Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA.
Cartilage breakdown, inflammation and pain are hallmark symptoms of osteoarthritis, and autologous blood products such as citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) have been developed as a regenerative approach to rebuild cartilage, inhibit inflammation and reduce pain. However, mechanisms of action of these blood derivatives are still not fully understood, in part due to the large number of components present in these medical products. In addition, the discovery of extracellular vesicles (EVs) and their involvement in intercellular communication mediated by cargo molecules like microRNAs (miRNAs) opened up a whole new level of complexity in understanding blood products. In this study we focused on the development of an isolation protocol for EVs from CPRP and hypACT that can also deplete lipoproteins, which are often co-isolated in EV research due to shared physical properties. Several isolation methods were compared in terms of particle yield from CPRP and hypACT. To gain insights into the functional repertoire conveyed via EV-associated miRNAs, we performed functional enrichment analysis and identified NFκB signaling strongly targeted by CPRP EV miRNAs, whereas hypACT EV miRNAs affect IL6- and TGFβ/SMAD signaling.
Hydroxymethylglutaryl-coenzyme A (HMG-coA) reductase inhibitors (statins) have been shown to overcome tyrosine kinase inhibitor (tKi) resistance in epithelial growth factor receptor (eGfR) mutated non-small cell lung cancer (nScLc) cells in vivo and in vitro. However, little is known about the putative induction of non-apoptotic cell death pathways by statins. We investigated the effects of pitavastatin and fluvastatin alone or in combination with erlotinib in three NSCLC cell lines and examined the activation of different cell death pathways. We assessed apoptosis via fluorometric caspase assay and poly (ADP-ribose) polymerase 1 (PARP) cleavage. Furthermore, annexinV/propidium iodide (PI) flow cytometry was performed. Small molecule inhibitors benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone (zVAD), necrostatin 1 (Nec1), ferrostatin 1 (Fer1), Ac-Lys-Lys-Norleucinal (Calp1) were used to characterise cell death pathway(s) putatively (co-)activated by pitavastatin/erlotinib co-treatment. Synergism was calculated by additivity and isobolographic analyses. pitavastatin and fluvastatin induced cell death in EGFR TKI resistant NSCLC cells lines A549, Calu6 and H1993 as shown by caspase 3 activation and PARP cleavage. Co-treatment of cells with pitavastatin and the EGFR TKI erlotinib resulted in synergistically enhanced cytotoxicity compared to pitavastatin monotherapy. flow cytometry indicated the induction of alternative regulated cell death pathways. However, only co-treatment with mevalonic acid (Mev) or the pan-caspase inhibitor zVAD could restore cell viability. The results show that cytotoxicity mediated by statin/erlotinib co-treatment is synergistic and can overcome erlotinib resistance in K-ras mutated nScLc and relies only on apoptosis. Lung cancer is the leading cause of cancer death worldwide and is commonly classified into small and non-small cell lung cancer (SCLC and NSCLC). NSCLC is subdivided into three major types: (1) squamous cell carcinomas (SCC), (2) adenocarcinomas and (3) large cell carcinomas. In particular, NSCLC accounts for more than 80% of total pulmonary malignancies 1-3. EGFR is overexpressed in over 50% of NSCLCs. Oncogenic mutations of the EGFR occur in up to 20% of adenocarcinomas 4. Targeting EGFR has played a central role in advancing NSCLC research, treatment and patient outcome over the last several years. Osmertinib, afatinib, gefitinib and erlotinib are approved EGFR tyrosine kinase inhibitors (EGFR-TKI) for the treatment of advanced EGFR mutated NSCLC. Erlotinib therapy resulted in a significant improvement in median progression free survival, quality of life, and related symptom control compared to chemotherapy in an EGFR mutated population of advanced and metastatic NSCLC patients (EURTAC trial 5 ; OPTIMAL trial 6 ; ENSURE trial 7). K-Ras mutations lead to primary resistance to EGFR TKIs and are found in 25-30% of adenocarcinomas 8. In addition to primary resistance, secondary resistance to EGFR TKIs in EGFR-mutant NSCLC patients is commonly
Gene-therapeutic approaches seek to alleviate inherited familial hypercholesterolemia by complementing the defective low-density lipoprotein receptor (LDLR) gene with its functional homologue. One much-sought avenue toward this aim involves transduction with a replication-defective pseudotyped lentiviral expression vector carrying the gene encoding LDLR (reviewed in reference 1). Along these lines, it had been attempted to produce such lentiviral vectors via third-generation equine infectious anemia virus (EIAV)-based packaging in human embryonic kidney (HEK293T) cells. However, initial attempts at producing vesicular stomatitis virus spike protein G (VSVG)-pseudotyped lentivirus by using such a three-plasmid transient-cotransfection protocol demonstrated a dramatic decrease in vector yield on increased cytomegalovirus (CMV)-promoter-driven LDLR expression (F. A. Al-Allaf, unpublished observation).To confirm the above observation and determine the underlying reason, virus was produced in HEK293 cells (2.5 ϫ 10 6 /10-cm 2 plate) via transient transfection with the self-inactivating vector construct pHR-CMV-EGFP (3.33 g; Addgene 14858) (2), as well as the packaging construct psPAX2 (5 g; Addgene 12260) and the envelope construct pMD2.G, encoding the VSVG surface protein (1.66 g; Addgene 12259) (both a kind gift from Didier Trono).To prevent eventual SV40ori-based replication of plasmids mediated by large-T antigen, we used HEK293 cells in all experiments; despite the absence of the large-T antigen, we observed high expression of the transgenes. Cells were then cotransfected with different amounts of plasmids encoding either LDLR (Origene RC200006) or, as controls, human intercellular adhesion molecule 1 (ICAM1; Origene RC200714) and human transferrin receptor (TfR1; Origene RC200980), all carrying the sequence coding for the C-terminal peptide DYKDDDDK (DDK tag). The total DNA transfected was kept constant by adding herring sperm DNA to make up 11.66 g. The growth medium (Dulbecco's modified Eagle's medium [DMEM], 10% fetal calf serum [FCS]) was replaced and supplemented with 5 mM Na-butyrate at 24 h posttransfection (hpt). The cell supernatant was harvested at 48 hpt and filtered through a membrane (0.22-m pore size), and the lentivirus vector was pelleted by ultracentrifugation for 1.5 h at 4°C and 40,000 ϫ g in a Ti70.1 Beckman rotor. Titer was assessed via infection of HeLa cells grown in 12-well plates at 37°C in 5% CO 2 ; the medium was replaced with 500 l infection medium (DMEM, 2% FCS), and then aliquots of the resuspended pellets described above were added. After incubation for 30 min, 1 ml infection medium supplemented with Polybrene (final concentration, 8 g/ml) was added and incubation continued for 72 h. The percentage of fluorescent cells-as a consequence of transduction with the enhanced green fluorescent protein (EGFP)-encoding viral vector-was determined by flow cytometry in a BD FACSCalibur using CellQuest software, and the number of transducing units (TU) per milliliter was calculated. The numbe...
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