Published online Sep 01, 2023.
https://doi.org/10.4174/astr.2023.105.3.157
Suppressive role of vascular endothelial growth factor on intestinal apoptosis in induced necrotizing enterocolitis in rats
Abstract
Purpose
Necrotizing enterocolitis (NEC) is a devastating disease that can cause mortality in preterm babies. NEC may develop through an apoptotic pathway that is known to be inhibited by vascular endothelial growth factor (VEGF). This study determined whether VEGF exerted a protective effect against the development of NEC and apoptosis in rats.
Methods
To determine the effect of VEGF in NEC rats, neonatal rats were randomized into 4 groups: the control group, the NEC group, the NEC + intraperitoneal VEGF (50 ng/kg) group (NEC + VEGF IP group), and the NEC + oral VEGF (50 ng/kg) group (NEC + VEGF OR group). NEC was induced by lipopolysaccharide/hypoxia and cold stress. The animals were sacrificed 72 hours later. After laparotomy, we obtained a region of the proximal small bowel from the ileocecal valve about 18 cm in length.
Results
The NEC histological grade, apoptosis histological score, and caspase-3 activity were lower in the NEC + VEGF IP and OR groups than in the NEC group. In the NEC + VEGF IP and OR groups, the messenger RNA expression of apoptotic and inflammatory genes, such as Bax, NF-κB, p53, Fas, FasL, and PAF-R, but not that of Bcl-2, was decreased, as was the Bax/Bcl-2 protein ratio. Histological analysis revealed that the apoptosis-blocking effect of VEGF was more effective in the NEC + VEGF IP group than in the NEC + VEGF OR group.
Conclusion
We identified apoptotic and inflammatory genes to confirm the preventive effect of VEGF pretreatment on NEC in rats. This study presents a novel approach to prevent apoptosis via VEGF pretreatment in rats with lipopolysaccharide/hypoxia-induced NEC.
INTRODUCTION
Necrotizing enterocolitis (NEC) is a devastating disease that can cause morbidity and mortality in preterm babies. Medical treatment is usually sufficient, but some severe cases require surgical treatments [1]. NEC affects the bowel in premature infants in the first few weeks of life [1]. Although the cause of NEC is not known, its incidence is lower in breastmilk-fed infants than in formula-fed infants [2]. In addition, maternal milk reduces the severity of experimental NEC in neonatal animal models [3]. The components of maternal milk that are responsible for protection against NEC remain unknown, but epidermal growth factor and vascular endothelial growth factor (VEGF) are among the most promising candidates for NEC prophylaxis [4]. Breastmilk from the first week postpartum has very high concentrations of VEGF (approximately 300 times adult serum levels), which acts as a mediator of infant development [5]. However, there is no definitive understanding of how VEGF in human breastmilk or breastfeeding itself produces the protective effect. A recent report has suggested that VEGF synergistically promotes angiogenesis in some cell culture systems, such as bovine endothelial cell cultures, in vitro [6]. Although the protective function of VEGF against apoptosis is known [7], there is no information regarding whether VEGF is effective at ameliorating or preventing NEC in rat models. In our study, we investigated whether the use of VEGF reduces the histological grade of NEC in rats.
METHODS
Ethics statement, and sample collection
The animal experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University (No. 10-0121) and were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of U.S. National Research Council. Studies were carried out on 4-day-old male Sprague-Dawley rats (Orient Bio). Tissue was collected and preserved in RNAlater (Qiagen) at −80 ℃ or processed into paraffin blocks.
Induction of the necrotizing enterocolitis model with lipopolysaccharide/hypoxia and pretreatment with vascular endothelial growth factor
For induction of NEC, postnatal day-3 rats were orally administered lipopolysaccharide (LPS; 5 mg/kg) and subjected to hypoxia stress (5% O2) and 4 ℃ cold stress. Before each feeding (3 times daily), the rats were subjected to 10 minutes of 4 ℃ cold stress and 10 minutes of hypoxia stress. Hypoxia stress was induced by inhalation of a gas mixture comprised of 5% O2 and 95% N2 and was verified with an oxygen gas detector. Rats were gavaged with 5 mg/kg/day LPS (Sigma-Aldrich Chemical Co.) mixed with a feed formula every day. These rats were pretreated with recombinant VEGF (4571-50, BioVision) by oral administration (50 ng/kg) or by intraperitoneal injection (50 ng/kg) before LPS feeding. The area above the ileocecal valve was obtained for the in vitro experiment.
Study design and recruitment
This study consists of 3 parts. The first experiment is to determine the timing of the maximal effect of VEGF when injected intraperitoneally or orally administrated to rats. The second is to determine the occurring timing of NEC when inducted into rats. And third, is, the main purpose of this study, to determine the protective effect of VEGF in NEC rats. In the first experiment, VEGF was administered to normal rats to determine when the maximum concentration was seen in intestinal tissue after VEGF administration. The tissue VEGF concentration was measured by sacrificing rats at 0, 12, 24, 48, 72, and 96 hours after administration of VEGF, which was given orally (OR group) and injected intraperitoneally (IP group). There were 36 rats in the first experiment (3 rats per 6 time points in 2 groups). In the second experiment, at 24, 48, and 72 hours after induction was compared to confirm when NEC occurred with the induction method in the next paragraph. There were 16 rats in the second experiment: 1 control and 5 rats per 3 time points. In the third experiment, it was found that 72 hours was effective considering the VEGF tissue concentration and NEC induction timing in the above 2 experiments, and the experiment was performed as follows. The rats were randomly assigned to 4 groups: group 1, the control (breastfed) group, n = 8; group 2, the NEC model group (gavage feeding/hypoxia/cold stress/LPS), n = 8; group 3, the NEC group with orally administered VEGF (gavage/hypoxia/cold stress/LPS/VEGF oral administration; NEC + VEGF OR group), n = 8; and group 4, the NEC group with intraperitoneally injected VEGF (gavage/hypoxia/cold stress/LPS/VEGF intraperitoneal injection; NEC + VEGF IP group), n = 8. All rats were euthanized by cervical dislocation and sacrificed at 72 hours.
Clinical status assessment
At 24, 48, and 72 hours after the start of the experiment, rats of different groups were assessed by 2 blinded investigators using clinical sickness scores [8]. Soon after sacrifice, dissection of the gastrointestinal tract from the duodenum to the rectum was performed. A macroscopic assessment was performed using a scoring system based on gut color, consistency, and degree of dilatation. Consistency was assessed with gut friability when dissected free from surrounding tissue.
Pathological assessment of necrotizing enterocolitis progress
Formalin-fixed sections of intestinal tissue were cut into 4 µm sections and stained with H&E for morphological evaluation. NEC progression was evaluated according to the Jilling scoring system [9].
Evaluation of apoptosis in samples
For apoptosis scoring, we applied TUNEL staining to formalin-fixed sections in accordance with the manufacturer’s instructions (Millipore). We assessed the apoptosis score of each sample according to the Jilling scoring system [9]. The tissue sections were treated by end-labeling with digoxigenin-11-dUDT and stained with peroxidase substrate (Dako) and Mayer’s hematoxylin (Sigma-Aldrich).
For analysis of caspase-3 activity in homogenized intestines, we used a caspase-3/CPP32 assay kit (K105-100) according to the manufacturer’s instructions (BioVision) and an enzyme-linked immunosorbent assay reader (xMark, Bio-Rad). Phosphate-buffered saline was injected as a control in the NEC IP and OR groups. The emission intensity of each sample is expressed as a percentage of that of the control group.
Reverse transcription and real-time polymerase chain reaction analysis
We evaluated the messenger RNA (mRNA) expression of apoptosis-associated genes from ileal lysates using realtime reverse transcription (RT) polymerase chain reaction (PCR). RNA was isolated using an RNA extraction-RNeasy Plus Mini Kit (Qiagen). The isolated RNA samples were converted to complementary DNA (cDNA) using an RT-iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed in triplicate with Bcl-2 (Rn99999125_m1), Bax (Rn02532082_g1), NF-κB (Rn01502266_m1), p53 (Rn00755717_m1), Fas (Rn00685720_m1), FasL (Rn00563754_m1), PAF-R (Rn02132919_s1), and GAPDH (Rn01775763_g1) quantitative PCR primers using a LightCycler 480 system (Roche Diagnostics). The data were standardized to GAPDH values for all samples.
Western blot analysis of recombinant vascular endothelial growth factor peak time and apoptotic proteins
To prepare protein extracts, frozen tissue was homogenized in PRO-PREP (iNtRON). The total protein concentration was quantified with a bicinchoninic acid protein assay kit (#23225, Thermo Fisher Scientific). The proteins were separated on a 10% polyacrylamide gel (Bio-Rad) at 100 V for 2 hours and transferred to immunoblot polyvinylidene difluoride membranes at 270 mA for 2 hours. Then, the membranes were incubated with the following polyclonal antibodies for 2 hours: β-Actin (#4967, Cell Signaling), VEGF (ab46154, Abcam), Bcl-2 (#2876, Cell Signaling), Bax (#2772, Cell Signaling), Fas (sc-1023, Santa Cruz), and FasL (N-20, Santa Cruz). The membranes were incubated for 1 hour with anti-rabbit immunoglobulin G (Cell Signaling). The proteins were visualized with a chemiluminescent horseradish peroxidase substrate (#32106, Thermo Fisher) and exposed to X-ray film.
Statistical analysis
To determine the means and standard deviations in the numerical data, descriptive statistics were used. Statistical analysis was performed by analysis of medians followed by the Mann-Whitney U-test with IBM SPSS Statistics ver. 25 (IBM Corp.). The level of significance was set to a two-sided P-value of <0.05.
RESULTS
Evaluation of vascular endothelial growth factor transcriptional activation, clinical sickness scores, and histological scores in a necrotizing enterocolitis-induced rat model
The protein levels of VEGF were evaluated by Western blot analysis following IP or OR administration of recombinant VEGF in neonatal rats. The VEGF protein showed 2- to 3-fold higher peaks at 24, 48, and 72 hours in the IP and OR groups than in the control group (Fig. 1). A macroscopic assessment of the gut was performed using a clinical sickness scoring system based on the following factors: color (0–2), consistency (0–2), and degree of dilatation (0–2). The clinical sickness score was higher in the NEC group at 72 hours (mean, 2.9; range, 2–4) than in the control group (mean, 0; range, 0–1; P < 0.001) (Fig. 2A–C). The NEC group histological score was increased significantly at 72 hours (P < 0.001) (Fig. 2D, F). The apoptosis score of the NEC group was higher (mean, 1.8; range, 1–2) than that of the control group (mean, 0; range, 0–1) at 72 hours (P < 0.001) (Fig. 2E, G). The time required for effective NEC model establishment was approximately 3 days (5 mg/kg).
Fig. 1
Tissues harvested from rats given oral/IP VEGF (50 ng/kg) at 0, 12, 24, 48, 72, and 96 hours were used for the detection of VEGF peak time using (A) Western blot analysis and (B) quantification. IP, intraperitoneal; VEGF, vascular endothelial growth factor; PC, positive control.
Fig. 2
(A) Postnatal rats were treated with oral lipopolysaccharide (5 mg/kg) and subjected to hypoxia stress (5% O2) and 4 ℃ cold stress. Before each feeding (3 times daily), the rats were subjected to cold stress and hypoxia stress. (B) Clinical status was assessed at 24, 48, and 72 hours after the start of the experiment using (C) clinical sickness scores. (D) Formalin-fixed sections of intestinal tissue were stained with H&E for morphologic evaluation (×100), and (F) the progression of necrotizing enterocolitis (NEC) was evaluated. For apoptosis scoring, (E) TUNEL staining was applied to formalin-fixed tissue (×100), and (G) apoptosis scoring was performed. All scoring was performed according to the methods of Jilling et al. [9]. CTL, control.
Histological comparison of necrotizing enterocolitis and apoptotic scores between NEC and NEC + VEGF rats
The NEC scores were significantly lower in both the NEC + VEGF IP and OR groups than in the NEC group (median, 2.37; range, 2–3). Additionally, the NEC score of the NEC + VEGF IP group (mean, 0.88; range, 0–1; P < 0.05) was lower than that of the NEC + VEGF OR group (mean, 1.5; range, 1–2; P < 0.05) (Fig. 3A, C). However, the apoptotic score was not significantly different among the NEC, OR, and IP groups (Fig. 3B, C).
Fig. 3
After vascular endothelial growth factor (VEGF) treatment. Formalin-fixed sections of intestinal tissue from the NEC + VEGF (A) IP and (C) OR groups were stained with H&E for morphologic evaluation. (B, C) For apoptosis scoring, TUNEL staining was applied to formalin-fixed sections, and apoptosis scoring was performed. All scoring was performed according to the methods of Jilling et al. [9]. NEC, necrotizing enterocolitis; IP, intraperitoneal; OR, oral.
Regulation of apoptotic gene mRNA expression by recombinant vascular endothelial growth factor
We measured Bcl-2, Bax, NF-κB, p53, Fas, FasL, and PAF-R mRNA expression. The expression of the antiapoptotic gene Bcl-2 in NEC rats was lower than that in control rats (P < 0.001; Fig. 4A); however, the expression was higher in NEC + VEGF IP and OR rats than in NEC rats (P < 0.003; Fig. 4A). In contrast, the mRNA expression of proapoptotic genes, such as Bax (Fig. 4B), NF-κB (Fig. 4C), p53 (Fig. 4D), and PAF-R (Fig. 4G), was higher in the NEC group than in the control group. Bcl-2, p53, Fas, FasL, and PAF-R mRNA expression was lower in NEC + VEGF OR rats than in NEC + VEGF IP rats (P < 0.02; Fig. 4B–G).
Fig. 4
The messenger RNA (mRNA) expression of genes associated with apoptosis was measured using real-time polymerase chain reaction (PCR). (A) Bcl-2, (B) Bax, (C) NF-κB, (D) p53, (E) Fas, (F) FasL, and (G) PAF-R were assessed. The expression levels are presented relative to the expression of the GAPDH gene. The standard deviations of the real-time PCR data are indicated with vertical bars. NEC, necrotizing enterocolitis; IP, intraperitoneal; OR, oral. *p < 0.01, **p < 0.001.
Regulation of apoptotic proteins by recombinant vascular endothelial growth factor
There was a significant 1.5-fold increase in the level of caspase-3 in the NEC group compared with the control group (Fig. 5A). Cleaved caspase-3 was significantly decreased in the NEC + VEGF IP and OR groups compared with the NEC group (Fig. 5A). Bcl-2 protein expression was unchanged in the NEC group relative to the control group. However, in the NEC + VEGF IP and OR groups, Bcl-2 protein expression was significantly increased (Fig. 5B). Bax protein expression was significantly higher in the NEC group than in the control group. However, the expression was lower in the NEC + VEGF IP and OR groups than in the NEC group (Fig. 5B). The Bax/Bcl-2 ratio was higher in the NEC group than in the control group. However, the Bax/Bcl-2 ratio was lower in the NEC + VEGF IP and OR groups than in the NEC group (P < 0.001; Fig. 5C).
Fig. 5
After vascular endothelial growth factor (VEGF) treatment, caspase-3 analysis, and Western blotting. (A) Caspase-3 was detected by enzyme-linked immunosorbent assay, and (B) proapoptotic and antiapoptotic proteins in NEC and NEC + VEGF rats were measured by Western blotting. (C) The Bax/Bcl-2 ratio was measured by Western blot analysis in triplicate. The band density is plotted as the Bax/Bcl-xL ratio. The mean ± standard deviation from 3 independent experiments is indicated with vertical bars. NEC, necrotizing enterocolitis; IP, intraperitoneal; OR, oral. **p < 0.001.
DISCUSSION
VEGF acts as a critical regulator of angiogenesis by initiating vessel growth and inhibiting endothelial cell apoptosis [10]. The protective effects of VEGF against apoptosis in the cerebral cortex and against lung injury have been evaluated [7, 11]. Currently, anti-VEGF therapies are used to counteract both the proangiogenic activity of VEGF and the antiapoptotic functions of VEGF, such as in tumor control [12] and the use of small interfering RNAs targeting human VEGF [13]. However, there have been only a few studies on the antiapoptotic effects of VEGF in intestinal epithelial cells, and the function of VEGF in these cells has not been determined [7]. In this study, we evaluated the protective effect of VEGF in rats for ameliorating NEC with LPS/hypoxia-induced NEC and found that pretreatment with VEGF by IP or OR administration.
Recently, VEGF has been reported to inhibit apoptosis of intestinal epithelial cells by promoting angiogenesis [14], and decreases in VEGF may be associated with NEC pathogenesis in humans and experimental models [15]. Apoptosis is a mechanism of cell death that is involved in the regulation of tissue homeostasis and is regulated by 2 major pathways: namely, the extrinsic and intrinsic pathways [16]. In our study, we evaluated the expression levels of Fas, FasL, caspase-3, Bcl-2, Bax, p53, and NF-κB, which are components of the extrinsic and intrinsic apoptosis pathways, in intestinal epithelial cells. The Bcl-2/Bax pathway is the main regulator of apoptosis; both Bcl-2 and Bax are downstream genes of the p53 gene, whose activation induces apoptosis [17]. Moreover, previous studies have suggested that NF-κB directly binds to the FasL promoter site and upregulates the FasL gene, damaging chromosomal DNA, which suggests that NF-κB induces apoptosis through the Fas/FasL pathway [18]. Bcl-2 overexpression abolishes apoptosis triggered by apoptotic stimuli, and the Bax/Bcl-2 ratio modulates susceptibility to apoptosis [19]. Additionally, the Bax/Bcl-2 ratio may upregulate caspase-3 expression [20]. Our results suggest that administered VEGF exerts potential antiapoptotic effects by affecting mRNA and protein expression in rats with induced NEC (Figs. 4, 5). In rats with LPS/hypoxia-induced NEC, the mRNA levels of Fas, FasL, NF-κB, Bcl-2, Bax, p53, and caspase-3 were greatly increased. However, in the NEC + VEGF IP and OR groups, mRNA expression was suppressed (Figs. 4, 5). This result suggests that VEGF blocked LPS/hypoxia-induced apoptosis in NEC rats simultaneously through the Bcl-2/Bax-dependent and Fas/FasL-dependent pathways and that VEGF exerted a preventive effect against apoptosis in rats with LPS/hypoxia-induced NEC.
NF-κBp65 is an important transcription factor that controls inflammatory genes and is activated by cytokines and oxygen metabolites [21]. NF-κB activation appears to be a common factor in the expression of inflammation-associated prosurvival genes, such as Bcl-2, Bcl-x, and VEGF [22]. Additionally, the platelet-activating factor (PAF) and its receptor (PAF-R) appear to play pivotal roles in several inflammatory processes [23]. We evaluated the effect of VEGF on inflammation by assessing the mRNA expression of NF-κB and PAF-R in NEC rats and found that VEGF suppressed the mRNA expression of the inflammation-associated genes PAF-R (Fig. 4G) and NF-κB (Fig. 4C). The upregulated inflammatory pathway in NEC rats was effectively suppressed by VEGF delivered via IP and OR administration.
Additionally, our results showed that the mRNA expression of apoptotic genes was decreased to a greater extent in the NEC + VEGF OR group than in the NEC + VEGF IP group (Fig. 4). This result was in contrast to our findings that the NEC histological score of the NEC + VEGF IP group was lower than that of the NEC + VEGF OR group, compared with that of the control group (Fig. 3). There have been many conflicting studies on IP and OR administration. One comparison of IP and OR administration using the micronucleus test with mitomycin C showed that higher values were obtained with IP administration than with OR administration [24], and another study showed that inhibition of colon cancer metastasis with lunasin in vivo was more effective with IP administration than with OR administration [25]. In contrast, amifostine has been found to be as effective when delivered by OR administration as when delivered by IP administration in reducing electron paramagnetic resonance decay in vivo [26]. A rapid method for determining effective administration routes for systemic incorporation of VEGF was investigated in this study to support the development of effective therapies. Our results show that the differences in the bioavailability of IP VEGF and OR VEGF affect mRNA expression. Further study is needed to clarify the upregulation of the mRNA expression of apoptosis-associated genes in the NEC + VEGF OR group compared with the NEC + VEGF IP group.
The importance of translation rather than transcription could explain the inconsistency between the mRNA and protein levels of Bcl-2. According to Schwanhausser et al. [27], the amount of protein is much more closely related to the speed of mRNA translation than to the amount of mRNA.
In summary, VEGF may exert a protective effect on intestinal tissue by inhibiting apoptosis in NEC rats. Our observation should encourage the development of VEGF in clinical trials for the treatment of infants with NEC.
Fund/Grant Support:This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2010-0022839).
Conflict of Interest:No potential conflict of interest relevant to this article was reported.
Author Contribution:
Conceptualization: HYK, SK.
Formal Analysis, Methodology: HBY, SHK, SK.
Investigation: HBY, HYK.
Project Administration: HYK.
Writing – Original Draft: HBY, HYK.
Writing – Review & Editing: All authors.
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