Download PDF
J Reconstr Microsurg 2009; 25(4): 219-225
DOI: 10.1055/s-0028-1104550
© Thieme Medical Publishers

Vascular Endothelial Growth Factor Upregulates Inducible Nitric Oxide Synthase Expression in the Muscle Flap Ischemia Model in the Rat

Zeng-Gan Chen1 , 2 , Babara Persons1 , Lin Lin1 , William C. Lineaweaver1 , Feng Zhang1
  • 1Division of Plastic Surgery, University of Mississippi Medical Center, Jackson, Mississippi
  • 2Department of Orthopedic Surgery, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
Further Information

Publication History

Publication Date:
01 December 2008 (online)

Also available at
    Permissions and Reprints

ABSTRACT

The purpose of this investigation was to elucidate the regulation of inducible nitric oxide synthase (iNOS) expression by vascular endothelial growth factor (VEGF) in a muscle flap ischemia model in rats. The gracilis muscle flap model was chosen. Sixty adult male Sprague Dawley rats were randomly divided into two groups (n = 30). After 4 hours ischemia, the experimental group received VEGF treatment, and the control group was given saline in the same fashion. At time intervals of 0, 2, 6, 12, and 18 hours (n = 6 for each time interval) after injection, tissue samples were biopsied for reverse transcriptase polymerase chain reaction, routine hematoxylin-eosin staining, and CD31 immunohistochemical staining. The result showed that iNOS expression is increased in the gracilis muscle flap ischemia model in rats compared with the control group within 6 hours after ischemia (p < 0.05). In the VEGF group, iNOS expression increased rapidly over the first 2 hours, but no statistically significant difference was observed at 12 and 18 hours between the two groups (p > 0.05). We concluded that the application of VEGF could maintain the structure of capillaries and upregulate iNOS expression during the first 6 hours after ischemia in the ischemic muscle flap of a rat model. These findings may provide the evidence to study the mechanism of VEGF in improving flap survival.

KEYWORDS

Vascular endothelial growth factor - inducible nitric oxide synthase - muscle flap

REFERENCES

  • 1 Lineaweaver W C, Buncke H J, Whitney T M, Clapson J B, Buncke G M, Alpert B S. Factors associated with microvascular flap failure and direction for research.  Plast Surg Forum. 1989;  12 252-253
    PubMedSearch in Google Scholar
  • 2 Hallock G G. Is there a “learning curve” for muscle perforator flaps?.  Ann Plast Surg. 2008;  60 146-149
    CrossrefPubMedSearch in Google Scholar
  • 3 De Santis G, Pinelli M. Microsurgical model of ischemia reperfusion in rat muscle: evidence of free radical formation by spin trapping.  Microsurgery. 1994;  15 655-659
    CrossrefPubMedSearch in Google Scholar
  • 4 Morris S F, Pang C Y, Zhong A et al.. Assessment of ischemia-induced reperfusion injury in the pig latissimus dorsi myocutaneous flap model.  Plast Reconstr Surg. 1993;  92 1162-1172
    CrossrefPubMedSearch in Google Scholar
  • 5 Pu L Q, Gadowski G R, Graham A M et al.. Enhanced revascularisation after angiogenic stimulation in a rabbit model of bilateral limb ischaemia.  Eur J Vasc Endovasc Surg. 1995;  9(2) 189-196
    CrossrefPubMedSearch in Google Scholar
  • 6 Lefer A M, Ma X L, Weyrich A S, Scalia R. Mechanism of the cardioprotective effect of transforming growth factor beta 1 in feline myocardial ischemia and reperfusion.  Proc Natl Acad Sci U S A. 1993;  90(3) 1018-1022
    CrossrefPubMedSearch in Google Scholar
  • 7 Kenny D, Coughlan M G, Pagel P S, Kampine J P, Warltier D C. Transforming growth factor beta 1 preserves endothelial function after multiple brief coronary artery occlusions and reperfusion.  Am Heart J. 1994;  127(6) 1456-1461
    CrossrefPubMedSearch in Google Scholar
  • 8 Antonini A, Zacchigna S, Papa G, Novati F, Pascone M, Giacca M. Improved survival of rat ischemic cutaneous and musculocutaneous flaps after VEGF gene transfer.  Microsurgery. 2007;  27 439-445
    CrossrefPubMedSearch in Google Scholar
  • 9 Frangoulis M, Georgiou P, Chrisostomidis C et al.. Rat epigastric flap survival and VEGF expression after local copper application.  Plast Reconstr Surg. 2007;  119 837-843
    CrossrefPubMedSearch in Google Scholar
  • 10 Zhang F, Brooks D, Chen W, Mustain W, Chen M B, Lineaweaver W C. Improvement of venous flap survival by application of vascular endothelial growth factor in a rat model.  Ann Plast Surg. 2006;  56 670-673
    CrossrefPubMedSearch in Google Scholar
  • 11 Zhang F, Yang F, Hu E C, Sones W, Lei M, Lineaweaver W C. Vascular endothelial growth factor gene therapy in improvement of skin paddle survival in a rat TRAM flap model.  J Reconstr Microsurg. 2005;  21 391-396
    Thieme ConnectPubMedSearch in Google Scholar
  • 12 Yim K, Lineaweaver W, Briones R et al.. Critical ischemia time of rat serratus anterior and gracilis muscles. In: Carraro U, Salmon S Basic and Applied Myology: Perspectives for the 90's. Padova, Italy; Unipress 1991: 373
    Search in Google Scholar
  • 13 Kryger Z, Dogan T, Zhang F et al.. Effects of VEGF administration following ischemia on survival of the gracilis muscle flap in the rat.  Ann Plast Surg. 1999;  43 172-178
    CrossrefPubMedSearch in Google Scholar
  • 14 Tzeng E, Kim Y M, Pitt B R, Lizonova A, Kovesdi I, Billiar T R. Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis.  Surgery. 1997;  122(2) 255-263
    CrossrefPubMedSearch in Google Scholar
  • 15 Flamme I, von Reutern M, Drexler H C, Syed-Ali S, Risau W. Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation.  Dev Biol. 1995;  171 399-414
    CrossrefPubMedSearch in Google Scholar
  • 16 Hippenstiel S, Krull M, Ikemann A, Risau W, Clauss M, Suttorp N. VEGF induces hyperpermeability by a direct action on endothelial cells.  Am J Physiol. 1998;  274 L678-L684
    PubMedSearch in Google Scholar
  • 17 Yang R, Thomas G R, Bunting S et al.. Effects of vascular endothelial growth factor on hemodynamics and cardiac performance.  J Cardiovasc Pharmacol. 1996;  27 838-844
    CrossrefPubMedSearch in Google Scholar
  • 18 Zachary I. Vascular endothelial growth factor.  Int J Biochem Cell Biol. 1998;  30(11) 1169-1174
    CrossrefPubMedSearch in Google Scholar
  • 19 Takeshita S, Zheng L P, Brogi E et al.. Therapeutic angiogenesis: a single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model.  J Clin Invest. 1994;  93 662-670
    CrossrefPubMedSearch in Google Scholar
  • 20 Bauters C, Asahara T, Zheng L P et al.. Recovery of disturbed endothelium-dependent flow in the collateral-perfused rabbit ischemic hindlimb after administration of vascular endothelial growth factor.  Circulation. 1995;  91 2802-2809
    PubMedSearch in Google Scholar
  • 21 Becit N, Ceviz M, Kocak H et al.. The effect of vascular endothelial growth factor on angiogenesis: an experimental study.  Eur J Vasc Endovasc Surg. 2001;  22 310-316
    CrossrefPubMedSearch in Google Scholar
  • 22 Walder C E, Errett C J, Bunting S et al.. Vascular endothelial growth factor augments muscle blood flow and function in a rabbit model of chronic hindlimb ischemia.  J Cardiovasc Pharmacol. 1996;  27 91-98
    CrossrefPubMedSearch in Google Scholar
  • 23 Harada K, Friedman M, Lopez J J et al.. Vascular endothelial growth factor administration in chronic myocardial ischemia.  Am J Physiol. 1996;  270 H1791-H1802
    PubMedSearch in Google Scholar
  • 24 Lopez J J, Laham R J, Stamler A et al.. VEGF administration in chronic myocardial ischemia in pigs.  Cardiovasc Res. 1998;  40 272-281
    CrossrefPubMedSearch in Google Scholar
  • 25 Padubidri A, Browne Jr E. Effect of vascular endothelial growth factor (VEGF) on survival of random extension of axial pattern skin flaps in the rat.  Ann Plast Surg. 1996;  37 604-611
    CrossrefPubMedSearch in Google Scholar
  • 26 Seify H, Bulky U, Jones G. Effect of vascular endothelial growth factor-induced angiogenesis on TRAM flap harvesting after abdominoplasty.  Plast Reconstr Surg. 2003;  111 1212-1216
    CrossrefPubMedSearch in Google Scholar
  • 27 Gurunluoglu R, Meirer R, Shafighi M, Huemer G M, Yilmaz B, Piza-Katzer H. Gene therapy with adenovirus-mediated VEGF enhances skin flap prefabrication.  Microsurgery. 2005;  25(5) 433-441
    CrossrefPubMedSearch in Google Scholar
  • 28 Neumeister M W, Song Y H, Mowlavi A, Suchy H, Mathur A. Effects of liposome-mediated gene transfer of VEGF in ischemic rat gracilis muscle.  Microsurgery. 2001;  21(2) 58-62
    CrossrefPubMedSearch in Google Scholar
  • 29 Banbury J, Siemionow M, Porvasnik S, Petras S, Browne E. Improved perfusion after subcritical ischemia in muscle flaps treated with vascular endothelial growth factor.  Plast Reconstr Surg. 2000;  106(7) 1541-1546
    CrossrefPubMedSearch in Google Scholar
  • 30 Khan A, Ashrafpour H, Huang N et al.. Acute local subcutaneous VEGF165 injection for augmentation of skin flap viability: efficacy and mechanism.  Am J Physiol Regul Integr Comp Physiol. 2004;  287 R1219-R1229
    CrossrefPubMedSearch in Google Scholar
  • 31 Papapetropoulos A, Garcia-Cardena G, Madri J A, Sessa W C. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.  J Clin Invest. 1997;  100 3131-3139
    CrossrefPubMedSearch in Google Scholar
  • 32 Krum J M, Mani N, Rosenstein J M. Angiogenic and astroglial response to vascular endothelial growth factor administration in adult rat brain.  Neuroscience. 2002;  110 589-604
    CrossrefPubMedSearch in Google Scholar
  • 33 Pettersson A, Nagy J A, Brown C S et al.. Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular growth factor.  Lab Invest. 2000;  80 99-115
    CrossrefPubMedSearch in Google Scholar
  • 34 Angel M F, Ramasastry S S, Swartz W M et al.. The critical relationship between free radicals and degrees of ischemia: evidence for tissue intolerance of marginal perfusion.  Plast Reconstr Surg. 1988;  81 233-239
    PubMedSearch in Google Scholar
  • 35 Kerrigan C L, Daniel R K. Critical ischemic time and the failing skin flap.  Plast Reconstr Surg. 1982;  69 986-989
    CrossrefPubMedSearch in Google Scholar
  • 36 May J W, Chait L A, O'Brien B M, Hurley J V. The no-reflow phenomenon in experimental free flaps.  Plast Reconstr Surg. 1978;  61 256-267
    CrossrefPubMedSearch in Google Scholar
  • 37 Milton S H. Experimental studies on island flaps: ischemia and delay.  Plast Reconstr Surg. 1972;  49 444-447
    PubMedSearch in Google Scholar
  • 38 Selye H. Ischemic necrosis: prevention by stress.  Science. 1967;  156 1262-1263
    CrossrefPubMedSearch in Google Scholar
  • 39 Willms-Kretschmer K, Majno G. Ischemia of the skin: electron microscopic study of vascular injury.  Am J Pathol. 1969;  54 327-353
    PubMedSearch in Google Scholar
  • 40 Strock P E, Majno G. Microvascular changes in acutely ischemic rat muscle.  Surg Gynecol Obstet. 1969;  129(6) 1213-1224
    PubMedSearch in Google Scholar
  • 41 Gordon L, Buncke H J, Townsend J J. Histological changes in skeletal muscle after temporary independent occlusion of arterial and venous supply.  Plast Reconstr Surg. 1978;  61(4) 576-579
    PubMedSearch in Google Scholar
  • 42 Erdmann D, Sweis R, Wong M S et al.. Vascular endothelial growth factor expression in pig latissimus dorsi myocutaneous flaps after ischemia reperfusion injury.  Plast Reconstr Surg. 2003;  111(2) 775-780
    CrossrefPubMedSearch in Google Scholar
  • 43 Patel P, Qi W N, Allen D M et al.. Inhibition of iNOS with 1400W improves contractile function and alters nos gene and protein expression in reperfused skeletal muscle.  Microsurgery. 2004;  24 324-331
    CrossrefPubMedSearch in Google Scholar
  • 44 Wang W Z, Fang X H, Stepheson L L, Khiabani K T, Zamboni W A. Acute microvascular action of vascular endothelial growth factor in skeletal muscle ischemia/reperfusion injury.  Plast Reconstr Surg. 2005;  115 1355-1365
    CrossrefPubMedSearch in Google Scholar
  • 45 Huang N, Khan A, Ashrafpour H et al.. Efficacy and mechanism of adenovirus-mediated VEGF-165 gene therapy for augmentation of skin flap viability.  Am J Physiol Heart Circ Physiol. 2006;  291 H127-H137
    CrossrefPubMedSearch in Google Scholar
  • 46 Ashrafpour H, Huang N, Neligan P C et al.. Vasodilator effect and mechanism of action of vascular endothelial growth factor in skin vasculature.  Am J Physiol Heart Circ Physiol. 2004;  286 H946-H955
    PubMedSearch in Google Scholar
  • 47 Kreidstein M L, Pang C Y, Carlsen L N, Xu N. Evidence for endothelium-dependent and endothelium-independent vasodilation in human skin flaps.  Can J Physiol Pharmacol. 1992;  70 1208-1216
    PubMedSearch in Google Scholar
  • 48 Nakaya Y, Mawatari K, Takahashi A, Harada N, Hata A, Yasui S. The phytoestrogen ginsensoside Re activates potassium channels of vascular smooth muscle cells through PI3K/Akt and nitric oxide pathways.  J Med Invest. 2007;  54(3–4) 381-384
    CrossrefPubMedSearch in Google Scholar
  • 49 Maxey T S, Fernandez L G, Reece T B, Keeling W B, Kron I L, Laubach V E. Endothelial nitric oxide synthase is essential for postpneumonectomy compensatory vasodilation.  Ann Thorac Surg. 2006;  81(4) 1234-1238
    CrossrefPubMedSearch in Google Scholar
  • 50 Sorenson J, Santhanam A V, Smith L A, Akiyama M, Sessa W C, Katusic Z S. Expression and function of recombinant S1179D endothelial NO synthase in human pial arteries.  Stroke. 2005;  36(1) 158-160
    CrossrefPubMedSearch in Google Scholar
  • 51 Stanley K P, Chicoine L G, Young T L et al.. Gene transfer with inducible nitric oxide synthase decreases production of urea by arginase in pulmonary arterial endothelial cells.  Am J Physiol Lung Cell Mol Physiol. 2006;  290(2) L298-L306
    PubMedSearch in Google Scholar
  • 52 Afsharimani B, Moezi L, Sadeghipour H et al.. Effect of chronic lithium administration on endothelium-dependent relaxation of rat mesenteric bed: role of nitric oxide.  Can J Physiol Pharmacol. 2007;  85(10) 1038-1046
    CrossrefPubMedSearch in Google Scholar
  • 53 Viol A W, Prewitt R L, Doviak M, Britt L D. Endotoxin releases a substance from the aorta that dilates an isolated arteriole by up-regulating INOS.  J Surg Res. 2005;  127(2) 106-111
    CrossrefPubMedSearch in Google Scholar

Feng ZhangM.D. Ph.D. 

Division of Plastic Surgery, University of Mississippi Medical Center

2500 North State Street, Jackson, MS 39216

Email: fzhang@surgery.umsmed.edu