IRAP deficiency attenuates diet-induced obesity in mice through increased energy expenditure.

UNLABELLED: Activation of the adipose renin-angiotensin system contributes to the development of obesity and metabolic syndrome. Insulin-regulated aminopeptidase (IRAP) has been identified a key regulator of GLUT4 transporter as well as angiotensin IV (AngIV) receptor (AT4R). Although AngII-AT1R axis appears as anorexigenic and as an effector of energy expenditure, the impact of AngIV-IRAP/AT4R axis on energy metabolism remains unknown. The aim was to determine the role of IRAP in energy metabolism in mice. METHODS AND RESULTS: In adipocyte culture, plasminogen activator inhibitor type 1 (PAI-1) expression levels were diminished in IRAP knockout (IRAP(-/-)) if compared with those of wild-type (C57Bl/6J, WT) mice. Mice were fed high-fat diet (32% fat) at age of 8 weeks. At the entry, body weight, body fat content, and parameters of saccharometabolism were similar between groups. However, IRAP(-/-) mice exhibited blunted body weight gain compared to that of WT mice, despite comparable food intake and physical activity. At 20weeks of age, IRAP(-/-) mice had 25% lower body weight than WT mice. Glucose and insulin tolerance tests revealed that the glucose disposal and the hypoglycemic effect of insulin were pronounced in IRAP(-/-) mice after a high fat diet. Indirect calorimetry demonstrated that whole-body oxygen consumption rates were significantly higher in IRAP(-/-) mice by 18% with mild hyperthermia. Analysis of brown adipose tissue (BAT) in IRAP(-/-) showed increased levels of uncoupling protein-1 (UCP-1) at basal level and adaptive thermogenesis was not impaired. CONCLUSIONS: IRAP deficiency may lead to suppression of PAI-1 expression in adipocytes and upregulation of UCP-1-mediated thermogenesis in BAT and increased energy expenditure to prevent the development of obesity, and these facts suggest a therapeutic potential of IRAP/AT4R blockade in diet-induced obesity.

Enhanced angiogenesis by transplantation of mesenchymal stem cell sheet created by a novel magnetic tissue engineering method.

OBJECTIVE: Therapeutic angiogenesis with cell transplantation represents a novel strategy for severe ischemic diseases. However, some patients have poor response to such conventional injection-based angiogenic cell therapy. Here, we investigated a therapeutic potential of mesenchymal stem cell (MSC) sheet created by a novel magnetite tissue engineering technology for reparative angiogenesis. METHODS AND RESULTS: Human MSCs incubated with magnetic nanoparticle-containing liposomes were cultured, and a magnet was placed on the reverse side. Magnetized MSCs formed multilayered cell sheets according to magnetic force. Nude mice were subjected to unilateral hind limb ischemia and separated into 3 groups. For the control group, saline was injected into ischemic tissue. In the MSC-injected group, mice received magnetized MSCs by conventional needle injections without sheet formula as a control cell group. In the MSC-sheet group, MSC sheet was layered onto the ischemic tissues before skin closure. Blood flow recovery and the extent of angiogenesis were assessed by a laser Doppler blood flowmetry and histological capillary density, respectively. The MSC-sheet group had a greater angiogenesis in ischemic tissues compared to the control and MSC-injected groups. The angiogenic and tissue-preserving effects of MSC sheets were attributable to an increased expression of vascular endothelial growth factor and reduced apoptosis in ischemic tissues. In cultured MSCs, magnetic labeling itself inhibited apoptosis via a catalase-like antioxidative mechanism. CONCLUSIONS: MSC sheet created by the novel magnetic nanoparticle-based tissue engineering technology would represent a new modality for therapeutic angiogenesis and tissue regeneration.

Cholesterol and triglyceride concentrations in lipoproteins as related to carotid intima-media thickness.

Since distinct cholesterol and triglyceride concentrations in major lipoproteins and their subclasses may be related to atherosclerosis, we investigated the relationship of cholesterol and triglyceride concentrations in lipoprotein subclasses and the severity of carotid intima-media thickness (IMT), a surrogate marker of subclinical atherosclerosis. We studied 116 apparently healthy Japanese men (53 ± 9 years) without a history of cardiovascular diseases who were not taking any medication. Carotid IMT was measured by means of high-resolution vascular ultrasound. Plasma cholesterol and triglyceride concentrations in major lipoproteins and their subclasses were determined by HPLC with gel permeation columns. By univariate analyses, carotid IMT was the most closely related to age (r = 0.528, P < 0.001), followed by smoking habit expressed as pack-year cigarette consumption (r = 0.409, P < 0.001). In addition to total cholesterol and LDL cholesterol, carotid IMT was significantly associated with cholesterol and triglyceride concentrations in several LDL and VLDL subclasses. Stepwise multiple linear regression analysis revealed that age (ß = 0.436, P < 0.001), smoking (pack-years) (ß = 0.225, P = 0.007), and large LDL cholesterol (ß = 0.175, P = 0.023) were independent predictors of determining carotid IMT (adjusted R(2) = 0.347, P < 0.001). These results indicate that large LDL cholesterol is an important, independent determinant of carotid IMT in healthy men.

Actin cytoskeleton regulates stretch-activated Ca2+ influx in human pulmonary microvascular endothelial cells.

During high tidal volume mechanical ventilation in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), regions of the lung are exposed to excessive stretch, causing inflammatory responses and further lung damage. In this study, the effects of mechanical stretch on intracellular Ca(2+) concentration ([Ca(2+)](i)), which regulates a variety of endothelial properties, were investigated in human pulmonary microvascular endothelial cells (HPMVECs). HPMVECs grown on fibronectin-coated silicon chambers were exposed to uniaxial stretching, using a cell-stretching apparatus. After stretching and subsequent unloading, [Ca(2+)](i), as measured by fura-2 fluorescence, was transiently increased in a strain amplitude-dependent manner. The elevation of [Ca(2+)](i) induced by stretch was not evident in the Ca(2+)-free solution and was blocked by Gd(3+), a stretch-activated channel inhibitor, or ruthenium red, a transient receptor potential vanilloid inhibitor. The disruption of actin polymerization with cytochalasin D inhibited the stretch-induced elevation of [Ca(2+)](i). In contrast, increases in [Ca(2+)](i) induced by thapsigargin or thrombin were not affected by cytochalasin D. Increased actin polymerization with sphingosine-1-phosphate or jasplakinolide enhanced the stretch-induced elevation of [Ca(2+)](i). A simple network model of the cytoskeleton was also developed in support of the notion that actin stress fibers are required for efficient force transmission to open stretch-activated Ca(2+) channels. In conclusion, mechanical stretch activates Ca(2+) influx via stretch-activated channels which are tightly regulated by the actin cytoskeleton different from other Ca(2+) influx pathways such as receptor-operated and store-operated Ca(2+) entries in HPMVECs. These results suggest that abnormal Ca(2+) homeostasis because of excessive mechanical stretch during mechanical ventilation may play a role in the progression of ALI/ARDS.

Therapeutic angiogenesis by transplantation of induced pluripotent stem cell-derived Flk-1 positive cells.

BACKGROUND: Induced pluripotent stem (iPS) cells are the novel stem cell population induced from somatic cells. It is anticipated that iPS will be used in the expanding field of regenerative medicine. Here, we investigated whether implantation of fetal liver kinase-1 positive (Flk-1+) cells derived from iPS cells could improve angiogenesis in a mouse hind limb model of ischemia. RESULTS: Flk-1+ cells were induced from iPS cells after four to five days of culture. Hind limb ischemia was surgically induced and sorted Flk-1+ cells were directly injected into ischemic hind limbs of athymic nude mice. Revascularization of the ischemic hind limb was accelerated in mice that were transplanted with Flk-1+ cells compared with control mice, which were transplanted with vehicle, as evaluated by laser Doppler blood flowmetry. Transplantation of Flk-1+ cells also increased expression of VEGF mRNA in ischemic tissue compared to controls. CONCLUSIONS: Direct local implantation of iPS cell-derived Flk-1+ cells would salvage tissues from ischemia. These data indicate that iPS cells could be valuable in the therapeutic induction of angiogenesis.

Ischemia-induced angiogenesis is impaired in aminopeptidase A deficient mice via down-regulation of HIF-1α.

Aminopeptidase A (APA; EC 3.4.11.7) is a transmembrane metalloprotease with several functions in tumor angiogenesis. To investigate the role of APA in the process of ischemia-induced angiogenesis, we evaluated the cellular angiogenic responses under hypoxic conditions and the process of perfusion recovery in the hindlimb ischemia model of APA-deficient (APA-KO; C57Bl6/J strain) mice. Western blotting of endothelial cells (ECs) isolated from the aorta of APA-KO mice revealed that the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein in response to hypoxic challenge was blunted. Regarding the proteasomal ubiquitination, a proteasome inhibitor MG-132 restored the reduced accumulation of HIF-1α in ECs from APA-KO mice similar to control mice under hypoxic conditions. These were associated with decreased growth factor secretion and capillary formation in APA-KO mice. In the hindlimb ischemia model, perfusion recovery in APA-KO mice was decreased in accordance with a significantly lower capillary density at 2weeks. Regarding vasculogenesis, no differences were observed in cell populations and distribution patterns between wild type and APA-KO mice in relation to endothelial progenitor cells. Our results suggested that Ischemia-induced angiogenesis is impaired in APA-KO mice partly through decreased HIF-1α stability by proteasomal degradation and subsequent suppression of HIF-1α-driven target protein expression such as growth factors. APA is a functional target for ischemia-induced angiogenesis.

Ablation of angiotensin IV receptor attenuates hypofibrinolysis via PAI-1 downregulation and reduces occlusive arterial thrombosis.

OBJECTIVE: Reduced fibrinolytic activity is associated with adverse cardiovascular events. Although insulin-regulated aminopeptidase (IRAP) was recently identified as the angiotensin (Ang) IV receptor (AT4R), the impact of AngIV-AT4R signaling distal to AngII on the activation of type-1 plasminogen activator inhibitor (PAI-1) in the fibrinolytic process and subsequent formation of thrombosis remains unclarified. METHODS AND RESULTS: To determine whether AngIV would inhibit fibrinolysis via PAI-1 activation and promote thrombosis, we evaluated the degree of fibrinolysis in thrombosis models and investigated the roles of AT4R after vascular injury using IRAP knockout mice (IRAP(-/-)). In endothelial cells from control mice (WT; C57Bl6/J), both AngII and AngIV treatments increased PAI-1 mRNA expression in a dose-dependent manner, whereas the response was blunted in endothelial cells from IRAP(-/-) mice. FeCl(3)-induced thrombosis was suppressed in the carotid arteries of IRAP(-/-) mice when compared with WT mice. Similarly, in a model of carotid artery ligation and cuff placement, IRAP(-/-) mice demonstrated accelerated fibrinolysis 7 days after surgery and reduced occlusive thrombosis with negative remodeling at 28 days. CONCLUSIONS: AngIV-AT4R signaling has a key role in fibrinolysis and the subsequent formation of arterial thrombosis after vascular injury. AT4R may be a novel therapeutic target against cardiovascular disease.

Mechanical stretch enhances IL-8 production in pulmonary microvascular endothelial cells.

In patients with acute respiratory distress syndrome, mechanical over-distension of the lung by a large tidal volume causes further damage and inflammation, called ventilator-induced lung injury (VILI), however, it is unclear how mechanical stretch affects the cellular functions or morphology in human pulmonary microvascular endothelial cells (HPMVECs). IL-8 has been proposed to play an important role in the progression of VILI by activating neutrophils. We demonstrated that HPMVECs exposed to cyclic uni-axial stretch produce IL-8 protein with p38 activation in strain- and time-dependent manners. The IL-8 synthesis was not regulated by other signal transduction pathways such as ERK1/2, JNK, or stretch-activated Ca(2+) channels. Moreover, cyclic stretch enhanced IL-6 and monocyte chemoattractant protein-1 production and reoriented cell perpendicularly to the stretch axis accompanied by actin polymerization. Taken together, IL-8 production by HPMVECs due to excessive mechanical stretch may activate neutrophilic inflammation, which leads to VILI.

Mechanisms underlying the impairment of ischemia-induced neovascularization in matrix metalloproteinase 2-deficient mice.

Matrix metalloproteinases (MMPs) have been implicated in the process of neovascularization. However, the exact roles of individual MMPs in vessel formation are poorly understood. To study the putative role of MMP-2 in ischemia-induced neovascularization, a hindlimb ischemia model was applied to MMP-2(+/+) and MMP-2(-/-) mice. Serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in MMP-2(-/-) young and old mice remained impaired throughout the follow-up period. At day 35, microangiography and anti-l-lectin immunohistochemical staining revealed lesser developed collateral vessels and capillary formation in both old and young MMP-2(-/-) mice compared with the age-matched MMP-2(+/+) mice. An aortic-ring culture assay showed a markedly impaired angiogenic response in MMP-2(-/-) mice, which was partially recovered by supplementation of the culture medium with recombinant MMP-2. Aorta-derived endothelial cells or bone marrow-derived endothelial progenitor cell (EPC)-like c-Kit(+) cells from MMP-2(-/-) showed marked impairment of invasive or/and proliferative abilities. At day 7, plasma and ischemic tissues of vascular endothelial growth factor protein were reduced in MMP-2(-/-). Flow cytometry showed that the numbers of EPC-like CD31(+)c-Kit(+) cells in peripheral blood markedly decreased in MMP-2-deficient mice. Transplantation of bone marrow-derived mononuclear cells from MMP-2(+/+) mice restored neovascularization in MMP-2(-/-) young mice. These data suggest that MMP-2 deficiency impairs ischemia-induced neovascularization through a reduction of endothelial cell and EPC invasive and/or proliferative activities and EPC mobilization.

[RAAS and angiogenesis].

Angiotensin II(Ang II) is a potent vasoconstrictor and exerts inflammation and cell proliferation, thereby promoting angiogenesis. There are two major subtypes of Ang II receptors, AT1R and AT2R. AT1R mediates proangiogenic effect through enhancement of inflammation and leukocytes infiltration, while AT2R mediates antiangiogenic effect through regulation of apoptosis. Recently, Ang IV receptor was identified as insulin-regulated aminopeptidase, however, its roles in angiogenesis remain unknown. Although AT1R antagonist, ARB, has been proved its protective effects for the heart, kidney, and brain under many pathological conditions, it remains unknown whether long-term administration of ARB could be beneficial for therapeutic angiogenesis in patients with ischemic heart disease or peripheral artery disease. Further investigations are required.

Usefulness of adiponectin to predict myocardial salvage following successful reperfusion in patients with acute myocardial infarction.

Adiponectin is an adipose-derived plasma protein that demonstrates beneficial actions on myocardial injury under ischemic conditions. Circulating endothelial progenitor cells are reported to associate with rescue of cardiac damage after acute myocardial infarction (AMI). We examined whether circulating adiponectin level affects myocardial function and injury in patients with AMI. A total of 48 patients who underwent successful reperfusion treatment after AMI were enrolled. Cardiac function and perfusion defect were assessed by scintigraphic images of iodine-123 beta-methyl iodophenyl pentadecanoic acid in the acute phase and technetium-99m tetrofosmin in the long-term phase. Plasma adiponectin levels were measured by enzyme-linked immunosorbent assay at day 7 after AMI. Plasma adiponectin levels associated positively with myocardial salvage index representing the proportion of initial perfusion defect rescued by reperfusion and recovery of ejection fraction in the long-term phase and negatively with final infarct size. A positive correlation was also observed between adiponectin levels and number of circulating CD34(+) cells as determined by flow cytometry and between myocardial salvage index and recovery of ejection fraction independently associated with circulating CD34(+) cell levels. In conclusion, plasma adiponectin levels predict improvement of cardiac damage and function after reperfusion therapy in patients with AMI, suggesting that adiponectin could serve as a biomarker for assessment of myocardial injury after AMI.

Angiotensin II receptor blocker inhibits neointimal hyperplasia through regulation of smooth muscle-like progenitor cells.

OBJECTIVES: Angiotensin II (ATII) type 1 receptor (AT1R) blocker (ARB) has been shown to inhibit neointimal formation. Bone marrow-derived mononuclear cells (BM-MNCs) give rise to smooth muscle (SM)-like cells at injured arterial wall and contribute to neointimal formation. However, role of the renin-angiotensin system in the homing process of SM-like cells during neointimal formation is unknown. MATERIAL AND METHODS: When human BM-MNCs and peripheral blood MNCs (PB-MNCs) were cultured under treatment with PDGF-BB and bFGF, these cells gave rise to SM-like cells with expression of alphaSMA, SMemb, and SM1 proteins. RT-PCR showed the expression of AT1R, ATII type 2 receptor (AT2R), alphaSMA, and SMemb mRNAs. ATII accelerated the differentiation of SM-like cells, which was inhibited by an ARB CV11974 (P<0.05). We then examined the effects of ATII, CV11974, and AT2R antagonist PD123319 on neointimal formation and BM-derived SM-like cell incorporation at injured arteries in vivo. BM from green fluorescence protein (GFP)-transgenic mice was transplanted to irradiated WT mice. GFP-BM chimera mice were subjected to wire injury on the left femoral artery. ATII (100 ng/kg/min) stimulated whereas CV11974 (1 mg/kg/d) inhibited neointimal formation. Number of GFP+ alphaSMA+ cells at neointima correlated with the intima/media ratio (r=0.69, P<0.05). CONCLUSION: BM-derived SM-like progenitor cells contributed to the neointimal formation after arterial injury. ATII accelerated whereas ARB suppressed this process. These are new aspects of the ARB-mediated inhibition of atherosclerotic disease progression.

Therapeutic angiogenesis using novel vascular endothelial growth factor-E/human placental growth factor chimera genes.

BACKGROUND: Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis but causes adverse side effects such as edema or tissue inflammation. VEGF-E, found in the genome of the Orf virus, specifically binds to VEGF receptor-2 and shows mitotic activity on endothelial cells. Recently, we created two forms of VEGF-E and human placental growth factor (PlGF) chimera genes (VEGF-E chimera #9 and VEGF-E chimera #33), which are humanized genes with VEGF-E function but showing less antigenicity. METHODS AND RESULTS: We examined potential proangiogenic activities of these chimera genes. Four types of expression plasmids (pCDNA3.1-LacZ, phVEGF-A, pVEGF-Echimera#9, and pVEGF-Echimera#33) were administered in a rat model of hindlimb ischemia. Either pVEGF-Echimera#9, pVEGF-Echimera#33, or phVEGF-A significantly increased the ratio of ischemic/normal hindlimb blood-flow compared with the control pCDNA3.1-LacZ treated group (by 1.5-fold, 1.5-fold, and 1.4-fold, respectively, P<0.05). Histochemical staining by alkaline phosphatase also revealed that either pVEGF-Echimera#9, pVEGF-Echimera#33, or phVEGF-A increased the capillary density compared with the pCDNA3.1-LacZ treated group (1.4-fold, 1.5-fold, and 1.5-fold, respectively, P<0.05). Furthermore, immunostaining for anti-ED1 revealed that fewer macrophages had infiltrated in both pVEGF-Echimera#9 and pVEGF-Echimera#33 groups compared with the phVEGF-A group (P<0.05). CONCLUSIONS: Novel VEGF-E/human PlGF chimera genes, pVEGF-Echimera#9, and pVEGF-Echimera#33 significantly stimulated angiogenesis in response to tissue ischemia to an almost identical extent to that induced by phVEGF-A with fewer tissue inflammation responses.

Risk stratification of patients with prior myocardial infarction and advanced left ventricular dysfunction by gated myocardial perfusion SPECT imaging.

BACKGROUND: The Multicenter Automatic Defibrillator Implantation Trial II (MADIT-II) has shown that the prophylactic implantable cardiac defibrillator improves the survival rate of patients with prior myocardial infarction and advanced left ventricular (LV) dysfunction. However, a more accurate noninvasive predictor should be found to identify subgroups at high risk, one that would allow implantable cardiac defibrillator therapy to be directed specifically to the patients who would benefit most. METHODS AND RESULTS: To elucidate whether technetium 99m tetrofosmin electrocardiogram-gated single photon emission computed tomography (SPECT) imaging at rest can determine the risk of arrhythmic death, 106 patients who met the MADIT-II criteria (LV ejection fraction 1 month earlier, and no sustained ventricular tachyarrhythmia) were recruited from a pool of 4628 consecutive patients who had undergone resting Tc-99m tetrofosmin SPECT imaging. By use of the endpoints of lethal arrhythmic events, which included documentation of sustained ventricular tachycardia, ventricular fibrillation, or diagnosis of sudden cardiac death, we performed follow-up for a mean of 30 months. Lethal arrhythmic events occurred in 14 patients. Patients with lethal arrhythmic events had a lower LV ejection fraction, greater LV end-systolic and end-diastolic volume indices, and a greater perfusion defect volume than the remaining patients. By receiver operating characteristic curve analysis, myocardial defect volume was the strongest predictor for the development of lethal arrhythmic events. CONCLUSION: Our results confirm that perfusion defect volume by Tc-99m tetrofosmin scintigraphy is the most pivotal predictor of the future occurrence of lethal arrhythmic events and of sudden cardiac death. Tc-99m tetrofosmin SPECT images may assist in identifying subsets of patients with a greater likelihood of arrhythmic death among patients with LV dysfunction.

The impact of the capability of circulating progenitor cell to differentiate on myocardial salvage in patients with primary acute myocardial infarction.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) are known to be involved in vasculogenesis and mobilized after acute myocardial infarction (AMI). To test the hypothesis that the angiogenic function of EPCs affects post-myocardial infarction (MI) myocardial salvage, we evaluated the number and potential differentiation of EPCs and compared these data with clinical parameters 6 months after MI. METHODS AND RESULTS: Consecutive 51 patients (age, 61+/-8 years, mean+/-SD) with primary AMI who were successfully treated with stenting were enrolled. EPC identified as CD45low, CD34+, CD133+, and VEGFR2+ was quantified by a flow cytometry. The potential of EPCs to differentiate into endothelial cells (EPC differentiation) was also confirmed by the upregulation of CD31 and VEGFR2 after 7 days of culture. According to the proportion of EPC fraction, patients were divided into 2 groups (cut-off value=median). Although no difference was seen in myocardial damage shown by mean peak CK leakage and mean area at risk between the differentiated group (n=26) and nondifferentiated group (n=25), the number of attached cell was greater in differentiated group than in the nondifferentiated group (P=0.023). Left ventricular function and ischemic damaged area were assessed by scintigraphic images of (123)I-BMIPP in the acute phase and (99m)Tc-tetrofosmin in the chronic phase. We found that a greater increase in myocardial salvage (P=0.0091), decrease in end-systolic volume (P=0.012), and recovery of ejection fraction (P=0.011) occurred in the group with differentiated EPCs than in the nondifferentiated group. CONCLUSIONS: In patients with primary AMI, the capability of EPCs to differentiate influences the functional improvement and infarct size reduction, indicating that manipulation of EPCs could be a novel therapeutic target to salvage ischemic damage.

Impact of low-density lipoprotein particle size on carotid intima-media thickness in patients with type 2 diabetes mellitus.

Small low-density lipoprotein (LDL) particles and modifications to LDL such as glycation and oxidation have been linked to the pathogenesis of atherosclerosis in patients with diabetes. We investigated whether LDL particle size, or the levels of glycated LDL or malondialdehyde-modified LDL (MDA-LDL) are associated with carotid intima-media thickness (IMT) in patients with type 2 diabetes mellitus. One hundred seventy-two patients with type 2 diabetes mellitus were enrolled. Carotid IMT was measured by high-resolution ultrasound, and LDL particle size and serum glycated LDL and MDA-LDL levels were determined. The 3 variables were significantly correlated with one another. Univariate analyses defined statistically significant correlations of carotid IMT with LDL size, hemoglobin A(1c), glycated LDL, MDA-LDL, high-density lipoprotein (HDL) cholesterol, and age. The strongest association of IMT was with LDL size (r = -0.406, P < .0001), followed by that with HDL cholesterol (r = -0.225, P = .004). A stepwise multiple regression analysis revealed that LDL size and HDL cholesterol are independent predictors of carotid IMT. Neither glycated LDL nor MDA-LDL had a significant independent contribution to the severity of carotid IMT in the multivariate model. Low-density lipoprotein particle size, but not the glycated LDL or MDA-LDL level, was independently associated with carotid IMT in patients with type 2 diabetes mellitus regardless of antidiabetic and lipid-lowering medications. These results suggest that the measurement of LDL size may be more useful than quantification of modified LDLs for assessing atherosclerosis in patients with type 2 diabetes mellitus. Small LDL particles may be the most important predictor for the risk of cardiovascular disease in diabetic patients.

l-Caldesmon regulates proliferation and migration of vascular smooth muscle cells and inhibits neointimal formation after angioplasty.

OBJECTIVE: Light-type caldesmon (l-CaD) is a potent cytostatic and antiangiogenic protein that regulates cell growth and survival via modulation of the cell shape and cytoskeleton. The aim of this study is to explore the potential value of l-CaD for use as a cytostatic agent to inhibit neointimal formation after angioplasty by suppressing vascular smooth muscle cell (VSMC) growth and migration. METHODS AND RESULTS: We tested the cytostatic function of l-CaD in cultured VSMCs using assays for apoptosis, cell proliferation, and migration, and evaluated the expression pattern of relevant signaling proteins (focal adhesion kinase [FAK] and mitogen-activated protein kinases) in VSMCs. Transfection of adenoviral vector encoding l-CaD (Ad-l-CaD) resulted in progressive loss of actin stress fibers and cell retraction. Enzyme-linked immunosorbent assay demonstrated that Ad-l-CaD transfection increased the apoptosis rate by 75% and reduced BrdU uptake by 49%. Furthermore, transfection of Ad-l-CaD inhibited migration of VSMCs induced by platelet-derived growth factor-BB (PDGF) by 36% (P<0.05). Immunoblotting analysis revealed that l-CaD overexpression reduced PDGF-induced phosphorylation of both FAK and extracellular signal regulated-kinase (ERK). In balloon-injured rat carotid arteries, Ad-l-CaD transfection inhibited neointimal formation by 37% (P<0.05) without delaying re-endothelialization at 14 days. CONCLUSIONS: Overexpression of l-CaD suppressed cell growth and survival in VSMCs and inhibited neointimal formation after experimental angioplasty, partly by regulating the cytoskeletal tension-FAK-ERK axis.

Combined therapy with PPARalpha agonist and L-carnitine rescues lipotoxic cardiomyopathy due to systemic carnitine deficiency.

OBJECTIVE: Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and are key regulators of fatty acid oxidation (FAO) in the heart. Systemic carnitine deficiency (SCD) causes disorders of FAO and induces hypertrophic cardiomyopathy with lipid accumulation. We hypothesized that activation of PPARalpha by fenofibrate, a PPARalpha agonist, in addition to conventional L-carnitine supplementation may exert beneficial effects on the lipotoxic cardiomyopathy in juvenile visceral steatosis (JVS) mouse, a murine model of SCD. METHODS: Both wild-type (WT) and JVS mice were fed a normal chow, 0.2% fenofibrate containing chow (FE), a 0.1% L-carnitine containing chow (CA) or a 0.1% L-carnitine + 0.2% fenofibrate containing chow (CA + FE) from 4 weeks of age. Four to 8 animals per group were used for each experiment and 9 to 11 animals per group were used for survival analysis. RESULTS: At 8 weeks of age, JVS mice exhibited marked ventricular hypertrophy, which was more attenuated by CA + FE than by CA or FE alone. CA + FE markedly reduced the high plasma and myocardial triglyceride levels and increased the low myocardial ATP content to control levels in JVS mice. In JVS mice, myocardial 1,2-diacylglycerol (DAG) was significantly increased and showed a distinct fatty acid composition with elevation of 18:1(n-7,9) and 18:2(n-6) fatty acids compared with that in WT mice. CA + FE significantly altered the fatty acid composition of DAG and inhibited the membrane translocation of cardiac protein kinase C beta2 in JVS mice. Furthermore, CA + FE prevented the progressive left ventricular dysfunction and dramatically improved the survival rate in JVS mice (survival rate at 400 days after birth: 89 vs. 0%, P < 0.0001). CONCLUSIONS: PPARalpha activation, in addition to l-carnitine supplementation, may rescue the detrimental lipotoxic cardiomyopathy in SCD by improving cardiac energy and lipid metabolism as well as systemic lipid metabolism.

Klotho gene polymorphism may be a genetic risk factor for atherosclerotic coronary artery disease but not for vasospastic angina in Japanese.

BACKGROUND: The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes, including atherosclerosis. We tested the hypothesis that the G-395A polymorphism of the klotho gene is associated with increased risk for 2 types of ischemic heart disease in Japanese. METHODS: The study population consisted of 197 patients with coronary heart disease (CAD) who had >75% luminal diameter narrowing, 77 patients with vasospastic angina (VSA) without significant fixed coronary artery disease, and 331 healthy control subjects. RESULTS: The frequency of the A allele carriers of the klotho gene was significantly higher in the CAD group than in the control group (29.9% vs. 19.0%). The unadjusted odds ratio for CAD in the A allele carriers compared with the control group was 1.82 (p=0.004) and a traditional risk-adjusted logistic regression model revealed that the A allele was an independent predictor of CAD (odds ratio, 1.76; p=0.03). In contrast, the frequency of the A allele carriers was not significantly different in the VSA group (23.4%; adjusted odds ratio, 1.18. CONCLUSIONS: The -395A polymorphism of the human klotho gene may be a genetic risk factor for IHD and not for VSA.

Upregulation of renal eNOS by high-sodium diet facilitates hypertension in doxorubicin-treated rats through enhanced oxidative stress.

It is known that renal nitric oxide (NO) is an important controller of urinary sodium excretion. A defect in the kidney's NO system could cause salt-sensitive hypertension. Since it has been demonstrated that doxorubicin binds to the reductase domain of endothelial NO synthase (eNOS) and generates superoxide in vitro, we tested our hypothesis that a high-sodium diet would upregulate the expression of eNOS and enhance oxidative stress in the kidney of doxorubicin-treated rats, resulting in a facilitation of hypertension. At 4 weeks after treatment with doxorubicin in Sprague-Dawley rats, the systolic blood pressure significantly increased only in the high-sodium diet group. The expressions of eNOS protein in the renal cortex and medulla were significantly higher in high-sodium groups than in normal-sodium groups, regardless of doxorubicin treatment. In rats treated with doxorubicin, a biomarker of oxidative damage 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunohistological staining of renal tissues showed strong staining of the proximal and distal tubules. In particular, rats with doxorubicin in the high-sodium diet group demonstrated a significant increase in urinary exertion of 8-OHdG as well as more prominently stained tubules against 8-OHdG antibody, but markedly lower urinary NO(x) excretion than in rats without doxorubicin, even than in the untreated, low-sodium group. In conclusion, these results indicate that the oxidative stress induced by doxorubicin impairs NO production in the kidney. As such, doxorubicin treatment appears to contribute to the development of salt-sensitive hypertension through reductive activation of upregulated eNOS by a high-sodium diet instead of NO production.