Exosome Secretion Research Articles

Research on the TSPAN6 regulating the secretion of ADSCs-Exos through syntenin-1 and promoting wound healing.

Exosomes (Exos) from adipose-derived stem cells (ADSCs) have a high inclusion content and low immunogenicity, which helps to control inflammation and accelerate the healing of wounds. Unfortunately, the yield of exosomes is poor, which raises the expense and lengthens the treatment period in addition to impairing exosomes' therapeutic impact. Thus, one of the key problems that needs to be resolved in the current exosome study is increasing the exosome yield. Tetraspanin-6 (TSPAN6) overexpression and knockdown models of ADSCs were constructed to determine the number of exosomes secreted by each group of cells as well as the number of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) within the cells. Subsequently, the binding region of the interaction between TSPAN6 and syntenin-1 was identified using the yeast two-hybrid assay, and the interaction itself was identified by immunoprecipitation. Finally, cellular and animal studies were conducted to investigate the role of each class of exosomes. When compared to the control group, the number of intracellular MVBs and ILVs was significantly larger, and the number of ADSCsTSPAN6+-Exos was more than three times higher. However, TSPAN6's ability to stimulate exosome secretion was reduced as a result of syntenin-1 knockdown. Additional yeast two-hybrid assay demonstrated that the critical structures for their interaction were the N-terminal, Postsynaptic density protein 95/Discs large protein/Zonula occludens 1 (PDZ1), and PDZ2 domains of syntenin-1, and the C-terminal of TSPAN6. In animal trials, the wound healing rate was best in the ADSCsTSPAN6+-Exos group, while cellular experiments demonstrated that ADSCsTSPAN6+-Exos better enhanced the proliferation and migration of human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs). TSPAN6 stimulates exosome secretion and formation, as well as the creation of MVBs and ILVs in ADSCs. Syntenin-1 is essential for TSPAN6's stimulation of ADSCs-Exos secretion. Furthermore, ADSCsTSPAN6+-Exos has a greater ability to support wound healing, angiogenesis, and the proliferation and migration of a variety of cells.

Implications of glioblastoma-derived exosomes in modifying the immune system: state-of-the-art and challenges.

Glioblastoma is a brain cancer with a poor prognosis. Failure of classical chemotherapy and surgical treatments indicates that new therapeutic approaches are needed. Among cell-free options, exosomes are versatile extracellular vesicles (EVs) that carry important cargo across barriers suchas the blood-brain barrier (BBB) to their target cells. Thismakes exosomes an interesting option for the treatment ofglioblastoma. Moreover, exosomes can comprise many therapeutic cargos, including lipids, proteins, and nucleic acids,sampled from special intercellular compartments of their origin cell. Cells exposed to various immunomodulatory stimulican generate exosomes enriched in specific therapeutic molecules. Notably, the secretion of exosomes could modify theimmune response in innate and adaptive immune systems. For instance, glioblastoma-associated exosomes (GBex) uptake bymacrophages could influence macrophage dynamics (e.g., shifting CD markers expression). Expression of critical immunoregulatory proteins such as cytotoxic T-lymphocyte antigen-1 (CTLA1) and programmed death-1 (PD-1) on GBex indicates the direct crosstalk of these nano-size vesicles with the immune system. The present study reviews the role of exosomes in immune system cells, including Bcells, Tcells, natural killer (NK) cells, and dendritic cells (DCs), as well as novel technologies in the field.

STAT3 Inhibitor Increases α-Synuclein in PFF-Treated Astroglia Cells by Dysregulating Autophagy and Potentially Affects Exosome Biogenesis.

Astrocytes are the most abundant cells in the brain and show neuroprotective function in CNS and reactive astrocytes are characteristic in neurodegenerative diseases. The JAK2-STAT3 pathway plays a crucial role in the process of astrocyte activation. However, as a hallmark of Parkinson's disease, the change in α-syn under the influence of STAT3 inhibitor and the underlying cellular mechanisms remain elusive. Here, we show that PFF can induce an increase in α-syn in SVG p12 cells, which is further enhanced after the inhibition of STAT3. The underlying mechanisms involve the downregulation of autophagy levels and a concurrent decrease in lysosomal function after inhibition of STAT3. Additionally, we observed inhibition of STAT3 resulted in reduced exosome secretion in SVG p12 cells. This is attributed to alterations in SNARE, leading to impaired fusion between MVBs and the cell membrane, subsequently causing the accumulation of intracellular MVBs. Taken together, our data demonstrates that inhibition of STAT3 decreases both the autophagy and lysosome function, which may increase MVBs production. However, we found a potentially decreased exosome production that may be implicated with SNARE complex and need further exploration.

Abstract 4125419: Very-Low-Density Lipoprotein Induces Neuronal Growth with Increased Tyrosine Hydroxylase Expression through Cardiomyocyte-Secreted Exosomes in Metabolic Syndrome

Introduction: Metabolic syndrome (MS) is an important predisposing factor for atrial fibrillation, and is highly related to the autonomic nervous system. Triglyceride-rich very-low-density lipoprotein (VLDL) has specific VLDL receptors (VLDLR), which are abundantly expressed in cardiomyocytes. Hypothesis: VLDL affects neuronal growth through cardiomyocyte-secreted exosomes in metabolic syndrome. Goals/Aims: To determine whether exosomes from VLDL-incubated cardiomyocytes can alter neuronal proliferation. Methods/ Approach: HL-1 cardiomyocytes were incubated with VLDL isolated from human subjects with MS, and exosomes were extracted at the end of the incubation period. VLDLR knockdown HL-1 cells were used to determine the role of VLDLR in exosome secretion. The molecular contents of exosomes were determined by mass spectrometry, which identified neurotropic proteins only detected in the exosomes of MS VLDL-incubated cardiomyocytes. We selected transmembrane protein 14B (TMEM14B) to investigate its effect on neuronal proliferation in P19 cells. Results: Regardless of the knockdown or overexpression of VLDLR in HL-1 cardiomyocytes, exosomes isolated from after VLDL incubation were shown to cause 2.062-fold and 2.084-fold expression of tyrosine hydroxylase (TH) in P19 cells, relative to P19 cells induced by non-VLDL-treated HL-1 cardiomyocyte-secreted exosomes. A similar trend could be seen with HL-1 cardiomyocytes without VLDLR editing, as VLDL-incubated HL-1 cardiomyocytes produced exosomes that could cause 1.926-fold increase in P19 TH expression, relative to treatment by exosomes from HL-1 cardiomyocytes without VLDL-incubation. The P19 cells incubated with TMEM14B demonstrated increased neurite length, with an average of 2.342-fold neurite length when compared with that of control cells. Conclusions: VLDL in MS induced HL-1 cardiomyocytes to secrete exosomes containing neurotrophic molecules and increased dendritic growth of neuronal cells. This effect was VLDLR-independent and was partially caused by TMEM14B in the exosomes.

Dual-Synergistic Nanomodulator Alleviates Exosomal PD-L1 Expression Enabling Exhausted Cytotoxic T Lymphocytes Rejuvenation for Potentiated iRFA-Treated Hepatocellular Carcinoma Immunotherapy.

The tumor immunosuppressive microenvironment (TME) induced by incomplete radiofrequency ablation (iRFA) in hepatocellular carcinoma (HCC) is a critical driver of tumor progression and metastasis. Herein, we proposed a therapeutic strategy aimed at remodeling the post-iRFA TME by targeting exosome biogenesis, secretion, and PD-L1 expression, thereby rejuvenating cytotoxic T lymphocyte function to mitigate the progression and metastasis of HCC. Leveraging the versatile properties of polydopamine nanomodulators, we have engineered a tailored delivery platform for GW4869 and amlodipine (AM), enabling precise and tumor-specific release of these therapeutic agents. Initially, GW4869, a neutral sphingomyelinase inhibitor, synergized with AM, an intracellular calcium modulator, to suppress exosome biogenesis and secretion. Subsequently, AM triggered the autophagic degradation of PD-L1. In vitro and in vivo experiments demonstrated that this synergistic approach significantly enhanced the robust activation and proliferation of various functional T-cell subsets following iRFA, particularly CD8+T cells, IFN-γ+ CD8+ cytotoxic T cells, natural killer cells, and innate lymphoid cells. Concurrently, it effectively reduced the infiltration of immunosuppressive cell types, including regulatory T cells and myeloid-derived suppressor cells. This favorable remodeling of the TME substantially inhibited the progression and metastasis of HCC post-iRFA. Collectively, our study presented a promising paradigm for enhancing HCC treatment efficacy by integrating radiofrequency ablation with advanced immune modulation strategies.

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes.

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Exosomal miR-1a-3p derived from glucocorticoid-stimulated M1 macrophages promotes the adipogenic differentiation of BMSCs in glucocorticoid-associated osteonecrosis of the femoral head by targeting Cebpz

BackgroundBy interacting with bone marrow mesenchymal stem cells (BMSCs) and regulating their function through exosomes, bone macrophages play crucial roles in various bone-related diseases. Research has highlighted a notable increase in the number of M1 macrophages in glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH). Nevertheless, the intricate crosstalk between M1 macrophages and BMSCs in the glucocorticoid-stimulated environment has not been fully elucidated, and the underlying regulatory mechanisms involved in the occurrence of GA-ONFH remain unclear.MethodsWe employed in vivo mouse models and clinical samples from GA-ONFH patients to investigate the interactions between M1 macrophages and BMSCs. Immunofluorescence staining was used to assess the colocalization of M1 macrophages and BMSCs. Flow cytometry and transcriptomic analysis were performed to evaluate the impact of exosomes derived from normal (n-M1) and glucocorticoid-stimulated M1 macrophages (GC-M1) on BMSC differentiation. Additionally, miR-1a-3p expression was altered in vitro and in vivo to assess its role in regulating adipogenic differentiation.ResultsIn vivo, the colocalization of M1 macrophages and BMSCs was observed, and an increase in M1 macrophage numbers and a decrease in bone repair capabilities were further confirmed in both GA-ONFH patients and mouse models. Both n-M1 and GC-M1 were identified as contributors to the inhibition of osteogenic differentiation in BMSCs to a certain extent via exosome secretion. More importantly, exosomes derived from GC-M1 macrophages exhibited a heightened capacity to regulate the adipogenic differentiation of BMSCs, which was mediated by miR-1a-3p. In vivo and in vitro, miR-1a-3p promoted the adipogenic differentiation of BMSCs by targeting Cebpz and played an important role in the onset and progression of GA-ONFH.ConclusionWe demonstrated that exosomes derived from GC-M1 macrophages disrupt the balance between osteogenic and adipogenic differentiation in BMSCs, contributing to the pathogenesis of GA-ONFH. Inhibiting miR-1a-3p expression, both in vitro and in vivo, significantly mitigates the preferential adipogenic differentiation of BMSCs, thus slowing the progression of GA-ONFH. These findings provide new insights into the regulatory mechanisms underlying GA-ONFH and highlight potential therapeutic targets for intervention.Graphical

Correction for Wu et al., "Enterovirus A71 Promotes Exosome Secretion by the Nonstructural Protein 3A Interacting with Rab27a".

Correction for Wu et al., "Enterovirus A71 Promotes Exosome Secretion by the Nonstructural Protein 3A Interacting with Rab27a".

Cystatin C alleviates unconjugated bilirubin-induced neurotoxicity by promoting bilirubin clearance from neurocytes via exosomes, dependent on hepatocyte UGT1A1 activity.

There is an urgent need to identify effective drugs for the treatment of nerve injury caused by unconjugated bilirubin (UCB). Our previous research found that cystatin C (CST3) alleviates UCB-induced neurotoxicity by promoting autophagy in nerve cells, but that autophagy inhibitors did not completely inhibit the effects of CST3. This study investigated whether CST3 could alleviate the neurotoxicity of UCB by promoting the secretion and transport of exosomes containing UCB to the liver for metabolism. It demonstrated that hyperbilirubinemia mice treated with CST3 had a higher number of serum exosomes than those in hyperbilirubinemia mice treated with phosphate-buffered saline. CST3-mediated protection against UCB-induced damage was abolished when autophagy and extracellular vesicle inhibitors were used in combination. The number of exosomes in the CST3 overexpression group was higher than that in the control group. Molecular docking experiments showed that UCB and CST3 had high docking score (-8.2). These results suggest that UCB may be excreted from cells by exosomes, and CST3 may promote this process by binding to UCB and entering the exosomes. We demonstrated that the effect of CST3 relied on liver cells with normal UDP-glucuronyl transferase1A1 (UGT1A1) activity in a coculture system of HT22 and L02 cells. CST3 levels were lower in exosomes secreted by L02 cells than in those secreted by human umbilical vein endothelial cells (HUVECs), whereas CST3 levels were higher in the culture supernatants of L02 cells than in the culture supernatants of HUVECs. This suggests that UCB exosomes in L02 cells may be released and internalized by CST3 and that UCB is then processed by UGT1A1 to conjugate UCB, thus reducing its toxicity. These results suggest that CST3 might alleviate UCB-induced neurotoxicity by promoting the clearance of UCB from cells via exosomes and that these effects are dependent on UGT1A1 activity in liver cells.

Cancer-associated fibroblast-secreted exosomal miR-454-3p inhibits lipid metabolism and ferroptosis in breast cancer by targeting ACSL4.

Cancer-associated fibroblasts (CAFs) participate in the development of the tumor microenvironment through the secretion of exosomes. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is an essential component of ferroptosis. However, the regulatory mechanism of ACSL4 in breast cancer remains unexplored. The study aimed to determine the influence of exosomal miR-454-3p from CAFs on lipid metabolism and ferroptosis. CAF-derived exosomes (CAF-exo) were isolated from breast cancer tissue of breast cancer patients and characterized using transmission electron microscopy (TEM) and Western blot. Luciferase reporter assay and RNA immunoprecipitation (RIP) were used to demonstrate the relationship between miR-454-3p and ACSL4. Cell viability and ferroptosis-related markers were detected by CCK-8 and Western blot. Malondialdehyde (MDA), glutathione (GSH), and iron levels were detected. Reverse transcription-quantitative PCR (RT-qPCR) and fluorescence in situ hybridization (FISH) were used to assess miR-454-3p expression. miR-454-3p and ACSL4 levels were abnormally expressed in breast cancer tissues. CAF-exo significantly enhanced cell viability and GSH levels and suppressed MDA, and iron levels. CAF-exo upregulated ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) expression, and reduced ACSL4 levels. miR-454-3p was strongly expressed in CAF-exo, and exosomal miR-454-3p suppressed lipid metabolism and ferroptosis in breast cancer cells. The effects of miR-454-3p inhibitor on lipid metabolism and ferroptosis were eliminated by ACSL4 knockdown. CAF-secreted exosomal miR-454-3p inhibited lipid metabolism and ferroptosis by targeting ACSL4 in breast cancer. This study revealed a novel molecular mechanism that offers a potential therapeutic intervention in breast cancer treatment.

Female-age-dependent changes in the lipid fingerprint of the mammalian oocytes.

Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes' lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/- mice were also used. Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. Here we demonstrated progressive changes in the LF of oocytes associated with the woman's age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. Due to differences in lipid content between different species' oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo's ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to Ł.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program "Excellence Initiative - Research University" at the Jagiellonian University in Krakow. The authors declare no competing interest. N/A.

Ca2+-SpVAMP2 pathway promotes exosome secretion to resist the infection of Vibrio parahaemolyticus in mud crab (Scylla paramamosain)

Exosomes play an important role in the innate immune system. Many types of cells can secrete exosomes in both normal and abnormal status. However, the mechanism of exosome secretion in invertebrates has not been reported. In this study, the mechanism of the secretion of exosomes regulated by the Ca2+ signaling pathway in the hemocytes of mud crabs (Scylla paramamosain) under infection with Vibrio parahaemolyticus was investigated. The results showed that the increase of exosome secretion in mud crabs infected with V. parahaemolyticus was caused by the increase in intracellular Ca2+ level. In addition, transcriptome sequencing and differential expression analysis revealed that SpVAMP2, a vesicle-associated membrane protein 2, was found to be involved in the exosome secretion regulated by Ca2+ signaling. Further research revealed that SpVAMP2 can inhibit MVB from entering the lysosome for degradation and promote the release of exosomes. Additionally, the secretion of exosomes upregulated the expression of anti-lipopolysaccharide factors and enhanced the survival rate of mud crabs after V. parahaemolyticus infection. This study clarified the exosome secretion regulated by the Ca2+-SpVAMP2 pathway, which, in turn, increased the innate immune response in mud crabs to resist the invasion of V. parahaemolyticus.

Impaired cardiomyocytes accelerate cardiac hypertrophy and fibrosis by delivering exosomes containing Shh/N-Shh/Gli1 in angiotensin II infused mice

BackgroundsHeart failure (HF) is characterized by progressive cardiac hypertrophy and fibrosis, yet the underlying pathological mechanisms remain unclear. Exosomes are pivotal in cellular communication and are key signaling carriers in HFs. This study investigated the roles of exosomes in HF. MethodsEight-week-old male mice were divided into three groups: a control group, an Ang II group receiving angiotensin II (Ang II) infusion for 4 weeks, and an Ang II + DMA group receiving Ang II and dimethyl amiloride (DMA) infusion. This study examined the associations between cardiac injury, exosomes, and their substrate Shh. Furthermore, we conducted cellular experiments to assess the effects of Ang II-induced injury in primary cardiomyocytes on other cardiomyocytes and fibroblasts, and to test the therapeutic effects of the exosome inhibitor DMA and the Shh signaling inhibitor cyclopamine (CPN). ResultsAng II-induced cardiac hypertrophy and fibrosis, which were accompanied by exosome secretion and Shh upregulation in vivo. DMA relieved these cardiac lesions. Furthermore, cellular experiments revealed that Ang II-induced cardiomyocytes hypertrophy and activated cardiac fibroblasts by promoting the release of exosomes containing Shh/N-Shh/Gli1. Both DMA and CPN nullified fibroblast activation and proliferation. ConclusionsAng II-induced cardiomyocyte injury leads to cardiac hypertrophy and fibrosis through the release of exosomes carrying Shh signaling. The suppression of exosome secretion or the Shh pathway could offer new strategies for treating HF.

Exosomes Derived from Rejuvenated Stem Cells Inactivate NLRP3 Inflammasome and Pyroptosis of Nucleus Pulposus Cells via the Transfer of Antioxidants.

Accumulating evidence supports the potential of exosomes as a promising therapeutic approach for intervertebral disc degeneration (IDD). Nevertheless, enhancing the efficiency of exosome treatment remains an urgent concern. This study investigated the impact of quercetin on the characteristics of mesenchymal stem cells (MSCs) and their released exosomes. Exosomes were obtained from quercetin pre-treated MSCs and quantified for the production based on nanoparticle tracking and western blot analysis. The molecules involved in the secretion and cargo sorting of exosomes were investigated using western blot and immunofluorescence analysis. Based on the in vitro biological analysis and in vivo histological analysis, the effects of exosomes derived from conventional or quercetin-treated MSCs on nucleus pulposus (NP) cells were compared. A significant enhancement in the production and transportation efficiency of exosomes was observed in quercetin-treated MSCs. Moreover, the exosomes derived from quercetin-treated MSCs exhibited a greater abundance of antioxidant proteins, specifically superoxide dismutase 1 (SOD1), which inhibit the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome in NP cells. Through in vitro and in vivo experiments, it was elucidated that exosomes derived from quercetin-treated MSCs possessed enhanced anti-inflammatory and antioxidant properties. Collectively, our research underscores an optimized therapeutic strategy for IDD utilizing MSC-derived exosomes, thereby augmenting the efficacy of exosomes in intervertebral disc regeneration.

NSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer.

nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer.

Abstract A066: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

Abstract The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. We investigated the effects of eliminating nSMase2 on early and advanced PDA, including its influence on the microenvironment. Employing the KPC mouse model of pancreatic cancer, we demonstrate that pancreatic epithelial nSMase2 ablation reduces neoplasia and promotes a PDA subtype switch from aggressive basal-like to classical. nSMase2 elimination prolongs survival of KPC mice, hinders vasculature development, and fosters a robust immune response. nSMase2 loss leads to recruitment of cytotoxic T cells, N1-like neutrophils, and abundant infiltration of anti-tumorigenic macrophages in the pancreatic preneoplastic microenvironment. Mechanistically, we demonstrate that nSMase2-expressing PDA cell small extracellular vesicles (sEVs) reduce survival of KPC mice; PDA cell sEVs generated independently of nSMase2 prolong survival of KPC mice and reprogram macrophages to a proinflammatory phenotype. Collectively, our study highlights previously unappreciated opposing roles for exosomes, based on biogenesis pathway, during PDA progression. Citation Format: Audrey M Hendley, Sudipta Ashe, Atsushi Urano, Martin Ng, Tuan A Phu, Xianlu Laura Peng, Changfei Luan, Anna-Marie Finger, Gun Ho Jang, Natanya R Kerper, David I Berrios, David Jin, Jonghyun Lee, Irene R Riahi, Oghenekevwe M Gbenedio, Christina Chung, Jeroen P Roose, Jen Jen Yeh, Steven Gallinger, Andrew V Biankin, Grainne M O’Kane, Vasilis Ntranos, David K Chang, David W Dawson, Grace E Kim, Valerie M Weaver, Robert L Raffai, Matthias Hebrok. nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A066.

Engineering exosomes derived from TNF-α preconditioned IPFP-MSCs enhance both yield and therapeutic efficacy for osteoarthritis

BackgroundThe pathogenesis of osteoarthritis (OA) involves the progressive degradation of articular cartilage. Exosomes derived from mesenchymal stem cells (MSC-EXOs) have been shown to mitigate joint pathological injury by attenuating cartilage destruction. Optimization the yield and therapeutic efficacy of exosomes derived from MSCs is crucial for promoting their clinical translation. The preconditioning of MSCs enhances the therapeutic potential of engineered exosomes, offering promising prospects for application by enabling controlled and quantifiable external stimulation. This study aims to address these issues by employing pro-inflammatory preconditioning of MSCs to enhance exosome production and augment their therapeutic efficacy for OA.MethodsThe exosomes were isolated from the supernatant of infrapatellar fat pad (IPFP)-MSCs preconditioned with a pro-inflammatory factor, TNF-α, and their production was subsequently quantified. The exosome secretion-related pathways in IPFP-MSCs were evaluated through high-throughput transcriptome sequencing analysis, q-PCR and western blot analysis before and after TNF-α preconditioning. Furthermore, exosomes derived from TNF-α preconditioned IPFP-MSCs (IPFP-MSC-EXOsTNF−α) were administered intra-articularly in an OA mouse model, and subsequent evaluations were conducted to assess joint pathology and gait alterations. The expression of proteins involved in the maintenance of cartilage homeostasis within the exosomes was determined through proteomic analysis.ResultsThe preconditioning with TNF-α significantly enhanced the exosome secretion of IPFP-MSCs compared to unpreconditioned MSCs. The potential mechanism involved the activation of the PI3K/AKT signaling pathway in IPFP-MSCs by TNF-α precondition, leading to an up-regulation of autophagy-related protein 16 like 1(ATG16L1) levels, which subsequently facilitated exosome secretion. The intra-articular administration of IPFP-MSC-EXOsTNF−α demonstrated superior efficacy in ameliorating pathological changes in the joints of OA mice. The preconditioning of TNF-α enhanced the up-regulation of low-density lipoprotein receptor-related protein 1 (LRP1) levels in IPFP-MSC-EXOsTNF−α, thereby exerting chondroprotective effects.ConclusionTNF-α preconditioning constitutes an effective and promising method for optimizing the therapeutic effects of IPFP-MSCs derived exosomes in the treatment of OA.Graphical

Construction of a prognostic model with exosome biogenesis- and release-related genes and identification of RAB27B in immune infiltration of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and fatal disease. Exosomes are extracellular vesicles that plays a vital rule in the progression and metastasis of PDAC. However, the specific mechanism of exosome biogenesis and release in the tumorigenesis and development of pancreatic cancer remains elusive. The aim of this study is to develop novel biomarkers and construct a reliable prognostic signature to accurately stratify patients and optimize clinical decision-making. Gene expression and clinical data were acquired from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Univariate Cox regression analysis, random forest analysis, least absolute shrinkage and selection operator (LASSO) regression analysis, and multivariate Cox regression analysis were used to construct the risk signature. The effectiveness of the model was validated by survival point plot, Kaplan-Meier survival analysis, and receiver operating characteristic (ROC) curve in training, testing and entire cohorts. Meanwhile, single sample gene set enrichment analysis (ssGSEA), ESTIMATE and CIBERSORT algorithm were utilized to assess the association of the risk signature with the immune status in the PDAC tumor microenvironment. We also performed functional enrichment, tumor mutation analysis, and DNA methylation analyses based on the risk signature. The function of the core gene was further verified by polymerase chain reaction (PCR), western blot, bicinchoninic acid (BCA), immunohistochemistry (IHC) and in vitro experiments including cell proliferation, migration, and apoptosis experiments. We constructed an exosome biogenesis- and release-related risk model which could serve as an effective and independent prognosis predictor for PDAC patients. The immune infiltration analysis revealed that our signature was related to the PDAC immune microenvironment, mainly associated with a lower proportion of natural killer (NK) cells and CD8+ T cells. Tissue microarray IHC confirmed the association of RAB27B with poor prognosis in PDAC. Knockdown of RAB27B expression promoted PDAC cells' apoptosis, while decreased cellular proliferation and migration. Also, knockdown of RAB27B expression led to reduced exosome secretion, while RAB27B overexpression promoted exosome secretion. The predictive signature can predict overall survival, help elucidate the mechanism of exosome biogenesis and release, and provide immunotherapy guidance for PDAC patients.

Cigarette smoke-induced exosomal miR-221-3p facilitates M1 macrophage polarization via the STAT3 pathway in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is marked by irreversible airflow limitations stemming from small airway constriction and lung emphysema. The advancement of COPD is greatly influenced by the M1 polarization of macrophages. The mechanisms governing macrophage polarization in inflammation conditions in COPD are not yet fully understood. To investigate the interplay between exosomes triggered by cigarette smoke and the polarization of macrophages, we utilized a combination of flow cytometry, quantitative real-time reverse transcription PCR, and western blot analysis. Our research reveals that cigarette smoke (CS) exposure induces the secretion of exosomes from human bronchial epithelial cells, with exosomal miR-221-3p identified as a key player in modulating the polarization of M1 macrophages. The evidence indicates that cigarette smoke promotes exosome secretion in these cells, with exosomal miR-221-3p targeting SOCS3 and regulating the STAT3 signaling pathway to facilitate M1 macrophage polarization. This research delves into the molecular pathways through which miR-221-3p facilitates the polarization of M1 macrophages, presenting a groundbreaking approach for potential targeted therapy in COPD.