Hiroshi Satonaka

AMP-Activated Protein Kinase Inhibits Angiotensin II–Stimulated Vascular Smooth Muscle Cell Proliferation

Background—AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that works as a metabolic sensor of cellular ATP levels. Here, we investigated whether AMPK signaling has a role in the regulation of the angiotensin II (Ang II)–induced proliferation signal in rat vascular smooth muscle cells (VSMCs). Methods and Results—Aminoimidazole-4-carboxamide-1-&bgr;-ribofuranoside (AICAR) activated AMPK in rat VSMCs and inhibited Ang II–induced extracellular signal–regulated kinase 1/2 phosphorylation but not that of p38 MAPK or Akt/PKB. Although Ang II activated AMPK, this activation was significantly inhibited by catalase, N-acetylcysteine, and diphenyleneiodonium chloride, an NADPH oxidase inhibitor. Moreover, the observation that AMPK was activated by H2O2 suggests that AMPK is redox sensitive. The Ang II type 1 receptor antagonist valsartan but not the Ang II type 2 receptor antagonist PD123319 significantly inhibited Ang II–induced AMPK activation, suggesting that Ang II–induced AMPK activation was Ang II type 1 receptor dependent. Whereas 3H-thymidine incorporation by VSMCs treated with Ang II was significantly inhibited when the cells were pretreated with 1 mmol/L AICAR, the inhibition of AMPK by dominant-negative AMPK overexpression augmented Ang II–induced cell proliferation. Subcutaneous injection of AICAR (1 mg/g body weight per day) for 2 weeks suppressed neointimal formation after transluminal mechanical injury of the rat femoral artery. Conclusions—Our findings indicate that Ang II–induced AMPK activation is synchronized with extracellular signal-regulated kinase signaling and that AMPK works as an inhibitor of the Ang II proliferative pathway. AMPK signaling might serve as a new therapeutic target of vascular remodeling in cardiovascular diseases.

Transcriptional Activation of the cyclin D1 Gene Is Mediated by Multiple Cis-Elements, Including SP1 Sites and a cAMP-responsive Element in Vascular Endothelial Cells

In an attempt to examine the mechanisms by which transcriptional activity of the cyclin D1 promoter is regulated in vascular endothelial cells (EC), we examined thecis-elements in the human cyclin D1 promoter, which are required for transcriptional activation of the gene. The results of luciferase assays showed that transcriptional activity of the cyclin D1 promoter was largely mediated by SP1 sites and a cAMP-responsive element (CRE). DNA binding activity at the SP1 sites, which was analyzed by electrophoretic mobility shift assays, was significantly increased in the early to mid G1 phase, whereas DNA binding activity at CRE did not change significantly. Furthermore, Induction of the cyclin D1 promoter activity in the early to mid G1phase depended largely on the promoter fragment containing the SP1 sites, whereas the proximal fragment containing CRE but not the SP1 sites was constitutively active. Finally, the increase in DNA binding and promoter activities via the SP1 sites was mediated by the Ras-dependent pathway. The results suggested that the activation of the cyclin D1 gene in vascular ECs was regulated by a dual system; one was inducible in the G1phase, and the other was constitutively active.

Role of Endogenous Adrenomedullin in the Regulation of Vascular Tone and Ischemic Renal Injury: Studies on Transgenic/Knockout Mice of Adrenomedullin Gene

Adrenomedullin (AM) is a potent depressor peptide whose vascular action is suggested to involve nitric oxide (NO) release. To explore the role of endogenous AM in vascular and renal function, we examined the effects of acetylcholine (ACh), AM, and AM receptor antagonists AM(22-52) and CGRP(8-37) on the renal perfusion pressure (RPP) of kidneys isolated from AM transgenic (TG)/heterozygote knockout (KO) mice and wild-type littermates (WT). Furthermore, we evaluated the renal function and histology 24 hours after bilateral renal artery clamp for 45 minutes in TG, KO, and WT mice. Baseline RPP was significantly lower in TG than in KO and WT mice (KO 93.4±4.6, WT 85.8±4.2, TG 72.4±2.4 mm Hg [mean±SE], P <0.01). ACh and AM caused a dose-related reduction in RPP, but the degree of vasodilatation was smaller in TG than that in KO and WT (%&Dgr;RPP 10−7 mol/L ACh: KO −48.1±3.9%, WT −57.5±5.6%, TG −22.8±4.8%, P <0.01), whereas NG-nitro-l-arginine methyl ester (L-NAME) caused greater vasoconstriction in TG (%&Dgr;RPP 10−4 mol/L: KO 33.1±3.3%, WT 55.5±7.2%, TG 152.6±21.2%, P <0.01). Both AM antagonists increased RPP in TG to a greater extent compared with KO and WT mice (%&Dgr;RPP 10−6 mol/L CGRP(8-37): KO 12.8±2.6%, WT 19.4±3.6%, TG 41.8±8.7%, P <0.01). In mice with ischemic kidneys, serum levels of urea nitrogen and renal damage scores showed smaller values in TG and greater values in KO mice (urea nitrogen: KO 104±5>WT 98±15>TG 38±7 mg/dL, P <0.05 each). Renal NO synthase activity was also greater in TG mice. However, the differences in serum urea nitrogen and renal damage scores among the 3 groups of mice were not observed in mice pretreated with L-NAME. In conclusion, AM antagonists increased renal vascular tone in WT as well as in TG, suggesting that endogenous AM plays a role in the physiological regulation of the vascular tone. AM is likely to protect renal tissues from ischemia/reperfusion injury through its NO releasing activity.

Angiotensin II Induces Myocyte Enhancer Factor 2- and Calcineurin/Nuclear Factor of Activated T Cell-Dependent Transcriptional Activation in Vascular Myocytes

It is well known that angiotensin II (Ang II) is implicated in the phenotypic modulation and hypertrophy of vascular smooth muscle cells (VSMCs). To study the mechanisms by which Ang II contributes to the pathological changes of VSMCs, we examined whether Ang II stimulated myocyte enhancer factor 2 (MEF2)- and calcineurin/nuclear factor of activated T cell (NFAT)-dependent transcriptional activation of genes in VSMCs. Ang II increased the DNA binding activity of MEF2A and its expression at the protein level. Ang II induced c-jun promoter activity, and this increase was inhibited by dominant-negative mutants of MEF2A and mitogen-activated protein kinase kinase 6 but not by calcineurin inhibitors. Ang II stimulated NFAT DNA binding activity and NFAT-dependent gene transcription, and these effects of Ang II were inhibited by calcineurin inhibitors. Furthermore, Ang II induced the promoter activity of the nonmuscle-type myosin heavy chain B gene, which we used as a marker of the dedifferentiated state of VSMCs, and this increase was inhibited by calcineurin inhibitors but not by the dominant-negative mutants of MEF2A or mitogen-activated protein kinase kinase 6. Finally, Ang II increased protein synthesis, and this increase was inhibited by infection with an adenovirus construct that expresses the dominant-negative mutant of MEF2A but not by calcineurin inhibitors. These results suggest that Ang II stimulates the MEF2- and calcineurin/NFAT-dependent pathways and that these pathways have distinct roles in VSMCs.

Blockade of Endogenous Cytokines Mitigates Neointimal Formation in Obese Zucker Rats

Background—It is well known that diabetes mellitus is a major risk factor for vascular diseases such as atherosclerosis and restenosis after angioplasty. It has become clear that advanced glycation end products (AGE) and their receptor (RAGE) are implicated in vascular diseases, especially in diabetes mellitus. Nevertheless, the mechanisms by which diabetes mellitus is often associated with vascular diseases remain unclear. Methods and Results—To study the role of endogenous cytokines such as tumor necrosis factor-&agr; (TNF-&agr;) and interleukin-6 in the development of vascular diseases and in the expression of RAGE, we used semapimod, a pharmacological inhibitor of cytokine production, and examined its effect on neointimal formation in the femoral artery of obese Zucker (OZ) rats. We also used an adenovirus construct expressing a dominant negative mutant of the receptor for TNF-&agr; (AdTNFR&Dgr;C) to block the action of endogenous TNF-&agr;. Semapimod significantly suppressed neointimal formation and RAGE expression in OZ rats compared with untreated OZ rats. This inhibitory effect of semapimod on neointimal formation was overcome by infection of an adenovirus expressing RAGE into the femoral artery of OZ rats. Furthermore, AdTNFR&Dgr;C infection significantly suppressed neointimal formation and RAGE expression in the femoral artery of OZ rats. Conclusions—These results suggest that endogenous cytokines, especially TNF-&agr;, were implicated in neointimal formation in OZ rats and that RAGE was a mediator of the effect of these cytokines on neointimal formation.

Ghrelin Improves Renal Function in Mice with Ischemic Acute Renal Failure

Growth hormone and IGF-1 have been suggested to have tissue-protective effects. Ghrelin is a stomach-derived growth hormone secretagogue. The effects of ghrelin on ischemia/reperfusion-induced renal failure in mice were examined. Ischemic acute renal failure was induced by bilateral renal artery clamping for 45 min and reperfusion for 24 h. Ghrelin (100 microg/kg mouse) or vehicle was injected subcutaneously six times before surgery and three times after surgery every 8 h. Twenty-four hours after reperfusion, the right kidney was isolated and perfused. Acetylcholine (ACh)- and adrenomedullin-induced endothelium-dependent vasorelaxation of renal vessels significantly improved in ghrelin-pretreated mice (%Delta renal perfusion pressure by 10(-7) M ACh -63.5 +/- 3.7 versus -41.2 +/- 5.5%; P < 0.05). This change was associated with significant increases of nitric oxide release in the kidneys of ghrelin-treated mice (10(-7) M ACh 35.5 +/- 5.8 versus 16.9 +/- 3.5 fmol/g kidney per min; P < 0.05). Serum concentration of urea nitrogen (53 +/- 7 versus 87 +/- 15 mg/dl; P < 0.05) and renal injury score were significantly lower in the ghrelin group (2.5 +/- 0.8 versus 5.3 +/- 1.5; P < 0.01). Tubular apoptotic index was significantly lower in the ghrelin group (5 +/- 5 versus 28 +/- 4; P < 0.05). Furthermore, the survival rate after the 60-min ischemic period was higher in the ghrelin group (80 versus 20%; P < 0.05). Ghrelin treatment significantly increased the serum level of IGF-1. However, such renal protective effects of ghrelin on ischemia/reperfusion injury were not observed in insulin receptor substrate-2 knockout mice. These results suggest that ghrelin may protect the kidneys from ischemia/reperfusion injury and that this effect is related to an improvement of endothelial function through an IGF-1-mediated pathway.

Calcineurin Promotes the Expression of Monocyte Chemoattractant Protein-1 in Vascular Myocytes and Mediates Vascular Inflammation

Abstract— Although the role of the calcineurin-dependent pathway in the development of cardiac hypertrophy has been intensively studied, little is known of its role in vascular inflammatory diseases such as atherosclerosis and restenosis after angioplasty. To help elucidate the role of calcineurin in vascular inflammation, we infected cultured vascular smooth muscle cells (VSMCs) with an adenovirus construct expressing a constitutively active mutant of calcineurin, and examined its effect on the expression of monocyte chemoattractant protein-1 (MCP-1). We also examined the role of calcineurin in vivo using a transluminal wire injury model of the rat femoral artery. Forced activation of calcineurin significantly increased the expression of MCP-1 both at the transcriptional and protein levels. Angiotensin II (Ang II) also significantly stimulated MCP-1 expression, and this increase was significantly inhibited by cyclosporin A (CyA). Constitutive activation of calcineurin stabilized MCP-1 mRNA without enhancing MCP-1 promoter activity. In accordance with the results, Ang II–induced increase of MCP-1 promoter activity was not suppressed by CyA. Ang II stabilized MCP-1 mRNA, and this effect of Ang II was diminished by CyA. CyA suppressed MCP-1 expression in the femoral artery after the transluminal mechanical injury. CyA also inhibited macrophage infiltration and neointimal formation in the wire-injured femoral arteries. These results suggested that calcineurin mediates vascular inflammation via stimulation of MCP-1 expression in VSMCs and macrophage infiltration.

Myocyte Enhancer Factor 2 Mediates Vascular Inflammation via the p38-Dependent Pathway

Although it has been established that myocyte enhancer factor 2 (MEF2) plays pivotal roles in the development of the cardiovascular system as well as skeletal muscle cells, little is known of its role in vascular inflammatory diseases such as atherosclerosis and restenosis after angioplasty. To investigate the role of MEF2 in vascular inflammation and that of p38 in the activation of MEF2, we infected cultured rat vascular smooth muscle cells (VSMCs) with an adenovirus construct expressing a dominant-negative mutant of MEF2A (MEF2ASA) or mitogen-activated protein kinase kinase 6 (MEK6AA), and examined their effects on the expression of monocyte chemoattractant protein-1 (MCP-1), which is known to play important roles in vascular inflammation. We also examined the role of MEF2 in vivo using a rat model of transluminal wire-induced injury of the femoral artery. Angiotensin II (Ang II)–induced expression of MCP-1 mRNA was significantly inhibited by infection with adenoviruses encoding MEF2ASA (AdMEF2ASA) or MEK6AA. Ang II–induced increase of MCP-1 promoter activity was also significantly suppressed by overexpression of MEF2ASA or MEK6AA. Ang II stimulated the transactivating function of MEF2A and this activation was inhibited by overexpression of MEK6AA. Infection with AdMEF2ASA suppressed MCP-1 expression in the femoral artery after the transluminal mechanical injury. AdMEF2ASA infection also inhibited macrophages infiltration and neointimal formation in the wire-injured femoral arteries. These results suggested that MEF2 activation via the p38-dependent pathway mediates vascular inflammation via stimulation of MCP-1 expression in VSMCs and macrophages infiltration.

A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell

The inhibition of apoptotic changes in vascular endothelial cells is important for preventing vascular damage from hypoxia. AMP-activated protein kinase (AMPK) has recently been identified as playing a role in vascular protection. Although the chemical reagent 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) has been used to stimulate AMPK activity, AICAR has been associated with several nonspecific reactions. We therefore constructed a new constitutively active mutant of AMPKα1 (NcaAMPK), which lacks the autoinhibitory domain in AMPKα1 and in which threonine 172 has been replaced with aspartate. We investigated whether NcaAMPK has an anti-apoptotic effect in vascular endothelial cells under anoxic conditions. NcaAMPK, or green fluorescent protein (GFP) as a control, was overexpressed in human umbilical vein endothelial cells (HUVECs). After HUVECs were incubated for 40 h under normoxic or anoxic conditions, we examined cell viability, caspase 3/7 activity, and expression and phosphorylation levels of apoptosis-related proteins. Cell viabilities under anoxic conditions were improved in NcaAMPK-overexpressing cells. Anoxia increased caspase 3/7 activity, but NcaAMPK reduced this increase significantly. NcaAMPK overexpression increased protein kinase B/Akt Ser473 and endothelial nitric oxide synthase Ser1177 phosphorylation, but pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) did not decrease the viability of NcaAMPK-overexpressing HUVECs. Furthermore, co-expression of a dominant-negative Akt reduced the improvement in cell viability and the suppression of poly (ADP-ribose) polymerase cleavage by NcaAMPK under anoxic conditions. In conclusion, NcaAMPK inhibited anoxia-induced apoptosis in vascular endothelial cells through Akt activation, suggesting that activation of AMPK might protect against ischemic vascular injury.

Effects of intracavernous administration of adrenomedullin on erectile function in rats

We have reported that adrenomedullin (AM)-induced vasodilation is at least in part nitric oxide (NO)-cGMP-dependent in the rat. Although it is well known that NO is much involved in the erectile function, it is controversial as to whether AM influences the erectile function. Thus, we examined the effects of AM on intracavernous pressure (ICP) during penile erection. The left carotid artery of rats was cannulated to monitor of mean arterial pressure (MAP). Bipolar electrodes were positioned on the cavernous nerve. The right cavernous body was cannulated with a needle connected to a pressure transducer to monitor ICP. Electrical stimulation (ES) increased ICP in a voltage-dependent manner. Elevation of ICP continued during ES. The intracavernous injection of 0.5 nmol AM significantly potentiated ES-induced increases in both maximal developed ICP/MAP and area under the curve (ICP trace; AUC). Since AM slightly lowered MAP, ICP was normalized by MAP. i.v. administration of N(omega)-nitro-L-arginine, a NO synthase inhibitor, markedly decreased AM/ES-induced ICP elevation. However, in the presence of E-4021, a cGMP-specific phosphodiesterase inhibitor, AM further increased both ICP/MAP and AUC. These results suggest that a NO-cGMP pathway is involved in the regulation of AM-induced rat cavernous vasorelaxation.