Growth of arterioles precedes that of capillaries in stretch-induced angiogenesis in skeletal muscle

Arteriolar growth accompanying capillary angiogenesis has been linked with hemodynamic factors resulting from increased blood flow. Here we describe t he growth of arterioles occurring in rat skeletal muscles stretched by an o verload due to the removal of agonist muscles, where blood flow was not inc reased, and we provide morphological evidence for the type of cells involve d 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 cryo stat sections to mark endothelial cells (Griffonia simplicifolia I, GSI lec tin), smooth muscle cells and pericytes (alpha smooth muscle actin, alpha S MA), and "mature" arterioles (smooth muscle myosin heavy chains). EHP muscl es were used for corresponding evaluation by confocal and electron microsco py. The number of capillaries surrounding muscle fibers was not significant ly 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 (C D) and capillary/fiber ratio (C/F) gradually increased (CD 778 <plus/minus> 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 alpha SMA-positive vess els around fibers increased after 1 week (2.16 +/- 0.09 vs 0.25 +/- 0.02 in controls) and was lower after weeks (1.42 +/- 0.24, P < 0.05, vs 1 week). Arteriolar density was higher at 1 (110.9 <plus/minus> 7.5 mm(-2)) and 2 we eks (70.7 +/- 12.1) with respect to controls (31.0 +/- 1.6 mm(-2)). The inc reased density was greater in alpha SMA-positive vessels <10 <mu>m in diame ter (controls 18.0 +/- 1.04, 1 week 77.2 +/- 4.5, 2 wk 42.2 +/- 9.0 mm(-2)) than in vessels >10 mum (13.0 +/- 0.8, 33.7 +/- 4.0, 29.5 +/- 4.7 mm(-2)). Electron microscopy showed "activated" (TEM fine structure) and proliferat ing (immunogold labeling for BrdU) fibroblasts in the vicinity of capillari es, some of which were embedded in the capillary basement membrane, consist ent with a transformation into pericytes and possibly later smooth muscle c ells. Confocal microscopy indicated that some mesenchymal cells became GSI positive and formed extended processes which contacted capillaries via tape red endings. Growth of arterioles in stretched muscles appears to involve p roliferation of fibroblasts, which may migrate toward capillaries and prece des any apparent increase in capillarization. (C) 2001 Academic Press.