Regulation of vascular smooth muscle cell proliferation by nuclear orphan receptor Nur77

Endothelium-derived nitric oxide (NO) is a cytoprotective molecule to prevent endothelial cells (ECs) from apoptosis. CREB-binding protein (CBP) is involved in the apoptotic pathway in several tumor cells, however, little is known whether CBP is associated with apoptosis in ECs and the apoptotic effect of CBP on ECs is regulated by NO. Therefore, the purpose of the present study was to investigate whether silencing CBP expression could affect the sensitivity of ECs toward apoptotic stimuli and determined the role of NO. In this study, we found that when CBP expression was silenced by RNA interference, ECs were more prone to apoptosis under serum deprivation, whereas the apoptosis was not significantly induced in the serum-containing condition. The increased apoptosis is paralleled by a reduction of NO, and the apoptosis was reversed by NO donors, suggesting an important role of NO. Furthermore, CBP silencing decreased NO production by downregulating the endothelial NO synthase (eNOS) expression in a dose-dependent manner. These results indicated that CBP silencing is associated with decreased eNOS expression and NO production, and therefore concomitantly increased the sensitivity of ECs toward apoptosis.     

Golgi apparatus fragmentation participates in oxidized low-density lipoprotein-induced endothelial cell injury

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial injury plays crucial roles in the development of arteriosclerosis (AS). Golgi apparatus (GA) fragmentation is involved in various pathological processes, including endothelial injury. However, the role of GA fragmentation in ox-LDL-induced endothelial injury has not been determined. In this study, human umbilical vein endothelial cells (HUVECs) subjected to ox-LDL were used as an in vitro AS model. Herein, we showed that ox-LDL restrained proliferation and induced apoptosis and GA fragmentation of HUVECs. Moreover, overexpression of GRASP65 significantly prevented ox-LDL-induced GA fragmentation and endothelial cell injury by enhancing cell viability, nitric oxide production, and endothelial NOS expression and reducing apoptosis. Mechanistically, ox-LDL resulted in the activation of the extracellular signal-regulated kinase (ERK) pathway in HUVECs. Inactivation of the ERK pathway by U0126 suppressed the phosphorylation of GRASP65, GA fragmentation, and endothelial cell injury induced by ox-LDL. In conclusion, ox-LDL triggers GA fragmentation in HUVECs via activating the ERK signaling pathway, which participates in endothelial injury during the development of AS.     

MicroRNA let-7c Inhibits Cell Proliferation and Induces Cell Cycle Arrest by Targeting CDC25A in Human Hepatocellular Carcinoma

Down-regulation of the microRNA let-7c plays an important role in the pathogenesis of human hepatocellular carcinoma (HCC). The aim of the present study was to determine whether the cell cycle regulator CDC25A is involved in the antitumor effect of let-7c in HCC. The expression levels of let-7c in HCC cell lines were examined by quantitative real-time PCR, and a let-7c agomir was transfected into HCC cells to overexpress let-7c. The effects of let-7c on HCC proliferation, apoptosis and cell cycle were analyzed. The in vivo tumor-inhibitory efficacy of let-7c was evaluated in a xenograft mouse model of HCC. Luciferase reporter assays and western blotting were conducted to identify the targets of let-7c and to determine the effects of let-7c on CDC25A, CyclinD1, CDK6, pRb and E2F2 expression. The results showed that the expression levels of let-7c were significantly decreased in HCC cell lines. Overexpression of let-7c repressed cell growth, induced cell apoptosis, led to G1 cell cycle arrest in vitro, and suppressed tumor growth in a HepG2 xenograft model in vivo. The luciferase reporter assay showed that CDC25A was a direct target of let-7c, and that let-7c inhibited the expression of CDC25A protein by directly targeting its 3ʹ UTR. Restoration of CDC25A induced a let-7c-mediated G1-to-S phase transition. Western blot analysis demonstrated that overexpression of let-7c decreased CyclinD1, CDK6, pRb and E2F2 protein levels. In conclusion, this study indicates that let-7c suppresses HCC progression, possibly by directly targeting the cell cycle regulator CDC25A and indirectly affecting its downstream target molecules. Let-7c may therefore be an effective therapeutic target for HCC.     

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91^phox subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91^phox subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91^phox subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.     

Expression and Preliminary Functional Profiling of the let-7 Family during Porcine Ovary Follicle Atresia

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.     

Identification of microRNAs That Regulate TLR2-Mediated Trophoblast Apoptosis and Inhibition of IL-6 mRNA

While infection-induced placental inflammation is a common mechanism of adverse pregnancy outcome, some pathogens can also trigger placental apoptosis, and Toll-like receptors (TLRs) mediate this response. Treatment of human first trimester trophoblast cells with bacterial peptidoglycan (PDG) reduces their constitutive secretion of IL-6 protein and induces apoptosis. This apoptotic response is dependent upon the cell’s expression of TLR1, TLR2 and TLR10, and their lack of TLR6, such that ectopic expression of TLR6 prevents PDG-induced apoptosis and restores IL-6 production. In this current study we have identified three microRNAs (miRs) that regulate TLR2-mediated responses in the human trophoblast. Herein we report that miR-329 plays a pivotal role in mediating PDG-induced trophoblast apoptosis and inhibition of IL-6 mRNA expression by targeting the NF-κB subunit, p65. TLR2 activation by PDG upregulates miR-329 expression and inhibits NF-κB p65 and IL-6 mRNA, and this is reversed by the presence of TLR6. Moreover, inhibition of miR-329 prevents PDG-induced inhibition of NF-κB p65 and IL-6 mRNA expression, and restores cell survival. In addition, we have found miR-23a and let-7c to directly regulate PDG-mediated inhibition of IL-6 mRNA. TLR2 activation by PDG upregulates miR23a and let-7c expression and this is reversed by the presence of TLR6. Furthermore, inhibition of both miR23a and let-7c prevents PDG-inhibition of trophoblast IL-6 mRNA expression. Together, our findings suggest that multiple miRs are involved in the molecular regulation of TLR2-mediated responses in the trophoblast towards gram-positive bacterial components.     

Genistein‐3′‐sodium sulphonate protects against lipopolysaccharide‐induced lung vascular endothelial cell apoptosis and acute lung injury via BCL‐2 signalling

Under septic conditions, Lipopolysaccharide (LPS)‐induced apoptosis of lung vascular endothelial cells (ECs) triggers and aggravates acute lung injury (ALI), which so far has no effective therapeutic options. Genistein‐3′‐sodium sulphonate (GSS) is a derivative of native soy isoflavone, which has neuro‐protective effects through its anti‐apoptotic property. However, whether GSS protects against sepsis‐induced lung vascular endothelial cell apoptosis and ALI has not been determined. In this study, we found that LPS‐induced Myd88/NF‐κB/BCL‐2 signalling pathway activation and subsequent EC apoptosis were effectively down‐regulated by GSS in vitro. Furthermore, GSS not only reversed the sepsis‐induced BCL‐2 changes in expression in mouse lungs but also blocked sepsis‐associated lung vascular barrier disruption and ALI in vivo. Taken together, our results demonstrated that GSS might be a promising candidate for sepsis‐induced ALI via its regulating effects on Myd88/NF‐κB/BCL‐2 signalling in lung ECs.     

PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax–caspase9–caspase3 pathway

This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24?h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24?h, cells were treated with ox-LDL for 24?h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.     

Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation

Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.     

Atherosclerosis-associated endothelial cell apoptosis by miRNA let7-b-mediated downregulation of HAS-2

MicroRNAs (miRNAs) play essential roles in the regulation and pathophysiology of various types of human diseases including atherosclerosis. Increasing numbers of miRNAs have been identified to be important regulators in the progression of atherosclerosis by regulating gene expression. However, functional miRNAs and the underlying mechanisms involved in atherosclerosis need fully elucidation. In the present study, the function of miRNA let-7b was investigated in human aortic endothelial cells (HAECs). The results showed that downregulation of let-7b in the high-fat diet mice and HAECs was inversely correlated with the expression level of HAS-2. upregulation of let-7b significantly reduced apoptosis of HAECs. The results also revealed that HAS-2 was a target gene of let-7b and HAS-2 reduction reversed the antiapoptotic effect of let-7b through regulation of the P13K/Akt pathway. These results together suggest the potential of regulating the let-7b expression and endothelial apoptosis against development and progression of atherosclerosis.     

Caspase- and p53-dependent apoptosis in breast carcinoma cells induced by a synthetic selenadiazole derivative

Selenadiazole derivative is one kind of synthetic organoselenium compounds with potent and broad-spectrum antitumor activity. In this study, we showed that anthrax [1,2-c] [1,2,5] selenadiazolo-6,11-dione (ASDO), an novel selenadiazole derivative, induced time- and dose-dependent apoptotic cell death in MCF-7 human breast carcinoma cells, as indicated by accumulation of sub-G1 cell population, DNA fragmentation, nuclear condensation, caspase activation and PARP cleavage. ASDO-induced apoptosis was significantly inhibited by a general caspase inhibitor z-VAD-fmk, demonstrating the important role of caspases in ASDO-induced apoptotic pathway. Treatment of MCF-7 cells with ASDO resulted in a rapid depletion of mitochondrial membrane potential and release of cytochrome c and Smac/Diablo through up-regulation of Bax, Bad and PUMA expression and down-regulation of Bcl-xl expression. Moreover, ASDO treatment up-regulated the expression levels of total p53 and its target gene p21Waf1. Silencing of p53 activation with RNA interference effectively blocked the ASDO-induced cell PARP cleavage, DNA fragmentation and caspase activation. Furthermore, ASDO-induced apoptosis was interestingly found to be independent of reactive oxygen species production. Taken together, we conclude that ASDO induces MCF-7 cell apoptosis through a p53-dependent and mitochondria-mediated pathway.     

Connexin43 siRNA promotes HUVEC proliferation and inhibits apoptosis induced by ox-LDL: an involvement of ERK signaling pathway

Oxidized low-density lipoprotein (ox-LDL), one of the most important risk factors of atherosclerosis, is a highly antigenic, potent chemoattractant that facilitates the development of atherosclerosis. Gap junctions also play an important in the development of atherosclerosis. In this study, we investigated the effects of ox-LDL on connexin43 and the mechanisms of connexin43 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cell (HUVEC), to clarify the role of connexin43 in atherosclerosis. Our results showed that ox-LDL significantly inhibited the growth and promoted apoptosis of HUVEC in a dose-dependent manner. Also, ox-LDL upregulated the expression of connexin43. Furthermore, knockdown connexin43 by siRNA promoted proliferation and inhibited apoptosis in ox-LDL-stimulated HUVEC. Moreover, the level of phosphor-ERK1/2 and connexin43 was remarkably attenuated by a ERK pathway inhibitor (PD98059). These results suggest that connexin43 siRNA promotes HUVEC proliferation and inhibits apoptosis induced by ox-LDL, and ERK signaling pathway appears to be involved in these processes.     

CD95 Is Part of a Let-7/p53/miR-34 Regulatory Network

The death receptor CD95 (APO-1/Fas) mediates apoptosis induction upon ligation by its cognate ligand CD95L. Two types of CD95 signaling pathways have been identified, which are characterized by the absence (Type I) or presence (Type II) of mitochondrial involvement. Micro(mi)RNAs are small noncoding RNAs that negatively regulate gene expression. They are important regulators of differentiation processes and are found frequently deregulated in many human cancers. We recently showed that Type I cells express less of the differentiation marker miRNA let-7 and, hence, likely represent more advanced tumor cells than the let-7 high expressing Type II cells. We have now identified miR-34a as a selective marker for cells that are sensitive to CD95-mediated apoptosis. Both CD95 and miR-34a are p53 target genes, and consequently, both the sensitivity of cancer cells to CD95-mediated apoptosis and the ability to respond to p53 mediated DNA genotoxic stress are linked. Interestingly, while miR-34a was found to positively correlate with the ability of cells to respond to genotoxic stress, let-7 was negatively correlated. The expression level of CD95 inversely correlated with the expression of let-7 suggesting regulation of let-7 expression by CD95. To test a link between p53 and miR-34a, we altered the expression of CD95. This affected the ability of cells to activate p53 and to regulate miR-34a. Our data point to a novel regulatory network comprising p53, CD95, let-7, and miR-34a that affects cancer cell survival, differentiation, and sensitivity to apoptotic signals. The possible relevance of this regulatory network for cancer stem cells is discussed.     

In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line

Abstract: Amyloid β-peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that Aβ25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25-35 were specific because Aβ1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1-40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25-35. Damage and death of ECs induced by Aβ25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.