Effects of a High-Fat Diet on Spontaneous Metastasis of Lewis Lung Carcinoma in Plasminogen Activator Inhibitor-1 Deficient and Wild-Type Mice

This study investigated the effects of a high-fat diet on spontaneous metastasis of Lewis lung carcinoma (LLC) in plasminogen activator inhibitor-1 deficient (PAI-1^−/−) and wild-type mice. The high-fat diet increased the number of pulmonary metastases by 60% (p<0.01), tumor cross-sectional area by 82% (p<0.05) and tumor volume by 130% (p<0.05) compared to the AIN93G diet. Deficiency in PAI-1 reduced the number of metastases by 35% (p<0.01) compared to wild-type mice. In mice fed the high-fat diet, PAI-1 deficiency reduced tumor cross-sectional area by 52% (p<0.05) and tumor volume by 61% (p<0.05) compared to their wild-type counterparts; however, PAI-1 deficiency affected neither area nor volume in mice fed the AIN93G diet. Adipose and plasma concentrations of PAI-1 were significantly higher in high-fat fed wild-type mice than in their AIN93G-fed counterparts. Adipose and plasma PAI-1 were not detectable in PAI-1^−/− mice regardless of the diet. Mice deficient in PAI-1 showed significantly greater plasma concentrations of monocyte chemotactic protein-1, tumor necrosis factor-α, leptin, vascular endothelial growth factor, tissue inhibitor of metalloproteinase-1 and insulin compared to wild-type mice, indicating a compensatory overproduction of inflammatory cytokines, angiogenic factors and insulin in the absence of PAI-1. We conclude that PAI-1 produced by the host, including that by adipose tissue, promotes high-fat enhanced metastasis of LLC.     

Loss of PINK1 Increases the Heart's Vulnerability to Ischemia-Reperfusion Injury

Objectives

Mutations in PTEN inducible kinase-1 (PINK1) induce mitochondrial dysfunction in dopaminergic neurons resulting in an inherited form of Parkinson’s disease. Although PINK1 is present in the heart its exact role there is unclear. We hypothesized that PINK1 protects the heart against acute ischemia reperfusion injury (IRI) by preventing mitochondrial dysfunction.

Methods and Results

Over-expressing PINK1 in HL-1 cardiac cells reduced cell death following simulated IRI (29.2±5.2% PINK1 versus 49.0±2.4% control; N = 320 cells/group P<0.05), and delayed the onset of mitochondrial permeability transition pore (MPTP) opening (by 1.3 fold; P<0.05). Hearts excised from PINK1+/+, PINK1+/− and PINK1−/− mice were subjected to 35 minutes regional ischemia followed by 30 minutes reperfusion. Interestingly, myocardial infarct size was increased in PINK1−/− hearts compared to PINK1+/+ hearts with an intermediate infarct size in PINK1+/− hearts (25.1±2.0% PINK1+/+, 38.9±3.4% PINK1+/− versus 51.5±4.3% PINK1−/− hearts; N>5 animals/group; P<0.05). Cardiomyocytes isolated from PINK1−/− hearts had a lower resting mitochondrial membrane potential, had inhibited mitochondrial respiration, generated more oxidative stress during simulated IRI, and underwent rigor contracture more rapidly in response to an uncoupler when compared to PINK1+/+ cells suggesting mitochondrial dysfunction in hearts deficient in PINK1.

Conclusions

We show that the loss of PINK1 increases the heart''s vulnerability to ischemia-reperfusion injury. This may be due, in part, to increased mitochondrial dysfunction. These findings implicate PINK1 as a novel target for cardioprotection.     

Folate dependence of hyperhomocysteinemia and vascular dysfunction in cystathionine beta-synthase-deficient mice

Hyperhomocysteinemia is a risk factor for stroke, myocardial infarction, and venous thrombosis. Moderate hyperhomocysteinemia is associated with impaired endothelial function, but the mechanisms responsible for endothelial dysfunction in hyperhomocysteinemia are poorly understood. We have used genetic and dietary approaches to produce hyperhomocysteinemia in mice. Heterozygous cystathionine beta-synthase-deficient mice (CBS +/-), which have a selective defect in homocysteine transsulfuration, and wild-type (CBS +/+) littermates were fed either a control diet or a diet that is relatively deficient in folic acid for 6 wk. Plasma total homocysteine was 5.3 +/- 0.7 microM in CBS +/+ mice and 6.4 +/- 0.6 microM in CBS +/- mice (P = 0.3) given the control diet. Plasma total homocysteine was 11.6 +/- 4.5 microM in CBS +/+ mice and 25.1 +/- 3.2 microM in CBS +/- mice (P = 0.004) given a low-folate diet. In mice fed the control diet, relaxation of aortic rings in response to the endothelium-dependent vasodilator acetylcholine did not differ significantly between CBS +/+ mice and CBS +/- mice. In contrast, in mice fed a low-folate diet, maximal relaxation to acetylcholine was markedly impaired in CBS +/- mice (58 +/- 9%) compared with CBS +/+ mice (84 +/- 4%) (P = 0.01). No differences in relaxation to the endothelium-independent vasodilator sodium nitroprusside were observed among the four groups of mice. These data indicate that CBS-deficient mice are predisposed to hyperhomocysteinemia during dietary folate deficiency, and moderate hyperhomocysteinemia is associated with marked impairment of endothelial function in mice.     

The anti-inflammatory effects of exercise training promote atherosclerotic plaque stabilization in apolipoprotein E knockout mice with diabetic atherosclerosis

Physical exercise is the cornerstone of cardiovascular disease treatment. The present study investigated whether exercise training affects atherosclerotic plaque composition through the modification of inflammatoryrelated pathways in apolipoprotein E knockout (apoE^−/−) mice with diabetic atherosclerosis. Forty-five male apoE^−/− mice were randomized into three equivalent (n=15) groups: control (CO), sedentary (SED), and exercise (EX). Diabetes was induced by streptozotocin administration. High-fat diet was administered to all groups for 12 weeks. Afterwards, CO mice were euthanatized, while the sedentary and exercise groups continued high-fat diet for 6 additional weeks. Exercising mice followed an exercise program on motorizedtreadmill (5 times/week, 60 min/session). Then, blood samples and atherosclerotic plaques in the aortic root were examined. A considerable (P<0.001) regression of the atherosclerotic lesions was observed in the exercise group (180.339±75.613×10³µm²) compared to the control (325.485±72.302×10³ µm²) and sedentary (340.188±159.108×10³µm²) groups. We found decreased macrophages, matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-8 and interleukin-6 (IL-6) concentrations (P<0.05) in the atherosclerotic plaques of the exercise group. Compared to both control and sedentary groups, exercise training significantly increased collagen (P<0.05), elastin (P<0.001), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) (P<0.001) content in the atherosclerotic plaques. Those effects paralleled with increased fibrous cap thickness and less internal elastic lamina ruptures after exercise training (P<0.05), while body-weight and lipid parameters did not significantly change. Plasma MMP-2 and MMP-3 concentrations in atherosclerotic tissues followed a similar trend. From our study we can conclude that exercise training reduces and stabilizes atherosclerotic lesions in apoE^−/− mice with diabetic atherosclerosis. A favorable modification of the inflammatory regulators seems to explain those beneficial effects.Key words: diabetes, atherosclerosis, exercise, matrix metalloproteinases, plaque stability.     

Role of pulmonary microvascular endothelial cell apoptosis in murine sepsis-induced lung injury in vivo

Background

Sepsis remains a common and serious condition with significant morbidity and mortality due to multiple organ dysfunction, especially acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Sepsis-induced ALI is characterized by injury and dysfunction of the pulmonary microvasculature and pulmonary microvascular endothelial cells (PMVEC), resulting in enhanced pulmonary microvascular sequestration and pulmonary infiltration of polymorphonuclear leukocytes (PMN) as well as disruption of the normal alveolo-capillary permeability barrier with leak of albumin-rich edema fluid into pulmonary interstitium and alveoli. The role of PMVEC death and specifically apoptosis in septic pulmonary microvascular dysfunction in vivo has not been established.

Methods

In a murine cecal ligation/perforation (CLP) model of sepsis, we quantified and correlated time-dependent changes in pulmonary microvascular Evans blue (EB)-labeled albumin permeability with (1) PMVEC death (propidium iodide [PI]-staining) by both fluorescent intravital videomicroscopy (IVVM) and histology, and (2) PMVEC apoptosis using histologic fluorescent microscopic assessment of a panel of 3 markers: cell surface phosphatidylserine (detected by Annexin V binding), caspase activation (detected by FLIVO labeling), and DNA fragmentation (TUNEL labeling).

Results

Compared to sham mice, CLP-sepsis resulted in pulmonary microvascular barrier dysfunction, quantified by increased EB-albumin leak, and PMVEC death (PI+ staining) as early as 2 h and more marked by 4 h after CLP. Septic PMVEC also exhibited increased presence of all 3 markers of apoptosis (Annexin V+, FLIVO+, TUNEL+) as early as 30 mins – 1 h after CLP-sepsis, which all similarly increased markedly until 4 h. The time-dependent changes in septic pulmonary microvascular albumin-permeability barrier dysfunction were highly correlated with PMVEC death (PI+; r = 0.976, p < 0.01) and PMVEC apoptosis (FLIVO+; r = 0.991, p < 0.01). Treatment with the pan-caspase inhibitor Q-VD prior to CLP reduced PMVEC death/apoptosis and attenuated septic pulmonary microvascular dysfunction, including both albumin-permeability barrier dysfunction and pulmonary microvascular PMN sequestration (p < 0.05). Septic PMVEC apoptosis and pulmonary microvascular dysfunction were also abrogated following CLP-sepsis in mice deficient in iNOS (Nos2^−/−) or NADPH oxidase (p47^phox−/− or gp91^phox−/−) and in wild-type mice treated with the NADPH oxidase inhibitor, apocynin.

Conclusions

Septic murine pulmonary microvascular dysfunction in vivo is due to PMVEC death, which is mediated through caspase-dependent apoptosis and iNOS/NADPH-oxidase dependent signaling.     

Does Early Mismatched Nutrition Predispose to Hypertension and Atherosclerosis,in Male Mice?

Background

A link between early mismatched nutritional environment and development of components of the metabolic syndrome later in life has been shown in epidemiological and animal data. The aim of this study was to investigate whether an early mismatched nutrition produced by catch-up growth after fetal protein restriction could induce the appearance of hypertension and/or atherosclerosis in adult male mice.

Methodology/Principal Findings

Wild-type C57BL6/J or LDLr−/− dams were fed a low protein (LP) or a control (C) diet during gestation. Catch-up growth was induced in LP offspring by feeding dams with a control diet and by culling the litter to 4 pups against 8 in controls. At weaning, male mice were fed either standard chow or an obesogenic diet (OB), leading to 4 experimental groups. Blood pressure (BP) and heart rate (HR) were assessed in conscious unrestrained wild-type mice by telemetry. Atherosclerosis plaque area was measured in aortic root sections of LDLr−/− mice. We found that: (1) postnatal OB diet increased significantly BP (P<0.0001) and HR (P<0.008) in 3-month old OB-C and OB-LP offspring, respectively; (2) that maternal LP diet induced a significant higher BP (P<0.009) and HR (P<0.004) and (3) an altered circadian rhythm in addition to higher plasma corticosterone concentration in 9 months-old LP offspring; (4) that, although LP offspring showed higher plasma total cholesterol than control offspring, atherosclerosis assessed in aortic roots of 6-mo old mice featured increased plaque area due to OB feeding but not due to early mismatched nutrition.

Conclusions/Significance

These results indicate a long-term effect of early mismatched nutrition on the appearance of hypertension independently of obesity, while no effect on atherosclerosis was noticed at this age.     

Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice

There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C₂C₁₂ myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C₂C₁₂ myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C₂C₁₂ myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm², P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice.     

Nanomechanics of the Endothelial Glycocalyx in Experimental Sepsis

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.     

Niacin Reduces Atherosclerosis Development in APOE*3Leiden.CETP Mice Mainly by Reducing NonHDL-Cholesterol

Objective

Niacin potently lowers triglycerides, mildly decreases LDL-cholesterol, and largely increases HDL-cholesterol. Despite evidence for an atheroprotective effect of niacin from previous small clinical studies, the large outcome trials, AIM-HIGH and HPS2-THRIVE did not reveal additional beneficial effects of niacin (alone or in combination with laropiprant) on top of statin treatment. We aimed to address this apparent discrepancy by investigating the effects of niacin without and with simvastatin on atherosclerosis development and determine the underlying mechanisms, in APOE*3Leiden.CETP mice, a model for familial dysbetalipoproteinemia (FD).

Approach and Results

Mice were fed a western-type diet containing cholesterol without or with niacin (120 mg/kg/day), simvastatin (36 mg/kg/day) or their combination for 18 weeks. Similarly as in FD patients, niacin reduced total cholesterol by -39% and triglycerides by −50%, (both P<0.001). Simvastatin and the combination reduced total cholesterol (−30%; −55%, P<0.001) where the combination revealed a greater reduction compared to simvastatin (−36%, P<0.001). Niacin decreased total cholesterol and triglycerides primarily by increasing VLDL clearance. Niacin increased HDL-cholesterol (+28%, P<0.01) and mildly increased reverse cholesterol transport. All treatments reduced monocyte adhesion to the endothelium (−46%; −47%, P<0.01; −53%, P<0.001), atherosclerotic lesion area (−78%; −49%, P<0.01; −87%, P<0.001) and severity. Compared to simvastatin, the combination increased plaque stability index [(SMC+collagen)/macrophages] (3-fold, P<0.01). Niacin and the combination reduced T cells in the aortic root (−71%, P<0.01; −81%, P<0.001). Lesion area was strongly predicted by nonHDL-cholesterol (R² = 0.69, P<0.001) and to a much lesser extent by HDL-cholesterol (R² = 0.20, P<0.001).

Conclusion

Niacin decreases atherosclerosis development mainly by reducing nonHDL-cholesterol with modest HDL-cholesterol-raising and additional anti-inflammatory effects. The additive effect of niacin on top of simvastatin is mostly dependent on its nonHDL-cholesterol-lowering capacities. These data suggest that clinical beneficial effects of niacin are largely dependent on its ability to lower LDL-cholesterol on top of concomitant lipid-lowering therapy.     

Connective Tissue Growth Factor (CTGF) Expression Modulates Response to High Glucose

Connective tissue growth factor (CTGF) is an important mediator of fibrosis; emerging evidence link changes in plasma and urinary CTGF levels to diabetic kidney disease. To further ascertain the role of CTGF in responses to high glucose, we assessed the consequence of 4 months of streptozotocin-induced diabetes in wild type (+/+) and CTGF heterozygous (+/−) mice. Subsequently, we studied the influence of glucose on gene expression and protein in mice embryonic fibroblasts (MEF) cells derived from wildtype and heterozygous mice. At study initiation, plasma glucose, creatinine, triglyceride and cholesterol levels were similar between non-diabetic CTGF+/+ and CTGF+/− mice. In the diabetic state, plasma glucose levels were increased in CTGF+/+ and CTGF+/− mice (28.2 3.3 mmol/L vs 27.0 3.1 mmol/L), plasma triglyceride levels were lower in CTGF+/− mice than in CTGF+/+ (0.7 0.2 mmol/L vs 0.5 0.1 mmol/L, p<0.05), but cholesterol was essentially unchanged in both groups. Plasma creatinine was higher in diabetic CTGF+/+ group (11.7±1.2 vs 7.9±0.6 µmol/L p<0.01), while urinary albumin excretion and mesangial expansion were reduced in diabetic CTGF+/− animals. Cortices from diabetic mice (both CTGF +/+ and CTGF +/−) manifested higher expression of CTGF and thrombospondin 1 (TSP1). Expression of nephrin was reduced in CTGF +/+ animals; this reduction was attenuated in CTGF+/− group. In cultured MEF from CTGF+/+ mice, glucose (25 mM) increased expression of pro-collagens 1, IV and XVIII as well as fibronectin and thrombospondin 1 (TSP1). In contrast, activation of these genes by high glucose was attenuated in CTGF+/− MEF. We conclude that induction of Ctgf mediates expression of extracellular matrix proteins in diabetic kidney. Thus, genetic variability in CTGF expression directly modulates the severity of diabetic nephropathy.     

Heat Shock Protein 22 (Hsp22) Regulates Oxidative Phosphorylation upon Its Mitochondrial Translocation with the Inducible Nitric Oxide Synthase in Mammalian Heart

Objectives

Stress-inducible heat shock protein 22 (Hsp22) confers protection against ischemia through induction of the inducible isoform of nitric oxide synthase (iNOS). Hsp22 overexpression in vivo stimulates cardiac mitochondrial respiration, whereas Hsp22 deletion in vivo significantly reduces respiration. We hypothesized that Hsp22-mediated regulation of mitochondrial function is dependent upon its mitochondrial translocation together with iNOS.

Methods and Results

Adenoviruses harboring either the full coding sequence of Hsp22 (Ad-WT-Hsp22) or a mutant lacking a N-terminal 20 amino acid putative mitochondrial localization sequence (Ad-N20-Hsp22) were generated, and infected in rat neonatal cardiomyocytes. Compared to β-Gal control, WT-Hsp22 accumulated in mitochondria by 2.5 fold (P<0.05) and increased oxygen consumption rates by 2-fold (P<0.01). This latter effect was abolished upon addition of the selective iNOS inhibitor, 1400W. Ad-WT-Hsp22 significantly increased global iNOS expression by about 2.5-fold (P<0.01), and also increased iNOS mitochondrial localization by 4.5 fold vs β-gal (P<0.05). Upon comparable overexpression, the N20-Hsp22 mutant did not show significant mitochondrial translocation or stimulation of mitochondrial respiration. Moreover, although N20-Hsp22 did increase global iNOS expression by 4.6-fold, it did not promote iNOS mitochondrial translocation.

Conclusion

Translocation of both Hsp22 and iNOS to the mitochondria is necessary for Hsp22-mediated stimulation of oxidative phosphorylation.     

Cystathionine γ-Lyase-deficient Mice Require Dietary Cysteine to Protect against Acute Lethal Myopathy and Oxidative Injury

Cysteine is considered a nonessential amino acid in mammals as it is synthesized from methionine via trans-sulfuration. However, premature infants or patients with hepatic failure may require dietary cysteine due to a lack of cystathionine γ-lyase (CTH), a key trans-sulfuration enzyme. Here, we generated CTH-deficient (Cth^−/−) mice as an animal model of cystathioninemia/cystathioninuria. Cth^−/− mice developed normally in general but displayed hypercystathioninemia/hyperhomocysteinemia though not hypermethioninemia. When fed a low cyst(e)ine diet, Cth^−/− mice showed acute skeletal muscle atrophy (myopathy) accompanied by enhanced gene expression of asparagine synthetase and reduced contents of glutathione in livers and skeletal muscles, and intracellular accumulation of LC3 and p62 in skeletal myofibers; they finally died of severe paralysis of the extremities. Cth^−/− hepatocytes required cystine in a culture medium and showed greater sensitivity to oxidative stress. Cth^−/− mice exhibited systemic vulnerability to oxidative injury, which became more prominent when they were fed the low cyst(e)ine diet. These results reveal novel roles of trans-sulfuration previously unrecognized in mice lacking another trans-sulfuration enzyme cystathionine β-synthase (Cbs^−/−). Because Cbs^−/− mice display hyperhomocysteinemia and hypermethioninemia, our results raise questions against the homocysteine-based etiology of CBS deficiency and the current newborn screening for homocysteinemia using Guthrie''s method, which detects hypermethioninemia.     

Improvement of Insulin Sensitivity by Isoenergy High Carbohydrate Traditional Asian Diet: A Randomized Controlled Pilot Feasibility Study

The prevalence of diabetes is rising dramatically among Asians, with increased consumption of the typical Western diet as one possible cause. We explored the metabolic responses in East Asian Americans (AA) and Caucasian Americans (CA) when transitioning from a traditional Asian diet (TAD) to a typical Western diet (TWD), which has not been reported before. This 16-week randomized control pilot feasibility study, included 28AA and 22CA who were at risk of developing type 2 diabetes. Eight weeks of TAD were provided to all participants, followed by 8 weeks of isoenergy TWD (intervention) or TAD (control). Anthropometric measures, lipid profile, insulin resistance and inflammatory markers were assessed. While on TAD, both AA and CA improved in insulin AUC (−960.2 µU/mL×h, P = 0.001) and reduced in weight (−1.6 kg; P<0.001), body fat (−1.7%, P<0.001) and trunk fat (−2.2%, P<0.001). Comparing changes from TAD to TWD, AA had a smaller weight gain (−1.8 to 0.3 kg, P<0.001) than CA (−1.4 to 0.9 kg, P = 0.001), but a greater increase in insulin AUC (AA: −1402.4 to 606.2 µU/mL×h, P = 0.015 vs CA: −466.0 to 223.5 µU/mL×h, P = 0.034) and homeostatic static model assessment-insulin resistance (HOMA-IR) (AA: −0.3 to 0.2, P = 0.042 vs CA: −0.1 to 0.0, P = 0.221). Despite efforts to maintain isoenergy state and consumption of similar energy, TAD induced weight loss and improved insulin sensitivity in both groups, while TWD worsened the metabolic profile. Trial Registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00379548","term_id":"NCT00379548"}}NCT00379548     

Association between Myocardial Triglyceride Content and Cardiac Function in Healthy Subjects and Endurance Athletes

Ectopic fat accumulation plays important roles in various metabolic disorders and cardiovascular diseases. Recent studies reported that myocardial triglyceride (TG) content measured by proton magnetic resonance spectroscopy (¹H-MRS) is associated with aging, diabetes mellitus, and cardiac dysfunction. However, myocardial TG content in athletes has not yet been investigated. We performed ¹H-MRS and cardiac magnetic resonance imaging in 10 male endurance athletes and 15 healthy male controls. Serum markers and other clinical parameters including arterial stiffness were measured. Cardiopulmonary exercise testing was also performed. There were no significant differences in clinical characteristics including age, anthropometric parameters, blood test results, or arterial stiffness between the two groups. Peak oxygen uptakes, end–diastolic volume (EDV), end–systolic volume (ESV), left ventricular (LV) mass, peak ejection rates and peak filling rates were significantly higher in the athlete group than in the control group (all P<0.02). Myocardial TG content was significantly lower in the athlete group than in the control group (0.60±0.20 vs. 0.89±0.41%, P<0.05). Myocardial TG content was negatively correlated with EDV (r = −0.47), ESV (r = −0.64), LV mass (r = −0.44), and epicardial fat volume (r = 0.47) (all P<0.05). In conclusion, lower levels of myocardial TG content were observed in endurance athletes and were associated with morphological changes related to physiological LV alteration in athletes, suggesting that metabolic imaging for measurement of myocardial TG content by ¹H-MRS may be a useful technique for noninvasively assessing the “athlete’s heart”.     

Vitamin E Attenuates the Progression of Non-Alcoholic Fatty Liver Disease Caused by Partial Hepatectomy in Mice

Background and Aim

The progression of non-alcoholic fatty liver disease (NAFLD) likely involves a ‘multiple hit’ mechanism. We hypothesized that partial hepatectomy, a procedure performed frequently in patients with NAFLD, would accelerate the progression of disease.

Methods

C57BL/6JolaHsd mice were fed a choline-deficient L-amino acid-defined diet (CD-AA) or a choline-sufficient L-amino acid-defined control diet (CS-AA). Part of the mice in the CD-AA group received a diet enriched in vitamin E (~20 mg /day). Two weeks after the start of the diet, mice underwent a partial hepatectomy or a sham operation.

Results

In the CD-AA group, NAFLD activity scores were significantly higher at 7 days after partial hepatectomy compared to the sham operated mice (3.7 ± 1.3 vs. 1.8 ± 0.7; P<0.05). In addition, TBARS, a measure for oxidative stress, in liver tissue of the CD-AA group were significantly higher at day 1, 3 and 7 after partial hepatectomy compared to the sham operated mice (P<0.05). Vitamin E therapy significantly reduced TBARS level at day 7 after partial hepatectomy compared to the CD-AA diet group (P< 0.05). Vitamin E suppletion reduced NAFLD activity score at day 7 after partial hepatectomy compared to the CD-AA group (2.3 ± 0.8 vs. 3.8 ± 1.0; P<0.05).

Conclusion

Partial hepatectomy accelerates the progression of NAFLD. Disease progression induced by partial hepatectomy is substantially attenuated by vitamin E.     

Two-Dimensional Echocardiography in the Assessment of Long-Term Prognosis in Patients with Pulmonary Arterial Hypertension

Objective

To investigate the relationship between cardiac diastolic dysfunction and outcomes in patients with pulmonary arterial hypertension (PAH) and to clarify the potential effect of two-dimensional echocardiography (2D-echo) on prognostic value in patients with PAH.

Methods

Patients diagnosed with PAH (as WSPH (World Symposia on Pulmonary Hypertension) classification I) confirmed by right heart catheterization (RHC), received targeted monotherapy or combination therapy. 2D-echo parameters, World Health Organization (WHO) functional classification and 6-minute walking distance (6MWD) were recorded. The clinical prognosis of patients was assessed by the correlation between echo parameters and clinical 6MWD using receiver operating characteristic (ROC) curve analysis.

Results

Fifty-eight patients were included. Left and right ventricular diastolic dysfunction (LVDD and RVDD) scores measured by 2D-echo had good correlation with 6MWD at baseline (r_LVDD  = −0.699; r_RVDD  = −0.818, both P<0.001) and at last follow-up (r_LVDD  = −0.701; r_RVDD  = −0.666, both P<0.001). Furthermore, bi-ventricular (LVDD+RVDD) scores measured by 2D-echo had a better correlation with 6MWD at baseline and last follow-up (r = −0.831; r = −0.771, both P<0.001). ROC curve analysis showed that the area under curves (AUCs) for LVDD score, RVDD score and (LVDD+RVDD) scores were 0.823 (P<0.0001), 0.737 (P = 0.0002), and 0.825 (P<0.0001), respectively. Compared with ROC analysis of other single parameters, cardiac diastolic function score was more accurate in predicting survival in patients with PAH.

Conclusion

LVDD score, RVDD score and (LVDD+RVDD) scores yielded a comprehensive quantitative assessment of LV and RV diastolic function that correlated moderately with clinical functional parameters and might be useful in the assessment of PAH.     

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis

The use of nicotinic acid to treat dyslipidemia is limited by induction of a “flushing” response, mediated in part by the interaction of prostaglandin D₂ (PGD₂) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr^−/−ApoE^−/− mice versus ApoE^−/− mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr^−/−ApoE^−/− mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE^−/− mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr^−/− mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.     

A novel approach to oral apoA-I mimetic therapy

Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR^−/−) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice (P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions (P < 0.0001); HDL cholesterol and PON were inversely correlated (P < 0.0001). After feeding WD + 6F: i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.     

Impairment of Myocardial Mitochondria in Viral Myocardial Disease and Its Reflective Window in Peripheral Cells

Background

Viral myocardial disease (VMD) is a common disease inducing heart failure. It has not been clear the roles of mitochondrial damage in the pathological changes of cardiomyocytes in VMD.

Methods

Myocardial tissues and lymphocytes were collected from 83 VMD patients. Control groups included 12 cases of healthy accidental death with myocardial autopsy and 23 healthy blood donors. The mouse model of viral myocarditis (VMC) was established by Coxsackie virus B₃ infection and myocardial tissues and skeletal muscle were collected. Mitochondrial DNA (mtDNA) deletion rate was quantitatively determined using polymerase chain reaction.

Results

There was significantly difference of myocardial mitochondrial DNA deletion rate between VMD or VMC group and control group (P<0.05). Moreover, the loss of mitochondrial membrane phospholipids was significantly different between VMD or VMC group and control group. In VMC mice, there were negative correlations between myocardial mtDNA³⁸⁶⁷ deletion rate and left ventricular peak systolic pressure (LVPSP) (r = −0.66, P<0.05), and between myocardial mtDNA³⁸⁶⁷ deletion rate and +dp/dt_max (r = −0.79, P<0.05), while there was positive correlation between myocardial mtDNA³⁸⁶⁷ deletion rate and −dp/dt_max (r = 0.80, P<0.05).

Conclusion

Mitochondrial damage is an important pathophysiological mechanism leading to myocardial injury and cardiac dysfunction. The mitochondrial damage in the skeletal muscle and lymphocytes reflect a “window” of myocardial mitochondrial damage.