Lipid-soluble cigarette smoking particles induce expression of inflammatory and extracellular-matrix-related genes in rat cerebral arteries

Petter Vikman, Cang-Bao Xu, Lars Edvinsson

Department of Clinical Sciences, Experimental Vascular Research, Lund, Sweden

Aims: Cigarette smoking is one of the strongest risk factors for stroke. However, the underlying molecular mechanisms that smoke leads to the pathogenesis of stroke are incompletely understood.

Methods: Dimethyl sulfoxide (DMSO)-soluble (lipid-soluble) cigarette smoking particles (DSP) were extracted from cigarette smoke (0.8 mg nicotine per cigarette; Marlboro^®). Rat cerebral arteries were isolated and organ cultured in the presence of DSP (0.2 μl/ml, equivalent to the plasma level in smokers) for 24 h. The expression of matrix metalloproteinase 9 and 13 (MMP9 and MMP13), angiotensin receptor 1 and 2 (AT₁ and AT₂), interleukin 6 and inducible nitric oxide synthase (iNOS) were investigated at mRNA level by real-time PCR and/or at protein level by immunohistochemistry. In addition, the activity of three mitogen-activated protein kinases (p38, ERK 1/2 and SAPK/JNK) and their downstream transcription factors (ATF-2, Elk-1 and c-Jun) were examined.

Results: We observed that compared with control (DMSO-treated cerebral arteries), the cerebral arteries treated by DSP exhibited enhanced expression of MMP13 and AT₁ receptors, but not of AT₂ receptors, at both mRNA and protein levels, suggesting that a transcriptional mechanism is most likely involved in the DSP effects. This is further supported by the findings that DSP induced phosphorylation of p38 mitogen-activated protein kinases inflammatory signal protein in parallel with activation of its downstream transcription factor ATF-2 and Elk-1. However, ERK 1/2 and SAPK/JNK activities were markedly expressed in the control (organ culture per se with DMSO), and DSP failed to further enhance the activation of ERK 1/2 and SAPK/JNK in the cerebral arteries.

Conclusions: DSP induces cerebral vessel inflammation with activation of p38 MAPK inflammatory signal and the downstream transcriptional factors (ATF-2 and Elk-1) in parallel with enhanced extracellular-matrix-related gene transcription and increased AT₁ receptor expression in the cerebral arteries, which are key events in stroke pathogenesis.

Keywords: cigarette smoking, vascular inflammation, signal transduction, extracellular matrix, MMP, stroke