Yasushi Kitaura

Vascular Endothelial Growth Factor–Expressing Mesenchymal Stem Cell Transplantation for the Treatment of Acute Myocardial Infarction

Objective—Vascular endothelial growth factor (VEGF) plays an important role in inducing angiogenesis. Mesenchymal stem cells (MSCs) may have potential for differentiation to several types of cells, including myocytes. We hypothesized that transplantation of VEGF-expressing MSCs could effectively treat acute myocardial infarction (MI) by providing enhanced cardioprotection, followed by angiogenic effects in salvaging ischemic myocardium. Methods and Results—The human VEGF165 gene was transfected to cultured MSCs of Lewis rats using an adenoviral vector. Six million VEGF-transfected and LacZ-transfected MSCs (VEGF group), LacZ-transfected MSCs (control group), or serum-free medium only (medium group) were injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. At 1 week after MI, MSCs were detected by X-gal staining in infarcted region. High expression of VEGF was immunostained in the VEGF group. At 28 days after MI, infarct size, left ventricular dimensions, ejection fraction, E wave/A wave ratio and capillary density of the infarcted region were most improved in the VEGF group, compared with the medium group. Immunohistochemically, &agr;-smooth muscle actin–positive cells were most increased in the VEGF group. Conclusions—This combined strategy of cell transplantation with gene therapy could be a useful therapy for the treatment of acute MI.

Evaluation of viral infection in the myocardium of patients with idiopathic dilated cardiomyopathy

OBJECTIVES The aim of this study was to evaluate the viral etiology of idiopathic dilated cardiomyopathy (DCM). BACKGROUND The demonstration of enteroviral genome in hearts with DCM has reinforced the importance of enteroviruses in the pathogenesis of DCM. However, there is uncertainty about the character and activity of enteroviruses detected in the myocardium. Recently, the association of hepatitis C virus or adenovirus with DCM has been reported. METHODS Myocardial specimens from 26 patients with idiopathic DCM, which were obtained at partial left ventriculectomy (PLV), were examined virologically. Strand-specific detection of enteroviral RNA was performed to differentiate active viral replication from latent persistence. Polymerase chain reaction was used to detect genomic sequences of hepatitis C virus, adenovirus, cytomegalovirus, influenza viruses, mumps virus, herpes simplex viruses, varicella-zoster virus and Epstein-Barr virus. RESULTS Plus-strand enteroviral RNA was detected in 9 (35%) of the 26 patients. Minus-strand enteroviral RNA was determined in seven (78%) of these nine plus-strand RNA-positive patients. Sequence analysis revealed that the enteroviruses detected were coxsackie B viruses, such as coxsackievirus B3 and B4. However, genetic material from other viruses was not detected. Six (86%) of seven minus-strand enteroviral RNA-positive patients died of cardiac insufficiency within the first six months after PLV. CONCLUSIONS Coxsackie B viruses were seen in hearts with idiopathic DCM. Active viral RNA replication appeared to be present in a significant proportion of these cases. Minus-strand coxsackieviral RNA in the myocardium can be a marker for poor clinical outcome after PLV. There was no evidence of persistent infection by other viruses in hearts with DCM.

Use of a whole blood rapid panel test for heart-type fatty acid-binding protein in patients with acute chest pain: comparison with rapid troponin T and myoglobin tests.

PURPOSE We sought to determine the clinical utility of a newly developed qualitative test to measure heart-type fatty acid-binding protein levels in blood for the early identification of myocardial infarction. METHODS We measured heart-type fatty acid-binding protein levels in 371 consecutive patients with acute chest pain and suspected myocardial infarction, and compared the performance of this test with those of troponin T and myoglobin tests. Levels of heart-type fatty acid-binding protein >or=6.2 ng/mL were considered as positive results. RESULTS A final diagnosis of acute myocardial infarction was made in 181 patients (49%). Of the 68 patients who presented within 2 hours of the onset of symptoms, 37 (54%) had a final diagnosis of myocardial infarction. The sensitivity of the rapid heart-type fatty acid-binding protein test was 89% (33/37), significantly higher than for troponin T (22% [8/37]; P<0.001) and myoglobin (38% [14/37]; P<0.001). However, the specificity of troponin T (94% [29/31]) was significantly better than for heart-type fatty acid-binding protein (52% [16/31]; P= 0.002) within 2 hours. The area under the receiver operating characteristic curve for heart-type fatty acid-binding protein levels was greater than that for myoglobin (0.72 vs. 0.61, P = 0.01) among patients who presented within 2 hours. CONCLUSION A novel whole blood rapid heart-type fatty acid-binding protein test can be useful in the early evaluation of patients who present with acute chest pain.

CD36 mediates long-chain fatty acid transport in human myocardium: Complete myocardial accumulation defect of radiolabeled long-chain fatty acid analog in subjects with CD36 deficiency

Long-chain fatty acids (LCFA) are the major energy substrate for heart and their oxidation is important for achieving maximal cardiac work. However, the mechanism of uptake of LCFA by myocardium has not been clarified. We previously reported that bovine myocardial LCFA transporter has a sequence homology to human CD36. Clinically, total defect of myocardial uptake of radiolabeled long-chain fatty acid analog [123I-BMIPP: Iodine-123 15-(p-iodophenyl)-(R,S)-methylpentadecanoic acid] has been reported in some restricted cases, but the etiology has not been clarified. In the present study, we analyzed CD36 expression and CD36 gene in subjects who showed total lack of myocardial 123I-BMIPP accumulation, and, vice versa, evaluated myocardial 123I-BMIPP uptake in subjects with CD36 deficiency. Four unrelated subjects were evaluated; Two were found to have negative myocardial LCFA accumulation by 123I-BMIPP scintigraphy, after which the expression of CD36 on their platelets and monocytes was analyzed. Remaining two subjects were identified as CD36 deficiency by screening, then 123I-BMIPP scintigraphy was performed. Expression of CD36 on platelets and monocytes was measured by flow cytometric analysis. The molecular defects responsible for CD36 deficiency was detected by allele-specific restriction enzyme analysis. CD36 expression was totally deficient in all 4 subjects on both platelets and monocytes. Two subjects were homozygous for a 478C→T mutation. One was heterozygous for the dinucleotide deletion of exon V and single nucleotide insertion of exon X, and remaining one was considered to be heterozygous for the dinucleotide deletion of exon V and an unknown gene abnormality. All cases demonstrated a completely negative accumulation of myocardial LCFA despite of normal myocardial perfusion, which was evaluated by thallium scintigraphy. In addition, all cases demonstrated apparently normal hepatic LCFA accumulation Thus, these findings suggested that CD36 acts as a major myocardial specific LCFA transporter in humans.

Ca2+/Calmodulin-Dependent Kinase IIδ Causes Heart Failure by Accumulation of p53 in Dilated Cardiomyopathy

Background— Dilated cardiomyopathy (DCM), characterized by dilatation and dysfunction of the left ventricle, is an important cause of heart failure. Many mutations in various genes, including cytoskeletal protein genes and contractile protein genes, have been identified in DCM patients, but the mechanisms of how such mutations lead to DCM remain unknown. Methods and Results— We established the mouse model of DCM by expressing a mutated cardiac &agr;-actin gene, which has been reported in patients with DCM, in the heart (mActin-Tg). mActin-Tg mice showed gradual dilatation and dysfunction of the left ventricle, resulting in death by heart failure. The number of apoptotic cardiomyocytes and protein levels of p53 were increased in the hearts of mActin-Tg mice. Overexpression of Bcl-2 or downregulation of p53 decreased the number of apoptotic cardiomyocytes and improved cardiac function. This mouse model showed a decrease in myofilament calcium sensitivity and activation of calcium/calmodulin-dependent kinase II&dgr; (CaMKII&dgr;). The inhibition of CaMKII&dgr; prevented the increase in p53 and apoptotic cardiomyocytes and ameliorated cardiac function. Conclusion— CaMKII&dgr; plays a critical role in the development of heart failure in part by accumulation of p53 and induction of cardiomyocyte apoptosis in the DCM mouse model.

Functional and histopathologic correlation in patients with dilated cardiomyopathy: An integrated evaluation by multivariate analysis

To correlate left ventricular function and histologic features in patients with dilated cardiomyopathy, precise indexes of hemodynamics and semiquantitative histologic data were combined for multivariate analysis. Right endomyocardial biopsy was performed at the time of cardiac catheterization. Five hemodynamic indexes were used for functional assessment: ejection fraction, ratio of end-systolic stress to volume index, end-diastolic stress, time constant (T) of left ventricular pressure fall, and end-systolic stress. Six histologic findings (disarray of myofibers, hypertrophy of myofibers, scarcity of myofibrils, nuclear changes of myofibers, vacuolization of myofibers and proliferation of collagen fibers) were graded from (-) to (4+). Each finding was assigned to category (-) or (+) according to the absence or presence of significant abnormality. Ordinary statistical analysis revealed that, although ejection fraction was lower in category (+) for proliferation of collagen fibers, ratio of end-systolic to volume index was reduced for category (+) of hypertrophy of myofibers. A significant correlation was present between hypertrophy of myofibers and proliferation of collagen fibers by Spearman rank correlation. When principal component analysis was applied to the hemodynamic data, two principal components could be extracted. Fishers discriminant analysis could clearly differentiate two categories (-) and (+) in the semiquantitative histologic finding of proliferation of collagen fibers. The analysis indicated that contractility was reduced with elevated afterload in that category (+). Thus, proliferation of collagen fibers may play a pivotal role in deteriorating contractility in patients with dilated cardiomyopathy.

Development of a simple whole blood panel test for detection of human heart-type fatty acid-binding protein.

OBJECTIVE For the diagnosis of acute myocardial infarction (AMI), we have developed a rapid and simple whole blood panel test for the detection of human heart-type fatty acid-binding protein (H-FABP) using one-step immunochromatography. METHODS AND RESULTS We have developed a whole blood panel test for rapid detection of human H-FABP using a one-step immunochromatography technique. The result of this panel test was not affected by the other contents of the blood such as bilirubin, hemoglobin and others. Furthermore, no cross-reactivity of the antibodies was found with other cardiac markers or other tissue-type FABPs. The result of this panel test was similar to the diagnostic cut-off value, 6.2 ng of H-FABP per mL of serum which was evaluated by the enzyme-linked immunosorbent assay (ELISA). CONCLUSION We have developed a simple one-step immunochromatography technique to detect H-FABP in whole blood sample. Further studies are required to identify the value of this point-of-care testing (POCT) as a diagnostic marker for AMI.

Angiotensin II receptor blockade prevents microangiopathy and preserves diastolic function in the diabetic rat heart

Background: Cardiac microangiopathy may be involved in the development of heart failure in diabetes mellitus. Objective: To evaluate the effect of angiotensin II receptor blockade on cardiac function and fine structures in diabetes. Methods: Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats (n = 30), a model of spontaneously developing diabetes mellitus, and their diabetes resistant counterparts (n = 20) were used. At 30 weeks of age, when the OLETF rats show hyperglycaemic obesity with hyperinsulinaemia, the animals were divided into two groups and given candesartan, an angiotensin II receptor blocker, 0.2 mg/kg/day, or vehicle for six weeks. Capillary density was evaluated in the left ventricular myocardium by electron microscopy, matrix metalloproteinase (MMP) activity by zymography, and cytokines by reverse transcriptase polymerase chain reaction. Results: Compared with the control rats, the OLETF rats at 36 weeks showed decreased peak negative dP/dt (mean (SD): 2350 (250) v 3492 (286) mm Hg/s) and increased cardiomyocyte diameter (24.3 (0.6) v 18.9 (0.6) μm) (both p < 0.05). Thickening of the capillary basement membranes and decreased capillary density were observed. Angiotensin receptor blockade improved almost all the haemodynamic variables, and the histological findings became similar to those of the controls. Angiotensin receptor blockade also activated MMP-2 and prevented an increase of inflammatory cytokines, especially interleukin (IL)-1β and IL-6, in the diabetic heart. Conclusions: Angiotensin II receptor blockade preserved left ventricular diastolic function. It was also potent at improving cardiomyocyte diameter and the thickening of the capillary basement membrane, increasing MMP-2 activity, and decreasing inflammatory cytokines. With all these changes, candesartan could contribute to cardioprotection in diabetes mellitus.

Role of gp91phox-containing NADPH oxidase in left ventricular remodeling induced by intermittent hypoxic stress.

Intermittent hypoxia due to sleep apnea syndrome is associated with cardiovascular diseases. However, the precise mechanisms by which intermittent hypoxic stress accelerates cardiovascular diseases are largely unclear. The aim of this study was to investigate the role of gp91(phox)-containing NADPH oxidase in the development of left ventricular (LV) remodeling induced by intermittent hypoxic stress in mice. Male gp91(phox)-deficient (gp91(-/-)) mice (n = 26) and wild-type (n = 39) mice at 7-12 wk of age were exposed to intermittent hypoxia (30 s of 4.5-5.5% O(2) followed by 30 s of 21% O(2) for 8 h/day during daytime) or normoxia for 10 days. Mean blood pressure and LV systolic and diastolic function were not changed by intermittent hypoxia in wild-type or gp91(-/-) mice, although right ventricular systolic pressure tended to be increased. In wild-type mice, intermittent hypoxic stress significantly increased the diameter of cardiomyocytes and interstitial fibrosis in LV myocardium. Furthermore, intermittent hypoxic stress increased superoxide production, 4-hydroxy-2-nonenal protein, TNF-alpha and transforming growth factor-beta mRNA, and NF-kappaB binding activity in wild-type, but not gp91(-/-), mice. These results suggest that gp91(phox)-containing NADPH oxidase plays a crucial role in the pathophysiology of intermittent hypoxia-induced LV remodeling through an increase of oxidative stress.

Chronic Hypoxia Accelerates the Progression of Atherosclerosis in Apolipoprotein E-Knockout Mice

The aim of this study was to investigate the effect of chronic hypoxia on the development and progression of atherosclerosis in apolipoprotein E-knockout (apoE-KO) mice. Male and female apoE-KO mice (6 weeks old) and age- and sex-matched wild-type mice were kept under hypoxic conditions (10.0±0.5% O2) in a gas chamber or in room air for 3 weeks. Aortic atherosclerotic plaque was not observed in wild-type mice under normoxic or hypoxic conditions. In the apoE-KO mice, however, hypoxia induced proliferation of smooth muscle cells and plaque formation in the aorta, which were not observed under normoxic conditions. Although sexual dimorphism of the response to hypoxia was not observed, these hypoxia-induced atherogenic changes were accompanied by a significant increase of plasma low density lipoprotein (LDL) cholesterol and NADPH-dependent vascular superoxide (O2−) production. Furthermore, matrix metalloproteinase (MMP)-9 was activated in the aorta of apoE-KO mice. In conclusion, chronic hypoxia accelerated the development of atherosclerosis in apoE-KO mice, along with increased O2− production and activated MMP-9 in the aorta.