Purpose: The hypolipidaemic agent, fenofibrate, has been shown to exert pleiotropic (lipid-independent) effects, of which the stimulation of the cardioprotective vasodilator, nitric oxide (NO), is particularly relevant. In this study, the NO generating properties of fenofibrate were investigated in cardiac microvascular endothelial cells (CMECs), with particular focus on the underlying role of NO synthase (NOS) isoforms.
Methods: CMECs were treated with 50μM fenofibrate for 1 and 24h. Intracellular NO levels were measured by mean diaminofluorescein diacetate (DAF-2/DA) fluorescence intensity (FACS analysis) and nitrite levels by the Griess-method (platereader analysis). The putative involvement of NOS isoforms was investigated by administering the pharmacological inhibitors, L-NMMA (100μM; selective for eNOS/nNOS) or 1400W (80μM; selective for iNOS). Relevant signalling proteins were investigated by means of Western blotting.
Results: DAF-2/DA measurements showed that fenofibrate increased NO-production significantly after 1 hour (Control 100%: Feno: 174.7%±11.03;p<0.05), but not after 24 hours. Similarly, nitrite levels were increased after 1h (Control: 2.1μM±0.32; Feno: 3.4μM±0.39;p<0.05), but not at 24h. Surprisingly, western blot data showed that 1h fenofibrate treatment did not affect the phospho/total eNOS ratio of either the Ser 1177 or Thr 495 residues. iNOS expression was not detected in fenofibrate-treated cells, as shown by qPCR and western blotting, and nNOS phosphorylation Ser 1417 remained unchanged. Although pharmacological inhibition of NOS decreased baseline NO-production at 1h (DAF-2/DA fluorescence control: 100% vs. L-NMMA: 85.9%±4.53; p<0.05, and control: 100% vs. 1400W: 85.49%±4.5; p<0.05), the inhibitors had no effect on fenofibrate-induced increases in NO-production.
Conclusion: Fenofibrate showed short-term NO generating properties, as demonstrated by two independent NO-measurement techniques. This effect was not sustained and disappeared at 24h. Previous studies (on different endothelial cell lines) showed involvement of eNOS Ser1177 phosphorylation. However, in our hands, involvement of this phosphorylation site could not be demonstrated. Furthermore, neither iNOS nor nNOS seemed to be affected by fenofibrate treatment. Our data suggest that short-term fenofibrate-induced NO-production in CMECs was NOS-independent, which is a novel finding.