Mechanism of cytotoxicity induced by the cigarette smoke extract (CSE) of heated tobacco products in vascular smooth muscle cells: A comparative study of the cytotoxic effects of CSE and the ferroptosis inducer, erastin.

Heated tobacco products (HTPs) are marketed worldwide as less harmful alternatives to combustible cigarettes; however, their cytotoxic mechanisms in vascular smooth muscle cells are poorly understood. Ferroptosis is defined as iron-dependent cell death caused by the accumulation of lipid peroxidation products. In this study, the cytotoxic effects of nicotine- and tar-free cigarette smoke extracts (CSE) derived from three types of HTPs and the ferroptosis inducer, erastin, on vascular smooth muscle A7r5 cells were compared. Cigarette smoke from all HTPs was generated according to the following puffing regime: 55 mL, puff volume; 30 s, puff interval; 2 s, puff duration; bell-shaped, puff profile; and no blocking of the ventilation holes. Erastin and CSE decreased mitochondrial metabolic activity and increased lactate dehydrogenase leakage. The cytotoxic effects of erastin were almost completely inhibited by the radical-trapping antioxidant, UAMC-3203; iron chelator, deferoxamine mesylate (DFO); 12/15-lipoxygenase (12/15-LOX) inhibitor, baicalein; and selective 15-LOX inhibitor, ML351. In contrast, CSE-induced cell damage was partially attenuated by UAMC-3203, baicalein, and ML351 but not by DFO. These results suggest that erastin induces ferroptosis via 15-LOX-mediated iron-dependent lipid peroxidation, whereas CSE causes iron-independent cell damage via 15-LOX-mediated lipid peroxidation-dependent and -independent mechanisms.

Comparison of cytotoxicity of cigarette smoke extract derived from heat-not-burn and combustion cigarettes in human vascular endothelial cells.

The present study compared the properties of mainstream smoke generated from heat-not-burn (HNB) cigarettes and a combustion cigarette (hi-lite™ brand). Three types of cigarette heating devices were used to generate cigarette smoke at different heating temperatures [Ploom S™ (200 °C), glo™ (240 °C), and IQOS™ (300-350 °C)]. Mainstream smoke was generated using the following puffing regimen: volume, 55 mL; duration, 3 s; and interval, 30 s. The rank order of particulate phase (nicotine and tar) amounts trapped on a Cambridge filter was Ploom S < glo < IQOS < hi-lite. Heated cigarette-derived smoke extract (hCSE) from the devices except for Ploom S, and burned CSE (bCSE) decreased mitochondrial metabolic activity (glo < IQOS < hi-lite) in human vascular endothelial cells. Furthermore, the cytotoxicity was reduced by removing the particulate phase from the mainstream smoke. Endothelial nitric oxide synthase activity was reduced by nicotine- and tar-free CSE of IQOS and hi-lite (IQOS < hi-lite), but not Ploom S and glo. These inhibitory effects were diminished by removing the carbonyl compounds from the mainstream smoke. These results indicated that the cytotoxicity of hCSE was lower than that of bCSE in vascular endothelial cells.

Fibrinolysis-resistant carbonylated fibrin detected in thrombi attached to the vascular wall of abdominal aortic aneurysms.

In this study, we investigated how carbonylation of fibrinogen by acrolein modified its indispensable function to enhance fibrinolysis after being converted to fibrin and contributed to generating a fibrinolysis-resistant fibrin clot. Acrolein-treated fibrinogen was subjected to tissue plasminogen activator-induced fibrinolysis assay and the effect of lysine residue carbonylation in fibrinogen on fibrinolysis was analyzed. The acrolein-treated fibrinogen-derived fibrin clot appeared more resistant to fibrinolysis and the N-acetyl 3-formyl-3,4-dehydropiperidino (FDP)-Lysine levels in the lysed solution were positively correlated with the duration of clot lysis. The lysine analog 6-amino hexanoic acid (6AHA), which mimics the C-terminal lysine of fibrin, was carbonylated and its enhancing effect on Glu1-plasminogen activation was evaluated. After incubation with acrolein, 6AHA was converted to N-acetyl FDP-6AHA, losing its ability to enhance Glu1-plasminogen activation. These results suggest that fibrinogen carbonylation by acrolein to generate N-acetyl FDP-Lysine resulted in the generation of fibrinolysis-resistant fibrin by attenuating the C-terminal lysine-dependent activation of the Glu1-plasminogen. In abdominal aortic aneurysms, fibrin(ogen) containing the acrolein adduct N-acetyl FDP-Lysine was detected in the vascular wall-attached thrombi. These results suggest that this mechanism is likely involved in the modification of fibrinolysis-resistant thrombi and to their persistence for a long period.

Cigarette Smoke Extract and Its Cytotoxic Factor Acrolein Inhibit Nitric Oxide Production in Human Vascular Endothelial Cells.

Acrolein (ACR), a highly reactive α,ß-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at serine (Ser)1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS.

Extracellular Ca2+ promotes nitric oxide production via Ca2+-sensing receptor-Gq/11 protein-endothelial nitric oxide synthase signaling in human vascular endothelial cells.

This study examined the possible involvement of Ca2+-sensing receptor (CaSR) in nitric oxide (NO) production in human vascular endothelial cells. Extracellular Ca2+ elevated the intracellular Ca2+ concentration, the endothelial NO synthase (eNOS) phosphorylation level, and NO release from the cells. These responses were inhibited by a CaSR antagonist and a Gq/11 protein inhibitor. Application of an endothelial cell suspension induced vasorelaxation in isolated rat thoracic aorta precontracted by phenylephrine. Adding an NO scavenger to the organ bath abolished this vasorelaxation response. These results suggest that extracellular Ca2+ promotes NO generation via CaSR- and Gq/11 protein-mediated eNOS activation.

Ca2+ signal is involved in endothelin-1-induced internalization of endothelin type A receptor expressed in Chinese hamster ovary cells.

Endothelin type A receptor (ETAR) is internalized upon agonist stimulation; however, the mechanism thereof remains controversial. In this study, we characterized the endothelin-1 (ET-1)-induced internalization of ETAR expressed in Chinese hamster ovary cells. ET-1 elicited ETAR internalization and increase in intracellular Ca2+ concentration. ET-1-induced ETAR internalization was completely inhibited by a reduction in intracellular and extracellular Ca2+ levels and partially suppressed by inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2), both of which are downstream molecules in ETAR signaling. These results suggest that Ca2+ mobilization, PKC, and ERK1/2 are involved in ET-1-induced ETAR internalization.

Annexin A2 is involved in activation of extracellular signal-regulated kinase upon endothelin-1 stimulation.

The overexpression of endothelin (ET)-1 or ET receptors (ETRs) is related to initiation and progression of tumor. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase C through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain uncertain. Here, we found that ETRs interacted with annexin A2, which is overexpressed in various cancers. Annexin A2 bound to ET type A receptor and ET type B receptor. Upon ET-1 stimulation, serine phosphorylation of annexin A2 increased, while there is no change in tyrosine phosphorylation of annexin A2. On the other hand, annexin A2 silencing suppressed activation of ERK upon ET-1 stimulation. These results suggest that interaction of ETRs and annexin A2 may play important roles in activation of extracellular signal-regulated kinase upon ET-1 stimulation.

Endothelin type B receptor interacts with the 78-kDa glucose-regulated protein.

Endothelin (ET)-1 is involved in the vascular system, cell proliferation and apoptosis. ET receptors consist of ET type A receptor (ETA R) and ET type B receptor (ETB R). ETA R and ETB R generally exhibit opposite responses, although many exceptions exist. In the present study, we attempted to identify ETA R- or ETB R-specific binding proteins to understand the differences in ETA R- and ETB R-mediated responses after ET-1 stimulation. The 78-kDa glucose-regulated protein (GRP78) showed a stronger binding affinity towards ETB R than towards ETA R. Moreover, GRP78 overexpression promoted ETB R-mediated ERK activation and GRP78 silencing suppressed ETB R-mediated ERK activation. Furthermore, ETB R can localize GRP78 to the cell periphery. These results suggest that the interaction of ETB R with GRP78 affects ERK activation and GRP78 localization.

Glutathione and cysteines suppress cytotoxicity of gas phase of cigarette smoke by direct reacting with unsaturated carbonyl compounds in the gas phase.

Unsaturated carbonyl compounds, such as acrolein (ACR) and methyl vinyl ketone (MVK), are environmental pollutants, and are contained in smoke, automobile exhaust, and heated oil. We have previously reported that major cytotoxic factors in the gas phase of cigarette smoke are ACR and MVK. ACR and MVK induce cell damage by reactive oxygen species generation via protein kinase C and NADPH oxidases, and antioxidants, such as glutathione (GSH) and N-acetylcysteine (NAC), can effectively suppress their cytotoxic activities. In this study, we attempted to elucidate the molecular mechanism(s) for suppression of ACR- and MVK-induced cytotoxic activities by these antioxidants. GSH, NAC, L- and D-cysteines completely suppressed cell damage induced by gas phase extract of cigarette smoke. The results of HPLC and mass spectrometry showed that GSH and NAC directly reacted with ACR and MVK. Cysteines and cysteine derivatives suppressed ACR-induced GAPDH carbonylation, a representative protein for carbonylation. The current results suggest that GSH, NAC, and cysteines directly reacted with ACR and MVK, and suppressed these unsaturated carbonyl compounds-induced cell damage by inhibition of protein carbonylation.

[Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells].

Insulin resistance is a condition where the sensitivity to insulin of the tissues expressing insulin receptor (InsR) is decreased due to a functional disturbance of InsR-mediated intracellular signaling. Insulin promotes the entry of glucose into the tissues and skeletal muscle is the most important tissue responsible for the insulin's action of decreasing blood glucose levels. Endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory peptide, induces insulin resistance through a direct action on skeletal muscle. However, the signaling pathways of ET-1-induced insulin resistance in skeletal muscle remain unclear. Here we show molecular mechanism underlying the inhibitory effect of ET-1 on insulin-stimulated Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during L6 myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously contract. In myotubes, insulin promoted Akt phosphorylation at Thr308 and Ser473, and [3H]-labelled 2-deoxy-D-glucose ([3H]2-DG) uptake. The insulin-facilitated Akt phosphorylation and [3H]2-DG uptake were inhibited by ET-1. The inhibitory effect of ET-1 was counteracted by blockade of ET type A receptor (ETAR), inhibition of Gq/11 protein, and siRNA knockdown of G protein-coupled receptor kinase 2 (GRK2). The exogenously overexpressed GRK2 directly bound to endogenous Akt and their association was facilitated by ET-1. In summary, activation of ETAR with ET-1 inhibits insulin-induced Akt phosphorylation and [3H]2-DG uptake in a Gq/11 protein- and GRK2-dependent manner in skeletal muscle. These findings indicate that ETAR and GRK2 are potential targets for insulin resistance.

Disruption of the structural and functional features of surfactant protein A by acrolein in cigarette smoke.

The extent to which defective innate immune responses contribute to chronic obstructive pulmonary disease (COPD) is not fully understood. Pulmonary surfactant protein A (SP-A) plays an important role in regulating innate immunity in the lungs. In this study, we hypothesised that cigarette smoke (CS) and its component acrolein might influence pulmonary innate immunity by affecting the function of SP-A. Indeed, acrolein-modified SP-A was detected in the lungs of mice exposed to CS for 1 week. To further confirm this finding, recombinant human SP-A (hSP-A) was incubated with CS extract (CSE) or acrolein and then analysed by western blotting and nanoscale liquid chromatography-matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry. These analyses revealed that CSE and acrolein induced hSP-A oligomerisation and that acrolein induced the modification of six residues in hSP-A: His39, His116, Cys155, Lys180, Lys221, and Cys224. These modifications had significant effects on the innate immune functions of hSP-A. CSE- or acrolein-induced modification of hSP-A significantly decreased hSP-A's ability to inhibit bacterial growth and to enhance macrophage phagocytosis. These findings suggest that CS-induced structural and functional defects in SP-A contribute to the dysfunctional innate immune responses observed in the lung during cigarette smoking.

Intracellular Ca2+ is an essential factor for cell damage induced by unsaturated carbonyl compounds.

The unsaturated carbonyl compounds are known as the environmental pollutants. Acrolein (ACR) and methyl vinyl ketone (MVK) are representative unsaturated carbonyl compounds. ACR is contained in smoke, automobile exhaust, industrial waste, and several foods. MVK is widely used as the industrial chemical. Although ACR and MVK are highly toxic, the molecular mechanism for their cytotoxicity has been unclear. We have previously reported that ACR and MVK are major cytotoxic compounds in the gas phase of cigarette smoke, and protein kinase C (PKC) inhibitor and NADPH oxidases inhibitor partially rescued cells from ACR- or MVK-induced cell death (Noya et al., Toxicology, 314, 1-10, 2013). PKC translocation, which is hallmark for PKC activation, and cell damage were induced by treatment of cultured cells with ACR or MVK. Intracellular Ca2+ chelator completely suppressed ACR- or MVK-induced PKC translocation to the cell membrane and cell damage, while extracellular Ca2+ chelator had no effects on ACR- and MVK-induced cytotoxicity. These results suggest that intracellular Ca2+ is an essential factor for cell damage caused by both PKC-dependent and PKC-independent pathways, and mobilization of Ca2+ from intracellular Ca2+ stores is induced by ACR or MVK.

Cigarette Smoke Extract Inhibits Platelet Aggregation by Suppressing Cyclooxygenase Activity.

The results of studies that were performed to determine whether cigarette smoking affects platelet function have been controversial, and the effects of nicotine- and tar-free cigarette smoke extract (CSE) on platelet function remain to be determined. The aim of this study was to determine the effect of CSE on platelet aggregation and to clarify the mechanism by which CSE affects platelet function. CSE inhibited murine platelet aggregation induced by 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619), a thromboxane (TX) A 2 receptor agonist, and that induced by collagen with respective IC 50 values of 1.05 ± 0.14% and 1.34 ± 0.19%. A similar inhibitory action of CSE was also observed in human platelets. CSE inhibited arachidonic acid-induced TXA 2 production in murine platelets with an IC 50 value of 7.32 ± 2.00%. Accordingly, the inhibitory effect of CSE on collagen-induced aggregation was significantly blunted in platelets lacking the TXA 2 receptor compared with the inhibitory effect in control platelets. In contrast, the antiplatelet effects of CSE in platelets lacking each inhibitory prostanoid receptor, prostaglandin (PG) I 2 receptor and PGE 2 receptor subtypes EP 2 and EP 4 , were not significantly different from the effects in respective control platelets. Among the enzymes responsible for TXA 2 production in platelets, the activity of cyclooxygenase (COX)-1 was inhibited by CSE with an IC 50 value of 1.07 ± 0.15% in an uncompetitive manner. In contrast, the activity of TX synthase was enhanced by CSE. The results indicate that CSE inhibits COX-1 activity and thereby decreases TXA 2 production in platelets, leading to inhibition of platelet aggregation.

A Standardized Method for the Preparation of a Gas Phase Extract of Cigarette Smoke.

The gas phase of cigarette smoke is important from the viewpoint of human health, because it can pass through alveolar epithelium and enter the circulation. There is no standard method for the preparation of a gas phase extract of cigarette smoke (CSE), although CSE is widely used for research instead of whole cigarette smoke. We have established a standard method for the preparation of CSE. One cigarette per trial is continuously combusted under a reduced pressure generated by an aspiration pump with a velocity of 1.050 L/min: the main stream of the smoke is passed through a Cambridge filter to remove tar, and subsequently, bubbled through a glass ball filter (pore size, 20-30 µm) into 15 mL of phosphate-buffered saline (PBS). To express the concentration of CSE, a virtual tar concentration is introduced, which is calculated assuming that tar trapped on the Cambridge filter is dissolved in the PBS. CSEs prepared from smaller numbers of cigarettes (original virtual tar concentration≤15 mg/mL) show similar concentration-response curves for cytotoxicity versus virtual tar concentrations. CSEs prepared from various brands of cigarettes and by different smoking regimes (continuous and puff smoking) show similar cytotoxic potency if the virtual tar concentrations are the same. In conclusion, using the standardized method for CSE preparation in combination with the virtual tar concentration, it becomes possible to simply and rapidly prepare standard CSEs with defined concentrations from any brand of cigarettes, which are toxicologically equivalent to CSE prepared by puff smoking.

Carbonyl Compounds in the Gas Phase of Cigarette Mainstream Smoke and Their Pharmacological Properties.

Cigarette mainstream smoke is composed of gas and tar phases and contains >4000 chemical constituents, including nicotine and tar. The substances in the gas phase but not in the tar phase can pass through the airway epithelial barrier, enter the systemic circulation via the pulmonary circulation, and increase systemic oxidative damage, leading to the development of cigarette smoking-related diseases such as atherosclerosis. Recently, we identified some stable carbonyl compounds, including acrolein (ACR) and methyl vinyl ketone (MVK), as major cytotoxic factors in nicotine- and tar-free cigarette smoke extract (CSE) of the gas phase. CSE, ACR, and MVK induce protein kinase C (PKC)-dependent activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) via NOX, causing plasma membrane damage and cell apoptosis. CSE, ACR, and MVK also trigger carbonylation of PKC, which is an irreversible oxidative modification. Cell damage and PKC carbonylation in response to treatment with CSE, ACR, or MVK are abolished by thiol-containing antioxidants such as N-acetyl-L-cysteine and reduced glutathione. Thus pharmacological modulation of PKC and NOX activities and the trapping of ROS are potential strategies for the prevention of diseases related to cigarette smoking.

Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.

Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.

Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells.

BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET-1 of insulin signalling. EXPERIMENTAL APPROACH: We used the rat L6 skeletal muscle cells fully differentiated into myotubes. Changes in the phosphorylation of Akt was assessed by Western blotting. Effects of ET-1 on insulin-stimulated glucose uptake was assessed with [(3) H]-2-deoxy-d-glucose ([(3) H]2-DG). The C-terminus region of GPCR kinase 2 (GRK2-ct), a dominant negative GRK2, was overexpressed in L6 cells using adenovirus-mediated gene transfer. GRK2 expression was suppressed by transfection of the corresponding short-interfering RNA (siRNA). KEY RESULTS: In L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr(308) and Ser(473) , which was suppressed by ET-1. The inhibitory effects of ET-1 were prevented by treatment with a selective ETA receptor antagonist and a Gq protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [(3) H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [(3) H]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [(3) H]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2. CONCLUSIONS AND IMPLICATIONS: Activation of ETA receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr(308) and Ser(473) and [(3) H]2-DG uptake in a GRK2-dependent manner in skeletal muscle cells. These findings suggest that ETA receptors and GRK2 are potential targets for overcoming insulin resistance.

Decreased proteasomal function accelerates cigarette smoke-induced pulmonary emphysema in mice.

Chronic obstructive pulmonary disease (COPD) is a disease common in elderly people, characterized by progressive destruction of lung parenchyma and chronic inflammation of the airways. The pathogenesis of COPD remains unclear, but recent studies suggest that oxidative stress-induced apoptosis in alveolar cells contributes to emphysematous lung destruction. The proteasome is a multicatalytic enzyme complex that plays a critical role in proteostasis by rapidly destroying misfolded and modified proteins generated by oxidative and other stresses. Proteasome activity decreases with aging in many organs including lungs, and an age-related decline in proteasomal function has been implicated in various age-related pathologies. However, the role of the proteasome system in the pathogenesis of COPD has not been investigated. Recently, we have established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity, showing age-related phenotypes. Using this model, we demonstrate here that decreased proteasomal function accelerates cigarette smoke (CS)-induced pulmonary emphysema. CS-exposed Tg mice showed remarkable airspace enlargement and increased foci of inflammation compared with wild-type controls. Importantly, apoptotic cells were found in the alveolar walls of the affected lungs. Impaired proteasomal activity also enhanced apoptosis in cigarette smoke extract (CSE)-exposed fibroblastic cells derived from mice and humans in vitro. Notably, aggresome formation and prominent nuclear translocation of apoptosis-inducing factor were observed in CSE-exposed fibroblastic cells isolated from Tg mice. Collective evidence suggests that CS exposure and impaired proteasomal activity coordinately enhance apoptotic cell death in the alveolar walls that may be involved in the development and progression of emphysema in susceptible individuals such as the elderly.