Efficacy of Barriers and Hypoxia-Inducible Factor Inhibitors

Journal of Minimally Invasive Gynecology (2007) 14, 591–599 Efficacy of barriers and hypoxia-inducible factor inhibitors to prevent CO2 pneumoperitoneum-enhanced adhesions in a laparoscopic mouse model Maria Mercedes Binda, PhD, Carlos Roger Molinas, MD, PhD, Adriana Bastidas, MD, Marc Jansen, MD, and Philippe Robert Koninckx, MD, PhD From the Department of Obstetrics and Gynecology, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium (Drs. Binda, Bastidas, and Koninckx); the Centre for Gynaecological Endoscopy (Cendogyn), Centro Médico La Costa, Asunción, Paraguay (Dr. Molinas); and Department of Surgery, University Clinic, RWTH Aachen, Germany (Dr. Jansen). KEYWORDS: Abstract Barriers; STUDY OBJECTIVE: To investigate the effects of hypoxia-inducible factor (HIF) inhibitors, flotation Flotation; agents, barriers, and a surfactant on pneumoperitoneum-enhanced adhesions in a laparoscopic mouse HIF; model. Hypoxia; DESIGN: Prospective randomized trial (Canadian Task Force classification I). Intraperitoneal SETTING: Department of Obstetrics and Gynecology, University Hospital Gasthuisberg, Catholic adhesion formation; University of Leuven. Laparoscopy; SUBJECTS: One hundred fourteen female BALB/c mice. Pneumoperitoneum; INTERVENTIONS: Adhesions were induced during laparoscopy in BALB/c female mice. Pneumo- Prevention; peritoneum was maintained for 60 minutes with humidified CO2. In 3 experiments the effects of HIF Surfactant inhibitors such as 17-allylamino 17-demethoxygeldanamycin, radicicol, rapamycin, and wortmanin, flotation agents such as Hyskon and carboxymethylcellulose, barriers such as Hyalobarrier gel and SprayGel, and surfactant such as phospholipids were evaluated. MEASUREMENTS AND MAIN RESULTS: Adhesions were scored after 7 days during laparotomy. Adhesion formation decreased with the administration of wortmannin (p Ͻ.01), phospholipids (p Ͻ.01), Hyalobarrier Gel (p Ͻ.01), and SprayGel (p Ͻ.01). CONCLUSIONS: These experiments confirm the efficacy of barriers and phospholipids to separate or lubricate damaged surfaces. They also confirm the role of mesothelial hypoxia in this model by the efficacy of the HIF inhibitor wortmannin. © 2007 AAGL. All rights reserved. Supported by Onderzoeks Toelagen Katholieke Universiteit Leuven (Leuven, Belgium grant TBA/00/27) and in part by Karl Storz Endoscopy (Tuttlingen, Germany). Presented, in part, at the 21st annual meeting of European Society of Human Reproduction and Embryology (ESHRE), June 19th–22nd, 2005, Copenhagen, Denmark, and at the XV annual congress of the International Society for Gynecological Endoscopy (ISGE), March 29th–April 1st, 2006, Buenos Aires, Argentina. Corresponding author: Maria Mercedes Binda, PhD, Department of Obstetrics and Gynecology, University Hospital Gasthuisberg, Herestraat 49 Bus 611, B3000 Leuven, Belgium. E-mail: [email protected] or [email protected] Submitted January 8, 2007. Accepted for publication April 2, 2007. 1553-4650/$ -see front matter © 2007 AAGL. All rights reserved. doi:10.1016/j.jmig.2007.04.002 592 Journal of Minimally Invasive Gynecology, Vol 14, No 5, September/October 2007 One of the complications of abdominal surgery adhesionis intra- prevention. In this article, the effects of HIF in- abdominal adhesion formation. Adhesions can cause hibitors,intes- flotation agents, barriers, and a surfactant were tinal obstruction, chronic pain, and infertility. Althoughinvestigated. postoperative adhesion formation remains an important clinical problem, its prevention is still inadequate and over- all poorly understood. Materials and methods Over recent years, 2 COpneumoperitoneum became known as a cofactor in postoperative adhesion1 formation. Mesothelial hypoxia was suggested as a mechanism,The be-laparoscopic mouse model for adhesion cause adhesions increased with duration2 pneumo-of CO formation peritoneum and with insufflation pressure, because similar effects were observed with helium pneumoperitoneum andExperimental setup, that is, animals, anesthesia, and ven- because the addition of 2% to 4% of oxygen2 totilation, both laparoscopicCO surgery, induction, and scoring of and helium pneumoperitoneum decreased adhesion forma-intraperitoneal adhesions, has previously been described tion.1 This hypothesis was supported by the absencein detail.of 1– 4,9,10,17–19,27Briefly, in the pneumoperitoneum- pneumoperitoneum-enhanced adhesions in mice deficientenhanced adhesions model, adhesions were induced during for factors which are up-regulated during hypoxia, laparoscopysuch as by creating a mechanical Pneumoperi-lesion. plasminogen activator inhibitor 1 (PAI-1),2 vascular endo- toneum was maintained for 60 minutes with pure and 3 thelial growth factor (VEGF), placental growthand factor,humidified CO2 at 15 mm Hg insufflation pressure. Gas hypoxia-inducible factor␣ and1 ␣2 (HIF-1␣ and HIF-2␣).4 and body temperatures were kept strictly at 37°C with a HIF is ␣an/␤ heterodimeric DNA-binding complex thatheated chamber (Figure 1). directs an extensive transcriptional response to hypoxia. HIF activity is induced during hypoxia through theAnimals stabilization and activation of its subunit␣ whereas HIF-1 during normoxia subunit HIF-1␣ is rapidly degraded by the ubiq-One hundred fourteen 9- to 10-week-old female BALB/c uitin-proteosome system.5 Inhibition of HIF activity can micebe weighing 20 g were used. Animals were kept under achieved by decreasing heat shock protein 90 (Hsp-90)standard or laboratory conditions, and they were fed a standard by blocking the phosphatidylinositol 3-kinase (PI3K) laboratorypath- diet with free access to food and water at any way. The molecular chaperone Hsp-90 is important totime. main- The study was approved by the Institutional Review tain the appropriate folding and conformation and toAnimal regu- Care Committee. late the balance of synthesis and degradation of its clients such as HIF-1␣.6 Therefore Hsp-90 inhibitors, such as radi-Anesthesia and ventilation cicol, geldanamycin, and its derivated 7-allylaminogeldana- mycin (17-AAG), decrease HIF-1␣ activity.6 In addition, Mice were anesthetized with intraperitoneal 0.08 mg/g 17-AAG and radicicol bind to the intrinsic ATPasa pentobarbital,activity intubated with a 20-gauge catheter, and me- in the N-terminal site of Hsp-90, resulting in degradationchanically of ventilated (Mouse Ventilator MiniVent, Type Hsp-90 client proteins by the ubiquitin proteosome 845;path- Hugo Sachs Elektronik-Harvard Apparatus GmbH, way. Another approach to decrease HIF activity is the inhibition of the PI3K/Akt pathway with inhibitors such as wortmannin and rapamycin7 because phosphorylation is in- volved in the HIF-1␣ subunit stabilization, as well as in the regulation of HIF-1 transcriptional activity.8 Prevention of adhesion formation after laparoscopic surgery has been poorly addressed. Several agents have been tested, such as antibodies against VEGFR1,9 crystal- loids,10,11 4% icodextrin,11 ferric hyaluronate 11–13gel, Se- pracoat,14 a cross-linked hyaluronan solution,15 and hyaluronate membrane,16 which were effective in different animal models. These observations, however, were gener- ally reported with only 1 drug in different models, different species, and with different scoring systems. A comprehensive quantitative evaluation of efficacy in 1 model is still lacking. These experiments are the second part of a series of experiments to evaluate most known substances in 1 model to obtain quantitative and comprehensive informationFigure on 1 Laparoscopic mouse model. Binda et al Efficacy of barriers and hypoxia-inducible factor inhibitors to prevent adhesions 593 March-Hugstetten, Germany) by use of humidified roomAccording air to the law of error propagation⌬Z/Z (whereϭ with a tidal volume ␮ofL 250at 160 strokes/min. Humid-⌬X/X ϩ⌬Y/Y) the accuracy yields some 4% CV or 1% for ified air for ventilation was used to prevent cooling,the sum as of 4 estimates. Therefore only 1 digit becomes occurs during ventilation with non-humidified17 air. significant.20 Laparoscopic surgery Products A midline incision was performed caudal to the xiphoid HIF inhibitors process, a 2-mm endoscope with a 3.3-mm external sheath for insufflation (Karl Storz, Tuttlingen, Germany) was in- The 17-allylamino 17-demethoxygeldanamycin (17-AAG) troduced into the abdominal cavity, and the incision was was donated by Kosan Biosciences (Hayward, CA). Radicicol, closed gas tight around the endoscope to avoid leakage. wortmannin, and rapamycin were boughtA.G. from Scien- Pneumoperitoneum was created with pure2 at 15CO mm Hg insufflation pressure using the Thermoflator Plus tific,(Karl Inc., (San Diego, CA). The doses administered were Storz) and a water valve to damper pressure changes.17-AAG The 20 mg/kg, radicicol 25 mg/kg, wortmannin 0.31 gas was humidified (Storz Humidifier 204320 33;mg/kg, Karl and rapamycin 3 mg/kg. Radicicol, rapamycin, 17- Storz), and the whole setup was kept in a chamberAAG, at and37°C wortmannin were dissolved in pure dimethylsul- phoxide (DMSO) at final concentrations of 7.5 mg/mL, 0.9 to obtain 2CO at 37°C and with 100% relative humidity. We used, as described previously, a controlled flow2 mg/mL,of CO 6 mg/mL, and 5 mg/mL, respectively. Afterward, through the abdominal cavity of 23 mL/min with awortmannin 26-gauge stock was diluted to 0.093 mg/mL in saline Ϫ needle, to ascertain a continuously 100%2 environment CO solution. Stocks were kept20°C at until they were used. by removing constantly any oxygen that might haveThe dif- doses used in

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