Abstract-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, 3.3ϫ10 10 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. T he klotho gene, identified by insertional mutagenesis in mice, is a suppressor of the expression of multiple aging phenotypes similar to age-related diseases in humans, such as arteriosclerosis, osteoporosis, infertility, pulmonary emphysema, and short lifespan. 1 Interestingly, expression of klotho mRNA in the kidney can be only faintly detected in the prenatal rat, and it is markedly augmented after 4 days of age. 2 Although the klotho gene has a role in phenotypic alterations in various organs, expression of klotho mRNA is predominantly observed in the kidney, 1 suggesting that the Klotho protein or its metabolites may function as humoral factors. Recent studies have shown that expression of renal klotho gene is regulated in animals 2 and in humans 3 in some diseased conditions. At present, however, the mechanism regulating klotho gene expression is poorly understood.In the present study, we have investigated the role of angiotensin (Ang) II in the regulation of renal klotho gene expression. In addition, to clarify the possible physiological role of the klotho gene in the Ang II-infused rats, exogenous klotho gene was delivered into Ang II-infused rats, and functional and histological changes in the kidney were analyzed. Methods Animal ModelsThe experiments were performed in accordance with the guidelines and practices established by the Animal Center for Biomedical Research, University of Tokyo, Faculty of Medicine. The rat Ang II hypertension model was induced in male Sprague-Dawley rats (Nippon Bio-Supply Center, Tokyo, Japan) by the continuous infusion of [Val 5 ]-Ang II (Sigma) at a dose of 0.7 mg/kg per day via an osmotic minipump (Alza) as described previously. 4 In some experiments, the selective angiotensin type 1 receptor antagonist losartan (25 mg/kg per day; a gift from Merck, Rahway, NJ) ...
Abstract-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 ϩ/Ϫ ) 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 ϩ/Ϫ and AM22-52-treated mice. Notably, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM ϩ/Ϫ 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.
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