The innate immune response to coxsackievirus B3 predicts progression to cardiovascular disease and heart failure in male mice

<p>Abstract</p> <p>Background</p> <p>Men are at an increased risk of dying from heart failure caused by inflammatory heart diseases such as atherosclerosis, myocarditis and dilated cardiomyopathy (DCM). We previously showed that macrophages in the spleen are phenotypically distinct in male compared to female mice at 12 h after infection. This innate immune profile mirrors and predicts the cardiac immune response during acute myocarditis.</p> <p>Methods</p> <p>In order to study sex differences in the innate immune response, five male and female BALB/c mice were infected intraperitoneally with coxsackievirus B3 (CVB3) or phosphate buffered saline and their spleens were harvested 12 h later for microarray analysis. Gene expression was determined using an Affymetrix Mouse Gene 1.0 ST Array. Significant gene changes were verified by quantitative real-time polymerase chain reaction or ELISA.</p> <p>Results</p> <p>During the innate immune response to CVB3 infection, infected males had higher splenic expression of genes which are important in regulating the influx of cholesterol into macrophages, such as phospholipase A₂(PLA₂) and the macrophage scavenger receptor compared to the infected females. We also observed a higher expression in infected males compared to infected females of squalene synthase, an enzyme used to generate cholesterol within cells, and Cyp2e1, an enzyme important in metabolizing cholesterol and steroids. Infected males also had decreased levels of the translocator protein 18 kDa (TSPO), which binds PLA₂and is the rate-limiting step for steroidogenesis, as well as decreased expression of the androgen receptor (AR), which indicates receptor activation. Gene differences were not due to increased viral replication, which was unaltered between sexes.</p> <p>Conclusions</p> <p>We found that, compared to females, male mice had a greater splenic expression of genes which are important for cholesterol metabolism and activation of the AR at 12 h after infection. Activation of the AR has been linked to increased cardiac hypertrophy, atherosclerosis, myocarditis/DCM and heart failure in male mice and humans.</p

Cardioprotective Effect of Beta-3 Adrenergic Receptor Agonism Role of Neuronal Nitric Oxide Synthase

ObjectivesThe aim of this study was to determine whether activation of β3-adrenergic receptor (AR) and downstream signaling of nitric oxide synthase (NOS) isoforms protects the heart from failure and hypertrophy induced by pressure overload.Backgroundβ3-AR and its downstream signaling pathways are recognized as novel modulators of heart function. Unlike β1- and β2-ARs, β3-ARs are stimulated at high catecholamine concentrations and induce negative inotropic effects, serving as a “brake” to protect the heart from catecholamine overstimulation.MethodsC57BL/6J and neuronal NOS (nNOS) knockout mice were assigned to receive transverse aortic constriction (TAC), BRL37344 (β3 agonist, BRL 0.1 mg/kg/h), or both.ResultsThree weeks of BRL treatment in wild-type mice attenuated left ventricular dilation and systolic dysfunction, and partially reduced cardiac hypertrophy induced by TAC. This effect was associated with increased nitric oxide production and superoxide suppression. TAC decreased endothelial NOS (eNOS) dimerization, indicating eNOS uncoupling, which was not reversed by BRL treatment. However, nNOS protein expression was up-regulated 2-fold by BRL, and the suppressive effect of BRL on superoxide generation was abrogated by acute nNOS inhibition. Furthermore, BRL cardioprotective effects were actually detrimental in nNOS–/– mice.ConclusionsThese results are the first to show in vivo cardioprotective effects of β3-AR–specific agonism in pressure overload hypertrophy and heart failure, and support nNOS as the primary downstream NOS isoform in maintaining NO and reactive oxygen species balance in the failing heart

A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and premature lethality. This effect is both extracellular signal-regulated kinase (ERK1/2) dependent and angiotensin-II type 1 receptor (AT1R) dependent. We have identified protein kinase C beta (PKCβ) as a critical mediator of this pathway and demonstrate that the PKCβ inhibitor enzastaurin, and the clinically available anti-hypertensive agent hydralazine, both normalize aortic growth in Marfan mice, in association with reduced PKCβ and ERK1/2 activation. Furthermore, patients with Marfan syndrome and other forms of inherited thoracic aortic aneurysm taking CCBs display increased risk of aortic dissection and need for aortic surgery, compared to patients on other antihypertensive agents. DOI: http://dx.doi.org/10.7554/eLife.08648.00

Disruption of Nrf2, a Key Inducer of Antioxidant Defenses, Attenuates ApoE-Mediated Atherosclerosis in Mice

Background: Oxidative stress and inflammation are two critical factors that drive the formation of plaques in atherosclerosis. Nrf2 is a redox-sensitive transcription factor that upregulates a battery of antioxidative genes and cytoprotective enzymes that constitute the cellular response to oxidative stress. Our previous studies have shown that disruption of Nrf2 in mice (Nrf2-/-) causes increased susceptibility to pulmonary emphysema, asthma and sepsis due to increased oxidative stress and inflammation. Here we have tested the hypothesis that disruption of Nrf2 in mice causes increased atherosclerosis. Principal Findings: To investigate the role of Nrf2 in the development of atherosclerosis, we crossed Nrf2-/- mice with apoliporotein E-deficient (ApoE-/- mice. ApoE-/- and ApoE-/- Nrf2-/- mice were fed an atherogenic diet for 20 weeks, and plaque area was assessed in the aortas. Surprisingly, ApoE-/- Nrf2-/- mice exhibited significantly smaller plaque area than ApoE-/- controls (11.5% vs 29.5%). This decrease in plaque area observed in ApoE-/- Nrf2-/- mice was associated with a significant decrease in uptake of modified low density lipoproteins (AcLDL) by isolated macrophages from ApoE-/- Nrf2-/- mice. Furthermore, atherosclerotic plaques and isolated macrophages from ApoE-/- Nrf2-/- mice exhibited decreased expression of the scavenger receptor CD36. Conclusions: Nrf2 is pro-atherogenic in mice, despite its antioxidative function. The net pro-atherogenic effect of Nrf2 may be mediated via positive regulation of CD36. Our data demonstrates that the potential effects of Nrf2-targeted therapies on cardiovascular disease need to be investigated.9 page(s

Effect of inhibition of glycosphingolipid synthesis on vascular and cardiac function: a novel therapeutic strategy

Empirical thesis.Includes bibliographical references.Chapter 1. Introduction -- Chapter 2. Literature review -- Chapter 3. Specific aims and experimental design -- Chapter 4. Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in Apo E(-/-) mice and rabbits fed a high fat and cholesterol diet -- Chapter 5. Prevention of cardiac hypertrophy by the use of a glycosphingolipid synthesis inhibitor in ApoE(-/-) mice -- Chapter 6. Improved Intervention of atherosclerosis and cardiac hypertrophy through Biodegradable polymer-encapsulated delivery of glycosphingolipid inhibitor -- Chapter 7. Molecular imaging of inflammation in the ApoE (-/-) mouse model of atherosclerosis with IodoDPA -- Chapter 8. General discussion -- Appendices.Background. Vascular dysfunction of conduit arteries is manifest as atherosclerosis and vascular stiffness, which are significant contributors to cardiovascular disease. Vascular dysfunction is characterized by thickening and stiffening of the vessel walls, and is accelerated by a sedentary lifestyle and advancing age. Despite the heavy public health burden of vascular dysfunction, no cure has been found. A recent study by Chatterjee et al., using a dietary model of atherosclerosis in rabbits, showed that atherogenesis was inhibited by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glycosphingolipid synthesis inhibitor. The work presented in this thesis focusses on determining the molecular mechanism by which D-PDMP affects vascular and cardiac function through modification of wall properties, by inhibition of atherosclerosis and associated pathological cardiac hypertrophy (LVH), in murine models of atherosclerosis. Novel imaging methods of assessing atherosclerosis are explored, as well as drug delivery systems of D-PDMP with the aim of improving therapeutic efficacy.Methods. Experiments were performed in ApoE (-/-) mice fed a high cholesterol and high fat diet, with and without D-PDMP, from 12 to 36 weeks, during the prevention phase and treated from 20 to 36 weeks for the intervention phase. Plaque area, intima-media wall thickness, cardiac hypertrophy and contractility were assessed using high-resolution ultrasound. Blood pressure (tail cuff) and aortic pulse wave velocity (PWV, measure of arterial stiffness) were measured. Tissues were harvested for further molecular and histopathological studies. In separate cohorts of mice, molecular imaging of inflammation was conducted using the radiotracer Iodo-DPA-713 and SPECT. Drug delivery of D-PDMP was explored using biodegradable polymer encapsulation.Results. Apo E(-/-) mice fed a hyperlipidemic diet showed marked accumulation of atherosclerotic plaque, increased PWV independent of blood pressure, decreased cardiac contractility, and increased LVH and fibrosis, compared to control and treated mice These effects were largely attenuated during the prevention phase, and reversed during the intervention phase of D-PDMP. Measurement of glycolipid glycotransferases showed that a hyperlipidemic diet increased the activity of glucosylceramide synthase and lactosylceramide synthase in the aortic wall and myocardium of Apo E (-/-) mice. Treatment with D-PDMP prevented these changes. Novel molecular imaging showed that IodoDPA is a promising, new imaging agent for assessing atherosclerosis, with specificity for the macrophage component of the lesions. Delivery of D-PDMP via biodegradable polymer encapsulation resulted in improved efficacy of glycolipid synthase inhibition, through increases in gastro-intestinal absorption and residence time, compared to non-encapsulated D-PDMP treatment method.Conclusion: D-PDMP is therapeutically an anti-atherogenic and cardio-protective treatment, with concomitant reduction of arterial stiffness. The work presented in this thesis has elucidated a mechanism of action through inhibition of glucosylceramide and lactosylceramide synthase. This work, and related studies, will enhance our understanding of the pathophysiological, molecular, and genetic mechanisms underlying the progression, prevention and intervention of cardiovascular diseases, using novel therapeutic and methodic strategies.Mode of access: World wide web1 online resource (xxi, 295 pages) colour illustration

Prevention of cardiac hypertrophy by the use of a glycosphingolipid synthesis inhibitor in ApoE-/- mice

ApoE-/- mice fed a high fat and high cholesterol (HFHC) diet (20% fat and 1.25% cholesterol) from 12 weeks of age to 36 weeks revealed an age-dependent increase in the left ventricular mass (LV mass) and decline in fractional shortening (FS%), which worsened with HFHC diet. These traits are indicative of maladaptive pathological cardiac hypertrophy and dysfunction. This was accompanied by loading of glycosphingolipids and increased gene expression of ANP, BNP in myocardial tissue. Masson's trichrome staining revealed a significant increase in cardiomyocyte size and fibrosis. In contrast, treatment with 5 and 10 μM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase, dose-dependently decreased the load of glycosphingolipids and preserved fractional shortening and maintained left ventricular mass to normal 12-week-old control levels over a 6 month treatment period. Our mechanistic studies showed that D-PDMP inhibited cardiac hypertrophy by inhibiting the phosphorylation of mitogen-activated protein kinase (MAPK). We propose that associating increased glycosphingolipid synthesis with cardiac hypertrophy could serve as a novel approach to prevent this phenotype in experimental animal models of diet-induced atherosclerotic heart disease.6 page(s

Anthracyclines used in the treatment of cancer: their harmful effects on the Reno-cardiovascular connection

Background: The molecular and cellular mechanisms corresponding to the compensatory and maladaptive hypertrophy and remodeling of the left ventricle with chronic doxorubicin (DOX) treatment are currently unclear. Non-invasive methods of determining these changes are still deficient. To investigate these changes, 8 groups of rats in 4 different studies including a control saline group of the same age, gender and strain were evaluated for cardiac morphology and function including: (1) DOX dose response using a cumulative dose of 7.5mg/kg, and 15mg/kg in 8-10 week old female Sprague-Dawley (SD) rats, (2) strain differences were investigated in response to a cumulative dose of 15mg/kg in 8-10 week old female Fisher (F344) rats compared to the SD rats treated with same dose, (3) the role of gender and aging were studied in response to DOX at a cumulative dose of 3mg/kg in male and female neonates, and (4) combined losartan and a cumulative dose of 15mg/kg of DOX in 8-10 week old female SD rats compared to controls of saline and 15mg/kg treated SD rats.Method: Onset of cardiac toxicity was assessed by echocardiography and the rat model of heart failure was developed when the fractional shortening declined &#8804; 40%. The mean arterial pressure and single-photon-emission computer tomography scanning and Tc-99m-HYNIC-Annexin V were performed at week 10 to analyze blood pressure and quantify apoptosis, respectively. All rats were euthanized at week 10 except for the neonates and two of the 7.5mg/kg-treated SD rats that were left alive for study of long -term cardiac side effects. The heart and kidney tissues were harvested for protein isolation and histopathological studies. Blood samples were collected for hematological and lipid profile analysis in all the rats. Results: A dose- and time-dependent increase in LVmass coincided with a parallel increase in MAP, kidney damage, expression of myocardial erbB2, heat shock protein 90 Akt, mTOR, GSK-3&#946;, TGF-&#946;, pSMAD2, and cardiomyocyte apoptosis in SD rats treated with 7.5mg/kg and 15mg/kg of DOX at week 10. The 7.5 kg/kg treatment showed adaptive hypertrophy whereas the 15mg/kg treatment group showed maladaptive hypertrophy. However decompensation was apparent by week 14 in other rats treated with 7.5mg/kg. LVmass, FS, MAP, kidney damage, red blood cells and blood lipid levels were not significantly altered in the F344 rats compared to the 15 mg/kg-treated SD rats. Losartan supplementation reduced the left ventricular hypertrophy, improved myocardial contractility, and reduced TGF-&#946; expression compared to the DOX-treated SD rats. The 3mg/kg of DOX in neonates induced cardiac toxicity and deaths in about 60% of males 50 weeks after treatment; the females instead developed mammary tumors. Conclusion: The results of this study suggest that age, gender, and strain differences are risks factors for doxorubicin-induced harmful reno-cardiovascular toxicity. The inhibition of TGF-&#946; expression by losartan can be used in prevention of chronic doxorubicin-induced cardiac toxicity without interfering with its anti-tumor activities

Phosphatase PTEN is critically involved in post-myocardial infarction remodeling through the Akt/interleukin-10 signaling pathway

The inflammatory cytokines interleukin (IL)-10 and tumor necrosis factor (TNF)-α play an important role in left ventricular (LV) remodeling after myocardial infarction (MI). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) inactivates protein kinase Akt and promotes cell death in the heart. However, it is not known whether PTEN promotes post-MI remodeling by regulating IL-10 and TNF-α. MI was induced in wild-type (WT) mice and Pten heterozygous mutant (HET) mice. Pten adenoviruses (adPten) or empty vectors (adNull) were injected into the peri-infarct area of WT mice. LV dilation was attenuated and fractional shortening was increased in HET mice compared to WT mice. Survival rate and fractional shortening were decreased in adPten mice compared to adNull mice. Leukocyte infiltration into the peri-infarct area was attenuated in HET mice and worsened in adPten mice. PTEN expression was upregulated in the infarcted heart of WT mice. Partial inactivation of PTEN increased the production of IL-10 and decreased the expression of TNF-α and matrix metalloproteinase (MMP)-2 and -9 after MI in HET mice. PTEN overexpression caused opposite effects in the infarcted heart. Moreover in the infarcted heart of HET mice, Akt inhibition decreased Stat3 phosphorylation and IL-10 expression, and blockade of the IL-10 receptor increased TNF-α and MMP-2 expression. Both Akt inhibition and IL-10 receptor blockade abolished the attenuation of post-MI remodeling in HET mice. In conclusion, PTEN is critically involved in post-MI remodeling through the Akt/IL-10 signaling pathway. Therefore, targeting PTEN may be an effective approach to post-MI remodeling.15 page(s

Inhibiting glycosphingolipid glycosyltransferase activity prevents cardiac hypertrophy in apoE-/- mice fed a western diet

Background: Although diet enriched in high fat and cholesterol can induce cardiac hypertrophy, little is known about the role of glycosphigolipids and glycosyltransferases contributing to this phenotype. Herein, using transgenic apo E-/- mice fed a high fat and cholesterol diet we show that treatment with a glycosphingolipid inhibitor blunts cardiac hypertrophy. Methods and Results: Male apoE-/- mice were fed a western diet (20% fat and 1.25% cholesterol) from 12 weeks of age till 36 weeks with and without D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) daily by oral gavage (vehicle alone, 5 mpk and 10mpk) . At 12 week, 20 week and 36 week, trans -thoracic echocardiography were performed in conscious mice using an ultrasound system (n=5 in each group). Mice were euthanized once maladaptive cardiac hypertrophy time point is reach. Tissues such as the heart were harvested for were harvested for histochemical, biochemical, and molecular analysis to ascertain the effects of treatment on glycosphingolipid glycosyltransferase activity, gene/protein expression. We observed an age-dependent increase in the left ventricular mass (LVmass) and decline in fractional shortening indicative of cardiac hypertrophy and dysfunction which worsens with western diet. This was accompanied by increased gene expression for atrial natriuretic protein, Brain-type natriuretic protein, and increased protein expression of transforming growth factor, MAPK and AKT-1. Masson’s trichrome staining revealed hypertrophy in cardiomyocytes and extensive fibrosis. In contrast, treatment with D-PDMP preserved fractional shortening and the left ventricular mass to control levels over a 6 month treatment period. Also, the level of various cardiac glycosphingolipids and glycosyltransferases were decreased to control levels. Conclusion: Treatment with a glycosphingolipid glycosyltransferase inhibitor blunts cardiac hypertrophy in apoE-/- mice fed a western diet.1 page(s