The relationship between plasma angiopoietin-like protein 4 levels, angiopoietin-like protein 4 genotype, and coronary heart disease risk

Abstract

Objective: To investigate the relationship between angiopoietin-like protein 4 (Angptl4) levels, coronary heart disease (CHD) biomarkers, and ANGPTL4 variants.

Methods and results: Plasma Angptl4 was quantified in 666 subjects of the Northwick Park Heart Study II using a validated ELISA. Seven ANGPTL4 single-nucleotide polymorphisms were genotyped, and CHD biomarkers were assessed in the whole cohort (N=2775). Weighted mean±SD plasma Angptl4 levels were 10.0±11.0 ng/mL. Plasma Angptl4 concentration correlated positively with age (r=0.15, P<0.001) and body fat mass (r=0.19, P=0.003) but negatively with plasma high-density lipoprotein cholesterol (r=-0.13, P=0.01). No correlation with triglycerides (TGs) was observed. T266M was independently associated with plasma Angptl4 levels (P<0.001) but was not associated with TGs or CHD risk in the meta-analysis of 5 studies (4061 cases/15 395 controls). E40K showed no independent association with plasma Angptl4 levels. In human embryonic kidney 293 and human hepatoma 7 cells compared with wild type, E40K and T266M showed significantly altered synthesis and secretion, respectively.

Conclusions: Circulating Angptl4 levels may not influence TG levels or CHD risk for the following reasons: (1) Angptl4 levels were not correlated with TGs; (2) T266M, although associated with Angptl4 levels, showed no association with plasma TGs; and (3) TG-lowering E40K did not influence Angptl4 levels. These results provide new insights into the role of Angptl4 in TG metabolism.

Figure 1. Distribution of plasma Angptl4 levels in a sub-cohort derived from NPHSII

The reciprocal (inverse transformation) was used to normalize the data. Median Angptl4 levels were 7.7 (inter quartile range 5.9 – 11.0) ng/ml. Weighted mean (±SD) plasma Angptl4 levels were 10.0 (±11.0) ng/ml n=666.

Figure 2. Evaluation of synthesis and secretion of wild type, T266M and E40K variants of Angptl4

HEK293 cells were transiently transfected with wild-type (WT), T266M and E40K variants of Angptl4. Angptl4 concentration was evaluated using ELISA 24h after transfection in cell lysate (Panel A) and media (Panel B) and adjusted to total cellular protein concentration. Panel C, HEK293 media collected 24h after transfection was concentrated and subjected to electrophoresis under reducing (+DTT) and non-reducing conditions (−DTT) as described in Methods section. Panel D, densitometry of the bands from the gel performed under reducing conditions (+DTT) using ImageJ software. The figures are representative of at least three independent experiments. * P<0.05, *** P<0.001, measured by t test.

Figure 3. Forest plot of the individual and combined odds ratio (OR) for T266M and CHD risk from 5 studies including 4061 CHD cases/15395 controls

Unadjusted ORs are estimated from an additive model of random effects as there was significant heterogeneity between studies (I-squared=65.9, P=0.02). Northwick Park Heart Study II (NPHSII), Whitehall II (WHII), European Prospective Investigation of Cancer-Norfolk, the British Women's Heart and Health Study (BWHHS), British Heart Foundation Family Heart Study (BHF-FHS).