Role of integrin-linked kinase for functional capacity of endothelial progenitor cells in patients with stable coronary artery disease

Number and function of endothelial progenitor cells (EPCs) are down-regulated in patients with coronary artery disease (CAD). Integrin-linked kinase (ILK) is a signal and adaptor protein that regulates survival of mature endothelial cells and vascular development.Here we show that EPC dysfunction in patients with CAD is paralleled by down-regulation of ILK while restoration of ILK expression rescues the migratory defect of CAD-EPCs. Human EPCs transduced with dominant-negative ILK (DN-ILK) display significantly reduced expression of CD34⁺/VEGFR-2⁺, DiI-Ac-LDL uptake, and Ulex europaeus lectin binding. Mechanistically, DN-ILK-transfected EPCs are characterized by decreased proliferation, while proliferation is increased in wild-type ILK-transfected EPCs. These effects are paralleled by changes in cyclin D1 expression, colony forming units, and cytoskeletal rearrangement. Functionally, ILK is necessary and sufficient for SDF-1-triggered migration and adhesion in EPCs.These data extend current knowledge about the role of ILK in EPC biology and implicate ILK as a therapeutic target in CAD.     

Osteopontin stimulates vascular smooth muscle cell migration by inducing FAK phosphorylation and ILK dephosphorylation

Focal adhesion kinase (FAK) and integrin-linked kinase (ILK) are both involved in integrin-mediated cell migration. However, the molecular mechanism, and the relationship between FAK and ILK activity in signaling transduction for the osteopontin (OPN)-induced migration of vascular smooth muscle cells (VSMCs) remain unclear. Here, we show that treating VSMCs with OPN could result in the dissociation of FAK with ILK by inducing phosphorylation of the former and dephosphorylation of the latter. Furthermore, we demonstrate that FAK phosphorylation induced by OPN is coupled with ILK dephosphorylation. We also provide evidence that ILK acts downstream of FAK in the signaling pathways that mediate OPN-induced VSMC migration. These findings suggest that FAK phosphorylation and ILK dephosphorylation play important roles in VSMC migration induced by OPN.     

Leukocyte antigen-related deficiency enhances insulin-like growth factor-1 signaling in vascular smooth muscle cells and promotes neointima formation in response to vascular injury

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.     

Integrin-linked kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition

The role of integrin-linked kinase (ILK) in transforming growth factor beta (TGFbeta)-mediated epithelial to mesenchymal transition was investigated. A stable transfection of dominant-negative ILK results in the prevention of TGFbeta-mediated E-cadherin delocalization. TGFbeta-mediated phosphorylation of Akt at Ser-473 was inhibited by dominant-negative ILK and PI3K inhibitors, LY294002 and wortmannin. Treatment with TGFbeta stimulated induction of Akt and ILK kinase activity in HaCat control cells. This increased ILK activity by TGFbeta was lowered by PI3K inhibitor, LY294002. In addition, PI3K inhibitor, dominant-negative Akt, and dominant-negative ILK could not block TGFbeta-mediated C-terminal phosphorylation of Smad2. Taken together, these data suggest that PI3K-ILK-Akt pathway that is independent of the TGFbeta-induced Smad pathway is required for TGFbeta-mediated epithelial to mesenchymal transition.     

Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

Integrin-linked kinase (ILK) is an intracellular effector of cell–matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing.     

Effects of statin treatment and withdrawal on angiotensin II-induced phosphorylation of p38 MAPK and ERK1/2 in cultured vascular smooth muscle cells

Abrupt discontinuation of 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase inhibitors (statins) is associated with increased cardiovascular risk. To investigate the molecular mechanisms determining the increased cardiovascular risk after statin withdrawal, we studied the effects of statin treatment and withdrawal on angiotensin II (AII) actions in rat aortic vascular smooth muscle cells (VSMC) in culture. In VSMC, AII stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and of p38 mitogen-activated protein kinase (p38 MAPK), with an EC50% of 0.86 and 3 nM, respectively. Maximal stimulation was observed after 5-10 min of exposure to AII. Pretreatment with 1-3 microM simvastatin for 24h inhibited AII-mediated stimulation of ERK1/2 and p38 MAPK phosphorylation; without affecting the levels on non-phosphorylated MAPK. Washout of simvastatin produced a rebound increase above control levels of AII-mediated phosphorylation of ERK1/2 and p38 MAPK. As previously reported for other agonists, the rebound increase of AII effects was observed from 1 to 3h after statin withdrawal, and was lost at later times. The basal levels of phosphorylation and the amount of non-phosphorylated kinases were unaffected by statin withdrawal. Similar effects were observed with lovastatin. Our results suggest that statins modulate AII effects in VSMC, and that transient increases in AII effects mediated via the MAPK pathway may play a role in the vascular dysfunction associated with statin withdrawal.     

Integrin-linked kinase (ILK) is highly expressed in first trimester human chorionic villi and regulates migration of a human cytotrophoblast-derived cell line

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.     

Effects of TH-142177 on angiotensin II-induced proliferation, migration and intracellular signaling in vascular smooth muscle cells and on neointimal thickening after balloon injury.

We investigated the effects of TH-142177 (N-n-butyl-N-[2'-(1-H-tetrazole-5-yl) biphenyl-4-yl]-methyl-(N-carboxy methyl-benzylamino)-acetamide), a novel selective antagonist of angiotensin II type 1-receptor (AT1-R) on angiotensin II (AII)-induced proliferation and migration of vascular smooth muscle cells (VSMC), and on neointimal formation in the rat carotid artery after balloon injury, and on the intracellular signaling by the stimulation of AT1-R. High affinity AII receptor sites were detected in rat VSMC by the use of [125I]Sar1,Ile8-AII. TH-142177 and losartan competed with [125I]Sar1,Ile8-AII for the binding sites in VSMC in a monophasic manner, although PD123177, a selective antagonist of angiotensin II type 2-receptor (AT2-R), had little inhibitory effect, demonstrating the predominant existence of AT1-R in rat VSMC. TH-142177 prevented AII-induced DNA synthesis and migration, with a significant inhibition of 74 and 55%, respectively, at the concentration of 100 nM. AII-induced activation of p21ras, mitogen-activated protein kinase (p42MAPK and p44MAPK), and protein kinase C was significantly (50-78%) inhibited by TH-142177 (100 nM), suggesting that the activation of these enzymes is mediated through the stimulation of AT1-R. Balloon-injured left carotid arteries in rats showed extensive neointimal thickening, and TH-142177 (3 mg/kg) brought out a marked decrease in the neointimal thickening after balloon injury. In conclusion, TH-142177 inhibited AII-induced proliferation and migration of rat VSMC and neointimal formation in the carotid artery after balloon injury, and these effects may be related, in part, to the suppression of ras, p42MAPK and p44MAPK, and protein kinase C activities through the blockade of AT1-R. Thus, TH-142177 may have therapeutic potential for the treatment of vascular diseases such as atherosclerosis and restenosis.     

Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1)

Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.     

Gene modification with integrin-linked kinase improves function of endothelial progenitor cells in pre-eclampsia in vitro

Integrin-linked kinase (ILK), a multifunctional serine-threonine protein kinase, has been shown to have implications for the treatment of ischemia vascular diseases by promoting angiogenesis in various tissues. However, whether this kinase has therapeutic potential in pre-eclampsia is not well studied. In this report, we determined the changes in the production and action of ILK on endothelial progenitor cells (EPCs) isolated from patients with pre-eclampsia. The effects of ILK transfection on proliferation, migration, and angiogenesis of EPCs were investigated. We showed that EPCs transfected with the ILK gene expressed high levels of ILK protein and mRNA. Transfection with ILK also enhanced the proliferative, migratory, and angiogenic capabilities of EPCs, and promoted the production of VEGF. These results suggest that ILK gene transfection is an effective approach to augment angiogenic properties of EPCs in vitro and providing basis for clinical cell-based gene therapy in patients with pre-eclampsia.     

Src介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移过程

为阐明酪氨酸激酶Src在整合素被骨桥蛋白(OPN)激活所触发的细胞黏附和迁移信号途径中所起的作用,应用Src特异性抑制剂PP2阻断Src,观察OPN诱导的血管平滑肌细胞(VSMC)黏附和迁移活性的改变,并利用免疫沉淀检查PP2对整合素下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化及其相互作用的影响。结果显示,PP2可明显抑制OPN诱导的VSMC黏附和伤口愈合(黏附和迁移活性分别为对照组的76.6%和33.8%);OPN可显著诱导FAK磷酸化(磷酸化水平达对照组的1.9倍),促进ILK去磷酸化,并使FAK与ILK的结合减少(降至对照组的46.4%)。10μmol/LPP2可明显抑制OPN诱导的FAK磷酸化、拮抗OPN诱导对ILK的去磷酸化作用、促进FAK与ILK之间的结合。研究结果表明,Src作为OPN-整合素-FAK信号途径中的信号分子,通过影响FAK和ILK的磷酸化以及两者之间的相互作用来调节VSMC的黏附和迁移活性。     

c-Jun N-terminal kinase attenuates TNFα signaling by reducing Nox1-dependent endosomal ROS production in vascular smooth muscle cells

Tumor necrosis factor-α (TNFα), a proinflammatory cytokine, causes vascular smooth muscle cell (VSMC) proliferation and migration and promotes inflammatory vascular lesions. Nuclear factor-kappa B (NF-κB) activation by TNFα requires endosomal superoxide production by Nox1. In endothelial cells, TNFα stimulates c-Jun N-terminal kinase (JNK), which inhibits NF-κB signaling. The mechanism by which JNK negatively regulates TNFα-induced NF-κB activation has not been defined. We hypothesized that JNK modulates NF-κB activation in VSMC, and does so via a Nox1-dependent mechanism. TNFα-induced NF-κB activation was TNFR1- and endocytosis-dependent. Inhibition of endocytosis with dominant-negative dynamin (DynK44A) potentiated TNFα-induced JNK activation, but decreased ERK activation, while p38 kinase phosphorylation was not altered. DynK44A attenuated intracellular, endosomal superoxide production in wild-type (WT) VSMC, but not in NADPH oxidase 1 (Nox1) knockout (KO) cells. siRNA targeting JNK1 or JNK2 potentiated, while a JNK activator (anisomycin) inhibited, TNFα-induced NF-κB activation in WT, but not in Nox1 KO cells. TNFα-stimulated superoxide generation was enhanced by JNK1 inhibition in WT, but not in Nox1 KO VSMC. These data suggest that JNK suppresses the inflammatory response to TNFα by reducing Nox1-dependent endosomal ROS production. JNK and endosomal superoxide may represent novel targets for pharmacologic modulation of TNFα signaling and vascular inflammation.     

Mutations in the Paxillin-binding Site of Integrin-linked Kinase (ILK) Destabilize the Pseudokinase Domain and Cause Embryonic Lethality in Mice

Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val³⁸⁶ and Thr³⁸⁷ were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILK-VT/GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and α-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins.     

Integrin-linked kinase regulates endothelial cell survival and vascular development

Integrin-linked kinase (ILK) is a phosphoinositide 3-kinase-dependent serine/threonine kinase that interacts with beta integrins. Here we show that endothelial cell (EC)-specific deletion of ILK in mice confers placental insufficiency with decreased labyrinthine vascularization, yielding no viable offspring. Deletion of ILK in zebra fish using antisense morpholino oligonucleotides results in marked patterning abnormalities of the vasculature and is similarly lethal. To dissect potential mechanisms responsible for these phenotypes, we performed ex vivo deletion of ILK from purified EC of adult mice. We observed downregulation of the active-conformation of beta1 integrins with a striking increase in EC apoptosis associated with activation of caspase 9. There was also reduced phosphorylation of the ILK kinase substrate, Akt. However, phenotypic rescue of ILK-deficient EC by wild-type ILK, but not by a constitutively active mutant of Akt, suggests regulation of EC survival by ILK in an Akt-independent manner. Thus, endothelial ILK plays a critical role in vascular development through integrin-matrix interactions and EC survival. These data have important implications for both physiological and pathological angiogenesis.     

Downregulation of integrin-linked kinase inhibits epithelial-to-mesenchymal transition and metastasis in bladder cancer cells

Integrin-linked kinase (ILK) is a multifunctional serine/threonine kinase in cytoplasm. Recent studies showed that cancer patients with increased ILK expression had low survival, poor prognosis and increased metastasis. Although the causes of ILK overexpression remain to be fully elucidated, accumulating evidence suggests that its oncogenic capacity derives from its regulation of several downstream targets that provide cells with signals that promote proliferation, survival and migration. However, the mechanisms underlying tumor metastasis by ILK is still not fully understood. Epithelial–mesenchymal transition (EMT) is a critical event of cancer cells that triggers invasion and metastasis. We recently reported that knockdown of ILK inhibited the growth and induced apoptosis in human bladder cancer cells. Therefore, we postulate that ILK might involve in EMT. Here we further investigate the function of ILK with RNA interference in bladder cancer cells. Knockdown of ILK impeded an EMT with low Vimentin, Snail, Slug and Twist as well as high E-cadherin expression in vivo and vitro. In addition, we found that knockdown of ILK inhibited cell proliferation, migration and invasion as well as changed cell morphology, adhesion and rearranged cytoskeleton in vitro. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3β, increased expression of nm23-H1, as well as reduced expression of MMP-2 and MMP-9 in vivo and vitro. Furthermore, downregulation of ILK could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. Finally, the effects of ILK siRNA on bladder cancer cell phenotype and invasiveness translate into suppression for tumorigenesis and metastasis in vivo. Taken together, our findings highlight that ILK signaling pathway plays a novel role in the development of bladder cancer through regulating EMT. ILK could be a promising diagnostic marker and therapeutic target for bladder cancer.     

Persistent migration of neuroblasts from the subventricular zone to the injured striatum mediated by osteopontin following intracerebral hemorrhage

We investigated intracerebral hemorrhage (ICH)-induced lateral migration of neuroblasts and the mechanism underlying this migration. ICH model was induced by collagenase injection into the striatum of adult wild-type and osteopontin (OPN) knockout mice. In the wild-type mice, the lateral migration of neuroblasts from the ipsislateral subventricular zone (SVZ) towards the hematoma started at day 3 and continued up to day 28 after ICH. In addition to migrating towards the hematoma, neuroblasts also migrated to the area of ipsilateral striatum remote to the hematoma. The migrating neuroblasts were closely associated with activated astrocytes and blood vessels in the injured striatum. Following ICH, the expression of OPN was up-regulated in the ipsilateral striatum from day 1 to day 28. In vitro , OPN treatment did not affect the proliferation of neural progenitors, but enhanced the trans-well and radial migration of neural progenitors. In vivo , OPN deficiency did not affect the proliferation of neural progenitors in the SVZ. However, following ICH a significant decrease in lateral neuroblast migration was observed in the OPN knockout mice compared with the wild-type mice. These results suggest that increased OPN expression in the injured striatum plays a significant role in the lateral migration of neuroblasts following ICH.     

Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways

Angiotensin II, a potent vasoconstrictor, is known to stimulate Ca2+ mobilization and Na+ influx in vascular smooth muscle cells (VSMC). The fact that the Na+/H+ exchange inhibitor, amiloride, blocks angiotensin II-stimulated Na+ influx and is itself a vasodilator suggests that Na+/H+ exchange may play a role in the angiotensin II-mediated effects on VSMC. We have used a pH-sensitive fluorescent dye to study Na+/H+ exchange in cultured rat aortic VSMC. Basal intracellular pH was 7.08 in physiological saline buffer. Angiotensin II stimulation caused an initial transient acidification, followed by a Na+-dependent alkalinization. Angiotensin II increased the rate of alkalinization with apparent threshold, half-maximal, and maximal effect of 0.01, 3, and 100 nM, respectively. Angiotensin II stimulation appeared to be mediated by a shift in the Km of the Na+/H+ exchanger for extracellular Na+. Since angiotensin II activates phospholipase C in VSMC, we tested the possibility that angiotensin II increased Na+/H+ exchange by activation of protein kinase C via stimulation of diacylglycerol formation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated Na+/H+ exchange in VSMC cultured for 24 h in serum-free medium, and the subsequent angiotensin II response was inhibited. However, VSMC grown in serum and treated for 24 h with TPA to decrease protein kinase C activity showed no inhibition of angiotensin II-stimulated Na+/H+ exchange. TPA caused no intracellular alkalinization of VSMC grown in serum, while the angiotensin II response was actually enhanced compared to VSMC deprived of serum for 24 h. We conclude that angiotensin II stimulates an amiloride-sensitive Na+/H+ exchange system in cultured VSMC which is mediated by protein kinase C-dependent and -independent mechanisms. Angiotensin II-mediated Na+ influx and intracellular alkalinization may play a role in excitation-response coupling in vascular smooth muscle.     

Sodium Tanshinone IIA Silate Inhibits High Glucose-Induced Vascular Smooth Muscle Cell Proliferation and Migration through Activation of AMP-Activated Protein Kinase

The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. AMPK additionally exerts several salutary effects on vascular function and improves vascular abnormalities. The current study sought to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation and migration under high glucose conditions mimicking diabetes without dyslipidemia, and establish the underlying mechanism. In this study, STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in VSMCs. VSMC proliferation was enhanced under high glucose (25 mM glucose, HG) versus normal glucose conditions (5.5 mM glucose, NG), and this increase was inhibited significantly by STS treatment. We utilized western blotting analysis to evaluate the effects of STS on cell-cycle regulatory proteins and found that STS increased the expression of p53 and the Cdk inhibitor, p21, subsequent decreased the expression of cell cycle-associated protein, cyclin D1. We further observed that STS arrested cell cycle progression at the G₀/G₁ phase. Additionally, expression and enzymatic activity of MMP-2, translocation of NF-κB, as well as VSMC migration were suppressed in the presence of STS. Notably, Compound C (CC), a specific inhibitor of AMPK, as well as AMPK siRNA blocked STS-mediated inhibition of VSMC proliferation and migration. We further evaluated its potential for activating AMPK in aortas in animal models of type 2 diabetes and found that Oral administration of STS for 10 days resulted in activation of AMPK in aortas from ob/ob or db/db mice. In conclusion, STS inhibits high glucose-induced VSMC proliferation and migration, possibly through AMPK activation. The growth suppression effect may be attributable to activation of AMPK-p53-p21 signaling, and the inhibitory effect on migration to the AMPK/NF-κB signaling axis.