Takato Hara

Proteoglycans are macromolecules consisting of a core protein and one or more glycosaminoglycan side chains. Proteoglycans synthesized by vascular endothelial cells modulate various functions such as anticoagulant activity and vascular permeability. We previously reported that some heavy metals interfere with proteoglycan expression, and that organic–inorganic hybrid molecules, such as metal complexes and organometallic compounds, serve as useful tools to analyze proteoglycan synthesis mechanisms. However, the effects of metal compounds lacking electrophilicity on proteoglycan synthesis remain unclear. Au₂₅(SG)₁₈, a nanoscale gold cluster consisting of a metal core protected by gold–glutathione complexes, exhibits extremely low intramolecular polarity. In this study, we investigated the effect of Au₂₅(SG)₁₈ on proteoglycan synthesis in vascular endothelial cells. Au₂₅(SG)₁₈ accumulated significantly in vascular endothelial cells at low cell density and suppressed the expression of perlecan, a major heparan sulfate proteoglycan in cells, by inactivating ADP-ribosylation factor 6 (Arf6). Additionally, Au₂₅(SG)₁₈ reduced the expression of biglycan, a small dermatan sulfate proteoglycan, in vascular endothelial cells at low cell density; however, the underlying mechanisms remain unclear. Overall, our findings suggest that organic–inorganic hybrid molecules regulate the activity of Arf6-mediated protein transport to the extracellular space and that perlecan is regulated through this mechanism, highlighting the importance of Arf6-mediated extracellular transport for maintaining vascular homeostasis.

Proteoglycans are macromolecules consisting of a core protein and one or more glycosaminoglycan side chains. Proteoglycans synthesized by vascular endothelial cells modulate various functions, such as anticoagulant activity and vascular permeability. We previously reported that some heavy metals interfere with proteoglycan expression and that organic–inorganic hybrid molecules, such as metal complexes and organometallic compounds, serve as useful tools to analyze proteoglycan synthesis mechanisms. However, the effects of metal compounds lacking electrophilicity on proteoglycan synthesis remain unclear. Au25(SG)18, a nanoscale gold cluster consisting of a metal core protected by gold–glutathione complexes, exhibits extremely low intramolecular polarity. In this study, we investigated the effect of Au25(SG)18 on proteoglycan synthesis in vascular endothelial cells. Au25(SG)18 accumulated significantly in vascular endothelial cells at low cell density and suppressed the expression of perlecan, a major heparan sulfate proteoglycan in cells, by inactivating ADP-ribosylation factor 6 (Arf6). Additionally, Au25(SG)18 reduced the expression of biglycan, a small dermatan sulfate proteoglycan, in vascular endothelial cells at low cell density; however, the underlying mechanisms remain unclear. Overall, our findings suggest that organic–inorganic hybrid molecules regulate the activity of Arf6-mediated protein transport to the extracellular space and that perlecan is regulated through this mechanism, highlighting the importance of Arf6-mediated extracellular transport for maintaining vascular homeostasis.

Perlecan, a major heparan sulfate proteoglycan in the vascular basement membrane, plays an essential role in maintaining endothelial barrier integrity, regulating fibroblast growth factor-2 signaling, and exerting anticoagulant activity. Although alterations in perlecan expression are implicated in the initiation and progression of atherosclerosis, the upstream regulatory mechanisms remain unclear. In this study, we investigated the effects of extracellular ATP on perlecan expression in vascular endothelial cells. ATP, but not ADP or adenosine, suppressed perlecan expression at both mRNA and protein levels in a time- and concentration-dependent manner. This suppression was recovered by knockdown of P2Y2 receptor (P2Y2R), but not by P2X4 receptor, P2X7 receptor, or P2Y1 receptor knockdown, indicating the selective involvement of P2Y2R. Mechanistically, ATP reduced Akt phosphorylation mediated by P2Y2R, and inhibition of Akt by inhibitors decreased perlecan expression, whereas inhibitors of phosphoinositide 3-kinase, mammalian target of rapamycin complex 1, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases did not exhibit this recovery effect. These results suggest that ATP downregulates perlecan synthesis via the P2Y2R-mediated inhibition of Akt signaling. Given that ATP is markedly elevated under pathological conditions, such as inflammation and platelet activation, suppression of perlecan synthesis is an important mechanism by which ATP promotes vascular disease progression.

Sensory disturbances and central nervous system symptoms are important in patients with Minamata disease. In the peripheral nervous system of these patients, motor nerves are not strongly injured, whereas sensory nerves are predominantly affected. In this study, we investigated the mechanisms underlying the sensory-predominant impairment of the peripheral nervous system caused by methylmercury. We found that the types of cell death in rat dorsal root ganglion (DRG) neurons caused by methylmercury included apoptosis, necrosis, and necroptosis. Methylmercury induced apoptosis in cultured rat DRG neurons but not in anterior horn neurons or Schwann cells. Additionally, methylmercury activated both caspase 8 and caspase 3 in DRG neurons. It increased the expression of tumor necrosis factor (TNF) receptor-1 and the phosphorylation of receptor-interacting protein kinase 3 (RIP3) and mixed-lineage kinase domain-like pseudokinase (MLKL). The expression of TNF-α was increased in macrophage-like RAW264.7 cells by methylmercury. The increase was suggested to be mediated by the NF-κB pathway. Moreover, methylmercury induced neurological symptoms, evaluated by a hindlimb extension response, were significantly less severe in TNF-α knockout mice. Based on these results and our previous studies, we propose the following hypothesis regarding the pathogenesis of sensory nerve-predominant damage by methylmercury: First, methylmercury accumulates within sensory nerve neurons and initiates cell death mechanisms, such as apoptosis, on a small scale. Second, cell death triggers the infiltration of macrophages into the sensory fibers. Third, the macrophages are stimulated by methylmercury and secrete TNF-α through the NF-κB pathway. Fourth, TNF-α induces cell death mechanisms, including necrosis, apoptosis through the caspase 8/3 pathway, and necroptosis through the TNFR1-RIP1-RIP3-MLKL pathway, activated by methylmercury in sensory neurons. Consequently, methylmercury exhibits potent cytotoxicity specific to the DRG/sensory nerve cells in the peripheral nervous system. This chain of events caused by methylmercury may contribute to sensory disturbances in patients with Minamata disease.

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.

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.

Vascular endothelial cell growth is essential for the repair of intimal injury. Perlecan, a large heparan sulfate proteoglycan, intensifies fibroblast growth factor-2 (FGF-2) signaling as a co-receptor for FGF-2 and its receptor, and promotes the proliferation of vascular endothelial cells. Previously, we reported that 2 µM of lead, a toxic heavy metal, downregulated perlecan core protein expression and then suppressed the growth of vascular endothelial cells. However, since the mechanisms involved in the repression of perlecan by lead remains unclear, we analyzed its detailed signaling pathway using cultured bovine aortic endothelial cells. Our findings indicate that 2 µM of lead inhibited protein tyrosine phosphatase (PTP) activity and induced cyclooxygenase-2 (COX-2) via phosphorylation of the epidermal growth factor receptor (EGFR) and its downstream extracellular signal-regulated kinases (ERK1/2). In addition, among the prostanoids regulated by COX-2, prostaglandin I2 (PGI2) specifically contributes to the downregulation of perlecan expression by lead. This study revealed an intracellular pathway—the EGFR-ERK1/2-COX-2-PGI2 pathway activated by inhibition of PTP by lead—as a pathway that downregulates endothelial perlecan synthesis. The pathway is suggested to serve as a mechanism for the repression of perlecan expression, which leads to a delay in cell proliferation by lead.

Transforming growth factor-β₁ (TGF-β₁) 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-β₁. Bovine aortic endothelial cells in a culture system were treated with TGF-β₁ to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-β₁, induction of RSS-producing enzymes by TGF-β₁, and intracellular signal pathways that mediate this induction. The results suggest that TGF-β₁ 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 β-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-β₁ regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-β₁, may modulate the regulation activity in 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.