Far-infrared irradiation increases the uptake of LDL cholesterol by downregulating PCSK9 through the activation of TRPV4 calcium channels in HepG2 cells.

This study investigated the effects of far-infrared (FIR) irradiation on low-density lipoprotein cholesterol (LDL-C) uptake by human hepatocellular carcinoma G2 (HepG2) cells via the regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9). FIR irradiation for 30 min significantly decreased PCSK9 expression (p < 0.01) in HepG2 cells. FIR irradiation substantially increased the low-density lipoprotein receptor (p < 0.0001) and LDL-C uptake (p < 0.01). Activation of transient receptor potential vanilloid (TRPV) channels mimicked the effects of FIR irradiation, significantly decreasing the protein expression of PCSK9 (p < 0.05). Conversely, inhibition of TRP channels using ruthenium red reversed the reduction in PCSK9 protein expression following FIR irradiation (p < 0.01). The specific activation of TRPV4 using 4α-PDD mimicked the effect of FIR irradiation (p < 0.01), whereas PCSK9 reduction by FIR irradiation was significantly reversed by the inhibition of TRPV4 using RN1734 (p < 0.05). These findings implied that FIR irradiation emitted from a ceramic lamp specifically increased TRPV4 activity. These findings provide insights into a novel therapeutic approach using FIR irradiation for LDL-C regulation and its implications for cardiovascular health.

Nitrosylation of ß2-Tubulin Promotes Microtubule Disassembly and Differentiated Cardiomyocyte Beating in Ischemic Mice.

BACKGROUND: Beating cardiomyocyte regeneration therapies have revealed as alternative therapeutics for heart transplantation. Nonetheless, the importance of nitric oxide (NO) in cardiomyocyte regeneration has been widely suggested, little has been reported concerning endogenous NO during cardiomyocyte differentiation. METHODS: Here, we used P19CL6 cells and a Myocardiac infarction (MI) model to confirm NO-induced protein modification and its role in cardiac beating. Two tyrosine (Tyr) residues of ß2-tubulin (Y106 and Y340) underwent nitrosylation (Tyr-NO) by endogenously generated NO during cardiomyocyte differentiation from pre-cardiomyocyte-like P19CL6 cells. RESULTS: Tyr-NO-ß2-tubulin mediated the interaction with Stathmin, which promotes microtubule disassembly, and was prominently observed in spontaneously beating cell clusters and mouse embryonic heart (E11.5d). In myocardial infarction mice, Tyr-NO-ß2-tubulin in transplanted cells was closely related with cardiac troponin-T expression with their functional recovery, reduced infarct size and thickened left ventricular wall. CONCLUSION: This is the first discovery of a new target molecule of NO, ß2-tubulin, that can promote normal cardiac beating and cardiomyocyte regeneration. Taken together, we suggest therapeutic potential of Tyr-NO-ß2-tubulin, for ischemic cardiomyocyte, which can reduce unexpected side effect of stem cell transplantation, arrhythmogenesis.

Nucleus-targeted delivery of nitric oxide in human mesenchymal stem cells enhances osteogenic differentiation.

Nitric oxide (NO) is an important gaseous signaling molecule in various physiological processes, which functions through interactions with its acceptor molecules located in organelles. NO has an extremely short half-life, making it challenging to experimentally achieve effective NO levels in organelles to study these interactions. Here we developed an organelle-specific, peptide-based NO delivery material that targets the nucleus. NO was attached to the SH group of a cysteine residue inserted into the N-terminus of a cell-penetrating peptide (CPP) conjugated to varying repeats of the nuclear localization signal (NLS), which we denoted NO-CysCPP-NLS, through S-nitrosylation. NO-CysCPP-NLS strongly induced osteogenic differentiation of mesenchymal stem cells. This delivery concept can be extended to cells other than stem cells to elucidate the effects of NO release in the nucleus. Furthermore, conjugation of NO to CysCPP fused to mitochondria- or lysosome-targeting signals can be used to deliver NO to other organelles such as mitochondria and lysosomes, respectively.

Bone Morphogenic Protein-2-Conjugated Three-Dimensional-Printed Poly (L-Lactic Acid) (PLLA) Scaffold is likely Promising as an Effective Bone Substitute.

Bone morphogenic protein-2 (BMP-2)-conjugated three-dimensional (3-D)-printed poly (L-lactic acid)(PLLA) scaffold is likely promising as an effective bone substitute for enhancing bone regeneration of massive bone defects caused by tumor resection, traumatic injury, or congenital diseases. The authors developed a new bone substitute using a novel strategy composed of 3-D-printed PLLA scaffolds through a sequential coating of catechol-conjugated alginate (C-AL), BMP-2, and collagen (CO). The 3-D-printed PLLA scaffold was successfully obtained with 5 mm of diameter, 1 mm of thickness, 400 µm of pore size, 187-230 µm of grid thickness, and 82% of porosity. Alkaline phosphatase (ALP) activity of the BMP-2-immobilized PLLA scaffold in MC3T3-E1 and W-20-17 cells was more increased than BMP-2 itself due to the controlled release of BMP-2 from the scaffold. Tenfold new bone formation for the BMP-2-immobilized PLLA scaffold was obtained by micro-CT analysis than PLLA scaffold without BMP-2 weeks after 4 weeks of transplantation model mouse. Further another big animal model study should be performed before clinical trials.

A COVID-19 mortality prediction model for Korean patients using nationwide Korean disease control and prevention agency database.

The experience of the early nationwide COVID-19 pandemic in South Korea led to an early shortage of medical resources. For efficient resource allocation, accurate prediction of the prognosis or mortality of confirmed patients is essential. Therefore, the aim of this study was to develop an accurate model for predicting COVID-19 mortality using epidemiolocal and clinical variables and for identifying a high-risk group of confirmed patients. Clinical and epidemiolocal variables of 4049 patients with confirmed COVID-19 between January 20, 2020 and April 30, 2020 collected by the Korean Disease Control and Prevention Agency were used. Among the 4049 total confirmed patients, 223 patients died, while 3826 patients were released from isolation. Patients who had the following risk factors showed significantly higher risk scores: age over 60 years, male sex, difficulty breathing, diabetes, cancer, dementia, change of consciousness, and hospitalization in the intensive care unit. High accuracy was shown for both the development set (n = 2467) and the validation set (n = 1582), with AUCs of 0.96 and 0.97, respectively. The prediction model developed in this study based on clinical features and epidemiological factors could be used for screening high-risk groups of patients and for evidence-based allocation of medical resources.

Valproic Acid-Induced CCN1 Promotes Osteogenic Differentiation by Increasing CCN1 Protein Stability through HDAC1 Inhibition in Tonsil-Derived Mesenchymal Stem Cells.

Our previous study found that the level of CCN1 increases as osteogenic differentiation progresses in tonsil-derived mesenchymal stem cells (TMSCs). This study investigated how CCN1 is regulated through HDAC inhibition in TMSCs and their relationship with osteogenesis. Valproic acid (VPA) (1-5 mM), a well-known histone deacetylase (HDAC) inhibitor, strongly inhibited TMSC proliferation without altering MSC-specific surface markers, CD14, 34, 45, 73, 90 and 105. However, CD146 expression increased at 5 mM VPA. VPA increased osteogenic differentiation of TMSCs but decreased adipogenesis and chondrogenesis, as evidenced by the cell-specific staining of differentiation. The former was validated by the increased osteocalcin (OCN). The changes in CCN1 by VPA was biphasic; it increased until 48 h and decreased thereafter. Knockdown of CCN1 by using siRNA inhibited the osteogenic effect of VPA. VPA had no effect on CCN1 mRNA expression, but inhibition of protein synthesis by cycloheximide showed that VPA slowed down the CCN1 protein degradation. Moreover, overexpression of HDAC1 completely inhibited VPA-induced CCN1. Our results indicate that VPA inhibits the HDAC1, inducing CCN1 protein stability rather than gene expression, thereby promoting osteogenic differentiation of TMSCs. These findings present the noble implication of VPA as an inhibitor of HDAC1 to facilitate CCN1-induced osteogenic differentiation of MSCs.

Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells.

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.

Mortality Risk within 14 Days after Coronavirus Disease 2019 Diagnosis in Dementia Patients: A Nationwide Analysis.

INTRODUCTION: The study evaluated the increased mortality risk within 14 days of coronavirus disease 2019 (COVID-19) diagnosis in dementia patients. METHODS: This retrospective study was conducted from February to April 2020 using the COVID-19 patients' database from the Korea Disease Control and Prevention Agency. The risk factors for early death within 14 days were determined using generalized logistic regression performed in a stepwise manner. Dementia patients diagnosed with COVID-19 were used for the study. The propensity score-matched cohort was included as controls. The differences in mortality within 14 days after COVID-19 diagnosis between the dementia patients and controls were evaluated. RESULTS: We enrolled 5,349 COVID-19 patients from the database; 224 had dementia as comorbidity. The mortality rate within 14 days after COVID-19 diagnosis in dementia patients and the controls was 23.7% versus 1.7%, respectively, before propensity score matching (PSM) (p < 0.001), and 23.7% versus 9.2% after PSM (p < 0.001). The hazard ratio (HR) for mortality within 14 days in COVID-19 patients with dementia was significant even after PSM (HR 5.104, 95% confidence interval 2.889-5.673, p < 0.001). The survival curve of dementia patients was steeply inclined within 14 days after COVID-19 diagnosis, resulting in 70.7% of all deaths in dementia patients. CONCLUSIONS: COVID-19 patients with dementia had a higher risk of early death within 14 days. Thus, prompt intervention is necessary for dementia patients after COVID-19 diagnosis.

Tonsil-derived mesenchymal stem cells incorporated in reactive oxygen species-releasing hydrogel promote bone formation by increasing the translocation of cell surface GRP78.

Controlling the senescence of mesenchymal stem cells (MSCs) is essential for improving the efficacy of MSC-based therapies. Here, a model of MSC senescence was established by replicative subculture in tonsil-derived MSCs (TMSCs) using senescence-associated ß-galactosidase, telomere-length related genes, stemness, and mitochondrial metabolism. Using transcriptomic and proteomic analyses, we identified glucose-regulated protein 78 (GRP78) as a unique MSC senescence marker. With increasing cell passage number, GRP78 gradually translocated from the cell surface and cytosol to the (peri)nuclear region of TMSCs. A gelatin-based hydrogel releasing a sustained, low level of reactive oxygen species (ROS-hydrogel) was used to improve TMSC quiescence and self-renewal. TMSCs expressing cell surface-specific GRP78 (csGRP78+), collected by magnetic sorting, showed better stem cell function and higher mitochondrial metabolism than unsorted cells. Implantation of csGRP78+ cells embedded in ROS-hydrogel in rats with calvarial defects resulted in increased bone regeneration. Thus, csGRP78 is a promising biomarker of senescent TMSCs, and the combined use of csGRP78+ cells and ROS-hydrogel improved the regenerative capacity of TMSCs by regulating GRP78 translocation.

Three-dimensional culture method enhances the therapeutic efficacies of tonsil-derived mesenchymal stem cells in murine chronic colitis model.

Tonsil-derived mesenchymal stem cells (TMSCs) showed therapeutic effects on acute and chronic murine colitis models, owing to their immunomodulatory properties; therefore, we evaluated enhanced therapeutic effects of TMSCs on a murine colitis model using three-dimensional (3D) culture method. The expression of angiogenic factors, VEGF, and anti-inflammatory cytokines, IL-10, TSG-6, TGF-ß, and IDO-1, was significantly higher in the 3D-TMSC-treated group than in the 2D-TMSC-treated group (P < 0.05). At days 18 and 30 after inducing chronic colitis, disease activity index scores were estimated to be significantly lower in the 3D-TMSC-treated group than in the colitis control (P < 0.001 and P < 0.001, respectively) and 2D-TMSC-treated groups (P = 0.022 and P = 0.004, respectively). Body weight loss was significantly lower in the 3D-TMSC-treated group than in the colitis control (P < 0.001) and 2D-TMSC-treated groups (P = 0.005). Colon length shortening was significantly recovered in the 3D-TMSC-treated group compared to that in the 2D-TMSC-treated group (P = 0.001). Histological scoring index was significantly lower in the 3D-TMSC-treated group than in the 2D-TMSC-treated group (P = 0.002). These results indicate that 3D-cultured TMSCs showed considerably higher therapeutic effects in a chronic murine colitis model than those of 2D-cultured TMSCs via increased anti-inflammatory cytokine expression.

Microstructured Surfaces for Reducing Chances of Fomite Transmission via Virus-Containing Respiratory Droplets.

Evaporation-induced particle aggregation in drying droplets is of significant importance in the prevention of pathogen transfer due to the possibility of indirect fomite transmission of the infectious virus particles. In this study, particle aggregation was directionally controlled using contact line dynamics (pinned or slipping) and geometrical gradients on microstructured surfaces by the systematic investigation of the evaporation process on sessile droplets and sprayed microdroplets laden with virus-simulant nanoparticles. Using this mechanism, we designed robust particle capture surfaces by significantly inhibiting the contact transfer of particles from fomite surfaces. For the proof-of-concept, interconnected hexagonal and inverted pyramidal microwall were fabricated using ultraviolet-based nanoimprint lithography, which is considered to be a promising scalable manufacturing process. We demonstrated the potentials of an engineered microcavity surface to limit the contact transfer of particle aggregates deposited with the evaporation of microdroplets by 93% for hexagonal microwall and by 96% for inverted pyramidal microwall. The particle capture potential of the interconnected microstructures was also investigated using biological particles, including adenoviruses and lung-derived extracellular vesicles. The findings indicate that the proposed microstructured surfaces can reduce the indirect fomite transmission of highly infectious agents, including norovirus, rotavirus, or SARS-CoV-2, via respiratory droplets.

Tonsil-derived mesenchymal stem cells enhance allogeneic bone marrow engraftment via collagen IV degradation.

BACKGROUND: Co-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified. METHODS: Mice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration. RESULTS: Mice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro. CONCLUSIONS: Collectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

Tip-Induced Strain Engineering of a Single Metal Halide Perovskite Quantum Dot.

Strain engineering of perovskite quantum dots (pQDs) enables widely tunable photonic device applications. However, manipulation at the single-emitter level has never been attempted. Here, we present a tip-induced control approach combined with tip-enhanced photoluminescence (TEPL) spectroscopy to engineer strain, bandgap, and the emission quantum yield of a single pQD. Single CsPbBrxI3-x pQDs are clearly resolved through hyperspectral TEPL imaging with ∼10 nm spatial resolution. The plasmonic tip then directly applies pressure to a single pQD to facilitate a bandgap shift up to ∼62 meV with Purcell-enhanced PL increase as high as ∼105 for the strain-induced pQD. Furthermore, by systematically modulating the tip-induced compressive strain of a single pQD, we achieve dynamical bandgap engineering in a reversible manner. In addition, we facilitate the quantum dot coupling for a pQD ensemble with ∼0.8 GPa tip pressure at the nanoscale estimated theoretically. Our approach presents a strategy to tune the nano-opto-electro-mechanical properties of pQDs at the single-crystal level.

Zearalenone Induces Endothelial Cell Apoptosis through Activation of a Cytosolic Ca2+/ERK1/2/p53/Caspase 3 Signaling Pathway.

Zearalenone (ZEN) is a mycotoxin that has been reported to damage various types of cells/tissues, yet its effects on endothelial cells (ECs) have never been investigated. Therefore, this study investigates the potential effects of ZEN using bovine aortic ECs (BAECs). In this study, we found that ZEN induced apoptosis of BAECs through increased cleavage of caspase 3 and poly ADP-ribose polymerase (PARP). ZEN also increased phosphorylation of ERK1/2 and p53, and treatment with the ERK1/2 or p53 inhibitor reversed ZEN-induced EC apoptosis. Transfection of BAECs with small interfering RNA against ERK1/2 or p53 revealed ERK1/2 as an upstream target of p53 in ZEN-stimulated apoptosis. ZEN increased the production of reactive oxygen species (ROS), yet treatment with the antioxidant did not prevent EC apoptosis. Similarly, blocking of estrogen receptors by specific inhibitors also did not prevent ZEN-induced apoptosis. Finally, chelation of cytosolic calcium (Ca2+) using BAPTA-AM or inhibition of endoplasmic reticulum (ER) Ca2+ channel using 2-APB reversed ZEN-induced EC apoptosis, but not by inhibiting ER stress using 4-PBA. Together, our findings demonstrate that ZEN induces EC apoptosis through an ERK1/2/p53/caspase 3 signaling pathway activated by Ca2+ release from the ER, and this pathway is independent of ROS production and estrogen receptor activation.

Far-infrared irradiation inhibits breast cancer cell proliferation independently of DNA damage through increased nuclear Ca2+/calmodulin binding modulated-activation of checkpoint kinase 2.

Far-infrared (FIR) irradiation is reported to inhibit cell proliferation in various types of cancer cells; the underlying mechanism, however, remains unclear. We explored the molecular mechanisms using MDA-MB-231 human breast cancer cells. FIR irradiation significantly inhibited cell proliferation and colony formation compared to hyperthermal stimulus, with no alteration in cell viability. No increase in DNA fragmentation or phosphorylation of DNA damage kinases including ataxia-telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase, and DNA-dependent protein kinase indicated no DNA damage. FIR irradiation increased the phosphorylation of checkpoint kinase 2 (Chk2) at Thr68 (p-Chk2-Thr68) but not that of checkpoint kinase 1 at Ser345. Increased nuclear p-Chk2-Thr68 and Ca2+/CaM accumulations were found in FIR-irradiated cells, as observed in confocal microscopic analyses and cell fractionation assays. In silico analysis predicted that Chk2 possesses a Ca2+/calmodulin (CaM) binding motif ahead of its kinase domain. Indeed, Chk2 physically interacted with CaM in the presence of Ca2+, with their binding markedly pronounced in FIR-irradiated cells. Pre-treatment with a Ca2+ chelator significantly reversed FIR irradiation-increased p-Chk2-Thr68 expression. In addition, a CaM antagonist or small interfering RNA-mediated knockdown of the CaM gene expression significantly attenuated FIR irradiation-increased p-Chk2-Thr68 expression. Finally, pre-treatment with a potent Chk2 inhibitor significantly reversed both FIR irradiation-stimulated p-Chk2-Thr68 expression and irradiation-repressed cell proliferation. In conclusion, our results demonstrate that FIR irradiation inhibited breast cancer cell proliferation, independently of DNA damage, by activating the Ca2+/CaM/Chk2 signaling pathway in the nucleus. These results demonstrate a novel Chk2 activation mechanism that functions irrespective of DNA damage.

Transient receptor potential vanilloid 2 mediates the inhibitory effect of far-infrared irradiation on adipogenic differentiation of tonsil-derived mesenchymal stem cells.

AIMS: Far-infrared (FIR) irradiation inhibits adipogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs) by activating Ca2+-dependent protein phosphatase 2B (PP2B), but it stimulates osteogenic differentiation in a PP2B-independent pathway. We investigated the potential involvement of transient receptor potential vanilloid (TRPV) channels, a well-known Ca2+-permeable channel, in the effects of FIR irradiation on adipogenic or osteogenic differentiation of TMSCs. METHODS: TMSCs, in the absence or presence of activators or inhibitors, were exposed to FIR irradiation followed by adipogenic or osteogenic differentiation, which was assessed using Oil red O or Alizarin red S staining, respectively. RT-PCR, qRT-PCR, and Western blotting were used to determine gene and protein expression of calcium channels and adipocyte-specific markers. RESULTS: Treatment with the calcium ionophore ionomycin simulated the inhibitory effect of FIR irradiation on adipogenic differentiation but had no effect on osteogenic differentiation, indicating the involvement of intracellular Ca2+ in adipogenic differentiation. Inhibition of pan-TRP channels using ruthenium red reversed the FIR irradiation-induced inhibition of adipogenic differentiation. Among the TRP channels tested, inhibition of the TRPV2 channel by tranilast or siRNA against TRPV2 attenuated the inhibitory effect of FIR irradiation on adipogenic differentiation, accompanied by a decrease in intracellular Ca2+ levels. By contrast, activation of the TRPV2 channel by probenecid simulated FIR irradiation-induced inhibition of adipogenic differentiation. Expectedly, the stimulatory effect of FIR irradiation on osteogenic differentiation was independent of the TRPV2 channel. CONCLUSION: Our data demonstrate that the TRPV2 channel is a sensor/receptor for the inhibited adipogenic differentiation of TMSCs associated with FIR irradiation.

Nuclear localization of endothelial nitric oxide synthase and nitric oxide production attenuates aphidicolin-induced endothelial cell death.

Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 µM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser1179 were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca2+ chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso-l-glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser1179, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.