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Background and Aims: Two weight gain prevention strategies, one targeting small changes to diet and physical activity and a second targeting large changes, significantly reduced weight gain in young adulthood. We examined whether weight gain prevention blunts genetic risk for body weight increase and/or high density lipoprotein cholesterol (HDL-C) lowering over two years., Methods and Results: Participants were 524 male and female young adults (mean age = 28.2, SD = 4.3; mean BMI = 25.5, SD = 2.6). Obesity-related SNPs accounting for ≥ 0.04% of the variance were genotyped and combined into a genetic risk score. For HDL-C, SNPs within CETP, LIPC and FADS2 were genotyped. The obesity-related genetic risk score did not predict change in BMI independently or in interaction with treatment arm. However, consistent with the prior literature, each copy of the HDL-C risk, C, allele at CETP rs3764261 was associated with lower HDL-C at baseline. Moreover, significant interaction between SNP and treatment arm for change in HDL-C was observed (p = 0.02). In the control group, HDL-C change was dependent upon rs3764261 (p = 0.004) with C allele carriers showing a continued reduction in HDL-C. In contrast, within the two intervention groups, HDL-C increased on average with no differential effect of rs3764261 (p > 0.24). Notably, even among carriers of the CC genotype, small and large change arms were associated with increased HDL-C and the control arm a reduction (p = 0.013)., Conclusions: The C allele at CETP rs3764261 is a strong risk factor for low HDL-C in young adulthood but weight gain prevention may mitigate this risk. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE: clinicaltrials.gov Identifier: NCT01183689, https://clinicaltrials.gov/., (Copyright © 2018 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Heme oxygenase (HO)-1 is a stress response protein that is regulated by oxidative stress. HO-1 catalyzes the generation of biliverdin, carbon monoxide, and iron from heme. Lipopolysaccharide (LPS) and interleukin (IL)-1beta induce HO-1 through the binding of nuclear proteins to AP-1 motifs in enhancer regions upstream from the transcription start site. The DNA binding activity of AP-1 proteins depends on the reduction of cysteines in their DNA-binding domains. We found that agents that disrupt free sulfhydryl groups abolish AP-1 binding activity in nuclear proteins obtained from rat aortic smooth muscle cells and macrophages stimulated with IL-1beta or LPS. Thioredoxin (TRX) may regulate the redox status of nuclear transcription factors in response to oxidative stimuli, thus we determined the role of TRX in the physiologic regulation of HO-1. TRX underwent nuclear translocation in cells stimulated with IL-1beta and LPS. We transfected macrophages with a heterologous promoter construct containing two AP-1 sites from an upstream enhancer region in the HO-1 promoter. Recombinant TRX induced promoter activity to a level analogous to that induced by LPS, and this TRX response was abolished by mutation of the AP-1 sites. An inhibitor of TRX reductase, used to prevent TRX translocation in the reduced state, decreased HO-1 induction by IL-1beta and LPS. These data provide the first evidence that TRX contributes to the induction of HO-1 by inflammatory mediators.

GATA4 is a transcriptional activator of cardiac-restricted promoters and is required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a multizinc finger protein that associates with GATA4 and represses GATA4-dependent transcription. To better understand the transcriptional repressor activity of FOG-2 we performed a functional analysis of the FOG-2 protein. The results demonstrated that 1) zinc fingers 1 and 6 of FOG-2 are each capable of interacting with evolutionarily conserved motifs within the N-terminal zinc finger of mammalian GATA proteins, 2) a nuclear localization signal (RKRRK) (amino acids 736-740) is required to program nuclear targeting of FOG-2, and 3) FOG-2 can interact with the transcriptional co-repressor, C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS). Surprisingly, however, this interaction with C-terminal-binding protein-2 is not required for FOG-2-mediated repression of GATA4-dependent transcription. Instead, we have identified a novel N-terminal domain of FOG-2 (amino acids 1-247) that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent transcription and thereby form the foundation for genetic studies designed to elucidate the role of FOG-2 in cardiac development.

Tricuspid atresia (TA) is a common form of congenital heart disease, accounting for 1-3% of congenital cardiac disorders. TA is characterized by the congenital agenesis of the tricuspid valve connecting the right atrium to the right ventricle and both an atrial septal defect (ASD) and a ventricular septal defect (VSD). Some patients also have pulmonic stenosis, persistence of a left-sided superior vena cava or transposition of the great arteries. Most cases of TA are sporadic, but familial occurrences with disease in multiple siblings have been reported. Gata4 is a zinc-finger transcription factor with a role in early cardiac development. Gata4-deficient mice fail to form a ventral heart tube and die of circulatory failure at embryonic day (E) 8.5 (refs 6,7). Zfpm2 (also known as Fog-2) is a multi-zinc-finger protein that is co-expressed with Gata4 in the developing heart beginning at E8.5 (refs 8-10). Zfpm2 interacts specifically with the N-terminal zinc finger of Gata4 and represses Gata4-dependent transcription. Here we use targeted mutagenesis to explore the role of Zfpm2 in normal cardiac development. Zfpm2-deficient mice died of congestive heart failure at E13 with a syndrome of tricuspid atresia that includes an absent tricuspid valve, a large ASD, a VSD, an elongated left ventricular outflow tract, rightward displacement of the aortic valve and pulmonic stenosis. These mice also display hypoplasia of the compact zone of the left ventricle. Our findings indicate the importance of Zfpm2 in the normal looping and septation of the heart and suggest a genetic basis for the syndrome of tricuspid atresia.

CD44 is a multifunctional cell adhesion molecule that participates in pathological states such as inflammation and tumorigenesis. CD44 is induced on vascular smooth muscle cells after arterial wall injury and may mediate their proliferation and migration into the neointima during arteriosclerosis. We have demonstrated elsewhere that the proinflammatory cytokine interleukin (IL)-1beta up-regulates CD44 mRNA and protein expression in cultured rat aortic smooth muscle cells (RASMC) by increasing gene transcription. By transient transfection of 5'-deletion constructs into RASMC, we show in the present study that a conserved AP-1 site 110 base pairs from the transcription start site of the mouse CD44 promoter is important for basal activity. Mutation of the AP-1 site significantly reduced induction of promoter activity by IL-1beta, and electrophoretic mobility shift assays demonstrated that Fos and c-Jun were present in the CD44 AP-1 binding complex after IL-1beta stimulation. In addition, cotransfection of the architectural transcription factor high mobility group (HMG)-I(Y) protein with c-Fos and c-Jun markedly increased trans-activation of the CD44 promoter. Taken together, our studies demonstrate that AP-1 proteins are a central regulatory component used by IL-1beta to modulate expression of CD44 during an inflammatory response in vascular smooth muscle cells and that transcription of CD44 by AP-1 proteins is enhanced by HMG-I(Y). -Foster, L. C., Wiesel, P., Huggins, G. S, Pañares, R., Chin, M. T., Pellacani, A., Perrella, M. A. Role of activating protein-1 and high mobility group-I(Y) protein in the induction of CD44 gene expression by interleukin-1beta in vascular smooth muscle cells.

Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arteriosclerosis. Architectural transcription factors of the high mobility group (HMG)-I family have been implicated in the control of cell proliferation and gene expression. We studied the pattern of HMG-I mRNA and protein expression in proliferating VSMCs. HMG-I(Y) and HMGI-C mRNAs were barely detectable by Northern analysis in samples prepared from uninjured rat carotid arteries. In contrast, these mRNAs were induced dramatically in carotid arteries 2 and 5-6 days after balloon injury. By in situ hybridization at 6 days after injury, the induced mRNAs localized to smooth muscle cells of the developing neointima, and immunocytochemical analysis showed that HMG-I(Y) protein was expressed in the nuclei of these cells. To confirm this association between HMG-I protein induction and cell growth, we assessed HMG-I(Y) and HMGI-C mRNA expression in rat aortic smooth muscle cells (RASMCs) in primary culture. The HMG-I mRNAs were barely detectable in quiescent RASMCs but were induced markedly by serum stimulation. This induction of mRNA by serum was time dependent and peaked at 9 h. Western blot analysis confirmed that HMG-I(Y) protein induction also occurred in vitro. To our knowledge, this is the first demonstration of induction of HMG-I protein expression in proliferating RASMCs in vivo and in vitro. This demonstration suggests that the HMG-I proteins may play an important role in smooth muscle cell proliferation., (Copyright 1999 Academic Press.)

Background: We tested the hypothesis that correction of hyperlipidemia improves coronary vasodilator response and maximal perfusion in myocardial regions having substantial impairment of pretreatment vasodilator capacity., Methods and Results: Measurements of myocardial blood flow were made with PET [13N]ammonia in 12 patients with ischemic heart disease (11 men; age, 65+/-8 years [mean+/-SD]) at rest and during adenosine at 70 and then 140 microg . kg-1 . min-1 for 5 minutes each before and approximately 4 months after simvastatin treatment (40 mg daily). Simvastatin reduced LDL (171+/-13 before versus 99+/-18 mg/dL after simvastatin, P<0.001) and increased HDL (39+/-8 versus 45+/-9 mg/dL, P<0.05). Myocardial segments were classified on the basis of pretreatment blood flow response to 140 microg . kg-1 . min-1 adenosine as normal (flow >/=2 mL . min-1 . g-1) or abnormal (flow <2 mL . min-1 . g-1). In normal segments, baseline myocardial blood flow (0.95+/-0.32) increased (P<0.001) at both low- (1.62+/-0.81) and high- (2.63+/-0.41) dose adenosine and was unchanged both at rest and with adenosine after simvastatin. In abnormal segments, myocardial blood flow at rest (0. 73+/-0.19) increased at low- (1.06+/-0.59, P<0.02) and high- (1. 29+/-0.33, P<0.01) dose adenosine. After simvastatin, myocardial blood flow increased more compared with pretreatment at both low- (1. 37+/-0.66, P<0.05 versus pretreatment) and high- (1.89+/-0.79, P<0. 01 versus pretreatment) dose adenosine., Conclusions: Short-term lipid-lowering therapy increases stenotic segment maximal myocardial blood flow by approximately 45%. The mechanism involves enhanced, flow-mediated dilation of stenotic epicardial conduit vessels and may account at least in part for the efficacy of lipid lowering in secondary prevention trials and in reducing ischemic episodes in ambulatory patients.

Heme oxygenase (HO)-1 generates CO, a gas with vasodilatory properties, during heme metabolism. HO-1 is expressed highly in vascular tissue after endotoxin stimulation, and generation of CO through the HO-1 pathway contributes to the hemodynamic compromise of endotoxic shock. Shock related to endotoxemia is an immune-mediated process that involves the generation of proinflammatory cytokines such as interleukin (IL)-1beta. Because transforming growth factor (TGF)-beta1 is a modulator of immune-mediated inflammatory responses and it blocks the hypotension of endotoxic shock, we determined whether TGF-beta1 could be used to reduce expression of HO-1 in vascular tissue and smooth muscle cells. In a rat model of endotoxic shock, lipopolysaccharide-induced HO-1 mRNA and protein expression was reduced by TGF-beta1 in highly vascularized tissue, such as heart and lung, by Northern and Western analysis. Furthermore, TGF-beta1 downregulated HO-1 mRNA after its induction by IL-1beta in vascular smooth muscle cells in culture. TGF-beta1 also decreased HO-1 but not HO-2 protein expression in these cells. TGF-beta1 decreased HO enzyme activity induced in IL-1beta treated vascular smooth muscle cells to a level not different from that in vehicle-treated cells. These studies suggest that this downregulation of HO-1 mRNA and protein expression and decrease in IL-1beta-induced HO enzyme activity may contribute to the beneficial effect of TGF-beta1 on endotoxic shock.