Proteomic basis of modulation of postischemic fibrosis by MSC exosomes.

After an ischemic event, there is activation of fibroblasts leading to scar formation. It is critical to limit the profibrotic remodeling and activate the reparative remodeling phase to limit cardiac diastolic dysfunction. Mesenchymal stem cell (MSC) exosomes offer significant protection against ischemia-related systolic dysfunction. Here, we studied if MSC exosomes would offer protection against profibrotic events in mouse hearts subjected to acute ischemia [1 h left coronary artery (LCA) occlusion] or chronic ischemia (7 days LCA occlusion). After acute ischemia, there was activation of inflammatory signals, more in the peri-infarct than in the infarct area, in the saline (vehicle)-treated mice. At the same time, there was expression of cardiac remodeling signals (vimentin, collagens-1 and -3, and fibronectin), more in the infarct area. Treatment with MSC exosomes before LCA ligation suppressed inflammatory signals during acute and chronic ischemia. Furthermore, exosome treatment promoted pro-reparative cardiac extracellular matrix (ECM) remodeling in both infarct and peri-infarct areas by suppressing fibronectin secretion and by modulating collagen secretion to reduce fibrotic scar formation through altered cellular signaling pathways. Proteomics study revealed intense expression of IL-1ß and activation of profibrotic signals in the saline-treated hearts and their suppression in MSC exosome-treated hearts. To our knowledge, this is the first report on the infarct and peri-infarct area proteomics of ischemic mice hearts to explain MSC exosome-mediated suppression of scar formation in the ischemic mouse hearts.

MSC exosome-mediated cardioprotection in ischemic mouse heart comparative proteomics of infarct and peri-infarct areas.

Mesenchymal stem cell (MSC) exosomes may limit cardiac injury, and even reverse cardiac damage in animal models of ischemia. To understand exosome-mediated improvement in cardiac function we examined the proteomic alternations in the MSC exosome-treated mice hearts subjected to left coronary artery (LCA) ligation, with particular emphasis on peri-infarct areas. At 7 days after LCA ligation, left ventricular end systolic thickness, infarct size and survival of mice were studied. Mass spectrometric analysis of infarct and peri-infarct areas was carried out. Expression of inflammatory markers (LOX-1 and NLRP3) and cell death markers (Bax, Bcl-2, Caspases 1 and 3 and GSDMD) were investigated by Western blots and immunofluorescence. Proteomic analysis of the infarct and peri-infarct areas in saline-treated hearts revealed differentially expressed proteins involved in inflammation and apoptotic cell death, while showing depletion of processes governing cell death. Exosome treatment significantly improved the proteomic profile in both infarct and peri-infarct areas, more so in the peri-infarct areas. The infarct size was smaller (9 ± 1%), and cardiac contractile function (fractional shortening) was preserved in the exosome-treated mice (28 ± 2%). Survival of exosome-treated mice was also better. White blood cell accumulation in and around the infarct area, expression of LOX-1 and NLRP3 inflammasome, and markers of cell death (cleaved Caspase-3, Caspase-1, GSDMD, Bcl-2 and Bax) were dramatically reduced by MSC exosome treatment (all p < 0.01). In cultured primary mouse cardiomyocytes, treatment with MSC exosomes essentially reversed inflammation-induced pro-apoptotic and inflammatory signals (p < 0.01). MSC exosomes exert their cardioprotective effects by suppressing inflammation and pro-apoptotic processes, particularly in the peri-infarct areas, resulting in preservation of cardiac function after LCA ligation.

Tumor-endothelial cell interaction in an experimental model of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a densely vascularized tumor that is highly dependent on angiogenic pathways to direct arterial blood flow to the growing neoplasm, though little is known about how the interaction of tumor and endothelial cells drives these processes and the degree of clinical importance. To this end, we examined the intercellular cross-talk between HepG2 (human HCC) and human endothelial progenitor cells (EPC) in a co-culture system that mimics some aspects of initial tumor parenchyma and stroma interactions. The results showed that the remote cell-to-cell (paracrine) interactions between HepG2 cells and EPC play a critical role in the differentiation and angiogenic activity of endothelial cells, possibly through intercellular signaling function of the exosomes released in the medium by HepG2 cells. The tumor-cell activated phenotype of EPC was associated with increased migration and elevated expression of ephrin-B2, and Delta-like 4 ligand (DLL4). Furthermore, ephrin-B2 was found to be overexpressed in HCC and cholangiocarcinoma tissue samples taken from humans. Overall, our results demonstrate that ephrin-B2 and Dll4 mediated co-dependence of HCC and EPC intercellular crosstalk in the initial stages of HCC establishment and development, a promising target for new clinical strategies.

Phosphorylation negatively regulates exosome mediated secretion of cryAB in glioma cells.

Exosomes mediate secretion of crystallin alphaB (cryAB), a well characterized molecular chaperone with anti-apoptotic activity. However, the mechanisms governing its packaging and secretion remained unexplored. In glioma cells, notwithstanding extensive phosphorylation of cryAB at Ser59 followed by Ser45 (Ser19 is largely unphosphorylated), we discovered that the majority of secreted exosomal cryAB is nonphosphorylated. Transient ectopic expression of a yellow fluorescent protein (YFP) tagged triple phosphomimic (3-SD) cryAB construct in cryAB absent glioma cells led to the formation of large cytosolic inclusions. Our findings demonstrate that mimicking phosphorylation significantly reduces cryAB secretion via exosomes. Moreover, decreased colocalization of 3-SD YFP-cryAB with multivesicular endosome (MVE) and exosome marker, CD63 or Rab27, a small GTPase regulating exocytosis of MVEs, suggests that phosphorylation deters packaging of cryAB in vesicles bound for secretion as exosomes. Additionally, we found that preventing O-GlcNAcylation on cryAB also curtailed its colocalization with CD63 and Rab27 resulting in reduced exosomal secretion. Thus, our study points to O-GlcNAcylation and lack of phosphorylation as being the selective processes involved in the packaging and secretion of cryAB via exosomes.

Inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha, upregulated in glioblastoma multiforme, raise the levels of CRYAB in exosomes secreted by U373 glioma cells.

In the brain, levels of inflammatory cytokines, interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α), are elevated under traumatic brain injury, neuroinflammatory conditions and glioblastoma multiforme (GBM). In GBM, the levels of small heat shock protein, CRYAB (HspB5) are also reported to be elevated, where it has been shown to exert anti-apoptotic activity. Interestingly, CRYAB is secreted via exosomes by various cells. In order to understand the relation between inflammatory cytokines and CRYAB, U373 glioma cells, were stimulated with proinflammatory cytokines, IL-1ß and TNF-α, and their effect on CRYAB levels in cells and secreted exosomes was studied. Our results show that U373 cells produce and secrete CRYAB via exosomes and that stimulation with IL-1ß and TNF-α significantly increase the levels of CRYAB in not only the cells but also in the secreted exosomes. In addition, cytokine stimulation of U373 cells brings about changes in the secreted exosomal proteome, many of which are involved in cancer progression.

Proteomic analysis of transcription factors involved in the alteration of ischemic mouse heart as modulated by MSC exosomes.

Mesenchymal stem cell (MSC) exosomes have been found to attenuate cardiac systolic and diastolic dysfunction in animal models of ischemia. Exosomes carry a plethora of active and inactive proteins as their cargo, which are readily available to the recipient cell for use in intracellular signaling pathways-depending on the stresses, such as ischemia or hypoxia. Among the exosomal proteins are the often-overlooked cargo of transcriptional regulators. These transcriptional regulators influence the transcriptome and subsequently the proteome of recipient cell. Here, we report the transcriptional factors and regulators differentially modulated and their potential role in modulating cardiac function in MSC exosome treated ischemic mice hearts. Our analysis shows ischemic stress modulating transcriptional regulators and factors such as HSF1 and HIF1A in the infarct and peri-infarct areas of ischemic hearts which is mitigated by MSC exosomes. Similarly, STAT3 and SMAD3 are also modulated by MSC exosomes. Interestingly, NOTCH1 and ß-catenin were detected in the ischemic hearts. The differential expression of these regulators and factors drives changes in various biological process governed in the ischemic cardiac cells. We believe these studies will advance our understanding of cardiac dysfunction occurring in the ischemic hearts and lay the groundwork for further studies on the modulation of cardiac function during ischemia by MSC exosomes.

Mesenchymal stem cell treatment for hyperactive immune response in patients with COVID-19.

The human immune system protects the body against invasive organisms and kicks into a hyperactive mode in COVID-19 patients, particularly in those who are critically sick. Therapeutic regimens directed at the hyperactive immune system have been found to be effective in the treatment of patients with COVID-19. An evolving potential treatment option is therapy with mesenchymal stem cells (MSCs) due to their regenerative and reparative ability in epithelial cells. Clinical trials have reported the safe usage of MSC therapy. Systemic effects of MSC treatment have included a reduction in pro-inflammatory cytokines and a decrease in the levels of CRP, IL-6, and lactase dehydrogenase, which function as independent biomarkers for COVID-19 mortality and respiratory failure.

Treatment of COVID-19 is becoming increasingly difficult because of new variants, such as Delta, and more recently Omicron. Each virus variant becomes smarter at being able to evade the body's immune system, vaccines and drug treatments. The biggest challenge in treating COVID-19 is when the body's immune system starts to become hyperactive. In such a scenario, the immune system releases the compounds that are supposed to be released in small doses all at once. Thus, overwhelming the body and causing many complications. One possible solution to this is the mesenchymal stem cell. Multiple clinical trials have shown that mesenchymal stem cells can heal all different cell types in the body and stop the hyperactive immune system.

The structural basis of effective LOX-1 inhibition.

Along with other scavenger receptors, splice variants of LOX-1 play an important role in modulating numerous subcellular mechanisms such as normal cell development, differentiation and growth in response to physiological stimuli. Thus, LOX-1 activity is a key regulator in determining the severity of many genetic, metabolic, cardiovascular, renal, and neurodegenerative diseases and/or cancer. Increased expression of LOX-1 precipitates pathological disorders during the aging process. Therefore, it becomes important to develop novel LOX-1 inhibitors based on its ligand binding polarity and/or affinity and disrupt the uptake of its ligand: oxidized low-density lipoproteins (ox-LDL). In this review, we shed light on the presently studied and developed novel LOX-1 inhibitors that may have potential for treatment of diseases characterized by LOX-1 activation.

Erratum: NLRP3 inflammasome via IL-1ß regulates PCSK9 secretion: Erratum.

[This corrects the article DOI: 10.7150/thno.45939.].

NLRP3 inflammasome via IL-1ß regulates PCSK9 secretion.

Background: Both PCSK9 and NLRP3 inflammasome play important roles in atherogenesis. This study was designed to test the hypothesis that NLRP3 inflammasome via IL-1ß induces PCSK9 secretion. The inter-twined relationship between NLRP3 inflammasome, IL-1ß and PCSK9 may be relevant in atherogenesis. Methods: We studied NLRP3 inflammasome-mediated PCSK9 secretion in mouse peritoneal macrophages and in a variety of tissues, such as liver, kidney and small intestine. Macrophages were derived from wild-type (WT) and a variety of gene deletion mice to define the mechanistic basis of NLRP3 inflammasome -mediated PCSK9 secretion. Additional studies were performed in high-fat diet fed mice. Results: We observed that NLRP3 and its downstream signals ASC, Caspase-1, IL-18, and IL-1ß all participate in PCSK9 secretion. IL-1ß seems to be more important than IL-18 in the induction of PCSK9 secretion. Further, there appears to be significant involvement of MAPKs in this process. Lastly, we observed that mice fed high fat diet have high expression of NLRP3 and a greater secretion of PCSK9 than mice fed a standard diet, and this increased secretion of PCSK9 in high fat diet-fed mice was attenuated in IL-1ß-/- mice. Conclusions: This study based on extensive in vitro and in vivo data provides evidence that NLRP3 inflammasome via IL-1ß plays an important role in determining PCSK9 secretion, particularly in the presence of high-fat diet.

Glioma-derived exosomes drive the differentiation of neural stem cells to astrocytes.

Exosomes appear to be effective inter-cellular communicators delivering several types of molecules, such as proteins and RNAs, suggesting that they could influence neural stem cell (NSC) differentiation. Our RNA sequencing studies demonstrated that the RNAs related to cell proliferation and astrocyte differentiation were upregulated in human mesenchymal stem cells (hMSC) when co-cultured with exosomes obtained from the culture medium of human glioma cells (U87). Metallothionein 3 and elastin genes, which are related to cell proliferation, increased 10 and 7.2 fold, respectively. Expression of genes for astrocyte differentiation, such as tumor growth factor alpha, induced protein 3 of the NOTCH1 family, colony stimulating factor and interleukin 6 of the STAT3 family and Hes family bHLH transcription factor 1 also increased by 2.3, 10, 4.7 and 2.9 fold, respectively. We further examined the effects of these exosomes on rat fetal neural stem cell (rNSC) differentiation using the secreted exosomes from U87 glioma cells or exosomes from U87 cells that were stimulated with interleukin 1ß (IL-1ß). The rNSCs, extracted from rat brains at embryonic day 14 (E14), underwent a culture protocol that normally leads to predominant (~90%) differentiation to ODCs. However, in the presence of the exosomes from untreated or IL-1ß-treated U87 cells, significantly more cells differentiated into astrocytes, especially in the presence of exosomes obtained from the IL-1ß-challenged glioma cells. Moreover, glioma-derived exosomes appeared to inhibit rNSC differentiation into ODCs or astrocytes as indicated by a significantly increased population of unlabeled cells. A portion of the resulting astrocytes co-expressed both CD133 and glial fibrillary acidic protein (GFAP) suggesting that exosomes from U87 cells could promote astrocytic differentiation of NSCs with features expected from a transformed cell. Our data clearly demonstrated that exosomes secreted by human glioma cells provide a strong driving force for rat neural stem cells to differentiate into astrocytes, uncovering potential pathways and therapeutic targets that might control this aggressive tumor type.

Molecular events in MSC exosome mediated cytoprotection in cardiomyocytes.

A host of hormonal-metabolic alterations take place following exposure of cardiomyocytes to hypoxia and other noxious stimuli. Here, we demonstrate that exposure of cultured rat cardiomyocytes to lipopolysaccharide (LPS) resulted in upregulation (~1.5 fold) of oxidized low-density lipoprotein receptor-1 (LOX-1). There was also a marked increase in apoptosis 12 hrs after LPS treatment with caspase-3 levels being significantly elevated (~1.3 fold) and a significant increase in LDH release at 24 hrs. Interestingly, there was a ~1.4-fold upregulation of LC-3 expression post-LPS treatment indicating development of autophagy, which probably is a compensatory response to combat cellular injury induced by LPS. Treatment with LPS also reduced the size and morphology of cardiomyocyte spheroids. In an attempt to limit LPS-induced injury, cardiomyocytes were treated with exosomes derived from mesenchymal stromal cells (MSCs). We noted a significant suppression of LOX-1 expression that in turn suppressed apoptosis as well as autophagic response and restored spheroid morphology. Mass spectrophotometric analysis of MSC exosomes revealed a cargo rich in proteins which are involved in pathways negatively modulating cell death and apoptosis while promoting cell survival. This is first report to our knowledge on the initial molecular events in MSC exosome mediated cytoprotection of stressed cardiomyocytes.

Glutaminase inhibitor CB-839 increases radiation sensitivity of lung tumor cells and human lung tumor xenografts in mice.

PURPOSE: The purpose of this study was to translate our in vitro therapy approach to an in vivo model. Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Studying lymph-node aspirates containing malignant lung tumor cells showed a strong correlation between glutamine consumption and glutathione (GSH) excretion. Subsequent experiments with A549 and H460 lung tumor cell lines provided additional evidence for glutamine's role in driving synthesis and excretion of GSH. Using stable-isotope-labeled glutamine as a tracer metabolite, we demonstrated that the glutamate group in GSH is directly derived from glutamine, linking glutamine utilization intimately to GSH syntheses. MATERIALS AND METHODS: To understand the possible mechanistic link between glutamine consumption and GSH excretion, we studied GSH metabolism in more detail. Inhibition of glutaminase (GLS) with BPTES, a GLS-specific inhibitor, effectively abolished GSH synthesis and excretion. Since our previous work, several novel GLS inhibitors became available and we report herein effects of CB-839 in A427, H460 and A549 lung tumor cells and human lungtumor xenografts in mice. RESULTS: Inhibition of GLS markedly reduced cell viability, producing ED50 values for inhibition of colony formation of 9, 27 and 217 nM in A427, A549 and H460, respectively. Inhibition of GLS is accompanied by ∼30% increased response to radiation, suggesting an important role of glutamine-derived GSH in protecting tumor cells against radiation-induced injury. In subsequent mouse xenografts, short-term CB-839 treatments reduced serum GSH by >50% and increased response to radiotherapy of H460-derived tumor xenografts by 30%. CONCLUSION: The results support the proposed mechanistic link between GLS activity and GSH synthesis and suggest that GLS inhibitors are effective radiosensitizers.

Hypoxia-derived exosomes induce putative altered pathways in biosynthesis and ion regulatory channels in glioblastoma cells.

Hypoxia, a hallmark characteristic of glioblastoma (GBM) induces changes in the transcriptome and the proteome of tumor cells. We discovered that hypoxic stress produces significant qualitative and quantitative changes in the protein content of secreted exosomes from GBM cells. Among the proteins found to be selectively elevated in hypoxic exosomes were protein-lysine 6-oxidase (LOX), thrombospondin-1 (TSP1), vascular derived endothelial factor (VEGF) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), well studied contributors to tumor progression, metastasis and angiogenesis. Our findings demonstrate that hypoxic exosomes induce differential gene expression in recipient glioma cells. Glioma cells stimulated with hypoxic exosomes showed a marked upregulation of small nucleolar RNA, C/D box 116-21 (SNORD116-21) transcript among others while significantly downregulated the potassium voltage-gated channel subfamily J member 3 (KCNJ3) message. This differential expression of certain genes is governed by the protein cargo being transferred via exosomes. Additionally, compared to normoxic exosomes, hypoxic exosomes increased various angiogenic related parameters vis-à-vis, overall tube length, branching intervals and length of isolated branches studied in tube formation assay with endothelial progenitor cells (EPCs). Thus, the intercellular communication facilitated via exosomes secreted from hypoxic GBM cells induce marked changes in the expression of genes in neighboring normoxic tumor cells and possibly in surrounding stromal cells, many of which are involved in cancer progression and treatment resistance mechanisms.

Modifying Dendritic Cell Activation with Plasmonic Nano Vectors.

Dendritic cells (DCs) can acquire, process, and present antigens to T-cells to induce an immune response. For this reason, targeting cancer antigens to DCs in order to cause an immune response against cancer is an emerging area of nanomedicine that has the potential to redefine the way certain cancers are treated. The use of plasmonically active silver-coated gold nanorods (henceforth referred to as plasmonic nano vectors (PNVs)) as potential carriers for DC tumor vaccines has not been presented before. Effective carriers must be able to be phagocytized by DCs, present low toxicity, and induce the maturation of DCs-an early indication of an immune response. When we treated DCs with the PNVs, we found that the cell viability of DCs was unaffected, up to 200 µg/ml. Additionally, the PNVs associated with the DCs as they were phagocytized and they were found to reside within intracellular compartments such as endosomes. More importantly, the PNVs were able to induce expression of surface markers indicative of DC activation and maturation, i.e. CD40, CD86, and MHC class II. These results provide the first evidence that PNVs are promising carriers for DC-based vaccines and warrant further investigating for clinical use.

In Vivo Flow Cytometry of Circulating Tumor-Associated Exosomes.

Circulating tumor cells (CTCs) demonstrated the potential as prognostic markers of metastatic development. However, the incurable metastasis can already be developed at the time of initial diagnosis with the existing CTC assays. Alternatively, tumor-associated particles (CTPs) including exosomes can be a more valuable prognostic marker because they can be released from the primary tumor long before CTCs and in larger amount. However, little progress has been made in high sensitivity detection of CTPs, especially in vivo. We show here that in vivo integrated photoacoustic (PA) and fluorescence flow cytometry (PAFFC) platform can provide the detection of melanoma and breast-cancer-associated single CTPs with endogenously expressed melanin and genetically engineered proteins or exogenous dyes as PA and fluorescent contrast agents. The two-beam, time-of-light PAFFC can measure the sizes of CTCs and CTPs and identify bulk and rolling CTCs and CTC clusters, with no influence on blood flow instability. This technique revealed a higher concentration of CTPs than CTCs at an early cancer stage. Because a single tumor cell can release many CTPs and in vivo PAFFC can examine the whole blood volume, PAFFC diagnostic platform has the potential to dramatically improve (up to 105-fold) the sensitivity of cancer diagnosis.