Circulating α-Klotho Counteracts Transforming Growth Factor-ß-Induced Sarcopenia.

α-Klotho is a longevity-related protein. Its deficiency shortens lifespan with prominent senescent phenotypes, including muscle atrophy and weakness in mice. α-Klotho has two forms: membrane α-Klotho and circulating α-Klotho (c-α-Klotho). Loss of membrane α-Klotho impairs a phosphaturic effect, thereby accelerating phosphate-induced aging. However, the mechanisms of senescence on c-α-Klotho loss remain largely unknown. Herein, with the aging of wild-type mice, c-α-Klotho declined, whereas Smad2, an intracellular transforming growth factor (TGF)-ß effector, became activated in skeletal muscle. Moreover, c-α-Klotho suppressed muscle-wasting TGF-ß molecules, including myostatin, growth and differentiation factor 11, activin, and TGF-ß1, through binding to ligands as well as type I and type II serine/threonine kinase receptors. Indeed, c-α-Klotho reversed impaired in vitro myogenesis caused by these TGF-ßs. Oral administration of Ki26894, a small-molecule inhibitor of type I receptors for these TGF-ßs, restored muscle atrophy and weakness in α-Klotho (-/-) mice and in elderly wild-type mice by suppression of activated Smad2 and up-regulated Cdkn1a (p21) transcript, a target of phosphorylated Smad2. Ki26894 also induced the slow to fast myofiber switch. These findings show c-α-Klotho's potential as a circulating inhibitor counteracting TGF-ß-induced sarcopenia. These data highlight the potential of a novel therapy involving TGF-ß blockade to prevent sarcopenia.

The Personal Trait of Spiritual Growth Is Correlated With the White Matter Integrity of the Brain.

Determining the relationship between the entire brain structure and individual differences is important in extending healthy life expectancy, which can be affected by brain atrophy. The entire brain structure has been gradually known to be correlated not only with age but also with individual differences, such as quality of life, general intelligence, and lifestyle. However, little attention has been paid to the relationship between the entire brain structure and personal traits. We herein focused on one personal trait, namely spiritual growth, and examined its relationship with the entire brain structure using two neuroimaging-derived measures, namely the gray matter Brain Healthcare Quotient (GM-BHQ), a measure of GM volume, and the fractional anisotropy Brain Healthcare Quotient (FA-BHQ), a measure of white matter (WM) integrity, in 229 healthy participants (53 female, 176 male). The results indicated no significant relationship between the GM-BHQ and spiritual growth, but there was a significant positive correlation between the FA-BHQ and spiritual growth after controlling for age, sex, and body mass index (BMI) with partial correlation analysis. Furthermore, multiple regression analysis revealed a significant positive correlation between the FA-BHQ and spiritual growth after controlling for physical characteristics, such as age, sex, and BMI, as well as other variables related to lifestyle that were collected using the Health-Promoting Lifestyle Profile. These results support the idea that there is a relationship between the entire WM brain structure and spiritual growth. Further studies are required to clarify the causal relationship between the entire WM brain structure and spiritual growth with some interventions to improve spiritual growth. Such studies will help extend healthy life expectancy from a new perspective of personal trait.

The Validity and Reliability of the Short Form of the Five Facet Mindfulness Questionnaire in Japan.

Background: A brief measure of dispositional mindfulness is important for applied research on mindfulness. Although short forms of the Five Facet Mindfulness Questionnaire (FFMQ), which measures the five aspects of mindfulness (i.e., observing, describing, acting with awareness, non-judging, and non-reactivity), have been developed worldwide, the validity and reliability of the Japanese version has not been examined. This study aims to examine the validity and reliability of the 24-item and 15-item versions of the FFMQ in Japan, which are the most widely used versions worldwide. Methods: Online surveys were conducted for 889 adults in Japan through an online survey company using self-reported questionnaires including the FFMQ to confirm the factor structure and validity. To examine construct validity, we examined the relationship between the short form of FFMQ and mind wandering, interoceptive awareness, experiential avoidance, cognitive fusion, openness, neuroticism, self-compassion, depression, and anxiety, which have been theoretically or empirically shown to be related to mindfulness. In addition, 137 adults responded to the FFMQ again, after four weeks, for the test-retest reliability. Results: The correlated five-factor and four-factor (excluding observing) models and the higher-order factor hierarchical model did not show sufficient goodness of fit, while the 24-item version showed acceptable fit when uncorrelated method factors loaded on by the positive and negative (reverse-scored) items were added. However, the 15-item version did not show acceptable fits for any of the models. Regarding reliability, the 24-item version showed acceptable values. In terms of the relationship between the original and the shortened version of the FFMQ, the 24-item version shared approximately 80% of the variance with the original one. In addition, although the wording effects of positive and negative items seemed to affect the correlations between the FFMQ and the other scales, the associations with related concepts were as predicted generally, supporting the construct validity of the short form of the FFMQ. Conclusion: In Japan, the 24-item version of FFMQ showed acceptable validity and reliability similar to the original version, and we recommend that the 24-item version be used.

Subjective judgments on direct and generative retrieval of autobiographical memory: The role of interoceptive sensibility and emotion.

Autobiographical remembering is a subjective experience, and whether retrieval is perceived to occur through involuntary or voluntary, direct or generative cognitive processes is also based on subjective intuition. The present study examined factors that may contribute to the subjective judgment that occurs when we perceive memories as being retrieved directly (i.e., a memory comes to mind directly and immediately) or through generative processes (i.e., recalling a memory with effort or by using additional information). We examined the hypothesis that internal awareness (interoceptive sensibility and mindfulness traits) contributes to the physical reaction and emotional impact of memories at retrieval, which then influence the subjective judgment that memories are retrieved directly. In two online experiments, participants were asked to recall specific memories following verbal cues and to judge the retrieval process (i.e., direct or generative). We demonstrated that emotional awareness, an interoceptive sensibility scale factor, consistently predicted a high probability of direct retrieval judgments independent of other predictors of direct retrieval, such as retrieval latency and cue concreteness. This effect was especially common for concrete cues. In Experiment 2 we demonstrated that emotional awareness predicted direct retrieval judgments through the mediation of retrieval impact (physical reaction and emotional impact). These results indicate the involvement of interoceptive processing in the direct retrieval of autobiographical memories. We discuss the role of interoception in memory retrieval and present interoceptive prediction error as a novel and potentially integrative account of our findings.

Differential Effects of Focused Attention and Open Monitoring Meditation on Autonomic Cardiac Modulation and Cortisol Secretion.

Mindfulness-based interventions (MBIs) have been used widely as a useful tool for the alleviation of various stress-related symptoms. However, the effects of MBIs on stress-related physiological activity have not yet been ascertained. MBIs primarily consist of focused-attention (FA) and open-monitoring (OM) meditation. Since differing effects of FA and OM meditation on brain activities and cognitive tasks have been mentioned, we hypothesized that FA and OM meditation have also differing effects on stress-related physiological activity. In this study, we examined the effects of FA and OM meditation on autonomic cardiac modulation and cortisol secretion. Forty-one healthy adults (aged 20-46 years) who were meditation novices experienced 30-min FA and OM meditation tasks by listening to instructions. During resting- and meditation-states, electrocardiogram transducers were attached to participants to measure the R-R interval, which were used to evaluate heart rate (HR) and perform heart rate variability (HRV) analyses. Saliva samples were obtained from participants pre- and post-meditation to measure salivary cortisol levels. Results showed that FA meditation induced a decrease in HR and an increase in the root mean square of successive differences (rMSDD). In contrast, OM meditation induced an increase in the standard deviation of the normal-to-normal interval (SDNN) to rMSSD ratio (SDNN/rMSSD) and a decrease in salivary cortisol levels. These results suggest that FA meditation elevates physiological relaxation, whereas OM meditation elevates physiological arousal and reduces stress.

Open monitoring meditation reduces the involvement of brain regions related to memory function.

Mindfulness meditation consists of focused attention meditation (FAM) and open monitoring meditation (OMM), both of which reduce activation of the default mode network (DMN) and mind-wandering. Although it is known that FAM requires intentional focused attention, the mechanisms of OMM remain largely unknown. To investigate this, we examined striatal functional connectivity in 17 experienced meditators (mean total practice hours = 920.6) during pre-resting, meditation, and post-resting states comparing OMM with FAM, using functional magnetic resonance imaging. Both FAM and OMM reduced functional connectivity between the striatum and posterior cingulate cortex, which is a core hub region of the DMN. Furthermore, OMM reduced functional connectivity of the ventral striatum with both the visual cortex related to intentional focused attention in the attentional network and retrosplenial cortex related to memory function in the DMN. In contrast, FAM increased functional connectivity in these regions. Our findings suggest that OMM reduces intentional focused attention and increases detachment from autobiographical memory. This detachment may play an important role in non-judgmental and non-reactive attitude during OMM. These findings provide new insights into the mechanisms underlying the contribution of OMM to well-being and happiness.

The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy.

Myostatin, a muscle-specific transforming growth factor-ß (TGF-ß), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-ß family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-ß1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.

Small molecules with similar structures exhibit agonist, neutral antagonist or inverse agonist activity toward angiotensin II type 1 receptor.

Small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism or inverse agonism toward a G-protein-coupled receptor (GPCR). Although each ligand may stabilize the receptor conformation in a different way, little is known about the precise conformational differences. We synthesized the angiotensin II type 1 receptor blocker (ARB) olmesartan, R239470 and R794847, which induced inverse agonism, antagonism and agonism, respectively, and then investigated the ligand-specific changes in the receptor conformation with respect to stabilization around transmembrane (TM)3. The results of substituted cysteine accessibility mapping studies support the novel concept that ligand-induced changes in the conformation of TM3 play a role in stabilizing GPCR. Although the agonist-, neutral antagonist and inverse agonist-binding sites in the AT(1) receptor are similar, each ligand induced specific conformational changes in TM3. In addition, all of the experimental data were obtained with functional receptors in a native membrane environment (in situ).

An inhibitor of transforming growth factor beta type I receptor ameliorates muscle atrophy in a mouse model of caveolin 3-deficient muscular dystrophy.

Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3(P104L)) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-ß) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-ß type I receptor (TßRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-ß1, -ß2, and -ß3 signaling. Here, we show that a TßRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-ß1, as well as CAV3(P104L). Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3(P104L) mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-ß family members in muscle. These data indicate that both TGF-ß-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TßRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-ß signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings.

A small difference in the molecular structure of angiotensin II receptor blockers induces AT1 receptor-dependent and -independent beneficial effects.

Angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) induce multiple pharmacological beneficial effects, but not all ARBs have the same effects and the molecular mechanisms underlying their actions are not certain. In this study, irbesartan and losartan were examined because of their different molecular structures (irbesartan has a cyclopentyl group whereas losartan has a chloride group). We analyzed the binding affinity and production of inositol phosphate (IP), monocyte chemoattractant protein-1 (MCP-1) and adiponectin. Compared with losartan, irbesartan showed a significantly higher binding affinity and slower dissociation rate from the AT1 receptor and a significantly higher degree of inverse agonism and insurmountability toward IP production. These effects of irbesartan were not seen with the AT1-Y113A mutant receptor. On the basis of the molecular modeling of the ARBs-AT1 receptor complex and a mutagenesis study, the phenyl group at Tyr(113) in the AT1 receptor and the cyclopentyl group of irbesartan may form a hydrophobic interaction that is stronger than the losartan-AT1 receptor interaction. Interestingly, irbesartan inhibited MCP-1 production more strongly than losartan. This effect was mediated by the inhibition of nuclear factor-kappa B activation that was independent of the AT1 receptor in the human coronary endothelial cells. In addition, irbesartan, but not losartan, induced significant adiponectin production that was mediated by peroxisome proliferator-activated receptor-γ activation in 3T3-L1 adipocytes, and this effect was not mediated by the AT1 receptor. In conclusion, irbesartan induced greater beneficial effects than losartan due to small differences between their molecular structures, and these differential effects were both dependent on and independent of the AT1 receptor.

Clinical and pharmacotherapeutic relevance of the double-chain domain of the angiotensin II type 1 receptor blocker olmesartan.

We previously reported that the angiotensin II type 1 (AT(1)) receptor blocker (ARB) olmesartan has two important interactions to evoke inverse agonism (IA). We refer to these interactions as the "double-chain domain (DCD)." Since the clinical pharmacotherapeutic relevance of olmesartan is still unclear, we examined these effects in rats and humans. We analyzed the effects at an advanced stage of renal insufficiency in Dahl salt-sensitive hypertensive rats (Study 1). Rats were fed a high-salt diet from age 9 weeks and arbitrarily assigned to three treatment regimens at age 16 to 21 weeks: olmesartan (2 mg/kg/day) with DCD, a compound related to olmesartan without DCD (6 mg/kg/day, R-239470) or placebo. We also compared the depressor effects of olmesartan to those of other ARBs in patients with essential hypertension (Study 2). Thirty essential hypertensive outpatients who had been receiving ARBs other than olmesartan were recruited for this study. Our protocol was approved by the hospital ethics committee and informed consent was obtained from all patients 12 weeks prior to switching from ARBs other than olmesartan to olmesartan. In Study 1, olmesartan induced a more prominent suppression of the ratio of urinary protein excretion to creatinine at age 21 weeks without lowering blood pressure among the three groups. In Study 2, the depressor effect of olmesartan was significantly stronger than those of other ARBs, which do not contain the DCD. These additive effects by olmesartan may be due to DCD.

Purulent pericarditis with Salmonella enteritidis in a patient with CD4/CD8 depression.

A 65-year-old man was admitted for high-grade fever with a shaking chill and general fatigue. Chest X-ray showed cardiomegaly, and echocardiography revealed a large amount of pericardial effusion. Emergency pericardiocentesis was performed, and Salmonella enteritidis was found in pericardial fluids. We diagnosed purulent pericarditis with S. enteritidis, and administered antibiotics. While high-grade fever resolved 10 days after beginning of treatment, effusive-constrictive pericarditis (ECP) without definite symptoms persisted for 2 months. Because of the improvement of his hemodynamic states on cardiac catheterization after 1 year, an operative procedure was not required. He was diagnosed as having CD4/CD8 depression without apparent diseases. There are few reports of pericarditis with S. enteritidis, and we believe this case might be only the second recorded case of ECP with S. enteritidis.

Conformational switch of angiotensin II type 1 receptor underlying mechanical stress-induced activation.

The angiotensin II type 1 (AT(1)) receptor is a G protein-coupled receptor that has a crucial role in the development of load-induced cardiac hypertrophy. Here, we show that cell stretch leads to activation of the AT(1) receptor, which undergoes an anticlockwise rotation and a shift of transmembrane (TM) 7 into the ligand-binding pocket. As an inverse agonist, candesartan suppressed the stretch-induced helical movement of TM7 through the bindings of the carboxyl group of candesartan to the specific residues of the receptor. A molecular model proposes that the tight binding of candesartan to the AT(1) receptor stabilizes the receptor in the inactive conformation, preventing its shift to the active conformation. Our results show that the AT(1) receptor undergoes a conformational switch that couples mechanical stress-induced activation and inverse agonist-induced inactivation.

Differential bonding interactions of inverse agonists of angiotensin II type 1 receptor in stabilizing the inactive state.

Although the sartan family of angiotensin II type 1 (AT(1)) receptor blockers (ARBs), which includes valsartan, olmesartan, and losartan, have a common pharmacophore structure, their effectiveness in therapy differs. Although their efficacy may be related to their binding strength, this notion has changed with a better understanding of the molecular mechanism. Therefore, we hypothesized that each ARB differs with regard to its molecular interactions with AT(1) receptor in inducing inverse agonism. Interactions between valsartan and residues Ser(105), Ser(109), and Lys(199) were important for binding. Valsartan is a strong inverse agonist of constitutive inositol phosphate production by the wild-type and N111G mutant receptors. Substituted cysteine accessibility mapping studies indicated that valsartan, but not losartan, which has only weak inverse agonism, may stabilize the N111G receptor in an inactive state upon binding. In addition, the inverse agonism by valsatan was mostly abolished with S105A/S109A/K199Q substitutions in the N111G background. Molecular modeling suggested that Ser(109) and Lys(199) bind to phenyl and tetrazole groups of valsartan, respectively. Ser(105) is a candidate for binding to the carboxyl group of valsartan. Thus, the most critical interaction for inducing inverse agonism involves transmembrane (TM) V (Lys(199)) of AT(1) receptor although its inverse agonist potency is comparable to olmesartan, which bonds with TM III (Tyr(113)) and TM VI (His(256)). These results provide new insights into improving ARBs and development of new G protein-coupled receptor antagonists.

Azobenzene derivatives carrying a nitroxide radical.

Several trans-azobenzene derivatives carrying a nitroxide (aminoxyl) radical (2a, 6a-12a) were prepared, and their photoisomerization reactions to the corresponding cis-isomers were investigated. Although no fruitful results could be obtained for the photoisomerizations of the derivatives with para-subsituents (9a-12a), the unsubstututed derivatives at the para-position (2a, 6a, 7a, 8a) were found to show photoisomerizations by irradiation to give the corresponding cis-isomers (2b, 6b, 7b, 8b), being isolated as relatively stable solid materials, and the change of the intermolecular magnetic interactions was apparently observed by the structural change for each photochromic couple.

Dominant-negative lox-1 blocks homodimerization of wild-type lox-1-induced cell proliferation through extracellular signal regulated kinase 1/2 activation.

C-type lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor-1 (Lox-1) belongs to the same family as natural killer cell receptors Ly49A and CD94 and functionally undergoes dimerization. Although Lys262 and Lys263 in the C terminus of bovine (b)Lox-1 play an important role in the uptake of Ox-LDL, mutation of these residues has not been suggested to be a potential source of the dominant-negative property. We hypothesize that dominant-negative human (h)Lox-1 forms a heterodimer with Lox-1-wild-type (WT) and blocks Lox-1-WT-induced cell signaling. Based on the use of molecular imaging techniques with laser scanning confocal microscopy and immunoprecipitation in an hLox-1-expressing Chinese hamster ovary cell system, homodimerization of hLox-1-WT was localized in the cell membrane, and Ox-LDL activated extracellular signal regulated kinase (ERK)1/2 without the translocation of hLox-1-WT. Lys266 and Lys267 of hLox-1, corresponding with Lys262 and Lys263 of bLox-1, were mutated (hLox1-K266A/K267A), and the mutant receptor inhibited hLox-1-WT-induced thymidine incorporation and ERK1/2 activation. Although Ox-LDL binds to the dominant-negative mutant receptor and is taken up by cytoplasm, ERK1/2 activation was blocked by heterodimerization with the mutant receptor and hLox-1-WT in the cell membrane. In addition, in human coronary artery smooth muscle cells, which express hLox-1-WT, we confirmed that the activation of ERK1/2 and [3H]-thymidine incorporation was caused by the addition of Ox-LDL, and these actions were blocked by hLox1-K266A/K267A. In conclusion, the present findings constitute the first evidence that strategies aimed at blocking cell-proliferative pathways at the receptor level could be useful for impairing Lox-1-induced cell proliferation.

Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor.

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.

Low-density lipoprotein oxidized to various degrees activates ERK1/2 through Lox-1.

Although the standard procedure for preparing extensively oxidized low-density lipoprotein (Ox-LDL) is to incubate it with 10muM CuSO(4) at 37 degrees C for 24h, it is not well known how important the degree of oxidation of LDL is for inducing cell signaling. Since Lox-1 (an Ox-LDL receptor) contributes to cell proliferation through extracellular-signal-regulated kinase (ERK)1/2 activation and subsequently induces plaque growth, we analyzed ERK activity using LDL with various degrees of oxidation, from minimally Ox-LDL, which is mainly in human plasma, to extensively Ox-LDL using capillary electrophoresis (cITP). The cITP was a suitable tool for evaluating the degree of oxidation of LDL for analyzing the optimal conditions for the oxidation of LDL by CuSO(4) to obtain LDL that was oxidized to a degree comparable to that in human plasma. In addition, both minimally and extensively Ox-LDL induced similar levels of ERK1/2 activation through Lox-1 in human coronary artery smooth muscle cells. These results indicate that both minimally and extensively Ox-LDL may be important for the progression of plaque growth through Lox-1. Since most previous reports have provided data only using extensively Ox-LDL, a re-evaluation is needed to analyze several signals that use LDL which has been oxidized to various degrees.

Pitavastatin-induced downregulation of CCR2 and CCR5 in monocytes is associated with the arrest of cell-cycle in S phase.

The pleiotropic effects of statin, including its anti-inflammatory effects, via chemokines may be independent of statin-induced cholesterol reduction. Therefore, we examined the effect of pitavastatin on cell proliferation and the association between chemokine receptors (CCR2 and CCR5) and their ligands, RANTES (regulated upon activation, normal T cell-expressed and secreted) and monocyte chemotactic protein-1 (MCP-1), in monocytes. Pitavastatin but not pravastatin inhibited cell proliferation in a dose-dependent manner and showed S-phase arrest associated with the downregulation of CCR2 and CCR5 expression in human monocytic tumor cells (U937 cells). Although the anti-proliferative effects of pitavastatin were not inhibited by lower concentrations of RANTES and MCP-1, overexpression of CCR2/CCR5 significantly blocked the anti-proliferation with a low concentration of RANTES or MCP-1. Pitavastatin upregulated p21(waf1) but not p27(kip1), and did not change the expression levels of cyclin D1 or cdk4. In addition, RANTES and MCP-1 upregulated cyclin D1 in the presence of pitavastatin. In conclusion, the anti-proliferative effect of pitavastatin, but not pravastatin, through the downregulation of CCR2/CCR5 may be a pleiotropic effect. This effect may be anti-atherogenic in monocytes.

Counteracting effects of high density lipoprotein-cholesterol subfractions on statin-induced growth arrest.

An important issue in atherosclerosis is the timing of intimal microvascular formation and its relation to the initiation of plaque formation. Monocytes and endothelial cells (ECs) are important cell components in these steps. Statins not only reduce atherogenic low density lipoprotein cholesterol, they also increase high density lipoprotein-cholesterol (HDL). Although a higher concentration of statin has an anti-proliferative effect, HDL is potentially mitogenic. Therefore, we examined the opposing effects of statin, Apo-AI, HDL and HDL fractions on cell proliferation and apoptosis in monocytes and on angiogenesis in ECs. A high concentration of simvastatin inhibited monocyte proliferation as evaluated by counting cell numbers using a hematocytometer. This inhibition was mainly blocked by the HDL3 subfraction. The same concentration of simvastatin induced apoptosis as assessed by the fluorescence-labeled annexin-V method through caspase-3 activation in monocytes. HDL inhibited simvastatin-induced apoptosis. In addition, HDL blocked simvastatin-inhibited angiogenesis in an in vitro model of EC tube formation. In conclusion, the compensatory balance between the effects of statin and HDL may play an important role in the formation of atherosclerotic lesions by affecting proliferation and apoptosis in monocytes and angiogenesis in ECs.