ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

When adherent cells, such as epithelial or endothelial cells, are detached and continuously maintained in suspension, they undergo a form of programmed cell death termed anoikis. We demonstrate that coincident with endothelial cell detachment, there is a dramatic rise in the intracellular level of reactive oxygen species (ROS). Reattachment to a solid surface rapidly attenuates the level of ROS. The mitochondria appear to be the major source of the detachment-induced rise in ROS. The change in the intracellular redox state appears to contribute to endothelial anoikis, because treatment with either the cell-permeant antioxidantN-acetylcysteine or the flavin protein inhibitor diphenylene iodonium is demonstrated to reduce oxidant levels and protect against subsequent cell death. Similarly, the endogenous intracellular level of ROS is shown to correlate with the extent of cell death. Finally, we demonstrate that the activities of both caspases and of the c-Jun N-terminal kinases are modulated by the rise in intracellular ROS levels. These results suggest that oxidants serve as signaling molecules and regulators of anoikis.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The goal of the present studies was to determine whether phospholipid oxidation products and/or plateletactivating factor (PAF) are mediators of early atherogenesis in vivo. Monocyte-endothelial cell interactions have been shown to play an important role in early atherogenesis. We and others have demonstrated that PAF and phospholipid oxidation products, present in atherosclerotic lesions, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 1-palmitoryl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC), mediate the activation of monocytes and/or endothelial cells in vitro. Previous studies have shown that the action of PAF and PAF-like ether-containing phospholipids was inhibited by WEB 2086. We now demonstrate that pretreatment of human aortic endothelial cells with WEB 2086 (10 μmol/L) and several other PAF antagonists before treatment with POVPC and PEIPC but not PGPC prevented the activation of the endothelial cells to bind monocytes. We present evidence to suggest that this inhibition is not mediated by the PAF receptor. The role of bioactive oxidized phospholipids in fatty streak formation was tested using C57BL/6J LDL R−/− mice fed a chow or Western diet for 5 weeks with or without WEB 2086 mixed with drinking water. Quantitative electrospray ionization mass spectrometry showed similar concentrations of WEB 2086 in the plasma of mice on both diets (≈4 to 5 μmol/L); this concentration was inhibitory in vitro. Administration of WEB 2086 did not affect the lipid composition of mouse plasma. However, fatty streak formation was reduced by 62% in animals fed a Western diet, whereas no change was observed in the small lesions of mice on a chow diet. These studies provide evidence that PAF and/or PAF-like phospholipid oxidation products are important mediators of atherosclerotic lesion development in vivo and that specific receptor antagonists for these molecules may represent a novel therapeutic modality.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

E1A can evoke G1 exit in cardiac myocytes and other cell types by displacing E2F transcription factors from tumor suppressor "pocket" proteins and by a less well-characterized p300-dependent pathway. Bypassing pocket proteins (through overexpression of E2F-1) reproduces the effect of inactivating pocket proteins (through E1A binding); however, pocket proteins associate with a number of molecular targets apart from E2F. Hence, pocket protein binding by E1A might engage mechanisms for cell cycle reentry beyond those induced by E2F-1. To test this hypothesis, we used adenoviral gene transfer to express various E2F-1 and E1A proteins in neonatal rat cardiac myocytes that are already refractory to mitogenic serum, in the absence or presence of several complementary cell cycle inhibitors-p16, p21, or dominant-negative cyclin-dependent kinase-2 (Cdk2). Rb binding by E2F-1 was neither necessary nor sufficient for G1 exit, whereas DNA binding was required; thus, exogenous E2F-1 did not merely function by competing for the Rb "pocket." E2F-1-induced G1 exit was blocked by the "universal" Cdk inhibitor p21 but not by p16, a specific inhibitor of Cdk4/6; p21 was permissive for E2F-1 induction of cyclins E and A, but prevented their stimulation of Cdk2 kinase activity. In addition, E2F-1-induced G1 exit was blocked by dominant-negative Cdk2. Forced expression of cyclin E induced endogenous Cdk2 activity but not G1 exit. Thus, E2F-1-induced Cdk2 function was necessary, although not sufficient, to trigger DNA synthesis in cardiac muscle cells. In contrast, pocket protein-binding forms of E1A induced G1 exit that was resistant to inhibition by p21, whereas G1 exit via the E1A p300 pathway was sensitive to inhibition by p21. Both E1A pathways-via pocket proteins and via p300-upregulated cyclins E and A and Cdk2 activity, consistent with a role for Cdk2 in G1 exit induced by E1A. However, p21 blocked Cdk2 kinase activity induced by both E1A pathways equally. Thus, E1A can cause G1 exit without an increase in Cdk2 activity, if the pocket protein-binding domain is intact. E1A also overrides p21 in U2OS cells, provided the pocket protein-binding domain is intact; thus, this novel function of E1A is not exclusive to cardiac muscle cells. In summary, E1A binding to pocket proteins has effects beyond those produced by E2F-1 alone and can drive S-phase entry that is resistant to p21 and independent of an increase in Cdk2 function. This suggests the potential involvement of other endogenous Rb-binding proteins or of alternative E1A targets.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Heart-type fatty acid binding protein (H-FABP), abundantly expressed in cardiac myocytes, has been postulated to facilitate the cardiac uptake of long-chain fatty acids (LCFAs) and to promote their intracellular trafficking to sites of metabolic conversion. Mice with a disrupted H-FABP gene were recently shown to have elevated plasma LCFA levels, decreased cardiac deposition of a LCFA analogue, and increased cardiac deoxyglucose uptake, which qualitatively establishes a requirement for H-FABP in cardiac LCFA utilization. To study the underlying defect, we developed a method to isolate intact, electrically stimulatable cardiac myocytes from adult mice and then studied substrate utilization under defined conditions in quiescent and in contracting cells from wild-type and H-FABP−/−mice. Our results demonstrate that in resting and in contracting myocytes from H-FABP−/−mice, both uptake and oxidation of palmitate are markedly reduced (between −45% and −65%), whereas cellular octanoate uptake, and the capacities of heart homogenates for palmitate oxidation and for octanoate oxidation, and the cardiac levels of mRNAs encoding sarcolemmal FA transporters remain unaltered. In contrast, in resting H-FABP−/−cardiac myocytes, glucose oxidation is increased (+80%) to a level that would require electrical stimulation in wild-type cells. These findings provide a physiological demonstration of a crucial role of H-FABP in uptake and oxidation of LCFAs in cardiac muscle cells and indicate that in H-FABP−/−mice the diminished contribution of LCFAs to cardiac energy production is, at least in part, compensated for by an increase in glucose oxidation.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Smooth muscle cells (SMCs) perform diverse functions that can be categorized as contractile and synthetic. A traditional model holds that these distinct functions are performed by the same cell, by virtue of its capacity for bidirectional modulation of phenotype. However, this model has been challenged, in part because there is no physiological evidence that an adult synthetic SMC can acquire the ability to contract. We sought evidence for this by cloning adult SMCs from human internal thoracic artery. One clone, HITB5, expressed smooth muscle α-actin, smooth myosin heavy chains, heavy caldesmon, and calponin and showed robust calcium transients in response to histamine and angiotensin II, which confirmed intact transmembrane signaling cascades. On serum withdrawal, these cells adopted an elongated and spindle-shaped morphology, random migration slowed, extracellular matrix protein production fell, and cell proliferation and [3H]thymidine incorporation fell to near 0. Cell viability was not compromised, however; in fact, apoptosis rate fell significantly. In this state, agonist-induced elevation of cytoplasmic calcium was even more pronounced and was accompanied by SMC contraction. Readdition of 10% serum completely returned HITB5 cells to a noncontractile, proliferative phenotype. Contractile protein expression increased after serum withdrawal, although modestly, which suggested that the switch to contractile function involved reorganization or sensitization of existing contractile structures. To our knowledge, the physiological properties of HITB5 SMCs provide the first direct demonstration that cultured human adult SMCs can convert between a synthetic, noncontracting state and a contracting state. HITB5 cells should be valuable for characterizing the basis of this critical transition.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The role of endogenous ADP-ribosylation in mediating the activation of the Ca2+-activated K+channels was determined in bovine coronary arteries. Endogenous ADP-ribosylation was examined by incubating coronary arterial homogenates or lysates of cultured coronary arterial smooth muscle cells with [adenylate-32P]NAD. Four32P-labeled proteins were observed at 51, 52, 80, and 124 kDa in the homogenates and lysates. This reaction was enhanced by the addition of 11,12-epoxyeicosatrienoic acid (11,12-EET), a cytochrome P450-derived eicosanoid, and GTP to the incubation. By Western blot analysis, 42- and 70-kDa proteins were recognized by specific antibodies against ADP-ribosyltransferase in the coronary arterial homogenates and smooth muscle cell lysate but not in the lysate of endothelial cells. The 52-kDa acceptor protein of endogenous ADP-ribosylation comigrated with a protein ADP-ribosylated by cholera toxin and was recognized and immunoprecipitated by an anti-GSα antibody. These results suggest that GSα is one of several acceptors of the ADP-ribose moiety. As shown by the patch-clamp technique, 11,12-EET stimulated the activation of the K+channels in the smooth muscle cells, and this activation was completely blocked by novobiocin, vitamin K1, 3-aminobenzamide, andm-iodobenzylguanidine, inhibitors of endogenous mono-ADP-ribosyltransferases. We conclude that endogenous mono-ADP-ribosyltransferases are present in smooth muscle from bovine coronary arteries. These enzymes transfer ADP-ribose to the cellular proteins such as GSα and may mediate intracellular signal transduction in coronary vascular smooth muscle. In the coronary circulation, the ADP-ribosylation signaling pathway may play an important role in mediating the activation of the K+channels induced by 11,12-EET.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5,5′-dimethyl-1-pyrroline-N-oxide, in the presence of NADH and succinate as a substrate for NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (P<0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (274±13 versus 136±9 nmol · min−1· mg−1protein,P<0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The development of congestive heart failure (CHF) is associated with left ventricle (LV) dilation and myocardial remodeling. The matrix metalloproteinases (MMPs) play a significant role in extracellular remodeling, and recent studies have demonstrated increased MMP expression and activity with CHF. Whether increased MMP activity directly contributes to the LV remodeling with CHF remains unknown. Accordingly, this study examined the effects of chronic MMP inhibition (MMPi) on LV size and function during the progression of CHF. Pigs were assigned to the following groups: (1) CHF, rapid pacing for 3 weeks at 240 bpm (n = 12); (2) CHF/MMPi, rapid pacing and concomitant MMPi (PD166793, 20 mg/kg per day [n = 10]), and (3) control (n = 11). With pacing CHF, LV fractional shortening was reduced (19±1 versus 45±1%), and end-diastolic dimension increased (5.67±0.11 versus 3.55±0.05 cm), compared with baseline values (P<0.05). In the CHF/MMPi group, LV endocardial shortening increased (25±2%) and the end-diastolic dimension was reduced (4.92±0.17 cm) compared with CHF-only values (P<0.05). LV midwall shortening was reduced to a comparable degree in the CHF-only and CHF/MMPi groups. LV peak wall stress increased 3-fold with pacing CHF compared with controls and was significantly reduced in the CHF/MMPi group. LV myocardial stiffness was unchanged with CHF but was increased in the CHF/MMPi group. LV myocyte length was increased with pacing CHF compared with controls (180±3 versus 125±4 μm,P<0.05) and was reduced in the CHF/MMPi group (169±4 μm,P<0.05). Basal-state myocyte shortening velocity was reduced with pacing CHF compared with controls (33±2 versus 66±1 μm/s,P<0.05) and was unchanged in the CHF/MMPi group (31±2 μm/s). Using an ex vivo assay system, myocardial MMP activity was increased with pacing CHF and was reduced with chronic MMPi. In summary, concomitant MMPi with developing CHF limited LV dilation and reduced wall stress. These results suggest that increased myocardial MMP activity contributes to LV myocardial remodeling in developing CHF.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The mechanism by which estrogens protect against atherosclerosis is not known. We evaluated in vivo whether there is a gender difference in monocyte adhesion and subendothelial migration in hypercholesterolemic rabbits and whether any gender differences observed are due to estradiol. Monocyte adhesion and subendothelial migration were assessed in a blinded fashion by analyzing a standardized segment of aorta using a scanning electron microscope. We also assessed whether estradiol modulates induction of vascular cell adhesion molecule-1 (VCAM-1) protein using Western blot and flow cytometric analyses. We observed that male rabbits develop more monocyte adhesion and subendothelial migration than do female rabbits during hypercholesterolemia. We also observed that oophorectomized rabbits given physiological estradiol supplementation demonstrate fewer adherent and subendothelial monocytes than do oophorectomized rabbits given placebo. VCAM-1 protein expression was increased in aortae obtained from hypercholesterolemic, oophorectomized animals supplemented with placebo, and this increase was attenuated by estradiol. Finally, in cultured rabbit aortic endothelial cells stimulated with lysophosphatidylcholine, we observed an increase in VCAM-1 protein that was inhibited in a concentration-dependent fashion by estradiol. We have demonstrated in vivo that there is a gender difference in monocyte adhesion to endothelial cells and transendothelial migration after hypercholesterolemia and that this gender difference is due in part to estradiol. Our results also suggest that estradiol inhibits monocyte adhesion by inhibiting expression of VCAM-1.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID