Therapeutic Effects and Repair Mechanism of HGF Gene Transfected Mesenchymal Stem Cells on Injured Endometrium via Activating Phosphorylated c-Met

There are many studies on the advantages of mesenchymal stem cells (MSCs) that could secret various paracrine factors in repairing endometrial injury. It is necessary to improve the stability and effectiveness of MSCs. Hepatocyte growth factor (HGF), as one of the cytokines secreted by MSCs, plays a signicant role in vascular repair and mesenchymal to epithelial transformation (MET). It can be deduced that HGF is closely related to the repair process of endometrium.Therefore, we aim to investigate the effect and mechanism of MSCs from umbilical cord transfected with HGF gene in the damaged mouse endometrium.

There are many studies on the advantages of mesenchymal stem cells (MSCs) that could secret various paracrine factors in repairing endometrial injury. It is necessary to improve the stability and effectiveness of MSCs. Hepatocyte growth factor (HGF), as one of the cytokines secreted by MSCs, plays a signi cant role in vascular repair and mesenchymal to epithelial transformation (MET). It can be deduced that HGF is closely related to the repair process of endometrium.Therefore, we aim to investigate the effect and mechanism of MSCs from umbilical cord transfected with HGF gene in the damaged mouse endometrium. The healthy endometrium is one of the essential factors in pregnancy, otherwise severe damage of endometrial basement layer in women of childbearing age can cause intrauterine adhesions (IUA) or endometrial thinning, leading to endometrial brosis, amenorrhea, infertility or abortion [1][2][3]. Several therapeutic methods have been tried to improve the regeneration of the endometrium. However, the complex nature of the uterine environment and function limits the possibilities of effective treatment. Therefore, it is an urgent gynecological need to explore novel methods to repair endometrial damage, improve its microenvironment and tolerance status, and enhance the pregnancy rate [4][5][6].

Methods
Previous studies have demonstrated that the administration of mesenchymal stem cells(MSCs)can promote the endometrial regeneration in morphology and function, and the mechanism of endosmetrial regeneration were explored. Accumulated evidences shows that the functional bene t of transplanted MSCs are probably due to the paracrine effect of cytokines produced by transplanted MSCs [7][8][9]. To maintain the paracrine effect of MSCs, researchers have combined materials or pretreated MSCs with chemical drugs to promote endometrial regeneration after damage [10][11][12]. However, it may be a more feasible way to enhance the expression of reparative genes in transplanted MSCs to synergistically restore endometrial function after injury.
Hepatocyte growth factor (HGF) is a reparative gene among the numerous cytokines secreted by MSCs [13,14]. HGF has a trophic effect on many types of cells, such as epithelial, endothelial and stromal cells, by promoting mitotic activity, growth and migration and preventing apoptosis [15]. It had been explored the combination of cell and gene therapy strategies using MSCs genetically modi ed to overexpress HGF as a single treatment in some disease model, such as myocardial infarction, hepatocirrhosis, healing of burn wounds, and proved that they have achieved better therapeutic effect then normal MSCs [16-18].
MSCs transfected with HGF can play a better effect in these diseases is precisely because of the function of HGF, which plays an important role in vascular repair and mesenchymal epithelial transformation (MET) process. Similarly, the key mechanisms of endometrial repair are the angiogenesis and MET process. Therefore, MSCs transfected with HGF can enhance the effect of endometrial repair theoretically.
In the current study, we transfected MSCs with HGF genes to investigate the bene cial effects in injured endometrial mouse model, and explore the mechanism of its reconstruction and regeneration.

Preparation of HGF gene transfected MSCs
Human umbilical cords were obtained from full-term cesarean section surgery in The First A liated

Establishment of the mouse endometrial injury model
Eight-week-old female C57BL/6 mouse(SPF level, Wei Tong Li Hua)were used in the study. They were housed ve to six mouse per cage at room temperature,with humidity 40-60% light/dark cycle 12h/12h, and free access to food and water. The mice were anesthetized and the uterine horns were exteriorized through a midline incision under sterile surgical conditions, then injected about 50ul 95% ethanol until lling one side of the uterine horns, rinsed with saline three time nextly, repositioned the uterus gently then sutured the wound and waited for anesthesia to wake up. Ethanol-treated experimental and saline-treated control mouse were examined after 5 days to validate the establishment of the endometrial injury model.

Groups and treatment
For the in-vivo experiment, 8-week-old female mice were randomly divided into four groups. Each group consisted of 15 mice, a total of 60 mice with synchronous estrous cycle. The groups are as follows: Control group (did not undergo any treatment), Saline group (saline injected post-injury group), MSCs group (MSC injected post-injury group), and HGF gene transfected MSCs (MSCs hgf ) group (HGF gene transfected MSCs injected post-injury group). Cells (2×10 5 ) were injected in a total volume of 200 ul, and were injected into the tail vein on the fth day after injury of mouse endometrium. Consider the half-life of living cells in vivo, we select to execute the mouse one week after cell transplantation and take out the uterine tissue. Additionally, we con rmed that all experiments and methods were performed in accordance with the relevant ethical guidelines and regulations, and they were approved by the Experimental Center of the First A liated Hospital of Anhui Medical University.

Hematoxylin and eosin (H&E) staining
One estrous period after treatment, the "Y" uterus was completely removed from the abdominal cavity. The tissues were para n embedded, sectioned (4um) and stained with hematoxylin and eosin (H&E). The morphological changes of mouse uterus were observed by inverted phase contrast microscope. The thickness of endometrial epithelium and the number of glands were recorded. Select 10 elds in each image to count.
Immuno uorescence analysis Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA from rat uteri was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocols; 1 µg of total RNA was subjected to reverse transcription of mRNA using oligo dT as a primer and a reverse transcription kit (Transgen Biotech, Beijing, China) to generate total cDNA.
The quantitative PCR was then carried using primers shown in Table 1 and faststart universal SYBR Green master (Toyobo, Osaka, Japan) and 7500 real-time PCR system (Applied Biosystems company, foster, California, USA) were used for quantitative PCR. β-actin is used for standardization. The experiment was repeated at least three times.  Statistical analysis SPSS software was used for statistical analysis. All data are expressed as standard deviation of at least three independent operations. The quantitative data are expressed as the mean standard deviation of at least three independent experiments. Statistically signi cant is P < 0.05. The asterisk (**) indicates P < 0.01.

Characteristics of HGF gene-transfected MSCs
To characterize the HGF gene transfected MSCs, we detected the morphology and cell proliferation of MSCs and HGF gene transfected MSCs. Figure 1A shows the cell morphology of MSCs and HGF gene transfected MSCs, They have the same shape of spindle cells. Under the same culture conditions, MSCs and HGF gene transfected MSCs were almost the same cell proliferation (Fig. 1B). To verify the characteristics of mesenchymal stem cells, we checked the positive markers, including CD73, CD90 and CD105, were both highly expressed in MSCs and HGF gene transfected MSCs (Fig. 1C). To assess the effect of HGF gene transfection, the concentration of HGF gene were detected. The concentration of HGF in the cell suspension of HGF gene transfected MSCs was higher than that of the non-transfected MSCs at 0, 1, 2, 3 and 4 day after transfection (Fig. 1D). Compared with the non-transfected MSCs, the HGF gene transfected MSCs progressively increased HGF level at 1, 2 and 3 day. Non-transfected MSCs gradually increased but still remained totally lower than 4 day. These results indicated that HGF gene transfected MSCs retain the characteristics of original MSCs, and signi cantly increased the expression of HGF gene in transfected MSCs.

HGF gene transfected MSCs recover the morphology of injured uterus
In order to verify the effect of HGF gene transfected MSCs, we established the mouse model of endometrial injury, and observed the morphological characteristics of mouse uterus after HGF gene transfected MSCs therapy. The results show that HGF gene transfected MSCs could improve the appearance and morphological characteristics of the uterus on the damaged side, and the appearance of the uterus is close to the normal side ( Fig. 2A). H & E staining showed that the endometrial structure of the HGF gene transfected MSCs group was relatively complete, the epithelial cells were closely arranged, and the glands were clearly visible. In the Saline group, the endometrium of the injured side was poor, the thickness of endometrium was obviously thinner, and the epithelium was discontinuous and incomplete (Fig. 2B). The thickness of endometrial epithelium and the number of endometrial glands in each group were counted. HGF gene transfected MSCs treatment group was signi cantly higher than those of the Saline group and MSCs group. Morphologically, HGF gene transfected MSCs is more effective than normal MSCs. (Fig. 2C, D).

HGF gene transfected MSCs improve proliferation and decrease apoptosis of endometrial cells
In order to verify the repair ability of HGF, we detected the proliferation and apoptosis of epithelial cells. It can be seen that HGF gene transfected MSCs can improve the proliferation ability of damaged endometrial epithelial cells (Fig. 3A). Through the statistical analysis of Ki-67 uorescence intensity, a relatively quantitative comparison can re ect the therapeutic effect of HGF gene transfected MSCs, who was signi cantly higher than those of the Saline group and MSCs group (Fig. 3B). Besides, HGF gene transfected MSCs can reduce the apoptosis of endometrial cells (Fig. 3C). Through the statistical analysis of TUNEL uorescence intensity, the therapeutic effect of HGF gene transfected MSCs can be quantitatively re ected. It is signi cant that the apoptotic cells in HGF gene transfected MSCs group were less than Saline group and MSCs group (Fig. 3D).

HGF gene transfected MSCs improves the angiogenesis of damaged endometrium
In order to verify the repair effect of HGF on the injured endometrial vessels, we detected the expression levels of two kinds of angiogenesis related mRNA: vascular endothelial growth factor (VEGF) and insulinlike growth factor 1 (IGF-1). It can be found that HGF gene-transfected MSCs can improve the angiogenesis of endometrial epithelial cells. The mRNA expression of VEGF and IGF-1 in uterus of each group was counted (Fig. 4A, B). It can be con rmed that HGF gene transfected MSCs have better vascular repair ability than normal MSCs.

HGF gene transfected MSCs promote the MET process in damaged endometrium
To observe the effect of HGF gene transfected MSCs on mesenchyma-lepithelial transition process in damaged endometrium, we used immuno uorescence to label epithelial marker E-cadherin and mesenchymal marker Vimentin respectively (Fig. 5A,B). By observing the expression of E-cadherin and Vimentin in epithelial and stromal regions, we quantitatively analyzed their uorescence intensity, and counted the ratio of E-cadherin and Vimentin intensity in each treatment group (Fig. 5C). The results showed that the process of MET in HGF gene transfected MSCs group was more active and the degree of epithelial transformation was higher. These results show that HGF plays an indispensable role in MET process.
HGF gene transfected MSCs plays a role by activating phosphorylated c-Met and downstream phosphorylated AKT pathway Finally, we explore how HGF can play its role. The rst step is to verify the HGF receptor c-Met, detect the expression level of c-Met total protein and its phosphorylation(p-c-Met)by Western blot, and then check the expression level of AKT and AKT phosphorylation p-AKT in its downstream pathway (Fig. 6A,B) The results showed that the expression of p-c-Met in HGF gene transfected MSCs group was the highest in acute in ammation stage after injury, and the expression of p-AKT in its downstream pathway was also the highest.

Discussion
In this study, we found that HGF gene transfected MSCs had better therapeutic effect compared with normal MSCs in mice model of endometrial damage. Increasing evidence suggests that the therapeutic effects of MSCs mainly depend on their capacity to secrete a variety of growth factors and cytokines [19], including HGF. However, exogenous HGF have a temporary effect on the tissue regeneration, due to its short half-life in the blood circulation [20]. In contrast, MSCs can persistently release HGF, which ensures a continuous expression of HGF in the organisms and overcomes the disadvantage of only injecting exogenous HGF. In that regard, genetic engineering of MSCs transfected with HGF gene can improve the intrinsic secretion of HGF, thus improving the endometrial repair potential of these cells in a context of gene therapy.
It is particularly important to generate the ideal HGF gene transfected MSCs. Even though some viral vectors were con rmed to be safe for clinical use, an improved method that can entirely avoid the integration of viral vectors is required. In this study, we generated HGF-secreting MSCs using minicircle vectors containing only the transgene expression cassette [21][22][23]. Minicircles are small circular plasmids that carry transgenes. The absence of the bacterial backbone and the non-integrating transfection system make minicircle vectors become an attractive delivery tool in vivo for future clinical applications. Genetically engineering HGF into MSCs by minicircles to treat injured endometrium would be optimal from both pharmacological and practical perspectives [24,25]. After transfected with HGF gene, we detected characteristics and secretion ability of cells to ensure its safety and effectiveness. By analyzing the cell activity of MSCs after HGF gene transfection and the characteristic expression of mesenchymal stem cell markers (CD73, CD90 and CD105), it was con rmed that HGF gene transfection would not change the inherent properties of MSC. At the same time, the ability of MSCs to secrete HGF before and after transfection was further tested. It was found that the ability of MSC to secrete HGF after transfection was signi cantly improved. In line with our expectations, HGF gene transfected MSCs can be used for transplantation therapy. In order to verify the therapeutic effect of MSCs transfected with HGF gene on damaged endometrium, we compared the treatment results of ordinary MSCs and HGF gene transfected MSCs in mouse model through biochemical, histological and molecular biological methods.
The results show that HGF gene transfected MSCs has a positive recovery effect on endometrial regeneration, which is more signi cant than ordinary MSCs.
Recent studies have shown that the endometrial tissue of patients with uterine trauma is characterized by vascular closure and microvascular damage, angiogenesis will appear in endometrial tissue after treatment, suggesting that angiogenesis of damaged endometrium may affect the regeneration of endometrium [26,27]. Meanwhile, HGF plays an indispensable role in angiogenesis [28,29]. In order to explore the mechanism of HGF gene transfected MSCs in the treatment of endometrial injury, we veri ed the ability of vascular regeneration after transplanted with normal MSCs and HGF gene transfected MSCs. In our experiment, the expression of VEGF and IGF-1 in the endometrium of mice treated with MSCs transfected with HGF gene was higher than that of mice treated with normal MSCs. This further suggests that HGF gene transfected MSCs can play a role in angiogenesis by increasing the amount of angiogenesis related factors.
Another role of HGF plays is about mesenchymal to epithelial transition (MET) process. First and foremost, it absolutely believed that HGF and its receptor c-Met related to the transformation of the epithelium and mesenchyme, most of these reports are about tumors or endometriosis [30][31][32][33]. The endometrium is the dynamic inner layer of the uterus, from a functional point of view, the endometrium is composed of functional layer and basement layer, and from the perspective of histology, composed of mesenchymal and epithelial cells that undergo monthly proliferation, differentiation, and shedding throughout the menstrual cycle in childbearing age women. In the functional layer of endometrium, the epithelium and mesenchyme are in a dynamic balance process. When the epithelial tissue of endometrium is damaged, the mesenchyme will transform to the epithelium, so as to maintain the integrity of the epithelium on the surface of endometrium [34,35]. Furthermore, MSCs transfected with HGF gene can promote the transformation of mesenchymal to epithelial. The expression of the related antigen, E-cadherin in epithelial cells and N-cadherin in mesenchymal cells can re ect the degree of MET [36]. Therefore, we detected the expression of protein to verify that MSCs transfected with HGF plays a role by binding to its receptor c-Met, activating the phosphorylation of c-Met receptor and activating the downstream AKT pathway. This process plays a leading role in both increasing angiogenesis and MET process [37][38][39]. However, preliminary detection of these two proteins only con rmed that HGF transfected MSCs plays a role in the repair of endometrium through angiogenesis and mesenchyme to epithelium transformation, and the more exact mechanism is still unclear.

Conclusion
In summary, the present study shows that cell therapy with HGF gene transfected MSCs signi cantly promotes endometrial repair in the morphological and molecular aspects, these effects are superior to treatment with normal MSCs. In addition, the enhanced therapeutic effects of HGF gene transfected MSCs were accompanied by increased levels of the proliferative cell, and decreased the apoptosis. Moreover, MSCs transfected with HGF gene can promote angiogenesis and MET process by activating pc-Met pathway after transplantation. These results suggest that cell therapy with HGF gene transfected MSCs may be a new and effective tool for the treatment of injured endometrium. The data that support the ndings of this study are available upon request from the corresponding author. The data are not publicly available due to privacy concerns or ethical reasons.