Thrombin and exercise similarly influence expression of cell cycle genes in cultured putative endothelial progenitor cells

Abstract

Acute exercise and exercise training may influence putative endothelial progenitor cell (EPC) number and colony forming units (CFU-ECs), although the mechanisms remain unclear. This study examined the effects of in vitro thrombin supplementation and acute exercise on CFU-EC gene expression, associated with cellular proliferation and differentiation. The effect of habitual physical activity was evaluated through analysis of EPCs from chronically high- and low-active men. Participants were healthy high- and low-active men (n=23), aged 55-80 yr. Circulating CD34+/VEGFR2+ number, CFU-ECs, plasma prothrombin fragment (F1+2), and thrombin-antithrombin III were measured at rest and after 30 min of exercise. Gene expression of cyclin A2, cyclin D1, p27, VE-cadherin, and VEGFR2 was assessed in postexercise CFU-ECs and resting CFU-ECs treated with 0, 1, 5, or 10 U/ml of thrombin. Outcomes were compared between high- and low-active participants. F1+2 and thrombin-antithrombin III, but not CD34+/VEGFR2+ number and CFU-ECs, increased with exercise. Exercise-induced changes in F1+2 correlated with changes in CD34+/VEGFR2+ number in both groups. Thrombin treatments and acute exercise increased cyclin A2 and cyclin D1 expression and decreased p27 expression. One unit per milliliter thrombin increased VEGFR2 and VE-cadherin expression, whereas 5 U/ml, 10 U/ml, and acute exercise did not elicit any changes. An exercise training effect was observed with greater decreases in p27 expression with 5 and 10 U/ml thrombin and greater increases in VEGFR2 and VE-cadherin expression with 1 U/ml thrombin in high-active men. Exercise-induced changes in putative EPC gene expression are associated with thrombin production and may be modulated by long-term exercise training.

Fig. 1.

Prothrombin fragment (F1+2; A) and thrombin-antithrombin III (TAT) production (B) in response to 30 min of vigorous exercise. Values are means ± SE. *Significant change from baseline in low-active group (P ≤ 0.05). †Significant change from baseline in high-active group (P ≤ 0.05).

Fig. 2.

Cell cycle gene expression in colony-forming unit-endothelial cells (CFU-ECs) supplemented with thrombin and after 30 min of vigorous exercise. Values are means ± SE. A: high-active: n = 6; low-active: n = 6. †Significant difference between groups (P ≤ 0.05). B: n = 12: high-active, n = 6; low-active, n = 6. All comparisons were relative to the control, 0 U/ml thrombin supplementation, set at 0.0. *Significant difference from control (P ≤ 0.05).

Fig. 3.

VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR2) gene expression in CFU-ECs supplemented with thrombin and after 30 min of vigorous exercise. Values are means ± SE. A: high-active: n = 6; low-active: n = 6. †Significant difference between groups (P ≤ 0.05). B: n = 12: high-active, n = 6; low-active, n = 6. All comparisons were relative to the control, 0 U/ml thrombin supplementation, set at 0.0. *Significant difference from control (P ≤ 0.05).

Fig. 4.

CFU-EC (A) and CD34+/VEGFR2⁺ (B) before and after 30 min of vigorous treadmill exercise. Values are means ± SE. A: high-active: n = 12; low-active: n = 7. High-active before exercise: 17.8 ± 3.9; after exercise: 26.4 ± 9.8 (P = 0.505). Low-active before exercise: 14.2 ± 4.1; after exercise: 17.0 ± 12.8 (P = 0.681). B: high-active: n = 12; low-active: n = 6. High-active before exercise: 84.0 ± 22.9; after exercise: 132.0 ± 59.7 (P = 0.326). Low-active before exercise: 41.3 ± 23.9; after exercise: 80.1 ± 84.4 (P = 0.471).

Fig. 5.

Relationship between the change in plasma F1+2 and circulating CD34+/VDGFR2⁺ with 30 min of vigorous exercise. Spearman correlation r = 0.698, P = 0.001. Variables presented as rank order reflect the nonparametric Spearman correlation test.