Acta Chirurgica Belgica ISSN: 0001-5458 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tacb20 Upregulation of VEGFR1 in a rat model of esophagogastric anastomotic healing Laura Christina Landes, Daniel Drescher, Evangelos Tagkalos, Peter P. Grimminger, René Thieme, Boris Jansen-Winkeln, Hauke Lang & Ines Gockel To cite this article: Laura Christina Landes, Daniel Drescher, Evangelos Tagkalos, Peter P. Grimminger, René Thieme, Boris Jansen-Winkeln, Hauke Lang & Ines Gockel (2017): Upregulation of VEGFR1 in a rat model of esophagogastric anastomotic healing, Acta Chirurgica Belgica, DOI: 10.1080/00015458.2017.1394673 To link to this article: http://dx.doi.org/10.1080/00015458.2017.1394673 Published online: 25 Oct 2017. Submit your article to this journal Article views: 2 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tacb20 Download by: [University of Florida] Date: 28 October 2017, At: 20:45 ACTA CHIRURGICA BELGICA, 2017 https://doi.org/10.1080/00015458.2017.1394673 ORIGINAL PAPER Upregulation of VEGFR1 in a rat model of esophagogastric anastomotic healing Laura Christina Landesa, Daniel Dreschera,b, Evangelos Tagkalosa, Peter P. Grimmingera Rene Thiemec , Boris Jansen-Winkelna,c , Hauke Langa and Ines Gockela,c , Downloaded by [University of Florida] at 20:45 28 October 2017 a Department of General, Visceral and Transplantation Surgery, University Medical Center of Mainz, Mainz, Germany; bDepartment of General and Visceral Surgery, St. Juliusspital, W€urzburg, Germany; cDepartment of Visceral, Transplant, Thoracic and Vascular Surgery, University Medical Center of Leipzig, Leipzig, Germany ABSTRACT ARTICLE HISTORY Introduction: Anastomotic leakage after gastrointestinal surgery is a significant cause of morbidity and mortality. Esophagogastric and colorectal anastomoses are vulnerable to leakage. Extended knowledge of growth factors and their receptors is needed to understand anatomic healing. Methods: The expression pattern of vascular growth factor receptor (VEGFR1-3), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFRa/b) and keratinocyte growth factor receptor (KGFR) were analyzed by semiquantitative-PCR in the rat intestinal tract and in esophagogastric anastomosis 5d after surgery. Results: VEGFR1, VEGFR2, EGFR, KGFR and PDGFRa expression was observed throughout the intestinal tract including esophagus, stomach, small bowl and colon. VEGFR3 was not found in gastric samples and PDGFRb expression was not detected in the small bowl. Semiquantitative analyses of the VEGFR1, PDGFRa and EGFR expression in esophagogastric anastomotic tissues revealed a 2-fold upregulation of the VEGFR1 in gastric samples, while no change was observed in the esophageal anastomotic side. Conclusion: Our results revealed a distinct expression pattern of the investigated growth factor receptors in rat intestinal tract. Showing higher expression levels of growth factor receptors at the gastric anastomotic tissue at the fifth postoperative day suggests a different contribution of the gastric and the esophageal side to the anastomotic healing. Received 20 September 2017 Accepted 9 October 2017 Introduction Anastomotic leakage after gastrointestinal surgery is a significant cause of morbidity and mortality [1–3]. Compared to small bowel or colon anastomoses, leakage of colorectal and esophagogastric anastomoses are more frequent and have been reported in up to 30% [1–6]. Anastomotic complications increase the risk of reoperation, prolonged interventional treatment, subsequent anastomotic stenosis formation and fistula [7–9]. The major factors influencing the healing of intestinal anastomoses are neoangiogenesis and collagen deposition [8,10]. Growth factors, in particular VEGFs, play a crucial role in stimulating angiogenesis, which involves multiple processes including vasodilatation, endothelial cell migration and proliferation, as well as basement membrane degradation . Studies in well-established experimental rodent models had shown a positive impact of administrated growth factors on anastomotic healing CONTACT Ines Gockel email@example.com Vascular Surgery, Liebigstraße 20, D-04103 Leipzig, Germany These authors supervised this manuscript jointly. ß 2017 The Royal Belgian Society for Surgery KEYWORDS Gastrointestinal anastomosis; growth factor receptors; anastomotic leakage; wound healing; experimental surgery; stomach [12–15]. To our knowledge, no methodic investigations on the expression profile of a brought range of growth factor receptors in the gastrointestinal tract of the rat is available. Our aim was to investigate the expression pattern of growth factor receptors and its postoperative alteration in an established rat model for esophagogastric anastomosis . Material and methods Tissue source and storage Tissue samples of 12 Brown-Norway rats (mean weight: 320 g; Charles River, Sulzfeld, Germany) were obtained from preliminary studies on esophagogastric anastomosis in rats and used for methodic investigations on the expression profile of growth factor receptors in esophageal, gastric, small bowel and colonic samples prior to surgery . Specimen consisted of a 2 cm segment of University Medical Center Leipzig, Department of Visceral, Transplant, Thoracic and Downloaded by [University of Florida] at 20:45 28 October 2017 2 L. C. LANDES ET AL. esophageal, gastric, small bowel or colonic tissues have been used. Samples were shock-frozen in liquid nitrogen and then stored at 80 C in cryovials for consecutive RNA-isolation as well as for RT- and PCR-analysis. Further, tissue samples of 18 animals, undergoing standardized operative procedures for esophagogastric anastomosis as reported before , were gained for the analyses of the postoperative expression pattern of growth factor receptors. An upper median laparotomy was done and the peritoneal cavity was opened. Gastrolysis was carried out by dissection of the stomach greater curvature. Short gastric vessels and the left gastric artery and vein were clipped, whereas the right gastric and gastroepiploic arteries and veins were preserved. Gastric tube formation was performed after resection of the proximal lesser curvature, forestomach and the esophagogastric junction. End-to-end intra-abdominal esophagogastric anastomosis was performed and the abdomen was closed. At the fifth postoperative day, a relaparotomy was performed and the intraabdominal aspects related to the preoperative surgical procedure were assessed, including the esophagogastric anastomosis and the gastric tube. Specimen consisted of a 5 mm peri-anastomotic segment of esophageal and gastric tissue. Samples were shock-frozen in liquid nitrogen and then stored at 80 C in cryovials for consecutive semiquantitative PCR analysis. The postoperative growth factor receptor expression levels were compared with the previous taken tissue samples (preoperative). Thereby, we took attention to sample the esophageal and gastric tissue from the same anatomical location in both trials. All animal studies were carried out in strict accordance with the recommendations for the care and use of animals and were approved by the local ethics committee of the ‘Landesuntersuchungsamt Rheinland-Pfalz’, Koblenz, Germany (reference number: G 10-1-002). Qualitative RT and PCR analysis Total RNA was isolated from specimen using a single-step extraction method (RNeasy Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA integrity was assessed by electrophoresis on 1.8% agarose gels. RNA concentrations were determined by spectrophotometry at A260 and purity was assessed by measuring the RNA to DNA ratio at A260/280. The oligonucleotide sequences used for amplification of growth factor receptors are shown in Table 1. Aliquots of 1.0 mg total RNA were reverse transcribed for 1 h at 37 C, using oligo-dt-nucleotides and the Omniscript RT kit (Qiagen, Hilden, Germany). For the amplification of target genes, 1 ml cDNA was used for amplification by the Taq PCR Core Kit (Qiagen, Hilden, Germany). PCRs were subjected to defined cycles of denaturation, annealing and extension (Table 2). PCR products were separated by gel electrophoresis, visualized under UV light (Gel Doc EZ Imager, Bio-Rad, Munich, Germany) and quantified by densitometry using Image Lab 3.0.1 (Bio-Rad, Munich, Germany). Semiquantitative PCR analysis For semiquantitative analysis, relative expression of VEGFR1, EGFR and PDGFRa were normalized by referring to the density of the respective beta-actin band. Results are shown as target gene/beta-actin ratios. Hematoxylin-eosin staining Esophageal and gastric specimens were fixed using 4% paraformaldehyde at day 5. After dehydration and embedding in paraffin, 5 mm slides were trimmed and a conventional hematoxylin–eosin staining was performed. Statistical analysis VEGFR1, EGFR and PDGFRa/beta-actin densitometric ratio values in preoperative and 5d postoperative tissue samples from esophagic and gastric Table 1. Oligonucleotide sequences used for the amplification of growth factor receptors. Sequence 50 ! 30 Primer sz-actin 400 VEGFR1 VEGFR2 VEGFR3 PDGFRa PDGFRb EGFR KGFR Sense Antisense atcgtgggccgcctaggcac caagggactctacacttgtc actcagacgacacagatacc ctgaggcagaatatcagtctggag atccattgtctctgtccccc tcaacagcctctacaccacc gtactgcactgccatcagtg tccatcaaccacacctacc ctcatagatgggcacagtgtg ccgaatagcgagcagatttc acatctcacccatcccaac agatgctcatacgtgtagttgtcc gctctcacacatttaccacacc actcctcagaaactcctcatcc ccgaggagcataaaggatta cgaaactgttacctgtctcc Table 2. Number of cycles for semiquantitative PCR analysis for VEGFR1, EGFR and PDGFRa in esophageal and gastric samples. Number of cycles Primer VEGFR-1 PDGFR-a EGFR Temperature ( C) Esophagus Stomach 55,5 60,5 56 31 31 33 35 35 38 ACTA CHIRURGICA BELGICA ansastomotic tissues was performed by an analysis of variance (two-way ANOVA corrected for multiple comparisons by Bonferroni) to assess whether is an statistical significant difference between the growth factor receptor expression levels in esophageal an gastric tissue samples at both investigated time points (preoperative and 5-d postoperative). All analyses were done using GraphPad Prism 6.0 (GraphPad Software Inc. San Diego, CA). All data are expressed as mean ± SEM. Results Downloaded by [University of Florida] at 20:45 28 October 2017 Qualitative mRNA expression of growth factors receptors VEGFR1-3, EGFR, PDGFRa/b and KGFR expression in rat esophageal, gastric, small bowel or colonic tissue samples revealed varying occurrence (Table 3). VEGFR1, VEGFR2, EGFR, PDGFRa and KGFR expression was observed in esophageal, gastric, small bowel and colonic samples. VEGFR3 only showed an expression in esophageal, small bowel and colonic samples, not in gastric samples. PDGFRb expression was seen in esophageal and gastric samples. 3 trend toward upregulation of all three growth factor receptors in the gastric samples at the 5th postoperative day (Figure 1). The mRNA levels of the VEGFR1 in gastric anastomotic tissue was significantly higher compared to its preoperative gastric tissue (gastric tissue: 0.19 ± 0.043 vs. 0.39 ± 0.061 and esophageal tissue: 0.16 ± 0.013 vs. 0.13 ± 0.039) (p ¼ .014) and to postoperative esophageal anastomotic tissue (p ¼ .031) (Figure 1(a)). However, not significant the expression of PDGFRa and EGFR tend to be increased in gastric anastomotic tissue (Figure 1(b,c)). HE-staining of esophageal and gastric anastomotic sides To determine tissue integrity the esophageal (Figure 2(a–c)) and gastric part of the anastomotic sides were stained with hematoxylin–eosin. No disturbances in tissue morphology could be observed, suggesting functional anastomoses and no signs of apoptosis or neuroses could be determined (Figure 2(b–d)). Discussion Semiquantitative mRNA expression of VEGFR1, EGFR and PDGFRa in rat model of esophagogastric anastomotic tissue Semiquantitative analyses of the expression profile of VEGFR1, EGFR and PDGFRa showed a distinct Table 3. Qualitative expression of growth factor receptors in gastrointestinal tract. Growth Factor Receptor VEGFR-1 VEGFR-2 VEGFR-3 PDGFR-a PDGFR-b EGFR KGFR Esophagus Stomach Small bowel Colon þ þ þ þ þ þ þ þ þ n.d. þ þ þ þ þ þ þ þ n.d. þ þ þ þ þ þ n.d. þ þ þ ¼ expression; n.d.¼ no expression detected. This is the first study analyzing the expression profile of a series of growth factor receptors in the intestinal tract of the rat (Table 2). Vascular endothelial growth factor receptors (VEGFR1-3) Vascular endothelial growth factors (VEGF-A, VEGF-B, VEGF-C, VEGF -D and VEGF-E) are secreted by many cell types that are involved in anastomotic healing, for example, macrophages, endothelial cells, fibroblasts or smooth muscle cells [10,11,17]. Their high-affinity receptors VEGFR1 (Flt-1) and VEGFR2 (KDR) are members of the receptor tyrosine Figure 1. VEGFR1, PDGFa and EGFR mRNA expression in the gastroesophageal anastomoses. An esophagogastric anastomosis had been performed in Brown-Norway rats and esophageal (eso) and gastric anastomotic samples had been recovered at the 5th postoperative day. The expression pattern of VEGFR1 (a), PDGFRa (b) and EGFR (c) was analyzed by semiquantitative RTPCR. The expression of VEGFR1 was significant increased in postoperative gastric anastomotic tissue compared to post-operative esophageal anastomotic tissue (a, p ¼ .014) and to preoperative gastric tissue (b, p ¼ .031). All data are shown as mean ± SEM. (neso ¼ 4; ngastric ¼ 7; two-way ANOVA corrected for multiple comparisons by Bonferroni). Downloaded by [University of Florida] at 20:45 28 October 2017 4 L. C. LANDES ET AL. kinase (RTK) family, containing extracellular ligandbinding and intracellular catalytic domains [11,17]. Receptor binding of VEGF results in RTK-autophosphorylation and a consecutive Mek1/2 and Erk1/2 activation via Raf or Ras [11,17]. VEGF induces chemotaxis, angiogenesis and the expression of matrix metalloproteinases [10,11,17]. VEGFR3 mediates primarily lymphangiogenesis . In this study, we reveal VEGFR1-3 expression in rat’s esophagus and VEGFR2 in the stomach of the rat. VEGFR1 and 2 expressions were detected in colonic samples confirming previously reported data [18,19]. Furthermore, we detected an expression of VEGFR1 and 2 in the rat’s small bowel. Investigations in animal anastomotic healing models using localized VEGF gene therapy or local administrated VEGF showed increased angiogenesis with subsequently improved anastomotic healing [10,14,20]. These findings encourage the application of VEGF in rats to enhance anastomotic healing promoting angiogenesis and the expression of matrix metalloproteinases. Platelet-derived growth factor receptors (PDGFRa/b) Platelet-derived growth factor homo or heterodimeric isoforms (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD) are major paracrine and autocrine growth factors for certain cells including smooth muscle cells, fibroblasts and endothelial cells [17,21]. External stimuli, such as low oxygen tension, increase synthesis. Classical target cells express both a- and b-receptors. Activation of PDGF receptors by ligand-binding leads to stimulation of cell growth, chemoattraction and to changes in cell shape and synthesis of matrix constituents [17,21]. Semiquantitative PCR analyses confirmed temporal expression of PDGF-BB in a colonic wound-healing model in rats from the 3rd to the 7th, reaching a basal levels 2 weeks postoperative . We detected expression of PDGFRa in the stomach, small bowel and colon as described before [22–24]. PDGFRb was found in esophageal and gastric samples. As to the best of our knowledge, no data were available describing PDGFRa/b expression in the rat esophagus before. Epidermal growth factor receptor (EGFR) EGFR, a tyrosine kinase transmembrane protein, is particularly expressed on epithelial and mesenchymal cells [17,25]. Intrinsic kinase activation is mediated by related ligands, including EGF, transforming growth factor-a (TGFa) and heparinbinding growth factor (HB-EGF) . The integrated biological responses to EGFR signaling include mitogenesis, cell motility, maintenance and repair . We could confirm EGFR expression in Figure 2. Histology of the esophageal and gastric side of gastroesophageal anastomoses in rats. Hematoxylin–eosin staining has been performed at esophageal (a and c) and gastric (b and d) specimens at the 5th postoperative day. Two representative animals are shown (animal 1 (a and b) animal 2 (c and d). Both show a normal tissue pattern without any signs of apoptosis or necrosis as well in the esophageal as in the gastric part of the gastroesophageal anastomoses. (Bars – 100 mm). ACTA CHIRURGICA BELGICA esophageal, gastric and colonic samples in accordance to prior reports [15,26,27]. But we also detected EGFR expression in small bowel tissue of the rat. 5 future trials and to investigate pharmaceuticals to improve esophagogastric anastomotic healing to overcome the frequent anastomotic leakage in esophagogastric surgery to reduce morbidity and mortality [3,4,6]. Downloaded by [University of Florida] at 20:45 28 October 2017 Keratinocyte growth factor receptor (KGFR) KGFR (FGFR2 IIIb), a splicing transcript variant of fibroblast growth factor receptor 2 (FGFR2), is expressed by many types of epithelial cells and activated by its respective ligands (FGF-1, FGF-3, FGF-7 (KGF) and FGF-10 that are predominantly synthesized by mesenchymal cells [13,17,28]. Activation leads to receptor endocytosis and intracellular accumulation of receptor–ligand complexes . KGFR-mediated processes include reparative mechanisms, such as mucosal repair improved wound closure and increased matrix deposition [13,26]. We detected KGFR expression in rat small intestine. Previously, KGFR expression was described in the esophagus and stomach  and in the colon, analyzing the influence of keratinocyte growth factor on the healing of left-sided colon anastomoses.  Semiquantitative expression of VEGFR1, EGFR and PDGFRa mRNA in rat model of esophagogastric anastomotic healing Previously reported data showed a positive impact of VEGF, PDGF and EGF expression on anastomotic healing in animal models, promoting angiogenesis, cell repair mechanisms and synthesis of matrix constituents [14,15,23]. The usage of localized over-expression models for anastomotic healing, resulting in improved angiogenesis, tissue blood flow and anastomotic strength, will prevent systemic admiration of recombinant VEGF, but ethical and safety issues need to be resolved before translation in humans . In our study, the corresponding receptors VEGFR1, EGFR and PDGFRa, respectively, revealed distinct expression in esophageal and gastric samples. Our findings had shown an upregulation of VEGFR1 in gastric anastomotic tissues confirming experimental investigations that revealed increased VEGFR1. However, there was no change in receptor expression at all in esophagic anastomotic tissue in pre- and postoperative samples. However, as we analyzed growth factor expression in an animal model there are certain limitations for the translation in humans. Nevertheless, the animal model presented here can be used for Conclusions For the first time, we are able to present an expression profile of a series of growth factor receptors in the rat intestine. Our results reveal an expression of VEGFR1, VEGFR2, VEGFR3, PDGFRa/sz and KGFR in the rat intestine. These results describe an experimental model for gastrointestinal anastomotic healing in rats. In particular, expression of VEGFR1 in esophageal and gastric tissue and its post-operative upregulation in gastric anastomotic tissue suggest a positive impact and a superior role of the gastric tissue in anastomotic wound healing. Disclosure statement There are no conflicts of interest or research funding concerning this study. All authors disclose any sponsorship or financial arrangement relating to their research. ORCID Peter P. Grimminger http://orcid.org/0000-0002-18599213 Ren e Thieme http://orcid.org/0000-0002-0537-3979 Boris Jansen-Winkeln http://orcid.org/0000-0002-39969391 Hauke Lang http://orcid.org/0000-0001-9303-2148 Ines Gockel http://orcid.org/0000-0001-7423-713X References     Bruce J, Krukowski ZH, Al-Khairy G, et al. Systematic review of the definition and measurement of anastomotic leak after gastrointestinal surgery. Br J Surg. 2001;88:1157–1168. van't Sant HP, Weidema WF, Hop WCJ, et al. Evaluation of morbidity and mortality after anastomotic leakage following elective colorectal surgery in patients treated with or without mechanical bowel preparation. Am J Surg. 2011;202:321–324. Walther B, Johansson J, Johnsson F, von HCS, et al. Cervical or thoracic anastomosis after esophageal resection and gastric tube reconstruction: a prospective randomized trial comparing sutured neck anastomosis with stapled intrathoracic anastomosis. Ann Surg. 2003;238:803–812. discussion 812–814. 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