Effects of Adrenomedullin and Glucagon-like Peptide on Distal Flap Necrosis and Vascularity: The Role of Receptor Systems and Nitric Oxide

Objective. Flap necrosis in the distal area due to the deficiency of blood circulation is a major complication in flap treatment. In many previous studies, some natural substances such as chlorogenic acid, adrenomedullin (ADM), and glucagon-like peptide-1 (GLP-1) have been used to improve flap viability via their vasodilator, angiogenic, and antioxidant effects. The aim of this study is to clarify the mechanism through the use of selective antagonists for calcitonin gene-related peptide (CGRP) receptors and GLP-1 receptors such as CGRP-(8-37), exendin-(9-39), respectively, in the flap healing effects of ADM and GLP-1.The role of nitric oxide (NO) was investigated in the mechanism as well. Materials and Methods. Seventy adult female Wistar rats (200 g–250 g) were used in the study. The cutaneous skin flap (8 cm x 3 cm) on the abdominal wall was raised based on the superficial inferior epigastric artery (SIEA). Single-dose substance injections were administered into the SIEA. Necrosis in the flap area was evaluated on postoperative day 7. The proportion of the necrosis area (necrosis area % = [necrosis area/flap area] x 100) and vascularity (vascular number/cm²) in the distal area were calculated. Results. The administrations of ADM or GLP-1 increased the vascularity and decreased the necrosis area in the distal flap region. The ADM receptor antagonist, CGRP-(8-37), did not prevent the positive effects of ADM on flap healing and vascularity. A GLP-1 receptor antagonist, exendin-(9-39), prevented the effect of GLP-1 on flap healing and vascularity. Nitric oxide mediated the beneficial effects of both peptides on flap healing. Conclusion. The CGRP receptors have no direct role, but NO acts as a mediator in the beneficial effect of ADM on flap healing. The GLP-1 specific receptors and NO act as important interagents for the effects of GLP-1 on flap healing.

Introduction

The most frequent complication of flap treatment is tissue necrosis, which generally occurs in the distal part of the flap area. Inadequate blood flow and disturbed venous drainage cause necrosis.¹ A variety of agents (eg, some growth factors, natural compounds, peptides, and vasodilatory agents) are used to prevent flap necrosis.^2-5 Increasing the microcirculation in the distal flap area is the most helpful treatment for flap healing.^3-6 Adrenomedullin (ADM) is a potent vasodilatory peptide that regulates the endothelial permeability.^7-9 In addition, ADM has angiogenic and anti-apoptotic effects on the endothelium and isolated tumor tissues.^10-14 It has been shown that ADM regulates local blood flow and increases collateral capillary development.^15-17 Glucagon-like peptide-1 (GLP-1) is a hormone secreted from the enteroendocrine L cells in the small intestine and has various biological effects such as increasing the gastrointestinal motility and secretion as well as dose-dependent vasodilatation in the femoral artery.^17-28 This effect of GLP-1 was directly blocked by the specific GLP-1 receptor antagonist exendin-(9-39). Nitric oxide (NO) and an endothelium-independent mechanism are responsible for the direct vascular effect of GLP-1.²⁹ In previous studies, the investigators demonstrated flap necrosis was prevented by the administration of some natural compounds and peptides such as chlorogenic acid, ADM, and GLP-1.^7,30 The aim of this study is to clarify the mechanisms of GLP-1 and ADM on the healing process of flap tissue.

Materials and Methods

Animal protocols. Seventy female Wistar rats (200 g–250 g) were used in this study. The rats were kept with ad lib food and water before the experiment. The ambient temperature was kept stable at 20°C–24°C and the room lighting was set at a 12-hour light/dark cycle. The study began after consent was obtained from the Uludag University Animal Experiments Local Ethical Committee.

Experimental skin flap model. The surgical procedures were performed under 2.5% to 3.5% isoflurane inhaler anesthesia. All rats were placed in the dorsal decubitus position before surgery, and the surgical area was manually depilated (Veet; Reckitt Benckiser, Parsippany, NJ). Following iodine antisepsis, the surgical area was covered with sterile drapes. A flap area of 8 cm x 3 cm in size was drawn with a pen on the abdominal wall and then divided into 4 equal areas per the distance to the pedicle (eFigure 1). The flap tissue was cutaneously removed based on the superficial inferior epigastric artery (SIEA). The sides of the flap, other than the pedicle, were again sutured in place. The femoral artery branches were dissected for intraarterial local injections. The substance injections were performed using a 30-gauge injector into the femoral artery after a vascular clamp was placed to the proximal femoral artery section and distal area after SIEA branching, leaving the SIEA as the single artery feeding the flap as described previously.⁷ The injection areas were closed and sutured. The animals were placed in separate cages with ad lib food and water.

Study plan. Animals were randomly divided into 10 subgroups with 6 to 7 rats in each. An axial pattern abdominal skin flap was created (8 cm x 3 cm) from the abdominal wall. Adrenomedullin and GLP-1 were administered at 166 pmol/500 µL, intraarterially; and 1.5 pmol/500 µL, intraarterially, respectively. These doses were selected as effective doses based on Etoz et al.⁷ Calcitonin gene-related peptide-(8-37) [CGRP-(8-37)] (200 pmol/500 µL, intraarterially) was used as a CGRP receptor antagonist to block the effects of ADM. Exendin-(9-39) (3.5 pmol/500 µL, intraarterially) was administered as a GLP-1 receptor antagonist to prevent the effects of GLP-1. The NO synthesis inhibitor L-NAME (30 pmol/500 µL; Intraarterially; Sigma-Aldrich, St Louis, MO) was injected to investigate the role of NO in the effects of ADM or GLP-1 on flap necrosis and revascularization. Saline (500 µL) was administered as a control. Adrenomedullin, GLP-1, exendin-(9–39), CGRP-(8–37), and L-NAME were dissolved in saline. Other chemicals were obtained from local commercial sources. All drugs were given as the final amount in 500 µL. Buprenorphine (0.05 mg/kg body weight) was injected subcutaneously for postoperative pain management.

Evaluation of flap survival. On postoperative day 7, the rats were anesthetized as described above. The pictures of the flap area were taken with a digital camera (Canon PowerShot A550, Tokyo, Japan) from 15 cm away. The images were evaluated in a blinded fashion. Each total flap tissue and necrosis area was measured using ImageJ Version 1.42q (National Institutes of Health, Bethesda, MD). The proportion of the necrosis area to the flap area was calculated with the following formula:

necrosis area % = (necrosis area/flap area) x 100.

Histopathological evaluation. The distal region of the flaps (1 cm below suture) was used for the histopathological evaluation. The flap tissue samples (2 cm x 1 cm) were collected and fixed in 10% buffered formalin solution until the tissues hardened. The tissue samples were embedded in paraffin wax, cut into 5-µm thick tissue sections, mounted on slides, and stained with hematoxylin and eosin (H&E). Each slide was quantitatively examined under a light microscope, and the vessels were counted on 200x magnification. Then, the numbers of capillaries were noted (eFigure 2). The vascularity was counted as the number of vessels per square centimeter. The evaluation of digital images and vessel counting were performed in a blinded manner.

Results

Role of CGRP receptors and NO on ADM for flap healing. The intraarterial application of ADM (166 pmol/500 µL single dose) decreased the necrosis formation observed in the distal flap area. The tissue section from the distal flap region also has shown more vascularization than the control group (P = .004). The CGRP receptor antagonist, CGRP-(8–37) (200 pmol/500 µL, intraarterially), did not prevent the beneficial effect of ADM on necrosis in the distal flap area. The use of CGRP-(8-37) alone did not have any significant effect on the necrosis formation and vascularity numbers. The NO synthesis inhibitor L-NAME (30 pmol/500 µL, intraarterially) significantly blocked the effect of ADM on the necrosis in the distal flap area. Administration of L-NAME alone did not have any significant effect in the necrosis development or vascularity numbers (eFigures 3A–3E, 4).

Role of GLP-1 receptors and NO on GLP-1 for flap healing. Intraarterially administered GLP-1 (1.5 pmol/500 µL single dose) decreased the widespread necrosis formation observed in the distal flap area. The vascularity in the tissue section from the distal flap region was significantly higher than the control group (P = .002). The flap tissue necrosis decreased the effects of GLP-1, and revascularization increased the effects of GLP-1, which was prevented by the GLP-1 receptor antagonist exendin-(9-39). Administration of exendin-(9-39) alone did not have any significant effect on the necrosis formation and vascularity numbers. The flap tissue decrease in necrosis and increase in revascularization in response to GLP-1 were prevented by the NO synthesis inhibitor L-NAME. Application of L-NAME alone did not have any significant effect on necrosis development and vascularity numbers (eFigures 3C–3G, 5).

Discussion

Skin flap operations are a crucial part of reconstructive surgery. Necrosis is often a complication during the flap healing process. Necrosis generally occurs in the distal flap region because of an inadequacy of the blood circulation and perfusion in this area.¹ A variety of substances (eg, vasodilatory agents or angiogenic factors) have been studied to minimize flap necrosis by enhancing blood flow. Reduced distal flap necrosis was demonstrated using the local or systemic administration of substances.^4-6 Previously, the authors showed that ADM or GLP-1 (in different doses) increased the blood flow and decreased the necrosis formation in the distal flap region.⁷ Adrenomedullin and GLP-1 are secreted in the body in many tissues, and both have very powerful vasodilator effects⁸ as well as increased tissue angiogenesis.^9-12 Adrenomedullin also increases vascular endothelial growth factor (VEGF) expression.³¹ The angiogenetic activity of ADM occurs through Akt, MAPK, and CRLR/RAMP2-CRLR/RAMP3 receptors and focal adhesion kinases in endothelial cells.^12,13 Adrenomedullin binds to plasma membrane receptors, which are similar to CGRP receptors.^8,9 A competitive antagonist for CGRP receptors is CGRP-(8-37).³² The preventive effect of ADM in flap necrosis was not decreased by the CGRP-(8-37) in this study. This finding indicates that ADM uses pathways other than the CGRP/ADM receptor complex to exert its healing effect on necrosis. Although ADM uses the cyclic adenosine 3’,5’-monophosphate pathway as the main secondary messenger in the target cell, the presence of other signal transmission pathways have also been detected, such as NO/cyclic guanosine monophosphate (cGMP) activation, inositol triphosphate activation, and adenosine and three phosphate-sensitive potassium channel activation.^13-17 The effect of ADM on the NO/cGMP pathway is responsible for the vasodilator effect as well as decreased oxidative stress, antiproliferative, antimitotic, and anti-apoptotic activity.^33,34

Another peptide (GLP-1) used in this study has vasorelaxant and angiogenetic effects.^18-20 Previously, the protective effects of both GLP-1 and exendin-4 were demonstrated against ischemia-reperfusion injury in a rat heart.^25,26 A dose-dependent, direct vasorelaxant effect of GLP-1 has also been shown on conduit vessels in a rat organ bath model, which is prevented by the specific GLP-1 receptor antagonist exendin-(9–39) and is independent of NO.²⁷ Golpon et al²⁸ showed GLP-1 causes the dose-, time-, and endothelium-dependent relaxation of pulmonary artery rings without affecting the tone of the aorta. Buyukcoskun et al³⁵ showed GLP-1 increases the gastric mucosal blood circulation. This effect of GLP-1 is dose-dependent and is blocked by the GLP-1 receptor antagonist exendin-(9-39). Previously, the authors have shown GLP-1 injection into the SIEA decreases necrosis and increases blood flow in the distal flap area.⁷ The effect of GLP-1 on flap necrosis was prevented by exendin-(9-39) in the present study as well. This result shows that the effect of GLP-1 on flap necrosis and revascularization was mediated by its specific receptors. Exogenous NO has been shown to have beneficial effects on wound healing. Nitric oxide therapy improves wound repair, but the exact mechanisms of NO activity on wound healing parameters are still unknown. The salutary effect of GLP-1 and ADM on flap necrosis was blocked by L-NAME, indicating NO plays an important role in the flap healing process.

Limitations

There are some limitations in this study. The authors have shown the increasing vascularity in the distal flap area with capillary counting following H&E staining. Additional histological staining methods, such as immunostained methods, should be added to evaluate angiogenesis in the flap tissue. Second, the level of blood flow to the distal flap area should be measured via a laser Doppler flowmeter. Third, VEFG, matrix metallopeptidase 9, nitric oxide synthase isoforms, and other growth factors should be evaluated in the healing tissue by molecular analysis techniques. The authors have planned further studies to improve these findings.

Conclusion

The results of this study have shown the therapeutic applicability of ADM and GLP-1 in reconstructive surgery as new approaches. These endogenously secreted peptides in the body might be more useful compared with synthetic agents for the management of flap treatment as they have fewer or no side effects. The current study confirms the authors’ previous findings that ADM or GLP-1 have salutary effects on flap healing. In the present study, they found specific receptors of peptides play important roles in the flap healing process. Nitric oxide acts as an important mediator in these effects. Further studies are necessary to clarify their mechanisms as well as possible alterations made by these agents on intercellular and extracellular signaling pathways, leading to improvement of wound healing. Therefore, new alternative treatments might be used to manage the possible complications of wounds.

Acknowledgments

From the Department of Physiology, Faculty of Medicine, Uludag University, Bursa, Turkey; Faculty of Medicine, Experimental Animals Breeding and Research Center, Uludag University; Department of Pathology, Veterinary Faculty, Uludag University; and Department of Biophysics, Faculty of Medicine, Uludag University

Address correspondence to:
Betul Cam, MD
Faculty of Medicine
Department of Physiology
Uludag University
Özlüce Mahallesi
16059 Nilüfer/Bursa
Turkey
mbetulcam@gmail.com

Disclosure: The authors disclose no financial or other conflicts of interest.

References

1. Myers B. Understanding flap necrosis. Plast Reconstr Surg. 1986;78(6):813–814. 2. Komorowska-Timek E, Timek TA, Brevetti LS, Zhang F, Lineaweaver WC, Buncke HJ. The effect of single administration of vascular endothelial growth factor or L-arginine on necrosis and vasculature of the epigastric flap in the rat model. Br J Plast Surg. 2004;57(4):317–325. 3. Khan A, Ashrafpour H, Huang N, et al. Acute local subcutaneous VEGF165 injection for augmentation of skin ﬂap viability: efﬁcacy and mechanism [published online ahead of print June 24, 2004]. Am J Physiol Regul Integr Comp Physiol. 2004;287(5):R1219–R1229. 4. Shafighi M, Olariu R, Brun C, et al. The role of androgens on hypoxia-inducible factor (HIF)-1α-induced angiogenesis and on the survival of ischemically challenged skin flaps in a rat model [published online ahead of print June 18, 2012]. Microsurgery. 2012;32(6):475–481. 5. Fromes Y, Liu JM, Kovacevic M, Bignon J, Wdzieczak-Bak-ala J. The tetrapeptide acetyl-serine-aspartyl-lysine-proline improves skin flap survival and accelerates wound healing. Wound Repair Regen. 2006;14(3):306–312. 6. Merz K, Schweizer R, Schlosser S, Giovanoli P, Emi D, Plock JA. Distinct microhemodynamic efficacy of arteriogenesis and angiogenesis in critically ischemic skin flaps [published online ahead of print November 4, 2011]. Microvasc Res. 2012;83(2):249–256. 7. Etoz BC, Buyukcoskun NI, Etoz A, Ozluk K. Preventing flap necrosis with adrenomedullin and glucagon-like peptide-1. Wounds. 2012;24(2):29–35. 8. Hinson JP, Kapas S, Smith DM. Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev. 2000;21(2): 138–167. 9. Brain SD, Grant AD. Vascular actions of calcitonin gene-related peptide and adrenomedullin. Physiol Rev. 2004;84(3):903–934. 10. Oehler MK, Hague S, Rees MC, Bicknell R. Adrenomedullin promotes formation of xenografted endometrial tumors by stimulation of autocrine growth and angiogenesis. Oncogene. 2002;21(18):2815–2821. 11. Iimuro S, Shindo T, Moriyama N, et al. Angiogenic effects of adrenomedullin in ischemia and tumor growth [published online ahead of print July 8, 2004]. Circ Res. 2004;95(4):415–423. 12. Fernandez-Sauze S, Delfino C, Mabrouk K, et al. Effects of adrenomedullin on endothelial cells in the multistep process of angiogenesis: involvement of CRLR/RAMP2 and CRLR/ RAMP3 receptors. Int J Cancer. 2004;108(6):797–804. 13. Kim W, Moon SO, Sung MJ, et al. Angiogenic role of adrenomedullin through activation of Akt, mitogen activated protein kinase, and focal adhesion kinase in endothelial cells [published online ahead of print August 1, 2003]. FASEB J. 2003;17(13):1937–1939. 14. Miyashita K, Itoh H, Sawada N, et al. Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo. FEBS Lett. 2003;544(1-3):86-92. 15. Hasbak P, Eskesen K, Lind H, Holst J, Edvinsson L. The vasorelaxant effect of adrenomedullin, proadrenomedullin N-terminal 20 peptide and amylin in human skin. Basic Clin Pharmacol Toxicol. 2006;99(2):162–167. 16. Shimekake Y, Nagata K, Ohta S, et al. Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J Biol Chem. 1995;270(9):4412–4417. 17. Matsunaga K, Iwasaki T, Yonetani Y, et al. Nitric oxide-dependent hypotensive effects of adrenomedullin in rats. Drug Dev Res. 1996;37(1):55–60. 18. Drucker DJ. Biological actions and therapeutic potential of the glucagon-like peptides. Gastroenterology. 2002;122(2):531–544. 19. Fehmann HC, Habener JF. Insulinotropic glucagon-like peptide-1 (7-37)/(7-36) amide: a new incretin hormone. Trends Endocrinol Metab. 1992;3(5):158–163. 20. Brubaker PL, Drucker DJ. Minireview: Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system [published online ahead of print March 24, 2004]. Endocrinology. 2004;145(6):2653–2659. 21. Perfetti R, Hui H. The role of GLP-1 in the life and death of pancreatic beta cells. Horm Metab Res. 2004;36(11-12):804–810. 22. Cornu M, Thorens B. GLP-1 protects β-cells against apoptosis by enhancing the activity of an IGF-2/IGF1-receptor autocrine loop. Islets. 2009;1(3):280–282. 23. Jiao S, Liu Z, Ren WH, et al. cAMP/protein kinase A signalling pathway protects against neuronal apoptosis and is associ¬ated with modulation of Kv2.1 in cerebellar granule cells [published online ahead of print November 29, 2006]. J Neurochem. 2007;100(4):979–991. 24. Wang X, Tang X, Li M, Marshall J, Mao Z. Regulation of neuroprotective activity of myocyte-enhancer factor 2 by cAMP-protein kinase A signaling pathway in neuronal survival [published online ahead of print February 25, 2005]. J Biol Chem. 2005;280(17):16705–16713. 25. Nikolaidis LA, Mankad S, Sokos GG, et al. Effects of glucagon-like peptide-1 in patient with acute myocardial infarction and left ventricular dysfunction after successful reperfusion [published online ahead of print February 23, 2004]. Circulation. 2004;109(8):962–965. 26. Sonne DP, Engstrøm T, Treiman M. Protective effects of GLP-1 analogues exendin-4 and GLP-1(9–39) amide against ischemia-reperfusion injury in rat heart [published online ahead of print October 13, 2007]. Regul Pept. 2008;146(1-3):243–249. 27. Nyström T, Gonon AT, Sjöholm A, Pernow J. Glucagon-like peptide-1 relaxes rat conduit arteries via an endothelium-independent mechanism. Regul Pept. 2005;125(1-3):173–177. 28. Golpon HA, Puechner A, Welte T, Wichert PV, Feddersen CO. Vasorelaxant effect of glucagon-like peptide-(7-36) amide and amylin on the pulmonary circulation of the rat. Regul Pept. 2001;102(2-3):81–86. 29. Donnelly D. The structure and function of the glucagon-like peptide-1 receptor and its ligands. Br J Pharmacol. 2012;166(1):27–41. 30. Bagdas D, Etoz BC, Gul Z, et al. Chlorogenic acid enhances abdominal skin flap survival based on epigastric artery in nondiabetic and diabetic rats. Ann Plast Surg. 2016;77(2):e21–e25. 31. Maki T, Ihara M, Fujita Y, et al. Angiogenic roles of adrenomedullin through vascular endothelial growth factor induction. Neuroreport. 2011;22(9):442–447. 32. Eto T. A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides. Peptides. 2001;22(11):1693–1711. 33. Michibata H, Mukoyama M, Tanaka I, et al. Autocrine/paracrine role of adrenomedullin in cultured endothelial and mesangial cells. Kidney Int. 1998;53(4):979–985. 34. Parameswaran N, Nambi P, Brooks DP, Spielman WS. Regulation of glomerular mesangial cell proliferation in culture by adrenomedullin. Eur J Pharmacol. 1999;372(1):85–95. 35. Buyukcoskun NI, Etoz BC, Gulec G, Ozluk K. Effect of peripherally-injected glucagon-like peptide-1 on gastric mucosal blood flow. Regul Pept. 2009;157(1-3):72–75.