Levels In Conditioned Media Research Articles

Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

Atherosclerosis is rare in internal thoracic arteries (ITA) even in patients with severe atherosclerotic coronary artery (ACA) disease. To explore cellular differences, ITA SMC from 3 distinct donors and ACA SMC from 3 distinct donors were grown to sub-confluence and growth arrested for 48 h. Proliferation and thrombospondin-1 (TSP1) production were determined using standard techniques. ITA SMC were larger, grew more slowly and survived more passages than ACA SMC. ACA SMC had a more pronounced proliferative response to 10% serum than ITA SMC. Both ACA SMC and ITA SMC proliferated in response to exogenous TSP1 (12.5 µg/ml and 25 µg/ml) and platelet derived growth factor-BB (PDGF-BB; 20 ng/ml) but TSP1- and PDGF-BB-induced proliferation were partially inhibited by anti-TSP1 antibody A4.1, microRNA-21(miR-21)-3p inhibitors and miR-21-5p inhibitors in each of the 3 ACA SMC lines, but not in any of the ITA SMC lines. PDGF-BB stimulated TSP1 production in ACA SMC but not in ITA SMC but there was no increase in TSP1 levels in conditioned media in either SMC type. In summary, there are significant differences in morphology, proliferative capacity and in responses to TSP1 and PDGF-BB in SMC derived from ITA compared to SMC derived from ACA.

Mesenchymal stem cells pretreated with interferon-gamma attenuate renal fibrosis by enhancing regulatory T cell induction

Mesenchymal stem cells (MSCs) exert their anti-inflammatory and anti-fibrotic effects by secreting various humoral factors. Interferon-gamma (IFN-γ) can enhance these effects of MSCs, and enhancement of regulatory T (Treg) cell induction is thought to be an underlying mechanism. However, the extent to which Treg cell induction by MSCs pretreated with IFN-γ (IFN-γ MSCs) ameliorates renal fibrosis remains unknown. In this study, we investigated the effects of Treg cell induction by IFN-γ MSCs on renal inflammation and fibrosis using an siRNA knockdown system. Administration of IFN-γ MSCs induced Treg cells and inhibited infiltration of inflammatory cells in ischemia reperfusion injury (IRI) rats more drastically than control MSCs without IFN-γ pretreatment. In addition, administration of IFN-γ MSCs more significantly attenuated renal fibrosis compared with control MSCs. Indoleamine 2,3-dioxygenase (IDO) expression levels in conditioned medium from MSCs were enhanced by IFN-γ pretreatment. Moreover, IDO1 knockdown in IFN-γ MSCs reduced their anti-inflammatory and anti-fibrotic effects in IRI rats by reducing Treg cell induction. Our findings suggest that the increase of Treg cells induced by enhanced secretion of IDO by IFN-γ MSCs played a pivotal role in their anti-fibrotic effects. Administration of IFN-γ MSCs may potentially be a useful therapy to prevent renal fibrosis progression.

A noncanonical splicing variant c.875-5 T > G in von Willebrand factor causes in-frame exon skipping and type 2A von Willebrand disease

IntroductionA novel variant involving noncanonical splicing acceptor site (c.875-5 T > G) in propeptide coding region of von Willebrand factor (VWF) was identified in a patient with type 2A von Willebrand disease (VWD), who co-inherited with a null variant (p.Tyr271*) and presented characteristic discrepancy of plasma level of VWF antigen and activity, and a selective reduction of both intermediate-molecular-weight (IMWMs) and high-molecular-weight VWF multimers (HMWMs). Materials and methodsVWF mRNA transcripts obtained from peripheral leukocytes and platelets of the patients were investigated to analyze the consequence of c.875-5 T > G on splicing. The impact of the variant on expression and multimer assembly was further analyzed by in vitro expression studies in AtT-20 cells. The intracellular processing of VWF mutant and the Weibel-Palade bodies (WPBs) formation was evaluated by immunofluorescence staining and electron microscopy. ResultsThe mRNA transcript analysis revealed that c.875-5 T > G variant led to exon 8 skipping and an in-frame deletion of 41 amino acids in the D1 domain of VWF (p.Ser292_Glu333delinsLys), yielding a truncated propeptide. Consistent with the patient's laboratory manifestations, the AtT-20 cells transfected with mutant secreted less VWF, with the VWF antigen level in conditioned medium 47 % of wild-type. A slight retention in the endoplasmic reticulum was observed for the mutant. Almost complete loss of IMWMs and HMWMs in the medium and impaired WPBs formation in the cell, indicating truncated VWF propeptide lost its chaperon-like function for VWF multimerization and tubular storage. ConclusionsThe VWF splicing site variant (c.875-5 T > G) causes propeptide truncation, severely compromising VWF multimer assembly and tubular storage.

Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction.

Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGFβ1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGFβ1 and GM-CSF and thereby alter airway epithelial barrier function. Human or rat LF were cultured ± ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFβ1 receptor type 1 (TGFβR1) inhibitor (SB431542) or a TGFβ1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGFβ1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA. AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and α-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGFβ1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFβ1 activity. Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFβ1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.

Pharmacologic inhibition of astrocyte ApoE release

AbstractBackgroundThe ApoE4 allele is the strongest known genetic risk factor for late onset Alzheimer’s disease. Despite this knowledge, therapeutic strategies seeking to modulate brain ApoE homeostasis remain largely unexplored. Several lines of evidence demonstrate that astrocytes function as the primary producers of ApoE within the brain. In this study, we examine how targeted disruption of astrocyte lipid metabolism and protein prenylation effect the secretion of ApoE from primary astrocyte cultures.MethodPrimary cell cultures of mouse astrocytes were grown from 1‐3 day old pups. Drugs were applied to cells in serum free conditions over a range of timepoints and doses. Astrocyte conditioned media was collected from cultures and ApoE and other proteins of interest were measured by western blot.ResultWe find that inhibiting HMG‐CoA reductase, a rate‐limiting enzyme in the cholesterol synthesis pathway, with simvastatin reduces astrocyte ApoE levels in conditioned media in a dose dependent manner, while secretion of other proteins remain preserved. Inhibition of downstream cholesterol synthesis through blockade of the enzyme FDFT1 with zaragozic acid, however, has no effect on total ApoE secretion. Mice with genetic disruption of brain cholesterol metabolism also have similar levels of ApoE in CSF samples compared to control. These data suggest the effect of HMGCR inhibition on ApoE is not mediated through cholesterol. It is known that HMGCR activity is also required for maintenance of cellular protein prenylation. We tested the prenylation inhibitor FGTI‐2734 and found that it is sufficient to block astrocyte ApoE secretion, suggesting that HMGCR inhibitors may exert their effect on ApoE through this mechanism. Recent work suggests that inflammatory astrocytes exert toxic effects via neurotoxic lipids carried by ApoE and another carrier protein, CLU. We find that inhibiting HMGCR in astrocytes does not prevent cytokine‐induced conversion to an inflammatory phenotype, but remains able to block ApoE releaseConclusionWe conclude that targeted inhibition of prenylation enzymes may provide a therapeutic strategy to modulate ApoE levels in the brain.

Abstract B010: The effects of tumor cell-secreted factors on endothelial cell junction phenotype

Abstract Triple negative breast cancer (TNBC) is subtype of breast cancer that has no expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC metastasizes to specific tissues such as brain and bone in a process termed organotropism. In this process, TNBC cells cross the vascular endothelium; transport across endothelial cells is, in part, regulated by cell-cell junctions. The TNBC cell secretome has been shown to contain factors such as vascular growth factor (VEGF) and matrix metalloproteinases (MMPs) that could play a role in altering endothelium permeability. Prior work by us has shown that VEGF in tumor conditioned media can alter endothelial cell junction phenotype in human brain microvascular endothelial cells. Given the various factors present in tumor conditioned media (TCM), we hypothesized that both VEGF and MMPs are present at distinct levels in conditioned media derived from brain- or bone-seeking TNBC cells and that these factors alter junction phenotype in human umbilical vein endothelial cells. We quantified levels of VEGF and MMPs 1 and 9 in tumor conditioned media from parental, brain-seeking, and bone-seeking MDA-MB-231 cells. Our results show that brain-seeking tumor cell conditioned media reduces VE-cadherin continuous junction coverage compared to conditioned media from parental or bone-seeking cells. VE-Cadherin junctions also have decreased junction widths in response to tumor conditioned treatment from brain-seeking MDAs. These results show that secreted factors from TNBC cells have a modest impact on endothelium junction integrity, and future work will quantify changes in endothelium permeability to test barrier function. This work will contribute to a further understanding of TNBC organotropic metastasis and more effective targeted therapeutics to prevent disease progression. Citation Format: Ariana Joy L. DeCastro, Lauren Griffith, Kimberly Stroka. The effects of tumor cell-secreted factors on endothelial cell junction phenotype [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B010.

HMGB1 Positive Feedback Loop Between Cancer Cells and Tumor-Associated Macrophages Promotes Osteosarcoma Migration and Invasion

Numerous studies have demonstrated the key roles of tumor-associated macrophages (TAMs) in osteosarcoma metastasis. Higher levels of high mobility group box 1 (HMGB1) promote osteosarcoma progression. However, whether HMGB1 is involved in the polarization of M2 macrophages into M1 macrophages in osteosarcoma remains largely unknown. Here, HMGB1 and CD206 mRNA expression levels were measured by a quantitative reverse transcription-polymerase chain reaction in osteosarcoma tissues and cells. HMGB1 and receptor for advanced glycation end products (RAGE) protein expression levels were measured by western blotting. Osteosarcoma migration was measured using transwell and wound-healing assays, while a transwell assay determined osteosarcoma invasion. Macrophage subtypes were detected using flow cytometry. HMGB1 expression levels were aberrantly enhanced in osteosarcoma tissues compared with normal tissues and were positively correlated with AJCC III and IV stages, lymph node metastasis, and distant metastasis. Silencing HMGB1 inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. Furthermore, reduced HMGB1 expression levels in conditioned media derived from osteosarcoma cells induced the polarization of M2 TAMs to M1 TAMs. In addition, silencing HMGB1 inhibited the liver and lung metastasis of tumors and reduced the expression levels of HMGB1, CD163, and CD206 in vivo. HMGB1 was found to regulate macrophage polarization through RAGE. Polarized M2 macrophages induced osteosarcoma migration and invasion, activating HMGB1 expression in osteosarcoma cells to form a positive feedback loop. In conclusion, HMGB1 and M2 macrophages enhanced osteosarcoma migration, invasion, and EMT through positive feedback regulation. These findings reveal the significance of tumor cell and TAM interactions in the metastatic microenvironment.

Abstract 168: DDR2 regulates angiogenesis via HSP-90 modulation in ovarian cancer

Abstract Ovarian cancer is often diagnosed in later stages and patients have low five-year survival. There is a need to develop better therapies for this patient population. Solid tumors like ovarian cancer respond to hypoxia by inducing the sprouting of new blood cells. This process is called angiogenesis. Angiogenesis leads to formation of disordered and immature vessels that precludes efficient drug delivery. Prior work has shown that overexpressing DDR2 in endothelial cells leads to increased angiogenic activity, measured by tube formation and sprouting. We show that DDR2 expression in ovarian cancer tumor cells modulates angiogenic activity in endothelial cells. Human umbilical vein endothelial cells (HUVECs) cultured in conditioned media from DDR2-expressing tumor cells (ES2 shSCRM) have 20% increase in tube length and 18% increase in sprout network area than HUVECs cultured in conditioned media from DDR2-depleted tumor cells (ES2 shDDR2) (P=0.032). This difference in HUVEC angiogenesis is mediated by vascular endothelial growth factor (VEGF). We observed a 16% decrease in VEGF levels in conditioned media from ES2 shDDR2 cells compared to media from ES2 shSCRM cells (P​​<0.0001). DDR2 WT mice bearing ID8 Trp53-/- BRCA2-/- tumors have 53% more CD31+ microvessels than DDR2 KO mice (P​​<0.0001). We also identified that DDR2’s role in angiogenesis is mediated by the Hsp70/90 chaperone machinery. In summary, we have identified that DDR2 regulates angiogenesis in ovarian cancer. Citation Format: Favour A. Akinjiyan, Ritu M. Dave, Gregory Longmore, Katherine C. Fuh. DDR2 regulates angiogenesis via HSP-90 modulation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 168.

Combination Treatment with an MDM2 Antagonist and a BET Inhibitor Targets Both Myelofibrosis Hematopoietic Stem/ Progenitor Cells and Their Tumor Promoting Microenvironment

Introduction: Myelofibrosis (MF) arises at the level of hematopoietic stem and progenitor cells (HSC/HPC) due to a sequence of mutational events that results in increased JAK/STAT signaling. JAK2 has served as a target for drug development and has led to the development of several novel small molecule inhibitors. Clinical trials of these agents have revealed their ability to decrease the degree of splenomegaly and to improve systemic symptoms of PMF patients, but not to substantially affect the progeny of the malignant clone or the rate of disease progression. MF HSC/HPCs and their progeny are known to elaborate a number pro-inflammatory mediators including lipocalin (LCN2) (Lu et al, Blood, 2015), and IL-8 which are capable of creating a tumor promoting microenvironment. LCN2 promotes osteoblast formation and marrow fibrosis (Lu et al, Blood, 2013) while IL8 promotes endothelial cell proliferation. We have previously reported that plasma LCN2 levels are increased in MF patients (Lu et al, ASH, 2016) and show now that plasma IL-8 levels are also dramatically increased (Figure 1). Furthermore the levels of IL-8 as shown by IHC were increased in splenic myeloid cells especially megakaryocytes and the IL-8 receptor CXCR2 was expressed at greater levels by MF splenic littoral cells that line the sinusoids as well as endothelial cells as compared to normal spleens. We hypothesize that effective treatment of MF would require a combination of drugs that directly deplete the malignant HSC/HPCs as well as dampen the tumor promoting microenvironment. In a previous study, we have found the MDM2 protein, a negative regulator of p53, are highly expressed in PV CD34+ cells from PV patients and can be targeted by nutlin therapy (Lu et al, Blood, 2013). In order to assess if nutlin therapy would deplete MF CD34+ cells, using flow cytometry method, we documented that MDM2 protein expression was significantly increased in MF CD34+ cells as compared to normal CD34+ cells and was higher than that in PV CD34+ cells (Figure 2). This finding suggests that MF malignant HSC/HPC might also be sensitive to the treatment with a nutlin. Furthermore, MF HSC/HPCs reside in the BM and extra medullary sites containing dysfunctional microenvironments which promote the predominance of the malignant stem cells. We assume that BET inhibitors might dampen the MF inflammatory milieu since BET proteins play a central role in regulation of inflammatory responses. BET proteins interact with transcription factors (TF) including NF-KB, P53 and c-Myc, and downregulate the transcriptional activation of numerous cytokines including IL-6 and IL-8. We then hypothesize that treatment with low doses of nutlin combined with JQ1, a BET inhibitor, will target MF malignant cells as well as correct MF HSC tissue specific supportive microenvironments by decreasing pro-inflammatory cytokine generation.Results: 1. Treatment with a combination of low dose RG7112 and JQ1 selectively decreased hematopoietic colony formation by MF CD34+ cells. Exposure to either low dose RG7112 (200nM) or JQ1 (50nM) alone for 2 days increased the degree of MF CD34+ cell apoptosis modestly, while a combination of the 2 drugs significantly induced MF CD34+ cell apoptosis (p<0.05). By contrast, treatment with each drug alone or in combination however did not significant induce apoptosis of normal CD34+ cells. Combination treatment of RG7112 and JQ1 inhibited total number of colonies generated by MF CD34+ cells by 60%, as compared to the treatment with each drug alone (p=0.004 and p=0.03, respectively). Combination treatment with RG7112 and JQ1 did not, however, decrease colony formation by normal HPC. In a small number of cases, combination therapy decreased the numbers of homozygous JAK2V617F+ colonies assayed from MF CD34+ cells (at range from 8% to 20%).2. Both RG7112 and JQ1 treatment decrease the elaboration of MF pro-inflammatory cytokines. Treatment of MF mononuclear cells (MNC) with RG7112 but not JQ1 decreased LCN2 levels in conditioned medium (CM) (at range from 5% to 39%, p=0.012); while treatment with JQ1 at 50nM strongly decreased the levels of IL-8 in MF MNC CM by 50% (p=0.0003).Summary: These data indicate that JQ1 combined with RG7112 might be a potential effective strategy for the treatment of MF. This drug combination will not only selectively target MF HSC/HPCs but also alter the MF HSC promoting microenvironment by inhibiting the elaboration of pro-inflammatory mediators. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recombinant Human Proteoglycan-4 Mediates Interleukin-6 Response in Both Human and Mouse Endothelial Cells Induced Into a Sepsis Phenotype.

Sepsis is a leading cause of death in the United States. Putative targets to prevent systemic inflammatory response syndrome include antagonism of toll-like receptors 2 and 4 and CD44 receptors in vascular endothelial cells. Proteoglycan-4 is a mucinous glycoprotein that interacts with CD44 and toll-like receptor 4 resulting in a blockade of the NOD-like receptor pyrin domain-containing-3 pathway. We hypothesized that endothelial cells induced into a sepsis phenotype would have less interleukin-6 expression after recombinant human proteoglycan 4 treatment in vitro. Enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction to measure interleukin-6 protein and gene expression. Research laboratory. Human umbilical vascular endothelial cells, human lung microvascular endothelial cells, and transgenic mouse (wild type) (Cd44 +/+/Prg4 +/+), Cd44 -/- (Cd44 tm1Hbg Prg4 +/+), Prg4 GT/GT (Cd44 +/+ Prg4 tm2Mawa/J), and double knockout (Cd44 tm1Hbg Prg4 tm2Mawa/J) lung microvascular endothelial cells. Cells were treated with 100 or 250 ng/mL lipopolysaccharide-Escherichia coli K12 and subsequently treated with recombinant human proteoglycan 4 after 30 minutes. Interleukin-6 levels in conditioned media were measured via enzyme-linked immunosorbent assay and gene expression was measured via reverse transcriptase-quantitative polymerase chain reaction with ΔΔ-Ct analysis. Additionally, human umbilical vascular endothelial cells and human lung microvascular endothelial cells were treated with 1:10 diluted plasma from 15 patients with sepsis in culture media. After 30 minutes, either 50 or 100 µg/mL recombinant human proteoglycan 4 was administered. Interleukin-6 protein and gene expression were assayed. Proteoglycan 4 levels were also compared between control and sepsis patient plasma. Human umbilical vascular endothelial cell, human lung microvascular endothelial cell, and mouse lung microvascular endothelial cell treated with lipopolysaccharide had significantly increased interleukin-6 protein compared with controls. Recombinant human proteoglycan-4 significantly reduced interleukin-6 in human and mouse endothelial cells. Interleukin-6 gene expression was significantly increased after lipopolysaccharide treatment compared with controls. This response was reversed by 50 or 100 µg/mL recombinant human proteoglycan-4 in 80% of sepsis samples in human umbilical vascular endothelial cells and in 60-73% in human lung microvascular endothelial cells. In Cd44 -/- genotypes of the mouse lung microvascular endothelial cells, recombinant human proteoglycan-4 significantly reduced interleukin-6 protein levels after lipopolysaccharide treatment, indicating that Cd44 is not needed for recombinant human proteoglycan-4 to have an effect in a toll-like receptor 4 agonist inflammation model. Patient sepsis samples had higher plasma levels of native proteoglycan-4 than controls. Recombinant human proteoglycan-4 is a potential adjunct therapy for sepsis patients and warrants future in vivo model studies.

Macrophage migration inhibitory factor modulates proliferation, cell cycle, and apoptotic activity in head and neck cancer cell lines

Background/purposeMacrophage migration inhibitory factor (MIF) is a multifunctional cytokine that contributes to the progression of several cancers. MIF overexpression has been reported in head and neck squamous cell carcinoma (HNSCC) patients. However, the exact role of MIF in HNSCC is not fully understood. Our aim was to evaluate the amount of secreted MIF and the role of MIF in the proliferation, cell cycle, and apoptosis in HNSCC cell lines.Materials and methodsGenetically matched HNSCC cell lines derived from primary (HN18 and HN30) and metastatic sites (HN17 and HN31) from the same patient were used in this study. The MIF levels in conditioned media from the HNSCC cell lines were evaluated using ELISA. The HNSCC cell lines were treated with recombinant MIF at concentrations 25, 50 and 100 ng/ml, and cell proliferation was evaluated by MTT assay. A proliferative dose of MIF was used to treat the cells then, cell cycle, and apoptotic status were determined by flow cytometry.ResultsThe HNSCC-secreted MIF concentration ranged from 49.33 to 973 pg/ml. Exogenous MIF (25 ng/ml) significantly increased HN18, HN30, and HN31 cell proliferation. Moreover, MIF induced cell cycle progression and inhibited apoptosis in these cells. However, MIF did not affect growth or apoptosis in HN17 cell.ConclusionMIF secreted from the HNSCC cell lines were evaluated. Exogenous MIF promotes various effects on proliferation, cell cycle, and apoptosis in HNSCC cells.

Linc-OIP5 in the breast cancer cells regulates angiogenesis of human umbilical vein endothelial cells through YAP1/Notch/NRP1 signaling circuit at a tumor microenvironment

BackgroundLincRNAs have been revealed to be tightly associated with various tumorigeneses and cancer development, but the roles of specific lincRNA on tumor-related angiogenesis was hardly studied. Here, we aimed to investigate whether linc-OIP5 in breast cancer cells affects the angiogenesis of HUVECs and whether the linc-OIP5 regulations are involved in angiogenesis-related Notch and Hippo signaling pathways.MethodsA trans-well system co-cultured HUVECs with linc-OIP5 knockdown breast cancer cell MDA-MB-231 was utilized to study the proliferation, migration and tube formation abilities of HUVECs and alterations of related signaling indicators in breast cancer cells and their conditioned medium through a series of cell and molecular experiments.ResultsOverexpressed linc-OIP5, YAP1, and JAG1 were found in breast cancer cell lines MCF7 and MDA-MB-231 and the expression levels of YAP1 and JAG1 were proportional to the breast cancer tissue grades. MDA-MB-231 cells with linc-OIP5 knockdown led to weakened proliferation, migration, and tube formation capacity of co-cultured HUVECs. Besides, linc-OIP5 knockdown in co-cultured MDA-MB-231 cells showed downregulated YAP1 and JAG1 expression, combined with a reduced JAG1 level in conditioned medium. Furthermore, a disrupted DLL4/Notch/NRP1 signaling in co-cultured HUVECs were also discovered under this condition.ConclusionHence, linc-OIP5 in MDA-MB-231 breast cancer cells may act on the upstream of the YAP1/Notch/NRP1 signaling circuit to affect proliferation, migration, and tube formation of co-cultured HUVECs in a non-cellular direct contact way through JAG1 in conditioned medium. These findings at least partially provide a new angiogenic signaling circuit in breast cancers and suggest linc-OIP5 could be considered as a therapeutic target in angiogenesis of breast cancers.

A Curcumin Analog Reduces Levels of the Alzheimer's Disease-Associated Amyloid-β Protein by Modulating AβPP Processing and Autophagy.

Alzheimer's disease (AD) is a devastating neurodegenerative disease with no cure currently available. A pathological hallmark of AD is accumulation and deposition of amyloid-β protein (Aβ), a ∼4 kDa peptide generated through serial cleavage of the amyloid-β protein precursor (AβPP) by β- and γ-secretases. Curcumin is a natural compound primarily found in the widely used culinary spice, turmeric, which displays therapeutic potential for AD. Recently, we reported the development of curcumin analogs and identified a lead compound, curcumin-like compound-R17 (CLC-R17), that significantly attenuates Aβ deposition in an AD transgenic mouse model. Here, we elucidated the mechanisms of this analog on Aβ levels and AβPP processing using cell models of AD. Using biochemical methods and our recently developed nanoplasmonic fiber tip probe technology, we showed that the lead compound potently lowers Aβ levels in conditioned media and reduces oligomeric amyloid levels in the cells. Furthermore, like curcumin, the lead compound attenuates the maturation of AβPP in the secretory pathway. Interestingly, it upregulated α-secretase processing of AβPP and inhibited β-secretase processing of AβPP by decreasing BACE1 protein levels. Collectively, our data reveal mechanisms of a promising curcumin analog in reducing Aβ levels, which strongly support its development as a potential therapeutic for AD.

Extracellular transglutaminase 2 induces myotube hypertrophy through G protein-coupled receptor 56

Skeletal muscle secretes biologically active proteins that contribute to muscle hypertrophy in response to either exercise or dietary intake. The identification of skeletal muscle-secreted proteins that induces hypertrophy can provide critical information regarding skeletal muscle health. Dietary provitamin A, β-carotene, induces hypertrophy of the soleus muscle in mice. Here, we hypothesized that skeletal muscle produces hypertrophy-inducible secretory proteins via dietary β-carotene. Knockdown of retinoic acid receptor (RAR) γ inhibited the β-carotene-induced increase soleus muscle mass in mice. Using RNA sequencing, bioinformatic analyses, and literature searching, we predicted transglutaminase 2 (TG2) to be an all-trans retinoic acid (ATRA)-induced secretory protein in cultured C2C12 myotubes. Tg2 mRNA expression increased in ATRA- or β-carotene-stimulated myotubes and in the soleus muscle of β-carotene-treated mice. Knockdown of RARγ inhibited β-carotene-increased mRNA expression of Tg2 in the soleus muscle. ATRA increased endogenous TG2 levels in conditioned medium from myotubes. Extracellular TG2 promoted the phosphorylation of Akt, mechanistic target of rapamycin (mTOR), and ribosomal p70 S6 kinase (p70S6K), and inhibitors of mTOR, phosphatidylinositol 3-kinase, and Src (rapamycin, LY294002, and Src I1, respectively) inhibited TG2-increased phosphorylation of mTOR and p70S6K. Furthermore, extracellular TG2 promoted protein synthesis and hypertrophy in myotubes. TG2 mutant lacking transglutaminase activity exerted the same effects as wild-type TG2. Knockdown of G protein-coupled receptor 56 (GPR56) inhibited the effects of TG2 on mTOR signaling, protein synthesis, and hypertrophy. These results indicated that TG2 expression was upregulated through ATRA-mediated RARγ and that extracellular TG2 induced myotube hypertrophy by activating mTOR signaling-mediated protein synthesis through GPR56, independent of transglutaminase activity.

Abstract 142: The EGFR signaling modulates in mesenchymal stem cells the expression of microRNAs involved in the interaction with breast cancer cells

Abstract Introduction. We previously demonstrated that the activation of the epidermal growth factor receptor (EGFR) in mesenchymal stem cells (MSCs) modulates the expression of a number of genes coding for secreted proteins that promote breast cancer cell progression. As different microRNAs (miRNAs) have been shown to be involved in the cross-talk between MSCs and tumor cells, we hypothesized that the EGFR might regulate in MSCs the expression of miRNAs that might affect breast cancer progression through paracrine circuits. To this aim, we analysed the whole small RNA transcriptome of MSCs stimulated or not with transforming growth factor α (TGFα), one of the main ligands of the EGFR. Materials and methods. Small RNA sequencing was performed using the SOLiD 5500xl System. The DAVID (Database for Annotation, Visualization and Integrated Discovery) database was used for pathway analysis. Exosomes containing miRNAs were isolated from conditioned media with the ExoQuick reagent. MiRNA expression levels in conditioned medium were analyzed by Real Time PCR. Anchorage-dependent growth assays and Boyden-chamber based colorimetric migration assays were used to assess the proliferation and the migration of breast cancer cells, respectively. Results. Following small RNA sequencing, we identified 36 miRNAs differentially expressed between untreated and TGFα-treated MSCs with a fold change (FC) &amp;lt;0.56 or FC ≥1.90 (Confidence Interval, CI, 95%) and a threshold of sequence counts≥150 in at least one sample. In particular, 18 miRNAs resulted up-regulated with FC≥1.90 and 18 miRNAs down-regulated with FC&amp;lt;0.56. Pathways analysis of the target genes of the differentially expressed miRNAs revealed a significant enrichment in different KEGG pathways involved in cancer development and progression, including TGFβ signaling, focal adhesion, Rap1 signaling, Hippo signaling, mTOR and RAS signaling. To study miRNAs involved in the TGFα-mediated interaction between MSCs and breast cancer cells, we collected conditioned media from MSCs and analysed the expression levels of selected miRNAs. Real Time PCR analysis revealed the presence of several miRNAs in the conditioned medium from MSCs, including miR-23c, miR379-3p and miR-432-5p. The biological activity of the identified miRNAs was explored in a panel of breast cancer cell lines. MiR-23c was found to regulate the proliferation and migration of triple negative MDA-MB-468 and MDA-MB-231 breast cancer cells. Conclusion. Collectively, our data suggest that the EGFR signaling regulate in MSCs a wide number of miRNAs that might be involved in breast cancer progression, providing novel information on the mechanisms that regulate the MSC-tumor cross-talk. Citation Format: Marianna Gallo, Marianeve Carotenuto, Cristin Roma, Francesca Bergantino, Pasqualino de Antonellis, Nicola Normanno, Antonella De Luca. The EGFR signaling modulates in mesenchymal stem cells the expression of microRNAs involved in the interaction with breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 142.

Resveratrol, an activator of SIRT1, improves ER stress by increasing clusterin expression in HepG2 cells.

Endoplasmic reticulum stress (ER stress) is involved in lipid metabolism and lipotoxicity and can lead to apoptosis. Resveratrol, a sirtuin 1 (SIRT1) agonist, prevents ER stress and improves ER stress-induced hepatic steatosis and cell death. Clusterin is a secreted chaperone and has roles in various physiological processes. However, changes in the expression of clusterin upon ER stress and the connection between SIRT1 and clusterin in protection against ER stress are not well known. In cells treated with tunicamycin, resveratrol increased the expression of clusterin mRNA and protein and the secreted clusterin protein level in conditioned medium. Resveratrol decreased protein expression of the ER stress markers, p-PERK, p-IRE1α, and CHOP, and increased the expression of the ER-associated degradation (ERAD) factors, SEL1L and HRD1, in tunicamycin-treated cells. However, no changes in the expression of these genes were observed in clusterin siRNA-transfected cells. Moreover, increased LAMP2 and LC3 expression and decreased Rubicon expression were observed in cells treated with resveratrol or secreted clusterin. These data suggest that SIRT1 activation by resveratrol attenuates ER stress by promoting protective processes such as ERAD and autophagy pathways and that these protective effects are mediated by clusterin.

Influence of glucose and insulin in human adipogenic differentiation models with adipose-derived stem cells.

ABSTRACT Autologous fat grafting represents an attractive source for tissue engineering applications in the field of reconstructive medicine. However, in adipogenic differentiation protocols for human adipose-derived stem cells, the concentration of glucose and insulin varies considerably. With the intent to gain maximum tissue augmentation, we focused on the late phase of adipogenesis. In this study, we modified the differentiation protocol for adipose-derived stem cells by prolongation of the induction period and the application highly concentrated glucose and insulin. Human adipose-derived stem cells were isolated from subcutaneous depots and differentiated in a standard induction medium for the first two weeks, followed by two weeks with varying glucose and insulin concentrations. Morphological changes assessed using Oil-Red-O staining were examined for corresponding alterations in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL). Furthermore, glucose and lactate levels in conditioned media were monitored over the period of differentiation. We found high-glucose media increasing the level of lipid accumulation and the size of single droplets whereas insulin significantly showed a dose-dependent negative effect on fat storage. However, whereas high glucose stimulated PPARγ transcription, expression levels in insulin-treated cells remained constant. Results permit assumptions that a high-glucose medium intensifies the degree of differentiation in mature adipocytes providing conditions to promote graft volume while we have identified highly concentrated insulin treatment as an inhibitor of lipid storage in the late adipogenic differentiation.

S100B regulates inflammatory response during osteoarthritis via fibroblast growth factor receptor 1 signaling.

The present study aimed to investigate the role of S100B in the inflammation process during osteoarthritis (OA). OA cartilage samples were collected for S100B expression analysis. S100B expression levels were significantly increased in patients with OA compared with the Controls (1.28±0.66 vs. 0.42±0.31; P=0.01) and were determined to be correlated with TNF-α (r=0.42; P=0.04) and IL-1β (r=0.73; P=0.001) expression levels. Orthopedic casting tape was used to immobilize the right knee at 180° extension of adult female New Zealand white rabbits for 4 weeks to establish an OA model. Cartilage specimens from the medial femoral condyle of these rabbits were used for histological confirmation and immunohistochemical analyses, whereas synovial fluid was used in ELISA assays for tumor necrosis factor (TNF)-α and interleukin (IL)-1β expression levels. Human synovial fibroblasts from the knee synovial tissues of normal patients with traumatic injury were transfected with S100B overexpression and knockdown plasmids and subjected to lipopolysaccharide (LPS) stimulation; subsequently, TNF-α and IL-1β expression levels in conditioned medium were determined by ELISA; S100B overexpression and knockdown significantly increased and decreased the TNF-α and IL-1β expression levels, respectively. Increased TNF-α (573.3±15.4 vs. 102.6±8.7 pg) and IL-1β (378.6±7.2 vs. 170.1±5.8 pg) expression levels were detected in OA model rabbits compared with the Control rabbits. Additionally, S100B, fibroblast growth factor (FGF)-1 and FGF receptor (FGFR)-1 mRNA and protein expression levels were increased in OA model rabbits compared with the Control group. FGFR1 knockdown significantly decreased TNF-α and IL-1β expression levels in LPS-stimulated S100B-overexpressing human synovial fibroblasts. S100B is involved in FGFR1 signaling-mediated inflammatory response during OA, which may be considered as a potential therapeutic target.

PO-267 Notch-dependent tumourigenic mechanism associated with exosome signalling

IntroductionTumour microenvironment (TME) plays a crucial role in tumours initiation, progression and response to therapy. It is becoming increasingly clear that one of the most important promoters of tumour growth and progression is a subpopulation of fibroblasts – cancer-associated fibroblasts (CAFs). There is emerging evidence that tumor-derived (TD) exosomes could stimulate TME fibroblasts to acquire an ‘activated’ phenotype that is usually characterised by α-smooth-muscle actin (α-SMA) expression and secretion of growth factors (TGF-β, etc.). Notch signalling pathway is involved in a variety of developmental processes and its alterations are known to play an oncogenic role in tumour progression. Notch activation can also lead to CAFs-mediated tumour growth stimulation, invasion and premetastatic niche formation. This study aims to explore Notch-dependent mechanism responsible for primary tumour cells remote stimulation of CAF phenotype formation during tumour progression.Material and methodsWestern-blot analysis was used for exosome markers evaluation, Notch signalling components and α-SMA expression levels in human colorectal adenocarcinoma cell line (HCT116) and primary human dermal fibroblasts (HF). The α-SMA+ HF proportion was estimated using immunofluorescence microscopy. Female BALB/c nude mice were used for all experiments in vivo. TGF-β levels in conditioned media were estimated by ELISA.Results and discussionsFibroblasts with full-length Notch1 surface exposure (HF Notch1+) were able to stimulate HCT116 xenograft growth when injected in mixture in contrast with HF Notch1-. Moreover, if HCT116 and HF Notch1+ were injected in different mouse sides tumour growth was also enchanced. Luciferase reporter gene system revealed Notch1 signalling transactivation in HF Notch1+ during HF and HCT116 cocultivation in vitro as well as separate HF Notch1+ and HCT116 injection in vivo correlated with increased proportion of α-SMA+ HF. Considering that Notch signalling is mediated via a cell-cell receptor-ligand interactions we proposed a possible exosome-mediated distant Notch1 signalling. TD exosomes analysis revealed Notch ligand Jagged1 enrichment in TD exosomes that could stimulate HF Notch1+ distant activation with subsequent TGF-β-mediated stimulation of tumour growth.ConclusionWe have shown a positive feedback loop between tumour and stromal cells based on exosome-dependent Notch signalling molecular mechanism driving tumour progression. The study was supported by Russian Science Foundation (RSCF), grant No. 14-15-00467.

Substance P ameliorates tumor necrosis factor-alpha-induced endothelial cell dysfunction by regulating eNOS expression in vitro.

The aim of this study was to explore the beneficial effects of SP on NO production and inflammation-induced vascular endothelium cell death. To mimic the inflammatory environment, TNF-α was treated with HUVECs, and SP was added prior to TNF-α to determine its protective effect. WST-1 assay was performed to detect cell viability. NO level in conditioned medium was measured by Griess Reagent System. The protein level of cleaved caspase-3, eNOS, and phosphorylated Akt was detected by Western blot analysis. TNF-α declined endothelial cell viability by downregulating Akt and NO production. TNF-α-induced cell death was reliably restored by NO, confirming the requirement of NO for cell survival. By contrast, pretreatment of SP attenuated TNF-α-induced cellular apoptosis, accompanied by an increase in the phosphorylation of Akt, eNOS expression, and NO production. Blockage of NK-1R, phosphorylated Akt or eNOS by CP-96345, A6730, or L-NAME entirely eliminated the effect of SP. SP can protect the vascular endothelium against inflammation-induced damage through modulation of the Akt/eNOS/NO signaling pathway.