Therapeutic angiogenesis for the critical limb ischemia decreases platelet activation.

Platelets are required for the recruitment of bone marrow-derived mononuclear cells (BMMNC) into ischemia-induced vasculature, which underlines their key role in angiogenesis. The difference in platelet immunophenotype between healthy controls and patients with critical limb ischemia (CLI) treated with therapeutic angiogenesis (TA) using BMMNC was assessed. The impact of TA on the expression of platelet membrane markers was studied as well. CLI patients (N = 26) and blood donors as controls (N = 21) were enrolled. Bone marrow (600 ± 50 ml) was centrifuged (3200 g, 20 min, 22 °C). BMMNC (100-120 ml) were separated by Optipress I and implanted to the ischemic limb using deep intramuscular injections. Flow cytometry was employed for the peripheral blood platelets immunophenotyping. CD41FITC, CD62PE, CD36FITC, CD29FITC antibodies were used. Patients were followed up prior to the procedure and at months 1, 3 and 6. The expression of CD41 was lower in CLI patients than in the controls. P-selectin (CD62P) was higher in CLI patients than in controls at the baseline and at month 6. It was significantly down-regulated at month 3, however not at months 1 and 6 compared to baseline. Platelet GPIV (CD36) was higher at the baseline, but not during the follow-up compared to the controls. ß1-integrin (CD29) progressively decreased during the follow-up as compared to the baseline value. Platelets in CLI express P-selectin, GPIV and ß1-integrin more abundantly than platelets of healthy subjects. TA down-regulates the expression of the respective markers. Possible mechanism could be higher clearance of the activated platelets in the ischemic tissues during angiogenesis.

Therapeutic angiogenesis improves fibrinolytic imbalance in patients with critical limb ischemia.

The mechanisms of fibrinolysis have been suggested to be linked to the pathogenesis of peripheral artery disease. The impact of therapeutic angiogenesis on the parameters of fibrinolysis was studied in critical limb ischemia (CLI). CLI patients (N = 29) and blood donors as controls (N = 29) were enrolled. Bone marrow (600 ±â€Š50 ml) was centrifuged (3200g, 20 min, 22°C), bone marrow-derived mononuclear cells (100-120 ml) were separated by Optipress I and implanted into the ischemic limb using intramuscular injections. ELISA was employed for the assessment of plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) levels. Patients were followed-up prior to the procedure and after 1, 3 and 6 months. All stage-IV patients (N = 22) had ischemic lesions. The lesions resolved in 10 patients. Five patients underwent major amputation; they all were stage-IV. Ischemic lesions persisted in seven patients beyond 6 months. The t-PA levels were higher in patients compared with the healthy controls both at baseline (P < 0.01) and after 6 months (P < 0.05). No significant changes were observed in the t-PA levels during the follow-up. PAI-1 was higher in patients than in the healthy individuals at baseline (P < 0.001) and at month 1 (P < 0.05). However, no difference in PAI-1 levels between the patients and the healthy individuals was found after 3 and 6 months. The PAI-1 levels were significantly downregulated during the follow-up compared with the baseline (P < 0.0001). Therapeutic angiogenesis for the CLI downregulates PAI-1 levels, thus having a systemic effect on fibrinolysis.