Figure 1.
DMS-induced and SMase-induced contraction in SHR and WKY carotid artery.
A) Original tracing of rat carotid artery segments exposed to DMS (10 µmol/L) or SMase (0.1 U/mL). Note reduced relaxing response to MCh (10 µmol/L) in SHR, indicating pronounced endothelial dysfunction. B) Maximal contractile responses to DMS and/or SMase in intact WKY and SHR vessels and C) SHR vessel responses in the presence of L-NAME or endothelium-denudation (-EC). Phenylephrine (Phe), methacholine (MCh), dimethylsphingosine (DMS), sphingomyelinase C (SMase), Nω-Nitro-L-arginine methyl ester (L-NAME). Data presented as mean ± SEM, n = 5–6, (*) p<0.05.
Table 1.
General characteristics of anaesthetized SHR and WKY rats and ex vivo carotid artery segments.
Figure 2.
Characterization of SMase-induced contraction in SHR carotid artery.
SMase-induced contraction, in absence and presence of the non-specific COX inhibitor indomethacin (Indo), COX-1 specific inhibitor SC560, COX-2 specific inhibitor NS398, the PLA2 inhibitors AACOF3 (cPLA2) and Luffariellolide (Luff; sPLA2), Bromoenol lactone (BEL; iPLA2), the thromboxane synthase inhibitor Ozagrel and the thromboxane receptor antagonist SQ29548 (SQ29). Data presented as mean ± SEM, n = 4–6, (*) p<0.05 compared to control SMase.
Figure 3.
Immunohistochemistry of relevant proteins in SHR carotid artery.
A) Immunohistochemical staining (left, typical staining images; 200× magnification) and quantification (right) of SHR or WKY carotid artery segments depicting cell nuclei staining (blue), with/without the von Willebrand Factor (vWF) endothelium marker (green) and cyclooxygenase-1 (COX-1; red). B) thromboxane synthase (TXAS; red) and C) calcium-independent phospholipase A2 (iPLA2; red). Please note the increased EC/VSMC iPLA2 expression ratio in SHR. Data presented as mean ± SEM, n = 5–6, (*) p<0.05, scale bars 100 µm.
Figure 4.
In vivo effects of DMS infusion in SHR and WKY.
Rats were treated with bolus injection and subsequent infusion of DMS (3 mg/kg followed by 6 mg/kg/hr) or vehicle (0.75% rat serum albumin in saline) during recording of mean arterial pressure (MAP). Data expressed as mean maximal change from baseline ± SEM, n = 6–8, (*) p<0.05.
Figure 5.
Liquid-chromatography-mass spectrometry measurements of total sphingolipid content.
Content was measured in A) SHR and WKY aorta homogenate and B) blood plasma. Data presented as mean ± SEM, n = 3–5 (for 6–10 pooled aortas) and 6–12 (for plasma samples), (*) p<0.05.
Figure 6.
Liquid-chromatography-mass spectrometry measurements of sphingolipid content in human blood plasma.
A) Quantification of total sphingolipid pools in human plasma. Human samples were collected from normotensive controls (BP<140/90 mmHg) and from patients with stage 2 and 3 hypertension (BP≥160/100). B) Total plasma ceramide levels in normotensives compared to stage 1 hypertensives (BP 140–159/90–99 mmHg) and stage 2+3 hypertensives. (Data presented as mean ± SEM, n = 18 for normotensive controls n = 12 for patients with stage 1 hypertension, n = 19 for stage 2+3 hypertension. (*) p<0.05.
Table 2.
Patient characteristics.