Cholesterol-independent Manner Research Articles

Atorvastatin ameliorates lipid overload-induced mitochondrial dysfunction and myocardial hypertrophy by decreasing fatty acid oxidation through inactivation of the p-STAT3/CPT1 pathway

Although statins are shown to have cardiac pleiotropic effects independent of lowering cholesterol, the underlying mechanism remains unclear. Mitochondrial dysfunction induced by increased fatty acid oxidation (FAO) is the culprit in the development of cardiac hypertrophy and dysfunction. This study was to explore whether the cardiac pleiotropic effects of atorvastatin were associated with FAO regulation, with a specific focus on carnitine palmitoyltransferase 1 (CPT1). High-fat diet (HFD)-fed mice and palmitic acid (PA)-stimulated neonatal rat primary cardiomyocytes (NRCMs) were treated with atorvastatin, with or without FAO modulators, signal transducer and activator of transcription 3 (STAT3) agonist, and inhibitor. Atorvastatin (3 mg/kg) did not reduce serum cholesterol levels in HFD-fed mice but ameliorated mitochondrial dysfunction and cardiac hypertrophy. In vitro, atorvastatin and the FAO inhibitor alleviated PA-induced mitochondrial dysfunction and cardiomyocyte hypertrophy. However, the FAO enhancer eliminated atorvastatin’s protective effects. Furthermore, atorvastatin decreased CPT1 and FAO levels and prevented STAT3 phosphorylation and nuclear translocation. STAT3 inhibitor had the same inhibitory effects as atorvastatin on CPT1, FAO levels, and cardiomyocyte hypertrophy, whereas STAT3 agonist disrupted these effects of atorvastatin. Our results demonstrate that atorvastatin decreases myocardial FAO by inactivating the p-STAT3/CPT1 signaling pathway, which improves lipid overload-induced mitochondrial dysfunction and cardiac hypertrophy in a cholesterol-independent manner. This is the first study to explore the cardiac pleiotropic effects of atorvastatin with respect to FAO. However, whether atorvastatin regulates FAO in the cardiac hypertrophy model induced by other variables has not been investigated in this work, and this is expected to be performed in the future.

Serotonin transporter regulation by cholesterol-independent lipid signaling

Serotonin neurotransmission is largely governed by the regulation of the serotonin transporter (SERT). SERT is modulated in part by cholesterol, but the role of cholesterol and lipid signaling intermediates in regulating SERT are unknown. Serotonergic neurons were treated with statins to decrease cholesterol and lipid signaling intermediates. Contrary to reported decreases in 5-HT uptake after cholesterol depletion, biochemical and imaging methods both showed that statins increased 5-HT uptake in a fluoxetine-dependent manner. Simvastatin lowered the Km without changing Vmax for 5-HT or SERT distribution to the plasma membrane. Cholesterol repletion did not block enhanced 5-HT uptake by simvastatin but the enhanced uptake was blocked by lipid isoprenylation intermediates farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Blockade of geranylgeranylation alone without statins also enhanced 5-HT uptake. Overall, this study revealed a specific neuronal effect of statin drugs and identified lipid signaling through geranylgeranylation within the isoprenylation pathway regulates SERT in a cholesterol-independent manner.

Investigation of the phosphatidylserine binding properties of the lipid biosensor, Lactadherin C2 (LactC2), in different membrane environments.

Lipid biosensors are robust tools used in both in vitro and in vivo applications of lipid imaging and lipid detection. Lactadherin C2 (LactC2) was described in 2000 as being a potent and specific sensor for phosphatidylserine (PS) (Andersen et al. Biochemistry 39:6200-6206, 2000). PS is an anionic phospholipid enriched in the inner leaflet of the plasma membrane and has paramount roles in apoptosis, cells signaling, and autophagy. The myriad roles PS plays in membrane dynamics make monitoring PS levels and function an important endeavor. LactC2 has functioned as a tantamount PS biosensor namely in the field of cellular imaging. While PS specificity and high affinity of LactC2 for PS containing membranes has been well established, much less is known regarding LactC2 selectivity for subcellular pools of PS or PS within different membrane environments (e.g., in the presence of cholesterol). Thus, there has been a lack of studies that have compared LactC2 PS sensitivity based upon the acyl chain length and saturation or the presence of other host lipids such as cholesterol. Here, we use surface plasmon resonance as a label-free method to quantitatively assess the apparent binding affinity of LactC2 for membranes containing PS with different acyl chains, different fluidity, as well as representative lipid vesicle mimetics of cellular membranes. Results demonstrate that LactC2 is an unbiased sensor for PS, and can sensitively interact with membranes containing PS with different acyl chain saturation and interact with PS species in a cholesterol-independent manner.

A screen for inducers of bHLH activity identifies pitavastatin as a regulator of p21, Rb phosphorylation and E2F target gene expression in pancreatic cancer.

The average survival for patients with Pancreatic Ductal Adenocarcinoma (PDA) is merely 6 months, underscoring the need for new therapeutic approaches. During PDA progression, pancreatic acinar cells lose activity of the ClassI/II bHLH factors that regulate quiescence. We previously found that promoting transcriptional activity of the Class I bHLH factor E47 in highly aggressive PDA cells induced stable growth arrest in vitro and in vivo. To translate these findings for clinical utility, we developed a high throughput screening platform to identify small molecule inducers of Class I/II bHLH activity. A screen of 4,375 known drugs identified 70 bHLH activators. Prominent among the hits were members of the statin class of HMG-CoA reductase inhibitors, cholesterol lowering drugs that are also being evaluated in cancer. Studies with pitavastatin in primary patient derived tumor cells and established PDA lines, revealed dose dependent growth inhibition. At the molecular level, pitavastatin induced expression of the cyclin dependent kinase (CDK) inhibitor p21 in a cholesterol independent manner, blocked repressive phosphorylation of the Retinoblastoma tumor suppressor protein at CDK targeted sites, and reduced expression of E2F target genes required for progression through the G1/S boundary. Together, the data provide new insight into mechanisms by which statins constrain proliferation in cancer and establish the effectiveness of a novel screening platform to identify small molecules of clinical relevance in pancreatic cancer.

Low levels of methyl β-cyclodextrin disrupt GluA1-dependent synaptic potentiation but not synaptic depression.

Methyl-β-cyclodextrin (MβCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol-enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MβCD. However, how MβCD changes synaptic function, such as N-methyl-d-aspartate receptor (NMDA-R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MβCD. At low concentrations (0.5 mg/mL), MβCD decreased basal synaptic transmission and miniature excitatory post-synaptic current without changing NMDA-R-mediated synaptic transmission and the paired-pulse facilitation ratio. Interestingly, low doses of MβCD failed to deplete cholesterol or affect α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R) and NMDA-R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MβCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2-lacking AMPA-R. MβCD successfully decreased NMDA-R-mediated long-term potentiation but did not affect the formation of either NMDA-R-mediated or group I metabotropic glutamate receptor-dependent long-term depression. MβCD inhibited de-depression without affecting de-potentiation. These results suggest that MβCD regulates GluA1-dependent synaptic potentiation but not synaptic depression in a cholesterol-independent manner.

Statin treatment affects cytokine release and phagocytic activity in primary cultured microglia through two separable mechanisms.

BackgroundAs the primary immune cells of the central nervous system, microglia contribute to development, homeostasis, and plasticity of the central nervous system, in addition to their well characterized roles in the foreign body and inflammatory responses. Increasingly, inappropriate activation of microglia is being reported as a component of inflammation in neurodegenerative and neuropsychiatric disorders. The statin class of cholesterol-lowering drugs have been observed to have anti-inflammatory and protective effects in both neurodegenerative diseases and ischemic stroke, and are suggested to act by attenuating microglial activity.ResultsWe sought to investigate the effects of simvastatin treatment on the secretory profile and phagocytic activity of primary cultured rat microglia, and to dissect the mechanism of action of simvastatin on microglial activity. Simvastatin treatment altered the release of cytokines and trophic factors from microglia, including interleukin-1-β, tumour necrosis factor-α, and brain derived neurotrophic factor in a cholesterol-dependent manner. Conversely, simvastatin inhibited phagocytosis in microglia in a cholesterol-independent manner.ConclusionsThe disparity in cholesterol dependence of cytokine release and phagocytosis suggests the two effects occur through distinct molecular mechanisms. These two pathways may provide an opportunity for further refinement of pharmacotherapies for neuroinflammatory, neurodegenerative, and neuropsychiatric disorders.

Co-administration of ezetimibe enhances proteinuria-lowering effects of pitavastatin in chronic kidney disease patients partly via a cholesterol-independent manner

Since co-administration of ezetimibe, a specific inhibitor of cholesterol absorption into the intestine, has been shown to augment lipid-lowering effects of statins, ezetimibe plus statins is a novel therapeutic strategy for the treatment of dyslipidemia in high-risk patients. Statins have been shown to ameliorate renal function and reduce proteinuria in patients with chronic kidney disease (CKD). However, effects of co-administration of ezetimibe with statins on renal damage and dysfunction in CKD patients remain unknown. In this study, we examined whether co-administration of ezetimibe with pitavastatin could augment renoprotective properties of pitavastatin in non-diabetic CKD patients with dyslipidemia. Total cholesterol, LDL-cholesterol and triglycerides levels were reduced more by co-administration of ezetimibe (10 mg/day) with pitavastatin (2 mg/day) ( n = 10) than by pitavastatin alone ( n = 10). In addition, ezetimibe plus pitavastatin treatment produced significant incremental reduction in proteinuria related to pitavastatin therapy alone. In univariate analyses, proteinuria was correlated with plasma levels of total cholesterol, LDL-cholesterol, triglycerides, HDL-cholesterol (inversely), asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, and urinary excretion levels of L-fatty acid binding protein (L-FABP), a marker of tubular injury and 8-hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker. Multiple stepwise regression analysis revealed that LDL-cholesterol ( p < 0.001) and urinary excretion levels of L-FABP ( p = 0.001) and 8-OHdG ( p < 0.001) were independently related to proteinuria ( R 2 = 0.969). Our present study demonstrated for the first time that co-administration of ezetimibe enhanced proteinuria-lowering effects of pitavastatin in non-diabetic CKD patients partly via a cholesterol-independent manner. Ezetimibe may have pleiotropic actions that could contribute to renoprotective properties of this lipid-lowering agent.

Cholesterol in the senile plaque: often mentioned, never seen

The lipid components of the senile plaque (SP) remain largely unknown. Senile plaques were said to be enriched in cholesterol in a few studies using the cholesterol probe filipin and a histoenzymatic method based upon cholesterol oxidase activity. We provide data that strongly suggest that these results are false-positive: the SPs were still stained in the absence of the enzyme cholesterol oxidase; filipin still labeled the plaques after lipid extraction. On the other hand, resorufin, the highly fluorescent end-product of the histoenzymatic method, bound with high affinity to the SPs and neurofibrillary tangles in a cholesterol-independent manner, and might serve as a new marker of amyloid. In conclusion, the probable cholesterol enrichment of the SPs has never been proven so far, and might necessitate non-histological methods to be ascertained.

High-Sensitivity C-Reactive Protein as a Predictor of All-Cause Mortality: Implications for Research and Patient Care

Based on more than a decade of evidence, high-sensitivity C-reactive protein (hsCRP)1 is in clinical use as an inexpensive adjunct to global cardiovascular risk prediction, as a tool to determine risk of future diabetes and metabolic syndrome, and as a method for monitoring efficacy of statin therapy. In the last decade, more than 20 prospective cohort studies have indicated that hsCRP concentrations independently associate with future risk of myocardial infarction, stroke, metabolic syndrome, and type 2 diabetes, and most studies of adequate sample size have found that hsCRP adds prognostic information to global risk prediction scores. On this basis, guidelines for use of hsCRP in clinical practice have been issued by the Centers for Disease Control and Prevention (1), and novel algorithms for risk prediction that incorporate hsCRP, such as the Reynolds Risk Score (www.reynoldsriskscore.com), have been shown to improve risk classification, particularly for “intermediate risk” patients who account for more than 70% of all vascular events (2). Further, as statins lower hsCRP in an LDL cholesterol–independent manner and as best clinical outcomes occur among statin-treated patients who not only reduce LDL cholesterol below 700 mg/L (70 mg/dL) but also reduce hsCRP below 2 mg/L, the concept of “dual goals” has been introduced into cardiovascular clinical practice (3)(4). Because of its relationships with insulin resistance, leptin, and cytokine function (5), as well as its role in endothelial dysfunction and inhibition of fibrinolysis (6)(7), CRP has also been of pathophysiologic interest as an intellectual bridge linking low-grade systemic inflammation to both diabetogenesis and atherogenesis. Given the consistency and generalizability of these data, it is …

373. Targeting the Central Nervous System by Intravenous Administration of a Targeting Lentiviral Vector

We have developed a targeting lentiviral vector using the modified Sindbis virus envelope protein (Morizono, K et al, Nat. Med. 11: 346|[ndash]|352, Morizono, K et al, J. Virol. 75: 8016|[ndash]|8020). The modified envelope, designated m168, can bind to the Fc region of antibodies, so viral binding and entry can redirected based upon the specificity of the conjugated antibody. We have used this vector to demonstrate targeted transduction of metastatic melanoma in the mouse lung by intravenous administration of the virus. Although the viral vector binds, enters and transduces a wide variety of cells via targeted cell surface molecules, certain cell types are resistant to transduction by the vector in spite of the abundant expression of the targeted molecules. To broaden the application of the vector to more tissues and cell types, we attempted to analyze and overcome the blockage of transduction by the vector. Because the resistant cells are permissive for transduction by VSV-G-pseudotyped lentiviral vectors, the blockage of transduction seems to be present at the step prior to reverse transcription. In addition, the targeting vector efficiently binds to target cells via targeted molecules, so the blockage most likely occurs at fusion between the target cell membrane and the viral envelope. We found that the amount of cholesterol on the target cell membrane controls the efficiency of viral fusion (entry); i.e., depletion of cholesterol from permissive cells decreases the transduction efficiency by the vector, and addition of cholesterol to resistant cells increases the transduction efficiency of the vector. To increase the transduction efficiency to the cells that contain low amounts of cholesterol, we introduced an additional mutation into the E1 gene of the m168 construct, which has been demonstrated to mediate viral fusion in a cholesterol-independent manner. The envelope with the E1 mutation, designated 2.2, dramatically increasedthe transduction efficiency to cholesterol-depleted cells. The 2.2-pseudotyped vector also more efficiently transduces the previously resistant cells in an antibody-dependent manner, including primary peripheral blood mononuclear cells, and human umbilical vein endothelial cells. The 2.2-pseudotyped vector was also tested in an in vivo setting. The virus conjugated with the anti-transferrin receptor was administered intravenously into mice to target blood-brain barrier capillary endothelial cells previously known to express abundant amounts of the transferrin receptor. The 2.2 pseudotype expressed the firefly luciferase transgene at the location of the brain and spine, in an antibody-dependent manner, as demonstrated by cooled coupled charged device imaging. We are trying to define the specific cell types transduced in the central nervous system (CNS) tissues by immunohistochemical analysis, as well as delivery of therapeutic molecules into the CNS.

Descenso de proteína C reactiva tras tratamiento con atorvastatina en diabéticos tipo 2 con dislipemia

Type 2 diabetes mellitus is associated with an augmented risk for cardiovascular disease. The levels of C-reactive protein (CRP), the prototypic marker of inflammation, are associated with an increased risk for cardiovascular events. The statins have direct anti-inflammatory effects. Thus, we tested the effects of atorvastatin on levels of CRP on patients with type 2 diabetes. We evaluated CRP in baseline and 6 months after onset of 20 mg daily atorvastatin therapy of 30 patients with type 2 diabetes with hyperlipidemia. Clinical and biochemical data were obtained. CRP-levels were significantly decreased after treatment with atorvastatin compared with baseline (median change: -4,99 mg/l; p < 0.001). We observed an correlation between CRP baseline with body mass index (r = 0.429; p = 0.018), serum fibrinogen (r = 0.607; p = 0.001) and microalbuminuria (r = 0.470; p = 0.01). Conversely, there was no significant correlation between CRP baseline with LDL cholesterol. The CRP reduction was significantly correlated with fasting glucose (r = -0.457; p = 0.019) and glycosylated hemoglobin at 6 months (r = -0.421; p = 0.03). These results confirm findings from previous studies that atorvastatin reduce CRP levels in a largely LDL cholesterol independent manner.

Statin use is associated with enhanced collateralization of severely diseased coronary arteries

Background The presence of coronary collateral vessels has been associated with improved clinical outcome in patients with coronary artery disease. Animal experiments have shown that hydroxymethyl glutaryl coenzyme A reductase inhibitors (statins) can promote angiogenesis in ischemic tissues in a cholesterol-independent manner. We hypothesized that statin therapy is associated with increased coronary collateral formation in patients with severe coronary artery disease. Methods and results Patients undergoing clinically indicated coronary angiography at the Tufts–New England Medical Center from September 2000 to April 2001 who had at least 1 major coronary artery occlusion, or a stenosis of ≥95% with Thrombolysis In Myocardial Infarction (TIMI) trial grade ≤1 anterograde flow on their angiograms, were included. Fifty-one patients were taking statins before admission, and 43 patients were not. Their angiograms were reviewed and coronary collaterals were graded from 0 to 3 according to the Cohen-Rentrop method. The statin-treated group had a significantly higher mean collateral score compared with the patients not taking statins (2.05 vs 1.52, P = .005). Multivariate analysis supported the significance of the effect of statin therapy on the collateral score. There was no relation between collateral score and low-density lipoprotein levels ( r = −0.06, P = .64). The statin-treated group also had a significantly higher left ventricular ejection fraction compared to the patients not taking statins (51% vs 44%, P < .05). Conclusions Statin therapy is associated with enhanced coronary collateral formation in patients with severely diseased coronary arteries.