Early inducible nitric oxide synthase 2 (NOS 2) activity enhances ischaemic skin flap survival Sunao Furuta 1 , Peter Vadiveloo 2 , Rosalind Romeo-Meeuw 2 , Wayne Morrison 2 , Alastair Stewart 3 & Geraldine Mitchell 2 1 Plastic and Reconstructive Surgery, Suwa Red Cross Hospital, Kogan-Douri, Suwa, Japan; 2 Bernard O’Brien Institute of Microsurgery and Department of Surgery, University of Melbourne, St. Vincent’s Hospital, Fitzroy, Melbourne, Victoria, Australia; 3 Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia Received 22 May 2003; accepted in revised form 20 December 2003 Key words: angiogenesis, fibroblast growth factor 2, mast cells, nitric oxide, nitric oxide synthase inhibitors, nitric oxide synthase 2)/) mice, skin-flap survival, vascular endothelial growth factor Abstract A functional skin-flap model of angiogenesis in the mouse was utilized to investigate ischaemic flap survival/ angiogenesis whilst under pharmacological or genetic inhibition of nitric oxide synthase (NOS). In this model, the epigastric artery was cauterized. Following a five-day angiogenic period an abdominal skin-flap supplied by the pre- existing epigastric artery was raised and resutured. After a further six days the outcome was determined by measuring the area of living skin-flap that was sustained by new vessel growth around the cauterized artery. Both pharmacological [S-methyl-isothiourea (SMT) given via i.p. injection for the five-day angiogenic period] and genetic inhibition of NOS 2 (using NOS 2)/) mice) caused a similar and significant fall in flap survival compared to saline- treated wild-type mice (P < 0.05). Delaying pharmacological NOS 2 inhibition for two days post-arterial cauterization increased flap survival in wild-type mice to that of saline-treated controls, whilst treating wild-type mice with SMT for only the first three days of angiogenesis produced a significant decrease in flap survival, similar to that of five-day SMT-treated wild types. Immunoreactivity for NOS 2 in NOS 2)/) knockout mice was absent in both pedicles and skin-flaps, whilst mast cells from both the pedicle and the skin-flap stained positively for NOS 2 in wild-type mice. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF 2) immunoreactivity was also strongly evident in mast cells of both wild-type and NOS 2)/) mice at both sites. These results point to a significant role for NOS 2 in promoting wound healing/angiogenesis in its early stages. Abbreviations: AET – aminoethylthiourea; AG – aminoguanide; FGF 2, bFGF – basic fibroblast growth factor; cNOS – constitutive nitric oxide synthase; CAM – chorioallantoic membrane; DAB – diaminobenzidine; eNOS – endothelial nitric oxide synthase; HDMVEC – human dermal microvascular endothelial cells; HUVECS – human umbilical vein endothelial cells; iNOS – NOS 2 inducible nitric oxide synthase; i.p. – intra-peritoneal; NOS 2)/) mice – nitric oxide synthase 2 knockout mice; NO – nitric oxide; NOS – nitric oxide synthase; L -NAME – nitro- L -arginine methyl ester; L -NIL – L -N 6 -(1-iminoethyl)lysine; L -NIO – nitro-imino-L -ornithine; nNOS – neuronal nitric oxide synthase; ONOO )) – peroxynitrite; PBS – phosphate buffered saline; SMT – S-methyl-isothiourea; SPSS – statistical package for the social sciences; VEGF – vascular endothelial growth factor; VEGFR-1(Flt-1) – vascular endothelial growth factor receptor-1; VEGFR-2 (KDR/flk-1) – vascular endothelial growth factor receptor-2; WT – wild-type Introduction Reconstructive surgical techniques and newer tissue- engineering techniques frequently employ vascularized flaps of fat/skin/muscle/bone to fill and functionally replace damaged tissues. These vascularized flaps of tissue also require permanent vascularization via angi- ogenesis from their recipient site. Recently Kane et al. [1] found that nitric oxide (NO) produced by different nitric oxide synthase (NOS) isoforms may either inhibit or promote ischaemic skin-flap survival in the rat. The ability of NO to regulate skin-flap survival may be related to the promotion or inhibition of angiogenesis. Thus, there is significant therapeutic potential for strategies that alter the production of this molecule in Correspondence to: Dr Geraldine Mitchell, Bernard O’Brien Institute of Microsurgery, 42 Fitzroy Street, Fitzroy, Victoria, 3065, Australia. Tel: +61-3-92884018; Fax: +61-3-9416-0926; E-mail: mitchelg@ svhm.org.au Angiogenesis 7: 33–43, 2004. 33 Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.