Development of a Novel Biomarker for the Progression of Idiopathic Pulmonary Fibrosis.

The progression of idiopathic pulmonary fibrosis (IPF) is diverse and unpredictable. We identified and validated a new biomarker for IPF progression. To identify a candidate gene to predict progression, we assessed differentially expressed genes in patients with advanced IPF compared with early IPF and controls in three lung sample cohorts. Candidate gene expression was confirmed using immunohistochemistry and Western blotting of lung tissue samples from an independent IPF clinical cohort. Biomarker potential was assessed using an enzyme-linked immunosorbent assay of serum samples from the retrospective validation cohort. We verified that the final candidate gene reflected the progression of IPF in a prospective validation cohort. In the RNA-seq comparative analysis of lung tissues, CD276, COL7A1, CTSB, GLI2, PIK3R2, PRAF2, IGF2BP3, and NUPR1 were up-regulated, and ADAMTS8 was down-regulated in the samples of advanced IPF. Only CTSB showed significant differences in expression based on Western blotting (n = 12; p < 0.001) and immunohistochemistry between the three groups of the independent IPF cohort. In the retrospective validation cohort (n = 78), serum CTSB levels were higher in the progressive group (n = 25) than in the control (n = 29, mean 7.37 ng/mL vs. 2.70 ng/mL, p < 0.001) and nonprogressive groups (n = 24, mean 7.37 ng/mL vs. 2.56 ng/mL, p < 0.001). In the prospective validation cohort (n = 129), serum CTSB levels were higher in the progressive group than in the nonprogressive group (mean 8.30 ng/mL vs. 3.00 ng/mL, p < 0.001). After adjusting for baseline FVC, we found that CTSB was independently associated with IPF progression (adjusted OR = 2.61, p < 0.001). Serum CTSB levels significantly predicted IPF progression (AUC = 0.944, p < 0.001). Serum CTSB level significantly distinguished the progression of IPF from the non-progression of IPF or healthy control.

CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames.

Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

Initial Tumor Necrosis Factor-Alpha and Endothelial Activation Are Associated with Hemorrhagic Complications during Extracorporeal Membrane Oxygenation.

Studies on inflammatory markers, endothelial activation, and bleeding during extracorporeal membrane oxygenation (ECMO) are lacking. Blood samples were prospectively collected after ECMO initiation from 150 adult patients who underwent ECMO for respiratory failure between 2018 and 2021. After excluding patients who died early (within 48 h), 132 patients were finally included. Their tumor necrosis factor-alpha (TNF-α), tissue factor (TF), soluble thrombomodulin (sTM), and E-selectin levels were measured. A Cox proportional hazards regression model was used to estimate the hazard ratio for hemorrhagic complications during ECMO. The 132 patients were divided into hemorrhagic (n = 23, H group) and non-complication (n = 109, N group) groups. The sequential organ failure assessment score, hemoglobin level, and ECMO type were included as covariates in all Cox models to exclude the effects of clinical factors. After adjusting for these factors, initial TNF-α, TF, sTM, E-selectin, and activated protein C levels were significantly associated with hemorrhagic complications (all p < 0.001). TNF-α, TF, and E-selectin better predicted hemorrhagic complications than the model that included only the aforementioned clinical factors (clinical factors only (area under the curve [AUC]: 0.804), reference; TNF-α (AUC: 0.914); TF (AUC: 0.915); E-selectin (AUC: 0.869)). Conclusions: TNF-α levels were significantly predictive of hemorrhagic complications during ECMO.

Cobalt-molybdenum-selenide nanoflowers for bifunctional visible light photocatalysis.

The renewability and zero carbon emissions of hydrogen make it a promising clean energy resource to meet future energy demands. Owing to its benefits, photocatalytic water-splitting has been extensively investigated for hydrogen production. However, the low efficiency poses a serious challenge to its implementation. Herein, we attempted to synthesize bimetallic transition metal selenides, namely Co/Mo/Se (CMS) photocatalysts, with varying atomic compositions (CMSa, CMSb, and CMSc) and investigated their photocatalytic water splitting efficiencies. The observed hydrogen evolution rates were as follows: 134.88 µmol g-1 min-1 for CoSe2, 145.11 µmol g-1 min-1 for MoSe2, 167.31 µmol g-1 min-1 for CMSa, 195.11 µmol g-1 min-1 for CMSb, and 203.68 µmol g-1 min-1 for CMSc. Hence, we deemed CMSc to be the most potent photocatalytic alternative among the compounds. CMSc was also tested for its efficiency towards degradation of triclosan (TCN), and results substantiated that CMSc succeeded degrading 98% TCN while CMSa and b were able to degrade 80 and 90% TCN respectively-the attained efficiency being exponentially higher than CoSe2 and MoSe2 taken for comparative analysis in addition to complete degradation of the pollutants leaving no harmful intermediaries during the process. Thus, CMSc shall be identified as a highly potential photocatalyst with respect to both environmental and energy applications.

Feasibility and acceptability of a digital tele-guided intervention targeting components of the addictive appetite model for bulimia nervosa and binge-eating disorder in Korea.

OBJECTIVE: We aimed to evaluate the feasibility, acceptability, and potential impact of a tele-guided digital-based intervention based on the addictive appetite model of recurrent binge eating. METHOD: Female college students with bulimia nervosa (BN) or binge-eating disorder (BED) (n = 22) received a 6-week guided intervention targeting addictive processes and emotion regulation. The feasibility of the intervention was evaluated, and the outcomes were assessed at baseline, the end of the intervention, and 1-month follow-up. RESULTS: Of the participants, 86.4% (n = 19) completed the intervention. The self-help materials were viewed 6.03 ± 3.06 times per week, and the duration of using the self-help materials was 113.16 ± 160.19 min/week. The intervention group experienced a significant reduction with a moderate effect on binge eating at the end of the intervention (Hedges' g = 0.58), and the effects lasted through follow-up (Hedges' g = 0.82). DISCUSSION: The results suggest that the digital intervention targeting a maintenance mechanism of recurrent binge eating was feasible and acceptable for patients with BN and BED, proving the potential for symptom improvement. PUBLIC SIGNIFICANCE: The addictive appetite model provides the framework for new interventions to improve treatments for BN and BED. This study found that the digital intervention based on the model was feasible and acceptable for patients with BN and BED.

Components of engagement in saying-is-believing exercises.

The saying-is-believing effect is an important step for changing students' attitudes and beliefs in a wise intervention. However, most studies have not closely examined the process of the saying-is-believing effect when individuals are engaged in the activity. Using a qualitative approach, the present study uses an engagement framework to investigate (a) components of engagement in the saying-is-believing effect; and (b) how differently students may engage in a saying-is-believing exercise. Semi-structured interviews were conducted with 14 undergraduates in a scholarship program for low-income transfer students from community college. Analysis using inductive and deductive approaches found that students varied on the extent to which they experienced the effectiveness of the saying-is-believing effect through affective, cognitive, and behavioral experiences. The study offers examples of how people can indeed differ in the extent to which they experience the saying-is-believing effect, and the implications for designing more effective interventions. Specifically, students' positive affective experiences from seeing the larger goal of creating videos may be important components for the saying-is-believing effect to work. Behavioral experiences, such as learning soft skills, academic skills learned indirectly from the intervention, and academic skills learned directly from the intervention were accompanied by both positive affective and cognitive experiences. Findings show the importance of students' differential engagement in saying-is-believing exercises both for building more effective wise interventions and interpreting heterogeneity in intervention effectiveness.

Social Isolation, Loneliness and Their Relationships with Mental Health Status in South Korea.

OBJECTIVE: This study aims to investigate the prevalence and psychological impact of social isolation and loneliness in South Korea. Loneliness and social isolation have been regarded as a risk to both physical and mental health. However, most studies have focused on the elderly; hence, there are limited studies on the characteristics of socially isolated or lonely people considering age. METHODS: A sample of 1,700 participants was selected from three major cities in South Korea. In-person interviews were conducted to evaluate loneliness, social isolation and mental health status. RESULTS: Among the participants, the prevalence of social isolation and loneliness was 17.8% and 4.1%, respectively. Males decreased the odds of loneliness (AOR 0.49, 95% CI=0.28-0.87), while increasing the odds of social isolation (AOR 1.44, 95% CI=1.12-1.86) after adjusting for age and sex. Greater depressive and social phobic symptoms were associated with increased odds of loneliness and social isolation. CONCLUSION: Social isolation and loneliness are prevalent among Koreans and associated with depression, social phobic symptoms, and suicidality. This study provides a foundation for further research to investigate nationwide prevalence and a more in-depth analysis of loneliness and social isolation.

Development and validation of the Korean version of the Reading the Mind in the Eyes Test.

The Reading the Mind in the Eyes Test (RMET) is one of the most widely used instruments for assessing the ability to recognize emotion. To examine the psychometric properties of the Korean version of the RMET and to explore the possible implications of poor performance on this task, 200 adults aged 19-32 years completed the RMET and the Korean version of the 20-item Toronto Alexithymia Scale (TAS-20K), the cognitive empathy domain of the Korean version of the Interpersonal Reactivity Index (IRI-C), and the Buss-Durkee Hostility Inventory-Aggression (BDHI-A). In the present study, confirmatory factor analyses confirmed that the hypothesized three-factor solution based on three different emotional valences of the items (positive, negative, or neutral) had a good fit to the data. The Korean version of the RMET also showed good test-retest reliability over a 4-week time interval. Convergent validity was also supported by significant correlations with subscales of the TAS-20K, and discriminant validity was identified by nonsignificant associations with IRI-C scores. In addition, no difference was found in RMET performance according to the sex of the photographed individuals or the sex or educational attainment of the participants. Individuals with poor RMET performance were more likely to experience alexithymia and aggression. The current findings will facilitate not only future research on emotion processing but also the assessment of conditions related to the decreased ability to decode emotional stimuli.

Public Acceptance of a Health Information Exchange in Korea.

OBJECTIVES: We assessed the public acceptance of a health information exchange (HIE) and examined factors that influenced the acceptance and associations among constructs of the Technology Acceptance Model (TAM). METHODS: We collected data from a survey of 1,000 individuals in Korea, which was administered through a structured questionnaire. We assessed the validity and reliability of the survey instrument with exploratory factor analysis and Cronbach's alpha coefficients. We computed descriptive statistics to assess the acceptance and performed regression analyses with a structural equation model to estimate the magnitude and significance of influences among constructs of TAM. RESULTS: Eighty-seven percent of the respondents were willing to use the technology, and the average level of agreement with the need for the technology was 4.16 on a 5-point Likert scale. The perception of ease of use of the technology significantly influenced perceptions of usefulness and attitudes about the need for HIE. Perceptions of usefulness influenced attitude and behavioral intention to use HIE, and attitude influenced intention. Age showed a wide range of influences throughout the model, and experience with offline-based information exchange and health status also showed noteworthy influences. CONCLUSIONS: The public acceptance of HIE was high, and influences posited by TAM were mostly confirmed by the study results. The study findings indicated a need for an education and communication strategy tailored by population age, health status, and prior experience with offline-based exchange to gain public buy-in for a successful introduction of the technology.

Preparation of glutaraldehyde-treated lipase-inorganic hybrid nanoflowers and their catalytic performance as immobilized enzymes.

The use of protein-inorganic hybrid nanoflowers for the immobilization of enzymes has received a significant degree of attention owing to their capability to retain high enzymatic activity and stability. However, the relative lack of reusability due to the weakness of the flower-like structure has limited their practical applications. Herein, we have developed a simple but efficient method to synthesize highly robust enzyme-inorganic hybrid nanoflowers, which relies on further crosslinking of the enzyme molecules entrapped in the hybrid nanoflowers by treatment with glutaraldehyde (GA). By employing lipase from Candida rugosa as a model enzyme with copper phosphate during 3days incubation followed by the additional GA treatment for only 1h, we could successfully synthesize GA-treated lipase nanoflowers having similar flower-like morphology and hydrolytic activity (ca. 95% compared with the free lipase) as conventionally synthesized lipase nanoflowers without GA treatment. Importantly, the conventional lipase nanoflowers seemed not to be reusable because they lost most of their activity (∼90%) after recycling 4 times, whereas GA-treated lipase nanoflowers exhibited higher retention of their initial activity (over 70%) after 4 reuses, which was also accompanied by an efficient maintenance of their flower-like morphology. Based on our results, we expect that this simple GA-mediated strategy to synthesize enzyme-inorganic hybrid nanoflowers can be readily extended to other enzymes for various biotechnological applications.

Cilostazol Modulates Autophagic Degradation of ß-Amyloid Peptide via SIRT1-Coupled LKB1/AMPKα Signaling in Neuronal Cells.

A neuroprotective role of autophagy mediates the degradation of ß-amyloid peptide (Aß) in Alzheimer's disease (AD). The previous study showed cilostazol modulates autophagy by increasing beclin1, Atg5 and LC3-II expressions, and depletes intracellular Aß accumulation. This study elucidated the mechanisms through which cilostazol modulates the autophagic degradation of Aß in neurons. In N2a cells, cilostazol (10-30 µM), significantly increased the expression of P-AMPKα (Thr 172) and downstream P-ACC (acetyl-CoA carboxylase) (Ser 79) as did resveratrol (SIRT1 activator), or AICAR (AMPK activator), which were blocked by KT5720, compound C (AMPK inhibitor), or sirtinol. Furthermore, phosphorylated-mTOR (Ser 2448) and phosphorylated-P70S6K (Thr 389) expressions were suppressed, and LC3-II levels were elevated in association with decreased P62/Sqstm1 by cilostazol. Cilostazol increased cathepsin B activity and decreased p62/SQSTM 1, consequently decreased accumulation of Aß1-42 in the activated N2aSwe cells, and these results were blocked by sirtinol, compound C and bafilomycin A1 (autophagosome blocker), suggesting enhanced autophagosome formation by cilostazol. In SIRT1 gene-silenced N2a cells, cilostazol failed to increase the expressions of P-LKB1 (Ser 428) and P-AMPKα, which contrasted with its effect in negative control cells transfected with scrambled siRNA duplex. Further, N2a cells transfected with expression vectors encoding pcDNA SIRT1 showed increased P-AMPKα expression, which mimicked the effect of cilostazol in N2a cells; suggesting cilostazol-stimulated expressions of P-LKB1 and P-AMPKα were SIRT1-dependent. Unlike their effects in N2a cells, in HeLa cells, which lack LKB1, cilostazol and resveratrol did not elevate SIRT1 or P-AMPKα expression, indicating cilostazol and resveratrol-stimulated expressions of SIRT1 and P-AMPKα are LKB1-dependent. In conclusion, cilostazol upregulates autophagy by activating SIRT1-coupled P-LKB1/P-AMPKα and inhibiting mTOR activation, thereby decreasing Aß accumulation.

Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-ß Peptide and APP-CTFß Levels in Neuronal Cells.

Autophagy is a vital pathway for the removal of ß-amyloid peptide (Aß) and the aggregated proteins that cause Alzheimer's disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aß and C-terminal APP fragment ß subunit (APP-CTFß) by up-regulating autophagy.When N2a cells were exposed to soluble Aß1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 µM) or resveratrol (20 µM) prevented these Aß1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aß overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aß and APP-CTFß expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aß and APP-CTFß were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aß was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aß induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aß clearance and increases cell viability.

Metabolic engineering of Escherichia coli for the biosynthesis of flavonoid-O-glucuronides and flavonoid-O-galactoside.

Most flavonoids are glycosylated and the nature of the attached sugar can strongly affect their physiological properties. Although many flavonoid glycosides have been synthesized in Escherichia coli, most of them are glucosylated. In order to synthesize flavonoids attached to alternate sugars such as glucuronic acid and galactoside, E. coli was genetically modified to express a uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) specific for UDP-glucuronic acid (AmUGT10 from Antirrhinum majus or VvUGT from Vitis vinifera) and UDP-galactoside (PhUGT from Petunia hybrid) along with the appropriate nucleotide biosynthetic genes to enable simultaneous production of their substrates, UDP-glucuronic acid and UDP-galactose. To engineer UDP-glucuronic acid biosynthesis, the araA gene encoding UDP-4-deoxy-4-formamido-L-arabinose formyltransferase/UDP-glucuronic acid C-4″ decarboxylase, which also used UDP-glucuronic acid as a substrate, was deleted in E. coli, and UDP-glucose dehydrogenase (ugd) gene was overexpressed to increase biosynthesis of UDP-glucuronic acid. Using these strategies, luteolin-7-O-glucuronide and quercetin-3-O-glucuronide were biosynthesized to levels of 300 and 687 mg/L, respectively. For the synthesis of quercetin 3-O-galactoside, UGE (encoding UDP-glucose epimerase from Oryza sativa) was overexpressed along with a glycosyltransferase specific for quercetin and UDP-galactose. Using this approach, quercetin 3-O-galactoside was successfully synthesized to a level of 280 mg/L.

Inhibition of HMGB1-induced angiogenesis by cilostazol via SIRT1 activation in synovial fibroblasts from rheumatoid arthritis.

High mobility group box chromosomal protein 1 (HMGB-1) released from injured cells plays an important role in the development of arthritis. This study investigated the anti-angiogenic effects of cilostazol in collagen-induced arthritis (CIA) of mice, and the underlying mechanisms involved. The expressions of HIF-1α, VEGF, NF-κB p65 and SIRT1 in synovial fibroblasts obtained from rheumatoid arthritis (RA) patients were assessed by Western blotting, and in vitro and in vivo angiogenesis were analyzed. Tube formations by human microvascular endothelial cells (HMVECs) were significantly increased by direct exposure to HMGB1 or to conditioned medium derived from HMGB1-stimulated RA fibroblasts, and these increases were attenuated by cilostazol, the latter of which was blocked by sirtinol. HMGB1 increased the expression of HIF-1α and VEGF and concomitantly increased nuclear NF-κB p65 and DNA binding activity, but these effects of HMGB1 were inhibited by cilostazol. SIRT1 protein expression was time-dependently decreased (3-24 hr) by HMGB1, which was recovered by pretreatment with cilostazol (1-30 µM) or resveratrol, accompanying with increased SIRT1 deacetylase activity. In the tibiotarsal joint tissues of CIA mice treated with vehicle, HIF-1α- and VEGF-positive spots and CD31 staining were markedly exaggerated, whereas SIRT1 immunofluorescence was diminished. These variables were wholly reversed in cilostazol (30 mg/kg/day)-treated mice. Furthermore, number of blood vessels stained by von Willebrand factor antibody was significantly lower in cilostazol-treated CIA mice. Summarizing, cilostazol activated SIRT1 and inhibited NF-κB-mediated transcription, thereby suppressing the expression of HIF-1α and VEGF. In addition, cilostazol caused HIF-1α deacetylation by enhancing SIRT1 activity and reduced VEGF production, thereby had an anti-angiogenic effect in vitro studies and in CIA murine model.

Cilostazol suppresses ß-amyloid production by activating a disintegrin and metalloproteinase 10 via the upregulation of SIRT1-coupled retinoic acid receptor-ß.

The accumulation of plaques of ß-amyloid (Aß) peptides, a hallmark of Alzheimer's disease, results from the sequential cleavage of amyloid precursor protein (APP) by activation of ß- and γ-secretases. However, the production of Aß can be avoided by alternate cleavage of APP by α-and γ-secretases. We hypothesized that cilostazol attenuates Aß production by increasing a disintegrin and metalloproteinase 10 (ADAM10)/α-secretase activity via SIRT1-coupled retinoic acid receptor-ß (RARß) activation in N2a cells expressing human APP Swedish mutation (N2aSwe). To evoke endogenous Aß overproduction, the culture medium was switched from medium containing 10% fetal bovine serum (FBS) to medium containing 1% FBS, and cells were cultured for 3∼24 hr. After depletion of FBS in media, N2aSwe cells showed increased accumulations of full-length APP (FL-APP) and Aß in a time-dependent manner (3-24 hr) in association with decreased ADAM10 protein expression. When pretreated with cilostazol (10-30 µM), FL-APP and Aß levels were significantly reduced, and ADAM10 and α-secretase activities were restored. Furthermore, the effect of cilostazol on ADAM10 expression was antagonized by pretreating Rp-cAMPS and sirtinol and by SIRT1-gene silencing. In the N2aSwe cells overexpressing the SIRT1 gene, ADAM10, and sAPPα levels were significantly elevated. In addition, like all-trans retinoic acid, cilostazol enhanced the protein expressions of RARß and ADAM10, and the cilostazol-stimulated ADAM10 elevation was significantly attenuated by LE135 (a RARß inhibitor), sirtinol, and RARß-gene silencing. In conclusion, cilostazol suppresses the accumulations of FL-APP and Aß by activating ADAM10 via the upregulation of SIRT1-coupled RARß.

Attenuation of ß-amyloid-induced tauopathy via activation of CK2α/SIRT1: targeting for cilostazol.

ß-Amyloid (Aß) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2-coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac-tau) and tau phosphorylation (P-tau) by inhibiting activation of P300 and GSK3ß. Aß was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aß accumulation was accompanied by increased Ac-tau and P-tau levels. Concomitantly, these cells showed increased P300 and GSK3ß P-Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3-30 µM) and resveratrol (20 µM). Moreover, decreased expression of SIRT1 and its activity by Aß were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 µM, PKA inhibitor), TBCA (20 µM, inhibitor of CK2), and sirtinol (20 µM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP-dependent protein kinase-linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau-related neurodegeneration in the AD brain.

Protection by cilostazol against amyloid-ß(1-40)-induced suppression of viability and neurite elongation through activation of CK2α in HT22 mouse hippocampal cells.

Amyloid-ß peptide (Aß) deposits in the brain are critical in the neurotoxicity induced by Aß. This study elucidates the underlying signaling pathway by which cilostazol protects HT22 neuronal cells from Aß(1-40) (3-30 µM)-induced deterioration of cell proliferation, viability, and neurite elongation. Cilostazol rescued HT22 cells from the apoptotic cell death induced by Aß toxicity through the downregulation of phosphorylated p53 (Ser15), Bax, and caspase-3 and the upregulation of Bcl-2 expression, which improved neuronal cell proliferation and viability. Furthermore, Aß(1-40) suppressed both phosphorylated CK2α protein expression and CK2 activity in the cytosol; these were concentration dependently recovered by cilostazol (3-30 µM). Cilostazol significantly increased the levels of GSK-3ß phosphorylation at Ser9 and ß-catenin phosphorylation at Ser675 in the cytosol and nucleus. Cilostazol effects were reversed by KT5720 (1 µM, PKA inhibitor) and TBCA (40 µM, inhibitor of CK2) and CK2α knockdown by siRNA transfection. Likewise, Aß-stimulated GSK-3ß phosphorylation at Tyr 216 was decreased by cilostazol in the control but not in the CK2α siRNA-transfected cells. Furthermore, the Aß (10 µM)-induced suppression of neurite elongation was recovered by cilostazol; this recovery was attenuated by inhibitors such as KT5720 and TBCA and blocked by CK2α knockdown. In conclusion, increased cAMP-dependent protein kinase-linked CK2α activation underlies the pharmacological effects of cilostazol in downregulating p53 phosphorylation at Ser15 and upregulating GSK-3ß phosphorylation at Ser9/ß-catenin phosphorylation at Ser675, thereby suppressing Aß(1-40)-induced neurotoxicity and improving neurite elongation.

Cilostazol enhances integrin-dependent homing of progenitor cells by activation of cAMP-dependent protein kinase in synergy with Epac1.

Recruitment and adhesion of exogenous endothelial progenitor cells (EPCs) or endogenously mobilized bone marrow mononuclear cells (BM MNCs) to the sites of ischemia is an important focus of cell therapy. This study sought to determine whether cilostazol enhances integrin-dependent homing of progenitor cells both in vitro and in vivo. In the in vitro experiments with human umbilical cord blood (HUCB)-derived EPCs, cilostazol (10 µM) stimulated up-regulation of integrins ß1, α1, and αv as well as 8-pCPT-2'-O-Me-cAMP (100 µM; 8-pCPT, Epac activator). Cilostazol and 8-pCPT significantly enhanced migration and adhesion of HUCB EPCs to a fibronectin-coated plate and endothelial cells, which were inhibited by KT5720 (PKA inhibitor, 1 µM) and GGTI-298 (Rap1 inhibitor, 20 µM). Cilostazol stimulated Epac1 expression and up-regulated the active Rap1, as did 8-pCPT, and they were suppressed by KT5720 (P < 0.001) and GGTI-298 (P < 0.001). 8-pCPT increased p-CREB expression and stimulated PKA activity, which was inhibited by KT5720, Rp-cAMPS, and GGTI-298. In addition, N(6)-benzoyl-cAMP (100 µM) increased Rap1 GTP expression, as did 8-pCPT; they were suppressed by Rp-cAMPS and GGTI-298. The in vivo experiments showed that cilostazol (30 mg/kg/day, orally for 7 days) significantly enhanced the integrin ß1 expression in the molecular layer and up-regulated homing of BM MNCs to the injured molecular layer with increased capillary density in mouse brain subjected to transient forebrain ischemia (n = 6, P < 0.001). In conclusion, cilostazol stimulated integrin expression and enhanced migration and adhesion of progenitor cells through cooperative activation of PKA and Epac signals; such activity may improve the efficacy of cell therapy for ischemic disease.

Cilostazol enhances neovascularization in the mouse hippocampus after transient forebrain ischemia.

Cilostazol is known to be a specific type III phosphodiesterase inhibitor, which promotes increased intracellular cAMP levels. We assessed the effect of cilostazol on production of angioneurins and chemokines and recruitment of new endothelial cells for vasculogenesis in a mouse model of transient forebrain ischemia. Pyramidal cell loss was prominently evident 3-28 days postischemia, which was markedly ameliorated by cilostazol treatment. Expression of angioneurins, including endothelial nitric oxide synthase, vascular endothelial growth factor, and brain-derived neurotrophic factor, was up-regulated by cilostazol treatment in the postischemic hippocampus. Cilostazol also increased Sca-1/vascular endothelial growth factor receptor-2 positive cells in the bone marrow and circulating peripheral blood and the number of stromal cell-derived factor-1alpha-positive cells in the molecular layer of the hippocampus, which colocalized with CD31. CXCR4 chemokine receptors were up-regulated by cilostazol in mouse bone marrow-derived endothelial progenitor cells, suggesting that cilostazol may be important in targeting or homing in of bone marrow-derived stem cells to areas of injured tissues. CD31-positive cells were colocalized with almost all bromodeoxyuridine-positive cells in the molecular layer, indicating stimulation of endothelial cell proliferation by cilostazol. These data suggest that cilostazol markedly enhances neovascularization in the hippocampus CA1 area in a mouse model of transient forebrain ischemia, providing a beneficial interface in which both bone marrow-derived endothelial progenitor cells and angioneurins influence neurogenesis in injured tissue. (c) 2010 Wiley-Liss, Inc.