Our objective has been to establish a pro-angiogenic role for exosomes in endometriosis and to determine whether a differential expression profile of cellular and exosomal microRNAs (miRNAs) exists in endometriosis. We performed an in vitro study of human primary endometrial stromal cells (ESCs) and human umbilical vein endothelial cells (HUVECs). We isolated and characterized exosomes from ESCs from five endometriosis patients and five phase-matched controls. Exosomes were characterized by transmission electron microscopy and NanoSight technology. MiRNA was assessed by deep sequencing and reverse transcription with quantitative polymerase chain reaction. Exosome uptake studies were achieved by means of confocal microscopy. The pro-angiogenic experiments were executed by treating HUVECs with ESC-derived exosomes. We observed differential profiles of exosomal miRNA expression between exosomes derived from endometriosis lesion cells and diseased eutopic stromal cells compared with exosomes derived from control ESCs. We also demonstrated autocrine cellular uptake of exosomes and paracrine functional angiogenic effects of exosomes on HUVECs. The results of this study support the hypothesis that exosomes derived from ESCs play autocrine/paracrine roles in the development of endometriosis, potentially modulating angiogenesis. The broader clinical implications are that Sampson’s theory of retrograde menstruation possibly encompasses the finding that exosomes work as intercellular communication modulators in endometriosis.

Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.

Current approaches to the treatment of ovarian cancer are limited because of the development of resistance to chemotherapy. Prohibitin (Phb1) is a possible candidate protein that contributes to development of drug resistance, which could be targeted in neoplastic cells. Phb1 is a highly conserved protein that is associated with a block in the G0/G1 phase of the cell cycle and also with cell survival. Our study was designed to determine the role of Phb1 in regulating cellular growth and apoptosis in ovarian cancer cells. Our results showed that Phb1 content is differentially overexpressed in papillary serous ovarian carcinoma and endometrioid ovarian adenocarcinoma when compared to normal ovarian epithelium and was inversely related to Ki67 expression. Immunofluorescence microscopy and Western analyses revealed that Phb1 is primarily associated with the mitochondria in ovarian cancer cells. Over‐expression of Phb1 by adenoviral Phb1 infection resulted in an increase in the percentage of ovarian cancer cells accumulating at G0/G1 phase of the cell cycle. Treatment of ovarian cancer cells with staurosporine (STS) induced apoptosis in a time‐dependent manner. Phb1 over‐expression induced cellular resistance to STS via the intrinsic apoptotic pathway. In contrast, silencing of Phb1 expression by adenoviral small interfering RNA (siRNA) sensitized ovarian cancer cells to STS‐induce apoptosis. Taken together, these results suggest that Phb1 induces block at G0/G1 phase of the cell cycle and promotes survival of cancer cells. Furthermore, silencing of the Phb1 gene expression may prove to be a valuable therapeutic approach for chemoresistant ovarian cancer by increasing sensitivity of cancer cells to apoptosis.

Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1/2 → Bcl/Bcl-xL pathway and may have important clinical implications.

Early-life exposure of the myometrium to endocrine-disrupting chemicals (EDCs) has been shown to increase the risk of uterine fibroid (UF) prevalence in adulthood. Vitamin D3 (VitD3) is an unique, natural compound that may reduce the risk of developing UFs. However, little is known about the role and molecular mechanism of VitD3 on exposed myometrial stem cells (MMSCs). We investigated the role and molecular mechanism underlying VitD3 action on DNA damage response (DDR) defects in rat MMSCs due to developmental exposure to diethylstilbestrol (DES), with the additional goal of understanding how VitD3 decreases the incidence of UFs later in life. Female newborn Eker rats were exposed to DES or a vehicle early in life; they were then sacrificed at 5 months of age (pro-fibroid stage) and subjected to myometrial Stro1+/CD44+ stem cell isolation. Several techniques were performed to determine the effect of VitD3 treatment on the DNA repair pathway in DES-exposed MMSCs (DES-MMSCs). Results showed that there was a significantly reduced expression of RAD50 and MRE11, key DNA repair proteins in DES-exposed myometrial tissues, compared to vehicle (VEH)-exposed tissues (p < 0.01). VitD3 treatment significantly decreased the DNA damage levels in DES-MMSCs. Concomitantly, the levels of key DNA damage repair members, including the MRN complex, increased in DES-MMSCs following treatment with VitD3 (p < 0.01). VitD3 acts on DNA repair via the MRN complex/ATM axis, restores the DNA repair signaling network, and enhances DDR. This study demonstrates, for the first time, that VitD3 treatment attenuated the DNA damage load in MMSCs exposed to DES and classic DNA damage inducers. Moreover, VitD3 targets primed MMSCs, suggesting a novel therapeutic approach for the prevention of UF development.