The rising roles of exosomes in the tumor microenvironment reprogramming and cancer immunotherapy.

Exosomes are indispensable for intercellular communications. Tumor microenvironment (TME) is the living environment of tumor cells, which is composed of various components, including immune cells. Based on TME, immunotherapy has been recently developed for eradicating cancer cells by reactivating antitumor effect of immune cells. The communications between tumor cells and TME are crucial for tumor development, metastasis, and drug resistance. Exosomes play an important role in mediating these communications and regulating the reprogramming of TME, which affects the sensitivity of immunotherapy. Therefore, it is imperative to investigate the role of exosomes in TME reprogramming and the impact of exosomes on immunotherapy. Here, we review the communication role of exosomes in regulating TME remodeling and the efficacy of immunotherapy, as well as summarize the underlying mechanisms. Furthermore, we also introduce the potential application of the artificially modified exosomes as the delivery systems of antitumor drugs. Further efforts in this field will provide new insights on the roles of exosomes in intercellular communications of TME and cancer progression, thus helping us to uncover effective strategies for cancer treatment.

Aflatoxin B1-exposed hepatocyte-derived extracellular vesicles: Initiating hepatic stellate cell-mediated liver fibrosis through a p53-Parkin-dependent mitophagy pathway.

Environmental aflatoxin B1 (AFB1) exposure has been proposed to contribute to hepatocellular carcinoma by promoting liver fibrosis, but the potential mechanisms remain to be further elucidated. Extracellular vesicles (EVs) were recognized as crucial traffickers for hepatic intercellular communication and play a vital role in the pathological process of liver fibrosis. The AFB1-exposed hepatocyte-derived EVs (AFB1-EVs) were extracted, and the functional effects of AFB1-EVs on the activation of hepatic stellate cells (HSCs) were explored to investigate the molecular mechanism of AFB1 exposure-induced liver fibrogenesis. Our results revealed that an environment-level AFB1 exposure induced liver fibrosis via HSCs activation in mice, while the AFB1-EVs mediated hepatotoxicity and liver fibrogenesis in vitro and in vivo. AFB1 exposure in vitro increased PINK1/Parkin-dependent mitophagy in hepatocytes, where upregulated transcription of the PARK2 gene via p53 nuclear translocation and mitochondrial recruitment of Parkin, and promoted AFB1-EVs-mediated mitochondria-trafficking communication between hepatocytes and HSCs. The knockdown of Parkin in HepaRG cells reversed HSCs activation by blocking the mitophagy-related AFB1-EVs trafficking. This study further revealed that the hepatic fibrogenesis of AFB1 exposure was rescued by genetic intervention with siPARK2 or p53's Pifithrin-α (PFTα) inhibitors. Furthermore, AFB1-EVs-induced HSCs activation was relieved by GW4869 pharmaceutic inhibition of EVs secretion. These results revealed a novel mechanism that AFB1 exposure-induced p53-Parkin signal axis regulated mitophagy-dependent hepatocyte-derived EVs to mediate the mitochondria-trafficking intercellular communication between hepatocytes and HSCs in the local hepatotoxic microenvironment to promote the activated HSCs-associated liver fibrogenesis. Our study provided insight into p53-Parkin-dependent pathway regulation and promised an advanced strategy targeting intervention to EVs-mediated mitochondria trafficking for preventing xenobiotics-induced liver fibrosis.

Mrc1+ macrophage-derived IGF1 mitigates crystal nephropathy by promoting renal tubule cell proliferation via the AKT/Rb signaling pathway.

Rationale: The present understanding of the cellular characteristics and communications in crystal nephropathy is limited. Here, molecular and cellular studies combined with single-cell RNA sequencing (scRNA-seq) were performed to investigate the changes in cell components and their interactions in glyoxylate-induced crystallized kidneys to provide promising treatments for crystal nephropathy. Methods: The transcriptomes of single cells from mouse kidneys treated with glyoxylate for 0, 1, 4, or 7 days were analyzed via 10× Genomics, and the single cells were clustered and characterized by the Seurat pipeline. The potential cellular interactions between specific cell types were explored by CellChat. Molecular and cellular findings related to macrophage-to-epithelium crosstalk were validated in sodium oxalate (NaOx)-induced renal tubular epithelial cell injury in vitro and in glyoxylate-induced crystal nephropathy in vivo. Results: Our established scRNA atlas of glyoxylate-induced crystalline nephropathy contained 15 cell populations with more than 40000 single cells, including relatively stable tubular cells of different segments, proliferating and injured proximal tubular cells, T cells, B cells, and myeloid and mesenchymal cells. In this study, we found that Mrc1+ macrophages, as a subtype of myeloid cells, increased in both the number and percentage within the myeloid population as crystal-induced injury progresses, and distinctly express IGF1, which induces the activation of a signal pathway to dominate a significant information flow towards injured and proliferating tubule cells. IGF1 promoted the repair of damaged tubular epithelial cells induced by NaOx in vitro, as well as the repair of damaged tubular epithelial cells and the recovery of disease outcomes in glyoxylate-induced nephrolithic mice in vivo. Conclusion: After constructing a cellular atlas of glyoxylate-induced crystal nephropathy, we found that IGF1 derived from Mrc1+ macrophages attenuated crystal nephropathy through promoting renal tubule cell proliferation via the AKT/Rb signaling pathway. These findings could lead to the identification of potential therapeutic targets for the treatment of crystal nephropathy.

Non-Classical Intercellular Communications: Basic Mechanisms and Roles in Biology and Medicine.

In multicellular organisms, interactions between cells and intercellular communications form the very basis of the organism's survival, the functioning of its systems, the maintenance of homeostasis and adequate response to the environment. The accumulated experimental data point to the particular importance of intercellular communications in determining the fate of cells, as well as their differentiation and plasticity. For a long time, it was believed that the properties and behavior of cells were primarily governed by the interactions of secreted or membrane-bound ligands with corresponding receptors, as well as direct intercellular adhesion contacts. In this review, we describe various types of other, non-classical intercellular interactions and communications that have recently come into the limelight-in particular, the broad repertoire of extracellular vesicles and membrane protrusions. These communications are mediated by large macromolecular structural and functional ensembles, and we explore here the mechanisms underlying their formation and present current data that reveal their roles in multiple biological processes. The effects mediated by these new types of intercellular communications in normal and pathological states, as well as therapeutic applications, are also discussed. The in-depth study of novel intercellular interaction mechanisms is required for the establishment of effective approaches for the control and modification of cell properties both for basic research and the development of radically new therapeutic strategies.

Integrating Bulk Transcriptome and Single-Cell RNA Sequencing Data Reveals the Landscape of the Immune Microenvironment in Thoracic Aortic Aneurysms.

Thoracic aortic aneurysm (TAA) is a life-threatening cardiovascular disease whose formation is reported to be associated with massive vascular inflammatory responses. To elucidate the roles of immune cell infiltration in the pathogenesis underlying TAA, we utilized multiple TAA datasets (microarray data and scRNA-seq data) and various immune-related algorithms (ssGSEA, CIBERSORT, and Seurat) to reveal the landscapes of the immune microenvironment in TAA. The results exhibited a significant increase in the infiltration of macrophages and T cells, which were mainly responsible for TAA formation among the immune cells. To further reveal the roles of immunocytes in TAA, we inferred the intercellular communications among the identified cells of aortic tissues. Notably, we found that in both normal aortic tissue and TAA tissue, the cells that interact most frequently are macrophages, endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells (VSMCs). Among the cells, macrophages were the most prominent signal senders and receivers in TAA and normal aortic tissue. These findings suggest that macrophages play an important role in both the physiological and pathological conditions of the aorta. The present study provides a comprehensive evaluation of the immune cell composition and reveals the intercellular communication among aortic cells in human TAA tissues. These findings improve our understanding of TAA formation and progression and facilitate the development of effective medications to treat these conditions.

17ß-Estradiol Enhances Porcine Meiosis Resumption from Autophagy-Induced Gap Junction Intercellular Communications and Connexin 43 Phosphorylation via the MEK/ERK Signaling Pathway.

Estrogen and its analogues are ubiquitous in agricultural environments, with large biological functions of oocyte development. Gap junction intercellular communications (GJICs) are the structural basis in cumulus-oocyte complexes (COCs) and regulate oocyte maturation and developmental material transport through a number of pathways. This study mainly determines the effect and potential mechanism of estrogen (17ß-estradiol) in regulating GJICs in porcine COCs. In our study, 17ß-estradiol increased porcine nuclear maturation in a time-dependent manner. The analysis revealed that 17ß-estradiol upregulated the autophagy in COCs during in vitro maturation. In contrast with the control, 17ß-estradiol decreased GJICs in a time-dependent manner between cumulus cells and oocytes, while it was consistent with the control group at 24 h. Carbenoxolone (CBX) blocks GJICs as a negative control group used in our system. Autophagy inhibitor autophinib decreased oocyte maturation, and the reduced nuclear maturation treated with autophinib was abolished by 17ß-estradiol. Besides, the upregulation effect of autophinib on GJICs and transzonal projections (TZPs) was decreased by 17ß-estradiol. 17ß-Estradiol could reduce serine 368 phosphorylation of connexin 43 (Cx43) protein by autophinib in porcine COCs. These results were dependent upon the MEK/ERK signaling pathway. Furthermore, 17ß-estradiol-induced GJICs and Cx43 phosphorylation were inhibited by autophinib or the MEK/ERK pathway inhibitors (Trametinib and FR 180204), indicating that 17ß-estradiol regulated GJICs through the MEK/ERK signaling pathway. In conclusion, 17ß-estradiol improves the autophagy-mediated nuclear maturation with downregulating GJICs and TZPs in porcine COCs. Such an effect occurs by phosphorylation of Cx43, which was regulated via the MEK/ERK signaling pathway.

Trends in the biological functions and medical applications of extracellular vesicles and analogues.

Natural extracellular vesicles (EVs) play important roles in many life processes such as in the intermolecular transfer of substances and genetic information exchanges. Investigating the origins and working mechanisms of natural EVs may provide an understanding of life activities, especially regarding the occurrence and development of diseases. Additionally, due to their vesicular structure, EVs (in small molecules, nucleic acids, proteins, etc.) could act as efficient drug-delivery carriers. Herein, we describe the sources and biological functions of various EVs, summarize the roles of EVs in disease diagnosis and treatment, and review the application of EVs as drug-delivery carriers. We also assess the challenges and perspectives of EVs in biomedical applications.

Exosomes Derived from Hydroquinone-transformed Human Bronchial Epithelial Cells Inhibited Recipient Cell Apoptosis by transferring miR-221.

OBJECTIVE: Although benzene is a confirmed environmental carcinogen, the mechanism of its carcinogenicity remains largely unclear. The suggested oncogene, miR-221, is elevated and plays important roles in various tumors, but its role in benzene-induced carcinogenesis remains unknown. METHODS: In the present study, we constructed hydroquinone (HQ, a representative metabolite of benzene with biological activity)-transformed malignant cell line (16HBE-t) and analyzed the level of miR-221 in it with qRT-PCR. Exosomes from 16HBE-t cells incubated with or without an miR-221 inhibitor were isolated by ultracentrifugation, characterized by transmission electron microscopy and laser scanning confocal microscope, and then transfected into 16HBE cells. The effects of exosomal miR-221 on apoptosis induced by HQ in recipient cells were determined using flow cytometry. RESULTS: The amount of miR-221 in 16HBE-t was significantly increased compared with controls. When recipient cells ingested exosomes derived from 16HBE-t, miR-221 was increased, and apoptosis induced by HQ was inhibited. Blocking miR-221 in 16HBE-t using an inhibitor did not significantly alter miR-221 or apoptosis in recipient cells. CONCLUSION: Exosomal miR-221 secreted by 16HBE-t inhibits apoptosis induced by HQ in normal recipient cells.

Diversity and intratumoral heterogeneity in human gallbladder cancer progression revealed by single-cell RNA sequencing.

BACKGROUND: Gallbladder cancer (GC) is a malignant disease characterized with highly cellular heterogeneity and poor prognosis. Determining the intratumoral heterogeneity and microenvironment (TME) can provide novel therapeutic strategies for GC. METHODS: We performed the single-cell RNA sequencing on the primary and lymph node metastatic gallbladder tumors and the adjacent normal tissues of five patients. The transcriptomic atlas and ligand-receptor-based intercellular communication networks of the single cells were characterized. RESULTS: The transcriptomic landscape of 24,887 single cells was obtained and characterized as 10 cellular clusters, including epithelial, neuroendocrine tumor cells, T&NK cells, B cells, RGS5+ fibroblasts, POSTN+ fibroblasts, PDGFRA+ fibroblasts, endothelial, myeloid cells, and mast cells. Different types of GC harbored distinct epithelial tumor subpopulations, and squamous cell carcinoma could be differentiated from adenocarcinoma cells. Abundant immune cells infiltrated into adenocarcinoma and squamous cell carcinoma, rather than neuroendocrine neoplasms, which showed significant enrichment of stromal cells. CD4+/FOXP3+ T-reg and CD4+/CXCL13+ T helper cells with higher exhausting biomarkers, as well as a dynamic lineage transition of tumor-associated macrophages from CCL20hi /CD163lo , CCL20lo /CD163hi to APOE+, were identified in GC tissues, suggesting the immunosuppressive and tumor-promoting status of immune cells in TME. Two distinct endothelial cells (KDR+ and ACKR1+), which were involved in angiogenesis and lymphangiogenesis, showed remarkable ligand-receptor interactions with primary GC cells and macrophages in gallbladder tumors. CONCLUSIONS: This study reveals a widespread reprogramming across multiple cell populations in GC progression, dissects the cellular heterogeneity and interactions in gallbladder TME, and provides potential therapeutic targets for GC.

Unidirectional intercellular communication on a microfluidic chip.

Cell co-culture serves as a standard method to study intercellular communication. However, random diffusion of signal molecules during co-culture may arouse crosstalk among different types of cells and hide directive signal-target responses. Here, a microfluidic chip is proposed to study unidirectional intercellular communication by spatially controlling the flow of the signal molecules. The chip contains two separated chambers connected by two channels where the culture media flows oppositely. A zigzag signal-blocking channel is designed to study the function of a specific signal. The chip is applied to study the unidirectional communication between tumor cells and stromal cells. It shows that the expression of α-smooth muscle actin (a marker of cancer-associated fibroblast (CAF)) of both MRC-5 fibroblasts and mesenchymal stem cells can be up-regulated only by the secreta from invasive MDA-MB-231 cells, but not from non-invasive MCF-7 cells. The proliferation of the tumor cells can be improved by the stromal cells. Moreover, transforming growth factor beta 1 is found as one of the main factors for CAF transformation via the signal-blocking function. The chip achieves unidirectional cell communication along X-axis, signal concentration gradient along Y-axis and 3D cell culture along Z-axis, which provides a useful tool for cell communication studies.

Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular Communication Between Cytotoxic T Cells and Target Tumor Cells.

Gap junctions (GJs) are clusters of intercellular connexin-formed channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacent cells. Numerous reports have described GJs as modulators of key immunological processes, including in anti-tumor immune responses. Here, we described a simple flow cytometry method to test in vitro antigen-dependent GJ-mediated cell-to-cell coupling between cytotoxic T cells and target melanoma cells.

Trends in the biological functions and medical applications of extracellular vesicles and analogues

Natural extracellular vesicles (EVs) play important roles in many life processes such as in the intermolecular transfer of substances and genetic information exchanges. Investigating the origins and working mechanisms of natural EVs may provide an understanding of life activities, especially regarding the occurrence and development of diseases. Additionally, due to their vesicular structure, EVs (in small molecules, nucleic acids, proteins, etc.) could act as efficient drug-delivery carriers. Herein, we describe the sources and biological functions of various EVs, summarize the roles of EVs in disease diagnosis and treatment, and review the application of EVs as drug-delivery carriers. We also assess the challenges and perspectives of EVs in biomedical applications.

Exosomes Derived from Hydroquinone-transformed Human Bronchial Epithelial Cells Inhibited Recipient Cell Apoptosis by transferring miR-221 / 生物医学与环境科学（英文）

Objective@#Although benzene is a confirmed environmental carcinogen, the mechanism of its carcinogenicity remains largely unclear. The suggested oncogene, miR-221, is elevated and plays important roles in various tumors, but its role in benzene-induced carcinogenesis remains unknown.@*Methods@#In the present study, we constructed hydroquinone (HQ, a representative metabolite of benzene with biological activity)-transformed malignant cell line (16HBE-t) and analyzed the level of miR-221 in it with qRT-PCR. Exosomes from 16HBE-t cells incubated with or without an miR-221 inhibitor were isolated by ultracentrifugation, characterized by transmission electron microscopy and laser scanning confocal microscope, and then transfected into 16HBE cells. The effects of exosomal miR-221 on apoptosis induced by HQ in recipient cells were determined using flow cytometry.@*Results@#The amount of miR-221 in 16HBE-t was significantly increased compared with controls. When recipient cells ingested exosomes derived from 16HBE-t, miR-221 was increased, and apoptosis induced by HQ was inhibited. Blocking miR-221 in 16HBE-t using an inhibitor did not significantly alter miR-221 or apoptosis in recipient cells.@*Conclusion@#Exosomal miR-221 secreted by 16HBE-t inhibits apoptosis induced by HQ in normal recipient cells.

A novel role of SIRT2 in regulating gap junction communications via connexin-43 in bovine cumulus-oocyte complexes.

SIRT2, the predominantly cytosolic sirtuin, plays important role in multiple biological processes, including metabolism, stress response, and aging. However, the function of SIRT2 in gap junction intercellular communications (GJICs) of cumulus-oocyte complexes (COCs) is not yet known. The purpose of the present study was to evaluate the effect and underlining mechanism of SIRT2 on GJICs in COCs. Here, we found that treatment with SIRT2 inhibitors (SirReal2 or TM) inhibited bovine oocyte nuclear maturation. Further analysis revealed that SIRT2 inactivation disturbed the GJICs of COCs during in vitro maturation. Correspondingly, both the Cx43 phosphorylation levels and MEK/MER signaling pathways were induced by SIRT2 inhibition. Importantly, SIRT2-mediated Cx43 phosphorylation was completely abolished by treatment with MEK1/2 inhibitor (Trametinib). Furthermore, treatment with SIRT2 inhibitors resulted in the high levels of MEK1/2 acetylation. Functionally, downregulating the MER/ERK pathways with inhibitors (Trametinib or SCH772984) could attenuate the closure of GJICs caused by SIRT2 inactivation in partly. In addition, inhibition of SIRT2 activity significantly decreased the membrane and zona pellucida localization of Cx43 by upregulating the levels of Cx43 acetylation. Taken together, these results demonstrated a novel role that SIRT2 regulates GJICs via modulating the phosphorylation and deacetylation of Cx43 in COCs.

Exosomal miR-221 derived from hydroquinone-transformed malignant human bronchial epithelial cells is involved in cell viability of recipient cells.

miR-221, an oncogenic microRNA, can promote cell proliferation and is highly expressed in various types of tumors. However, the role of exosomal miR-221 in benzene-caused carcinogenesis remains elusive. Our study was designed to investigate whether exosomes secreted by the hydroquinone (HQ; an active metabolite of benzene)-transformed malignant cells can transmit miR-221 to normal recipient cells and its possible effects on cell viability. Our investigation revealed that expression levels of miR-221 were significantly increased in HQ-transformed malignant cells relative to normal controls. Furthermore, exposure of control cells to exosomes that were derived from HQ-transformed malignant cells increased miR-221 levels and promoted their proliferation. Analyses of the biological potency of exosomes derived from HQ-transformed malignant cells in which miR-221 levels were decreased using an inhibitor, showed that both miR-221 levels and proliferation of recipient cells were decreased, but still were higher than those of normal 16HBE cells. Our study indicates that exosomal miR-221 derived from HQ-transformed malignant human bronchial epithelial cells is involved in the proliferation of recipient cells.

Age-related schwannomatosis with potential exosome-mediated contribution to prostate hyperplasia: a case report and mini-review.

A 72-year-old Caucasian man incurring a prostate hypertrophy presented with a right forearm nodule, the growth of which appeared to parallel the rise in his blood prostate-specific antigen (PSA) level. Echographic examination was consistent with a median-nerve schwannoma, and was confirmed upon magnetic resonance imaging (MRI). Excision of the nodule was readily performed without significant neural damage, and its schwannoma nature was confirmed upon immunohistochemistry analysis. Importantly, blood PSA dropped abruptly from ≈13 to ≈5 ng/ml within 2 months postschwannoma resection, a swift drastic reduction unachievable with oral dutasteride alone. However, 6 weeks later, a new nodule became apparent on the back of the left knee and was identified as a second schwannoma, thereby suggesting that its growth could have been stimulated by the resection of the first schwannoma, as previously described for vestibular schwannomas. The second schwannoma was in fact two: the bigger one was in the common fibular nerve and the smaller one in the tibial nerve. Both echography and MRI results were confirmed upon surgical resection of the bigger knee schwannoma. Although the third schwannoma has not yet been resected and formally characterized, we face a schwannomatosis case with an unexpected potential exosome-mediated stimulating effect on PSA secretion (PSA immunohistochemistry was negative on both schwannomas). On the other hand, preliminary genomic analysis showed a deficient balance for chromosome 22, the very chromosome carrying the three main genes involved in schwannomatosis. This age-related schwannomatosis case is thus discussed in light of the following: age-related DNA repair deficiency culminating in loss of chromosome/heterozygosity; CpG methylation/demethylation-based epigenetic aging; age-related functional decline of the immune system responsible for inefficient elimination of abnormal cells and subsequent tumorigenic cell turn-over; exosome-mediated pathologic intercellular communications; and prostate-invading brain neural progenitors as pathologic peripheral nervous system (PNS) cells.

How does Helicobacter pylori cause gastric cancer through connexins: An opinion review.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium with a number of virulence factors, such as cytotoxin-associated gene A, vacuolating cytotoxin A, its pathogenicity island, and lipopolysaccharide, which cause gastrointestinal diseases. Connexins function in gap junctional homeostasis, and their downregulation is closely related to gastric carcinogenesis. Investigations into H. pylori infection and the fine-tuning of connexins in cells or tissues have been reported in previous studies. Therefore, in this review, the potential mechanisms of H. pylori-induced gastric cancer through connexins are summarized in detail.

[Lectin populations of NK cells against tumors coupled to viral infections (review of literature).]

Analysis of the human NK (natural killers) cells and their functionally different populations in connection to tumor processes accompanied with viral infections is presented. Receptor lectins (non-immunoglobulin proteins and their complexes recognizing polysaccharides, glycoconjugates and glycopattern-containing molecules) play important role in regulation of innate immunity. Communicative diversity of NK-cell populations (in which lectins cofunction to other receptors) is directed against tumors and viruses. Effectiveness and selectivity of action of NK cell populations can be increased in cooperation together with adaptive immunity. Evaluations of occurrence, redistribution (also under influence of cytokines) and contribution of NK-populations (depending on lectin receptors recognition coupled to multifunctions of receptors) in respect of increasing antitumor and antiviral immune responces are given. The data indicate extended prospects of lectin receptors (coupled to other type receptors) containing NK populations of the network compartment of innate immunity upon realization of different variants of organism protection in cooperation with cellular and humoral immunity. Such NK populations are the basis for further intercellular interactions. Innate immunity Cross-Talk, involving the leader NK cell populations acting according to humoral immunity mechanisms, acts on duty regime (importance for therapy of chronic pathology) that results in providing optimal combined antitumor and antiviral cytokine and cytotoxic responses according to the principle of action as «network-in-network¼. The influence network of lectin, Ig-like, cytotoxic, other regulator NK populations (also throuph redistribution of production of cytokines by immunocompetent cells) is perspective for forming early prolongated antitumor and antiviral processes of different types in organism. It is of importance to consider CD diversity of receptor repertuar of lectin, Ig-like and other NK populations revealing different ontogenesis as well as to seach patient key NK-populations to select and construct personally (or for contingents in cases of epidemiological significance) optimal therapeutic/prophylactic NK populations (like variants of CAR-T). Aforementioned data indicate perspectiveness of NK cell populations in development of new antitumor/antiviral effective and selective vaccine strategies, preparations and regimes of their applications. Probiotic lectins reveal features of perspective ligands cofunctioning to network of NK cell populations.

Extracellular vesicles in bone: "dogrobbers" in the "eternal battle field".

Throughout human life, bone is constantly in a delicate dynamic equilibrium of synthesis and resorption, hosting finely-tuned bone mineral metabolic processes for bone homeostasis by collaboration or symphony among several cell types including osteoclasts (OCs), osteoblasts (OBs), osteocytes (OYs), vascular endothelial cells (ECs) and their precursors. Beyond these connections, a substantial level of communication seems to occur between bone and other tissues, and together, they form an organic unit linked to human health and disease. However, the current hypothesis, which includes growth factors, hormones and specific protein secretion, incompletely explains the close connections among bone cells or between bone and other tissues. Extracellular vesicles (EVs) are widely-distributed membrane structures consisting of lipid bilayers, membrane proteins and intravesicular cargo (including proteins and nucleic acids), ranging from 30 nm to 1000 nm in diameter, and their characters have been highly conserved throughout evolution. EVs have targeting abilities and the potential to transmit multidimensional, abundant and complicated information, as powerful and substantial "dogrobbers" mediating intercellular communications. As research has progressed, EVs have gradually become thought of as "dogrobbers" in bone tissue-the "eternal battle field" -in a delicate dynamic balance of destruction and reconstruction. In the current review, we give a brief description of the major constituent cells in bone tissues and explore the progress of current research on bone-derived EVs. In addition, this review also discusses in depth not only potential directions for future research to breakthrough in this area but also problems existing in current research that need to be solved for a better understanding of bone tissues.

G protein-coupled receptor 30 mediates meiosis resumption and gap junction communications downregulation in goat cumulus-oocyte complexes by 17ß-estradiol.

Estrogen plays a critical role in the regulation of gap junctions between oocytes and granulosa cells in mammalian ovaries. G protein-coupled receptor 30 (GPR30) was identified as a membrane estrogen receptor, mediating rapid, nongenomic signaling events that might be responsible for the regulation of oocyte meiosis resumption and gap junction intercellular communications (GJICs). The present study aimed to determine the expression and localization of GPR30 and its role in oocyte meiotic progression and GJICs in goat cumulus-oocyte complexes (COCs). Immunofluorescence experiments revealed that GPR30 was primarily located in the plasma membrane of cumulus cells and oocytes in goats. 17ß-estradiol could promote oocyte meiotic progression, which was blocked by G15 (a selective GPR30 antagonist) but not ICI182780 (a nuclear estrogen receptor inhibitor) in the early stage of in vitro culture. The effect of 17ß-estradiol on the GJICs was quantified by lucifer yellow (LY) microinjection and calcein-AM fluorescent dye diffusion. 17ß-estradiol treatment of goat COCs resulted in rapid downregulation of GJICs. The transfer of calcein from cumulus cells to oocytes could be significantly inhibited by carbenoxolone (a known gap junction blocker), 17ß-estradiol or G1 (a GPR30 agonist), and this inhibition could be reversed by G15 but not ICI182780, indicating that GPR30 mediates the effect of 17ß-estradiol on the rapid downregulation of GJICs. 17ß-estradiol also stimulated the serine 368 phosphorylation of connexin 43 (Cx43) when COCs were in vitro cultured for 4 h, 6 h, and 8 h. More importantly, 17ß-estradiol or G1 could separately promote the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and Cx43 significantly when COCs were cultured for 4 h. Furthermore, both ERK1/2 and Cx43 phosphorylation could be inhibited by G15 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or by the ERK1/2 inhibitor PD98059, indicating that EGFR-ERK1/2 signaling was involved in these events. These results supported the hypothesis that GPR30 mediated 17ß-estradiol-stimulated meiotic resumption and GJIC reduction in goat COCs. Thus, the present study provides novel insights into elucidating the mechanisms for steroid hormone action in the regulation of oocyte maturation.