Propionyl-L-carnitine reduces proliferation and potentiates Bax-related apoptosis of aortic intimal smooth muscle cells by modulating nuclear factor-kappaB activity

Propionyl-l-carnitine (PLC) has been introduced among the therapeutic approaches of peripheral arterial disease, and more recently, an increase of intimal cell apoptosis has been demonstrated to contribute to its effectiveness in rabbit carotid postinjury myointimal hyperplasia prevention. How PLC mediates these effects on vascular smooth muscle cells (SMCs) remains poorly understood. We investigated the role of NF-kappaB in PLC-induced arterial remodeling. In vivo, daily PLC treatment 15 days after injury resulted in a reduction of relative rat aortic intimal volume, an increase of apoptosis, Bax up-regulation without changing the Bcl-2 level, and a reduction of NF-kappaB, vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and survivin in myointimal thickening compared with controls. In the presence of 10% serum, a reduced G(1) --> S phase progression preceded PLC-induced intimal cell apoptosis; in 0.1% serum cultures, in a dose-dependent manner, PLC rapidly induced intimal cell apoptosis and reduced p65, p50, IAP-1, and IAP-2 expression. Inhibiting NF-kappaB activation through SN50 increased apoptotic rate and Bax expression in intimal but not in medial SMCs, and successive PLC treatment failed to induce a further increase in apoptotic rate. Bax antisense oligodeoxynucleotide reduced PLC-induced intimal cell apoptosis and cytochrome c release. The PLC-induced attenuation of NF-kappaB activity in intimal cells was also due to the increase of IkappaB-alpha bioavailability, as the result of a parallel induction of IkappaB-alpha synthesis and reduction of phosphorylation and degradation. Collectively, these findings document that NF-kappaB activity inhibition contributes to PLC-induced proliferative arrest and Bax-related apoptosis of intimal SMCs.     

Adhesion Molecule-Mediated Hippo Pathway Modulates Hemangioendothelioma Cell Behavior

Hemangioendotheliomas are categorized as intermediate-grade vascular tumors that are commonly localized in the lungs and livers. The regulation of this tumor cell''s proliferative and apoptotic mechanisms is ill defined. We recently documented an important role for Hippo pathway signaling via endothelial cell adhesion molecules in brain microvascular endothelial cell proliferation and apoptosis. We found that endothelial cells lacking cell adhesion molecules escaped from contact inhibition and exhibited abnormal proliferation and apoptosis. Here we report on the roles of adherens junction molecule modulation of survivin and the Hippo pathway in the proliferation and apoptosis of a murine hemangioendothelioma (EOMA) cell. We demonstrated reduced adherens junction molecule (CD31 and VE-cadherin) expression, increased survivin and Ajuba expression, and a reduction in Hippo pathway signaling resulting in increased proliferation and decreased activation of effector caspase 3 in postconfluent EOMA cell cultures. Furthermore, we confirmed that YM155, an antisurvivin drug that interferes with Sp1-survivin promoter interactions, and survivin small interference RNA (siRNA) transfection elicited induction of VE-cadherin, decreased Ajuba expression, increased Hippo pathway and caspase activation and apoptosis, and decreased cell proliferation. These findings support the importance of the Hippo pathway in hemangioendothelioma cell proliferation and survival and YM155 as a potential therapeutic agent in this category of vascular tumors.     

Angiopoietin 1, PDGF-B, and TGF-beta gene regulation in endothelial cell and smooth muscle cell interaction

The vascular wall is mainly composed of endothelial cells (ECs) and smooth muscle cells (SMCs). The crosstalking between these two cell types is critical in the vascular maturation process. Genetic studies suggest that the Tie2/angiopoietin 1 (Ang1) pathway regulates vascular remodeling. However, the molecular mechanism is unclear. PDGF is a potent chemoattractant for SMCs, and TGF-beta regulates SMC differentiation. Here, we examined gene regulation. PDGF-B stimulation upregulated Ang1 expression in SMCs through the PI3K and PKC pathways. PDGF-B stimulation also produced an acute induction of TGF-beta expression in SMCs through the MAPK/ERK pathway. Interestingly, TGF-beta negatively regulated Ang1 expression induced by the PDGF-B stimulation in SMCs. Reciprocally, we observed that stimulation of ECs with either Ang1 or TGF-beta slightly downregulated PDGF expression. A combination of both TGF-beta with Ang1 produced much stronger downregulation of PDGF. Our data showed complex gene regulations that include both positive and negative regulations between ECs and SMCs to maintain vascular homeostasis.     

Stem cell marker expression,proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.     

LncRNA GAS5 regulates vascular smooth muscle cell cycle arrest and apoptosis via p53 pathway

Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.     

Fibronectin protects prostate cancer cells from tumor necrosis factor-alpha-induced apoptosis via the AKT/survivin pathway

Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84--> Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via beta1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-alpha-induced apoptosis. Evidence is provided that expression of beta1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-alpha-induced apoptosis. In contrast, the beta1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-alpha in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis.     

Reduced Intimal Thickening Following αvβ 3 Blockade is Associated with Smooth Muscle Cell Apoptosis

The adhesion integrin αvβ3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of αvβ 3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of αvβ 3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of αvβ 3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of αvβ 3. At both 2 and 4wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of αvβ 3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing αvβ 3. These findings may explain the reduction in restenosis observed clinically following,93 integrin blockade.     

Reduced intimal thickening following alpha(v)beta3 blockade is associated with smooth muscle cell apoptosis

The adhesion integrin alpha(v)beta3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of alpha(v)beta3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of alpha(v)beta3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of alpha(v)beta3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of alpha(v)beta3. At both 2 and 4 wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of alpha(v)beta3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing alpha(v)beta3. These findings may explain the reduction in restenosis observed clinically following beta3 integrin blockade.     

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.     

CCN1 induces apoptosis in esophageal adenocarcinoma through p53-dependent downregulation of survivin

Many cancer drugs have been developed to control tumor growth by inducing cancer cell apoptosis. However, several intracellular barriers could fail this attempt. One of these barrier is high expression of survivin. Survivin can interfere caspase activation and thereby abort apoptosis. In this study, we found that CCN1 suppressed the survivin expression in tumor cells of esophageal adenocarcinoma (EAC) and thus allowed apoptosis to finish. Furthermore, we demonstrated that this downregulation was dependent on p53 phosphorylation at Ser20, and CCN1 induced EAC cell apoptosis through the activation of p53.     

Survivin down-regulation plays a crucial role in 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor-induced apoptosis in cancer

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HRIs) are widely used to reduce serum cholesterol in patients with hypercholesterolemia. Previous studies have shown that HRIs can induce apoptosis in colon cancer cells. In this study, we investigated the mechanisms underlying the apoptosis-inducing effect of HRIs in greater detail. The HRI lovastatin induced apoptosis in the human colon cancer cell line SW480 by blocking the cholesterol synthesis pathway. Immunoblot analysis of antiapoptotic molecules, including survivin, XIAP, cIAP-1, cIAP-2, Bcl-2, and Bcl-X(L), revealed that only survivin expression was decreased by lovastatin. Survivin down-regulation by RNA interference induced apoptosis, and survivin overexpression rendered the cells resistant to lovastatin-induced growth inhibition. These results indicate that survivin down-regulation contributes substantially to the proapoptotic properties of lovastatin. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate, two downstream intermediates in the cholesterol synthesis pathway, simultaneously reversed survivin down-regulation and the blocking of Ras isoprenylation by lovastatin. Ras isoprenylation is important for the activation of Ras-mediated signaling, including the activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The PI3-kinase inhibitor down-regulated survivin in SW480 cells. In addition, lovastatin blocked Ras activation and Akt phosphorylation. We conclude that survivin down-regulation is crucial in lovastatin-induced apoptosis in cancer cells and that lovastatin decreases survivin expression by inhibiting Ras-mediated PI3-kinase activation via the blocking of Ras isoprenylation.     

PKB phosphorylation and survivin expression are cooperatively regulated by disruption of microfilament cytoskeleton

Changes in cell shape can lead to detachment and cell death, and the disruption in the actin cytoskeletal network, as one marker of cell shape changes, can itself induce apoptosis. In this study, the effects of cytochalasin B on the apoptosis-related proteins, protein kinase B and survivin were investigated. Apoptosis induced by disruption of microfilaments with cytochalasin B was found, although it happened at a low level, to simultaneously occur with G2/M arrest in 50% of the cytochalasin B-treated cells. During apoptosis, PKB phosphorylation and survivin expression was decreased by cytochalasin B, and the decline in survivin expression were preceded by PKB dephosphorylation, which implicated that survivin may be a target of PKB protein. The G2/M arrest of cytochalasin B-treated cells may be the direct function of cytochalasin B to microfilaments or the subsequent inhibition of survivin expression, or both. These results suggest that PKB/survivin signaling pathway may be responsible for the apoptosis induced by the disruption of actin cytoskeleton.     

Thimerosal-Induced Apoptosis in Mouse C2C12 Myoblast Cells Occurs through Suppression of the PI3K/Akt/Survivin Pathway

Background

Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells.

Methodology/Principal Findings

The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125–500 nM) for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt^ser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml) increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells.

Conclusions/Significance

Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway.     

Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type‐Iα (PKG‐Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG‐Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti‐apoptotic Bcl‐2‐related proteins in NCI‐H460 and A549 non‐small cell lung cancer (NSCLC) cells. 1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxalin‐1‐one (ODQ), which blocks endogenous NO‐induced activation of cGMP/PKG‐Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c‐IAP1, livin, and survivin. DT‐2 (inhibitor of PKG‐Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c‐IAP1, livin, and survivin expression. Gene knockdown of PKG‐Iα expression using small‐interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c‐IAP1, livin, survivin, and Mcl‐1 expression. Inhibition of PKG‐Iα kinase activity with DT‐2 dramatically enhanced pro‐apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT‐2 and cisplatin increased apoptosis compared with cisplatin or DT‐2 alone, showing a synergistic effect. The data suggest that the PKG‐Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c‐IAP1, livin, survivin, and Mcl‐1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin. J. Cell. Biochem. 113: 3587–3598, 2012. © 2012 Wiley Periodicals, Inc.     

Survivin is involved in the anti-apoptotic effect of edaravone in PC12 cells

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a newly developed hydroxy radical scavaging agent which has been widely used for protection against ischemia-reperfusion injury is highly effective in preventing cell apoptosis. However, the exact intracellular mechanism(s) underlying the protective action of edaravone is not clear. We observed that in PC12 cells cultured under serum deprivation (DEPV) condition, the levels of survivin were positively correlated with the anti-apoptotic action of edaravone. Survivin RNA interference (RNAi) increased DEPV-induced PC12 cell apoptosis, whereas the anti-apoptotic effect of edaravone was blunted by survivin RNAi. Moreover, survivin overexpression provided protection against DEPV-induced PC12 cell apoptosis. Inhibition of ERK and PI₃-K/AKT prevented edaravone’s ability to decrease apoptosis and increase survivin. In conclusion, the present study provides the first direct evidence that survivin involves in the anti-apoptotic effects of edaravone via a pathway involving ERK and PI₃-K/AKT.