Serum Starvation Research Articles

NFATc1 Regulation of Dexamethasone-Induced TGFB2 Expression Is Cell Cycle Dependent in Trabecular Meshwork Cells.

Although elevated TGFβ2 levels appear to be a causative factor in glaucoma pathogenesis, little is known about how TGFβ2 expression is regulated in the trabecular meshwork (TM). Here, we investigated if activation of the cytokine regulator NFATc1 controlled transcription of TGFβ2 in human TM cells by using dexamethasone (DEX) to induce NFATc1 activity. The study used both proliferating and cell cycle arrested quiescent cells. Cell cycle arrest was achieved by either cell-cell contact inhibition or serum starvation. β-catenin staining and p21 and Ki-67 nuclear labeling were used to verify the formation of cell-cell contacts and activity of the cell cycle. NFATc1 inhibitors cyclosporine A (CsA) or 11R-VIVIT were used to determine the role of NFATc1. mRNA levels were determined by RT-qPCR. DEX increased TGFβ2 mRNA expression by 3.5-fold in proliferating cells but not in quiescent cells or serum-starved cells, and both CsA and 11R-VIVIT inhibited this increase. In contrast, the expression of other DEX/NFATc1-induced mRNAs (myocilin and β3 integrin) occurred regardless of the proliferative state of the cells. These studies show that NAFTc1 regulates TGFβ2 transcription in TM cells and reveals a previously unknown connection between the TM cell cycle and modulation of gene expression by NFATc1 and/or DEX in TM cells.

Chemical Modifications and Design Influence the Potency of Huntingtin Anti-Gene Oligonucleotides.

Huntington's disease is a neurodegenerative, trinucleotide repeat (TNR) disorder affecting both males and females. It is caused by an abnormal increase in the length of CAG•CTG TNR in exon 1 of the Huntingtin gene (HTT). The resultant, mutant HTT mRNA and protein cause neuronal toxicity, suggesting that reduction of their levels would constitute a promising therapeutic approach. We previously reported a novel strategy in which chemically modified oligonucleotides (ONs) directly target chromosomal DNA. These anti-gene ONs were able to downregulate both HTT mRNA and protein. In this study, various locked nucleic acid (LNA)/DNA mixmer anti-gene ONs were tested to investigate the effects of varying ON length, LNA content, and fatty acid modification on HTT expression. Altering the length did not significantly influence the ON potency, while LNA content was critical for activity. Utilization of palmitoyl-modified LNA monomers enhanced the ON activity relatively to the corresponding nonmodified LNA under serum starvation conditions. Furthermore, the number of palmitoylated LNA monomers and their positioning greatly affected ON potency. In addition, we performed RNA sequencing analysis, which showed that the anti-gene ONs affect the "immune system process, mRNA processing, and neurogenesis." Furthermore, we observed that for repeat containing genes, there is a higher tendency for antisense off-targeting. Taken together, our findings provide an optimized design of anti-gene ONs that could potentially be developed as DNA-targeting therapeutics for this class of TNR-related diseases.

Measuring cell cycle changes in RBL-2H3 cells upon treatment with n-alcohols.

N-alcohols are model general anesthetics. Past work showed that these hydrophobic molecules partition into membranes and alter miscibility transition temperatures. Past work have also found that factors that impacted rat basophilic leukemia (RBL-2H3) cell growth, such as serum starvation or cell density, also impacted miscibility transition temperatures of vesicles isolated from cells. This poster presents early investigations into if and how n-alcohols modulate cell growth. Preliminary measurements found that cell counts of RBL-2H3 cells are lower after a 24-hour incubation with 1-Butanol and greater after a 24-hour incubation with 1-Hexadecanol. To explore further, we employed a flow cytometry assay of the cell cycle using an EdU-Click reagent that incorporates into S-phase cells and DAPI stain that labels all DNA, allowing for the differentiation of a population of cells into their cell cycle phase. Using this assay, we found a dose dependent increase in cells in G0/G1 phases of the cell cycle after treatment with n-Butanol, without impact on cell size. Ongoing work is exploring the impact of other n-alcohols and possible causes for slow-down of cells through the G1 checkpoint.

Serum starvation-based method of ovarian cancer cell dormancy induction and termination in vitro.

Awakening and growth reinitiation by dormant cells may contribute to epithelial ovarian cancer (EOC) relapse. The links between these phenomena are loose because of the limited stock of compelling models of EOC dormancy. Here, we show a simple and convenient dormancy research protocol based on serum starvation. This study was conducted on established EOC cell lines A2780, OVCAR-3, and SKOV-3, as well as on primary EOC cells. Cell growth arrest and proliferation were monitored by assessing the Ki67 antigen, PKH26 fluorescence, and cell cycle distribution. In addition, cells were tested for ERK1/2/p38 MAPK activity ratio, apoptosis, and senescence. The study showed that 72-h serum starvation induces G0/G1 growth arrest of a significant fraction of cells, accompanied by reduced Ki67 and ERK1/2/p38 MAPK activity ratio, without signs of apoptosis or cellular senescence. Moreover, providing cells with 72 h of a medium enriched in 5% serum allows the culture to regain its proliferative potential. At the same time, we attempted to induce and terminate dormancy with Mitomycin C addition and withdrawal, which were unsuccessful. In conclusion, serum starvation is a convenient way to reliably induce dormancy in EOC cells, allowing them to be efficiently awakened for further mechanistic research in vitro.

Vitronectin acts as a key regulator of adhesion and migration in human umbilical cord-derived MSCs under different stress conditions

To improve mesenchymal stem cell (MSC)-based therapy efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under microenvironmental stress conditions. We observed that human Wharton's jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-κβ was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, formed under serum starvation stress. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. VTN is established as a key player which possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.

Serum Starvation Accelerates Intracellular Metabolism in Endothelial Cells.

Periods of low energy supply are challenging conditions for organisms and cells during fasting or famine. Although changes in nutrient levels in the blood are first sensed by endothelial cells, studies on their metabolic adaptations to diminished energy supply are lacking. We analyzed the dynamic metabolic activity of human umbilical vein endothelial cells (HUVECs) in basal conditions and after serum starvation. Metabolites of glycolysis, the tricarboxylic acid (TCA) cycle, and the glycerol pathway showed lower levels after serum starvation, whereas amino acids had increased levels. A metabolic flux analysis with 13C-glucose or 13C-glutamine labeling for different time points reached a plateau phase of incorporation after 30 h for 13C-glucose and after 8 h for 13C-glutamine under both experimental conditions. Notably, we observed a faster label incorporation for both 13C-glucose and 13C-glutamine after serum starvation. In the linear range of label incorporation after 3 h, we found a significantly faster incorporation of central carbon metabolites after serum starvation compared to the basal state. These findings may indicate that endothelial cells develop increased metabolic activity to cope with energy deficiency. Physiologically, it can be a prerequisite for endothelial cells to form new blood vessels under unfavorable conditions during the process of angiogenesis in vivo.

Claspin is Required for Growth Recovery from Serum Starvation through Regulating the PI3K-PDK1-mTOR Pathway in Mammalian Cells.

Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation, which requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. In the absence of Claspin, cells do not proceed into S phase and eventually die partially in a ROS- and p53-dependent manner. Claspin directly interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4EBP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 physically interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. Thus, Claspin plays a novel role as a key regulator for nutrition-induced proliferation/survival signaling by activating the mTOR pathway. The results also suggest a possibility that Claspin may serve as a common mediator that receives signals from different PI3K-related kinases and transmit them to specific downstream kinases.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.

Glycerol monolaurate inhibits Francisella novicida growth and is produced intracellularly in an ISG15-dependent manner.

Glycerol Monolaurate (GML) is a naturally occurring fatty acid monoester with antimicrobial properties. Francisella tularensis is an agent of bioterrorism known for its unique lipopolysaccharide structure and low immunogenicity. Here we assessed whether exogenous GML would inhibit the growth of Francisella novicida . GML potently impeded Francisella growth and survival in vitro . To appraise the metabolic response to infection, we used GC-MS to survey the metabolome, and surprisingly, observed intracellular GML production following Francisella infection. Notably, the ubiquitin-like protein ISG15 was necessary for increased GML levels induced by bacterial infection, and enhanced ISG15 conjugation correlated with GML levels following serum starvation.

N-Functionalized fluorophores: detecting urinary albumin and imaging lipid droplets.

A series of novel N-sulfonyl pyridinium fluorophores were designed, synthesized, and explored in terms of their ability to bind with serum albumins. Upon binding the fluorophores with BSA, noticeable emission wavelength or intensity changes accompanied by color changes were observed. Competitive binding studies revealed that the fluorophore selectively binds to the warfarin site, but the binding affinity also depends on the nature of the scaffold. Additionally, the fluorophores were employed to detect spiked serum albumin in artificial urine. Cellular imaging experiments indicated that the fluorophores accumulate within lipid droplets (LDs), suggesting their potential as promising biomarkers for lipid droplets. Furthermore, the fluorescence intensity, number, and size of LDs increased upon serum starvation.

Continuous nutrient supply culture strategy controls multivesicular endosomes pathway and anti-photo-aging miRNA cargo loading of extracellular vesicles

Extracellular vesicle (EV) therapy recently had shown significant efficacy in various diseases. Serum starvation culture (SC) is one of the most widely used methods for collecting EVs. However, SC may cause inadvertent effects and eventually dampen the therapeutic potential of EVs. Therefore, we developed a novel method for EV collection, continuous nutrient supply culture (CC), which can provide an optimal condition for mesenchymal stem cells (MSCs) by continuously supplying essential nutrients to MSCs. By comparing with SC strategy, we revealed that CC could maintain CC-MSCs in a normal autophagy and apoptotic state, which reduced the shunting of EV precursors in cells and useless information material carried by EVs. In CC-MSCs, the expression levels of endosomal sorting complexes required for transport (ESCRT) and targeting GTPase27 (Rab27) were upregulated compared to those in SC-MSCs. Besides, we analyzed the membrane transport efficiency of EV formation, which demonstrated the CC strategy could promote the formation of EV precursors and the release of EVs. In addition, miRNA analysis revealed that CC-EVs were enriched with anti-chronic inflammatory factors, which could inhibit the nuclear factor kappa-B (NF-κB) pathway, mitigate chronic inflammation, and effectively repair skin photo-aging damage.

LINC01615 maintains cell survival in adaptation to nutrient starvation through the pentose phosphate pathway and modulates chemosensitivity in colorectal cancer

Numerous mechanisms involved in promoting cancer cell survival under nutrient starvation have been described. Long noncoding RNAs (lncRNAs) have emerged as critical players in colorectal cancer (CRC) progression, but the role of lncRNAs in the progression of CRC under nutrient starvation has not been well clarified. Here, we identified a lncRNA, LINC01615, that was significantly upregulated in response to serum starvation. LINC01615 can contribute to the adaptation of CRC cells to serum-deprived conditions and enhance cell survival under similar conditions. LINC01615 activated the pentose phosphate pathway (PPP) under serum starvation, manifested as decreased ROS production and enhanced nucleotide and lipid synthesis. Glucose-6-phosphate dehydrogenase (G6PD) is a key rate-limiting enzyme of the PPP, and LINC01615 promoted G6PD expression by competitively binding with hnRNPA1 and facilitating G6PD pre-mRNA splicing. Moreover, we also found that serum starvation led to METTL3 degradation by inducing autophagy, which further increased the stability and level of LINC01615 in a m6A-dependent manner. LINC01615 knockdown combined with oxaliplatin achieved remarkable antitumor effects in PDO and PDX models. Collectively, our results demonstrated a novel adaptive survival mechanism permitting tumor cells to survive under limiting nutrient supplies and provided a potential therapeutic target for CRC.

Kampo formula hochu-ekki-to (Bu-Zhong-Yi-Qi-Tang, TJ-41) ameliorates muscle atrophy by modulating atrogenes and AMPK in vivo and in vitro

BackgroundMuscle disuse results in loss of skeletal muscle mass and function. Hochu-ekki-to (TJ-41; Bu-Zhong-Yi-Qi-Tang in Chinese) is an herbal medicinal formulation used to treat patients with frailty, fatigue and appetite loss. It has been suggested that two atrogenes, atrogin-1 and muscle Ring finger 1 (MuRF1), are ubiquitin ligases involved in disuse-induced muscle atrophy and that 5’ adenosine monophosphate-activated protein kinase (AMPK) is involved in skeletal muscle metabolism. Effects of TJ-41 on disuse-induced muscle atrophy are unclear.MethodsWe subjected differentiated C2C12 myotubes to serum starvation, then examined the effects of TJ-41 on atrogenes expression, AMPK activity and the morphology of the myotubes. Male C57BL/6J mice were subjected to tail-suspension to induce hindlimb atrophy. We administered TJ-41 by gavage to the control group and the tail-suspended group, then examined the effects of TJ-41 on atrogene expression, AMPK activity, and the muscle weight.ResultsSerum starvation induced the expression of atrogin-1 and MuRF1 in C2C12 myotubes, and TJ-41 significantly downregulated the expression of atrogin-1. Tail-suspension of the mice induced the expression of atrogin-1 and MuRF1 in skeletal muscle as well as its muscle atrophy, whereas TJ-41 treatment significantly downregulated the expression of atrogin-1 and ameliorated the loss of the muscle weight. In addition, TJ-41 also activated AMPK and inactivated Akt and mTOR in skeletal muscle in vivo.ConclusionTJ-41 inhibited atrogenes in an Akt-independent manner as well as activating AMPK in skeletal muscles in vivo, further implying the therapeutic potential of TJ-41 against disuse-induced muscle atrophy and other atrogenes-dependent atrophic conditions.

The rise of viperin: the emerging role of viperin in cancer progression.

Viperin, an IFN-regulated gene product, is known to inhibit fatty acid β-oxidation in the mitochondria, which enhances glycolysis and lipogenesis during viral infections. Yet, its role in altering the phenotype of cancer cells has not been established. In this issue of the JCI, Choi, Kim, and co-authors report on a role of viperin in regulating metabolic alterations in cancer cells. The authors showed a correlation between clinical outcomes and viperin expression levels in multiple cancer tissues and proposed that viperin expression was upregulated in the tumor microenvironment via the JAK/STAT and PI3K/AKT/mTOR/HIF-1α pathways. Functionally, viperin increased lipogenesis and glycolysis in cancer cells by inhibiting fatty acid β-oxidation. Viperin expression also enhanced cancer stem cell properties, ultimately promoting tumor initiation in murine models. This study proposes a protumorigenic role for viperin and identifies HIF-1α as a transcription factor that increases viperin expression under serum starvation and hypoxia.

Autophagy protects retinal pigment epithelial cells from hydroquinone‐induced toxicity

AbstractPurpose: Age‐related macular degeneration (AMD) is a complex disease and the most common cause of blindness among the elderly in the Western world. Dysfunctional cellular clearance and increased oxidative stress are known to play a role in retinal pigment epithelial (RPE) cell death and dysfunction, which secondarily leads to photoreceptor degeneration and a loss of central vision. Smoking, which increases oxidative stress on a cellular level is a known risk factor for AMD. Here we assess the role of autophagy in the survival of ARPE‐19 cells exposed to the cigarette smoke component hydroquinone and in the secretion of the vascular endothelial growth factor (VEGF).Methods: We exposed ARPE‐19 cells to 100 μM hydroquinone following either 1 h or 24 h of serum‐starvation, which induced autophagic clearance. At the same time, autophagy was inhibited either at the early or the late stage using 3‐Methyladenine or bafilomycin A1, respectively. Cytotoxicity was assessed using the lactate dehydrogenase (LDH) or the MTT assay. VEGF secretion was measured from the medium using ELISA.Results: Hydroquinone exposure reduced cell viability to 50% of control when hydroquinone was added following a 1 h serum starvation. Inversely, if cells were starved for 24 h prior to hydroquinone exposure, no toxicity was observed. Inhibition of autophagy at the early but not the late stage significantly reduced the survival of cells starved for 24 h prior to hydroquinone exposure. At the same time, autophagy inhibition significantly reduced VEGF secretion to the medium.Conclusions: Autophagy activation effectively prevented oxidative stress‐induced cytotoxicity in RPE cells, highlighting the importance of functional autophagy in RPE cells and in the prevention of AMD.

Melatonin suppresses serum starvation-induced autophagy of ovarian granulosa cells in premature ovarian insufficiency

ObjectivesPremature ovarian insufficiency (POI) refers to the decline and cessation of ovarian functions in women under 40 years of age. Melatonin (MT) acts as a protective for the ovary. This study elucidated the role of MT in autophagy of granulosa cells (GCs) in POI via modulating the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway.MethodsThe expression levels of microRNA (miR)-15a-5p, signal transducer and activator of transcription 3 (Stat3), and relevant hormones in the clinically collected serum samples of POI patients and healthy controls were examined. Human ovarian granulosa-like tumor cells (KGN) underwent serum starvation (SS) treatment to induce POI cell models and then received MT treatment. The expression levels of miR-15a-5p, Stat3, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR in KGN cells were tested via quantitative real-time polymerase chain reaction and Western blotting. KGN cell viability was assessed by MTT assay and the protein levels of autophagy-related markers Beclin-1, microtubule-associated protein light chain 3 II/I, and p62 were detected by Western blotting. The binding relation between miR-15a-5p and Stat3 was verified via the dual-luciferase reporter gene assay. Functional rescue experiments were performed to probe the underlying role of miR-15a-5p/Stat3/the PI3K-Akt-mTOR pathway in KGN cell autophagy.ResultsmiR-15a-5p was increased whilst Stat3 was decreased in the serum of POI patients and SS-induced KGN cells. MT inhibited miR-15a-5p and Stat3, activated the PI3K-Akt-mTOR pathway, and repressed cell autophagy in SS-induced KGN cells. miR-15a-5p targeted and repressed Stat3 expression. Upregulation of miR-15a-5p or downregulation of Stat3 or the PI3K-Akt-mTOR pathway promoted KGN cell autophagy.ConclusionMT suppressed miR-15a-5p and activated Stat3 and the PI3K-Akt-mTOR pathway, finally impeding SS-induced autophagy of GCs.

Abstract 15292: Il21R-AS1 Long Non-Coding RNA as Potential Regulator in Therapeutic Angiogenesis in Peripheral Arterial Disease

Background: Peripheral arterial disease (PAD) is the major complication of systemic atherosclerosis. The clinical complications are intermittent claudication (IC) or its more severe form, critical limb ischemia (CLI). Potential therapeutic agents, to treat PAD failed in clinical studies. Vascular endothelial growth factor (VEGF) induced significant edema with no clinical benefit. Still today, no medical therapy exists to improve perfusion in PAD. Our lab has been studying the role of the interleukin-21 receptor (IL21R) in therapeutic angiogenesis. IL21 is undergoing clinical studies but its ability to be a therapeutic may be limited. IL21R transcribes a single human long non-coding RNA, the IL21R- antisense 1 (IL21R-AS1), with no mice homolog. Its role in PAD is not established yet. Hypothesis: IL21R-AS1 can serve as an activator of IL21R signaling to mediate hypoxia-induced angiogenesis without increasing vascular permeability. Methods and Results: Plasma from patients with IC, CLI, and non-PAD controls was obtained from whole blood. IL21R-AS1 was elevated in the plasma of patients with CLI. IL21R-AS1 expression was higher in endothelial cells (ECs) under hypoxia (2% O 2 ) serum starvation (HSS), an in vitro model of PAD. To reduce the expression of IL21R-AS1 cells were transfected with gapmeR, and for the overexpression, IL21-AS1 pCMV-TnT plasmid was used. The knockdown of the IL21R-AS1 decreased tube formation in angiogenesis assay performed on growth factor reduced matrigel, while the overexpression of the lncRNA was pro-angiogenic in hypoxic endothelial cells. We detected IL21R-AS1 in the cytoplasm and showed its translocation to the cell surface in HSS. More importantly, in ECs under HSS, we detected IL21R-AS1 in IL21R immunoprecipitation samples; suggesting a direct interaction between IL21R-AS1 and the IL21R. Both IL21 treatment and IL21R-AS1 overexpression increased transendothelial resistance (TER) in hypoxic ECs, in contrast to the permeability induced by VEGF as measured by ECIS (Endothelial Cell Impedance Sensing). Conclusion: Collectively our data suggest that the lncRNA IL21R-AS1 serves as an activator of the IL21R that can mediate angiogenesis but without inducing vascular leakage.

Abstract 14080: LINC02397 in Skeletal Muscle: A Marker of, and Therapeutic Target for, Chronic Limb Threatening Ischemia

Background: Chronic limb threatening ischemia (CLTI) is the most severe complication of peripheral arterial disease (PAD) and the leading cause of amputations for US adults. The response to reduced blood flow within ischemic muscle plays a role in determining disease outcome in PAD, but the response mechanisms have not been fully elucidated. Recently, long non-coding RNAs (lncRNAs) have been shown to regulate gene expression and modify disease. The objective of this study is to identify and characterize lncRNA that is differentially expressed in ischemic muscle in CLTI to serve as a therapeutic target in PAD. Methods: RNA sequencing (n=6/group) and whole transcriptome shotgun sequencing (WTSS n=15/group) from human muscle tissue in CLTI and healthy adults (HA) were performed to identify differentially expressed lncRNA candidates. qPCR (n=10/group) was performed on separate muscle samples to validate the findings. In-vitro experiments were conducted to examine which major cell type in muscle tissue likely expressed the candidate lncRNAs which were subsequently knocked down in cell types with the highest expression to determine function by qPCR, metabolic assay, and flow cytometry. Results: RNA-seq and WTSS analysis identified LINC02397, and qPCR confirmed LINC02397 was expressed significantly higher in CLTI patients (cT count 27.3±1.7 vs 34.1±1.4 in HA, p <0.0001). Differentiation of monocytes isolated from human PBMCs to macrophages (a process triggered by inflammatory stimuli) increased expression of LINC02397 by 2 4 , the highest expression level among major cell types in muscle. In monocytes (THP-1 cells), PMA treatment induced a macrophage-like phenotype, and LINC02397 expression increased in conditions that trigger inflammatory activation: PMA, LPS, and hypoxia serum starvation. LINC02397 knockdown significantly downregulated inflammatory cytokines IL-1β and IL-6, CD86+ cells, and glycolysis. LPS-activated monocyte media increased the proliferative capacity of endothelial cells after knockdown, suggesting potential promotion of angiogenesis. Conclusions: LINC02397 is uniquely expressed in skeletal muscle of CLTI patients and may serve as a promising therapeutic via its ability to limit inflammation and promote angiogenesis.

PMON184 Ligand-dependent interaction between phosphatidylcholine transfer protein and PPARδ: therapeutic implications for metabolic syndrome

Abstract Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is a soluble lipid binding protein that transports phosphatidylcholine (PC) between cellular membranes. PC-TP knockout mice are more sensitive to glucose and insulin, have increased beta oxidation and improved liver health compared to wild-type mice. To better understand the protective metabolic effects associated with PC-TP deletion, we investigated how hepatic PC-TP affects lipid metabolism, sensitivity to glucose and insulin, and energy homeostasis in a mouse model of diet-induced obesity. We generated a liver-specific PC-TP knockout mouse (L-Pctp-/-), which gains less weight and accumulates less liver fat compared to wild-type mice when challenged with a high fat diet. Hepatic deletion of PC-TP also reduces levels of triglycerides and phospholipids in skeletal muscle, suggesting that PC-TP in the liver influences lipid metabolism in the muscle. Gene expression analyses suggest that the observed metabolic changes are related to transcriptional activity of peroxisome proliferative activating receptor (PPAR) family members. An in-cell protein complementation screen between lipid transfer proteins and PPARs uncovered a novel, direct interaction between PC-TP and PPARδ that was not observed for other PPAR family members. We confirmed the PC-TP-PPARδ interaction in Huh7 hepatocytes, where it was found to repress PPARδ-mediated transactivation. Mutations of PC-TP residues implicated in PC binding and transfer combined with a reduction of exogenously supplied lipids through serum starvation relieves PC-TP mediated PPAR repression. Together our data points to a ligand sensitive PC-TP– PPARδ interaction that suppresses PPAR activity. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Tuning pro-survival effects of human induced pluripotent stem cell-derived exosomes using elastin-like polypeptides

Exosome-based regenerative therapies are potentially easier to manufacture and safer to apply compared to cell-based therapies. However, many questions remain about how to bio-manufacture reproducible and potent exosomes using animal-free reagents. Here we evaluate the hypothesis that designer biomaterial substrates can be used to alter the potency of exosomes secreted by human induced pluripotent stem cells (iPSCs). Two animal-free designer matrices were fabricated based on recombinant elastin-like polypeptides (ELPs): one including a cell-adhesive RGD ligand and a second with a non-adhesive RDG peptide. While iPSCs cultured on these two substrates and Matrigel-coated controls had similar levels of proliferation, the RDG-ELP substrate significantly increased protein expression of stemness markers OCT4 and SOX2 and suppressed spontaneous differentiation compared to those on RGD-ELP. The pro-survival potency of iPSC-derived exosomes was evaluated using three distinct stress tests: serum starvation in murine fibroblasts, hypoxia in human endothelial cells, and hyperosmolarity in canine kidney cells. In all three cases, exosomes produced by iPSCs grown on RDG-ELP substrates had similar pro-survival effects to those produced using iPSCs grown on Matrigel, while use of RGD-ELP substrates led to significantly reduced exosome potency. These data demonstrate that recombinant substrates can be designed for the robust bio-manufacturing of iPSC-derived, pro-survival exosomes.