Activation of neutral sphingomyelinase 2 through hyperglycemia contributes to endothelial apoptosis via vesicle-bound intercellular transfer of ceramides

Background Pro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development. Local cellular ceramide accumulation mediates endothelial apoptosis, especially in type 2 diabetes mellitus, which is a major cardiovascular risk factor. In recent years, large extracellular vesicles (lEVs) have been identified as an important means of intercellular communication and as regulators of cardiovascular health and disease. A potential role for lEVs as vehicles for ceramide transfer and inductors of diabetes-associated endothelial apoptosis has never been investigated. Methods and Results A mass-spectrometric analysis of human coronary artery endothelial cells (HCAECs) and their lEVs revealed C16 ceramide (d18:1–16:0) to be the most abundant ceramide in lEVs and to be significantly increased in lEVs after hyperglycemic injury to HCAECs. The increased packaging of ceramide into lEVs after hyperglycemic injury was shown to be dependent on neutral sphingomyelinase 2 (nSMase2), which was upregulated in glucose-treated HCAECs. lEVs from hyperglycemic HCAECs induced apoptosis in the recipient HCAECs compared to native lEVs from untreated HCAECs. Similarly, lEVs from hyperglycemic mice after streptozotocin injection induced higher rates of apoptosis in murine endothelial cells compared to lEVs from normoglycemic mice. To generate lEVs with high levels of C16 ceramide, ceramide was applied exogenously and shown to be effectively packaged into the lEVs, which then induced apoptosis in lEV-recipient HCAECs via activation of caspase 3. Intercellular transfer of ceramide through lEVs was confirmed by use of a fluorescently labeled ceramide analogue. Treatment of HCAECs with a pharmacological inhibitor of nSMases (GW4869) or siRNA-mediated downregulation of nSMase2 abrogated the glucose-mediated effect on apoptosis in lEV-recipient cells. In contrast, for small EVs (sEVs), hyperglycemic injury or GW4869 treatment had no effect on apoptosis induction in sEV-recipient cells. Conclusion lEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides. Graphical abstract

Chip-Based Sensing of the Intercellular Transfer of Cell Surface Proteins: Regulation by the Metabolic State

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are anchored at the surface of mammalian blood and tissue cells through a carboxy-terminal GPI glycolipid. Eventually, they are released into incubation medium in vitro and blood in vivo and subsequently inserted into neighboring cells, potentially leading to inappropriate surface expression or lysis. To obtain first insight into the potential (patho)physiological relevance of intercellular GPI-AP transfer and its biochemical characterization, a cell-free chip- and microfluidic channel-based sensing system was introduced. For this, rat or human adipocyte or erythrocyte plasma membranes (PM) were covalently captured by the TiO2 chip surface operating as the acceptor PM. To measure transfer between PM, donor erythrocyte or adipocyte PM were injected into the channels of a flow chamber, incubated, and washed out, and the type and amount of proteins which had been transferred to acceptor PM evaluated with specific antibodies. Antibody binding was detected as phase shift of horizontal surface acoustic waves propagating over the chip surface. Time- and temperature-dependent transfer, which did not rely on fusion of donor and acceptor PM, was detected for GPI-APs, but not typical transmembrane proteins. Transfer of GPI-APs was found to be prevented by α-toxin, which binds to the glycan core of GPI anchors, and serum proteins in concentration-dependent fashion. Blockade of transfer, which was restored by synthetic phosphoinositolglycans mimicking the glycan core of GPI anchors, led to accumulation in the chip channels of full-length GPI-APs in association with phospholipids and cholesterol in non-membrane structures. Strikingly, efficacy of transfer between adipocytes and erythrocytes was determined by the metabolic state (genotype and feeding state) of the rats, which were used as source for the PM and sera, with upregulation in obese and diabetic rats and counterbalance by serum proteins. The novel chip-based sensing system for GPI-AP transfer may be useful for the prediction and stratification of metabolic diseases as well as elucidation of the putative role of intercellular transfer of cell surface proteins, such as GPI-APs, in (patho)physiological mechanisms.

Intercellular transfer of mitochondrial DNA carrying metastasis-enhancing pathogenic mutations from high- to low-metastatic tumor cells and stromal cells via extracellular vesicles

Background Mitochondrial DNA (mtDNA) carrying certain pathogenic mutations or single nucleotide variants (SNVs) enhances the invasion and metastasis of tumor cells, and some of these mutations are homoplasmic in tumor cells and even in tumor tissues. On the other hand, intercellular transfer of mitochondria and cellular components via extracellular vesicles (EVs) and tunneling nanotubes (TNTs) has recently attracted intense attention in terms of cell-to-cell communication in the tumor microenvironment. It remains unclear whether metastasis-enhancing pathogenic mutant mtDNA in tumor cells is intercellularly transferred between tumor cells and stromal cells. In this study, we investigated whether mtDNA with the NADH dehydrogenase subunit 6 (ND6) G13997A pathogenic mutation in highly metastatic cells can be horizontally transferred to low-metastatic cells and stromal cells in the tumor microenvironment. Results When MitoTracker Deep Red-labeled high-metastatic Lewis lung carcinoma A11 cells carrying the ND6 G13997A mtDNA mutation were cocultured with CellLight mitochondria-GFP-labeled low-metastatic P29 cells harboring wild-type mtDNA, bidirectional transfer of red- and green-colored vesicles, probably mitochondria-related EVs, was observed in a time-dependent manner. Similarly, intercellular transfer of mitochondria-related EVs occurred between A11 cells and α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs, WA-mFib), macrophages (RAW264.7) and cytotoxic T cells (CTLL-2). Intercellular transfer was suppressed by inhibitors of EV release. The large and small EV fractions (L-EV and S-EV, respectively) prepared from the conditioned medium by differential ultracentrifugation both were found to contain mtDNA, although only S-EVs were efficiently incorporated into the cells. Several subpopulations had evidence of LC3-II and contained degenerated mitochondrial components in the S-EV fraction, signaling to the existence of autophagy-related S-EVs. Interestingly, the S-EV fraction contained a MitoTracker-positive subpopulation, which was inhibited by the respiration inhibitor antimycin A, indicating the presence of mitochondria with membrane potential. It was also demonstrated that mtDNA was transferred into mtDNA-less ρ0 cells after coculture with the S-EV fraction. In syngeneic mouse subcutaneous tumors formed by a mixture of A11 and P29 cells, the mitochondria-related EVs released from A11 cells reached distantly positioned P29 cells and CAFs. Conclusions These results suggest that metastasis-enhancing pathogenic mtDNA derived from metastatic tumor cells is transferred to low-metastatic tumor cells and stromal cells via S-EVs in vitro and in the tumor microenvironment, inferring a novel mechanism of enhancement of metastatic potential during tumor progression.

Intercellular transfer of exosomal wild type EGFR triggers osimertinib resistance in non-small cell lung cancer

Background Epidermal growth factor receptor (EGFR)-mutated lung cancer constitutes a major subgroup of non-small cell lung cancer (NSCLC) and osimertinib is administrated as first-line treatment. However, most patients with osimertinib treatment eventually relapse within one year. The underlying mechanisms of osimertinib resistance remain largely unexplored. Methods Exosomes isolation was performed by differential centrifugation. Co-culture assays were conducted to explore the alteration of drug sensitivity by cell viability and apoptosis assays. Immunofluorescence and flow cytometry were performed to visualize the formation or absorption of exosomes. Exosomes secretion was measured by Nanoparticle Tracking Analysis or ELISA. The xenograft tumor model in mice was established to evaluate the effect of exosomes on osimertinib sensitivity in vivo. Results Intercellular transfer of exosomal wild type EGFR protein confers osimertinib resistance to EGFR-mutated sensitive cancer cells in vitro and in vivo. Co-culture of EGFR-mutated sensitive cells and EGFR-nonmutated resistant cells promoted osimertinib resistance phenotype in EGFR-mutated cancer cells, while depletion of exosomes from conditioned medium or blockade of exosomal EGFR by neutralizing antibody alleviated this phenotype. Mechanistically, osimertinib promoted the release of exosomes by upregulated a Rab GTPase (RAB17). Knockdown of RAB17 resulted in the decrease of exosomes secretion. Moreover, exosomes could be internalized by EGFR-mutated cancer cells via Clathrin-dependent endocytosis and then the encapsulated exosomal wild type EGFR protein activated downstream PI3K/AKT and MAPK signaling pathways and triggered osimertinib resistance. Conclusions Intercellular transfer of exosomal wild type EGFR promotes osimertinib resistance in NSCLC, which may represent a novel resistant mechanism of osimertinib and provide a proof of concept for targeting exosomes to prevent and reverse the osimertinib resistance.

Inhibition of Extracellular Vesicle-Associated MMP2 Abrogates Intercellular Transfer of Hepatic miR-122 to Tissue Macrophages and Curtails Liver Inflammation

AbstractmicroRNA-122 (miR-122), a liver specific regulatory RNA, plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 is also a proinflammatory microRNA and when exported to tissue resident macrophage induces expression of inflammatory cytokines there. We found intercellular transfer of miR-122 in lipid exposed liver plays a role in liver inflammation. Exploring the mechanism of intercellular miR-122 transfer from hepatic cells, we detected MMP2 on the membrane of extracellular vesicles derived from hepatic cells which proved to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. Matrix metalloprotease 2 or MMP2 is a metalloproteinase that plays a key role in shaping and remodelling the extracellular matrix of human tissue by targeting degradation of matrix proteins. MMP2 was found to increase the movement of the EVs along the extracellular matrix to enhance their uptake in recipient cells. Inhibition of MMP2 restricts functional transfer of hepatic miRNAs across the hepatic and non-hepatic cell boundaries. By targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer.Abstract FigureHuman hepatocytes on exposure to high lipid export out miRNAs including proinflammatory miR-122.Extracellular miR-122 is taken up by tissue macrophages to get them activated to produce inflammatory cytokines.MMP2 present on the surface of the EVs released by hepatocyte is essential for miRNA transfer to macrophage cellsInhibition of MMP2 prevents miR-122 transfer to macrophage and stops activation of recipient macrophage.

ACE2 in tumor cells and tumor vasculature: Negligible intercellular transfer from cancer cells into endothelial cells

Cancer patients are more susceptible to severe coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Angiotensin-converting enzyme 2 (ACE2) is the functional host receptor for SARS-CoV-2 entering into human cells. Bioinformatics’ analyses have revealed that ACE2 is upregulated in some cancer cells. In the present study, we evaluated ACE2 protein expression levels in several common malignancies compared to non-cancerous normal tissues. ACE2 expression was elevated in colorectal adenocarcinoma, pancreatic adenocarcinoma, gastric adenocarcinoma, and papillary renal cell carcinoma cancer. Yet, it was suppressed in chromophobe renal cell carcinoma, testicular germ cell tumors, and papillary thyroid carcinoma. Two tumor tissue microarrays were used to evaluate the prognostic value of ACE2 expression in patients with gastric adenocarcinoma, and colorectal adenocarcinoma without COVID-19. No significant survival benefit was found for patients with overexpression of ACE2 in the tumor. In the tumor vasculature, ACE2 expression was observed in only 54% of the tumor micro-vessels. Using an in vitro co-culture of endothelial cells and tumor cells overexpressing fusion protein ACE2-red fluorescent protein, we did not observe any clear and convincing intercellular transfer of ACE2 from cancer cells into endothelial cells. In summary, alteration of ACE2 expression was found in common malignancies, but there is no evidence of intercellular transfer of ACE2 from cancer cells to endothelial cells.

Exosomal-mediated transfer of OIP5-AS1 enhanced cell chemoresistance to trastuzumab in breast cancer via up-regulating HMGB3 by sponging miR-381-3p

Background Long noncoding RNA OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) was confirmed to involve in the malignancy of breast cancer. However, whether exosomal OIP5-AS1 is implicated in trastuzumab resistance remains unclear. Methods The IC50 value of cells to trastuzumab, cell proliferation, migration, and apoptosis was analyzed by cell counting kit-8 assay, colony formation assay, transwell assay, or flow cytometry, respectively. The expression of OIP5-AS1 and microRNA (miR)-381-3p was detected using quantitative real-time polymerase chain reaction. Exosomes were isolated by ultracentrifugation and qualified by nanoparticle tracking analysis software. Western blot was used to detect the protein levels of tumor susceptibility gene 101 (TSG101), CD81, CD63, or high-mobility group protein B3 (HMGB3). The interaction between miR-381-3p and OIP5-AS1 or HMGB3 was confirmed by dual-luciferase reporter assay and pull-down assay. In vivo experiments were conducted using murine xenograft models. Results OIP5-AS1 was elevated in trastuzumab-resistant breast cancer cells, and OIP5-AS1 knockdown rescued trastuzumab sensitivity. Extracellular OIP5-AS1 was packaged into exosomes, which were secreted by trastuzumab-resistant cells, and could be absorbed by trastuzumab-sensitive cells in breast cancer. Importantly, intercellular transfer of OIP5-AS1 via exosomes enhanced trastuzumab resistance in vitro. OIP5-AS1 was a sponge of miR-381-3p; besides, miR-381-3p targeted HMGB3. Murine xenograft analysis showed exosomal OIP5-AS1 induced trastuzumab resistance in vivo. Exosomal OIP5-AS1 was dysregulated in the serum of breast cancer patients and might be a promising diagnostic biomarker in trastuzumab resistance. Conclusion Intercellular transfer of OIP5-AS1 by exosomes enhanced trastuzumab resistance in breast cancer via miR-381-3p/HMGB3 axis, indicating a potential therapeutic strategy to boost the effectiveness of trastuzumab in resistant breast cancer patients.