Synthetic growth hormone-releasing hormone agonist ameliorates the myocardial pathophysiology characteristic of heart failure with preserved ejection fraction.

AIMS: To test the hypothesis that the activation of the growth hormone-releasing hormone (GHRH) receptor signalling pathway within the myocardium both prevents and reverses diastolic dysfunction and pathophysiologic features consistent with heart failure with preserved ejection fraction (HFpEF). Impaired myocardial relaxation, fibrosis, and ventricular stiffness, among other multi-organ morbidities, characterize the phenotype underlying the HFpEF syndrome. Despite the rapidly increasing prevalence of HFpEF, few effective therapies have emerged. Synthetic agonists of the GHRH receptors reduce myocardial fibrosis, cardiomyocyte hypertrophy, and improve performance in animal models of ischaemic cardiomyopathy, independently of the growth hormone axis. METHODS AND RESULTS: CD1 mice received 4- or 8-week continuous infusion of angiotensin-II (Ang-II) to generate a phenotype with several features consistent with HFpEF. Mice were administered either vehicle or a potent synthetic agonist of GHRH, MR-356 for 4-weeks beginning concurrently or 4-weeks following the initiation of Ang-II infusion. Ang-II-treated animals exhibited diastolic dysfunction, ventricular hypertrophy, interstitial fibrosis, and normal ejection fraction. Cardiomyocytes isolated from these animals exhibited incomplete relaxation, depressed contractile responses, altered myofibrillar protein phosphorylation, and disturbed calcium handling mechanisms (ex vivo). MR-356 both prevented and reversed the development of the pathological phenotype in vivo and ex vivo. Activation of the GHRH receptors increased cAMP and cGMP in cardiomyocytes isolated from control animals but only cAMP in cardiac fibroblasts, suggesting that GHRH-A exert differential effects on cardiomyocytes and fibroblasts. CONCLUSION: These findings indicate that the GHRH receptor signalling pathway(s) represents a new molecular target to counteract dysfunctional cardiomyocyte relaxation by targeting myofilament phosphorylation and fibrosis. Accordingly, activation of GHRH receptors with potent, synthetic GHRH agonists may provide a novel therapeutic approach to management of the myocardial alterations associated with the HFpEF syndrome.

S-nitrosoglutathione reductase (GSNOR) deficiency accelerates cardiomyocyte differentiation of induced pluripotent stem cells.

INTRODUCTION: Induced pluripotent stem cells (iPSCs) provide a model of cardiomyocyte (CM) maturation. Nitric oxide signaling promotes CM differentiation and maturation, although the mechanisms remain controversial. AIM: The study tested the hypothesis that in the absence of S-nitrosoglutathione reductase (GSNOR), a denitrosylase regulating protein S-nitrosylation, the resultant increased S-nitrosylation accelerates the differentiation and maturation of iPSC-derived cardiomyocytes (CMs). METHODS AND RESULTS: iPSCs derived from mice lacking GSNOR (iPSCGSNOR-/-) matured faster than wildtype iPSCs (iPSCWT) and demonstrated transient increases in expression of murine Snail Family Transcriptional Repressor 1 gene (Snail), murine Snail Family Transcriptional Repressor 2 gene (Slug) and murine Twist Family BHLH Transcription Factor 1 gene (Twist), transcription factors that promote epithelial-to-mesenchymal transition (EMT) and that are regulated by Glycogen Synthase Kinase 3 Beta (GSK3ß). Murine Glycogen Synthase Kinase 3 Beta (Gsk3ß) gene exhibited much greater S-nitrosylation, but lower expression in iPSCGSNOR-/-. S-nitrosoglutathione (GSNO)-treated iPSCWT and human (h)iPSCs also demonstrated reduced expression of GSK3ß. Nkx2.5 expression, a CM marker, was increased in iPSCGSNOR-/- upon directed differentiation toward CMs on Day 4, whereas murine Brachyury (t), Isl1, and GATA Binding Protein (Gata4) mRNA were decreased, compared to iPSCWT, suggesting that GSNOR deficiency promotes CM differentiation beginning immediately following cell adherence to the culture dish-transitioning from mesoderm to cardiac progenitor. CONCLUSION: Together these findings suggest that increased S-nitrosylation of Gsk3ß promotes CM differentiation and maturation from iPSCs. Manipulating the post-translational modification of GSK3ß may provide an important translational target and offers new insight into understanding of CM differentiation from pluripotent stem cells. ONE SENTENCE SUMMARY: Deficiency of GSNOR or addition of GSNO accelerates early differentiation and maturation of iPSC-cardiomyocytes.

High-Throughput Screening Identifies MicroRNAs Regulating Human PCSK9 and Hepatic Low-Density Lipoprotein Receptor Expression.

Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3'-UTR) of human microRNA library screen, we identified microRNAs targeting the 3' untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3'-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.

Growth hormone-releasing hormone agonists ameliorate chronic kidney disease-induced heart failure with preserved ejection fraction.

Therapies for heart failure with preserved ejection fraction (HFpEF) are lacking. Growth hormone-releasing hormone agonists (GHRH-As) have salutary effects in ischemic and nonischemic heart failure animal models. Accordingly, we hypothesized that GHRH-A treatment ameliorates chronic kidney disease (CKD)-induced HFpEF in a large-animal model. Female Yorkshire pigs (n = 16) underwent 5/6 nephrectomy via renal artery embolization and 12 wk later were randomized to receive daily subcutaneous injections of GHRH-A (MR-409; n = 8; 30 µg/kg) or placebo (n = 8) for 4 to 6 wk. Renal and cardiac structure and function were serially assessed postembolization. Animals with 5/6 nephrectomy exhibited CKD (elevated blood urea nitrogen [BUN] and creatinine) and faithfully recapitulated the hemodynamic features of HFpEF. HFpEF was demonstrated at 12 wk by maintenance of ejection fraction associated with increased left ventricular mass, relative wall thickness, end-diastolic pressure (EDP), end-diastolic pressure/end-diastolic volume (EDP/EDV) ratio, and tau, the time constant of isovolumic diastolic relaxation. After 4 to 6 wk of treatment, the GHRH-A group exhibited normalization of EDP (P = 0.03), reduced EDP/EDV ratio (P = 0.018), and a reduction in myocardial pro-brain natriuretic peptide protein abundance. GHRH-A increased cardiomyocyte [Ca2+] transient amplitude (P = 0.009). Improvement of the diastolic function was also evidenced by increased abundance of titin isoforms and their ratio (P = 0.0022). GHRH-A exerted a beneficial effect on diastolic function in a CKD large-animal model as demonstrated by improving hemodynamic, structural, and molecular characteristics of HFpEF. These findings have important therapeutic implications for the HFpEF syndrome.

The Presence of Cholesteryl Ester Transfer Protein (CETP) in Endothelial Cells Generates Vascular Oxidative Stress and Endothelial Dysfunction.

Endothelial dysfunction precedes atherosclerosis and is an independent predictor of cardiovascular events. Cholesterol levels and oxidative stress are key contributors to endothelial damage, whereas high levels of plasma high-density lipoproteins (HDL) could prevent it. Cholesteryl ester transfer protein (CETP) is one of the most potent endogenous negative regulators of HDL-cholesterol. However, whether and to what degree CETP expression impacts endothelial function, and the molecular mechanisms underlying the vascular effects of CETP on endothelial cells, have not been addressed. Acetylcholine-induced endothelium-dependent relaxation of aortic rings was impaired in human CETP-expressing transgenic mice, compared to their non-transgenic littermates. However, endothelial nitric oxide synthase (eNOS) activation was enhanced. The generation of superoxide and hydrogen peroxide was increased in aortas from CETP transgenic mice, while silencing CETP in cultured human aortic endothelial cells effectively decreased oxidative stress promoted by all major sources of ROS: mitochondria and NOX2. The endoplasmic reticulum stress markers, known as GADD153, PERK, and ARF6, and unfolded protein response effectors, were also diminished. Silencing CETP reduced endothelial tumor necrosis factor (TNF) α levels, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression, diminishing monocyte adhesion. These results support the notion that CETP expression negatively impacts endothelial cell function, revealing a new mechanism that might contribute to atherosclerosis.

The Anti-atherogenic Role of Exercise Is Associated With the Attenuation of Bone Marrow-Derived Macrophage Activation and Migration in Hypercholesterolemic Mice.

An early event in atherogenesis is the recruitment and infiltration of circulating monocytes and macrophage activation in the subendothelial space. Atherosclerosis subsequently progresses as a unresolved inflammatory disease, particularly in hypercholesterolemic conditions. Although physical exercise training has been a widely accepted strategy to inhibit atherosclerosis, its impact on arterial wall inflammation and macrophage phenotype and function has not yet been directly evaluated. Thus, the aim of this study was to investigate the effects of aerobic exercise training on the inflammatory state of atherosclerotic lesions with a focus on macrophages. Hypercholesterolemic LDL-receptor-deficient male mice were subjected to treadmill training for 8 weeks and fed a high-fat diet. Analyses included plasma lipoprotein and cytokine levels; aortic root staining for lipids (oil red O); macrophages (CD68, MCP1 and IL1ß); oxidative (nitrotyrosine and, DHE) and endoplasmic reticulum (GADD) stress markers. Primary bone marrow-derived macrophages (BMDM) were assayed for migration activity, motility phenotype (Rac1 and F-actin) and inflammation-related gene expression. Plasma levels of HDL cholesterol were increased, while levels of proinflammatory cytokines (TNFa, IL1b, and IL6) were markedly reduced in the exercised mice. The exercised mice developed lower levels of lipid content and inflammation in atherosclerotic plaques. Additionally, lesions in the exercised mice had lower levels of oxidative and ER stress markers. BMDM isolated from the exercised mice showed a marked reduction in proinflammatory cytokine gene expression and migratory activity and a disrupted motility phenotype. More importantly, bone marrow from exercised mice transplanted into sedentary mice led to reduced atherosclerosis in the recipient sedentary mice, thus suggesting that epigenetic mechanisms are associated with exercise. Collectively, the presented data indicate that exercise training prevents atherosclerosis by inhibiting bone marrow-derived macrophage recruitment and activation.

A novel cardiomyogenic role for Isl1+ neural crest cells in the inflow tract.

The degree to which populations of cardiac progenitors (CPCs) persist in the postnatal heart remains a controversial issue in cardiobiology. To address this question, we conducted a spatiotemporally resolved analysis of CPC deployment dynamics, tracking cells expressing the pan-CPC gene Isl1 Most CPCs undergo programmed silencing during early cardiogenesis through proteasome-mediated and PRC2 (Polycomb group repressive complex 2)-mediated Isl1 repression, selectively in the outflow tract. A notable exception is a domain of cardiac neural crest cells (CNCs) in the inflow tract. These "dorsal CNCs" are regulated through a Wnt/ß-catenin/Isl1 feedback loop and generate a limited number of trabecular cardiomyocytes that undergo multiple clonal divisions during compaction, to eventually produce ~10% of the biventricular myocardium. After birth, CNCs continue to generate cardiomyocytes that, however, exhibit diminished clonal amplification dynamics. Thus, although the postnatal heart sustains cardiomyocyte-producing CNCs, their regenerative potential is likely diminished by the loss of trabeculation-like proliferative properties.

LDL Receptor Pathway Regulation by miR-224 and miR-520d.

MicroRNAs (miRNA) have emerged as important post-transcriptional regulators of metabolic pathways that contribute to cellular and systemic lipoprotein homeostasis. Here, we identify two conserved miRNAs, miR-224, and miR-520d, which target gene networks regulating hepatic expression of the low-density lipoprotein (LDL) receptor (LDLR) and LDL clearance. In silico prediction of miR-224 and miR-520d target gene networks showed that they each repress multiple genes impacting the expression of the LDLR, including the chaperone molecules PCSK9 and IDOL that limit LDLR expression at the cell surface and the rate-limiting enzyme for cholesterol synthesis HMGCR, which is the target of LDL-lowering statin drugs. Using gain- and loss-of-function studies, we tested the role of miR-224 and miR-520d in the regulation of those predicted targets and their impact on LDLR expression. We show that overexpression of miR-224 or miR-520d dose-dependently reduced the activity of PCSK9, IDOL, and HMGCR 3'-untranslated region (3'-UTR)-luciferase reporter constructs and that this repression was abrogated by mutation of the putative miR-224 or miR-520d response elements in the PCSK9, IDOL, and HMGCR 3'-UTRs. Compared to a control miRNA, overexpression of miR-224 or miR-520d in hepatocytes inhibited PCSK9, IDOL, and HMGCR mRNA and protein levels and decreased PCSK9 secretion. Furthermore, miR-224 and miR-520d repression of PCSK9, IDOL, and HMGCR was associated with an increase in LDLR protein levels and cell surface expression, as well as enhanced LDL binding. Notably, the effects of miR-224 and miR-520d were additive to the effects of statins in upregulating LDLR expression. Finally, we show that overexpression of miR-224 in the livers of Ldlr +/- mice using lipid nanoparticle-mediated delivery resulted in a 15% decrease in plasma levels of LDL cholesterol, compared to a control miRNA. Together, these findings identify roles for miR-224 and miR-520d in the posttranscriptional control of LDLR expression and function.

Lack of mitochondrial NADP(H)-transhydrogenase expression in macrophages exacerbates atherosclerosis in hypercholesterolemic mice.

The atherosclerosis prone LDL receptor knockout mice (Ldlr-/-, C57BL/6J background) carry a deletion of the NADP(H)-transhydrogenase gene (Nnt) encoding the mitochondrial enzyme that catalyzes NADPH synthesis. Here we hypothesize that both increased NADPH consumption (due to increased steroidogenesis) and decreased NADPH generation (due to Nnt deficiency) in Ldlr-/- mice contribute to establish a macrophage oxidative stress and increase atherosclerosis development. Thus, we compared peritoneal macrophages and liver mitochondria from three C57BL/6J mice lines: Ldlr and Nnt double mutant, single Nnt mutant and wild-type. We found increased oxidants production in both mitochondria and macrophages according to a gradient: double mutant > single mutant > wild-type. We also observed a parallel up-regulation of mitochondrial biogenesis (PGC1a, TFAM and respiratory complexes levels) and inflammatory (iNOS, IL6 and IL1b) markers in single and double mutant macrophages. When exposed to modified LDL, the single and double mutant cells exhibited significant increases in lipid accumulation leading to foam cell formation, the hallmark of atherosclerosis. Nnt deficiency cells showed up-regulation of CD36 and down-regulation of ABCA1 transporters what may explain lipid accumulation in macrophages. Finally, Nnt wild-type bone marrow transplantation into LDLr-/- mice resulted in reduced diet-induced atherosclerosis. Therefore, Nnt plays a critical role in the maintenance of macrophage redox, inflammatory and cholesterol homeostasis, which is relevant for delaying the atherogenesis process.

Mesenchymal Stem Cell Secretion of SDF-1α Modulates Endothelial Function in Dilated Cardiomyopathy.

BACKGROUND: Endothelial dysfunction contributes to the pathophysiology of dilated cardiomyopathy (DCM). Allogeneic but not autologous mesenchymal stem cells (MSCs) improve endothelial function in DCM patients. We hypothesized that these effects are modulated by release of stromal derived factor-1α (SDF-1α). METHODS: Plasma TNFα and endothelial progenitor cell-colony forming units (EPC-CFUs) were assessed at baseline and 3-months post-injection in a subset of POSEIDON-DCM patients that received autologous (n = 11) or allogeneic (n = 10) MSCs. SDF-1α secretion by MSCs, endothelial cell (EC) TNFα mRNA expression, and levels of reactive oxygen species (ROS) in response to SDF-1α were measured in vitro. RESULTS: As previously shown, DCM patients (n = 21) had reduced EPC-CFUs at baseline (3 ± 3), which were restored to normal by allogeneic MSCs 3-months post-treatment (Δ10 ± 4). DCM patients had elevated baseline plasma TNFα (n = 15, 22 ± 9.4 pg/mL). Allogeneic MSCs (n = 8) decreased, and autologous MSCs (n = 7) increased, plasma TNFα (-7.1 ± 3.1 vs. 22.2 ± 17.1 pg/mL, respectively; P = 0.0005). In culture, autologous MSCs (n = 11) secreted higher levels of SDF-1α than allogeneic MSCs (n = 6) [76.0 (63.7, 100.9) vs. 22.8 (7.2, 43.5) pg/mL, P = 0.0002]. SDF-1α and plasma TNFα negatively correlated with EPC-CFUs in both treatment groups (R = -0.7, P = 0.0004). ECs treated with 20 ng SDF-1α expressed lower levels of TNFα mRNA than cells treated with 100 ng (0.7 ± 0.2 vs. 2.1 ± 0.3, P = 0.0008). SDF-1α at low but not high concentration inhibited the generation of ROS. CONCLUSION: MSC secretion of SDF-1α inversely correlates with EPC-CFU production in DCM patients and therefore may be a modulator of MSC therapeutic effect in this clinical setting. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT01392625, identifier NCT01392625.

Laboratory validation of an LC-MS/MS method for the detection of ractopamine, clenbuterol and salbutamol in bovine and swine muscle at sub-µg kg-1 regulatory limits.

Ractopamine (RAC), is a ß-adrenergic agonist increasingly used in the swine and cattle industry. This compound redirects nutrients to favour leanness rather than fat deposition, improves growth and carcass traits gaining higher economic benefit to producers. Countries around the world are split over whether to allow the use of RAC in meat production. Clenbuterol (CLB) and salbutamol (SLB) are anillinic and phenolic ß-agonists, respectively, with the same capacity of producing economic benefits for the meat sector. However, they are prohibited because of the potentially adverse reactions they can cause in consumers. The three ß-agonist compounds have been included in the Brazilian National Regulatory Survey and consequentially there is an eminent need for reliable methods capable of detecting those substances at the same time and reduce analytical costs. Therefore, an LC-MS/MS method for the simultaneous determination of residual RAC, CLB and SAL in swine and cattle muscle was developed and validated with quantification levels respecting the action levels established for Brazil which are 0.1, 0.2 and 5 µg kg-1 for RAC, CLB and SAL, respectively. Samples were quantified using RAC-d5, CLB-d9 and SLB-d6 as internal standards. The validation was performed according to European Union Decision 2002/657, which includes criteria (CCα, CCß, recovery, repeatability, reproducibility and calibration curve). The method meets the Brazilian regulatory requirement that establishes criteria and procedures for the determination of parameters such as CCα, CCß, precision and recovery. CCα values were 0.02, 0.21 and 5.42 µg kg-1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples; CCß values were 0.03, 0.22 and 5.8 µg kg-1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples. Average recoveries fortified with 0.05-7.5 µg kg-1 of the studied ß-agonist leads around 95%. The method was demonstrated to be suitable for the determination of RAC, CLB and SLB in swine and cattle muscle samples.

Apolipoprotein CIII Overexpression-Induced Hypertriglyceridemia Increases Nonalcoholic Fatty Liver Disease in Association with Inflammation and Cell Death.

Nonalcoholic fatty liver disease (NAFLD) is the principal manifestation of liver disease in obesity and metabolic syndrome. By comparing hypertriglyceridemic transgenic mice expressing apolipoprotein (apo) CIII with control nontransgenic (NTg) littermates, we demonstrated that overexpression of apoCIII, independent of a high-fat diet (HFD), produces NAFLD-like features, including increased liver lipid content; decreased antioxidant power; increased expression of TNFα, TNFα receptor, cleaved caspase-1, and interleukin-1ß; decreased expression of adiponectin receptor-2; and increased cell death. This phenotype is aggravated and additional NAFLD features are differentially induced in apoCIII mice fed a HFD. HFD induced glucose intolerance together with increased gluconeogenesis, indicating hepatic insulin resistance. Additionally, the HFD led to marked increases in plasma TNFα (8-fold) and IL-6 (60%) in apoCIII mice. Cell death signaling (Bax/Bcl2), effector (caspase-3), and apoptosis were augmented in apoCIII mice regardless of whether a HFD or a low-fat diet was provided. Fenofibrate treatment reversed several of the effects associated with diet and apoCIII expression but did not normalize inflammatory traits even when liver lipid content was fully corrected. These results indicate that apoCIII and/or hypertriglyceridemia plays a major role in liver inflammation and cell death, which in turn increases susceptibility to and the severity of diet-induced NAFLD.

ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression.

Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and it's absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation.

Lacking of estradiol reduces insulin exocytosis from pancreatic ß-cells and increases hepatic insulin degradation.

Low levels of plasma estrogens are associated with weight-gain, android fat distribution, and a high prevalence of obesity-related comorbidities such as glucose intolerance and type II diabetes. The mechanisms underlying the association between low levels of estrogens and impaired glucose homeostasis are not completely understood. To begin to test this, we used three-month-old female C57BL/6J mice that either underwent ovariectomy (OVX) or received a sham surgery (Sham), and we characterized glucose homeostasis. In a subsequent series of experiments, OVX mice received estradiol treatment (OVX+E2) or vehicle (OVX) for 6 consecutive days. As has been previously reported, lack of ovarian hormones resulted in dysregulated glucose homeostasis. To begin to explore the mechanisms by which this occurs, we characterized the impact of estrogens on insulin secretion and degradation in these mice. Insulin secretion and plasma insulin levels were lower in OVX mice. OVX mice had lower levels of pancreatic Syntaxin 1-A (Synt-1A) protein, which is involved in insulin extrusion from the pancreas. In the liver, OVX mice had higher levels of insulin-degrading enzyme (IDE) and this was associated with higher insulin clearance. Estradiol treatment improved glucose intolerance in OVX mice and restored insulin secretion, as well as normalized the protein content of pancreatic Synt-1A. The addition of estrogens to OVX mice reduced IDE protein to that of Sham mice. Our data suggest loss of ovarian estradiol following OVX led to impaired glucose homeostasis due to pancreatic ß-cell dysfunction in the exocytosis of insulin, and upregulation of hepatic IDE protein content resulting in lower insulinemia, which was normalized by estradiol replacement.

Chronic use of pravastatin reduces insulin exocytosis and increases ß-cell death in hypercholesterolemic mice.

We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr(-/-) mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr(-/-) mice were treated with pravastatin (400mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, p<0.05) of pravastatin treatment. Although reducing insulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases ß-cell death. These findings suggest that prolonged use of statins may have a diabetogenic effect.

Genetic Evidence Supports a Major Role for Akt1 in VSMCs During Atherogenesis.

RATIONALE: Coronary artery disease, the direct result of atherosclerosis, is the most common cause of death in Western societies. Vascular smooth muscle cell (VSMC) apoptosis occurs during the progression of atherosclerosis and in advanced lesions and promotes plaque necrosis, a common feature of high-risk/vulnerable atherosclerotic plaques. Akt1, a serine/threonine protein kinase, regulates several key endothelial cell and VSMC functions including cell growth, migration, survival, and vascular tone. Although global deficiency of Akt1 results in impaired angiogenesis and massive atherosclerosis, the specific contribution of VSMC Akt1 remains poorly characterized. OBJECTIVE: To investigate the contribution of VSMC Akt1 during atherogenesis and in established atherosclerotic plaques. METHODS AND RESULTS: We generated 2 mouse models in which Akt1 expression can be suppressed specifically in VSCMs before (Apoe(-/-)Akt1(fl/fl)Sm22α(CRE)) and after (Apoe(-/-)Akt1(fl/fl)SM-MHC-CreER(T2E)) the formation of atherosclerotic plaques. This approach allows us to interrogate the role of Akt1 during the initial and late steps of atherogenesis. The absence of Akt1 in VSMCs during the progression of atherosclerosis results in larger atherosclerotic plaques characterized by bigger necrotic core areas, enhanced VSMC apoptosis, and reduced fibrous cap and collagen content. In contrast, VSMC Akt1 inhibition in established atherosclerotic plaques does not influence lesion size but markedly reduces the relative fibrous cap area in plaques and increases VSMC apoptosis. CONCLUSIONS: Akt1 expression in VSMCs influences early and late stages of atherosclerosis. The absence of Akt1 in VSMCs induces features of plaque vulnerability including fibrous cap thinning and extensive necrotic core areas. These observations suggest that interventions enhancing Akt1 expression specifically in VSMCs may lessen plaque progression.

Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis.

Atherosclerosis is the major cause of death and disability in diabetic and obese subjects with insulin resistance. Akt2, a phosphoinositide-dependent serine-threonine protein kinase, is highly express in insulin-responsive tissues; however, its role during the progression of atherosclerosis remains unknown. Thus, we aimed to investigate the contribution of Akt2 during the progression of atherosclerosis. We found that germ-line Akt2-deficient mice develop similar atherosclerotic plaques as wild-type mice despite higher plasma lipids and glucose levels. It is noteworthy that transplantation of bone marrow cells isolated from Akt2(-/-) mice to Ldlr(-/-) mice results in marked reduction of the progression of atherosclerosis compared with Ldlr(-/-) mice transplanted with wild-type bone marrow cells. In vitro studies indicate that Akt2 is required for macrophage migration in response to proatherogenic cytokines (monocyte chemotactic protein-1 and macrophage colony-stimulating factor). Moreover, Akt2(-/-) macrophages accumulate less cholesterol and have an alternative activated or M2-type phenotype when stimulated with proinflammatory cytokines. Together, these results provide evidence that macrophage Akt2 regulates migration, the inflammatory response and cholesterol metabolism and suggest that targeting Akt2 in macrophages might be beneficial for treating atherosclerosis.

Long-term therapeutic silencing of miR-33 increases circulating triglyceride levels and hepatic lipid accumulation in mice.

Plasma high-density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR-33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short-term anti-miR-33 treatment has been previously studied, the long-term effect of miR-33 antagonism in vivo remains to be elucidated. Here, we show that long-term therapeutic silencing of miR-33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high-fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR-33 increases the expression of genes involved in fatty acid synthesis such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR-33 antisense oligonucleotides. We also report that anti-miR-33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP-responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR-33 when mice are fed a high-fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR-33 in non-human primates before we can translate this therapy to humans.