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Background: Neuromuscular blockade was shown to improve surgical conditions. However, the risk of residual neuromuscular blockade upon extubation prevents anaesthesiologists from maintaining complete paralysis. For this reason, deep NMB is still underused in anaesthesia. This review focused on answering six questions revolving around the use of deep NMB versus moderate NMB., Methods: This was a non-exhaustive narrative review based on 6 selected relevant questions: does deep NMB 1) improve surgical conditions? 2) reduce surgical complications? 3) facilitate a reduction in intraoperative pneumoperitoneum pressure (PnP)? 4) does a reduction in intraoperative PnP impact clinical outcomes? 5) does the combination of deep NMB and lower PnP improve respiratory parameters? 6) improve OR efficiency or readmission rates?, Results: This review highlights some of the key studies that have demonstrated potential benefits of deep NMB, but it also included reports showing no benefit, highlighting that the evidence is not unequivocal. Deep NMB does in fact improve surgical conditions, but whether this improvement translates into improved clinical outcomes is far from concluded. Indeed, there is an increased risk or residual curarisation, especially if patients are not monitored and reversed appropriately. The most important benefit of deep NMB may be the prevention of unacceptable surgical working conditions. The other potential major benefits are the reduction in PnP and reduction in pain. Deep NMB must be used with appropriate monitoring., Conclusion: Deep NMB was associated with an improvement in surgical conditions, reduction in PnP, pain, and complications; but further research is needed to definitively prove this relationship., (Copyright © 2021 Société française d'anesthésie et de réanimation (Sfar). Published by Elsevier Masson SAS. All rights reserved.)

We have previously shown that palmitate causes ER stress in primary cardiomyocytes and this was associated with a diffuse lipid staining histology. In contrast, oleate, which was non-toxic, led to the formation of abundant, clearly delineated lipid droplets. The aberrant lipid histology in palmitate treated cells led us to hypothesize that perhaps there was an impairment in lipid droplet formation, which could lead to accumulation of lipids in the ER and consequent ER stress. To test this hypothesis we treated H9C2s (a cardiomyoblast cell line) with either 300µM oleate or palmitate for 8h. We found that palmitate resulted in significantly less lipid droplet abundance despite elevated intracellular lipid accumulation. Next we showed that palmitate was packaged primarily as diacylglycerol (DAG), in contrast oleate formed primarily triacylglycerol (TAG). Furthermore, the palmitate induced DAG accumulated mostly in the ER, while oleate treatment resulted in accumulation of TAG primarily in lipid droplets. The palmitate-induced accumulation of lipid in the ER was associated with a strong ER stress response. Interestingly, we found that ER stress induced by either palmitate, tunicamycin, or thapsigargin led to the degradation of Plin2, an important lipid droplet binding protein. In contrast palmitate had little effect on either Plin3 or Plin5. Furthermore, we found that acute MG132 administration significantly attenuated palmitate mediated ER stress and cell death. This protection was associated with a moderate attenuation of Plin2 degradation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background/aims: Diabetic hearts exhibit intracellular lipid accumulation. This suggests that the degree of fatty acid oxidation (FAO) in these hearts is insufficient to handle the elevated lipid uptake. We previously showed that palmitate impaired the rate of FAO in primary rat neonatal cardiomyocytes. Here we were interested in characterizing the site of FAO impairment induced by palmitate since it may shed light on the metabolic dysfunction that leads to lipid accumulation in diabetic hearts., Methods: We measured fatty acid oxidation, acetyl-CoA oxidation, and carnitine palmitoyl transferase (Cpt1b) activity. We measured both forward and reverse aconitase activity, as well as NAD+ dependent isocitrate dehydrogenase activity. We also measured reactive oxygen species using the 2', 7'-Dichlorofluorescin Diacetate (DCFDA) assay. Finally we used thin layer chromatography to assess diacylglycerol (DAG) levels., Results: We found that palmitate significantly impaired mitochondrial β-oxidation as well as citric acid cycle flux, but not Cpt1b activity. Palmitate negatively affected net aconitase activity and isocitrate dehydrogenase activity. The impaired enzyme activities were not due to oxidative stress but may be due to DAG mediated PKC activation., Conclusion: This work demonstrates that palmitate, a highly abundant fatty acid in human diets, causes impaired β-oxidation and citric acid cycle flux in primary neonatal cardiomyocytes. This metabolic defect occurs prior to cell death suggesting that it is a cause, rather than a consequence of palmitate mediated lipotoxicity. This impaired mitochondrial metabolism can have important implications for metabolic diseases such as diabetes and obesity., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Here we sought to evaluate the effect of palmitate on cytokine and PPAR activity/expression. We investigated the effect of BSA conjugated palmitate and oleate on PPAR activity, PPAR-α and δ expression, as well as the expression of cytokines and key factors responsible for β-oxidation by qRT-PCR and western blotting in primary rat neonatal cardiomyocytes (NCMs). Furthermore we evaluated the effect of anti-inflammatory actions of AICAR and PPAR agonists on cytokine expression and cell death in palmitate treated NCMs. We found that palmitate caused down regulation of PPARs and increased cytokine expression and cell death, all of which was significantly attenuated by the co-administration of either AICAR or PPAR agonists. This work supports the pro-inflammatory actions of intracellular lipid and provides further insight into the pathological mechanism of cardiac lipotoxicity as occurs in diabetic hearts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Apoptosis is a hallmark of multiple etiologies of heart failure, including dilated cardiomyopathy. Since microRNAs are master regulators of cardiac development and key effectors of intracellular signaling, they represent novel candidates for understanding the mechanisms driving the increased dysfunction and loss of cardiomyocytes during cardiovascular disease progression. To determine the role of cardiac miRNAs in the apoptotic response, we used microarray technology to monitor miRNA levels in a validated murine phospholambam mutant model of dilated cardiomyopathy. 24 miRNAs were found to be differentially expressed, most of which have not been previously linked to dilated cardiomyopathy. We showed that individual silencing of 7 out of 8 significantly down-regulated miRNAs (mir-1, -29c, -30c, -30d, -149, -486, -499) led to a strong apoptotic phenotype in cell culture, suggesting they repress pro-apoptotic factors. To identify putative miRNA targets most likely relevant to cell death, we computationally integrated transcriptomic, proteomic and functional annotation data. We showed the dependency of prioritized target abundance on miRNA expression using RNA interference and quantitative mass spectrometry. We concluded that down regulation of key pro-survival miRNAs causes up-regulation of apoptotic signaling effectors that contribute to cardiac cell loss, potentially leading to system decompensation and heart failure.

Background: Endoplasmic reticulum (ER) resident protein 44 (ERp44) is a member of the protein disulfide isomerase family, is induced during ER stress, and may be involved in regulating Ca(2+) homeostasis. However, the role of ERp44 in cardiac development and function is unknown. The aim of this study was to investigate the role of ERp44 in cardiac development and function in mice, zebrafish, and embryonic stem cell (ESC)-derived cardiomyocytes to determine the underlying role of ERp44., Methods and Results: We generated and characterized ERp44(-/-) mice, ERp44 morphant zebrafish embryos, and ERp44(-/-) ESC-derived cardiomyocytes. Deletion of ERp44 in mouse and zebrafish caused significant embryonic lethality, abnormal heart development, altered Ca(2+) dynamics, reactive oxygen species generation, activated ER stress gene profiles, and apoptotic cell death. We also determined the cardiac phenotype in pressure overloaded, aortic-banded ERp44(+/-) mice: enhanced ER stress activation and increased mortality, as well as diastolic cardiac dysfunction with a significantly lower fractional shortening. Confocal and LacZ histochemical staining showed a significant transmural gradient for ERp44 in the adult heart, in which high expression of ERp44 was observed in the outer subepicardial region of the myocardium., Conclusions: ERp44 plays a critical role in embryonic heart development and is crucial in regulating cardiac cell Ca(2+) signaling, ER stress, ROS-induced oxidative stress, and activation of the intrinsic mitochondrial apoptosis pathway., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Calnexin (CNX) is an endoplasmic reticulum (ER) quality control chaperone that has been implicated in ER stress. ER stress is a prominent pathological feature of various pathologic conditions, including cardiovascular diseases. However, the role of CNX and ER stress has not been studied in the heart. In the present study, we aimed to characterize the role of CNX in cardiomyocyte physiology with respect to ER stress, apoptosis, and cardiomyocyte Ca(2+) cycling. We demonstrated significantly decreased CNX mRNA and protein levels by LentiVector mediated transduction of targeting shRNAs. CNX silenced cardiomyocytes exhibited ER stress as evidenced by increased GRP78 and ATF6 protein levels, increased levels of spliced XBP1 mRNA, ASK-1, ERO1a, and CHOP mRNA levels. CNX silencing also led to significant activation of caspases-3 and -9. This activation of caspases was associated with hallmark morphological features of apoptosis including loss of sarcomeric organization and nuclear integrity. Ca(2+) imaging in live cells showed that CNX silencing resulted in Ca(2+) transients with significantly larger amplitudes but decreased frequency and Ca(2+) uptake rates in the basal state. Interestingly, 5 mM caffeine stimulated Ca(2+) transients were similar between control and CNX silenced cardiomyocytes. Finally, we demonstrated that CNX silencing induced the expression of the L-type voltage dependent calcium channel (CAV1.2) but reduced the expression of the sarcoplasmic reticulum ATPase (SERCA2a). In conclusion, this is the first study to demonstrate CNX has a specific role in cardiomyocyte viability and Ca(2+) cycling through its effects on ER stress, apoptosis and Ca(2+) channel expression., (© 2013 Wiley Periodicals, Inc.)

In-depth proteomic analyses offer a systematic way to investigate protein alterations in disease and, as such, can be a powerful tool for the identification of novel biomarkers. Here, we analyzed proteomic data from a transgenic mouse model with cardiac-specific overexpression of activated calcineurin (CnA), which results in severe cardiac hypertrophy. We applied statistically filtering and false discovery rate correction methods to identify 52 proteins that were significantly different in the CnA hearts compared to controls. Subsequent informatic analysis consisted of comparison of these 52 CnA proteins to another proteomic dataset of heart failure, three available independent microarray datasets, and correlation of their expression with the human plasma and urine proteome. Following this filtering strategy, four proteins passed these selection criteria, including myosin heavy chain 7, insulin-like growth factor-binding protein 7, annexin A2, and desmin. We assessed expression levels of these proteins in mouse plasma by immunoblotting, and observed significantly different levels of expression between healthy and failing mice for all four proteins. We verified antibody cross-reactivity by examining human cardiac explant tissue by immunoblotting. Finally, we assessed protein levels in plasma samples obtained from four unaffected and four heart failure patients and demonstrated that all four proteins increased between twofold and 150-fold in heart failure. We conclude that MYH7, IGFBP7, ANXA2, and DESM are all excellent candidate plasma biomarkers of heart failure in mouse and human., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mass spectrometry-based targeted proteomic assays are experiencing a surge in awareness due to the diverse possibilities arising from the re-application of traditional LC-SRM technology. The FDA-approved quantitative LC-SRM-pipeline in drug discovery motivates the use to quantitatively validate putative proteomic biomarkers. However, complexity of biological specimens bears a huge challenge to identify, in parallel, specific peptides and proteins of interest from large biomarker candidate lists. Methods have been devised to increase scan speeds, improve detection specificity and verify quantitative SRM-features. In contrast, high-resolution mass spectrometers could be used to improve reliability and precision of targeted proteomics assays. Here, we present a new method for identifying, quantifying and reporting peptides in high-resolution targeted proteomics experiments performed on an orbitrap hybrid instrument using stable isotope-labeled internal reference peptides. This high precision targeted peptide monitoring (TPM) method has unique advantages over existing techniques, including the need to only detect the most abundant product ion of a given target for confident peptide identification using a scoring function that evaluates assay performance based on 1) m/z-mass accuracy, 2) retention time accuracy of observed species relative to prediction, and 3) retention time accuracy relative to internal reference peptides. Further, we show management of multiplexed precision TPM-assays using sentinel peptide standards. This article is part of a Special Issue entitled: From protein structures to clinical applications., (Copyright © 2012. Published by Elsevier B.V.)

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs) and has been shown to have potent antiapoptotic properties. Because the mechanism by which cryAB prevents apoptosis has not been fully characterized, we examined its protective effects at the cellular level by silencing cryAB in mouse neonatal CMs using lentivector-mediated transduction of short hairpin RNAs. Subcellular fractionation of whole hearts showed that cryAB is cytosolic under control conditions, and after H(2)O(2) exposure, it translocates to the mitochondria. Phosphorylated cryAB (PcryAB) is mainly associated with the mitochondria, and any residual cytosolic PcryAB translocates to the mitochondria after H(2)O(2) exposure. H(2)O(2) exposure caused increases in cryAB and PcryAB levels, and cryAB silencing resulted in increased levels of apoptosis after exposure to H(2)O(2). Coimmunoprecipitation assays revealed an apparent interaction of both cryAB and PcryAB with mitochondrial voltage-dependent anion channels (VDAC), translocase of outer mitochondrial membranes 20 kDa (TOM 20), caspase 3, and caspase 12 in mouse cardiac tissue. Our results are consistent with the conclusion that the cardioprotective effects of cryAB are mediated by its translocation from the cytosol to the mitochondria under conditions of oxidative stress and that cryAB interactions with VDAC, TOM 20, caspase 3, and caspase 12 may be part of its protective mechanism.

Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified α-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.

Rationale: Expression of the vasoactive peptide Urotensin II (UII) is elevated in a number of cardiovascular diseases., Objective: Here, we sought to determine the effect of UII receptor (UT) gene deletion in a mouse model of atherosclerosis., Methods and Results: UT knockout (KO) mice were crossed with ApoE KO mice to generate UT/ApoE double knockout (DKO) mice. Mice were placed on a high-fat Western-type diet for 12 weeks. We evaluated the degree of atherosclerosis and hepatic steatosis by histology. In addition, serum glucose, insulin, and lipids were determined. DKO mice exhibited significantly increased atherosclerosis compared to ApoE KO mice (P<0.05). This was associated with a significant increase in serum insulin and lipids (P<0.001) but a decrease in hepatic steatosis (P<0.001). UT gene deletion led to a significant increase in systolic pressure and pulse pressure. RT-PCR and immunoblot analyses showed significant reductions in hepatic scavenger receptors, nuclear receptors, and acyl-CoA:cholesterol acyltransferase (ACAT1) expression in DKO mice. UII induced a significant increase in intracellular cholesteryl ester formation in primary mouse hepatocytes, which was blocked by the MEK inhibitor, PD98059. Hepatocytes of UTKO mice showed a significant reduction in lipoprotein uptake compared to wild-type mice., Conclusions: We propose that UT gene deletion in an ApoE-deficient background promotes downregulation of ACAT1, which in turn attenuates hepatic lipoprotein receptor-mediated uptake and lipid transporter expression. As the liver is the main organ for uptake of lipoprotein-derived lipids, DKO leads to an increase in hyperlipidemia, with a concomitant decrease in hepatic steatosis, and consequently increased atherosclerotic lesion formation. Furthermore, the hypertension associated with UT gene deletion is likely to contribute to the increased atherosclerotic burden.

Urotensin II (UII) and its receptor UT are upregulated in the pathological setting of various cardiovascular diseases including atherosclerosis. However, their exact role in atherosclerosis remains to be determined. In the present study we used four strains of mice; wild-type (WT), UT(+) (a transgenic strain expressing human UT driven by the alpha-smooth muscle-specific, SM22, promoter), ApoE knockout (ko), and UT(+)/ApoE ko. All animals were fed high fat diet for 12 weeks. Western blot analysis revealed a significant increase in aortic UT expression in UT(+) relative to WT mice (P<0.05). Aortas of ApoE ko mice expressed comparable UT protein level to that of UT(+). Immunohistochemistry revealed the presence of strong expression of UT and UII proteins in the atheroma of UT(+), ApoE ko and UT(+)/ApoE ko mice, particularly in foam cells. Serum cholesterol and triglyceride levels were significantly increased in ApoE ko and in UT(+)/ApoE ko but not in UT(+) mice when compared to WT mice (P<0.0001). Analysis of aortas showed a significant increase in atherosclerotic lesion in the UT(+), ApoE ko and UT(+)/ApoE ko compared to WT mice (P<0.05). Oral administration of the UT receptor antagonist SB-657510A (30 microg/Kg/day gavage) for 10 weeks in a group of ApoE ko mice fed on high fat diet resulted in a significant reduction of lesion (P<0.001). SB-657510A also significantly reduced ACAT-1 protein expression in the atherosclerotic lesion of ApoE ko mice (P<0.05). The present findings demonstrate an important role for UT in the pathogenesis of atherosclerosis. The use of UT receptor antagonists may provide a beneficial tool in the management of this debilitating disease process.

Recent advances in mass spectrometry and bioinformatics have provided the means to characterize complex protein landscapes from a wide variety of organisms and cell types. Development of standard proteomes exhibiting all of the proteins involved in normal physiology will facilitate the delineation of disease mechanisms. Here, we examine the wild-type cardiac proteome using data obtained from a subcellular fractionation protocol in combination with a multidimensional protein identification proteomics approach. We identified 4906 proteins which were allocated to either cytosolic, microsomal, mitochondrial matrix or mitochondrial membrane fractions with relative abundance values in each fraction. We subjected these proteins to hierarchical clustering, gene ontology terms analysis, immunoblotting, comparison to publicly available protein databases, comparison to 4 distinct cardiac transcriptomes, and finally, to 6 other related proteomic data sets. This study provides an exhaustive analysis of the cardiac proteome and is the first large-scale investigation of the subcellular location for over 2000 unannotated proteins. With the use of a subtractive transcriptomics approach, we have also extended our analysis to identify 'cardiac selective' factors in our proteome. Finally, using specific filtering criteria, we identified proteotypic peptides for subsequent use in targeted studies of both mouse and human. Therefore, we offer this as a major contribution to the advancement of the field of proteomics in cardiovascular research.

Cells contain a large yet, constant genome, which contains all the coding information necessary to sustain cellular physiology. However, proteins are the end products of genes, and hence dictate the phenotype of cells and tissues. Therefore, proteomics can provide key information for the elucidation of physiological and pathophysiological mechanisms by identifying the protein profile from cells and tissues. The relatively novel techniques used for the study of proteomics thus have the potential to improve diagnostic, prognostic, as well as therapeutic avenues. In this review, we first discuss the benefits of animal models over the use of human samples for the proteomic analysis of human disease. Next, we aim to demonstrate the potential of proteomics in the elucidation of disease mechanisms that may not be possible by other conventional technologies. Following this, we describe the use of proteomics for the analysis of PTM and protein interactions in animal models and their relevance to the study of human disease. Finally, we discuss the development of clinical biomarkers for the early diagnosis of disease via proteomic analysis of animal models. We also discuss the development of standard proteomes and relate how this data will benefit future proteomic research. A comprehensive review of all animal models used in conjunction with proteomics is beyond the scope of this manuscript. Therefore, we aimed to cover a large breadth of topics, which together, demonstrate the potential of proteomics as a powerful tool in biomedical research., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Urotensin-II (U-II), a cyclic undecapeptide, and its receptor, UT, have been linked to vascular and cardiac remodeling. In patients with coronary artery disease (CAD), it has been shown that U-II plasma levels are significantly greater than in normal patients and the severity of the disease is increased proportionally to the U-II plasma levels. We showed that U-II protein and mRNA levels were significantly elevated in the arteries of patients with coronary atherosclerosis in comparison to healthy arteries. We observed U-II expression in endothelial cells, foam cells, and myointimal and medial vSMCs of atherosclerotic human coronary arteries. Recent studies have demonstrated that U-II acts in synergy with mildly oxidized LDL inducing vascular smooth muscle cell (vSMC) proliferation. Additionally, U-II has been shown to induce cardiac fibrosis and cardiomyocyte hypertrophy leading to cardiac remodeling. When using a selective U-II antagonist, SB-611812, we demonstrated a decrease in cardiac dysfunction including a reduction in cardiomyocyte hypertrophy and cardiac fibrosis. These findings suggest that U-II is undoubtedly a potential therapeutic target in treating cardiovascular remodeling.

Treatment for symptomatic atherosclerosis is being carried out by balloon mediated angioplasty, with or without stent implantation, more and more frequently. Although advances with the development of drug eluting stents have improved prognosis, restenosis is still the most limiting factor for this treatment modality. Urotensin-II (UII), a small pleiotropic vasoactive peptide is increasingly being recognized as a contributory factor in cardiovascular diseases. We qualitatively evaluated UII immunoreactivity (IR) in three models of balloon angioplasty mediated restenosis. Specifically, we performed balloon angioplasty in the ilio-femoral arteries of New Zealand White Rabbits (NZWR) fed either a normal chow or high fat diet. In addition, UIIIR was also assessed in stent implanted abdominal aortae of NZWR fed a high fat diet. UII was constitutively expressed in the endothelium of all arterial segments evaluated. Abundant expression of UII was associated with lesion progression, particularly in myointimal cells, and less so in medial smooth muscle cells (SMC). The strongest UII-IR was observed in foam cells of animals fed a high fat diet. We demonstrate abundant expression of UII in regenerating endothelial cells and myointimal cells in vascular lesions following balloon mediated angioplasty and stent implantation in both animals fed a normal chow and high fat diet.

Urotensin-II (U-II) is a vasoactive factor with pleiotropic effects. U-II exerts its activity by binding to a G-protein-coupled receptor termed UT. U-II and its receptor are highly expressed in the cardiovascular system. Increased U-II plasma levels have been reported in patients with cardiovascular disease of varying etiologies. We and others have shown that U-II and UT expression is elevated in both clinical and experimental heart failure and atherosclerosis. U-II induces cardiac fibrosis by increasing fibroblast collagen synthesis. In addition, U-II induces cardiomyocyte hypertrophy and increased vascular smooth muscle cell proliferation. We have shown that U-II antagonism using a selective U-II blocker, SB-611812 reduces neointimal thickening and increases lumen diameter in a rat restenosis model of carotid artery angioplasty. These findings suggest an important role for U-II in cardiovascular dysfunction and remodeling.

It is now well established that urotensin-II (UII) levels are increased in several cardiovascular diseases. We previously demonstrated that UII and the UII receptor (UT) protein levels are significantly increased in the hearts of both humans and rats with congestive heart failure (CHF). We have also recently demonstrated that UII blockade, with a selective UII antagonist, improves heart function in a rat model of ischemic CHF. Here, we evaluated the attenuation of cardiac remodeling associated with UII antagonism in the same rat model of ischemic CHF. Animals were administered a specific UT receptor antagonist, SB-611812 (30 mg/kg/day, gavage), or vehicle 30 min prior to coronary artery ligation followed by daily treatment for 8 weeks. Myocardial interstitial fibrosis was analyzed by Masson's trichrome and picrosirius red staining. RT-PCR analysis was utilized for mRNA expression studies. We used Western blotting to assess levels of collagen types I and III. Mitogenic activity of UII on cultured neonatal cardiac fibroblasts was also evaluated. Following coronary ligation, SB-611812 significantly attenuated both myocardial and endocardial interstitial fibrosis, and reduced collagen type I:III ratio (P<0.01). UII induced proliferation of cardiac fibroblasts and this mitogenic effect was significantly inhibited with 1 microM of SB-611218 (P<0.05). We demonstrate here that selective blockade of UT reduces diastolic dysfunction by decreasing myocardial fibrosis post-coronary ligation in vivo, and inhibits UII-mediated fibroblast proliferation in vitro.

Expression of urotensin II (UII) is significantly elevated in the hearts of patients with congestive heart failure (CHF). Recent reports have also shown increased plasma levels of UII in patients with CHF, and these levels correlated with the severity of disease. We therefore hypothesized that blockade of UII signaling would improve cardiac function in a rat model of CHF. CHF was induced in rats by ligating the left coronary artery. Animals were randomized to either treatment with a specific UT receptor antagonist, SB-611812 (30 mg/kg/day, UID by gavage), or vehicle, starting either 30 min prior to coronary ligation (early treatment) or 10 days after ligation (delayed treatment). Treatment drug or vehicle was administered daily thereafter for 8 weeks. We measured cardiac function and evaluated the levels of mRNA expression for mediators of CHF. In addition, we evaluated UII and UT protein levels using immunohistochemistry and Western blotting. Cardiomyocyte hypertrophy was evaluated by measuring cardiomyocyte cross-sectional area. Animals with CHF showed increased UII and UT expression as evidenced by immunohistochemistry and Western blotting. Treatment with the SB-611812 significantly reduced overall mortality, left ventricular end-diastolic pressure by 72%, lung edema by 71%, right ventricular systolic pressure by 92%, central venous pressure by 59%, cardiomyocyte hypertrophy by 54%, and ventricular dilatation by 79% (P < 0.05). Therefore, blockade of the UT receptor reduced mortality and improved cardiac function in this model of myocardial infarction and CHF, suggesting an important role for UII in the pathogenesis of this condition.

TLRs are essential mediators of host defense against infection via recognition of unique microbial structures. Recent observations indicate that TLR4, the principal receptor for bacterial LPS, may also be activated by noninfectious stimuli including host-derived molecules and environmental oxidant stress. In mice, susceptibility to ozone-induced lung permeability has been linked to the wild-type allele of TLR4, whereas deficiency of TLR4 predisposes to lethal lung injury in hyperoxia. To precisely characterize the role of lung epithelial TLR4 expression in the host response to oxidant stress, we have created an inducible transgenic mouse model that targets the human TLR4 signaling domain to the airways. Exposure of induced transgenic mice to hyperoxia revealed a significant reduction in pulmonary apoptosis compared with controls. This phenotype was associated with sustained up-regulation of antiapoptotic molecules such as heme oxygenase-1 and Bcl-2, yet only transient activation of the transcription factor NF-kappaB. Specific in vivo knockdown of pulmonary heme oxygenase-1 or Bcl-2 expression by intranasal administration of short interfering RNA blocked the effect of TLR4 signaling on hyperoxia-induced lung apoptosis. These results define a novel role for lung epithelial TLR4 as a modulator of cellular apoptosis in response to oxidant stress.

Urotensin II (U-II), a novel vasoactive peptide, possesses a wide range of cardiovascular effects. U-II binds a seven transmembrane spanning G-protein coupled receptor termed GPR14. In the present study, we have characterized U-II expression in both carotid and aortic atherosclerotic plaques. Using immunohistochemistry we demonstrated U-II immunoreactivity in endothelial, smooth muscle and inflammatory cells of both carotid and aortic plaques, with a clear propensity for intimal staining. Using quantitative real-time RT-PCR we observed both increased U-II and GPR14 mRNA expression in tissue extracts from abdominal aortic aneurysms. We also extended our PCR analysis to include leukocyte expression of U-II and GPR14. We found that lymphocytes were by far the largest producers of U-II mRNA. In contrast monocytes and macrophages were the largest producers of GPR14 mRNA, with relatively little expression in foam cells, lymphocytes, and platelets. Our findings qualitatively and quantitatively demonstrate increased expression of U-II in atherosclerosis with a large degree of inflammatory cell involvement. These findings suggest a possible role for U-II in the pathophysiology of atherosclerosis.

Atherosclerosis is a major risk factor for both myocardial infarction and stroke. A key aspect of this disease is the imbalance of vasoactive factors. In this concise review, we focus on the role of endothelin-1 in the atherosclerotic process and other vasculopathies. Previously, we have demonstrated that there is a correlation between the expression of endothelin and the underlying atherosclerotic lesion. Immunoreactivity was observed for both ET-1 and ECE-1 in endothelial cells, smooth muscle cells, and macrophages within lesions. Endothelin's role in atherosclerosis must extend from its varying physiological activities, including vasoconstriction, mitogenesis, neutrophil adhesion, and platelet aggregation, and hypertrophy, as well as its propensity to induce the formation of reactive oxygen species. We also discuss regulation of endothelin by angiotensin II, reactive oxygen species, thrombin, aging, and LDL in the cardiovascular system. Finally, we demonstrate the role of endothelin in pulmonary hypertension and transplant associated vasculopathy.