The effect of passive movement training on angiogenic factors and capillary growth in human skeletal muscle

Abstract

The effect of a period of passive movement training on angiogenic factors and capillarization in skeletal muscle was examined. Seven young males were subjected to passive training for 90 min, four times per week in a motor-driven knee extensor device that extended one knee passively at 80 cycles min₋₁. The other leg was used as control. Muscle biopsies were obtained from m. v. lateralis of both legs before as well as after 2 and 4 weeks of training. After the training period, passive movement and active exercise were performed with both legs, and muscle interstitial fluid was sampled from microdialysis probes in the thigh. After 2 weeks of training there was a 2-fold higher level of Ki-67 positive cells, co-localized with endothelial cells, in the passively trained leg which was paralleled by an increase in the number of capillaries around a fibre (P <0.05). Capillary density was higher than pre-training at 4 weeks of training (P <0.05). The training induced an increase in the mRNA level of endothelial nitric oxide synthase (eNOS), the angiopoietin receptor Tie-2 and matrix metalloproteinase (MMP)-9 in the passively trained leg and MMP-2 and tissue inhibitor of MMP (TIMP)-1 mRNA were elevated in both legs. Acute passive movement increased (P <0.05) muscle interstitial vascular endothelial growth factor (VEGF) levels 4- to 6-fold above rest and the proliferative effect, determined in vitro, of the muscle interstitial fluid ~16-fold compared to perfusate. The magnitude of increase was similar for active exercise. The results demonstrate that a period of passive movement promotes endothelial cell proliferation and angiogenic factors and initiates capillarization in skeletal muscle.

Figures

Figure 1. Capillarization and presence of proliferating endothelial cells in skeletal muscle before and after passive training of the leg

Capillary-to-fibre ratio (C:F, A), no. of capillaries around each fibre (B), capillary density (cap mm−2, C), mean fibre area (μm2, D), and Ki-67 positive cells per fibre (Ki-67/fibre, E) before and after 2 weeks and 4 weeks of passive training in the untrained (filled bars) and passively trained (open bars) leg. Values are means ±s.e.m. (n= 7). †P < 0.05 vs. week 0; #P < 0.05 vs. untrained leg week 2; ‡P < 0.05 vs. passively trained leg week 4; §P < 0.05 vs. week 2.

Figure 2. Interstitial concentration of VEGF protein in skeletal muscle at rest, and during passive movement and exercise

The concentration of VEGF protein was measured in the dialysate and the concentration in the interstitium was estimated by determination of relative loss of tritium labelled adenosine for each probe. Microdialysate samples were collected at rest, during two bouts of passive movement (Pass 1 and Pass 2, respectively), and exercise at 10 and 30 W in the untrained (filled bars) and passively trained (open bars) leg. Values are means ±s.e.m. (n= 7). †P < 0.05 vs. rest untrained leg; ‡P < 0.05 vs. rest passively trained leg.

Figure 3. Effect of skeletal muscle microdialysate on proliferation of cultured endothelial cells

Proliferation of human umbilical vein endothelial cells (HUVECs) determined by incorporation of bromodeoxyurdine (BrdU), after addition of skeletal muscle microdialysate from the untrained (filled bars) and passively trained (open bars) leg. Microdialysate samples were collected at rest, during two bouts of passive movement (Pass 1 and Pass 2, respectively) and during exercise at 10 or 30 W. Values are means ±s.e.m. *P < 0.05 vs. PBS; †P < 0.05 vs. rest untrained leg; ‡P < 0.05 vs. rest passively trained leg; §P < 0.05 vs. Pass 1, Pass 2, and 30 W passively trained leg (n= 7). PBS: proliferation of endothelial cells with addition of the microdialysis perfusate consisting of PBS.

Figure 4. Content of VEGF, eNOS, MMP-2, MMP-9, TIMP1-, TIMP-2, Tie-2, Ang-1 and Ang-2 mRNA in human skeletal muscle tissue with passive movement training of the leg

The mRNA content of VEGF (A), eNOS (B), MMP-2 (C), MMP-9 (D), TIMP-1 (E), TIMP-2 (F),Tie-2 (G), Ang-1 (H), Ang-2 (I), and the ratio Ang-2/Ang-1 (J) were determined in skeletal muscle tissue before, and after 2 and 4 weeks of passive training in the untrained (filled bars) and passively trained (open bars) leg. Muscles biopsies were obtained from m.v. lateralis. mRNA levels were determined with real-time PR-PCR, and data are presented relative to GAPDH mRNA. Values are means ±s.e.m. (n= 7). A, &P < 0.05 vs. rest passively trained leg; B, *P < 0.005 vs. rest passively trained leg, †P < 0.05 vs. 2 weeks passively trained leg; C, #P= 0.05 vs. rest; D, §P= 0.001 vs. rest untrained leg, ‡P= 0.001 vs. 2 weeks untrained leg, *P < 0.05 vs. rest passively trained leg; E, §P < 0.005 vs. rest untrained leg, £P < 0.05 vs. 4 weeks passively trained leg, #P < 0.05 vs. rest; G, *P < 0.05 vs. rest passively trained leg, #P < 0.05 vs. rest.

Figure 5. Thigh blood flow, arterio-venous oxygen difference, and oxygen uptake during passive movement of the leg

Thigh blood flow (l min−1, A), arterio-venous oxygen difference (ml l−1, B), and oxygen uptake (ml min−1, C) were determined at rest and during 30 s, 5 min, and 10 min of passive movement of the leg. Blood flow values were time-matched with blood sample measurements using a linear connection of consecutive data points. Values are means ±s.e.m. (n= 6). †P < 0.05 vs. rest.