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 . 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 ; 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 . Differentiation and migration of these cells are mediated by various signaling pathways, such as Wnt/β-catenin , Notch  and Hedgehog . 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 . Goblet cells produce epithelial-protective mucins that are involved in intestinal wound healing . In the same fashion, Paneth cells’ absolute number increases in the crypts, contributing to intestinal adaptation process by producing stimulating factors .
The extend of intestinal adaptation ranges between cases and it is believed that it is proportional to the amount of intestine loss . 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  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 . 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 . 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 .
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 . It is secreted from non-parenchymal cells as an inactive form (pro-HGF), which can be activated by proteolysis . 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 . 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 . 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 . 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 . 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  that ensure coordinated control of different cellular activities such as gene transcription and adhesion through their interaction with multiple target proteins .
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 . 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 , but many studies have shown that is also involved in an anti-apoptotic signaling elicited by HGF .
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 . In fact, teduglutide, the GLP-2 analog, has been approved for treatment in patients with SBS . Growth factor (GH), insulin-like growth factor 1 (IGF-1) , epidermal growth factor (EGF) , and glycagon-like peptide 1 (GLP-1)  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 . 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 . 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 . Moreover, Kinoshita et al reported that HGF mRNA levels are increased in response of extended hepatectomy in rats  and Huh et al highlighted the importance of HGF activation in liver regeneration after extended hepatectomy . 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 . 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 . 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  and about 4% of all deaths in patients with SBS can be directly related with it . 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 . 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 . 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.