永井 良三

A 15-year-old Japanese man was referred for evaluation of heart failure. Conventional heart failure therapy had little effect, and severe left ventricular dysfunction as well as elevated erythrocyte sedimentation rate persisted. Magnetic resonance angiography showed aortic dilatation with wall thickening characteristic of Takayasu's arteritis. An endomyocardial biopsy specimen revealed infiltration of natural killer cells and gamma delta T lymphocytes, which play major roles in vascular injury of Takayasu's arteritis. Prednisolone administration provided great benefits to cardiac function. These findings suggest that autoimmune cytotoxic mechanisms similar to those in arterial tissue may contribute to cardiac impairment in Takayasu's arteritis.

Changes in the expression levels of several genes have been described in aortic aneurysm specimens, however, the spectrum of diverse molecular alterations remains to be elucidated. We attempted to identify key molecules that modulate the pathogenesis of aortic aneurysm, using a complimentary DNA microarray carrying approximately 13,000 human genes. Segments of thoracic aortic aneurysms (TAA) and adjacent normal thoracic aortic tissues without aneurysmal changes (NTA) were obtained from 20 patients undergoing graft surgery. RNA obtained from five pairs of TAA and NTA samples was compared to determine aneurysm-specific alterations using microarray. Further, the expression levels of several genes of interest were verified in the remaining specimens by real-time reverse transcription-polymerase chain reaction (RT-PCR). In microarray assays, several types of the matrix metalloproteinases were upregulated as reported previously. Also, 220 genes suggested to be involved in protein degradation, inflammation, apoptosis, stress response, intracellular signaling, and other processes were significantly upregulated. Many of these genes have not been previously implicated in cardiovascular disease. The real time RT-PCR independently confirmed that the expression levels of MMP-2, MMP-9, ADAMTS-1, and caspase 4 were consistently increased in TAA. The results indicate that many genes are involved in a complicated manner in the pathogenesis of TAA. Investigation of these genes will help clarify the pathogenesis of this disease, and may lead to the discovery of novel therapeutic targets.

OBJECTIVE: Transforming growth factor-beta1 (TGF-beta1) controls the expression of numerous genes, including smooth muscle cell (SMC)-specific genes and extracellular matrix protein genes. Here we investigated whether c-Src plays a role in TGF-beta1 signaling in mouse embryonic fibroblast C3H10T1/2 cells. METHODS AND RESULTS: TGF-beta1 induction of the SMC contractile protein SM22alpha gene expression was inhibited by PP1 (an inhibitor of Src family kinases) or by C-terminal Src kinase (a negative regulator of c-Src). Induction of SM22alpha by TGF-beta1 was markedly attenuated in SYF cells (c-Src(-), Yes(-), and Fyn(-)) compared with Src(++) cells (c-Src(++), Yes(-), and Fyn(-)). PP1 also inhibited the TGF-beta1-induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-beta1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF-beta1-induced expression of SM22alpha. Furthermore, we demonstrate that TGF-beta1 induction of the plasminogen activator inhibitor-1 (PAI-1) gene, which is known to be dependent on Smad but not on SRF, is inhibited by PP1 and apocynin. CONCLUSIONS: Our results suggest that TGF-beta1 activates c-Src and generates hydrogen peroxide through NAD(P)H oxidase, and these signaling pathways lead to the activation of specific sets of genes, including SM22alpha and PAI-1. TGF-beta1 controls the expression of numerous genes, including SM22alpha and PAI-1. We investigated whether c-Src plays a role in TGF-beta1 signaling. TGF-beta1 induction of such genes was significantly reduced in Src family tyrosine kinase-deficient cells, and Csk and pharmacological inhibitors for Src family kinases or antioxidants inhibit the effects of TGF-beta1. These results indicate that c-Src and hydrogen peroxide are required for TGF-beta1 signaling.

Sudden cardiac death is defined as an unpredictable death within 24 hours. It is estimated to occur with a frequency of more than 50,000 per year in Japan. The inherited arrhythmogenic diseases associated with the transmembranous ionic channels, anchoring proteins or intracellular calcium regulating proteins are thought to be responsible for sudden cardiac death in infants, children, and young adults who have structurally normal hearts. Recent genetic analyses have identified congenital diseases such as the long-QT syndrome (LQTS), the Jervell and Lange-Nielsen syndrome (JLNS), the Brugada syndrome (BrS), the short-QT syndrome (SQTS), the arrhythmogenic right ventricular cardiomyopathy type 2 (ARVC2), and the catecholamine-induced polymorphic ventricular tachycardia (CPVT) /familial polymorphic ventricular tachycardia (FPVT). Loss of function in the slow component of the delayed rectifier potassium current (I(Ks)) channels (KCNQ1, KCNE1), the rapid component of the potassium current (I(Kr)) channels (KCNH2, KCNE2) and the inward rectifier potassium current (I(Kl), Kir2.1) channel (KCNJ2) is linked to the LQTSs (type 1, 2, 5, 6, and 7 (Andersen syndrome)) and the JLNSs (type 1 and 2). Changes of function in the alpha-subunit of cardiac sodium channels (SCN5A) is also linked to the LQTS type 3 and the BrS. A mutation in the ankyrin-B, anchoring proteins, has been identified as cause of the LQTS type 4. The SQTS is caused by gain of function in the KCNH2. Further, the missense mutations in the gene encoding ryanodine receptor 2 (RyR2) or calsequestrin 2 (CASQ2) that regulate intra-cardiac calcium handling is possibly implicated in the ARVC2 and the CPVT/FPVT. Herein, we present a review of the literature regarding the genetic mechanisms of the inherited arrhythmogenic diseases.

KAATSU training is a novel method for strength training to induce muscle strength and hypertrophy. The purpose of the present study was to investigate the hemodynamic and autonomic nervous responses to the restriction of femoral blood flow by KAATSU. Ultrasonography, echocardiography and impedance cardiography were performed in ten healthy male volunteers aged 34 ± 1.5 before (pre), during and after (post) pressurization on both legs with KAATSU belts placed around proximal portion of both legs. The parameters measured were as follows; the superficial femoral arterial blood flow, left ventricular end-diastolic/systolic dimension (LVDd/LVDs), cardiac output (CO), stroke volume (SV), diameter of inferior vena cava (IVC), heart rate (HR), mean blood pressure (mBP), total peripheral resistance (TPR) and heart rate variability (HRV). The pressurization on both legs with KAATSU suppressed venous blood flow, and markedly induced pooling of blood into the legs with pressure-dependent reduction of femoral arterial blood flow. The application of 200 mmHg KAATSU decreased femoral arterial blood flow, LVDd, CO, SV and IVC significantly. HR tended to increase, and TPR increased significantly, but mBP did not change significantly. In addition, high frequency (HF_RR), a marker of parasympathetic activity, decreased during KAATSU, while LF_RR/HF_RR, a quantitative marker of sympathetic autonomic nervous activity, increased significantly. These results indicate that the application of KAATSU on both legs induces venous pooling in the legs, and then inhibits venous return. The reduction of venous return causes a decrease of IVC diameter, cardiac size and stroke volume with an increase in TPR and LF_RR/HF_RR. Thus, the KAATSU training appears to become a useful method for potential countermeasure like lower body negative pressure (LBNP) against orthostatic intolerance for long-term bed rest or space flight as well as strength training to induce muscle strength and hypertrophy.

Cardiovascular function and frequencies of onset of cardiovascular diseases show circadian oscillation. Furthermore, cultured vascular endothelial cells and cardiomyocytes showed circadian oscillation of clock genes expression, suggesting the existence of the peripheral clock in cardiovascular systems. To elucidate the functional relevance of the peripheral clock in cardiovascular system, we first tried to identify the target genes of the peripheral clock. We demonstrated that PAI-1 mRNA levels exhibited circadian variation and that CLOCK and BMAL increased PAI-1gene expression mediated through the E-box sites. The circadian oscillation of PAI-1 gene expression is responsible for the decreased fibrinolytic activity that attributes to the morning onset of myocardial infarction. We further identified several target genes of the peripheral clock by cDNA microarray analysis using RNA from vascular endothelial cells. We are analyzing the function of these genes in cardiovascular system. We next generated transgenic mice in which Cry1, a negative regulator of the molecular clock, is overexpressed specifically in vascular endothelial cells to analyze the function of the peripheral clock separately from the central clock. Taken together, these results suggest that cardiovascular systems have their own peripheral clocks and at least in part, they may regulate the circadian oscillation of cardiovascular function directly. These results potentially provide a molecular basis for the circadian variation of cardiovascular function and novel strategies for the treatment of cardiovascular diseases. <b>[Jpn J Physiol 55 Suppl:S25 (2005)]</b>

OBJECTIVE: Hex (hematopoietically expressed homeobox), a member of homeobox family of transcription factors, has been implicated in the vascular development because of its expression in hemangioblast, a hypothetical stem cell that gives rise to both angioblasts and hematopoietic lineages. In the present study, we examined the role of Hex in the differentiation of vascular smooth muscle cells. METHODS AND RESULTS: We constructed adenovirus expressing Hex, to which we refer to as AxCA/Hex, and transduced murine embryonic fibroblasts, 10T1/2 cells. Northern blot analyses showed that Hex increased the mRNA levels of smooth muscle alpha-actin and SM22alpha but not of calponin and smooth muscle myosin heavy chain. Transient transfection assays showed that Hex activates the transcription from the SM22alpha promoter in a CArG box-dependent manner. Electrophoretic mobility shift assays demonstrate that Hex is not able to bind to CArG box, but binding of serum responsive factor (SRF) to CArG box is enhanced in AxCA/Hex-transduced cells. Recombinant Hex protein produced by in vitro translation system augmented the binding activity of SRF to CArG box. Immunoprecipitation experiments revealed the physical association between Hex and SRF. CONCLUSIONS: Hex induces transcription of the SM22alpha gene by facilitating the interaction between SRF and its cognate binding site in pluripotent embryonic fibroblasts. This study demonstrates that Hex, a hematopoietically expressed homeobox protein, induces transcription of the SM22alpha gene by facilitating the interaction between SRF and its cognate binding site in embryonic fibroblasts. These findings will provide the clue for understanding the mechanisms by which bone marrow-derived SMC precursor cells undergo differentiation.

Endothelial PAS domain protein-1 (EPAS1) regulates transcription of the genes encoding erythropoietin and vascular endothelial growth factor, which are important for maintaining oxygen homeostasis. We have previously shown that plasminogen activator inhibitor-1 (PAI-1) gene expression is induced by hypoxia. In this study, we sought to determine whether PAI-1 gene expression is directly regulated by EPAS1 in cancer cells because activities of proteases and their inhibitors are tightly regulated for tumor invasion. Hypoxia increased the PAI-1 mRNA levels in human adenocarcinoma A549 cells. Overexpression of EPAS1 significantly increased the PAI-1 mRNA and protein levels. Transient transfection assays revealed that EPAS1 increased PAI-1 gene transcription through a sequence containing 5'-CACGTACA-3' located at -194 (we refer to it as site HREPAI-1) and GT-box located at -78. Electrophoretic gel mobility shift assays revealed that HREPAI-1 serves as a binding site for EPAS1, and Sp1 constitutively binds to GT-box. In conclusion, PAI-1 expression is induced by EPAS1 through HREPAI-1 and through an Sp1-binding site. These results indicate that the PAI-1 gene is a direct target of EPAS1 and suggest the role of EPAS1 and Sp1 in the hypoxic response of cancer cells.