Original Communications| Volume 129, ISSUE 3, P341-350, March 2001

Nitric oxide synthase isoform expression in a porcine model of granulation tissue formation


      Background. This study was designed to determine whether the nitric oxide (NO) pathway is involved in wound granulation tissue formation. Methods. A section of the pig abdominal wall (excluding the skin) was excised, creating an incisional hernia. The resulting defect was repaired with silicone sheeting in a manner that mimics a temporary abdominal wall closure. During the 14-day experimental period, porcine omentum adhered to the peritoneal edges of the defect and a highly vascularized granulation tissue formed on both sides of the sheeting. Granulation tissue thickness and wound fluid volume were monitored by ultrasonography and epigastric artery flow velocity was monitored by color Doppler flow analysis at days 2, 4, 7, 9, 11, and 14. Fluid was serially harvested from the wound compartment at days 2, 4, 7, 9, 11, and 14 for nitrite/ nitrate (NOx) analysis. Finally, granulation tissue was harvested at day 14 for immunohistochemical and molecular analyses. Results. There was a significant increase in granulation tissue thickness and wound fluid volume during the 14-day study period. Blood flow to the wound increased significantly by day 4 and returned toward baseline by day 14. Wound fluid NOx levels significantly increased from days 7 to 11 and then decreased to near baseline values by day 14. Wound fluid arginine levels significantly decreased when compared with peritoneal fluid and plasma levels at day 14, while wound fluid ornithine levels significantly increased. Immunohistochemical analysis of granulation tissue at day 14 revealed nitric oxide synthase (NOS) 2 was present in the majority of the cells in the granulation tissue. NOS 3 was expressed in endothelial cells only, and NOS 1 expression was not observed in the granulation tissue. Conclusions. This study suggests that NO, NOS 2, and arginine may play critical roles in granulation tissue formation and wound healing. Arginase and NOS 2 may compete for available arginine as a substrate, thereby limiting later NO production in favor of sustained ornithine synthesis. (Surgery 2001;129:341-50.)
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Surgery
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Howdieshell TR
        • Rieger C
        • Gupta V
        • Callaway D
        • McNeil P
        Normoxic wound fluid contains high levels of vascular endothelial growth factor.
        Ann Surg. 1998; 228: 707-715
        • Howdieshell TR
        • Yeh K
        • Hawkins JL
        • Cue JI
        Temporary abdominal wall closure in trauma patients: indications, technique, and results.
        World J Surg. 1995; 19: 154-158
        • Kone BC
        • Baylis C
        Biosynthesis and homeostatic roles of nitric oxide in the kidney.
        Am J Physiol. 1997; 272: F561-F578
        • Yamasaki K
        • Edington HD
        • McClosky C
        • Tzeng E
        • Lizonova A
        • Kovesdi I
        • et al.
        Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer.
        J Clin Invest. 1998; 101: 967-971
        • Thornton RJ
        • Schaffer MR
        • Witte MB
        • Moldawer LL
        • MacKay LS
        • Abouhamze A
        • et al.
        Enhanced collagen accumulation following direct transfection of the nitric oxide synthase gene in cutaneous wounds.
        Biochem Biophys Res Comm. 1998; 246: 654-659
        • Pollock JS
        • Forstermann U
        • Mitchell JA
        • Warner TD
        • Schmidt HH
        • Nakane M
        • et al.
        Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells.
        Proc Natl Acad Sci U S A. 1991; 88: 10480-10484
        • Nishida K
        • Harrison D
        • Navas J
        • Fisher A
        • Dockery S
        • Uematsu M
        • et al.
        Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.
        J Clin Invest. 1992; 90: 2092-2096
        • Xu XP
        • Pollock JS
        • Tanner MA
        • Myers PR
        Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells.
        Cardiovasc Res. 1995; 30: 841-847
        • Inoue N
        • Venema RC
        • Sayegh HS
        • Ohara Y
        • Murphy TJ
        • Harrison DG
        Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-beta 1.
        Arteriolscler Thromb Vasc Biol. 1995; 15: 1255-1261
        • Papaetropoulos A
        • Garcia-Cardena G
        • Madri JA
        • Sessa WC
        Nitric oxide contributes to the angiogenic properties of vascular endothelial growth factor in human cells.
        J Clin Invest. 1997; 29: 2923-2930
      1. Pinkney MN. Ultrasonography: an introduction to normal structure and functional anatomy. In: Curry RA, Tempkin BB, editors. Instrumentation. Philadelphia: WB Saunders; p. 8-19.

        • Ziche M
        • Morbidelli L
        • Masini E
        • Amerini S
        • Granger HJ
        • Maggi CA
        • et al.
        Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P.
        J Clin Invest. 1994; 94: 2036-2044
        • Morbidelli L
        • Chang CH
        • Douglas JG
        • Granger HJ
        • Ledda F
        • Ziche M
        Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium.
        Am J Physiol. 1996; 270: H411-H415
        • Drapier JC
        • Hirling H
        • Wietzerbin J
        • Kaldy P
        • Kuhn LC
        Biosynthesis of nitric oxide activates iron regulatory factor in macrophages.
        EMBO J. 1993; 12: 3643-3649
        • Lander HM
        • Sehajpal PK
        • Novogrodsy A
        Nitric oxide signaling: a possible role for G proteins.
        J Immunol. 1993; 150: 1509-1516
        • Schaffer M
        • Tantry U
        • Gross SS
        • Wasserburg HL
        • Barbul A
        Nitric oxide regulates wound healing.
        J Surg Res. 1996; 63: 237-240
        • Schaffer M
        • Efron PA
        • Thorton FJ
        • Klingel K
        • Gross SS
        • Barbul A
        Nitric oxide, an autocrine regulator of wound fibroblast synthetic function.
        J Immunol. 1997; 158: 2382-2389
        • Bulgrin JP
        • Shabani M
        • Chakravarthy D
        • Smith DJ
        Nitric oxide synthesis is suppressed in steroid-impaired and diabetic wounds.
        Wounds. 1995; 7: 48-57
        • Moncada S
        The l-arginine: nitric oxide pathway.
        Acta Physiol Scand. 1992; 145: 201-227
        • Ignarro LJ
        Biosynthesis and metabolism of endothelium-derived nitric oxide.
        Annu Rev Pharmacol Toxicol. 1990; 30: 535-560
        • Vodovotz Y
        • Bogdan C
        • Paik J
        • Xie QW
        • Nathan C
        Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor β.
        J Exp Med. 1993; 17x: 605-613
        • Pinsky DJ
        • Cai B
        • Yang X
        • Rodriguez C
        • Sciacca RR
        • Cannon PJ
        The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor β.
        J Clin Invest. 1995; 95: 677-685
        • Wu G
        • Morris SM
        Arginine metabolism: nitric oxide and beyond.
        Biochem J. 1998; 336: 1-17
        • Griffith OW
        • Stuehr DJ
        Nitric oxide synthases: properties and catalytic mechanism.
        Annu Rev Physiol. 1995; 57: 707-736
        • Albina JE
        • Abate JA
        • Mastrofrancesco B
        Role of ornithine as a proline precursor in healing wounds.
        J Surg Res. 1993; 55: 97-102
        • Hecker M
        • Sessa WC
        • Harris HJ
        • Anggard EE
        • Vane JR
        The metabolism of l-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: cultured endothelial cells recycle l-citrulline to l-arginine.
        Proc Natl Acad Sci U S A. 1990; 87: 8612-8616
        • Marzinzig M
        • Nussler AK
        • Marzinzig E
        • Barthlen W
        • Morris Jr, SM
        • Ruckner UB
        Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols.
        Nitric Oxide. 1997; 1: 177-189
        • Hattori Y
        • Campbell EB
        • Gross SS
        Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle.
        J Biol Chem. 1994; 269: 9405-9408
        • Nussler AK
        • Billiar TR
        • Liv ZZ
        • Morris Jr, SM
        Coinduction of nitric oxide synthase and argininosuccinate synthetase in a murine macrophage cell line.
        J Biol Chem. 1994; 269: 257-1261
        • Chiung C
        • Liao JC
        • Kuo L
        Arginase modulates nitric oxide production in activated ophages.
        Am J Physiol. 1998; 274: H342-H348