text:
Open Access This article is
- freely available
- re-usable
Cells 2019, 8(4), 356; https://doi.org/10.3390/cells8040356
Activation of Nrf2/HO-1 Pathway and Human Atherosclerotic Plaque Vulnerability: An In Vitro and In Vivo Study
Centro Cardiologico Monzino, I.R.C.C.S., 20138 Milan, Italy
Department of Cardiovascular & Thoracic Sciences, Fondazione Policlinico Universitario A. Gemelli, I.R.C.C.S., Università Cattolica del Sacro Cuore, 00168 Rome, Italy
Author to whom correspondence should be addressed.
Received: 27 March 2019 / Accepted: 15 April 2019 / Published: 16 April 2019
Reactive oxygen species (ROS) induce nuclear factor erythroid 2–related factor 2 (Nrf2) activation as an adaptive defense mechanism, determining the synthesis of antioxidant molecules, including heme-oxygenase-1 (HO-1). HO-1 protects cells against oxidative injury, degrading free heme and inhibiting ROS production. HO-1 is highly expressed in macrophages during plaque growth. Macrophages are morpho-functionally heterogeneous, and the prevalence of a specific phenotype may influence the plaque fate. This heterogeneity has also been observed in monocyte-derived macrophages (MDMs), a model of macrophages infiltrating tissue. The study aims to assess oxidative stress status and Nrf2/HO-1 axis in MDM morphotypes obtained from healthy subjects and coronary artery disease (CAD) patients, in relation to coronary plaque features evaluated in vivo by optical coherence tomography (OCT). We found that MDMs of healthy subjects exhibited a lower oxidative stress status, lower Nrf2 and HO-1 levels as compared to CAD patients. High HO-1 levels in MDMs were associated with the presence of a higher macrophage content, a thinner fibrous cap, and a ruptured plaque with thrombus formation, detected by OCT analysis. These findings suggest the presence of a relationship between in vivo plaque characteristics and in vitro MDM profile, and may help to identify patients with rupture-prone coronary plaque.
Keywords:oxidative stress; nuclear factor erythroid 2–related factor 2; heme-oxygenase-1; macrophages; plaque vulnerability; optical coherence tomography
1. Introduction
The progression of coronary atherosclerotic plaque and its destabilization with plaque rupture and thrombus formation are the key mechanisms of acute myocardial infarction (AMI) [1,2]. Post-mortem reports have demonstrated that the vulnerable plaque is characterized by a large lipid/necrotic core, a thin fibrous cap, and a great amount of resident macrophages [3]. Macrophages are versatile cells and, in relation to microenvironmental stimuli, they respond by activating different signal transduction pathways, expressing several receptors, and acquiring specific phenotypes. At coronary atherosclerotic plaque level, macrophage population is also characterized by morphological and functional heterogeneity that may enhance plaque growth and/or rupture [4].
An increasing body of evidence suggests that oxidative stress is closely associated with the atherosclerotic process and plaque instability [5,6] through different pathological mechanisms, including endothelial dysfunction, lipid oxidation, expression of adhesion molecules, and monocyte recruitment [7,8,9]. In response to oxidative stress stimuli, cells implement several defense mechanisms and, among them, the activation of nuclear erythroid factor 2 – related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway was reported to be associated with atherosclerosis [10,11]. Under unstressed conditions, Nrf2 is constitutively expressed and sequestrated in the cytoplasm by Keap1 (Kelch-like erythroid cell-derived protein with cap 'n' collar homology-associated protein 1), inducing its proteasomal degradation. On the other hand, under an oxidative stress stimulus, the complex Keap1/Nrf2 dissociates itself, and Nrf2 translocates into the nucleus. At this level, Nrf2 binds the antioxidant responsive element and promotes the transcription of proteins with antioxidant activity. Among them, HO-1 plays a fundamental role in the antioxidant mechanism within the cell by degrading the prooxidant heme to carbon monoxide, biliverdin, and ferrous ion [11]. In addition, HO-1 induction partially inhibits nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity representing a mechanism of cytoprotection against oxidative stress [12].
HO-1 expression is induced by various proatherogenic stimuli and risk factors for cardiovascular diseases [13,14,15]. HO-1 is highly expressed in atherosclerotic plaques, mainly localized in macrophages and foam cells [16], where its antioxidant and anti-inflammatory properties could be fundamental to counteract the development of early stage lesions [17]. In particular, HO-1 reduces the immune cell recruitment and infiltration [18], regulates the macrophage polarization also driving a phenotypic shift towards an anti-inflammatory phenotype [19,20], and inhibits the maturation of dendritic cells [21], thus affecting lesion formation.
HO-1 may also affect plaque progression and its anti-atherogenic role was highlighted in in vitro and in vivo models. Indeed, the induction of HO-1 in co-cultures of human aortic endothelial cells and smooth muscle cells inhibited the monocyte oxidized low density lipoprotein (oxLDL)-dependent chemotaxis [22]. Accordingly, the evidence of an augmented atherogenesis after HO-1 inhibition and an attenuation in the development of atherosclerotic lesion after HO-1 induction confirms this protective role. Indeed, accelerated and more advanced atherosclerotic plaques were described in HO-1 knockout mice [23]. Moreover, the inhibition of HO-1 expression in hyperlipidemic rabbits [24] or in LDL-receptor deficient mice fed with high-fat diet [25] resulted in greater atherosclerotic lesions and increased plasma and tissue lipid peroxide levels.
Despite these preclinical studies, HO-1 levels, higher than those of healthy subjects, were observed in lymphocytes and monocytes isolated from coronary artery disease (CAD) patients. In particular, its expression was higher in patients with AMI than in those with stable angina (SA) [26]. More recently, Cheng et al. showed an increased HO-1 expression in carotid atherosclerotic lesions with a vulnerable phenotype. Of note, the HO-1 levels positively correlated with plaque macrophage and lipid content, and they inversely correlated with stable plaque features, like the presence of intra-plaque smooth muscle cells and collagen [16]. Overall, animal and human studies suggest that HO-1 reflects the severity of atherosclerosis, indicating that a high level of this protein in vulnerable plaque macrophages may represent an antioxidant response, aiming at counteracting the oxidative damage inside atherosclerotic plaque [16,27].
Since resident macrophages are not easily obtainable and manageable, macrophages obtained from a spontaneous differentiation of monocytes (MDMs) are considered to be a good in vitro model to study tissue macrophages. We have previously reported the co-existence of two main and different macrophage morphotypes (round and spindle cells) after 7-day culture of human monocytes isolated from healthy subjects [28]. Similarly to tissue macrophages heterogeneity [4], different MDM morphotypes showed different functional properties: in particular, round-shaped cells were reminiscent of M2 macrophage phenotype with anti-inflammatory and reparative characteristics. On the contrary, spindle-shaped cells showed a pro-inflammatory profile resembling M1 macrophages [28].
In our more recent study, we demonstrated that the peculiar morpho-phenotype profile of MDMs isolated from CAD patients is associated with the characteristics of coronary vulnerable plaque, as assessed by optical coherence tomography (OCT) [29]. This accurate intracoronary imaging technique allows the visualization and the characterization of the atherosclerotic plaque [30,31] providing its detailed architecture and highlighting the rupture prone plaques.
Currently, no data have been provided on the association between HO-1 levels and macrophage phenotype in CAD patients. In this work, we investigated HO-1 levels and the activation of Nrf2/HO-1 axis in different MDM morphotypes obtained from healthy subjects and CAD patients, also in relation to coronary plaque morphology and activity, as analyzed in vivo by OCT.
2. Materials and Methods
2.1. Study Population
Thirty consecutive CAD patients undergoing coronary angiography, due to SA or AMI, as their first manifestation of ischemic heart disease, were enrolled at Centro Cardiologico Monzino, Milan, Italy. SA was defined as angina on effort with a stable pattern of symptoms for at least the last six months prior to admission; AMI diagnosis encompassed patients presenting with non-ST-elevation (NSTEMI) or ST-elevation-acute myocardial infarction (STEMI). NSTEMI was defined as chest pain at rest in the last 48 h preceding the admission associated with evidence of transient ST-segment depression on 12-lead electrocardiogram and normal (unstable angina) or elevated (NSTEMI) serum troponin I (TnI) levels. The diagnosis of STEMI was based on typical symptoms lasting more than 30 min and new ST-segment elevation at the J point in ≥2 contiguous leads. Exclusion criteria were: Previous history of CAD; severe chronic heart failure, severe heart valve disease, acute and chronic infections, liver diseases, neoplasia, evidence of immunologic disorders, recent (<3 months) surgical procedures or trauma and use of anti-inflammatory or immunosuppressive drugs and antioxidant supplements. All AMI included patients underwent percutaneous coronary intervention, specifically within 24 h from admission for NSTEMI and within 12 h of symptom onset for STEMI patients. OCT assessment was performed both in SA and in AMI in order to investigate coronary plaque features. The control group consisted of 10 healthy subjects, with neither history of CAD, nor cardiovascular risk factors, nor inflammatory disorders, and specifically not taking any cardiovascular therapy. The ethics committee approved the study protocol, and all participants provided written informed consent to participate in the study. The study was performed according to the Declaration of Helsinki.
2.2. Monocyte Isolation and Culture
Venous blood samples were drawn from the antecubital vein of healthy subjects and CAD patients when fasting into tubes containing ethylenediaminetetraacetic acid (EDTA) (9.3 mM; Vacutainer Systems, Becton Dickinson, USA). Mononuclear cells were isolated by Ficoll-Paque Plus (GE Healthcare, Milan, Italy) density centrifugation and plated (2 × 106/mL) in 35 mm well plates (Primaria, Falcon, Como, Italy) as previously described [11]. After 90 min, non-adherent cells were removed and adherent cells were cultured over 7 days at 37 °C (5% CO2) in Medium 199 (Lonza, Milan, Italy) supplemented with 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% autologous serum without replacement of the medium throughout the entire culture period. MDM morphology was examined by phase contrast microscopy (Axiovert 200 M; Zeiss, Milan, Italy) at 20× or 40× magnification. MDMs were defined spindle/elongated when a length > 70 µm and a width < 30 µm were detected, and round MDMs when width and length were similar and >30 to 40 µm. Cells, whose morphology and dimension did not satisfy the above criteria, were classified as undefined.
2.3. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis
For the determination of reduced (GSH) and oxidized (GSSG) glutathione levels, MDMs were washed with phosphate buffer saline (PBS) and detached by gentle scraping. After centrifugation (400× g, 10 min), the supernatant was removed and MDMs were lysed in PBS containing 0.1 µg of leupeptin, 0.2 M benzamidine, and 1 µg of trypsin inhibitor. Lysed cells were mixed in a 1:1 (v/v) ratio with 10% trichloroacetic acid (TCA) containing 1 mM EDTA. After centrifugation (10,000× g, 10 min) at room temperature (RT), the supernatant was diluted 1:20 with 0.1% formic acid. The analysis was performed using LC-MS/MS method as previously described [32]. Liquid chromatography was performed on Luna analytical PFP column (100 × 2.0 mm, 3 µm) using an Accela HPLC pump system (Thermo Fisher Scientific, Milan, Italy). Mass spectrometric analysis was performed using a TSQ Quantum Access (Thermo Fisher Scientific) triple quadrupole mass spectrometer coupled with electrospray ionization (ESI) operated in positive mode.
2.4. Western Blot Analysis
Western blot analysis was performed on MDMs total lysate, and on MDMs cytosolic and nuclear fractions.
To obtain MDMs total lysate, cells were harvested and lysated in a buffer composed by 20 mM Tris, 4% sodium dodecyl sulfate (SDS) and 20% glycerol, containing 1 mM sodium orthovanadate, 1 mM NaF, 1 µg/mL leupeptin hemisulfate, 1 mM benzamidine hydrochloride, 1 mM EDTA, 10 µg/mL soybean trypsin inhibitor, 0.5 mM pefabloc, 0.5 mM dithiothreitol (DTT) [33].
For the isolation of cytosolic fraction, MDMs were harvested in 50 µL of buffer containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and then Triton X-100 (0.2% final concentration) was added. Cells were centrifuged at 15,000× g for 1 min at 4 °C to separate cytosols from nuclei [34]. The supernatant containing cytosolic fraction was used for the analysis and the pellet was used for nuclear fraction extraction.
The pellet nuclear fraction was washed with a buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA) containing 0.5 mM PMSF, and 0.5 mM DTT and centrifuged at 15,000× g (10 min, 4 °C) [34]. The supernatant containing nuclear fraction was used for the analysis.
SDS-polyacrylamide gel electrophoresis (PAGE) was performed as previously described [33]. After blotting, membranes carrying MDMs total lysates were incubated overnight at 4 °C with primary antibodies directed against HO-1 (1:250) (catalogue number: ab13248; Abcam, Milan, Italy) or Nrf2 (1:200) (Santa Cruz Biotechnology; catalogue number: sc-722, Milan, Italy).
Membranes carrying MDMs cytosolic and nuclear fraction samples were incubated overnight at 4 °C with primary antibodies directed against Nrf2 (1:200) (Santa Cruz Biotechnology). After incubation with horseradish peroxidase-conjugated anti-mouse (1:10,000, catalogue number: 715-035-151) or anti-rabbit secondary antibody (1:5000, catalogue number: 111-035-003) (Jackson ImmunoResearch Labs Inc., Li StarFISH, Milan, Italy), as appropriate, for 1 h at RT, protein bands were detected by chemiluminescence. β-Actin was used as internal standard for control of protein load.
2.5. Immunofluorescence Analysis
Immunofluorescence was performed as previously described [35]. Fixed MDMs were incubated overnight at 4 °C with a monoclonal rabbit anti-human HO-1 antibody (1:100) (Abcam), or with a polyclonal rabbit anti-human Nrf2 antibody (1:200) (Santa Cruz Biotechnology). Detection was performed with Alexa Fluor 488 (1:200, catalogue number: A11034, 60 min at RT) (Life Technologies Italia, Monza, Italy). Nuclei were visualized by Hoechst 33258 (1:10,000, catalogue number: B2883) (Sigma-Aldrich, Milan, Italy). Negative control experiments were performed by omitting the primary antibodies. Fluorescence quantification was performed as previously described [33]. Data are expressed as mean ± SD of fluorescence intensity/µm2 for each MDM morphotype, subtracted of the negative control value obtained in the absence of primary antibody. Multiple fields of view (at least three fields, 400× magnification) were captured for each culture.
2.6. OCT Image Acquisition and Analysis
OCT examination was performed in AMI patients at culprit lesion and in SA patients at the minimal lumen area (MLA) site. Some plaque characteristics were determined such as the measurement of the thickness of fibrous cap, lipid content, and macrophage accumulation. Fibrous cap thickness was defined as the minimum distance from the coronary artery lumen to the inner border of the lipid pool and a thin-cap fibroatheroma (TCFA) was defined as a minimal fibrous cap thickness ≤ 65 µm; thick-cap fibroatheroma was a plaque with a minimal fibrous cap thickness > 65 μm. The max lipid arc was measured on the frame with the largest lipid core by visual screening. A plaque showing two or more lipid containing quadrants was considered a lipid-rich plaque. A lipid plaque with fibrous cap discontinuity and cavity formation inside the plaque was defined as rupture plaque. A thrombus was defined as an irregular mass protruding into the lumen with a measured dimension > 250 μm.
The macrophage infiltration (MØI) in the analyzed lesions by OCT, has been assessed as previously reported [36], according to the International Working Group for Intravascular Optical Coherence Tomography (IWG-IVOCT) Consensus standards [37]. Briefly, macrophages were qualitatively identified on raw OCT data within a 300 × 125 μm2 (lateral x axial) region of interest (ROI). In particular, macrophages have been visualized by OCT imaging as signal-rich, distinct, or confluent punctate regions that exceed the intensity of background speckle noise and generate a backward shadowing. For caps having a thickness < 125 μm2, the depth of the ROI was matched to the cap thickness. Median filtering was performed with a 3 × 3 square kernel to remove speckle noise. In plaques with MØI, quantitative evaluation of macrophage content was obtained by measuring the normalized standard deviation (NSD) known to have a high degree of positive correlation with histological measurements of macrophage content, by using a dedicated software provided by S. Jude medical [38,39]. In particular, NSD was measured for each pixel within each cap using a 125 μm2 window centered at the pixel location:where NSD (x,y) is the normalized standard deviation of the OCT signal at pixel location (x,y), Smax is the maximum OCT image value, and Smin is the minimum OCT image value. Pixels within the (125 × 125) μm2 window that did not overlap with the segmented cap were excluded.
NSD (x,y) = [σ (x,y)125 μm2/(Smax-Smin)] × 100
2.7. Statistical Analysis
Continuous variables are presented as mean ± SD, variables not normally distributed are presented as median and interquartile ranges (IQR), and categorical variables as absolute numbers and percentages. Comparisons between 'healthy subjects' vs. 'CAD patients' groups were performed using independent samples t-Test for normally distributed variables and Wilcoxon rank-sum test for not normally distributed variables.
Comparisons between 'healthy subjects' vs. 'SA', 'NSTEMI', and 'STEMI' groups were performed using ANOVA Test for normally distributed variables and Kruskal–Wallis Test for not normally distributed variables. Categorical variables were compared using Fisher's exact test. Post-hoc testing of main effects was performed using Bonferroni adjustment for multiple comparisons (α/[number of comparisons]). Correlations between variables were determined using Spearman's rank test. Trends of variation from healthy subjects to STEMI patients were assessed by general linear models. All tests were two-sided, and a p value of less than 0.05 was required for statistical significance. All calculations were computed by using SAS software package v9.4 (SAS Institute Inc., Cary, NC, USA).
3. Results
3.1. Clinical Features
Demographic and clinical characteristics of the enrolled subjects are shown in Table 1. CAD patients had higher body mass index (BMI) and they were more frequently males. Moreover, as expected, in CAD group there was a prevalence of subjects with cardiovascular risk factors as smoking, diabetes, dyslipidemia, hypertension, and family history of cardiovascular disease. Despite there being more subjects with dyslipidemia among CAD patients, the LDL cholesterol values were similar between patients and healthy subjects, as the result of pharmacological treatment. Furthermore, CAD patients had higher levels of glycaemia and of C-reactive protein (hs-CRP), an inflammatory marker.
Of the 30 consecutive CAD patients, 10 (33.3%) had a diagnosis of SA, whereas 20 (66.6%) of AMI (10 NSTEMI (33.3%) and 10 STEMI (33.3%)). Among CAD patients, those with STEMI showed a higher BMI, higher levels of hs-CRP, creatinine, glycaemia, total cholesterol, triglycerides, TnI, and creatine phosphokinase-MB (CK-MB). In addition, lower high-density lipoprotein (HDL) cholesterol levels were observed in AMI patients. No difference in admission therapy was observed among CAD patients.
3.2. Oxidative Stress Status
The levels of GSH and GSSG, whose ratio is a recognized index of oxidative stress, were measured in MDMs obtained from the study population. The results are showed in Figure 1. The GSH/GSSG ratio was significantly lower in CAD patients as compared to healthy subjects. The analysis of the different clinical presentations of CAD revealed a progressive decrease of GSH/GSSG ratio in MDMs going from SA, NSTEMI, to STEMI patients (ptrend < 0.005).
3.3. HO-1 and Nrf2 Expression
MDMs of CAD patients displayed higher levels of HO-1 protein as compared to those observed in healthy subjects (0.12 ± 0.09 vs. 0.06 ± 0.02, p < 0.05) (Figure 2a). Moreover, the immunofluorescence analysis showed higher protein levels in spindle compared to round cells in all study patients' groups. (Figure 2b) with a significant increase in both MDM morphotypes of STEMI patients. A progressive increase was also shown going from healthy subjects to STEMI patients (ptrend round < 0.0002, ptrend spindle < 0.0001; Figure 2c).
This behavior was mirrored by the levels of the transcription factor Nrf2. Higher levels of total Nrf2 were detected in CAD patients as compared to healthy subjects (1.49 ± 0.73 vs. 0.51 ± 0.62, p < 0.01) (Figure 3a). In addition, the evaluation of Nrf2 levels in cytoplasmic and nuclear fractions demonstrated a significant increase of Nrf2 translocation into the nucleus in CAD patients as compared to those of healthy subjects (Figure 3b). The immunofluorescence analysis of the MDM morphotypes evidenced significantly higher Nrf2 protein levels in both spindle and round MDMs of AMI patients (NSTEMI and STEMI) as compared to those of healthy subjects (Figure 3c). Furthermore, an increasing trend in protein levels was detected in both MDM morphotypes paralleling the severity of the clinical presentations (ptrend round < 0.02, ptrend spindle = 0.06) (Figure 3d).
3.4. Association Between In Vivo Plaque Morphology and HO-1 Levels in MDMs
The coronary plaque characteristics in CAD patients are illustrated in Table 2.
Patients with high levels of HO-1 in both MDM morphotypes more frequently displayed a TCFA (p = 0.049 and p = 0.015, spindle and round, respectively) (Figure 4a), a ruptured plaque (p = 0.001 and p = 0.036, spindle and round, respectively) (Figure 4b), and presence of thrombi (p = 0.0005 and p = 0.028, spindle and round, respectively) (Figure 4c).
In addition, in both MDM morphotypes we observed significant positive correlations between HO-1 levels and macrophage content (NSD) (spindle: r = 0.62, p = 0.003; round: r = 0.59, p = 0.005) (Figure 5a). Moreover, borderline positive correlations were observed between HO-1 levels and max lipid arc (spindle: r = 0.34, p = 0.06; round: r = 0.43, p = 0.02) (Figure 5b).
4. Discussion
In the present study we demonstrate higher HO-1 protein levels and the activation of its related Nrf2/HO-1 pathway in MDMs obtained from CAD patients as compared to those obtained from healthy subjects. For the first time, we analyzed this pathway in the different MDM morphotypes. Moreover, we show a positive association between HO-1 levels in MDMs obtained in vitro and the vulnerable coronary plaque features, as detected in vivo by OCT.
HO-1 is a stress response protein that is expressed in several cell types, including macrophages. It is induced by several stimuli inducing oxidative stress, such as cardiovascular risks factors [13,14,15], hypoxia [40], and GSH depletion [41]. In line with in animal models studies [42,43], our previous data obtained in whole blood from CAD patients demonstrated a reduction of GSH levels and a related imbalance of the GSH/GSSG ratio, that is commonly used as an index of oxidative status [44,45]. Here, we detect a progressive decrease of this marker in MDMs obtained from SA, NSTEMI, and STEMI patients, indicating an increase of oxidative stress status in patients with a worse prognosis.
Augmented oxidative stress levels activate the Nrf2/HO-1 pathway as one of the cellular protective mechanisms. The important protective role of HO-1 against human atherosclerosis has been highlighted in population genetic studies that evidenced a polymorphism in the promoter region of human HO-1 gene associated with atherosclerosis predisposition [46,47,48]. In the clinical setting HO-1 deficiency is a very rare condition, but the autopsy case report of HO-1 deficient boy showed hyperlipidemia, presence of foam cells in the liver, and of fatty streak and fibrous plaque in the aorta. These are several lines of evidence that outline the role of HO-1 against atherogenesis [49].
Nevertheless, in our experimental conditions, MDMs from CAD patients expressed a progressive increase of HO-1 levels in both morphotypes, going from healthy to STEMI patients. This is in line with previous studies that infer an adaptive defense role of HO-1 against atherosclerosis [50]. The mechanisms underlying the cardioprotective function of HO-1 involve the antioxidant action of its products [51,52], the reduction in leukocyte recruitment and infiltration, and the suppression of the pro-inflammatory response of immune cells [18].
The increase in HO-1 levels observed in our study CAD population goes in parallel with the increased levels of Nrf2, the transcription factor involved in cell protection from oxidant stressors. Several results strengthen the anti-atherogenic role of Nrf2 in preserving vascular integrity and endothelial function [53], potentially through the release of NO [54] and the protection from cell apoptosis [55]. Moreover, the activation of Nrf2 protects human coronary artery endothelial cells against oxidative challenge [56]. In the present study, we also demonstrate the activation of Nrf2 pathway by its translocation into the nucleus. Indeed, CAD MDMs show higher levels in the nucleus as compared to healthy MDMs. In turn, the latter exhibit very low and feeble protein levels in the nucleus, and high protein levels in the cytosol. This is in line with Nrf2 cytosolic localization in basal condition, where Nrf2 is associated to Keap-1 protein, which induces its ubiquitination and proteasomal degradation. The increase of the Nrf2 stability and its translocation into the nucleus in response to stress signals activates the antioxidant gene transcription [57].
This defense mechanism is evidenced also in atherosclerotic plaques by the presence of high levels of HO-1. Notably, its levels in both carotid [16] and coronary [58] lesions positively correlated with atherosclerotic grade and plaque vulnerability. In addition, Li et al. showed increased levels of HO-1 in lymphocytes and monocytes associated with the severity of the pathology [59]. Accordingly, in our study, patients with the highest levels of HO-1 in MDMs, more frequently showed a vulnerable coronary plaque featured by a TCFA, and an increase in macrophage and lipid content. In addition, in our study, higher levels of HO-1 are detected in MDMs of CAD patients that presented with ruptured plaque and with the presence of thrombus as compared to those with non-ruptured plaque and without thrombi formation. This finding may be explained in light of previous studies reporting a positive correlation between HO-1 and matrix metalloproteinase-9 levels and a negative correlation between the presence of smooth muscle cells and collagen deposition [16]. Moreover, it has been observed that MMP-9 is abundant in carotid plaques characterized by the presence of intraplaque hemorrhage [60].
Overall, these results highlight the activation of Nrf2/HO-1 pathway as an antioxidant response mechanism in MDMs from CAD patients and point out that HO-1 levels may reflect coronary plaque vulnerability. This association could help in identifying patients with rupture-prone plaque and suggests Nrf2/HO-1 pathway as a new potential therapeutic target to counteract plaque progression.
Author Contributions
S.E., V.C., F.C., G.M. and E.T. proposed and supervised the project. S.F., S.E. and V.C. designed the experiments. S.F., B.P., A.D.M. and C.M.M. performed the in vitro experiments. N.C. and G.M. were involved in clinical evaluation and subject enrolment. F.F. (Franco Fabbiocchi), F.F. (Francesco Fracassi) and G.N. performed the in vivo analyses. S.B. performed the statistical analysis. S.F., S.E., V.C., wrote the manuscript. S.F., B.P., A.D.M., S.E., V.C., G.N., F.F. (Franco Fabbiocchi), F.F. (Francesco Fracassi), N.C. and G.M. contributed to data analysis and interpretation of the results. All authors reviewed the manuscript.
This work was supported by the Italian Ministry of Health, Rome, Italy (Ricerca Corrente 2014: CC13; 2015: CC13; 2016: CC18). Co-funding provided by the contribution of the Italian "5 × 1000" tax (2010, 2011, 2012).
The authors thank Fabrizio Veglia for his help in the statistical analysis revision. The authors also thank Alessandra Terragni for her assistance in English language editing.
Conflicts of Interest
The authors declare no conflict of interest
- Schaar, J.A.; Muller, J.E.; Falk, E.; Virmani, R.; Fuster, V.; Serruys, P.W.; Colombo, A.; Stefanadis, C.; Ward Casscells, S.; Moreno, P.R.; et al. Terminology for high-risk and vulnerable coronary artery plaques. Report of a meeting on the vulnerable plaque, June 17 and 18, 2003, Santorini, Greece. Eur. Heart J. 2004, 25, 1077–1082. [Google Scholar] [CrossRef] [PubMed]
- Schwartz, S.M.; Galis, Z.S.; Rosenfeld, M.E.; Falk, E. Plaque rupture in humans and mice. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 705–713. [Google Scholar] [CrossRef]
- Vancraeynest, D.; Pasquet, A.; Roelants, V.; Gerber, B.L.; Vanoverschelde, J.L. Imaging the vulnerable plaque. J. Am. Coll. Cardiol. 2011, 57, 1961–1979. [Google Scholar] [CrossRef] [PubMed]
- Chinetti-Gbaguidi, G.; Colin, S.; Staels, B. Macrophage subsets in atherosclerosis. Nat. Rev. Cardiol. 2015, 12, 10–17. [Google Scholar] [CrossRef]
- Yokoyama, M. Oxidant stress and atherosclerosis. Curr. Opin. Pharmacol. 2004, 4, 110–115. [Google Scholar] [CrossRef] [PubMed]
- Uno, K.; Nicholls, S.J. Biomarkers of inflammation and oxidative stress in atherosclerosis. Biomark. Med. 2010, 4, 361–373. [Google Scholar] [CrossRef] [PubMed]
- Higashi, Y.; Matsuoka, H.; Umei, H.; Sugano, R.; Fujii, Y.; Soga, J.; Kihara, Y.; Chayama, K.; Imaizumi, T. Endothelial function in subjects with isolated low HDL cholesterol: Role of nitric oxide and circulating progenitor cells. Am. J. Physiol. Endocrinol. Metab. 2010, 298, E202–E209. [Google Scholar] [CrossRef] [PubMed]
- Peluso, I.; Morabito, G.; Urban, L.; Ioannone, F.; Serafini, M. Oxidative stress in atherosclerosis development: The central role of LDL and oxidative burst. Endocr. Metab. Immune Disord. Drug Targets 2012, 12, 351–360. [Google Scholar] [CrossRef] [PubMed]
- Lee, C.F.; Ullevig, S.; Kim, H.S.; Asmis, R. Regulation of Monocyte Adhesion and Migration by Nox4. PLoS ONE 2013, 8, e66964. [Google Scholar] [CrossRef]
- Wang, L.J.; Lee, T.S.; Lee, F.Y.; Pai, R.C.; Chau, L.Y. Expression of heme oxygenase-1 in atherosclerotic lesions. Am. J. Pathol. 1998, 152, 711–720. [Google Scholar] [PubMed]
- Morita, T. Heme oxygenase and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 1786–1795. [Google Scholar] [CrossRef] [PubMed]
- Luo, M.; Tian, R.; Lu, N. Nitric oxide protected against NADPH oxidase-derived superoxide generation in vascular endothelium: Critical role for heme oxygenase-1. Int. J. Biol. Macromol. 2019, 126, 549–554. [Google Scholar] [CrossRef] [PubMed]
- Ndisang, J.F.; Zhao, W.; Wang, R. Selective regulation of blood pressure by heme oxygenase-1 in hypertension. Hypertension 2002, 40, 315–321. [Google Scholar] [CrossRef] [PubMed]
- Fukano, Y.; Oishi, M.; Chibana, F.; Numazawa, S.; Yoshida, T. Analysis of the expression of heme oxygenase-1 gene in human alveolar epithelial cells exposed to cigarette smoke condensate. J. Toxicol. Sci. 2006, 31, 99–109. [Google Scholar] [CrossRef] [PubMed]
- Ishii, T.; Itoh, K.; Ruiz, E.; Leake, D.S.; Unoki, H.; Yamamoto, M.; Mann, G.E. Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages: Activation by oxidatively modified LDL and 4-hydroxynonenal. Circ. Res. 2004, 94, 609–616. [Google Scholar] [CrossRef] [PubMed]
- Cheng, C.; Noordeloos, A.M.; Jeney, V.; Soares, M.P.; Moll, F.; Pasterkamp, G.; Serruys, P.W.; Duckers, H.J. Heme oxygenase 1 determines atherosclerotic lesion progression into a vulnerable plaque. Circulation 2009, 119, 3017–3027. [Google Scholar] [CrossRef]
- Araujo, J.A.; Zhang, M.; Yin, F. Heme oxygenase-1, oxidation, inflammation, and atherosclerosis. Front. Pharmacol. 2012, 3, 119. [Google Scholar] [CrossRef] [PubMed]
- Durante, W. Protective role of heme oxygenase-1 against inflammation in atherosclerosis. Front. Biosci. (Landmark Ed.) 2011, 16, 2372–2388. [Google Scholar] [CrossRef]
- Naito, Y.; Takagi, T.; Higashimura, Y. Heme oxygenase-1 and anti-inflammatory M2 macrophages. Arch. Biochem. Biophys. 2014, 564, 83–88. [Google Scholar] [CrossRef]
- Zhang, M.; Nakamura, K.; Kageyama, S.; Lawal, A.O.; Gong, K.W.; Bhetraratana, M.; Fujii, T.; Sulaiman, D.; Hirao, H.; Bolisetty, S.; et al. Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion injury. JCI Insight 2018, 3. [Google Scholar] [CrossRef] [PubMed]
- Chauveau, C.; Remy, S.; Royer, P.J.; Hill, M.; Tanguy-Royer, S.; Hubert, F.X.; Tesson, L.; Brion, R.; Beriou, G.; Gregoire, M.; et al. Heme oxygenase-1 expression inhibits dendritic cell maturation and proinflammatory function but conserves IL-10 expression. Blood 2005, 106, 1694–1702. [Google Scholar] [CrossRef] [PubMed]
- Ishikawa, K.; Navab, M.; Leitinger, N.; Fogelman, A.M.; Lusis, A.J. Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. J. Clin. Investig. 1997, 100, 1209–1216. [Google Scholar] [CrossRef] [PubMed]
- Yet, S.F.; Layne, M.D.; Liu, X.; Chen, Y.H.; Ith, B.; Sibinga, N.E.; Perrella, M.A. Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodeling. FASEB J. 2003, 17, 1759–1761. [Google Scholar] [CrossRef] [PubMed]
- Ishikawa, K.; Sugawara, D.; Goto, J.; Watanabe, Y.; Kawamura, K.; Shiomi, M.; Itabe, H.; Maruyama, Y. Heme oxygenase-1 inhibits atherogenesis in Watanabe heritable hyperlipidemic rabbits. Circulation 2001, 104, 1831–1836. [Google Scholar] [CrossRef] [PubMed]
- Ishikawa, K.; Sugawara, D.; Wang, X.; Suzuki, K.; Itabe, H.; Maruyama, Y.; Lusis, A.J. Heme oxygenase-1 inhibits atherosclerotic lesion formation in LDL-receptor knockout mice. Circ. Res. 2001, 88, 506–512. [Google Scholar] [CrossRef] [PubMed]
- Chen, S.M.; Li, Y.G.; Wang, D.M. Study on changes of heme oxygenase-1 expression in patients with coronary heart disease. Clin. Cardiol. 2005, 28, 197–201. [Google Scholar] [CrossRef] [PubMed]
- Schaer, C.A.; Schoedon, G.; Imhof, A.; Kurrer, M.O.; Schaer, D.J. Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin. Circ. Res. 2006, 99, 943–950. [Google Scholar] [CrossRef]
- Eligini, S.; Crisci, M.; Bono, E.; Songia, P.; Tremoli, E.; Colombo, G.I.; Colli, S. Human monocyte-derived macrophages spontaneously differentiated in vitro show distinct phenotypes. J. Cell. Physiol. 2013, 228, 1464–1472. [Google Scholar] [CrossRef] [PubMed]
- Eligini, S.; Cosentino, N.; Fiorelli, S.; Fabbiocchi, F.; Niccoli, G.; Refaat, H.; Camera, M.; Calligaris, G.; De Martini, S.; Bonomi, A.; et al. Biological profile of monocyte-derived macrophages in coronary heart disease patients: Implications for plaque morphology. Sci. Rep. under review.
- Jang, I.K.; Bouma, B.E.; Kang, D.H.; Park, S.J.; Park, S.W.; Seung, K.B.; Choi, K.B.; Shishkov, M.; Schlendorf, K.; Pomerantsev, E.; et al. Visualization of coronary atherosclerotic plaques in patients using optical coherence tomography: comparison with intravascular ultrasound. J. Am. Coll. Cardiol. 2002, 39, 604–609. [Google Scholar] [CrossRef]
- Jang, I.K.; Tearney, G.J.; MacNeill, B.; Takano, M.; Moselewski, F.; Iftima, N.; Shishkov, M.; Houser, S.; Aretz, H.T.; Halpern, E.F.; et al. In vivo characterization of coronary atherosclerotic plaque by use of optical coherence tomography. Circulation 2005, 111, 1551–1555. [Google Scholar] [CrossRef]
- Squellerio, I.; Caruso, D.; Porro, B.; Veglia, F.; Tremoli, E.; Cavalca, V. Direct glutathione quantification in human blood by LC-MS/MS: comparison with HPLC with electrochemical detection. J. Pharm. Biomed. Anal. 2012, 71, 111–118. [Google Scholar] [CrossRef] [PubMed]
- Eligini, S.; Colli, S.; Basso, F.; Sironi, L.; Tremoli, E. Oxidized low density lipoprotein suppresses expression of inducible cyclooxygenase in human macrophages. Arterioscler. Thromb. Vasc. Biol. 1999, 19, 1719–1725. [Google Scholar] [CrossRef] [PubMed]
- Eligini, S.; Brambilla, M.; Banfi, C.; Camera, M.; Sironi, L.; Barbieri, S.S.; Auwerx, J.; Tremoli, E.; Colli, S. Oxidized phospholipids inhibit cyclooxygenase-2 in human macrophages via nuclear factor-kappaB/IkappaB- and ERK2-dependent mechanisms. Cardiovasc. Res. 2002, 55, 406–415. [Google Scholar] [CrossRef]
- Eligini, S.; Brioschi, M.; Fiorelli, S.; Tremoli, E.; Banfi, C.; Colli, S. Human monocyte-derived macrophages are heterogenous: Proteomic profile of different phenotypes. J. Proteomics 2015, 124, 112–123. [Google Scholar] [CrossRef]
- Scalone, G.; Niccoli, G.; Refaat, H.; Vergallo, R.; Porto, I.; Leone, A.M.; Burzotta, F.; D'Amario, D.; Liuzzo, G.; Fracassi, F.; et al. Not all plaque ruptures are born equal: An optical coherence tomography study. Eur. Heart J. Cardiovasc. Imaging 2017, 18, 1271–1277. [Google Scholar] [CrossRef] [PubMed]
- Tearney, G.J.; Regar, E.; Akasaka, T.; Adriaenssens, T.; Barlis, P.; Bezerra, H.G.; Bouma, B.; Bruining, N.; Cho, J.M.; Chowdhary, S.; et al. Consensus standards for acquisition, measurement, and reporting of intravascular optical coherence tomography studies: A report from the International Working Group for Intravascular Optical Coherence Tomography Standardization and Validation. J. Am. Coll. Cardiol. 2012, 59, 1058–1072. [Google Scholar] [CrossRef] [PubMed]
- Tearney, G.J.; Yabushita, H.; Houser, S.L.; Aretz, H.T.; Jang, I.K.; Schlendorf, K.H.; Kauffman, C.R.; Shishkov, M.; Halpern, E.F.; Bouma, B.E. Quantification of macrophage content in atherosclerotic plaques by optical coherence tomography. Circulation 2003, 107, 113–119. [Google Scholar] [CrossRef]
- Di Vito, L.; Agozzino, M.; Marco, V.; Ricciardi, A.; Concardi, M.; Romagnoli, E.; Gatto, L.; Calogero, G.; Tavazzi, L.; Arbustini, E.; et al. Identification and quantification of macrophage presence in coronary atherosclerotic plaques by optical coherence tomography. Eur. Heart J. Cardiovasc. Imaging 2015, 16, 807–813. [Google Scholar] [CrossRef]
- Neubauer, J.A.; Sunderram, J. Heme oxygenase-1 and chronic hypoxia. Respir. Physiol. Neurobiol. 2012, 184, 178–185. [Google Scholar] [CrossRef]
- Ewing, J.F.; Maines, M.D. Glutathione depletion induces heme oxygenase-1 (HSP32) mRNA and protein in rat brain. J. Neurochem. 1993, 60, 1512–1519. [Google Scholar] [CrossRef]
- Biswas, S.K.; Newby, D.E.; Rahman, I.; Megson, I.L. Depressed glutathione synthesis precedes oxidative stress and atherogenesis in Apo-E(-/-) mice. Biochem. Biophys. Res. Commun. 2005, 338, 1368–1373. [Google Scholar] [CrossRef]
- Ozsarlak-Sozer, G.; Sevin, G.; Ozgur, H.H.; Yetik-Anacak, G.; Kerry, Z. Diverse effects of taurine on vascular response and inflammation in GSH depletion model in rabbits. Eur. Rev. Med. Pharmacol. Sci. 2016, 20, 1360–1372. [Google Scholar]
- Cavalca, V.; Veglia, F.; Squellerio, I.; Marenzi, G.; Minardi, F.; De Metrio, M.; Cighetti, G.; Boccotti, L.; Ravagnani, P.; Tremoli, E. Glutathione, vitamin E and oxidative stress in coronary artery disease: Relevance of age and gender. Eur. J. Clin. Investig. 2009, 39, 267–272. [Google Scholar] [CrossRef]
- Eligini, S.; Porro, B.; Lualdi, A.; Squellerio, I.; Veglia, F.; Chiorino, E.; Crisci, M.; Garlasche, A.; Giovannardi, M.; Werba, J.P.; et al. Nitric oxide synthetic pathway in red blood cells is impaired in coronary artery disease. PLoS ONE 2013, 8, e66945. [Google Scholar] [CrossRef]
- Kaneda, H.; Ohno, M.; Taguchi, J.; Togo, M.; Hashimoto, H.; Ogasawara, K.; Aizawa, T.; Ishizaka, N.; Nagai, R. Heme oxygenase-1 gene promoter polymorphism is associated with coronary artery disease in Japanese patients with coronary risk factors. Arterioscler. Thromb. Vasc. Biol. 2002, 22, 1680–1685. [Google Scholar] [CrossRef]
- Chen, Y.H.; Lin, S.J.; Lin, M.W.; Tsai, H.L.; Kuo, S.S.; Chen, J.W.; Charng, M.J.; Wu, T.C.; Chen, L.C.; Ding, Y.A.; et al. Microsatellite polymorphism in promoter of heme oxygenase-1 gene is associated with susceptibility to coronary artery disease in type 2 diabetic patients. Hum. Genet. 2002, 111, 1–8. [Google Scholar] [CrossRef]
- Schillinger, M.; Exner, M.; Mlekusch, W.; Ahmadi, R.; Rumpold, H.; Mannhalter, C.; Wagner, O.; Minar, E. Heme oxygenase-1 genotype is a vascular anti-inflammatory factor following balloon angioplasty. J. Endovasc. Ther. 2002, 9, 385–394. [Google Scholar] [CrossRef] [PubMed]
- Kawashima, A.; Oda, Y.; Yachie, A.; Koizumi, S.; Nakanishi, I. Heme oxygenase-1 deficiency: The first autopsy case. Hum. Pathol. 2002, 33, 125–130. [Google Scholar] [CrossRef]
- Wu, M.L.; Ho, Y.C.; Yet, S.F. A central role of heme oxygenase-1 in cardiovascular protection. Antioxid. Redox Signal. 2011, 15, 1835–1846. [Google Scholar] [CrossRef]
- Kawamura, K.; Ishikawa, K.; Wada, Y.; Kimura, S.; Matsumoto, H.; Kohro, T.; Itabe, H.; Kodama, T.; Maruyama, Y. Bilirubin from heme oxygenase-1 attenuates vascular endothelial activation and dysfunction. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 155–160. [Google Scholar] [CrossRef]
- Balla, G.; Jacob, H.S.; Balla, J.; Rosenberg, M.; Nath, K.; Apple, F.; Eaton, J.W.; Vercellotti, G.M. Ferritin: A cytoprotective antioxidant strategem of endothelium. J. Biol. Chem. 1992, 267, 18148–18153. [Google Scholar]
- Zakkar, M.; Van der Heiden, K.; Luong le, A.; Chaudhury, H.; Cuhlmann, S.; Hamdulay, S.S.; Krams, R.; Edirisinghe, I.; Rahman, I.; Carlsen, H.; et al. Activation of Nrf2 in endothelial cells protects arteries from exhibiting a proinflammatory state. Arterioscler. Thromb. Vasc. Biol. 2009, 29, 1851–1857. [Google Scholar] [CrossRef] [PubMed]
- Buckley, B.J.; Marshall, Z.M.; Whorton, A.R. Nitric oxide stimulates Nrf2 nuclear translocation in vascular endothelium. Biochem. Biophys. Res. Commun. 2003, 307, 973–979. [Google Scholar] [CrossRef]
- Ungvari, Z.; Bagi, Z.; Feher, A.; Recchia, F.A.; Sonntag, W.E.; Pearson, K.; de Cabo, R.; Csiszar, A. Resveratrol confers endothelial protection via activation of the antioxidant transcription factor Nrf2. Am. J. Physiol. Heart Circ. Physiol. 2010, 299, H18–H24. [Google Scholar] [CrossRef] [PubMed]
- Donovan, E.L.; McCord, J.M.; Reuland, D.J.; Miller, B.F.; Hamilton, K.L. Phytochemical activation of Nrf2 protects human coronary artery endothelial cells against an oxidative challenge. Oxid. Med. Cell. Longev. 2012, 2012, 132931. [Google Scholar] [CrossRef] [PubMed]
- Nguyen, T.; Nioi, P.; Pickett, C.B. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J. Biol. Chem. 2009, 284, 13291–13295. [Google Scholar] [CrossRef] [PubMed]
- Song, J.; Sumiyoshi, S.; Nakashima, Y.; Doi, Y.; Iida, M.; Kiyohara, Y.; Sueishi, K. Overexpression of heme oxygenase-1 in coronary atherosclerosis of Japanese autopsies with diabetes mellitus: Hisayama study. Atherosclerosis 2009, 202, 573–581. [Google Scholar] [CrossRef] [PubMed]
- Li, Y.G.; Wang, D.M.; Chen, S.M.; Tan, X.R.; Fang, X.Y.; Wu, J.W.; Zhang, G.H.; Mai, R.Q. Haem oxygenase-1 expression and coronary heart disease--association between levels of haem oxygenase-1 expression and angiographic morphology as well as the quantity of coronary lesions. Acta Cardiol. 2006, 61, 295–300. [Google Scholar] [CrossRef] [PubMed]
- Choudhary, S.; Higgins, C.L.; Chen, I.Y.; Reardon, M.; Lawrie, G.; Vick, G.W., 3rd; Karmonik, C.; Via, D.P.; Morrisett, J.D. Quantitation and localization of matrix metalloproteinases and their inhibitors in human carotid endarterectomy tissues. Arterioscler. Thromb. Vasc. Biol. 2006, 26, 2351–2358. [Google Scholar] [CrossRef]
Figure 1. GSH/GSSG evaluation in monocyte-derived macrophages (MDMs). MDMs were obtained from CAD patients and healthy subjects. Data are expressed as mean ± SD and derive from independent cultures obtained from 10 healthy subjects and 30 CAD patients (SA n = 10; NSTEMI n = 10; STEMI n = 10. * p < 0.05 vs. healthy subjects.
Figure 2. HO-1 levels in CAD patients and healthy subjects. (a) The protein levels of HO-1 were detected by western blot analysis. β-actin was used as a control of protein loading. Densitometry is shown in the bar graph. Data are expressed as mean ± SD and derive from MDMs obtained from 10 healthy subjects and 17 CAD patients. (b) Representative images of HO-1 in round and spindle MDMs obtained from healthy subjects and CAD patients (400× original magnification), nuclei were visualized by Hoechst 33258. (c) Quantitative analysis of HO-1 in round and spindle MDMs. Data are expressed as mean ± SD of fluorescence intensity/µm2 (at least three fields, 400× magnification, were analyzed) and data derive from independent cultures obtained from 10 healthy subjects and 30 CAD patients (SA n = 10; NSTEMI n = 10; STEMI n = 10. # p < 0.05 vs. round; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. healthy subjects.
Figure 3. Nrf2 levels in CAD patients and healthy subjects. (a,b) Nrf2 levels in (a) total cellular lysate, (b) nuclear and cytosolic compartments were detected by western blot analysis. β-actin was used as a control of protein loading. Densitometry is shown in the bar graph. Data are expressed as mean ± SD and derive (a) from MDMs obtained from 10 healthy subjects and 17 CAD patients; (b) from MDMs obtained from 5 healthy subjects and 5 CAD patients. (c) Representative images of Nrf2 in round and spindle MDMs obtained from healthy subjects and CAD patients (400× original magnification), nuclei were visualized by Hoechst 33258. (d) Quantitative analysis of Nrf2 in round and spindle MDMs. Data are expressed as mean ± SD of fluorescence intensity/µm2 (at least three fields, 400× magnification, were analyzed) and derive from independent cultures obtained from 10 healthy subjects and 30 CAD patients (SA n = 10; NSTEMI n = 10; STEMI n = 10). * p < 0.05, ** p < 0.01, vs. healthy subjects.
Figure 4. In vivo plaque features and HO-1 levels in MDM morphotypes. (a) Association between HO-1 levels and thin cap fibroatheroma (TCFA), (b) fibrous cap integrity, (c) presence of thrombi detected by means of optical coherence tomography (OCT). Data are expressed as median and IQR. * p < 0.05, ** p < 0.01.
Figure 5. Correlations between in vivo plaque features and HO-1 levels in MDM morphotypes. Correlation between HO-1 levels in round and spindle MDMs and (a) macrophage content (NSD) and (b) max lipid arc, detected by means of OCT in in vivo plaque.
|Healthy Subjects
(n = 10)
(n = 30)
|p Value Healthy Subjects vs. CAD °°
(n = 10)
(n = 10)
(n = 10)
|ANOVA p Value °
|Age (years)
|61.5 ± 10
|63.8 ± 12.1
|70.3 ± 7.2
|61.0 ± 11.9
|60.0 ± 14.8
|Male sex, n (%)
|5 (50)
|26 (86.7)
|8 (80)
|9 (90)
|8 (80)
|0.8179 ‡
|Body mass index (kg/m2)
|23.5 ± 1.6
|29.3 ± 4.6
|28.0 ± 4.5 *
|28.1 ± 3.7 *
|32.3 ± 4.6 *
|Clinical characteristics
|Current smoking, n (%)
|18 (60)
|7 (70)
|6 (60)
|5 (50)
|0.5884 ‡
|Diabetes mellitus, n (%)
|16 (53.3)
|5 (50)
|5 (50)
|6 (60)
|0.6593 ‡
|Dyslipidemia, n (%)
|16 (53.3)
|7 (70)
|5 (50)
|4 (40)
|0.4704 ‡
|Hypertension, n (%)
|14 (46.7)
|4 (40)
|5 (50)
|5 (50)
|0.9004 ‡
|Family history of CAD, n (%)
|17 (56.7)
|4 (40)
|9 (90) #
|4 (40)
|50.1 ± 8.8
|48 ± 9.3
|51.3 ± 9.8
|51.0 ± 7.7
|Laboratory data
|7.6 ± 2.9
|9.2 ± 3.9
|8.9 ± 2.4
|9.1 ± 5.1
|9.7 ± 4.0
|4.5 ± 0.8
|5.1 ± 2.0
|4.6 ± 0.6
|5.1 ± 0.6
|5.8 ± 3.6
|Neutrophil count (× 109/L)
|4.8 ± 2.3
|6.2 ± 3.5
|5.8 ± 2.3
|6.1 ± 4.7
|6.9 ± 3.4
|Lymphocyte count (× 109/L)
|2.1 ± 1.2
|2.0 ± 0.9
|2.3 ± 1.2
|1.9 ± 0.7
|1.8 ± 0.9
|Eosinophil count (× 109/L)
|0.1 ± 0.1
|0.2 ± 0.2
|0.2 ± 0.1
|0.1 ± 0.1
|0.2 ± 0.4
|Monocyte count (× 109/L)
|0.5 ± 0.2
|0.6 ± 0.3
|0.6 ± 0.2
|0.6 ± 0.4
|0.7 ± 0.4
|Basophil count (× 109/L)
|0.03 ± 0.02
|0.01 ± 0.00
|0.01 ± 0.02
|0.01 ± 0.03 *
|0.01 ± 0.02
|Platelets (× 109/L)
|248 ± 61.9
|230.8 ± 83.4
|213.7 ± 49.8
|252.2 ± 106.0
|223.7 ± 85.6
|hs-CRP (mg/L)
|1.9 (1.4–2.3)
|4.9 (2.0–21.0)
|2.1 (1.6–2.1) ǂ #
|6.7 (1.6–17.0) *
|38.6 (6.0–75.0) *
|Creatinine (mg/dL)
|1 ± 0.1
|1.0 ± 0.5
|0.8 ± 0.3 #
|0.8 ± 0.3 #
|1.4 ± 0.5
|Glycaemia (mg/dL)
|93.5 ± 12.2
|140.2 ± 42.8
|116.4 ± 27.1 #
|130.3 ± 33.4 #
|178.8 ± 43.7 *
|Total cholesterol (mg/dL)
|187.7 ± 22.1
|204.4 ± 42.6
|181.1 ± 34.1 #
|207.9 ± 42.8
|226.1 ± 41.7
|LDL (mg/dL)
|112.6 ± 26
|122.4 ± 41.9
|102.0 ± 23.5
|130.4 ± 47.5
|135.2 ± 46.2
|HDL (mg/dL)
|41.1 ± 5.3
|48.83 ± 14.9
|61.6 ± 16.7 *,ǂ,#
|44.5 ± 10.5
|43.3 ± 9.3
|Triglycerides (mg/dL)
|143.8 ± 31.6
|161.2 ± 55.5
|117.9 ± 42.7 ǂ#
|176.7 ± 59.6
|190.3 ± 32.2
|Peak TnI (μg/dL)
|1 (0.0–29.4)
|1.2 (0.5–1.4) #
|29.7 (25.0–163.0)
|<0.0001 §
|Peak CK-MB (μg/dL)
|11.1 (2.1–110.0)
|2.1 (1.5–2.1)
|12.3 (2.5–28.0) #
|281 (110.0–521.0) #
|<0.0001 §
|Angiographic data
|Culprit or treated vessel, n (%)
|0.1489 ‡
|14 (46.7)
|3 (30)
|8 (80)
|3 (30)
|10 (30.3)
|4 (40)
|1 (10)
|5 (50)
|6 (20)
|3 (30)
|1 (10)
|2 (20)
|Multivessel disease, n (%)
|17 (56.7)
|8 (80)
|4 (40)
|5 (50)
|0.3276 ‡
|Admission therapy
|ASA, n (%)
|11 (36.7)
|3 (30)
|5 (50)
|3 (30)
|0.3192 ‡
|Beta-Blockers, n (%)
|8 (26.7)
|0.1650 ‡
|2 (20)
|5 (50)
|1 (10)
|0.2319 ‡
|ACE-inhibitors, n (%)
|9 (30)
|0.0810 ‡
|5 (50)
|2 (20)
|2 (20)
|0.3192 ‡
|Statins, n (%)
|10 (30.3)
|5 (50)
|2 (20)
|3 (30)
|0.3459 ‡
SA: Stable angina; CAD: Coronary artery disease; LVEF: Left ventricular ejection fraction; WBC: White blood cells; RBC: Red blood cells; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; hs-CRP: High-sensitive C-reactive protein: TnI: Troponin-I; CK-MB: Creatine phosphokinase-MB; LAD: Left anterior descending; LCX: Left circumflex; RCA: Right coronary artery; ASA: Aspirin; ACE-inhibitors, angiotensin-converting enzyme-inhibitors; NA: Not assessed. Data are expressed as mean ± SD or median and interquartile range. * p < 0.05 vs. healthy subjects; ǂ p < 0.05 vs. NSTEMI; # p < 0.05 vs. STEMI; ° by ANOVA test, except: § Kruskal–Wallis Test, ‡ Fisher exact test; °° by Independent t-test, except: † Wilcoxon rank-sum test, ‡ Fisher exact test.
|CAD (n = 30)
|Lipid plaque, n (%)
|26 (86.67)
|Fibrous plaque, n (%)
|1 (3.33)
|Calcific plaque, n (%)
|3 (10)
|Plaque rupture, n (%)
|15 (50)
|MLA, mm2 (IQR)
|1.70 (1.43–2.58)
|TCFA, n (%)
|15 (50)
|Thrombus, n (%)
|14 (46.67)
|Lipid quadrants, n
|2.70 ± 1.02
|Lipid arc, degree ° (IQR)
|163 (133.5–280)
|Max lipid arc, degree °
|206.37 ± 87.10
|Macrophage infiltration detection, n (%)
|21 (70)
|Macrophage NSD
|6.24 ± 1.16
MLA: Minimal lumen area; TCFA: Thin-cap fibroatheroma; NSD: Normalized standard deviation. Data are expressed as mean ± SD or median and IQR.
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


text:
- Navy SEAL Eddie Gallagher is on trial for allegedly murdering a prisoner of war. Another SEAL just confessed to the killing in bombshell testimony
- It sounds more and more like the Lakers didn't realize the salary cap ramifications of their Anthony Davis trade, and now they're 'scrambling'
- A teenager's jaw shattered after a vape pen exploded in his mouth, once again raising concerns about e-cigarettes
- The safest US bonds are offering their worst returns since the 2016 presidential election — and that's a major warning sign the economy is in trouble
EVER WONDERED WHAT animal kills the most humans?
Bill Gates posted this infographic on his blog last week that shows a surprising number one.
"The number of mosquito-caused deaths really is a mind-blowing thing. Other than humans killing humans during periods of war, most years, the mosquito wins," the Mosquito Week trailer says.
The reason?
Mosquitoes carry terrible diseases, including: malaria (which kills more than 600,000 people every year), dengue fever virus, Rift Valley fever virus, yellow fever, chikungunya virus, West Nile virus, Lymphatic filariasis, and Japanese encephalitis.Source: thegatesnotes/YouTube
These mosquito-borne infections don't just kill — they debilitate millions of people. Sometimes these people can't work and can't support themselves. Billions of dollars in productivity is lost in places where these debilitating infections run rampant — fueled by mosquitoes.
The deadliest of all is malaria, a parasitic infection that mosquitoes inject directly into our bloodstream when they bite. The parasites then travel to the victim's liver, where they multiply and reproduce. Their babies travel the bloodstream, destroying the red blood cells.
Usually the infection doesn't kill, but it's so abundant in so much of the equatorial world that more than 200 million people are infected every year, and more than 700,000 die from the disease.
Here are the areas of the world where Malaria is the worst, from the US Centre for Disease Control:
The site is doing a whole series of posts for "Mosquito Week" including a post about "What It Feels Like To Have Malaria".
Read: Cases of malaria in Ireland rise for the third year in a row
Read: Over half of Africa remains at "moderate to high" risk of malaria

subset: commoncrawl


text:
La Mer Blue Heart Benefits World Oceans Day If there's something La Mer is understood for it's sumptuous merchandise. Fans of the emblem are obsessive about its skin-transforming components. Now, La Mer is giving again to what helped make it so well liked by the limited-edition La Mer Blue Heart …Read More »
What Is Carb Rinsing? The Benefits For Runners
What Is Carb Rinsing? The Benefits For Runners There is not anything extra pleasant than taking a large gulp of that electrolyte-filled sports activities drink right through that rapid run. Imagine feeling thirsty and realizing that refreshment is only a sip away. Then consider seeing a runner move by way …Read More »
Ice Bath Benefits for Brain and Body
Ice Bath Benefits for Brain and Body You've more than likely noticed me on Instagram playing surviving a soak in my bloodless plunge bathtub, and sharing about my shuttle to Finland the place we actually jumped right into a freezing river. I believed I must give an explanation for about …Read More »
Vitamin C Benefits for the Skin — Dermatologist Advice
Vitamin C Benefits for the Skin — Dermatologist Advice Vitamin C serums are mythical for a explanation why: They actually paintings. Uneven pores and skin tone, tough texture, high quality traces, zits scars, common dullness — you'll title with regards to any not unusual complexion worry and there is a …Read More »
Are There Health Benefits from Burning Sage?
Are There Health Benefits from Burning Sage? May 21, 2019 — Acupuncturist Claretha Yeager often makes use of smudging — or burning sage — to assist rid her sufferers of destructive feelings. Yeager, a physician of acupuncture and Chinese medication, and a reiki grasp/instructor, says the sage smoke unburdens …Read More »
Walking During Work Meetings Brings Benefits
Walking During Work Meetings Brings Benefits May 20, 2019 — Holding paintings conferences whilst taking a stroll open air provides an a variety of benefits, researchers file. Not best does it supply a lot wanted workout for people who find themselves frequently tied to their desks for all the …Read More »
Low-Dose Aspirin's Risks Outweigh Benefits
Low-Dose Aspirin's Risks Outweigh Benefits By Amy Norton &#zero13; &#zero13; HealthDay Reporter&#zero13; &#zero13; TUESDAY, May 14, 2019 (HealthDay News) — Let's say you are one of the crucial tens of millions of older adults who takes a low-dose aspirin religiously, within the trust that it is going to guard in …Read More »
Health Benefits of Spices | Indian Spices & Their Benefits
Health Benefits of Spices | Indian Spices & Their Benefits Good chefs will swear via their spices proper right down to the remaining pinch. And meals enthusiasts will flip up their noses at dishes that don't have that little bit of 'dal chini' or 'garam masala'. The newest to enroll …Read More »
Lavender Uses, Benefits & How to Grow It
Lavender Uses, Benefits & How to Grow It It most effective takes a short lived scroll thru my house therapies and DIY recipes to know that lavender is via a long way considered one of my favourite herbs. Not most effective is is lovely and aromatic within the lawn, it has …Read More »
Does doing steady-state cardio immediately after HIIT kill its benefits? : Fitness
Does doing steady-state cardio immediately after HIIT kill its advantages? : Fitness I've a HIIT regimen that I combine in three times per week, after which will observe it up with a part hour at the ARC instructor (period mode) at my gymnasium. Does following HIIT up with a sluggish …Read More »


text:
At ILRI, African swine fever research began in early 2000 with a focus on the epidemiology and socio-economic impacts of the disease. ILRI scientists and partners are currently working on developing vaccines for the disease using the CRISPR-cas genome editing system. Continue reading
Category Archives: Vaccines
German agroecological livestock research investments are benefiting both people and the planet
ILRI was honoured this week (3 Sep 2018) to host a high-level German delegation including Maria Flachsbarth, parliamentary state secretary to Germany's federal minister for economic cooperation and development, and Stefan Schmitz, deputy director of the Federal Ministry of Economic Cooperation and Development (BMZ), as well as senior staff of the Kenya Ministry of Agriculture, Livestock, Fisheries and Irrigation, including Andrew Tuimur, chief administrative secretary, and Ann Onyango, agriculture secretary; and representatives from several other CGIAR centres working in Kenya, including Tony Simons, director general of ICRAF, and representatives from the Nairobi-based International Centre of Insect Physiology and Ecology (icipe). Continue reading
Deadly African swine fever arrives in China, the world's largest producer of pigs
Long feared, it's now finally happened. African swine fever (ASF), an infectious and highly lethal viral disease of pigs, has for the first time reared its head in China. Just two weeks ago, African swine fever was confirmed as the cause of death of pigs on a small farm in Shenyang City, in Liaoning Province, located in the northeast, bordering North Korea and the Yellow Sea. Continue reading
Reducing use of antimicrobial drugs in livestock in low-income countries: Two scientists reflect on options
Delia Grace Randolph, a veterinary epidemiologist and food safety expert who co-leads the Animal and Human Health program at ILRI, was interviewed recently by Wilton Park, a non-profit discussion centre in the UK. The event at which Grace spoke was a workshop held 11–13 Apr 2018 in West Sussex on Innovations to reduce the use of antimicrobials in food-producing animals in low- and middle-income countries. Continue reading
Vaccinating farm animals helps drive up disposable incomes
East Coast fever causes annual losses estimated at more than USD300 million and more than one million cattle deaths. Recent research-for-development approaches have accelerated the uptake of the ITM vaccine and demonstrated how its use is driving increased incomes for small-scale livestock farmers in Kenya and Tanzania. Continue reading
Improving control of a chicken disease supports the livelihoods of Kenya's women
Poultry farming in Kenya is growing rapidly and remains largely dominated by women, who typically invest most of their earnings in feeding their families and educating their children. That is why controlling major poultry diseases is so important. Continue reading
Introducing English and Swahili instructional videos on the patterns, signs, symptoms and control of Rift Valley fever
Originally posted on ILVAC:
Written by Bernard Bett Several outbreaks of Rift Valley fever in livestock and people have occurred in eastern Africa over the last three months or so. In the first week of June 2018, local media reported at least five fatal human cases in Kenya's northern Wajir County. More suspected cases in…


text:
On May 2, more than 40 friends and partners of the PIR Center gathered at the Permanent Mission of Russia to the UN. Among them were heads and high representatives of delegations participating in the third session of the Preparatory Committee of the NPT Review Conference, the world's largest experts in the field of non-proliferation, graduates of PIR Center programs working in New York, young scientists and master students of double degree in the field of non-proliferation.
"Can it really be true that PIR Center is a quarter of a century old today? It would be a great occasion for a grand celebration, wouldn't it. But I still cannot quite believe it.
When me and a tiny team of my associates were establishing PIR Center in the spring of 1994, working in a small room on the corner of Tverskaya Ulitsa and Strastny Boulevard overlooking the Pushkin statue in the very heart of Moscow, I could hardly imagine that this great institution would live long enough to see the new century and indeed the new millennium. If someone told me back at the time that it would mark its 25th anniversary in Moscow, Geneva and New York, or that greetings would be pouring in from all over the world to what is now a highly reputable international nongovernmental organization, I probably wouldn't believe them. After all, how many fly-by-night NGOs have we all seen over the years – here today, gone tomorrow?," Vladimir Orlov, PIR Center Founder.
"Today a member of the PIR Center Executive Board, a member of the IMEMO (Institute of World Economy and International Relations) Directorate, Army General, Ambassador Extraordinary and Plenipotentiary, Hero of Russia Vyacheslav Trubnikov celebrates his 75th birthday. Vyacheslav Ivanovich, having reached the greatest heights of public service, you remained open to new ideas, plans and actions. We are happy to work with you for almost ten years! You are a source of soft, but mighty and attracting power, which is especially important in relations with the new generation of specialists – those who will construct a new world. Many well-known and beginning diplomats and military men, scientists and journalists throughout Eurasia are grateful for your inspiration and support, for your wise and precise judgement. We wish you good health, optimism and high spirits! We look forward to new meetings with you! Keep it up!" – PIR Center Director Albert Zulkharneev.
Summer School 2010 Speakers
AKHTAMZYAN Ildar A., Associate Professor, Moscow State Institute of International Relations (MGIMO) of the Ministry of Foreign Affairs of Russia, PIR Center Advisory Board Member
BUZHINSKY Evgeny P., General-Lieutenant, PIR Center Consultant
DANILOV Dmitry A., Head of the Security Section, Institute of Europe, Russian Academy of Sciences
DURAY Michele, Head, Outreach Countries Section, Public Diplomacy Division, NATO HQ
EVSTAFIEV Gennady M., Lieutenant-general (ret), Senior Vice-President, PIR Center, author of Nuclear Nonproliferation Encyclopedia
FEDOROV Alexander V., Expert of the Foreign Intelligence Service, PIR Center Advisory Board Member
GRIGORIEV Leonid M., President, Institute for Energy and Finance
GUDOVSKY Waclaw, Deputy Executive Director, International Science and Technology Center (Moscow)
KALININA Natalia I. Senior Research Officer, Center for International Security Studies at the Institute of World Economy and International Relations (IMEMO), Russian Academy of Science, PIR Center Executive Board member
KORTUNOV Sergey V., Head of the Department of World Politics, State University – Higher School ofEconomics
KOZYULIN Vadim B., Director of Conventional Arms Project, PIR Center
MARGELOV Mikhail V., Head of the Committee for Foreign Affairs, Council of the Federation
NAUMKIN Vitaly V., Institute of Oriental Studies, Russian Academy of Sciences
NIKONOV Vyacheslav A., Executive Director, Russkiy Mir Foundation
OGANESYAN Armen G., Advisor of the Minister of Foreign Affairs of the Russian Federation, Chief Editor,International Affairs Journal
ORLOV Vladimir A., President, PIR Center, Editor-in-Chief of the Security Index Journal, President of Centre Russe D'etudes Politiques (Geneve)
PALAZHCHENKO Pavel R., Chief of the International Relations and Media Contacts Service, International Non-Governmental Foundation for Socio-Economic and Political Studies (Gorbachev Foundation)
POLIKANOV Dmitry V., Vice President, PIR Center, Editor of the international edition of the Security Indexjournal, Trialogue Club International Chairman
PSHAKIN Gennady M., Director, Analytical Center for Non-proliferation, Institute for Physics and Power Engineering
ROGACHYOV Ilya I., Director, Department on New Threats and Challenges, Ministry of Foreign Affairs of theRussian Federation
SATANOVSKY Evgeny Y., President, Institute for Middle East Studies, PIR Center Advisory Board Member
SHLYKOV Vitaly V., Chair of the Public Council Commission on Security Policy and Public Expertise of Federal Law and Other Legal Acts Drafts, Ministry of Defense of the Russian Federation, Advisory Board member of the PIR Center
STEPANOVA Ekaterina A., the Leader of Research group on unconventional security threats at the Institute ofWorld Economy and International Relations (IMEMO), Security Index Editorial Board Member, PIR Center Executive Board Member
SULSER Urs, Defence Attace, Federal Department of Defence, Civil Protection and Sports DDPS (Swiss Armed Forces, Embassy of Switzerland)
TIMERBAEV Roland M., Ambassador Extraordinary and Plenipotentiary, Chairman of the PIR Center Executive Board
TOLORAYA Georgy D., Director of Research Programs, Center for Modern Korea Studies IMEMO, Russian Academy of Sciences, Regional Director on Asia and Africa, Chief of the Department of Regional Projects Management, Russkiy Mir Foundation
TYURYUKANOVA Elena V., Leading Research Fellow, Institute of Social and Economic Studies of Population,Russian Academy of Sciences
UBEEV Alexey V., Chief Deputy, Foreign Affairs Department, Rosatom State Corporation
VITKOVSKAYA Galina S., Senior Research Fellow, Institute of Economic Forecasting, Russian Academy of Sciences
VORONKOV Vladimir I., Director of Department for Pan-European Cooperation, Ministry of Foreign Affairs of the Russian Federation
VYAZEMSKY Yuri P., Chair of the Department of World Literature and Culture, MGIMO
YAKUSHEV Mikhail V., Chairman, Coordination Center for TLD RU, PIR Center Advisory Board Member,Security Index Editorial Board Member
ZAKHAROV Sergey V., Deputy Head, Institute of Demography, State University - Higher School of Economics


text:
Edition 671, 19 September 2016
- Women in STEM and Entrepreneurship $8 million Grants and Support
- The 2016-2017 online ACPET Catalogue is open for entries!
In Focus
What's On
- ACPET Cairns 2016 Forum - Hon Yvette D'Ath MP is confirmed to attend
- Raising the Bar - speakers and volunteers
- 2016 WorldSkills Australia National Competition, Melbourne
- Victorian VET RTO Breakfast Series
International Engagement
More updates in your state
- Queensland (3)
- New South Wales (1)
- Australian Capital Territory (2)
- Victoria (7)
- Northern Territory (2)
- Western Australia (3)
- South Australia (2)
- Tasmania (3)
2016 WorldSkills Australia National Competition, Melbourne
Monday, September 19 2016
Have you ever wondered what it takes to build a beautiful bathroom, how a garment is created for the runway or how industrial robots are built to assist with day-to-day jobs? You can find out at the 2016 WorldSkills Australia National Competition, Melbourne – the largest trades and skills showcase in the country.
Held from Thursday, 6 to Saturday, 8 October 2016, the National Competition will provide visitors with a unique inside look into trade and skill-based professions. Close to 500 young apprentices, trainees and students from all corners of Australia will converge on Melbourne Showgrounds to compete for the coveted title of 'National Champion' in over 50 skill categories including Vehicle Painting, Bricklaying, Hairdressing, Programming, Cookery, Welding and many more.
Date/Time: 6th October - 8th October
9:00 am - 4:00 pm
Where: Melbourne Showgrounds
Epsom Road, Ascot Vale
For more information or to register for the National Competition visit:


text:
Gave til kjæresten 2017 Bank deposits and liquid securities amounted to NOK 808.3 million and corresponded.4 of total assets.Nov 2014.NET developer might notice the omission of certain types they are used.Start the playrec of you Boombox: Funky Express, the Best of Groove and Soul 24/7Read more
Dieses Video zur Überprüfung melden: Dein Bericht hilft uns, SpankBang zu verbessern.Watch this hd video now, you will never see ads again!Special Show, visualizar mais, horário, domingo, segunda-feira.Grande, enorme, caralho, pequeno Mediano Grande Enorme Circuncidado Circuncidado Não circuncidado Etnia Asiática Caucasiano Hispânico Mulato Tipo deRead more
Masters of sex sesong 1 episode 1 online
Test dine kunnskaper om samfunn og kultur i Norge i perioden, og lær noe nytt!Jok, jeg har sendt deg til hvor mye for å lade cam jenter til flash bunnen av sola.Er ikke "mor" fornøyd med det samme hun går inn i bobilen er detRead more
This site actively cooperates with LAW enforcement IN ALL instances OF suspected illegal USE OF THE service, especially IN THE case OF underage usage OF THE service.
Chatters Searched Keywords: incest chat rooms (2546 sexchat (1629) free sex chat (970) lesbian sex chat (891) ceht sex (592).Looking for big cock guys to do sex with you?Watch free nude cam shows by amateur webcam models from over the world and free live sex chat with them.You can also spin the roulette wheel and let hazard choosing for you.Hot black women are ready to chat sexy and really horny to make you cam jenter gjør fetish handlinger masturbate in front of your webcam.At this time XRoulette give you the opportunity to choose between 8 different chat rooms to find random persons for chatting by live webcam according to your sexual preferences.Join the, asian Roulette sex cam chat, mature Chatroulette, meet naked mature womens in front of their webcam in the MilfRoulette sexchat.Join the, teen Roulette sex cam chat, asian Chatroulette.
Welcome to t a full website dedicated.
It is really fun and worth to spend your valuable time in our chat site.Join the, soft Roulette sex cam chat.Mature Roulette sex cam chat, soft Chatroulette, the SoftRoulette room bring you to passionate sex chat with random girls in front of their webcam.Enter in the xRoulette room to get total random sexchats with sexy strangers in front of their webcam.Most of the peoples are feel shy to do sex with whores or sluts in real life before they start doing with you life partner.Run the GayRoulette room to chat with sexy gay mans who love hindi gratis sex chat to exhibit naked in front of their cam.
Sexy cam girls are in here and you can fun with them any time anywhere from this world.
Choose the AsianRoulette room to meet horny Asian girls who search to be friend with sexy foreigners.


text:
Job with Intermountain Healthcare Primary Children's Hospital in supervisory position where I can utilize the skills I
learned with my BIS degree in Health Promotion, Health Administration, and Child and Family Studies.
Weber State University- Ogden, UT
Bachelor of Integrated Studies- December 2018
Areas of emphasis: Health Promotion- GPA 3.7, Health Administration-GPA 4.0, Child and Family Studies-
Capstone Project:
I wrote a book containing coping strategies for children ages 0-18. These strategies can be useful when
experiencing pain, anxiety, stress, or feelings of depression and sadness. The objective of my project was
to give children a resource to help control and improve their mental health.
Educational Highlight:
Deans Honor Roll- 4 semesters
Work Experience:
Phlebotomist II February 2016- Present
Intermountain Healthcare- TOSH Murray, Utah
Performed venipunctures and capillary sticks on adult and pediatric patients
Process all samples and prepare them for testing
Assist in training phlebotomy students and new hires
Proficient in Microsoft Word, PowerPoint, and Excel
Interpersonal skills and team player
Effective Communicator


text:
Escape mass tourism! At Villa Finca el Drago you will find that special place that is unlike any other. Our residence has six apartments for rent, which are discreetly placed over 8.500 square meters of lush and well kept gardens. Here you can enjoy your privacy, being tucked away in an area with few other guests. Many of our clients are surprised by the Finca's quality, atmosphere and beauty.
Your apartment is fully furnished, well equipped and functional. On arrival, we will greet you, show you the facilities like WIFI, laundry room, etc. Ask our professional staff for restaurant recommendations or for directions to the beach, markets, stores, malls, spa services, etc. A small team is always available. We clean every other day, but can come daily if requested. We'll do our best to take care of any special needs. Enjoy the well kept garden. Play some tennis, enjoy a sauna, go swimming, rest under palm trees or watch sunsets over the ocean from our gazebo with a good cup of tea.
The Villa Finca el Drago is located in the Orotava valley, in Tenerife's green and fruitful north, where Alexander von Humboldt conducted botanical, geological and astronomical research at the age of 30 making him world famous. From here you can follow his path up to spectacular moon-like lava mountains, the cañadas around Pico del Teide, or by watching the sunrise over the islands, following his path down for a healthy beach walk on the black sand banks of the Atlantic Ocean.


text:
Abstract Turtle Logo
Abstract Turtle Logo
Designer's description
This logo design is perfect if you need abstract logos, circle logos, turtle logos or nature logos. Start editing this Abstract Turtle logo for your business or team.
Tags that describe this logo:nature, abstract, circle, ocean, sea, reef, turtle, relaxing
All payments are made securely through Stripe and PayPal and have 128-bit SSL encryption
Other logos you might like
Generate logo designs for any industry
Choose an industry or keyword below and we'll start creating logos in seconds...
Download your Logo
Where should we send the design files?
Email address required
By proceeding you accept our Terms of Use and Privacy Policy


text:
Talk about taking up a topic pertinent to the current state of our nation, this attempt hits probably the most important issue of lack of proper sanitation which does a fine job in taking up a harmless situation to highlight the issue. Brilliantly done music brings out the plight and the humor that usually links up to the issue is also portrayed well.
Despite the disclaimer at the start and though it may be true that hard measures are needed, the title and tagline seems very insensitive to religious beliefs. The apt mention of grave mismatches in number of places of worships or liquor shops in comparison to number of restrooms is quite welcome though. Natural dialogues and a realistic performance from the lead actor is another boost that the short film receives in addition to the impressive camera work – a purposeful show with great intent on the whole!
Purposeful Intent
Watch it here
Info/Cast & Crew
|Running Time:
|11 min
|Directed by:
|Guru subbu
|Written by:
|Guru subbu
|Vishnu, Haniffa, Vijayalakshmi, Subirtha, Deepan Kumar, Saravanan Jackie, Ram Kumar
|Music by:
|Karthik aacharya
|Shot by:
|Jagdeesh sridhar ravichandran
|Editing by:
|Venkatesh B


text:
MediaBeacon is a provider of digital asset management solutions for advertising, pre-press, printing, and financial industries. The company specializes in software and web development. The company also specializes in transaction-focused websites.
Company Info
- HQ Location
- United States
- Year Founded
- 1989
- Employees
- 50
- Website
- www.mediabeacon.com
Products by MediaBeacon
Company Headquarters
Related News
Jun 24
Why the Future of Customer Experience Lies in Predictive Technology
Predictive technology helps companies use historical and behavioral data to anticipate decisions their customers will make in the future.
Jun 24
How a Data-Driven Company Culture Can Enhance Digital Experiences
Companies across all industries are under increased pressure to deliver enhanced digital experiences.
Jun 24
Building vs. Buying an Office 365 Provisioning Tool: 5 Things to Consider
How to decide if you should buy or build an Office 365 provisioning tool.
Jun 24
Words Still Matter (Even in IT)
Words matter. When will we ever learn?
Jun 24
9 Takeaways from the Digital Workplace Experience Conference 2019
Here we look at the key takeaways from CMSwire's annual Digital Workplace Experience in Chicago.


text:
Haiti Tourism Police
Related Photos:
Related Articles:
Jason Derulo in Haiti a second time within a few months
Haiti Police launches "Operation Welcome to Haiti," NO Motorcycles allowed near the Airport!
Increased Security Measures near Haiti Port-au-Prince International Airport
POLITOUR Haiti - Tourism Police First Dispatched in Nothern Haiti
POLITOUR - New Haiti Tourism Police Officers have just Graduated
Messages and comments from our readers...
True or False: Many Haitians want government jobs but most only want the gov paycheck
Virtually every Haitian you speak to who spent two minutes campaigning for a Senator or Depute want a job in ONA, OAVCT or other government...
PetroCaribe: Petro challengers announces Haiti street protest Sunday June 09
The PetroCaribe street protest are about to restart in Haiti. Petro challengers regrouped under a structure called "Nou Pap Domi" announced a new...
Happy Easter 2019 to you and your Family
I wanted to take this moment to wish you and your family an happy Easter 2019. I really hope that this Easter brings you lots of joy and happiness...
Haitian Photo Stories...
Naomi Osaka during the 2019 Italian Open Third Round Press Conference
Here is a photo of Haitian-Japanese No. 1 Tennis player Naomi Osaka during the 2019 Italian Open Third Round Press Conference
PHOTO: Wyclef Jean in graduation Uniforms
Here is a photo of Haitian Rap superstar Wyclef Jean in graduation uniforms
Currency: The Haitian gourdes is being abused by the U.S. Dollar
There is no other way to put it... With over 90 Haitian gourdes for one U.S. dollar, this is abuse. But whose fault it is? right before the fall of...


text:
Share on ThriftyFunThis guide contains the following solutions. Have something to add? Please share your solution!
Create pig, mouse, cat, dog, or other animal from those inexpensive plastic Easter eggs. Great for children's Easter baskets.
Approximate Time: About 30 minutes
By Rachel's Mom from Wilkesboro, NC
Using either two part or hinged plastic eggs you and your children can make several of these cute bunnies. Try each design for a bit of variety. This guide contains fun to make plastic egg bunny crafts.


text:
This is Pat Kane's Typepad Profile.
Join Typepad and start following Pat Kane's activity
Pat Kane
Author of The Play Ethic, one half of Hue And Cry, father, partner
Interests: The future, love, sex, funkiness, great technology, classic life-changing songs, the multitude, longevity, Scottish culture/independence/prospects
Recent Activity
By Pat Kane. First published on JULY 14TH, 2018 THE other day I watched John Cleese – the head of the comedy institution known as Monty Python – announce on Newsnight that he was leaving Britain, because of the "awfulness" of Brexit and the British press. Cleese harrumphed away about "friendly races" and "I only lived here two weeks last year", sounding sadly like one of the bumptious blimps he used to satirise. The backdrop on Newsnight was a tidily animated tumble of various Pythonesque symbols: random legs, flying fish, antique pointing hands, coconuts, an old TV. All too comfortable.... Continue reading
By Pat Kane. First published on AUGUST 21ST, 2018 THE pelters from above were, indeed, near cosmic. "Back from holiday to find that science fiction still dominates one half of Scottish politics", harrumphed Tory MSP Adam Tomkins in a tweet 12 months ago. The image he'd attached was a front cover from this very publication, which had mocked up an orbiting Space Shuttle with a saltire on its hull. The picture floated above this headline: "Independence and Beyond: new report outlines how an indy Scottish Space Agency and spaceport could be set up to give huge boost to our economy".... Continue reading
By Pat Kane. First published on JULY 28TH, 2018 IF he wasn't acting in everything already, you might imagine a pensive Benedict Cumberbatch, chewed ballpoint hovering over his list of suspects, murmuring to himself in a shabby, dust-moted office: "Sheridan, Militant … Jones, Plaid … ah yes, McTavish, Scots Nat ..." Such is the bad spy drama evoked by this week's disclosure from the National Archives: that during the 80s, MI5 maintained a list of 1420 civil service employees identified as "subversive", who were closely monitored and kept away from certain jobs and roles. Among the Trots, communists (the majority),... Continue reading
PK column in The National: This is what an independent Scotland can learn from the Wings/BBC copyright row
By Pat Kane. First published on AUGUST 4TH, 2018 THE dramatist Peter Arnott is famous for his definition of Scotland as "not a country, but an argument". Our major Scottish news story this week is, to some degree, about how we can even have that argument – if what is argued over suddenly disappears into a digital and legal void. The BBC's triggering of YouTube's takedown of two indy-supporting bloggers and their video archives – Stuart Campbell (aka Wings Over Scotland) and Peter Curran (aka Moridura) – has been a stramash for connoisseurs of the form. But like most decent... Continue reading
PK column in The National: The philosophy of politics highlights the dilemmas of nationalist identity
By Pat Kane. First published on AUGUST 11TH, 2018 IT'S been fun reading the snash around Chris McEleny's case against the MoD. He's pursuing them for their poor and punitive treatment of him as an employee when he announced his candidacy for the SNP depute leader position in 2016. A Glasgow employment tribunal, presided over by Judge Frances Eccles, ruled this week that he could make a case under the Equality Act 2010. This allows certain "philosophical and religious beliefs" to be understood as "protected characteristics". That is, they can't be taken into consideration as grounds for dismissal or harsh... Continue reading
By Pat Kane. First published on AUGUST 18TH, 2018 AS the Queen of Soul, she would expect nothing less than candour. So, on the passing of one of my lifetime inspirations, the elemental Aretha Franklin, I will try to appreciate her as honestly as I can. Like Sinatra's, or McCartney's, or Bowie's, Franklin's voice makes possible what any singer afterwards even imagines they could produce. It clears original ground. She fused together the best of the gospel tradition, and the timing of jazz and blues, to perfection. Might this be because few children ever had a musical upbringing like hers?... Continue reading
By Pat Kane. First published on AUGUST 25TH, 2018 "THIS is going to be the most amazing ride of your life". The wee man with the piercing blue eyes leaned towards me, with some urgency. His offer was, to be specific, the position of associate editor (culture and books) at the Sunday Herald, eight months before its launch-date on February 7, 1999. Andrew Jaspan had already been the editor who'd turned around Scotland on Sunday's fortunes in the 90s, and then left for stints at the Observer and the Big Issue. His mysterious middle-distance stares, and jack-knife conversational turns, were... Continue reading
By Pat Kane. First published on SEPTEMBER 1ST, 2018 FOR long-term America-watchers like me – often stranded between love of, and frustration with, the whole inferno – it is a strange thrill to see the US grapple with the idea of an "American socialism". What could possibly have triggered such a weird, indeed un-American activity? Well, perhaps the election of some out-and-out American socialists. The long-time "independent democratic socialist senator from Vermont" Bernie Sanders blasted the s-word into the centre of American political discourse through his explosive run for the Democrats' presidential ticket against Hillary Clinton in 2016. Bernie's "Revolution... Continue reading
PK column in The National: A Celtic bridge project could keep us sane as the chaos of Brexit unfolds
By Pat Kane. First published on SEPTEMBER 8TH, 2018 THERE'S no doubting the power, symbolic or otherwise, of a mighty bridge. Earlier this week, we saw the initial sketches of a proposal to link up Scotland and Northern Ireland. The shorter option is between the Mull of Kintyre and Torr Head; the longer, between Portpatrick and Larne. The pencilled impression of the "Celtic Bridge" suggests a design that's like a copy-and-paste of the Queensferry Crossing over the Forth. Like that bridge, many "cable stays" peak and trough their way across the Irish Sea. But the artist puts it all in... Continue reading
By Pat Kane. First published on SEPTEMBER 15TH, 2018 WE'RE in the middle of a great and substantive debate about play in Scotland (if we can suspend the party-political tribalism for a moment). On one side are those who want to bring formal tests in a school setting to five-year-olds (P1), in order to best monitor their progress. On the other are those who believe play-based kindergarten, from three to seven, would be the best developmental start for future learners. I'm on the latter side (being on the board of Upstart Scotland, the campaign group behind #playnottests), and I could... Continue reading
By Pat Kane. First published on JUNE 8TH, 2019 IT'S a storm in a cortado cup, admittedly. But the demise of the paper edition of the Scottish Review of Books, as a consequence of Creative Scotland rejecting their grant application, is a dark and concentrated swirl of contemporary Scottish issues all on its own. Adjust your vintage tortoiseshell specs, re-roll your black poloneck, and read on. For one thing, such language! Creative Scotland is "metamorphosing into a manifesto-waving, policy-driven arm of an increasingly authoritarian state", wrote the co-editor of the SRB, Rosemary Goring. The nub of this charge seems to... Continue reading
By Pat Kane. First published on SEPTEMBER 22ND, 2018 YOU don't know what you've got till it's gone. The last few times I've ventured into Glasgow, head lost in a podcast, I've absently let my feet take their usual route: Off at Charing Cross, a nod to the Mitchell Library, and then head up the Street of Dreams ... Except I forget: the upheaval of the roadworks; the weird, permanently Sunday calm of the deserted pavements; and of course, the charred hulks of the ABC and the GSA. And then the dull and dusty front of the CCA. You can... Continue reading
By Pat Kane. First published on SEPTEMBER 29TH, 2018 In these highly polarised times, we should note when there seems to be unity across the parties. The effects of online abuse on female participation in politics appears to be one of those overlaps. In the last seven days, both Theresa May and UK Labour's shadow education secretary, Angela Rayner, have highlighted how much social media, and its hordes of screen-jabbing misogynists, dishearten and discourage women politicians – both actual and aspiring. A few months ago, Nicola Sturgeon announced a fund of £500,000 to promote political engagement among young women. Sturgeon... Continue reading
By Pat Kane. First published on OCTOBER 6TH, 2018 THE advance tremors are already rattling the tea-cups. The seismic event, this coming Monday, is the latest Intergovernmental Panel on Climate Change. Which, if it was an Arctic survey boat (to change metaphors), would be at the bottom of the sea, so leaky has it become. According to the latest PDF to slip out from the biggest scientific peer-review exercise ever conducted, the message is stark. We must aim to keep global warming below 1.5% (that's the percentage above pre-industrial eras) by 2050. We're already at 1% above. Unamended, current behaviour... Continue reading
PK column in The National: It's time to ask some serious questions ... about the BBC's Question Time
By Pat Kane. First published on OCTOBER 13TH, 2018 THE tweets went round, a little less desultory than usual: "I suppose this seems like a BBC Question Time (#bbcqt) I might watch this week." And indeed, Thursday's show – its cast-list scattered all over the HolyroodParliament chamber – looked as if it might rub cliches together vigorously enough to catch some flame. A strangulated editor of the Spectator, a Duracell-powered young Scots Tory, the near-papal Michael Russell, a Celebrity Who's Clearly Getting Out Of Here, and Val McDermid (representing literature and humans). Plus a "diverse" audience which we now know... Continue reading
By Pat Kane. First published on OCTOBER 20TH, 2018 YOU think you're looking into the heart of darkness – and then, over there, something even worse distracts you. If you've come upon the barbaric details of the murder of the journalist Jamal Khashoggi, in the Saudi Arabian consulate in Istanbul, then no need to repeat them here. Your soul deserves some respite. But suffice to say, and combined with his valiant final column for the Washington Post, Khashoggi's death might well have you running up the "free speech" flag above any other this week. "What the Arab world needs most... Continue reading
PK column in The National: Meat eaters can hardly take the moral high ground when it comes to hunting
By Pat Kane. First published on OCTOBER 27TH, 2018 O MIGHTY American huntress, in your pseudo-Gulf-War combats, bearing your sniper-level high-tech rifle, picking unsuspecting goats off the cliffs of Islay, taking selfies with your bro-friend, grinning over your grotesquely glass-eyed spoils. The modern Artemis, you ain't. But what are you, exactly? The immediate answer is that Larysa Switlyk, whose Twitter celebrations of her killing spree on the lovely island have triggered pearl-clutching on an epidemic scale, was legitimately hiring a sporting service provided by a local Islay landowner. This is something we do (or let happen) in Caledonia. That is,... Continue reading
PK column in The National: Impending visa trouble for artists is nothing but sheer cultural vandalism
By Pat Kane. First published on NOVEMBER 3RD, 2018 IT'S beginning to feel stuffy and constricted in here. The two leading African acts (from Mali and Senegal) who have pulled out of this year's Celtic Connections music festival, due to the "hassle and stress" of trying to secure permission to enter the UK, are only the latest in a recent and disheartening wave. This August's Edinburgh International Book Festival – a global republic of letters, if ever there was one – had 12 authors with visas refused, from predominantly African and Middle Eastern countries. And in the same festival period,... Continue reading
By Pat Kane. First published on NOVEMBER 10TH, 2018 OUR news-cycle always seems to find plenty of time for errant celebrities, literary award winners or ratings-topping reality-TV formats. So it's extraordinary to note how poorly covered the launch of the computer game Red Dead Redemption 2 has been. It has literally marked up the biggest single opening weekend in entertainment history. The game has grossed $725 million, with 17 million units sold in 10 days (as much as the first game sold in total). And the pace is hardly slowing. The result of four years of work, involving two thousand... Continue reading
PK column in The National: Outlaws and kings: Striking a balance in reclaiming our national identity
By Pat Kane. First published on NOVEMBER 17TH, 2018 I HAD to check a few times on Netflix, but yes it's there. In its opening credits, David Mackenzie's fascinating film about the degradation and redemption of Robert the Bruce spells its title precisely this way: Outlaw/King. To my mind, that slash (and yes, there are many other forms of slashing in this gorefest) makes intellectually explicit what is the movie's implicit theme. If you are a national community residing within a bigger polity, to what extent should its leaders seek progress (or even maintenance) within the rules – or by... Continue reading
Posted Jun 2, 2019 at Thoughtland
By Pat Kane. First published on JUNE 1ST, 2019 NOSTALGIA is a feeling that lingers inside you, like a cloud of smoke you can't dispel. It's an interesting question to ask yourself: what do you feel nostalgic about? Spontaneously, my own response is: radical bookshops. As a GlasgowUniversity student in the 80s, I used to drift between about six of them, studding the town centre and the West End. Pre-internet, the whole, wide, struggling world poured into these places, where the booksellers sat like wispy-bearded magi. When I was music-touring, I even established an informal map of them: they turned... Continue reading
Posted Jun 2, 2019 at Thoughtland
By Pat Kane. First published on NOVEMBER 24TH, 2018 I DON'T have much connection to the Scottish fishing industry, but I do have this. My late ex-father-in-law, Jim McAlpine – a quiet but fiercely proud father of three fiercely unquiet daughters – was a seaman engineer. Firstly in the Merchant Navy, then on the Greenock shipyards and, in his final years, on his own wee boat, moored at a marina on the Firth of Clyde. The highlight of every Christmas was when Jim was just smeeked enough to start clearing his throat, signalling the arrival of his party piece: Ewan... Continue reading
By Pat Kane. First published on DECEMBER 1ST, 2018 THE future bears down upon us... in the form of a single-decker passenger bus, sedately proceeding along the old Forth Road Bridge. That's the means whereby self-driving vehicles will introduce themselves to Scottish roads in 2021. Five driverless buses will run a 14-mile route between Fife and Edinburgh – 10,000 journeys a week across the bridge, 40 passengers a bus. And eventually, one assumes, not a grumpy, taciturn human driver in sight. But not immediately. The capacity for autonomous vehicles to freak people out, on a number of levels, means that... Continue reading
By Pat Kane. First published on DECEMBER 8TH, 2018 I'M one of those non-engaged observers who has been slowly turning his attention to transgender issues. And it's mostly because of the sound of previously equable colleagues, exploding all over their own columns in outrage. Rather than launch into theory and policy, let me begin from a personal angle. On all matters of human diversity, I tend to proceed from our long-standing family axiom, "it takes all kinds to make a world". Working-class Scottish life, in my 50-something experience, has always had skeins of the non-normative running through it. The florid... Continue reading
By Pat Kane. First published on MAY 25TH, 2019 FOR those of a certain vintage, the news that the comedian Craig Ferguson has abandoned the TV studios of Los Angeles, for the sandstone-and-trimmed-bush bourgeois of the West End of Glasgow, is strangely comforting. From Bing Hitler bouncing off the walls of the Sub Club, to this silver fox ambling down Byres Road, with the vintage microphones and starlet parades of late-night US TV in between, is one of those Scottish journeys to take some pleasure in. However, we also live in a global village. And Ferguson must surely have known... Continue reading


text:
|City of Simi Valley
|Type of Government:
|State & Local
|Posted Date:
|Jun 11, 2017
|Due Date:
|Jun 14, 2017
|Bid Source:
|Please Login to View Page
|Contact information:
|Please Login to View Page
|Bid Documents:
|Please Login to View Page
|Due Date
Cochran Street Bridge Widening From Fig Street to Ralston Avenue Bridge No. 52C115 (Specification No. SV 16-03)
June 14, 2017
Sarah Sheshebor
(805) 583-6792
With Free Trial, you can:
You will have a full access to bids, website, and receive daily bid report via email and web.
Opportunity History Complete View Original Synopsis Presolicitation Mar 29, 2019 12:13 pm Changed
Department of the Army
Opportunity History Original Synopsis Apr 19, 2019 9:09 pm Solicitation Number : AdvNtc_ByvwP1C-SP
Department of Energy
Opportunity History Complete View Original Synopsis Presolicitation Mar 21, 2019 3:46 pm Changed
Department of Agriculture
Opportunity History Original Synopsis Jun 14, 2019 9:56 am Solicitation Number : 140F0519R0016
Department of the Interior


text:
I would have Tyler back every time if possible. I very rarely give the TOP points for any thing or any body.
Cleaned & checked
Earl is a terrific technician, very knowledgeable and thorough.
Your technician was extremely polite and courteous. He did the job well.
Went well. Tech was on time. He answered all questions and performed his job well.
Earl is very knowledgeable, efficient, polite and explains things clearly.
Trane Heatpump split
Ac cooling service
Summer check
Club member
Summer check
Tune up on 11 year old split rheem
Tune up on 5 year old split hp train
Trane ac cooling service
Rheem ac cooling service
Rheem ac cooling service
Carrier air conditioner maintenance
Tune up on 10 year old split gas rheem
Alaskification of a Rheem heat pump for an Alaskan AC Club Member
Unit acting up