Pediatr Surg Int (2014) 30:327–332 DOI 10.1007/s00383-013-3460-z
ORIGINAL ARTICLE
The effect of vascular endothelial growth factor overexpression in experimental necrotizing enterocolitis Hande Ozgun Karatepe • Huseyin Kilincaslan • Mustafa Berber Ahmet Ozen • Hulya Ercan Saricoban • Duran Ustek • Ahu Sarbay Kemik • Mine Adas • Filiz Bakar
•
Accepted: 20 December 2013 / Published online: 1 January 2014 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose Necrotizing enterocolitis (NEC) is a serious condition, predominantly observed in premature infants. We used an experimental NEC model to investigate the effects of vascular endothelial growth factor (VEGF) cloned into a plasmid. Materials and methods Twenty-four newborn Wistar albino rats were randomized equally into three groups as follows: control, NEC and NEC?VEGF. NEC was induced by hyperosmolar enteral formula feeding, exposure to hypoxia/reoxygenation and cold stress. In the NEC?VEGF group, VEGF (1 lg) incorporated into plasmid (2 lg) was administered subcutaneously once daily for a total of 3 days starting on the first day of the NEC procedure. All rats were sacrificed on the 4th day of life, and the specimens were harvested for histopathological and biochemical
H. O. Karatepe M. Berber A. Ozen H. E. Saricoban F. Bakar Department of Pediatrics, Faculty of Medicine, Yeditepe University, Istanbul, Turkey H. Kilincaslan (&) Department of Pediatric Surgery, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey e-mail: [email protected] D. Ustek Department of Genetics, Faculty of Medicine, Istanbul University, Istanbul, Turkey A. S. Kemik Department of Biochemistry, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey M. Adas Department of Endocrinology, Okmeydani Education and Research Hospital, Istanbul, Turkey
examinations [including tissue oxidative stress (malondialdehyde and nitric oxide), inflammation (myeloperoxidase, interleukin-6 and tumor necrosis factor alpha) and apoptosis (caspase-3 activity) parameters]. Results In the NEC?VEGF group, tissue malondialdehyde, nitric oxide, interleukin-6, tumor necrosis factor alpha levels and caspase-3 activity were significantly decreased. In addition, the myeloperoxidase level was increased compared to that of the NEC group (p \ 0.05). Histopathologically, VEGF overexpression enhanced angiogenesis, alleviated villous atrophy and tissue edema (p \ 0.05). Conclusion VEGF overexpression with plasmids seems to be a promising approach in the management of NEC. Keywords Necrotizing enterocolitis Overexpression Oxidative damage Vascular endothelial growth factor Plasmid
Introduction Necrotizing enterocolitis (NEC) is a surgical disease of newborns characterized by inflammation and necrosis that starts at the mucosa and may involve all layers of the intestines. The majority of NEC cases (62–94 %) are premature infants. The risk of NEC increases with decreased gestational age and birth weight. The incidence of NEC in neonatal intensive care units ranges from 1 to 5 % [1–6]. Gastrointestinal ischemia, formula feeding and bacterial invasion have been proposed as major etiological factors for the development of NEC. Premature infants are more likely to be exposed to stressors such as hypotension, hypothermia, hypoxia, anemia and umbilical
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catheterization [7, 8]. Mesenteric circulation is unfavorably affected by these stressors, and intestinal mucosal damage can easily occur [1–3]. The formation of new blood vessels, which is necessary for the repair of damaged tissue, is called angiogenesis [9]. In NEC, the angiogenic process starts simultaneously with destructive changes and is stimulated by several angiogenic mediators such as vascular endothelial growth factor (VEGF). VEGF is a glycoprotein that exerts powerful angiogenic and mitogenic effects, especially on endothelial cells. In addition, VEGF has anti-apoptotic and antiinflammatory effects [10–13]. Transfer of cloned growth factor genes into ischemic tissues is a novel approach to angiogenesis induction. This can be done by cloning the specific gene into a plasmid and transfer it into a target cell. Plasmids are the simplest tools used in gene transfer and are preferred because they have relatively lower toxicities while providing transient expressions without chromosomal integration [14]. The etiology of NEC has not been fully elucidated, and there is no common treatment protocol. The mainstay of current therapy is based on minimization of intestinal loss. To this end, various agents have been used to increase the intestinal blood supply and decrease inflammation; however, sufficiently effective therapy has not been introduced into current clinical practice [15–17]. The aim of the present study was to investigate the impact of VEGF overexpression on oxidative stress and histopathological damage in an experimental NEC model.
Materials and methods Experimental animals This study was approved by the Ethics Committee of the Istanbul University Faculty of Medicine. Three pregnant, time-mated Wistar albino rats were allowed to deliver spontaneously. We used 25 Wistar albino newborn rats each weighing 6–9 g. One rat in the NEC group died on the first day and was replaced by a new rat. No rats died in the NEC?VEGF and control groups. The rats were randomized equally into three groups of eight animals as follows: Group 1: the control group, left with their mother to breast feed freely Group 2: NEC group Group 3: NEC?VEGF group In Groups 2 and 3, the pups were immediately separated from their mothers to avoid any protective effect of the mother’s milk and were placed in humidified incubators at 37 °C.
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Plasmid preparation Total RNA extracted from cultured human cells was isolated using an RNA purification kit (Qiagen Co, US). Following reverse transcription, polymerase chain reaction (PCR) was used to amplify oligonucleotides with restriction sites at the 50 and 30 ends. The PCR fragments were recovered from a 1 % low-melting agarose gel, and DNA was eluted from the sliced agarose gel using a gel extraction kit (Qiagen). A 50 ng DNA vector and 1 ll of PCR product were incubated with 1 ml (3 l/ll) of 29 T4 DNA ligase buffer at 4 °C overnight. Heat shock transformation was applied to 100 ll of DH5 alpha competent cells and 2 ll of ligation reaction. This solution was spread on agar plates containing 100 mg/ml of ampicillin. The plates were incubated overnight at 37 °C. PCR-tested and selected positive clones were confirmed by sequencing (Refgen Co, Turkey). Plasmids were purified using the Endofree Plasmid kit (Qiagen, USA), and human VEGF cDNA-plasmid constructs were produced. An empty pcDNA3.1 plasmid was used as the control group [18, 19]. NEC model In Groups 2 and 3, we induced experimental NEC using the procedure described by Guven et al. [20]. These rat pups were fed 3 times daily with 0.2 ml of special rodent formula [15 g Similac 60/40 (Ross Pediatrics, Columbus, Ohio, USA)]. To induce NEC, the rats were subjected to 100 % CO2 inhalation for 10 min, cold exposure at ?4 °C for 5 min, and 97 % O2 delivery for 5 min twice daily over three successive days. In Group 3, VEGF (1 lg) incorporated into plasmid (2 lg) was administered subcutaneously in 0.5 ml normal saline solution once daily for three consecutive days simultaneously with the start of the induction of the NEC model. All pups were sacrificed on the fourth day of life. For each rat, a midline laparotomy was performed followed by removal of a 2-cm segment of the terminal ileum. Each segment was divided into three sections, which were used for histopathological, VEGF measurement and other biochemical analyses. In the biochemical analysis, tissue oxidative stress [malondialdehyde (MDA) and nitric oxide (NO)], inflammation [myeloperoxidase (MPO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-a)] and apoptosis (caspase-3 activity) were studied. Histopathological analysis Following fixation in a 10 % formaldehyde solution, the tissue samples were imbedded in paraffin blocks. From
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these blocks, 4-lm sections were obtained and stained with hematoxylin and eosin. Histopathological examinations were performed with a light microscope under various (109, 209) magnifications (Olympus, BX51, Japan) by an experienced pathologist blinded to the groups. Villous atrophy, edema, inflammation and ischemia were graded as follows: none (0); mild (1); moderate (2); and severe (3). Angiogenesis was scored according to number of vessels in light microscope high power field (2009): none (0), mild (1) \5, moderate (2) 5–14 and strong (3) C15. VEGF measurements Samples were homogenized using a Potter type glass homogenizers (Heidolphy-RZR 2021, Germany). The homogenate was centrifuged at 13,200 revolutions per minute for 30 min at 4 °C, and the aqueous lipid-free supernatant was removed. The tissue VEGF and VEGF receptor (1 and 2) levels were examined using ELISA (Quantikine, R&D Systems, USA). VEGF and VEGF receptor levels were recorded as pg/ml per mg protein. Biochemical analysis The terminal ileum specimens in physiological saline were homogenized using Potter type glass homogenizers. Homogenates were centrifuged at 1,500 rpm at 4° for 15 min, and the supernatants were hydrolyzed for 10–18 h with the addition of equal proportions of 50 mM hydrochloric acid.
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0.026 % ortho-dianisidine dihydrochloride plus 0.018 % H2O2 were added to the homogenate. The reactions were followed for 30 min at 450 nm at room temperature. Sodium azide (0.1 mM) was used to confirm the specificity of the reaction. All analyses were conducted in at least three independent trials. The MPO values were reported in unit/mg protein. TNF-a and IL-6 measurements TNF-a and IL-6 levels were measured using an immunoenzymatic ELISA technique (Quantikine, R&D Systems, USA). The minimum detectable values were 0.12 pg/ml for TNF-a and 0.03 pg/ml for IL-6. Caspase-3 activity Caspase-3 activity was measured using the method described by Jonges et al. [24]. Values were recorded in pmol/ml. Statistical evaluation Intergroup comparisons were performed using one-way ANOVA and Tukey’s post hoc multiple comparison tests. Histopathological scores were compared using Kruskal– Wallis and Mann–Whitney U tests with a post hoc Bonferroni correction. A p \ 0.05 was considered statistically significant. SPSS for Windows, version 16.0 (Statistical Package for Social Sciences, Inc.) was used in the calculations.
Results MDA measurements The MDA levels were determined in a supernatant homogenized to a ratio of 1/10 in a 1.15 % weight per volume cold KCl solution using the thiobarbituric acid method reported by Uchiyama and Mihara [21]. The results were expressed as nmol/mg protein. NO measurements Tissue NO levels were measured using the Griess reagent according to a method previously described by Moshage [22]. Nitrate was converted to nitrite by a nitrate reductase reaction. Nitrogen components that reacted with the Griess reagent became purple. Zinc sulfate was added to the homogenates, which were centrifuged at 10,000 rpm for 5 min. Measurements were performed using an azo chromatographic spectrophotometer set at a wavelength of 450 nm. NO values were reported in mmol/mg.
Feeding intolerance and abdominal distention were observed in the NEC group. After macroscopic evaluation, intestines in the NEC group revealed more fragile, edematous, and discolored mucosa than the control and NEC?VEGF groups. The biochemical test results and histopathologic scores are shown in Tables 1 and 2, respectively. VEGF and VEGF receptors Tissue VEGF, VEGF receptor 1 and VEGF receptor 2 levels were statistically significantly increased in the NEC?VEGF group when compared to the other groups (p \ 0.05). Apoptosis
MPO measurements The MPO activity was measured as described by Kruidenier et al. [23] with some modifications. The tissue homogenates were incubated in 0.5 % hexadecyl-trimethylammonium bromide (pH 5.5). Afterwards,
Apoptosis was evaluated by assessing caspase-3 activity levels. The NEC group had higher levels of caspase-3 activity than the other two groups (p \ 0.05).
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Table 1 Biochemical results and comparisons Control
NEC
VEGF treatment
p values Control–NEC
Control–VEGF treatment
NEC–VEGF treatment
51.42 ± 4.51
23.70 ± 5.57
94.82 ± 4.53
0.01
0.01
\0.001
VEGF 1 receptor (pg/ml)
531.66 ± 34.47
633.78 ± 42.29
953.43 ± 27.78
\0.001
\0.001
\0.001
VEGF 2 receptor (pg/ml) Caspase-3 (pmol/ml)
4,009.33 ± 271.25 11.56 ± 1.25
3,315 ± 801.21 50.47 ± 5.19
7,377. 96 ± 1,313.74 24.50 ± 4.63
\0.001 \0.001
\0.001 \0.001
\0.001 \0.001
0.87 ± 1.26
3.67 ± 0.17
1.53 ± 0.32
0.01
0.01
\0.001
VEGF (pg/ml)
MDA (nmol/mg) NO (mmol/mg)
93.68 ± 2.57
286.37 ± 19.45
144.00 ± 20.52
\0.001
\0.001
\0.001
TNF-a (pg/ml)
0.21 ± 0.05
0.64 ± 0.10
0.38 ± 0.04
\0.001
\0.001
\0.001
IL-6 (pg/ml)
0.20 ± 0.04
0.78 ± 0.08
0.60 ± 0.07
\0.001
\0.001
\0.001
MPO (u/mg)
0.13 ± 0.01
0.33 ± 0.04
0.48 ± 0.06
\0.001
\0.001
0.01
Table 2 Mean histopathologic scores and p values for pairwise comparisons between groups for histopathologic findings Control
NEC
VEGF treatment
p values Control–NEC
Control–VEGF treatment
NEC–VEGF treatment
Villous atrophy
0
2
0.5
\0.001
0.075
\0.001
Edema
0.25
2
1
\0.001
0.01
\0.001
Angiogenesis
2.75
0.25
1.5
\0.001
0.007
0.003
Ischemia
0
1.5
1.12
\0.001
0.001
0.32
Inflammation
0
0.75
0.62
0.003
0.009
0.60
Oxidative damage The untreated NEC group had higher MDA and NO levels than the other two groups (p \ 0.05). The MDA levels were significantly lower in the NEC?VEGF group than in the NEC group (p \ 0.05). Inflammation Statistically significant differences were detected in the TNF-a, IL-6 and MPO levels (p \ 0.05). When compared to the NEC group, the NEC?VEGF group had lower TNFa and IL-6 levels and higher MPO levels (p \ 0.05). Histopathological evaluation There was significantly higher angiogenic activity in the NEC?VEGF group. In addition, this group had lower edema and villous atrophy than the NEC group (p \ 0.05). No statistically significant differences in inflammation and ischemia were observed between the NEC and NEC?VEGF groups. The villous atrophy grade did not differ between the NEC?VEGF group and the control group (p = 0.20). The NEC group had higher edema, villous atrophy, inflammation and
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Fig. 1 Significant atrophy and shortening of the intestinal villi are observed in the NEC group
ischemia and lower angiogenesis than the control group. Normal intestinal villi were observed in the control group. Significant atrophy and shortening of the intestinal villi in the NEC group and normal and atrophic villus structures in the NEC?VEGF group are shown in Figs. 1 and 2, respectively. Angiogenesis can be seen in Fig. 3.
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Fig. 2 A normal (arrow) and an atrophic villus (filled arrow) structures are observed in the NEC?VEGF group
Discussion NEC is a necrotizing intestinal disease that frequently affects premature newborns. The mortality rate of NEC ranges from 10 to 50 %. The persistence of high mortality and morbidity rates in NEC emphasizes the importance of identifying effective therapeutic agents [1–5]. Histopathological NEC-like intestinal damage was induced in experimental animal models using newborn rats exposed to hypoxia and cold stress. A positive correlation has been demonstrated between destructive changes and the duration of hypoxia and cold stress [6, 7]. Previous studies investigating treatments for NEC have predominantly focused on inhibiting hypoxia, inflammation and bacterial colonization. Agents such as dexamethasone, immunoglobulin A, enteral antibiotics, saturated fatty acids, lactoferrin, arginine, probiotics and growth factors have been investigated in experimental studies [9–
Fig. 3 Normal vascular structures are seen in the control group. In the NEC group, severe mucosal and submucosal congestion, partial hemorrhagic areas and villous shortening can be seen. In the
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12, 16, 17]. Maynard et al. [11] demonstrated that epidermal growth factor had a protective effect and explained that these effects were associated with the regulation of intestinal autophagy. VEGF is also a growth factor and a powerful stimulator of angiogenesis. Adas et al. [25] demonstrated that VEGF cloned into a plasmid had a healing effect on ischemic colonic anastomoses in rats, and VEGF overexpression was used for ischemic tissues in diabetic extremities. The positive effect of VEGF was related to its angiogenic activity [26]. Regarding NEC, Ba´nya´sz et al. [27] found that the carrier state for the VEGF-2578 mutant allele, which predisposes to low VEGF production, may enhance the susceptibility to NEC. There have been no studies in the literature that address direct application of VEGF or VEGF overexpression for the management of NEC. In this study, VEGF was cloned into a plasmid to increase its effectiveness. Gene therapy is the transfer of genetic material into target cells using a vector (i.e., a virus or a plasmid). Recently, investigations into plasmid-mediated gene delivery have increased. Cloned proteins delivered in conjugation with plasmids affect the specific DNA genome and exert more powerful and specific effects than the molecules’ direct application. We preferred plasmid-mediated gene transfer because it provides more controlled and transient gene expression. Compared with other vectors, it has fewer disadvantages such as activation of immune response and limited cloning capacity [13, 14]. In this study, we hypothesized that VEGF overexpression would be beneficial in the treatment of NEC via powerful stimulation of angiogenesis, attenuation of inflammation, ischemia, villous atrophy, edema, tissue oxidative stress and apoptotic parameters. Histopathological examination demonstrated that VEGF overexpression enhanced angiogenesis, and alleviated villous atrophy and tissue edema. Our results revealed macroscopic evidence
NEC?VEGF group, decreased congestion, increased vascularization and longer villous structures are seen
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for ischemia, and biochemical evidence for decreased oxidative stress and inflammation. However, histopathologically, we were unable to show statistically significant reduction in ischemia, which may be explained by the short study duration or limited number of animals. In conclusion, VEGF overexpression with plasmids seems to be a promising approach for the management of NEC and long-term studies should be conducted in the future. Acknowledgments tion to this study.
We thank to Gulcin Kamali for their contribu-
Conflict of interest The authors declare that they have no conflicts of interest.
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12. Wu SF, Caplan M, Lin HC (2012) Necrotizing enterocolitis: old problem with new hope. Pediatr Neonatol 53(3):158–163 13. van Montfrans C, te Velde AA, van Deventer SJ, Rodriguez Pena MS (2004) Gene therapy in the treatment of intestinal inflammation. Int J Colorectal Dis 19:79–86 (Review) 14. Giacca M (2007) Virus-mediated gene transfer to induce therapeutic angiogenesis: where do we stand? Int J Nanomedicine 2(4):527–540 (Review) 15. Hunter CJ, Upperman JS, Ford HR, Camerini V (2008) Understanding the susceptibility of the premature infant to necrotizing enterocolitis (NEC). Pediatr Res 63:117–123 (Review) 16. Rowland KJ, Choi PM, Warner BW (2013) The role of growth factors in intestinal regeneration and repair in necrotizing enterocolitis. Semin Pediatr Surg 22(2):101–111 (Review) 17. Downard CD, Renaud E, St Peter SD, Abdullah F, Islam S, Saito JM, Blakely ML, Huang EY, Arca MJ, Cassidy L, Aspelund G (2012) Treatment of necrotizing enterocolitis: an American Pediatric Surgical Association Outcomes and Clinical Trials Committee systematic review. J Pediatr Surg 47(11):2111–2122 (Review) 18. Patil SD, Rhodes DG, Burgess DJ (2005) DNA-based therapeutics and DNA delivery systems: a comprehensive review. AAPS J 7:E61–E77 (Review) 19. Scotland G, Beard M, Erdogan S, Huston E, McCallum F, MacKenzie SJ, Peden AH, Pooley L, Rena NG, Ross AH, Yarwood SJ, Houslay MD (1998) Intracellular compartmentalization of PDE4 cyclic AMP-specific phosphodiesterases. Methods 14(1):65–79 20. Guven A, Gundogdu G, Vurucu S, Uysal B, Oztas E, Ozturk H, Korkmaz A (2009) Medical ozone therapy reduces oxidative stress and intestinal damage in an experimental model of necrotizing enterocolitis in neonatal rats. J Pediatr Surg 44:1730–1735 21. Uchiyama M, Mihars M (1978) Determination of malondialdehyde precursor in tissues by thiobarbituric acid. Ann Biochem 86:271–278 22. Moshage H (2009) Simple and reliable measurement of nitric oxide metabolites in plasma. Clin Chem 55:1881–1882 23. Kruidenier L, Kuiper I, Van Duijn W, Mieremet-Ooms MA, van Hogezand RA, Lamers CB, Verspaget HW (2003) Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease. J Pathol 201(1):17–27 24. Jonges LE, Nagelkerke JF, Ensink NG, van der Velde EA, Tollenaar RA, Fleuren GJ, van de Velde CJ, Morreau H, Kuppen PJ (2001) Caspase-3 activity as a prognostic factor in colorectal carcinoma. Lab Invest 81(5):681–688 25. Adas G, Percem A, Adas M, Kemik O, Arikan S, Ustek D, Cakiris A, Abaci N, Kemik AS, Kamali G, Karahan S, Akcakaya A, Karatepe O (2011) VEGF-A and FGF gene therapy accelerate healing of ischemic colonic anastomoses (experimental study). Int J Surg 9:467–471 26. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M (2008) Growth factors and cytokines in wound healing. Wound Repair Regen 16:585–601 27. Ba´nya´sz I, Bokodi G, Va´sa´rhelyi B, Treszl A, Derzbach L, Szabo´ A, Tulassay T, Vannay A (2006) Genetic polymorphisms for vascular endothelial growth factor in perinatal complications. Eur Cytokine Netw 17:266–270
ORIGINAL ARTICLE
The effect of vascular endothelial growth factor overexpression in experimental necrotizing enterocolitis Hande Ozgun Karatepe • Huseyin Kilincaslan • Mustafa Berber Ahmet Ozen • Hulya Ercan Saricoban • Duran Ustek • Ahu Sarbay Kemik • Mine Adas • Filiz Bakar
•
Accepted: 20 December 2013 / Published online: 1 January 2014 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose Necrotizing enterocolitis (NEC) is a serious condition, predominantly observed in premature infants. We used an experimental NEC model to investigate the effects of vascular endothelial growth factor (VEGF) cloned into a plasmid. Materials and methods Twenty-four newborn Wistar albino rats were randomized equally into three groups as follows: control, NEC and NEC?VEGF. NEC was induced by hyperosmolar enteral formula feeding, exposure to hypoxia/reoxygenation and cold stress. In the NEC?VEGF group, VEGF (1 lg) incorporated into plasmid (2 lg) was administered subcutaneously once daily for a total of 3 days starting on the first day of the NEC procedure. All rats were sacrificed on the 4th day of life, and the specimens were harvested for histopathological and biochemical
H. O. Karatepe M. Berber A. Ozen H. E. Saricoban F. Bakar Department of Pediatrics, Faculty of Medicine, Yeditepe University, Istanbul, Turkey H. Kilincaslan (&) Department of Pediatric Surgery, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey e-mail: [email protected] D. Ustek Department of Genetics, Faculty of Medicine, Istanbul University, Istanbul, Turkey A. S. Kemik Department of Biochemistry, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey M. Adas Department of Endocrinology, Okmeydani Education and Research Hospital, Istanbul, Turkey
examinations [including tissue oxidative stress (malondialdehyde and nitric oxide), inflammation (myeloperoxidase, interleukin-6 and tumor necrosis factor alpha) and apoptosis (caspase-3 activity) parameters]. Results In the NEC?VEGF group, tissue malondialdehyde, nitric oxide, interleukin-6, tumor necrosis factor alpha levels and caspase-3 activity were significantly decreased. In addition, the myeloperoxidase level was increased compared to that of the NEC group (p \ 0.05). Histopathologically, VEGF overexpression enhanced angiogenesis, alleviated villous atrophy and tissue edema (p \ 0.05). Conclusion VEGF overexpression with plasmids seems to be a promising approach in the management of NEC. Keywords Necrotizing enterocolitis Overexpression Oxidative damage Vascular endothelial growth factor Plasmid
Introduction Necrotizing enterocolitis (NEC) is a surgical disease of newborns characterized by inflammation and necrosis that starts at the mucosa and may involve all layers of the intestines. The majority of NEC cases (62–94 %) are premature infants. The risk of NEC increases with decreased gestational age and birth weight. The incidence of NEC in neonatal intensive care units ranges from 1 to 5 % [1–6]. Gastrointestinal ischemia, formula feeding and bacterial invasion have been proposed as major etiological factors for the development of NEC. Premature infants are more likely to be exposed to stressors such as hypotension, hypothermia, hypoxia, anemia and umbilical
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catheterization [7, 8]. Mesenteric circulation is unfavorably affected by these stressors, and intestinal mucosal damage can easily occur [1–3]. The formation of new blood vessels, which is necessary for the repair of damaged tissue, is called angiogenesis [9]. In NEC, the angiogenic process starts simultaneously with destructive changes and is stimulated by several angiogenic mediators such as vascular endothelial growth factor (VEGF). VEGF is a glycoprotein that exerts powerful angiogenic and mitogenic effects, especially on endothelial cells. In addition, VEGF has anti-apoptotic and antiinflammatory effects [10–13]. Transfer of cloned growth factor genes into ischemic tissues is a novel approach to angiogenesis induction. This can be done by cloning the specific gene into a plasmid and transfer it into a target cell. Plasmids are the simplest tools used in gene transfer and are preferred because they have relatively lower toxicities while providing transient expressions without chromosomal integration [14]. The etiology of NEC has not been fully elucidated, and there is no common treatment protocol. The mainstay of current therapy is based on minimization of intestinal loss. To this end, various agents have been used to increase the intestinal blood supply and decrease inflammation; however, sufficiently effective therapy has not been introduced into current clinical practice [15–17]. The aim of the present study was to investigate the impact of VEGF overexpression on oxidative stress and histopathological damage in an experimental NEC model.
Materials and methods Experimental animals This study was approved by the Ethics Committee of the Istanbul University Faculty of Medicine. Three pregnant, time-mated Wistar albino rats were allowed to deliver spontaneously. We used 25 Wistar albino newborn rats each weighing 6–9 g. One rat in the NEC group died on the first day and was replaced by a new rat. No rats died in the NEC?VEGF and control groups. The rats were randomized equally into three groups of eight animals as follows: Group 1: the control group, left with their mother to breast feed freely Group 2: NEC group Group 3: NEC?VEGF group In Groups 2 and 3, the pups were immediately separated from their mothers to avoid any protective effect of the mother’s milk and were placed in humidified incubators at 37 °C.
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Plasmid preparation Total RNA extracted from cultured human cells was isolated using an RNA purification kit (Qiagen Co, US). Following reverse transcription, polymerase chain reaction (PCR) was used to amplify oligonucleotides with restriction sites at the 50 and 30 ends. The PCR fragments were recovered from a 1 % low-melting agarose gel, and DNA was eluted from the sliced agarose gel using a gel extraction kit (Qiagen). A 50 ng DNA vector and 1 ll of PCR product were incubated with 1 ml (3 l/ll) of 29 T4 DNA ligase buffer at 4 °C overnight. Heat shock transformation was applied to 100 ll of DH5 alpha competent cells and 2 ll of ligation reaction. This solution was spread on agar plates containing 100 mg/ml of ampicillin. The plates were incubated overnight at 37 °C. PCR-tested and selected positive clones were confirmed by sequencing (Refgen Co, Turkey). Plasmids were purified using the Endofree Plasmid kit (Qiagen, USA), and human VEGF cDNA-plasmid constructs were produced. An empty pcDNA3.1 plasmid was used as the control group [18, 19]. NEC model In Groups 2 and 3, we induced experimental NEC using the procedure described by Guven et al. [20]. These rat pups were fed 3 times daily with 0.2 ml of special rodent formula [15 g Similac 60/40 (Ross Pediatrics, Columbus, Ohio, USA)]. To induce NEC, the rats were subjected to 100 % CO2 inhalation for 10 min, cold exposure at ?4 °C for 5 min, and 97 % O2 delivery for 5 min twice daily over three successive days. In Group 3, VEGF (1 lg) incorporated into plasmid (2 lg) was administered subcutaneously in 0.5 ml normal saline solution once daily for three consecutive days simultaneously with the start of the induction of the NEC model. All pups were sacrificed on the fourth day of life. For each rat, a midline laparotomy was performed followed by removal of a 2-cm segment of the terminal ileum. Each segment was divided into three sections, which were used for histopathological, VEGF measurement and other biochemical analyses. In the biochemical analysis, tissue oxidative stress [malondialdehyde (MDA) and nitric oxide (NO)], inflammation [myeloperoxidase (MPO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-a)] and apoptosis (caspase-3 activity) were studied. Histopathological analysis Following fixation in a 10 % formaldehyde solution, the tissue samples were imbedded in paraffin blocks. From
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these blocks, 4-lm sections were obtained and stained with hematoxylin and eosin. Histopathological examinations were performed with a light microscope under various (109, 209) magnifications (Olympus, BX51, Japan) by an experienced pathologist blinded to the groups. Villous atrophy, edema, inflammation and ischemia were graded as follows: none (0); mild (1); moderate (2); and severe (3). Angiogenesis was scored according to number of vessels in light microscope high power field (2009): none (0), mild (1) \5, moderate (2) 5–14 and strong (3) C15. VEGF measurements Samples were homogenized using a Potter type glass homogenizers (Heidolphy-RZR 2021, Germany). The homogenate was centrifuged at 13,200 revolutions per minute for 30 min at 4 °C, and the aqueous lipid-free supernatant was removed. The tissue VEGF and VEGF receptor (1 and 2) levels were examined using ELISA (Quantikine, R&D Systems, USA). VEGF and VEGF receptor levels were recorded as pg/ml per mg protein. Biochemical analysis The terminal ileum specimens in physiological saline were homogenized using Potter type glass homogenizers. Homogenates were centrifuged at 1,500 rpm at 4° for 15 min, and the supernatants were hydrolyzed for 10–18 h with the addition of equal proportions of 50 mM hydrochloric acid.
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0.026 % ortho-dianisidine dihydrochloride plus 0.018 % H2O2 were added to the homogenate. The reactions were followed for 30 min at 450 nm at room temperature. Sodium azide (0.1 mM) was used to confirm the specificity of the reaction. All analyses were conducted in at least three independent trials. The MPO values were reported in unit/mg protein. TNF-a and IL-6 measurements TNF-a and IL-6 levels were measured using an immunoenzymatic ELISA technique (Quantikine, R&D Systems, USA). The minimum detectable values were 0.12 pg/ml for TNF-a and 0.03 pg/ml for IL-6. Caspase-3 activity Caspase-3 activity was measured using the method described by Jonges et al. [24]. Values were recorded in pmol/ml. Statistical evaluation Intergroup comparisons were performed using one-way ANOVA and Tukey’s post hoc multiple comparison tests. Histopathological scores were compared using Kruskal– Wallis and Mann–Whitney U tests with a post hoc Bonferroni correction. A p \ 0.05 was considered statistically significant. SPSS for Windows, version 16.0 (Statistical Package for Social Sciences, Inc.) was used in the calculations.
Results MDA measurements The MDA levels were determined in a supernatant homogenized to a ratio of 1/10 in a 1.15 % weight per volume cold KCl solution using the thiobarbituric acid method reported by Uchiyama and Mihara [21]. The results were expressed as nmol/mg protein. NO measurements Tissue NO levels were measured using the Griess reagent according to a method previously described by Moshage [22]. Nitrate was converted to nitrite by a nitrate reductase reaction. Nitrogen components that reacted with the Griess reagent became purple. Zinc sulfate was added to the homogenates, which were centrifuged at 10,000 rpm for 5 min. Measurements were performed using an azo chromatographic spectrophotometer set at a wavelength of 450 nm. NO values were reported in mmol/mg.
Feeding intolerance and abdominal distention were observed in the NEC group. After macroscopic evaluation, intestines in the NEC group revealed more fragile, edematous, and discolored mucosa than the control and NEC?VEGF groups. The biochemical test results and histopathologic scores are shown in Tables 1 and 2, respectively. VEGF and VEGF receptors Tissue VEGF, VEGF receptor 1 and VEGF receptor 2 levels were statistically significantly increased in the NEC?VEGF group when compared to the other groups (p \ 0.05). Apoptosis
MPO measurements The MPO activity was measured as described by Kruidenier et al. [23] with some modifications. The tissue homogenates were incubated in 0.5 % hexadecyl-trimethylammonium bromide (pH 5.5). Afterwards,
Apoptosis was evaluated by assessing caspase-3 activity levels. The NEC group had higher levels of caspase-3 activity than the other two groups (p \ 0.05).
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Table 1 Biochemical results and comparisons Control
NEC
VEGF treatment
p values Control–NEC
Control–VEGF treatment
NEC–VEGF treatment
51.42 ± 4.51
23.70 ± 5.57
94.82 ± 4.53
0.01
0.01
\0.001
VEGF 1 receptor (pg/ml)
531.66 ± 34.47
633.78 ± 42.29
953.43 ± 27.78
\0.001
\0.001
\0.001
VEGF 2 receptor (pg/ml) Caspase-3 (pmol/ml)
4,009.33 ± 271.25 11.56 ± 1.25
3,315 ± 801.21 50.47 ± 5.19
7,377. 96 ± 1,313.74 24.50 ± 4.63
\0.001 \0.001
\0.001 \0.001
\0.001 \0.001
0.87 ± 1.26
3.67 ± 0.17
1.53 ± 0.32
0.01
0.01
\0.001
VEGF (pg/ml)
MDA (nmol/mg) NO (mmol/mg)
93.68 ± 2.57
286.37 ± 19.45
144.00 ± 20.52
\0.001
\0.001
\0.001
TNF-a (pg/ml)
0.21 ± 0.05
0.64 ± 0.10
0.38 ± 0.04
\0.001
\0.001
\0.001
IL-6 (pg/ml)
0.20 ± 0.04
0.78 ± 0.08
0.60 ± 0.07
\0.001
\0.001
\0.001
MPO (u/mg)
0.13 ± 0.01
0.33 ± 0.04
0.48 ± 0.06
\0.001
\0.001
0.01
Table 2 Mean histopathologic scores and p values for pairwise comparisons between groups for histopathologic findings Control
NEC
VEGF treatment
p values Control–NEC
Control–VEGF treatment
NEC–VEGF treatment
Villous atrophy
0
2
0.5
\0.001
0.075
\0.001
Edema
0.25
2
1
\0.001
0.01
\0.001
Angiogenesis
2.75
0.25
1.5
\0.001
0.007
0.003
Ischemia
0
1.5
1.12
\0.001
0.001
0.32
Inflammation
0
0.75
0.62
0.003
0.009
0.60
Oxidative damage The untreated NEC group had higher MDA and NO levels than the other two groups (p \ 0.05). The MDA levels were significantly lower in the NEC?VEGF group than in the NEC group (p \ 0.05). Inflammation Statistically significant differences were detected in the TNF-a, IL-6 and MPO levels (p \ 0.05). When compared to the NEC group, the NEC?VEGF group had lower TNFa and IL-6 levels and higher MPO levels (p \ 0.05). Histopathological evaluation There was significantly higher angiogenic activity in the NEC?VEGF group. In addition, this group had lower edema and villous atrophy than the NEC group (p \ 0.05). No statistically significant differences in inflammation and ischemia were observed between the NEC and NEC?VEGF groups. The villous atrophy grade did not differ between the NEC?VEGF group and the control group (p = 0.20). The NEC group had higher edema, villous atrophy, inflammation and
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Fig. 1 Significant atrophy and shortening of the intestinal villi are observed in the NEC group
ischemia and lower angiogenesis than the control group. Normal intestinal villi were observed in the control group. Significant atrophy and shortening of the intestinal villi in the NEC group and normal and atrophic villus structures in the NEC?VEGF group are shown in Figs. 1 and 2, respectively. Angiogenesis can be seen in Fig. 3.
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Fig. 2 A normal (arrow) and an atrophic villus (filled arrow) structures are observed in the NEC?VEGF group
Discussion NEC is a necrotizing intestinal disease that frequently affects premature newborns. The mortality rate of NEC ranges from 10 to 50 %. The persistence of high mortality and morbidity rates in NEC emphasizes the importance of identifying effective therapeutic agents [1–5]. Histopathological NEC-like intestinal damage was induced in experimental animal models using newborn rats exposed to hypoxia and cold stress. A positive correlation has been demonstrated between destructive changes and the duration of hypoxia and cold stress [6, 7]. Previous studies investigating treatments for NEC have predominantly focused on inhibiting hypoxia, inflammation and bacterial colonization. Agents such as dexamethasone, immunoglobulin A, enteral antibiotics, saturated fatty acids, lactoferrin, arginine, probiotics and growth factors have been investigated in experimental studies [9–
Fig. 3 Normal vascular structures are seen in the control group. In the NEC group, severe mucosal and submucosal congestion, partial hemorrhagic areas and villous shortening can be seen. In the
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12, 16, 17]. Maynard et al. [11] demonstrated that epidermal growth factor had a protective effect and explained that these effects were associated with the regulation of intestinal autophagy. VEGF is also a growth factor and a powerful stimulator of angiogenesis. Adas et al. [25] demonstrated that VEGF cloned into a plasmid had a healing effect on ischemic colonic anastomoses in rats, and VEGF overexpression was used for ischemic tissues in diabetic extremities. The positive effect of VEGF was related to its angiogenic activity [26]. Regarding NEC, Ba´nya´sz et al. [27] found that the carrier state for the VEGF-2578 mutant allele, which predisposes to low VEGF production, may enhance the susceptibility to NEC. There have been no studies in the literature that address direct application of VEGF or VEGF overexpression for the management of NEC. In this study, VEGF was cloned into a plasmid to increase its effectiveness. Gene therapy is the transfer of genetic material into target cells using a vector (i.e., a virus or a plasmid). Recently, investigations into plasmid-mediated gene delivery have increased. Cloned proteins delivered in conjugation with plasmids affect the specific DNA genome and exert more powerful and specific effects than the molecules’ direct application. We preferred plasmid-mediated gene transfer because it provides more controlled and transient gene expression. Compared with other vectors, it has fewer disadvantages such as activation of immune response and limited cloning capacity [13, 14]. In this study, we hypothesized that VEGF overexpression would be beneficial in the treatment of NEC via powerful stimulation of angiogenesis, attenuation of inflammation, ischemia, villous atrophy, edema, tissue oxidative stress and apoptotic parameters. Histopathological examination demonstrated that VEGF overexpression enhanced angiogenesis, and alleviated villous atrophy and tissue edema. Our results revealed macroscopic evidence
NEC?VEGF group, decreased congestion, increased vascularization and longer villous structures are seen
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for ischemia, and biochemical evidence for decreased oxidative stress and inflammation. However, histopathologically, we were unable to show statistically significant reduction in ischemia, which may be explained by the short study duration or limited number of animals. In conclusion, VEGF overexpression with plasmids seems to be a promising approach for the management of NEC and long-term studies should be conducted in the future. Acknowledgments tion to this study.
We thank to Gulcin Kamali for their contribu-
Conflict of interest The authors declare that they have no conflicts of interest.
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