Nalmefene, an opioid receptor modulator, aggravates atherosclerotic plaque formation in apolipoprotein E knockout mice by enhancing oxidized low-density lipoprotein uptake in macrophages.

Nalmefene, an antagonist of mu- and delta-opioid receptors and a partial agonist of kappa-opioid receptors, has shown promise in reducing alcohol consumption among patients with alcohol dependence. Opioid receptors play pivotal roles in various physiological processes, including those related to peripheral inflammatory diseases such as colitis and arthritis, as well as functions in the immune system and phagocytosis. Atherosclerosis, a chronic inflammatory disease, progresses through the phagocytosis and uptake of oxidized low-density lipoprotein (oxLDL) by macrophages in atherosclerotic plaques. Despite this knowledge, it remains unclear whether nalmefene influences the formation of atherosclerotic plaques and increases the risk of serious cardiovascular events. This study aims to elucidate the impact of nalmefene on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice and peritoneal macrophages in vitro. In this experiment, 8-week-old male ApoE KO mice were fed a high-fat diet intraperitoneally administered either vehicle (saline) or nalmefene (1 mg and 3 mg kg-1 day-1) for 21 days. Oil red O-staining and immunohistochemistry with an anti-MOMA2 (monocyte/macrophage) antibody showed that a dose-dependent increase in atherosclerotic plaque formation and augmentation of macrophage-rich plaque formation in ApoE-KO mice. Further investigations focused on the effects of nalmefene on the expression of scavenger receptor CD36 in RAW264.7 cells, conducted through western blotting analysis. Nalmefene demonstrated a significant increase in CD36 protein expression in RAW264.7 cells. To explore the impact on oxidized LDL uptake in peritoneal macrophages, cells were treated with nalmefene (300 µg/mL) for 24 h, followed by the addition of DiI-labeled oxLDL (DiI-oxLDL) for 4 h. Nalmefene significantly enhanced DiI-oxLDL uptake in macrophages. Additionally, treatment with nalmefene (300 µg/mL) for 24 h decreased the mRNA expression of mu-, delta-, and kappa-opioid receptors in RAW264.7 cells. In conclusion, nalmefene may augment oxLDL uptake by macrophages through increased CD36 expression and decreased opioid receptor, thereby contributing to atherosclerotic plaque formation and vulnerability. Consequently, the use of nalmefene may be associated with an elevated risk of cardiovascular events.

Menahydroquinone-4 may play a key role in regulating CCL5 expression induced by epidermal growth factor receptor inhibitors.

Epidermal growth factor receptor (EGFR) inhibitors frequently cause severe skin rash as a side effect, which is a critical burden for patients who continuously receive drug treatments. Several recent clinical trials have shown that vitamin K is effective against these side effects; however, the underlying mechanisms remain unclear. EGFR inhibitors induce C-C motif chemokine ligand 5 (CCL5) in dermopathy. We hypothesized that menahydroquinone-4 (MKH), the active form of menaquinone-4 (MK-4, vitamin K2(20)), supplied by biosynthesis or external delivery, is essential for the suppressive effect on CCL5. The aim of this study was to explore the underlying mechanisms governing the relieving effects of MKH against skin rashes caused by EGFR inhibitors. The responses generated by EGFR inhibitors and the effect of MKH derivatives (two ester derivatives and MK-4) on them were evaluated using human skin cell lines (HaCaT and HSC-1). EGFR inhibitors downregulated UbiA prenyltransferase domain-containing protein-1 (UBIAD1, MKH synthetase) expression and MKH biosynthesis. Knockdown of UBIAD1 or γ-glutamyl carboxylase and treatment with warfarin upregulated CCL5 expression. MKH derivatives suppressed the CCL5 expression induced by EGFR inhibitors. Our data strongly suggest that MKH is involved in suppressing CCL5 expression and alleviating the skin damage caused by EGFR inhibitors.

Association of T cell infiltration and morphological change of thymus gland with the aggravation of pulmonary emphysema in testosterone deficiency.

Chronic obstructive pulmonary disease is an inflammatory lung disease characterized by chronic bronchitis and emphysema. Our previous study revealed that testosterone depletion induced T cell infiltration in the lungs and aggravated pulmonary emphysema in orchiectomized (ORX) mice exposed to porcine pancreatic elastase (PPE). However, the association between T cell infiltration and emphysema remains unclear. The aim of this study was to determine whether thymus and T cells are involved in the exacerbation of PPE-induced emphysema in ORX mice. The weight of thymus gland in ORX mice was significantly greater than that of sham mice. The pretreatment of anti-CD3 antibody suppressed PPE-induced thymic enlargement and T cell infiltration in the lungs in ORX mice, resulting in improved expansion of the alveolar diameter, a marker of emphysema exacerbation. These results suggest that increased thymic function due to testosterone deficiency and the associated increased pulmonary infiltration of T cells may trigger the development of emphysema.

Cationic Ester Prodrugs of Curcumin with N,N-dimethyl Amino Acid Promoieties Improved Poor Water Solubility and Intestinal Absorption.

PURPOSE: Although curcumin (Cur) has powerful pharmacological effects, its use in medicine has not been established yet. The oral bioavailability (BA) of Cur is limited because of its poor water solubility. The purpose of this study was to confirm whether cationic N,N-dimethyl amino acid esters of Cur could act as prodrugs and improve its water solubility and oral bioavailability. METHODS: Two N,N-dimethyl amino acid esters of Cur were synthesized. The hydrolysis profile of the esters was evaluated using rat and human microsomes. A pharmacokinetic study after oral administration of the Cur ester derivatives was performed in rats and compared to the administration of suspended or dissolved Cur formulation. The anti-inflammatory effects of the Cur derivatives were evaluated using macrophage RAW 264.7 stimulated with lipopolysaccharide. RESULTS: Cur ester derivatives showed > 200 mM water solubility. The derivatives were reconverted to the parent compound (Cur) after cleavage of the ester bonds by microsomal esterase, indicating that the compounds could act as Cur prodrugs. The Cur prodrugs enhanced the absolute oral bioavailability of Cur by a 9- and threefold increase of suspended and dissolved Cur administration, respectively, thereby improving intestinal absorption. Cur prodrugs strongly attenuated COX2, iNOS, and ERK phosphorylation. CONCLUSIONS: The cationic N,N-dimethyl amino acid ester prodrugs of Cur improved the water solubility of Cur and enhanced oral bioavailability in rats. These Cur prodrugs may be good candidates for developing medicinal options previously unavailable due to the poor water solubility and oral BA of Cur.

Delivery of the reduced form of vitamin K2(20) to NIH/3T3 cells partially protects against rotenone induced cell death.

Mitochondria generate energy through the action of the electron transport chain (ETC) and ATP synthase. Mitochondrial malfunction can lead to various disorders, including neurodegenerative diseases. Several reports have shown that menaquinone-4 (MK-4, vitamin K2(20)), a safe drug for osteoporosis, may improve mitochondrial function. Here, we hypothesized that the efficient delivery of menahydroquinone-4 (MKH), an active form of MK-4, could exert a supporting effect. We verified the effects of MKH delivery on mitochondrial dysfunction by using MK-4 and MKH ester derivatives in NIH/3T3 mouse fibroblast cells treated with mitochondrial inhibitors. MK-4 and MKH derivatives suppressed cell death, the decline in mitochondrial membrane potential (MMP), excessive reactive oxygen species (ROS) production, and a decrease in intrinsic coenzyme Q9 (CoQ9) induced by rotenone (ROT, complex I inhibitor). MK-4 and MKH derivatives delivered MKH to NIH/3T3 cells, acting as an effective MKH prodrug, proving that the delivered MKH may reflect the mitigation effects on ROT-induced mitochondrial dysfunction. MKH prodrugs are also effective against 3-nitropropionic acid (3-NP, complex II inhibitor) and carbonyl cyanide-m-chlorophenylhydrazone (CCCP, uncoupler)-induced cell death. In conclusion, MKH delivery may mitigate mitochondrial dysfunction by maintaining MMP, ROS, and CoQ9, indicating that MKH prodrugs may be good candidates for treating mitochondrial disorders.

Moxifloxacin induces aortic aneurysm and dissection by increasing osteopontin in mice.

Fluoroquinolones are one of the most frequently prescribed antibiotics. However, their use increases the risk of Aortic aneurysm and dissection (AAD). The mechanism underlying this effect remains unclear. AAD are caused by weakening of the aortic wall and loss of vascular smooth muscle cells. Osteopontin is involved in the occurrence and development of AAD. The aim of the present study was to examine the role of moxifloxacin, a fluoroquinolone, in the occurrence of AAD using a moderate-severity AAD mouse model. Four-week-old male C57BL/6J mice were fed a high-fat diet. At 8 weeks of age, the mice were infused with saline or angiotensin II (1000 ng kg-1 min-1) via osmotic minipumps for 4 weeks, and then orally administered water (vehicle) or moxifloxacin (30 and 100 mg kg-1 day-1) for another 3 weeks. Moxifloxacin (30 and 100 mg kg-1 day-1) induced AAD and elastin degradation in aortic tissues, as revealed by hematoxylin and eosin staining and elastica-van Gieson staining. Additionally, immunohistochemical staining and Western blot analyses showed that moxifloxacin 100 mg kg-1 day-1 decreased the protein expression of smooth muscle protein 22α, one of the markers of the contractile phenotype of vascular smooth muscle cells, in aortic tissues compared to vehicle and moxifloxacin 30 mg kg-1 day-1. Furthermore, moxifloxacin (100 mg kg-1 day-1) increased the protein expression of osteopontin and matrix metalloproteinases-2 in the aortic tissues when compared to control. Moxifloxacin may induce the onset of AAD and weakening of the aortic media by increasing the expression of osteopontin and matrix metalloproteinase-2 and decreasing that of smooth muscle protein 22α in aortic tissue.

Cationic N,N-Dimethylglycine Ester Prodrug of 2R-α-Tocotrienol Promotes Intestinal Absorption via Efficient Self-Micellization with Intrinsic Bile Acid Anion.

The intestinal absorption of hydrophobic compounds is severely influenced by their transportation rate through the unstirred water layer in the intestinal lumen. A member of the vitamin E family, α-Tocotrienol (α-T3) has remarkable pharmacological effects, but its intestinal absorption is hampered due to its hydrophobicity. Here, we prepared three ester derivatives of 2R-α-T3, and we selected a suitable prodrug compound using rat plasma and liver microsomes. The micellization profile of the selected compound in the presence of taurocholic acid (TCA) was evaluated. After gastrostomy administration of the prodrug candidate or α-T3 solution containing TCA, AUC values were determined for α-T3 in plasma obtained from bile duct-ligated rats. Among the three types in the efficiency of the reconversion to the parent drug, α-T3 N,N-dimethylglycinate (α-T3DMG) was the best prodrug; α-T3DMG formed mixed micelles via ion pairs with anionic TCA. The solubility of α-T3DMG in n-octanol/water depended on its ratio to TCA. The AUC after α-T3DMG administration to ligated rats was 2-fold higher than that after α-T3 administration, suggesting a smooth interaction with intrinsic bile acids. In conclusion, utilization of the prodrug synthesized using N,N-dimethylglycine ester may be a beneficial approach to promote intestinal absorption of α-T3 via self-micellization with intrinsic bile acid.

Growth Inhibitory Effects of Ester Derivatives of Menahydroquinone-4, the Reduced Form of Vitamin K2(20), on All-Trans Retinoic Acid-Resistant HL60 Cell Line.

The first-choice drug for acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA), frequently causes drug-resistance and some adverse effects. Thus, an effective and safe agent for ATRA-resistant APL is needed. Menaquinone-4 (MK-4, vitamin K2(20)), used for osteoporosis treatment, does not have serious adverse effects. It has been reported that MK-4 has growth-inhibitory effects on HL60 cells by inducing apoptosis via the activation of Bcl-2 antagonist killer 1 (BAK). However, the effect of MK-4 on ATRA-resistant APL has not been reported. Here, we show that ester derivatives of menahydroquinone-4 (MKH; a reduced form of MK-4), MKH 1,4-bis-N,N-dimethylglycinate (MKH-DMG) and MKH 1,4-bis-hemi-succinate (MKH-SUC), exerted strong growth-inhibitory effects even on ATRA-resistant HL60 (HL-60R) cells compared with ATRA and MK-4. MKH delivery after MKH-SUC treatment was higher than that after MK-4 treatment, and the results indicated apoptosis induced by BAK activation. In contrast, for MKH-DMG, reconversion to MKH was slow and apoptosis was not observed. We suggest that the ester forms, including monoesters of MKH-DMG, exhibit another mechanism independent of apoptosis. In conclusion, the MKH derivatives (MKH-SUC and MKH-DMG) inhibited not only HL60 cells but also HL-60R cells, indicating a potential to overcome ATRA resistance.

Testosterone deficiency promotes the development of pulmonary emphysema in orchiectomized mice exposed to elastase.

Testosterone deficiency is commonly observed in male patients with chronic obstructive pulmonary disease (COPD), which is characterized by chronic inflammation of the airways and pulmonary emphysema. Although clinical trials have indicated that testosterone replacement therapy can improve respiratory function in patients with COPD, the role of testosterone in the pathogenesis of COPD remains unclear. The aim of this study was to explore the effect of testosterone deficiency on the development of pulmonary emphysema in orchiectomized (ORX) mice exposed to porcine pancreatic elastase (PPE). ORX mice developed more severe emphysematous changes 21 d after PPE inhalation than non-ORX mice. Testosterone propionate supplementation significantly reduced PPE-induced emphysematous changes in ORX mice. PPE exposure also increased the number of neutrophils and T cells in bronchoalveolar lavage fluid (BALF) of mice that had undergone ORX and sham surgery. T cell counts were significantly higher in the BALF of ORX mice than of sham mice. Testosterone supplementation reduced the infiltration of T cells into BALF and alleviated emphysematous changes in the lungs of ORX mice. Our findings suggest that testosterone, a male-specific hormone, may suppress the development of pulmonary emphysema through the regulation of T cell-mediated immunity.

Evaluation of photostability and phototoxicity of esterified derivatives of ubiquinol-10 and their application as prodrugs of reduced coenzyme Q10 for topical administration.

Ubiquinol-10 (UqH-10), the fully reduced form of ubiquinone-10 (Uq-10, coenzyme Q10 ), is an antioxidant and is involved in energy production. However, physicochemical disadvantages, such as rapid oxidation, water-insolubility, photoinstability, and phototoxicity, limit its application. We previously reported that UqH-10 1,4-bis-N,N-dimethylglycinate improved the oxidation susceptibility and poor bioavailability of UqH-10 in rats. Herein, we evaluated the photochemical properties of UqH-esterified derivatives (N,N-dimethylglycinate, hemi-succinate, ethylsuccinate, and hemi-glutarate). Photostability was examined by irradiation using artificial sunlight and monochromatic light. The concentration of each compound was determined using LC-MS/MS. Phototoxicity was assessed by singlet oxygen and superoxide assays. Delivery of UqH-10 via UqH-esters to the HaCaT human keratinocyte cell line was determined using LC-MS/MS. UqH-esters showed higher photostability to artificial sunlight than Uq-10 and UqH-10. Uq-10 and UqH-10 were rapidly degraded by monochromatic light at 279 nm, whereas UqH-esters were more stable. UVA and/or UVB irradiation generated high levels of singlet oxygen and superoxide in Uq-10, whereas UqH-esters were unreactive. Additionally, UqH-esters effectively delivered UqH-10 to HaCaT cells following efficient uptake in their ester forms and ester bond hydrolysis in the cells. In conclusion, UqH-ester derivatives exhibit higher photostability and lower phototoxicity compared with Uq-10 and UqH-10.

Atypical Antipsychotic Drug Ziprasidone Protects against Rotenone-Induced Neurotoxicity: An In Vitro Study.

Schizophrenia is a severe, chronic mental illness characterized by delusions, hallucinations, negative symptoms, and cognitive dysfunction. Recently, several studies have demonstrated that the pathogenesis of schizophrenia involves mitochondrial dysfunction and oxidative stress. However, the effect of antipsychotic drugs for these events has been poorly investigated. In the present study, we evaluated the neuroprotective effect of an atypical antipsychotic drug, ziprasidone (ZPD), on rotenone (ROT)-induced neurotoxicity involving oxidative stress in PC12 cells. Our data showed that ZPD treatment promoted the translocation of NF-E2-related factor-2 (Nrf2) from cytoplasm to nucleus and activated the expression of its target genes NAD(P)H quinone oxidoreductase (NQO-1), catalase (CAT), and heme oxygenase (HO-1). Additionally, ZPD prevented ROT-induced cell death and intracellular reactive oxygen species production. Interestingly, the use of serotonin 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4 (4-(2-phtalimido) butyl) piperazine (NAN-190) completely blocked the protective effect of ZPD against ROT-induced cell death. Our results demonstrate the neuroprotective effect of ZPD against ROT-induced neurotoxicity and suggest that ZPD may be a potential candidate for the prevention of mitochondrial dysfunction and oxidative stress in schizophrenia.

Ester derivatives of phyllohydroquinone effectively deliver the active form of vitamin K1 topically, owing to their non-photosensitivity.

Topical application of phylloquinone (PK) is beneficial to the skin; however, its topical use is limited in Europe owing to its photosensitive properties such as photodegradation and phototoxicity. We evaluated the suitability of ester derivatives of phyllohydroquinone (PKH), the active form of PK, for topical application to overcome the abovementioned problems of PK. We used the PKH derivatives PKH-1,4-bis-N,N-dimethylglycinate hydrochloride (PKH-DMG) and PKH-1,4-bis-hemisuccinate (PKH-SUC) for our studies. Photostability was determined by measuring the residual concentration after irradiation with artificial sunlight and multi-wavelength light. Phototoxicity after ultraviolet A (UVA) irradiation was assessed by measuring drug-induced singlet oxygen and intracellular reactive oxygen species (ROS) generation, and cell viability of a human epidermal keratinocyte cell line (HaCaT). Delivery of PKH into HaCaT cells was assessed by measuring PK epoxide (PKO) levels. The PKH derivatives showed higher photostability than PK. After UVA irradiation, PK induced high singlet oxygen levels and intracellular ROS generation, and reduced cell viability, whereas the PKH derivatives showed no effects. The PKH derivatives increased intracellular PKO levels. AUCPKO(0-72 h) values after PKH-DMG and PKH-SUC treatments were 0.741- and 22.9-fold higher than that after PK treatment, respectively. In conclusion, PKH derivatives act as PKH prodrugs and are suitable for topical application without the need for special protection from light.

Hesperidin blocks varenicline-aggravated atherosclerotic plaque formation in apolipoprotein E knockout mice by downregulating net uptake of oxidized low-density lipoprotein in macrophages.

Varenicline is a widely used and effective drug for smoking cessation. We have previously reported experimental evidence suggesting that varenicline increases the risk of cardiovascular events. Varenicline progresses atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice. This adverse effect is likely due to enhanced net uptake of oxidized low-density lipoprotein (oxLDL) in macrophages as a result of increased scavenger receptors and decreased cholesterol efflux transporters. However, a regimen has not yet been presented for avoidance or amelioration of the risk for varenicline-induced cardiovascular events. The aim of this study was to examine the effect of hesperidin, a citrus flavonoid, on varenicline-aggravated atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice. Hesperidin inhibited the aggravating effect of varenicline in the whole aorta, aortic arch, and aortic root of ApoE KO mice. In addition, hesperidin protected against varenicline-enhanced oxLDL net uptake by blocking the increased expression of CD36 and LOX-1 scavenger receptors and decreased expression of ABCA1 and ABCG1 cholesterol efflux transporters in RAW 264.7 cells. Our findings suggest that hesperidin can avoid or ameliorate the risk for cardiovascular events induced by varenicline treatment.

Novel Cationic Prodrug of Ubiquinol-10 Enhances Intestinal Absorption via Efficient Formation of Nanosized Mixed-Micelles with Bile Acid Anions.

The aim of this study was to develop a prodrug of ubiquinol-10 (UqH-10), the active form of ubiquinone-10 (Uq-10), for oral delivery. Bioavailability of UqH-10 is hampered by its high susceptibility to oxidation and water-insolubility. We prepared three novel N,N-dimethylglycine ester derivatives of UqH-10, including a 1-monoester (UqH-1-DMG), 4-monoester (UqH-4-DMG), and 1,4-bis-ester (UqH-DMG), and assessed their physicochemical properties in vitro and in vivo. UqH-DMG spontaneously formed an aqueous micelle solution comprising 20 nm particles at 36.5 °C. Cationic UqH-DMG formed nano-sized (5 nm) mixed-micelles with taurocholic acid. Reconversion of the derivatives to UqH-10 was accelerated in human liver microsomes. The oral bioavailability of UqH-10 after administration of UqH-derivatives or Uq-10 was determined in fasted and postprandial rats secreting normal and high levels of bile, respectively. In fasted rats, plasma UqH-10 after UqH-derivatives administration reached Cmax at 2-3 h and after Uq-10 administration, it remained low. The AUC0-24h of UqH-10 after UqH-derivatives administration was 2-3-fold higher than that after Uq-10 administration. In postprandial rats, the Tmax of UqH-10 after UqH-derivatives administration was an hour earlier than after Uq-10 administration. In conclusion, cationic UqH-derivatives are convenient prodrugs that enhance UqH-10 bioavailability by forming nanosized mixed-micelles with intestinal bile acids.

Varenicline aggravates atherosclerotic plaque formation in nicotine-pretreated ApoE knockout mice due to enhanced oxLDL uptake by macrophages through downregulation of ABCA1 and ABCG1 expression.

Varenicline is a widely used and effective drug for smoking cessation. We previously reported that varenicline aggravates atherosclerosis in apolipoprotein E knockout (ApoE KO) mice. However, it remains unknown whether varenicline affects cardiovascular events in patients with nicotine addiction. Here, we examined the effect of varenicline on atherosclerotic plaque formation in nicotine-pretreated ApoE KO mice and oxidized low-density lipoprotein (oxLDL) uptake in nicotine-treated peritoneal macrophages. Varenicline caused significant progression of plaque formation in the whole aorta and aortic root and further accelerated the increased formation of a macrophage-rich plaque area in the aortic root in nicotine-pretreated ApoE KO mice. Varenicline (10 µM) enhanced oxLDL uptake in peritoneal macrophages. Furthermore, this treatment significantly further lowered the decreased protein levels of ATP-binding cassette (ABC) transporter without affecting the expression of scavenger receptors LOX-1 and CD36 in RAW264.7 cells treated with 100 nM nicotine. Varenicline enhanced nicotine-induced oxLDL uptake in macrophages through decreased expression of cholesterol efflux transporters ABCA1 and ABCG1 and thereby progressed atherosclerotic plaque formation. Taken together, we tentatively conclude that nicotine exposure before and/or during varenicline treatment can aggravate varenicline-increased atherosclerotic plaque formation and progression. Therefore, this enhanced risk requires special consideration when prescribing varenicline to smoker patients.

Feeding-produced subchronic high plasma levels of uric acid improve behavioral dysfunction in 6-hydroxydopamine-induced mouse model of Parkinson's disease.

The development of Parkinson's disease (PD) involves the degeneration of dopaminergic neurons caused by oxidative stress. Accumulating clinical evidence indicates that high blood levels of uric acid (UA), an intrinsic antioxidative substance, are associated with reduced risk of PD. However, this hypothesis has not been confirmed by in-vivo experiments. The present study investigated the effects of UA on behavioral abnormalities in the development of PD. We used unilateral 6-hydroxydopamine-lesioned mice, which were fed on a diet containing 1% UA and 2.5% potassium oxonate (an uricase inhibitor) to induce hyperuricemia. A significant elevation in UA levels was found in groups that were fed a UA diet. The 6-hydroxydopamine-lesioned mice showed impaired rotarod performance and increased apomorphine-induced contralateral rotations. These behavioral abnormalities were significantly reversed by feeding a UA diet for 1 week before and 5 weeks after surgery (subchronic hyperuricemia). These behavioral improvements occurred in parallel with recovery of tyrosine hydroxylase protein levels in the lesioned striatal side. The present study with a dietary hyperuricemia mice model confirms that UA exerts a neuroprotective effect on dopaminergic neuronal loss, improving motor dysfunction and ameliorating PD development.

Varenicline is a smoking cessation drug that blocks alveolar expansion in mice intratracheally administrated porcine pancreatic elastase.

Smoking cessation is the most effective treatment in patients with emphysema and lung inflammation. The aim of the present study was to examine the effect of varenicline, a smoking cessation drug, on emphysema in porcine pancreatic elastase (PPE)-inhaled mice. PPE-inhaled mice were treated with varenicline and an α7 nicotinic acetylcholine receptor (nAChR) antagonist, methyllycaconitine (MLA) for 5 and 21 days. Varenicline markedly ameliorated alveolar expansion and inflammatory response in bronchoalveolar lavage fluid in PPE-inhaled mice. These blocking effects were inhibited by MLA. Our findings demonstrate that varenicline likely has an anti-inflammatory property including reduced inflammatory cell recruitment in lung tissue to protect PPE-induced alveolar expansion via α7 nAChR.

Varenicline promotes endothelial cell migration by lowering vascular endothelial-cadherin levels via the activated α7 nicotinic acetylcholine receptor-mitogen activated protein kinase axis.

Varenicline is a widely used and effective drug for smoking cessation. Despite its efficacy, varenicline increases the risk of cardiovascular disease. We previously demonstrated that varenicline aggravates atherosclerotic plaque formation in apolipoprotein E knockout mice. However, little is known about its effects in vascular endothelial cells. Therefore, we examined whether varenicline promotes migration of human umbilical vein endothelial cells (HUVECs) using the Boyden chamber assay. Varenicline (100µM) markedly promoted migration of HUVECs and decreased expression of vascular endothelial (VE)-cadherin, an endothelial adhesion molecule. Extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) signaling were markedly activated by varenicline. Methyllycaconitine (MLA; 100nM), an α7 nicotinic acetylcholine receptor (nAChR) antagonist, but not dihydro-ß-erythroidine hydrobromide (DHßE; 20µM) blocked varenicline-stimulated migration and varenicline-activated ERK, p38 and JNK signaling in HUVECs. MLA (100nM), PD98059 (an ERK inhibitor; 20µM), SB203580 (a p38 inhibitor; 20µM) and SP600125 (a JNK inhibitor; 20µM) also blocked cell migration and varenicline-induced downregulation of VE-cadherin expression in HUVECs. These findings suggest that varenicline promotes HUVEC migration by lowering VE-cadherin expression due to activated ERK/p38/JNK signaling through α7 nAChR. These processes probably contribute to varenicline-aggravated atherosclerotic plaque. Hence, an increased risk of cardiovascular events upon varenicline treatment might occur and must be considered in patients with cardiovascular diseases.

Varenicline enhances oxidized LDL uptake by increasing expression of LOX-1 and CD36 scavenger receptors through α7 nAChR in macrophages.

Varenicline is a widely used and effective drug for smoking cessation. It is a partial agonist of the α4ß2 nicotinic acetylcholine receptor (nAChR) and full agonist of α7 nAChR. We have reported that varenicline aggravates formation of atherosclerotic plaques through α7 nAChR in apolipoprotein E knockout mice. However, little is known about its effects on macrophages in atherosclerotic plaques. Here, we ascertained whether varenicline promotes oxidized low-density lipoprotein (oxLDL) uptake in mouse peritoneal macrophages in vitro and clarified its mechanism. We investigated the effects of varenicline (1-10µM) on expression of scavenger receptors (lectin-like oxidized LDL receptor-1 (LOX-1), cluster of differentiation (CD) 36 and scavenger receptor class A (SR-A)) in RAW264.7 cells. Expression of protein and mRNA was determined by western blotting and real-time quantitative reverse transcription-polymerase chain reaction, respectively. Effects of varenicline (10µM) on oxLDL uptake were examined by counting the number of macrophages stained with oil red O and hematoxylin. Varenicline significantly increased expression of the protein and mRNA of LOX-1 and CD36, but not SR-A, in RAW264.7 cells, and increased oxLDL uptake in macrophages. These effects of varenicline were blocked significantly by an α7 nAChR antagonist, methyllycaconitine (MLA) (50nM), but not by an α4ß2 nAChR antagonist, dihydro-ß-erythroidine hydrobromide (DHßE) (1µM). These data suggest that varenicline promotes oxLDL uptake by upregulating expression of LOX-1 and CD36 through α7 nAChR in macrophages. We found that varenicline significantly activated extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-kappa B (NF-κB) signaling pathways in RAW264.7 cells. This activation was blocked by MLA but not DHßE. Therefore, ERK1/2-NF-κB signaling pathway is highly likely to be responsible for varenicline-induced upregulation of LOX-1 and CD36 expression through α7 nAChR in macrophages. These processes probably contribute to varenicline-aggravated atherosclerotic plaque formation. Hence, an increased risk of cardiovascular events upon varenicline treatment could occur, and must be considered in patients (especially those suffering from cardiovascular diseases).

Elevated permeability of the blood-brain barrier in mice intratracheally administered porcine pancreatic elastase.

Chronic obstructive pulmonary disease (COPD) shows progressive, irreversible airflow limitation induced by emphysema and lung inflammation. The aim of the present study was to determine if COPD conditions induce blood-brain barrier (BBB) dysfunction. We found that the intratracheal administration of porcine pancreatic elastase (PPE; 3 U) induced alveolar enlargement, increased neutrophil number in bronchoalveolar lavage fluid, and decreased blood oxygen saturation in mice at 21 days after inhalation. In parallel with these lung damages, BBB permeability to sodium fluorescein and Evans blue albumin was markedly increased. Our findings demonstrate that COPD conditions are associated with risk for BBB impairment.