Fatty Acid binding protein 4 is associated with carotid atherosclerosis and outcome in patients with acute ischemic stroke

Abstract

Background and purpose: Fatty acid binding protein 4 (FABP4) has been shown to play an important role in macrophage cholesterol trafficking and associated inflammation. To further elucidate the role of FABP4 in atherogenesis in humans, we examined the regulation of FABP4 in carotid atherosclerosis and ischemic stroke.

Methods: We examined plasma FABP4 levels in asymptomatic (n = 28) and symptomatic (n = 31) patients with carotid atherosclerosis, as well as in 202 subjects with acute ischemic stroke. In a subgroup of patients we also analysed the expression of FABP4 within the atherosclerotic lesion. In addition, we investigated the ability of different stimuli with relevance to atherosclerosis to regulate FABP4 expression in monocytes/macrophages.

Results: FABP4 levels were higher in patients with carotid atherosclerosis, both systemically and within the atherosclerotic lesion, with particular high mRNA levels in carotid plaques from patients with the most recent symptoms. Immunostaining of carotid plaques localized FABP4 to macrophages, while activated platelets and oxidized LDL were potent stimuli for FABP4 expression in monocytes/macrophages in vitro. When measured at the time of acute ischemic stroke, high plasma levels of FABP4 were significantly associated with total and cardiovascular mortality during follow-up, although we did not find that addition of FABP4 to the fully adjusted multivariate model had an effect on the prognostic discrimination for all-cause mortality as assessed by c-statistics.

Conclusions: FABP4 is linked to atherogenesis, plaque instability and adverse outcome in patients with carotid atherosclerosis and acute ischemic stroke.

Figure 1. Increased plasma levels of FABP4 in patients with carotid atherosclerotic plaques.

Plasma levels of FABP4 were measured by enzyme immunoassay in patients with asymptomatic (n = 28) and symptomatic (n = 31) carotid plaques and healthy controls (n = 18). Data are mean±SEM. **p<0.001 compared to controls.

Figure 2. Increased FABP4 expression in atherosclerotic carotid plaques from symptomatic patients.

mRNA levels of FABP4 in atherosclerotic carotid plaques were measured in 12 patients with asymptomatic carotid plaques, in 25 patients with symptoms within the last months (<1 month) and in 17 patients with symptoms 1–6 months prior to collection. mRNA levels were quantified by real-time RT-PCR. The expression of β-actin was used as endogenous control. Data are mean ± SEM. *p<0.05 compared to asymptomatic patients.

Figure 3. FABP4 is co-localized to macrophages within carotid atherosclerotic plaques.

Panel A shows immunostaining of FABP4 in symptomatic carotid atherosclerotic plaques (n = 2) primarily located to macrophage-rich areas. Representative images obtained with 10x and 40x objective. Panel B shows double immunofluorescent staining of FABP4 (green fluorescence), CD68 (macrophages, red fluorescence) and nucleus (DAPI, blue fluorescence) from symptomatic carotid atherosclerotic plaques (n = 2). The lower panel is a merge of the three pictures above. Panel C shows the correlations of mRNA levels in atherosclerotic plaques between FABP4, ADFP and CD68, respectively.

Figure 4. Oxidized LDL and platelets enhances FABP4 in macrophages.

Panel A shows mRNA expression of FABP4 in THP-1 cells during PMA differentiation (left) and in THP-1 macrophages stimulated with oxidized LDL (20 µg/ml), TNFα (5 ng/ml) or both for 18 hours. mRNA levels were quantified with the use of real-time RT-PCR. The expression of β-actin was used as endogenous control. Panel B shows the release of FABP4 protein into the cell medium, as determined by enzyme immunoassay, in THP-1 monocytes that had been pre-incubated with rhTNFα (5 ng/ml) for 96 hours before being incubated with LPS (5 ng/ml), a TLR2 agonist (Pam3Cys, 1 µg/ml), isoproterenol (20 µM), rh-IL-1β (1 ng/ml) and platelet releasate from un-stimulated (UPRL) and thrombin-activated platelets (SPRL) for additional 20 hours. Data are mean ± SEM relative to values in un-stimulated cells (control). *p<0.05 and **p<0.001 versus control (or 0 hours [h] in panel A, left).

Figure 5. High plasma levels of FABP4 are associated with long-term mortality in patients with acute stroke.

Panel A shows ROC curve analysis for the predictive value of FABP4 for all-cause and CV mortality. AUC and 95% CI are given. Panel B shows Kaplan-Meier curves with the cumulative incidence of all-cause and CV mortality during the entire study [median follow-up 4.4 years (interquartile range: 3.7 to 4.9 years)], according to tertiles of FABP4 at admission. Panel C shows the restricted cubic spline analysis of FABP4 in relation to all-cause mortality. Panel D shows multivariate analyses of FABP4 as an independent predictor of mortality in patients with acute stroke. *3^rd tertile vs. lower 2. Panel E shows the increase in hazard ratios (HR) for the prediction of all-cause and CV mortality when combing tertiles of FABP4 and SSS score (inverse for SSS, ie. increasing severity with higher tertiles). Tertile 1 was set as reference and the combination of T3 for both parameters is shown.