Growth of Arterioles Precedes That of Capillaries in Stretch-Induced Angiogenesis in Skeletal Muscle

Abstract

Arteriolar growth accompanying capillary angiogenesis has been linked with hemodynamic factors resulting from increased blood flow. Here we describe the growth of arterioles occurring in rat skeletal muscles stretched by an overload due to the removal of agonist muscles, where blood flow was not increased, and we provide morphological evidence for the type of cells involved in this growth. Rat extensor digitorum longus (EDL) and extensor hallucis proprius (EHP) were overloaded by unilateral extirpation of their agonist, tibialis anterior. EDL muscles were taken for immunohistochemistry in cryostat sections to mark endothelial cells (Griffonia simplicifolia I, GSI lectin), smooth muscle cells and pericytes (alpha smooth muscle actin, αSMA), and “mature” arterioles (smooth muscle myosin heavy chains). EHP muscles were used for corresponding evaluation by confocal and electron microscopy. The number of capillaries surrounding muscle fibers was not significantly different after 1 week of stretch but was higher after 2 weeks (5.15 ± 0.2 vs 4.3 ± 0.2 in controls, P < 0.05). Similarly, capillary density (CD) and capillary/fiber ratio (C/F) gradually increased (CD 778 ± 86 at 2 weeks vs 593 ± 35 mm−2 in controls, C/F 2.07 ± 0.13 vs 1.38 ± 0.06, respectively). In contrast, the number of αSMA-positive vessels around fibers increased after 1 week (2.16 ± 0.09 vs 0.25 ± 0.02 in controls) and was lower after 2 weeks (1.42 ± 0.24, P < 0.05, vs 1 week). Arteriolar density was higher at 1 (110.9 ± 7.5 mm−2) and 2 weeks (70.7 ± 12.1) with respect to controls (31.0 ± 1.6 mm−2). The increased density was greater in αSMA-positive vessels <10 μm in diameter (controls 18.0 ± 1.04, 1 week 77.2 ± 4.5, 2 wk 42.2 ± 9.0 mm−2) than in vessels >10 μm (13.0 ± 0.8, 33.7 ± 4.0, 29.5 ± 4.7 mm−2). Electron microscopy showed “activated” (TEM fine structure) and proliferating (immunogold labeling for BrdU) fibroblasts in the vicinity of capillaries, some of which were embedded in the capillary basement membrane, consistent with a transformation into pericytes and possibly later smooth muscle cells. Confocal microscopy indicated that some mesenchymal cells became GSI positive and formed extended processes which contacted capillaries via tapered endings. Growth of arterioles in stretched muscles appears to involve proliferation of fibroblasts, which may migrate toward capillaries and precedes any apparent increase in capillarization.