Simvastatin improves the homing of BMSCs via the PI3K/AKT/miR-9 pathway.

Bone marrow-derived mesenchymal stem cells (BMSCs) have great therapeutic potential for many diseases. However, the homing of BMSCs to injury sites remains a difficult problem. Recent evidence indicates that simvastatin stimulates AKT phosphorylation, and p-AKT affects the expression of chemokine (CXC motif) receptor-4 (CXCR4). Therefore, simvastatin may improve the expression of CXCR4 in BMSCs, and microRNAs (miRs) may participate in this process. In this study, we demonstrated that simvastatin increased both the total and the surface expression of CXCR4 in BMSCs. Stromal cell-derived factor-1α (SDF-1α)-induced migration of BMSCs was also enhanced by simvastatin, and this action was inhibited by AMD 3100(a chemokine receptor antagonist for CXCR4). The PI3K/AKT pathway was activated by simvastatin in this process, and LY294002 reversed the overexpression of CXCR4 caused by simvastatin. MiR-9 directly targeted CXCR4 in rat BMSCs, and simvastatin decreased miR-9 expression. P-AKT affected the expression of miR-9; as the phosphorylation of AKT increased, miR-9 expression decreased. In addition, LY294002 increased miR-9 expression. Taken together, our results indicated that simvastatin improved the migration of BMSCs via the PI3K/AKT pathway. MiR-9 also participated in this process, and the phosphorylation of AKT affected miR-9 expression, suggesting that simvastatin might have beneficial effects in stem cell therapy.

Recurrent paraganglioma of the vulva: A rare case report and review of the literature.

Purpose: Vulva paragangliomas are rare and usually misdiagnosed or missed, especially in juveniles. Our aim was to summarize the clinical characteristics and treatments of vulva paragangliomas. Methods and results: We present a case of a 17-year-old Chinese patient with functional paraganglioma from the vulva that was misdiagnosed as clear cell carcinoma. She had suffered from severe headaches, palpitations, sweating, pallor and hypertension. The vaginal wall was invaded by this mass. The tumour was surgically removed smoothly. However, the disease recurred 7 years after surgery, and the patient was treated again. Personalized genetic testing was performed while recovering, and the results suggested that the patient had a germline mutation in the Succinate Dehydrogenase subunit B (SDHB) gene. Now, the patient has been discharged successfully, her blood pressure has returned to normal and some of her clinical symptoms disappeared. A review of the literature concerning the topic is also presented, there have been only 2 cases of paraganglioma of the vulva and 11 cases of vaginal paraganglioma since 1955. Conclusion: Our case describes a recurrent vulvovaginal paraganglioma with SDHB gene mutation and the largest tumor diameter to date. The diagnosis and treatment process of this case can provide reference for the management of other similar patients.

Exosomal lncRNA HOTAIR Promotes the Progression and Angiogenesis of Endometriosis via the miR-761/HDAC1 Axis and Activation of STAT3-Mediated Inflammation.

Background: Long non-coding RNA (lncRNA) and exosomes are implicated in endometriosis development. We measured the expression of an exosomal lncRNA, homeobox transcript antisense RNA (HOTAIR), and explored its molecular mechanism in endometriosis progression. Methods: Expression of HOTAIR and microRNA (miR)-761 in different endometrial tissues was measured. Exosomes were isolated from a culture medium of endometrial stromal cells (ESCs). RT-qPCR was used to measure HOTAIR expression in different exosome types. CCK-8, Edu, wound healing, transwell assays, flow cytometry and tube formation were used to detect the role of exosomal HOTAIR on ESCs and human umbilical vein endothelial cells (HUVECs). The relationship among miR-761, HOTAIR, and histone deacetylase 1 (HDAC1) was verified by dual-luciferase reporter assay. ESCs were transfected with miR-761 mimics or HDAC1 small interfering RNA (si-RNA) to ascertain if alterations in expression of miR-761 or HDAC1 could reverse the effect of exosomal HOTAIR. Then, we detected the effect of the HOTAIR/miR-761/HDAC1 axis on signal transducer and activator of transcription 3 (STAT3)-mediated inflammation. In vivo experiments were conducted to verify in vitro results. Results: HOTAIR expression was upregulated and miR-761 expression was downregulated in ectopic endometrium tissues. HOTAIR was packaged into exosomes and transported from ESCs to surrounding cells. Exosomal HOTAIR promoted the proliferation, migration, and invasion, and inhibited the apoptosis of ESCs. Angiogenesis of HUVECs was enhanced after cultured with exosomal HOTAIR. HOTAIR acted as a competing endogenous RNA to downregulate miR-761 and increase HDAC1 expression. miR-761 overexpression or HDAC1 knockdown reversed the role of exosomal HOTAIR on ESCs and HUVECs. The HOTAIR/miR-761/HDAC1 axis could activate STAT3-related proinflammatory cytokines and stattic (inhibitor of phosphorylated-STAT3) could reverse the effect of HOTAIR on ESCs and HUVECs. In vivo experiments suggested that exosomal HOTAIR promoted the growth of endometrial lesions in vivo. Conclusion: Exosomal HOTAIR promoted the progression and angiogenesis of endometriosis by regulating the miR-761/HDAC1 axis and activating STAT3-mediated inflammation in vitro and in vivo, which may provide promising treatment for endometriosis.

Exosomes from Human Omental Adipose-Derived Mesenchymal Stem Cells Secreted into Ascites Promote Peritoneal Metastasis of Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) patients frequently develop peritoneal metastasis, especially in the human omentum. However, the mechanism underlying this propensity remains unknown. A previous study found that human omental adipose-derived mesenchymal stem cells are potentially involved in ovarian cancer growth and metastasis, but the results were inconsistent and even contradictory. In addition, the underlying mechanisms of visceral adipose metastasis remain poorly understood. Here, our goal is to clarify the role and mechanism of human omental adipose-derived mesenchymal stem cells (HO-ADSCs) in EOC cancer growth and metastasis. We first found that human omental tissue conditioned medium (HO-CM) enhances EOC cell function. Subsequent coculture studies indicated that HO-ADSCs increase the growth, migratory and invasive capabilities of ovarian cancer cells. Then, we demonstrated that exosomes secreted by HO-ADSCs (HO-ADSC exosomes) enhanced ovarian cancer cell function, and further mechanistic studies showed that the FOXM1, Cyclin F, KIF20A, and MAPK signaling pathways were involved in this process. In addition, subcutaneous tumorigenesis and peritoneal metastatic xenograft experiments provided evidence that HO-ADSC exosomes promote ovarian cancer growth and metastasis in vivo. Finally, our clinical studies provided evidence that ascites from ovarian cancer patients enhance EOC cell line proliferation, migration, and invasion in vitro. The present study indicated that HO-ADSC exosomes are secreted into ascites and exert a tumor-promoting effect on EOC growth and metastasis, providing a new perspective and method to develop future novel therapeutic strategies for the treatment of ovarian cancer.

miR-126-3p containing exosomes derived from human umbilical cord mesenchymal stem cells promote angiogenesis and attenuate ovarian granulosa cell apoptosis in a preclinical rat model of premature ovarian failure.

BACKGROUND: In our previous research, we found that overexpression of miR-126-3p in human umbilical cord MSCs (hucMSCs) promoted human umbilical vein endothelial cells angiogenic activities through exosome-mediated mechanisms. The present study aimed to investigate the role of miR-126-3p-modified hucMSCs derived exosomes (miR-126-3p-hucMSCs-exosomes) on the treatment of premature ovarian failure (POF). METHODS: Primary hucMSCs were isolated from human umbilical cords and identified by differentiation experiments and flow cytometry. miR-126-3p-hucMSCs were obtained by miR-126-3p lentivirus infection. miR-126-3p-hucMSCs-exosomes were purified by ultracentrifugation method and characterized by transmission electron microscopy and western blot analysis. Primary rat ovarian granulosa cells (OGCs) were collected from ovarian tissues and identified by cell immunohistochemistry. The effects of miR-126-3p-hucMSCs-exosomes and miR-126-3p on OGCs function were determined by cell proliferation and apoptosis assays in a cisplatin induced POF cell model. The levels of suitable target genes were analyzed by PCR and Western blot analysis and subsequent Dual-Luciferase reporter assay. The signal pathway was also analyzed by western blot analysis. A cisplatin-induced POF rat model was used to validate the therapeutic effects of miR-126-3p-hucMSCs-exosomes to treat POF. Ovarian function was evaluated by physical, enzyme-linked immunosorbent assay, and histological examinations in chemotherapy-treated rats. The angiogenesis and apoptosis of ovarian tissues were assessed by immunohistochemical staining and Western blots. RESULTS: Primary hucMSCs and miR-126-3p-hucMSCs-exosomes and primary rat OGCs were successfully isolated and identified. The cellular uptake experiments indicated that miR-126-3p-hucMSC-exosomes can be internalized into OGCs in vitro. Annexin V-FITC/PI staining and EDU assays revealed that both miR-126-3p-hucMSCs-exosomes and miR-126-3p promoted proliferation and inhibited apoptosis of OGCs damaged by cisplatin. PCR and western blot analysis and subsequent dual-luciferase reporter assay verified that miR-126-3p targets the sequence in the 3' untranslated region of PIK3R2 in OGCs. Further analysis showed that PI3K/AKT/mTOR signaling pathway took part in miR-126-3p/PIK3R2 mediated proliferation and apoptosis in OGCs. In rat POF model, administration of miR-126-3p-hucMSCs-exosomes increased E2 and AMH levels, increased body and reproductive organ weights and follicle counts, and reduced FSH levels. But more importantly, immunohistochemistry results indicated miR-126-3p-hucMSCs-exosomes significantly promoted ovarian angiogenesis and inhabited apoptosis in POF rats. Additionally, the analysis of angiogenic-related factors and apoptosis-related factors showed miR-126-3p-hucMSCs-exosomes had pro-angiogenesis and anti-apoptosis effect in rat ovaries. CONCLUSIONS: Our findings revealed that hucMSCs-derived exosomes carrying miR-126-3p promote angiogenesis and attenuate OGCs apoptosis in POF, which highlighted the potential of exosomes containing miR-126-3p as an effective therapeutic strategy for POF treatment.

Prostaglandin E1 reduces apoptosis and improves the homing of mesenchymal stem cells in pulmonary arterial hypertension by regulating hypoxia-inducible factor 1 alpha.

BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with oxidative stress and affects the survival and homing of transplanted mesenchymal stem cells (MSCs) as well as cytokine secretion by the MSCs, thereby altering their therapeutic potential. In this study, we preconditioned the MSCs with prostaglandin E1 (PGE1) and performed in vitro and in vivo cell experiments to evaluate the therapeutic effects of MSCs in rats with PAH. METHODS: We studied the relationship between PGE1 and vascular endothelial growth factor (VEGF) secretion, B-cell lymphoma 2 (Bcl-2) expression, and C-X-C chemokine receptor 4 (CXCR4) expression in MSCs and MSC apoptosis as well as migration through the hypoxia-inducible factor (HIF) pathway in vitro. The experimental rats were randomly divided into five groups: (I) control group, (II) monocrotaline (MCT) group, (III) MCT + non-preconditioned (Non-PC) MSC group, (IV) MCT + PGE1-preconditioned (PGE1-PC) MSC group, and (V) MCT+PGE1+YC-1-PCMSC group. We studied methane dicarboxylic aldehyde (MDA) levels, MSC homing to rat lungs, mean pulmonary artery pressure, pulmonary artery systolic pressure, right ventricular hypertrophy index, wall thickness index (%WT), and relative wall area index (%WA) of rat pulmonary arterioles. RESULTS: Preconditioning with PGE1 increased the protein levels of HIF-1 alpha (HIF-1α) in MSCs, which can reduce MSC apoptosis and increase the protein levels of CXCR4, MSC migration, and vascular endothelial growth factor secretion. Upon injection with PGE1-PCMSCs, the pulmonary artery systolic pressure, mean pulmonary artery pressure, right ventricular hypertrophy index, %WT, and %WA decreased in rats with PAH. PGE1-PCMSCs exhibited better therapeutic effects than non-PCMSCs. Interestingly, lificiguat (YC-1), an inhibitor of the HIF pathway, blocked the effects of PGE1 preconditioning. CONCLUSIONS: Our findings indicate that PGE1 modulates the properties of MSCs by regulating the HIF pathway, providing insights into the mechanism by which PGE1 preconditioning can be used to improve the therapeutic potential of MSCs in PAH.

Exosomes derived from human umbilical cord mesenchymal stem cells inhibit vein graft intimal hyperplasia and accelerate reendothelialization by enhancing endothelial function.

BACKGROUND: In our previous research, we found that mesenchymal stem cell (MSC) transplantation therapy can inhibit intimal hyperplasia and enhance endothelial function in arterialized vein grafts in rats. However, whether MSC-derived exosomes (MSC-exosomes) can reduce neointimal formation and its possible mechanism is still unclear. METHODS: The primary human umbilical cord MSCs (hucMSCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry and immunofluorescence. The exosomes derived from hucMSCs (hucMSC-exosomes) were identified by transmission electron microscopy and western blots. hucMSC-exosomes were intravenously injected into a rat model of vein grafting, and its effect on vein grafts reendothelialization and intimal hyperplasia was assessed by physical, histological, immunohistochemistry, and immunofluorescence examinations. The effects of hucMSC-exosomes on endothelial cells were evaluated by integrated experiment, EdU staining, scratch assay, and Transwell assay. The expression levels of key gene and pathways associated with the biological activity of vascular endothelial cells were evaluated following the stimulation of hucMSC-exosomes. RESULTS: We successfully isolated and characterized primary hucMSCs and hucMSC-exosomes and primary HUVECs. We verified that the systemic administration of hucMSC-exosomes accelerates reendothelialization and decreases intimal hyperplasia of autologous vein graft in a rat model. We also identified that hucMSC-exosomes can be uptaken by endothelial cells to stimulate cell proliferative and migratory activity in vitro. Furthermore, we detected that vascular endothelial growth factor (VEGF) plays an important part in hucMSC-exosome-mediated proliferation and migration in HUVECs. In addition, we also provided evidence that the signalling pathways of PI3K/AKT and MAPK/ERK1/2 take part in hucMSC-exosome-induced VEGF regulation. CONCLUSION: Our data suggest that hucMSC-exosomes exert a vasculoprotective role in the setting of vein graft disease, which may provide a new clue to protect against vein graft failure in the future.

miRNA-126-3p carried by human umbilical cord mesenchymal stem cell enhances endothelial function through exosome-mediated mechanisms in vitro and attenuates vein graft neointimal formation in vivo.

BACKGROUND: The aim of this study was to determine whether the combination of MSC implantation with miRNA-126-3p overexpression would further improve the surgical results after vein grafting. METHODS: human umbilical cord MSCs (hucMSCs) and human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords and characterized by a series of experiments. Lentivirus vector encoding miRNA-126-3p was transfected into hucMSCs and verified by PCR. We analyzed the miRNA-126-3p-hucMSC function in vascular endothelial cells by using a series of co-culture experiments. miRNA-126-3p-hucMSCs-exosomes were separated from cell culture supernatants and identified by WB and TEM. We validated the role of miRNA-126-3p-hucMSCs-exosomes on HUVECs proliferative and migratory and angiogenic activities by using a series of function experiments. We further performed co-culture experiments to detect downstream target genes and signaling pathways of miRNA-126-3p-hucMSCs in HUVECs. We established a rat vein grafting model, CM-Dil-labeled hucMSCs were injected intravenously into rats, and the transplanted cells homing to the vein grafts were detected by fluorescent microscopy. We performed historical and immunohistochemical experiments to exam miRNA-126-3p-hucMSC transplantation on vein graft neointimal formation and reendothelialization in vitro. RESULTS: We successfully isolated and identified primary hucMSCs and HUVECs. Primary hucMSCs were transfected with lentiviral vectors carrying miRNA-126-3p at a MOI 75. Co-culture studies indicated that overexpression of miRNA-126-3p in hucMSCs enhanced HUVECs proliferation, migration, and tube formation in vivo. We successfully separated hucMSCs-exosomes and found that miRNA-126-3p-hucMSCs-exosomes can strengthen the proliferative, migratory, and tube formation capacities of HUVECs. Further PCR and WB analysis indicated that, SPRED-1/PIK3R2/AKT/ERK1/2 pathways are involved in this process. In the rat vein arterialization model, reendothelialization analysis showed that transplantation with hucMSCs modified with miRNA-126-3p had a higher reendothelialization of the vein grafts. The subsequent historical and immunohistochemical examination revealed that delivery with miRNA-126-3p overexpressed hucMSCs significantly reduced vein graft intimal hyperplasia in rats. CONCLUSION: These results suggest hucMSC-based miRNA-126-3p gene therapy may be a novel option for the treatment of vein graft disease after CABG.

Upregulation of miR-126-3p promotes human saphenous vein endothelial cell proliferation in vitro and prevents vein graft neointimal formation ex vivo and in vivo.

Poor long-term patency of vein grafts remains an obstacle in coronary artery bypass grafting (CABG) surgery using an autologous saphenous vein graft. Recent studies have revealed that miR-126-3p promotes vascular integrity and angiogenesis. We aimed to identify the role of miR-126-3p in the setting of vein graft disease and investigate the value of miR-126-3p agomir as a future gene therapy in vein graft failure. Expression analysis of circulating miR-126-3p in plasma from CABG patients established the basic clues that miR-126-3p participates in CABG. The in vitro results indicated that elevated miR-126-3p expression significantly improved proliferation and migration in human saphenous vein endothelial cells (HSVECs) by targeting sprouty-related protein-1 (SPRED-1) and phosphatidylinositol-3-kinase regulatory subunit 2 (PIK3R2), but not in human saphenous vein smooth muscle cells (HSVSMCs). Moreover, the therapeutic potential of miR-126-3p agomir was demonstrated in cultured human saphenous vein (HSV) ex vivo. Finally, local delivery of miR-126-3p agomir was confirmed to enhance reendothelialization and attenuate neointimal formation in a rat vein arterialization model. In conclusion, we provide evidence that upregulation of miR-126-3p by agomir possesses potential clinical value in the prevention and treatment of autologous vein graft restenosis in CABG.