ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The growth-promoting effect of mechanical stress on vascular smooth muscle cells (VSMCs) has been implicated in the progress of vascular disease in hypertension. Extracellular signal-regulated kinases (ERKs) have been implicated in cellular responses, such as vascular remodeling, induced by mechanical stretch. However, it remains to be determined how mechanical stretch activates ERKs. The cytoskeleton seems the most likely candidate for force transmission into the interior of the cell. Therefore, we examined (1) whether the cytoskeleton involves mechanical stretch-induced signaling, (2) whether Rho is activated by stretch, and (3) whether Rho mediates the stretch-induced signaling in rat cultured VSMCs. Mechanical stretch activated ERKs, with a peak response observed at 20 minutes, followed by a significant increase in DNA synthesis. Treatment with the ERK kinase-1 inhibitor, PD98059, inhibited the stretch-induced increase in DNA synthesis. Cytochalasin D, which selectively disrupts the network of actin filaments, markedly inhibited stretch-induced ERK activation. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to mechanical stretch. Botulinum C3 exoenzyme, which inactivates Rho p21 (known to participate in the reorganization of the actin cytoskeleton), attenuated stretch-induced ERK activation. Inhibition of Rho kinase (p160ROCK) also suppressed stretch-induced ERK activation dose dependently. Our results suggest that mechanotransduction in VSMCs is dependent on intact actin filaments, that Rho is activated by stretch, and that Rho/p160ROCK mediates stretch-induced ERK activation and vascular hyperplasia.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

PP1 has previously been described as an inhibitor of theSrc-family kinases p56Lckand FynT. We have therefore decided to use PP1 to determine the functional role ofSrcin platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 againstSrcwas examined. PP1/AGL1872 potently inhibited recombinant p60c-srcin vitro andSrc-dependent tyrosine phosphorylation in p60c-srcF572-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition ofSrckinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF α-receptor was susceptible, albeit to a lesser extent than PDGF β-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for theSrcfamily of tyrosine kinases. It is also a potent inhibitor of the PDGF β-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Angiotensin II and hypertension increase vascular oxidant stress. We examined how these might affect expression of the extracellular superoxide dismutase (ecSOD), a major form of vascular SOD. In mice, angiotensin II infusion (1.1 mg/kg for 7 days) increased systolic blood pressure from 107±3 to 152±9 mm Hg and caused a 3-fold increase in ecSOD, but there was no change in the cytosolic Cu/Zn SOD protein, as determined by Western blot analysis. This was associated with a similar increase in ecSOD mRNA as assessed by RNase protection assay and was prevented by losartan. Induction of ecSOD by angiotensin II was not due to hypertension alone, because hypertension caused by norepinephrine (5.6 mg · kg−1· d−1) had no effect on ecSOD. Similarly, exposure of mouse aortas to angiotensin II (100 nmol/L) in organoid culture increased ecSOD by ≈2-fold. In the organoid culture, angiotensin II-induced upregulation of ecSOD was prevented by losartan (10 μmol/L) and PD985059 (30 μmol/L), a specific inhibitor of p42/44 MAP kinase kinase. Angiotensin II activates the NADH/NADPH oxidase; however, diphenyleneiodonium chloride (10 μmol/L), an inhibitor of this oxidase, did not prevent p42/44 MAP kinase phosphorylation or ecSOD induction by angiotensin II. Finally, in human aortic smooth muscle cells, angiotensin II moderately increased transcriptional rate (as assessed by nuclear run-on analysis) but markedly increased ecSOD mRNA stability. Thus, angiotensin II increases ecSOD expression independent of hypertension, and this increase involves both an increase in ecSOD transcription and stabilization of ecSOD mRNA. This effect of angiotensin II on ecSOD expression may modulate the oxidative state of the vessel wall in pathological processes in which the renin-angiotensin system is activated.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 μg/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 μmol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Disturbed sarcoplasmic reticulum (SR) Ca2−content may underlie the altered force-frequency and postrest contractile behavior in failing human myocardium. We used rapid cooling contractures (RCCs) to assess SR Ca2+content in ventricular muscle strips isolated from nonfailing and end-stage failing human hearts. With an increase in rest intervals (1 to 240 s; 37°C), nonfailing human myocardium (n=7) exhibited a parallel increase in postrest twitch force (at 240 s by 121±44%;P<0.05) and RCC amplitude (by 69±53%;P<0.05). In contrast, in failing myocardium (n=30), postrest twitch force decreased at long rest intervals and RCC amplitude declined monotonically with rest (by 25 ± 9% and 53 ± 9%, respectively;P<0.05). With an increase in stimulation frequencies (0.25 to 3 Hz), twitch force increased continuously in nonfailing human myocardium (n=7) by 71 ± 17% (at 3 Hz;P<0.05) and RCC amplitude increased in parallel by 247 ± 55% (P<0.05). In contrast, in failing myocardium (n=26), twitch force declined by 29 ± 7% (P<0.05) and RCC amplitude increased only slightly by 36 ± 14% (P<0.05). Paired RCCs were evoked to investigate the relative contribution of SR Ca2+uptake and Na+/Ca2+exchange to cytosolic Ca2+removal during relaxation. SR Ca2+uptake (relative to the Na+/Ca2+exchange) increased significantly in nonfailing but not in failing human myocardium as stimulation rates increased. We conclude that the negative force-frequency relation in failing human myocardium is due to an inability of SR Ca2+content to increase sufficiently at high frequencies and thus cannot overcome the frequency-dependent refractoriness of SR Ca2+release. The rest-dependent decay in twitch force in failing myocardium is due to rest-dependent decline in SR Ca2+content. These alterations could be secondary to depressed SR Ca2+-ATPase combined with enhanced cytosolic Ca2+extrusion via Na+/Ca2+exchange.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant α-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the α-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous α-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain α-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC α-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The aim of the present study was to investigate whether and which cardiac growth factors are involved in human hypertrophy, whether growth factor synthesis is influenced by overload type and/or by the adequacy of the hypertrophy, and the relationships between cardiac growth factor formation and ventricular function. Cardiac growth factor formation was assessed by measuring aorta-coronary sinus concentration gradient in patients with isolated aortic stenosis (n = 26) or regurgitation (n = 15) and controls (n = 12). Gene expression and cellular localization was investigated in ventricular biopsies using reverse transcriptase-polymerase chain reaction and in situ hybridization. Cardiac hypertrophy with end-systolic wall stress <90 kdyne/cm2was associated with a selective increased formation of insulin-like growth factor (IGF)-I in aortic regurgitation and of IGF-I and endothelin (ET)-1 in aortic stenosis. mRNA levels for IGF-I and preproET-1 were elevated and mainly expressed in cardiomyocytes. At stepwise analysis, IGF-I formation was correlated to the mean velocity of circumferential fiber shortening (r=0.86,P<0.001) and ET-1 formation to relative wall thickness (r=0.82,P<0.001). When end-systolic wall stress was >90 kdyne/cm2, IGF-I and ET-1 synthesis by cardiomyocytes was no longer detectable, and only angiotensin (Ang) II was generated, regardless of the type of overload. The mRNA level for angiotensinogen was high, and the mRNA was exclusively expressed in the interstitial cells. Ang II formation was positively correlated to end-systolic stress (r=0.89,P<0.001) and end-diastolic stress (r=0.84,P<0.001). Multivariate stepwise analysis selected end-systolic stress as the most predictive variable and left ventricular end-diastolic pressure as the independent variable for Ang II formation (r=0.93,P<0.001). In conclusion, the present results indicate that the course of human left ventricular hypertrophy is characterized by the participation of different cardiac growth factors that are selectively related both to the type of hemodynamic overload and to ventricular function.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID