Polymorphisms of norepinephrine transporter and adrenergic receptor alpha1D are associated with the response to beta-blockers in dilated cardiomyopathy.

Recent clinical trials have clearly demonstrated that the administration with beta-blockers decreases the mortality in the patients with chronic heart failure (CHF). However, significant heterogeneity exists in the effectiveness of beta-blockers among individual cases. We focused on 39 polymorphisms in 16 genes related to adrenergic system and investigated their association with the response to beta-blockers among 80 patients with CHF owing to idiopathic dilated cardiomyopathy. The polymorphisms of NET T-182C (P=0.019), ADRA1D T1848A (P=0.023) and ADRA1D A1905G (P=0.029) were associated with the improvement of left ventricular fractional shortening (LVFS) by beta-blockers. Furthermore, combined genotype analysis of NET T-182C and ADRA1D T1848A revealed a significant difference in LVFS improvement among genotype groups (P=0.011). These results suggest that NET (T-182C) and ADRA1D (T1848A and A1905G) polymorphisms are predictive markers of the response to beta-blockers. Genotyping of these polymorphisms may provide clinical insights into an individual difference in the response to the beta-blocker therapy in CHF.

Novel quantitative assessment of myocardial perfusion by harmonic power Doppler imaging during myocardial contrast echocardiography.

OBJECTIVE: To test the hypothesis that the power of the received signal of harmonic power Doppler imaging (HPDI) is proportional to the bubble concentration under conditions of constant applied acoustic pressure, and to determine whether a new quantitative method can overcome the acoustic field inhomogeneity during myocardial contrast echocardiography (MCE) and identify perfusion abnormalities caused by myocardial infarction. METHODS: The relation between Levovist concentration and contrast signal intensity (CI) of HPDI was investigated in vitro under conditions of constant acoustic pressure. MCE was performed during continuous infusion of Levovist with intermittent HPDI every sixth cardiac cycle in 11 healthy subjects and 25 patients with previous myocardial infarction. In the apical views myocardial CI (CI(myo)) was quantified in five myocardial segments. The CI from the left ventricular blood pool adjacent to the segment was also measured in dB and subtracted from the CI(myo) (relative CI (RelCI)). RESULTS: CI had a logarithmic correlation and the calculated signal power a strong linear correlation with Levovist concentration in vitro. Thus, a difference in CI of X dB indicates a microbubble concentration ratio of 10(X/10). In normal control subjects, CI(myo) differed between the five segments (p < 0.0001), with a lower CI(myo) in deeper segments. However, RelCI did not differ significantly between segments (p = 0.083). RelCI was lower (p < 0.0001) in the 39 infarct segments (mean (SD) -18.6 (2.8) dB) than in the 55 normal segments (mean (SD) -15.1 (1.6) dB). RelCI differed more than CI(myo) between groups. CONCLUSIONS: The new quantitative method described can overcome the acoustic field inhomogeneity in evaluation of myocardial perfusion during MCE. RelCI represents the ratio of myocardium to blood microbubble concentrations and may correctly reflect myocardial blood volume fraction.

Aromatase is abundantly expressed by neonatal rat penis but downregulated in adulthood.

Although synthesis of estrogen by male gonads has been well documented for over half a century, it is only recently that the role of estrogen in male reproductive events has gained appreciation. We recently reported abundant expression of estrogen receptor (ER)-alpha and -beta in different cell types of the rat penis, whose levels diminished with advancing age. The present study, which builds on data from the ER study, was designed to determine whether the penis is capable of generating its own local estrogen by examining evidence of the expression of aromatase, a microsomal enzymatic complex which irreversibly converts androgens to estrogens, using immunohistochemistry, Western blotting, in situ hybridization and real-time PCR analyses. Secondly, the effects of sex steroid hormones on penile aromatase were examined. Discrete aromatase immunoreactive cells were localized in primordial corpus cavernosum, corpus spongiosus and os penis, blood vessels and sensory corpuscle of glans penis. In situ hybridization signals corresponded with immunohistochemical findings. Western blot, enzyme immunoassay and real-time PCR analyses of rat penile samples revealed an age-dependent expression of aromatase and estrogen, with levels at week 1 almost resembling those of the ovary, but they decreased sharply by week 8, and decreased further by week 35. This expression pattern was strikingly similar to that of ER-alpha reported previously. Testosterone and diethylstilbesterol administered prenatally upregulate levels of aromatase mRNA and protein, and estrogen postnatally. Dihydrotestosterone upregulated aromatase mRNA and protein, but not estrogen. We conclude that estrogen acts via ER in a paracrine and/or autocrine manner to regulate penile events, particularly during development, and that estrogen synthesis is regulated by estrogen and androgens.

Effect of flow disturbances remaining at the beginning of diastole on intraventricular diastolic flow and colour M-mode Doppler echocardiograms.

A computational model of the fluid dynamics of intraventricular flow was used to investigate the importance of the effects of flow disturbances existing within the left ventricle (LV) at the onset of diastole on a diastolic flow field. The simulation started with a quiescent flow state; it continued for a number of cardiac cycles to obtain a cyclically repeatable flow. After the flow became periodic, the initial diastolic flow was not quiescent: flow disturbances, remnants of a systolic flow, were present within the LV. Nevertheless, they faded away during an acceleration phase of diastole and almost ceased by the end of this phase. Consequently, a flow field during a deceleration phase of diastole, characterised by the formation of a vortex ring, was hardly affected by the initial flow disturbances. The propagation velocity of a colour M-mode Doppler echocardiogram obtained by scanning velocity along the LV long axis was 0.58 m s(-1) in the case where diastolic flow was initially quiescent and 0.56 m s(-1) in the case where flow disturbances existed at the beginning of diastole. These results indicated that the colour M-mode Doppler echocardiographic technique captures flow dynamics produced purely by ventricular expansion, with little influence from initial diastolic flow disturbances.

A cardiac sodium channel mutation identified in Brugada syndrome associated with atrial standstill.

Mutations in the cardiac Na+ channel gene SCN5A are responsible for multiple lethal ventricular arrhythmias including Brugada syndrome and congenital long QT syndrome. Here we report a case of Brugada syndrome with ST elevation in the right precordial and inferior leads accompanied by atrial standstill and spontaneous ventricular fibrillation. Atrial standstill and J wave elevation were provoked by procainamide. Genetic analysis revealed a missense mutation (R367H) in SCN5A. The resultant mutant Na+ channel was nonfunctional when expressed heterologously in Xenopus oocytes. Our study suggests that genetic defects in SCN5A may be associated with atrial standstill in combination with ventricular arrhythmias.

Diminished penile expression of vascular endothelial growth factor and its receptors at the insulin-resistant stage of a type II diabetic rat model: a possible cause for erectile dysfunction in diabetes.

Erectile dysfunction (ED) is commonly experienced in men with diabetes mellitus. Vascular endothelial growth factor (VEGF) has been extensively documented for its pathogenic significance in different complications of diabetes. We hypothesized that expressions of VEGF, its receptors and its signaling pathway Akt may be drastically altered in diabetic penile tIssues and their alterations may modulate penile expression of the molecules that are believed to play a role in diabetic ED. Otsuka Long-Evans Fatty (OLETF) rats, a type II (non-insulin-dependent) diabetes mellitus, were used at the insulin-resistant stage of type II diabetes (20 weeks of age). We determined protein and mRNA expressions of VEGF, its receptors, Akt, nitric oxide synthase isoforms, and apoptosis-related molecules in the penis using immunohistochemistry, Western blotting, in situ hybridization, and real-time quantitative PCR analyses. The penile sections were also submitted to the Tdt-mediated dUTP nick end labeling assay for apoptosis. OLETF rats showed marked reductions in penile expression of VEGF, its two receptors and Akt. In OLETF rat penises, endothelial and neuronal nitric oxide synthase isoforms were expressed less abundantly. Furthermore, while anti-apoptotic markers, Bcl-2 and phosphorylated Bad, were down-regulated, pro-apoptotic markers, active caspase-3 and Bax, were up-regulated, resulting in the appearance of apoptotic cells in the penile tIssues of OLETF rats. The VEGF signaling system would work less well in diabetic penile tIssues as a result of the reduced expression, leading to diminished endothelial production of nitric oxide and apoptosis-related erectile tIssue damage. We propose that the abnormalities of the VEGF signaling system in the penis may play a role in the pathophysiology of diabetic ED.

Computational study on the evolution of an intraventricular vortical flow during early diastole for the interpretation of color M-mode Doppler echocardiograms.

A computational fluid dynamics study of intraventricular flow during early diastole was carried out using a 3D model of the human left ventricle (LV). It was found that a vortical flow formed under the aortic orifice and then grew in size and extended laterally along the ventricular wall towards the posterior side. With further expansion of the LV, it developed into an annular vortex asymmetrically enlarged on the side of the aortic orifice, narrowing the passage of blood inflow and thus causing a shift of the high-velocity portion of inflow towards the apex. This appeared as an elongation of the aliasing area when the velocity of the inflow was expressed as a spatiotemporal map in the same manner as a color M-mode Doppler (CMD) echocardiogram. Based on these findings, it was concluded that the shape of the aliasing area in a CMD echocardiogram shows the change in the velocity of blood inflow affected by the development of an annular vortex formed in the LV.

Transient left ventricular dysfunction in ischaemic myocardium after stress: comparative study with exercise and pharmacological stress gated myocardial single photon emission computed tomography.

In ischaemic heart disease patients, transient left ventricular dysfunction is observed due to post-exercise stunning. The aim of this study was to determine whether transient left ventricular dysfunction could also be seen after short-acting pharmacological stress (adenosine triphosphate). A 1 day rest/stress gated myocardial single photon emission computed tomography was performed on 362 patients suspected of having ischaemic heart disease by exercise (n=199) or short-acting pharmacological stress (n=163). Left ventricular ejection fraction were estimated both at rest and stress. Based on perfusion findings, patients were subdivided into ischaemia, fixed defect and normal group. For the ischaemia and fixed defect group, left ventricular ejection fraction after stress was significantly decreased compared with the resting value by exercise stress (ischaemia group, 57.5+/-11.0 vs 60.4+/-10.4; fixed defect group, 47.7+/-16.7 vs 49.6+/-16.8; P<0.01), but not by pharmacological stress (ischaemia group, 55.8+/-13.4 vs 57.1+/-13.8; fixed defect group, 50.8+/-13.5 vs 50.6+/-13.1; P=NS). In the normal group, left ventricular ejection fraction after stress was not significantly changed by either exercise (65.7+/-10.4 vs 66.8+/-10.2; P=NS) or pharmacological stress (63.0+/-11.7 vs 64.0+/-12.1; P=NS). It is concluded that a transient decrease in left ventricular ejection fraction after stress was observed following post-exercise, not following a short-acting pharmacological stress in patients showing perfusion abnormalities. Transient left ventricular dysfunction may be the result of post-exercise stunning, not from subendocardial hypoperfusion induced by short-acting pharmacological stress.

Long-acting calcium channel blocker benidipine suppresses expression of angiogenic growth factors and prevents cardiac remodelling in a Type II diabetic rat model.

AIMS/HYPOTHESIS: Calcium channel blockers, widely used for the treatment of hypertension and angina, could prevent cardiovascular complications in patients with diabetes. They can improve cardiac remodelling in animal models of a variety of cardiovascular diseases. Here, we examined the therapeutic effect of benidipine, a long-acting calcium channel blocker, on cardiac remodelling in Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a Type II (non-insulin-dependent) diabetes mellitus model. METHODS: The methods for morphometric analysis included double staining for coronary capillaries, dye-binding staining for collagen content and Masson's trichrome staining for perivascular fibrosis. Immunohistochemical and in situ hybridization techniques were used for detecting protein and mRNA expressions for vascular endothelial growth factors (VEGF), basic fibroblast growth factors (bFGF) and TGF-beta(1), endothelial nitric oxide synthase (eNOS), and anti- and pro-apoptotic markers. RESULTS: OLETF rats showed an increased coronary capillary density, a reduced venular capillary proportion, an increased cardiac collagen content and prominent cardiac perivascular fibrosis. In OLETF rat hearts, significant increases in vascular expressions for VEGF, bFGF and TGF- beta(1) were found. Furthermore, the apoptosis signalling pathways, involving eNOS and apoptotic markers, were markedly altered, and coronary endothelial cell apoptosis was lower. These alterations with the exception of eNOS expression were significantly blocked by benidipine treatment. CONCLUSION/INTERPRETATION: The suppressive effect of benidipine on overproduction of angiogenic growth factors could prevent cardiac angiogenesis and fibrosis, resulting in an improvement of cardiac remodelling in diabetes. As VEGF and bFGF potently block endothelial cell apoptosis execution, physiological apoptosis revived by benidipine treatment could also contribute to coronary vessel regression.

Assessment of newly developed perfluorocarbon emulsion: oxygen carrying capacity as the blood substitute in vivo.

This study provides the evaluation of oxygen carrying capacity of the novel perfluorocarbon emulsion (Neo-PFC) produced by the new emulsifying technology named High Pressure Process. For the performance comparison of oxygen carrying abilities of Neo-PFC and a representative PFC emulsion, the oxidation states of cerebral tissues in substituted animals were measured by near-infrared spectrometry. After the 70% exchange transfusion of whole blood of rats by Neo-PFC and Fluosol-DA, fractional inspired oxygen (FiO2) was gradually decreased from 100% to 0%. As the control experiments, the blood was substituted by Krebs Ringer bicarbonate buffer containing 3% BSA. When the blood of rats was substituted by Neo-PFC, Cyt. ox., a terminal enzyme in mitochondrial respiratory chain maintained fully oxidized state with FiO2 values between 100 to 40%. By contrast, in the models substituted by Fluosol-DA and BSA-buffer. Cyt. ox. was gradually reduced with FiO2 values below 60% and 80%, respectively. This specific advantage of Neo-PFC was explained by its higher oxygen solubility in arterial blood. The novel PFC emulsion prepared by the new emulsifying technology is a potential basis for blood substitutes.

Allograft inflammatory factor-1 augments production of interleukin-6, -10 and -12 by a mouse macrophage line.

Mouse allograft inflammatory factor-1 (AIF-1) cDNA was cloned and the AIF-1-specific monoclonal antibodies were established to examine its tissue distribution. The mouse AIF-1 was highly conserved among all reported AIF-1 from a variety of species, from invertebrates to mammals, and the cloned cDNA was in good accordance with putative expressed regions of genomic sequences in the mouse major histocompatibility complex (MHC) class III region. The messages of mouse AIF-1 were abundantly expressed in the testis, moderately in the spleen and lymph nodes and slightly in the liver and thymus of normal BALB/c mice. Immunohistological examination revealed that differentiating germ cells in the testis and presumably macrophages in the red pulp of the spleen were positive for AIF-1. To analyse the function of the AIF-1, a macrophage cell line, RAW 264.7, was transfected with mouse AIF-1 cDNA. Upon stimulation with bacterial lipopolysaccharide, the transfectants that overexpressed AIF-1 showed marked morphological changes and produced significantly large amounts of interleukin (IL)-6, IL-10 and IL-12p40 but not IL-12p70 compared with control cells. No difference was noted in production of tumour necrosis factor-alpha, transforming growth factor-beta1 and IL-1alpha. These results suggest that AIF-1 plays an important role in cells of a monocyte/macrophage lineage upon stimulation with inflammatory stimuli by augmenting particular cytokine production.

Phenotypic modulation of vascular smooth muscle cells induced by unsaturated lysophosphatidic acids.

The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one is critically involved in the development and progression of atherosclerosis. Although many cytokines and growth factors have been reported as atherogenic factors, the critical pathogens for inducing atherosclerosis remain unknown, largely because proper examining systems of them have not been developed. We recently established primary culture systems for visceral SMCs and VSMCs in which both SMCs, when cultured on laminin with insulin-like growth factor-I, show a differentiated phenotype, as indicated by a spindle-like shape, ligand-induced contractility, and a high level of SMC differentiation marker gene expression. In this study, we searched for critical dedifferentiation factors for these SMCs using our culture system. We found that polar lipids extracted from human serum markedly induced VSMC dedifferentiation, and this activity was solely present in the lysophosphatidic acid (LPA) fraction. Among several LPA species detected in human serum lipids, unsaturated LPAs were identified as major contributors to the induction of VSMC dedifferentiation. Signaling and phenotype analyses revealed that unsaturated LPA-induced VSMC dedifferentiation is mediated through the coordinated activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. Thus, this report demonstrates the first finding that unsaturated LPAs, but not saturated LPAs, specifically induce VSMC phenotypic modulation, suggesting that these molecules could function as atherogenic factors.

Clinical significance of noninvasive assessment of left ventricular diastolic function by Doppler echocardiography.

Transmitral flow velocity pattern obtained by the pulsed Doppler technique reflects left ventricular (LV) diastolic function, but an increase in left atrial pressure pseudonormalizes the flow pattern and masks diastolic dysfunction. We propose an index to assess LV diastolic function using color M-mode Doppler echocardiography and the base-line shift technique. This index, flow propagation velocity, represents the average velocity of early diastolic LV filling flow from the mitral orifice to mid-ventricle. In patients with ischemic heart disease and dilated cardiomyopathy including those with pseudonormalized transmitral flow pattern, flow propagation velocity had a good correlation with the time constant of early diastolic LV pressure decay (Tau), indicating that flow propagation velocity is a useful noninvasive parameter of diastolic function which does not pseudonormalize. We also found a significant correlation between flow propagation velocity and Tau in hypertrophic cardiomyopathy patients in contrast to inadequate or no correlation between each of the conventional parameters and Tau. In addition, our recent data suggest that flow propagation velocity was distinctly decreased even in the patients with hypertension who did not show significant increase in LV mass index. Flow propagation velocity is a unique noninvasive parameter of LV diastolic function, which can sensitively and accurately detect the diastolic impairment in patients with different types of cardiac diseases with various loading conditions.

Vascular smooth muscle cell proliferation is effectively suppressed by the non-specific growth factor inhibitor suramin.

The purpose of this study was to investigate the effects of the non-specific growth factor inhibitor suramin on smooth muscle cell proliferation in vitro and in vivo. Cultured vascular smooth muscle cells (VSMC) were stimulated by platelet-derived growth factor (PDGF) and cellular DNA synthesis assessed by [3H]-thymidine uptake. Suramin dose-dependently inhibited DNA synthesis in VSMC, and 100 microM of suramin completely suppressed the PDGF-AB-induced cellular DNA synthesis. Rabbit carotid arteries were injured by the balloon catheter, and then suramin locally delivered using a porous balloon catheter over ten minutes. Three weeks after the vascular injury, the extent of intimal thickening was compared between the suramin-treated and control rabbits. The neointimal formation triggered by balloon-mediated vascular injury was suppressed significantly and dose-dependently by locally infused suramin, and the intima to media area ratios of the control and 1 mM suramin-treated animals were 48.8+/-14.9 and 12.2+/-6.0%, respectively (p < 0.01. n = 6 for each group). These results suggest that one time local administration of suramin was sufficient to suppress neointimal formation after balloon-mediated vascular injury, and that pharmacological intervention targeting the growth factor's signaling pathways could be a promising approach to prevent smooth muscle cell proliferation in various proliferative vascular diseases.

Low-dose dobutamine electrocardiograph-gated myocardial SPECT for identifying viable myocardium: comparison with dobutamine stress echocardiography and PET.

UNLABELLED: The identification of severely dysfunctional but viable myocardium is of particular importance for the selection of patients with depressed left ventricular function who will benefit from coronary revascularization. Assessment of inotropic reserve with dobutamine has recently been used for this purpose. This study compared the accuracy of low-dose dobutamine stress gated myocardial SPECT (DS SPECT) with the accuracy of dobutamine stress echocardiography (DSE) and resting perfusion SPECT for the identification of viable myocardium in patients with previous myocardial infarction. METHODS: Resting and low-dose dobutamine (7.5 microg/kg/min) gated (99m)Tc-tetrofosmin SPECT and echocardiography and resting (18)F-FDG PET were prospectively studied in 23 patients with previous myocardial infarction and severely depressed regional function. Twenty-one of them were successfully studied with each technique. The left ventricular wall was divided into 14 segments to assess wall motion using a 5-point scale. PET viability was defined as FDG uptake >/= 50% of the maximum uptake in a region with normal wall motion. For DS SPECT and DSE studies, viable myocardium was defined as hypokinetic areas with > or = 1 point improvement in wall motion. For resting perfusion SPECT, viable myocardium was defined as hypokinetic areas with a relative uptake > or = 50% of the maximum uptake. RESULTS: Of a total of 294 segments, 55 had severe resting dyskinesis. Thirty-four segments were identified as viable on FDG PET, and 21 segments were identified as nonviable. Eleven segments were inadequately visualized with DSE, including 5 segments in the apex. Sensitivities (78% vs. 76%) and specificities (94% vs. 100%) were similar for DSE and DS SPECT, with a concordance of 86% (kappa = 0.72). DS SPECT and perfusion SPECT did not significantly differ with respect to sensitivities (76% vs. 85%, respectively). However, specificity was significantly higher for DS SPECT than for perfusion SPECT (100% vs. 52%, respectively, P < 0.05). CONCLUSION: This study indicated that DS SPECT correlates well with DSE in the assessment of viability. In addition, gated SPECT can evaluate regional wall motion, even in areas inadequately assessed by echocardiography. DS SPECT may also provide additional information for identifying viable myocardium, which is often overestimated by routine perfusion scans.

Successful gene therapy via intraarticular injection of adenovirus vector containing CTLA4IgG in a murine model of type II collagen-induced arthritis.

We previously constructed an adenovirus vector carrying a gene encoding a soluble form of fusion protein, consisting of the extracellular portion of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglobulin G1 (Adex1CACTLA4IgG). Murine type II collagen-induced arthritis (CIA) was treated with Adex1CACTLA4IgG. A single intraarticular injection of 1 x 10(5) PFU was able to support serum CTLA4IgG at more than 10 microg/ml for at least 12 weeks and was able to inhibit the CIA clinically and histologically. In contrast, intravenous, intramuscular, or subcutaneous injection of 1 x 10(5) PFU was unable to support a significant level of serum CTLA4IgG and thus was unable to inhibit the development of arthritis. Thus, we demonstrated that (1) a low-dose intraarticular injection of Adex1CACTLA4IgG was effective in delaying the onset of CIA and reducing the severity of arthritis; (2) an intraarticular (knee joint) injection of Adex1CACTLA4IgG effectively blocked the development of arthritis in distal paws; (3) the inhibitory effect of Adex1CACTLA4IgG lasted at least up to 20 weeks; (4) although serum CTLA4IgG at more than 10 microg/ml persisted for at least 12 weeks, mice treated by intraarticular injection of Adex1CACTLA4IgG were not anergic to adenovirus and were able to mount antibody responses against various antigens.

Preparation and characterization of SNO-PEG-hemoglobin as a candidate for oxygen transporting material.

Acellular hemoglobin (Hb) derivates developed as oxygen carriers are known to cause hypertensive reactions due to their nitric oxide (NO) scavenging action. To modulate this undesired activity, we have developed a new Hb derivative, s-nitrosylated polyethylene glycol (PEG)-modified hemoglobin (SNO-PEG-Hb), which can deliver oxygen and NO. After human Hb was modified with PEG to increase its molecular weight, the free sulfhydryl groups of Hb were s-nitrosylated with s-nitrosoglutathione. Administration of unmodified Hb into anesthetized rats caused a hypertensive reaction, while s-nitrosylated Hb derivatives such as SNO-Hb and SNO-PEG-Hb did not raise blood pressure. The plasma half-lives of heme and NO bound to SNO-PEG-Hb were 11.5 and 2.4 hours respectively, indicating that the s-nitrosylated Hb derivative may act as a slow-releasing agent for NO. Based on these findings, SNO-PEG-Hb is a useful candidate for a blood substitute and tool for oxygen therapeutics.

Angiotensin-converting enzyme inhibition attenuates hypofibrinolysis and reduces cardiac perivascular fibrosis in genetically obese diabetic mice.

BACKGROUND: Obesity and insulin resistance are associated with accelerated macrovascular and microvascular coronary disease, cardiomyopathic phenomena, and increased concentrations and activity in blood of plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of fibrinolysis. METHODS AND RESULTS: To determine whether hypofibrinolysis in blood and tissues and its potential sequelae could be attenuated pharmacologically, we studied genetically modified obese mice. By 10 weeks of age, obese mice exhibited increases in left ventricular weight and glucose and immunoreactive insulin in blood. PAI-1 activity in blood measured spectrophotometrically was significantly elevated as well. The difference compared with values in lean controls widened by 20 weeks of age. Perivascular fibrosis in coronary arterioles and small coronary arteries was evident in obese mice 10 and 20 weeks of age, paralleling increases in PAI-1 and tissue factor expression evident by immunohistochemical image analysis, in situ hybridization, and reverse transcription-polymerase chain reaction. Inhibition of ACE activity initiated in obese mice 10 weeks of age and continued for 20 weeks arrested the increase in PAI-1 activity in blood and in cardiac PAI-1 and tissue factor mRNA as well as coronary perivascular fibrosis. CONCLUSIONS: Thus, inhibition of proteo(fibrino)lysis and augmented tissue factor expression in the heart precede and may contribute to the coronary perivascular fibrosis seen with obesity and insulin resistance. Furthermore, inhibition of ACE activity can attenuate all 3 phenomena.

Critical role of Rho-kinase and MEK/ERK pathways for angiotensin II-induced plasminogen activator inhibitor type-1 gene expression.

Plasminogen activator inhibitor type-1 (PAI-1) plays an integral role not only in the regulation of fibrinolytic activity but also in the pathogenesis of atherosclerosis and hypertension. We investigated the signaling pathways of angiotensin II (Ang II) leading to PAI-1 gene expression. Ang II increased the PAI-1 mRNA and protein levels in a time- and dose-dependent manner through the Ang II type 1 receptor in vascular smooth muscle cells. PAI-1 gene promoter activity measured by luciferase assay was significantly increased by Ang II. PAI-1 mRNA stability was also increased by Ang II. Ang II-induced PAI-1 mRNA upregulation was inhibited by BAPTA-AM, genistein, and AG1478, suggesting that intracellular calcium, tyrosine kinase, and epidermal growth factor receptor transactivation are involved. Furthermore, PD98059, an inhibitor of extracellular signal-regulated kinase (ERK) kinase (MEK), almost completely suppressed Ang II-induced PAI-1 upregulation. Adenovirus-mediated overexpression of the dominant-negative form of Rho-kinase or Y27632, a Rho-kinase inhibitor, also completely prevented PAI-1 induction by Ang II without affecting Ang II-induced ERK activation. These data suggest that activation of MEK/ERK and Rho-kinase pathways plays a pivotal role in PAI-1 gene upregulation by Ang II. The Rho-kinase pathway may be a novel target to inhibit Ang II signaling, and its inhibition may be useful in the treatment of hypertension as well as atherosclerosis.

Mixed allogeneic chimerism with wild-type strains ameliorates atherosclerosis in apolipoprotein E-deficient mice.

Atherosclerosis involves inflammatory processes between vascular tissues and hematocytes with a hyperlipidemic background. To examine whether variations of hematocytes constitute one of the genetic components in atherosclerosis, irradiated apolipoprotein E (apoE)-deficient (apoE(-/-)) mice with hypercholesterolemia and preexisting atherosclerotic lesions were reconstituted with mixed bone marrow cells (BMC) from syngeneic and wild-type (apoE(+/+); atherosclerosis-resistant SJL or -susceptible B10.S) mice. Stable mixed allogeneic chimeras with small amounts of serum apoE were established without any detrimental complications. Compared with untreated apoE(-/-) mice or apoE(-/-) mice transplanted with syngeneic BMC alone, significant reduction of the cholesterol level and significant lesion regression were observed in the mixed chimeras. Furthermore, mixed chimeras given SJL BMC showed marked reductions in numbers of lesions compared with those reconstituted with B10.S BMC. Cholesterol levels in the former SJL chimeras, however, were significantly higher than those in the latter B10.S chimeras. These findings indicate that the resistance of SJL to atherosclerosis resides in the bone marrow-derived cells.