APE1/Ref-1 Inhibits Adipogenic Transcription Factors during Adipocyte Differentiation in 3T3-L1 Cells.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. However, the effect of APE1/Ref-1 on adipogenic transcription factor regulation remains unknown. In this study, we investigated the effect of APE1/Ref-1 on the regulation of adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression significantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-α and peroxisome proliferator-activated receptor (PPAR)-γ, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-α, PPAR-γ, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-α, PPAR-γ, and aP2 during adipocyte differentiation. These results suggest that APE1/Ref-1 inhibits adipocyte differentiation by regulating adipogenic transcription factors, suggesting that APE1/Ref-1 is a potential therapeutic target for regulating adipocyte differentiation.

CRIF1 siRNA-Encapsulated PLGA Nanoparticles Suppress Tumor Growth in MCF-7 Human Breast Cancer Cells.

Mitochondrial oxidative phosphorylation (OXPHOS) system dysfunction in cancer cells has been exploited as a target for anti-cancer therapeutic intervention. The downregulation of CR6-interacting factor 1 (CRIF1), an essential mito-ribosomal factor, can impair mitochondrial function in various cell types. In this study, we investigated whether CRIF1 deficiency induced by siRNA and siRNA nanoparticles could suppress MCF-7 breast cancer growth and tumor development, respectively. Our results showed that CRIF1 silencing decreased the assembly of mitochondrial OXPHOS complexes I and II, which induced mitochondrial dysfunction, mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential depolarization, and excessive mitochondrial fission. CRIF1 inhibition reduced p53-induced glycolysis and apoptosis regulator (TIGAR) expression, as well as NADPH synthesis, leading to additional increases in ROS production. The downregulation of CRIF1 suppressed cell proliferation and inhibited cell migration through the induction of G0/G1 phase cell cycle arrest in MCF-7 breast cancer cells. Similarly, the intratumoral injection of CRIF1 siRNA-encapsulated PLGA nanoparticles inhibited tumor growth, downregulated the assembly of mitochondrial OXPHOS complexes I and II, and induced the expression of cell cycle protein markers (p53, p21, and p16) in MCF-7 xenograft mice. Thus, the inhibition of mitochondrial OXPHOS protein synthesis through CRIF1 deletion destroyed mitochondrial function, leading to elevated ROS levels and inducing antitumor effects in MCF-7 cells.

High Salt Intake Recruits Tonic Activation of NR2D Subunit-Containing Extrasynaptic NMDARs in Vasopressin Neurons.

In addition to producing a classical excitatory postsynaptic current via activation of synaptic NMDA receptors (NMDARs), glutamate in the brain also induces a tonic NMDAR current (INMDA) via activation of extrasynaptic NMDARs (eNMDARs). However, since Mg2+ blocks NMDARs in nondepolarized neurons, the potential contribution of eNMDARs to the overall neuronal excitatory/inhibitory (E/I) balance remains unknown. Here, we demonstrate that chronic (7 d) salt loading (SL) recruited NR2D subunit-containing NMDARs to generate an Mg2+-resistant tonic INMDA in nondepolarized [Vh (holding potential) -70 mV] vasopressin (VP; but not oxytocin) supraoptic nucleus (SON) neurons in male rodents. Conversely, in euhydrated (EU) and 3 d SL mice, Mg2+-resistant tonic INMDA was not observed. Pharmacological and genetic intervention of NR2D subunits blocked the Mg2+-resistant tonic INMDA in VP neurons under SL conditions, while an NR2B antagonist unveiled Mg2+-sensitive tonic INMDA but not Mg2+-resistant tonic INMDA In the EU group VP neurons, an Mg2+-resistant tonic INMDA was not generated by increased ambient glutamate or treatment with coagonists (e.g., d-serine and glycine). Chronic SL significantly increased NR2D expression but not NR2B expression in the SON relative to the EU group or after 3 d under SL conditions. Finally, Mg2+-resistant tonic INMDA selectively upregulated neuronal excitability in VP neurons under SL conditions, independent of ionotropic GABAergic input. Our results indicate that the activation of NR2D-containing NMDARs constitutes a novel mechanism that generates an Mg2+-resistant tonic INMDA in nondepolarized VP neurons, thus causing an E/I balance shift in VP neurons to compensate for the hormonal demands imposed by a chronic osmotic challenge.SIGNIFICANCE STATEMENT The hypothalamic supraoptic nucleus (SON) consists of two different types of magnocellular neurosecretory cells (MNCs) that synthesize and release the following two peptide hormones: vasopressin (VP), which is necessary for regulation of fluid homeostasis; and oxytocin (OT), which plays a major role in lactation and parturition. NMDA receptors (NMDARs) play important roles in shaping neuronal firing patterns and hormone release from the SON MNCs in response to various physiological challenges. Our results show that prolonged (7 d) salt loading generated a Mg2+-resistant tonic NMDA current mediated by NR2D subunit-containing receptors, which efficiently activated nondepolarized VP (but not OT) neurons. Our findings support the hypothesis that NR2D subunit-containing NMDARs play an important adaptive role in adult brain in response to a sustained osmotic challenge.

Dual Function of Secreted APE1/Ref-1 in TNBC Tumorigenesis: An Apoptotic Initiator and a Regulator of Chronic Inflammatory Signaling.

The simultaneous regulation of cancer cells and inflammatory immune cells in the tumor microenvironment (TME) can be an effective strategy in treating aggressive breast cancer types, such as triple-negative breast cancer (TNBC). Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multi-functional nuclear protein that can be stimulated and then secreted. The extracellular APE1/Ref-1 causes a reduction in disulfide bonds in cytokine receptors, resulting in their conformational changes, thereby inhibiting inflammatory signaling. Furthermore, the secreted APE1/Ref-1 in response to acetylation has been shown to bind to a receptor for the advanced glycation end product (RAGE), initiating the apoptotic cell death of TNBC in vitro and in vivo. This study used PPTLS-APE1/Ref-1 in an adenovirus vector (Ad-PPTLS-APE1/Ref-1) for the constant expression of extracellular APE1/Ref-1, and our results demonstrated its dual function as an apoptotic initiator and inflammation regulator. Injecting MDA-MB 231 orthotopic xenografts with the Ad-PPTLS-APE1/Ref-1 inhibited tumor growth and development in response to acetylation. Moreover, Ad-PPTLS-APE1/Ref-1 generated reactive oxygen species (ROS), and tumor tissues derived from these xenografts exhibited apoptotic bodies. Compared to normal mice, a comparable ratio of anti- and pro-inflammatory cytokines was observed in the plasma of Ad-PPTLS-APE1/Ref-1-injected mice. Mechanistically, the disturbed cytokine receptor by reducing activity of PPTLS-APE1/Ref-1 inhibited inflammatory signaling leading to the inactivation of the p21-activated kinase 1-mediated signal transducer and activator of transcription 3/nuclear factor-κB axis in tumor tissues. These results suggest that the regulation of inflammatory signaling with adenoviral-mediated PPTLS-APE1/Ref-1 in tumors modulates the secretion of pro-inflammatory cytokines in TME, thereby inhibiting aggressive cancer cell progression, and could be considered as a promising and safe therapeutic strategy for treating TNBCs.

Ref-1 protects against FeCl3-induced thrombosis and tissue factor expression via the GSK3ß-NF-κB pathway.

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl3 solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl3 aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3ß-NF-κB signaling pathway. Ref1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.

Bestrophin1-mediated tonic GABA release from reactive astrocytes prevents the development of seizure-prone network in kainate-injected hippocampi.

Tonic extrasynaptic GABAA receptor (GABAA R) activation is under the tight control of tonic GABA release from astrocytes to maintain the brain's excitation/inhibition (E/I) balance; any slight E/I balance disturbance can cause serious pathological conditions including epileptic seizures. However, the pathophysiological role of tonic GABA release from astrocytes has not been tested in epileptic seizures. Here, we report that pharmacological or genetic intervention of the GABA-permeable Bestrophin-1 (Best1) channel prevented the generation of tonic GABA inhibition, disinhibiting CA1 pyramidal neuronal firing and augmenting seizure susceptibility in kainic acid (KA)-induced epileptic mice. Astrocyte-specific Best1 over-expression in KA-injected Best1 knockout mice fully restored the generation of tonic GABA inhibition and effectively suppressed seizure susceptibility. We demonstrate for the first time that tonic GABA from reactive astrocytes strongly contributes to the compensatory shift of E/I balance in epileptic hippocampi, serving as a good therapeutic target against altered E/I balance in epileptic seizures.

CRIF1 deficiency induced mitophagy via p66shc-regulated ROS in endothelial cells.

Inhibition of mitochondrial protein CR6 interacting factor 1 (CRIF1) disturbs mitochondrial function, depolarizes membrane potential, and increases reactive oxygen species (ROS) levels in endothelial cells. Impaired mitochondrial function accompanied by oxidative damage is a major contributor to the initiation of mitophagy. We hypothesized that CRIF1 deficiency-induced harmful effects may promote mitophagy, and explored the mechanism underlying this effect in human umbilical vein endothelial cells (HUVECs). Our results showed that CRIF1 downregulation not only induced the mitophagy-related markers LC3 (LC3-II/Ⅰ), PTEN-induced putative kinase 1 (PINK1) and parkin, but also stimulated redox enzyme p66shc expression. Scavenging mitochondrial ROS markedly blunted the CRIF1 deficiency-induced increase in p66shc expression. In addition, knockdown of p66shc inhibited the CRIF1 deletion-triggered mitochondrial ROS increase, membrane potential depolarization, and mitochondrial fusion. The restoration of mitochondrial dysfunction by p66shc downregulation also decreased CRIF1 deficiency-induced mitophagy, by elevating the levels of LC3-II/Ⅰ, PINK1 and parkin. These findings suggest that CRIF1 deficiency induces mitophagy via p66shc-regulated ROS in endothelial cells.

Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions.

Elevated expression of human enhancer filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors. This process requires HEF1 localization to focal adhesions (FAs). Although the association of HEF1 with FAs is considered to play a role in cancer cell migration, the mechanism targeting HEF1 to FAs remains unclear. Moreover, up-regulation of Polo-like kinase 1 (Plk1) positively correlates with human cancer metastasis, yet how Plk1 deregulation promotes metastasis remains elusive. Here, we report that casein kinase 1δ (CK1δ) phosphorylates HEF1 at Ser-780 and Thr-804 and that these phosphorylation events promote a physical interaction between Plk1 and HEF1. We found that this interaction is critical for HEF1 translocation to FAs and for inducing migration of HeLa cells. Plk1-docking phosphoepitopes were mapped/confirmed in HEF1 by various methods, including X-ray crystallography, and mutated for functional analysis in HeLa cells. In summary, our results reveal the role of a phosphorylation-dependent HEF1-Plk1 complex in HEF1 translocation to FAs to induce cell migration. Our findings provide critical mechanistic insights into the HEF1-Plk1 complex-dependent localization of HEF1 to FAs underlying the metastatic process and may therefore contribute to the development of new cancer therapies.

ATP Binding Cassette Transporter A1 is Involved in Extracellular Secretion of Acetylated APE1/Ref-1.

Acetylation of nuclear apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is associated with its extracellular secretion, despite the lack of an N-terminal protein secretion signal. In this study, we investigated plasma membrane targeting and translocation of APE1/Ref-1 in HEK293T cells with enhanced acetylation. While APE1/Ref-1 targeting was not affected by inhibition of the endoplasmic reticulum/Golgi-dependent secretion, its secretion was reduced by inhibitors of ATP-binding cassette (ABC) transporters, and siRNA-mediated down-regulation of ABC transporter A1. The association between APE1/Ref-1 and ABCA1 transporter was confirmed by proximal ligation assay and immunoprecipitation experiments. An APE1/Ref-1 construct with mutated acetylation sites (K6/K7R) showed reduced co-localization with ABC transporter A1. Exposure of trichostatin A (TSA) induced the acetylation of APE1/Ref-1, which translocated into membrane fraction. Taken together, acetylation of APE1/Ref-1 is considered to be necessary for its extracellular targeting via non-classical secretory pathway using the ABCA1 transporter.

Isocitrate dehydrogenase 2 deficiency induces endothelial inflammation via p66sh-mediated mitochondrial oxidative stress.

Isocitrate dehydrogenase 2 (IDH2) is an essential enzyme in the mitochondrial antioxidant system, which produces nicotinamide adenine dinucleotide phosphate, and thereby defends against oxidative stress. We have shown that IDH2 downregulation results in mitochondrial dysfunction and reactive oxygen species (ROS) generation in mouse endothelial cells. The redox enzyme p66shc is a key factor in regulating the level of ROS in endothelial cells. In this study, we hypothesized that IDH2 knockdown-induced mitochondrial dysfunction stimulates endothelial inflammation, which might be regulated by p66shc-mediated oxidative stress. Our results showed that IDH2 downregulation led to mitochondrial dysfunction by decreasing the expression of mitochondrial oxidative phosphorylation complexes I, II, and IV, reducing oxygen consumption, and depolarizing mitochondrial membrane potential in human umbilical vein endothelial cells (HUVECs). The dysfunction not only increased mitochondrial ROS levels but also activated p66shc expression in HUVECs and IDH2 knockout mice. IDH2 deficiency increased intercellular adhesion molecule (ICAM)-1 expression and mRNA levels of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, and interleukin [IL]-1ß) in HUVECs. The mRNA expression of ICAM-1 in endothelial cells and plasma levels of TNF-α and IL-1ß were also markedly elevated in IDH2 knockout mice. However, p66shc knockdown rescued IDH2 deficiency-induced mitochondrial ROS levels, monocyte adhesion, ICAM-1, TNF-α, and IL-1ß expression in HUVECs. These findings suggest that IDH2 deficiency induced endothelial inflammation via p66shc-mediated mitochondrial oxidative stress.

Angiotensin II facilitates neointimal formation by increasing vascular smooth muscle cell migration: Involvement of APE/Ref-1-mediated overexpression of sphingosine-1-phosphate receptor 1.

Angiotensin II (Ang II) is implicated in the development of cardiovascular disorders including hypertension and atherosclerosis. However, the role of Ang II in the interaction between apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) and sphingosine-1-phosphate (S1P) signals in relation to vascular disorders remains to be clarified. This study aimed to determine whether APE/Ref-1 plays a role in epigenetic regulation of the S1P receptor (S1PR) in response to Ang II in vascular smooth muscle cell (VSMC) migration and vascular neointima formation. Ang II augmented the expression of S1PR1 in aortic smooth muscle cells of Sprague Dawley rats (RASMCs), which was attenuated by Ang II receptor (AT) 1 inhibitors, antioxidants, and APE/Ref-1 knockdown with small interference RNA. Ang II stimulation produced H2O2, and exogenous H2O2 elevated S1PR1 expression in RASMCs. Moreover, Ang II caused translocation of cytoplasmic APE/Ref-1 into the nucleus in RASMCs. H3 histone acetylation and APE/Ref-1 binding at the S1PR1 promoter were increased in RASMCs treated with Ang II. In addition, Ang II induced migration in RASMCs, which was suppressed by AT1 and S1PR1 inhibitors. The expression of S1PR1, and colocalization of APE/Ref-1 and acetylated histone H3 in vascular neointima, were greater in Ang II-infused rats compared with a control group. These findings demonstrate that Ang II stimulates the epigenetic regulation of S1PR1 expression via H2O2-mediated APE/Ref-1 translocation, which may consequently be involved in Ang II-induced VSMC migration and vascular neointima formation. Therefore, APE/Ref-1-mediated overexpression of S1PR1 may be implicated in the vascular dysfunction evoked by Ang II.

Anthocyanin-Rich Extract from Red Chinese Cabbage Alleviates Vascular Inflammation in Endothelial Cells and Apo E-/- Mice.

Anthocyanins, the most prevalent flavonoids in red/purple fruits and vegetables, are known to improve immune responses and reduce chronic disease risks. In this study, the anti-inflammatory activities of an anthocyanin-rich extract from red Chinese cabbage (ArCC) were shown based on its inhibitory effects in cultured endothelial cells and hyperlipidemic apolipoprotein E-deficient mice. ArCC treatment suppressed monocyte adhesion to tumor necrosis factor-α-stimulated endothelial cells. This was validated by ArCC's ability to downregulate the expression and transcription of endothelial adhesion molecules, determined by immunoblot and luciferase promoter assays, respectively. The regulation of adhesion molecules was accompanied by transcriptional inhibition of nuclear factor-κB, which restricted cytoplasmic localization as shown by immunocytochemistry. Administration of ArCC (150 or 300 mg/kg/day) inhibited aortic inflammation in hyperlipidemic apolipoprotein E-deficient mice, as shown by in vivo imaging. Immunohistochemistry and plasma analysis showed that the aortas from these mice exhibited markedly lower leukocyte infiltration, reduced plaque formation, and lower concentrations of blood inflammatory cytokines than those observed in the control mice. The results suggest that the consumption of anthocyanin-rich red Chinese cabbage is closely correlated with lowering the risk of vascular inflammatory diseases.

APE1/Ref-1 Inhibits Phosphate-Induced Calcification and Osteoblastic Phenotype Changes in Vascular Smooth Muscle Cells.

Vascular calcification plays a role in the pathogenesis of atherosclerosis, diabetes, and chronic kidney disease; however, the role of apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) in inorganic phosphate (Pi)-induced vascular smooth muscle cell (VSMC) calcification remains unknown. In this study, we investigated the possible role of APE1/Ref-1 in Pi-induced VSMC calcification. We observed that Pi decreased endogenous APE1/Ref-1 expression and promoter activity in VSMCs, and that adenoviral overexpression of APE1/Ref-1 inhibited Pi-induced calcification in VSMCs and in an ex vivo organ culture of a rat aorta. However, a redox mutant of APE1/Ref-1(C65A/C93A) did not reduce Pi-induced calcification in VSMCs, suggesting APE1/Ref-1-mediated redox function against vascular calcification. Additionally, APE1/Ref-1 overexpression inhibited Pi-induced intracellular and mitochondrial reactive oxygen species production, and APE1/Ref-1 overexpression resulted in decreased Pi-induced lactate dehydrogenase activity, pro-apoptotic Bax levels, and increased anti-apoptotic Bcl-2 protein levels. Furthermore, APE1/Ref-1 inhibited Pi-induced osteoblastic differentiation associated with alkaline phosphatase activity and inhibited Pi-exposure-induced loss of the smooth muscle phenotype. Our findings provided valuable insights into the redox function of APE1/Ref-1 in preventing Pi-induced VSMC calcification by inhibiting oxidative stress and osteoblastic differentiation, resulting in prevention of altered osteoblastic phenotypes in VSMCs.

Elevation of Serum APE1/Ref-1 in Experimental Murine Myocarditis.

Myocarditis is an inflammatory disease of the myocardium that causes cardiogenic shock and death. However, endomyocardial biopsy that is, the gold standard for a diagnosis is limited. Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a multifunctional protein, which is involved in DNA-based excision repair pathway, and in redox signaling, its changes are observed in various cardiovascular diseases including hypertension and coronary artery disease. We analyzed serum APE1/Ref-1 in experimental murine myocarditis. To induce myocarditis, coxsackievirus B3 was injected intraperitoneally to BALB/c mice. The serum APE1/Ref-1, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and troponin I were measured. The histology and virus titers measurements were performed. The troponin I and inflammation were significantly elevated at day 3, peaked to day 7 and decreased at day 10. The NT-proBNP and virus titers were significantly peaked at day 3, and dropped at day 7 and 10. The serum APE1/Ref-1 was gradually raised and its elevation is still maintained until a later time, namely day 10. Also, its level was positively correlated with myocardial inflammation, reflecting severity of myocardial injury. We suggest that serum APE1/Ref-1 can be used to assess for myocardial injury in viral myocarditis without endomyocardial biopsy.

Protein kinase C beta II upregulates intercellular adhesion molecule-1 via mitochondrial activation in cultured endothelial cells.

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCßII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCßII gene transfer and pharmacological inhibitors, the role of PKCßII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCßi (10 nM), a selective inhibitor of PKCßII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCßi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCßII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCßII using adenoviral PKCßII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCßII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCßII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCßII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.

Deficiency of endothelium-specific transcription factor Sox17 induces intracranial aneurysm.

BACKGROUND: Intracranial aneurysm (IA) is a common vascular disorder that frequently leads to fatal vascular rupture. Although various acquired risk factors associated with IA have been identified, the hereditary basis of IA remains poorly understood. As a result, genetically modified animals accurately modeling IA and related pathogenesis have been lacking, and subsequent drug development has been delayed. METHODS AND RESULTS: The transcription factor Sox17 is robustly expressed in endothelial cells of normal intracerebral arteries. The combination of Sox17 deficiency and angiotensin II infusion in mice induces vascular abnormalities closely resembling the cardinal features of IA such as luminal dilation, wall thinning, tortuosity, and subarachnoid hemorrhages. This combination impairs junctional assembly, cell-matrix adhesion, regeneration capacity, and paracrine secretion in endothelial cells of intracerebral arteries, highlighting key endothelial dysfunctions that lead to IA pathogenesis. Moreover, human IA samples showed reduced Sox17 expression and impaired endothelial integrity, further strengthening the applicability of this animal model to clinical settings. CONCLUSIONS: Our findings demonstrate that Sox17 deficiency in mouse can induce IA under hypertensive conditions, suggesting Sox17 deficiency as a potential genetic factor for IA formation. The Sox17-deficient mouse model provides a novel platform to develop therapeutics for incurable IA.

TonEBP suppresses adipocyte differentiation via modulation of early signaling in 3T3-L1 cells.

TonEBP belongs to the Rel family of transcription factors and plays important roles in inflammation as well as kidney homeostasis. Recent studies suggest that TonEBP expression is also involved in differentiation of several cell types such as myocytes, chondrocytes, and osteocytes. In this study, we investigated the roles of TonEBP during adipocyte differentiation in 3T3-L1 cells. TonEBP mRNA and protein expression was dramatically reduced during adipocyte differentiation. Sustained expression of TonEBP using an adenovirus suppressed the formation of lipid droplets as well as the expression of FABP4, a marker of differentiated adipocytes. TonEBP also inhibited the expression of PPARγ, a known master regulator of adipocytes. RNAi-mediated knock down of TonEBP promoted adipocyte differentiation. However, overexpression of TonEBP did not affect adipogenesis after the initiation of differentiation. Furthermore, TonEBP expression suppressed mitotic clonal expansion and insulin signaling, which are required early for adipocyte differentiation of 3T3-L1 cells. These results suggest that TonEBP may be an important regulatory factor in the early phase of adipocyte differentiation.

Nafamostat mesilate promotes endothelium-dependent vasorelaxation via the Akt-eNOS dependent pathway.

Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has anticoagulant and anti-inflammatory properties. The intracellular mediator and external anti-inflammatory external signal in the vascular wall have been reported to protect endothelial cells, in part due to nitric oxide (NO) production. This study was designed to examine whether NM exhibit endothelium dependent vascular relaxation through Akt/endothelial nitric oxide synthase (eNOS) activation and generation of NO. NM enhanced Akt/eNOS phosphorylation and NO production in a dose- and time-dependent manner in human umbilical vein endothelial cells (HUVECs) and aorta tissues obtained from rats treated with various concentrations of NM. NM concomitantly decreased arginase activity, which could increase the available arginine substrate for NO production. Moreover, we investigated whether NM increased NO bioavailability and decreased aortic relaxation response to an eNOS inhibitor in the aorta. These results suggest that NM increases NO generation via the Akt/eNOS signaling pathway, leading to endothelium-dependent vascular relaxation. Therefore, the vasorelaxing action of NM may contribute to the regulation of cardiovascular function.

Facilitation of AMPA receptor-mediated steady-state current by extrasynaptic NMDA receptors in supraoptic magnocellular neurosecretory cells.

In addition to classical synaptic transmission, information is transmitted between cells via the activation of extrasynaptic receptors that generate persistent tonic current in the brain. While growing evidence supports the presence of tonic NMDA current (INMDA) generated by extrasynaptic NMDA receptors (eNMDARs), the functional significance of tonic INMDA in various brain regions remains poorly understood. Here, we demonstrate that activation of eNMDARs that generate INMDA facilitates the α-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptor (AMPAR)-mediated steady-state current in supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs). In low-Mg(2+) artificial cerebrospinal fluid (aCSF), glutamate induced an inward shift in Iho lding (IGLU) at a holding potential (Vholding) of -70 mV which was partly blocked by an AMPAR antagonist, NBQX. NBQX-sensitive IGLU was observed even in normal aCSF at Vholding of -40 mV or -20 mV. IGLU was completely abolished by pretreatment with an NMDAR blocker, AP5, under all tested conditions. AMPA induced a reproducible inward shift in Iholding (IAMPA) in SON MNCs. Pretreatment with AP5 attenuated IAMPA amplitudes to ~60% of the control levels in low-Mg(2+) aCSF, but not in normal aCSF at Vholding of -70 mV. IAMPA attenuation by AP5 was also prominent in normal aCSF at depolarized holding potentials. Memantine, an eNMDAR blocker, mimicked the AP5-induced IAMPA attenuation in SON MNCs. Finally, chronic dehydration did not affect IAMPA attenuation by AP5 in the neurons. These results suggest that tonic INMDA, mediated by eNMDAR, facilitates AMPAR function, changing the postsynaptic response to its agonists in normal and osmotically challenged SON MNCs.

Enhanced astroglial GABA uptake attenuates tonic GABAA inhibition of the presympathetic hypothalamic paraventricular nucleus neurons in heart failure.

γ-Aminobutyric acid (GABA) generates persistent tonic inhibitory currents (Itonic) and conventional inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus (PVN) via activation of GABAA receptors (GABAARs). We investigated the pathophysiological significance of astroglial GABA uptake in the regulation of Itonic in the PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM). The Itonic of PVN-RVLM neurons were significantly reduced in heart failure (HF) compared with sham-operated (SHAM) rats. Reduced Itonic sensitivity to THIP argued for the decreased function of GABAAR δ subunits in HF, whereas similar Itonic sensitivity to benzodiazepines argued against the difference of γ2 subunit-containing GABAARs in SHAM and HF rats. HF Itonic attenuation was reversed by a nonselective GABA transporter (GAT) blocker (nipecotic acid, NPA) and a GAT-3 selective blocker, but not by a GAT-1 blocker, suggesting that astroglial GABA clearance increased in HF. Similar and minimal Itonic responses to bestrophin-1 blockade in SHAM and HF neurons further argued against a role for astroglial GABA release in HF Itonic attenuation. Finally, the NPA-induced inhibition of spontaneous firing was greater in HF than in SHAM PVN-RVLM neurons, whereas diazepam induced less inhibition of spontaneous firing in HF than in SHAM neurons. Overall, our results showed that combined with reduced GABAARs function, the enhanced astroglial GABA uptake-induced attenuation of Itonic in HF PVN-RVLM neurons explains the deficit in tonic GABAergic inhibition and increased sympathetic outflow from the PVN during heart failure.