Tomoya Fujie

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants associated with cardiovascular diseases; however, the mechanisms underlying PFAS-induced vascular endothelial injury remain incompletely understood. In this study, we systematically evaluated the effects of 15 PFAS on endothelial morphology and cell viability with different carbon-chain lengths and functional groups in cultured bovine aortic endothelial cells. Morphological observations and MTT assays revealed that perfluorononanoic acid, perfluorodecanoic acid (PFDA), and perfluorooctane sulfonate (PFOS) markedly reduced cell viability, with estimated concentrations producing a 50% reduction in viability of 60.9, 34.7, and 87.3 µM, respectively, whereas the other tested PFAS did not reduce viability by 50% at concentrations up to 100 µM in bovine aortic endothelial cells. Among the perfluoroalkyl carboxylic acids, the reduction in cell viability increased with increasing carbon-chain length. Among perfluoroalkyl sulfonates, PFOS caused the greatest reduction in cell viability, whereas perfluorodecanesulfonate did not induce clear endothelial damage. Comparative analyses across multiple cell types showed that PFDA reduced cell viability broadly across all cell types examined, whereas PFOS caused a greater reduction in cell viability in bovine-derived cell types examined than in human- or porcine-derived cell types examined. Since PFDA and PFOS were the most cytotoxic compounds among perfluoroalkyl carboxylic acids and perfluoroalkyl sulfonates, respectively, in bovine aortic endothelial cells, they were selected to compare cell death signaling. In both PFOS- and PFDA-treated cells, the selected apoptosis- and pyroptosis-related markers were not altered under the tested conditions. PFDA was associated with increases in phosphorylated RIP3 and phosphorylated MLKL, whereas PFOS increased MLKL expression without detectable RIP3 activation. Inhibition experiments further suggested that necroptosis-related signaling contributes, in part, to PFOS- and PFDA-induced endothelial injury in vascular endothelial cells. These findings suggest that PFAS-induced vascular endothelial injury depends on molecular structure and cell type, and may involve distinct necroptosis-related signaling patterns. However, it should be noted that the PFAS concentrations used in this study were higher than those typically detected in environmental and human exposure settings.

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.

Purinergic signaling plays an important role in vascular biology by vascular tone, inflammation, and remodeling through extracellular nucleotides that activate the P1 and P2 receptors. However, the expression patterns of these receptors in commonly used vascular cell models are not well characterized. In this study, we examined purinergic receptor expression in bovine aortic endothelial cells (BAECs), bovine aortic smooth muscle cells (BASMCs), and human vascular endothelial EA.hy926 cells. In BAECs, ADORA2A, ADORA2B, P2X4R, P2X7R, P2Y1R, P2Y2R, P2Y4R, P2Y6R, and P2Y11R were expressed, whereas the other purinergic receptors were not. BASMCs expressed ADORA2A, ADORA2B, P2X4R, P2X5R, P2Y1R, P2Y2R, P2Y6R, and P2Y11R. EA.hy926 cells expressed ADORA2A, ADORA2B, P2X4R, P2Y2R, P2Y6R, and P2Y11R. These results showed distinct expression profiles of purinergic receptors across different cell types. BAECs exhibited a purinergic receptor expression pattern similar to that of primary human vascular endothelial cells, suggesting that BAECs are a suitable model for studying purinergic signaling in vascular endothelial cells.

Methylmercury is the causative organometallic compound of Minamata disease. Pathological changes in the cerebrum of patients are localized along the deep sulci and fissures of the cerebral cortex such as the calcarine fissure. It has been suggested that the occurrence of brain edema is important for the cerebral damage caused by methylmercury. Previously, we found that methylmercury induces vascular endothelial growth factor-A in cultured human brain microvascular pericytes, which may increase permeability of the brain microvasculature. In the present study, our findings suggest that this induction is mediated via the epidermal growth factor receptor–p38 mitogen-activated protein kinase–cyclooxygenase-2–protein kinase A pathway in cultured human brain microvascular pericytes. These results partly support our hypothesis that methylmercury causes neurotoxicity by activation of intracellular signaling pathways in various cell types, including neurons, macrophages, vascular endothelial cells, and pericytes.

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.

Cadmium is a known contributing factor for cardiovascular diseases, such as atherosclerosis and hypertension. Although zinc protects against cadmium cytotoxicity in vascular endothelial cells, the detailed mechanisms, especially the differences in the roles of a metal transporter Zrt- and Irt-like protein 8 (ZIP8) and metallothionein (MT), remain unclear. ZIP8 is involved in the uptake of cadmium, whereas MT is induced by and sequesters it. Zinc protects bovine aortic endothelial cells from cadmium cytotoxicity in a culture system in a concentration-dependent manner. Zinc significantly decreased intracellular cadmium accumulation. Cadmium elevated the expression of ZIP8 mRNA, and zinc significantly suppressed this increase in a concentration-dependent manner. However, the expression of MT was not induced by zinc alone but by cadmium; the induction of MT by cadmium was suppressed by zinc at lower concentrations, but intensified by zinc at higher concentrations. However, even when MT induction was strongly suppressed by siRNA-mediated knockdown, cadmium cytotoxicity was reduced by zinc at both lower and higher concentrations. In the absence of zinc, cadmium cytotoxicity was increased by metal response element-binding transcription factor-1 (MTF-1) siRNA-mediated knockdown of MT-1/2. Consequently, it has been suggested that zinc protects the vascular endothelial cells through a concentration-dependent mechanism. Specifically, the decrease in ZIP8 expression is crucially important for the protective effect of zinc at low concentrations against cadmium cytotoxicity in vascular endothelial cells, whereas both the decrease in ZIP8 and the induction of MT contribute to the protection by zinc at high concentrations.

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Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.