Angiotensin II induces vascular endothelial growth factor synthesis in mesenchymal stem cells

Mesenchymal stem cells (MSCs) transplantation has been proposed as a promising means for ischemic heart disease. Vascular endothelial growth factor (VEGF) has been demonstrated to play an important role in MSCs transplantation. Angiotensin II (AngII), the most important effector peptide of the renin-angiotensin system (RAS), is also an angiogenesis factor. However, the effects of AngII on VEGF expression in MSCs and the related signaling cascades were unknown. In this experiment, we first demonstrated that incubation of MSCs with AngII-induced a rapid increase in VEGF mRNA expression and protein synthesis. However, these effects were abolished by prior treatment with AngII type 1 (AT₁) receptor antagonist losartan while not AngII type 2 (AT₂) receptor antagonist PD123319. The addition of either the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 or Akt inhibitor LY294002 also led to a marked inhibition of the AngII-induced VEGF mRNA and protein production. Taken together, these results suggested that AngII stimulated the synthesis of VEGF in MSCs through ERK1/2 and Akt pathway via AT₁ receptor.     

Arrestin-dependent Angiotensin AT1 Receptor Signaling Regulates Akt and mTor-mediated Protein Synthesis

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT₁ receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT₁ receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT₁ receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar¹-Ile⁴-Ile⁸]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT₁ receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT₁ receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.     

Critical role of cAMP-response element-binding protein for angiotensin II-induced hypertrophy of vascular smooth muscle cells

We reported previously an important role of cyclic AMP-response element (CRE) for the induction of interleukin-6 gene expression by angiotensin II (AngII). We examined signaling pathways that are responsible for AngII-induced phosphorylation of CRE-binding protein (CREB) at serine 133 that is a critical marker for the activation in rat vascular smooth muscle cells (VSMC). AngII time dependently induced phosphorylation of CREB with a peak at 5 min. The AngII-induced phosphorylation of CREB was blocked by CV11974, an AngII type I receptor antagonist, suggesting that AngII type I receptor may mediate the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase (ERK) by PD98059 or inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 partially inhibited AngII-induced CREB phosphorylation. A protein kinase A inhibitor, H89, also partially suppressed AngII-induced CREB phosphorylation. Inhibition of epidermal growth factor-receptor by AG1478 suppressed the AngII-induced CREB phosphorylation as well as activation of ERK and p38MAPK. Overexpression of the dominant negative form of CREB by an adenovirus vector suppressed AngII-induced c-fos expression and incorporation of [(3)H]leucine to VSMC. These findings suggest that AngII may activate multiple signaling pathways involving two MAPK pathways and protein kinase A, all of which contribute to the activation of CREB. Transactivation of epidermal growth factor-receptor is also critical for AngII-induced CREB phosphorylation. Activation of CREB may be important for the regulation of gene expression and hypertrophy of VSMC induced by AngII.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck

p21-activated kinase (PAK) has been shown to be an upstream mediator of JNK in angiotensin II (AngII) signaling. Little is known regarding other signaling molecules involved in activation of PAK and JNK by AngII. Rho family GTPases Rac and Cdc42 have been shown to enhance PAK activity by binding to p21-binding domain of PAK (PAK-PBD). In vascular smooth muscle cells (VSMC) AngII stimulated Rac1 binding to GST-PAK-PBD fusion protein. Pretreatment of VSMC by genistein inhibited AngII-induced Rac1 activation, whereas Src inhibitor PP1 had no effect. Inhibition of protein kinase C by phorbol 12,13-dibutyrate pretreatment also decreased AngII-mediated activation of Rac1. The adaptor molecule Nck has been shown previously to mediate PAK activation by facilitating translocation of PAK to the plasma membrane. In VSMC AngII stimulated translocation of Nck and PAK to the membrane fraction. Overexpression of dominant-negative Nck in Chinese hamster ovary (CHO) cells, stably expressing the AngII type I receptor (CHO-AT1), inhibited both PAK and JNK activation by AngII, whereas it did not affect ERK1/2. Finally, dominant-negative Nck inhibited AngII-induced DNA synthesis in CHO-AT1 cells. Our data provide evidence for Rac1 and Nck as upstream mediators of PAK and JNK in AngII signaling and implicate JNK in AngII-induced growth responses.     

The ROS derived mitochondrial respirstion not from NADPH oxidase plays key role in Celastrol against angiotensin II-mediated HepG2 cell proliferation

Angiotensin II (AngII) is an important factor that promotes the proliferation of cancer cells, whereas celastrol exhibits a significant antitumor activity in various cancer models. Whether celastrol can effectively suppress AngII mediated cell proliferation remains unknown. In this study, we studied the effect of celastrol on AngII-induced HepG2 cell proliferation and evaluated its underlying mechanism. The results revealed that AngII was able to significantly promote HepG2 cell proliferation via up-regulating AngII type 1 (AT₁) receptor expression, improving mitochondrial respiratory function, enhancing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, increasing the levels of reactive oxygen species (ROS) and pro-inflammatory cytokines. The excess ROS from mitochondrial dysfunction is able to cause the apoptosis of tumor cells via activating caspase3 signal pathway. In addition, the reaction between NO and ROS results in the formation of peroxynitrite (ONOO^?), and then promoting cell damage. celastrol dramatically enhanced ROS generation, thereby causing cell apoptosis through inhibiting mitochodrial respiratory function and boosting the expression levels of AngII type 2 (AT₂) receptor without influencing NADPH oxidase activity. PD123319 as a special inhibitor of AT₂R was able to effectively decreased the levels of inflammatory cytokines and endothelial nitric oxide synthase (eNOS) activity, but only partially attenuate the effect of celastrol on AnII mediated HepG2 cell proliferation. Thus, celastrol has the potential for use in liver cancer therapy. ROS derived from mitochondrial is an important factor for celastrol to suppress HepG2 cell proliferation.     

TGFβ-induced GRK2 expression attenuates AngII-regulated vascular smooth muscle cell proliferation and migration

Through diametric actions, the transforming growth factor β (TGFβ) and Angiotensin II (AngII) play important roles in regulating various biological responses such as cell proliferation and migration. Signaling initiated by TGFβ and AngII occurs through two structurally and functionally distinct receptor super families, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Previously, we identified the G protein-coupled receptor kinase-2 (GRK2), a key regulatory factor in the desensitization of GPCRs, as a direct downstream target of the TGFβ signaling cascade. GRK2 acts through a negative feed-back loop mechanism to terminate TGFβ-induced smad signaling. To investigate the impact of TGFβ-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). In this study, we show that activation of the TGFβ signaling cascade in VSMCs results in increased GRK2 expression levels, which consequently inhibits AngII-induced ERK phosphorylation and antagonizes AngII-induced VSMC proliferation and migration. Moreover, the inhibitory effect of TGFβ on AngII signaling occurs at the Mek-Erk interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFβ signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation and that GRK2 is a key factor mediating the cross-talk between these two receptor super families.     

CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells

We have shown earlier a requirement for Ca²⁺ and calmodulin (CaM) in the H₂O₂-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H₂O₂-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKIIα. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H₂O₂-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H₂O₂, calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281–309) of CaMKII and with siRNA of CaMKIIα attenuated the H₂O₂-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H₂O₂-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKIIα siRNA abolished the H₂O₂-induced IGF-1R phosphorylation. H₂O₂ treatment also induced Thr²⁸⁶ phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H₂O₂ on ERK1/2, PKB, and IGF-1R phosphorylation.     

Src Is Required for Mechanical Stretch-Induced Cardiomyocyte Hypertrophy through Angiotensin II Type 1 Receptor-Dependent β-Arrestin2 Pathways

Angiotensin II (AngII) type 1 receptor (AT1-R) can be activated by mechanical stress (MS) without the involvement of AngII during the development of cardiomyocyte hypertrophy, in which G protein-independent pathways are critically involved. Although β-arrestin2-biased signaling has been speculated, little is known about how AT1-R/β-arrestin2 leads to ERK1/2 activation. Here, we present a novel mechanism by which Src kinase mediates AT1-R/β-arrestin2-dependent ERK1/2 phosphorylation in response to MS. Differing from stimulation by AngII, MS-triggered ERK1/2 phosphorylation is neither suppressed by overexpression of RGS4 (the negative regulator of the G-protein coupling signal) nor by inhibition of Gαq downstream protein kinase C (PKC) with GF109203X. The release of inositol 1,4,5-triphosphate (IP₃) is increased by AngII but not by MS. These results collectively suggest that MS-induced ERK1/2 activation through AT1-R might be independent of G-protein coupling. Moreover, either knockdown of β-arrestin2 or overexpression of a dominant negative mutant of β-arrestin2 prevents MS-induced activation of ERK1/2. We further identifies a relationship between Src, a non-receptor tyrosine kinase and β-arrestin2 using analyses of co-immunoprecipitation and immunofluorescence after MS stimulation. Furthermore, MS-, but not AngII-induced ERK1/2 phosphorylation is attenuated by Src inhibition, which also significantly improves pressure overload-induced cardiac hypertrophy and dysfunction in mice lacking AngII. Finally, MS-induced Src activation and hypertrophic response are abolished by candesartan but not by valsartan whereas AngII-induced responses can be abrogated by both blockers. Our results suggest that Src plays a critical role in MS-induced cardiomyocyte hypertrophy through β-arrestin2-associated angiotensin II type 1 receptor signaling.     

Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

Angiotensin II (AngII), a peptide hormone released by adipocytes, can be catabolized by adipose angiotensin-converting enzyme 2 (ACE2) to form Ang(1–7). Co-expression of AngII receptors (AT₁ and AT₂) and Ang(1–7) receptors (Mas) in adipocytes implies the autocrine regulation of the local angiotensin system upon adipocyte functions, through yet unknown interactive mechanisms. In the present study, we reveal the adipogenic effects of Ang(1–7) through activation of Mas receptor and its subtle interplays with the antiadipogenic AngII-AT1 signaling pathways. Specifically, in human and 3T3-L1 preadipocytes, Ang(1–7)-Mas signaling promotes adipogenesis via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways, and Ang(1–7)-Mas antagonizes the antiadipogenic effect of AngII-AT₁ by inhibiting the AngII-AT₁-triggered MAPK kinase/ERK pathway. The autocrine regulation of the AngII/AT₁-ACE2-Ang(1–7)/Mas axis upon adipogenesis has also been revealed. This study suggests the importance of the local regulation of the delicately balanced angiotensin system upon adipogenesis and its potential as a novel therapeutic target for obesity and related metabolic disorders.     

Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the G(q)-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca(2+)-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca(2+) ionophore without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270. and EGF markedly activate p38MAPK, whereas but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.     

Metalloprotease inhibitor blocks angiotensin II-induced migration through inhibition of epidermal growth factor receptor transactivation

In vascular smooth muscle cells (VSMCs), angiotensin II (AngII) induces transactivation of the EGF receptor (EGFR) which involves a metalloprotease that stimulates processing of heparin-binding EGF from its precursor. However, the identity and pharmacological sensitivity of the metalloprotease remain unclear. Here, we screened the effects of several metalloprotease inhibitors on AngII-induced EGFR transactivation in VSMCs. We found that an N-phenylsulfonyl-hydroxamic acid derivative [2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropinamide] (BiPS), previously known as matrix metalloprotease (MMP)-2/9 inhibitor, markedly inhibited AngII-induced EGFR transactivation, whereas the MMP-2 or -9 inhibition by other MMP inhibitors failed to block the transactivation. BiPS markedly inhibited AngII-induced ERK activation and protein synthesis without affecting AngII-induced intracellular Ca2+ elevation. VSMC migration induced by AngII was also inhibited not only by an EGFR inhibitor but also by BiPS. Thus, BiPS is a specific candidate to block AngII-induced EGFR transactivation and subsequent growth and migration of VSMCs, suggesting its potency to prevent vascular remodeling.     

PGC-1α limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species

AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22^phox, p40^phox, p47^phox and p67^phox) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis.     

Activation of PKCbeta(II) and PKCtheta is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation

Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKCβ_II and PKCθ from cytosol to plasma membrane, and inhibition of PKCβ_II and PKCθ decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKCβ_II and PKCθ, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, and LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKCβ_II and PKCθ. Inhibition of PKCβ_II or PKCθ, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKCθ in VSMC proliferation is unique.     

Hypertrophic response to angiotensin II is mediated by protein kinase D-extracellular signal-regulated kinase 5 pathway in human aortic smooth muscle cells

Angiotensin II plays a critical role in hypertrophy of vascular smooth muscle cells, however, the molecular underpinnings remain unclear. The present study indicated that AT₁/PKC/PKD pathway was able to regulate downstream ERK5, affecting pro-hypertrophic responses to Ang II. Ang II-stimulated phosphorylation of ERK5 in a time- and dose-dependent manner in human aortic smooth muscle cells (HASMCs). The pharmacological inhibitors for AT₁ and PKCs significantly inhibited Ang II-induced ERK5 activation, suggesting the involvement of the AT₁/PKC pathway. In particular, PKD was critical for Ang II-induced ERK5 activation since silencing PKD by siRNA markedly inhibited Ang II-induced ERK5 activation. Consequently, we found that Losartan, Gö 6983 and PKD siRNA significantly attenuated ERK5 activated translocation and hypertrophy of HASMCs by Ang II. Taken together, we demonstrated for the first time that Ang II activates ERK5 via the AT₁/PKC/PKD pathway and revealed a critical role of ERK5 in Ang II-induced HASMCs hypertrophy.     

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

Angiotensin II induces matrix metalloproteinase-9 expression via a nuclear factor-kappaB-dependent pathway in vascular smooth muscle cells

Angiotensin II (AngII) is widely recognized as a critical regulator of the development of atherosclerosis. Matrix metalloproteinases (MMPs) are thought to participate in plaque destabilization through degradation of the extracellular matrix. In the present study, we investigated the potential mechanism of AngII-induced MMP-9 expression in vascular smooth muscle cells (VSMC). AngII upregulated the expression of MMP-9 significantly in VSMC obtained from rat aorta. RNAi-mediated knockdown of p65 and losartan, an inhibitor of AngII receptors subtype-1 (AT₁), could abolish AngII-induced MMP-9 expression. In addition, AngII induced the NF-κB binding activity via AT₁ and AT₂ receptors in VSMC, and AngII-induced activation of NF-κB is not associated with significant downregulation of IκB. In summary, this study demonstrates that AngII stimulates NF-κB nuclear translocation in VSMC via AT₁ and AT₂. AngII increases the expression of MMP-9 in VSMC, and AT₁ and NF-κB pathways have an important role in this response.     

Angiotensin II,as well as 5-hydroxytriptamine,is a potent vasospasm inducer of saphenous vein graft for coronary artery bypass grafting in patients with diabetes mellitus

Diabetes mellitus (DM) is an important risk factor for adverse outcomes of coronary artery bypass grafting. The bypass grafts harvested from patients with DM tend to go into spasm after their implantation into the coronary circulation. To clarify the contribution of 5-hydroxytriptamine (5-HT) and angiotensin II (AngII) in the bypass graft spasm, we examined the contractile reactivity to 5-HT or AngII of isolated human endothelium-denuded saphenous vein (SV) harvested from DM and non-DM patients. The 5-HT-induced constriction of the SV was significantly augmented in the DM group than in the non-DM group, which is similar to our previous report. AngII-induced constriction of the SV was also significantly augmented in the DM group than the non-DM group. Especially in the non-DM group, the AngII-induced maximal vasoconstriction was markedly lower than the 5-HT-induced one. Meanwhile, the increasing rates of AngII-induced vasoconstriction in the DM group to the non-DM group were significantly greater than those of 5-HT-induced vasoconstriction. These results indicate that 5-HT is a potent inducer of SV graft spasm in both DM and non-DM patients, while AngII is a potent inducer of SV graft spasm only in patients with DM. Furthermore, the protein level of AngII AT₁ receptor (AT₁R), but not the protein level of 5-HT_2A receptor, in the membrane fraction of the SV smooth muscle cells of DM patients was significantly increased as compared with that of the non-DM patients. These results suggest that the mechanism for hyperreactivity to AngII in the SV from DM patients is due to, at least in part, the increase in the amount of AT₁R on membrane of the SV smooth muscle cells.