Etanercept restores vasocontractile sensitivity
affected by mesenteric ischemia reperfusion
S. Erpulat Ozis, MD,
aTamila Akhayeva, PhD,
bSahika Guner, PhD,
cSibel S. Kilicoglu, MD,
dand Arzu Pampal, MD
e,*
a
Department of General Surgery, Faculty of Medicine, TOBB-ETU University, Ankara, Turkey bDepartment of Pharmacology, Astana Medical University, Astana, Kazakhstan
cDepartment of Medical Pharmacology, Faculty of Medicine, Ufuk University, Ankara, Turkey dDepartment of Histology and Embryology, Faculty of Medicine, Ufuk University, Ankara, Turkey eDepartment of Pediatric Surgery, Faculty of Medicine, Ufuk University, Ankara, Turkey
a r t i c l e i n f o
Article history:
Received 22 August 2017 Received in revised form 23 November 2017 Accepted 3 January 2018 Available online 9 February 2018 Keywords:
Etanercept
Tumor necrosis factor-a receptor Mesenteric ischemic reperfusion
injury Transactivation
a b s t r a c t
Background: The aim of the study is to evaluate in vivo and in vitro effects of etanercept, a soluble tumor necrosis factor receptor, on the contractile responses of superior mesenteric artery in an experimental mesenteric ischemia and reperfusion model.
Material and methods: After obtaining animal ethics committee approval, 24 Sprague eDawley rats were allocated to three groups. Control group (Gr C, n ¼ 6) underwent a sham operation, whereas ischemia/reperfusion and treatment groups underwent 90 min ischemia and 24-h reperfusion (Gr I/R, n¼ 12; Gr I/RþE, n ¼ 6). The treatment group received 5 mg/kg etanercept intravenously at the beginning of reperfusion. At the end of reperfu-sion, all animals were sacrificed, and third branch of superior mesenteric artery was dissected for evaluation of contractile responses. In vitro effects of etanercept on vaso-contractile responses were also evaluated. The excised ileums were analyzed under light microscope. Two-way analysis of variance following Bonferroni post hoc test was used for evaluation of contractile responses.
Results: Endothelin-1 and phenylephrine-mediated vasocontractile sensitivity were found increased in Gr I/R when compared with Gr C. Both intravenous administration and organ bath incubation of etanercept decreased the sensitivity of contractile agents for Gr I/R. Mucosal injury, lamina propria disintegration, and denuded villous tips were observed in Gr I/R, whereas the epithelial injury and the subepithelial edema were found to be milder in Gr I/RþE.
Conclusions: Etanercept can be a promising agent in mesenteric ischemic reperfusion injury as it does not only inhibit inflammation by blocking tumor necrosis factor-a in circulation but also restores vascular contractility during reflow. These findings support an unex-plained recuperative effect of drug beyond its anti-inflammatory effects.
ª 2018 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Pediatric Surgery, Faculty of Medicine, Ufuk University, Dr Ridvan Ege Hastanesi Cocuk Cerrahisi, AD Konya Yolu No:86-88 06500 Balgat, Ankara, Turkey. Tel.:þ90 5325792778; fax: þ90 3122872390.
E-mail address:ademirtola@yahoo.com(A. Pampal).
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Introduction
Ischemic reperfusion injury (IRI) is a complex process involving a number of cytokines, chemokines, complement factors, lipid mediators, and oxygen radicals. The ischemic phase of the injury is related to the parenchymal damage due to intracellular acidosis resultant of hypoxia and adenosine triphosphate depletion. The postischemic reperfusion para-doxically causes additional injury due to the inflammatory response at tissue level. Also, postischemic reperfusion is generally related to capillary perfusion failure due to endo-thelial cell swelling, intravascular hemoconcentration, capil-lary narrowing as a result of increased interstitial pressure due to edema, and especially the sensitivity to vasoconstric-tion.1Mesenteric IRI is a life-threatening problem that occurs
in a variety of clinical situations like mesenteric arterial em-bolism, mesenteric arterial or venous thrombosis, intestinal strangulation, and intestinal transplantation. Even though the resultant of local and systemic effects of the mesenteric IRI is related to numerous mediators, tumor necrosis factor (TNF)-a has shown to have a pivotal role in this setting. TNF-a triggers the production of cytokines in which return amplifies and propagates its biological effects. Overproduction of TNF-a is related to endothelial dysfunction, inflammatory genes in-duction, immune cells recruitment and activation, apoptosis, and cellular survival.2
Etanercept is a recombinant soluble TNF receptor that binds TNF-a and inhibits TNF-a-endogenous TNF receptors interac-tion. It is mostly known for its anti-inflammatory effects and is generally used to block the inflammatory and autoimmune effects of TNF-a in clinical practice. It has the Food Drug and Administration approval in the United States to treat rheu-matoid arthritis, juvenile rheurheu-matoid arthritis, psoriatic arthritis, plaque psoriasis, and ankylosing spondylitis.
Previous studies have demonstrated that IRI increases a1-adrenoceptor and endothelin-1 (ET-1) receptoremediated vasocontractile responsiveness in various arteries.3,4 Thus,
we wanted to question whether TNF-a has any role in vaso-contractile responsiveness during IRI and if so, do TNF-a blockers restore the altered vasocontractile responses due to IRI. This study is designed to evaluate in vivo and in vitro effects of etanercept, the soluble TNF receptor, on contractile re-sponses of superior mesenteric artery in an experimental model of mesenteric IRI.
Material and methods
The study protocol was approved by the animal ethics com-mittee (2013-7-53/Ankara University Animal Experiments Local Ethics Committee) and performed according to the guidelines of the Research Committee of the Faculty of Med-icine at Ankara University. The study comprised 24, 12 wk- to 14-wk-old male SpragueeDawley rats weighing 300 to 360 g (mean: 335 g). All animals were kept under controlled tem-perature (21 2C) and humidity (55.5%), with a 14 h light and
10 h dark cycle. They were fed with commercial food, and they had free access to water. There were no water and light re-strictions throughout the experiment. All animals received
humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Re-sources published by the National Institutes of Health. Two surgeons performed all surgical procedures in absolute sterile conditions. Every surgical intervention was performed under anesthesia using intraperitoneal injection of ketamine hy-drochloride (Ketalar, Eczacibasi, Turkey) and xylazine hydro-chloride (Alfazyne, Ege Vet, Turkey) with doses of 80-100 mg kg1and 10-12.5 mg kg1, respectively.
Twenty-four SpragueeDawley rats were allocated to 3 groups. The rats in control group (Gr C, n¼ 6) underwent a median laparotomy and dissection of superior mesenteric artery with no further intervention. The rats in ischemia reperfusion group (Gr I/R, n¼ 12) underwent a median lapa-rotomy, dissection of superior mesenteric artery, and occlu-sion of the artery adjacent to its root by a microclamp for 90 min. After clamping the artery, the paleness of the jeju-noileal segments along with pulseless mesenteric artery confirmed the ischemia. The rats in the treatment group (Gr I/ RþE, n ¼ 6) underwent the same surgical procedure and received etanercept intravenously. The caudal caval veins of the rats were cannulated with 24G catheter, and 5 mg/kg of etanercept in distilled water was started to infuse via catheter at the beginning of reperfusion. The infusion of each animal was completed in an hour. All animals were sacrificed at the 24th h of reperfusion. Bowel mesentery as well as ileum was excised. Third branch of superior mesenteric artery (SMA) was immediately dissected to evaluate the contractile responses. In vitro effects of etanercept on vasocontractile responses were also investigated by using organ bath incubation of the drug for animals in Gr I/R. The excised ileums were analyzed under light microscope.
Pharmacological evaluation
To dissect the SMA apart from superior mesenteric vein and evaluate the mesenteric arterial branching, bowel mesentery was separated from bowel segments, placed in an ice-cold, modified Krebs’ solution composed of NaCl (118 mM/L), KCl (4.7 mM/L), NaH2PO4 (1.2 mM/L), NaHCO3 (25 mM/L), MgSO4.7H2O (1.2 mM/L), glucose (11.2 mM/L), and CaCl2 (2.5 mM/L). Connective tissue of the mesenteric artery was cleaned out and cut into four segments using Cold Lighted Loop (Leica). One stainless steel wire was passed through arterial lumen and attached to chamber of Myograph System (610M; Danish Myo Technology, Aarhus, Denmark) in a temperature-controlled environment that was 37C and aerated with a mixture of 95% O2and 5% CO2. Second wire was also passed parallel to the wire and fixed transducer side of chamber. Parallel wires were separated from each other through micrometric screw. After 40 min of incubation period, all rings were gradually adjusted to a pressure of 100 mm Hg. To reach 100 mm Hg tension, all rings were stretched in four steps, each step lasting for 2 min. All rings were prestimulated with 100 mM KCl and 1mM Noradrenalin mixture to check vascular contractile property and washed at least three times to return to baseline tension. Contractile experiments were
recorded using data computerized acquisition system (MP35 BIOPAC Systems; MAY Commat Inc, CA)
Histopathological evaluation
The histopathological analysis of the study was carried out in histology and embryology departments of Ufuk and Ankara Universities, Faculty of Medicine. For light microscopic anal-ysis, ileal section of the intestinal tissue was removed from each rat and fixed in 10% neutral buffered formalin solution for 1 wk. Tissues were washed and dehydrated with rising concentrations of ethanol. After dehydration, specimens were put into xylene to obtain transparency and were then infil-trated with and embedded in paraffin. Embedded tissues were cut into sections of 6-mm thickness by Leica RM 2125 RT. Systematically, randomly selected sections were stained with hematoxylin-eosin dye. Histopathological examinations were performed by a histologist blinded to the study design and photographed with an Olympus DP71.
Statistical analysis
Statistical analyses were performed using GraphPad Prism software (Trial 6). Two-way analysis of variance following Bonferroni post hoc test was used for phenylephrine (Phe) and ET-1-mediated concentration-response curves (CRCs). The P value< 0.05 was deemed to indicate statistical significance.
Results
Pharmacological evaluation
The effects of in vitro etanercept treatment on Phe-induced vasocontractile responses in control group
First of all, the possible “nonselective” effects of etanercept on contractile responses were investigated. To this aim, the Phe-induced vasocontractile responses were evaluated in pres-ence of three etanercept doses (0.001mM, 0.01 mM, and 0.1 mM) in Gr C. After 20 min of etanercept incubation period, Phe-induced CRCs were obtained from Gr C. Our results showed that Phe-induced CRCs only shifted to the right side at the highest etanercept dose (pD2 for Gr C, 5.98 0.09 versus in vitro etanercept (0.1mM) incubation 5.66 0.12, P > 0.05) (Fig. 1). Even though the difference was not statistically significant, it was thought as a supportive evidence for a possible relation-ship between TNF-a and a1-adrenergic receptor signaling. Furthermore, Phe-induced vasocontraction might be changed by etanercept even in the absence of its anti-inflammatory effects that required long term.
The effects of in vitro and in vivo etanercept treatment on Phe-induced vasocontractile responses in ischemia reperfusion and control groups
After 90-min ischemia and 24-h reperfusion, superior mesen-teric arteries of Gr I/R were obtained to evaluate contractile responsiveness. Our findings revealed that Phe-induced CRCs were significantly shifted to the left side in Gr I/R (pD2 for Gr I/R: 6.46 0.1 versus Gr C: 5.98 0.1, P < 0.05) (Fig. 2A). These find-ings indicated thata1-adrenergic receptor sensitivity to Phe
was increased 4.2-fold in Gr I/R. Furthermore, maximal con-tractile responses in Gr I/R increased approximately 30% when compared with Gr C (P< 0.05) (Fig. 2B).
Etanercept-dependent in vivo and in vitro effects were also investigated in Gr I/R. For this purpose, the animals treated with etanercept after I/R (Gr I/RþE) and the arteries of Gr I/R were incubated with etanercept (0.01mM and 0.1 mM) in the organ bath for 20 min (As these doses were shown to be effective in Gr C). Our results showed that Phe-induced CRCs were signifi-cantly shifted to the right side by both in vitro and in vivo eta-nercept treatments (pD2 for in vitro etaeta-nercept [0.01 mM] incubation to Gr I/R, 5.79 0.05, P < 0.01, in vitro etanercept [0.1mM] incubation to Gr I/R 5.92 1.2, P < 0.05; Gr I/R þ E, 5.63 0.17, P < 0.01 versus Gr I/R) (Fig. 3). Both in vitro and in vivo etanercept treatments decreased maximal contractile re-sponses (Fig. 3). These findings indicated that in vivo and in vitro treatment of etanercept corrected both vascular sensitivity and contractile amplitude in Gr I/R. Even though there was no sig-nificant difference between pD2 values in all etanercept-treated Gr I/R, in vivo treatment was seemed more effective than in vitro due to the pattern of the CRC. In vivo etanercept treatment caused a decrease in the slope of the Phe-induced CRCs. Furthermore, HillSlope value of the CRCs in in vivo treatment was found lower than other I/R groups (Gr I/R: 1.79, Gr I/RþE: 1.29, in vitro etanercept [0.01mM] incubation to Gr I/R:4.94, in vitro etanercept [0.1 mM] incubation to Gr I/R:2.36). It meant that in vivo etanercept was able to repress a wide range of Phe-induced CRCs. Actually, in vivo etanercept treatment, as shown to be more effective on vasocontraction, might be related to its anti-inflammatory effects that emerged long term.
The effects of in vitro and in vivo etanercept treatment on ET-1-induced vasocontractile responses in ischemia reperfusion and control groups
We also investigated ET-1-induced vasocontractile responses in Gr C, I/R, I/RþE, and in vitro etanercept incubation to I/R. For Fig. 1e In vitro etanercept treatment effects on
vasocontraction in control group. Vasocontraction of superior mesenteric arteries induced by cumulative concentration of selectivea1-adrenoceptor agonist Phe in the presence and absence of etanercept. Each dose of etanercept was added to organ bath 20 min before the agonist-induced response. The contraction responses were calculated as the percentage of KCl-induced contractions. Each point shows the mean ± SEM.
this aim, broad ranges of ET-1 concentrations (1 pM-3mM) were used. Our findings revealed that ET-1-induced CRCs were fit two-site models for mesenteric arteries.
The first phase of the CRCs showed a high potency component, whereas the second phase indicated a low po-tency component. ET-1 had two pD2 values to indicate high and low components (pD2 values in Gr C; high 8.73 0.53 and low 7.54 0.25). The first phase of ET-1-induced CRCs was significantly shifted to the left side in Gr I/R (pD2 values for Gr I/R, high 10.15 0.13 versus Gr C, P < 0.01). Vascular sensitivity to the first phase of ET-1-induced response increased approximately 10-fold for Gr I/R when compared with Gr C. For that reason, pD2 value of high potency component in Gr I/R
was higher than that of Gr C. The second phase of CRCs moderately shifted to the left side, but there was no statisti-cally significant difference between Gr I/R and Gr C in terms of pD2 values (pD2 values for Gr I/R, low 7.97 0.27 versus Gr C, P> 0.05). Interestingly, in vitro treatment did not affect ET-1-induced CRCs in Gr I/R. pD2 values for both high and low po-tency component could not be changed by in vitro treatment (pD2 values for in vitro etanercept incubation to Gr I/R high 9.98 0.14 and low 8.28 0.31) (Fig. 4). However, ET-1-induced CRCs in Gr I/R were close to control values with in vivo treat-ment (pD2 for Gr I/RþE: high 9.51 0.32 and low 7.42 0.22). In vivo treatment was found more effective on high potency component of the CRCs than low potency component. In fact, our results indicated that first phase of CRCs shifted more to the left side than second phase of CRCs in I/R state. Therefore, in vivo treatment was expected to be more effective on the first phase of ET-1-induced vasocontractile response.
Histopathological findings
In all of the specimens belonging to the control group (Group C), the histologic features showed regular appearance of ileal tissue (Fig. 5A). When we examined the specimens of the ischemia reperfusion group (group Gr I/R), we observed that the epithelial tissue lining the apical surface of the villi was degenerated and desquamated. The tips of the villi were blunt, and lamina propria was disintegrated in the absence of the covering epithelium. Inflammatory cells’ infiltration extending through the deep lamina propria layer was observed. The subepithelial edema was clear at the villous tips (Fig. 5B). The treatment group (group I/RþE), showed an ileal histomorphology close to Gr C. Generally, the villus integrity was well preserved in the whole tissue resembling the surface epithelium lining the villi and the uniform pattern of the lamina propria. Only a few number of villi presented the existence of subepithelial spaces at villous tips. (Fig. 5C).
Fig. 2e The effects of ischemia and reperfusion on vascular contractility. Vasocontraction of superior mesenteric arteries induced by cumulative concentration of selectivea1-adrenoceptor agonist Phe in Gr C and I/R. (A) Bar graph showing the maximal contraction of Phe-induced responses in Gr C and I/R. (B) The contraction responses were calculated as the percentage of KCl-induced contractions. Each point shows the mean ± SEM. *,P < 0.05, **, P < 0.01, and ***, P < 0.001 statistically significant differenceversus Gr C.
Fig. 3e The effects of in vivo and in vitro etanercept treatment on vascular contractility in Gr I/R.
Vasocontraction of superior mesenteric arteries induced by cumulative concentration of selectivea1-adrenoceptor agonist Phe in Gr I/R, I/RDE, and in vitro etanercept-incubated group. Each dose of etanercept was added to organ bath 20 min before the agonist-induced response. The contraction responses were calculated as the
percentage of KCl-induced contractions. Each point shows the mean ± SEM. *,P < 0.05, **, P < 0.01, and ***, P < 0.001 statistically significant differenceversus Gr C.
Discussion
The aim of the study was to evaluate the in vivo and in vitro effects of etanercept, a soluble TNF-a receptor, on contractile responses of SMA in an experimental model of mesenteric ischemia and reperfusion. We found that the either way of the drug decreased vasocontractile sensitivity to Phe and ET-1 that was increased during IRI.
Vascular tonus depends on an ongoing balance between vasodilator and vasoconstrictor facts. An insult to this balance results in an impairment in vasomotricity. Previous studies have demonstrated an altered vasomotricity following IRI. The endothelium-dependent NO-mediated vascular relaxa-tion, the endothelium-independent vascular relaxarelaxa-tion, and vascular contraction were announced to be affected following IRI.3-9In terms of intestinal IRI, as the end organ injury is
re-ported to be aggravated with vasoconstriction of vessels, we aimed to identify the vasocontractile activity of SMA. We found that botha-adrenoceptor-mediated vasocontractile sensitivity and endothelium-dependent vasocontractile sensitivity are increased during mesenteric IRI. These findings are compatible with others who studied vasocontractile responses of SMA following mesenteric IRI. Ko¨ksoy et al.3studied vasomotor
ef-fects of 120 and 240 min reperfusion following 60 min ischemia of SMA on conduit arteries. They stated affected contractile responsiveness of SMA to Phe. They demonstrated temporary increase in the Phe-mediated maximal contractile response of
SMA at the 120th min of reperfusion despite ongoing tissue damage. Martinez-Revelles et al.4also studied ET-1 mediated
vasocontractile activity of SMA following IRI. They performed 24-h reperfusion following 90 min clamping of SMA. They stated that potentiated vasoconstrictor responses to ET-1 following mesenteric IRI while Phe and KCl responses remained unaffected. They attributed these vasocontractile responses selective to ET-1 due to the changes either in the number or the sensitivity of ET-B receptor located at vascular smooth muscle (VSM) and the decrease in endothelial nitric oxide bioavailability following IRI.
The endothelium is known to be the most affected site of the vessel during IRI.6,10Endothelial dysfunction seems to be
the consequence of reperfusion but not ischemia and lasts for a long time. This dysfunction is hypothesized to be related to overproduction of free radicals, consumption of arginine in metabolic pathways other than NO synthesis, or NO synthase cofactor deficiencies. TNF-a seems to be one of the key factors during this process. Its increase during reperfusion provokes more production of TNF-a, stimulates production of free rad-icals, and decreases production of NO. Several studies demonstrated blocking either production or circulation of TNF-a provides beneficial effects during the early endothelial dysfunction period. In this study, as we demonstrated the increased vasocontractile activity after IRI, we wanted to question whether TNF-a has any role in this activity. We showed that both in vivo and in vitro administration of eta-nercept blocked TNF-a and restored the altered vaso-contractile responses due to IRI. Interestingly, even a 20-min period of etanercept incubation was shown to correct the increased sensitivity of a1-adrenergic receptoremediated contractile responses in IRI. A possible explanation of this acute beneficial effect of etanercept is the probable involve-ment of TNF-a in the a1-adrenergic receptor signaling. In fact, it is known that stimulation of one receptor family can affect different receptor families even in the absence of its ligand. This phenomenon is called transactivation.11-14 As an example, stimulation ofa1-adrenergic receptor by its ligand can also activate any other receptor family such as receptor tyrosine kinases.11It is known thata1-adrenergic receptor is a
member of G protein-coupled receptor family (GPCR). Different froma1-adrenergic receptor, TNF-a receptors are the members of TNF receptor super family including 23 different members. For that reason, it could be expected to exist a cross-talk relationship between a1-adrenergic receptor and TNF-a receptor signaling.
A disintegrin and metalloprotease 17 (ADAM17) is known to be involved in TNF-a signaling. ADAM17 is also called TNF-a converting enzyme. ADAM17 cleavages tethered pro- TNF-a to cell membrane surface and releases active form of TNF-a into circulation. ADAM17 not only activates TNF-a signaling but also stimulates various cytokines, receptors, and ligands with its sheddase activity.15One of the transactivation mech-anisms is GPCR-dependent ADAM17 activation that causes transactivation of TNF-a, receptor tyrosine kinase, and various cytokine receptors.15,16 a-1 adrenergic receptoredependent
ADAM17 activation in epithelial cells was shown.17 In this
study, we showed that a-1 adrenergic receptoredependent vasocontraction was changed by TNF-a blocker etanercept in control and I/R groups. Our results suggested that ADAM17 Fig. 4e The effects of in vivo and in vitro etanercept
treatment on ET-1-induced vascular contractility.
Vasocontraction of superior mesenteric arteries induced by cumulative concentration of ET-1 in Gr I/R, I/RDE, and in vitro etanercept-incubated group. Each dose of etanercept was added to organ bath 20 min before the agonist-induced response. The contraction responses were calculated as the percentage of baseline value. Each point shows the mean ± SEM. *,P < 0.05 statistically significant differenceversus Gr C, **, P < 0.01 statistically significant differenceversus Gr C.
could mediate a cross-talk relationship betweena1-adrenergic receptor and TNF-a receptor signaling. For that reason, in vitro etanercept was restored vascular sensitivity toa1-adrenergic receptor in short term. In vivo treatment, shown to be more effective than in vitro, might also represent both long-term anti-inflammatory effects and short-term blocking effects on transactivation mechanisms.
In addition toa-1 adrenergic receptor, ET receptor is also a member of the GPCR family. Two-type ET receptors were found in vascular tissue. ETA subtype presents in VSM while ETB exists in both endothelium and VSM. Even though acti-vation of ETB receptors mediates both vasorelaxation and vasocontraction response in the vessels, ET-1-dependent stimulation leads to vasocontractile response in mesenteric artery.18,19In this study, we used a broad range of the ET-1
concentrations to detect these two-phases of CRCs. A num-ber of studies proposed that the first phase indicates an ETB-mediated response in endothelium and the second phase indicates both ETA- and ETB-dependent responses in VSM and showed that the first phase was more affected by IRI.18,19
Our findings suggested that ETB-dependent endothelium
responses could deteriorate in IRI and in vivo treatment especially repaired the first phase in vasocontractile response, whereas in vitro treatment did not. For that reason, it required long time for etanercept to restore ET-1-induced vaso-contractile responses. These effects are probably due to its anti-inflammatory effects.
The histopathological findings of the study revealed an ileal histomorphology in the etanercept-treated group close to the control group. Either blocking or inhibiting TNF-a was shown to be effective on the tissue level in experimental mesenteric IRI models.20,21 Our findings also support these
findings. According to the findings of the study, we think that not only the blockage of circulating TNF-a but also the resto-ration in vasocontractile sensitivity are responsible of better histomorphology seen in the treatment group.
Lack of measurement of circulating TNF-a levels in each group can be thought as a limitation of this study. But our aim was to evaluate the local effect of the etanercept, we showed drug’s effectiveness as early as 20 min after its administration. We need not measure the circulatory TNF-a levels with the probable result of decreased levels in Gr I/RþE.
Fig. 5e Histopathologic view of ileum in all groups. This panel of the rat ileum is stained with hematoxylin-eosin. “A” micrographs on the first line is Group C, “B” micrographs on the second line is Group I/R, and “C” micrographs on the third line is Group I/RDE. (A) The normal appearance of the ileum villi (V) surrounding lumen. The goblet cells (G) existing in the lining epithelium on the surface of the villi and the lamina propria (LP) under the epithelium, tunica mucosa (M), tunica submucosa (SM), tunica muscularis (MC) and tunica serosa (S) are in regular aspect. Intraepithelial lymphocytes (Ly), fibroblasts (Fb) and plasmocytes (Pl) represent the regular histologic aspect. (B) Desquamation of the epithelial tissue (arrow head) on the tips of the villi and the disintegration of the lamina propria (*) represent the injury of the tunica mucosa in the ischemia reperfusion group. (C) Treated group showing the ileal histology close to control group with a mild injury limited in the mucosa. (Color version of figure is available online.)
The findings of this study revealed that mesenteric ischemia reperfusion affects the vascular contractility, and TNF-a has a major role during these processes. The findings of this study also supported that etanercept restores this vascular sensitivity during reflow. Taking together, we consider etanercept to be a promising agent in the clinical setting. Because the drug does not only seem to inhibit inflammation by blocking TNF-a during IRI but also it seems to restore vascular contractility during reflow.
Conclusion
Etanercept can be a promising agent in mesenteric IRI by its dual effects of inhibition of inflammation by blocking TNF-a in cir-culation and restoration of vascular contractility during reflow. These effects are probably the consequences of cytokine-mediated and transactivation-dependent mechanisms.
Acknowledgment
Authors’ contributions: S.E.O. contributed for study concep-tion and design. T.A. contributed for study design, acquisiconcep-tion, and analysis of pharmacological data. S.G. contributed for analysis and interpretation of the pharmacological data and manuscript preparation. S.S.S. contributed for analysis and interpretation of the histological data. A.P. contributed for study conception, design, and manuscript preparation.
Disclosure
The authors report no proprietary or commercial interest in any product or concept discussed in this article.
r e f e r e n c e s
1. Menger MD, Vollmar B. Pathomechanisms of ischemia-reperfusion injury as the basis for novel preventive strategies: is it time for the introduction of pleiotropic compounds? Transplant Proc. 2007;39:485e488.
2. Sprague AH, Khalil RA. Inflammatory cytokines in vascular dysfunction and vascular disease. Biochem Pharmacol. 2009;78:539e552.
3. Koksoy C, Uydes-Dogan BS, Kuzu MA, Aydemir-Koksoy A, Demirpence E, Kesenci M. Effects of intestinal ischemia-reperfusion on major conduit arteries. J Invest Surg. 2000;13:35e43.
4. Martı´nez-Revelles S, Caracuel L, Ma´rquez-Martı´n A, et al. Increased endothelin-1 vasoconstriction in mesenteric resistance arteries after superior mesenteric ischaemia-reperfusion. Br J Pharmacol. 2012;165:937e950.
5. Kedzierski RM, Yanagisawa M. Endothelin system: the double-edged sword in health and disease. Annu Rev Pharmacol Toxicol. 2001;41:851e876.
6. Gourdin MJ, Bree B, De Kock M. The impact of ischaemia-reperfusion on the blood vessel. Eur J Anaesthesiol. 2009;26:537e547.
7. Seal JB, Gewertz BL. Vascular dysfunction in ischemia-reperfusion injury. Ann Vasc Surg. 2005;19:572e584.
8. Soydan G, Cekic¸ EG, Tuncer M. Endothelial dysfunction in the mesenteric artery and disturbed nonadrenergic
noncholinergic relaxation of the ileum due to intestinal ischemia-reperfusion can be prevented by sildenafil. Pharmacology. 2009;84:61e67.
9. Uddman E, Mo¨ller S, Adner M, Edvinsson L. Cytokines induce increased endothelin ET(B) receptor-mediated contraction. Eur J Pharmacol. 1999;376:223e232.
10. Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest.
1997;100:2153e2157.
11. Prenzel N, Zwick E, Daub H, et al. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature. 1999;6764:884e888. 12. Guner S, Akhayeva T, Gurdal H. The pattern of
5-hydroxytryptamine receptor subtypes mediated epidermal growth factor receptor transactivation in rat aorta (selected oral presentation). Proc Br Pharmacol Soc pA2. 2014;12:259. abst.
13. Guner S, Ozakca I, Kandilci HB, Zoto-Mustafayeva T, Duman-Dalkılıc B, Gurdal H. 5-Hydroxytryptamine mediated vasocontraction and Caþ2/Calmodulin and Src- kinase dependent epidermal growth factor receptor transactivation in rat aorta. FASEB J. 2014;28(1 Suppl):1065e1067.
14. Ulu N, Henning RH, Guner S, et al. Intracellular Transactivation of epidermal growth factor receptor by a1A-adrenoceptor is mediated by phosphatidylinositol 3-kinase independently of activation of extracellular signal regulated kinases 1/2 and serine-threonine kinases in Chinese hamster ovary cells. J Pharmacol Exp Ther. 2013;347:47e56.
15. Gooz M. ADAM-17: the enzyme that does it all. Crit Rev Biochem Mol Biol. 2010;45:146e169.
16. Cattaneo F, Guerra G, Parisi M, et al. Cell-surface receptors transactivation mediated by g protein-coupled receptors. Int J Mol Sci. 2014;15:19700e19728.
17. Chen L, Hodges RR, Funaki C, et al. Effects of alpha1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells. Am J Physiol Cell Physiol. 2006;291:C946eC956.
18. Adner M, Cantera L, Ehlert F, Nilsson L, Edvinsson L. Plasticity of contractile endothelin-B receptors in human arteries after organ culture. Br J Pharmacol. 1996;119:1159e1166.
19. Schneider MP, Boesen EI, Pollock DM. Contrasting actions of endothelin ET(A) and ET(B) receptors in cardiovascular disease. Annu Rev Pharmacol Toxicol. 2007;47:731e759. 20. Ko¨ksoy C, Kuzu MA, Kuzu I, Ergu¨n H, Gu¨rhan I. Role of tumour
necrosis factor in lung injury caused by intestinal ischaemia-reperfusion. Br J Surg. 2001;88:464e468.
21. Yang Q, Zheng FP, Zhan YS, et al. Tumor necrosis factor-a mediates JNK activation response to intestinal ischemia-reperfusion injury. World J Gastroenterol.
