We recently isolated a Krüppel-like zinc-finger transcription factor 5 (KLF5; also known as BTEB2 and IKLF), which is markedly induced in activated vascular smooth-muscle cells and fibroblasts. Here we describe our analysis of the in vivo function of KLF5 using heterozygous KLF5-knockout mice (Klf5(+/-)). In response to external stress, Klf5(+/-) mice showed diminished levels of arterial-wall thickening, angiogenesis, cardiac hypertrophy and interstitial fibrosis. Also, angiotensin II induced expression of KLF5, which in turn activated platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta (TGF-beta) expression. In addition, we determined that KLF5 interacted with the retinoic-acid receptor (RAR), that synthetic RAR ligands modulated KLF5 transcriptional activity, and that in vivo administration of RAR ligands affected stress responses in the cardiovascular system in a KLF5-dependent manner. KLF5 thus seems to be a key element linking external stress and cardiovascular remodeling.

Based on the findings that the azo functional group has excellent properties as the hypoxia-sensor moiety, we developed hypoxia-sensitive near-infrared fluorescent probes in which a large fluorescence increase is triggered by the cleavage of an azo bond. The probes were used for fluorescence imaging of hypoxic cells and real-time monitoring of ischemia in the liver and kidney of live mice.

Novel near-infrared (NIR) fluorescent probes for nitric oxide (NO) have been designed, synthesized, and evaluated. Their NIR fluorescence was increased in an NO concentration-dependent manner under physiological conditions, and their reaction efficiency with NO was at least 53 times higher than that of a widely used NO probe, DAF-2. They were confirmed to function in isolated intact rat kidneys. Because NIR light can penetrate deeply into tissues, these probes may have potential for in vivo NO imaging.

Background-Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. Levels of AM are markedly increased in the fetoplacental circulation during pregnancy, although its function there remains unknown. To clarify the physiological functions of AM, we chose a gene-targeting strategy in mice. Methods and Results-Targeted null mutation of the AM gene is lethal in utero: the mortality rate among AM Ϫ/Ϫ embryos was Ͼ80% at E13.5. The most apparent abnormality in surviving AM Ϫ/Ϫ embryos at E13.5 to E14.0 was severe hemorrhage, readily observable under the skin and in visceral organs. Hemorrhage was not detectable at E12.5 to E13.0, although the yolk sac lacked well-developed vessels. Electron microscopic examination showed endothelial cells to be partially detached from the basement structure at E12.5 in vitelline vessels and hepatic capillaries, which allowed efflux of protoerythrocytes through the disrupted barrier. The basement membrane was not clearly recognizable in the aorta and cervical artery, and the endothelial cells stood out from the wall of the lumen, only partially adhering to the basement structure. AM ϩ/Ϫ mice survived to adulthood but exhibited elevated blood pressures with diminished nitric oxide production. Conclusions-AM is indispensable for the vascular morphogenesis during embryonic development and for postnatal regulation of blood pressure by stimulating nitric oxide production.

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Abstract-␣-Calcitonin gene-related peptide (␣CGRP) is a pleiotropic neuropeptide implicated in a variety of physiological processes. To better understand the biological functions of ␣CGRP, we developed an ␣CGRP-null mouse model using a gene targeting approach. Recordings of mean arterial pressure (MAP) and heart rate (HR) showed that basal MAP and HR were significantly higher in both anesthetized and conscious, unrestrained ␣CGRP-null mice than in corresponding wild-type mice. The elevated MAP in ␣CGRP-null mice was shown to be the result of elevated peripheral vascular resistance by ␣-adrenergic blockade with prazosin and by transthoracic echocardiogram, which revealed no significant differences between ␣CGRP-null and wild-type mice in the stroke volume, fractional shortening, and ejection fraction. Moreover, evaluation of autonomic nervous activity by measuring HR after pretreatment of atropine and/or atenolol and by analyzing arterial baroreceptor reflexes showed sympathetic nervous activity to be significantly elevated in ␣CGRP-null mice; elevated levels of urinary catecholamine metabolites and decreased HR variability in mutant mice were also consistent with that finding. These findings suggest that ␣CGRP contributes to the regulation of cardiovascular function through inhibitory modulation of sympathetic nervous activity. Key Words: calcitonin gene-related peptide Ⅲ gene targeting Ⅲ blood pressure Ⅲ autonomic nervous system Ⅲ hypertension C alcitonin gene-related peptide (CGRP) is a 37-amino acid vasoactive neuropeptide produced by tissue-specific alternative splicing of the primary transcript of the calcitonin/ ␣CGRP gene. 1 While calcitonin (CT), which controls calcium homeostasis, is expressed almost exclusively in the C cells of the thyroid gland, ␣CGRP is widely distributed in the central and peripheral nervous systems in mammals. In addition, a second CGRP isoform, ␤CGRP, is encoded by a different gene locus and is expressed almost exclusively in specific neuronal sites. 2 These two CGRP isoforms-␣ and ␤ in rat and I and II in humans-exhibit overlapping biological activities in most vascular beds. 3 Within the nervous system, CGRP immunoreactivity has been detected in spinal cord motor neurons, dorsal root ganglia, and motor nerve endings. 4 Other neuropeptides, the tachykinins, which include substance P (SP) and neurokinin A (NKA), exhibit similar expression patterns to ␣CGRP in several regions of the nervous system. ␣CGRP and tachykinins coexist in primary afferent neurons, forming the part of the so-called nonadrenergic, noncholinergic (NANC) nervous system, 5 and their release from sensory pain fibers has been implicated in the perception of pain.NANC neurons containing ␣CGRP are also widely distributed among autonomic fibers innervating the vasculature; for example, they are found in blood vessels at the junction of the adventitia and the media passing into the muscle layer. 5 Moreover, ␣CGRP is a potent vasodilator, 6 and several investigators have claimed that ␣CGRP may play a key role in regu...

Abstract-To study the mechanisms by which adrenomedullin (AM) induces endothelium-dependent vasorelaxation, we examined whether AM-induced endothelium-dependent vasodilation was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent pathway in rat aorta, because it was recently reported that PI3K/Akt was implicated in the activation of endothelial NO synthase. AM-induced vasorelaxation in thoracic aorta with intact endothelium was inhibited by pretreatment with PI3K inhibitors to the same level as that in endothelium-denuded aorta. AM elicited Akt phosphorylation in a time-and dose-dependent manner. AM-induced Akt phosphorylation was inhibited by pretreatment with a calmodulin-dependent protein kinase inhibitor as well as with PI3K inhibitors. When an adenovirus construct expressing a dominant-negative Akt mutant (Ad/dnAkt) was injected into abdominal aortas so that the mutant was expressed predominantly in the endothelium layer, AM-induced vasodilation was diminished to the same level as that in endothelium-denuded aortas. Finally, AM-induced cGMP production, which was used as an indicator for NO production, was suppressed by PI3K inhibition or by Ad/dnAkt infection into the endothelium. These results suggested that AM induced Akt activation in the endothelium via the Ca 2ϩ /calmodulin-dependent pathway and that this was implicated in the production of NO, which in turn induced endothelium-dependent vasodilation in rat aorta. Key Words: adrenomedullin Ⅲ phosphatidylinositol 3-kinase Ⅲ Akt Ⅲ nitric oxide Ⅲ gene transfer V ascular endothelial cells (ECs) are critically implicated in the modulation of vascular tone by producing a variety of vasoactive substances. Among them, endothelin has a vasoconstrictive activity, whereas NO and prostacyclin possess a vasorelaxant activity. The modulation of vascular tone by ECs is known to be perturbed in pathophysiological states such as hypertension, diabetes mellitus, and hyperlipidemia. 1 A novel peptide, which increases the level of cAMP in platelets, was recently isolated from human adrenal medulla and was dubbed adrenomedullin (AM). 2 AM is a 52-amino acid peptide with a potent vasorelaxant activity and natriuretic activity. [3][4][5] Although AM was originally isolated from human pheochromocytoma tissue, it is now well known that AM is also produced by vascular ECs, vascular smooth muscle cells, and macrophages, 6 -8 suggesting its role as a local mediator in normal and/or diseased vessels. Although little is known of the intracellular signaling pathways that are activated by AM, it is reported that AM increases intracellular cAMP and [Ca 2ϩ ] i . 9 AM also activates extracellular signalregulated kinase (ERK), which appears to be involved in AM-induced cell proliferation, 10 -12 although AM seems to inhibit cell proliferation in some cells, 13 depending on cell types. It was originally postulated that AM-induced vasorelaxation might be mediated by its effect on intracellular cAMP. However, we have recently shown that AM induced vasorelaxation, at least p...

Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.