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Background: Pressure ulcer is common in the bedridden elderly with high mortality and lack of effective treatment. In this study, human-adipose-derived-stem-cells-hyaluronic acid gel (hADSCs-HA gel) was developed and applied topically to treat pressure ulcers, of which efficacy and paracrine mechanisms were investigated through in vivo and in vitro experiments., Methods: Pressure ulcers were established on the backs of C57BL/6 mice and treated topically with hADSCs-HA gel, hADSCs, hyaluronic acid, and normal saline respectively. The rate of wound closure was observed continuously during the following 14 days and the wound samples were obtained for Western blot, histopathology, immunohistochemistry, and proteomic analysis. Human dermal fibroblasts (HDFs) and human venous endothelial cells (HUVECs) under normal or hypoxic conditions were treated with conditioned medium of human ADSCs (ADSC-CM), then CCK-8, scratch test, tube formation, and Western blot were conducted to evaluate the paracrine effects of hADSCs and to explore the underlying mechanism., Results: The in vivo data demonstrated that hADSCs-HA gel significantly accelerated the healing of pressure ulcers by enhancing collagen expression, angiogenesis, and skin proliferation. The in vitro data revealed that hADSCs strengthened the proliferation and wound healing capabilities of HDFs and HUVECs, meanwhile promoted collagen secretion and tube formation through paracrine mode. ADSC-CM was also proved to exert protective effects on hypoxic HDFs and HUVECs. Besides, the results of proteomic analysis and Western blot elucidated that lipid metabolism and PPARβ/δ pathway mediated the healing effect of hADSCs-HA gel on pressure ulcers., Conclusion: Our research showed that topical application of hADSCs-HA gel played an important role in dermal regeneration and angiogenesis. Therefore, hADSCs-HA gel exhibited the potential as a novel stem-cell-based therapeutic strategy of treating pressure ulcers in clinical practices., Competing Interests: The authors declare no competing interests in this work., (© 2024 Jin et al.)

After injury, mammalian spinal cords develop scars to confine the lesion and prevent further damage. However, excessive scarring can hinder neural regeneration and functional recovery 1,2 . These competing actions underscore the importance of developing therapeutic strategies to dynamically modulate scar progression. Previous research on scarring has primarily focused on astrocytes, but recent evidence has suggested that ependymal cells also participate. Ependymal cells normally form the epithelial layer encasing the central canal, but they undergo massive proliferation and differentiation into astroglia following certain injuries, becoming a core scar component 3-7 . However, the mechanisms regulating ependymal proliferation in vivo remain unclear. Here we uncover an endogenous κ-opioid signalling pathway that controls ependymal proliferation. Specifically, we detect expression of the κ-opioid receptor, OPRK1, in a functionally under-characterized cell type known as cerebrospinal fluid-contacting neuron (CSF-cN). We also discover a neighbouring cell population that expresses the cognate ligand prodynorphin (PDYN). Whereas κ-opioids are typically considered inhibitory, they excite CSF-cNs to inhibit ependymal proliferation. Systemic administration of a κ-antagonist enhances ependymal proliferation in uninjured spinal cords in a CSF-cN-dependent manner. Moreover, a κ-agonist impairs ependymal proliferation, scar formation and motor function following injury. Together, our data suggest a paracrine signalling pathway in which PDYN + cells tonically release κ-opioids to stimulate CSF-cNs and suppress ependymal proliferation, revealing an endogenous mechanism and potential pharmacological strategy for modulating scarring after spinal cord injury., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Idiopathic pulmonary fibrosis (IPF) is a devastating condition characterized by progressive lung scarring and uncontrolled fibroblast proliferation, inevitably leading to organ dysfunction and mortality. Although elevated iron levels have been observed in patients and animal models of lung fibrosis, the mechanisms linking iron dysregulation to lung fibrosis pathogenesis, particularly the role of macrophages in orchestrating this process, remain poorly elucidated. Here we evaluate iron metabolism in macrophages during pulmonary fibrosis using both in vivo and in vitro approaches. In murine bleomycin- and amiodarone-induced pulmonary fibrosis models, we observed significant iron deposition and lipid peroxidation in pulmonary macrophages. Intriguingly, the ferroptosis regulator glutathione peroxidase 4 (GPX4) was upregulated in pulmonary macrophages following bleomycin instillation, a finding corroborated by single-cell RNA sequencing analysis. Moreover, macrophages isolated from fibrotic mouse lungs exhibited increased transforming growth factor (TGF)-β1 expression that correlated with lipid peroxidation. In vitro, iron overload in bone marrow-derived macrophages triggered lipid peroxidation and TGF-β1 upregulation, which was effectively suppressed by ferroptosis inhibitors. When cocultured with iron-overloaded macrophages, lung fibroblasts exhibited heightened activation, evidenced by increased α-smooth muscle actin and fibronectin expression. Importantly, this profibrotic effect was attenuated by treating macrophages with a ferroptosis inhibitor or blocking TGF-β receptor signaling in fibroblasts. Collectively, our study elucidates a novel mechanistic paradigm in which the accumulation of iron within macrophages initiates lipid peroxidation, thereby amplifying TGF-β1 production, subsequently instigating fibroblast activation through paracrine signaling. Thus, inhibiting iron overload and lipid peroxidation warrants further exploration as a strategy to suppress fibrotic stimulation by disease-associated macrophages. NEW & NOTEWORTHY This study investigates the role of iron in pulmonary fibrosis, specifically focusing on macrophage-mediated mechanisms. Iron accumulation in fibrotic lung macrophages triggers lipid peroxidation and an upregulation of transforming growth factor (TGF)-β1 expression. Coculturing iron-laden macrophages activates lung fibroblasts in a TGF-β1-dependent manner, which can be mitigated by ferroptosis inhibitors. These findings underscore the potential of targeting iron overload and lipid peroxidation as a promising strategy to alleviate fibrotic stimulation provoked by disease-associated macrophages.

Telmisartan, an angiotensin II type 1 receptor (AT1R) blocker, exhibits broad anti-tumor activity. However, in vitro, anti-proliferative effects are shown at doses far beyond the therapeutic plasma concentration. Considering the role of tumor microenvironment in glioma progression, glioma-astrocyte co-cultures were employed to test the anti-tumor potential of low-dose telmisartan. When a high dose was required for a direct anti-proliferative effect on glioma cell lines, a low dose significantly inhibited glioma cell proliferation and migration in the co-culture system. Under co-culture conditions, upregulated IL-6 expression in astrocytes played a critical role in glioma progression. Silencing IL-6 in astrocytes or IL-6R in glioma cells reduced proliferation and migration. Telmisartan (5 μM) inhibited astrocytic IL-6 expression, and its anti-tumor effects were reversed by silencing IL-6 or IL-6R and inhibiting signal transducer and activator of transcription 3 (STAT3) activity in glioma cells. Moreover, the telmisartan-driven IL-6 downregulation was not imitated by losartan, an AT1R blocker with little capacity of peroxisome proliferator-activated receptor-gamma (PPARγ) activation, but was eliminated by a PPARγ antagonist, indicating that the anti-glioma effects of telmisartan rely on its PPARγ agonistic activity rather than AT1R blockade. This study highlights the importance of astrocytic IL-6-mediated paracrine signaling in glioma growth and the potential of telmisartan as an adjuvant therapy for patients with glioma, especially those with hypertension., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Transforming growth factor-beta 1 (TGF-β1) is a critical regulator of skeletal homeostasis and has diverse effects on osteoblastogenesis. To date, the mechanisms behind the intriguing inhibitory effect of TGF-β1 on osteoblast maturation are not fully understood. Here, we demonstrate a novel mechanism by which TGF-β1 modulates osteoblast maturation through the lysosomal protease legumain. We observed that addition of TGF-β1 to osteogenic cultures of human bone marrow derived mesenchymal stromal (stem) cells enhanced legumain activity and secretion, in-spite of decreased legumain mRNA expression, suggesting post-transcriptional regulation. We further showed that osteogenic cells internalize and activate prolegumain, associated with inhibited osteoblast maturation, revealing legumain as a paracrine regulator of osteoblast maturation. Interestingly, TGF-β1 treatment exacerbated legumain internalization and activity, and showed an additive effect on legumain-induced inhibition of osteoblast maturation. Importantly, pharmacological inhibition of legumain abolished the inhibitory effect of TGF-β1 on osteoblast maturation. Our findings reveal that TGF-β1 inhibits osteoblast maturation by stimulating secretion and activity of endogenous legumain, as well as enhancing internalization and activation of extracellular prolegumain. Therefore, our study provides a deeper understanding of the complex regulation of osteoblastogenesis and unveils a novel TGF-β1-legumain axis in regulation of osteoblast maturation, offering novel insights for possible therapeutic interventions related to bone diseases associated with aberrant TGF-β1 signaling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Forbord, Lunde, Bosnjak-Olsen, Johansen, Solberg and Jafari.)

Rotator cuff injuries present a clinical challenge for repair due to current limitations in functional regeneration of the native tendon-to-bone enthesis. A biomaterial that can regionally instruct unique tissue-specific phenotypes offers potential to promote enthesis repair. We have recently demonstrated the mechanical benefits of a stratified triphasic biomaterial made up of tendon- and bone-mimetic collagen scaffold compartments connected via a continuous hydrogel, and we now explore the potential of a biologically favorable enthesis hydrogel for this application. Here we report in vitro behavior of human mesenchymal stem cells (hMSCs) within thiolated gelatin (Gel-SH) hydrogels in response to chondrogenic stimuli as well as paracrine signals derived from MSC-seeded bone and tendon scaffold compartments. Chondrogenic differentiation media promoted upregulation of cartilage and entheseal fibrocartilage matrix markers COL2 , COLX , and ACAN as well as the enthesis-associated transcription factors SCX , SOX9 , and RUNX2 in hMSCs within Gel-SH. Similar effects were observed in response to TGF-β3 and BMP-4, enthesis-associated growth factors known to play a role in entheseal development and maintenance. Conditioned media generated by hMSCs seeded in tendon- and bone-mimetic collagen scaffolds influenced patterns of gene expression regarding enthesis-relevant growth factors, matrix markers, and tendon-to-bone transcription factors for hMSCs within the material. Together, these findings demonstrate that a Gel-SH hydrogel provides a permissive environment for enthesis tissue engineering and highlights the significance of cellular crosstalk between adjacent compartments within a spatially graded biomaterial.

Glucagon is critical for the maintenance of blood glucose, however nutrient regulation of pancreatic α-cells remains poorly understood. Here, we identified a role of leucine, a well-known β-cell fuel, in the α-cell-intrinsic regulation of glucagon release. In islet perifusion assays, physiologic concentrations of leucine strongly inhibited alanine- and arginine-stimulated glucagon secretion from human and mouse islets under hypoglycemic conditions. Mechanistically, leucine dose-dependently reduced α-cell cAMP, independently of Ca2+, ATP/ADP, or fatty acid oxidation. Leucine also reduced α-cell cAMP in islets treated with somatostatin receptor 2 antagonists or diazoxide, compounds that limit paracrine signaling from β/δ-cells. Studies in dispersed mouse islets confirmed an α-cell-intrinsic effect. The inhibitory effect of leucine on cAMP was mimicked by glucose, α-ketoisocaproate, succinate, and the glutamate dehydrogenase activator BCH and blocked by cyanide, indicating a mechanism dependent on mitochondrial metabolism. Glucose dose-dependently reduced the impact of leucine on α-cell cAMP, indicating an overlap in function; however, leucine was still effective at suppressing glucagon secretion in the presence of elevated glucose, amino acids, and the incretin GIP. Taken together, these findings show that leucine plays an intrinsic role in limiting the α-cell secretory tone across the physiologic range of glucose levels, complementing the inhibitory paracrine actions of β/δ-cells., (© 2024 by the American Diabetes Association.)

Ethnopharmacological Relevance: Wenshen Xiaozheng Tang (WXT), a traditional Chinese medicine (TCM) decoction, is effective for treating endometriosis. However, the effect of WXT on endometrium-derived mesenchymal stem cells (eMSCs) which play a key role in the fibrogenesis of endometriosis requires further elucidation., Aims of the Study: The aim of this study was to clarify the potential mechanism of WXT in improving fibrosis in endometriosis by investigating the regulation of WXT on differentiation and paracrine of eMSCs., Materials and Methods: The nude mice with endometriosis were randomly divided into model group, WXT group and mifepristone group. After 21 days of treatment, the lesion volume was calculated. Fibrosis in the lesions was evaluated by Masson staining and expression of fibrotic proteins. The differentiation of eMSCs in vivo was explored using a fate-tracking experiment. To further clarify the regulation of WXT on eMSCs, primary eMSCs from the ectopic lesions of endometriosis patients were isolated and characterized. The effect of WXT on the proliferation and differentiation of ectopic eMSCs was examined. To evaluate the role of WXT on the paracrine activity of ectopic eMSCs, the conditioned medium (CM) from ectopic eMSCs pretreated with WXT was collected and applied to treat ectopic endometrial stromal cells (ESCs), after which the expression of fibrotic proteins in ectopic ESCs was assessed. In addition, transcriptome sequencing was used to investigate the regulatory mechanism of WXT on ectopic eMSCs, and western blot and ELISA were employed to determine the key mediator., Results: WXT impeded the growth of ectopic lesions in nude mice with endometriosis and reduced collagen deposition and the expression of fibrotic proteins fibronectin, collagen I, α-SMA and CTGF in the endometriotic lesions. The fate-tracking experiment showed that WXT prevented human eMSCs from differentiating into myofibroblasts in the nude mice. We successfully isolated eMSCs from the lesions of patients with endometriosis and demonstrated that WXT suppressed proliferation and myofibroblast differentiation of ectopic eMSCs. Moreover, the expression of α-SMA, collagen I, fibronectin and CTGF in ectopic ESCs was significantly down-regulated by the CM of ectopic MSCs pretreated with WXT. Combining the results of RNA sequencing, western blot and ELISA, we found that WXT not only reduced thrombospondin 4 expression in ectopic eMSCs, but also decreased thrombospondin 4 secretion from ectopic eMSCs. Thrombospondin 4 concentration-dependently upregulated the expression of collagen I, fibronectin, α-SMA and CTGF in ectopic ESCs, indicating that thrombospondin 4 was a key mediator of WXT in inhibiting the fibrotic process in endometriosis., Conclusion: WXT improved fibrosis in endometriosis by regulating differentiation and paracrine signaling of eMSCs. Thrombospondin 4, whose release from ectopic eMSCs is inhibited by WXT, may be a potential target for the treatment of endometriosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation., Methods: The transforming growth factor-β1 (TGF-β1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts., Results: Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-β1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue., Conclusions: Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism., (© 2024. The Author(s).)

Background and Aims: Patients with pancreatic ductal adenocarcinoma (PDA) have not yet benefitted from the revolution in cancer immunotherapy due in large part to a dominantly immunosuppressive tumor microenvironment. MEK inhibition combined with autophagy inhibition leads to transient tumor responses in some patients with PDA. We examined the functional effects of combined MEK and autophagy inhibition on the PDA immune microenvironment and the synergy of combined inhibition of MEK and autophagy with CD40 agonism (aCD40) against PDA using immunocompetent model systems., Methods: We implanted immunologically "cold" murine PDA cells orthotopically in wide type C57BL/6J mice. We administered combinations of inhibitors of MEK1/2, inhibitors of autophagy, and aCD40 and measured anticancer efficacy and immune sequelae using mass cytometry and multiplexed immunofluorescence imaging analysis to characterize the tumor microenvironment. We also used human and mouse PDA cell lines and human macrophages in vitro to perform functional assays to elucidate the cellular effects induced by the treatments., Results: We find that coinhibition of MEK (using cobimetinib) and autophagy (using mefloquine), but not either treatment alone, activates the STING/type I interferon pathway in tumor cells that in turn activates paracrine tumor associated macrophages toward an immunogenic M1-like phenotype. This switch is further augmented by aCD40. Triple therapy (cobimetinib + mefloquine + aCD40) achieved cytotoxic T-cell activation in an immunologically "cold" mouse PDA model, leading to enhanced antitumor immunity., Conclusions: MEK and autophagy coinhibition coupled with aCD40 invokes immune repolarization and is an attractive therapeutic approach for PDA immunotherapy development., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Glioblastoma multiforme (GBM) is a highly invasive brain tumour, characterized by its ability to secrete factors promoting its virulence. Brain endothelial cells (BECs) in the GBM environment are physiologically modulated. The present study investigated the modulatory effects of normoxically and hypoxically induced glioblastoma U-87 cell secretions on BECs., Methods: Conditioned media (CM) were derived by cultivating U-87 cells under hypoxic incubation (5% O 2 ) and normoxic incubation (21% O 2 ). Treated bEnd.3 cells were evaluated for mitochondrial dehydrogenase activity, mitochondrial membrane potential (ΔΨm), ATP production, transendothelial electrical resistance (TEER), and endothelial tight-junction (ETJ) gene expression over 96 h., Results: The coculture of bEnd.3 cells with U-87 cells, or exposure to either hypoxic or normoxic U-87CM, was associated with low cellular viability. The ΔΨm in bEnd.3 cells was hyperpolarized after hypoxic U-87CM treatment ( p < 0.0001). However, normoxic U-87CM did not affect the state of ΔΨm. BEC ATP levels were reduced after being cocultured with U-87 cells, or with hypoxic and normoxic CM ( p < 0.05). Suppressed mitochondrial activity in bEnd.3 cells was associated with increased transendothelial permeability, while bEnd.3 cells significantly increased the gene expression levels of ETJs ( p < 0.05) when treated with U-87CM., Conclusions: Hypoxic and normoxic glioblastoma paracrine factors differentially suppressed mitochondrial activity in BECs, increasing the BECs' barrier permeability.

Doxorubicin (Dox), an important anthracycline, is a potent anticancer agent that is used for treating solid tumors and hematologic malignancies. However, its clinical use is hampered by cardiac cardiotoxicity. This study aimed to investigate the cardioprotective potential of miR-199a-3p. Continuous Dox treatment not only markedly induced cardiomyocyte senescence but also resulted in a growing number of senescence-associated secretory phenotype (SASP) cardiomyocytes, frequently leading to heart senescence. This study showed that miR-199a-3p was downregulated in cardiomyocytes when exposed to Dox. The cardiac-specific overexpression of miR-199a-3p promoted cell cycle re-entry and cell proliferation, resulting in relief from cardiac senescence. Also, the elevation of miR-199a-3p inhibited the generation of SASP, thus, hampering the spread of senescence. In cardiomyocytes, the modulation of miR-199a-3p changed the levels of senescence-related protein GATA4. The ectopic expression of GATA4 blunted the anti-senescence effect of miR-199a-3p. Together, the data supported a role for miR-199a-3p during Dox cardiotoxicity. The elevation of miR-199a-3p might provide a dual therapeutic advantage in Dox cardiotoxicity therapy by simultaneously preventing cardiac senescence and reducing the spread of senescence.

The limited ability of mammalian adult cardiomyocytes to proliferate following an injury to the heart, such as myocardial infarction, is a major factor that results in adverse fibrotic and myocardial remodeling that ultimately leads to heart failure. The continued high degree of heart failure-associated morbidity and lethality requires the special attention of researchers worldwide to develop efficient therapeutics for cardiac repair. Recently, various strategies and approaches have been developed and tested to extrinsically induce regeneration and restoration of the myocardium after cardiac injury have yielded encouraging results. Nevertheless, these interventions still lack adequate success to be used for clinical interventions. This review highlights and discusses both cell-based and cell-free therapeutic approaches as well as current advancements, major limitations, and future perspectives towards developing an efficient therapeutic method for cardiac repair.

Dimeric translationally controlled tumor protein (dTCTP), also known as histamine-releasing factor, amplifies allergic responses and its production has been shown to increase in inflammatory diseases such as allergic asthma. Despite the critical role of dTCTP in allergic inflammation, little is known about its production pathways, associated cellular networks, and underlying molecular mechanisms. In this study, we explored the dTCTP-mediated inflammatory networks and molecular mechanisms of dTCTP associated with lipopolysaccharides (LPS)-induced severe asthma. LPS stimulation increased dTCTP production by mast cells and dTCTP secretion during degranulation, and extracellular dTCTP subsequently increased the production of pro-inflammatory molecules, including IL-8, by airway epithelial cells without affecting mast cell activation. Furthermore, dimeric TCTP-binding peptide 2 (dTBP2), a dTCTP inhibitor peptide, selectively blocked the dTCTP-mediated signaling network from mast cells to epithelial cells and decreased IL-8 production through IkB induction and nuclear p65 export in airway epithelial cells. More importantly, dTBP2 efficiently attenuated LPS-induced severe airway inflammation in vivo, resulting in decreased immune cell infiltration and IL-17 production and attenuated dTCTP secretion. These results suggest that dTCTP produced by mast cells exacerbates airway inflammation through activation of airway epithelial cells in a paracrine signaling manner, and that dTBP2 is beneficial in the treatment of severe airway inflammation by blocking the dTCTP-mediated inflammatory cellular network., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment.

Increased expression and activity of the Ca 2+ channel transient receptor potential channel 6 (TRPC6) is associated with focal segmental glomerulosclerosis, but therapeutic strategies to target TRPC6 are currently lacking. Nitric oxide (NO) is crucial for normal glomerular function and plays a protective role in preventing glomerular diseases. We investigated if NO prevents podocyte injury by inhibiting injurious TRPC6-mediated signaling in a soluble guanylate cyclase (sGC)-dependent manner and studied the therapeutic potential of the sGC stimulator Riociguat. Experiments were performed using human glomerular endothelial cells and podocytes. Podocyte injury was induced by Adriamycin incubation for 24 h, with or without the NO-donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP), the sGC stimulator Riociguat or the TRPC6 inhibitor Larixyl Acetate (LA). NO and Riociguat stimulated cGMP synthesis in podocytes, decreased Adriamycin-induced TRPC6 expression, inhibited the Adriamycin-induced TRPC6-mediated Ca 2+ influx and reduced podocyte injury. The protective effects of Riociguat and NO were blocked when sGC activity was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or when TRPC6 activity was inhibited by LA. Our data demonstrate a glomerular (e)NOS-NO-sGC-cGMP-TRPC6 pathway that prevents podocyte injury, which can be translated to future clinical use by, e.g., repurposing the market-approved drug Riociguat.

Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However, the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations, therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC), we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-α. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion, we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 You, Stelzl, Joseph, Aldo, Maxwell, Dekel, Liao, Whirledge and Mor.)

Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Active cancer-associated fibroblasts (CAFs) are major components of the tumor microenvironment, which promote carcinogenesis and modulate response to therapy. Therefore, targeting these cells or reducing their paracrine pro-carcinogenic effects could be of great therapeutic value. To this end, we sought to investigate the effect of eugenol, a natural phenolic molecule, on active breast CAFs. We have shown that decitabine (5-Aza-2'-deoxycytidine, DAC) and eugenol inhibit the expression of the DNA methyltransferase genes DNMT1 and DNMT3A at both the protein and mRNA levels in breast CAF cells. While the effect of eugenol was persistent, DAC had only a transient inhibitory effect on the mRNA level of both DNMT genes. Furthermore, eugenol and DAC suppressed the invasive/migratory and proliferative potential of CAF cells as well as their paracrine pro-carcinogenic effects both in vitro and in humanized orthotopic tumor xenografts. Interestingly, these inhibitory effects of decitabine and eugenol were mediated through E2F1 downregulation. Indeed, ectopic expression of E2F1 upregulated both genes and attenuated the effects of eugenol. Additionally, we provide clear evidence that eugenol, like DAC, strongly modulates the methylation pattern in active CAF cells, through methylating several oncogenes and demethylating various important tumor suppressor genes, which affected their mRNA expression levels. Importantly, the E2F1 promoter was also hypermethylated and the gene downregulated in response to eugenol. Together, these findings show that the active features of breast CAF cells can be normalized through eugenol-dependent targeting of DNMT1/DNMT3A and the consequent modulation in gene methylation., (© 2021 Wiley Periodicals LLC.)

COPD is characterized by irreversible lung tissue damage. We hypothesized that lung-derived mesenchymal stromal cells (LMSCs) reduce alveolar epithelial damage via paracrine processes, and may thus be suitable for cell-based strategies in COPD. We aimed to assess whether COPD-derived LMSCs display abnormalities. LMSCs were isolated from lung tissue of severe COPD patients and non-COPD controls. Effects of LMSC conditioned-medium (CM) on H 2 O 2 -induced, electric field- and scratch-injury were studied in A549 and NCI-H441 epithelial cells. In organoid models, LMSCs were co-cultured with NCI-H441 or primary lung cells. Organoid number, size and expression of alveolar type II markers were assessed. Pre-treatment with LMSC-CM significantly attenuated oxidative stress-induced necrosis and accelerated wound repair in A549. Co-culture with LMSCs supported organoid formation in NCI-H441 and primary epithelial cells, resulting in significantly larger organoids with lower type II-marker positivity in the presence of COPD-derived versus control LMSCs. Similar abnormalities developed in organoids from COPD compared to control-derived lung cells, with significantly larger organoids. Collectively, this indicates that LMSCs' secretome attenuates alveolar epithelial injury and supports epithelial repair. Additionally, LMSCs promote generation of alveolar organoids, with abnormalities in the supportive effects of COPD-derived LMCS, reflective of impaired regenerative responses of COPD distal lung cells.

Multicellular spheroids show three-dimensional (3D) organization with extensive cell-cell and cell-extracellular matrix interactions. Owing to their native tissue-mimicking characteristics, mesenchymal stem cell (MSC) spheroids are considered promising as implantable therapeutics for stem cell therapy. Herein, we aim to further enhance their therapeutic potential by tuning the cultivation parameters and thus the inherent niche of 3D MSC spheroids. Significantly increased expression of multiple pro-regenerative paracrine signaling molecules and immunomodulatory factors by MSCs was observed after optimizing the conditions for spheroid culture. Moreover, these alterations in cellular behaviors may be associated with not only the hypoxic niche developed in the spheroid core but also with the metabolic reconfiguration of MSCs. The present study provides efficient methods for manipulating the therapeutic capacity of 3D MSC spheroids, thus laying solid foundations for future development and clinical application of spheroid-based MSC therapy for regenerative medicine.

Recent epidemiological studies reported cases of cholangiocarcinoma in workers exposed to 1,2-dichloropropane (1,2-DCP) in an offset proof printing factory in Japan. The present study investigated the effects of 1,2-DCP on the expression of histone family member X (H2AX) phosphorylated on Ser 139 (γ-H2AX), a marker of DNA double strand break, in human immortalized cholangiocytes MMNK-1 cells. Mono-cultures of MMNK-1 cells and co-cultures of MMNK-1 cells with THP-1 macrophages were exposed to 1,2-DCP at concentrations of 100 and 500 μM for 24 h. Expression of γ-H2AX was visualized by immunofluorescence staining. Exposure to 1,2-DCP had no effect on the expression of γ-H2AX in mono-cultured MMNK-1 cells, but significantly increased the number of nuclear foci stained by γ-H2AX in MMNK-1 cells co-cultured with THP-1 macrophages. Exposure to 1,2-DCP also significantly increased the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in co-cultured MMNK-1 cells. The results suggest that macrophages play a critical role by producing cytokines in 1,2-DCP-induced DNA double strand break in MMNK-1 cells., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Intervertebral disc degeneration is an irreversible process associated with accumulation of senescent nucleus pulposus (NP) cells. This study investigates the hypothesis that Tumor necrosis factor-α (TNF-α)-treated senescent NP cells propagate senescence of neighboring healthy cells via a paracrine effect that involves p-Stat3 signaling and the cytokine interleukin-6 (IL-6). NP cells isolated from bovine caudal intervertebral disc (IVD) were treated with TNF-α to induce senescence which was confirmed by demonstrating upregulation of senescence-associated β-galactosidase and p16. This was correlated with downregulation of NP-associated markers, Aggrecan, Col2A1, and Sox9. Direct contact and non-contact co-culture of healthy and senescent cells showed that TNF-α-treated cells increased the senescence in healthy cells via a paracrine effect. The senescent cells have a secretory phenotype as indicated by increased gene and protein levels of IL-6. Phosphorylated Signal Transducer and Activator of Transcription 3 (pStat3) levels were also high in treated cells and appeared to upregulate IL-6 as inhibition of Stat3 phosphorylation by StatticV downregulated IL-6 mRNA expression in cells and protein levels in the culture media. All trans retinoic acid, an IL-6 inhibitor, also decreased the secretion of IL-6 and reduced the paracrine effect of senescent cells on healthy cells. Decreased pStat3 levels and inhibition of IL-6 secretion did not fully restore NP gene expression of Col2A1 but importantly, appeared to cause senescent cells to undergo apoptosis and cell death. This study demonstrated the paracrine effect of senescent NP cells which involves Stat3 and IL-6 and may explain why senescent NP cells accumulate in IVD with age. The role of pSTAT3 and IL-6 in mediating NP senescence requires further study as it may be a novel strategy for modulating the senescent-inducing effects of TNF-α., (© 2021 Federation of American Societies for Experimental Biology.)

Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-β signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.

Rationale: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies., Aim: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research., Methods and Results: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor β3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs., Conclusion: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.

Vascular calcification is common in chronic kidney disease; however, the extent to which such condition can affect the renal microvasculature and the neighboring cell types is unclear. Our induced-calcification model in renal proximal tubular (PT) cells exhibited endoplasmic reticulum (ER) stress and oxidative damage, leading to apoptosis. Here, we utilized such calcification in mouse vascular smooth muscle (MOVAS-1) cells as a vascular calcification model, because it exhibited reactive oxygen species (ROS) generation, ER and oxidative stress, inflammatory, and apoptotic gene expressions. To demonstrate whether the vascular calcification condition can dictate the function of the adjacent PT cell layer, we utilized a Transwell multilayer culture system by combining those MOVAS-1 cells in the bottom chamber and polarized PT cells in the upper chamber to show the dimensional cross-signaling effect. Interestingly, calcification of MOVAS-1 cells, in this co-culture, induced H 2 O 2 and lactate dehydrogenase (LDH) release leading to store-operated Ca 2+ entry, ROS generation, and activation of oxidative, inflammatory, and apoptotic gene expressions in PT cells through paracrine signaling. Interestingly, application of tannic acid (TA) to either calcified MOVAS-1 or uncalcified PT cells diminished such detrimental pathway activation. Furthermore, the TA-mediated protection was much higher in the PT cells when applied on the calcified MOVAS-1 cells, and the delayed the pathological effects in neighboring PT cells can well be via paracrine signaling. Together, these results provide evidence of vascular calcification-induced PT cell damage, and the protective role of TA in preventing such pathological consequences, which can potentially be used as a nephroprotective remedy., (Published by Elsevier Masson SAS.)

3,5,6-Trichloro-2-pyridinol (TCP) is an important biomarker and one of the final metabolites of chlorpyrifos (CPF). TCP inhibits secretion of sex hormones. Similar to CPF, TCP can bind to sex steroid hormone receptors and decrease the secretion of sex hormones. However, little attention has been paid to the ability of TCP and CPF to interfere with androgen receptor (AR) in Sertoli cells. This study aimed to explain how TCP promotes the inhibitory effect of CPF on the paracrine function of Sertoli cells. Western blotting indicated that after 20 weeks of exposure, expression of AR in testes was significantly reduced by CPF. An in vitro assay measured the cytotoxicity of CPF, TCP and diethylphosphate (DEP) on viability of Sertoli cells by Cell Counting Kit-8. CPF cytotoxicity was greater than that of TCP, and TCP cytotoxicity was greater than that of DEP at concentrations of 1000 μmol/L. Western blotting indicated that TCP and CPF both decreased expression of AR and cAMP-response element binding protein phosphorylation, while DEP had no effect in Sertoli cells, which are important in regulating paracrine function of Sertoli cells. The fluorescence measurements and docking studies revealed that testosterone, CPF and TCP showed four types of intermolecular interactions with AR, highlighting alkyl bonds with some of the same amino acids. Compared with testosterone, CPF and TCP also showed significant synergistic interaction with AR. CPF interacted with more amino acids and interaction energy than TCP did. This research elucidates TCP in the antiandrogenic effect of CPF on the paracrine function and suggests that TCP or chemicals with a trichloropyridine structure must be considered during reproductive toxicity assessment of potential environmental pollutants., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis.

The resolution of inflammation is closely linked with tissue repair. Recent studies have revealed that macrophages suppress inflammatory reactions by producing lipid mediators, called specialized proresolving mediators (SPMs); however, the biological significance of SPMs in tissue repair remains to be fully elucidated in the heart. In this study, we focused on maresin-1 (MaR1) and examined the reparative effects of MaR1 in cardiomyocytes. The treatment with MaR1 increased cell size in cultured neonatal rat cardiomyocytes. Since the expression of fetal cardiac genes was unchanged by MaR1, physiological hypertrophy was induced by MaR1. SR3335, an inhibitor of retinoic acid-related orphan receptor α (RORα), mitigated MaR1-induced cardiomyocyte hypertrophy, consistent with the recent report that RORα is one of MaR1 receptors. Importantly, in response to MaR1, cardiomyocytes produced IGF-1 via RORα. Moreover, MaR1 activated phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and wortmannin, a PI3K inhibitor, or triciribine, an Akt inhibitor, abrogated MaR1-induced cardiomyocyte hypertrophy. Finally, the blockade of IGF-1 receptor by NVP-AEW541 inhibited MaR-1-induced cardiomyocyte hypertrophy as well as the activation of PI3K/Akt pathway. These data indicate that MaR1 induces cardiomyocyte hypertrophy through RORα/IGF-1/PI3K/Akt pathway. Considering that MaR1 is a potent resolving factor, MaR1 could be a key mediator that orchestrates the resolution of inflammation with myocardial repair.

In obesity, high levels of TNF-α in the bone marrow microenvironment induce the bone marrow-mesenchymal stem cells (BM-MSCs) towards a pro-adipogenic phenotype. Here, we investigated the effect of obesity on the migratory potential of BM-MSCs and their fate towards the adipose tissues. BM-MSCs were isolated from male C57Bl/06 mice with high-fat diet-induced obesity. The migratory potential of the BM-MSCs, their presence in the subcutaneous (SAT) and the visceral adipose tissues (VAT), and the possible mechanisms involved were investigated. Obesity did not affect MSC content in the bone marrow but increased the frequency of MSCs in blood, SAT, and VAT. In these animals, the SAT adipocytes presented a larger area, without any changes in adipokine production or the Sdf-1α gene expression. In contrast, in VAT, obesity increased leptin and IL-10 levels but did not modify the size of the adipocytes. The BM-MSCs from obese animals presented increased spontaneous migratory activity. Despite the augmented expression of Cxcr4, these cells exhibited decreased migratory response towards SDF-1α, compared to that of BM-MSCs from lean mice. The PI3K-AKT pathway activation seems to mediate the migration of BM-MSCs from lean mice, but not from obese mice. Additionally, we observed an increase in the spontaneous migration of BM-MSCs from lean mice when they were co-cultured with BM-HCs from obese animals, suggesting a paracrine effect. We concluded that obesity increased the migratory potential of the BM-MSCs and induced their accumulation in VAT, which may represent an adaptive mechanism in response to chronic nutrient overload.

Bone marrow mononuclear cell therapy improves cardiac repair after myocardial infarction (MI), in-part through signaling to resident cardiac cells, such as fibroblasts, which regulate scar formation. The efficacy of cell therapy declines with age, as aging of both donor and recipient cells decreases repair responses. Autophagy regulates the microenvironment by both extracellular vesicle (EV)-dependent and independent secretion pathways. We hypothesized that age-related autophagy changes in bone marrow cells (BMCs) alter paracrine signaling, contributing to lower cell therapy efficacy. Here, we demonstrate that young Sca-1 + BMCs exhibited a higher LC3II/LC3I ratio compared to old Sca-1 + BMCs, which was accentuated when BMCs were cultured under hypoxia. To examine the effect on paracrine signaling, old cardiac fibroblasts were cultured with conditioned medium (CM) from young and old Sca-1 + BMCs. Young, but not old CM, enhanced fibroblast proliferation, migration, and differentiation, plus reduced senescence. These beneficial effects were lost when autophagy or EV secretion in BMCs was blocked pharmacologically, or by siRNA knockdown of Atg7 . Therefore, both EV-dependent and -independent paracrine signaling from young BMCs is responsible for paracrine stimulation of old cardiac fibroblasts. In vivo , bone marrow chimerism of old mice with young BMCs increased the number of LC3b + cells in the heart compared to old mice reconstituted with old BMCs. These data suggest that the deterioration of autophagy with aging negatively impacts the paracrine effects of BMCs, and provide mechanistic insight into the age-related decline in cell therapy efficacy that could be targeted to improve the function of old donor cells.

The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell-specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows., Competing Interests: The authors declare no competing interest.

Chemotherapy (CTX) remains the standard of care for most aggressive tumours, including breast cancer (BC). In BC chemotherapeutic regimens, the maximum tolerated dose of cytotoxic drugs is administered at regular intervals, and cancer cells can re-grow or adapt during the resting periods between cycles. The impact of the tumour microenvironment on the fate of cancer cells after CTX remains poorly understood. Here, we show that paracrine signalling from CTX-treated cancer cells to stromal fibroblasts can drive cancer cell recovery after cytotoxic drug withdrawal. Interferon β1 (IFNβ1) secreted by cancer cells following treatment with high doses of CTX instigates the acquisition of an anti-viral state in stromal fibroblasts. This state is associated with an expression pattern here referred to as interferon signature (IFNS), which encompasses several interferon-stimulated genes (ISGs), including numerous pro-inflammatory cytokine genes. This crosstalk is an important driver of the expansion of BC cells after CTX, and IFNβ1 blockade in tumour cells abrogated their fibroblast-dependent recovery potential. Analysis of human breast carcinomas supported a link between CTX-induced IFNS in tumour stroma and poor response to CTX treatment. First, IFNβ1 expression in human breast carcinomas was found to inversely correlate with recurrence free survival (RFS). Second, using laser capture microdissection data sets, we show a higher expression of IFNS in the stromal tumour compartment compared to the epithelial one and this signature was found to be more prominent in more aggressive subtypes of BC (basal-like), pointing to a pro-tumorigenic role of this signature. Moreover, IFNS was associated with higher recurrence rates and a worse outcome in BC patients. Our study unravels a novel form of paracrine communication between cancer cells and fibroblasts that ultimately results in CTX resistance. Targeting this axis has the potential to improve CTX outcomes in patients with BC., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of COVID-19, is reported to increase the rate of mortality worldwide. COVID-19 is associated with acute respiratory symptoms as well as blood coagulation in the vessels (thrombosis), heart attack and stroke. Given the requirement of angiotensin converting enzyme 2 (ACE2) receptor for SARS-CoV-2 entry into host cells, here we discuss how the downregulation of ACE2 in the COVID-19 patients and virus-induced shift in ACE2 catalytic equilibrium, change the concentrations of substrates such as angiotensin II, apelin-13, dynorphin-13, and products such as angiotensin (1-7), angiotensin (1-9), apelin-12, dynorphin-12 in the human body. Substrates accumulation ultimately induces inflammation, angiogenesis, thrombosis, neuronal and tissue damage while diminished products lead to the loss of the anti-inflammatory, anti-thrombotic and anti-angiogenic responses. In this review, we focus on the viral-induced imbalance between ACE2 substrates and products which exacerbates the severity of COVID-19. Considering the roadmap, we propose multiple therapeutic strategies aiming to rebalance the products of ACE2 and to ameliorate the symptoms of the disease., (Copyright © 2021. Published by Elsevier Masson SAS.)

IL-33 is a member of the IL-1 family. By binding to its receptor ST2 (IL-33R) on mast cells, IL-33 induces the MyD88-dependent activation of the TAK1-IKK2 signalling module resulting in activation of the MAP kinases p38, JNK1/2 and ERK1/2, and of NFκB. Depending on the kinases activated in these pathways, the IL-33-induced signalling is essential for production of IL-6 or IL-2. This was shown to control the dichotomy between RORγt + and Helios + T regs , respectively. SCF, the ligand of c-Kit (CD117), can enhance these effects. Here, we show that IL-3, another growth factor for mast cells, is essential for the expression of ICOS-L on BMMCs, and costimulation with IL-3 potentiated the IL-33-induced IL-6 production similar to SCF. In contrast to the enhanced IL-2 production by SCF-induced modulation of the IL-33 signalling, IL-3 blocked the production of IL-2. Consequently, IL-3 shifted the IL-33-induced T reg dichotomy towards RORγt + T regs at the expense of RORγt - Helios + T regs . However, ICOS-L expression was downregulated by IL-33. In line with that, ICOS-L did not play any important role in the T reg modulation by IL-3/IL-33-activated mast cells. These findings demonstrate that different from the mast cell growth factor SCF, IL-3 can alter the IL-33-induced and mast cell-dependent regulation of T reg subpopulations by modulating mast cell-derived cytokine profiles., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Mesenchymal stromal cells (MSCs) secreting multiple growth factors and immunomodulatory cytokines are promising for regenerative medicine. To further enhance their secretory activity, efforts have emerged to tether nanosized carriers of secretory stimuli, named nanostimulators, to the MSC surface by forming nonchemical bonds. Despite some successes, there is a great need to improve the retention of nanostimulators during transport through a syringe needle, where high shear stress exerted on the cell surface separates them. To this end, we hypothesize that poly(lactic- co -glycolic acid)- block -hyaluronic acid (PLGA-HA) conjugated with integrin-binding RGD peptides, denoted PLGA-HA-RGD, can form nanostimulators that remain on the cell surface stably during the injection. The resulting HA-CD44 and RGD-integrin bonds would synergistically increase the adhesion strength of nanostimulators. Interestingly, nanostimulators prepared with PLGA-HA-RGD show 3- to 6-fold higher retention than those made with PLGA-HA. Therefore, the PLGA-HA-RGD nanostimulators induced MSCs to secrete 1.5-fold higher vascular endothelial growth factors and a 1.2-fold higher tissue inhibitor of matrix metalloproteinase-1 as compared to PLGA-HA nanostimulators. Consequently, MSCs tethered with PLGA-HA-RGD nanostimulators served to stimulate endothelial cell activities to form a blood vessel-like endothelial lumen with increased length and number of junctions. The nanostimulator design strategy would also be broadly applicable to regulate, protect, and home a broad array of therapeutic or immune cells by tethering carriers with bioactive molecules of interest.

Background: When vascular endothelial cells are subjected to external stimuli, paracrine hormones and cytokines act on adjacent cells. The regulation of the biological behaviour of cells is closely related to the maintenance of organ function and the occurrence and development of disease. However, it is unclear whether vascular endothelial cells affect the biological behaviour of cells involved in wound repair through autocrine and paracrine mechanisms and ultimately play a role in wound healing. We aimed to verify the effect of the autocrine and paracrine functions of vascular endothelial cells on wound healing., Materials and Methods: ELISA was used to detect platelet-derived growth factor, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor in human umbilical vascular endothelial cell-conditioned medium (HUVEC-CM). Different concentrations of HUVEC-CM were used to treat different stem cells. CCK-8 and scratch assays were used to detect the proliferation and migration ability of each cell. A full-thickness dorsal skin defect model was established in mice, and skin wound healing was observed after the local injection of HUVEC-CM, endothelial cell medium (ECM), or normal saline. H&E staining and immunofluorescence were used to observe the gross morphology of the wound tissue, the epithelial cell migration distance, and the expression of CD3 and CD31., Results: HUVEC-CM promotes the proliferation and migration of epidermal stem cells, skin fibroblasts, bone marrow mesenchymal stem cells, and HUVECs themselves. Furthermore, HUVEC-CM can promote angiogenesis in mouse skin wounds and granulation tissue formation and can accelerate wound surface epithelialization and collagen synthesis, thereby promoting wound healing., Conclusion: Our results clearly suggest that it is practicable and effective to promote wound healing with cytokines secreted by vascular endothelial cells in a mouse model., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2021 Yang Lu et al.)

Conventionally, Cancer-associated fibroblasts (CAFs) are considered as an inducer of chemoresistance in cancer cells. However, the underlying mechanism by which carcinomas induce chemoresistance in CAFs through tumor-stroma cross-talk is largely unknown. Henceforth, we uncovered a network of paracrine signals between carcinoma and CAFs that drives chemoresistance in CAFs. Acquired tamoxifen and 5-Fu resistant cell lines MCF-7 and MDA-MB-468 respectively showed higher apoptotic resistance compared to the parental cell. Besides, chemoresistant breast cancer cells showed overexpression of TGF-β1 and have the higher potential to induce CAF phenotype in the normal dermal fibroblasts in a paracrine manner through the TGF-β1 cytokine, compared to their parental cell. Moreover, the chemoresistant cancer cells augmented the EMT markers with a reduction of E-cadherin in the CAFs. Importantly we found out that the TGF- β1 enriched conditioned media from both of the resistant cells triggered chemoresistance in the CAFs by p44/42 MAPK signaling axis. Mechanistically, pharmacological and genetic blockade of TGF-β1 inhibits p44/42 MAPK activation with the subsequent restoration of chemosensitivity in the CAFs. Altogether we ascertained that chemoresistant cancer cells have tremendous potential to modulate the CAFs compared to the parental counterpart. Targeting TGF-β1 and p44/42 MAPK signaling in the future may help to abrogate the chemoresistance in the CAFs., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Age-related changes in the dermis can play a primary role in tumor initiation promoting the unrestrained proliferation of precancerous keratinocytes (KCs) through cytokines and GF secretion. We found a high percentage of epithelial-to-mesenchymal transition-like colonies raising in primary human KC cultures from old subjects after treatment with aged fibroblast supernatants (SPNs). Continuous extracellular signals were required for maintaining these changes. Conversely, the secretome did not induce epithelial-to-mesenchymal transition-like colonies in KCs from young subjects. SPN-treated aged KCs displayed the activation of pathways involved in the disjunction of cell‒cell adhesion, extracellular matrix remodeling, manifestation of a mesenchymal phenotype, and dedifferentiation programs. Moreover, they recovered proliferation and clonogenic ability and showed enhanced migration. We identified an age-related increase of the BDNF secretion from fibroblasts as well as of the expression of its receptor TrkB in KCs. BDNF treatment of aged KCs induced TrkB phosphorylation and recapitulated the modifications promoted by aged fibroblast SPN. Furthermore, the treatment with a specific antibody against BDNF or a TrkB antagonist inhibited the paracrine signaling preventing SPN-mediated morphological and molecular changes. Finally, BDNF induced signs of matrix invasion in a three-dimensional organotypic model. Therefore, we demonstrate that aged fibroblast SPN promotes phenotypic plasticity in KCs from the elderly through BDNF-TrkB axis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Epigenetic reprogramming and autophagy have critical roles in differentiation of stem cells. However, very little is known about how epigenetic modifications are mediated and how they contribute to autophagy and differentiation in human cardiac stem cells (hCSCs). Previously, we have reported that intracellular matrix metalloproteinase-9 (MMP9), a collagenase, mediates cell death in hCSCs. Here, we investigated whether intracellular MMP9 mediates epigenetic modifications and autophagy in hCSCs. We created MMP9KO hCSCs and treated them with 5-azacytidine, an inhibitor of DNA methylation, and bafilomycin A1, an inhibitor of autophagosome degradation, and evaluated epigenetic modifications, autophagic flux, and differentiation. Our results showed compromised epigenetic modifications, reduced autophagy, and impaired differentiation in MMP9KO hCSCs. Remarkably, paracrine MMP9 supplementation restored epigenetic modifications but further reduced autophagy in MMP9KO hCSCs. We conclude that intracellular MMP9 is a critical mediator of epigenetic modifications and autophagy in hCSCs. Furthermore, the endocrine and paracrine effects of MMP9 vary for regulating autophagy in hCSCs. These novel roles of MMP9 are valuable for stem cell therapy., (©AlphaMed Press 2021.)

Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used small-molecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.

Obese subjects have an increased risk of developing triple-negative breast cancer (TNBC), in part associated with the chronic low-grade inflammation state. On the other hand, epidemiological data indicates that increased consumption of polyphenol-rich fruits and vegetables plays a key role in reducing incidence of some cancer types. Here, we tested whether green tea-derived epigallocatechin-3-gallate (EGCG) could alter adipose-derived mesenchymal stem cell differentiation into adipocytes, and how this impacts the secretome profile and paracrine regulation of the TNBC invasive phenotype. Here, cell differentiation was performed and conditioned media (CM) from preadipocytes and mature adipocytes harvested. Human TNBC-derived MDA-MB-231 real-time cell migration was performed using the exCELLigence system. Differential gene arrays and RT-qPCR were used to assess gene expression levels. Western blotting was used to assess protein expression and phosphorylation status levels. In vitro vasculogenic mimicry (VM) was assessed with Matrigel. EGCG was found to inhibit the induction of key adipogenic biomarkers, including lipoprotein lipase, adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. Increased TNBC-derived MDA-MB-231 cell chemotaxis and vasculogenic mimicry were observed in response to mature adipocytes secretome, and this was correlated with increased STAT3 phosphorylation status. This invasive phenotype was prevented by EGCG, the JAK/STAT inhibitors Tofacitinib and AG490, as well as upon STAT3 gene silencing. In conclusion, dietary catechin-mediated interventions could, in part through the inhibition of adipogenesis and modulation of adipocytes secretome profile, prevent the onset of an obesogenic environment that favors TNBC development.

This paper attempts to explain how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus causes the complications that make coronavirus disease 2019 (COVID-19) a serious disease in specific patient subgroups. It suggests that cortisol-associated activation of the mineralocorticoid receptor (MR) in epithelial and endothelial cells infected with the virus stimulates the release of adenosine 5'-triphosphate (ATP), which then acts back on purinergic receptors. In the lung this could produce the nonproductive cough via purinergic P2X3 receptors on vagal afferent nerves. In endothelial cells it could stimulate exocytosis of Weibel-Palade bodies (WPBs) that contain angiopoietin-2, which is important in the pathogenesis of acute respiratory distress syndrome (ARDS) by increasing capillary permeability and von Willebrand factor (VWF), which mediates platelet adhesion to the endothelium and hence clotting. Angiopoietin-2 and VWF levels both are markedly elevated in COVID-19-associated ARDS. This paper offers an explanation for the sex differences in SARS-CoV-2 complications and also for why these are strongly associated with age, race, diabetes, and body mass index. It also explains why individuals with blood group A have a higher risk of severe infection than those with blood group O. Dexamethasone has been shown to be of benefit in coronavirus ARDS patients and has been thought to act as an anti-inflammatory drug. This paper suggests that a major part of its effect may be due to suppression of cortisol secretion. There is an urgent need to trial the combination of dexamethasone and an MR antagonist such as spironolactone to more effectively block the MR and hence the exocytosis of WPBs., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society.)

Targeted therapies for gastrointestinal stromal tumor (GIST) are modestly effective, but GIST cannot be cured with single agent tyrosine kinase inhibitors. In this study, we sought to identify new therapeutic targets in GIST by investigating the tumor microenvironment. Here, we identified a paracrine signaling network by which cancer-associated fibroblasts (CAFs) drive GIST growth and metastasis. Specifically, CAFs isolated from human tumors were found to produce high levels of platelet-derived growth factor C (PDGFC), which activated PDGFC-PDGFRA signal transduction in GIST cells that regulated the expression of SLUG, an epithelial-mesenchymal transition (EMT) transcription factor and downstream target of PDGFRA signaling. Together, this paracrine induce signal transduction cascade promoted tumor growth and metastasis in vivo. Moreover, in metastatic GIST patients, SLUG expression positively correlated with tumor size and mitotic index. Given that CAF paracrine signaling modulated GIST biology, we directly targeted CAFs with a dual PI3K/mTOR inhibitor, which synergized with imatinib to increase tumor cell killing and in vivo disease response. Taken together, we identified a previously unappreciated cellular target for GIST therapy in order to improve disease control and cure rates.

Objective: Adipose tissue-derived stem cells (ASCs) might play an important role in adipose microenvironment remodelling during tissue expansion through their response to hypoxia. We examined the cytokine profiles of hypoxic visceral ASCs (hypox-visASCs) from subjects with different metabolic risk, the interactions between cytokines as well as the impact of TNFα-induced death in the behavior of surviving hypoxic subcutaneous ASCs (hypox-subASCs) both at bulk population and single-cell level., Materials/methods: Visceral adipose tissue was processed to isolate the ASCs from 33 subjects grouped into normal weight, obese with and without metabolic syndrome. Multiplex assay was used to simultaneously measure multiple inflammatory, anti-inflammatory and angiogenic cytokines in hypox-visASCs from these patients and to elucidate cytokine profiles of hypox-subASCs upon stimulation with IL1β or TNFα and after TNFα-induced death. qPCR and single-cell RNA-sequencing were also performed to elucidate transcriptional impact in surviving hypox-subASCs after TNFα-induced apoptosis., Results: Hypox-visASCs from subjects without metabolic syndrome showed greater secretion levels of inflammatory, anti-inflammatory and angiogenic cytokines compared with those from patients with metabolic syndrome. While IL-1β stimulation was sufficient to increase the secretion levels of these cytokines in hypox-subASCs, TNFα-induced apoptosis also increased their levels and impacted on the expression levels of extracellular matrix proteins, acetyl-CoA producing enzymes and redox-balance proteins in surviving hypox-subASCs. TNFα-induced apoptosis under different glucose concentrations caused selective impoverishment of cell clusters and differentially influenced gene expression profiles of surviving hypox-subASCs., Conclusions: Immunoregulatory and angiogenic functions of hypox-visASCs from patients with metabolic syndrome could be insufficient to promote healthy adipose tissue expansion. TNFα-induced apoptosis may impact on functionality of hypox-subASC populations, whose differential metabolic sensitivity to death could serve to manipulate individual populations selectively in order to elucidate their role in shaping adipose heterogeneity and treating metabolic disorders., Competing Interests: Declaration of competing interest No potential conflicts of interest relevant to this article were reported., (Copyright © 2020. Published by Elsevier Inc.)

Cancer-associated fibroblasts (CAFs) are one of the most abundant and critical components of the tumor stroma. CAFs can impact many important steps of cancerogenesis and may also influence treatment resistance. Some of these effects need the direct contact of CAFs and cancer cells, while some involve paracrine signals. In this study, we investigated the ability of head and neck squamous cell carcinomas (HNSCC) patient-derived CAFs to promote or inhibit the colony-forming ability of HNSCC cells. The effect of cisplatin on this promoting or inhibiting influence was also studied. The subsequent analysis focused on changes in the expression of genes associated with cancer progression. We found that cisplatin response in model HNSCC cancer cells was modified by coculture with CAFs, was CAF-specific, and different patient-derived CAFs had a different "sensitizing ratio". Increased expression of VEGFA , PGE2S , COX2 , EGFR , and NANOG in cancer cells was characteristic for the increase of resistance. On the other hand, CCL2 expression was associated with sensitizing effect. Significantly higher amounts of cisplatin were found in CAFs derived from patients who subsequently experienced a recurrence. In conclusion, our results showed that CAFs could promote and/or inhibit colony-forming capability and cisplatin resistance in HNSCC cells via paracrine effects and subsequent changes in gene expression of cancer-associated genes in cancer cells.

Tetraspanin CD9 is widely expressed on various cell types, such as cancer cells and mesenchymal stem cells (MSCs), and/or cell-released exosomes. It has been reported that exosomal CD9 plays an important role in intercellular communications involved in cancer cell migration and metastasis. However, reports on the effect of the CD9 of MSCs or MSC-derived exosomes on cancer cell migration are still lacking. In this study, using a transwell migration assay, we found that both dextran-coated iron oxide nanoparticles (dex-IO NPs) and ionomycin stimulated exosomal CD9 expression in human MSCs (hMSCs); however, hMSCs could not deliver them to melanoma cells to affect cell migration. Interestingly, a reduced migration of melanoma cell line was observed when the ionomycin-incubated hMSC-conditioned media but not dex-IO NP-labeled hMSC-conditioned media were in the bottom chamber. In addition, we found that dex-IO NPs decreased cellular CD9 expression in hMSCs but ionomycin increased this. Simultaneously, we found that ionomycin suppressed the expression and secretion of the chemokine CCL21 in hMSCs. The silencing of CD9 demonstrated an inhibitory role of cellular CD9 in CCL21 expression in hMSCs, suggesting that ionomycin could upregulate cellular CD9 to decrease CCL21 expression and secretion of hMSCs, which would reduce the migration of B16F10, A549 and U87MG cancer cell lines due to chemoattraction reduction of CCL21. The present study not only highlights the important role of bone marrow-derived hMSCs' CD9-mediated CCL21 regulation in cancer bone metastasis but also suggests a new distinct pharmaceutical strategy for prevention or/and therapy of cancer metastasis.

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with dysregulated interleukin (IL)-6 and autophagy. Although such disturbances are increasingly recognized in patients with SLE and animal models of the disease, little is known about the specific role of IL-6 and autophagy in SLE macrophages. Here, we investigated alterations in the IL-6 axis and autophagy in macrophages derived from patients with SLE and determined whether IL-6 modulates autophagy using human macrophage models. Serum IL-6 detected by ELISA was higher in SLE patients (n = 19) than in normal controls (n = 19, p < 0.001). Levels of the IL-6 receptor (IL-6R) and autophagic markers LC3B and p62 in SLE and normal macrophages were assessed by real-time PCR, western blotting, and immunofluorescence. Compared with normal macrophages, SLE macrophages not only overexpressed IL-6Rs but also exhibited impaired autophagic degradation as evidenced by elevated levels of LC3B and p62. In vitro analyses using macrophage models revealed that prolonged exposure to exogenous recombinant human IL-6 induced a marked impairment of autophagic degradation indicated by elevated levels of LC3B and p62 in both primary macrophages and transformed macrophages. Pretreatment with tocilizumab, a humanized anti-IL-6R monoclonal antibody, restored autophagic degradation and reversed p62 accumulation in a paracrine manner in macrophages. These findings demonstrate that SLE involves IL-6-induced impairment of autophagic degradation through augmentation of IL-6R in human macrophages., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Macrophages help defend the host against Mycobacterium tuberculosis (Mtb), the major cause of tuberculosis (TB). Once phagocytized, Mtb resists killing by macrophages, replicates inside them, and leads to their death, releasing Mtb that can infect other cells. We found that the death of Mtb-infected mouse macrophages in vitro does not appear to proceed by a currently known pathway. Through genome-wide CRISPR-Cas9 screening, we identified a critical role for autocrine or paracrine signaling by macrophage-derived type I IFNs in the death of Mtb-infected macrophages in vitro, and blockade of type I IFN signaling augmented the effect of rifampin, a first-line TB drug, in Mtb-infected mice. Further definition of the pathway of type I IFN-mediated macrophage death may allow for host-directed therapy of TB that is more selective than systemic blockade of type I IFN signaling., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Zhang et al.)

An essential requirement for the success of in vitro maturation (IVM) of the oocyte is to provide an optimal microenvironment similar to in vivo conditions. Recently, somatic cell-based coculture or supplementation of a conditioned medium during IVM has been performed to obtain better quality of oocytes, because they mimic the in vivo reproductive tract by secreting paracrine factors. In this study, human adipose-derived stem cells (ASC) and their conditioned medium (ASC-CM) were applied to IVM of porcine oocytes to evaluate the effectiveness of ASC on oocyte development and subsequent embryo development. In results, both ASC and ASC-CM positively influence on oocyte maturation and embryo development by regulating growth factor receptors ( VEGF , FGFR , and IGFR ), apoptosis ( BCL2 ), cumulus expansion ( PTGS2 , HAS2 , and TNFAIP6 ), and oocyte maturation-related genes ( GDF9 and BMP15 ). In particular, the fluorescence intensity of GDF9 and BMP15 was markedly upregulated in the oocytes from the ASC-CM group. Furthermore, significantly high levels of growth factors/cytokine including VEGF, bFGF, IGF-1, IL-10, and EGF were observed in ASC-CM. Additionally, the ASC-CM showed active scavenging activity by reducing the ROS production in a culture medium. Consequently, for the first time, this study demonstrated the effect of human ASC-CM on porcine oocyte development and the alteration of mRNA transcript levels in cumulus-oocyte complexes.