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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 883 records · Page 49
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Exosomes derived from imatinib-resistant chronic myeloid leukemia cells mediate a horizontal transfer of drug-resistant trait by delivering miR-365

Highlights: • Imatinib resistant CML cells-derived exosomes are internalized into sensitive CML cells. • Imatinib resistant CML cells-derived exosomes confer drug resistance trait to sensitive CML cells. • Exosomes mediate horizontal transfer of miR-365 between imatinib resistant CML cells and sensitive ones. • MiR-365 lowers chemosensitivity and apoptosis in imatinib-sensitive CML cells. Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem/progenitor cells. Majority of patients can be effectively treated with tyrosine kinase inhibitors (TKIs) such as imatinib, but a portion of patients will develop drug resistance. Accumulated evidences have identified exosomes in cancer as promoters of tumor progression. Herein, we found that exosomes derived from imatinib resistant CML cells can be internalized into sensitive CML cells and confer drug-resistance traits. We also demonstrated a significant higher level of miR-365 in exosomes derived from drug-resistant CML cells compared with those from sensitive ones using microarray and qRT-PCR. The imatinib sensitive CML cells transfected with pre-miR-365 displayed lower chemosensitivity and apoptosis rate compared with controls. We further confirmed that exosomal transfer of miR-365 induced drug resistance by inhibiting expression of pro-apoptosis protein in sensitive CML cells. In conclusion, our study reveals that exosomes mediate a horizontal transfer of drug-resistant trait in chronic myeloid leukemia cell by delivering miR-365.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The release of microparticles and mitochondria from RAW 264.7 murine macrophage cells undergoing necroptotic cell death in vitro

Highlights: • Macrophages undergoing necroptotic cell death release microparticles. • Microparticles from necroptotic and apoptotic macrophages have similar properties. • Microparticle preparations contain both nuclear and mitochondrial DNA. • Microparticle preparations contain mitochondria staining with MitoTracker Green. • Purified mitochondria behave as microparticles by flow cytometry. Microparticles (MPs) are small membrane-bound vesicles released from activated or dying cells. As shown previously, LPS stimulation of the RAW 264.7 macrophage cell line can induce MP release, with the caspase inhibitor Z-VAD increasing the extent of this process. Since combined treatment of cells with LPS and Z-VAD can induce necroptosis, we explored particle release during this form of cell death using flow cytometry to assess particle size, binding of annexin V and staining for DNA with propidium iodide (PI) and SYTO 13. The role of necroptosis was assessed by determining the effects of necrostatin, an inhibitor of RIP1, a kinase regulating this form of cell death. These studies demonstrated that, during necroptosis, RAW 264.7 cells release MPs that resemble those released from cells treated with staurosporine to induce apoptosis. The particles contained DNA as determined by binding of PI and SYTO 13, with PCR analysis demonstrating both chromosomal and mitochondrial DNA. The presence of mitochondria in the MP preparations was demonstrated by staining with MitoTracker Green. Flow cytometry indicated that purified mitochondria have properties of MPs. Together, these studies indicate that cells undergoing necroptosis can release MPs and that mitochondria can be components of MP preparations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

E4BP4 inhibits AngII-induced apoptosis in H9c2 cardiomyoblasts by activating the PI3K-Akt pathway and promoting calcium uptake

Highlights: • E4BP4 expression in H9c2 significantly attenuates AngII-induced apoptosis. • E4BP4 inhibits AngII-induced IGF-II expression & caspase cleavage through PI3K-Akt pathway. • E4BP4 enhances calcium reuptake into SR by up-regulating p-PKA and p-PLB proteins. • E4BP4 thus acts as a survival factor in H9c2, regulating IGF-II and calcium cycling. The bZIP transcription factor E4BP4 is a survival factor that is known to be elevated in diseased heart and promote cell survival. In this study the role of E4BP4 on angiotensin-II (AngII)-induced apoptosis has been examined in in vitro cell model. H9c2 cardiomyoblast cells that overexpressed E4BP4 were exposed to AngII to observe the cardio-protective effects of E4BP4 on hypertension related apoptosis. The results from TUNEL assays revealed that E4BP4 significantly attenuated AngII-induced apoptosis. Further analysis by Western blot and RT-PCR showed that E4BP4 inhibited AngII-induced IGF-II mRNA expression and cleavage of caspase-3 through the PI3K-Akt pathway. In addition, E4BP4 enhanced calcium reuptake into the sacroplasmic reticulum by down-regulating PP2A and by up-regulating the phosphorylation of PKA and PLB proteins. Our findings indicate that E4BP4 functions as a survival factor in cardiomyoblasts by inhibiting IGF-II transcription and by regulating calcium cycling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Alteration in microRNA-25 expression regulate cardiac function via renin secretion

Highlights: • Overexpression of miR-25 cause cardiomyocyte fibrosis and apoptosis, inhibition of miR-25 cause high blood pressure by activate RAS. • miR-25 expression in heart failure varies by stage, and its function pattern present “time-space” characteristic. • Excessive miR-25 cause heart dysfunction is mainly due to target gene alteration, whereas inhibition of miR-25 do not have this effect. • A novel mechanism of miR-25 is increased miR-25 directly cause heart impairment by target alteration and decreased miR-25 indirectly cause heart dysfunction by induced high blood pressure. Heart failure arises from diverse cardiovascular diseases, including hypertension, ischemic disease and atherosclerosis, valvular insufficiency, myocarditis, and contractile protein mutations. MicroRNAs are dysregulated in heart failure, but identification of the specific microRNAs involved remains incomplete. Here, we evaluate miR-25 expression in the peripheral blood from healthy, dilated cardiomyopathy (DCM), remote infarct (OMI), hypertensive heart disease (HHD), and HHD resulting in heart failure (HHDF) using q-PCR. Interestingly, we discovered miR-25 expression in humans is initially decreased at the onset of heart failure but is later increased in end-stage heart failure. We also show that overexpression of miR-25 in normal mice causes cardiomyocyte fibrosis and apoptosis. However, inhibition of miR-25 in normal mice led to activate renin-angiotensin system (RAS) and high blood pressure, mild heart dilation. Notably, the miR-25 cluster knock-out mice was also characterized high blood pressure and no obvious cardiac function alteration. RNA sequencing showed the alteration of miR-25 target genes in angomir-treated mice, including the renin secretion signal related gene. In vitro, cotransfection with the miR-25 antagomir repressed luciferase activity from a reporter construct containing the Pde3a and Cacnalc untranslated region. In summary, miR-25 expression during different stages of heart disease, offers a new perspective for the role of miR-25 function in heart failure.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MiR-155 targets PTCH1 to mediate endothelial progenitor cell dysfunction caused by high glucose

Highlights: • MiR-155 was upregulated in EPCs of patients with DFU. • High glucose–induced upregulation of miR-155 in EPCs. • MiR-155 exacerbated EPCs dysfunction induced by high glucose. • MiR-155 targeted PTCH1 in EPCs. • MiR-155 impaired EPCs function via PTCH1 suppression. Endothelial progenitor cells (EPCs) are involved in diabetes-associated complications, including diabetic foot ulcer (DFU). Recent reports showed that miR-155 downregulation promotes wound healing in diabetic rats and ameliorates endothelial injury induced by high glucose, but its role in DFU is unknown. We found that miR-155 was overexpressed in EPCs from patients with DFU and in high glucose–induced EPCs from healthy people. Reductions in cell viability, migration, tube formation and nitric oxide production, as well as increases in lactated hydrogenase, cell apoptosis, and reactive oxygen species induced by high glucose, were enhanced by miR-155 overexpression and restrained by miR-155 inhibition. Additionally, dual-luciferase reporter assay demonstrated that miR-155 directly targeted the 3′ untranslated region of patched-1 (PTCH1), a receptor of the sonic hedgehog signaling pathway, and downregulated the mRNA and protein expression of PTCH1. qRT-PCR and Western blot results revealed that the PTCH1 was downregulated in EPCs treated with high glucose. Silencing PTCH1 by PTCH1 siRNA alleviated the protective effect of anti-miR-155 on high glucose–induced EPC dysfunction. Our results indicate that miR-155 worsened high glucose–induced EPC function by downregulating PTCH1. These findings suggest that miR-155 may be a potential therapeutic target for DFU.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Systems biology approach to study the role of miRNA in promoter targeting during megakaryopoiesis

Highlights: • miRNAs could regulate Cyclins and CDKs by targeting their promoters. • Perl programming was used to identify putative targets of miRNA in promoters. • miR-1273g-3p and miR-619-5p showed significance with longest seed sequence matches. • Through CDK10, CDK11 and Cyclin, these two miRNAs could regulate cell cycle during megakaryopoiesis. The distinct process of megakaryopoiesis requires occurrence of endomitosis for polyploidization of the megakaryocytes. Although, Cyclins, CDKs and have been described to regulate endomitosis, the exact mechanism still remains an enigma. miRNA which were otherwise known as post transcriptional gene silencers are now emerging with various non-canonical functions including gene regulation at pre-transcriptional level by miRNA binding at promoter region. Out of the many processes they regulate, miRNA have been manifested to play a role in megakaryocyte differentiation. In this study an attempt has been made to identify miRNA that could regulate cell cycle genes (Cyclins and CDKs) by targeting their promoters, during megakaryopoiesis. A new computational algorithm was implemented using Perl programming to identify putative targets of miRNA in CDK and Cyclin promoters. Perl script was also used to check nuclear localizing miRNA based on the presence of a consensus sequence. Real-time PCR was performed to analyze the expression of miRNA and their predicted targets in Dami vs. PMA treated Dami cells. Putative targets of miRNAs with longest, high complementarity matches in CDK/Cyclin promoters were obtained. We identified two significant miRNA, miR-1273g-3p and miR-619-5p with longest seed sequence matches. We further identified three main targets (CDK10, CDK11, Cyclin F) through which these two miRNA could regulate cell cycle during megakaryopoiesis. Our results reinforce the role of promoting targeting miRNA in regulation of cell cycle through certain CDK/Cyclins to support the process of endomitosis during megakaryopoiesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Dysfunction of cholesterol sensor SCAP promotes inflammation activation in THP-1 macrophages

Highlights: • The dysfunction of SCAP interplays with inflammatory responses. • The effect above is independent of SCAP's well-known role in regulating cholesterol homeostasis. • The dysfunction of SCAP mediated the activation of NF-κB signaling pathway. Crosstalk occurs between dyslipidemia and chronic inflammation, which are both precipitants of atherosclerosis. Sterol regulatory element binding proteins cleavage-activating protein (SCAP) plays a key role in regulating cholesterol homeostasis. The present study investigated the effects of SCAP dysfunction on the expression of inflammatory cytokines and lipid metabolism in THP-1 macrophages. Intracellular cholesterol content was assessed by Oil Red O staining and quantitative assays. The expression of SCAP, HMGCR, pro-IL-1β and N-SREBP2, p65(N) in the nucleus were examined by real-time quantitative RT-PCR and Western blotting. The level of secretary proteins IL-1β, TNF-α and MCP-1 in the supernatants were determined by ELISA. The translocation of SCAP from the endoplasmic reticulum (ER) to the Golgi was detected by confocal microscopy. Our results demonstrated that over-expression of SCAP significantly increased the expression of HMGCR, pro-IL-1β in the cytoplasm, and mature IL-1β, TNF-α, MCP-1 in the supernatants, while knocking down SCAP dramatically decreased the expression of these molecules. Betulin effectively suppressed the accumulation of intracellular cholesterol in the SCAP over-expressed THP-1 macrophages, but did not affect the expression of inflammatory cytokines, indicating that the pro-inflammatory effect of SCAP was independent of its routine role in regulating cholesterol homeostasis. Furthermore, we investigated the molecular mechanisms mediating the crosstalk between dyslipidemia and inflammatory responses. Knocking down SCAP attenuated LPS-induced IκB phosphorylation and reduced the nuclear level of p65, while over-expression of SCAP increased the nuclear level of p65. Knocking down p65 abolished the proinflammatory effect represented by elevated expression of the inflammatory mediators in the SCAP over-expressed THP-1 macrophages, suggesting that SCAP dysfunction stimulated inflammatory responses via activating the NF-κB signaling pathway. In conclusion, the cholesterol sensor SCAP plays a role in regulating the expression of inflammatory factors such as IL-1β, TNF-α, and MCP-1 in THP-1 macrophages. SCAP mediates the inflammatory response via activating the NF-κB pathway. This new function of SCAP is independent of its role in lipid metabolism.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The change of nuclear LC3 distribution in acute myeloid leukemia cells

Highlights: • AML cells with Ara-c or Sorafenib resulted in autophagy enhancement. • Nuclear LC3 moved into the cytoplasm when autophagy was enhanced. • Sirt1 and DOR as regulatory molecules for the observed nuclear LC3 shift. • Sirt1 or DOR affected the expression of Beclin1, HMGB1, and AMPK. • Cytotoxicity of drugs was enhanced when nuclear LC3 shifting was blocked. Making sure the change of nuclear LC3 distribution in the autophagy of acute myeloid leukemia (AML) cell and finding out the regulation mechanism may lead to a breakthrough for killing AML cells. Western blots were performed to assess the expression of autophagy proteins. Changes in the LC3 distribution were monitored by immunofluorescence assays together with western blots, and the expression levels of Sirt1, DOR, Beclin1, HMGB1, and AMPK mRNA were detected via fluorescent quantitative PCR. The effects of Sirt1 and DOR on cell proliferation and survival were analyzed by MTT, flow cytometry, and western blotting assays. We found that treating AML cells with Ara-c or Sorafenib resulted in autophagy enhancement, and when autophagy was enhanced, nuclear LC3 moved into the cytoplasm. Notably, when autophagy was inhibited by blocking the nuclear LC3 shift, the cytotoxicity of drugs was enhanced. Our results also identified Sirt1 and DOR as regulatory molecules for the observed nuclear LC3 shift, and these molecules further affected the expression of Beclin1, HMGB1, and AMPK. Our results suggest the distribution of nuclear LC3 can be a novel way for further studying death of AML cells,and the regulatory molecules may be new targets for treating AML.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

TRAF6 mediates high glucose-induced endothelial dysfunction

Highlights: • TRAF6 mediated high glucose-induced endothelial dysfunction. • NF-κB- and AP-1-dependent signaling served as mechanism. • Targeting TRAF6 may delay progression of vascular diseases during diabetes mellitus and atherosclerosis. To investigate the role of tumor necrosis factor-associated factor 6 (TRAF6) in high glucose-induced endothelial cell dysfunction. Human aortic endothelial cells (HAECs) were cultured in high glucose medium, and TRAF6 expression was assayed by quantitative real-time Polymerase Chain Reaction (PCR) and western blotting. The effect of TRAF6 on in vitro endothelial cell viability, apoptosis, migration, and endothelial–monocyte adhesion was investigated by gene knockdown. The expression of TRAF6 and related adhesion molecules was assayed in a mouse streptozotocin-induced type I diabetes model. The signaling pathways associated with TRAF6 effects on endothelial cells were investigated in high glucose HAEC cultures. Culture of HAECs in high glucose medium significantly increased TRAF6 mRNA and protein expression in a time dependent manner. High glucose markedly reduced HAEC viability, apoptosis, and migration, and these effects was significantly reversed by TRAF6 knockdown. High glucose significantly increased intercellular adhesion of THP-1 monocytic cells and HAECs via upregulation of ICAM-1 and VCAM-1 expression, and TRAF6 knockdown attenuated the effect on THP-1 cell adhesion. TRAF6, ICAM-1, and VCAM-1 expression were increased in aorta tissue of mice with streptozotocin-induced diabetes. The free radical scavenger N-acetyl-L-cysteine attenuated TRAF6 expression in HAECs cultured in high glucose medium, and TRAF6 knockdown inhibited high glucose-induced IκB-α degradation and JNK phosphorylation. TRAF6 mediated high glucose-induced endothelial dysfunction via NF-κB- and AP-1-dependent signaling. Targeting TRAF6 may delay progression of vascular diseases during diabetes mellitus and atherosclerosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer

Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA-143 (miR-143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild-type KRAS gene, to paclitaxel-mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR-143 replenishment is inhibiting pre-metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR-143 as predicted by TargetScan were evaluated by qRT-PCR in LoVo cells which were performed mock transfection or miR-143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro-metastatic behavior was assayed by gain- and loss-of-function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR-143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR-143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA-mediated silencing in LoVo cells induced and repressed, respectively, pro-mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Matrix stiffness regulates the differentiation of tendon-derived stem cells through FAK-ERK1/2 activation

Highlights: • TDSC proliferation was enhanced with the increasing matrix stiffness. • More F-actin stress fibers formed in TDSCs while matrix stiffness increased. • Higher matrix stiffness inhibited the differentiation of TDSCs. • FAK or ERK1/2 activation was involved in the regulatory effect. Tendon derived stem cells (TDSCs) were vital in tendon homeostasis. Nevertheless, the regulation of TDSCs differentiation in tendinopathy is unclear. Matrix stiffness modulated stem cells differentiation, and matrix stiffness of tendinopathic tissues decreased significantly. In order to clarify the role of matrix stiffness in TDSCs differentiation, they were cultured on the gelatin hydrogels with the stiffness from 2.34 ± 1.48 kPa to 24.09 ± 14.03 kPa. The effect of matrix stiffness on TDSCs proliferation and differentiation were investigated with CCK8 assay, immunofluorescences, real time PCR and western blot. It was found the proliferation of TDSCs increased and more stress fibers formed with increasing matrix stiffness. The differentiation of TDSCs into tenogenic, chondrogenic, and osteogenic lineages were inhibited on stiff hydrogel evidenced by reduced expression of tenocyte markers THBS4, TNMD, SCX, chondrocyte marker COL2, and osteocyte markers Runx2, Osterix, and ALP. Furthermore, the phosphorylation of FAK and ERK1/2 were enhanced when TDSCs grew on stiff hydrogel. After FAK or ERK1/2 was inhibited, the effect of matrix stiffness on differentiation of TDSCs was inhibited as well. The above results indicated matrix stiffness modulated the proliferation and differentiation of TDSCs, and the regulation effect could correlate to the activation of FAK or ERK1/2.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Diacylglycerol kinase γ predicts prognosis and functions as a tumor suppressor by negatively regulating glucose transporter 1 in hepatocellular carcinoma

Highlights: • DGKγ expression was downregulated in hepatocellular carcinoma (HCC). • Lower DGKγ expression in HCC tumor tissues was an independent prognostic factor for poor post-surgical overall survival. • Cell proliferation and migration of HCC cells were suppressed by DGKγ ectopic expression. • DGKγ overexpression led to down regulation of GLUT1 protein level and AMPK activity. Diacylglycerol kinases (DGK) are a family of enzymes catalyzing the transformation of diacylglycerol into phosphatidic acid, which have been recognized as key regulators in cell signaling pathways. The role of DGKγ in human malignancies has seldom been studied. In this study, we investigated the role of DGKγ in hepatocellular carcinoma (HCC). We found that DGKγ was down-regulated in HCC tumor tissues and cell lines as compared to that in non-tumor tissues. The prognostic value of DGKγ expression was evaluated by Cox regression and Kaplan-Meier analyses. Lower DGKγ expression in tumor tissues was an independent prognostic factor for poor post-surgical overall survival. By using HDACs inhibitors treatment and ChIP-PCR, we discovered that histone H3 and H4 deacetylation mainly contributed to the downregulation of DGKγ expression. Functional studies revealed that ectopic expression of DGKγ inhibited cell proliferation and cell migration in HCC cells. Mechanism studies showed that DGKγ overexpression led to down regulation of GLUT1 protein level and AMPK activity, which result in glucose uptake suppression as well as lactate and ATP production declination. The decrease of GLUT1 level could be partially rescued by treatments with either DGK inhibitor and lysosome inhibitor, indicating DGKγ may down-regulate GLUT1 through its kinase activity and lysosome degradation process. Together, this study demonstrated that DGKγ plays a tumor suppressor role in HCC by negatively regulating GLUT1. DGKγ could be a novel prognostic indicator and therapeutic target for HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Inactivated STAT5 pathway underlies a novel inhibitory role of EBF1 in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (CLL) is a disease caused by gradual accumulation of functionally incompetent lymphocytes. The majority of CLL cases are accompanied by chemoresistance. Early B cell factor 1 (EBF1) is a crucial contributor to B-cell lymphopoiesis. This study is to explore the effect of EBF1 on CLL cell progression and its involvement in regulating the signal transducers and activators of transcription 5 (STAT5) pathway. We conducted a correlation analysis between EBF1 and the clinical characteristics of CLL patients. Subsequently, EBF1 was overexpressed by transfection with EBF1 overexpression plasmid and the STAT5 pathway was also blocked by treatment with SH-4-54 in isolated CD20{sup +} B lymphocytes to investigate their roles in the regulation of cellular functions. STAT5, Janus kinase 2 (JAK2) expression and their phosphorylation levels were determined by quantitative PCR and Western blot analyses. The in vivo effects of EBF1 on tumor growth were evaluated using a xenotransplant model. Downregulation of EBF1 was observed in CD20{sup +} B lymphocytes of CLL patients. EBF1 overexpression disrupted the activation of STAT5 pathway, as evidenced by decreased expression and phosphorylation levels of STAT5 and JAK2. Furthermore, overexpression of EBF1 repressed viability and cell cycle entry, and increased apoptosis of CD20{sup +} B lymphocytes by inhibiting the STAT5 pathway. Finally, EBF1 exerted antitumor effects in nude mice. Overall, our study elucidates the inhibitory role of EBF1 in CLL through inactivation of the STAT5 pathway, which may provide new targets for CLL treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Endothelium-specific endothelin-1 expression promotes pro-inflammatory macrophage activation by regulating miR-33/NR4A axis

The hallmark of atherogenesis is characterized as endothelial dysfunction and subsequent macrophage activation. Although our previous study has demonstrated that endothelin-1 (ET-1) plays an important role in atherogenesis, the underlying mechanism remains deeply investigation. Enhanced atherosclerotic plaques were observed in endothelium-specific ET-1 overexpression ApoE{sup −/-} mice (eET-1/ApoE{sup −/-}) concomitant with increased secretion of pro-inflammatory adhesion molecules and cytokines. The conditional media used for culturing human umbilical vein endothelial cells (HUVECs) with AdET-1 infection and subjected to OX-LDL stimulation, was collected and utilized for bone marrow-derived macrophages (BMDMs) culturing. RT-PCR analysis showed increased genes expression related to classical M1 macrophages but decreased alternative activated M2 macrophages genes expression in macrophage culturing with the conditional media. Furthermore, consistent regulations of macrophage polarization were observed using isolated exosomes from the conditional media. More importantly, we noticed that miR-33 was enriched in the exosomes derived by HUVECs with AdET-1 infection, while bioinformatics analysis further indicated that miR-33 directly targeted NR4A and miR-33/NR4A axis was required for the effect of endothelial-specific ET-1 overexpression on pro-inflammatory macrophage activation. By contrast, such effects could be reversed by ET-1 knockdown. Taken together, our study indicated that the exosomes derived by HUVECs with AdET-1 infection can transfer miR-33 to macrophages and subsequently promote pro-inflammatory macrophage activation by directly targeting to NR4A. These evidences clearly revealed that miR-33/NR4A axis was the important mechanism underlying the effect of ET-1 on macrophage activation and indicated that ET-1 may act as a promising target for atherosclerosis management.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Noonan syndrome patient-specific induced cardiomyocyte model carrying SOS1 gene variant c.1654A>G

Noonan syndrome (NS) is a dominant autosomal genetic disorder, associated with mutations in several genes that exhibit multisystem abnormal development including cardiac defects. NS associated with the Son of Sevenless homolog 1 (SOS1) gene mutation attributes to the development of cardiomyopathy and congenital heart defects. Since the treatment option for NS is very limited, an in vitro disease model with SOS1 gene mutation would be beneficial for exploring therapeutic possibilities for NS. We reprogrammed cardiac fibroblasts obtained from a NS patient and normal control skin fibroblasts (C-SF) into induced pluripotent stem cells (iPSCs). We identified NS-iPSCs carry a heterozygous single nucleotide variation in the SOS1 gene at the c.1654A > G. Furthermore, the control and NS-iPSCs were differentiated into induced cardiomyocytes (iCMCs), and the electron microscopic analysis showed that the sarcomeres of the NS-iCMCs were highly disorganized. FACS analysis showed that 47.5% of the NS-iCMCs co-expressed GATA4 and cardiac troponin T proteins, and the mRNA expression levels of many cardiac related genes, studied by qRT-PCR array, were significantly reduced when compared to the control C-iCMCs. We report for the first time that NS-iPSCs carry a single nucleotide variation in the SOS1 gene at the c.1654A>G were showing significantly reduced cardiac genes and proteins expression as well as structurally and functionally compromised when compared to C-iCMCs. These iPSCs and iCMCs can be used as a modeling platform to unravel the pathologic mechanisms and also the development of novel drug for the cardiomyopathy in patients with NS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Ergothioneine alleviates senescence of fibroblasts induced by UVB damage of keratinocytes via activation of the Nrf2/HO-1 pathway and HSP70 in keratinocytes

Highlights: • EGT activated Nrf2/HO-1 pathway and HSP70 in UVB-irradiated keratinocytes. • EGT alleviated ROS production, apoptosis, and inflammation in UVB-irradiated keratinocytes. • EGT suppressed senescence and collagen imbalance in fibroblasts by inhibiting the keratinocyte-secreted factors. • EGT suppresses photoaging by UVB irradiation in a keratinocyte/fibroblast co-culture system. Ultraviolet B (UVB) irradiation induces skin damage and photoaging through several deleterious effects, including generation of reactive oxygen species (ROS), apoptosis of epidermal cells, inflammation, and collagen degradation in fibroblasts. Ergothioneine (EGT) is a naturally occurring amino acid with potential biological properties. We evaluated whether EGT protects against UVB-induced photoaging using a keratinocyte/fibroblast co-culture system. Keratinocytes were pretreated with EGT, irradiated with UVB, and co-cultured with fibroblasts. In keratinocytes, ROS production and apoptosis were assessed. We also analyzed the Nrf2/HO-1 pathway, HSP70, proapoptotic proteins, and paracrine cytokines by Western blotting and real-time PCR. Collagen degradation-related genes and senescence were also assessed in fibroblasts. EGT pretreatment of keratinocytes significantly inhibited downregulation of the Nrf2/HO-1 pathway and HSP70, and protected keratinocytes by suppressing production of ROS and cleavage of proapoptotic proteins, including caspase-8 and PARP. Furthermore, EGT significantly reduced the paracrine cytokines, including IL-1β, IL-6, and TNF-α. In co-cultures of fibroblasts with EGT-treated keratinocytes, the expression levels of collagen degradation-related genes and fibroblast senescence were significantly decreased; however, synthesis of procollagen type I was significantly increased. Our results confirm that EGT suppresses the modification of collagen homeostasis in fibroblasts by preventing downregulation of the Nrf2/HO-1 pathway and HSP70 in keratinocytes following UVB irradiation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Upregulation of PPPDE1 contributes to anorectal malformations via the mitochondrial apoptosis pathway during hindgut development in rats

Congenital anorectal malformations (ARMs) are among the most prominent deformities of the gastrointestinal tract; however, their precise aetiology remains obscure. Immunohistochemistry demonstrated that, in the ARM group, the PPPDE1-positive cells were widely distributed in the hindgut epithelial tissue from GD13 to GD16. Immunofluorescence revealed that most TUNEL-, Bax-, and Cytochrome C (Cyt C)-positive cells overlapped with PPPDE1-positive cells in the urorectal septum (URS). Western blotting and quantitative real-time RT-PCR revealed that PPPDE1 levels were significantly higher in the ARM group from GD13 to GD14 (p < 0.05). IEC-6 cells were transfected with PPPDE1 overexpression plasmid/NC (negative control) or si-PPPDE1/si-NC. Flow cytometry analysis and CCK-8 assay (used to detect apoptosis and proliferation, respectively), as well as western blotting, showed that the levels of PPPDE1 were positively correlated with the pro-apoptotic molecules Bax and Cyt C. Accordingly, aberrantly high expression of PPPDE1 caused a spatiotemporal imbalance in foetal rats with ARMs during hindgut development. Therefore, the upregulation of PPPDE1 may promote epithelial apoptosis and reduce proliferation in the hindgut via the mitochondrial apoptotic pathway. This could affect the fusion of the URS and cloacal membrane, ultimately inhibiting the hindgut development and resulting in ARMs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes

Highlights: • Cortical granule exocytosis is sensitive to tetanus toxin in mouse oocytes. • VAMP1 and VAMP3 proteins participate in cortical granule exocytosis. • This work confirms that cortical granule exocytosis is mediated by SNARE complex. Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin –which specifically cleavages VAMP1, VAMP2 or VAMP3– inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 –but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.

60 APPLIED LIFE SCIENCES↗