Polydeoxyribonucleotide (PDRN) restores blood flow in an experimental model of peripheral artery occlusive disease Alessandra Bitto, MD, Francesca Polito,

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Polydeoxyribonucleotide (PDRN) restores blood flow in an experimental model of peripheral artery occlusive disease Alessandra Bitto, MD, Francesca Polito, PhD, Domenica Altavilla, PhD, Letteria Minutoli, MD, Alba Migliorato, PhD, Francesco Squadrito, MD Journal of Vascular Surgery Volume 48, Issue 5, Pages 1292-1300 (November 2008) DOI: 10.1016/j.jvs.2008.06.041 Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 1 A, Blood flow evaluated by laser Doppler at 10 minutes, and at 7, 14, and 21 days after hind limb ischemia (HLI) in the various treatment groups. Rats were administered the vehicle or intraperitoneal polydeoxyribonucleotide (PDRN), at 2, 4, 8, or 16 mg/kg daily, starting immediately after surgery. PDRN (8 and 16 mg/kg) restored blood flow in treated rats. The blood flow of ischemic hind limb is expressed as a relative amount compared with the uninjured limb. *P < .005 vs HLI + vehicle. B, Percentage of blood flow vs animals undergoing sham operations (sham HLI) evaluated by laser Doppler at 7, 14, and 21 days after hind limb ischemia. Rats were administered the vehicle or intraperitoneal PDRN (2, 4, 8, and 16 mg/kg daily) starting immediately after surgery. PDRN (8 and 16 mg/kg) restored blood flow in treated animals. *P < .005 vs HLI + vehicle. C, Blood flow evaluated by laser Doppler at day 21 after HLI in the experimental groups. The blood flow of ischemic hind limb was expressed as a relative amount compared with the uninjured limb. *P < .005 vs HLI + vehicle. Error bars in the panels represent the standard deviation. DMPX, 3,7-dimethyl-1-propargilxanthine. Journal of Vascular Surgery 2008 48, 1292-1300DOI: (10.1016/j.jvs.2008.06.041) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 2 Laser Doppler images at (Row A) 10 minutes, and at (Row B) 7, (Row C) 14, and (Row D) 21 days after hind limb ischemia (HLI). Polydeoxyribonucleotide (PDRN), at 8 mg/kg, restored blood flow in treated animals. DMPX, 3,7-dimethyl-1-propargilxanthine. Journal of Vascular Surgery 2008 48, 1292-1300DOI: (10.1016/j.jvs.2008.06.041) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 3 A, Western blots for vascular endothelial growth factor (VEGF) at 7, 14, and 21 days after hind limb ischemia (HLI).Upper panel, Representative autoradiography highlighting VEGF expression. Lower panel, quantitative data representing the mean ± SD of seven experiments. *P < .005 and #P < .0001 vs HLI + vehicle. B, Expression of messenger RNA for VEGF at 7, 14, and 21 days after HLI. Bars represent the mean ± SD of seven experiments. *P < .005 and #P < .0001 vs HLI + vehicle. DMPX, 3,7-dimethyl-1-propargilxanthine; PDRN polydeoxyribonucleotide. Journal of Vascular Surgery 2008 48, 1292-1300DOI: (10.1016/j.jvs.2008.06.041) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 4 Histologic evaluation of adductor muscles after hind limb ischemia (HLI) at day 21 (original magnification ×20). A, Sham HLI animal adductor muscle showing normal muscular architecture. B, HLI animal adductor muscle showing edema with marked cellular infiltration and necrosis. C, Adductor muscle from HLI rats treated with intraperitoneal polydeoxyribonucleotide (PDRN), at 8-mg/kg daily, showing reduced edema and cellular infiltration with no sign of necrosis. D, The means ± SD of the histologic score obtained from seven animals. *P < .005 vs HLI + vehicle. E, Tissue edema evaluated in adductor muscles at day 7 after ischemia. PDRN (8 mg/kg) diminished the degree of edema in treated animals. *P < .005 vs HLI + vehicle. F, Malondialdehyde levels evaluated in adductor muscles of both legs at day 7 after ischemia. PDRN diminished the degree of edema in treated animals. #P < .05 vs HLI. DMPX, 3,7-dimethyl-1-propargilxanthine. Journal of Vascular Surgery 2008 48, 1292-1300DOI: (10.1016/j.jvs.2008.06.041) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 5 Immunohistologic evaluation of adductor muscles after hind limb ischemia (HLI) at day 14 (original magnification ×40). A, Adductor muscle from HLI animal stained with platelet-endothelial cell adhesion molecule 1 (PECAM-1). Arrowhead shows very slight positive capillary between muscle fibers. B, Adductor muscle from HLI animal treated with polydeoxyribonucleotide (PDRN; 8 mg/kg) stained with PECAM-1. Arrowheads show positive capillaries between muscle fibers. *P < .005 vs HLI + vehicle. C, The mean ± SD number of capillaries/mm2 are presented; each bar is the mean of seven animals. *P < .005 vs HLI + vehicle. D, Adductor muscle from HLI animal stained with vascular endothelial growth factor (VEGF). Arrowheads show positive cells between muscle fibers. E, Adductor muscle from HLI animal treated with PDRN (8 mg/kg) stained with VEGF. Arrowheads show positive cells between muscle fibers. Journal of Vascular Surgery 2008 48, 1292-1300DOI: (10.1016/j.jvs.2008.06.041) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions