Implications Of Angiogenic Factors And Thrombospondin-1 Underlying Capillary Regression In Chronically Unloaded Rat Soleus Muscle

Abstract

Capillary regression is induced by chronic unloading of skeletal muscles. The mechanisms involved, however, are not fully understood. At least two independent mechanisms can be considered, i.e., adaptations in the levels of specific angiogenic factors such as vascular endothelial growth factor (VEGF) and angiopoietin, and of thrombospondin-1 (TSP-1), an inhibitor of angiogenesis that induces apoptosis in endothelial cells. PURPOSE: The aim of present study was to determine the responses of pro- and anti-angiogenic factors in rat soleus capillaries after 2 weeks of hindlimb unloading (HU). METHODS: Male Wistar rats (8-9 weeks of age) were assigned randomly into a control or HU group. Frozen transverse sections from the soleus mid-belly were stained for alkaline phosphatase which is present in the capillary endothelium. Apoptotic endothelial cells were identified by von Willebrand factor and DNA fragmentation (TUNEL) using immunofluorescent staining. VEGF, KDR, Flt-1, angiopoietin-1 (Ang-1), Ang-2, Tie-2, HIF 1 alpha, and TSP-1 mRNAs were determined by TaqMan probe-based real-time PCR reactions. RESULTS: The capillary-to-fiber ratio was 23% lower in HU than control rats. TUNEL-positive endothelial cells were observed only in HU rats and TSP levels were higher in HU than control rats, suggesting capillary regression with vascular endothelial cell apoptosis. mRNA levels of angiogenic factors VEGF and Ang-1, their receptors, and HIF 1 alpha were lower in HU than control rats whereas Ang-2 levels were unaffected. As a result, the Ang-2/Ang-1 ratio increased, suggesting that the vasculature was destabilized. Ang-2 expressed in the absence of VEGF also results in vessel regression. CONCLUSIONS: These data indicate that a decrease in angiogenic factors and an increase in TSP-1 expression may play an important role in capillary regression associated with chronic unloading. Supported by Grants-in-Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, 19650148, and 19300196.