Hepcidin destabilizes atherosclerotic plaque via overactivating macrophages after erythrophagocytosis.

OBJECTIVE: To explore a direct and causal relationship between vascular hepcidin and atherosclerotic plaque stability. METHODS AND RESULTS: Accelerated atherosclerotic lesions were established by perivascular collar placement in apolipoprotein E-deficient (ApoE(-/-)) mice. Adenoviral overexpression of hepcidin in the carotid artery during plaque formation enhanced intraplaque macrophage infiltration and suppressed the contents of collagen and vascular smooth muscle cells, whereas hepcidin shRNA treatment exerts opposite effects. The overexpression or knockdown of hepcidin did not affect plaque lipid deposition but increased or decreased oxidized low-density lipoprotein (ox-LDL) levels within intraplaque macrophages. In cultured macrophages, ox-LDL not only increased reactive oxygen species formation, inflammatory cytokine production, and apoptosis but also upregulated hepcidin expression. However, hepcidin did not exaggerate the ox-LDL-induced activation of macrophages until an onset of erythrophagocytosis. Whereas hepcidin was critical for the upregulation of L-ferritin and H-ferritin in both ox-LDL-treated erythrophagocytosed macrophages and atherosclerotic plaques, the adding of iron chelators suppressed the intracellular lipid accumulation, reactive oxygen species formation, inflammatory cytokine expression, and apoptosis in erythrophagocytosed macrophages. CONCLUSIONS: Hepcidin promotes plaque destabilization partly by exaggerating inflammatory cytokine release, intracellular lipid accumulation, oxidative stress, and apoptosis in the macrophages with iron retention.

Ubiquitin carboxyl-terminal hydrolase L1, a novel deubiquitinating enzyme in the vasculature, attenuates NF-kappaB activation.

OBJECTIVE: We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library. UCHL1 is expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson's disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor-kappaB (NF-kappaB) inactivation in ECs and vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: From Northern blot and immunohistochemical analysis, the UCHL1 gene was endogenously expressed in vascular ECs, VSMCs, and brain tissue. Expression of UCHL1 was markedly increased in the neointima of the balloon-injured carotid artery and was also present in atherosclerotic lesions from human carotid arteries. Overexpression of the UCHL1 gene significantly attenuated tumor necrosis factor (TNF)-alpha-induced NF-kappaB activity in vascular cells and increased inhibitor of kappa B-alpha (IkappaB-alpha), possibly through the attenuation of IkappaB-alpha ubiquitination, leading to decreased neointima in the balloon-injured artery. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF-kappaB activity in VSMCs. CONCLUSIONS: These data suggest that UCHL1 may partially attenuate vascular remodeling through inhibition of NF-kappaB activity.

Effect of azelnidipine on angiotensin II-mediated growth-promoting signaling in vascular smooth muscle cells.

The detailed mechanism of the effects of extracellular Ca2+ entry blockade on angiotensin II (Ang II) type 1 (AT1) receptor-mediated growth-promoting signals in vascular smooth muscle cells (VSMCs) is not fully understood. Ang II stimulation caused biphasic activation of growth-promoting signals, reaching a peak at 5 to 10 min followed by a decrease and a second peak at around 2 to 4 h. Addition of PD98059 (2'-amino-3'-methoxyflavone), a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, or AG490 [alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide], a Janus-activated kinase 2 (Jak2) inhibitor, even 4 h after Ang II treatment inhibited [3H]thymidine incorporation. The calcium channel blocker azelnidipine attenuated the later peaks of extracellular signal-regulated kinase (ERK), tyrosine kinase 2, Jak2 activation, and phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3. Interestingly, azelnidipine increased rather than decreased the later ERK peaks in cells treated with small interfering RNA against mitogen-activated protein kinase phosphatase-1. Ang II-mediated [3H]thymidine incorporation was inhibited dose dependently by azelnidipine and also by azelnidipine, plus olmesartan, whereas olmesartan or azelnidipine alone at such lower doses did not affect [3H]thymidine incorporation. These data provide new insight into the manner in which calcium channels exert an essential action in the AT1 receptor-mediated growth-promoting actions in VSMCs.

Fluvastatin enhances the inhibitory effects of a selective AT1 receptor blocker, valsartan, on atherosclerosis.

We investigated the effects of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor (statin) on the inhibitory effects of an angiotensin II type-1 receptor (AT1) blocker on atherosclerosis and explored cellular mechanisms. We gave apolipoprotein E null mice a high-cholesterol diet for 10 weeks and measured atherosclerotic plaque area and lipid deposition. Neither 1 mg/kg per day of valsartan nor 3 mg/kg per day of fluvastatin had any effect on blood pressure or cholesterol concentration; however, both drugs decreased plaque area and lipid deposition after 10 weeks. We then reduced the doses of both drugs to 0.1 mg/kg per day and 1 mg/kg per day, respectively. At these doses, neither drug had an effect on atherosclerotic lesions. When both drugs were combined at these doses, a significant reduction in atherosclerotic lesions was observed. Similar inhibitory effects of valsartan or fluvastatin on the expressions of nicotinamide-adenine dinucleotide/nicotinamide-adenine dinucleotide phosphate oxidase subunits p22phox and p47phox, production of superoxide anion, the expression of monocyte chemoattractant protein-1, and intercellular adhesion molecule-1 expression were observed. These results suggest that concomitant AT1 receptor and cholesterol biosynthesis blockade, particularly when given concomitantly, blunts oxidative stress and inflammation independent of blood pressure or cholesterol-related effects.

Roles of angiotensin type 1 and 2 receptors in pregnancy-associated blood pressure change.

BACKGROUND: Activation of the renin-angiotensin system with increased levels of renin and angiotensin (Ang) II in pregnancy has been reported, but the vascular responsiveness to Ang II seems to be decreased, thereby keeping maternal blood pressure (BP) constant. We postulated that the balance of angiotensin type 1 (AT1) and angiotensin type 2 (AT2) receptor expression, which would exert antagonistic actions on vasoconstriction and cell growth, might control BP in pregnancy. METHODS: Using wild type (C57BL/6J), AT1a receptor null and AT2 receptor null mice, we examined the changes in BP, expression and localization of AT1 and AT2 receptors in placenta, umbilical cord, and uterus by immunohistochemical staining and urinary albumin measurement during pregnancy. RESULTS: Wild type mice did not show any significant change in BP throughout pregnancy. The BP in AT1a receptor null mice declined significantly in the second trimester of pregnancy, whereas BP in AT2 receptor null mice increased significantly in the third trimester. We did not observe any significant differences in albuminuria, litter size, or body weight of neonates among the three groups. Vascular smooth muscle cells in blood vessels of the umbilical cord and placenta specifically expressed AT2 receptors, which are minimally expressed in adult vessels. In contrast, AT1 receptors were dominantly expressed in the cytotrophoblast and chorionic plate as well as blood vessels in placenta and umbilical cord. CONCLUSIONS: Our results suggested that disturbance of the balance of the AT1 and AT2 receptors could trigger pregnancy induced hypertension.

Trans-inactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP.

Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic beta(2) receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade.

Angiotensin II type-1 receptor blocker valsartan enhances insulin sensitivity in skeletal muscles of diabetic mice.

Angiotensin II has been shown to contribute to the pathogenesis of insulin resistance; however, the mechanism is not well understood. The present study was undertaken to investigate the potential effect of an angiotensin II type-1 (AT1) receptor blocker, valsartan, to improve insulin resistance and to explore the signaling basis of cross-talk of the AT1 receptor- and insulin-mediated signaling in type 2 diabetic KK-Ay mice. Treatment of KK-Ay mice with valsartan at a dose of 1 mg/kg per day, which did not influence systolic blood pressure, significantly increased insulin-mediated 2-[3H]deoxy-d-glucose (2-[3H]DG) uptake into skeletal muscle and attenuated the increase in plasma glucose concentration after a glucose load and plasma concentrations of glucose and insulin. In contrast, insulin-mediated 2-[3H]DG uptake into skeletal muscle was not influenced in AT2 receptor null mice, and an AT2 receptor blocker, PD123319, did not affect 2-[3H]DG uptake and superoxide production in skeletal muscle of KK-Ay mice. Moreover, we observed that valsartan treatment exaggerated the insulin-induced phosphorylation of IRS-1, the association of IRS-1 with the p85 regulatory subunit of phosphoinositide 3 kinase (PI 3-K), PI 3-K activity, and translocation of GLUT4 to the plasma membrane. It also reduced tumor necrosis factor-alpha (TNF-alpha) expression and superoxide production in skeletal muscle of KK-Ay mice. Specific AT1 receptor blockade increases insulin sensitivity and glucose uptake in skeletal muscle of KK-Ay mice via stimulating the insulin signaling cascade and consequent enhancement of GLUT4 translocation to the plasma membrane.

Calcium channel blocker azelnidipine enhances vascular protective effects of AT1 receptor blocker olmesartan.

The present studies were undertaken to investigate the potential effect of a calcium channel blocker (CCB) to enhance the inhibitory effect of an angiotensin (Ang) II type 1 (AT1) receptor blocker (ARB) on vascular injury and the cellular mechanism of the effect of CCB on vascular remodeling. In polyethylene cuff-induced vascular injury of the mouse femoral artery, proliferation of vascular smooth muscle cells (VSMCs) and neointimal formation associated with activation of extracellular signal-regulated kinase (ERK), and tyrosine-phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3, inflammatory response assessed by monocyte chemoattractant protein-1 and tumor necrosis factor-alpha expression, as well as oxidative stress such as expression of NADH/NADPH oxidase p22(phox) subunit and superoxide production, were less in AT1a receptor null mice. Administration of nonhypotensive doses of a CCB, azelnidipine (0.5 or 1 mg/kg per day) attenuated these parameters in wild-type and AT1a receptor null mice. Coadministration of lower doses of an ARB, olmesartan (0.5 mg/kg per day), and azelnidipine (0.1 mg/kg per day), which did not affect vascular remodeling, significantly inhibited these parameters in wild-type mice. Moreover, the effective dose of azelnidipine (1 mg/kg per day) exaggerated the inhibitory action of olmesartan at effective doses of 1 or 3 mg/kg per day on VSMC proliferation in the injured arteries. These results suggest that azelnidipine could inhibit vascular injury at least partly independent of the inhibition of AT1 receptor activation and that azelnidipine could exaggerate the vascular protective effects of olmesartan, suggesting clinical possibility that the combination of CCB and ARB could be more effective in the treatment of vascular diseases.

Regulation of inhibitory protein-kappaB and monocyte chemoattractant protein-1 by angiotensin II type 2 receptor-activated Src homology protein tyrosine phosphatase-1 in fetal vascular smooth muscle cells.

In the present study we examined the effects of angiotensin II (Ang II) type 2 (AT(2)) receptor stimulation on AT(1) receptor-mediated monocyte chemoattractant protein-1 (MCP-1) expression and the possible mechanisms of AT(2) receptor-mediated signaling in cultured rat fetal vascular smooth muscle cells, which express both AT(1) and AT(2) receptors. Ang II stimulation induced MCP-1 mRNA expression as well as an increase in nuclear factor-kappaB (NF-kappaB) binding to the corresponding cis DNA element of the MCP-1 promoter region and a decrease in the cytosolic inhibitory protein-kappaB (IkappaB) protein level via AT(1) receptor stimulation, whereas stimulation of the AT(2) receptor decreased Ang II-induced MCP-1 expression, NF-kappaB DNA binding, and IkappaB degradation, suggesting that activation of the AT(2) receptor attenuated AT(1) receptor-mediated MCP-1 expression via a decrease in NF-kappaB DNA binding and an increase in IkappaB stability. Moreover, we demonstrated that AT(2) receptor stimulation attenuated TNFalpha-mediated NF-kappaB activation and MCP-1 expression. A tyrosine phosphatase inhibitor, orthovanadate, attenuated the AT(2) receptor-mediated increase in IkappaB protein. Moreover, we observed that two IkappaB subunits (IkappaBalpha and IkappaBbeta) were tyrosine-phosphorylated after Ang II stimulation. Transfection of a dominant-negative Src homology protein tyrosine phosphatase-1 mutant into vascular smooth muscle cells inhibited the AT(2) receptor-mediated increase in IkappaB, leading to a significant increase in AT(1) receptor-induced NF-kappaB activation and MCP-1 expression. Taken together, our results demonstrated that AT(2) receptor stimulation attenuated MCP-1 expression via IkappaB stabilization, and Src homology protein tyrosine phosphatase-1 might play a critical role in the transcriptional regulation of MCP-1 expression through the control of IkappaB protein stability.

Nicotine enhances angiotensin II-induced mitogenic response in vascular smooth muscle cells and fibroblasts.

OBJECTIVE: The pathogenetic mechanism of tobacco-related cardiovascular diseases is still not well defined. We examined the potential possibility of an interaction between nicotine, a major component of cigarette smoke, and angiotensin II (Ang II), which plays an important role in the pathogenesis of cardiovascular diseases characterized by Ang II type 1 (AT1) receptor-mediated abnormal growth of vascular smooth muscle cells (VSMC) and fibroblasts. METHODS AND RESULTS: Nicotine or Ang II-stimulated [3H]thymidine incorporation and c-fos expression in adult rat aortic VSMC and adventitial fibroblast. The nicotine-induced DNA synthesis was not affected by valsartan, an AT1 receptor-specific blocker, or PD123319, an Ang II type 2 (AT2) receptor-specific antagonist. Nicotine or Ang II stimulation rapidly increased extracellular signal-regulated kinase (ERK) activation, tyrosine- and serine-phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3, and p38 mitogen-activated protein kinase (p38 MAPK), in both cell types. Interestingly, co-administration of nicotine and Ang II at lower doses, which did not affect cell growth, induced DNA synthesis and c-fos expression accompanied by enhancement of ERK, STAT, and p38MAPK activity. PD98059, a mitogen-activated protein kinase/ERK kinase inhibitor, or SB23058, a p38MAPK inhibitor, significantly attenuated the vasotrophic effect of nicotine and Ang II. CONCLUSIONS: These results suggest that nicotine exerts a growth-promoting effect on vascular cells and enhances the Ang II-induced vasotrophic effect, which is at least partly mediated by the activation of ERK, STAT, and p38MAPK.

Regulation of collagen synthesis in mouse skin fibroblasts by distinct angiotensin II receptor subtypes.

We examined the possibility of whether angiotensin (Ang) II type 1 (AT1) and type 2 (AT2) receptor stimulation differentially regulates collagen production in mouse skin fibroblasts. Both AT1 and AT2 receptors were expressed in neonatal skin fibroblasts prepared from wild-type mice to a similar degree, and the AT1a receptor was exclusively expressed as opposed to the AT1b receptor. In wild-type fibroblasts, Ang II increased collagen synthesis accompanied by an increase in expression of tissue inhibitor of metalloproteinase (TIMP)-1, and these increases were inhibited by valsartan, an AT1 receptor blocker, but augmented by PD123319, an AT2 receptor antagonist. Ang II decreased basal and IGF-I-induced collagen production and inhibited TIMP-1 expression in neonatal skin fibroblasts prepared from AT1a knockout (KO) mice. These Ang II-mediated inhibitory effects on collagen production and TIMP-1 expression observed in AT1a KO fibroblasts were attenuated by the addition of PD123319 or a tyrosine phosphatase inhibitor, sodium orthovanadate, but not affected by a serine/threonine phosphatase inhibitor, okadaic acid. Moreover, we demonstrated that transfection of a catalytically inactive, dominant negative SHP-1 (Src homology 2-containing protein-tyrosine phosphatase-1) mutant inhibited the Ang II-mediated inhibitory effect on both collagen synthesis and TIMP-1 expression in AT1a KO fibroblasts. These results suggest that AT1a receptor stimulation increases collagen production in skin fibroblasts at least in part due to the inhibition of collagen degradation via the increase in TIMP-1 expression, whereas AT2 receptor stimulation exerts inhibitory effects on TIMP-1 expression, which is mediated at least partially by the activation of SHP-1, thereby possibly inhibiting collagen production.

Important role of nitric oxide in the effect of angiotensin-converting enzyme inhibitor imidapril on vascular injury.

To examine the possible role of the bradykinin-NO system in the action of ACE inhibitors, we studied the effects of imidapril, an ACE inhibitor, on inflammatory vascular injury by using AT1a-receptor-deficient (AT1aKO) mice. A polyethylene cuff was placed around the femoral artery of AT1aKO mice and wild-type (WT; C57BL/6J) mice. Neointimal area in cross sections of the artery was measured 14 days after cuff placement. A low dose of imidapril (1 mg/kg per day), which did not affect blood pressure, was administered by gavage. Expression of monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-alpha was detected by immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR) 7 days after the operation. Neointimal formation, vascular smooth muscle cell proliferation, and expression of MCP-1 and TNF-alpha were attenuated in the injured artery in AT1aKO mice compared with those in WT mice. Imidapril inhibited neointimal formation, DNA synthesis of vascular smooth muscle cells, and expression of MCP-1 and TNF-alpha in AT1aKO mice as well as in WT mice. In addition, imidapril increased tissue cGMP content after cuff placement. These inhibitory effects of imidapril were significantly reduced or abolished by a bradykinin receptor antagonist, Hoechst 140, or an NO synthase inhibitor, L-NAME, both in WT and AT1aKO mice. Treatment with imidapril did not change AT2 receptor and ACE expression detected by RT-PCR in the injured artery. These results indicate that not only blockade of angiotensin II production but also activation of the bradykinin-NO system plays an important role in the beneficial effects of imidapril on vascular remodeling.

Fluvastatin enhances the inhibitory effects of a selective angiotensin II type 1 receptor blocker, valsartan, on vascular neointimal formation.

BACKGROUND: The present studies were undertaken to investigate the potential effect of a hydroxymethylglutaryl coenzyme A reductase inhibitor (statin) to enhance the inhibitory effect of an angiotensin (Ang) II type 1 (AT1) receptor blocker (ARB) on vascular neointimal formation and to explore the cellular mechanism of cross-talk of the AT1 receptor and statin in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Neointimal formation and the proliferation of VSMCs induced by cuff placement around the femoral artery were significantly inhibited by treatment with an ARB, valsartan, at a dose of 0.1 mg x kg(-1) x d(-1) and with fluvastatin at a dose of 1 mg x kg(-1) x d(-1), which did not influence mean arterial blood pressure or plasma cholesterol level, whereas valsartan or fluvastatin alone at these doses did not affect neointimal formation or the proliferation of VSMCs. Pretreatment with fluvastatin (approximately 5 micromol/L) for 24 hours significantly inhibited Ang II (1 micromol/L)-mediated DNA synthesis and c-fos promoter activity in cultured VSMCs. Moreover, pretreatment of VSMCs with fluvastatin significantly inhibited Ang II-mediated extracellular signal-regulated kinase (ERK) activation and tyrosine- and serine-phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3. AT1 receptor-mediated recruitment of Rac-1 to Janus kinase (Jak) family/STATs was also inhibited by fluvastatin. Consistent with these in vitro results, phosphorylation of ERK, STAT1, and STAT3 was attenuated by the coadministration of valsartan and fluvastatin even at low doses in vivo. CONCLUSION: These results suggest that the cholesterol-independent inhibition of AT1 receptor-mediated VSMC proliferation by statins may contribute to the beneficial effects of statins combined with an ARB on vascular diseases.

Role of AT2 receptor in the brain in regulation of blood pressure and water intake.

The effects of intracerebroventricular (ICV) injection of angiotensin II (ANG II) on blood pressure and water intake were examined with the use of ANG II receptor-deficient mice. ICV injection of ANG II increased systolic blood pressure in a dose-dependent manner in wild-type (WT) mice and ANG type 2 AT(2) receptor null (knockout) (AT(2)KO) mice; however, this increase was significantly greater in AT(2)KO mice than in WT mice. The pressor response to a central injection of ANG II in WT mice was inhibited by ICV preinjection of the selective AT(1) receptor blocker valsartan but exaggerated by the AT(2) receptor blocker PD-123319. ICV injection of ANG II also increased water intake. It was partly but significantly suppressed both in AT(2)KO and AT(1)aKO mice. Water intake in AT(2)/AT(1)aKO mice did not respond to ICV injection of ANG II. Both valsartan and PD-123319 partly inhibited water intake in WT mice. These results indicate an antagonistic action between central AT(1)a and AT(2) receptors in the regulation of blood pressure, but they act synergistically in the regulation of water intake induced by ANG II.

Effect of estrogen and AT1 receptor blocker on neointima formation.

The present study explored the possibility that estrogen may enhance the inhibitory effect of an angiotensin (Ang) II type 1 (AT1) receptor blocker on neointima formation in vascular injury, and investigated the signaling mechanism involved in their actions. Polyethylene cuff placement around the femoral artery of mice induced neointima formation and increased bromodeoxyuridine (BrdU) incorporation into vascular smooth muscle cells. These changes were significantly smaller in female mice than in male mice. Ovariectomy enhanced neointima formation and BrdU incorporation in the injured artery, which were reversed by 17beta-estradiol (80 microg/kg per day) replacement. Treatment with a selective AT1 receptor blocker, olmesartan (3 mg/kg per day), significantly inhibited neointima formation and BrdU incorporation, whereas the inhibitory effects of olmesartan were more marked in intact female mice than in male or ovariectomized mice. Phosphorylation of extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription (STAT) 1, and STAT3 was increased in the injured artery. These increases were significantly smaller in intact female mice than in male or ovariectomized mice. Olmesartan or estrogen attenuated the phosphorylation of ERK and STAT in the injured artery, whereas these inhibitory effects were greater in intact female mice. Lower doses of olmesartan (0.5 mg/kg per day) or 17beta-estradiol (20 microg/kg per day) did not influence neointima formation, BrdU incorporation, and ERK and STAT phosphorylation in ovariectomized mice, whereas coadministration of olmesartan and 17beta-estradiol at these doses attenuated these parameters. These results indicate that estrogen and an AT1 receptor blocker synergistically attenuate vascular remodeling, which is at least partly via inhibition of ERK and STAT activity.

ACE inhibitor improves insulin resistance in diabetic mouse via bradykinin and NO.

Improvement of insulin resistance by ACE inhibitors has been suggested; however, this mechanism has not been proved. We postulated that activation of the bradykinin-nitric oxide (NO) system by an ACE inhibitor enhances glucose uptake in peripheral tissues by means of an increase in translocation of glucose transporter 4 (GLUT4), resulting in improvement of insulin resistance. Administration of an ACE inhibitor, temocapril, significantly decreased plasma glucose and insulin concentrations in type 2 diabetic mouse KK-Ay. Mice treated with temocapril showed a smaller plasma glucose increase after glucose load. We demonstrated that temocapril treatment significantly enhanced 2-[3H]-deoxy-D-glucose (2-DG) uptake in skeletal muscle but not in white adipose tissue. Administration of a bradykinin B2 receptor antagonist, Hoe140, or an NO synthase inhibitor, L-NAME, attenuated the enhanced glucose uptake by temocapril. Moreover, we observed that translocation of GLUT4 to the plasma membrane was significantly enhanced by temocapril treatment without influencing insulin receptor substrate-1 phosphorylation. In L6 skeletal muscle cells, 2-DG uptake was increased by temocaprilat, and Hoe140 inhibited this effect of temocaprilat but not that of insulin. These results suggest that temocapril would improve insulin resistance and glucose intolerance through increasing glucose uptake, especially in skeletal muscle at least in part through enhancement of the bradykinin-NO system and consequently GLUT4 translocation.

Angiotensin II subtype 2 receptor activation inhibits insulin-induced phosphoinositide 3-kinase and Akt and induces apoptosis in PC12W cells.

In the present study, we identified novel negative cross-talk between the angiotensin II subtype 2 (AT2) receptor and insulin receptor signaling in the regulation of phosphoinositide 3-kinase (PI3K), Akt, and apoptosis in rat pheochromocytoma cell line, PC12W cells, which exclusively express AT2 receptor. We demonstrated that insulin-mediated insulin receptor substrate (IRS)-2-associated PI3K activity was inhibited by AT2 receptor stimulation, whereas IRS-1-associated PI3K activity was not significantly influenced. AT2 receptor stimulation did not change insulin-induced tyrosine phosphorylation of IRS-2 or its association with the p85alpha subunit of PI3K, but led to a significant reduction of insulin-induced p85alpha phosphorylation. AT2 receptor stimulation increased the association of a protein tyrosine phosphatase, SHP-1, with IRS-2. Moreover, we demonstrated that AT2 receptor stimulation inhibited insulin-induced Akt phosphorylation and that insulin-mediated antiapoptotic effect was also blocked by AT2 receptor activation. Overexpression of a catalytically inactive dominant negative SHP-1 markedly attenuated the AT2 receptor- mediated inhibition of IRS-2-associated PI3K activity, Akt phosphorylation, and antiapoptotic effect induced by insulin. Taken together, these results indicate that AT2 receptor-mediated activation of SHP-1 and the consequent inhibition IRS-2-associated PI3K activity contributed at least partly to the inhibition of Akt phosphorylation, thereby inducing apoptosis.

Tumor necrosis factor-alpha inhibits growth factor-mediated cell proliferation through SHP-1 activation in endothelial cells.

Src homology 2-containing protein-tyrosine phosphatase 1 (SHP-1) is known to regulate signal transduction through the dephosphorylation of tyrosine kinases. In this study, we addressed the role of SHP-1 under tumor necrosis factor-alpha (TNF-alpha) stimulation in endothelial cells. The addition of recombinant vascular endothelial growth factor (50 ng/mL) or epidermal growth factor (50 ng/mL) significantly increased thymidine incorporation and c-fos promoter activity, whereas TNF-alpha (5 ng/mL) attenuated these effects in human or bovine aortic endothelial cells. In bovine aortic endothelial cells, we confirmed endogenous SHP-1 expression and that TNF-alpha activated SHP-1. Importantly, overexpression of dominant-negative SHP-1 attenuated the effect of TNF-alpha on thymidine incorporation and c-fos promoter activity. In addition, TNF-alpha attenuated vascular endothelial growth factor- and epidermal growth factor-induced extracellular signal-regulated kinase phosphorylation, whereas overexpression of dominant-negative SHP-1 prevented this inhibitory effect of TNF-alpha. Taken together, our results suggested that TNF-alpha inhibited growth factor-mediated cell proliferation through SHP-1 activation.

Estrogen activates phosphatases and antagonizes growth-promoting effect of angiotensin II.

Accumulating evidence suggests that estrogen exerts cardioprotective effects and protects against neointima formation in response to vascular injury in vivo, whereas angiotensin (Ang) II stimulation via the Ang II type 1 (AT(1)) receptor exaggerates vascular injury. We postulate that estrogen treatment antagonizes the AT(1) receptor-mediated growth-promoting effects in vascular smooth muscle cells (VSMCs). The present in vitro study was designed to explore this possibility and to establish the cellular mechanism whereby estrogen attenuates the growth of VSMCs. Primary cultures of VSMCs derived from male adult Sprague-Dawley rats express exclusively AT(1) receptors. Treatment with Ang II enhanced proliferation of VSMC and c-fos expression, whereas 17beta-estradiol (E2) attenuated these vasotrophic effects of Ang II. We also demonstrated that E2 attenuated AT(1) receptor-mediated extracellular signal-regulated kinase activation and that this effect of E2 was restored by pretreatment with vanadate or okadaic acid. Moreover, we demonstrated that E2 enhanced SHP-1 activity, rapidly reaching a peak after 3 minutes of E2 stimulation, whereas E2 transactivated mitogen-activated protein kinase phosphatase-1 expression, showing a peak after 60 minutes of E2 treatment. SHP-1 activation was not influenced by actinomycin D treatment, whereas E2-mediated mitogen-activated protein kinase phosphatase-1 expression was attenuated. Taken together, our results suggest a novel mechanism of vasoprotection by which estrogen antagonizes the effect of the AT(1) receptor via the activation and induction of phosphatases through nongenomic as well as genomic signaling.