Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also has proliferative properties on various cell types, including endothelial and vascular smooth muscle cells. A potential pathophysiological role for ET-1 in lower limb ischaemia has been suggested since this peptide and its receptors are associated with microvessels in calf muscle biopsies from patients with tourniquet-induced ischaemia (Tsui et al. 2001) and critical limb ischaemia (Dashwood et al. 2000). Here we have identified and quantified microvessels and ET-1 immunoreactivity in muscle biopsies from patients undergoing total knee replacement (TKR) and amputation for crtitical limb ischaemia (CLI).

Following local ethics committee approval and patients’ informed consent, quadriceps muscle biopsies were obtained from patients undergoing TKR at the start of the operation (control), and 1 h following tourniquet application (acute ischaemia). Calf muscle biopsies were also obtained from patients undergoing amputation for CLI (chronic ischaemia). Frozen biopsy sections (10 µm) were used for the immunohistochemical identification of ET-1 using a monoclonal anti-ET-1 antibody (Peninsula Labs) and the ABC peroxidase method (Dako Labs) with DAB as the chromogen. Endothelial cells were identified using CD31 and vascular smooth muscle cells identified using anti-α-smooth muscle actin (anti-αSMA). Quantitative assessment of microvessels within muscle biopsies was performed by counting the number of CD31-positive staining cells that fell within a grid measuring 100 µm Ω 150 µm at Ω 40 magnification.

The number of microvessels was increased approximately 2.5-fold on CLI biopsies (n = 12) compared with control biopsies (n = 6; P = 0.0009, Mann-Whitney), whereas there was no change following a period of acute ischaemia (n = 6), see Fig. 1.CD31 and anti-αSMA staining co-localised, confirming the identity of the microvessels. A high proportion of these vessels also exhibited positive ET-1 immuno-staining. Angiogenesis has been shown to occur in severely ischaemic, but not mildly ischaemic, rat skeletal muscles (Brown et al. 1999). Our results show that a similar pattern occurs clinically, where there is an increase in the number of microvessels in chronically ischaemic, but not in acutely ischaemic skeletal muscle. The association of ET-1 with these microvessels suggests that this peptide may be involved in the pathophysiology of CLI via its potent constrictor action. Alternatively, it is possible that ET-1 may play a role in promoting new vessel growth in an attempt to restore muscle perfusion in these patients.

Figure 1. Quantification of microvessels within muscle sections (means ± S.E.M.).

Brown, M.D., Kent, J., Kelsall, C.J. & Hudlicka, O. (1999). J. Vasc. Res. 36, 335.

Dashwood, M.R., Atwal, A.S., Hamilton, G. & Baker, D.M. (2000). J. Physiol. 525.P, 9P.

Tsui, J.C.S., Baker, D.M., Garlick, N.I. & Dashwood, M.R. (2001). J. Physiol. 531.P, 19P.