Decreased metalloprotease 9 induction, cardiac fibrosis, and higher autophagy after pressure overload in mice lacking the transcriptional regulator p8.

Left ventricular remodeling, including the deposition of excess extracellular matrix, is key to the pathogenesis of heart failure. The stress-inducible transcriptional regulator p8 is increased in failing human hearts and is required both for agonist-stimulated cardiomyocyte hypertrophy and for cardiac fibroblasts matrix metalloprotease-9 (MMP9) induction. In the heart, upregulation of autophagy is an adaptive response to stress and plays a causative role in cardiomyopathies. We have recently shown that p8 ablation in cardiac cells upregulates autophagy and that, in vivo, loss of p8 results in a decrease of cardiac function. Here we investigated the effects of p8 genetic deletion in mediating adverse myocardial remodeling. Unstressed p8-/- mouse hearts manifested complex alterations in the expression of fibrosis markers. In addition, these mice displayed elevated autophagy and apoptosis compared with p8+/+ mice. Transverse aortic constriction (TAC) induced left ventricular p8 expression in p8+/+ mice. Pressure overload caused left ventricular remodeling in both genotypes, however, p8-/- mice showed less cardiac fibrosis induction. Consistent with this, although MMP9 induction was attenuated in the p8-/- mice, induction of MMP2 and MMP3 were strikingly upregulated while TIMP2 was downregulated. Left ventricular autophagy increased after TAC and was significantly higher in the p8-/- mice. Thus p8-deletion results in reduced collagen fibrosis after TAC, but in turn, is associated with a detrimental higher increase in autophagy. These findings suggest a role for p8 in regulating in vivo key signaling pathways involved in the pathogenesis of heart failure.

Deficiency of the transcriptional regulator p8 results in increased autophagy and apoptosis, and causes impaired heart function.

Autophagy is a cytoprotective pathway used to degrade and recycle cytoplasmic content. Dysfunctional autophagy has been linked to both cancer and cardiomyopathies. Here, we show a role for the transcriptional regulator p8 in autophagy. p8 RNA interference (RNAi) increases basal autophagy markers in primary cardiomyocytes, in H9C2 and U2OS cells, and decreases cellular viability after autophagy induction. This autophagy is associated with caspase activation and is blocked by atg5 silencing and by pharmacological inhibitors. FoxO3 transcription factor was reported to activate autophagy by enhancing the expression of autophagy-related genes. P8 expression represses FoxO3 transcriptional activity, and p8 knockdown affects FoxO3 nuclear localization. Thus, p8 RNAi increases FoxO3 association with bnip3 promoter, a known proautophagic FoxO3 target, resulting in higher bnip3 RNA and protein levels. Accordingly, bnip3 knockdown restores cell viability and blocks apoptosis of p8-deficient cells. In vivo, p8 -/- mice have higher autophagy and express higher cardiac bnip3 levels. These mice develop left ventricular wall thinning and chamber dilation, with consequent impaired cardiac function. Our studies provide evidence of a p8-dependent mechanism regulating autophagy by acting as FoxO3 corepressor, which may be relevant for diseases associated with dysregulated autophagy, as cardiovascular pathologies and cancer.

Deficiency of transcriptional regulator p8 induces autophagy and causes impaired cardiac function.

Through autophagy cells adapt to nutrient availability, recycle cellular material and eliminate toxic proteins and damaged cellular organelles. Dysregulation of autophagy is implicated in the pathogenesis of various diseases, including cancer, neurodegeneration and cardiomyopathies. The transcription factor FoxO3 activates autophagy by enhancing the expression of several genes. We find a role for the transcriptional regulator p8 in controling autophagy by repressing FoxO3 transcriptional activity. p8 silencing increases the association of FoxO3 with the bnip3 promoter, a known pro-autophagic FoxO3 target, and results in increasead basal autophagy and decreased cellular viability. Likewise, p8 overexpression inhibits Bnip3 upregulation after autophagy activation. Thus, p8 appears to antagonize the promotion of autophagy mediated by the FoxO3-Bnip3 axis. Consistent with this, bnip3 knockdown restores viability in p8-deficient cells. In vivo, hearts from p8-/- mice have higher basal autophagy and bnip3 levels. These mice develop left ventricular wall thinning and chamber dilation, with consequent impaired cardiac function.

Role of SREBP-1 in the development of parasympathetic dysfunction in the hearts of type 1 diabetic Akita mice.

RATIONALE: Diabetic autonomic neuropathy (DAN), a major complication of diabetes mellitus, is characterized, in part, by impaired cardiac parasympathetic responsiveness. Parasympathetic stimulation of the heart involves activation of an acetylcholine-gated K+ current, I(KAch), via a (GIRK1)2/(GIRK4)2 K+ channel. Sterol regulatory element binding protein-1 (SREBP-1) is a lipid-sensitive transcription factor. OBJECTIVE: We describe a unique SREBP-1-dependent mechanism for insulin regulation of cardiac parasympathetic response in a mouse model for DAN. METHODS AND RESULTS: Using implantable EKG transmitters, we demonstrated that compared with wild-type, Ins2(Akita) type I diabetic mice demonstrated a decrease in the negative chronotropic response to carbamylcholine characterized by a 2.4-fold decrease in the duration of bradycardia, a 52+/-8% decrease in atrial expression of GIRK1 (P<0.01), and a 31.3+/-2.1% decrease in SREBP-1 (P<0.05). Whole-cell patch-clamp studies of atrial myocytes from Akita mice exhibited a markedly decreased carbamylcholine stimulation of I(KAch) with a peak value of -181+/-31 pA/pF compared with -451+/-62 pA/pF (P<0.01) in cells from wild-type mice. Western blot analysis of extracts of Akita mice demonstrated that insulin treatment increased the expression of GIRK1, SREBP-1, and I(KAch) activity in atrial myocytes from these mice to levels in wild-type mice. Insulin treatment of cultured atrial myocytes stimulated GIRK1 expression 2.68+/-0.12-fold (P<0.01), which was reversed by overexpression of dominant negative SREBP-1. Finally, adenoviral expression of SREBP-1 in Akita atrial myocytes reversed the impaired I(KAch) to levels in cells from wild-type mice. CONCLUSIONS: These results support a unique molecular mechanism for insulin regulation of GIRK1 expression and parasympathetic response via SREBP-1, which might play a role in the pathogenesis of DAN in response to insulin deficiency in the diabetic heart.

Tissue specific MR contrast media role in the differential diagnosis of cirrhotic liver nodules.

State-of-the-art magnetic resonance (MR) imaging using tissue specific contrast media facilitates detection and characterization in most cases of hepatic nodules. According to the currently used nomenclature, in liver cirrhosis there are only two major types of hepatocellular nodular lesions: regenerative lesions and dysplastic or neoplastic lesions. The purpose of this clinical imaging review is to provide information on the properties of tissue-specific MR contrast agents and on their usefulness in the demonstration of the pathologic changes that take place at the level of the hepatobiliary and reticuloendothelial systems during the carcinogenesis in liver cirrhosis.

Combined cardiac-neurosurgical treatment of acute aortic dissection, stroke, and coma.

Coma or stroke with secondary brain malperfusion is usually considered a strong contraindication for emergent surgical treatment of acute aortic dissection. Herein, we present the case of a 30-year-old woman who presented with sudden left hemiplegia and level-7 coma on the Glasgow Coma Scale. Transthoracic echocardiography showed type A aortic dissection. Although the patient was unable to communicate, her family approved an emergency Bentall operation. She regained consciousness but developed anisocoria and Glasgow Coma Scale level-4 coma 30 hours after the operation. Computed tomography showed massive cerebral infarction with hernia of the uncus gyri hippocampi. Emergency surgical cerebral decompression was performed. The patient survived; after 1 year, she had full mental acuity and minor left motor sequelae.

Spiral computed tomography and magnetic resonance angiography evaluation in Budd-Chiari syndrome.

Budd-Chiari syndrome is caused by the obstruction of the hepatic venous outflow at the level of the hepatic venules, large hepatic veins, and inferior vena cava up to the confluence with the right atrium. When it is untreated, the mortality rate for patients is high. Because the clinical presentation of this syndrome is nonspecific, imaging investigation--computed tomography and magnetic resonance--are important diagnostic steps. Contrast-enhanced multiphase spiral computed tomography (CT) and magnetic resonance (MR) angiography permits morphologic and functional assessment of parenchymatous liver changes in this particular entity. In this review, we present the spectrum of vascular and hepatic parenchymal abnormalities in Budd-Chiari syndrome observed on multiphase contrast enhanced spiral CT and MR angiography.

Parasympathetic response in chick myocytes and mouse heart is controlled by SREBP.

Parasympathetic stimulation of the heart, which provides protection from arrhythmias and sudden death, involves activation of the G protein-coupled inward rectifying K+ channel GIRK1/4 and results in an acetylcholine-sensitive K+ current, I KACh. We describe a unique relationship between lipid homeostasis, the lipid-sensitive transcription factor SREBP-1, regulation of the cardiac parasympathetic response, and the development of ventricular arrhythmia. In embryonic chick atrial myocytes, lipid lowering by culture in lipoprotein-depleted serum increased SREBP-1 levels, GIRK1 expression, and I KACh activation. Regulation of the GIRK1 promoter by SREBP-1 and lipid lowering was dependent on interaction with 2 tandem sterol response elements and an upstream E-box motif. Expression of dominant negative SREBP-1 (DN-SREBP-1) reversed the effect of lipid lowering on I KACh and GIRK1. In SREBP-1 knockout mice, both the response of the heart to parasympathetic stimulation and the expression of GIRK1 were reduced compared with WT. I KACh, attenuated in atrial myocytes from SREBP-1 knockout mice, was stimulated by SREBP-1 expression. Following myocardial infarction, SREBP-1 knockout mice were twice as likely as WT mice to develop ventricular tachycardia in response to programmed ventricular stimulation. These results demonstrate a relationship between lipid metabolism and parasympathetic response that may play a role in arrhythmogenesis.

Computer tomographic evaluation of digestive tract non-Hodgkin lymphomas.

Computer Tomographic (CT) study is crucial for defining distribution, characteristics and staging of primary gastrointestinal lymphomas. The presence of multifocal sites, the wall thickening with diffuse infiltration of the affected gastrointestinal (GI) segment in association with regional adenopathies, permit the orientation of the CT diagnosis for primary GI lymphomas. The gold standard for diagnosis remains, in all cases of digestive tract non-Hodgkin lymphomas (NHL), the histological examination, which allows a tissue diagnosis, performed preferably by transmural biopsy.

Gastrointestinal stromal tumors: retrospective analysis of the computer-tomographic aspects.

PURPOSE: To describe the computer-tomographic (CT) aspects of gastrointestinal stromal tumors (GISTs) in correlation to their histology. MATERIAL AND METHODS: The medical records of all patients at our hospital with a histologic diagnosis of GIST between January 2002 and June 2006, and investigated before surgery by CT, were reviewed. Two radiologists with knowledge of the diagnosis reviewed the CT findings. RESULTS: Amongst 15 cases of GISTs, 9 cases involved the stomach and 4 cases the small intestine. Location of the primary tumor could not be determined for 2 of 15 tumors, because of the presence of extensive peritoneal metastases. Most primary tumors were predominantly extraluminal (13 cases) while two were clearly endoluminal. The mean diameter of the primary tumor was 8 cm. The tumor margin was well defined in 12 patients and irregular in 3 cases. Central fluid attenuation was present in 11 tumors, while central gas was seen in two cases. Metastases were seen in 2 cases at presentation and in another 2 patients during follow-up. Spread was exclusive to the liver or peritoneum. Visceral obstruction was absent even in extensive peritoneal metastatic disease. Ascites was an unusual finding. CONCLUSIONS: CT plays an important role not only in the detection and the localization but also in the evaluation of the extension and follow-up of theses tumors. Using only CT aspects, we can only suspect the diagnosis to GISTs. Often other soft-tissue tumors with gastrointestinal involvement can mimic GISTs. In all cases histological diagnosis is essential.

Hepatic perfusion disorders: Computer-tomographic and magnetic resonance imaging.

The liver has a unique dual blood supply from the hepatic artery (25%) and the portal vein (75%). Helical computer tomography (CT) and also magnetic resonance imaging (MRI) are suitable techniques for hepatic imaging. Helical CT and MR angiography allow single breath-hold scanning without motion artifacts. This article illustrates helical CT and MRI findings of different types of hepatic perfusion disorders. Because of rapid image acquisition, three-phase (hepatic arterial phase, portal venous phase and parenchymal phase) CT or MR-angiography evaluation of the hepatic parenchyma is possible, improving perfusion disorders evaluation, tumors detection and characterization in a single study. We classified hepatic perfusion abnormalities in: portal disorders, arterial disorders, hepatic veins abnormalities, intrahepatic vascular communication, hepatic lesions and perfusion disorders and other causes. Differential diagnosis and pitfalls of these entities must be known for a correct diagnosis of focal hepatic lesions.

Simvastatin potentiates tumor necrosis factor alpha-mediated apoptosis of human vascular endothelial cells via the inhibition of the geranylgeranylation of RhoA.

HMG-CoA reductase inhibitors (statins) are widely used in the treatment and prevention of atherosclerosis. Here we demonstrate that the HMG-CoA reductase inhibitor simvastatin potentiates TNFalpha-mediated apoptosis and TNFalpha signaling in human umbilical vein endothelial cells (HUVECs). While 2.5 microM simvastatin or 40 ng/ml TNFalpha alone had only a small effect on apoptosis in HUVECs, co-incubation with simvastatin and TNFalpha markedly increased apoptosis in a time- and dose-dependent manner as measured by FACS analysis of propidium iodide-stained cells. Geranylgeraniol, which serves as a substrate for the geranylgeranylation of small GTP binding proteins such as RhoA, which is required for the function and membrane localization of Rho, reversed the effect of simvastatin on apoptosis. GGTI, an inhibitor of protein geranylgeranylation, mimicked the effect of simvastatin on apoptosis and interfered with the membrane localization of RhoA. Furthermore, simvastatin increased the expression of the TNFalpha type I receptor (TNFalphaRI) with a dose dependence and a dependence on geranylgeranylation similar to that demonstrated for the potentiation of TNFalpha-mediated apoptosis. Adenoviral expression of a dominant-negative RhoA mimicked the effect of simvastatin on the expression of TNFalphaRI, while adenoviral expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on the expression of TNFalphaRI. Simvastatin also potentiated TNFalpha signaling as determined by increased TNFalpha-mediated E-selectin expression. These data support the conclusion that TNFalpha signaling is under the negative control of RhoA and that statins potentiate TNFalpha signaling at least in part via interference with RhoA inhibition of TNFalpha type I receptor expression.

Transforming growth factor beta regulates the expression of the M2 muscarinic receptor in atrial myocytes via an effect on RhoA and p190RhoGAP.

Transforming growth factor beta (TGFbeta) signaling is involved in the development and regulation of multiple organ systems and cellular signaling pathways. We recently demonstrated that TGFbeta regulates the response of atrial myocytes to parasympathetic stimulation. Here, TGFbeta(1) is shown to inhibit expression of the M(2) muscarinic receptor (M(2)), which plays a critical role in the parasympathetic response of the heart. This effect is mimicked by overexpression of a dominant negative mutant of RhoA and by the RhoA kinase inhibitor Y27632, whereas adenoviral expression of a dominant activating-RhoA reverses TGFbeta inhibition of M(2) expression. TGFbeta(1) also mediates a decrease in GTP-bound RhoA and a reciprocal increase in the expression of the RhoA GTPase-activating protein, p190RhoGAP, whereas total RhoA is unchanged. Inhibition of M(2) promoter activity by TGFbeta(1) is mimicked by overexpression of p190RhoGAP, whereas a dominant negative mutant of p190RhoGAP reverses this effect of TGFbeta(1). In contrast to atrial myocytes, in mink lung epithelial cells, in which TGFbeta signaling through activation of RhoA has been previously identified, TGFbeta(1) stimulated an increase in GTP-bound RhoA in association with a reciprocal decrease in the expression of p190RhoGAP. Both effects demonstrated a similar dose dependence on TGFbeta(1). Thus TGFbeta regulation of M(2) muscarinic receptor expression is dependent on RhoA, and TGFbeta regulation of p190RhoGAP expression may be a cell type-specific mechanism for TGFbeta signaling through RhoA.

CT and MRI of acquired portal venous system anomalies.

In this educational presentation, we offer an overview of acquired anomalies of the portal venous system explored by biphasic helical CT and MRI. Portosystemic collateral vessels, cavernous transformation of the portal vein, intrahepatic vascular shunts, aneurysms of the portal venous system, thrombosis of the portal venous system, and gas in the portal venous system will be discussed. For liver surgery and interventional procedures it is necessary to have a correct mapping of normal anatomy, variants, and different pathologies involving the portal venous system.

Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis.

Human umbilical vein endothelial cells (HUVECs) have played a major role as a model system for the study of the regulation of endothelial cell function and the role of the endothelium in the response of the blood vessel wall to stretch, shear forces, and the development of atherosclerotic plaques and angiogenesis. Here, we use HUVECs and human microvascular endothelial cells to study the role of the HMG-CoA reductase inhibitor, simvastatin, and the small GTP-binding protein Rho in the regulation of angiogenesis. Simvastatin inhibited angiogenesis in response to FGF-2 in the corneal pocket assay of the mouse and in vascular endothelial growth factor (VEGF)-stimulated angiogenesis in the chick chorioallontoic membrane. Furthermore, simvastatin inhibited VEGF-stimulated tube formation by human dermal microvascular endothelial cells and the formation of honeycomb-like structures by HUVECs. The effect was dose-dependent and was not secondary to apoptosis. Geranylgeranyl-pyrophosphate (GGPP), a product of the cholesterol metabolic pathway that serves as a substrate for the posttranslational lipidation of RhoA, was required for membrane localization, but not farnesylpyrophosphate (FPP), the substrate for the lipidation of Ras. Furthermore, GGTI, a specific inhibitor of GGPP, mimicked the effect of simvastatin of tube formation and the formation of honeycombs whereas FTI, a specific inhibitor of the farnesylation of Ras, had no effect. Adenoviral expression of a DN-RhoA mutant mimicked the effect of simvastatin on tube formation and the formation of honeycombs, whereas a dominant activating mutant of RhoA reversed the effect of simvastatin on tube formation. Finally, simvastatin interfered with the membrane localization of RhoA with a dose-dependence similar to that for the inhibition of tube formation. Simvastatin also inhibited the VEGFstimulated phosphorylation of the VEGF receptor KDR, and the tyrosine kinase FAK, which plays a role in cell migration. These data demonstrate that simvastatin interfered with angiogenesis via the inhibition of RhoA. Data supporting a role for angiogenesis in the development and growth of atherosclerotic plaques suggest that this antiangiogenic effect of Statins might prevent the progression of atherosclerosis via the inhibition of plaque angiogenesis.

Modulator recognition factor 1, an AT-rich interaction domain family member, is a novel corepressor for estrogen receptor alpha.

Cardiovascular tissues are important targets of estrogen action. Vascular cells express the two known estrogen receptors (ERs), ERalpha and ERbeta, ligand-activated transcription factors that regulate gene transcription through interactions with both coactivator and corepressor molecules. To isolate ERalpha coregulators in vascular cells, we performed a yeast two-hybrid screen for ERalpha-interacting proteins using a human aorta library. Here we report the identification of modulator recognition factor 1 (MRF1) as an ERalpha-interacting corepressor protein. Full-length MRF1 binds to both the N terminus and the C terminus of ERalpha. ERalpha and MRF1 coimmunoprecipitate in an estradiol-independent manner, and recombinant ERalpha binds to both full-length and COOH-terminal MRF1 in the absence of estradiol. MRF1 also interacts in a ligand-dependent manner with thyroid receptor alpha, retinoid X receptor alpha, and androgen receptor, and in a ligand-independent manner with ERbeta and the retinoic acid receptor. MRF1 RNA is highly expressed in aorta, heart, skeletal muscle, and liver. MRF1 has intrinsic repressor activity in an in vitro GAL reporter assay. Transient transfection studies show that MRF1 represses transcription by ERalpha activated by estradiol in a dose-dependent manner, as well as by the selective ER modulators 4-hydroxy-tamoxifen and raloxifene. MRF1 repression is not influenced by pharmacological inhibition of histone deacetylase. These data identify MRF1 as a repressor of ERalpha-mediated transcriptional activation and support a role for MRF1 in regulating ER-dependent gene expression in cardiovascular and other cells.