex biological relationships between pathways (such as angiogenesis and inflammation), which are involved in the development of chronic diseases.

TREM-1 SNP rs2234246 regulates TREM-1 protein and mRNA levels and is associated with plasma levels of L-selectin

High levels of TREM-1 are associated with cardiovascular and inflammatory diseases risks and the most recent studies have showed that TREM-1 deletion or blockade is associated with up to 60% reduction of the development of atherosclerosis. So far, it is unknown whether the levels of TREM-1 protein are genetically regulated. Moreover, TREM family receptors have been suggested to regulate the cellular adhesion process. The goal of this study was to investigate whether polymorphisms within TREM-1 are regulating the variants of serum TREM-1 levels and the expression levels of their mRNA. Furthermore, we aimed to point out associations between polymorphisms on TREM-1 and blood levels of selectins. Among the 10 SNPs studied, the minor allele T of rs2234246, was associated with increased sTREM-1 in the discovery population (p-value = 0.003), explaining 33% of its variance, and with increased levels of mRNA (p-value = 0.007). The same allele was associated with increased soluble L-selectin levels (p-value = 0.011). The higher levels of sTREM-1 and L-selectin were confirmed in the replication population (p-value = 0.0007 and p-value = 0.018 respectively). We demonstrated for the first time one SNP on TREM-1, affecting its expression levels. These novel results, support the hypothesis that TREM-1 affects monocytes extravasation and accumulation processes leading to atherogenesis and atherosclerotic plaque progression, possibly through increased inflammation and subsequent higher expression of sL-selectin.

Pro- and anti-angiogenic VEGF mRNAs in autoimmune thyroid diseases

Abstract The aim of this study was to assess the relationships between five different splice isoforms of VEGF mRNA and its plasma levels in individuals treated for autoimmune thyroid diseases (AITD); mainly Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). In a population from Tunisia, levels of thyroid hormones and antibodies were quantified simultaneously with plasma VEGF and VEGF mRNA isoforms after a period of 6 months of patients’ treatment. Plasma VEGF was measured in 110 AITD patients (21 GD and 89 HT patients). VEGF isoforms (VEGF121, VEGF165, VEGF145 and VEGF189 pro-angiogenic isoforms and VEGF165b anti-angiogenic isoform) in peripheral blood mononuclear cells were quantified in 71 patients (20 GD and 51 HT patients) and 86 healthy controls. Decreased levels of VEGF189 mRNA were observed in AITD compared to controls. VEGF165 was increased in GD patients compared to controls and the VEGF165b was increased in HT patients compared to GD. We observed increased levels of VEGF165b in hypothyroid AITD patients after treatment. We have also shown that the VEGF145 isoform levels were determined by FT4 in all patients and by the thyroid status after 6 months of treatment only in HT patients. An association was observed for VEGF165 mRNA levels with anti-TPO antibodies in all patients. Finally, FT4 was associated with VEGF plasma levels but only in healthy controls. In conclusion, this descriptive study highlights the specificity of VEGF mRNA isoforms in AITD, a fact underlining the need for novel clinical trials and the development of personalised theranostic approaches.

Plasma VEGF-related polymorphisms are implied in autoimmune thyroid diseases

Abstract Autoimmune thyroid diseases (AITD), including Graves’ disease (GD) and Hashimoto thyroiditis (HT), are complex multifactorial diseases. Vascular endothelial growth factor (VEGF) is implicated in various inflammatory diseases, especially autoimmune diseases. Our aim was to elucidate the relationships between plasma VEGF levels and four genome-wide association study-identified single nucleotide polymorphisms (SNPs) related to VEGF with AITD in Tunisian patients. A total of 364 healthy controls and 389 patients with AITD were genotyped for the SNPs rs6921438, rs4416670, rs6993770 and rs10738760. Levels of thyroid hormones and antibodies were quantified simultaneously with plasma VEGF after a period of six months of treatment. We found that the minor alleles of rs10738760 and rs6921438 are associated with the presence of GD. A allele of rs10738760 polymorphism is associated with increased plasma levels of free tri-iodothyronin (FT3) while no relationship was found with circulating VEGF plasma levels after six months of treatment. We also showed that the T allele of rs4416670 polymorphism was associated with increased risk of hyperthyroidism in patients treated for six months, independently of their initial diagnosis. There was no significant association between the SNPs and the risk for HT compared with controls. This study shows that AITD are influenced by 3 SNPs linked to VEGF circulating levels. Whereas rs10738760 appeared specific to GD and FT3 production after six months of treatment, rs6921438 and rs4416670 were implicated in the risk for GD. This study opens new ways to test pharmacogenomics concepts in the future especially in GD in which recurrence prognosis is still challenging.

The polymorphism rs6918289 located in the downstream region of the TREM2 gene is associated with TNF-α levels and IMT-F

Triggering receptor expressed on myeloid cells 2 (TREM2) is known for its anti-inflammatory properties during the immune response, and influences negatively on TNF-α expression levels. Genetic epidemiology studies have identified polymorphisms located in the TREM2 gene associated with neurodegenerative and chronic inflammatory diseases. TREM2 levels have been observed to affect plasma levels of TNF-α and plaque stability in symptomatic and asymptomatic patients with carotid stenosis. In this study, we investigated polymorphisms located in the TREM2 gene region and association with TNF-α levels and the intima media thickness of the femoral artery. The discovery population from the STANISLAS Family Study comprised of 809 individuals, whereas the replication population utilized an independent cohort of French origin (n = 916). Our results suggest that the minor allele (T) of SNP rs6918289 is positively associated with elevated plasma levels of TNF-α in discovery and replication populations (P = 0.0026, SE = 0.04 and P = 0.023, SE = 0.09, respectively), including femoral artery thickness in the discovery cohort (P = 0.026, SE = 0.009). Results indicate that rs6918289 may be considered as a risk factor for inflammatory diseases and could be used in stratified medicine with patients diagnosed with chronic inflammatory-related conditions, such as atherosclerosis.