Normoxic Culture Conditions Research Articles

Anshen Shumai Decoction inhibits post-infarction inflammation and myocardial remodeling through suppression of the p38 MAPK/c-FOS/EGR1 pathway.

Anshen Shumai Decoction (ASSMD) is traditionally employed to manage coronary artery disease arrhythmias. Its protective efficacy against myocardial infarction remains to be elucidated. This investigation employed a rat model of myocardial infarction, achieved through the ligation of the left anterior descending (LAD) coronary artery, followed by a 28-day administration of ASSMD. The study observed the decoction's mitigative impact on myocardial injury, with gene regulation effects discerned through transcriptomic analysis. Furthermore, ASSMD's influence on cardiomyocyte apoptosis and fibrotic protein secretion was assessed using an embryonic rat cardiomyocyte cell line (H9c2) under hypoxic conditions and rat cardiac fibroblasts subjected to normoxic culture conditions with TGF-β. A functional rescue assay involving overexpression of FOS and Early Growth Response Factor 1 (EGR1), combined with inhibition of the p38 Mitogen-activated Protein Kinase (MAPK) pathway, was conducted. Results indicated that ASSMD significantly curtailed cardiomyocyte apoptosis and myocardial fibrosis in infarcted rats, primarily by downregulating FOS and EGR1 gene expression and inhibiting the upstream p38 MAPK pathway. These actions of ASSMD culminated in reduced expression of pro-apoptotic, collagen, and fibrosis-associated proteins, conferring myocardial protection and anti-fibrotic effects on cardiac fibroblasts.

Synovial Membrane Is a Major Producer of Extracellular Inorganic Pyrophosphate in Response to Hypoxia.

Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-β1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-β1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-β1 in normoxic or hypoxic (5% O2) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of Ank and Enpp1 in TGF-β1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an Ank promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-β1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-β1. Hypoxic conditions enhanced Ank promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of Enpp1 and Ank. These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.

Abstract 6005: Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions

Abstract Traditional cancer cell lines used as surrogate models in vitro and in vivo have made significant contributions to cancer research and drug discovery. While cell culture and animal studies are critical steps in determining the efficacy, pharmacodynamics, and mechanism of action of novel anti-cancer drugs, it is well-known that cells grown in 2D culture systems have modified growth characteristics and different responses to chemotherapeutic drugs compared to tumors in vivo as a result of differing structural and environmental conditions that fail to replicate in vivo tumor microenvironments. AVATAR bioreactors (Xcell Biosciences, San Francisco, CA) were invented to address these limitations by providing discrete control over oxygen and hyperbaric pressure levels to better mimic the in vivo environment and allow cultured cells to be grown in conditions which mimic the human body. To better serve preclinical research needs, we generated new tumor cell models by long-term acclimating (LTA) existing cancer cells in the AVATAR incubator system using 1% O2 and 2 PSI conditions similar to the conditions expected in the tumor microenvironment (TME). Six different cancer cell lines of diverse origins including pancreas (MIA PaCa-2), breast (BT-474), colon (HCT116), prostate (LNCaP, PC-3) and melanoma (A-375) were adapted for 2-3 months with phenotypic changes recorded. Key marker changes were commonly observed in LTA cells compared to parental lines. For example, in HCT116 colorectal cancer cells, TME culture conditions induced a higher expression level of E-cadherin in both protein and mRNA levels (2.2-fold change), indicating a mesenchymal-epithelial transition of the cell after adaptation. This coincides with the observation that LTA adapted HCT116 cell bodies had a more angular cell morphology as compared to parental cells grown under conventional normoxic culture conditions. In-depth gene-expression analysis using a Nanostring tumor signaling pathway panel identified several common and tissue-specific gene and pathway changes. These included VEGFA, EGF receptor signaling pathway (EGFR, E-Cadherin, GRB7, c-Fos), TGF pathway (IGFBP3, TGFB1, TGFB2, SMAD7), and extracellular matrix signaling (LAMB3, ITGA5, ITGB3, ITGB8, PDGFA, CCND2). Current studies are focusing on drug response testing in 2D and 3D cultures based on above-mentioned gene and signaling pathway changes. Cell proliferation and migration capacity of xenograft LTA cells will be evaluated for the purposes of creating novel murine models for drug screening applications. Citation Format: Yewei Xing, Abriel Czachorowski, James Lim, Scott Wise. Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6005.

Sarcoma protein kinase inhibition alleviates liver fibrosis by promoting hepatic stellate cells ferroptosis

Abstract Liver fibrosis is a type of chronic pathological liver damage involving liver tissue hypoxia and abnormal extracellular matrix deposits. Hepatic stellate cells (HSCs) activation is critical for liver fibrosis. Currently, inhibiting HSCs activation or inducing HSCs ferroptosis is considered an effective strategy for the treatment of liver fibrosis. Sarcoma protein kinase (Src) is an important member of the tyrosine protein kinase family. Hypoxia causes Src phosphorylation at tyrosine 416 (Tyr 416), and inhibiting Src activation can alleviate liver fibrosis. There is currently little research on the relationship between Src activation and ferroptosis in liver fibrosis. 1-(1,1-Dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP1) is an inhibitor of Src activation at Tyr 416. Therefore, in this study we treated HSC-T6 cells with PP1 under normoxic and hypoxic culture conditions; moreover, PP1 was also used to treat a carbon tetrachloride-induced mouse liver fibrosis model. We explored whether inhibiting Src activation could alleviate liver fibrosis by promoting HSCs ferroptosis in vitro and in vivo. In vitro experiments showed that inhibiting Src activation in HSC-T6 cells significantly reduced hypoxia-inducible factor-1α (HIF-1α) expression and HSC-T6 cells activation, and ferroptosis was significantly increased. In vivo experiments revealed that inhibiting Src activation in fibrotic livers reduced HIF-1α expression; meanwhile, ferroptosis was promoted, and liver fibrosis was alleviated. Therefore, inhibiting Src activation, which increases HSCs ferroptosis, can alleviate liver fibrosis.

Extracellular Vesicles Derived from Osteogenic-Differentiated Human Bone Marrow-Derived Mesenchymal Cells Rescue Osteogenic Ability of Bone Marrow-Derived Mesenchymal Cells Impaired by Hypoxia.

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O2) suppressed the osteogenic differentiation of human bone marrow-derived mesenchymal cells (hBMSC) in vitro and simultaneously increased reactive oxygen species (ROS) production. We assumed that cellular origin and cargo of extracellular vesicles (EVs) affect the osteogenic differentiation capacity of hBMSCs cultured under different oxygen pressures. Proteomic analysis revealed that EVs isolated from osteogenic differentiated hBMSC cultured under hypoxia (hypo-osteo EVs) or under normoxia (norm-osteo EVs) contained distinct protein profiles. Extracellular matrix (ECM) components, antioxidants and pro-osteogenic proteins were decreased in hypo-osteo EVs. The proteomic analysis in our previous study revealed that under normoxic culture conditions, pro-osteogenic proteins and ECM components have higher concentrations in norm-osteo EVs than in EVs derived from naïve hBMSCs (norm-naïve EVs). When selected for further analysis, five anti-hypoxic proteins were significantly upregulated (response to hypoxia) in norm-osteo EVs. Three of them are characterized as antioxidant proteins. We performed qRT-PCR to verify the corresponding gene expression levels in the norm-osteo EVs' and norm-naïve EVs' parent cells cultured under normoxia. Moreover, we observed that norm-osteo EVs rescued the osteogenic ability of naïve hBMSCs cultured under hypoxia and reduced hypoxia-induced elevation of ROS production in osteogenic differentiated hBMSCs, presumably by inducing expression of anti-hypoxic/ antioxidant and pro-osteogenic genes.

EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

BackgroundTriple-negative breast cancer (TNBC) cells’ secretome can induce a pro-inflammatory phenotype in human adipose-derived mesenchymal stem cells (hADMSC). This can be prevented by the green tea polyphenol epigallocatechin-3-gallate (EGCG). The impact of EGCG on the paracrine regulation that the extracellular vesicles (EVs) specifically exert within the TNBC secretome remains unknown.MethodsEVs were obtained from a TNBC-derived serum-starved MDA-MB-231 cell model treated or not with EGCG under normoxic or hypoxic (< 1% O2) culture conditions. RNA-Seq analysis was used to assess the EVs’ genetic content. The modulation of inflammatory and senescence markers in hADMSC was evaluated by RT-qPCR using cDNA arrays and validated by immunoblotting. A protein profiler phospho-kinase array was used to explore signaling pathways.ResultsWhile hypoxic culture conditions did not significantly alter the genetic content of MDA-MB-231-secreted EVs, the addition of EGCG significantly modified EVs genetic material at low oxygen tension. Gene expression of cancer-associated adipocyte pro-inflammatory markers CXCL8, CCL2 and IL-1β was increased in hADMSC treated with EVs. Concomitantly, EVs isolated from MDA-MB-231 treated with EGCG (EGCG-EVs) downregulated CCL2 and IL-1β, while inducing higher expression of CXCL8 and IL-6 levels. EVs activated CHK-2, c-Jun, AKT and GSK-3β signaling pathways in hADMSC, whereas EGCG-EVs specifically reduced the latter two as well as the serum starvation-induced senescence markers p21 and β-galactosidase. Finally, the mitochondrial content within the TNBC cells-derived EVs was found reduced upon EGCG treatment.ConclusionThis proof of concept study demonstrates that the chemopreventive properties of diet-derived polyphenols may efficiently target the paracrine regulation that TNBC cells could exert upon their surrounding adipose tissue microenvironment.

Engineered bone marrow as a clinically relevant ex vivo model for primary bone cancer research and drug screening

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-y survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 wk for study. We interrogate the influence of the anatomical implantation site on eBM tissue quality, test ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, culture OS cells within these constructs, and compare them to human OS samples. We show that eBM stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.

M6A RNA MODIfiCATION AND A DRIVER OF CELLULAR PLASTICITY AND INTRA-TUMOURAL HETEROGENEITY IN GLIOBLASTOMA

Abstract AIMS The immense cellular plasticity of glioblastoma is a barrier to successful therapy. N6-methyladenosine (m6A) methylation of RNA is increasingly recognised as a key driver of cellular differentiation. Crucially, it is a reversible and dynamic modification that alters cellular phenotype via the regulation of mRNA metabolism. We hypothesise that m6A methylation mediates glioblastoma plasticity, regulating transcriptional networks and thus influencing cellular adaptation under different microenvironmental conditions. Our aims are to assess whether total m6A levels and m6A-regulatory gene expression are responsive to change in oxygenation levels and cellular confluency. Furthermore, we aim to characterise the spatial distribution of m6A in 3D cell culture models. METHOD We have investigated m6A levels in patient-derived cell lines from the invasive region of adult glioblastoma. Mass spectrometry and anti-m6A immunofluorescence confocal microscopy was used to quantify total m6A levels. We have compared total m6A levels in normoxic versus hypoxic culture conditions and in low versus high confluency 2D cell culture, including cell migration during a scratch assay. We have measured expression levels of key m6A-regulatory genes using rtPCR. RESULTS Total m6A levels and regulatory gene expression were not significantly different in normoxic versus hypoxic conditions. However, m6A levels were decreased in high confluency versus low confluency conditions. We will further characterise the spatial distribution of m6A in 3D cell culture models using immunofluorescence confocal microscopy. CONCLUSIONS We demonstrate a degree of variability in m6A levels in response to micro-environmental conditions, such as cellular confluency. These findings indicate a role for m6A in tumoral heterogeneity and cellular adaptation.

Abstract 589: Long-term acclimation of target cell lines to tumor microenvironment culture condition provides mechanistic insights into cell therapy effectiveness

Abstract The tumor microenvironment (TME) has several characteristics that distinguish it from normal tissue, including elevated interstitial fluid pressures and hypoxia. To study the effects of TME culture conditions, commonly used target cell lines were serially passaged under hyperbaric and hypoxic conditions for a period of eight weeks. Conventionally expanded parental cell lines (normoxia) were compared against TME-acclimated tumor cell lines (TACTLs; hyperbaric and hypoxic), using RNAseq, ATACseq, and flow cytometry analysis. Commonly used target cell lines for cell therapy development were selected for TME acclimation (SKOV3, A549, JEKO1, and NALM6). TACTLs were generated using an AVATAR system, with culture conditions set at 1% O2 and 2.0 PSI, and serially passaged twice a week for a minimum of 8 weeks. Cell doubling times were measured weekly and compared against parental lines maintained under normoxic culture conditions in a conventional CO2 incubator. In-depth characterization was performed on TME-Acclimated Tumor Cell Lines (TACTLs), utilizing a multi-omic approach. Differential gene expression analysis was performed using RNAseq datasets, and surface biomarkers/targets used for cell therapy development were assessed via nanostring and flow cytometry. Growth kinetics and cell doubling times of TACTLs were initially inhibited during the first two weeks of culture under TME conditions, but eventually reached parity with their normoxia maintained parental cell lines at 6 to 8 weeks, signaling a successful adaptation of the tumor lines to low oxygen and hyperbaric conditions. RNAseq analysis revealed upregulation in glycolytic pathways and epithelial-to-mesenchymal signaling, accompanied by altered metabolic profiles. Surface target expression showed increased expression of checkpoint ligands, such as PD-L1. Cell therapy targets, such as ROR1 were also upregulated in A549 and JEKO1 cell lines passaged under TME. Preliminary drug screening experiments were conducted using checkpoint inhibitors and CAR-T candidates on TACTLs, revealing significant changes in half maximal inhibitory concentration (IC50) when compared to parental tumor lines maintained under normoxia. In summary, acclimation of tumor cell lines to TME culture conditions can provide unique mechanistic insights that can facilitate drug development efforts. Future studies will incorporate TACTLs for CDX tumor models with the goal of identifying cell therapies that work effectively in the tumor microenvironment. Citation Format: James Lim, Albert Wong, Ann Lu, Candy Garcia, Evan Massi, Yewei Xing, Ningchun Liu, Scott Wise. Long-term acclimation of target cell lines to tumor microenvironment culture condition provides mechanistic insights into cell therapy effectiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 589.

H2O2 transit through the mitochondrial intermembrane space promotes tumor cell growth in vitro and in vivo

Cancer cells experience increased levels of oxidant stress as a consequence of oncogene activation, nucleotide biosynthesis, and growth factor receptor signaling. Mitochondria contribute to this redox stress by generating reactive oxygen species (ROS) along the electron transport chain, which are released to the matrix and the intermembrane space (IMS). Assessing the contribution of mitochondrial ROS in cancer cells is technically difficult, as electron transport chain inhibitors can increase or decrease ROS generation, while they also block oxidative phosphorylation and ATP synthesis. Mitochondria-targeted antioxidant compounds can scavenge ROS in the matrix compartment but do not act on ROS released to the IMS. We assessed the importance of mitochondrial ROS for tumor cell proliferation, survival, and for tumor xenograft growth by stably expressing a hydrogen peroxide (H2O2) scavenger, peroxiredoxin-5, in the mitochondrial IMS (IMS-Prdx5) in 143B osteosarcoma and HCT116 colorectal cancer cell lines. IMS-Prdx5 attenuates hypoxia-induced ROS signaling as assessed independently in cytosol and IMS, HIF-1α stabilization and activity, and cellular proliferation under normoxic and hypoxic culture conditions. It also suppressed tumor growth invivo. Stable expression of nondegradable HIF-1α only partially rescued proliferation in IMS-Prdx5-expressing cells, indicating that mitochondrial H2O2 signaling contributes to tumor cell proliferation and survival through HIF-dependent and HIF-independent mechanisms.

Cationic amino acid transporter-1 (CAT-1) promotes fibroblast-like synoviocyte proliferation and cytokine secretion by taking up L-arginine in rheumatoid arthritis

BackgroundAbnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA.MethodsThe concentrations of amino acids and cytokines in the synovial fluid of RA (n = 9) and osteoarthritis (OA, n = 9) were detected by LC–MS/MS and CBA assay, respectively. The mRNA and protein expression of cationic amino acid transporter-1 (CAT-1) were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion, and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo.ResultsL-rginine was upregulated in the synovial fluid of RA patients and was positively correlated with the elevation of the cytokines IL-1β, IL-6, and IL-8. Further examination demonstrated that CAT-1 was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration, and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice.ConclusionCAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.

Migratory chondroprogenitors retain superior intrinsic chondrogenic potential for regenerative cartilage repair as compared to human fibronectin derived chondroprogenitors

Cell-based therapy for articular hyaline cartilage regeneration predominantly involves the use of mesenchymal stem cells and chondrocytes. However, the regenerated repair tissue is suboptimal due to the formation of mixed hyaline and fibrocartilage, resulting in inferior long-term functional outcomes. Current preclinical research points towards the potential use of cartilage-derived chondroprogenitors as a viable option for cartilage healing. Fibronectin adhesion assay-derived chondroprogenitors (FAA-CP) and migratory chondroprogenitors (MCP) exhibit features suitable for neocartilage formation but are isolated using distinct protocols. In order to assess superiority between the two cell groups, this study was the first attempt to compare human FAA-CPs with MCPs in normoxic and hypoxic culture conditions, investigating their growth characteristics, surface marker profile and trilineage potency. Their chondrogenic potential was assessed using mRNA expression for markers of chondrogenesis and hypertrophy, glycosaminoglycan content (GAG), and histological staining. MCPs displayed lower levels of hypertrophy markers (RUNX2 and COL1A1), with normoxia-MCP exhibiting significantly higher levels of chondrogenic markers (Aggrecan and COL2A1/COL1A1 ratio), thus showing superior potential towards cartilage repair. Upon chondrogenic induction, normoxia-MCPs also showed significantly higher levels of GAG/DNA with stronger staining. Focused research using MCPs is required as they can be suitable contenders for the generation of hyaline-like repair tissue.

Hypoxia Promotes Adipose-Derived Stem Cells to Protect Human Dermal Microvascular Endothelial Cells Against Hypoxia/Reoxygenation Injury

Microcirculation is important for regulating ischemia-reperfusion (I/R) injury associated with skin flap transplantation surgery. We investigated whether co-culture with adipose-derived stem cells (ADSCs) could protect human dermal microvascular endothelial cells (HDMECs) from I/R injury by inhibiting cell apoptosis and enhancing cell proliferation. We also investigated the effects of hypoxic preconditioning on ADSCs. HDMECs were divided into four groups, control, HDMECs in normoxic culture conditions; hypoxia/reoxygenation (H/R), HDMECs in a hypoxic incubator for 8 h then in saturated aerobic culture medium for 24 h; H/R+ADSC(N), HDMECs treated similar to the H/R group then co-cultured with normoxic ADSCs; and H/R+ADSC(H), HDMECs treated similar to the H/R group then co-cultured with hypoxia preconditioned ADSCs. The rate of HDMECs apoptosis significantly increased in the H/R group, but decreased upon co-culture with ADSCs for 24 h, especially in the H/R + ADSC(H) group. Co-culture with ADSCs, especially hypoxia preconditioned ADSCs, significantly enhanced cell proliferation ability compared with that of the H/R group after 48 h and 72 h, but not after 24 h. Vascular endothelial growth factor levels were significantly higher in the H/R+ADSC(N) and H/R+ADSC(H) groups than in the H/R group. ADSCs attenuated H/R injury in endothelial cells by promoting proliferation ability and reducing apoptosis, with an increase in Vascular endothelial growth factor level, especially in the context of hypoxic preconditioning. This approach suggests the potential for an easy and safe method to reduce I/R injury associated with skin flap transplantation surgery.

Modulating electrospun polycaprolactone scaffold morphology and composition to alter endothelial cell proliferation and angiogenic gene response.

The aim of this study was to look at how the composition and morphology of polymer scaffolds could be altered to create an optimized environment for endothelial cells. Four polycaprolactone (PCL) scaffolds were electrospun with increasing fibre diameters ranging from 1.64 μm to 4.83 μm. The scaffolds were seeded with human umbilical vein endothelial cells (HUVEC) and cultured for 12 days. PCL scaffolds were then electrospun incorporating decellularized bovine aorta ECM and cultured in a hypoxic environment. We noted deeper cell infiltration on the largest fibre diameter compared to the other three scaffolds which resulted in an increase in the gene expression of CD31; a key angiogenic marker. Increased cell viability and cell proliferation were also noted on the largest fibre. Furthermore, we noted that the incorporation of extracellular matrix (ECM) had minimal effect on cell viability, both in normoxic and hypoxic culture conditions. Our results showed that these environments had limited influences on hypoxic gene expression. Interestingly, the major findings from this study was the production of excretory ECM components as shown in the scanning electron microscopy (SEM) images. The results from this study suggest that fibre diameter had a bigger impact on the seeded HUVECs than the incorporation of ECM or the culture conditions. The largest fibre dimeter (4.83 μm) is more suitable for seeding of HUVECs.

Transient and Stable Overexpression of Extracellular Superoxide Dismutase Is Positively Associated with the Myogenic Function of Human Skeletal Muscle-Derived Stem/Progenitor Cells.

In the present study, the genetic modification of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) was investigated to identify the optimal protocol for myogenic cell preparation for use in post-infarction heart therapy. We used two types of modifications: GFP-transfection (using electroporation) and SOD3 transduction (using a lentiviral vector). SkMDS/PCs were cultured under different in vitro conditions, including standard (21% oxygen) and hypoxic (3% oxygen), the latter of which corresponded to the prevailing conditions in the post-infarction heart. Transfection/transduction efficacy, skeletal myogenic cell marker expression (CD56), cellular senescence, and apoptosis, as well as the expression of antioxidant (SOD1, SOD2, and SOD3), anti-aging (SIRT1 and FOXO), anti-apoptotic (BCL2), and myogenic (MyoD and MyoG) genes, were evaluated. The percentage of GFP-positive SkMDS/PCs was determined as an indicator of the efficacy of transfection, which reached 55%, while transduction showed better efficiency, reaching approximately 85% as estimated by fluorescence microscopy. The CD56-positive SkMDS/PCs were present in approximately 77% of the tested cells after transient transfection and approximately 96% after transduction. Under standard in vitro culture conditions, the ability of the differentiated, transfected SkMDS/PCs to form myotubes was greater than that of the wild type (WT) cell population (p < 0.001), while the cells transduced with the SOD3 gene exhibited an increase in cell fusion under both standard (p < 0.05) and hypoxic conditions (p < 0.001). In transduced SkMDS/PCs, we observed a positive influence of SOD3 overexpression on cell ageing and apoptosis. We observed an increase in the percentage of young cells under standard (p < 0.05) and hypoxic (p < 0.001) in vitro culture conditions, with a notable decrease in the percentage of senescent and advanced senescent cells in the SOD3-overexpressing cell population detected compared to that observed for the untransduced muscle-derived cells. A lower percentage of apoptotic cells was observed for transduced SkMDS/PCs than that for WT cells under hypoxic in vitro culture conditions. In transiently transfected SkMDS/PCs, we observed significantly higher gene expression levels of SOD2 (almost 40-fold) (p < 0.001) and FOXO (p < 0.05) (approximately 3-fold) under both normoxic and hypoxic culture conditions and of BCL2 under hypoxia compared to those observed in untreated cells (WT). In addition, myogenic genes showed a significant increase in MyoD (almost 18-fold) expression under standard culture conditions (p < 0.0001) and decreased MyoG expression (approximately 2-fold) after transfection (p < 0.05) compared with that detected in the WT skeletal muscle-derived cell control. Taken together, these results demonstrate that SOD3-tranduced skeletal muscle-derived cells may have potential for use in the regenerative treatment of the post-infarction heart.

Role of Oxygen Supply in Macrophages in a Model of Simulated Orthodontic Tooth Movement.

Apart from periodontal ligament fibroblasts, immune cells like macrophages also play an important mediating role in orthodontic tooth movement (OTM). Upon orthodontic force application to malpositioned teeth, macrophages in the periodontal ligament get exposed to both mechanical strain and hypoxic conditions (via a compression of blood vessels). In this study, we assessed the relative impact of orthodontically induced mechanical strain and hypoxic conditions on macrophages for the mediation and regulation of OTM. Macrophages were stimulated with physiological orthodontic compressive forces of 2 g/cm2 for 4 h and 24 h on gas-impermeable or gas-permeable cell culture plates under normoxic or hypoxic cell culture conditions. We quantified expression of genes involved in inflammation (Tnf, Il-6, and Cox-2), extracellular remodelling (Mmp-9), and angiogenesis (Vegf) by RT-qPCR. Furthermore, we analysed HIF-1α, prostaglandin-E2, and VEGF protein expression via immunoblotting or ELISA. Mechanical strain and oxygen supply both differentially affected expression of genes and proteins involved in inflammation and angiogenesis. In this context, we found that HIF-1α protein levels were elevated by combined mechanical strain and hypoxic conditions, whereas gas-permeable plates providing sufficient oxygen supply prevented HIF-1α stabilization at the protein level after pressure application on macrophages. Our results thus indicate that macrophages involved in the mediation of OTM are affected by and respond differently to hypoxic conditions and mechanical compressive strain, which occur concomitantly during OTM, than periodontal ligament fibroblasts (PDLF), thus indicating different roles of these cells in the regulation of OTM at the cellular-molecular level. We further observed that contrary to PDLF HIF-1α stabilization in macrophages is rather induced via the decreased oxygen supply associated with OTM than via mechanotransduction by mechanical strain.

AB0153 ADIPOSE-DERIVED STROMAL/STEM CELLS FROM SYSTEMIC SCLEROSIS PATIENTS SUCCESSFULLY EXERT A PARACRINE ANTI-FIBROTIC ACTIVITY AND INDUCE A PRO-ANGIOGENIC PHENOTYPE OF SCLERODERMA FIBROBLASTS IN VITRO

Background:Adipose-derived stromal/stem cells (ADSCs) are multipotential non-hematopoietic progenitor cells with anti-inflammatory, immunomodulatory and regenerative effects. They have the advantage of accessibility and potent pro-angiogenic effects when compared with other stem cells, such as bone-marrow derived stem cells. Recent studies have shown that autologous fat grafting may be effective in the treatment of fibrotic and vascular complications in systemic sclerosis (SSc), despite a pro-fibrotic signature.Objectives:Aim of the study was to better characterize the proliferative and secretive profile of ADSCs in normoxic and hypoxic conditions, and to evaluate the mechanisms by which ADSCs exert these observed clinical effects.Methods:Adipose tissue samples were obtained by liposuction from 12 SSc patients and 10 healthy donors (HD). ADSCs were purified according to their adherence to the plastic and characterized to express specific MSC surface antigens by flow cytometry analysis. Proliferation of ADSCs from SSc patients and normal controls was evaluated in normoxic and hypoxic conditions. Fibroblasts and ADSCs derived from SSc patients were co-cultured in direct and indirect culture systems and compared to HD. Fibroblasts proliferation, mRNA expression and protein secretion of VEGF and known fibrotic mediators including TGF β-1, TGFR, CTGF, Collagen type I (Col I) were analyzed in the same conditions.Results:Normoxic and hypoxic culture conditions did not modify the proliferation rate of both normal and SSc ADSCs. Hypoxia significantly increased mRNA expression of VEGF by HD and SSc ADSCs but had no effect on the mRNA expression of pro-fibrotic mediators, ie TGFβ and TGFR. Normal and SSc fibroblast proliferation was significantly reduced in both co-culture systems (p &lt; 0.001) and by treatment with normoxic and hypoxic conditioned medium (CM) (p=0.001 and p=0.002). In the same conditions, mRNA expression and protein secretion of TGF-β1, CTGF and Col I were significantly reduced (p = 0.003, p =0.02, p=0.04). These results were confirmed when normal and SSc fibroblasts were cultured in the presence of ADSCs normoxic and hypoxic CM (p=0.02 and p=0.01). Furthermore, a significant increase in mRNA expression and production of VEGF was observed in SSc fibroblasts cultured in the presence of normoxic and hypoxic CM (p = 0.002 and p&lt; 0.0001, respectively).Conclusion:We found that treatment with the medium from normoxic and hypoxic preconditioned ADSCs has an anti-fibrotic effect through both the inhibition of fibroblast proliferation and key mediators of fibrosis. The increased expression of VEGF by SSc fibroblasts in the presence of normoxic and, even more, hypoxic ADSCs CM, suggests that ADSCs can induce a paracrine pro-angiogenic phenotype, even in fibroblasts with a pro-fibrotic signature. Altogether these data show that ADSCs may exert their anti-fibrotic and pro-angiogenetic effects on SSc fibroblasts by the secretion of paracrine factors, partly explaining the mechanisms underlining beneficial clinical results of fat graft in SSc patients.Disclosure of Interests:Nicoletta Del Papa: None declared, Maurizio Lorini: None declared, Vincenzo Carbonelli: None declared, Antonina Minniti: None declared, Francesca Pignataro: None declared, Wanda Maglione: None declared, Gabriele Di Luca: None declared, Nicola Montano: None declared, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp &amp; Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UCB

Simulated Stroke in a Mouse Neuronal Cell Culture Model

INTRODUCTIONDespite many efforts, stroke remains a cause of debilitating illness and death worldwide. The fact that not only ischemia itself, but also the return of blood flow aggravates cellular dysfunction, illustrates a clinical barrier. Hence, catheter‐based interventions, the therapy of ischemic strokes, can add detrimental tissue damage, described as ischemia/reperfusion (I/R) injury. It is therefore crucial to develop potential pharmacological strategies that attenuate reperfusion injury and improve neurological function. Animal in‐vivo and in‐vitro models are vital to determine potential targets for pharmacological therapies. While in‐vivo models better mimic the complexity of the clinical context, in‐vitro studies allow to focus on the pathophysiology and can be performed on individual cell types. This study aimed to establish an in‐vitro stroke model by investigating the effect of simulated I/R injury on mouse brain isolated neurons.METHODSNeurons were plated and grown to confluency. To simulate stroke, cells were then randomized to either control/normoxic (21% O2, 5% CO2, 74% N2) or hypoxic conditions in a hypoxic chamber (0.01% O2, 5% CO2, 94.99% N2) in serum‐ and glucose‐free media. Durations of hypoxia for 2.5, 5 and 10h were compared. After 2h of reoxygenation in regular media and normoxic culture conditions following hypoxia/normoxia, samples were assayed for cell number/viability, cytotoxicity by lactate dehydrogenase (LDH) release and mitochondrial viability. Statistics: Mann‐Whitney Rank Sum Test and regression analysis; alpha=0.05.RESULTSHypoxia significantly impaired cell number/viability after 5 and 10h, whereas 2.5h of hypoxia did not. A large decrease of cell number/viability was observed after 10h of hypoxia compared to 5h and 2.5h. The same observations were made for mitochondrial viability, apart from a slightly lower decrease after 5h hypoxia compared to the cell number/viability. Inverse relations were found for LDH release, except for an already significant increase in cytotoxicity after 2.5h of hypoxia.CONCLUSIONSOur data demonstrate that hypoxic injury in this model is time‐dependent. While 2.5h did not result in sufficient damage and 10h injured neurons too excessively, 5h hypoxia led to a moderate enough damage, still allowing for potential attenuation through protective strategies. Therefore, 5h hypoxia, followed by 2h reperfusion, appears to be a suitable duration for an in‐vitro mouse neuron stroke model to test new treatments.Support or Funding InformationThis work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&amp;D Service.

Scaffold- and serum-free hypertrophic cartilage tissue engineering as an alternative approach for bone repair.

Human adipose stem/stromal cell (ASC) spheroids were used as a serum-free in vitro model to recapitulate the molecular events and extracellular matrix organization that orchestrate a hypertrophic cartilage phenotype. Induced-ASC spheroids (ø=450µm) showed high cell viability throughout the period of culture. The expression of collagen type X alpha 1 chain (COLXA1) and matrix metallopeptidase 13 (MMP-13) was upregulated at week 2 in induced-ASC spheroids compared with week 5 (P<.001) evaluated by quantitative real-time PCR. In accordance, secreted levels of IL-6 (P<.0001), IL-8 (P<.0001), IL-10 (P<.0001), bFGF (P<.001), VEGF (P<.0001), and RANTES (P<.0001) were the highest at week 2. Strong in situ staining for collagen type X and low staining for TSP-1 was associated with the increase of hypertrophic genes expression at week 2 in induced-ASC spheroids. Collagen type I, osteocalcin, biglycan, and tenascin C were detected at week 5 by in situ staining, in accordance with the highest expression of alkaline phosphatase (ALPL) gene and the presence of calcium deposits as evaluated by Alizarin Red O staining. Induced-ASC spheroids showed a higher force required to compression at week 2 (P<.0001). The human ASC spheroids under serum-free inducer medium and normoxic culture conditions were induced to a hypertrophic cartilage phenotype, opening a new perspective to recapitulate endochondral ossification in vivo.

Imatinib inhibits the regrowth of human colon cancer cells after treatment with 5-FU and cooperates with vitamin D analogue PRI-2191 in the downregulation of expression of stemness-related genes in 5-FU refractory cells

Conventional cytotoxic drugs preferentially eliminate differentiated cancer cells but spare relatively more resistant stem-like cancer cells capable to initiate recurrence. Due to cancer cell plasticity, the stem-like phenotype can be also acquired by cancer cells refractory to treatment with cytotoxic drugs. We investigated whether drugs inhibiting receptor tyrosine kinases could be used to target human colon cancer cells initiating cancer regrowth following conventional cytotoxic treatment. The moderately differentiated cell line HT-29 and poorly differentiated cell line HCT-116 were exposed to 5-fluorouracil (5-FU). Cells that resisted the exposure to 5-FU were subsequently treated with imatinib or sunitinib. Both drugs reduced clonogenicity of 5-FU-refractory cells under normoxic and hypoxic culture conditions. The expression of numerous stemness-related genes was upregulated in cancer cells following the exposure to 5-FU, and remained at a high level in 5-FU-refractory cells undergoing renewal under normoxia, but decreased spontaneously under hypoxia. Imatinib downregulated the expression of stemness-related genes in cells undergoing renewal under normoxia. A combination of imatinib with PRI-2191, an analogue of 1,25-dihydroxyvitamin D3, downregulated stemness-related genes in HCT-116/5-FU cells more efficiently than imatinib alone. A synthetic analogue of 1,25-dihydroxyvitamin D2 (PRI-1906) abolished the effect of imatinib on gene expression in HCT-116/5-FU cells undergoing renewal under normoxia. Sunitinib promoted shift of phenotype of HT-29/5-FU cells undergoing renewal toward stem-like one. It suggests that the phenotype shift toward stemness induced by sequential sunitinib treatment following 5-FU treatment could increase a risk of cancer recurrence. In contrast to sunitinib, imatinib could be used both to interfere with cancer regrowth after conventional chemotherapy and to downregulate the expression of stemness-related genes in residual colon cancer cells capable to initiate cancer recurrence. The findings suggest that imatinib could also be combined with vitamin D analogue PRI-2191 to prevent recurrence more efficiently than imatinib alone and to compensate for vitamin D deficiency resulting from imatinib treatment.