Effect of FADS1 SNPs rs174546, rs174547 and rs174550 on blood fatty acid profiles and plasma free oxylipins.

Introduction: Previous studies have indicated that activity of fatty acid desaturase 1 (FADS1), is involved in cardiometabolic risk. Recent experimental data have shown that FADS1 knockdown can promote lipid accumulation and lipid droplet formation in liver cells. In this study, we aimed to characterize whether different FADS1 genotypes affect liver fat content, essential fatty acid content and free oxylipin mediators in the blood. Methods: We analyzed the impact of FADS1 single-nucleotide polymorphisms (SNPs) rs174546, rs174547, and rs174550 on blood fatty acids and free oxylipins in a cohort of 85 patients from an academic metabolic medicine outpatient center. Patients were grouped based on their genotype into the homozygous major (derived) allele group, the heterozygous allele group, and the homozygous minor (ancestral) allele group. Omega-3 polyunsaturated fatty acids (n-3 PUFA) and omega-6 polyunsaturated fatty acids (n-6 PUFA) in the blood cell and plasma samples were analyzed by gas chromatography. Free Oxylipins in plasma samples were analyzed using HPLC-MS/MS. Liver fat content and fibrosis were evaluated using Fibroscan technology. Results: Patients with the homozygous ancestral (minor) FADS1 genotype exhibited significantly lower blood levels of the n-6 PUFA arachidonic acid (AA), but no significant differences in the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). There were no significant differences in liver fat content or arachidonic acid-derived lipid mediators, such as thromboxane B2 (TXB2), although there was a trend toward lower levels in the homozygous ancestral genotype group. Discussion: Our findings suggest that FADS1 genotypes influence the blood levels of n-6 PUFAs, while not significantly affecting the n-3 PUFAs EPA and DHA. The lack of significant differences in liver fat content and arachidonic acid-derived lipid mediators suggests that the genotype-related variations in fatty acid levels may not directly translate to differences in liver fat or inflammatory lipid mediators in this cohort. However, the trend towards lower levels of certain lipid mediators in the homozygous ancestral genotype group warrants further investigation to elucidate the underlying mechanisms of different FADS1 genotypes and potential implications for cardiometabolic risk.

Essential Polyunsaturated Fatty Acids in Blood from Patients with and without Catheter-Proven Coronary Artery Disease.

Coronary artery disease (CAD) is the leading cause of death worldwide. Statins reduce morbidity and mortality of CAD. Intake of n-3 polyunsaturated fatty acid (n-3 PUFAs), particularly eicosapentaenoic acid (EPA), is associated with reduced morbidity and mortality in patients with CAD. Previous data indicate that a higher conversion of precursor fatty acids (FAs) to arachidonic acid (AA) is associated with increased CAD prevalence. Our study explored the FA composition in blood to assess n-3 PUFA levels from patients with and without CAD. We analyzed blood samples from 273 patients undergoing cardiac catheterization. Patients were stratified according to clinically relevant CAD (n = 192) and those without (n = 81). FA analysis in full blood was performed by gas chromatography. Indicating increased formation of AA from precursors, the ratio of dihomo-gamma-linolenic acid (DGLA) to AA, the delta-5 desaturase index (D5D index) was higher in CAD patients. CAD patients had significantly lower levels of omega-6 polyunsaturated FAs (n-6 PUFA) and n-3 PUFA, particularly EPA, in the blood. Thus, our study supports a role of increased EPA levels for cardioprotection.

Secure and optimized detection of PNPLA3 rs738409 genotype by an improved PCR-restriction fragment length polymorphism method.

The PNPLA3 reference single-nucleotide polymorphism rs738409 has been identified as a predisposing factor for nonalcoholic fatty liver disease. A simple method based on PCR and restriction fragment length polymorphism (RFLP) analysis had been published to detect the nonpathogenic allele PNPLA3 rs738409 variant. The presence of the pathogenic variant was deduced by the indigestibility of the corresponding PCR product with BtsCI recognizing the nonpathogenic allele. However, one cannot exclude that an enzymatic reaction does not occur for other, more trivial, reasons. For safe and secure detection of the pathogenic PNPLA3 rs738409, we have further developed the PCR-restriction fragment length polymorphism method by adding a second restriction enzyme digest, clearly identifying the correct PNPLA3 alleles and in particular the pathogenic variant.

Recombinant adeno-associated virus-based gene transfer of cathelicidin induces therapeutic neovascularization preferentially via potent collateral growth.

Therapeutic neovascularization is a concept well validated in animal models, however, without clear-cut success in clinical studies. To achieve prolonged transgene expression, recombinant adeno-associated virus (rAAV) was used in a chronic ischemic hind-limb model and the human antimicrobial peptide cathelicidin (LL-37/hCAP-18) was used as proangiogenic factor. Seven days after femoral artery excision, 0.5 x 10(11) rAAV particles encoding for green fluorescent protein (rAAV.GFP), cathelicidin (rAAV.cath), or vascular endothelial growth factor A (rAAV.VEGF-A) were retroinfused into the anterior tibial vein of rabbits (n = 5 per group). In addition, one rAAV.cath-treated group obtained a constant infusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin into the ischemic tissue starting on day 7. On day 7 and day 35 angiography of both hind limbs was performed for collateral quantification and frame count score (cinedensitometry). Capillary-to-muscle fiber ratios were obtained on day 35. Compared with controls, application of rAAV.cath induced a gain of perfusion (153 +/- 12 vs. 107 + 9% of day 7 controls) via increased collateral growth (length index, 161 +/- 14 vs. 97 +/- 9%, controls), but no significant capillary growth (1.16 +/- 0.09 vs. 0.99 +/- 0.08, controls). Wortmannin application completely abolished the effects of rAAV.cath, indicating the involvement of the PI3K signal pathway. In conclusion, rAAV-mediated cathelicidin expression is capable of inducing functionally relevant neovascularization, preferentially by collateral growth. The rAAV-based vectors as long-expressing vector expression systems and cathelicidin as proangiogenic factor provide a promising new combination in the treatment of peripheral artery disease.

In vitro selection of viral vectors with modified tropism: the adeno-associated virus display.

Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor-specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.