Chika Yamamoto

Cell Density-dependent Regulation of Proteoglycan Synthesis by Transforming Growth Factor-β1 in Cultured Bovine Aortic Endothelial Cells

The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-β1(TGF-β1) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-β1on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-β1 also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [3H]glucosamine, [35S]sulfate, or35S-labeled amino acids in the presence of TGF-β1. The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan M rand composition were analyzed by Sepharose CL-6B chromatography, and the core protein M r was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-β1 on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-β1 induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of ∼50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of ∼400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-β1 cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-β1 promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.

The cytotoxicity of organobismuth compounds with certain molecular structures can be diminished by replacing the bismuth atom with an antimony atom in the molecules.

Organic-inorganic hybrid molecules, which are composed of an organic structure and metal(s), are indispensable for synthetic chemical reactions; however, their toxicity has been incompletely understood. In the present study, we discovered two cytotoxic organobismuth compounds whose cytotoxicity diminished upon replacement of the intramolecular bismuth atom with an antimony atom. The intracellular accumulation of the organobismuth compounds was much higher than that of the organoantimony compounds with the corresponding organic structures. We also showed that both the organic structure and bismuth atom are required for certain organobismuth compounds to exert their cytotoxic effect, suggesting that the cytotoxicity of such a compound is a result of an interaction between the organic structure and the bismuth atom. The present data suggest that organobismuth compounds with certain molecular structures exhibit cytotoxicity via an interaction between the molecular structure and the bismuth atom, and this cytotoxicity can be diminished by replacing the bismuth atom with an antimony atom, resulting in lower intracellular accumulation.

Endothelin modulation of tissue plasminogen activator release from human vascular endothelial cells in culture

To clarify a possible involvement of the vasoconstrictive peptide endothelin in the regulation of endothelial cell-mediated fibrinolytic system, confluent cultures of vascular endothelial cells from human umbilical vein were incubated in serum-free medium in the presence of endothelin-1 at 100 nM and below, and tissue plasminogen activator antigen (t-PA:Ag) in the medium was determined by enzyme immunoassay. Endothelin-l at 1 nM and above significantly decreased the release of t-PA:Ag from the endothelial cells after a 24 h incubation. The t-PA:Ag release was also decreased by either endothelin-2 or endothelin-3 at 10 nM. The activity of lactate dehydrogenase in the medium was not changed by endothelin-l at 100 nM and below, suggesting that the peptide did not cause nonspecific cell damage. The decrease in the t-PA: Ag release induced by endothelin-1 occurred in the presence or absence of 8-bromo cyclic AMP, which is an active congener of cyclic AMP; 3-isobutyl-l-methylxanthine, which is an inhibitor of phosphodiesterase; and forskolin, which is a stimulator of adenylate cyclase. These results strongly indicated that cyclic AMP which is known to down-regulate t-PA;Ag release was not involved in the endothelin-l effect. However, endothelin-l failed to decrease the t-PA:Ag release in the presence of either calcium ionophore A23187 or EGTA; the ionophore itself markedly decreased the release. The cytosolic calcium accumulation was significantly increased by endothelin-l. These results suggest that endothelin-l decreases the release of t-P A:Ag from human endothelial cells through an excess accumulation of intracellular, especially cytosolic calcium which would be mediated by an extracellular, calcium-dependent mechanism.

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.

Induction of metallothionein isoforms by copper diethyldithiocarbamate in cultured vascular endothelial cells

Metallothionein (MT) plays a central role in cellular defense against heavy metals and oxidative stress. Since the induction of MT requires the activation of metal response element (MRE)-binding transcription factor-1 (MTF-1) by binding of zinc ions, inorganic zinc is regarded as a typical MT inducer. However, in a previous report, we showed that inorganic zinc could not induce MT in vascular endothelial cells. While it is suggested that endothelial MT presents mechanisms different from those of other cell types, these remain unclear. In this study, we investigated whether the induction of endothelial MT expression involves the Nrf2-ARE pathway using copper(II) bis(diethyldithiocarbamate), termed Cu10, using a culture system of bovine aortic endothelial cells. Cu10 induced MT-1/2 protein expression and increased the expression of mRNAs for MT-1A, MT-1E, and MT-2, MT isoforms expressed in the cells. Cu10 activated not only the MTF-1-MRE, but also the Nrf2-ARE pathway. MTF-1 knockdown resulted in the repression of Cu10-induced MT-1 and -2 expression. Cu10-induced MT-1 expression was down-regulated by Nrf2 knockdown. However, MT-2 expression was not affected by Nrf2 knockdown. These results suggest that the expression of endothelial MT is up-regulated by the Nrf2-ARE pathway as well as by the MTF-1-MRE pathway. Moreover, MT-1 regulation mechanisms differ from that of MT-2. Specifically, the present data support the hypothesis that MT-1 participates in the biological defense system, while MT-2 mainly regulates intracellular zinc metabolism.

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.

Biglycan Intensifies ALK5-Smad2/3 Signaling by TGF-β1 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‐β1 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‐β1 or the transforming growth factor‐β1 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‐β1 signaling pathway that mediates the suppression of syndecan‐4 expression in vascular endothelial cells. J. Cell. Biochem. 118: 1087–1096, 2017.

Zinc diethyldithiocarbamate as an inducer of metallothionein in cultured vascular endothelial cells

Vascular endothelial cells are in direct contact with blood. Inorganic zinc is thought to be incapable of inducing metallothionein, which protects cells from heavy metal toxicity and oxidative stress, in vascular endothelial cells. Here, we aimed to further characterize the induction of metallothionein in vascular endothelial cells. Our results confirmed that inorganic zinc could not induce metallothionein in vascular endothelial cells. Moreover, ZnSO4 could not activate both the metal response element (MRE) transcription factor 1 (MTF-1)/MRE and Nrf2/antioxidant response element (ARE) pathways and was incapable of inducing metallothionein. In addition, bis(L-cysteinato)zincate(II), a zinc complex that activates the MTF-1/MRE pathway, increased MRE promoter activity but failed to induce metallothionein, suggesting that vascular endothelial metallothionein was not induced only by activation of the MTF-1/MRE pathway. Further analysis of a library of zinc complexes showed that zinc(II) bis(diethyldithiocarbamate) activated the MTF-1/MRE pathway but not the Nrf2/ARE pathway, increased MT-1A, MT-1E, and MT-2A mRNA levels, and induced metallothionein proteins. These data indicated that zinc complexes may be excellent tools to analyze metallothionein induction in vascular endothelial cells.

Transforming Growth Factor-β1 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‐β1 (TGF‐β1) 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‐β1 first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β 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‐β1. 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‐β1, 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‐β1. J. Cell. Biochem. 118: 2009–2017,2017.

Copper diethyldithiocarbamate as an inhibitor of tissue plasminogen activator synthesis in cultured human coronary endothelial cells

Recent developments have shown that organic-inorganic hybrid molecules have the potential to provide useful tools for analyzing biological systems. In the case of fibrinolysis, which is the phenomenon whereby fibrin is degraded by plasmin that has been converted from plasminogen via tissue plasminogen activator (t-PA) secreted from vascular endothelial cells, we hypothesized that there may be organic-inorganic hybrid molecules that could be used to analyze the mechanisms by which endothelial fibrinolysis is regulated. In our present study, we found that a copper complex - copper diethyldithiocarbamate (Cu10) - reduces t-PA activity in a conditioned medium of cultured human coronary endothelial cells by inhibiting the t-PA synthesis without changing the synthesis of plasminogen activator inhibitor type 1, which is a t-PA inhibitor. Copper sulfate, the Cu10 ligand, and zinc/iron complexes with the same Cu10 ligand, did not exhibit such biological activity. These results indicate that Cu10 has the potential to provide a useful tool for finding alternative pathways that downregulate endothelial t-PA synthesis.