Skeletal Muscle Microvascular Changes in Response to Short-Term Blood Flow Restricted Training-Exercise-Induced Adaptations and Signs of Perivascular Stress

Jakob L Nielsen, Ulrik Frandsen, Kasper Y Jensen, Tatyana A Prokhorova, Line B Dalgaard, Rune D Bech, Tobias Nygaard, Charlotte Suetta, Per Aagaard, Jakob L Nielsen, Ulrik Frandsen, Kasper Y Jensen, Tatyana A Prokhorova, Line B Dalgaard, Rune D Bech, Tobias Nygaard, Charlotte Suetta, Per Aagaard

Abstract

Aim: Previous reports suggest that low-load muscle exercise performed under blood flow restriction (BFR) may lead to endurance adaptations. However, only few and conflicting results exist on the magnitude and timing of microvascular adaptations, overall indicating a lack of angiogenesis with BFR training. The present study, therefore, aimed to examine the effect of short-term high-frequency BFR training on human skeletal muscle vascularization. Methods: Participants completed 3 weeks of high-frequency (one to two daily sessions) training consisting of either BFR exercise [(BFRE) n = 10, 22.8 ± 2.3 years; 20% one-repetition maximum (1RM), 100 mmHg] performed to concentric failure or work-matched free-flow exercise [(CON) n = 8, 21.9 ± 3.0 years; 20% 1RM]. Muscle biopsies [vastus lateralis (VL)] were obtained at baseline, 8 days into the intervention, and 3 and 10 days after cessation of the intervention to examine capillary and perivascular adaptations, as well as angiogenesis-related protein signaling and gene expression. Results: Capillary per myofiber and capillary area (CA) increased 21-24 and 25-34%, respectively, in response to BFRE (P < 0.05-0.01), while capillary density (CD) remained unchanged. Overall, these adaptations led to a consistent elevation (15-16%) in the capillary-to-muscle area ratio following BFRE (P < 0.05-0.01). In addition, evaluation of perivascular properties indicated thickening of the perivascular basal membrane following BFRE. No or only minor changes were observed in CON. Conclusion: This study is the first to show that short-term high-frequency, low-load BFRE can lead to microvascular adaptations (i.e., capillary neoformation and changes in morphology), which may contribute to the endurance effects previously documented with BFR training. The observation of perivascular membrane thickening suggests that high-frequency BFRE may be associated with significant vascular stress.

Keywords: angiogenesis; capillary; hypoxia; vascular remodeling; vascular stress.

Copyright © 2020 Nielsen, Frandsen, Jensen, Prokhorova, Dalgaard, Bech, Nygaard, Suetta and Aagaard.

Figures

FIGURE 1
FIGURE 1
Representative skeletal muscle cross sections displaying immunoreactivity for NG2 (red), CD31 (green), and 4’,6-diamidino-2-phenylindole (DAPI) (nuclear stain) (1A,1B) and Laminin (2A–2C). 1A,1B: Samples are from blood flow restriction exercise (BFRE) at baseline (Pre, 1A) and 8 days into the intervention (Mid8, 1B). Note the increase in the area of CD31C structures and myofibers as well as a similar number of CD31C structures, suggesting a stable capillary density (CD) and an increase in capillary/fiber and capillary cross-sectional area. 2A: Normal laminin morphology, baseline (Pre); 2B/2C: lowly/highly increased perivascular laminin immunoreactivity relative to baseline (2A).
FIGURE 2
FIGURE 2
Capillaries per myofiber, capillary density (CD, capillaries per mm2), capillary cross-sectional area (μm2), and capillary-to-muscle area ratio at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and 10 days after cessation of training (Post3 and Post10, respectively). (A) Capillaries per myofiber, (B) CD (capillaries per mm2), (C) average capillary area, and (D) capillary-to-muscle area ratio. Pre to Mid/post differences: *P < 0.05, **P < 0.01, ***P < 0.001. Baseline-specific group difference: P < 0.05. Values are means ± SD; BFR: n = 10 at Mid8 n = 9; CON: n = 7.
FIGURE 3
FIGURE 3
Protein expression related to angiogenesis signaling at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and 10 days after cessation of training (Post3 and Post10, respectively). Dark gray bars denotes blood flow-restricted group. (A) Vascular endothelial growth factor A (VEGF-A), (B) VEGF-D, (C) VEGF receptor 2 (VEGF-R2), (D) VEGF-R3, (E) angiopoietin-1 (ANGPT1), (F) ANGPT2, and (G) urokinase-type plasminogen activator receptor (uPAR). Pre to Mid/post differences: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Values are means ± SD; BFR: n = 10 at Mid8 n = 9.
FIGURE 4
FIGURE 4
Protein expression related to vascular/extracellular matrix remodeling at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and 10 days after cessation of training (Post3 and Post10, respectively). Dark gray bars denotes blood flow-restricted group. (A) Tissue inhibitor of matrix metalloproteinase (TIMP)-1, (B) TIMP-2, (C) matrix metalloproteinase (MMP)-1, (D) MMP-9, (E) granulocyte colony-stimulating factor (G-CSF), and (F) granulocyte-macrophage colony-stimulating factor (GM-CSF). Pre to Mid/post differences: ∗P < 0.05. Values are means ± SD; BFR: n = 10 at Mid8 n = 9.
FIGURE 5
FIGURE 5
mRNA expression related to angiogenesis and vascular/matrix remodeling at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and 10 days after cessation of training (Post3 and Post10, respectively). Dark gray bars denotes blood flow-restricted group. (A) Vascular endothelial growth factor A (VEGF-A), (B) hypoxia-inducible factor-1α (HIF-1α), (C) VEGF receptor 2 (VEGF-R2), (D) heme oxygenase (HMOX)-1, (E) matrix metalloproteinase (MMP)-2, and (F) MMP-9. Pre to Mid/post differences: ∗P < 0.05, ∗∗P < 0.01. Values are geometric means ± SEM; BFR: n = 10 at Mid8 n = 9.

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