The Effect of Hepatocyte Growth Factor on Intestinal Adaption in an Experimental Model of Short Bowel Syndrome

doi:10.21203/rs.3.rs-1617696/v1

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Research Article

The Effect of Hepatocyte Growth Factor on Intestinal Adaption in an Experimental Model of Short Bowel Syndrome

https://doi.org/10.21203/rs.3.rs-1617696/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 11 Jan, 2023

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Purpose

Nowadays, the standard therapy for patients with short bowel syndrome is parenteral nutrition (PN). Various growth factors have been tested in order to achieve weaning from prolonged PN administration. We evaluated the effect of hepatocyte growth factor (HGF) on structural intestinal adaptation and cell proliferation in a rat model of SBS.

Methods

Thirty Sprague-Dawley rats were divided into three groups; group A rats (sham) underwent bowel transection, group B rats underwent a 75% bowel resection and group C rats underwent the same procedure but were treated postoperatively with HGF. Histopathologic parameters of intestinal adaptation were determined, while a microarray and a rt-PCR analyses of ileal RNA was also performed.

Results

Treatment with HGF resulted in significant increase in body weight, while the jejunal and ileal villus height and crypt depth were increased in HGF rats (36%, P < 0.05 and 27%, P < 0.05 respectively). Enterocyte proliferation was also significantly increased in HGF rats (21% P < 0.05). Microarray and quantitative rt-PCR analyses showed that the genes hgfac, rac 1, cdc42 and akt 1 were more than two-fold up-regulated after HGF treatment.

Conclusion

HGF emerges as a growth factor that enhances intestinal adaptation. The future use of HGF may potentially reduce the requirement for PN in SBS patients.

short bowel syndrome

hepatocyte growth factor

intestinal adaptation

cell proliferation

gene up-regulation

Short bowel syndrome (SBS) is a rather uncommon condition of malabsorption and malnutrition resulting from the loss of intestinal absorptive surface area. In adults, SBS may be defined as a length of functional bowel less than 2 meters; in infants is defined as the loss of 75% of the functional length which is expected for the gestational age [1]. The most prominent causes of SBS are mesenteric ischemia in adults and necrotizing enterocolitis in neonates [2]; other conditions which can lead to SBS are volvulus, intestinal atresia, radiation enteritis and malignancies. To date, parenteral nutrition (PN) is the standard therapy for patients with SBS; it has been reported that 10–15,000 patients in Europe have PN-dependent SBS, with similar estimations in the USA [3]. However, the probability of weaning from PN five years is only 55% and depends on functionality of remnant small bowel [4].

An important mechanism for weaning from PN in patients with SBS is intestinal adaptation. Intestinal adaptation occurs shortly after massive loss of bowel competence and its degree depends on many factors, such us the length of remaining bowel, the underlying disease and the activation of cytokines and hormones. Intestinal adaptation consists of proliferation of enterocytes and their differentiation, along with growth of other epithelial and mesenchymal cells, resulting in increase in both villus height and crypt depth. In that way, not only the absorptive surface area is increased, but also the peptide, glucose and electrolyte uptake is facilitated [5].

Intestinal adaptation is mediated by many cytokines and growth factors. Therefore, over the past decades, a lot of animal and clinical studies utilizing hormones and growth factors for augmentation of intestinal adaptation have been implemented, aiming the further increase in nutrition absorption and thus earlier weaning from PN in patients with severe SBS. Growth factor (GH), insulin-like growth factor 1 (IGF-1) and glycagon-like peptide 2 (GLP-2) have been extensively studied, with promising results [6]; in fact, GH and GLP-2 analog, teduglutide, have been approved for treatment in SBS [7]. Hepatocyte growth factor has been scarcely investigated for intestinal adaptation, although it is a multipotent cytokine, with mitogenic and morphogenic activity in many viscera [8].

Hepatocyte growth factor (HGF) and its receptor, c-MET, are expressed in various organs; the product of activation, c-MET tyrosine kinase, recruits signaling effectors, inducing multiple cell activities such as mitogenesis, cell survival and anti-fibrosis [9]. However, little is known about the role of HGF in intestinal adaptation. On the contrary, it is widely accepted that activation of HGF/c-MET signaling pathway is crucial for liver regeneration, especially in cases of liver diseases, via eliciting the proliferation of hepatocytes [10]. Τhis hepatotropic role of HGF may be of high significance in patients with SBS who develop intestinal failure-associated liver disease (IFALD), one of the most devastating complications of SBS. In this experimental study, we examine the effect of HGF administration on intestinal tissue after massive small bowel resection. We examine the impact of the treatment with HGF on the parameters of intestinal adaptation and we investigate the mechanisms of these results.

Animals

The study has been approved by the Veterinary Department of the Region of Attica under the code 441/22-01-2016 and the local ethics committee under the code 1281/1-2-16. Animal care complied with the Guide for the Care and Use of Laboratory Animals. The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were consulted throughout the conduct of this study [11]. Thirty Sprague-Dawley, non-genetic modified female rats, obtained from Hellenic Pasteur Institute, weighing 200–250 g were kept in stainless steel cages in a controlled environment (22°C with a 12-h light/dark cycle) for at least 7 days. The animals had free access to water and standard rat chow. The rats were divided in 3 groups: group A (sham), consisted of 10 rats who underwent small bowel transection and reanastomosis; group B (MSBR), consisted of 10 rats who underwent 75% small bowel resection followed by end-to-end jejuno-ileal anastomosis; group C (MSBR – HGF), consisted of 10 rats who underwent the same procedure, but were treated postoperatively with HGF. The number of animals per group was dedicated using IBM SPSS Sample Power 3.0.

Experimental protocol

All surgical procedures were performed after appropriate animal preparation and under sterile conditions. The animals received general anesthesia with ketamine (50 mg/kg) and xylazine (14 mg/kg) administered intramuscularly and the abdomen was opened with a midline incision. Group A rats underwent small bowel transection with reanastomosis, 7 cm proximal to ileocecal valve. In group B and group C animals, a 75% resection of the small intestine was performed, preserving the vascular arcade and leaving 5 cm of jejunum distal to Treitz ligament and 7 cm of ileum proximal to ileocecal valve. The intestinal continuity was restored by an end-to-end single layer anastomosis using interrupted 5/0 monofilament silk sutures (SMI, Belgium). Finally the abdominal cavity was closed in two layers with interrupted 3/0 Vicryl sutures. Immediately after operation, the rats were resuscitated with an intraperitoneal injection of 3 ml 0.9% saline solution.

Postoperatively, the animals had access only to water for the first 2 days following operation. From the 3rd postoperative day rats were fed with standard (10% kcal fat) diet and water ad libitum. Starting from the 4th postoperative day, the animals of group C were treated with HGF administered intraperitoneally at a dose of 250 µg/kg/day. General condition, body weight and food intake were monitored on a daily basis. At 15th postoperative day the animals were anesthetized with ketamine (50 mg/kg) and afterwards were sacrificed applying open pneumothorax. After assuring that no peritonitis was developed, the remnant small bowel was measured, using a suture extending from Treitz ligament to ileocecal valve. Then, the entire small bowel was resected, cut longitudinally, rinsed with saline, weighted and harvested. Bowel weight was expressed as weight per unit of bowel length per 100g body weight (mg/cm/100g) in order to reduce bias.

Morphometry

For the histopathological examination we used samples from both jejunum (1 cm distal to the Treitz ligament) and ileum (1 cm proximal to the ileocecal valve). The samples were fixed for 24 hours in 10% formalin solution, and then were dehydrated with ethanol and xylene and embedded in paraffin cubes. Subsequently, 3.5 µm thick sections from paraffin cubes were cut using a microtome and stained with hematoxylin-eosin staining. The sections were examined microscopically using an light microscope (Nikon Eclipse 80i, Nikon Corp, Tokyo) equipped with a digital camera (Nikon Corp.) under 10x4 original magnification. The images were then analyzed with the Image Pro Plus 5 software (Media Cybernetics, Baltimore, MD, USA). From each histological section we measured ten villi and crypts in µm and the mean height or depth was recorded.

Immunohistochemistry

The rate of enterocyte proliferation was assessed with Ki-67 antibody (MIB-1, Ki-67 concentrated and prediluted rabbit monoclonal antibody; dilution 1:50; Biocare Medical,Walnut Creek, CA). From the prepared paraffin cubes, additional 3.5 µm thick sections were cut and placed on poly-L-lysine coated slides. The sections were then be deparaffined, hydrated and placed in Tris / EDTA buffer, pH 9. After being rinsed with water for 5 minutes, the sections were incubated in 3% hydrogen peroxide for 10 minutes in a dark room, in order to inhibit the endogenous peroxidase activity and then were rinsed again with water and TBST for 5 minutes. The sections were thereafter incubated with Ki67 primary monoclonal antibody (MIB-1), rinsed with TBST three times and then were incubated with rabbit HRP-labeled polymer (Envision + System-HRP, DakoCytomation, Glostrup, Denmark) for 45 minutes. After another immersion in TBST for 5 minutes, the placement of DAB chromogen for 5 minutes and another rinsing with TBST followed. Hematoxylin was used to anneal the sections for 40 seconds, which thereafter was rinsed with plenty of water for 5 minutes. Finally, the sections were dehydrated with graded alcohol solutions, immersed in xylene, and covered with a gasket using GLC fixing agent. The proliferation index (PI) was defined as the number of proliferating crypt cells (stained positively for Ki-67) per 10 crypts.

The apoptosis rate was determined with monoclonal antibody caspase 3 (caspase-3 cleaved concentrated polyclonal antibody; dilution 1:100; Biocare Medical,Walnut Creek, CA). Additional 3.5 µm thick sections were cut and, using a combination of the streptovidin–biotin–peroxidase method and microwave antigen retrieval on formalin-fixed, paraffin-embedded tissues according to the manufacturer’s protocols were further analyzed. Based on the above immunohistochemical staining, the apoptotic index (AI) was defined as the number of apoptotic cells per 10 villi.

Microarray analysis

From each rat, an additional part from ileum was rinsed with saline and using a glass slide, the intestinal mucosa was collected in a RNA stabilization reagent (RNAlater® Thermo Fisher Scientific, Waltham, MA, USA) and the sample was immediately frozen. The RNA isolation was performed according to the TRI Reagent® protocol (Sigma-Aldrich, St. Louis, MO, USA) and the concentration of RNA in the sample was determined by spectrophotometer. Then, part from the total RNA was amplified into cRNA and biotinylated by in vitro transcription using the TargetAmp Nano-g Biotin-aRNA labeling kit for the Illumina system (picentre Biotechnologies, San Diego, CA, USA) according to the manufacturer's protocol, with 200 ng of total RNA as input material. Biotinylated cRNAs was purified, fragmented, and subsequently hybridized to an Illumina RatRef-12 Expression BeadChip V1 (Illumina inc., San Diego, CA, USA) for genome-wide expression analysis containing 21,910 probes selected primarily from the NCBI RefSeq database (Release 16) according to the Direct Hybridization assay. The hybridized chip capable of querying 12 samples in parallel was stained with streptavidin-Cy3 (Amersham™, Sigma-Aldrich, St. Louis, MO, USA) and scanned with an Illumina BeadArray 500GX Reader. The scanned images were imported into GenomeStudio (Illumina Inc.) for extraction and quality control, generating an output file for statistical analysis.

Real-time PCR

The remnant purified RNA was redissolved and DNase (Thermo Fisher Scientific, Waltham, MA, USA) was used to remove DNA traces. The RNA was transcribed into cDNA according to the PrimeScript RT Reagent kit gDNA eraser protocol (Takara, Kioto, Japan). The cDNA, along with genes which were upregulated or downregulated with mor than 2-fold change, were amplified by Real-Time Polymerase Chain Reaction (PCR) according to the Quantitect GRE protocol PCR kit (Qiagen, Hilden, NRW, Germany) on Applied Biosystems 7500 fast. GAPDH was used as a housekeeping gene.

Statistical analysis

All data are expressed as means ± standard error (SE). The one-way ANOVA test was used for comparison, followed by Tukey’s test for pairwise comparison, while the Prism software was used (GraphPad Software, San Diego, CA) was used for statistical analysis. The statistical significance was defined as p < 0.05. Microarray data were analyzed by using the Partek Genomics Suite software program. To evaluate the magnitude of differential gene expression the displacement of each detected transcript’s mean expression value was measured between the two groups. A standard regression analysis was performed on the MSBR and MSBR - HGF groups to test whether the mean transcription level differed from that of the Sham group.

Gross morphology

The fluctuation of body weight after surgery in each study group is presented in Fig. 1. Sham (group A) animals maintained their body weight during the first 4 postoperative days, with gradual increase thereafter. Group B (MSBR) animals showed rapid weight loss during the first 4 postoperative days, with gradual increase thereafter. Of note, weight gain rate in group B animals (MSBR) was faster, although not statistical significant; at day 15th, group B rats gained 17.5% of their weight at day 4, when sham (group A) rats gained 15.5% of their weight at day 4 (p = 0.09). Rats treated with HGF demonstrated an even faster increased weight gain rate compared not only to sham (group A) rats (21.8% vs 15.5% at day 15th, p < 0.01) but also to group B (MSBR) rats (21.8% vs 17.5% at day 15th, p < 0.05).

The intestinal weight at the 15th postoperative day differed also significantly between three study groups (Table 1). Group B (MSBR) animals had statistically significantly increased intestinal weight compared to sham (group A) animals (94 ± 7 vs 49 ± 4, mg/cm/100 g, p < 0.05). HGF-treated rats showed additional significant increase in intestinal weight, not only compared to sham (group A) rats (144 ± 12 vs 49 ± 4, mg/cm/100 g, p < 0.001), but also compared to group B (MSBR) rats (144 ± 12 vs 94 ± 7 mg/cm/100 g, p < 0.05).

Table 1

Effect of HGF on villus height and crypt depth
	Group A	Group B	Group C
Villus height (µm)
Jejunum	419 ± 40	562 ± 38*	765 ± 46*⁺
Ileum	290 ± 33	442 ± 35*	539 ± 4*⁺
Crypt depth (µm)
Jejunum	177 ± 7	209 ± 24*	274 ± 18*⁺
Ileum	163 ± 8	194 ± 5*	212 ± 5*
Group A: sham rats who underwent bowel transection, Group B: rats who underwent massive small bowel resection, Group C: rats who underwent massive small bowel resection and treated postoperatively with HGF
*p < 0.05 vs Sham
⁺p < 0.05 vs MSBR

Intestinal adaptation

The histopathological analysis showed that massive small bowel resection was correlated with enhanced intestinal adaptation (Table 1). Rats of group B (MSBR) demonstrated significant increase in villus height compared to sham (group A) rats in both jejunum (562 ± 38 µm vs 419 ± 40 µm, p < 0.05) and ileum (442 ± 35 µm vs 290 ± 33 µm, p < 0.05). Similarly, group B rats demonstrated also significant increase in crypt depth compared to sham (group A) rats in both jejunum (209 ± 24 µm vs 177 ± 7 µm, p < 0.05) and ileum (194 ± 5 µm vs 163 ± 8 µm, p < 0.05). It is however noteworthy that the treatment with HGF resulted in further augmentation of intestinal adaptation (Tabe 1); rats treated with HGF (group C) had higher mean villus height compared to MSBR rats (group B) in both jejunum (765 ± 46 µm vs 562 ± 38 µm p < 0.01) and ileum (539 ± 4 µm vs 442 ± 35 µm, p < 0.05). Furthermore, MSBR-HGF rats demonstrated significant increase in crypt depth compared to MSBR rats (group B) in jejunum (274 ± 18 µm vs 209 ± 24 µm, p < 0.05), but not in ileum (212 ± 5µm vs 194 ± 5 µm, p = 0.07).

Enterocyte proliferation/apoptosis

The animals of group B (MSBR) demonstrated a higher proliferation rate in both jejunum (160 ± 9 vs 135 ± 2 Ki-67 positive stained cells / 10 crypts, p < 0.05) and ileum (166 ± 10 vs 133 ± 5 Ki-67 positive stained cells / 10 crypts, p < 0.05) compared to group A (sham) animals (Table 2). Rats treated with HGF (group C) demonstrated though even higher proliferation rate compared not only to sham (group A) rats (p < 0.001), but also to group B (MSBR) rats, in both jejunum (208 ± 13 vs 160 ± 9 Ki-67 positive stained cells / 10 crypts, p < 0.01) and ileum (213 ± 14 vs 166 ± 10 Ki-67 positive stained cells / 10 crypts, p < 0.01). Regarding the apoptosis rate, group B (MSBR) animals had higher rate of apoptosis in jejunum compared to sham (group A) animals (1.7 ± 0.3 vs 1.1 ± 0.2 Caspase-3 positive stained cells/ 10 Villi, p = 0.07), while the HGF-treated rats (group C) had statistically significant lower apoptosis rate compared to group B (MSBR) animals (1.3 ± 0.2 vs 1.7 ± 0.3 Caspase-3 positive stained cells/ 10 Villi, p < 0.05) but not compared to sham (group A) rats (1.3 ± 0.2 vs 1.1 ± 0.2 Caspase-3 positive stained cells/ 10 Villi, p = 0.09). Similar results were found in ileal samples (Table 2), as the group B rats (MSBR) presented statistically significantly higher apoptosis rate compare to sham animals (2.2 ± 0.3 vs 1.5 ± 0.2 Caspase-3 positive stained cells/ 10 Villi, p < 0.05) and concomitantly rats treated with HGF demonstrated lower apoptosis rate compared to group B (MSBR) rats (p < 0.001).

Table 2

a: Effect of HGF on cellular proliferation/apoptosis
Cellular proliferation	Sham	MSBR	MSBR - HGF
Ki-67 cells/ 10 crypts
Jejunum	135 ± 2	160 ± 9*	208 ± 13*⁺
Ileum	133 ± 5	166 ± 10*	213 ± 14*⁺
Cellular apoptosis	Sham	MSBR	MSBR - HGF
Caspase-3 Cells/ 10 Villi
Jejunum	1.1 ± 0.2	1.7 ± 0.3*	1.3 ± 0.2⁺
Ileum	1.5 ± 0.2	2.2 ± 0.3*	1,6 ± 0.3⁺
Group A: sham rats who underwent bowel transection, Group B: rats who underwent massive small bowel resection, Group C: rats who underwent massive small bowel resection and treated postoperatively with HGF
*p < 0.05 vs Sham
⁺p < 0.05 vs MSBR

Microarray analysis

From the total amount of 21,910 probes, 165 genes were differentially expressed in animals of group B (MSBR) compared to sham animals. From these, 91 genes were up-regulated and 74 genes were down-regulated, while 10 genes were more than two-fold regulated. In HGF-treated rats (group C), a total of 212 genes were differentially expressed, with 127 up-regulated genes and 85 down-regulated genes; 16 genes were more than two-fold regulated. Functional clustering analysis revealed increased expression in signaling pathways involved in aminoacid (tryptophan, glycine, serine and threonine) and fatty acid metabolism, as well as propanoate, ascorbate and aldarate metabolism and biosynthesis of steroids. Next we investigated 22 genes related to HGF/c-MET signaling pathway (Table 3). In animals of group B (MSBR), 7 genes were up-regulated with a relative change in gene expression level of 10% or more, 12 genes remained practically unchanged and 3 genes were down-regulated. In HGF-treated animals (group C), 16 genes were up-regulated, 5 genes remained unchanged and 1 gene was down-regulated.

Table 3

Changes in HGF/c-MET pathway-related genes in MSBR and MSBR – HGF animals compared to sham animals by microarray analysis.
Gene symbol	Gene ID	Definition	Fold changes (MSBR/Sham)	Fold changes (MSBR-HGF/Sham)
Hgfac	ILMN_65182	Hepatocyte Growth Factor Activator	↓1.06312	↑ 2.16277
Pxn_predicted	ILMN_59825	Paxillin	↓1.02722	↑ 1.12877
Rac1	ILMN_56272	Ras-related C3 botulinum toxin substrate 1	↑1.07334	↑2.47382
Cdc42	ILMN_66990	Cell Division Cycle 42 gene	↑1.05503	↑2.22894
Cdc42ep5_predicted	ILMN_49668	CDC42 Effector Protein 5	↑1.05252	↑1.82796
Gab1_predicted	ILMN_54311	GRB2-Associated Binding Protein 1	↓1.08883	↓1.08055
Crkl	ILMN_57075	V-Crk Avian Sarcoma Virus CT10 Oncogene Homolog-Like	↓1.03078	↓1.04937
Dock6_predicted	ILMN_70295	Dedicator of cytokinesis 6 (Zir1)	↑1.16058	↑1.03797
Dock9	ILMN_60189	Dedicator of cytokinesis 9	↑1.07774	↑1.0348
Stat3	ILMN_58556	Signal Transducer And Activator Of Transcription 3 (Acute-Phase Response Factor)	↑1.58407	↑2.19494
Rap1b	ILMN_65723	Ras-related protein Rap-1b	↑1.15574	↑1.59333
Rap1a	ILMN_48993	Ras-related protein Rap-1a	↓1.05049	↑1.06713
Prap1	ILMN_66279	Proline-Rich Acidic Protein 1	↓1.18027	↑1.17729
Akt1	ILMN_47983	serine-threonine protein kinase AKT1	↓1.21519	↑2.18446
Dag1	ILMN_49612	Dystroglycan 1 (Dystrophin-Associated Glycoprotein 1)	↑1.27953	↓1.31317
Ets2	ILMN_62425	V-Ets Avian Erythro-blastosis Virus E26 Oncogene Homolog 2	↑1.08077	↑1.52014
Jun	ILMN_51512	Jun Proto-Oncogene	↑1.26899	↑1.37949
c-fos	ILMN_64186	Proto-Oncogene C-Fos	↑1.5262	↑1.75456
Vegfa	ILMN_65567	Vascular endothelial growth factor a	↓1.15799	↑1.76655
Vegfb	ILMN_59695	Vascular endothelial growth factor b	↑1.06517	↑1.30657
Vegfc	ILMN_59695	Vascular endothelial growth factor c	↑1.21701	↑ 1.32068
Spint1	ILMN_47993	Serine Peptidase Inhibitor, Kunitz Type 1	↓1.02528	↑1.22424
↓ down-regulation vs sham animals
↑ up-regulation vs sham animals

Real-time PCR

Of the 22 genes associated with the HGF/c-MET pathway, 5 genes were more than two-fold up regulated (p < 0.05) after treatment with HGF, according to microarray analysis. These genes were: hgfac (Hepatocyte Growth Factor Activator), rac 1 (Ras-related C3 botulinum toxin substrate 1), cdc42 (Cell Division Cycle 42 gene), stat-3 (Signal Transducer And Activator Of Transcription 3 (Acute-Phase Response Factor) and akt 1 (serine-threonine protein kinase AKT1); these genes were further investigated with real-time quantitative PCR. The results confirmed the fold-changes compared to sham (group A) animals (p < 0.05). Interesting is though, that all genes except stat-3 were statistically significantly more up-regulated in the animals treated with HGF (group C) compared to animals which had undergone massive small bowel resection (group B) (p < 0.05). All study results can be found in our institutional Databank.

In this study we investigated the possible effect of hepatocyte growth factor (HGF) on intestinal adaptation after massive small bowel resection. In the literature is well reported that treatment with hormones and growth factors such as GLP-2, IGF and EGF can augment the intestinal adaptation process; yet, little has been known about HGF. The experimental model that we used mimics the massive small bowel loss which is prominent in short bowel syndrome and has been used repeatedly in the past [12–14]. Our results indicate that HGF has a positive effect on all parameters of intestinal adaptation. Moreover, admission of HGF provokes the encoding and secretion of specific proteins which play important roles in the intestinal adaptation process.

Shortly after a massive loss of intestinal length, the intestine undergoes an adaptive response in order to increase the functionality of the remnant mass. In humans, intestinal adaptation begins 16h after loss of intestinal mass, and is mainly characterized by increased cellular proliferation [15]. The second phase of intestinal adaptation starts 1–2 weeks after intestinal loss and is characterized by cellular differentiation resulting in increase of absorptive area, and glucose and electrolyte uptake [16]; this phase will take place for 4–6 weeks. At the beginning, the mesenchymal cells, which are located under the epithelium in the crypts, are proliferating and condensing there. As these cells start to differentiate, they migrate toward the central lumen to form the villi; therefore each villus contains epithelial cells from the adjacent crypts [17]. Differentiation and migration of these cells are mediated by various signaling pathways, such as Wnt/β-catenin [18], Notch [19] and Hedgehog [20]. This hyperplasia results in increased absorptive competence per unit of intestinal length, as the new epithelial cells contribute to amino acid, glucose and electrolyte uptake; moreover, the villi are elongated and the crypts are deepened, thus the absorptive surface area is increased. At the same time, the number of goblet cells in the villi is increased, probably through switch of some enterocytes [15]. Goblet cells produce epithelial-protective mucins that are involved in intestinal wound healing [21]. In the same fashion, Paneth cells’ absolute number increases in the crypts, contributing to intestinal adaptation process by producing stimulating factors [15].

The extend of intestinal adaptation ranges between cases and it is believed that it is proportional to the amount of intestine loss [22]. Therefore, it comes as no surprise that massive small bowel resection enhanced almost all parameters of intestinal adaptation. Although the animals which underwent massive small bowel resection experienced a rapid weight loss the first 4 days postoperatively, they had greater body ant intestinal weight gain compared to sham animals, due to increased nutrient uptake. On a microscopic level. this increase is feasible due to increased absorption surface. These findings are consistent with the findings from our previous study [12] and from other similar studies [13, 23] and prove the efficacy of our experimental model. Interestingly, the chances in morphometry were more prominent in ileal samples; this is consistent with previous findings suggesting that distal ileum adapts faster and demonstrates greater morphological changes [24, 25]. The proliferation and apoptosis rate were also higher in ileum compared to jejunum, finding that is consistent with previous studies, which have shown that proliferation and apoptosis is relatively more evident in ileum than in jejunum [26, 27].

A finding of special interest was that the massive intestinal resection triggered the up-regulation of many genes belonging to the HGF/c-MET system, suggesting the participation of this pathway in the stages of intestinal adaptation [28]. From them, of specific interest was the upregulation of stat-3 gene. STAT-3 is a pleiotropic transcription factor that regulates cell stress response and promotes wound healing [29]. STAT3 is phosphorylated by receptor-associated Janus kinases (JAK) in response to cytokines and growth factors, including HGF, and translocate to the cell nucleus where they act as transcription activators [30].

Hepatocyte growth factor, also known as scatter factor, was first described in 1984 in two independent studies [31, 32] and its cDNA was firstly cloned in 1989 [33]. It is secreted from non-parenchymal cells as an inactive form (pro-HGF), which can be activated by proteolysis [34]. The proteolysis is mediated by various proteases, including HGF activator (HGFA), coagulation factors XII and XI, kallikrein, hepsin and plasminogen activators. In its active form is a heterodimer composed of a 69-kD α-chain and a 34-kD β-chain [35]. The α-chain contains a N-terminal hairpin domain and four subsequent kringle domains, and the β-chain contains a domain similar to serine proteases, though without proteolytic function. The kringle domain is also similar to this found in serine proteases for thrombolysis, thus HGF has a 38% amino acid homology to plasminogen [35]. HGF receptor, tyrosine kinase c-Met, is the product of the proto-oncogene c-met and is expressed in the parenchymal cells of many viscera, such as liver, intestine, kidney and bone marrow [36]. In its active form is an α/β disulphide- linked heterodimer with a 50-kD α-chain and a 145-kD β-chain. The extracellular part has three domain types; a large semaphoring (Sema) domain, the PSI domain and the IPT domain which connects the PSI domain with the transmembrane helix. Intracellularly, the c-MET receptor contains a tyrosine kinase catalytic domain flanked by distinctive juxtamembrane and carboxy-terminal sequences [9]. The binding of HGF to c- Met induces the activation of this tyrosine kinase, which recruits signaling effectors eliciting paths of cell proliferation, cell survival, cell differentiation and morphogenesis.

Since HGF/c-MET signaling activates a wide range of different cellular pathways, we hypothesized that HGF promotes intestinal tissue regrowth after massive bowel resection. The animals treated with HGF had significantly greater body and intestinal weight gain compared not only to sham animals but also to animals underwent massive small bowel resection. Villus height and crypt depth were also significantly increased compared to untreated animals. Moreover, cellular proliferation was enhanced while apoptosis was delineated in HGF-treated animals resulting in greater augmentation of intestinal hyperplasia. This augmentation can be explained by the regulation of genes associated with cell proliferation, cell survival, cell differentiation and migration. Microarray analysis showed that a total of 212 genes were differentially regulated after administration of HGF while real-time PCR showed a two-fold increase of hgfac, rac1, CDC42, akt1 and stat-3. Of particular interest is though the two-fold increase of rac 1 and cdc 42. Both CDC42 and Rac 1 are involved in regulation of the signaling pathways that control diverse cellular functions including cell morphology, migration, cell-cell adhesion, endocytosis and cell cycle progression. Furthermore, CDC42 and Rac1 are key signaling switches to actin cytoskeleton reorganization [37] that ensure coordinated control of different cellular activities such as gene transcription and adhesion through their interaction with multiple target proteins [38].

The up-regulation of akt 1 after treatment with HGF is also a remarkable finding. The PI3-kinase/Akt signaling pathway has a significant anti-apoptotic role; akt phosphorylates the IκB kinase, which results in IκB degradation and allows NF-kB to enter the nucleus and activate transcription of anti-apoptotic genes [39]. Another possible mechanism of the anti-apoptotic effect after HGF administration is the regulation of MAPK pathway. HGF/c-MET elicits the activity of RAS and via the Gab1/Grb2/SOS cascade, the MAPK effector kinase and ultimately MAPK are activated. MAPK pathway promotes cell scattering and tubulogenesis [40], but many studies have shown that is also involved in an anti-apoptotic signaling elicited by HGF [29].

The idea of using growth factors for augmentation of intestinal adaptation is not new; several studies, both experimental and clinical, have delineate the role of glycagon-like peptide 2 (GLP-2) in intestinal adaptation [6]. In fact, teduglutide, the GLP-2 analog, has been approved for treatment in patients with SBS [7]. Growth factor (GH), insulin-like growth factor 1 (IGF-1) [7], epidermal growth factor (EGF) [40], and glycagon-like peptide 1 (GLP-1) [6] have been also extensively studied, with decent results in terms of intestinal adaptation. On the contrary, HGF has not been examined in that extent. An interesting study from Kato et al conducted that treatment with HGF increased galactose and glycine absorption, as well as overall mucosal mass in rats after massive small bowel resection [41]. From the same institution, Katz et al reported similar results with significant increase of morphological parameters of intestinal adaptation after HGF treatment in an experimental model of short bowel syndrome [42]. In our study, the effect of HGF in intestinal adaptation was correlated with specific genetic pathways, providing more information about the HGF mechanisms on intestinal tissue.

Although the role of HGF in intestinal adaptation has not been extensively studied, its role in other viscera and especially in lungs, heart and liver has been decently delineated. Its antiapoptotic effect on damaged cardiomyocytes after myocardial infarction as well as its antifibrotic and antiinflammatory activity make HGF a promising agent for myocardial infarction therapy [43]. Moreover, Kinoshita et al reported that HGF mRNA levels are increased in response of extended hepatectomy in rats [44] and Huh et al highlighted the importance of HGF activation in liver regeneration after extended hepatectomy [8]. Today we know that HGF/c-MET is essential for preventing cell death of hepatocytes, yet, the endogenous HGF levels are insufficient for total liver injury reversal and a supplementary therapy is required [10]. More specifically, HGF acts as an antinecrotic factor, which inhibits cholestatic hepatitis and also acts as an antifibrotic factor which inhibits the hepatic fibrosis process by stimulating the hepatocyte proliferation [45]. This hepatotropic role of HGF may be of high significance in patients with short bowel syndrome who develop IFALD. IFALD is one of the most frequent complications in patients with short bowel syndrome receiving PN, as it can occur in up to 64% of all cases [46] and about 4% of all deaths in patients with SBS can be directly related with it [47]. IFALD is characterized by elevated liver function tests, cholestatsis (most prominent in children), steatosis (most prominent in adults) and even fibrosis. To date, the possible therapeutic role of HGF in IFALD has not been adequately studied; however, in an experimental model of chronic cholestatic liver injury in rats, HGF treatment attenuated hepatic inflammation by diminishing the levels of Il-6 and TNF-α, while preserved the hepatic ductal structure [48]. Liu et al also conducted that in an intestinal ischemia-reperfusion - induced liver injury model, HGF/c-MET pathway mediated mesenchymal stem cells which ultimately attenuated the increase of liver function tests and improved the histopathological changes [49]. Therefore, utilization of HGF in patients with short bowel syndrome may be greatly beneficial, as not only enhances intestinal adaptation, but also has a hepatoprotective effect. Regarding the safety of HGF gene therapy, potential deleterious effects, such as hepatic or intestinal malignancy can be avoided, as the HGF treatment would last a limited time. The cessation of gene expression beyond this time point can be considered to constitute an inherent safety feature of HGF gene transfer. All in all, no major clinical study with long-term follow-up questioning the long-term safety of HGF therapy has been undertaken, and more large, randomized trials are needed in order to establish not only the efficacy, but also the safety of HGF therapy.

In conclusion, treatment with HGF enhances intestinal adaptation after massive small bowel resection. Cell proliferation is augmented and therefore the intestinal absorption surface area is lengthened. Our analysis showed that the activation of HGF/c-MET pathway results in up-regulation of specific genes which are related with cell proliferation and differentiation.

Author contributions

All authors contributed to the study conception and design. Animal care and operative procedures were performed by George Bagias, Evangelos Misiakos and Evangelos Giamarelos - Bourboulis. Tissue preparation and histological analysis were performed by Stratigoula Sakellariou. Microarray analysis was performed by Igor Sukhotnik and rt-PCR analysis was performed by George Bagias. Data collection and analysis were performed by George Bagias, Anestis Charalampopoulos and Nick Zavras. The first draft of the manuscript was written by George Bagias and Evangelos Misiakos, Dimitrios Schizas and Emmanouil Pikoulis commented on previous versions of the manuscript and conducted the final version. All authors read and approved the final manuscript.

Declaration of Interest

The authors have no conflicts of interest to declare that are relevant to the content of this article

Funding

No funding was received for conducting this study.

Ethics approval

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The Effect of Hepatocyte Growth Factor on Intestinal Adaption in an Experimental Model of Short Bowel Syndrome

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Intestinal adaptation

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