Abstract: BACKGROUND: Irisin is a myokine induced by exercise. In addition to its metabolic regulatory effects, it can also improve endothelial function. However, its relationship with endothelial progenitor cells has not yet been fully clarified. 
OBJECTIVE: To investigate the effects of irisin induced by regular exercise on the function of bone marrow endothelial progenitor cells in spontaneously hypertensive rats and to reveal the role of the phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in this process. 
METHODS: Thirty 10-week-old male spontaneously hypertensive rats were randomly divided into a hypertensive control group and a hypertensive exercise group using a random number table. Fifteen age- and sex-matched Wistar-Kyoto rats served as the normotensive group. The normotensive group and hypertensive control group were kept quietly in cages, while the hypertensive exercise group underwent voluntary wheel running exercise for 5 days per week over 8 weeks. Seventy-two hours after the last intervention, tail artery blood pressure was measured using plethysmography. Blood samples were collected from the abdominal aorta, and plasma irisin concentration was measured by enzyme-linked immunosorbent assay. The thoracic aorta was collected to assess endothelium-dependent vasodilation (representing endothelial function) via vascular ring experiments. The gastrocnemius muscle was collected to detect capillary density using CD31 immunohistochemical staining, to measure irisin levels by enzyme-linked immunosorbent assay, and to determine fibronectin type III domain-containing protein 5 mRNA and protein expression by real-time quantitative polymerase chain reaction and western blot, respectively. The left tibia was isolated to measure irisin content and fibronectin type III domain-containing protein 5 expression. Bone marrow was collected from the femur and right tibia to isolate and culture endothelial progenitor cells. Matrigel tube formation assay was used to assess in vitro angiogenesis capacity. A nude rat model of carotid artery endothelial denudation was established to evaluate the in vivo vascular endothelial repair capacity of endothelial progenitor cells (a carotid endothelium injury model was established by tail vein injection of endothelial progenitor cell suspension. Vascular staining using Evans blue and the area of vascular re-endothelialization was observed). Protein expression of the phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway was measured by western blot. Recombinant human irisin was co-incubated with bone marrow endothelial progenitor cells. Endothelial progenitor cell function was assessed after treatment with the endothelial nitric oxide synthase inhibitor L-nitroarginine methyl ester. Protein expression levels of protein kinase B and endothelial nitric oxide synthase were measured after treatment with the phosphatidylinositol-3-kinase inhibitor LY-294002 or the protein kinase B inhibitor GSK-690693. 
RESULTS AND CONCLUSION: (1) Exercise reduced blood pressure, improved vascular endothelial function, and promoted angiogenesis (increased capillary density) in spontaneously hypertensive rats (P < 0.05). (2) Exercise increased irisin levels in the circulation, skeletal muscle, and bone (P < 0.05); however, fibronectin type III domain-containing protein 5 mRNA expression was only upregulated in skeletal muscle (P < 0.05). (3) Exercise enhanced the angiogenic and vascular endothelial repair capacity of bone marrow endothelial progenitor cells and activated the phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway (P < 0.05). (4) Exogenous irisin also improved the function of bone marrow endothelial progenitor cells in spontaneously hypertensive rats (P < 0.05). Endothelial progenitor cell function decreased after treatment with the endothelial nitric oxide synthase inhibitor L-nitroarginine methyl ester (P < 0.05). After treatment with phosphatidylinositol-3-kinase or protein kinase B inhibitors, the protein expression levels of protein kinase B and endothelial nitric oxide synthase were downregulated (P < 0.05). To conclude, irisin induced by regular exercise improves the function of bone marrow endothelial progenitor cells in spontaneously hypertensive rats via the phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway, thereby correcting vascular endothelial dysfunction and promoting angiogenesis. These findings suggest that irisin, as a potential "exercise mimetic," may provide health benefits to hypertensive patients who are unable to engage in physical activity by mimicking the protective effects of exercise on the cardiovascular system.

Key words: exercise, irisin, hypertension, endothelial progenitor cells, signaling pathway, fibronectin type III domain-containing protein 5