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Alterations In Endothelin-1 And Nitric Oxide Pathways In Non-Ischaemic Muscle Of Patients With Atherosclerosis

Janice Tsui¹, Michael R Dashwood¹, Sidney G Shaw², George Hamilton¹, Daryll M Baker¹∙
¹Royal Free Hospital, London, United Kingdom; ²University of Bern, Bern, Switzerland

Objectives: Endothelin-1 (ET-1) and nitric oxide (NO) are endothelial-derived chemicals which are important in endothelial function. Changes in these pathways have been shown in ischaemic muscle of patients with peripheral arterial disease (PAD) with increased ET-1 tissue levels, ET receptor upregulation and increased NO synthase (NOS) protein levels. This study investigated whether changes in these systems occur in skeletal muscle of patients with atherosclerotic disease without clinically evident PAD. Methods: Following ethical committee approval and patients’ informed consent, muscle biopsies were taken from 10 patients with no clinically significant atherosclerotic disease undergoing orthopaedic surgery (control) and 10 patients undergoing coronary artery bypass graft surgery with no clinical evidence of PAD (atherosclerotic). ET-1 pathway was studied using immunohistochemistry, real-time RT-PCR for ET-1, ETA and ETB receptor mRNA levels and ET receptor subtype autoradiography. NO pathway was investigated using NOS immunohistochemistry, real-time RT-PCR and Western blotting and NOS activity was assessed with the citrulline assay. The Mann-Whitney test was used for statistical analyses. Results: Positive ET-1 immunoreactivity was demonstrated in both control and atherosclerotic muscle sections, associated with microvessels. ETB receptor mRNA levels were reduced in atherosclerotic biopsies (p = 0.017). No significant differences in ET-1 and ETA receptor mRNA expression or in ET receptor binding were found between the 2 groups. All 3 NOS isoforms were found in muscle sections from both groups, associated with muscle fibres and microvessels. Higher NOS I and III mRNA levels were found in atherosclerotic biopsies (p = 0.004 and 0.002) whilst no change in NOS II mRNA occurred. No significant changes in NOS protein levels or NOS activity were found. Conclusions: Downregulation of ETB receptor mRNA occurs in non-ischaemic muscle from patients with atherosclerosis. Transcriptional activation of NOS I and III was also demonstrated. These results demonstrate that dysfunction of these endothelial pathways occur systemically in atherosclerotic disease and may eventually lead to clinical disease. Modification of these pathways may alter disease progression.