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Astaxanthin is a carotenoid that is present in high quantities in the meat of fish like salmon and the shells of shrimp and crab. It exhibits free radical scavenging antioxidant activity when consumed di- etarily. Astaxanthin is absorbed by the small intestine before exerting its antioxidant effect; however, a portion of dietary intake remains unabsorbed in the digestive tract and reaches the large intes- tines . We hypothesized that astaxanthin may exert its antioxidant action in the large intestine to influence the gut microbiota. In this review we introduce the results of two studies of astaxanthin. Firstly, a clinical trial targeting post-menopausal women screened for high oxidative stress burden. Astaxanthin was administered orally for eight weeks in order to examine its effects and safety, and subjects were surveyed for any changes in subjective symptoms. Secondly, in a mouse model, real time PCR (polymerase chain reaction) was used to examine the ability of astaxanthin to prevent changes in the enteric flora induced by a high-fat diet. When fat intake increases due to changes in diet, the equilibrium between the various species that constitute the intestinal flora is altered. As a result, degenerative changes in lifestyle-related disease and aging of the host are promoted. Here we find that the intake of astaxanthin was able to inhibit these changes in the gut microbiota of mice induced by a high-fat diet. Even in humans, it is highly probably that the unabsorbed astaxanthin that remains in the intestinal tract exerts a positive effect against disturbance of the intestinal flora caused by a high-fat diet., Astaksantin je karotenoid prisutan u velikom količinama u mesu riba poput lososa i oklopima kozica i rakova. Kada se uzima kao hrana ispoljava svoju sposobnost hvatanja slobodnih radikala i antioksidativnog djelovanja. Prije no što započne svoje antioksidativno djelovanje apsorbira se preko tankog crijeva, međutim , dio ostaje neapsorbiran i dospijeva u debelo crijevo. Razmatrali smo mogućnost da astaksantin pomoću svojih antioksidativnih svojstava može utjecati na crijevnu mikrobiotu. U okviru ovog preglednog rada osvrnut ćemo se na rezultate dvaju istraživanja. Prvo je kliničko ispitivanje usmjereno na postmenopauzalne žene koje su prošle screening na opterećenost oksidativnim stresom. Astaksantin su dobivale oralno kroz 8 tjedana kako bi se procijenili njegovi učinci i sigurnost, a ispitanice su anketirane vezano za promjene u subjektivnim simptomima. Dru- gi, mišji model, koristio je PCR (Lančanu reakciju polimeraze) u pravom vremenu kako bi ispitao potencijal astaksantina u sprječavanju promjena u crijevnoj flori induciranih dijetom bogatom mas- tima. Pri dijeti bogatoj mastima povećava se unos masti što narušava ravnotežu raznih vrsta koje sačinjavaju intestinalnu floru. Posljedična neravnoteža rezultira degenerativnim promjenama i boles- tima vezanima za način života. U ovom istraživanju nalazimo da unos astaksantina može spriječiti promjena u mikrobioti miševa izazvane prehranom bogatom mastima. Čak i u ljudi je za vjerovati da neapsorbirani astaksantin koji ostaje u debelom crijevu ima pozitivan učinak protiv poremećaja uzrokovanih prehranom bogatom mastima.

In the anti-aging medicine, we recommend to assess the skin aging by five categories: wrinkle age, spot age, yellow tint age, elasticity age, and moisture age. Photo-aging (oxidative stress) and glycative stress are major causes of age-related deterioration in the skin. Gly- cative stress finally causes skin accumulation of advanced glycation end products(AGEs), inducing yellow tint, and cross linkage between collagen fibers inducing less elastic skin. Oxidative stress causes skin dark spots through the various processes; excess pigment forma- tion and DNA damages. It also causes wrinkle formation associated with matrix metallopro- teinase(MMP) activation and degeneration of collagen and elastin fibers. Study of oxidative and glycative stress may help identify new anti-aging treatments so that we can achieve the skin rejuvenation.

We synthesized several candidates of 24(S)-hydroxycholesterol (24S-OHC) esters, which are involved in neuronal cell death, through catalysis with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1). We studied the regioselectivity of the acylation of the secondary alcohol at the 3- or 24-position of 24S-OHC. The appropriate saturated and unsaturated long-chain fatty acids were esterified with the protected 24S-OHC and then de-protected to afford the desired esters at a satisfactory yield. We then confirmed by HPLC monitoring that the retention times of four esters of 24S-OHC, namely 3-oleate, 3-linoleate, 3-arachidonoate and 3-docosahexaenoate, were consistent with those of 24S-OHC esters observed in 24S-OHC-treated SH-SY5Y cells.

Lysophosphatidylcholine (LPC) and oxysterols which are major components in oxidized low-density lipoprotein have been shown to possess an opposite effect on the expression of sterol regulatory element-binding protein-2 (SREBP-2) target genes in endothelial cells. In this study, we aimed at elucidating the mechanisms of activation of SREBP-2 by LPC and evaluating the effects of LPC and 25-hydroxycholesterol (25-HC) on the release of inflammatory cytokines. Human umbilical vein endothelial cells were treated with LPC or oxysterols including 25-HC. LPC activated SREBP-2 within 15 min, resulting in induction of expression of SREBP-2 target genes which were involved in intracellular cholesterol homeostasis. The rapid activation of SREBP-2 was caused by enhanced efflux of intracellular cholesterol, which was evaluated using (14)C-acetate. The LPC-induced activation of SREBP-2 was inhibited by addition of 25-HC. In contrast, both LPC and 25-HC increased release of interleukin-6 (IL-6) and IL-8, respectively and additively. In conclusion, LPC activated SREBP-2 via enhancement of cholesterol efflux, which was suppressed by 25-HC. The release of inflammatory cytokines such as IL-6 and IL-8 in endothelial cells was SREBP-2-independent. LPC and 25-HC may act competitively in cholesterol homeostasis but additively in inflammatory cytokine release.

Histological images from the longitudinal section of four diseased coronary arteries were used, for the first time, to study the pulsatile blood flow distribution within the lumen of the arteries by means of computational fluid dynamics (CFD). Results indicate a strong dependence of the hemodynamics on the morphology of atherosclerotic lesion. Distinctive flow patterns appear in different stenosed regions corresponding to the specific geometry of any artery. Results show that the stenosis affects the wall shear stress (WSS) locally along the diseased arterial wall as well as other adjacent walls. The maximum magnitude of WSS is observed in the throat of stenosis. Moreover, high oscillatory shear index (OSI) is observed along the stenosed wall and the high curvature regions. The present study is capable of providing information on the shear environment in the longitudinal section of the diseased coronary arteries, based on the models created from histological images. The computational method may be used as an effective way to predict plaque forming regions in healthy arterial walls.

Fluid shear stress plays a critical role in the regulation of vascular biology and its pathology, such as atherosclerosis, via modulation of redox balance. Both pro-atherogenic (either oscillatory or turbulent, nonunidirectional) shear stress and anti-atherogenic (either steady or pulsatile, unidirectional laminar) shear stress stimulate production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are involved in signal transduction of gene expression. Nonunidirectional shear stress induces pro-atherogenic genes encoding adhesion molecules and chemokines in a manner dependent on production of both superoxide and nitric oxide. Steady or pulsatile laminar shear stress induces expression of genes encoding cytoprotective enzymes for glutathione biosynthesis and detoxification, which are regulated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). We show that pulsatile laminar shear stress (PLSS)-induced expression of adhesion molecules and chemokines was enhanced in human umbilical vein endothelial cells (HUVEC) treated with Nrf2 siRNA and arterial endothelial cells isolated from Nrf2 knockout mice. Hence, we propose the hypothesis that PLSS maintains the endothelium in an anti-atherogenic state via intracellular antioxidant levels increased as a result of Nrf2 activation, thereby preventing excess ROS/RNS production required for pro-atherogenic gene expression.

Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO2•−) production via NADPH oxidase and c-Jun NH2-terminal kinase (JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (±3 dyn/cm2) induced JNK activation, which peaked at 1 h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO2•− production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O2•− and mtO2•− production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 (JNK inhibitor) significantly attenuated OSS-mediated mtO2•− production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO2•− production via NADPH oxidase and JNK activation relevant for vascular oxidative stress. Antioxid. Redox Signal. 15, 1379–1388.

Selenoprotein P (SeP) is a liver-derived selenium (Se)-supply protein to maintain Se levels in peripheral tissues. The safety window of Se is narrow, even below the levels required for intoxication, supra-nutritional intake of Se has been reported to cause an increase in the risk of type 2 diabetes. Previous studies suggested that Se-supply activity of excess SeP was related to deterioration of insulin secretion and glucose metabolism. Thus, the inhibition of Se-supply via suppression of SeP binding to cells might improve insulin secretion and glucose metabolism impaired by excess SeP. We established neutralizing antibodies against human SeP. Among them, anti-human SeP monoclonal antibody (mAb) AE2 was identified to have a strong neutralizing activity, and its administration to mice significantly improved glucose intolerance and reduction of insulin secretion, which were induced by intraperitoneal administration of human SeP. In addition, mAb AE2 significantly suppressed SeP-induced impairment of insulin secretion and cell viability of insulinoma MIN6 cells. In this presentation, we will discuss a molecular basis for the development of therapeutic agents for type 2 diabetes by targeting SeP.

Glycation, the nonenzymatic reaction between proteins and excess blood sugar, is implicated in multiple disorders and occurs via the formation and accumulation of advanced glycation end products (AGEs). In our previous studies, we demonstrated that the red-leaf variant of the Persicaria hydropiper sprout (Japanese red water pepper, Benitade) is one of the potent plants that inhibit formation of AGEs. In this study, we aimed to identify antiglycative compounds in Benitade. Benitade extracts were prepared with hot water, then fractionated by using high-performance liquid chromatography (HPLC). The antiglycative efficacy of each fraction was evaluated by measuring the formation of fluorescent AGEs (Ex 370 nm/Em 440 nm). Two fractions, which contained peaks at 26.4 min and 31.8 min, showed potent antiglycative efficacy. When we hydrolyzed these peaks, they shifted to 32.5 and 41.4 min, which are the same retention times as cyanidin and quercetin, respectively. Based on thin-layer chromatography, both compounds contained galactose. Finally, ultrahigh-performance liquid chromatography/quadrupole-time of flight mass spectrometry (UHPLC-QqTOF-MS) analyses were performed to determine the structure of those compounds. Overall, we identified two glycosides, cyanidin 3-O-galactoside (idaein) and quercetin 3-O-galactoside (hyperin), as representative antiglycative compounds in Benitade.

Zebrafish is an emerging model system for cardiac conduction and regeneration. Zebrafish heart regenerates after 20% ventricular resection within 60 days. Whether cardiac conduction phenotype correlated with cardiomyocyte regeneration remained undefined. Longitudinal monitoring of the adult zebrafish heart (n = 12) was performed in terms of atrial contraction (PR intervals), ventricular depolarization (QRS complex) and repolarization (heart rated corrected QTc interval). Baseline electrocardiogram (ECG) signals were recorded one day prior to resection and twice per week over 59 days. Immunostaining for gap junctions with anti-Connexin-43 antibody was compared between the sham (n = 5) and ventricular resection at 60 days post-resection (dpr) (n = 7). Heart rate variability, QTc prolongation and J-point depression developed in the resected group but not in the sham. Despite a trend toward heart rate variability in response to ventricular resection, the differences between the resected and sham fish were, by and large, statistically insignificant. At 10 dpr, J-point depression was statistically significant (sham: -0.179 +/- 0.061 mV vs. ventricular resection: -0.353 +/- 0.105 mV, p0.01, n = 7). At 60 days, histology revealed either cardiomyocyte regeneration (n = 4) or scar tissues (n = 3). J-point depression was no longer statistically significant at 59 dpr (sham: -0.114 +/- 0.085 mV; scar tissue: -0.268 +/- 0.178 mV, p0.05, n = 3; regeneration: -0.209 +/- 0.119 mV, p0.05, n = 4). Despite positive Connexin-43 staining in the regeneration group, QTc intervals remained prolonged (sham: 325 +/- 42 ms, n = 5; scar tissues: 534 +/- 51 ms, p0.01, n = 3; regeneration: 496 +/- 31 ms, p0.01, n = 4). Thus, we observed delayed electric repolarization in either the regenerated hearts or scar tissues. Moreover, early regenerated cardiomyocytes lacked the conduction phenotypes of the sham fish.

Objective— Oxidized low-density lipoprotein (oxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. However, the signal pathways and molecular mechanism remain unknown. In this study, we investigated the role of manganese superoxide dismutase (Mn-SOD) on oxLDL-induced apoptosis via c-Jun NH2-terminal kinase (JNK)-mediated ubiquitin/proteasome pathway. Methods and Results— OxLDL induced JNK phosphorylation that peaked at 30 minutes in human aortic endothelial cells. Fluorescence-activated cell sorting analysis revealed that oxLDL increased mitochondrial superoxide production by 1.88±0.19-fold and mitochondrial membrane potential by 18%. JNK small interference RNA (siJNK) reduced oxLDL-induced mitochondrial superoxide production by 88.4% and mitochondrial membrane potential by 61.7%. OxLDL did not affect Mn-SOD mRNA expression, but it significantly reduced Mn-SOD protein level, which was restored by siJNK. Immunoprecipitation by ubiquitin antibody revealed that oxLDL increased ubiquitination of Mn-SOD, which was inhibited by siJNK. OxLDL-induced caspase-3 activities were also attenuated by siJNK but were enhanced by Mn-SOD small interfering RNA. Furthermore, overexpression of Mn-SOD abrogated oxLDL-induced caspase-3 activities. Conclusion— OxLDL-induced JNK activation regulates mitochondrial redox status and Mn-SOD protein degradation via JNK-dependent ubiquitination, leading to endothelial cell apoptosis.

Mitochondrial dysfunction is intimately involved in cardiovascular diseases. Mitochondrial membrane potential (DeltaPsi(m)) is coupled with oxidative phosphorylation to drive ATP synthesis. In this study, we examined the effect of physiological pulsatile shear stress (PSS) on DeltaPsi(m) and the role of Mn-SOD expression on DeltaPsi(m). Confluent human aortic endothelial cells (HAEC) were exposed to PSS, and DeltaPsi(m) was monitored using tetramethylrhodamine methyl ester (TMRM(+)), a mitochondrial membrane potential probe. PSS significantly increased DeltaPsi(m) and the change in DeltaPsi(m) was a dynamic process. DeltaPsi(m) returned to baseline level after PSS for 2h followed by static state for 4h. Mitochondrial Mn-SOD expression and activities were also significantly up-regulated in response to PSS. Silencing Mn-SOD attenuated PSS-mediated DeltaPsi(m) increase while adding Mn-SOD mimetic, MnTMPyP, increased DeltaPsi(m) to the similar extent as induced by PSS. Our findings suggest that PSS-increased mitochondrial DeltaPsi(m), in part, via Mn-SOD up-regulation.

Exposure of particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultra fine particles (UFP) from diesel vehicle engines have been shown to be pro-atherogenic in apoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induced vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intra-cellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O2·-) production in human aortic endothelial cells (HAEC). Flow cytometry (FACS) showed that UFP increased MitoSOX Red intensity specific for mitochondrial superoxide. Protein carbonyl content is increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated hemeoxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pre-treatment with antioxidant, N-acetyl cysteine (NAC), significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP stimulated O2·- production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation play an important role in UFP-induced oxidative stress and stress response gene expression.

We have previously reported that antioxidant response element (ARE)-regulated genes, such as heme oxygenase 1 (HO-1), sequestosome 1 (SQSTM1), and NAD(P)H quinone oxidoreductase 1 (NQO1), are induced in human umbilical vein endothelial cells (HUVEC) upon exposure to laminar shear stress. In the present study, we have confirmed a critical role for NF-E2-related factor 2 (Nrf2) in the induction of gene expression in HUVEC exposed to laminar shear stress. Although the mRNA levels of Nrf2 were unchanged during exposure to shear stress, the protein levels of Nrf2 were markedly increased. Small interfering RNA (SiRNA) against Nrf2 significantly attenuated the expression of Nrf2-regulated genes such as HO-1, SQSTM1, NQO1, glutamate-cysteine ligase modifier subunit (GCLM), and ferritin heavy chain. Nrf2 was rapidly degraded in cells treated with cycloheximide under static conditions, but shear stress decreased the rate of Nrf2 degradation. Incubation with the thiol antioxidant N-acetylcysteine strongly inhibited both the Nrf2 accumulation and the expression of Nrf2-regulated genes such as HO-1, GCLM, and SQSTM1. Nitric oxide (NO) production was increased with the strength of shear stress but neither the inhibitor of endothelial NO synthase (eNOS) nor the siRNA against eNOS affected the expression of Nrf2-regulated genes. A xanthine oxidase inhibitor oxypurinol and the flavoprotein inhibitor diphenyleneiodonium, which inhibits NAD(P)H oxidase and mitochondrial respiratory chain, markedly suppressed the expression of these genes. Moreover, diphenylpyrenlphosphine, a reducing compound of lipid hydroperoxides, also significantly suppressed Nrf2-regulated gene expression. Taken together, these findings suggest that shear stress stabilizes Nrf2 protein via the lipid peroxidation elicited by xanthine oxidase and flavoprotein mediated generation of superoxide, resulting in gene induction by the Nrf2-ARE signaling pathway.

24(S)-Hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, has been known to play an important role in maintaining cholesterol homeostasis in the brain and has been proposed as a possible biomarker of neurodegenerative disease. Recent studies have revealed diverse functions of 24S-OHC and gained increased attention. For example, 24S-OHC at sublethal concentrations has been found to induce an adaptive response via activation of the liver X receptor signaling pathway, thereby protecting neuronal cells against subsequent oxidative stress. It has also been found that physiological concentrations of 24S-OHC suppress amyloid-β production via downregulation of amyloid precursor protein trafficking in neuronal cells. On the other hand, high concentrations of 24S-OHC have been found to induce a type of nonapoptotic programmed cell death in neuronal cells expressing little caspase-8. Because neuronal cell death induced by 24S-OHC has been found to proceed by a unique mechanism, which is different from but in some ways similar to necroptosis-necroptosis being a type of programmed necrosis induced by tumor necrosis factor α-neuronal cell death induced by 24S-OHC has been called "necroptosis-like" cell death. 24S-OHC-induced cell death is dependent on the formation of 24S-OHC esters but not on oxidative stress. This review article discusses newly reported aspects of 24S-OHC in neuronal cell death and sheds light on the possible importance of controlling 24S-OHC levels in the brain for preventing neurodegenerative disease.

Lysophosphatidylcholine (lysoPC) evokes diverse biological responses in vascular cells including Ca(2+) mobilization, production of reactive oxygen species, and activation of the mitogen-activated protein kinases, but the mechanisms linking these events remain unclear. Here, we provide evidence that the response of mitochondria to the lysoPC-dependent increase in cytosolic Ca(2+) leads to activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase through a redox signaling mechanism in human umbilical vein endothelial cells. ERK activation was attenuated by inhibitors of the electron transport chain proton pumps (rotenone and antimycin A) and an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), suggesting that mitochondrial inner membrane potential plays a key role in the signaling pathway. ERK activation was also selectively attenuated by chain-breaking antioxidants and by vitamin E targeted to mitochondria, suggesting that transduction of the mitochondrial hydrogen peroxide signal is mediated by a lipid peroxidation product. Inhibition of ERK activation with MEK inhibitors (PD98059 or U0126) diminished induction of the antioxidant enzyme heme oxygenase-1. Taken together, these data suggest a role for mitochondrially generated reactive oxygen species and Ca(2+) in the redox cell signaling path-ways, leading to ERK activation and adaptation of the pathological stress mediated by oxidized lipids such as lysoPC.

24(S)-Hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, is known to play an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces a type of non-apoptotic programmed necrosis in neuronal cells expressing little caspase-8. Necroptosis has been characterized as a type of programmed necrosis in which activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) is involved in the signaling pathway. In the present study, we investigated the involvement of these three proteins in 24S-OHC-induced cell death. We found that RIPK1 but neither RIPK3 nor MLKL was expressed in human neuroblastoma SH-SY5Y cells, while all three proteins were expressed in human T lymphoma caspase-8-deficient Jurkat (Jurkat(Cas8-/-)) cells. In Jurkat(Cas8-/-) cells, tumor necrosis factor α (TNFα)-induced cell death was significantly suppressed by treatment with respective inhibitors of RIPK1, RIPK3, and MLKL. In contrast, only RIPK1 inhibitor showed significant suppression of 24S-OHC-induced cell death, and even this was less prominent than was observed in TNFα-induced cell death. In Jurkat(Cas8-/-) cells, knockdown of either RIPK1 or RIPK3 caused moderate but significant suppression of 24S-OHC-induced cell death, but no such effect was observed as a result of knockdown of MLKL. Collectively, these results suggest that, for both SH-SY5Y cells and Jurkat(Cas8-/-) cells, 24S-OHC-induced cell death is dependent on RIPK1 but not on MLKL. We therefore conclude that, in the absence of caspase-8 activity, 24S-OHC induces a necroptosis-like cell death which is RIPK1-dependent but MLKL-independent.

Atherosclerosis is the underlying cause of cardiovascular events, such as heart attack and stroke, and preferentially occurs in arterial regions exposed to disturbed flow ( d-flow ) by mechanisms involving broad changes in gene expression. While microRNAs (miRNAs) are known to regulate various aspects of cardiovascular biology, their role in atherosclerosis has not been directly demonstrated. We used partial carotid ligation surgery to induce d-flow in the Left carotid artery (LCA) of mouse while the contralateral right carotid artery (RCA) continued to experience stable flow (s-flow). We devised a technique to quickly extract endothelial-enriched RNA from these carotids and performed miRNA array and subsequent validations. Also, using a cone-and-plate shear device, we exposed the immortalized mouse aortic endothelial cells to either laminar or oscillatory shear stress. Use used pre-miR-712 to induce and anti-miR-712 to knockdown the expression of miR-712 in our system. We identified mechanosensitive miRNAs using a mouse partial carotid ligation model and endothelial miRNA array. Of those mechanosensitive miRNAs identified, miR-712 was the most shear-sensitive miRNA upregulated by d-flow both in vivo and in vitro . We found that miR-712 is derived from the internal transcribed spacer 2 (ITS2) region of pre-ribosomal RNA ( RN45S gene) in a XRN1 exonuclease-dependent, but DGCR8-independent manner, suggesting that it is an atypical miRNA derived from an unexpected source. Using gain-of-function (pre-miR-712) and loss-of-function (anti-miR-712) approaches and target-binding assays showed that miR-712 directly downregulated the tissue inhibitor of metalloproteinase 3 (TIMP3) expression. This in turn activated downstream metalloproteinases (MMPs and ADAM family) and stimulated pro-atherogenic responses, endothelial tubule formation and sprouting, in a flow-dependent manner. Treatment with anti-miR-712 prevented atherosclerosis in two different models of murine atherosclerosis using ApoE -/- mice: a chronic conventional western-diet or an acute carotid partial ligation model on a high-fat diet. Targeting mechanosensitive "athero-miRs" with anti-miR-based approaches may provide a new treatment paradigm in atherosclerosis.

25-hydroxycholesterol (25-HC) is a potent suppressor of cholesterol synthesis gene transcription in cultured cells. A high affinity binding protein for 25-HC, oxysterol-binding protein (OSBP), has been identified from tissue cytosol. OSBP translocates from the cytosol to the Golgi apparatus membranes after addition of 25-HC to cell cultures and is thought to mediate 25-HC action on cholesterol metabolism through association to the Golgi apparatus. However, direct evidence to prove this hypothesis was lacking. In this study, we knocked down expression of OSBP by using duplex siRNAs specific for OSBP to examine the relationship between OSBP and 25-HC-induced inhibition of cholesterol synthesis gene transcription. We found that decreasing OSBP expression by approximately 90% did not affect 25-HC-induced inhibition of transcription of 3-hydoxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and squalene epoxidase to any extent. Exogenous lysophosphatidylcholine (LPC), which is known to cause the efflux of cellular cholesterol into the medium and to increase cholesterol synthesis, was found to rescue the 25-HC-induced down-regulation of sterol regulated genes, while LPC did not affect 25-HC-induced association of OSBP with the Golgi apparatus. These results suggest that inhibition of cholesterol biosynthesis genes by 25-HC is OSBP-independent.

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. Endothelial cell activation is mediated by many different extra-cellular signals, which result in overlapping yet distinct patterns of gene expression. Here we show, in DNA microarray analyses, that vascular endothelial growth factor (VEGF) and thrombin result in dramatic and rapid upregulation of Down syndrome critical region (DSCR)-1 gene encoding exons 4-7, a negative feedback regulator of calcium-calcineurin-NF-AT signaling. VEGF- and thrombin-mediated induction of DSCR-1 involves the cooperative binding of NF-ATc and GATA-2/3 to neighboring consensus motifs in the upstream promoter. Constitutive expression of DSCR-1 in endothelial cells markedly impaired NF-ATc nuclear localization, proliferation, and tube formation. Under in vivo conditions, overexpression of DSCR-1 reduced vascular density in matrigel plugs and melanoma tumor growth in mice. Taken together, these findings support a model in which VEGF- and thrombin-mediated induction of endothelial cell proliferation triggers a negative feedback loop consisting of DSCR-1 gene induction and secondary inhibition of NF-AT signaling. As a natural brake in the angiogenic process, this negative pathway may lend itself to therapeutic manipulation in pathological states.

Large-scale clinical trials have demonstrated significant reductions in cardiovascular events following statin therapy. The observed benefit of statin therapy, however, may be greater in these trials than is to be expected from lowering lipid levels alone. In order to clarify the mechanism by which statins prevent cardiovascular events in vascular wall cells, we investigated the changes in gene expression profiles after incubation with atorvastatin or pitavastatin in cultured human umbilical vein endothelial cells using DNA microarrays. Statins affected the expression levels of genes involved in inflammation, coagulation, and vascular constriction. The mRNA levels for interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) decreased after statin treatment. Statins reduced mRNA levels of plasminogen activator inhibitor-1 (PAI-1) and increased the mRNA levels of thrombomodulin. Statins reduced the mRNA levels of endothelin-1 and increased the mRNA levels of nitric oxide synthase-3 (eNOS). These results show that, statins are clinically effective because of their ability to change the gene expression profile of endothelial cells thereby preventing vascular events.

Objective— Fluid shear stress intimately regulates vasculogenesis and endothelial homeostasis. The canonical Wnt/β-catenin signaling pathways play an important role in differentiation and proliferation. In this study, we investigated whether shear stress activated angiopoietin-2 (Ang-2) via the canonical Wnt signaling pathway with an implication in vascular endothelial repair. Approach and Results— Oscillatory shear stress upregulated both TOPflash Wnt reporter activities and the expression of Ang-2 mRNA and protein in human aortic endothelial cells accompanied by an increase in nuclear β-catenin intensity. Oscillatory shear stress–induced Ang-2 and Axin-2 mRNA expression was downregulated in the presence of a Wnt inhibitor, IWR-1, but was upregulated in the presence of a Wnt agonist, LiCl. Ang-2 expression was further downregulated in response to a Wnt signaling inhibitor, DKK-1, but was upregulated by Wnt agonist Wnt3a. Both DKK-1 and Ang-2 siRNA inhibited endothelial cell migration and tube formation, which were rescued by human recombinant Ang-2. Both Ang-2 and Axin-2 mRNA downregulation was recapitulated in the heat-shock–inducible transgenic Tg(hsp70l:dkk1-GFP ) zebrafish embryos at 72 hours post fertilization. Ang-2 morpholino injection of Tg (kdrl:GFP ) fish impaired subintestinal vessel formation at 72 hours post fertilization, which was rescued by zebrafish Ang-2 mRNA coinjection. Inhibition of Wnt signaling with IWR-1 also downregulated Ang-2 and Axin-2 expression and impaired vascular repair after tail amputation, which was rescued by zebrafish Ang-2 mRNA injection. Conclusions— Shear stress activated Ang-2 via canonical Wnt signaling in vascular endothelial cells, and Wnt-Ang-2 signaling is recapitulated in zebrafish embryos with a translational implication in vascular development and repair.

Atherosclerotic plaque rupture and thrombosis remains the leading cause of death in the United States. We investigated the role of CD39, a potent ecto-enzymatic regulator of platelet activation and leukocyte trafficking, in atherosclerosis. We generated mice deficient in CD39 on a hyperlipidemic, apoE -/- background and noted a two-fold higher plaque burden in when compared to apoE -/- controls ( P =0.003). We noted higher levels of circulating markers of platelet activation, soluble P-selectin (39%) and RANTES (60%), in the CD39-deficient mice ( P =0.003 and P =0.015, respectively, n=7-11). CD39-haploinsufficient mice had 1.8-fold greater enhanced platelet reactivity in response to ADP compared to controls ( P =0.03, n=3-8). Macrophages from CD39-deficient mice had significantly higher lipoprotein uptake in vitro . Correspondingly, CD39 overexpression in RAW cells inhibited scavenger receptor expression and lipoprotein uptake. Altered fluid mechanics contribute to atherosclerosis, with non-laminar shear stress enhancing regional plaque formation as seen in arterial bifurcations. We examined coronal sections of aortas from apoE −/− mice and observed that CD39 is poorly expressed in the endothelium in regions of turbulent blood flow, where plaque develops, supporting our hypothesis that endothelial CD39 can be induced by fluid phase shear forces. HUVEC treated with physiologic laminar shear stress (LS) (15 dynes/cm 2 ) had a 5.9-fold increase in CD39 protein ( P =0.004, N=3-7) and a concordant increase in nucleotidase activity ( P =0.03 N=3) compared to static controls (SS). We identified Krüppel like factor 2 (KLF2) as an upstream candidate for transcriptional regulation of CD39 induction by fluid shear forces. Silencing KLF2 in vitro led to a 55% decrease in CD39 mRNA induction with LS vs SS controls ( P =0.002, N=3-4). Chromatin immunoprecipitation revealed that KLF2 binds to the CD39 ( P =0.01, N=3) and this binding was further enhanced under laminar shear stress ( P =0.0007, N=3). These data show that CD39, an anti-thrombotic, anti-inflammatory enzyme is a critical regulator of atherosclerosis by modulating platelet, macrophage and endothelial function and mechanistically identify KLF2 as a direct, upstream regulator of CD39 expression.

The effect of intracellular oxidative stress on the development of cell transformation was studied. Mouse embryo C3H/10T1/2 fibroblasts pre-treated with benzo[a] pyrene, developed transformed foci on exposure to free radical generators, such as 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 3-morpholinosydnonimine hydrochloride (SIN-1). These compounds generate peroxyl radicals and peroxynitrite, respectively. Neither AAPH nor SIN-1 alone induced transformation. The level of intracellular antioxidants, such as alpha-tocopherol and glutathione (GSH), decreased with time of exposure to the free radical generators, whereas the addition of exogenous alpha-tocopherol, GSH and ebselen showed a reduction in the frequency of transformation. An early event during exposure to AAPH and SIN-1 was the generation of acrolein, a highly mutagenic lipid peroxidation product, which was suppressed by the addition of alpha-tocopherol. Furthermore, it was confirmed that acrolein induced the transformation of cells which were pre-treated with benzo[a]pyrene but not of the untreated cells. These results suggest that acrolein may act as an important mediator of cell transformation under oxidative stress.

MicroRNAs (miRNAs) regulate cardiovascular biology and disease, but the role of flow-sensitive microRNAs in atherosclerosis is still unclear. Here we identify miRNA-712 (miR-712) as a mechanosensitive miRNA upregulated by disturbed flow (d-flow) in endothelial cells, in vitro and in vivo. We also show that miR-712 is derived from an unexpected source, pre-ribosomal RNA, in an exoribonuclease-dependent but DiGeorge Syndrome Critical Region-8 (DGCR8)-independent manner, suggesting that it is an atypical miRNA. Mechanistically, d-flow-induced miR-712 downregulates tissue inhibitor of metalloproteinase-3 (TIMP3) expression, which in turn activates the downstream matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) and stimulate pro-atherogenic responses, endothelial inflammation and permeability. Furthermore, silencing miR-712 by anti-miR-712 rescues TIMP3 expression and prevents atherosclerosis in murine models of atherosclerosis. Finally, we report that human miR-205 shares the same “seed sequence” as murine-specific miR-712, and also targets TIMP3 in a flow-dependent manner. Targeting these mechanosensitive “athero-miRs” may provide a new treatment paradigm in atherosclerosis.

Recently, we have shown that disturbed flow, characterized by low and oscillatory shear stress, caused by a partial ligation of mouse left carotid artery (LCA) rapidly induces atherosclerosis. Using the partial ligation model and genome-wide microarray study with aortic endothelial RNAs obtained directly from the flow-disturbed carotid arteries, we previously identified mechanosensitive genes in mouse endothelial RNA including LIM domain only 4 ( lmo4 ). Here we report that LMO4 is a shear-sensitive protein that regulates endothelial inflammation. Lmo4 was up-regulated by disturbed flow in mouse LCA compared to the contralateral right CA (RCA) exposed to stable flow. At protein levels, LMO4 expression was significantly higher not only in LCA in our surgical model but also in the lesser curvature (flow-disturbed and athero-prone region of mouse aortic arch) compared to the greater curvature (stable-flow and ather-protected region). In addition, immunohistochemical staining of LMO4 in human coronary arteries revealed that its expression is detectable only in intimal endothelial cells, but not in medial cells. While LMO4 is known as a potential oncogene and associated with growth, migration and invasion of breast cancer cells, its role in cardiovascular system is not known to our knowledge. We tested a hypothesis that LMO4 is a mechanosensitive gene and plays a critical role in regulation of endothelial cell biology. LMO4 protein expression was robustly induced by oscillatory shear stress (OS) compared to laminar shear (LS) in human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with siRNA against LMO4 significantly inhibited OS-induced inflammation and migration, but not apoptosis and cell cycle progression. Further, LMO4 siRNA treatment significantly blunted expression of VCAM-1 and interleukin-8 induced by OS in endothelial cells. These results suggest that LMO4 is a shear-induced gene that plays a critical role in OS-induced endothelial inflammation and migration, and potentially in atherosclerosis.

Disturbed flow-mediated PKCζ–PIASy association is critical for p53 SUMOylation and induces p53 nuclear export and endothelial cell apoptosis., Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow–mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow–mediated peroxynitrite (ONOO−) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53–Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO− increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301–410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ–PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53−/− mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ–PIASy interaction during d-flow–mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

Background Epidemiological evidence supports the association between exposure to ambient particulate matter (PM) and cardiovascular diseases. Chronic exposure to ultrafine particles (UFP; D p Results UFP2 contained a higher level of redox active organic compounds and metals on a per PM mass basis than UFP1. While both UFP1 and UFP2 induced superoxide production and up-regulated stress response genes such as heme oxygenease-1 (HO-1), OKL38, and tissue factor (TF), only UFP2 induced the expression of pro-inflammatory genes such as IL-8 (2.8 ± 0.3-fold), MCP-1 (3.9 ± 0.4-fold), and VCAM (6.5 ± 1.1-fold) (n = 3, P < 0.05). UFP2-exposed HAEC also bound to a higher number of monocytes than UFP1-exposed HAEC (Control = 70 ± 7.5, UFP1 = 106.7 ± 12.5, UFP2 = 137.0 ± 8.0, n = 3, P < 0.05). Adenovirus NF-κB Luciferase reporter assays revealed that UFP2, but not UFP1, significantly induced NF-κB activities. NF-κB inhibitor, CAY10512, significantly abrogated UFP2-induced pro-inflammatory gene expression and monocyte binding. Conclusion While UFP1 induced higher level of oxidative stress and stress response gene expression, only UFP2, with higher levels of redox active organic compounds and metals, induced pro-inflammatory responses via NF-κB signaling. Thus, UFP with distinct chemical compositions caused differential response patterns in endothelial cells.

The advent of microelectromechanical systems (MEMS) sensors has enabled real-time wall shear stress (WSS) measurements with high spatial and temporal resolution in a 3-D bifurcation model. To optimize intravascular shear stress assessment, we evaluated the feasibility of catheter/coaxial wire-based MEMS sensors in the abdominal aorta of the New Zealand white (NZW) rabbits. Theoretical and computational fluid dynamics (CFD) analyses were performed. Fluoroscope and angiogram provided the geometry of aorta, and the Doppler ultrasound system provided the pulsatile flow velocity for the boundary conditions. The physical parameters governing the shear stress assessment in NZW rabbits included (1) the position and distance from which the MEMS sensors were mounted to the terminal end of coaxial wire or the entrance length, (L(e)), (2) diameter ratios of aorta to the coaxial wire (D(aorta) /D(coaxial wire)=1.5-9.5), and (3) the range of Reynolds numbers (116-1550). At an aortic diameter of 2.4mm and a maximum Reynolds number of 212 (a mean Reynolds number of 64.2), the time-averaged shear stress (tau(ave)) was computed to be 10.06 dyn cm(-2) with a systolic peak at 33.18 dyn cm(-2). In the presence of a coaxial wire (D(aorta)/D(coaxial wire)=6 and L(e)=1.18 cm), the tau(ave) value increased to 15.54 dyn cm(-2) with a systolic peak at 51.25 dyn cm(-2). Real-time intravascular shear stress assessment by the MEMS sensor revealed an tau(ave) value of 11.92 dyn cm(-2) with a systolic peak at 47.04 dyn cm(-2). The difference between CFD and experimental tau(ave) was 18.5%. These findings provided important insights into packaging the MEMS sensors to optimize in vivo shear stress assessment.

Fluid shear stress modulates vascular production of endothelial superoxide anion (O2·−) and nitric oxide (·NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress (τave) of 23 dyn/cm2 upregulated Mn-SOD mRNA expression at a higher level than did OSS at τave = 0.02 dyn/cm2 ± 3.0 dyn·cm−2·s−1 and at 1 Hz (PSS by 11.3 ± 0.4-fold vs. OSS by 5.0 ± 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration ( P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level ( P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration.

This paper describes a polymer-based shear stress sensor built on catheter for in vivo measurements and potential application in atherosclerosis diagnosis. MEMS shear stress sensor with backside wire bonding has been used to address in vitro applications for micro-scale hemodynamics with high temporal and spatial resolution. However, to assess shear stress in the tortuous and dynamic arterial circulation, we had to develop a new generation of polymer- and catheter-based sensors that are both flexible and deployable. The individual sensor was packaged near the tip of a catheter for intravascular shear stress analysis. The wire bonding and electrode leads were insulated by a film of Parylene C and were connected to the external circuit along the guide-wire. The sensor was deployed through the catheter into the aorta of New Zealand White (NZW) rabbits by the femoral cut-down procedure. Based on the heat transfer principle, the device was able to detect small temperature perturbation in response to the pulsatile flow at ∼200 beats/minutes in the rabbits. The sensor was calibrated in the presence of rabbit blood flow at 37.8°C. We demonstrated the feasibility of translating a polymerbased device for dynamic intravascular measurement with a potential for clinical applications in detecting coronary artery disease and stroke.Copyright © 2008 by ASME

Hemodynamics, specifically, fluid shear stress, modulates the focal nature of atherosclerosis. Shear stress induces vascular oxidative stress via the activation of membrane-bound NADPH oxidases present in vascular smooth muscle cells, fibroblasts, and phagocytic mononuclear cells. Shear stress acting on the endothelial cells at arterial bifurcations or branching points regulates both NADPH oxidase and nitric oxide (NO) synthase activities. The former is considered a major source of oxygen-centered radicals (i.e., superoxide anion [O2(.-)]) that give rise to oxidative stress; the latter is a source of nitrogen-centered radicals (i.e., nitric oxide [NO]) that give rise to nitrative/nitrosative stress. In addition to conventional biochemical analyses, the emerging microelectromechanical systems (MEMS) provide spatial and temporal resolutions to investigate the mechanisms whereby the characteristics of shear stress regulate the biological activities of endothelial cells at the complicated arterial geometry. In parallel, the development of MEMS liquid chromatography (LC) provides a new venue to measure circulating oxidized low-density lipoprotein (ox-LDL) particles as a lab-on-a chip platform. Nanowire-based field effect transistors further pave the way for a high throughput approach to analyze the LDL redox state. Integration of MEMS with oxidative biology is synergistic in assessing vascular oxidative stress. The MEMS LC provides an emerging lab-on-a-chip platform for ox-LDL analysis. In this context, this chapter has integrated expertise from the fields of vascular biology and oxidative biology to address the dynamics of inflammatory responses.

Antioxidants possess potent ability to regulate gene expression beyond their specific antioxidant activity. Genomic analysis reveals that three phenolic antioxidants, probucol, BO-653, and tBHQ, all of which have a phenoxyl group with one or two tert-butyl groups at the ortho-position, inhibit both the mRNA and protein levels of proteasome alpha-subunits in human endothelial cells. The chemical structure required for the gene regulation was studied by using derivatives of BO-653 and other antioxidants. It was found that the phenoxyl group and tert-butyl group at the ortho-position of the compounds were critical for down-regulation of the proteasome gene. Two antioxidant responsive elements (AREs) were identified in the promoter region of proteasome alpha subunit 3 (PSMA3). Results from promoter truncation analysis revealed that the proximal ARE region was necessary for the down-regulation of the expression of PSMA3. Electrophoretic mobility shift assays revealed that BO-653-mediated induction of DNA-binding to an upstream promoter region of PSMA3 containing the ARE motif was blocked by antibody against c-Jun but not Nrf2. These results indicate that the suppression of the proteasome alpha subunits expression by phenolic antioxidants is strictly dependent on both their chemical structure and the ARE consensus region in the promoter, which may be negatively regulated by AP-1.

Objective— Liver X–activated receptor α (LXRα) regulates multiple genes controlling cholesterol metabolism and transport. To clarify its role in atherogenesis, we established a monoclonal antibody recognizing native human LXRα protein and studied the expression pattern in human atherosclerotic lesions. Methods and Results— A novel monoclonal antibody PPZ0412 was raised against the ligand-binding domain of LXRα, which can be used for immunostaining of human LXRα protein. LXRα protein was detected in the nucleus of macrophages in the liver, spleen, or lung and also in hepatocytes and adipocytes. In atherosclerotic lesions, the LXRα protein was detected in macrophages positive for scavenger receptor class A and/or CD68. Conclusions— In the human body, the LXRα protein is highly expressed in macrophage lineage cells and foam cells in atherosclerotic lesions and is identified as a target for intervention in atherosclerotic disease.

Objective— A diverse range of lipid oxidation products detected in oxidized low-density lipoprotein (oxLDL) and atherosclerotic lesions are capable of eliciting biological responses in vascular cells. We performed DNA microarray experiments to explore novel responses of human umbilical vein endothelial cells (HUVECs) to oxLDL and its components. Methods and Results— cDNA microarray analysis showed that oxLDL, lysophosphatidylcholine (LysoPC), 4-hydroxy-2-nonenal, and oxysterols altered gene expression specifically, but some genes were commonly induced in HUVECs. Solute carrier family 3 member 2 and family 7 member 5, encoding the heavy chain of the cell surface antigen 4F2 (4F2hc) and the L-type amino acid transporter 1 (LAT1), respectively, were induced by oxLDL and many oxidation products. LAT1 requires 4F2hc to form a heterodimeric functional complex to transport neutral amino acids into the cell. LysoPC increased membrane protein levels of LAT1 confirmed by Western blot analysis and also uptake of L-[ 14 C]leucine, which was inhibited by a competitive inhibitor for LAT1. The release of interleukin 6 (IL-6) and IL-8 was increased in LysoPC-treated cells and was attenuated by the LAT1 inhibitor. Conclusions— These findings suggest that an increase in uptake of neutral amino acids induced by LysoPC results in enhancement of inflammatory responses of endothelial cells.

In addition to a lipid-lowering effect, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have an effect on the expression levels of many genes. In order to elucidate the range of this effect as comprehensively as possible, we investigated the changes in gene expression profiles brought about by atorvastatin or pitavastatin in cultured human umbilical vein endothelial cells (HUVEC), cultured human coronary artery smooth muscle cells (HCASMC) and cultured human hepatocarcinoma Hep G2 cells by means of DNA microarrays. Among the 6146 genes in the array, statins affected the expression levels of genes involved in coagulation, vascular constriction and cell growth in a cell-type specific manner. In HUVEC, they induced integrin beta4 and thrombomodulin profoundly, and profoundly suppressed pentraxin 3 both at 8 and 24 hours. In HCASMC, the statins induced thrombomodulin and urokinase inhibitor, and potently suppressed the cysteine-rich angiogenic inducer 61 and cyclin B. Many genes related to the cell cycle and/or growth were also regulated in HUVEC and HCASMC by the statins. These results indicate that many aspects of the pleiotropic effect can be mediated by transcriptional control by statins. Genes newly identified by this study may be useful in statin therapy.

24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, has an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces necroptosis in human neuroblastoma SH-SY5Y cells. In the present study, we investigated the mechanisms by which 24S-OHC-induced cell death occurs. We found that lipid droplets formed at the early stages in the treatment of SH-SY5Y cells with 24S-OHC. These lipid droplets could be almost completely eliminated by treatment with a specific inhibitor or by siRNA knockdown of acyl-CoA:cholesterol acyltransferase 1 (ACAT1). In association with disappearance of lipid droplets, cell viability was recovered by treatment with the inhibitor or siRNA for ACAT1. Using gas chromatography–mass spectrometry, we confirmed that 24S-OHC-treated cells exhibited accumulation of 24S-OHC esters but not of cholesteryl esters and confirmed that accumulation of 24S-OHC esters was reduced when ACAT1 was inhibited. 24S-OHC induced apoptosis in T-lymphoma Jurkat cells, which endogenously expressed caspase-8, but did not induce apoptosis in SH-SY5Y cells, which expressed no caspase-8. In Jurkat cells treated with the pan-caspase inhibitor ZVAD and in caspase-8-deficient Jurkat cells, 24S-OHC was found to induce caspase-independent cell death, and this was partially but significantly inhibited by Necrostatin-1. Similarly, knockdown of receptor-interacting protein kinase 3, which is one of the essential kinases for necroptosis, significantly suppressed 24S-OHC-induced cell death in Jurkat cells treated with ZVAD. These results suggest that 24S-OHC can induce apoptosis or necroptosis, which of the two is induced being determined by caspase activity. Regardless of the presence or absence of ZVAD, 24S-OHC treatment induced the formation of lipid droplets and cell death in Jurkat cells, and this was suppressed by treatment with ACAT1 inhibitor. Collectively, these results suggest that it is ACAT1-catalyzed 24S-OHC esterification and the resulting lipid droplet formation that is the initial key event which is responsible for 24S-OHC-induced cell death.