Browsing by Subject "Exosomes"
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Item Analysis of Keratinocytic Exosomes from Diabetic and Nondiabetic Mice by Charge Detection Mass Spectrometry(American Chemical Society, 2022) Brown, Brooke A.; Guda, Poornachander R.; Zeng, Xuyao; Anthony, Adam; Couse, Andrew; Barnes, Lauren F.; Sharon, Edie M.; Trinidad, Jonathan C.; Sen, Chandan K.; Jarrold, Martin F.; Ghatak, Subhadip; Clemmer, David E.; Surgery, School of MedicineUnresolved inflammation compromises diabetic wound healing. Recently, we reported that inadequate RNA packaging in murine wound-edge keratinocyte-originated exosomes (Exoκ) leads to persistent inflammation [Zhou, X. ACS Nano 2020, 14(10), 12732-12748]. Herein, we use charge detection mass spectrometry (CDMS) to analyze intact Exoκ isolated from a 5 day old wound-edge tissue of diabetic mice and a heterozygous nondiabetic littermate control group. In CDMS, the charge (z) and mass-to-charge ratio (m/z) of individual exosome particles are measured simultaneously, enabling the direct analysis of masses in the 1-200 MDa range anticipated for exosomes. These measurements reveal a broad mass range for Exoκ from ∼10 to >100 MDa. The m and z values for these exosomes appear to fall into families (subpopulations); a statistical modeling analysis partially resolves ∼10-20 Exoκ subpopulations. Complementary proteomics, immunofluorescence, and electron microscopy studies support the CDMS results that Exoκ from diabetic and nondiabetic mice vary substantially. Subpopulations having high z (>650) and high m (>44 MDa) are more abundant in nondiabetic animals. We propose that these high m and z particles may arise from differences in cargo packaging. The veracity of this idea is discussed in light of other recent CDMS results involving genome packaging in vaccines, as well as exosome imaging experiments. Characterization of intact exosome particles based on the physical properties of m and z provides a new means of investigating wound healing and suggests that CDMS may be useful for other pathologies.Item Cancer-associated fibroblast exosomes regulate survival and proliferation of pancreatic cancer cells(SpringerNature, 2017-03-30) Richards, Katherine E.; Zeleniak, Ann E.; Fishel, Melissa L.; Wu, Junmin; Littlepage, Laurie E.; Hill, Reginald; Department of Pediatrics, IU School of MedicineCancer associated fibroblasts (CAFs) comprise the majority of the tumor bulk of pancreatic adenocarcinomas (PDACs). Current efforts to eradicate these tumors focus predominantly on targeting the proliferation of rapidly growing cancer epithelial cells. We know that this is largely ineffective with resistance arising in most tumors following exposure to chemotherapy. Despite the long-standing recognition of the prominence of CAFs in PDAC, the effect of chemotherapy on CAFs and how they may contribute to drug resistance in neighboring cancer cells is not well characterized. Here we show that CAFs exposed to chemotherapy play an active role in regulating the survival and proliferation of cancer cells. We found that CAFs are intrinsically resistant to gemcitabine, the chemotherapeutic standard of care for PDAC. Further, CAFs exposed to gemcitabine significantly increase the release of extracellular vesicles called exosomes. These exosomes increased chemoresistance-inducing factor, Snail, in recipient epithelial cells and promote proliferation and drug resistance. Finally, treatment of gemcitabine-exposed CAFs with an inhibitor of exosome release, GW4869, significantly reduces survival in co-cultured epithelial cells, signifying an important role of CAF exosomes in chemotherapeutic drug resistance. Collectively, these findings show the potential for exosome inhibitors as treatment options alongside chemotherapy for overcoming PDAC chemoresistance.Item Circulating Extracellular Vesicles Carrying Sphingolipid Cargo for the Diagnosis and Dynamic Risk Profiling of Alcoholic Hepatitis(Wolters Kluwer, 2021) Sehrawat, Tejasav S.; Arab, Juan P.; Liu, Mengfei; Amrollahi, Pouya; Wan, Meihua; Fan, Jia; Nakao, Yasuhiko; Pose, Elisa; Navarro-Corcuera, Amaia; Dasgupta, Debanjali; Liao, Chieh-Yu; He, Li; Mauer, Amy S.; Avitabile, Emma; Ventura-Cots, Meritxell; Bataller, Ramon A.; Sanyal, Arun J.; Chalasani, Naga P.; Heimbach, Julie K.; Watt, Kymberly D.; Gores, Gregory J.; Gines, Pere; Kamath, Patrick S.; Simonetto, Douglas A.; Hu, Tony Y.; Shah, Vijay H.; Malhi, Harmeet; Medicine, School of MedicineBackground and aims: Alcoholic hepatitis (AH) is diagnosed by clinical criteria, although several objective scores facilitate risk stratification. Extracellular vesicles (EVs) have emerged as biomarkers for many diseases and are also implicated in the pathogenesis of AH. Therefore, we investigated whether plasma EV concentration and sphingolipid cargo could serve as diagnostic biomarkers for AH and inform prognosis to permit dynamic risk profiling of AH subjects. Approach and results: EVs were isolated and quantified from plasma samples from healthy controls, heavy drinkers, and subjects with end-stage liver disease (ESLD) attributed to cholestatic liver diseases and nonalcoholic steatohepatitis, decompensated alcohol-associated cirrhosis (AC), and AH. Sphingolipids were quantified by tandem mass spectroscopy. The median plasma EV concentration was significantly higher in AH subjects (5.38 × 1011 /mL) compared to healthy controls (4.38 × 1010 /mL; P < 0.0001), heavy drinkers (1.28 × 1011 /mL; P < 0.0001), ESLD (5.35 × 1010 /mL; P < 0.0001), and decompensated AC (9.2 × 1010 /mL; P < 0.0001) disease controls. Among AH subjects, EV concentration correlated with Model for End-Stage Liver Disease score. When EV counts were dichotomized at the median, survival probability for AH subjects at 90 days was 63.0% in the high-EV group and 90.0% in the low-EV group (log-rank P value = 0.015). Interestingly, EV sphingolipid cargo was significantly enriched in AH when compared to healthy controls, heavy drinkers, ESLD, and decompensated AC (P = 0.0001). Multiple sphingolipids demonstrated good diagnostic and prognostic performance as biomarkers for AH. Conclusions: Circulating EV concentration and sphingolipid cargo signature can be used in the diagnosis and differentiation of AH from heavy drinkers, decompensated AC, and other etiologies of ESLD and predict 90-day survival permitting dynamic risk profiling.Item Editorial: Role of extracellular vesicles (EVs) in pathogenesis, diagnosis, therapeutic delivery, treatment and theranostic applications in cancer(Frontiers Media, 2023-09-15) Shahbazi, Reza; Kalishwaralal, Kalimuthu; Paul, Manash K.; Anto, Ruby John; Medicine, School of MedicineItem Exosomal Transfer of DNA Methyl-Transferase mRNA Induces an Immunosuppressive Phenotype in Human Monocytes(Wolters Kluwer, 2022) Wisler, Jon R.; Singh, Kanhaiya; McCarty, Adara; Harkless, Ryan; Karpurapu, Manjula; Hernandez, Edward; Mukherjee, Debasmita; Abouhashem, Ahmed S.; Christman, John W.; Sen, Chandan K.; Surgery, School of MedicineIntroduction: Survivors of sepsis exhibit persistent immunosuppression. Epigenetic events may be responsible for some of these immunosuppressive changes. During sepsis circulating exosomes contain large quantities of DNA methyltransferase (DNMT) mRNAs. We hypothesized that exosomes directly transfer DNMT mRNAs to recipient monocytes with resultant methylation events and immunosuppression. Methods: Exosomes containing DNMT mRNA were generated by stimulating monocytes with LPS. Confocal microscopy was used to determine uptake kinetics in the presence of pharmacologic inhibition. Expression and packaging of specific DNMT mRNA was controlled using DNMT siRNAs. Whole genome and gene specific methylation was assessed using bisulfite sequencing. Ingenuity pathway analysis was performed to determine the biological function of significance of differentially methylated regions. Results: Exosomes effectively transferred DNMT mRNA to recipient monocytes. Pharmacologic inhibition of exosome uptake prevented this increase in DNMT mRNA expression. Recipient monocytes exhibited hypermethylation changes and gene suppression. siRNAs decreased the packaging of DNMT mRNAs and prevented TNFα gene suppression, restoring immunocompetence. Conclusion: These data support a role for exosome-mediated transfer of DNMT mRNA with resultant methylation and gene silencing. Pharmacologic uptake inhibition or targeted siRNA mediated DNMT gene silencing prevented DNMT mRNA transfer and maintained the cell's ability to express TNFα in response to LPS. This highlights the potential therapeutic value of targeting these exosome-mediated epigenetic events to maintain the host immune response during sepsis.Item Human extracellular microvesicles from renal tubules reverse kidney ischemia-reperfusion injury in rats(PLOS, 2018-08-27) Dominguez II, James M.; Dominguez, Jesus H.; Xie, Danhui; Kelly, Katherine J.; Medicine, School of MedicineHypoxic acute kidney injury, a major unresolved problem, initiates, or aggravates, renal functional and structural decline. There is no treatment for hypoxic acute renal injury and its sequelae. We tested the hypothesis that human kidney tubular cells, or their extracellular vesicles (exosomes), prevent renal injury when infused intravenously 24 hours after 50 minutes of bilateral renal ischemia in Nude rats. Cells and their exosomes were from harvested human kidneys declined for transplantation. Injections of either cells or exosomes, given after 24 and 48 hours of reperfusion, preserved renal function and structure in both treatment groups. However, exosomes were superior to cells; and maintained renal vascular and epithelial networks, prevented renal oxidant stress, and apoptosis; and restrained activation of pro-inflammatory and pro-fibrogenic pathways. Exosomes worked in 24 hours, consistent with functional rather than regenerative activity. Comprehensive proteomic analysis identified 6152 renal proteins from all cellular compartments; and 628 were altered by ischemia at all cell levels, while 377 were significantly improved by exosome infusions. We conclude that renal damage from severe ischemia was broad, and human renal exosomes prevented most protein alterations. Thus, exosomes seem to acutely correct a critical and consequential abnormality during reperfusion. In their absence, renal structure and cells transition to a chronic state of fibrosis and extensive renal cell loss.Item Inflammation-associated microRNA changes in circulating exosomes of heart failure patients(BMC, 2017-12-19) Beg, Faheemullah; Wang, Ruizhong; Saeed, Zeb; Devaraj, Srikant; Masoor, Kamalesh; Nakshatri, Harikrishna; Surgery, School of MedicineObjective MiR-486 and miR-146a are cardiomyocyte-enriched microRNAs that control cell survival and self-regulation of inflammation. These microRNAs are released into circulation and are detected in plasma or in circulating exosomes. Little is known whether heart failure affects their release into circulation, which this study investigated. Results Total and exosome-specific microRNAs in plasma of 40 heart failure patients and 20 controls were prepared using the miRVana Kit. We measured exosomal and total plasma microRNAs separately because exosomes serve as cargos that transfer biological materials and alter signaling in distant organs, whereas microRNAs in plasma indicate the level of tissue damage and are mostly derived from dead cells. qRT-PCR was used to quantify miR-486, miR-146a, and miR-16. Heart failure did not significantly affect plasma miR-486/miR-16 and miR-146a/miR-16 ratio, although miR-146a/miR-16 showed a trend of elevated expression (2.3 ± 0.79, p = 0.27). By contrast, circulating exosomal miR-146a/miR-16 ratio was higher in heart failure patients (2.46 ± 0.51, p = 0.05). miR-146a is induced in response to inflammation as a part of inflammation attenuation circuitry. Indeed, Tnfα and Gm-csf increased miR-146a but not miR-486 in the cardiomyocyte cell line H9C2. These results, if confirmed in a larger study, may help to develop circulating exosomal miR-146a as a biomarker of heart failure.Item Label-Free Nanoplasmonic-Based Short Noncoding RNA Sensing at Attomolar Concentrations Allows for Quantitative and Highly Specific Assay of MicroRNA-10b in Biological Fluids and Circulating Exosomes(American Chemical Society, 2015-11-24) Joshi, Gayatri K.; Deitz-McElyea, Samantha; Liyanage, Thakshila; Lawrence, Katie; Mali, Sonali; Sardar, Rajesh; Korc, Murray; Department of Medicine, IU School of MedicineMicroRNAs are short noncoding RNAs consisting of 18-25 nucleotides that target specific mRNA moieties for translational repression or degradation, thereby modulating numerous biological processes. Although microRNAs have the ability to behave like oncogenes or tumor suppressors in a cell-autonomous manner, their exact roles following release into the circulation are only now being unraveled and it is important to establish sensitive assays to measure their levels in different compartments in the circulation. Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility. The sensor was used to specifically detect microRNA-10b at the attomolar (10(-18) M) concentration in pancreatic cancer cell lines, derived tissue culture media, human plasma, and media and plasma exosomes. In addition, for the first time, our label-free and nondestructive sensing technique was used to quantify microRNA-10b in highly purified exosomes isolated from patients with pancreatic cancer or chronic pancreatitis, and from normal controls. We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls. Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.Item Mesenchymal Stem Cell-Derived Exosomal microRNAs in Cardiac Regeneration(MDPI, 2023-12-11) Bhaskara, Meghana; Anjorin, Olufisayo; Wang, Meijing; Surgery, School of MedicineMesenchymal stem cell (MSC)-based therapy is one of the most promising modalities for cardiac repair. Accumulated evidence suggests that the therapeutic value of MSCs is mainly attributable to exosomes. MSC-derived exosomes (MSC-Exos) replicate the beneficial effects of MSCs by regulating various cellular responses and signaling pathways implicated in cardiac regeneration and repair. miRNAs constitute an important fraction of exosome content and are key contributors to the biological function of MSC-Exo. MSC-Exo carrying specific miRNAs provides anti-apoptotic, anti-inflammatory, anti-fibrotic, and angiogenic effects within the infarcted heart. Studying exosomal miRNAs will provide an important insight into the molecular mechanisms of MSC-Exo in cardiac regeneration and repair. This significant information can help optimize cell-free treatment and overcome the challenges associated with MSC-Exo therapeutic application. In this review, we summarize the characteristics and the potential mechanisms of MSC-derived exosomal miRNAs in cardiac repair and regeneration.Item A microRNA signature in circulating exosomes is superior to exosomal glypican-1 levels for diagnosing pancreatic cancer(Elsevier, 2017-05-01) Lai, Xianyin; Wang, Mu; Deitz McElyea, Samantha; Sherman, Stuart; House, Michael; Korc, Murray; Department of Biochemistry & Molecular Biology, IU School of MedicinePancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that often presents clinically at an advanced stage and that may be confused with chronic pancreatitis (CP). Conversely, CP may be misdiagnosed as PDAC leading to unwarranted pancreas resection. Therefore, early PDAC diagnosis and clear differentiation between PDAC and CP are crucial for improved care. Exosomes are circulating microvesicles whose components can serve as cancer biomarkers. We compared exosomal glypican-1 (GPC1) and microRNA levels in normal control subjects and in patients with PDAC and CP. We report that exosomal GPC1 is not diagnostic for PDAC, whereas high exosomal levels of microRNA-10b, (miR-10b), miR-21, miR-30c, and miR-181a and low miR-let7a readily differentiate PDAC from normal control and CP samples. By contrast with GPC1, elevated exosomal miR levels decreased to normal values within 24 h following PDAC resection. All 29 PDAC cases exhibited significantly elevated exosomal miR-10b and miR-30c levels, whereas 8 cases had normal or slightly increased CA 19-9 levels. Thus, our exosomal miR signature is superior to exosomal GPC1 or plasma CA 19-9 levels in establishing a diagnosis of PDAC and differentiating between PDAC and CP.