Toshihiro Amaki

In Vivo klotho Gene Transfer Ameliorates Angiotensin II-Induced Renal Damage

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal klotho gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the klotho gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal klotho gene expression. Long-term infusion of angiotensin II downregulated renal klotho gene expression at both the mRNA and protein levels. This angiotensin II-induced renal klotho downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse klotho gene (ad-klotho, 3.3×1010 plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. Ad-klotho gene transfer, but not ad-lacZ gene transfer, caused an improvement of creatinine clearance, decrease in urinary protein excretion, and amelioration of histologically demonstrated tubulointerstitial damage induced by angiotensin II administration. Our data suggest that downregulation of the renal klotho gene may have an aggravative role in the development of renal damage induced by angiotensin II, and that induction of the klotho gene may have therapeutic possibilities in treating angiotensin II-induced end organ damage.

Angiogenic Effects of Adrenomedullin in Ischemia and Tumor Growth

Adrenomedullin (AM) is a novel vasodilating peptide involved in the regulation of circulatory homeostasis and implicated in the pathophysiology of cardiovascular disease. We tested the hypothesis that AM also possesses angiogenic properties. Using laser Doppler perfusion imaging, we found that AM stimulated recovery of blood flow to the affected limb in the mouse hind-limb ischemia model. AM exerted this effect in part by promoting expression of vascular endothelial growth factor (VEGF) in the ischemic limb, and immunostaining for CD31 showed the enhanced flow to reflect increased collateral capillary density. By enhancing tumor angiogenesis, AM also promoted the growth of subcutaneously transplanted sarcoma 180 tumor cells. However, heterozygotic AM knockout mice (AM+/−) showed significantly less blood flow recovery with less collateral capillary development and VEGF expression than their wild-type littermates. Similarly, mice treated with AM22-52, a competitive inhibitor of AM, showed reduced capillary development, and growth of sarcoma 180 tumors was inhibited in AM+/− and AM22-52–treated mice. Notably, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM+/− and AM22-52–treated mice. In cocultures of endothelial cells and fibroblasts, AM enhanced VEGF-induced capillary formation, whereas in cultures of endothelial cells AM enhanced VEGF-induced Akt activation. These results show that AM possesses novel angiogenic properties mediated by its ability to enhance VEGF expression and Akt activity. This may make AM a useful therapeutic tool for relieving ischemia; conversely, inhibitors of AM could be useful for clinical management of tumor growth.

Diagnostic implications of circulating oxidized low density lipoprotein levels as a biochemical risk marker of coronary artery disease

OBJECTIVES To examine the diagnostic performance of circulating oxidized low density lipoprotein levels as a biochemical risk marker of coronary heart disease. DESIGN AND METHODS 361 patients with coronary artery disease and 710 healthy volunteers as normal controls were examined. Receiver-operating characteristics curve analysis in addition to statistical analysis (univariate, multivariate) were done to determine the usefulness of the assay. RESULTS Patients with coronary artery disease showed significantly elevated circulating oxidized low density lipoprotein levels. Males less than 70 years of age showed a significant association between oxidized low density lipoprotein levels and coronary artery disease. Receiver-operating characteristics curve analysis showed superior performance (e.g., sensitivity, specificity) of oxidized low density lipoprotein as a diagnostic marker of coronary artery disease as compared against other lipid markers (total cholesterol, triglyceride, high density lipoprotein, lipoprotein (a), and total cholesterol to high density lipoprotein ratio) with optimal performance in younger males. CONCLUSIONS Oxidized low density lipoprotein levels may be a promising biochemical risk marker of atherosclerotic disease, especially in young males.

Regulation of angiogenesis by the aging suppressor gene klotho

Advanced age is a major risk factor of peripheral artery disease. We examined the effects of the aging-suppressor gene klotho on angiogenesis in response to ischemia by introducing ischemic hindlimb model in mice heterozygously deficient for the klotho gene and in wild type mice. Blood flow recovery as assessed by laser doppler perfusion imaging and angiogenesis as assessed by density of PECAM-1/CD31-positive positive capillaries were markedly impaired in mice heterozygously deficient for the klotho gene (both <0.05). Our findings show that the aging-suppressor gene klotho affects angiogenesis and the possibility that age-related impairment of angiogenesis might be regulated by the klotho gene. Our results present a new possibility of therapeutic angiogenesis for patients of advanced age.

Neoendothelialization after peripheral blood stem cell transplantation in humans: a case report of a Tokaimura nuclear accident victim.

OBJECTIVES Neoendothelialization by circulating endothelial progenitor cells has been a topic of recent research. The extent and scale of this process in humans is not well understood. We examined the extent of neoendothelialization of the aorta and peripheral arteries in the case of a patient who underwent peripheral blood stem cell transplantation for acute radiation syndrome. METHODS Human tissue samples from the aorta and peripheral arteries were obtained at autopsy. Endothelial cells were isolated, confirmed by von Willebrand factor immunostaining, and then subjected to fluorescent in situ hybridization analysis using X- and Y-chromosome specific probes to examine neoendothelialization by donor cells as possible in this case in which the donor and recipient were of different genders. RESULTS The aorta showed almost 25% of all endothelial cells to be replaced by donor-origin endothelial cells. The peripheral arteries were also replaced but to a lesser extent. DISCUSSION The present study provides evidence that peripheral blood is a source of endothelial progenitor cells in humans. Neoendothelialization of the aorta occurs to a significant extent under certain conditions suggesting the potential for exploitation of therapeutic neovascularization by transplantation of circulating endothelial progenitor cells.

Phenotypic Modulation of Vascular Smooth Muscle Cells

Abstract: The smooth muscle myosin heavy chain (MHC) gene and its isoforms are excellent molecular markers that reflect smooth muscle phenotypes. The SMemb/Nonmuscle Myosin Heavy Chain B (NMHC‐B) is a distinct MHC gene expressed predominantly in phenotypically modulated SMCs (synthetic‐type SMC). To dissect the molecular mechanisms governing phenotypic modulation of SMCs, we analyzed the transcriptional regulatory mechanisms underlying expression of the SMemb gene. We previously reported two transcription factors, BTEB2/IKLF and Hex, which transactivate the SMemb gene promoter based on the transient reporter transfection assays. BTEB2/IKLF is a zinc finger transcription factor, whereas Hex is a homeobox protein. BTEB2/IKLF expression in SMCs is downregulated with vascular development in vivo but upregulated in cultured SMCs and in neointima in response to vascular injury after balloon angioplasty. BTEB2/IKLF and Hex activate not only the SMemb gene but also other genes activated in synthetic SMCs including plasminogen activator inhibitor‐1 (PAI‐1), iNOS, PDGF‐A, Egr‐1, and VEGF receptors. Mitogenic stimulation activates BTEB2/IKLF gene expression through MEK1 and Egr‐1. Elevation of intracellular cAMP is also important in phenotypic modulation of SMCs, because the SMemb promoter is activated under cooperatively by cAMP‐response element binding protein (CREB) and Hex.

Circulating malondialdehyde modified LDL is a biochemical risk marker for coronary artery disease

Oxidatively modified low density lipoprotein (OxLDL) plays an important role in the development of atherosclerosis as its uptake by macrophages and smooth muscle cells leads to formation of foam cells which is a critical step in the evolution of the pathological state.1,2 Circulating OxLDL concentrations may therefore reflect the state of pathological atherosclerosis, and be a possible biochemical risk marker for coronary artery disease (CAD). Numerous efforts have been directed at detecting OxLDL concentrations in the circulation for this reason, but technical difficulties have hampered detection of minute amounts of OxLDL. To overcome these limitations, we focused on circulating malondialdehyde modified LDL (MDA-LDL), a chemical modification thought to reflect naturally occurring oxidation of LDL,3,4 and developed a sensitive immunoassay of circulating MDA-LDL concentrations. The diagnostic performance of MDA-LDL in CAD was compared against known lipid markers. This comparison revealed, for the first time, that MDA-LDL is superior, thus suggesting that MDA-LDL may be a promising tool for the biochemical detection of CAD. Consenting patients with CAD defined as having greater than 75% stenosis in one or more arteries on coronary angiography were enrolled, as were normal control subjects which included patients with normal coronary angiograms, and subjects who were admitted for regular health examinations and had: (1) no history of CAD; (2) normal renal function; (3) normal ECG and chest x ray. Blood was drawn under fasting conditions and centrifuged within four hours. Stabilising reagent containing sucrose and EDTA was added and samples were stored at −20°C until the time of …