BNP as a New Biomarker of Cardiac Thyroid Hormone Function.

BACKGROUND: Cardiac re-expression of fetal genes in patients with heart failure (HF) suggests the presence of low cardiac tissue thyroid hormone (TH) function. However, serum concentrations of T3 and T4 are often normal or subclinically low, necessitating an alternative serum biomarker for low cardiac TH function to guide treatment of these patients. The clinical literature suggests that serum Brain Natriuretic Peptide (BNP) levels are inversely associated with serum triiodo-L-thyronine (T3) levels. The objective of this study was to investigate BNP as a potential serum biomarker for TH function in the heart. METHODS: Two animal models of thyroid hormone deficiency: (1) 8-weeks of propyl thiouracil-induced hypothyroidism (Hypo) in adult female rats were subsequently treated with oral T3 (10 µg/kg/d) for 3, 6, or 14 days; (2) HF induced by coronary artery ligation (myocardial infarction, MI) in adult female rats was treated daily with low dose oral T3 (5 µg/kg/d) for 8 or 16 wks. RESULTS: Six days of T3 treatment of Hypo rats normalized most cardiac functional parameters. Serum levels of BNP increased 5-fold in Hypo rats, while T3 treatment normalized BNP by day 14, showing a significant inverse relationship between serum BNP and free or total T3 concentrations. Myocardial BNP mRNA was increased 2.5-fold in Hypo rats and its expression was decreased to normal values by 14 days of T3 treatment. Measurements of hemodynamic function showed significant dysfunction in MI rats after 16 weeks, with serum BNP increased by 4.5-fold and serum free and total T3 decreased significantly. Treatment with T3 decreased serum BNP while increasing total T3 indicating an inverse correlation between these two biologic factors (r 2 = 0.676, p < 0.001). Myocardial BNP mRNA was increased 5-fold in MI rats which was significantly decreased by T3 over 8 to 16 week treatment periods. CONCLUSIONS: Results from the two models of TH dysfunction confirmed an inverse relationship between tissue and serum T3 and BNP, such that the reduction in serum BNP could potentially be utilized to monitor efficacy and dosing of T3 treatment. Thus, serum BNP may serve as a reliable biomarker for cardiac TH function.

Effects of CP-900691, a novel peroxisome proliferator-activated receptor α, agonist on diabetic nephropathy in the BTBR ob/ob mouse.

Piperidine-based peroxisome proliferator-activated receptor-α agonists are agents that are efficacious in improving lipid, glycemic, and inflammatory indicators in diabetes and obesity. This study sought to determine whether CP-900691 ((S)-3-[3-(1-carboxy-1-methyl-ethoxy)-phenyl]-piperidine-1-carboxylic acid 4-trifluoromethyl-benzyl ester; CP), a member of this novel class of agents, by decreasing plasma triglycerides, could prevent diabetic nephropathy in the Black and Tan, BRachyuric (BTBR) ob/ob mouse model of type 2 diabetes mellitus. Four-week old female BTBR WT and BTBR ob/ob mice received either regular chow or one containing CP (3 mg/kg per day) for 14 weeks. CP elevated plasma high-density lipoprotein, albuminuria, and urinary excretion of 8-epi PGF(2α), a product of the nonenzymatic metabolism of arachidonic acid and whose production is elevated in oxidative stress, in BTBR WT mice. In BTBR ob/ob mice, CP reduced plasma triglycerides and non-esterified fatty acids, fasting blood glucose, body weight, and plasma interleukin-6, while concomitantly improving insulin resistance. Despite these beneficial metabolic effects, CP had no effect on elevated plasma insulin, 8-epi PGF(2α) excretion, and albuminuria, and surprisingly, did not ameliorate the development of diabetic nephropathy, having no effect on the accumulation of renal macrophages, glomerular hypertrophy, and increased mesangial matrix expansion. In addition, CP did not increase plasma high-density lipoprotein in BTBR ob/ob mice, while paradoxically increasing total cholesterol levels. These findings indicate that 8-epi PGF(2α), possibly along with hyperinsulinemia and inflammatory and dysfunctional lipoproteins, is integral to the development of diabetic nephropathy and should be considered as a potential target of therapy in the treatment of diabetic nephropathy.

Insights into the arginine paradox: evidence against the importance of subcellular location of arginase and eNOS.

Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes overt cardiovascular disease. Increased expression of Arginase has been proposed as a mechanism to account for diminished NO production. Arginases consume l-arginine, the substrate for endothelial nitric oxide synthase (eNOS), and l-arginine depletion is thought to competitively reduce eNOS-derived NO. However, this simple relationship is complicated by the paradox that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. One mechanism proposed to explain this is compartmentalization of intracellular l-arginine into distinct, poorly interchangeable pools. In the current study, we investigated this concept by targeting eNOS and Arginase to different intracellular locations within COS-7 cells and also BAEC. We found that supplemental l-arginine and l-citrulline dose-dependently increased NO production in a manner independent of the intracellular location of eNOS. Cytosolic arginase I and mitochondrial arginase II reduced eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting arginase I to disparate regions of the cell did not differentially modify eNOS activity. Arginase-dependent suppression of eNOS activity was reversed by pharmacological inhibitors and absent in a catalytically inactive mutant. Arginase did not directly interact with eNOS, and the metabolic products of arginase or downstream enzymes did not contribute to eNOS inhibition. Cells expressing arginase had significantly lower levels of intracellular l-arginine and higher levels of ornithine. These results suggest that arginases inhibit eNOS activity by depletion of substrate and that the compartmentalization of l-arginine does not play a major role.

Reversibility of structural and functional damage in a model of advanced diabetic nephropathy.

The reversibility of diabetic nephropathy remains controversial. Here, we tested whether replacing leptin could reverse the advanced diabetic nephropathy modeled by the leptin-deficient BTBR ob/ob mouse. Leptin replacement, but not inhibition of the renin-angiotensin-aldosterone system (RAAS), resulted in near-complete reversal of both structural (mesangial matrix expansion, mesangiolysis, basement membrane thickening, podocyte loss) and functional (proteinuria, accumulation of reactive oxygen species) measures of advanced diabetic nephropathy. Immunohistochemical labeling with the podocyte markers Wilms tumor 1 and p57 identified parietal epithelial cells as a possible source of regenerating podocytes. Thus, the leptin-deficient BTBR ob/ob mouse provides a model of advanced but reversible diabetic nephropathy for further study. These results also suggest that restoration of lost podocytes is possible but is not induced by RAAS inhibition, possibly explaining the limited efficacy of RAAS inhibitors in promoting repair of diabetic nephropathy.

Acyl-CoA synthetase 1 is induced by Gram-negative bacteria and lipopolysaccharide and is required for phospholipid turnover in stimulated macrophages.

The enzyme acyl-CoA synthetase 1 (ACSL1) is induced by peroxisome proliferator-activated receptor α (PPARα) and PPARγ in insulin target tissues, such as skeletal muscle and adipose tissue, and plays an important role in ß-oxidation in these tissues. In macrophages, however, ACSL1 mediates inflammatory effects without significant effects on ß-oxidation. Thus, the function of ACSL1 varies in different tissues. We therefore investigated the signals and signal transduction pathways resulting in ACSL1 induction in macrophages as well as the consequences of ACSL1 deficiency for phospholipid turnover in LPS-activated macrophages. LPS, Gram-negative bacteria, IFN-γ, and TNFα all induce ACSL1 expression in macrophages, whereas PPAR agonists do not. LPS-induced ACSL1 expression is dependent on Toll-like receptor 4 (TLR4) and its adaptor protein TRIF (Toll-like receptor adaptor molecule 1) but does not require the MyD88 (myeloid differentiation primary response gene 88) arm of TLR4 signaling; nor does it require STAT1 (signal transducer and activator of transcription 1) for maximal induction. Furthermore, ACSL1 deletion attenuates phospholipid turnover in LPS-stimulated macrophages. Thus, the regulation and biological function of ACSL1 in macrophages differ markedly from that in insulin target tissues. These results suggest that ACSL1 may have an important role in the innate immune response. Further, these findings illustrate an interesting paradigm in which the same enzyme, ACSL1, confers distinct biological effects in different cell types, and these disparate functions are paralleled by differences in the pathways that regulate its expression.

Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells.

Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E2 (PGE2) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE2. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE2 secretion. Thus, ACSL4 modulates PGE2 release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall.

Neuregulin/ErbB signaling regulates cardiac subtype specification in differentiating human embryonic stem cells.

RATIONALE: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) exhibit either a "working" chamber or a nodal-like phenotype. To generate optimal hESC-CM preparations for eventual clinical application in cell-based therapies, we will need to control their differentiation into these specialized cardiac subtypes. OBJECTIVE: To demonstrate intact neuregulin (NRG)-1ß/ErbB signaling in hESC-CMs and test the hypothesis that this signaling pathway regulates cardiac subtype abundance in hESC-CM cultures. METHODS AND RESULTS: All experiments used hESC-CM cultures generated using our recently reported directed differentiation protocol. To support subsequent action potential phenotyping approaches and provide a higher-throughput method of determining cardiac subtype, we first developed and validated a novel genetic label that identifies nodal-type hESC-CMs. Next, control hESC-CM preparations were compared to those differentiated in the presence of exogenous NRG-1ß, an anti-NRG-1ß neutralizing antibody, or the ErbB antagonist AG1478. We used 3 independent approaches to determine the ratio of cardiac subtypes in the resultant populations: direct action potential phenotyping under current-clamp, activation of the aforementioned genetic label, and subtype-specific marker expression by RT-PCR. Using all 3 end points, we found that inhibition of NRG-1ß/ErbB signaling greatly enhanced the proportion of cells showing the nodal phenotype. CONCLUSIONS: NRG-1ß/ErbB signaling regulates the ratio of nodal- to working-type cells in differentiating hESC-CM cultures and presumably functions similarly during early human heart development. We speculate that, by manipulating NRG-1ß/ErbB signaling, it will be possible to generate preparations of enriched working-type myocytes for infarct repair, or, conversely, nodal cells for potential use in a biological pacemaker.

BTBR Ob/Ob mutant mice model progressive diabetic nephropathy.

There remains a need for robust mouse models of diabetic nephropathy (DN) that mimic key features of advanced human DN. The recently developed mouse strain BTBR with the ob/ob leptin-deficiency mutation develops severe type 2 diabetes, hypercholesterolemia, elevated triglycerides, and insulin resistance, but the renal phenotype has not been characterized. Here, we show that these obese, diabetic mice rapidly develop morphologic renal lesions characteristic of both early and advanced human DN. BTBR ob/ob mice developed progressive proteinuria beginning at 4 weeks. Glomerular hypertrophy and accumulation of mesangial matrix, characteristic of early DN, were present by 8 weeks, and glomerular lesions similar to those of advanced human DN were present by 20 weeks. By 22 weeks, we observed an approximately 20% increase in basement membrane thickness and a >50% increase in mesangial matrix. Diffuse mesangial sclerosis (focally approaching nodular glomerulosclerosis), focal arteriolar hyalinosis, mesangiolysis, and focal mild interstitial fibrosis were present. Loss of podocytes was present early and persisted. In summary, BTBR ob/ob mice develop a constellation of abnormalities that closely resemble advanced human DN more rapidly than most other murine models, making this strain particularly attractive for testing therapeutic interventions.

Histone deacetylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells.

Recent evidence indicates that mouse and human embryonic stem cells (ESCs) are fixed at different developmental stages, with the former positioned earlier. We show that a narrow concentration of the naturally occurring short-chain fatty acid, sodium butyrate, supports the extensive self-renewal of mouse and human ESCs, while promoting their convergence toward an intermediate stem cell state. In response to butyrate, human ESCs regress to an earlier developmental stage characterized by a gene expression profile resembling that of mouse ESCs, preventing precocious Xist expression while retaining the ability to form complex teratomas in vivo. Other histone deacetylase inhibitors (HDACi) also support human ESC self-renewal. Our results indicate that HDACi can promote ESC self-renewal across species, and demonstrate that ESCs can toggle between alternative states in response to environmental factors.

Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-gamma-independent mechanism in human arterial smooth muscle cells and macrophages.

Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator-activated receptor (PPAR)-gamma, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acyl-CoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-gamma-independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-gamma-independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis.

Aggressive very low-density lipoprotein (VLDL) and LDL lowering by gene transfer of the VLDL receptor combined with a low-fat diet regimen induces regression and reduces macrophage content in advanced atherosclerotic lesions in LDL receptor-deficient mice.

Very low-density lipoprotein (VLDL) and LDL plasma levels are associated with cardiovascular mortality. Whereas VLDL/LDL lowering causes regression of early atherosclerotic lesions, less is known about the effects of aggressive lipid lowering on regression of advanced complex lesions. We therefore investigated the effect of VLDL/LDL lowering on pre-existing lesions in LDL receptor-deficient mice. Mice fed a high-fat diet for 16 weeks developed advanced lesions with fibrous caps, necrotic cores, and cholesterol clefts in the brachiocephalic artery. After an additional 14 weeks on a low-fat diet, plasma cholesterol levels decreased from 21.0 +/- 2.6 to 8.4 +/- 0.6 mmol/L, but lesions did not regress. Levels of VLDL/LDL were further lowered by using a helper-dependent adenovirus encoding the VLDL receptor (HD-Ad-VLDLR) under control of a liver-selective promoter. Treatment with HD-Ad-VLDLR together with a low-fat diet regimen resulted in reduced lesion size (cross-sectional area decreased from 146,272 +/- 19,359 to 91,557 +/- 15,738 microm2) and an 89% reduction in the cross-sectional lesion area occupied by macrophages compared to controls. These results show that aggressive VLDL/LDL lowering achieved by hepatic overexpression of VLDLR combined with a low-fat diet regimen induces regression of advanced plaques in the brachiocephalic artery of LDL receptor-deficient mice.

Oleate and linoleate enhance the growth-promoting effects of insulin-like growth factor-I through a phospholipase D-dependent pathway in arterial smooth muscle cells.

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.