Impaired ceramide signalling in spontaneously hypertensive rat vascular smooth muscle: a possible mechanism for augmented cell proliferation

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Abstract
In hypertension, the vascular wall undergoes morphological changes that alter mechanical responses to vasoactive substances. Ceramide is a recently identified second messenger synthesized in response to cytokines such as tumour necrosis factor α (TNF-α). It has been previously demonstrated that vascular smooth muscle cells (VSMC) from genetically hypertensive rats proliferate at a higher rate than those of normotensive origin. We tested the hypothesis that the ceramide pathway is impaired in VSMC from spontaneously hypertensive rats (SHR). VSMC were isolated from aortae of SHR and from Wistar–Kyoto (WKY) rats. Ceramide levels were measured under baseline and agonist-stimulated conditions and cell proliferation was monitored. Cell proliferation was determined by cell counting. Ceramide levels were determined via radioactive labelling, high-performance thin-layer chromatography and phosphorimaging. Relative mRNA levels of neutral sphingomyelinase were determined using semi-quantitative polymerase chain reaction (PCR). Basal ceramide levels in untreated cells were lower in cells from SHR compared to WKY rats. During chronic treatment with TNF-α, ceramide levels increased in WKY rat cells but remained unchanged in cells from SHR. TNF-α treatment had an inhibitory effect on WKY rat VSMC proliferation, but stimulated proliferation in cells from SHR. Short-term incubation with TNF-α resulted in a greater increase in ceramide in cells from WKY rats than those from SHR. Semiquantitative PCR analysis indicated that neutral sphingomyelinase mRNA may be reduced in SHR VSMC. We conclude that ceramide synthesis is impaired in vascular smooth muscle from SHR and may contribute to increased VSMC proliferation in hypertension.