Endothelium-dependent contractile factor release is related to sphingolipid alterations in essential hypertension

Léon Spijkers1, Rob Akker van den1, Ben Janssen2, Jacques Debets2, Jo DeMey2, Erik Stroes1, Bert-Jan Born van den1, Dayanjan Wijesinghe3, Charles Chalfant3, Luke MacAleese4, Gert Eijkel4, Ron Heeren4, Astrid Alewijnse1, Stephan Peters1. 1Academic Medical Center; Pharmacology & Pharmacotherapy, 1105AZ; Amsterdam, Netherlands, 2Maastricht University; Pharmacology & Toxicology, 6211 LK; Maastricht, Netherlands, 3Virginia Commonwealth University; Biochemistry, 23298-0551; Richmond Virginia, United States, 4FOM; AMOLF, 1098 XG; Amsterdam, Netherlands.

We and others have previously shown that sphingolipids, a family of bioactive membrane lipids, can modulate endothelium-derived relaxing factor signalling. Since hypertension is associated with endothelial dysfunction, we investigated whether hypertension is associated with altered vascular sphingolipid biology and/or sphingolipid levels. In isolated carotid arteries from spontaneously hypertensive rats (SHR), pharmacological modulation of sphingolipid metabolism by means of exogenously applied sphingomyelinase (SMase, 0.1U/ml) or application of the sphingosine kinase inhibitor dimethylsphingosine (DMS, 10µM), induced marked transient contractions (2.1 ± 0.1 mN/mm and 1.4±0.4 mN/mm respectively; n = 10). These contractions were virtually absent in vessels from normotensive Wistar Kyoto rats (WKY; 0.6±0.1mN/mm and 0.0±0.0 mN/mm respectively; n = 9). Notably, the contractions were fully abolished by prior endothelium removal or by the cyclooxygenase inhibitor indomethacin (10µM; 0.0±0.0 mN/mm, n = 7). To investigate which enzyme was mainly responsible for generating the COX substrate arachidonic acid, several phospholipase A2 inhibitors were applied, of which the iPLA2 inhibitor bromoenol lactone (25µM) almost completely inhibited SMase-induced contractions (0.2±0.1 mN/mm; n = 6). In addition, the thromboxane synthase inhibitor ozagrel and the TP-receptor antagonists SQ29,548 concentration-dependently inhibited SMase-induced contraction, indicating that the contractions in SHR were mediated by thromboxane A2. In accordance with aforementioned, DMS (10µM) augmented endothelin-1-induced contractions in isolated aorta of SHR (6.8±0.4 mN/mm and 5.1±0.4 mN/mm Emax in the presence or absence of DMS respectively; n = 8-10, p<0.05), but not in WKY (10.2±0.3 mN/mm and 9.5±0.6 mN/mm). This DMS-induced augmentation was also endothelium-dependent and sensitive to COX inhibition and TP receptor antagonism, thus likely via augmented endothelin-1-induced thromboxane A2 production.

Lipidomics analysis by mass spectrometry revealed significantly elevated levels of ceramide in arterial tissue and blood plasma of SHR (691±43pmol aorta, 645±25pmol plasma) compared to WKY (419±27pmol aorta, 513±19 pmol plasma) (n = 6, p<0.05 SHR vs WKY). In order to investigate whether similar changes in sphingolipid biology may play a role in human hypertension we measured sphingolipid levels in plasma from normo-and hypertensive humans. We found that also plasma from humans with essential hypertension displayed elevated ceramide levels corresponding with the grade of hypertension severity (total ceramide: 183±11pmol normotension, 207±19pmol stage 1 hypertension, 243±23pmol stage 2+3 hypertension; n = 18,12,19 respectively, p<0.05 normotension vs stage 2+3 hypertension).

In summary, we show that sphingolipids modulate the release and action of endothelium-derived contracting factors in hypertension and that (essential) hypertension is associated with marked alterations in sphingolipid biology and sphingolipid (plasma)levels.

This study was performed within the framework of Top Institute Pharma project T2-108.