Increased Proangiogenic Activity of Mobilized CD34+ Progenitor Cells of Patients With Acute ST-Segment-Elevation Myocardial Infarction: Role of Differential MicroRNA-378 Expression.

OBJECTIVE: Proangiogenic effects of mobilized bone marrow-derived stem/progenitor cells are essential for cardiac repair after myocardial infarction. MicroRNAs (miRNA/miR) are key regulators of angiogenesis. We investigated the differential regulation of angio-miRs, that is, miRNAs regulating neovascularization, in mobilized CD34+ progenitor cells obtained from patients with an acute ST-segment-elevation myocardial infarction (STEMI) as compared with those with stable coronary artery disease or healthy subjects. APPROACH AND RESULTS: CD34+ progenitor cells were isolated from patients with STEMI (on day 0 and day 5), stable coronary artery disease, and healthy subjects (n=27). CD34+ progenitor cells of patients with STEMI exhibited increased proangiogenic activity as compared with CD34+ cells from the other groups. Using a polymerase chain reaction-based miRNA-array and real-time polymerase chain reaction validation, we identified a profound upregulation of 2 known angio-miRs, that are, miR-378 and let-7b, in CD34+ cells of patients with STEMI. Especially, we demonstrate that miR-378 is a critical regulator of the proangiogenic capacity of CD34+ progenitor cells and its stimulatory effects on endothelial cells in vitro and in vivo, whereas let-7b upregulation in CD34+ cells failed to proof its effect on endothelial cells in vivo. CONCLUSIONS: The present study demonstrates a significant upregulation of the angio-miRs miR-378 and let-7b in mobilized CD34+ progenitor cells of patients with STEMI. The increased proangiogenic activity of these cells in patients with STEMI and the observation that in particular miR-378 regulates the angiogenic capacity of CD34+ progenitor cells in vivo suggest that this unique miRNA expression pattern represents a novel endogenous repair mechanism activated in acute myocardial infarction.

AngiomiR-126 expression and secretion from circulating CD34(+) and CD14(+) PBMCs: role for proangiogenic effects and alterations in type 2 diabetics.

Several peripheral blood mononuclear cell (PBMC)-derived cell populations can promote angiogenesis, and differences in CD34(+) or CD14(+) surface expression have been used to separate PBMC subpopulations in this respect. AngiomiRs, microRNAs regulating angiogenesis, are key regulators of angiogenic processes. The present study examines differential angiomiR expression/secretion from CD34(+)/CD14(+), CD34(+)/CD14(-), CD34(-)/CD14(+), and CD34(-)/CD14(-) PBMC subsets and their relevance for different proangiogenic properties. Notably, both circulating human CD34(+)/14(+) and CD34(+)/14(-) PBMC subsets and their supernatants exerted more potent proangiogenic effects compared with CD34(-) PBMC subsets. MiR-126 was identified as most differentially expressed angiomiR in CD34(+) compared with CD34(-) PBMC subsets, determined by miR-array and RT-PCR validation. Modulation of miR-126 by anti-miR-126 or miR-mimic-126 treatment resulted in significant loss or increase of proangiogenic effects of CD34(+) PBMCs. MiR-126 levels in supernatants of CD34(+) PBMC subsets were substantially higher compared with CD34(-) PBMC subsets. MiR-126 was secreted in microvesicles/exosomes, and inhibition of their release impaired CD34(+) PBMCs proangiogenic effects. Notably, high-glucose treatment or diabetes reduced miR-126 levels of CD34(+) PBMCs, associated with impaired proangiogenic properties that could be rescued by miR-mimic-126 treatment. The present findings provide a novel molecular mechanism underlying increased proangiogenic effects of CD34(+) PBMCs, that is, angiomiR-126 expression/secretion. Moreover, an alteration of angiomiR-126 expression in CD34(+) PBMCs in diabetes provides a novel pathway causing impaired proangiogenic effects.

Endothelial overexpression of LOX-1 increases plaque formation and promotes atherosclerosis in vivo.

AIMS: Lectin-like oxLDL receptor-1 (LOX-1) mediates the uptake of oxidized low-density lipoprotein (oxLDL) in endothelial cells and macrophages. However, the different atherogenic potential of LOX-1-mediated endothelial and macrophage oxLDL uptake remains unclear. The present study was designed to investigate the in vivo role of endothelial LOX-1 in atherogenesis. METHODS AND RESULTS: Endothelial-specific LOX-1 transgenic mice were generated using the Tie2 promoter (LOX-1TG). Oxidized low-density lipoprotein uptake was enhanced in cultured endothelial cells, but not in macrophages of LOX-1TG mice. Six-week-old male LOX-1TG and wild-type (WT) mice were fed a high-cholesterol diet (HCD) for 30 weeks. Increased reactive oxygen species production, impaired endothelial nitric oxide synthase activity and endothelial dysfunction were observed in LOX-1TG mice as compared with WT littermates. LOX-1 overexpression led to p38 phosphorylation, increased nuclear factor κB activity and subsequent up-regulation of vascular cell adhesion molecule-1, thereby favouring macrophage accumulation and aortic fatty streaks. Consistently, HCD-fed double-mutant LOX-1TG/ApoE(-/-) displayed oxidative stress and vascular inflammation with higher aortic plaques than ApoE(-/-) controls. Finally, bone marrow transplantation experiments showed that endothelial LOX-1 was sufficient for atherosclerosis development in vivo. CONCLUSIONS: Endothelial-specific LOX-1 overexpression enhanced aortic oxLDL levels, thereby favouring endothelial dysfunction, vascular inflammation and plaque formation. Thus, LOX-1 may serve as a novel therapeutic target for atherosclerosis.

Novel insights into the critical role of bradykinin and the kinin B2 receptor for vascular recruitment of circulating endothelial repair-promoting mononuclear cell subsets: alterations in patients with coronary disease.

BACKGROUND: Endothelial injury is considered critical for progression of atherosclerosis and its complications in coronary artery disease (CAD). The endothelial-supportive effects of bradykinin have mainly been attributed to activation of the resident endothelium. Here we newly investigate the role of bradykinin and its B2 receptor for the recruitment and functional activation of circulating mononuclear cell subsets with endothelial-repair promoting capacity, such as CD34(+)CXCR4(+)cells, at sites of arterial injury. METHODS AND RESULTS: Bradykinin-B2-receptor (B2R) blockade by icatibant substantially impaired recruitment of circulating CD34(+)CXCR4(+) mononuclear cells (expressing high levels of B2R) to endothelial cells in vitro and to injured arterial wall in vivo, whereas recruitment of CD14(hi) monocytes (expressing low levels of B2R) was unchanged. Moreover, the capacity of genetically B2R-deficient bone marrow cells to promote endothelial repair in vivo was markedly impaired as compared with wild-type bone marrow cells. B2R expression was reduced on CD34(+)CXCR4(+)mononuclear cells and endothelial repair-promoting early outgrowth cells, but not on CD14(hi)monocytes, from CAD patients as compared with healthy subjects. B2R stimulation induced CD18 activation in early outgrowth cells of healthy subjects, but not in early outgrowth cells of CAD patients. Adenoviral B2R overexpression enhanced in vivo vascular recruitment and rescued impaired endothelial repair capacity of early outgrowth cells from CAD patients. CONCLUSIONS: We newly report that bradykinin/B2R signaling may promote endothelial repair after arterial injury by selective recruitment and functional activation of B2R-expressing circulating mononuclear cell subsets. In CAD patients, B2R downregulation on endothelial repair-promoting circulating mononuclear cells substantially impairs the bradykinin-dependent endothelial repair, representing a novel mechanism promoting endothelial injury in CAD patients.

Altered activation of endothelial anti- and proapoptotic pathways by high-density lipoprotein from patients with coronary artery disease: role of high-density lipoprotein-proteome remodeling.

BACKGROUND: Endothelial dysfunction and injury are thought to play an important role in the progression of coronary artery disease (CAD). High-density lipoprotein from healthy subjects (HDL(Healthy)) has been proposed to exert endothelial antiapoptotic effects that may represent an important antiatherogenic property of the lipoprotein. The present study therefore aimed to compare effects of HDL(CAD) and HDL(Healthy) on the activation of endothelial anti- and proapoptotic pathways and to determine which changes of the lipoprotein are relevant for these processes. METHODS AND RESULTS: HDL was isolated from patients with stable CAD (HDL(sCAD)), an acute coronary syndrome (HDL(ACS)), and healthy subjects. HDL(Healthy) induced expression of the endothelial antiapoptotic Bcl-2 protein Bcl-xL and reduced endothelial cell apoptosis in vitro and in apolipoprotein E-deficient mice in vivo. In contrast, HDL(sCAD) and HDL(ACS) did not inhibit endothelial apoptosis, failed to activate endothelial Bcl-xL, and stimulated endothelial proapoptotic pathways, in particular, p38-mitogen-activated protein kinase-mediated activation of the proapoptotic Bcl-2 protein tBid. Endothelial antiapoptotic effects of HDL(Healthy) were observed after inhibition of endothelial nitric oxide synthase and after delipidation, but not completely mimicked by apolipoprotein A-I or reconstituted HDL, suggesting an important role of the HDL proteome. HDL proteomics analyses and subsequent validations and functional characterizations suggested a reduced clusterin and increased apolipoprotein C-III content of HDL(sCAD) and HDL(ACS) as mechanisms leading to altered effects on endothelial apoptosis. CONCLUSIONS: The present study demonstrates for the first time that HDL(CAD) does not activate endothelial antiapoptotic pathways, but rather stimulates potential endothelial proapoptotic pathways. HDL-proteome remodeling plays an important role for these altered functional properties of HDL. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in coronary disease.

Deletion of the activated protein-1 transcription factor JunD induces oxidative stress and accelerates age-related endothelial dysfunction.

BACKGROUND: Reactive oxygen species are major determinants of vascular aging. JunD, a member of the activated protein-1 family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to reactive oxygen species homeostasis in the vasculature remains unknown. METHODS AND RESULTS: Endothelium-dependent vasorelaxation was impaired in young and old JunD(-/-) mice (6 and 22 months old) compared with age-matched wild-type mice. JunD(-/-) mice displayed an age-independent decline in endothelial nitric oxide release and endothelial nitric oxide synthase activity and increased mitochondrial superoxide formation and peroxynitrite levels. Furthermore, vascular expression and activity of the free radical scavengers manganese and extracellular superoxide dismutase and aldehyde dehydrogenase 2 were reduced, whereas the NADPH oxidase subunits p47phox, Nox2, and Nox4 were upregulated. These redox changes were associated with premature vascular aging, as shown by reduced telomerase activity, increased ß-galactosidase-positive cells, upregulation of the senescence markers p16(INK4a) and p53, and mitochondrial disruption. Interestingly, old wild-type mice showed a reduction in JunD expression and transcriptional activity resulting from promoter hypermethylation and binding with tumor suppressor menin, respectively. In contrast, JunD overexpression blunted age-induced endothelial dysfunction. In human endothelial cells, JunD knockdown exerted a similar impairment of the O2(-)/nitric oxide balance that was prevented by concomitant NADPH inhibition. In parallel, JunD expression was reduced in monocytes from old versus young healthy subjects and correlated with mRNA levels of scavenging and oxidant enzymes. CONCLUSIONS: JunD provides protection in aging-induced endothelial dysfunction and may represent a novel target to prevent reactive oxygen species-driven vascular aging.

Gene silencing of the mitochondrial adaptor p66(Shc) suppresses vascular hyperglycemic memory in diabetes.

RATIONALE: Hyperglycemic memory may explain why intensive glucose control has failed to improve cardiovascular outcomes in patients with diabetes. Indeed, hyperglycemia promotes vascular dysfunction even after glucose normalization. However, the molecular mechanisms of this phenomenon remain to be elucidated. OBJECTIVE: The present study investigated the role of mitochondrial adaptor p66(Shc) in this setting. METHODS AND RESULTS: In human aortic endothelial cells (HAECs) exposed to high glucose and aortas of diabetic mice, activation of p66(Shc) by protein kinase C ßII (PKCßII) persisted after returning to normoglycemia. Persistent p66(Shc) upregulation and mitochondrial translocation were associated with continued reactive oxygen species (ROS) production, reduced nitric oxide bioavailability, and apoptosis. We show that p66(Shc) gene overexpression was epigenetically regulated by promoter CpG hypomethylation and general control nonderepressible 5-induced histone 3 acetylation. Furthermore, p66(Shc)-derived ROS production maintained PKCßII upregulation and PKCßII-dependent inhibitory phosphorylation of endothelial nitric oxide synthase at Thr-495, leading to a detrimental vicious cycle despite restoration of normoglycemia. Moreover, p66(Shc) activation accounted for the persistent elevation of the advanced glycated end product precursor methylglyoxal. In vitro and in vivo gene silencing of p66(Shc), performed at the time of glucose normalization, blunted ROS production, restored endothelium-dependent vasorelaxation, and attenuated apoptosis by limiting cytochrome c release, caspase 3 activity, and cleavage of poly (ADP-ribose) polymerase. CONCLUSIONS: p66(Shc) is the key effector driving vascular hyperglycemic memory in diabetes. Our study provides molecular insights for the progression of diabetic vascular complications despite glycemic control and may help to define novel therapeutic targets.

Loss of angiomiR-126 and 130a in angiogenic early outgrowth cells from patients with chronic heart failure: role for impaired in vivo neovascularization and cardiac repair capacity.

BACKGROUND: MicroRNAs are key regulators of angiogenic processes. Administration of angiogenic early outgrowth cells (EOCs) or CD34(+) cells has been suggested to improve cardiac function after ischemic injury, in particular by promoting neovascularization. The present study therefore examines regulation of angiomiRs, microRNAs involved in angiogenesis, in angiogenic EOCs and circulating CD34(+) cells from patients with chronic heart failure (CHF) and the role for their cardiac repair capacity. METHODS AND RESULTS: Angiogenic EOCs and CD34(+) cells were isolated from patients with CHF caused by ischemic cardiomyopathy (n=45) and healthy subjects (n=35). In flow cytometry analyses, angiogenic EOCs were largely myeloid and positive for alternatively activated M2 macrophage markers. In vivo cardiac neovascularization and functional repair capacity were examined after transplantation into nude mice with myocardial infarction. Cardiac transplantation of angiogenic EOCs from healthy subjects markedly increased neovascularization and improved cardiac function, whereas no such effect was observed after transplantation of angiogenic EOCs from patients with CHF. Real-time polymerase chain reaction analysis of 14 candidate angiomiRs, expressed in angiogenic EOCs, revealed a pronounced loss of angiomiR-126 and -130a in angiogenic EOCs from patients with CHF that was also observed in circulating CD34(+) cells. Anti-miR-126 transfection markedly impaired the capacity of angiogenic EOCs from healthy subjects to improve cardiac function. miR-126 mimic transfection increased the capacity of angiogenic EOCs from patients with CHF to improve cardiac neovascularization and function. CONCLUSIONS: The present study reveals a loss of angiomiR-126 and -130a in angiogenic EOCs and circulating CD34(+) cells from patients with CHF. Reduced miR-126 expression was identified as a novel mechanism limiting their capacity to improve cardiac neovascularization and function that can be targeted by miR-126 mimic transfection.

Dietary α-linolenic acid inhibits arterial thrombus formation, tissue factor expression, and platelet activation.

OBJECTIVE: Plant-derived α-linolenic acid (ALA) may constitute an attractive cardioprotective alternative to fish-derived n-3 fatty acids. However, the effect of dietary ALA on arterial thrombus formation remains unknown. METHODS AND RESULTS: Male C57Bl/6 mice were fed a high-ALA or low-ALA diet for 2 weeks. Arterial thrombus formation was delayed in mice fed a high-ALA diet compared with those on a low-ALA diet (n=7; P<0.005). Dietary ALA impaired platelet aggregation to collagen and thrombin (n=5; P<0.005) and decreased p38 mitogen-activated protein kinase activation in platelets. Dietary ALA impaired arterial tissue factor (TF) expression, TF activity, and nuclear factor-κB activity (n=7; P<0.05); plasma clotting times and plasma thrombin generation did not differ (n=5; P=not significant). In cultured human vascular smooth muscle and endothelial cells, ALA inhibited TF expression and activity (n=4; P<0.01). Inhibition of TF expression occurred at the transcriptional level via the mitogen-activated protein kinase p38 in smooth muscle cells and p38, extracellular signal-regulated kinases 1 and 2, and c-Jun N-terminal kinases 1 and 2 in endothelial cells. CONCLUSIONS: ALA impairs arterial thrombus formation, TF expression, and platelet activation and thereby represents an attractive nutritional intervention with direct dual antithrombotic effects.

Dietary α-linolenic acid diminishes experimental atherogenesis and restricts T cell-driven inflammation.

AIMS: Epidemiological studies report an inverse association between plant-derived dietary α-linolenic acid (ALA) and cardiovascular events. However, little is known about the mechanism of this protection. We assessed the cellular and molecular mechanisms of dietary ALA (flaxseed) on atherosclerosis in a mouse model. METHODS AND RESULTS: Eight-week-old male apolipoprotein E knockout (ApoE(-/-)) mice were fed a 0.21 % (w/w) cholesterol diet for 16 weeks containing either a high ALA [7.3 % (w/w); n = 10] or low ALA content [0.03 % (w/w); n = 10]. Bioavailability, chain elongation, and fatty acid metabolism were measured by gas chromatography of tissue lysates and urine. Plaques were assessed using immunohistochemistry. T cell proliferation was investigated in primary murine CD3-positive lymphocytes. T cell differentiation and activation was assessed by expression analyses of interferon-γ, interleukin-4, and tumour necrosis factor α (TNFα) using quantitative PCR and ELISA. Dietary ALA increased aortic tissue levels of ALA as well as of the n-3 long chain fatty acids (LC n-3 FA) eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. The high ALA diet reduced plaque area by 50% and decreased plaque T cell content as well as expression of vascular cell adhesion molecule-1 and TNFα. Both dietary ALA and direct ALA exposure restricted T cell proliferation, differentiation, and inflammatory activity. Dietary ALA shifted prostaglandin and isoprostane formation towards 3-series compounds, potentially contributing to the atheroprotective effects of ALA. CONCLUSION: Dietary ALA diminishes experimental atherogenesis and restricts T cell-driven inflammation, thus providing the proof-of-principle that plant-derived ALA may provide a valuable alternative to marine LC n-3 FA.

Acetaminophen increases blood pressure in patients with coronary artery disease.

BACKGROUND: Because traditional nonsteroidal antiinflammatory drugs are associated with increased risk for acute cardiovascular events, current guidelines recommend acetaminophen as the first-line analgesic of choice on the assumption of its greater cardiovascular safety. Data from randomized clinical trials prospectively addressing cardiovascular safety of acetaminophen, however, are still lacking, particularly in patients at increased cardiovascular risk. Hence, the aim of this study was to evaluate the safety of acetaminophen in patients with coronary artery disease. METHODS AND RESULTS: The 33 patients with coronary artery disease included in this randomized, double-blind, placebo-controlled, crossover study received acetaminophen (1 g TID) on top of standard cardiovascular therapy for 2 weeks. Ambulatory blood pressure, heart rate, endothelium-dependent and -independent vasodilatation, platelet function, endothelial progenitor cells, markers of the renin-angiotensin system, inflammation, and oxidative stress were determined at baseline and after each treatment period. Treatment with acetaminophen resulted in a significant increase in mean systolic (from 122.4±11.9 to 125.3±12.0 mm Hg P=0.02 versus placebo) and diastolic (from 73.2±6.9 to 75.4±7.9 mm Hg P=0.02 versus placebo) ambulatory blood pressures. On the other hand, heart rate, endothelial function, early endothelial progenitor cells, and platelet function did not change. CONCLUSIONS: This study demonstrates for the first time that acetaminophen induces a significant increase in ambulatory blood pressure in patients with coronary artery disease. Thus, the use of acetaminophen should be evaluated as rigorously as traditional nonsteroidal antiinflammatory drugs and cyclooxygenase-2 inhibitors, particularly in patients at increased cardiovascular risk. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00534651.

Histamine H1 receptor promotes atherosclerotic lesion formation by increasing vascular permeability for low-density lipoproteins.

OBJECTIVE: Enhanced endothelial permeability leading to intimal accumulation of low-density lipoproteins (LDL) stimulates the formation of atherosclerotic lesions. Histamine is known to increase vascular permeability. Whether this affects the formation of atherosclerotic lesions, however, remains elusive. METHODS AND RESULTS: Apolipoprotein E-null (ApoE(-/-)) mice treated with a histamine H1 receptor but not an H2 receptor antagonist developed 40% fewer atherosclerotic lesions in the aorta than placebo-treated controls. Similarly, genetic deletion of the H1 but not the H2 receptor resulted in a 60% reduction of lesions compared with ApoE(-/-) controls. The H1 receptor enhanced LDL permeability and lipid accumulation in the aorta, whereas plasma lipoprotein levels remained unaltered. In contrast, the H1 receptor did not affect proliferation and migration of vascular smooth muscle cells. Bone marrow transplantation confirmed that the formation of atherosclerotic lesions depended on the H1 receptor in vascular cells, whereas its presence in bone marrow-derived cells was irrelevant for plaque development. Mice expressing the H1 receptor exhibited higher levels of the chemokine (C-C motif) ligand 5 and higher numbers of macrophages and T-helper lymphocytes in plaques, higher numbers of circulating lymphocytes, and larger spleens. CONCLUSION: These data indicate that H1 but not H2 receptor activation drives the formation of atherosclerotic lesions through an increased vascular permeability for LDL, which is associated with an enhanced secondary aortic and systemic inflammation. These data open novel perspectives for the prevention and treatment of atherosclerotic vascular disease.

PDGF-CC induces tissue factor expression: role of PDGF receptor alpha/beta.

Tissue factor (TF) is the principal trigger of the coagulation cascade and involved in arterial thrombus formation. Platelet-derived growth factor CC (PDGF-CC) is a recently discovered member of the PDGF family released upon platelet activation. This study assesses the impact of PDGF-CC on TF expression in human cells. PDGF-CC concentration-dependently induced TF expression by 2.5-fold in THP-1 cells, by 2.0-fold in human peripheral blood monocytes, by 1.4-fold in vascular smooth muscle cells, and by 2.6-fold in microvascular endothelial cells, but did not affect TF expression in aortic endothelial cells. A similar pattern was observed with PDGF-BB. In contrast, PDGF-AA did not alter TF expression in THP-1 cells. TF whole cell activity was induced following stimulation with PDGF-BB and PDGF-CC in THP-1 cells. Real-time polymerase chain reaction revealed that PDGF-CC induced TF mRNA. PDGF-CC transiently activated p42/44 MAP kinase [extracellular signal-regulated kinase (ERK)], while phosphorylation of the MAP kinases c-Jun NH(2)-terminal kinase (JNK) and p38 remained unaffected. PD98059, a specific inhibitor of ERK phosphorylation, but not the p38 inhibitor SB203580 or the JNK inhibitor SP600125 prevented PDGF-CC induced TF expression in a concentration-dependent manner. The effect of PDGF-CC was antagonized by both PDGF receptor alpha and PDGF receptor beta neutralizing antibodies; in contrast, PDGF-BB was only inhibited by PDGF receptor beta blocking antibody. PDGF receptor alpha and PDGF receptor beta inhibition prevented PDGF-CC-induced ERK phosphorylation. PDGF-CC induces TF expression via activation of alpha/beta receptor heterodimers and an ERK-dependent signal transduction pathway.

Inhibition of protein kinase Cbeta prevents foam cell formation by reducing scavenger receptor A expression in human macrophages.

BACKGROUND: Low-density lipoprotein (LDL) uptake by monocyte-derived macrophages is a crucial step in foam cell formation and early atherosclerotic lesion. Increasing evidence supports the theory that activation of protein kinase Cbeta (PKCbeta) is involved in many mechanisms promoting atherosclerosis. Thus, we investigated whether inhibition of PKCbeta prevents foam cell formation. METHODS AND RESULTS: The differentiation of human primary monocytes or the monocytic THP-1 cell line into monocyte-derived macrophages was induced by phorbol 12-myristate 13-acetate (PMA; 0.1 mmol/L), a potent activator of PKC. Incubation of monocyte-derived macrophages with DiI-modified LDL (acetylated LDL and oxidized LDL, 10 mug/mL) led to lipoprotein uptake. Interestingly enough, the nonselective inhibitor of PKCbeta(1) and PKCbeta(2), LY379196 (5x10(-7) to 10(-5) mol/L), blunted LDL uptake in monocyte-derived macrophages as shown by flow cytometry. Specific siRNA-mediated knockdown of PKCbeta exerted a similar effect. Furthermore, PMA alone and in the presence of modified LDL induced scavenger receptor A mRNA and protein expression, which was abolished by LY379196. CGP53353, a selective inhibitor of PKCbeta(2), did not affect LDL uptake, nor did it prevent scavenger receptor A upregulation. Incubation of monocyte-derived macrophages with PMA/LDL increased PKCbeta(1) phosphorylation at the Thr-642 residue, which was blunted by LY379196. However, the expression of CD68, a marker of activated macrophages, was not affected by LY379196. Moreover, LY379196 did not affect lipopolysaccharide-induced CD14 degradation, tumor necrosis factor-alpha release, or superoxide anion production, ruling out any effect of PKCbeta inhibition on innate immunity. CONCLUSIONS: Nonspecific inhibition of PKCbeta prevents LDL uptake in macrophages. These findings suggest that PKCbeta inhibitors may represent a novel class of antiatherosclerotic drugs.

Anacetrapib, but not evacetrapib, impairs endothelial function in CETP-transgenic mice in spite of marked HDL-C increase.

BACKGROUND AND AIMS: High-density lipoprotein cholesterol (HDL-C) is inversely related to cardiovascular risk. HDL-C raising ester transfer protein (CETP) inhibitors, are novel therapeutics. We studied the effects of CETP inhibitors anacetrapib and evacetrapib on triglycerides, cholesterol and lipoproteins, cholesterol efflux, paraoxonase activity (PON-1), reactive oxygen species (ROS), and endothelial function in E3L and E3L.CETP mice. METHODS: Triglycerides and cholesterol were measured at weeks 5, 14 and 21 in E3L.CETP mice on high cholesterol diet and treated with anacetrapib (3 mg/kg/day), evacetrapib (3 mg/kg/day) or placebo. Cholesterol efflux was assessed ex-vivo in mice treated with CETP inhibitors for 3 weeks on a normal chow diet. Endothelial function was analyzed at week 21 in isolated aortic rings, and serum lipoproteins assessed by fast-performance liquid chromatography. RESULTS: Anacetrapib and evacetrapib increased HDL-C levels (5- and 3.4-fold, resp.) and reduced triglycerides (-39% vs. placebo, p = 0.0174). Total cholesterol levels were reduced only in anacetrapib-treated mice (-32%, p = 0.0386). Cholesterol efflux and PON-1 activity (+45% and +35% vs. control, p < 0.005, resp.) were increased, while aortic ROS production was reduced with evacetrapib (-49% vs. control, p = 0.020). Anacetrapib, but not evacetrapib, impaired endothelium dependent vasorelaxation (p < 0.05). In contrast, no such effects were observed in E3L mice for all parameters tested. CONCLUSIONS: Notwithstanding a marked rise in HDL-C, evacetrapib did not improve endothelial function, while anacetrapib impaired it, suggesting that CETP inhibition does not provide vascular protection. Anacetrapib exerts unfavorable endothelial effects beyond CETP inhibition, which may explain the neutral results of large clinical trials in spite of increased HDL-C.

Globotriaosylsphingosine accumulation and not alpha-galactosidase-A deficiency causes endothelial dysfunction in Fabry disease.

BACKGROUND: Fabry disease (FD) is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GLA) resulting in the accumulation of globotriaosylsphingosine (Gb3) in a variety of tissues. While GLA deficiency was always considered as the fulcrum of the disease, recent attention shifted towards studying the mechanisms through which Gb3 accumulation in vascular cells leads to endothelial dysfunction and eventually multiorgan failure. In addition to the well-described macrovascular disease, FD is also characterized by abnormalities of microvascular function, which have been demonstrated by measurements of myocardial blood flow and coronary flow reserve. To date, the relative importance of Gb3 accumulation versus GLA deficiency in causing endothelial dysfunction is not fully understood; furthermore, its differential effects on cardiac micro- and macrovascular endothelial cells are not known. METHODS AND RESULTS: In order to assess the effects of Gb3 accumulation versus GLA deficiency, human macro- and microvascular cardiac endothelial cells (ECs) were incubated with Gb3 or silenced by siRNA to GLA. Gb3 loading caused deregulation of several key endothelial pathways such as eNOS, iNOS, COX-1 and COX-2, while GLA silencing showed no effects. Cardiac microvascular ECs showed a greater susceptibility to Gb3 loading as compared to macrovascular ECs. CONCLUSIONS: Deregulation of key endothelial pathways as observed in FD vasculopathy is likely caused by intracellular Gb3 accumulation rather than deficiency of GLA. Human microvascular ECs, as opposed to macrovascular ECs, seem to be affected earlier and more severely by Gb3 accumulation and this notion may prove fundamental for future progresses in early diagnosis and management of FD patients.

Deletion of L-selectin increases atherosclerosis development in ApoE-/- mice.

Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel(-/-)) animals were crossed with mice lacking Apolipoprotein E (ApoE(-/-)). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE(-/-)L-sel(-/-)) mice and the corresponding ApoE(-/-) controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE(-/-)L-sel(-/-) mice as compared to ApoE(-/-) controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE(-/-)L-sel(-/-) animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE(-/-)L-sel(-/-) and ApoE(-/-) mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE(-/-)L-sel(-/-) as compared to ApoE(-/-) mice. Leukocyte rolling on lesions in the aorta was similar in ApoE(-/-)L-sel(-/-) and ApoE(-/-) animals. ApoE(-/-)L-sel(-/-) mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE(-/-) controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis.

Impaired endothelial repair capacity of early endothelial progenitor cells in prehypertension: relation to endothelial dysfunction.

Prehypertension is a highly frequent condition associated with an increased cardiovascular risk. Endothelial dysfunction is thought to promote the development of hypertension and vascular disease; however, underlying mechanisms remain to be further determined. The present study characterizes for the first time the in vivo endothelial repair capacity of early endothelial progenitor cells (EPCs) in patients with prehypertension/hypertension and examines its relation with endothelial function. Early EPCs were isolated from healthy subjects and newly diagnosed prehypertensive and hypertensive patients (n=52). In vivo endothelial repair capacity of EPCs was examined by transplantation into a nude mouse carotid injury model. EPC senescence was determined (RT-PCR of telomere length). NO and superoxide production of EPCs were measured using electron spin resonance spectroscopy analysis. CD34(+)/KDR(+) mononuclear cells and circulating endothelial microparticles were examined by fluorescence-activated cell sorter analysis. Endothelium-dependent and -independent vasodilations were determined by high-resolution ultrasound. In vivo endothelial repair capacity of EPCs was substantially impaired in prehypertensive/hypertensive patients as compared with healthy subjects (re-endothelialized area: 15+/-3%/13+/-2% versus 28+/-3%; P<0.05 versus healthy subjects). Senescence of EPCs in prehypertension/hypertension was substantially increased, and NO production was markedly reduced. Moreover, reduced endothelial repair capacity of early EPCs was significantly related to an accelerated senescence of early EPCs and impaired endothelial function. The present study demonstrates for the first time that in vivo endothelial repair capacity of early EPCs is reduced in patients with prehypertension and hypertension, is related to EPC senescence and impaired endothelial function, and likely represents an early event in the development of hypertension.