Improvement in asymmetric dimethylarginine and oxidative stress in patients with limb salvage after autologous mononuclear stem cell application for critical limb ischemia

Abstract

Background: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, acts as an inhibitor of angiogenesis and is associated with an increased risk of cardiovascular mortality. Administration of stem cells may affect endogenous mechanisms that regulate ADMA production and metabolism. The aim of the present study was to analyze ADMA concentration and changes in oxidative stress in patients with advanced critical limb ischemia (CLI) after bone marrow-derived mononuclear cell (BM-MNC) therapy.

Methods: Fifty patients (age 64 ± 11 years, 44 males, 6 females) with advanced CLI (Rutherford category 5 or 6) not eligible for revascularization were treated by intramuscular (n = 25) or intra-arterial (n = 25) injection of 40 ml BM-MNC concentrate. Patients with limb salvage and improved wound healing after 6 months were considered responders to cell therapy. The concentrations of markers of oxidative stress and angiogenesis were analyzed before, and at 3 and 6 months after BM-MNC delivery.

Results: At 6-month follow-up, four patients died of reasons unrelated to stem cell therapy. Among the survivors, 80% (37/46) showed limb salvage and improved wound healing. At 6 months follow-up, ADMA concentration significantly decreased in patients with limb salvage (1.74 ± 0.66 to 0.90 ± 0.49 μmol/L, p < 0.001), in parallel with decreased tumor necrosis factor (TNF)-α (2.22 ± 0.16 to 1.94 ± 0.38 pg/ml, p < 0.001), and increased reduced glutathione (6.96 ± 3.1 to 8.67 ± 4.2 μmol/L, p = 0.02), superoxide dismutase activity (168 ± 50 to 218 ± 37 U/L, p = 0.002), and coenzyme Q10 concentration (468 ± 182 to 598 ± 283 μg/L, p = 0.02). The number of delivered BM-MNCs significantly correlated with the decrease in ADMA concentration at 3 months (p = 0.004, r = -0.48) and the decrease in TNF-α concentration at 6 months (p = 0.03, r = -0.44) after cell delivery. ADMA or TNF-α improvement did not correlate with the number of applied CD34⁺ cells, C-reactive protein concentration, leukocyte count, or the dose of atorvastatin.

Conclusions: The therapeutic benefit of BM-MNC therapy is associated with reduced ADMA levels and oxidative stress. Regulation of the ADMA-nitric oxide axis and improved antioxidant status may be involved in the beneficial effects of stem cell therapy.

Trial registration: The study was approved and retrospectively registered by ISRCTN registry, ISRCTN16096154 . Registered on 26 July 2016.

Keywords: Angiogenesis; Asymmetric dimethylarginine; Cell therapy; Critical limb ischemia; Oxidative stress.

Fig. 1

Oxidative stress and stem cell dysfunction. ADMA asymmetric dimethylarginine, CVS cardiovascular, DDAH dimethylarginine dimethylaminohydrolase, EPC endothelial progenitor cell, NO nitric oxide, NOS endothelial nitric oxide synthase, ROS reactive oxygen species

Fig. 2

Decrease in ADMA concentration in patients with limb salvage after BM-MNC application. p < 0.001, 180 days vs. baseline. ADMA asymmetric dimethylarginine

Fig. 3

Decrease in ADMA concentration in relation to the number of delivered BM-MNCs at 3 months after cell delivery. ADMA asymmetric dimethylarginine, BM-MNC bone marrow-derived mononuclear cell