Cadmium Induces Vascular Endothelial Cell Detachment by Downregulating Claudin-5 and ZO-1 Levels.

Cadmium is a contributing factor to cardiovascular diseases and highly toxic to vascular endothelial cells. It has a distinct mode of injury, causing the de-endothelialization of regions in the monolayer structure of endothelial cells in a concentration-dependent manner. However, the specific molecules involved in the cadmium toxicity of endothelial cells remain unclear. The purpose of this study was to identify the specific molecular mechanisms through which cadmium affects endothelial detachment. Cadmium inhibited the expression of claudin-5 and zonula occludens (ZO)-1, which are components of tight junctions (strongest contributors to intercellular adhesion), in a concentration- and time-dependent manner. Compared to arsenite, zinc, and manganese, only cadmium suppressed the expression of both claudin-5 and ZO-1 molecules. Moreover, the knockdown of claudin-5 and ZO-1 exacerbated cadmium-induced endothelial cell injury and expansion of the detachment area, whereas their overexpression reversed these effects. CRE-binding protein inhibition reduced cadmium toxicity, suggesting that CRE-binding protein activation is involved in the cadmium-induced inhibition of claudin-5 and ZO-1 expression and endothelial detachment. These findings provide new insights into the toxicological mechanisms of cadmium-induced endothelial injury and risk of cardiovascular disease.

Sb-Phenyl-N-methyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine Induces Perlecan Core Protein Synthesis in Cultured Vascular Endothelial Cells.

Vascular endothelial cells synthesize and secrete perlecan, a large heparan sulfate proteoglycan that increases the anticoagulant activity of vascular endothelium by inducing antithrombin III and intensifying fibroblast growth factor (FGF)-2 activity to promote migration and proliferation in the repair process of damaged endothelium during the progression of atherosclerosis. However, the exact regulatory mechanisms of endothelial perlecan expression remain unclear. Since organic-inorganic hybrid molecules are being developed rapidly as tools to analyze biological systems, we searched for a molecular probe to analyze these mechanisms using a library of organoantimony compounds and found that the Sb-phenyl-N-methyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine (PMTAS) molecule promotes the expression of perlecan core protein gene without exhibiting cytotoxicity in vascular endothelial cells. In the present study, we characterized proteoglycans synthesized by cultured bovine aortic endothelial cells using biochemical techniques. The results indicated that PMTAS selectively induced perlecan core protein synthesis, without affecting the formation of its heparan sulfate chain, in vascular endothelial cells. The results also implied that this process is independent of the endothelial cell density, whereas in vascular smooth muscle cells, it occurred only at high cell density. Thus, PMTAS would be a useful tool for further studies on the mechanisms underlying perlecan core protein synthesis in vascular cells, which is critical in the progression of vascular lesions, such as those during atherosclerosis.

Induction of ZIP8, a ZIP transporter, via NF-κB signaling by the activation of IκBα and JNK signaling in cultured vascular endothelial cells exposed to cadmium.

Cadmium is an environmental pollutant that adversely affects various organs in the human body and is a well-known risk factor for cardiovascular diseases. These disorders are caused by the dysfunction of the vascular endothelial cells that cover the luminal surface of blood vessels. The ZIP transporter ZIP8 is one of the primary importers of cadmium, and its expression appears to be important for the sensitivity of vascular endothelial cells to cadmium. In the present study, we investigated the influence of ZIP8 on cadmium-induced cytotoxicity in vascular endothelial cells, the induction of ZIP8 expression by cadmium, and its action mechanism in vascular endothelial cells. The study revealed that: (1) cadmium cytotoxicity in vascular endothelial cells was potentiated by the overexpression of ZIP8, and the intracellular accumulation of cadmium in the cells was increased; (2) cadmium highly induced the expression of ZIP8, but not other ZIPs; (3) lead and methylmercury moderately induced ZIP8 expression, but the other tested metals did not; (4) the induction of ZIP8 expression by cadmium was mediated by both NF-κB and JNK signaling, and the accumulation of NF-κB in the nucleus was regulated by JNK signaling. Particularly, it was found that cadmium activated NF-κB to transfer it into nuclei and activated JNK to stabilize NF-κB in nuclei, resulting in the induction of ZIP8 expression. This induction appears to be crucial for cadmium cytotoxicity in vascular endothelial cells.

Involvement of CD73 and A2B Receptor in Radiation-Induced DNA Damage Response and Cell Migration in Human Glioblastoma A172 Cells.

Glioblastoma is the most common malignant tumor of the central nervous system and is treated with a combination of surgery, radiation and chemotherapy. However, the tumor often acquires radiation resistance, which is characterized by an increased DNA damage response (DDR). Here, we show that CD73, which generates extracellular adenosine from ATP, and A2B receptor, which is activated by adenosine, are involved in the γ-radiation-induced DDR and the enhanced migration ability of human glioblastoma cell line A172. To investigate DDR, we evaluated ataxia telangiectasia mutated (ATM) activation and focus formation of histone H2A isoform γ (γH2AX) and p53-binding protein 1 (53BP1) in the nucleus of A172 cells after γ-irradiation. Antagonists of A2B receptor and CD73, or knockdown with small interfering RNA (siRNA), suppressed γ-radiation-induced DDR and promoted γ-radiation-induced cell death, as well as suppressing γ-radiation-induced cell migration and actin remodeling. These results suggest that activation of A2B receptor by extracellular adenosine generated via CD73 promotes γ-radiation-induced DDR, leading to recovery from DNA damage, and also enhances cell migration and actin remodeling. The CD73-A2B receptor pathway may be a promising target for overcoming radiation resistance and the acquisition of malignant phenotypes during radiotherapy of glioblastoma.

TGF-ß1 Potentiates the Cytotoxicity of Cadmium by Induction of a Metal Transporter, ZIP8, Mediated by the ALK5-Smad2/3 and ALK5-Smad3-p38 MAPK Signal Pathways in Cultured Vascular Endothelial Cells.

Vascular endothelial cells cover the luminal surface of blood vessels in a monolayer and play a role in the regulation of vascular functions, such as the blood coagulation-fibrinolytic system. When the monolayer is severely or repeatedly injured, platelets aggregate at the damaged site and release transforming growth factor (TGF)-ß1 in large quantities from their α-granules. Cadmium is a heavy metal that is toxic to various organs, including the kidneys, bones, liver, and blood vessels. Our previous study showed that the expression level of Zrt/Irt-related protein 8 (ZIP8), a metal transporter that transports cadmium from the extracellular fluid into the cytosol, is a crucial factor in determining the sensitivity of vascular endothelial cells to cadmium cytotoxicity. In the present study, TGF-ß1 was discovered to potentiate cadmium-induced cytotoxicity by increasing the intracellular accumulation of cadmium in cells. Additionally, TGF-ß1 induced the expression of ZIP8 via the activin receptor-like kinase 5-Smad2/3 signaling pathways; Smad3-mediated induction of ZIP8 was associated with or without p38 mitogen-activated protein kinase (MAPK). These results suggest that the cytotoxicity of cadmium to vascular endothelial cells increases when damaged endothelial monolayers that are highly exposed to TGF-ß1 are repaired.

Arsenite Inhibits Tissue-Type Plasminogen Activator Synthesis through NRF2 Activation in Cultured Human Vascular Endothelial EA.hy926 Cells.

Chronic arsenic exposure is known to be related to the progression of atherosclerosis. However, the pathogenic mechanisms of arsenic-induced atherosclerosis have not been fully elucidated. Because disruption of the blood coagulation/fibrinolytic system is involved in the development of arteriosclerosis, we investigated the effect of arsenite on fibrinolytic activity in human vascular endothelial EA.hy926 cells in the present study. Fibrinolysis depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) secreted from vascular endothelial cells. We found that arsenite reduced fibrinolytic t-PA activity by inhibiting its synthesis without affecting PAI-1 production. The inhibitory effect of arsenite on t-PA expression was partially recovered by the reactive oxygen species (ROS) scavenger Trolox. The nuclear factor erythroid 2 related factor 2 (NRF2) pathway is known to be activated by arsenite via ROS production. We confirmed that arsenite activated the NRF2 pathway, and arsenite-induced inhibition of fibrinolytic t-PA activity was abrogated in NRF2-knockdown EA.hy926 cells. These results suggest that arsenite inhibits the fibrinolytic activity of t-PA by selectively suppressing its synthesis via activation of the NRF2 pathway in vascular endothelial cells.

Synthesis of Reactive Sulfur Species in Cultured Vascular Endothelial Cells after Exposure to TGF-ß1: Induction of Cystathionine γ-Lyase and Cystathionine ß-Synthase Expression Mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 Pathways.

Transforming growth factor-ß1 (TGF-ß1) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-ß1. Bovine aortic endothelial cells in a culture system were treated with TGF-ß1 to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-ß1, induction of RSS-producing enzymes by TGF-ß1, and intracellular signal pathways that mediate this induction. The results suggest that TGF-ß1 increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine ß-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-ß1 regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-ß1, may modulate the regulation activity in vascular endothelial cells.

Effects of Substitution on Cytotoxicity of Diphenyl Ditelluride in Cultured Vascular Endothelial Cells.

Among organic-inorganic hybrid molecules consisting of organic structure(s) and metal(s), only few studies are available on the cytotoxicity of nucleophilic molecules. In the present study, we investigated the cytotoxicity of a nucleophilic organotellurium compound, diphenyl ditelluride (DPDTe), using a cell culture system. DPDTe exhibited strong cytotoxicity against vascular endothelial cells and fibroblasts along with high intracellular accumulation but showed no cytotoxicity and had less accumulation in vascular smooth muscle cells and renal epithelial cells. The cytotoxicity of DPDTe decreased when intramolecular tellurium atoms were replaced with selenium or sulfur atoms. Electronic state analysis revealed that the electron density between tellurium atoms in DPDTe was much lower than those between selenium atoms of diphenyl diselenide and sulfur atoms of diphenyl disulfide. Moreover, diphenyl telluride did not accumulate and exhibit cytotoxicity. The cytotoxicity of DPDTe was also affected by substitution. p-Dimethoxy-DPDTe showed higher cytotoxicity, but p-dichloro-DPDTe and p-methyl-DPDTe showed lower cytotoxicity than that of DPDTe. The subcellular distribution of the compounds revealed that the compounds with stronger cytotoxicity showed higher accumulation rates in the mitochondria. Our findings suggest that the electronic state of tellurium atoms in DPDTe play an important role in accumulation and distribution of DPDTe in cultured cells. The present study supports the hypothesis that nucleophilic organometallic compounds, as well as electrophilic organometallic compounds, exhibit cytotoxicity by particular mechanisms.

ATP and ADP enhance DNA damage repair in γ-irradiated BEAS-2B human bronchial epithelial cells through activation of P2X7 and P2Y12 receptors.

Agents that promote DNA repair may be useful as radioprotectants to minimize side effects such as radiation pneumonia caused by damage to normal cells during radiation therapy to treat lung cancer. We have reported that extracellular nucleotides and nucleosides are involved in the P2 or P1 receptor-mediated DNA damage response (DDR) after γ-irradiation. Here, we investigated the effects of ATP, UTP, GTP, ITP and their metabolites on the γH2AX/53BP1 focus formation in nuclei (a measure of γ-irradiation-induced DDR) and the survival of γ-irradiated immortalized human bronchial epithelial (BEAS-2B) cells. Fluorescence immunostaining showed that ATP and ADP increase DDR and DNA repair, and exhibit radioprotective effects as evaluated by colony formation assay. These effects of ATP or ADP were blocked by inhibitors of P2X7 or P2Y12 receptor, respectively, and by ERK1/2 inhibitor. ATP and ADP enhanced phosphorylation of ERK1/2 by suppressing MKP-1 and MKP-3 expression after γ-irradiation. These results indicate that ATP and ADP exhibit radioprotective effects by phosphorylation of ERK1/2 via activation of P2X7 and P2Y12 receptors, respectively, to promote γ-irradiation-induced DDR and DNA repair. ATP and ADP appear to be candidates for radioprotectants to reduce damage to non-cancerous cells during lung cancer radiotherapy by promoting DDR and DNA repair.

Cell Density-Dependent Fibroblast Growth Factor-2 Signaling Regulates Syndecan-4 Expression in Cultured Vascular Endothelial Cells.

Syndecan-4 is a member of the syndecan family of transmembrane heparan sulfate proteoglycans, and is involved in cell protection, proliferation, and the blood coagulation-fibrinolytic system in vascular endothelial cells. Heparan sulfate chains enable fibroblast growth factor-2 (FGF-2) to form a complex with its receptor and to transduce the cell growth signal. In the present study, bovine aortic endothelial cells were cultured, and the intracellular signal pathways that mediate the regulation of syndecan-4 expression in dense and sparse cultures by FGF-2 were analyzed. We demonstrated the cell density-dependent differential regulation of syndecan-4 expression. Specifically, we found that FGF-2 upregulated the synthesis of syndecan-4 in vascular endothelial cells via the MEK1/2-ERK1/2 pathway in dense cell cultures, with only a transcriptional induction of syndecan-4 at a low cell density via the Akt pathway. This study highlights a critical mechanism underlying the regulation of endothelial cell functions by proteoglycans.

Transcriptional Induction of Cystathionine γ-Lyase, a Reactive Sulfur-Producing Enzyme, by Copper Diethyldithiocarbamate in Cultured Vascular Endothelial Cells.

As toxic substances can enter the circulating blood and cross endothelial monolayers to reach parenchymal cells in organs, vascular endothelial cells are an important target compartment for such substances. Reactive sulfur species protect cells against oxidative stress and toxic substances, including heavy metals. Reactive sulfur species are produced by enzymes, such as cystathionine γ-lyase (CSE), cystathionine ß-synthase, 3-mercaptopyruvate sulfurtransferase, and cysteinyl-tRNA synthetase. However, little is known about the regulatory mechanisms underlying the expression of these enzymes in vascular endothelial cells. Bio-organometallics is a research field that analyzes biological systems using organic-inorganic hybrid molecules (organometallic compounds and metal coordinating compounds) as molecular probes. In the present study, we analyzed intracellular signaling pathways that mediate the expression of reactive sulfur species-producing enzymes in cultured bovine aortic endothelial cells, using copper diethyldithiocarbamate (Cu10). Cu10 selectively upregulated CSE gene expression in vascular endothelial cells independent of cell density. This transcriptional induction of endothelial CSE required both the diethyldithiocarbamate scaffold and the coordinated copper ion. Additionally, the present study revealed that ERK1/2, p38 MAPK, and hypoxia-inducible factor (HIF)-1α/HIF-1ß pathways mediate transcriptional induction of endothelial CSE by Cu10. The transcription factors NF-κB, Sp1, and ATF4 were suggested to act in constitutive CSE expression, although the possibility that they are involved in the CSE induction by Cu10 cannot be excluded. The present study used a copper complex as a molecular probe to reveal that the transcription of CSE is regulated by multiple pathways in vascular endothelial cells, including ERK1/2, p38 MAPK, and HIF-1α/HIF-1ß. Bio-organometallics appears to be an effective strategy for analyzing the functions of intracellular signaling pathways in vascular endothelial cells.

Copper(II) Bis(diethyldithiocarbamate) Induces the Expression of Syndecan-4, a Transmembrane Heparan Sulfate Proteoglycan, via p38 MAPK Activation in Vascular Endothelial Cells.

Proteoglycans synthesized by vascular endothelial cells are important for regulating cell function and the blood coagulation-fibrinolytic system. Since we recently reported that copper(II) bis(diethyldithiocarbamate) (Cu(edtc)2) modulates the expression of some molecules involving the antioxidant and blood coagulation systems, we hypothesized that Cu(edtc)2 may regulate the expression of proteoglycans and examined this hypothesis using a bovine aortic endothelial cell culture system. The experiments showed that Cu(edtc)2 induced the expression of syndecan-4, a transmembrane heparan sulfate proteoglycan, in a dose- and time-dependent manner. This induction required the whole structure of Cu(edtc)2-the specific combination of intramolecular copper and a diethyldithiocarbamate structure-as the ligand. Additionally, the syndecan-4 induction by Cu(edtc)2 depended on the activation of p38 mitogen-activated protein kinase (MAPK) but not the Smad2/3, NF-E2-related factor2 (Nrf2), or epidermal growth factor receptor (EGFR) pathways. p38 MAPK may be a key molecule for inducing the expression of syndecan-4 in vascular endothelial cells.

Transforming Growth Factor-ß1 Modulates the Expression of Syndecan-4 in Cultured Vascular Endothelial Cells in a Biphasic Manner.

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor-ß1 (TGF-ß1 ) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine-rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF-ß1 first upregulates and then downregulates the expression of syndecan-4, a transmembrane heparan sulfate proteoglycan, via the TGF-ß receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF-ß1 . Involvement of the downstream signaling pathways of ALK5-the Smad and MAPK pathways-in syndecan-4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3-p38 MAPK pathway mediates the early upregulation of syndecan-4 by TGF-ß1 , whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF-ß1 . J. Cell. Biochem. 118: 2009-2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Biglycan Intensifies ALK5-Smad2/3 Signaling by TGF-ß1 and Downregulates Syndecan-4 in Cultured Vascular Endothelial Cells.

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. A small leucine-rich dermatan sulfate proteoglycan, biglycan, is one of the predominant types of proteoglycans synthesized by vascular endothelial cells; however, the physiological functions of biglycan are not completely understood. In the present study, bovine aortic endothelial cells in culture were transfected with small interfering RNAs for biglycan, and the expression of other proteoglycans was examined. Transforming growth factor-ß1 signaling was also investigated, because the interaction of biglycan with cytokines has been reported. Biglycan was found to form a complex with either transforming growth factor-ß1 or the transforming growth factor-ß1 type I receptor, ALK5, and to intensify the phosphorylation of Smad2/3, resulting in a lower expression of the transmembrane heparan sulfate proteoglycan, syndecan-4. This is the first report to clarify the function of biglycan as a regulatory molecule of the ALK5-Smad2/3 TGF-ß1 signaling pathway that mediates the suppression of syndecan-4 expression in vascular endothelial cells. J. Cell. Biochem. 118: 1087-1096, 2017. © 2016 Wiley Periodicals, Inc.

Induction of Syndecan-4 by Organic-Inorganic Hybrid Molecules with a 1,10-Phenanthroline Structure in Cultured Vascular Endothelial Cells.

Organic-inorganic hybrid molecules constitute analytical tools used in biological systems. Vascular endothelial cells synthesize and secrete proteoglycans, which are macromolecules consisting of a core protein and glycosaminoglycan side chains. Although the expression of endothelial proteoglycans is regulated by several cytokines/growth factors, there may be alternative pathways for proteoglycan synthesis aside from downstream pathways activated by these cytokines/growth factors. Here, we investigated organic-inorganic hybrid molecules to determine a variant capable of analyzing the expression of syndecan-4, a transmembrane heparan-sulfate proteoglycan, and identified 1,10-phenanthroline (o-Phen) with or without zinc (Zn-Phen) or rhodium (Rh-Phen). Bovine aortic endothelial cells in culture were treated with these compounds, and the expression of syndecan-4 mRNA and core proteins was determined by real-time reverse transcription polymerase chain reaction and Western blot analysis, respectively. Our findings indicated that o-Phen and Zn-Phen specifically and strongly induced syndecan-4 expression in cultured vascular endothelial cells through activation of the hypoxia-inducible factor-1α/ß pathway via inhibition of prolyl hydroxylase-domain-containing protein 2. These results demonstrated an alternative pathway involved in mediating induction of endothelial syndecan-4 expression and revealed organic-inorganic hybrid molecules as effective tools for analyzing biological systems.

Copper diethyldithiocarbamate as an activator of Nrf2 in cultured vascular endothelial cells.

The interest in organic-inorganic hybrid molecules as molecular probes for biological systems has been growing rapidly. Such hybrid molecules exhibit unique biological activities. Herein, copper(II) bis(diethyldithiocarbamate) (Cu10) was found to activate the transcription factor NF-E2-related factor 2 (Nrf2), which is responsible for regulating antioxidant and phase II xenobiotic enzymes, in vascular endothelial cells. The copper complex rapidly accumulated within cells and induced nuclear translocation of Nrf2, leading to upregulation of the expression of downstream proteins without cytotoxic effects. However, while copper bis(2-hydroxyethyl)dithiocarbamate activated Nrf2, copper ion, diethyldithiocarbamate ligand with or without zinc or iron failed to exhibit this activity. Intracellular accumulation of Cu10 was higher than that of Cu(II) and Cu(I). While the accumulation of copper(II) bis(dimethyldithiocarbamate) was reduced by small interfering RNA (siRNA)-mediated knockdown of the copper transporter CTR1, the knockdown did not affect Cu10 accumulation, indicating that Cu10 rapidly enters vascular endothelial cells via CTR1-independent mechanisms. In addition, copper and iron complexes with other ligands tested could not activate Nrf2, suggesting that the intramolecular interaction between copper and dithiocarbamate ligand is important for the activation of the transcription factor. Cu10 induced the expression of heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and γ-glutamylcysteine synthetase, downstream proteins of Nrf2. It was suggested that Cu10-induced activation of Nrf2 was due to proteasome inhibition as well as binding to Kelch-like ECH-associated protein 1. Since the effects of Cu10 on vascular endothelial cells are unique and diverse, the copper complex may be a good molecular probe to analyze the functions of the cells.

Partial contribution of the Keap1-Nrf2 system to cadmium-mediated metallothionein expression in vascular endothelial cells.

Cadmium is an environmental electrophile that modifies protein reactive thiols such as Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear factor-erythroid 2-related factor 2 (Nrf2). In the present study, we investigated a role of the Keap1-Nrf2 system in cellular response to cadmium in vascular endothelial cells. Exposure of bovine aortic endothelial cells to cadmium resulted in modification of Keap1 and Nrf2 activation, thereby up-regulating not only its typical downstream proteins but also metallothionein-1/2. Experiments with siRNA-mediated knockdown of Nrf2 or Keap1 supported participation of the Keap1-Nrf2 system in the modulation of metallothionein-1/2 expression. Furthermore, chromatin immunoprecipitation assay showed that Nrf2 was recruited to the antioxidant response element of the promoter region of the bovine metallothionein-2 gene in the presence of cadmium. These results suggest that the transcription factor Nrf2 plays, at least in part, a role in the changes in metallothionein expression mediated by exposure to cadmium.

Heparan sulfate chains potentiate cadmium cytotoxicity in cultured vascular endothelial cells.

The monolayer of vascular endothelial cells, which is rich in heparan sulfate chains, is an important target of cadmium cytotoxicity. To investigate the effects of heparan sulfate chains on cadmium cytotoxicity, bovine aortic endothelial cells were cultured in the presence of cadmium, with or without exogenous heparan sulfate. The following results were obtained: (1) Heparan sulfate chains potentiated cadmium cytotoxicity. (2) Such a potentiation did not occur in bovine aortic smooth muscle cells. (3) Heparin chains as well as heparan sulfate chains potentiated cadmium cytotoxicity, while other glycosaminoglycan chains failed to exhibit such an activity. (4) The disaccharide units of heparan sulfate chains did not potentiate cadmium cytotoxicity in the endothelial cells. (5) Heparan sulfate chains did not potentiate mercury and arsenite cytotoxicity. (6) Fibroblast growth factor-2 (FGF-2) also potentiated cadmium cytotoxicity in the endothelial cells. (7) Heparan sulfate chains significantly increased intracellular cadmium accumulation by inducing the expression of metallothionein. Taken together, these results suggest that heparan sulfate chains activate FGF-2, which in turn elevates the expression and/or activity of metal transporter(s) that facilitate cadmium influx from the extracellular space into the cytoplasm.

Transcriptional Induction of Metallothionein by Tris(pentafluorophenyl)stibane in Cultured Bovine Aortic Endothelial Cells.

Vascular endothelial cells cover the luminal surface of blood vessels and contribute to the prevention of vascular disorders such as atherosclerosis. Metallothionein (MT) is a low molecular weight, cysteine-rich, metal-binding, inducible protein, which protects cells from the toxicity of heavy metals and active oxygen species. Endothelial MT is not induced by inorganic zinc. Adequate tools are required to investigate the mechanisms underlying endothelial MT induction. In the present study, we found that an organoantimony compound, tris(pentafluorophenyl)stibane, induces gene expression of MT-1A and MT-2A, which are subisoforms of MT in bovine aortic endothelial cells. The data reveal that MT-1A is induced by activation of both the MTF-1-MRE and Nrf2-ARE pathways, whereas MT-2A expression requires only activation of the MTF-1-MRE pathway. The present data suggest that the original role of MT-1 is to protect cells from heavy metal toxicity and oxidative stress in the biological defense system, while that of MT-2 is to regulate intracellular zinc metabolism.

Protection of cultured vascular endothelial cells against cadmium cytotoxicity by simultaneous treatment or pretreatment with manganese.

Cadmium is a heavy metal that pollutes the environment and foods and is a risk factor for vascular disorders. We have previously demonstrated that pretreatment of vascular endothelial cells with zinc and copper protects the cells against cadmium cytotoxicity. In contrast, cadmium cytotoxicity was potentiated in cells following exposure to lead, thereby indicating that in vascular endothelial cells, cadmium cytotoxicity can be differentially modified by the co-occurrence of other heavy metals. In this study, we revealed that simultaneous treatment or pretreatment with manganese protects vascular endothelial cells against cadmium cytotoxicity. Intracellular accumulation of cadmium was observed to be reduced by simultaneous treatment with manganese, although not by pretreatment. The mRNA expression of metal transporters that regulate the uptake of both cadmium and manganese (ZIP8, ZIP14, and DMT1) remained unaffected by either simultaneous treatment or pretreatment with manganese, and simultaneous treatment with manganese suppressed the cadmium-induced expression of metallothionein but pretreatment with manganese did not exhibit such suppressive effect. Thus, the protection of vascular endothelial cells against cadmium cytotoxicity conferred by simultaneous treatment with manganese is assumed to be partially attributed to a reduction in the intracellular accumulation of cadmium, whereas the effects of pretreatment with manganese are independent of both the reduced intracellular accumulation of cadmium and the induction of metallothionein. These observations accordingly indicate that the protective effects of manganese are mediated via alternative (as yet unidentified) mechanisms.