Evidence indicates that the therapeutic potential of statins expands beyond lipidic asset control and might include reparative neovascularization. However, there is no general consensus on the latter point. Since nitric oxide (NO) functions as a final mediator for various angiogenic growth factors, we tested the hypothesis that NO-releasing pravastatin derivative, NCX 6550, would enhance the therapeutic activity of parent compound in a murine model of limb ischemia.

Studies were performed in 2-month old CD1 male mice. Procedures were approved by the institutional ethical committee and complied with the Italian law for the use of experimental animals. After basal measurements of tail-cuff systolic blood pressure (SBP) and heart rate (HR), mice were randomly allocated into 3 treatment groups (n=20 each): pravastatin (10mg/kg b.w./day), NCX6550 (equimolar dose), regular chow (controls). Three days after entering the treatment, mice were anaesthetized with tribromoethanol (880mmolkg^-1ip) and left hindlimb ischemia was induced by electro-coagulation of femoral artery. Skin blood flow (BF) was measured sequentially by laser Doppler flowmetry and ischemic to nonischemic foot BF ratio was used as an index of perfusion recovery. SBP was re-measured at 7 and 14 days.

Final clinical outcome was determined by counting the number of necrotic or lost nails of the ischemic foot. At 14 days from ischemia, hindlimbs of anaesthetized mice were perfusion-fixed. Both adductors were coded and histologically processed. Capillary density (n_cap/mm²) and capillary to myofiber ratio (n_cap/n_fib) of H&E-stained transverse muscular sections were determined. The data are expressed as means ± SEM. Differences were statistically analyzed by ANOVA or ² test. A value of p <0.05 was considered statistically significant.

SBP and HR did not differ among groups. In contralateral muscles, capillary density and capillary to myofiber ratio was not affected by treatments. In contrast, reparative capillarization response to ischemia was potentiated by NCX6550 (1116±53 vs 852±32 n_cap/mm² in controls and 1.80±0.06 vs 1.29±0.06 n_cap/ n_fib in controls; p<0.01 for both comparisons). Conversely, pravastatin was unable to enhance spontaneous angiogenesis. The rate of skin BF recovery did not differ among groups. However, clinical outcome was significantly improved by NCX6550, as a complete prevention of auto-amputation was observed (vs. 14% in controls and 6% in pravastatin, respectively). In addition, NCX6550 reduced the number of necrotic or lost nails (0.47±0.31 vs 2.07±0.59 in controls and vs 1.47±0.42 in pravastatin group; p<0.05 for both comparisons).

In this study we demonstrate that NCX 6550 promotes reparative neovascularization without altering controlateral microcirculation and ameliorates clinical outcome in mice with experimentally-induced limb ischemia. These findings suggest that NCX 6550 may be regarded as a candidate drug for patients with atherosclerotic occlusive vascular disease.