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Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases worldwide. Understanding the effects of cultivar mixture on disease control and population genetic structure of Pst could promote the effective application of cultivar mixtures. This study designed nine cultivar mixtures composed of different component cultivars with field experiments in Zitong County, Sichuan Province in two seasons from 2016 to 2018. Diseased leaves were collected in each plot of treatment in the spring and, in total, 835 and 568 isolates were obtained and genotyped with 10 pairs of simple sequence repeat markers in the 2 years, respectively. The observed and theoretical values of the area under the disease progress curve (AUDPC), wheat yield, Pst genotypic diversity, and the frequency of Pst dominant genetic groups were compared among cultivar mixtures. With the 2 years’ experiments, there were three and two cultivar mixtures, respectively, showing significant effect on the relative reduction of AUDPC; there were no significant effects on the relative yield increase in all mixtures in the 2 years. There were four and five cultivar mixtures showing positive effects on the relative increase of Pst genotypic diversity in the 2 years, respectively. Moreover, all cultivar mixtures could decrease the frequencies of Pst dominant genetic groups on highly susceptible component cultivar compared with their theoretical frequencies. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a destructive wheat disease in China. The Gansu–Ningxia region (GN) is a key area for pathogen over-summering in China, and northwestern Hubei (HB) is an important region for pathogen over-wintering, serving as a source of inoculum in spring epidemic regions. The spatiotemporal population genetic structure of Pst in HB and the pathogen population exchanges between GN and HB are important for estimating the risk of interregional epidemics. Here, 567 isolates from GN and HB were sampled from fall 2016 to spring 2018 and were genotyped using simple sequence repeat markers. The genotypic and genetic diversity of Pst subpopulations in HB varied among seasons and locations. Greater genetic diversification levels were found in the spring compared with fall populations using principal coordinate analysis and Bayesian assignments. In total, there were 17 common genotypes among the 208 determined, as shown by a small overlap of genotypes in the principal coordinate analysis and dissimilar Bayesian assignments in both regions, which revealed the limited genotype exchange between the populations of GN and HB.

In this work, carboxylated carbon dots (C-CDs) with higher oxidation degrees or hydroxylated CDs (H-CDs) with lower oxidation degrees were applied to fabricate CDs capped silver nanoparticles (denoted as C-CDs@AgNPs and H-CDs@AgNPs, respectively) via a one-step strategy. These two types of as-prepared AgNPs have similar size, morphology and surface charge. Interestingly, both AgNPs exhibit a higher toxicity against Gram-negative bacteria Escherichia coli (E. coli) than against Gram-positive bacteria Staphylococcus aureus (S. aureus), and the AgNPs decorated with H-CDs with lower oxidation degrees can eliminate both bacteria more effectively. Subsequently, the possible reason for this fascinating phenomenon has been explored clearly in the perspective of nano/cell interface interactions, which has rarely been considered in detail in previous work. The species specific antibacterial activity can be understood by the typical structural difference of the two bacteria, i.e. the presence of hydrophilic lipopolysaccharides (LPS) which facilitate the adsorption of AgNPs onto the surface of E. coli and the absence of hydrophilic LPS and the existence of thicker peptidoglycan (PG) on S. aureus which prevent the adsorption of AgNPs. Interestingly, the adsorption capacity of H-CDs@AgNPs towards E. coli is also much larger than that of C-CDs@AgNPs, while their different abilities to eliminate S. aureus can be interpreted by the faster leakage rate of Ag+ from the H-CDs@AgNPs. The above adsorption capacity has been quantified through the association constants between AgNPs and LPS or PG determined by UV-vis absorption spectroscopy. The above mechanism can also be illustrated by the microscopic analysis and permeability changes of the two bacteria induced by the AgNPs. Our results may provide valuable insights into optimization of other AgNPs for security and practical disinfection application.

To comprehensively annotate miRNAs and their targets in tea plant, Camellia sinensis, we sequenced small and messenger RNAs of 9 samples of Camellia sinensis var. assamica (YK-10), a diploid elite cultivar widely grown in southwest China. In order to identify targets of miRNAs, we sequenced two degradome sequencing profiles from leaves and roots of YK-10, respectively. By analyzing the small RNA-Seq profiles, we newly identified 137 conserved miRNAs and 23 species specific miRNAs in the genome of YK-10, which significantly improved the annotation of miRNAs in tea plant. Approximately 2000 differently expressed genes were identified when comparing RNA-Seq profiles of any two of the three organs selected in the study. Totally, more than 5000 targets of conserved miRNAs were identified in the two degradome profiles. Furthermore, our results suggest that a few miRNAs play roles in the biosynthesis pathways of theanine, caffeine and flavonoid. These results enhance our understanding of small RNA guided gene regulations in different organs of tea plant.

MicroRNAs (miRNAs) are small non-coding RNAs that are critical in post-transcriptional regulation. Macaca mulatta is an important nonhuman primate that is often used in basic and translational researches. However, the annotation of miRNAs in Macaca mulatta is far from complete, and there are no reports of miRNA editing events in Macaca mulatta, although editing may affect the biogenesis or functions of the miRNAs. To improve miRNA annotation and to reveal editing events of miRNAs in Macaca mulatta, we generated 12 small RNA profiles from eight tissues and performed comprehensive analysis of these profiles. We identified 479 conserved pre-miRNAs that have not been reported in Macaca mulatta and 17 species specific miRNAs. Furthermore, we identified 3386 editing sites with significant editing levels from 471 pre-miRNAs after analyzing the 12 self-generated and 58 additional published sRNA-seq profiles from 17 different types of organs or tissues. In addition to 16 conserved A-to-I editing sites, we identified five conserved C-to-U editing sites in miRNAs of Macaca mulatta and Homo sapiens. We also identified 11 SNPs in the miRNAs of Macaca mulatta. The analysis of the potential targets of 69 miRNAs with editing or mutation events in their seed regions suggest that these editing or mutation events severely changed their targets and their potential functions. These results significantly increase our understanding of miRNAs and their mutation/editing events in Macaca mulatta.

Background MicroRNAs (miRNAs) are small non-coding RNAs that play important roles by regulating other genes. Rosa rugosa Thunb. is an important ornamental and edible plant, yet there are only a few studies on the miRNAs and their functions in R. rugosa. Results We sequenced 10 samll RNA profiles from the roots, petals, pollens, stamens, and leaves and 4 RNA-seq profiles in leaves and petals to analysis miRNA, phasiRNAs and mRNAs in R. rugosa. In addition, we acquired a degradome sequencing profile from leaf of R. rugosa to identify miRNA and phasiRNA targets using the SeqTar algorithm. We have identified 321 conserved miRNA homologs including primary transcripts for 25 conserved miRNAs, and 22 novel miRNAs. We identified 592 putative targets of the conserved miRNAs or tasiRNAs that showed significant accumulations of degradome reads. We found differential expression patterns of conserved miRNAs in five different tissues of R. rugosa. We identified three hundred and thirty nine 21 nucleotide (nt) PHAS loci, and forty nine 24 nt PHAS loci, respectively. Our results suggest that miR482 triggers generations of phasiRNAs by targeting nucleotide-binding, leucine-rich repeat (NB-LRR) disease resistance genes in R. rugosa. Our results also suggest that the deregulated genes in leaves and petals are significantly enriched in GO terms and KEGG pathways related to metabolic processes and photosynthesis. Conclusions These results significantly enhanced our knowledge of the miRNAs and phasiRNAs, as well as their potential functions, in R. rugosa. Electronic supplementary material The online version of this article (10.1186/s12864-018-5325-2) contains supplementary material, which is available to authorized users.

Fibrosis is emerging as a hallmark of metabolically dysregulated white adipose tissue (WAT) in obesity. Although adipose tissue fibrosis impairs adipocyte plasticity, little is known about how aberrant extracellular matrix (ECM) remodeling of WAT is initiated during the development of obesity. Here we show that treatment with the antidiabetic drug metformin inhibits excessive ECM deposition in WAT of ob/ob mice and mice with diet-induced obesity, as evidenced by decreased collagen deposition surrounding adipocytes and expression of fibrotic genes including the collagen cross-linking regulator LOX. Inhibition of interstitial fibrosis by metformin is likely attributable to the activation of AMPK and the suppression of transforming growth factor-β1 (TGF-β1)/Smad3 signaling, leading to enhanced systemic insulin sensitivity. The ability of metformin to repress TGF-β1-induced fibrogenesis is abolished by the dominant negative AMPK in primary cells from the stromal vascular fraction. TGF-β1-induced insulin resistance is suppressed by AMPK agonists and the constitutively active AMPK in 3T3L1 adipocytes. In omental fat depots of obese humans, interstitial fibrosis is also associated with AMPK inactivation, TGF-β1/Smad3 induction, aberrant ECM production, myofibroblast activation, and adipocyte apoptosis. Collectively, integrated AMPK activation and TGF-β1/Smad3 inhibition may provide a potential therapeutic approach to maintain ECM flexibility and combat chronically uncontrolled adipose tissue expansion in obesity.

Agricultural runoff is a major contributor of nutrients to downstream freshwater and marine waters that, in turn, cause harmful algal blooms. “Wetlaculture,” a new landscape management approach defined as “integrating wetland nutrient removal with recycling of these nutrients to agriculture,” has the dual goals of reducing the need for fertilizer in agriculture while mitigating fluxes of nutrients to downstream aquatic ecosystems with created or restored wetlands. A mesocosm compound consisted of twenty-eight 380 L, 1-m2 tubs filled with local hydric soil was initiated in mid-2016 near a highly eutrophic Buckeye Lake in central Ohio, USA, to investigate the wetlaculture approach in an experimental fashion. In October 2016, each mesocosm tub was inserted in the ground and planted with the sedge Schoenoplectus tabernaemontani. The mesocosms were assigned to four hydrologic treatments involving two water depths and two hydraulic loading rates (HLRs). Nearby river water containing significant agricultural runoff was added weekly into a water feed tank system to provide weekly hydraulic loadings to the mesocosms. Inflow and outflow water samples from each wetland mesocosm were collected and analyzed for five species of nitrogen and phosphorus. By August 2019, 17 plant species, including the planted S. tabernaemontani, were present in the wetland mesocosms. The wetland mesocosms soon became nutrient sinks with average removal efficiencies of total phosphorus (TP) and total nitrogen (TN) of 38 ± 2.5% and 44 ± 1%, respectively, through the three-year experiment. The combination of a high HLR (30 cm week−1) and 10 cm of standing water achieved the best nitrogen removal efficiencies while highest phosphorus removal occurred with the combination of a high HLR (30 cm week−1) and no standing water. This paper discusses the following integrative findings: • Seasonal flooding had positive impacts on removal efficiency of NOx and a negative impact on the removal efficiency of TP and TKN. • No standing water resulted in a higher removal efficiency of TP and TKN than standing water, results consistent with previous studies. • Macrophyte plant community richness was influenced more by water depth than by hydraulic loading rate. Wetlands with 10 cm of standing water instead of saturated soils developed lower species richness but were more dominated by true wetland (OBL and FACW) species. • Study scale does matter. Annual nutrient accumulation rates in the mesocosms (1 m2) soils were lower than in the full-scale (10,000-m2) multi-year wetlands at the nearby Olentangy River Wetland Research Park (ORWRP) in central Ohio. Annual accumulation rates of phosphorus and nitrogen in the full-scale wetlands ranged from 3.3 to 3.5 g-P m−2 yr−1 and 16.4 to 19.5 g-N m−2 yr−1, while in our mesocosms, the annual rates ranged from 0.66 to 1.40 g-P m−2 yr−1 and 7.85 to 15.08 g-N m−2 yr−1. Our experimental wetland mesocosms could only function in the non-freezing months and have been in operation for only three years.

This study focused on Puccinia striiformis f. sp. tritici (Pst), the causal pathogen of wheat stripe rust in China. The central and eastern China are the main wheat-producing areas in the country, and the occurrence of wheat stripe rust can cause severe yield losses. To determine the population genetic structure and potential pathways of migration among regional populations of the pathogen, 264 isolates were sampled during spring 2017 and genotyped using 12 simple sequence repeat (SSR) loci. The highest genetic diversity was detected in Hubei Province, indicating its potential role as one of the source populations for the rust epidemics. A high gene flow was detected among regional populations, supporting migration of Pst among regions. The significant decrease in genotypic and genetic diversity from Hubei to Shandong suggested that the migration direction was from central to eastern China. Analysis of molecular variance (AMOVA), Bayesian assignment, and principle coordinate analysis all indicated two major groups, one was mainly distributed in Hubei and the another was mainly in the eastern region. Shared genotypes, gene flow among regions, and Bayesian assignment tests revealed that the migration of Pst from central to eastern of China during spring occurred mainly through stepwise movement.

Activation of thromboxane receptors (TPr) may promote atherosclerosis by enhancing oxidative stress and the inflammation. This study examined the role of Nox1, an NADPH-oxidase subunit, in the enhancement of interleukin (IL)-1β-induced monocyte adhesion by TPr. In cultured rat aortic vascular smooth muscle cells (VSMCs), U46619, a stable thromboxane A2 mimetic, together with interleukin-1β significantly enhanced Nox1 mRNA expression, as well as adhesion of THP-1 monocytes. Activation of TPr also enhanced IL-1β-induced vascular cell adhesion molecule (VCAM)-1 expression, but inhibited inducible nitric oxide synthase (iNOS) expression. Silencing Nox1 expression by siRNA prevented the U46619 enhancement of IL-1β-induced monocyte adhesion, but had no significant effect on VCAM-1 or iNOS expression. Furthermore, monocyte adhesion was inhibited by superoxide dismutase, enhanced by a specific iNOS inhibitor, L-N6-(1-iminoethyl)-lysine, but not influenced by catalase. U46619 inhibited IL-1β-induced cyclic GMP production, and the inhibition was partially prevented by superoxide dismutase. In conclusion, activation of TPr enhances IL-1β-induced Nox1 expression in VSMCs, which is responsible for the up-regulation of monocyte adhesion. The effect of Nox1 is independent of the changes in VCAM-1 and iNOS expression, but depends upon the inactivation of nitric oxide via generation of superoxide anion.

Elevated plasma homocysteine (Hcy) is a risk factor for cardiovascular disease. While Hcy has been shown to promote endothelial dysfunction by decreasing the bioavailability of nitric oxide and increasing oxidative stress in the vasculature, the effects of Hcy on cardiomyocytes remain less understood. In this study we explored the effects of hyperhomocysteinemia (HHcy) on myocardial function ex vivo and examined the direct effects of Hcy on cardiomyocyte function and survival in vitro. Studies with isolated hearts from wild type and HHcy mice (heterozygous cystathionine-beta synthase deficient mice) demonstrated that HHcy mouse hearts had more severely impaired cardiac relaxation and contractile function and increased cell death following ischemia reperfusion (I/R). In isolated cultured adult rat ventricular myocytes, exposure to Hcy for 24 hours impaired cardiomyocyte contractility in a concentration-dependent manner, and promoted apoptosis as revealed by terminal dUTP nick-end labeling and cleaved caspase-3 immunoblotting. These effects were associated with activation of p38 MAPK, decreased expression of thioredoxin (TRX) protein, and increased production of reactive oxygen species (ROS). Inhibition of p38 MAPK by the selective inhibitor SB203580 (5 µM) prevented all of these Hcy-induced changes. Furthermore, adenovirus-mediated overexpression of TRX in cardiomyocytes significantly attenuated Hcy-induced ROS generation, apoptosis, and impairment of myocyte contractility. Thus, Hcy may increase the risk for CVD not only by causing endothelial dysfunction, but also by directly exerting detrimental effects on cardiomyocytes.

The development of cardiovascular fibrosis is associated with chronic inflammation, where activation of nuclear factor κB (NF-κB) signaling may play a critical role. NF-κB activation is tightly regulated by the cellular inhibitor of κB (IκB) family of proteins, such as IκBα and IκBβ. IκBα and IκBβ display different regulation kinetics in response to inflammatory stimulation. The present study tested the hypothesis that IκBα and IκBβ may have different roles in modulating cardiovascular inflammation and fibrosis, using a model of angiotensin II infusion-induced hypertension in wild-type mice and IκBβ knock-in mice, in which the IκBα gene is replaced by IκBβ cDNA (AKBI). In WT mice, subcutaneous angiotensin II infusion for 7 days induced increased perivascular and interstitial collagen deposition and fibrotic lesions, associated with myocardial interstitial hemosiderin accumulation and extensive macrophage infiltration. These effects of angiotensin II were dramatically limited in AKBI mice. Replacement of IκBα with IκBβ significantly attenuated angiotensin II infusion–induced expression of interleukin 1β, interleukin 6, monocyte chemotactic protein 1, collagen I and III, fibronectin, and tissue inhibitor of metalloproteinase 1 in the hearts. Furthermore, using cultured vascular smooth muscle cells, we demonstrated that interleukin 1β–induced NF-κB activation and monocyte chemotactic protein 1, vascular cell adhesion molecule 1, and tissue inhibitor of metalloproteinase 1 expressions were suppressed in the AKBI cells because of the replacement of IκBα with IκBβ. These results indicate that NF-κB has an essential role in mediating the cardiovascular inflammatory response to angiotensin II and suggest that targeting the balance of IκBα and IκBβ expression might be a novel therapeutic modality in preventing fibrosis in hypertensive cardiovascular disease.

Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1β-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-κB, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1β-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1β, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE(-/-)) mice. VCAM-1 and iNOS expression were higher in ApoE(-/-) than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE(-/-) mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells through influencing ERK-NF-κB crosstalk, which may contribute to angiotensin II-induced inflammatory disorders related to cardiovascular diseases.

Patients with primary (AL) cardiac amyloidosis suffer from progressive cardiomyopathy with a median survival of less than 8 months and a 5-year survival of

The sarcoplasmic reticulum Ca2+ ATPase (SERCA) is redox-regulated by post-translational thiol modifications of cysteine-674 to regulate smooth muscle relaxation and migration. To detect oxidation of cysteine-674 that irreversibly prevents redox-regulation, a polyclonal, sequence-specific antibody was developed towards a peptide containing cysteine-674 sulfonic acid. The antibody stained intact 110 kDa SERCA in pig cardiac SR that was oxidized in vitro by peroxynitrite in a sequence-specific manner, and histochemically stained atherosclerotic pig and rabbit aorta. Surprisingly, immuno-blots of the pig aorta failed to stain intact 110 kDa SERCA protein, but rather, higher molecular mass aggregates and lower molecular mass bands. Of the latter bands at 70 and 60 kDa, the largest were observed in diabetic, hyperlipidemic pigs, and coincided with the most positive histochemical staining. The 70 and 60 kDa molecular mass bands also coincided with the majority of the protein detected by a monoclonal total anti-SERCA antibody, which detected the intact 110 kDa protein in normal pigs. Mass spectrometry identified SERCA in all the major bands detected by the sulfonic acid antibody as well as the oxidation of cysteine-674 in the 70 kDa band. These studies demonstrate a sequence-specific antibody that detects partial degradation products of SERCA, which represent the majority of the protein in some diabetic hypercholesterolemic pig aortae. In addition, the results suggest an association between irreversible oxidation of SERCA and its degradation, and that an important portion of the oxidized protein in tissue samples may be partially degraded.

Activation of NF-κB requires the phosphorylation and degradation of its associated inhibitory proteins, IκB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1β to induce persistent activation of NF-κB in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-κB activation. In cultured rat VSMCs, IL-1β activated ERK and induced degradation of both IκBα and IκBβ, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-κB p65. RSK1, a downstream kinase of ERK, was associated with an IκBβ/NF-κB complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1β decreased IκBβ in the RSK1/IκBβ/NF-κB complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1β-induced IκBβ decrease without influencing ether ERK phosphorylation or the earlier IκBα degradation. By using recombinant wild-type and mutant IκBβ proteins, both active ERK2 and RSK1 were found to directly phosphorylate IκBβ, but only active RSK1 phosphorylated IκBβ on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IκBβ and contributes to the persistent activation of NF-κB by IL-1β.

Although reactive oxygen species (ROS) appear to play a central role in mediating myocardial hypertrophy in response to hemodynamic overload, little is known about the molecular targets by which ROS regulate growth signaling. In cardiac myocytes, we tested the hypothesis that mechanical strain causes cellular hypertrophy via ROS-dependent post-translational modification of Ras leading to activation of the Raf/Mek/Erk growth pathway. Cyclic mechanical strain increased Ras activity by 1.5 to 1.6-fold. Adenoviral overexpression of the N17 dominant negative mutant of Ras inhibited strain-stimulated Erk activation and protein synthesis. Strain-stimulated Ras activation was inhibited by overexpression of catalase, indicating that it is redox-dependent. Strain caused S-glutathiolation of Ras, which was inhibited by catalase overexpression and reversed by DTT. MALDI-TOF mass spectrometry demonstrated that in myocytes subjected to strain there was S-glutathiolation of Ras at Cys118. Adenoviral overexpression of a mutated Ras in which Cys118 was substituted with serine inhibited strain-stimulated S-glutathiolation of Ras, Erk activation and protein synthesis. Overexpression of glutaredoxin-1 likewise inhibited strain-stimulated Ras S-glutathiolation, Ras activation, Erk activation and protein synthesis. These findings indicate that mechanical strain causes ROS-dependent S-glutathiolation of Ras at Cys118, leading to myocyte hypertrophy via activation of the Raf/Mek/Erk pathway.

Arachidonic acid metabolites, some of which may activate thromboxane A 2 receptors (TPr) and contribute to the development of diabetes complications, including nephropathy, are elevated in diabetes. This study determined the effect of blocking TPr with S18886 or inhibiting cyclooxygenase with aspirin on oxidative stress and the early stages of nephropathy in streptozotocin-induced diabetic apolipoprotein E −/− mice. Diabetic mice were treated with S18886 (5 mg · kg −1 · day −1 ) or aspirin (30 mg · kg −1 · day −1 ) for 6 weeks. Neither S18886 nor aspirin affected hyperglycemia or hypercholesterolemia. There was intense immunohistochemical staining for nitrotyrosine in diabetic mouse kidney. In addition, a decrease in manganese superoxide dismutase (MnSOD) activity was associated with an increase in MnSOD tyrosine-34 nitration. Tyrosine nitration was significantly reduced by S18886 but not by aspirin. Staining for the NADPH oxidase subunit p47 phox , inducible nitric oxide synthase, and 12-lipoxygenase was increased in diabetic mouse kidney, as were urine levels of 12-hydroxyeicosatetraenoic acid and 8-iso-prostaglandin F 2α . S18886 attenuated all of these markers of oxidant stress and inflammation. Furthermore, S18886 significantly attenuated microalbuminuria in diabetic mice and ameliorated histological evidence of diabetic nephropathy, including transforming growth factor-β and extracellular matrix expression. Thus, in contrast to inhibiting cyclooxygenase, blockade of TPr may have therapeutic potential in diabetic nephropathy, in part by attenuating oxidative stress.

Background— S18886 is an orally active thromboxane A 2 (TXA 2 ) receptor (TP) antagonist in clinical development for use in secondary prevention of thrombotic events in cardiovascular disease. We previously showed that S18886 inhibits atherosclerosis in apolipoprotein E–deficient (apoE −/− ) mice by a mechanism independent of platelet-derived TXA 2 . Atherosclerosis is accelerated by diabetes and is associated with increased TXA 2 and other eicosanoids that stimulate TP. The purpose of this study was to determine whether S18886 lessens the enhanced atherogenesis in diabetic apoE −/− mice. Methods and Results— Diabetes mellitus was induced in apoE −/− mice with streptozotocin and was treated or not with S18886 (5 mg · kg −1 · d −1 ). After 6 weeks, aortic lesion area was increased >4-fold by diabetes in apoE −/− mice, associated with similar increases in serum glucose and cholesterol. S18886 largely prevented the diabetes-related increase in lesion area without affecting the hyperglycemia or hypercholesterolemia. S18886 prevented deterioration of endothelial function and endothelial nitric oxide synthase expression, as well as increases in intimal markers of inflammation associated with diabetes. In human aortic endothelial cells in culture, S18886 also prevented the induction of vascular cell adhesion molecule-1 and prevented the decrease in endothelial nitric oxide synthase expression caused by high glucose. Conclusions— The TP antagonist inhibits inflammation and accelerated atherogenesis caused by diabetes, most likely by counteracting effects on endothelial function and adhesion molecule expression of eicosanoids stimulated by the diabetic milieu.

Angiotensin II is implicated in pathophysiological processes associated with vascular injury and repair, which include regulating the expression of numerous NF-κB-dependent genes. The present study examined the effect of angiotensin II on interleukin-1β-induced NF-κB activation and the subsequent expression of inducible NO synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) in cultured rat vascular smooth muscle cells. Neither NF-κB activation nor iNOS or VCAM-1 expression was induced in cells treated with angiotensin II alone. However, when added together with interleukin-1β, angiotensin II, through activation of the AT1 receptor, inhibited iNOS expression and enhanced VCAM-1 expression induced by the cytokine. The inhibitory effect of angiotensin II on iNOS expression was associated with a down-regulation of the sustained activation of extracellular signal-regulated kinase (ERK) and NF-κB by interleukin-1β, whereas the effect on VCAM-1 was independent of ERK activation. The effect of angiotensin II on iNOS was abolished by inhibition of p38 mitogen-activated protein kinase (MAPK) with SB203580, but not by inhibition of PI3 kinase with wortmannin or stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) with JNK inhibitor II. Thus, angiotensin II, by a mechanism that requires the participation of p38 MAPK, differentially regulates the expression of NF-κB-dependent genes in response to interleukin-1β stimulation by controlling the duration of activation of ERK and NF-κB.

In cultured rat vascular smooth muscle cells, sustained activation of ERK is required for interleukin-1beta to persistently activate NF-kappaB. Without ERK activation, interleukin-1beta induces only acute and transient NF-kappaB activation. The present study examined whether the temporal control of NF-kappaB activation by ERK could differentially regulate the expression of NF-kappaB-dependent genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1), and manganese-containing superoxide dismutase (Mn-SOD). Treatment of vascular smooth muscle cells with interleukin-1beta induced the expression of iNOS, COX-2, VCAM-1, and Mn-SOD in a time-dependent manner, but with different patterns. Either PD98059 or U0126, selective inhibitors of MEK, or overexpression of a dominant negative MEK-1 inhibited interleukin-1beta- induced ERK activation and the expression of iNOS and COX-2 but had essentially no effect on the expression of VCAM-1 and Mn-SOD. The expression of these genes was inhibited when NF-kappaB activation was down-regulated by MG132, a proteasome inhibitor, or by overexpression of an I-kappaBalpha mutant that prevented both the transient and the persistent activation of NF-kappaB. Inhibition of ERK did not affect interleukin-1beta-induced I-kappaBalpha phosphorylation and degradation but attenuated I-kappaBbeta degradation. Thus, although NF-kappaB activation was essential for interleukin-1beta induction of each of the proteins studied, gene expression was differentially regulated by ERK and by the duration of NF-kappaB activation. These results reveal a novel functional role for ERK as an important temporal regulator of NF-kappaB activation and NF-kappaB-dependent gene expression.

Objective— Activation of extracellular signal-regulated kinases (ERKs) is required for interleukin-1β to persistently activate nuclear factor (NF)-κB and concomitantly express inducible NO synthase (iNOS) in rat vascular smooth muscle cells (VSMCs). The present study examined whether platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) could influence the VSMC response to interleukin-1β via an ERK-related signaling pathway. Methods and Results— Treatment of VSMCs with PDGF or EGF alone potently induced ERK phosphorylation and DNA synthesis but did not induce NF-κB activation or iNOS expression. However, either PDGF or EGF markedly enhanced interleukin-1β-induced persistent NF-κB activation and iNOS expression but did not affect the early and transient NF-κB activation. Growth factor-induced DNA synthesis was attenuated in the presence of interleukin-1β. Inhibition of ERK phosphorylation with selective inhibitors (PD98059 or U0126) attenuated interleukin-1β-induced persistent NF-κB activation and iNOS expression in either the absence or presence of the growth factors. Conclusions— These results indicate that interleukin-1β-induced expression of NF-κB-dependent genes, such as iNOS, is potentiated in the presence of growth factors through a mechanism requiring ERK-dependent enhanced NF-κB activation, and the results also suggest that NF-κB activation is not required for PDGF or EGF to trigger DNA synthesis in VSMCs.

Abstract — The role of extracellular signal-regulated kinase (ERK) was studied in the signaling pathway by which interleukin-1β (IL-1β) increases the expression of inducible NO synthase (iNOS) in rat vascular smooth muscle cells. IL-1β induced a rapid and transient activation of nuclear factor-κB (NF-κB), followed by a prolonged activation of NF-κB that was required to induce iNOS expression. Either PD98059 or U0126, selective inhibitors of ERK activation, did not influence IL-1β-induced early activation but effectively reduced the prolonged activation of NF-κB and significantly reduced IL-1β induction of iNOS. Transfection with antisense, but not sense, phosphorothioate-modified oligodeoxynucleotides directed toward ERK also reduced IL-1β-induced prolonged NF-κB activation and iNOS expression. IκBβ, but not IκBα degradation, induced by IL-1β was markedly attenuated when ERK activation was inhibited and could be partially responsible for the persistent NF-κB activation. These data suggest that ERK activity is required for persistent NF-κB activation by IL-1β that is necessary for iNOS gene expression.

Abstract —We have reported previously that N -acetyl- l -cysteine facilitated interleukin-1β–induced nitric oxide synthase (iNOS) expression in rat vascular smooth muscle cells. The present study compares the effect of N -acetyl- l -cysteine with other antioxidants and tested the possibility that N -acetyl- l -cysteine potentiates iNOS induction by a mechanism involving activation of p44/42 mitogen-activated protein kinases (MAPKs). The effect of N -acetyl- l -cysteine on potentiating interleukin-1β–induced nitrite production and iNOS expression was mimicked either by the enantiomers, l -cysteine and d -cysteine, or by a non–thiol-containing antioxidant, l -ascorbic acid. Interleukin-1β activated p44/42 MAPK, and this activation was enhanced in the presence of N -acetyl- l -cysteine. Inhibition of p44/42 MAPK phosphorylation by the selective inhibitor PD98059 clearly inhibited iNOS expression induced by interleukin-1β either in the absence or in the presence of N -acetyl- l -cysteine. These observations, combined with previous results, indicate that p44/42 MAPK activation is required for interleukin-1β induction of iNOS and that N -acetyl- l -cysteine may act as a reducing agent and facilitate interleukin-1β–induced iNOS expression through a reduction/oxidation-related mechanism involving potentiation of cytokine activation of the p44/42 MAPK signaling pathway.

Abstract The effect of parathyroid hormone–related protein on interleukin-1β–induced nitric oxide production was studied in rat vascular smooth muscle cells. Interleukin-1β time- and dose-dependently enhanced the production of nitrite, a stable metabolite of nitric oxide. Parathyroid hormone–related protein(1-34) alone up to 10 −7 mol/L had no obvious effect, but significantly increased the cytokine-induced nitrite production. RNA analysis revealed that the synergistic effect of parathyroid hormone–related protein(1-34) resulted from a potentiation of the expression of inducible nitric oxide synthase and GTP-cyclohydrolase I, the rate-limiting enzyme in the synthesis of tetrahydrobiopterin, which is a cofactor of nitric oxide synthase. The increased nitric oxide release induced by interleukin-1β or interleukin-1β with parathyroid hormone–related protein(1-34) was completely inhibited by coincubation with 3×10 −3 mol/L N G -monomethyl-l-arginine, a competitive inhibitor of nitric oxide synthase, or with 10 −3 mol/L 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP-cyclohydrolase I. Endothelin-1 potentiated interleukin-1β induction of nitric oxide, which might be mediated by endogenous parathyroid hormone–related protein. Neutralization of exogenous or endogenous parathyroid hormone–related protein with antibody attenuated the synergistic effect of parathyroid hormone–related protein, but did not affect interleukin-1β induction of nitric oxide. These results suggest that locally produced parathyroid hormone–related protein acts as a synergistic regulator upregulating interleukin-1β–induced nitric oxide synthesis in the cardiovascular system, and thereby may affect vascular tone and/or vascular remodeling after vascular injury in some pathological processes such as atherosclerosis and hypertension.

Background Aberrantly elevated sterol regulatory element binding protein (SREBP), the lipogenic transcription factor, contributes to the development of fatty liver and insulin resistance in animals. Our recent studies have discovered that AMP-activated protein kinase (AMPK) phosphorylates SREBP at Ser-327 and inhibits its activity, represses SREBP-dependent lipogenesis, and thereby ameliorates hepatic steatosis and atherosclerosis in insulin-resistant LDLR−/− mice. Chronic inflammation and activation of NLRP3 inflammasome have been implicated in atherosclerosis and fatty liver disease. However, whether SREBP is involved in vascular lipid accumulation and inflammation in atherosclerosis remains largely unknown. Principal Findings The preclinical study with aortic pouch biopsy specimens from humans with atherosclerosis and diabetes shows intense immunostaining for SREBP-1 and the inflammatory marker VCAM-1 in atherosclerotic plaques. The cleavage processing of SREBP-1 and -2 and expression of their target genes are increased in the well-established porcine model of diabetes and atherosclerosis, which develops human-like, complex atherosclerotic plaques. Immunostaining analysis indicates an elevation in SREBP-1 that is primarily localized in endothelial cells and in infiltrated macrophages within fatty streaks, fibrous caps with necrotic cores, and cholesterol crystals in advanced lesions. Moreover, concomitant suppression of NAD-dependent deacetylase SIRT1 and AMPK is observed in atherosclerotic pigs, which leads to the proteolytic activation of SREBP-1 by diminishing the deacetylation and Ser-372 phosphorylation of SREBP-1. Aberrantly elevated NLRP3 inflammasome markers are evidenced by increased expression of inflammasome components including NLPR3, ASC, and IL-1β. The increase in SREBP-1 activity and IL-1β production in lesions is associated with vascular inflammation and endothelial dysfunction in atherosclerotic pig aorta, as demonstrated by the induction of NF-κB, VCAM-1, iNOS, and COX-2, as well as by the repression of eNOS. Conclusions These translational studies provide in vivo evidence that the dysregulation of SIRT1-AMPK-SREBP and stimulation of NLRP3 inflammasome may contribute to vascular lipid deposition and inflammation in atherosclerosis.

The effects of CV-11974, a potent nonpeptide antagonist of the angiotensin II (AII) type-1 receptor (AT1), on cytosolic free calcium concentration ([Ca2+]i), hyperplasia, and hypertrophy of cultured vascular smooth muscle cells (VSMC) from rat aorta were studied. [Ca2+]i was measured by fura 2, and hyperplasia and hypertrophy were determined by incorporation of [3H]thymidine and [3H]leucine, respectively. CV-11974 had no effect on [Ca2+]i itself, but suppressed 10(-7) M AII-induced increase in [Ca2+]i dose dependently at concentrations from 10(-10) M and completely at 10(-7) M. CV-11974 suppressed both Ca2+ release from intracellular Ca2+ stores and Ca2+ influx from the extracellular space. However, CV-11974 had no effect on the increases in [Ca2+]i induced by prostaglandin F2 alpha (PGF2 alpha), a potent vasoconstrictor, or ionomycin, a Ca2+ ionophore. These results indicate that the suppressive effects of CV-11974 act on the binding of AII and its specific receptors. AII 10(-7) M increased the synthesis of DNA and protein to 1.5 and 1.7 times the control values, respectively. CV-11974 had no effect on synthesis of DNA or protein, but suppressed the AII-stimulated synthesis of DNA and protein dose dependently at concentrationsor = 10(-8) and 10(-10) M, respectively and completely at 10(-6) M. These results indicate that AII increases [Ca2+]i and synthesis of DNA and protein in VSMC through activation of AT1. CV-11974 showed no partial agonistic effects on AII. Thus, CV-11974 may act not only as an antihypertensive agent, but also as an inhibitor of vascular injury stimulated by AII.

To define the physiological roles of elastase in the vascular wall, we examined whether elastase at low concentrations can modulate the proliferation of vascular smooth muscle cells (VSMC). Elastase itself at low concentrations from 1 to 50 ng/ml inhibited DNA synthesis dose-dependently in VSMC. However, phenylmethylsulfonyl fluoride-inactivated elastase failed to induce this inhibition. OP-41483, a stable analogue of prostacyclin, inhibited DNA synthesis and stimulated accumulation of cAMP in VSMC. Preincubation of VSMC for 24 h with 50 ng/ml elastase enhanced both inhibition of DNA synthesis and the accumulation of cAMP induced by OP-41483. Preincubation of VSMC with 12-O-tetradecanoylphorbol 13-acetate, an activator of protein kinase C (PKC), also enhanced cAMP accumulation induced by OP-41483. On the other hand, elastase failed to enhance OP-41483-induced cAMP accumulation in PKC down-regulated cells. Furthermore, coincubation with chelerythrine, an inhibitor of PKC, inhibited the enhancement of cAMP accumulation induced by preincubation with elastase. These results suggest that elastase at low concentrations can enhance the inhibition of VSMC proliferation induced by prostacyclin through the activation of protein kinase C.

Our purpose was to determine if high-fat diet and treatment with a polyphenol regulate the acetylation of lysine-382 of p53, the site regulated by sirtuin-1, and apoptosis in the endothelium of the atherosclerotic lesion-prone mouse aortic arch. In cultured endothelial cells, 2 atherogenic stimuli, hydrogen peroxide and tumor necrosis factor-α, increased the acetylation of p53 lysine-382, and caspase-3 cleavage, an indicator of apoptotic signaling. The polyphenol, S17834, significantly prevented these changes. In low-density lipoprotein receptor-deficient mice, a high-fat diet increased, and treatment with S17834 attenuated early atherosclerotic lesions on the lesser curvature of the aortic arch. In wild-type C57BL6 mice fed the same diet, no atherosclerotic lesions were observed in this lesion-prone area, but p53 acetylation and caspase-3 cleavage increased in the endothelium. In high-fat fed mice, S17834 increased sirtuin-1 protein in the lesion-prone endothelium and prevented both the increase in p53 acetylation and caspase-3 cleavage without affecting blood lipids. These results indicate that high-fat diet increases and S17834 decreases the acetylation of p53 in lesion-prone aortic endothelial cells of normal mice independently of blood lipids, suggesting that the polyphenol may regulate endothelial cell p53 acetylation and apoptosis via local actions.

Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser428, and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKβ, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.

Objective— Activation of thromboxane receptors (TPr) is implicated in atherosclerosis and inflammation. This study examined how activation of TPr modulates IL-1β–induced vascular cell adhesion molecule (VCAM)-1 expression in aortic vascular smooth muscle cells (VSMCs). Methods and Results— In VSMCs, activation of TPr with U46619, a stable thromboxane A 2 mimetic, alone did not induce VCAM-1 expression, but enhanced that caused by IL-1β. The enhancement of VCAM-1 expression caused by U46619 occurred at the transcriptional level and was inhibited either by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, or by overexpression of a dominant-negative JNK1, but not by SB203580, a p38 mitogen-activated protein kinase inhibitor. The activation of JNK by U46619 resulted in enhanced phosphorylation and nuclear translocation of c-Jun associated with an enhanced activation of activator protein (AP)-1, which were abolished by SQ29548, a TPr antagonist, or the JNK inhibitor. Treatment of the cells with U46619 alone did not induce NF-κB activation. Furthermore, U46619 enhanced IL-1β–induced THP-1 monocyte binding to VSMCs, which was inhibited by SQ29548 or SP600125. Conclusion— This study demonstrates that activation of TPr upregulates IL-1β–induced VCAM-1 expression by enhancing the activation of JNK pathway that leads to enhanced AP-1 activation.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. The mechanisms leading to motor neuron degeneration in ALS are unclear. However, there is evidence for involvement of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in ALS, notably in mutant SOD1 mediated models of ALS. Stress induced phosphorylation of the eIF2 alpha subunit by eukaryotic translation initiation factor 2-alpha kinase 3 Perk activates the UPR. Guanabenz is a centrally acting alpha2 adrenergic receptor agonist shown to interact with a regulatory subunit of the protein phosphatase, Pp1/Gadd34, and selectively disrupt the dephosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eif2alpha). Here we demonstrate that guanabenz is protective in fibroblasts expressing G93A mutant SOD1 when they are exposed to tunicamycin mediated ER stress. However, in contrast to other reports, guanabenz treatment accelerated ALS-like disease progression in a strain of mutant SOD1 transgenic ALS mice. This study highlights challenges of pharmacological interventions of cellular stress responses in whole animal models of ALS.

Hypertension caused by angiotensin II is characterized by an increase in tissue oxidant stress as evidenced by increased quantities of reactive oxygen and nitrogen species. Manganese superoxide dismutase (MnSOD) is a key mitochondrial antioxidant enzyme that is inactivated in conditions of oxidant stress by reacting with peroxynitrite to form 3-nitrotyrosine in its active site. The increase in 3-nitrotyrosine content in MnSOD in the kidney of angiotensin II-infused rats was assessed in this study by immunohistochemistry, Western blotting, immunoprecipitation, and HPLC with UV detection (HPLC-UV). MnSOD activity decreased approximately 50% in angiotensin II-infused rat kidneys (24 +/- 4.6 vs. 11 +/- 5.2 U/mg) without a change in protein expression. Immunohistochemical staining showed 3-nitrotyrosine predominantly in distal tubules and collecting duct cells in the angiotensin II-infused rat kidneys. By two-photon microscopy, 3-nitrotyrosine colocalized with MnSOD. Total 3-nitrotyrosine content in kidney homogenates was increased in angiotensin II-infused rat kidney [3.2 +/- 1.9 (sham treated) vs. 9.5 +/- 2.3 ng/mg protein by HPLC-UV detection]. With tracer amounts of tyrosine-nitrated recombinant MnSOD, the most sensitive technique to detect tyrosine nitration of MnSOD was immunoprecipitation from tissue with anti-MnSOD antibody, followed by detection of 3-nitrotyrosine by Western blotting or HPLC. By HPLC, 3-nitrotyrosine content of kidney MnSOD increased 13-fold after angiotensin II infusion, representing an increase from approximately one-twentieth to one-fifth of the total 3-nitrotyrosine content in sham-treated and angiotensin II-infused rat kidney, respectively. Angiotensin II-induced hypertension is accompanied by increased tyrosine nitration of MnSOD, which, because it inactivates the enzyme, may contribute to increased oxidant stress in the kidney.

Abstract —The effect of N -acetyl- l -cysteine on interleukin-1β–induced nitric oxide synthase expression was studied in rat vascular smooth muscle cells to determine if the reduction/oxidation state would modulate cytokine-induced changes. Interleukin-1β induced the production of nitrite, a stable metabolite of nitric oxide in a time- and dose-dependent manner. Cytokine-induced nitrite production was enhanced by the addition of N -acetyl- l -cysteine in a dose-dependent manner, with a >50% increase produced by the addition of 1 mmol/L N- acetyl- l -cysteine. There was no influence on nitrite production when the cells were treated with N -acetyl- l -cysteine alone. Northern and Western blot analyses revealed that the upregulation of interleukin-1β–induced nitric oxide production by N -acetyl- l -cysteine resulted from an enhanced expression of inducible nitric oxide synthase. Interferon-γ or tumor necrosis factor-α when used alone had no influence on nitrite production in the absence or presence of N- acetyl- l -cysteine. Nitrite accumulation was higher by the cells treated with interleukin-1β combined with either interferon-γ or tumor necrosis factor-α compared with those treated with interleukin-1β alone. N -Acetyl- l -cysteine upregulated nitrite production and inducible nitric oxide synthase expression induced by combination treatment with interleukin-1β and either interferon-γ or tumor necrosis factor-α. However, N -acetyl- l -cysteine had no significant influence in cytokine-induced activation of nuclear factor-κB or signal transducer and activator of transciption-1, as assessed by electrophoretic mobility shift assays. These results demonstrate that N- acetyl- l -cysteine possibly acted as a thiol-containing reducing agent and facilitated the expression of inducible nitric oxide synthase in rat vascular smooth muscle cells by cytokines through a mechanism that is independent of nuclear factor-κB or signal transducer and activator of transciption-1.

The parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor has been reported to be expressed in many tissues, including vascular smooth-muscle cells (VSMCs), but it has not been identified in vascular endothelial cells. To determine whether vascular endothelial cells can express the PTH/PTHrP receptor, its gene expression was examined in simian virus 40-transformed rat lung vascular endothelial cells (TRLECs) by the reverse-transcription polymerase chain reaction (RT-PCR). Results in TRLEC, with rat VSMCs and kidney as controls, showed identical 741-bp products. Furthermore, incubation with PTHrP[1-34] reduced the thrombin-stimulated endothelin-1 (ET-1) expression in TRLECs. Our results demonstrate that vascular endothelial cells can express the PTH/PTHrP receptor and therefore are also a target tissue for PTHrP.

Background Inflammatory cytokines play an important role in the pathogenesis of heart failure. We have recently found the cytokine TWEAK (tumor necrosis factor (TNF)-like weak inducer of apoptosis), a member of the TNF superfamily, to be increased in patients with cardiomyopathy and result in the development of heart failure when overexpressed in mice. The molecular mechanisms underlying TWEAK-induced cardiac pathology, however, remain unknown. Methodology and Critical Finding Using mouse models of elevated circulating TWEAK levels, established through intravenous injection of adenovirus expressing TWEAK or recombinant TWEAK protein, we find that TWEAK induces a progressive dilated cardiomyopathy with impaired contractile function in mice. Moreover, TWEAK treatment is associated with decreased expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) and genes required for mitochondrial oxidative phosphorylation, which precede the onset of cardiac dysfunction. TWEAK-induced downregulation of PGC1α requires expression of its cell surface receptor, fibroblast growth factor-inducible 14 (Fn14). We further find that TWEAK downregulates PGC1α gene expression via the TNF receptor-associated factor 2 (TRAF2) and NFκB signaling pathways. Maintaining PGC1α levels through adenoviral-mediated gene expression is sufficient to protect against TWEAK-induced cardiomyocyte dysfunction. Conclusion Collectively, our data suggest that TWEAK induces cardiac dysfunction via downregulation of PGC1α, through FN14-TRAF2-NFκB-dependent signaling. Selective targeting of the FN14-TRAF2-NFκB-dependent signaling pathway or augmenting PGC1α levels may serve as novel therapeutic strategies for cardiomyopathy and heart failure.

Abstract The effect of human parathyroid hormone–related protein, a powerful vasodilator, on endothelin-1 production in cultured bovine pulmonary arterial endothelial cells was studied. Treatment with parathyroid hormone–related protein(1-34) at concentrations of 10 −9 to 10 −6 mol/L for 24 hours caused dose-dependent suppression of the secretion of endothelin-1, with maximal suppression at 10 −7 mol/L to 74% of the control value. This inhibitory effect was completely abolished by coincubation with 100 ng/mL pertussis toxin, an inhibitor of GTP binding protein. Furthermore, addition of N G -monomethyl- l -arginine, an inhibitor of nitric oxide synthase, at 10 −3 mol/L significantly blocked the suppressive effect of parathyroid hormone–related protein(1-34) on endothelin-1 secretion, and further addition of 5×10 −3 mol/L l -arginine significantly attenuated the blocking effect of N G -monomethyl- l -arginine. Parathyroid hormone–related protein(1-34) at 10 −7 mol/L resulted in an approximately fivefold increase in intracellular cGMP level. Northern blot analysis revealed that parathyroid hormone–related protein(1-34) inhibited both basal and thrombin-induced endothelin-1 gene expression. These findings suggest that the vasodilating property of parathyroid hormone–related protein may be mediated in part through its inhibitory effect on endothelin-1 production, which is probably mediated through nitric oxide and cGMP in endothelial cells. Thus, a feedback regulatory mechanism may exist between parathyroid hormone–related protein and endothelin-1 in the vascular wall.

We investigated the release of endothelin-1 (ET) from rat mesenteric arteries to clarify its pathophysiological role in the sustained hypertension of spontaneously hypertensive rats (SHR) following nephrectomy and the regulatory mechanism of the ET release which might be modified by vascular angiotensins and bradykinins. Nephrectomy increased the plasma level of ET and enhanced the ET release in both SHR and Wistar-Kyoto rats (WKY). CV-11974, an angiotensin II receptor antagonist, did not affect the ET release from arteries of nephrectomized rats. On the contrary, infusion of captopril, a converting enzyme inhibitor, further enhanced the ET release in both intact and nephrectomized rats. These findings suggest that the release of ET from mesenteric arteries may be regulated by bradykinins, but not by angiotensins. This pressor substance does not contribute to the sustained hypertension because the enhanced production of ET observed in both SHR and WKY. However, there is a possibility that the exaggerated responsiveness of vascular ET may in part account for local vascular tone and vascular remodeling in renal dysfunction.

International audience; AMPK has emerged as a critical mechanism for salutary effects of polyphenols on lipid metabolic disorders in type 1 and type 2 diabetes. Here we demonstrate that AMPK interacts with and directly phosphorylates sterol regulatory element binding proteins (SREBP-1c and -2). Ser372 phosphorylation of SREBP-1c by AMPK is necessary for inhibition of proteolytic processing and transcriptional activity of SREBP-1c in response to polyphenols and metformin. AMPK stimulates Ser372 phosphorylation, suppresses SREBP-1c cleavage and nuclear translocation, and represses SREBP-1c target gene expression in hepatocytes exposed to high glucose, leading to reduced lipogenesis and lipid accumulation. Hepatic activation of AMPK by the synthetic polyphenol S17834 protects against hepatic steatosis, hyperlipidemia, and accelerated atherosclerosis in diet-induced insulin-resistant LDL receptor-deficient mice in part through phosphorylation of SREBP-1c Ser372 and suppression of SREBP-1c- and -2-dependent lipogenesis. AMPK-dependent phosphorylation of SREBP may offer therapeutic strategies to combat insulin resistance, dyslipidemia, and atherosclerosis.