Vascular remodeling by placenta-derived mesenchymal stem cells restores ovarian function in ovariectomized rat model via the VEGF pathway

Abstract

Angiogenesis plays an important role in damaged organ or tissue and cell regeneration and ovarian development and function. Primary ovarian insufficiency (POI) is a prevalent pathology in women under 40. Conventional treatment for POI involves hormone therapy. However, due to its side effects, an alternative approach is desirable. Human mesenchymal stem cells (MSCs) from various sources restore ovarian function; however, they have many limitations as stem cell sources. Therefore, it is desirable to study the efficacy of placenta-derived MSCs (PD-MSCs), which possess many advantages over other MSCs, in a rat model of ovarian dysfunction. Here, we investigated the restorative effect of PD-MSCs on injured ovaries in ovariectomized (OVX) rats and the ability of intravenous transplantation (Tx) of PD-MSCs (5 × 10⁵) to enhance ovarian vasculature and follicular development. ELISA analysis of serum revealed that compared to the non-transplantation (NTx) group, the Tx group showed significantly increased levels of anti-Müllerian hormone, follicle stimulating hormone, and estradiol (E2) (*P < 0.05). In addition, histological analysis showed more mature follicles and less atresia and restoration of expanded blood vessels in the ovaries of the OVX PD-MSC Tx group than those of the NTx group (*P < 0.05). Furthermore, folliculogenesis-related gene expression was also significantly increased in the PD-MSC Tx group (*P < 0.05). Vascular endothelial growth factor (VEGF) and VEGF receptor 2 expressions were increased in the ovaries of the OVX PD-MSC Tx group compared to the NTx group through PI3K/AKT/mTOR and GSK3β/β-catenin pathway activation. Interestingly, ex vivo cocultivation of damaged ovaries and PD-MSCs or treatment with recombinant VEGF (50 ng/ml) increased folliculogenic factors and VEGF signaling pathways. Notably, compared to recombinant VEGF, PD-MSCs significantly increased folliculogenesis and angiogenesis (*P < 0.05). These findings suggest that VEGF secreted by PD-MSCs promotes follicular development and ovarian function after OVX through vascular remodeling. Therefore, these results provide fundamental data for understanding the therapeutic effects and mechanism of stem cell therapy based on PD-MSCs and provide a theoretical foundation for their application for obstetrical and gynecological diseases, including infertility and menopause.

Fig. 1. Homing of PD-MSCs to the ovary and ovarian follicle counts.

a Immunofluorescence staining confirmed that PKH67 (green) was expressed near follicles within the ovarian tissues of the PD-MSC group but was not expressed within those of the NTx group. Nuclei were stained with DAPI (blue). Arrow, PKH67 fluorescence. F, ovarian follicle. Scale bar: 25 μm. b The mRNA expression level of hAlu in ovarian tissue samples confirmed the successful localization and expression of PD-MSCs at the ovaries in the Tx group, whereas the opposite was observed in the normal and NTx groups. *vs. the normal group (P < 0.05). c H&E staining of ovarian tissues. d The graph of the percentage of follicles in different stages of folliculogenesis indicates an increased number of total follicles in all stages of development in the Tx group compared to the NTx group. The ovarian tissues were collected 2 weeks after the transplantation. Nor, normal group. NTx, non-transplantation group. Tx, transplantation group. 1w, week 1. 2w, week 2. 3w, week 3. 5w, week 5.

Fig. 2. Upregulation of folliculogenic factors at the mRNA and protein levels after PD-MSC transplantation.

a mRNA expression levels of Nanos3, Lhx8, Lin28α, Nobox, Bmp15, and Egfr were decreased in the NTx groups compared to the normal group and increased in the Tx groups compared to the NTx groups with some exceptions. b Western blot analysis showed an overall increase in the expression level in the Tx groups compared to the NTx groups with some exceptions. c Immunohistochemistry for LHX8 showed higher folliculogenic activity after Tx. PD-MSCs, treatment group. *vs. the normal group (P < 0.05). #, vs. NTx (P < 0.05). Nor, normal group. NTx, non-transplantation group. Tx, transplantation group. 1w, week 1. 2w, week 2. 3w, week 3. 5w, week 5.

Fig. 3. Measurements of the blood vessel luminal area and wall thickness.

a H&E staining of ovarian sections. Arrow, blood vessel. Scale bar: 1.5 mm. b The number of blood vessels within the ovaries of different groups appeared to be similar. c The luminal area of the blood vessels was increased in the NTx group compared to the normal group but was restored in the Tx group. d The same pattern was also observed for wall thickness. However, the number of blood vessels within the ovarian tissues remained relatively constant. *vs. the normal group (P < 0.05). # vs. the NTx group (P < 0.05). Nor normal group, NTx non-transplantation group, Tx transplantation group.

Fig. 4. Increased mRNA and protein expression levels of Vegf and Vegfr2.

a Immunohistochemistry staining showed high expression of VEGFR2 in antral follicles of Tx group compared to the NTx group. b Protein levels were also upregulated in most of the Tx groups compared to the NTx groups. c mRNA expression levels of VEGF and VEGFR2 were increased in the 2- and 3-week Tx groups. d Immunofluorescence showed high expression of VEGFR2 in blood vessel epithelial cells in the ovaries. V, blood vessel. Scale bar: 25 μm. *vs. the normal group (P < 0.05). # vs. the NTx group (P < 0.05). Nor normal group, NTx non-transplantation group, Tx transplantation group.

Fig. 5. Activation of the PI3K/AKT/mTOR and GSK3β/β-catenin pathways.

a Activation of the PI3K/AKT/mTOR pathway was induced by high expression of the phosphorylated forms of AKT, mTOR, and PI3K(p110α). b Protein levels of the phosphorylated form of GSK3β were increased. c Immunofluorescence showed high expression of CD31 (green), an epithelial cell marker, and β-catenin (red), and nuclear translocation of β-catenin merged with DAPI (blue). Scale bar: 25 μm. d, e Proportion of nuclear-translocated β-catenin was significantly higher in the Tx group than the NTx group. Arrowhead, β-catenin localized in the nucleus. Arrow, β-Catenin localized in the cytoplasm. *vs. the normal group (P < 0.05). #, vs. the NTx group (P < 0.05). Nor normal group, NTx non-transplantation group, Tx transplantation group.

Fig. 6. Enhancement of folliculogenic factors after cocultivation with PD-MSCs or treatment with recombinant VEGF ex vivo.

a The scheme of the ex vivo experiment. Ovaries were harvested from a wild-type rat, cut in half, and treated with either PD-MSCs or recombinant VEGF. b The mRNA expression levels of folliculogenic factors were upregulated after cocultivation with PD-MSCs or treatment with recombinant VEGF (50 ng/ml). *vs. no treatment (P < 0.05). #, vs. the 24-h group (P < 0.05). $, vs. the PD-MSC-cocultivated group.

Fig. 7. Activation of VEGF signaling pathways and increase in the number of mature follicles.

a Enhanced expression of soluble VEGF in the media after cocultivation or VEGF treatment. b H&E-stained ovarian tissues. c The PI3K/AKT/mTOR and GSK3β/β-catenin pathways were highly activated after cocultivation or VEGF treatment. Scale bar (H&E): 500 μm. Scale bar (VEGFR2): 100 μm. d The number of mature follicle was increased in the PD-MSC-cocultivated group and VEGF-treated group.

Fig. 8. Graphical abstract of the effect of PD-MSCs on folliculogenesis.

Secreted VEGF from PD-MSCs initiates the activation of endogenous VEGF signaling through the PI3K/AKT and GSK3β/β-catenin pathways, leading to vascular remodeling of ovarian blood vessels and, eventually, enhanced folliculogenesis.