Physiological angiogenesis is a graded, not threshold, response.

Angiogenesis may be induced in skeletal muscle by metabolic or mechanical factors, but whether an in vivo stimulus threshold applies for physiological angiogenesis is unknown. We compared three models of muscle overload inducing varying degrees of stretch on angiogenesis. Rat extensor digitorum longus (EDL) was overloaded by (a) extirpation of the synergist tibialis anterior (TA), (b) sectioning the distal tendon of the TA, or (c) release of the TA tendon by sectioning the retaining ligament. EDL samples were taken after 4, 7, 14 and 28 days to quantify capillary supply (alkaline phosphatase staining), and co-labelling for cell proliferation (using PCNA). The gradation of overload was confirmed by Western analysis of SERCA and CPT expression (1.6- to 7.2-fold and 8.3- to 33.9-fold changes, respectively), and the force characteristics of EDL. There was a significant increase in the number of new myonuclei only in the extirpated group after 7 days, while there was a graded increase in capillary-linked PCNA density (PCNAcap) among groups compared to controls. However, extirpation caused significant increase in PCNAcap after 7 days, whereas tenotomy showed a more modest and delayed increase at 14 days, and ligament transection induced no significant change. Muscle capillary supply followed a similar trend to that of PCNA, whereas the pro-angiogenic VEGF and Flk-1 protein levels were both up-regulated to a similar extent in all three experimental models 7–14 days after surgery. These results are consistent with the hypothesis that overload-induced angiogenesis is primarily a mechanical response, and that it is graded according to stimulus intensity.