Monocyte chemotactic protein (MCP)-1 (CCL2) and its receptor (CCR2) are elevated in chronic heart failure facilitating lung monocyte infiltration and differentiation which may contribute to lung fibrosis.

BACKGROUND: Dyspnea, the cardinal manifestation of chronic heart failure (CHF), may reflect both pulmonary oedema and pulmonary remodeling resulting in tissue stiffening. Emerging evidence suggests that predominance of distinct phenotypes of alveolar and recruited macrophages, designated M1 and M2, may regulate the course of inflammatory tissue repair and remodeling in the lung. METHODS: In a CHF rat model, we found fibrotic reinforcement of the extracellular matrix with an increase in monocyte chemotactic protein (MCP)-1/CCL2 in bronchoalveolar lavage (BAL), corresponding to a 3-fold increase in recruited macrophages. In this clinical cross sectional study, we aimed to examine potential mediators of leukocyte activation and lung infiltration in parallel BAL and blood from CHF patients compared to non-CHF controls. RESULTS: Mini-BAL and peripheral blood samples were obtained from hospitalized CHF, acute decompensated CHF and non-CHF patients. CHF patients and decompensated CHF patients demonstrated increases from non-CHF patients in BAL MCP-1, as well as the M2 macrophage cytokines interleukin-10 and transforming growth factor-ß. BAL and plasma MCP-1 were significantly correlated; however, MCP-1 was 20-fold higher in epithelial lining fluid in BAL, indicative of an alveolar chemotactic gradient. An increase in transglutaminase 2 positive M2 macrophages in parallel with a decrease in the MCP-1 receptor, CC chemokine receptor 2 (CCR2), was apparent in BAL cells of CHF patients compared to non-CHF. CONCLUSION: These data suggest a pathway of MCP-1 mediated M2 macrophage prevalence in the lungs of CHF patients which may contribute to pulmonary fibrotic remodeling and consequent increased severity of dyspnea.

Blueberry extract attenuates norepinephrine-induced oxidative stress and apoptosis in H9c2 cardiac cells.

Enhanced sympathetic system activation mediated by norepinephrine (NE) contributes to adverse cardiac remodeling leading to oxidative stress and cell death, progressing to heart failure. Natural antioxidants may help maintain redox balance, attenuating NE-mediated cardiac cell damage. In the present study, we evaluated the effect of a blueberry extract (BBE) on H9c2 cardiac cells exposed to NE on cell death, oxidative stress status and its major signaling pathways. H9c2 cells were pre-incubated with 50 µg/ml of BBE for 4 h and maintained in the presence of 100 µM NE for 24 h. NE exposure resulted in increased caspase 3/7 activity. This was associated with reduced protein expression of antioxidants catalase, superoxide dismutase and glutathione peroxidase and increase in 4-hydroxynonenal adduct formation. NE led to increased activity of Protein kinase B (Akt), Forkhead box O3a and AMP-activated protein kinase alpha and decreased activity of Signal transducer and activator of transcription 3. BBE prevented caspases activation and abrogated NE-induced increase in oxidative stress, as well as attenuated the increase in Akt. Based on these findings, it is concluded that BBE promoted cardioprotection of H9c2 cells in an in vitro model of NE-induced oxidative damage, suggesting a cardioprotective role for BBE in response to NE exposure.

Circadian mutant mice with obesity and metabolic syndrome are resilient to cardiovascular disease.

Obesity and metabolic syndrome commonly underlie cardiovascular disease. ClockΔ19/Δ19 mice fed a normal diet develop obesity and metabolic syndrome; however, it is not known whether they develop or are resilient to cardiovascular disease. We found that ClockΔ19/Δ19 mice do not develop cardiac dysfunction, despite their underlying conditions. Moreover, in contrast to wild-type controls fed a high-fat diet (HFD), ClockΔ19/Δ19 HFD mice still do not develop cardiovascular disease. Indeed, ClockΔ19/Δ19 HFD mice have preserved heart weight despite their obesity, no cardiomyocyte hypertrophy, and preserved heart structure and function, even after 24 wk of a HFD. To determine why ClockΔ19/Δ19 mice are resilient to cardiac dysfunction despite their underlying obesity and metabolic conditions, we examined global cardiac gene expression profiles by microarray and bioinformatics analyses, revealing that oxidative stress pathways were involved. We examined the pathways in further detail and found that 1) SIRT-dependent oxidative stress pathways were not directly involved in resilience; 2) 4-hydroxynonenal (4-HNE) increased in wild-type HFD but not ClockΔ19/Δ19 mice, suggesting less reactive oxygen species in ClockΔ19/Δ19 mice; 3) cardiac catalase (CAT) and glutathione peroxidase (GPx) increased, suggesting strong antioxidant defenses in the hearts of ClockΔ19/Δ19 mice; and 4) Pparγ was upregulated in the hearts of ClockΔ19/Δ19 mice; this circadian-regulated gene drives transcription of CAT and GPx, providing a molecular basis for resilience in the ClockΔ19/Δ19 mice. These findings shed new light on the circadian regulation of oxidative stress and demonstrate an important role for the circadian mechanism in resilience to cardiovascular disease.NEW & NOTEWORTHY We examined whether obesity and metabolic syndrome underlie the development of cardiac dysfunction in circadian mutant ClockΔ19/Δ19 mice. Surprisingly, we demonstrate that although ClockΔ19/Δ19 mice develop metabolic dysfunction, they are protected from cardiac hypertrophy, left ventricular remodeling, and diastolic dysfunction, in contrast to wild-type controls, even when challenged with a chronic high-fat diet. These findings shed new light on the circadian regulation of oxidative stress pathways, which can mediate resilience to cardiovascular disease.

Blueberry extract attenuates doxorubicin-induced damage in H9c2 cardiac cells 1.

The objective of this study was to analyze the cardioprotective roles of 3 wild blueberry genotypes and one commercial blueberry genotype by measuring markers of oxidative stress and cell death in H9c2 cardiac cells exposed to doxorubicin. Ripe berries of the 3 wild blueberry genotypes were collected from a 10-year-old clearcut forest near Nipigon, Ontario, Canada (49°1'39″N, 87°52'21″W), whereas the commercial blueberries were purchased from a local grocery store. H9c2 cardiac cells were incubated with 15 µg gallic acid equivalent/mL blueberry extract for 4 h followed by 5 µM doxorubicin for 4 h, and oxidative stress and active caspase 3/7 were analyzed. The surface area as well as total phenolic content was significantly higher in all 3 wild blueberry genotypes compared with the commercial species. Increase in oxidative stress due to doxorubicin exposure was attenuated by pre-treatment with all 3 types of wild blueberries but not by commercial berries. Furthermore, increase in caspase 3/7 activity was also attenuated by all 3 wild genotypes as well. These data demonstrate that wild blueberry extracts can attenuate doxorubicin-induced damage to H9c2 cardiomyocytes through reduction in oxidative stress and apoptosis, whereas the commercial blueberry had little effect.

Sensitive Electrochemical Detection of Nitric Oxide Release from Cardiac and Cancer Cells via a Hierarchical Nanoporous Gold Microelectrode.

The importance of nitric oxide (NO) in many biological processes has garnered increasing research interest in the design and development of efficient technologies for the sensitive detection of NO. Here we report on a novel gold microelectrode with a unique three-dimensional (3D) hierarchical nanoporous structure for the electrochemical sensing of NO, which was fabricated via a facile electrochemical alloying/dealloying method. Following the treatment, the electrochemically active surface area (ECSA) of the gold microelectrode was significantly increased by 22.9 times. The hierarchical nanoporous gold (HNG) microelectrode exhibited excellent performance for the detection of NO with high stability. On the basis of differential pulse voltammetry (DPV) and amperometric techniques, the obtained sensitivities were 21.8 and 14.4 µA µM-1 cm-2, with detection limits of 18.1 ± 1.22 and 1.38 ± 0.139 nM, respectively. The optimized HNG microelectrode was further utilized to monitor the release of NO from different cells, realizing a significant differential amount of NO generated from the normal and stressed rat cardiac cells as well as from the untreated and treated breast cancer cells. The HNG microelectrode developed in the present study may provide an effective platform in monitoring NO in biological processes and would have a great potential in the medical diagnostics.

Effects of Simulated 5-Ion Galactic Cosmic Radiation on Function and Structure of the Mouse Heart.

Missions into deep space will expose astronauts to the harsh space environment, and the degenerative tissue effects of space radiation are largely unknown. To assess the risks, in this study, male BALB/c mice were exposed to 500 mGy 5-ion simulated GCR (GCRsim) at the NASA Space Radiation Laboratory. In addition, male and female CD1 mice were exposed to GCRsim and administered a diet containing Transforming Growth Factor-beta (TGF-ß)RI kinase (ALK5) inhibitor IPW-5371 as a potential countermeasure. An ultrasound was performed to investigate cardiac function. Cardiac tissue was collected to determine collagen deposition, the density of the capillary network, and the expression of the immune mediator toll-like receptor 4 (TLR4) and immune cell markers CD2, CD4, and CD45. In male BALB/c mice, the only significant effects of GCRsim were an increase in the CD2 and TLR4 markers. In male CD1 mice, GCRsim caused a significant increase in total collagens and a decrease in the expression of TLR4, both of which were mitigated by the TGF-ß inhibitor diet. In female CD1 mice, GCRsim caused an increase in the number of capillaries per tissue area in the ventricles, which may be explained by the decrease in the left ventricular mass. However, this increase was not mitigated by TGF-ß inhibition. In both male and female CD1 mice, the combination of GCRsim and TGF-ß inhibition caused changes in left ventricular immune cell markers that were not seen with GCRsim alone. These data suggest that GCRsim results in minor changes to cardiac tissue in both an inbred and outbred mouse strain. While there were few GCRsim effects to be mitigated, results from the combination of GCRsim and the TGF-ß inhibitor do point to a role for TGF-ß in maintaining markers of immune cells in the heart after exposure to GCR.

Effects of proton and oxygen ion irradiation on cardiovascular function and structure in a rabbit model.

PURPOSE: Astronauts on missions beyond low Earth orbit will be exposed to galactic cosmic radiation, and there is concern about potential adverse cardiovascular effects. Most of the research to identify cardiovascular risk of space radiation has been performed in rodent models. To aid in the translation of research results to humans, the current study identified long-term effects of high-energy charged particle irradiation on cardiovascular function and structure in a larger non-rodent animal model. MATERIALS AND METHODS: At the age of 12 months, male New Zealand white rabbits were exposed to whole-body protons (250 MeV) or oxygen ions (16O, 600 MeV/n) at a dose of 0 or 0.5 Gy and were followed for 12 months after irradiation. Ultrasonography was used to measure in vivo cardiac function and blood flow parameters at 10- and 12-months post-irradiation. At 12 months after irradiation, blood cell counts and blood chemistry values were assessed, and cardiac tissue and aorta were collected for histological as well as molecular and biochemical analyses. Plasma was used for metabolomic analysis and to quantify common markers of cardiac injury. RESULTS: A small but significant decrease in the percentage of circulating lymphocytes and an increase in neutrophil percentage was seen 12 months after 0.5 Gy protons, while 16O exposure resulted in an increase in monocyte percentage. Markers of cardiac injury, cardiac troponin I (cTnI) and N-Terminal pro-B-type Natriuretic Peptide were modestly increased in the proton group, and cTnI was also increased after 16O. On the other hand, metabolomics on plasma at 12 months revealed no changes. Both types of irradiation demonstrated alterations in cardiac mitochondrial morphology and an increase in left ventricular protein levels of inflammatory cell marker CD68. However, changes in cardiac function were only mild. CONCLUSION: Low dose charged particle irradiation caused mild long-term changes in inflammatory markers, cardiac function, and structure in the rabbit heart, in line with previous studies in mouse and rat models.

Effects of low-dose oxygen ions on cardiac function and structure in female C57BL/6J mice.

PURPOSE: Astronauts in space vehicles beyond low-Earth orbit will be exposed to high charge and energy (HZE) ions, and there is concern about potential adverse effects on the cardiovascular system. Thus far, most animal studies that assess cardiac effects of HZE particles have included only males. This study assessed the effects of oxygen ions (16O) as a representative ion of the intravehicular radiation environment on the heart of female mice. MATERIALS AND METHODS: Female C57BL/6 J mice at 6 months of age were exposed to 16O (600 MeV/n) at 0.25-0.26 Gy/min to a total dose of 0, 0.1, or 0.25 Gy. Cardiac function and abdominal aorta blood velocity were measured with ultrasonography at 3, 5, 7, and 9 months after irradiation. At 2 weeks, 3 months, and 9 months, cardiac tissue was collected to assess collagen deposition and markers of immune cells. RESULTS: Ultrasonography revealed increased left ventricle mass, diastolic volume and diameter but there was no change in the abdominal aorta. There was no indication of cardiac fibrosis however, a 75 kDa peptide of left ventricular collagen type III and α-smooth muscle cell actin were increased suggesting some remodeling had occurred. Left ventricular protein levels of the T-cell marker CD2 was significantly increased at all time points, while the neutrophil marker myeloperoxidase was decreased at 2 weeks and 9 months. CONCLUSIONS: These results taken together suggest 16O ion exposure did not result in cardiac fibrosis or cardiac dysfunction in female mice. However, it does appear mild cardiac remodeling occurs in response to HZE radiation.

Mitigation of late cardiovascular effects of oxygen ion radiation by γ-tocotrienol in a mouse model.

PURPOSE: While there is concern about degenerative tissue effects of exposure to space radiation during deep-space missions, there are no pharmacological countermeasures against these adverse effects. γ-Tocotrienol (GT3) is a natural form of vitamin E that has anti-oxidant properties, modifies cholesterol metabolism, and has anti-inflammatory and endothelial cell protective properties. The purpose of this study was to test whether GT3 could mitigate cardiovascular effects of oxygen ion (16O) irradiation in a mouse model. MATERIALS AND METHODS: Male C57BL/6 J mice were exposed to whole-body 16O (600 MeV/n) irradiation (0.26-0.33 Gy/min) at doses of 0 or 0.25 Gy at 6 months of age and were followed up to 9 months after irradiation. Animals were administered GT3 (50 mg/kg/day s.c.) or vehicle, on Monday - Friday starting on day 3 after irradiation for a total of 16 administrations. Ultrasonography was used to measure in vivo cardiac function and blood flow parameters. Cardiac tissue remodeling and inflammatory infiltration were assessed with histology and immunoblot analysis at 2 weeks, 3 and 9 months after radiation. RESULTS: GT3 mitigated the effects of 16O radiation on cardiac function, the expression of a collagen type III peptide, and markers of mast cells, T-cells and monocytes/macrophages in the left ventricle. CONCLUSIONS: GT3 may be a potential countermeasure against late degenerative tissue effects of high-linear energy transfer radiation in the heart.

Lasting Effects of Low to Non-Lethal Radiation Exposure during Late Gestation on Offspring's Cardiac Metabolism and Oxidative Stress.

Ionizing radiation (IR) is known to cause fetal programming, but the physiological effects of low-dose IR are not fully understood. This study examined the effect of low (50 mGy) to non-lethal (300 and 1000 mGy) radiation exposure during late gestation on cardiac metabolism and oxidative stress in adult offspring. Pregnant C57BL/6J mice were exposed to 50, 300, or 1000 mGy of gamma radiation or Sham irradiation on gestational day 15. Sixteen weeks after birth, 18F-Fluorodeoxyglucose (FDG) uptake was examined in the offspring using Positron Emission Tomography imaging. Western blot was used to determine changes in oxidative stress, antioxidants, and insulin signaling related proteins. Male and female offspring from irradiated dams had lower body weights when compared to the Sham. 1000 mGy female offspring demonstrated a significant increase in 18F-FDG uptake, glycogen content, and oxidative stress. 300 and 1000 mGy female mice exhibited increased superoxide dismutase activity, decreased glutathione peroxidase activity, and decreased reduced/oxidized glutathione ratio. We conclude that non-lethal radiation during late gestation can alter glucose uptake and increase oxidative stress in female offspring. These data provide evidence that low doses of IR during the third trimester are not harmful but higher, non-lethal doses can alter cardiac metabolism later in life and sex may have a role in fetal programming.

Dose threshold for radiation induced fetal programming in a mouse model at 4 months of age: Hepatic expression of genes and proteins involved in glucose metabolism and glucose uptake in brown adipose tissue.

Exposure to ionizing radiation contributing to negative health outcomes is a widespread concern. However, the impact of low dose and sub-lethal dose radiation (SLDR) exposures remain contentious, particularly in pregnant women who represent a vulnerable group. The fetal programming hypothesis states that an adverse in utero environment or stress during development of an embryo or fetus can result in permanent physiologic changes often resulting in progressive metabolic dysfunction with age. To assess changes in gene expression profiles of glucose/insulin signaling and lipid metabolism caused by radiation exposure in utero, pregnant C57Bl/6J mice were irradiated using a dose response ranging from low dose to SLDR and compared to a Sham-irradiated group. mRNA expression analysis in 16 week old offspring (n = 84) revealed that genes involved in metabolic function including glucose metabolism, insulin signaling and lipid metabolism were unaffected by prenatal radiation exposures up to 300 mGy. However, female offspring of dams exposed to 1000 mGy had upregulated expression of genes contributing to insulin resistance and gluconeogenesis. In a second cohort of mice, the effects of SLDR on fetal programming of hepatic SOCS3 and PEPCK protein expression were assessed. 4 month old female offspring of dams irradiated at 1000 mGy had: 1) increased liver weights, 2) increased hepatic expression of proteins involved in glucose metabolism and 3) increased 18F-fluorodeoxyglucose (FDG) uptake in interscapular brown adipose tissue (IBAT) measured by positron emission tomography (PET) (n = 25). The results of this study indicate that prenatal radiation exposure does not affect metabolic function up to 300 mGy and 1000 mGy may be a threshold dose for sex-specific alterations in glucose uptake and hepatic gene and protein expression of SOCS3, PEPCK, PPARGC1A and PPARGC1B. These findings suggest that SLDR doses alter glucose uptake in IBAT and hepatic gene and protein expression of offspring and these changes may progress with age.

Bucindolol Modulates Cardiac Remodeling by Attenuating Oxidative Stress in H9c2 Cardiac Cells Exposed to Norepinephrine.

The increased circulation of norepinephrine, found in the diseased heart as a result of sympathetic nervous system overactivation, is responsible for its cardiotoxic effects including pathological hypertrophy, cell death, and oxidative stress. Bucindolol is a third generation adrenergic blocker, which acts on the ß1 and ß2 receptors, and has additional α1 antagonist activity. Thus, the aim of this study was to investigate the action of bucindolol on oxidative stress, hypertrophy, cell survival, and cell death signaling pathways in H9c2 cardiac cells exposed to norepinephrine. H9c2 cells were incubated with 10 µM norepinephrine for 24 h in the presence or absence of bucindolol (10 µM) treatment for 8 h. Western blot was used to determine the expression of proteins for hypertrophy/survival and death signaling pathways. Flow cytometry was used to assess cell death via caspase-3/7 activity and propidium iodide and reactive oxygen species via measuring the fluorescence of CM-H2DCFDA. Norepinephrine exposure resulted in an increase in oxidative stress as well as cell death. This was accompanied by an increased protein expression of LC3B-II/I. The protein kinase B/mammalian target of the rapamycin (Akt/mTOR) pathway which is involved in cardiac remodeling process was activated in response to norepinephrine and was mitigated by bucindolol. In conclusion, bucindolol was able to modulate cardiac remodeling which is mediated by oxidative stress.