Impact of inhalation exposure to cigarette smoke on the pathogenesis of pulmonary hypertension primed by monocrotaline in rats.

Extensive, long-term exposure to cigarette smoke (CS) was recently suggested to be a risk factor for pulmonary hypertension, although further validation is required. The vascular effects of CS share similarities with the etiology of pulmonary hypertension, including vascular inflammation and remodeling. Thus, we examined the influence of CS exposure on the pathogenesis of monocrotaline (MCT)-induced pulmonary hypertension, hypothesizing that smoking might accelerate the development of primed pulmonary hypertension. CS was generated from 3R4F reference cigarettes, and rats were exposed to CS by inhalation at total particulate matter concentrations of 100-300 µg/L for 4 h/day, 7 days/week for 4 weeks. Following 1 week of initial exposure, rats received 60 mg/kg MCT and were sacrificed and analyzed after an additional 3 weeks of exposure. MCT induced hypertrophy in pulmonary arterioles and increased the Fulton index, a measure of right ventricular hypertrophy. Additional CS exposure exacerbated arteriolar hypertrophy but did not further elevate the Fulton index. No significant alterations were observed in levels of endothelin-1 and vascular endothelial growth factor, or in hematological and serum biochemical parameters. Short-term inhalation exposure to CS exacerbated arteriolar hypertrophy in the lung, although this effect did not directly aggravate the overworked heart under the current experimental conditions.

NADPH Oxidase 1 Mediates Acute Blood Pressure Response to Angiotensin II by Contributing to Calcium Influx in Vascular Smooth Muscle Cells.

BACKGROUND: Reactive oxygen species (ROS) and calcium ions (Ca2+) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca2+ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca2+ signaling. METHODS: Vascular smooth muscle cells were primary cultured from rat aorta. Ca2+ and ROS were monitored mainly using fura-2 and HyPer family probes' respectively. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques. RESULTS: Ang II-induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca2+, and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca2+ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-Gßγ-PI3Kγ (phosphoinositide 3-kinase γ) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-Gαq/11-PLC (phospholipase C)-PKCß (protein kinase C ß) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice. CONCLUSIONS: NOX1 mediates Ca2+ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.

A mixture of chloromethylisothiazolinone and methylisothiazolinone impairs rat vascular smooth muscle by depleting thiols and thereby elevating cytosolic Zn2+ and generating reactive oxygen species.

Chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT) are biocidal preservatives and the active ingredients in Kathon CG, which contains ca. 1.5% mixture of CMIT and MIT at a ratio of 3:1 (CMIT/MIT). CMIT/MIT was misused as humidifier disinfectant products, which caused serious health problems in Korea. Here, the vascular effects of CMIT/MIT were investigated to evaluate claims of putative cardiovascular toxicity observed in humidifier disinfectant users. CMIT/MIT did not affect the basal tension of the rat thoracic aorta up to 2.5 µg/mL in myograph experiments. Instead, pretreatment with CMIT/MIT impaired phenylephrine- or 5-hydroxytryptamine-induced vasoconstriction in a range of 0.5-2.5 µg/mL, which was largely irreversible and not recovered by washing out the CMIT/MIT. Similarly, the application of CMIT/MIT to pre-contracted aorta caused a gradual loss of tension. In primary cultured vascular smooth muscle cells (VSMCs), CMIT/MIT caused thiol depletion, which in turn led to cytosolic Zn2+ elevation and reactive oxygen species (ROS) formation. CMIT/MIT-induced shrinkage, detachment, and lysis of VSMCs depending on the concentration and the treatment time. All events induced by CMIT/MIT were prevented by a thiol donor N-acetylcysteine (NAC). Cytolysis could be inhibited by a Zn2+ chelator TPEN and a superoxide scavenger TEMPOL, whereas they did not affect shrinkage and detachment. In accordance with these results, CMIT/MIT-exposed aortas exhibited dissociation and collapse of tissue in histology analysis. Taken together, CMIT/MIT causes functional impairment and tissue damage to blood vessels by depleting thiol and thereby elevating cytosolic Zn2+ and generating ROS. Therefore, exposure to CMIT/MIT in consumer products may be a risk factor for cardiovascular disorders.

Cytosolic zinc mediates the cytotoxicity of thiol-reactive electrophiles in rat vascular smooth muscle cells.

The aberrant increase or dysregulation of cytosolic Zn2+ concentration ([Zn2+]cyt) has been associated with cellular dysfunction and cytotoxicity. In this study, we postulated that Zn2+ mediates the cytotoxicity of thiol-reactive electrophiles. This notion was grounded on earlier research, which revealed that thiol-reactive electrophiles may disrupt Zn2+-binding motifs, consequently causing Zn2+ to be released from Zn2+-binding proteins, and leading to a surge in [Zn2+]cyt. The thiol-reactive electrophiles N-ethylmaleimide (NEM) and diamide were observed to induce an increase in [Zn2+]cyt, possibly through the impairment of Zn2+-binding motifs, and subsequent stimulation of reactive oxygen species (ROS) formation, resulting in cytotoxicity in primary cultured rat vascular smooth muscle cells. These processes were negated by the thiol donor N-acetyl-L-cysteine and the Zn2+ chelator TPEN. Similar outcomes were detected with co-treatment involving Zn2+ and Zn2+ ionophores such as pyrithione or disulfiram. Moreover, TPEN was found to inhibit cytotoxicity triggered by short-term exposure to various thiol-reactive electrophiles including hydrogen peroxide, acrylamide, acrylonitrile, diethyl maleate, iodoacetic acid, and iodoacetamide. In conclusion, our findings suggest that cytosolic Zn2+ acts as a universal mediator in the cytotoxic effects produced by thiol-reactive electrophiles.

Erratum: Publisher Correction: Short-term inhalation exposure to cigarette smoke induces oxidative stress and inflammation in lungs without systemic oxidative stress in mice.

[This corrects the article DOI: 10.1007/s43188-023-00223-y.].

Short-term inhalation exposure to cigarette smoke induces oxidative stress and inflammation in lungs without systemic oxidative stress in mice.

Smoking is a well-established risk factor for various pathologies, including pulmonary diseases, cardiovascular disorders, and cancers. The toxic effects of cigarette smoke (CS) are mediated through multiple pathways and diverse mechanisms. A key pathogenic factor is oxidative stress, primarily induced by excessive formation of reactive oxygen species. However, it remains unclear whether smoking directly induces systemic oxidative stress or if such stress is a secondary consequence. This study aimed to determine whether short-term inhalation exposure to CS induces oxidative stress in extrapulmonary organs in addition to the lung in a murine model. In the experiment, 3R4F reference cigarettes were used to generate CS, and 8-week-old male BALB/c mice were exposed to CS at a total particulate matter concentration of either 0 or 800 µg/L for four consecutive days. CS exposure led to an increase in neutrophils, eosinophils, and total cell counts in bronchoalveolar lavage fluid. It also elevated levels of lactate dehydrogenase and malondialdehyde (MDA), markers indicative of tissue damage and oxidative stress, respectively. Conversely, no significant changes were observed in systemic oxidative stress markers such as total oxidant scavenging capacity, MDA, glutathione (GSH), and the GSH/GSSG ratio in blood samples. In line with these findings, CS exposure elevated NADPH oxidase (NOX)-dependent superoxide generation in the lung but not in other organs like the liver, kidney, heart, aorta, and brain. Collectively, our results indicate that short-term exposure to CS induces inflammation and oxidative stress in the lung without significantly affecting oxidative stress in extrapulmonary organs under the current experimental conditions. NOX may play a role in these pulmonary-specific events.

Cigarette Smoke-Induced Reactive Oxygen Species Formation: A Concise Review.

Smoking is recognized as a significant risk factor for numerous disorders, including cardiovascular diseases, respiratory conditions, and various forms of cancer. While the exact pathogenic mechanisms continue to be explored, the induction of oxidative stress via the production of excess reactive oxygen species (ROS) is widely accepted as a primary molecular event that predisposes individuals to these smoking-related ailments. This review focused on how cigarette smoke (CS) promotes ROS formation rather than the pathophysiological repercussions of ROS and oxidative stress. A comprehensive analysis of existing studies revealed the following key ways through which CS imposes ROS burden on biological systems: (1) ROS, as well as radicals, are intrinsically present in CS, (2) CS constituents generate ROS through chemical reactions with biomolecules, (3) CS stimulates cellular ROS sources to enhance production, and (4) CS disrupts the antioxidant system, aggravating the ROS generation and its functions. While the evidence supporting these mechanisms is chiefly based on in vitro and animal studies, the direct clinical relevance remains to be fully elucidated. Nevertheless, this understanding is fundamental for deciphering molecular events leading to oxidative stress and for developing intervention strategies to counter CS-induced oxidative stress.

GPR40 agonist inhibits NLRP3 inflammasome activation via modulation of nuclear factor-κB and sarco/endoplasmic reticulum Ca2+-ATPase.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a multi-protein intracellular complex that activates proinflammatory cytokines, including interleukin (IL)-1ß and IL-18. Inflammasome activation is related to metabolic inflammation, such as the progression of non-alcoholic steatohepatitis. Fasiglifam (TAK875), a selective G-protein coupled receptor 40 (GPR40) agonist with high affinity, significantly improves glucose-dependent insulin secretion and weight gain without hypoglycemia. Interestingly, we found that two GPR40 agonists, TAK875 and AMG1638, suppressed activation of the NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs). TAK875 inhibited inflammasome activation by blocking formation of apoptosis-associated speck-like protein containing a CARD (ASC), an inflammasome component. TAK875 also suppressed NLRP3 inflammasome-induced pyroptosis of BMDMs. Moreover, nuclear factor-kappa B (NF-κB)-dependent priming of the NLRP3 inflammasome was partially inhibited by TAK875 and AMG1638. The intracellular Ca2+ increase caused by ATP, nigericin (pore-forming toxin), or endoplasmic reticulum stress activates the NLRP3 inflammasome. Pre-exposure of BMDMs to TAK875 suppressed the ATP-induced intracellular Ca2+ increase, which was reversed by thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor. Oral administration of mice with TAK875 suppressed the increase in serum IL-1ß in mice treated with lipopolysaccharide/D-galactosamine in vivo. These findings indicate that the free fatty acid-sensing GPR40 plays a key role in the NLRP3 inflammasome pathway.

Cigarette Smoke Extract Produces Superoxide in Aqueous Media by Reacting with Bicarbonate.

The toxicity of cigarette smoke (CS) is largely attributed to its ability to generate reactive oxygen species (ROS). Reportedly, CS generates superoxide in cell culture systems by stimulating the cells to produce superoxide and through direct chemical reactions with components of the culture media. In this study, we investigated CS-induced superoxide formation in biocompatible aqueous media and its characteristics. Cigarette smoke extract (CSE) and total particulate matter (TPM) were prepared from the mainstream smoke of 3R4F reference cigarettes. CSE and TPM generated superoxide in Hank's balanced salt solution (HBSS), Dulbecco's modified Eagle media (DMEM), and blood plasma, but not in distilled water and phosphate-buffered saline. Each constituent of HBSS in solution was tested, and bicarbonate was found to be responsible for the superoxide generation. More than half of the superoxide formation was abolished by pretreating CSE or TPM with peroxidase, indicating that the substrates of peroxidase, presumably peroxides and peroxy acids, mainly contributed to the superoxide production. In conclusion, the presence of bicarbonate in experimental conditions should be considered carefully in studies of the biological activity of CS. Furthermore, the local amount of bicarbonate in exposed tissues may be a determinant of tissue sensitivity to oxidative damage by CS.

Inhalation exposure to cigarette smoke induces endothelial nitric oxide synthase uncoupling and enhances vascular collagen deposition in streptozotocin-induced diabetic rats.

Smoking is an acknowledged risk factor for vascular disorders, and vascular complication is a main outcome of diabetes. Hence, we investigated the impact of cigarette smoke on blood vessels in diabetes, postulating that smoking might aggravate diabetic vascular impairment. Sprague-Dawley rats were divided into four groups: control, cigarette smoke-exposed, diabetic, and cigarette smoke-exposed diabetic groups. Streptozotocin-induced diabetic rats were exposed to cigarette smoke by inhalation at total particulate matter concentration of 200 µg/L for 4 h/day, 5 day/week for a total of 4 weeks. Diabetes caused structural change of aorta, but additional cigarette smoke exposure did not induce further alteration. Collagen, a marker for fibrosis, was increased in media of diabetic aorta, and this increase was augmented by cigarette smoke. Cigarette smoke induced endothelial nitric oxide synthase (eNOS) uncoupling in the diabetic group. Malondialdehyde was increased and glutathione was decreased in blood from diabetes, but these effects were not exaggerated by cigarette smoke. Cigarette smoke caused NADPH oxidase (NOX) 2 expression in diabetic aorta and enhanced diabetes-induced NOX4 expression in aorta. Taken together, cigarette smoke exposure can aggravate vascular fibrosis and induce eNOS uncoupling in diabetes under experimental condition, suggesting that smoking might exacerbate diabetic vascular impairments.

Application of Fisetin to the Quantitation of Serum Albumin.

Fisetin (3,3',4',7-tetrahydroxyflavone) is a widely distributed natural flavonol. It interacts with albumin, and thereby generates a fluorescence signal quantitatively. Based on such optical characteristics, we postulated that fisetin was applicable to the quantitation of albumin as an indicator. To establish the fisetin-based albumin assay, we examined the optical properties of fisetin and fisetin-albumin complex. The assay conditions were fine-tuned to fit for the actual concentration of serum albumin and to generate an optimal signal with a high signal-to-background ratio. The reaction between fisetin and albumin was linear in a wide range of concentrations. Non-protein serum components did not interfere with the reaction. The reactivity of fisetin was apparently specific for albumin among serum proteins. Both plasma and serum were compatible with the assay. The samples could be stored in a refrigerator or a freezer without the loss of reactivity toward fisetin. The generation and decay rates of the signal were acceptable for manual handling. The recovery of fortified albumin in serum was confirmed and the assay was validated with human sera. Fisetin-based albumin assay is suitable for clinical laboratory testing, considering the simple and short procedure, high specificity and sensitivity, linearity over a wide range of albumin concentrations, and, presumably, potential automatability.