DMSO-soluble smoking particles up-regulate the vascular endothelin receptors through AMPK-SIRT1 and MAPK pathways.

Smoking is a well-known risk factor for cardiovascular diseases. However, the mechanisms behind how smoking leads to vasospasm are not fully elucidated. Endothelin receptors are involved in the pathogenesis of cardiovascular diseases. This study examined whether cigarette smoke could induce up-regulation of vascular endothelin receptors through AMPK-SIRT1 and MAPK pathways. The results show that DMSO-soluble smoking particles (DSP) up-regulated the protein expressions of endothelin receptors and the contractile responses. Furthermore, the inhibition of MAPK or activation of AMPK-SIRT1 markedly attenuated DSP-enhanced vasoconstriction and the protein expression of endothelin receptors. The in vivo study showed that cigarette smoke increased the blood pressure of the rats and down-regulated p-AMPKα, SIRT1, and up-regulated endothelin receptors, p-ERK1/2, and p-P38 protein expressions. However, treatment with resveratrol attenuated vasoconstriction, endothelin receptor proteins expression, and blood pressure in vivo. In conclusion, this suggests that cigarette smoke up-regulates the vascular endothelin receptors through AMPK-SIRT1 and MAPK pathways.

Sorafenib not only impairs endothelium-dependent relaxation but also promotes vasoconstriction through the upregulation of vasoconstrictive endothelin type B receptors.

As a VEGF-targeting agent, sorafenib has been used to treat a number of solid tumors but can easily lead to adverse vascular effects. To elucidate the underlying mechanism, rat mesenteric arteries were subjected to organ cultured in the presence of different concentrations of sorafenib (0, 3, 6 and 9 mg/L) with or without inhibitors (U0126, 10-5 M; SB203580, 10-5 M; SP200126, 10-5 M) of MAPK kinases, and then acetylcholine- or sodium nitroprusside-induced vasodilation and sarafotoxin 6c-induced vasoconstriction were monitored by a sensitive myograph. The NO synthetases, the nitrite levels, the endothelial marker CD31,the ETB and ETA receptors and the phosphorylation of MAPK kinases were studied. Next, rats were orally administrated by sorafenib for 4 weeks (7.5 and 15 mg/kg/day), and their blood pressure, plasma ET-1, the ETB and ETA receptors and the phosphorylation of MAPK kinases in the mesenteric arteries were investigated. The results showed that sorafenib impairs endothelium-dependent vasodilation due to decreased NO levels and the low expression of eNOS and iNOS. Weak staining for CD31 indicated that sorafenib induced endothelial damage. Moreover, sorafenib caused the upregulation of vasoconstrictive ETB receptors, the enhancement of ETB receptor-mediated vasoconstriction and the activation of JNK/MAPK. Blocking the JNK, ERK1/2 and p38/MAPK signaling pathways by using the inhibitors significantly abolished ETB receptor-mediated vasoconstriction. Furthermore, it was observed that the oral administration of sorafenib caused an increase in blood pressure and plasma ET-1, upregulation of the ETB receptor and the activation of JNK in the mesenteric arteries. In conclusion, sorafenib not only impairs endothelium-dependent vasodilatation but also enhances ETB receptor-mediated vasoconstriction, which may be the causal factors for hypertension and other adverse vascular effects in patients treated with sorafenib.

The roles of endothelin and its receptors in cigarette smoke-associated pulmonary hypertension with chronic lung disease.

Chronic exposure to cigarette smoke is the major risk factor for the development of pulmonary hypertension (PH) with chronic lung disease (i.e. PH group III). The pathogenesis of smoke-associated PH group III in chronic obstructive pulmonary disease (COPD) involves cigarette smoke exposure-induced damage to lung tissue and dysfunction of pulmonary system with increased synthesis and release of endothelin-1 (ET-1), hypoxia, inflammation, pulmonary vascular remodeling. Many studies have demonstrated that cigarette smoke exposure induces activation of mitogen-activated protein kinase (MAPK) signal pathway that leads to up-regulation of ET-1 and its receptors with the receptor-mediated enhanced contraction, proliferation of pulmonary vascular smooth muscle cells, pulmonary vascular remodeling, elevated pulmonary arterial pressure and finally PH group III. This mini-review article aims to summarize the current state of understanding on the roles of cigarette smoke-induced up-regulation of ET-1 and its receptors in the development of PH group III. Understanding the underlying molecular mechanisms that cigarette smoke exposure leads to PH group III may provide a novel strategy for the treatment.

Development and Characterization of A New Dimethicone Nanoemulsion and its Application for Electronic Gastroscopy Examination.

PURPOSE: Although the effective and safe medical defoamers, dimethicone (DM) and simethicone (SM) are widely used in electronic gastroscope examination (EGE), their preparations are presented in the form of suspensions or emulsions, these are untransparent or milk-like in appearance and can easily cause misdiagnosis as a result of an unclear field of vision if the doctor does not master the amount of defoamer or operates incorrectly. At the same time, it is also difficult to wash out the camera and pipeline, due to the large oil droplets of preparations. The purpose of this study was to develop a new clear and transparent oil in water (O/W) DM nanoemulsions (DMNs) and observe the effect of application in EGE. METHODS: The oil phase was chosen for its antifoaming activity and viscosity. The emulsifier and co-emulsifier were selected according to the solubility of the oil phase in them. The water titration method was used to make the pseudoternary phase diagrams of nanoemulsions and optimize the prescription composition. DM-in-water nanoemulsion was prepared by the low energy method and evaluated for appearance, antifoaming ability, droplet size, and stability. The effect of DMNs utilized in EGEs was also observed. RESULTS: The optimal formulation of DMNs contained CRH-40 as an emulsifier, PEG-400 as a co-emulsifier, DM as oil phase with the viscosity of 10 mPa.s, and their proportion was 4.5:4.5:1, respectively. DMNs obtained the average particle size of 67.98 nm with the polydispersity index (PDI) of 0.332, and 57.14% defoaming rate. The result of using an EGE showed that DMNs were superior in comparison to the emulsions with regard to the defoaming effect, visual clarity, and easy cleanup. CONCLUSION: DMNs were found to provide excellent visual clarity to its other preparations. The novel DMNs is a promising substitute for DM emulsions or suspensions in EGEs.

Toll-like receptor protein 4 monoclonal antibody inhibits mmLDL-induced endothelium-dependent vasodilation dysfunction of mouse mesenteric arteries.

Minimally modified low-density lipoprotein (mmLDL) is a risk factor for cardiovascular disease. This study was designed to investigate the effect of a Toll-like receptor 4 monoclonal antibody (TLR4 mAb) on mmLDL-induced endothelium-dependent vasodilation (EDV) impairment in mouse mesenteric arteries and to explore the underlying mechanism. Animals were divided into a normal control group, an mmLDL treatment group, and a TLR4 mAb intervention group. The serum concentrations of IL-1ß and TNF-α were detected using enzyme-linked immunosorbent assays (ELISAs). EDV function was measured using a microvascular tension tracing method. The protein levels and mRNA expression of IL-1ß and TNF-α in vascular tissue were detected using western blot analysis and reverse transcription polymerase chain reaction, respectively. TLR4 mAb improved mmLDL-induced EDV functional impairment in a dose-dependent manner. TLR4 mAb significantly upregulated KCa3.1 and KCa2.3 channel protein levels and downregulated TNF-α and IL-1ß expression. These effects were possibly associated with the competitive antagonism of TLR4 mAb on the TLR4 signaling pathway and the downstream NF-κB p65 and p38 MAPK pathways, which are activated by mmLDL. In conclusion, pretreatment with TLR4 mAb lessens mmLDL-induced EDV dysfunction and inhibits overexpression of inflammatory factors. Regulation of the TLR4 pathway, as well as its downstream NF-κB p65 and p38 MAPK pathways, may be an effective strategy for the prevention and treatment of cardiovascular diseases.

Cigarette Smoke Particles-Induced Airway Hyperreactivity in Vivo and in Vitro.

Cigarette smoke is a well-known strong risk factor for inducing airway hyperreactivity (AHR), but the underlying molecular mechanisms are not fully understood. In the present study, mouse in-vivo and in-vitro models were used to study effects of dimethyl sulfoxide (DMSO)-extracted cigarette smoke particles (DSP) on the airway, and to explore the underlying molecular mechanisms that are involved in DSP-induced AHR. In mouse in-vivo model, DSP (0.75, 1.5 or 3 µL/mL) was administered intranasally daily for 7 d. At the end of this period, lung functions were measured with flexiVent™. The results showed that the mice exhibited AHR in a dose-dependent manner following methacholine inhalation in vivo. In mouse in-vitro organ culture model, exposure of mouse tracheal segments to DSP (0.1 µL/mL) with or without the following pharmacological inhibitors: specific c-Jun-N-terminal kinase (JNK) inhibitor SP600125 (10 µM) or the anti-inflammatory drug dexamethasone (1 µM). DSP-induced bradykinin receptor-mediated airway contraction with increased mRNA and protein expressions for bradykinin B1 and B2 receptors could be significantly reduced by SP600125 or dexamethasone. In conclusion, the present study demonstrates that DSP could induce AHR in vivo and in vitro. In addition to this, the upregulation of bradykinin receptors in airway is most likely one of the underlying molecular mechanisms involved.

Minimally modified low-density lipoprotein upregulates the ETB and α1 receptors in mouse mesenteric arteries in vivo by activating the PI3K/Akt pathway.

OBJECTIVES: The current study aimed to explore whether minimally modified low-density lipoprotein (mmLDL) via tail vein injection upregulates the ETB and α1 receptors in mouse mesenteric arteries by activating the PI3K/Akt pathway. METHODS: The contraction curves of the mesenteric arteries caused by sarafotoxin 6c (S6c, ETB receptor agonist) and phenylephrine (PE, α1 receptor agonist) were measured by a myograph system. Serum oxLDL was detected using enzyme-linked immunosorbent assays. The levels of the ETB receptor, the α1 receptor, PI3K, p-PI3K and p-Akt were detected using real-time polymerase chain reaction and Western blot analyses. KEY FINDINGS: Minimally modified low-density lipoprotein noticeably enhanced the contraction effect curves of S6c and PE, with significantly increased Emax values (P < 0.01), compared to those of the control group. This treatment significantly increased the mRNA expression and protein levels of the ETB and α1 receptors and the protein levels of p-PI3K and p-Akt in the vessel wall (P < 0.01). LY294002 inhibited the effect of mmLDL. CONCLUSIONS: An increase in mmLDL activated the PI3K/Akt pathway, which upregulated the expression of the ETB and α1 receptors and enhanced the ETB and α1- receptor-mediated contractile function.

MicroRNA-770 affects proliferation and cell cycle transition by directly targeting CDK8 in glioma.

BACKGROUND: MicroRNAs play crucial roles in tumorigenesis and tumor progression. miR-770 has been reported to be downregulated in several cancers and affects cancer cell proliferation, apoptosis, metastasis and drug resistance. However, the role and underlying molecular mechanism of miR-770 in human glioma remain unknown and need to be further elucidated. METHODS: The expression of miR-770 in glioma tissues and cell lines was measured by quantitative real-time PCR (qRT-PCR) to explore the association of miR-770 expression with clinicopathological characteristics. The expression of CDK8 was detected by qRT-PCR and Western blotting in glioma tissues. A target prediction program and a dual-luciferase reporter assay were used to confirm that CDK8 is a target gene of miR-770. MTT and cell counting assays were used to assess the effect of miR-770 on glioma cell proliferation. The cell cycle distribution and apoptosis were examined by flow cytometry. CDK8 siRNA and overexpression were used to further confirm the function of the target gene. RESULTS: We demonstrated that miR-770 expression was downregulated in human glioma tissues and cell lines. The overexpression of miR-770 inhibited glioma cell proliferation and cell cycle G1-S transition and induced apoptosis. The inhibition of miR-770 facilitated cell proliferation and G1-S transition and suppressed apoptosis. miR-770 expression was inversely correlated with CDK8 expression in glioma tissues. CDK8 was confirmed to be a direct target of miR-770 by using a luciferase reporter assay. The overexpression of miR-770 decreased CDK8 expression at both the mRNA and protein levels, and the suppression of miR-770 increased CDK8 expression. Importantly, CDK8 silencing recapitulated the cellular and molecular effects observed upon miR-770 overexpression, and CDK8 overexpression eliminated the effects of miR-770 overexpression on glioma cells. Moreover, both exogenous expression of miR-770 and silencing of CDK8 resulted in suppression of the Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our study demonstrates that miR-770 inhibits glioma cell proliferation and G1-S transition and induces apoptosis through suppression of the Wnt/ß-catenin signaling pathway by targeting CDK8. These findings suggest that miR-770 plays a significant role in glioma progression and serves as a potential therapeutic target for glioma.

Lipid-soluble Cigarette Smoke Particles Induced Vascular Endothelin Type A Receptor Up-Regulation through Activation of ERK1/2 Signal Pathways.

Abnormal contraction of vessels termed 'vasospasm' is associated with various cardiovascular diseases. Smoking is a well-known risk factor that increases vasospasm. However, the molecular mechanisms by which smoking leads to vasospasm and cardiovascular disease are not fully understood. This study was designed to examine whether DMSO-extracted cigarette smoke particles (DSP) could induce up-regulation of vascular endothelin type A (ETA ) receptors, and whether ETA receptor is up-regulated through activation of extracellular regulated protein kinases 1 and 2 (ERK1/2) signal pathways. Mesenteric arterial segments from rats were cultured in the presence of DSP, water-extracted cigarette smoke particles (WSP) or equivalent concentration of nicotine for up to 24 hr. The results showed that DSP, but not WSP or nicotine, induced ETA receptor up-regulation with increased ETA receptor-mediated contraction (myograph, p < 0.001). Simultaneously, the expression of ETA receptor mRNA (real-time PCR, p < 0.001) and protein (immunohistochemistry) were enhanced in the smooth muscle cells, suggesting that the lipid-soluble substances contained in cigarette smoke were responsible for the effects of DSP. Actinomycin D (a general transcriptional inhibitor) decreased ETA receptor mRNA expression and attenuated receptor-mediated contraction (p < 0.001), while DSP accelerated ETA receptor mRNA degradation (p < 0.01) and promoted the translation of ETA receptor mRNA into protein. Furthermore, the up-regulation of ETA receptors was significantly attenuated by inhibition of ERK1/2 signal pathways (p < 0.001). In conclusion, DSP most likely activate ERK1/2 signal pathway-mediated transcriptional and post-transcriptional (translational) mechanisms that lead to vascular ETA receptor up-regulation, which might contribute to vasospasm and the development of smoking-associated cardiovascular diseases.

The ERK1/2 pathway participates in the upregulation of the expression of mesenteric artery α1 receptors by intravenous tail injections of mmLDL in mice.

Minimally modified low density lipoprotein (mmLDL) is a risk factor for cardiovascular diseases. However, no studies examining the effect of mmLDL on vascular smooth muscle receptors have been released. The current study investigated the effect of mmLDL on the mesenteric artery α1 adrenoceptor and the molecular mechanisms. Mice were divided into the normal saline (NS), mmLDL, and mmLDL+U0126 groups. In the mmLDL+U0126 group, the animals were subjected to an intravenous tail injection of mmLDL and an intraperitoneal injection of U0126. Vascular tension caused by noradrenaline (NA) in mesenteric arteries was measured with a sensitive myograph system. The serum levels of oxLDL, TNF-α, and IL-1ß were detected using enzyme-linked immunosorbent assays. The expressions of the α1 adrenoceptor, the α2 adrenoceptor, TNF-α, IL-1ß, and pERK1/2 were detected using real-time polymerase chain reactions and Western blot analysis. Compared with the NS group, the mmLDL group exhibited a noticeably enhanced NA shrinkage dose-response curve and a significantly increased Emax value (P<0.01). Prazosin (α1 adrenoceptor antagonist) caused a noticeable right shift of the dose-response curve. U0126 inhibited the increases in the serum levels and vessel wall expression of IL-1ß and TNF-α and enhanced the NA shrinkage dose-response curve caused by mmLDL, as observed by a significantly decreased Emax value (P<0.01). It inhibited the increased α1 adrenoceptor expression caused by mmLDL. The serum levels of IL-1ß and TNF-α demonstrated a positive correlation with the NA-induced maximum shrinkage percentage. U0126 inhibited the mmLDL-induced increase in the pERK1/2 protein level in the vessel wall. In conclusion, mmLDL increased the serum levels of IL-1ß and TNF-α in vivo by activating the ERK1/2 pathway, which resulted in α1 receptor-mediated vasoconstriction and an increase in the expression of α1 adrenoceptor. The results of this study may provide new ideas for the prevention and cure of cardiovascular diseases in the future.

MAPK/NF-κB-dependent upregulation of kinin receptors mediates airway hyperreactivity: a new perspective for the treatment.

Airway hyperreactivity (AHR) is a major feature of asthmatic and inflammatory airways. Cigarette smoke exposure, and bacterial and viral infections are well-known environmental risk factors for AHR, but knowledge about the underlying molecular mechanisms on how these risk factors lead to the development of AHR is limited. Activation of intracellular mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) and their related signal pathways including protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and protein kinase A (PKA) signaling pathways may result in airway kinin receptor upregulation, which is suggested to play an important role in the development of AHR. Environmental risk factors trigger the production of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukins (ILs) that activate intracellular MAPK- and NF-κB-dependent inflammatory pathways, which subsequently lead to AHR via kinin receptor upregulation. Blockage of intracellular MAPK/NF-κB signaling prevents kinin B1 and B2 receptor expression in the airways, resulting in a decrease in the response to bradykinin (kinin B2 receptor agonist) and des-Arg9-bradykinin (kinin B1 receptor agonist). This suggests that MAPK- and NF-κB-dependent kinin receptor upregulation can provide a novel option for treatment of AHR in asthmatic as well as in other inflammatory airway diseases.

Secondhand smoke exposure causes bronchial hyperreactivity via transcriptionally upregulated endothelin and 5-hydroxytryptamine 2A receptors.

BACKGROUND: Cigarette smoke exposure is strongly associated with airway hyperreactivity (AHR) which is the main characteristic seen in asthma. The intracellular MAPK signaling pathways are suggested to be associated with the airway damage to the AHR. In the present study, we hypothesize that secondhand cigarette smoke (SHS) exposure upregulates the bronchial contractile receptors via activation of the Raf/ERK/MAPK pathway. METHODOLOGY/PRINCIPAL FINDINGS: Rats were exposed to SHS for 3 h daily for up to 8 weeks. The receptor agonists-induced bronchial contractile reactivity was analyzed with a sensitive myograph system. The mRNA transcription and protein translation of the target receptors and the kinases in Raf/ERK/MAPK pathway were investigated by real-time PCR, Western blotting and immunofluorescence, respectively. Compared with exposure to fresh air, SHS induced enhanced bronchial contractile responses mediated by the 5-hydroxytryptamine 2A (5-HT(2A)) receptors as well as the endothelin type B (ET(B)) and type A (ET(A)) receptors. The response curves were shifted toward the left with an increased maximal contraction (E(max)) demonstrating that SHS induced AHR. Additionally, the mRNA and protein levels of the 5-HT(2A), ET(B) and ET(A) receptors were increased. Furthermore, SHS exposure increased the phosphorylation of Raf-1 and ERK1/2, but it did not alter p38 or JNK. A Raf-1 inhibitor (GW5074) suppressed the SHS-induced increase in the expression of 5-HT(2A) and ET(A) receptors and the receptor-mediated AHR. CONCLUSIONS/SIGNIFICANCE: Our findings show that SHS exposure induces transcriptional upregulation of the 5-HT(2A), ET(B) and ET(A) receptors in rat bronchial smooth muscle cells, which mediates AHR. The Raf/ERK/MAPK pathway is involved in SHS-associated receptor upregulation and AHR.

Cigarette smoke and platelet-activating factor receptor dependent adhesion of Streptococcus pneumoniae to lower airway cells.

BACKGROUND: Exposure to cigarette smoke (CS) is associated with increased risk of pneumococcal infection. The mechanism for this association is unknown. We recently reported that the particulate matter from urban air simulates platelet-activating factor receptor (PAFR)-dependent adhesion of pneumococci to airway cells. We therefore sought to determine whether CS stimulates pneumococcal adhesion to airway cells. METHODS: Human alveolar (A549), bronchial (BEAS2-B), and primary bronchial epithelial cells (HBEpC) were exposed to CS extract (CSE), and adhesion of Streptococcus pneumoniae determined. The role of PAFR in mediating adhesion was determined using a blocker (CV-3988). PAFR transcript level was assessed by quantitative real-time PCR, and PAFR expression by flow cytometry. Lung PAFR transcript level was assessed in mice exposed to CS, and bronchial epithelial PAFR expression assessed in active-smokers by immunostaining. RESULTS: In A549 cells, CSE 1% increased pneumococcal adhesion (p<0.05 vs control), PAFR transcript level (p<0.01), and PAFR expression (p<0.01). Pneumococcal adhesion to A549 cells was attenuated by CV-3988 (p<0.001). CSE 1% stimulated pneumococcal adhesion to BEAS2-B cells and HBEpC (p<0.01 vs control). CSE 1% increased PAFR expression in BEAS2-B (p<0.01), and in HBEpC (p<0.05). Lung PAFR transcript level was increased in mice exposed to CS in vivo (p<0.05 vs room air). Active smokers (n=16) had an increased percentage of bronchial epithelium with PAFR-positive cells (p<0.05 vs never smokers, n=11). CONCLUSION: CSE stimulates PAFR-dependent pneumococcal adhesion to lower airway epithelial cells. We found evidence that CS increases bronchial PAFR in vivo.

Cigarette smoke upregulates rat coronary artery endothelin receptors in vivo.

BACKGROUND: Cigarette smoking is a strong cardiovascular risk factor and endothelin (ET) receptors are related to coronary artery diseases. The present study established an in vivo secondhand smoke (SHS) exposure model and investigated the hypothesis that cigarette smoke induces ET receptor upregulation in rat coronary arteries and its possible underlying mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Rats were exposed to SHS for 200 min daily for 8 weeks. The coronary arteries were isolated and examined. The vasoconstriction was studied by a sensitive myograph. The expression of mRNA and protein for receptors was examined by real-time PCR, Western blot and immunofluorescence. Compared to fresh air exposure, SHS increased contractile responses mediated by endothelin type A (ET(A)) and type B (ET(B)) receptors in coronary arteries. In parallel, the expression of mRNA and protein for ET(A) and ET(B) receptors of smoke exposed rats were higher than that of animals exposed to fresh air, suggesting that SHS upregulates ET(A) and ET(B) receptors in coronary arteries in vivo. Immunofluorescence staining showed that the enhanced receptor expression was localized to the smooth muscle cells of coronary arteries. The protein levels of phosphorylated (p)-Raf-1 and p-ERK1/2 in smoke exposed rats were significantly higher than in control rats, demonstrating that SHS induces the activation of the Raf/ERK/MAPK pathway. Treatment with Raf-1 inhibitor GW5074 suppressed SHS-induced enhanced contraction mediated by ET(A) receptors, and inhibited the elevated mRNA and protein levels of ET(A) and ET(B) receptors caused by SHS. The results of correlation and regression analysis showed that phosphorylation of Raf and ERK1/2 were independent determinants to affect protein expression of ET(B) and ET(A) receptors. CONCLUSIONS/SIGNIFICANCE: Cigarette smoke upregulates ET(B) and ET(A) receptors in rat coronary artery, which is associated with the activation of the Raf/ERK/MAPK pathway.

Activation of nuclear factor-κB pathway is responsible for tumor necrosis factor-α-induced up-regulation of endothelin B2 receptor expression in vascular smooth muscle cells in vitro.

The endothelin B2 (ET(B2)) receptors are induced in vascular smooth muscle cells (VSMCs) in cardiovascular diseases. We tested if in vitro short-term exposure to the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) could up-regulate ET(B2) receptors in rat mesenteric arteries, and if this effect is through activation of intracellular nuclear factor-κB (NF-κB) pathway. The mesenteric arteries were dissected from male Sprague-Dawley rats and the endothelium was removed. The arteries were co-incubated with TNF-α in serum-free Dulbecco's modified Eagle's medium. Real-time reverse transcription-PCR, Western blot and immunohistochemical staining were employed to assess the mRNA/protein expression of ET(B2) receptors and activation of NF-κB pathway. The results showed that, during organ culture, TNF-α concentration-dependently enhanced ET(B2) receptors expression at both mRNA and protein levels, paralleled with activation of NF-κB pathway in VSMC. The up-regulated ET(B2) receptor expression and NF-κB activation could be effectively suppressed by general transcriptional inhibitor actinomycin D, or either of the selective IκB kinase inhibitors wedelolactone and IMD-0354. Conclusively, the activation of intracellular NF-κB pathway is responsible for the up-regulation of ET(B2) receptors induced by short-term exposure to TNF-α. This could partly explain the toxic effects of TNF-α on VSMCs that account for cardiovascular diseases.

Secondhand smoke exposure induces Raf/ERK/MAPK-mediated upregulation of cerebrovascular endothelin ETA receptors.

BACKGROUND: Cigarette smoking enhances the risk of stroke. However, the underlying molecular mechanisms are largely unknown. The present study established an in vivo rat secondhand cigarette smoking (SHS) model and examined the hypothesis that SHS upregulates endothelin receptors with increased cerebrovascular contraction via the Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) pathway. RESULTS: Rats were exposed to SHS for up to 8 weeks. The cerebral artery vasoconstriction was recorded by a sensitive myograph. The mRNA and protein expressions for endothelin receptors in cerebral arteries were studied by real-time PCR and Western blot. Compared to fresh air exposed rats, cerebral arteries from SHS rats exhibited stronger contractile responses (P < 0.05) mediated by endothelin type A (ETA) receptors. The expressions of mRNA and protein for ETA receptors in the cerebral arteries from SHS rats were higher (P < 0.05) than that in control. SHS did not affect endothelin type B (ETB) receptor-mediated contractions, mRNA or protein levels. The results suggest that SHS upregulates ETA, but not ETB receptors in vivo. After SHS exposure, the mRNA levels of Raf-1 and ERK1/2, the protein expression of phosphorylated (p)-Raf-1 and p-ERK1/2 were increased (P < 0.05). Raf-1 inhibitor, GW5074 suppressed the enhanced ETA receptor-mediated contraction, mRNA and protein levels induced by SHS. In addition, GW5074 inhibited the SHS-caused increased mRNA and phosphorylated protein levels of Raf-1 and ERK1/2, suggesting that SHS induces activation of the Raf/ERK/MAPK pathway. CONCLUSIONS: SHS upregulates cerebrovascular ETA receptors via the Raf/ERK/MAPK pathway, which provides novel understanding of mechanisms involved in SHS-associated stroke.

Infliximab alleviates inflammation and ex vivo airway hyperreactivity in asthmatic E3 rats.

Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of asthma, and neutralization of TNF-α is an effective therapy for inflammatory diseases. The present study tested the idea that a TNF-α antibody, infliximab, may be useful in the management of asthma. E3 rats were immunized with ovalbumin (OVA)/alum and received infliximab intra-peritoneally. Two weeks later, OVA-PBS was instilled intranasally daily for 7 days. Bronchoalveolar lavage fluids (BALFs), serum and lung homogenates were collected for analysis of cells and inflammatory mediators. Contractile responses of lobar-bronchus segments to agonists were functionally tested. Pulmonary tissues were investigated using histological examination. The results showed that the sensitized 'model E3 rats' exhibited an increase in the total amount of inflammatory cells, primarily eosinophils, in BALF and pulmonary tissue, as well as epithelial damage. Serum levels of IgE increased and so did the levels of nitric oxide, inducible nitric oxide synthase, TNF-α and IL-4, IL-5 and IL-13 in lung homogenate and serum. Furthermore, the contractile responses in bronchi induced by endothelin-1, sarafotoxin 6c and bradykinin increased and isoprenaline-induced relaxations decreased. All these changes induced by the sensitization procedure were reduced by the infliximab treatment. The results suggest that infliximab prevents the development of local airway inflammation and antagonizes changes of the bronchial smooth muscle receptor phenotype, thereby blocking the development of airway smooth muscle hyperreactivity of asthmatic rats.

Alteration in contractile G-protein coupled receptor expression by moist snuff and nicotine in rat cerebral arteries.

The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression of vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET(B), serotonin 5-HT(1B), and thromboxane A(2) TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein kinases (MAPK). However, the effects of moist tobacco on the expression of GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine was kept at plasma level of snus users (25ng nicotine/ml). A high dose (250ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET(B) receptor agonist sarafotoxin 6c, 5-HT(1B) receptor agonist 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET(B), 5-HT(1B), and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25ng nicotine/ml) lowered the 5-HT(1B) receptor-mediated contraction. Furthermore, DSS shifted the TP receptor-mediated contraction curve left-wards with a stronger contraction. High dose of nicotine (250ng nicotine/ml) increased the ET(B) receptor-mediated contraction. The combined 5-HT(1B) and 5-HT(2A) receptor-mediated contraction was increased, and both the 5-CT and TxA2 induced contractions were left-ward shifted by WSS, DSS, or nicotine (250ng nicotine/ml). Only the DSS group showed that the increase of 5-HT(1B) receptor-mediated contraction occurred at the transcriptional level, demonstrated by an increased mRNA expression for the receptor. Thus, snus and nicotine alter the GPCR expression in the cerebral arteries, which may be involved in cerebral vascular disease.

Cigarette smoke extract promotes human vascular smooth muscle cell proliferation and survival through ERK1/2- and NF-κB-dependent pathways.

Tobacco use is one of the major risk factors of cardiovascular disease. The underlying molecular mechanisms that link cigarette smoke to cardiovascular disease remain unclear. The present study was designed to examine the effects of dimethyl sulfoxide (DMSO)-soluble smoke particles (DSPs) on human aortic smooth muscle cell (HASMC) cultures, and to explore the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and nuclear factor-kappaB (NF-κB) signal mechanisms involved. Serum-starved HASMCs were treated with DSPs for up to 48 h. DSPs promoted cell proliferation in a concentration-dependent manner from 0.05 to 0.2 µl/ml. Activation of ERK1/2 and NF-κB was seen after exposure to DSPs. This occurred in parallel with the increase in cell population, bromodeoxyuridine incorporation, and cyclinD1/cyclin-dependent kinase 4 expression. Blocking phosphorylation of ERK1/2 by MAPK inhibitors U0126 and PD98059, and inhibiting activation of NF-κB by IkappaB (IκB) kinase inhibitors wedelolactone or IMD-0354, abolished the DSP effects. However, either a p38 inhibitor (SB203580) or an inhibitor of lipopolysaccharide (polymyxin B), or nicotinic receptor blockers (mecamylamine and alpha-bungarotoxin), did not inhibit a DSP-induced increase in the cell population. DSPs increased the expression of intercellular adhesion molecule 1 and the release of interleukin-6 in HASMCs, both of which were inhibited by ERK1/2 or NF-κB pathway inhibitors. Furthermore, cell apoptosis and necrosis were found in serum-starved HASMCs. DSPs decreased cell death and increased B-cell leukemia/lymphoma 2 expression. Blocking phosphorylation of ERK1/2 or NF-κB attenuated DSP-induced cell death inhibition. Cigarette smoke particles stimulate HASMC proliferation and inhibit cell death. The intracellular signal mechanisms behind this involve activation of ERK1/2 and NF-κB pathways.

Up-regulation of endothelin receptors induced by cigarette smoke--involvement of MAPK in vascular and airway hyper-reactivity.

Cigarette smoke exposure is well known to cause cardiovascular and airway diseases, both of which are leading causes of death and disability in the world. However, the molecular mechanisms that link cigarette smoke to cardiovascular and airway diseases are not fully understood. Vascular and airway hyper-reactivity plays an important role in the pathogenesis of cardiovascular and airway diseases. Recent studies have demonstrated that endothelin receptor up-regulation mediates vascular and airway hyper-reactivity in response to endothelin-1 (ET-1, endothelin receptor agonist) in cardiovascular and airway diseases. In the vasculature and airways, the main functional consequences of up-regulated endothelin receptors by cigarette smoke exposure are enhanced contraction and proliferation of the smooth muscle cells, which subsequently result in abnormal contraction (spasm) and adverse proliferation (remodeling) of the vasculature and airways. The structural alteration by adverse remodeling involves changes in cell growth, cell death, cell migration, and production or degradation of the extracellular matrix. This review focuses on cigarette smoke exposure that induces activation of intracellular mitogen-activated protein kinase (MAPK) and subsequently results in the up-regulation of endothelin receptors in the vasculature and airways, which mediates vascular and airway hyper-reactivity, one of the important pathogenic characteristics of cardiovascular and airway diseases. Understanding the molecular mechanisms of how cigarette smoke causes up-regulation of endothelin receptors in the vasculature and airways may provide new strategies for the treatment of cigarette smoke-associated cardiovascular and lung diseases.