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Objective

Pleiotrophin (PTN) is a cytokine that is expressed by monocytes/macrophages in ischemic tissues and that promotes neovascularization, presumably by stimulating proliferation of local endothelial cells. However, the effect of PTN on monocytes/macrophages remains unknown. We investigated the role of PTN in regulating the phenotype of monocytes/macrophages.

Methods and Results

RT-PCR, real-time PCR, and fluorescence-activated cell sorter analysis revealed that the expression of PTN by monocytic cells led to a downregulation of CD68, c-fms, and CD14 monocytic cell markers and an upregulation of FLK-1, Tie-2, vascular endothelial-cadherin, platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor, CD34, GATA-2, and GATA-3 endothelial cell markers. Fibrin gel assays showed that the treatment of mouse and human monocytic cells with PTN led to the formation of tube-like structures. In vivo studies showed that PTN-expressing monocytic cells incorporated into the blood vessels of the quail chorioallantoic membrane. The intracardial injection of PTN-expressing monocytic cells into chicken embryos showed that cells integrated only into the developing vasculature. Finally, the injection of PTN-expressing monocytes into a murine ischemic hindlimb model significantly improved perfusion of the ischemic tissue.

Conclusions

PTN expression by monocytes/macrophages led to a downregulation of their monocytic cell markers and an upregulation of endothelial cell characteristics, thus inducing the transdifferentiation of monocytes into functional endothelial cells.

Vascular inflammation is an important factor which can promote diabetic complications. In this study, the inhibitory effects of aqueous extract from Prunella vulgaris (APV) on high glucose (HG)-induced expression of cell adhesion molecules in human umbilical vein endothelial cells (HUVEC) are reported. APV decreased HG-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. APV also dose-dependently inhibited HG-induced adhesion of HL-60 monocytic cells. APV suppressed p65 NF-κB activation in HG-treated cells. APV significantly inhibited the formation of intracellular reactive oxygen species (ROS). HG-stimulated HUVEC secreted gelatinases, however, APV inhibited it. APV induced Akt phosphorylation as well as activation of heme oxygenase-1 (HO-1), eNOS, and nuclear factor E2-related factor 2 (Nrf2), which may protect vascular inflammation caused by HG. In conclusion, APV exerts anti-inflammatory effect via inhibition of ROS/NF-κB pathway by inducing HO-1 and eNOS expression mediated by Nrf2, thereby suggesting that Prunella vulgaris may be a possible therapeutic approach to the inhibition of diabetic vascular diseases.

Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the “HSP60 pathway.” It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the “auto-immune hypothesis of atherosclerosis.”

Highlights

► Cigarette smoke alters the structure and function of mitochondria. ► Cigarette smoke potently induces HSP60 expression and translocation. ► Secondhand smokers are particularly prone to cigarette smoke-induced atherosclerosis.

Background

Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β2-integrin activation and function in neutrophilic transmigration through endothelium.

Methods and results

Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β2-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β2-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.

Conclusion

This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β2-integrins on the cell surface. Blocking this activation of β2-integrins might be an important target in cigarette smoke induced neutrophilic diseases.

Smoking is a major risk factor for heart disease, but the molecular effects of cigarette smoke on vascular cells are poorly understood. In this study, we demonstrate that matrix metalloproteinase-1 (MMP-1), a collagenase expressed in atherosclerosis and aneurysms but not in the normal vessel wall, is induced in the aortic endothelium of rabbits exposed to cigarette smoke. In vitro cigarette smoke extract (CSE) and one of its components, acrolein, inhibit the mammalian target of rapamycin (mTOR)/p70S6K pathway in human endothelial cells, and chemical inhibition of this pathway by rapamycin resulted in elevated MMP-1. Moreover, the tissue inhibitor of metalloproteases-3 (TIMP-3), a major regulator of angiogenesis, is significantly downregulated in aortic endothelial cells treated with CSE, acrolein, or rapamycin. These data indicate that inhibition of mTOR by cigarette smoke components is a key event in the modulation of endothelial MMP-1 and TIMP-3 expression. Our study suggests that circulating smoke components, including acrolein, contribute to vascular diseases through enhanced MMP-1 and decreased TIMP-3 secretion in the endothelium, potentially leading to impaired angiogenesis, matrix disruption, and vessel injury.

Background

Chronic elevation of glucose level activates vascular inflammation and increases endothelial adhesiveness to monocytes, an early sign of atherogenesis. This study aimed to elucidate the detailed mechanisms of high-glucose-induced endothelial inflammation, and to investigate the potential effects of Ginkgo biloba extract (GBE), an antioxidant herbal medicine, on such inflammation.

Materials and methods

Human aortic endothelial cells were cultured in high glucose or mannitol as osmotic control for 4 days. The expression of cytokines and adhesion molecules and the adhesiveness of endothelial cells to monocytes were examined. The effects of pretreatment of GBE or N-acetylcysteine, an antioxidant, were also investigated.

Results

Either high glucose or mannitol significantly increased reactive oxygen species (ROS) production, interleukin-6 secretion, intercellular adhesion molecule-1 (ICAM-1) expression, as well as endothelial adhesiveness to monocytes. The high-glucose-induced endothelial adhesiveness was significantly reduced either by an anti-ICAM-1 antibody or by an interleukin-6 neutralizing antibody. Interleukin-6 (5 ng/ml) significantly increased endothelial ICAM-1 expression. Piceatannol, a signal transducer and activator of transcription (STAT) 1/3 inhibitor, but not fludarabine, a STAT1 inhibitor, suppressed high-glucose-induced ICAM-1 expression. Pretreatment with GBE or N-acetylcysteine inhibited high-glucose-induced ROS, interleukin-6 production, STAT1/3 activation, ICAM-1 expression, and endothelial adhesiveness to monocytes.

Conclusions

Long-term presence of high glucose induced STAT3 mediated ICAM-1 dependent endothelial adhesiveness to monocytes via the osmotic-related redox-dependent interleukin-6 pathways. GBE reduced high-glucose-induced endothelial inflammation mainly by inhibiting interleukin-6 activation. Future study is indicated to validate the antioxidant/anti-inflammatory strategy targeting on interleukin-6 for endothelial protection in in vivo and clinical hyperglycemia.

Pulmonary accumulation of neutrophils is typical for active smokers who are also predisposed to multiple inflammatory and infectious lung diseases. We show that human neutrophil exposure to cigarette smoke extract (CSE) leads to an atypical cell death sharing features of apoptosis, autophagy and necrosis. Accumulation of tar-like substances in autophagosomes is also apparent. Before detection of established cell death markers, CSE-treated neutrophils are effectively recognized and non-phlogistically phagocytosed by monocyte-derived macrophages. Blockade of LOX-1 and scavenger receptor A, but not MARCO or CD36, as well as pre-incubation with oxLDL, inhibited phagocytosis, suggesting that oxLDL-like structures are major phagocytosis signals. Specific lipid (β-carotene and quercetin), but not aqueous, antioxidants increased the pro-phagocytic effects of CSE. In contrast to non-phlogistic phagocytosis, degranulation of secondary granules, as monitored by lactoferrin release, was apparent on CSE exposure, which is likely to promote pulmonary inflammation and tissue degradation. Furthermore, CSE-exposed neutrophils exhibited a compromised ability to ingest the respiratory pathogen, Staphylococcus aureus, which likely contributes to bacterial persistence in the lungs of smokers and is likely to promote further pulmonary recruitment of neutrophils. These data provide mechanistic insight into the lack of accumulation of apoptotic neutrophil populations in the lungs of smokers and their increased susceptibility to degradative pulmonary diseases and bacterial infections.

Background

Cigarette smoking is responsible for 5 million deaths worldwide each year, and is a major risk factor for cardiovascular and lung diseases. Cigarette smoke contains a complex mixture of over 4000 chemicals containing 1015 free radicals. Studies show smoke is perceived by cells as an inflammatory and xenobiotic stimulus, which activates an immune response. The specific cellular mechanisms driving cigarette smoke-induced inflammation and disease are not fully understood, although the innate immune system is involved in the pathology of smoking related diseases.

Methodology/Principle findings

To address the impact of smoke as an inflammagen on the innate immune system, THP-1 cells and Human PBMCs were stimulated with 3 and 10% (v/v) cigarette smoke extract (CSE) for 8 and 24 hours. Total RNA was extracted and the transcriptome analysed using Illumina BeadChip arrays. In THP-1 cells, 10% CSE resulted in 80 genes being upregulated and 37 downregulated by ≥1.5 fold after 8 hours. In PBMCs stimulated with 10% CSE for 8 hours, 199 genes were upregulated and 206 genes downregulated by ≥1.5 fold. After 24 hours, the number of genes activated and repressed by ≥1.5 fold had risen to 311 and 306 respectively. The major pathways that were altered are associated with cell survival, such as inducible antioxidants, protein chaperone and folding proteins, and the ubiquitin/proteosome pathway.

Conclusions

Our results suggest that cigarette smoke causes inflammation and has detrimental effects on the metabolism and function of innate immune cells. In addition, THP-1 cells provide a genetically stable alternative to primary cells for the study of the effects of cigarette smoke on human monocytes.

Background

Fas ligand (FasL) induces apoptosis in Fas-bearing target cells, such as leukocytes, and up-regulation of FasL expression on the endothelium may contribute to anti-inflammatory reactions that attenuate leukocyte extravasation during inflammation. Since oxidants generated during inflammation and cigarette smoking may modulate endothelial function, we examined the effect of H2O2 and cigarette smoke on endothelial FasL expression.

Methods

Human umbilical vein endothelial cells (HUVECs) were exposed to nontoxic concentrations of H2O2 and cigarette smoke extracts (CSE). Membrane FasL expression was assessed by immunostaining with anti-FasL antibody followed by either monolayer-cell-based spectrofluorimetry or flow cytometry. Soluble FasL in culture supernatants was measured by enzyme-linked immunosorbent assay. For the cytotoxic assay, HUVECs were exposed to H2O2 and co-cultured with neutrophils. Neutrophils were stained by a peroxidase/diaminobenzidine-based reaction, and apoptosis was evaluated on the basis of nuclear morphology after Giemsa staining. To analyze in vitro FasL expression in arteries, rat thoracic aortas were incubated with H2O2, and paraffin-embedded sections were prepared for immunohistochemistry with anti-FasL antibody.

Results

Exposure of HUVECs to H2O2 dose-dependently increased their levels of both membrane and soluble forms of FasL expression. CSE exposure also caused increased levels of FasL expression, but the increase was partially inhibited by the addition of catalase. When co-cultured with neutrophils, HUVECs exposed to H2O2 significantly promoted neutrophil apoptosis. Rat thoracic aortas incubated with H2O2 exhibited increased FasL expression on their endothelium.

Conclusion

Low levels of oxidative stress increase FasL expression on endothelial cells, thereby potentially reducing leukocyte extravasation and tissue damage.

The dietary polyphenolic compound resveratrol, by activating the protein deacetylase enzyme silent information regulator 2/sirtuin 1 (SIRT1), prolongs life span in evolutionarily distant organisms and may mimic the cytoprotective effects of dietary restriction. The present study was designed to elucidate the effects of resveratrol on cigarette smoke-induced vascular oxidative stress and inflammation, which is a clinically highly relevant model of accelerated vascular aging. Cigarette smoke exposure of rats impaired the acetylcholine-induced relaxation of carotid arteries, which could be prevented by resveratrol treatment. Smoking and in vitro treatment with cigarette smoke extract (CSE) increased reactive oxygen species production in rat arteries and cultured coronary arterial endothelial cells (CAECs), respectively, which was attenuated by resveratrol treatment. The smoking-induced upregulation of inflammatory markers (ICAM-1, inducible nitric oxide synthase, IL-6, and TNF-α) in rat arteries was also abrogated by resveratrol treatment. Resveratrol also inhibited CSE-induced NF-κB activation and inflammatory gene expression in CAECs. In CAECs, the aforementioned protective effects of resveratrol were abolished by knockdown of SIRT1, whereas the overexpression of SIRT1 mimicked the effects of resveratrol. Resveratrol treatment of rats protected aortic endothelial cells against cigarette smoking-induced apoptotic cell death. Resveratrol also exerted antiapoptotic effects in CSE-treated CAECs, which could be abrogated by knockdown of SIRT1. Resveratrol treatment also attenuated CSE-induced DNA damage in CAECs (comet assay). Thus resveratrol and SIRT1 exert antioxidant, anti-inflammatory, and antiapoptotic effects, which protect the endothelial cells against the adverse effects of cigarette smoking-induced oxidative stress. The vasoprotective effects of resveratrol will likely contribute to its anti-aging action in mammals and may be especially beneficial in patho-physiological conditions associated with accelerated vascular aging.

Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of naïve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased naïve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.

Previous studies have shown that ambient ultrafine particles with diameters less than 100 nm (UFPs) can pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant dysfunction of lung endothelial cells. However, no studies have addressed the potential combined effects of UFPs and cigarette smoke on vascular endothelial cells. We hypothesized that co-exposure to UFPs and cigarette smoke extract (CSE) may cause combined effects on activation of endothelial cells and dysfunction of endothelium by oxidative stress through activation of NADPH oxidase. We determined the effects of UFPs with or without CSE on mouse pulmonary microvascular endothelial cells (MPMVEC) obtained from C57BL/6J (wild-type) and gp91phox knock-out mice (gp91phox is one of the key components of NADPH oxidase, one of ROS generators). Our results showed that exposure of MPMVEC from wild-type mice to UFPs or CSE, at a non-toxic dose, induced reactive oxygen species (ROS) generation, increased phosphorylation of p38 and Erk1/2, and up-regulated early growth response -1 (Egr-1) and IL-6 genes. These effects were significantly enhanced when cells were co-exposed to both UFPs and CSE. However, exposure of MPMVEC from gp91phox knock-out mice did not induce the above effects. Furthermore, UFPs- and/or CSE-induced Egr-1 mRNA upregulation was attenuated significantly when cells were pre-treated with p38 specific inhibitor, SB 203580, or MEK1/2 inhibitor, PD98059, and Egr-1 siRNA treatment abolished UFPs- and/or CSE- induced overexpression of IL-6. Our results suggest that UFPs and/or CSE caused activation of NADPH oxidase, resulting in ROS generation that led to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs and upregulation of Egr-1. Those effects may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that co-exposure to UFPs and CSE causes enhanced injury to endothelial cells.

Cigarette smoke (CS) is the major cause of lung cancer and contributes to the development of other malignancies. Attempts have been made to construct reduced toxicity cigarettes, presumed to have diminished genotoxic potential. One such product on the market is the tobacco and nicotine free (T&N-free) cigarette type made from lettuce and herbal extracts. We have recently developed a sensitive assay of the genotoxicity of CS based on cytometric analysis of induction of the DNA damage response (DDR) in normal human pulmonary endothelial or A549 pulmonary adenocarcinoma cells. In the present study, we observed that exposure of A549 cells to CS from T&N-free cigarettes induced a smoke-dose dependent DDR as evidenced by phosphorylation (activation) of the Ataxia telangiectasia mutated (ATM) protein kinase and of the histone H2AX (γH2AX). the extent of DDR induced by T&N-free smoke was distinctly greater than that induced by comparable doses of CS from reference cigarettes (2R4F) containing tobacco and nicotine. The pattern of DDR induced by T&N-free smoke was similar to that of 2R4F cigarettes in terms of the cell cycle phase specificity and involvement of reactive oxygen species (ROS). The data also imply that similar to 2R4F exposure of cells to T&N-free smoke leads to formation of double-strand DNA breaks (DSBs) resulting from collapse of replication forks upon collision with the primary ssDNA lesions induced by smoke. Since DSBs are potentially carcinogenic our data indicate that smoking tobacco and nicotine-free cigarettes is at least as hazardous as smoking cigarettes containing tobacco and nicotine.

Cigarette smoking is one of the major causes of carcinogenesis. Direct genotoxicity induced by cigarette smoke leads to initiation of carcinogenesis. Nongenotoxic (epigenetic) effects of cigarette smoke also act as modulators altering cellular functions. These two effects underlie the mechanisms of tumor promotion and progression. While there is no lack of general reviews on the genotoxic and carcinogenic potentials of cigarette smoke in lung carcinogenesis, updated review on the epigenetic effects and molecular mechanisms of cigarette smoke and carcinogenesis, not limited to lung, is lacking. We are presenting a comprehensive review of recent investigations on cigarette smoke, with special attentions to nicotine, NNK, and PAHs. The current understanding on their molecular mechanisms include (1) receptors, (2) cell cycle regulators, (3) signaling pathways, (4) apoptosis mediators, (5) angiogenic factors, and (6) invasive and metastasis mediators. This review highlighted the complexity biological responses to cigarette smoke components and their involvements in tumorigenesis.

Accumulating evidence suggests a direct role for cigarette smoke in pulmonary vascular remodeling, which contributes to the development of pulmonary hypertension. However, the molecular mechanisms underlying this process remain poorly understood. Platelet-derived growth factor (PDGF) is a potential mitogen and chemoattractant implicated in several biological processes, including cell survival, proliferation, and migration. In this study, we investigated the effect of cigarette smoke extract (CSE) on cell proliferation of rat pulmonary artery smooth muscle cells (rPASMCs). We found that stimulation of rPASMCs with CSE significantly increased cell proliferation and promoted cell cycle progression from G1 phase to the S and G2 phases. CSE treatment also significantly upregulated the mRNA and protein levels of PDGFB and PDGFRβ. Our study also revealed that Rottlerin, an inhibitor of PKCδ signaling, prevented CSE-induced cell proliferation, attenuated the increase of S and G2 phase populations induced by CSE treatment, and downregulated PDGFB and PDGFRβ mRNA and protein levels in rPASMCs exposed to CSE. Collectively, our data demonstrated that CSE-induced cell proliferation of rPASMCs involved upregulation of the PKCδ-PDGFB pathway.

Purpose:

Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease (COPD). Recent studies propose a link between endoplasmic reticulum (ER) stress and emphysema, demonstrated by increased ER stress markers under smoking conditions. Here, we investigate whether cigarette smoke-induced ER stress is cell specific and correlates with acute and chronic cigarette smoke exposure.

Methods:

Gene and protein expression changes in human primary lung cell cultures following cigarette smoke extract (CSE) exposure were monitored by qPCR and Western blot analysis. Mice and guinea pigs were exposed to cigarette smoke and ER stress markers examined in whole lung homogenates. Inflammatory cells from the bronchoalveolar lavage fluid of 10 days smoke exposed mice were also examined.

Results:

Cigarette smoke induced a trend increase in the ER stress response through an activating transcription factor 4 (ATF4) mediated induction of C/EBP homologous protein (CHOP) in primary small airway epithelial cells. Bronchial epithelial cells and macrophages responded similarly to CSE. Wild-type mice and guinea pigs exposed to acute levels of cigarette smoke exhibited increased levels of CHOP but not at significant levels. However, after long-term chronic cigarette smoke exposure, CHOP expression was reduced. Interestingly, inflammatory cells from smoke exposed mice had a significant increase in CHOP/ATF4 expression.

Conclusion:

A trend increase in CHOP levels appear in multiple human lung cell types following acute cigarette smoke exposure in vitro. In vivo, inflammatory cells, predominately macrophages, demonstrate significant cigarette smoke-induced ER stress. Early induction of CHOP in cigarette smoke may play a pivotal role in early induction of lung disease, however in vivo long-term cigarette smoke exposure exhibited a reduction in the ER stress response.

1. ABSTRACT

Cigarette smoking is the major cause of preventable morbidity and mortality in the United States and constitutes a major risk factor for atherosclerotic vascular disease, including coronary artery disease and stroke. Increasing evidence supports the hypothesis that oxidative stress and inflammation provide the pathophysiological link between cigarette smoking and CAD. Previous studies have shown that cigarette smoke activates leukocytes to release reactive oxygen and nitrogen species (ROS/RNS) and secrete pro-inflammatory cytokines, increases the adherence of monocytes to the endothelium and elicits airway inflammation. Here we present an overview of the direct effects of water-soluble cigarette smoke constituents on endothelial function, vascular ROS production and inflammatory gene expression. The potential pathogenetic role of peroxynitrite formation, and downstream mechanisms including poly (ADP-ribose) polymerase (PARP) activation in cardiovascular complications in smokers are also discussed.

Platelets shed microparticles not only upon activation, but also upon ageing by an apoptosis-like process (apoptosis-induced platelet microparticles, PMap). While the activation-induced microparticles have widely been studied, not much is known about the (patho)physiological consequences of PMap formation. Flow cytometry and scanning electron microscopy demonstrated that PMap display activated integrins and interact to form microparticle aggregates. PMap were chemotactic for monocytic cells, bound to these cells, an furthermore stimulated cell adhesion and spreading on a fibronectin surface. After prolonged incubation, PMap promoted cell differentiation, but inhibited proliferation. Monocyte membrane receptor analysis revealed increased expression levels of CD11b (integrin αMβ2), CD14 and CD31 (platelet endothelial cell adhesion molecule-1), and the chemokine receptors CCR5 and CXCR4, but not of CCR2. This indicated that PMap polarized the cells into resident M2 monocytes. Cells treated with PMap actively consumed oxidized low-density lipoprotein (oxLDL), and released matrix metalloproteinases and hydrogen peroxide. Further confirmation for the differentiation towards resident professional phagocytes came from the finding that PMap stimulated the expression of the (ox)LDL receptors, CD36 and CD68, and the production of proinflammatory and immunomodulating cytokines by monocytes. In conclusion, interaction of PMap with monocytic cells has an immunomodulating potential. The apoptotic microparticles polarize the cells into a resident M2 subset, and induce differentiation to resident professional phagocytes.

Confluent cultures of aortic endothelial cells contain two different cell-cell adhesion mechanisms distinguished by their requirement for calcium during trypsinization and adhesion. A hybridoma clone was isolated producing a monoclonal antibody Ec6C10, which inhibits Ca2(+)- dependent adhesion of endothelial cells. There was no inhibition of Ca2(+)-independent adhesion of endothelial cells and only a minor effect on Ca2(+)-dependent adhesion of smooth muscle cells. Immunoblotting analysis shows that the antibody Ec6C10 recognizes a protein in endothelial but not epithelial cells with an apparent molecular weight of 135,000 in reducing conditions and 130,000 in non- reducing conditions. Monoclonal antibody Ec6C10 reacts with an antigen at the cell surface as shown by indirect immunofluorescence of confluent endothelial cells in a junctional pattern outlining the cobblestone morphology of the monolayer. Removal of extracellular calcium increased the susceptibility of the antigen recognized by antibody Ec6C10 to proteolysis by trypsin. The role of the Ca2(+)- dependent cell adhesion molecule in organization of the dense peripheral microfilament band in confluent endothelial cells was examined by adjusting the level of extracellular calcium to modulate cell-cell contact. Addition of the monoclonal antibody Ec6C10 at the time of the calcium switch inhibited the extent of formation of the peripheral F-actin band. These results suggest an association between cell-cell contact and the peripheral F-actin band potentially through the Ca2(+)-dependent CAM.

DNA polymorphisms in endothelial nitric oxide synthase (eNOS) gene have been shown to be associated with constitutive eNOS expression and coronary artery disease (CAD). In the present study we explored the hypothesis whether genotype-dependent effects can be maintained in vitro during replication, or the effect is conditional on in vivo biological environments. Human umbilical vein endothelial cells (HUVEC) were collected and cultured from 89 normal deliveries of Mexican Americans. The cells were treated with or without cigarette smoking extracts (CSE) and genotypes of eNOS polymorphisms were determined by PCR. We measured the levels of eNOS by ELISA and its binding proteins including heat-shock protein 90 (Hsp-90) and caveolin-1 by Western blotting. The rare C allele for the promoter T786C polymorphism (0.2), and the rare 4 × 27-bp repeat allele in the intron 4 (0.30) were different from those reported in other populations. Yet, the rare T allele in the exon 7 (G894T polymorphism) was similar as others. After four passages in vitro, both the in-tron 4 and promoter polymorphisms maintained significant effects on eNOS mRNA levels in HUVECs (P < 0.05). However, the effects on eNOS protein and enzyme activity were less consistent. Although primary smokers had significantly lower eNOS protein levels (P < 0.05), the in vitro CSE treatment on cultured HUVECs only resulted in a significant reduction in NO levels as measured by the stable metabolites of nitrite/nitrate (P < 0.001). Neither Hsp-90 nor caveolin-1—important eNOS regulators—appears to mediate the genotype-smoking effects on eNOS expression although HUVECs did produce more Hsp-90 when exposed to CSE. Our study demonstrates that endothelial cells maintain genotype-dependent expression even after the deprivation of in vivo environment. However, the cigarette smoking–genotype interaction may require such in vivo conditions to be manifested.

Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of β2 integrin in the unretracted tails.

Background

Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.

Methods

HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni's t test

Results

CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.

Conclusion

Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.

Exposure to cigarette smoke is a risk factor contributing to the severity of respiratory tract infections associated with respiratory syncytial virus (RSV). Stimulation of airway epithelial cells by either RSV or cigarette smoke condensate (CSC) has been shown to induce secretion of the proinflammatory chemokines. However, the effect of coexposure of airway epithelial cells to CSC and RSV on inducible chemokine production has not been previously investigated. The results of this study indicate that CSC costimulation significantly increased RSV-induced interleukin-8 (IL-8) and monocyte chemoattactant protein-1 gene and protein expression when compared with each stimulus alone. Promoter deletion studies identified the interferon stimulatory response element (ISRE) of the IL-8 promoter as a critical region responsible for the synergistic increase of IL-8 gene transcription during mixed exposure. CSC costimulation enhanced RSV-induced activation of interferon regulatory factor (IRF)-1 and IRF-7, which bind to the ISRE site. CSC also furthered RSV-induced activation of the transcription factor nuclear factor kappa B (NF-κB), as shown by increased NF-κB DNA binding to its specific site of the IL-8 promoter and increased NF-κB–driven gene transcription. Therefore, our data demonstrate that a combined exposure to CSC and RSV synergistically increases chemokine expression in airway epithelial cells, suggesting that CSC contributes to an exuberant immune response to RSV by stimulating overlapping signal transduction pathways.

Cigarette smoking is one of the most important and preventable risk factors for atherosclerosis. However, because of the complex composition of cigarette smoke, the detailed pathophysiological mechanisms are not fully understood. Based on controversial reports on the pro-atherogenic activity of cigarette smoke condensate, also called tar fraction (CSC), we decided to analyse the effects of CSC on the viability of endothelial cells in vitro. The results of this study show that low concentrations of the hydrophobic tar fraction induces DNA damage resulting in a P53-dependent and BCL-XL-inhibitable death cascade. Western blot analyses showed that this cascade is caspase-independent and immunofluorescence analysis have shown that the apoptotic death signalling is mediated by the release of apoptosis-inducing factor. Higher CSC concentrations also induce apoptotic-like signalling but the signalling cascade is then redirected to necrosis. Despite the fact that CSC induces a profound increase in cellular reactive oxygen species production, antioxidants exhibit only a minimal cell death protective effect. Our data indicates that not only hydrophilic constituents of cigarette smoke extract, but also CSC is harmful to endothelial cells. The mode and the outcome of CSC-induced cell death signalling are highly concentration dependent: lower concentrations induce caspase-independent apoptosis-like cell death, whereas incubation with higher concentrations interrupts apoptotic signalling and induces necrosis.

Rationale: Impaired endothelial cell–dependent vasodilation, inflammation, apoptosis, and proliferation are manifestations of endothelial dysfunction in chronic obstructive pulmonary disease (COPD). Prostacyclin (PGI2) is a major product of the cyclooxygenase pathway with potent vasodilatory and antimitogenic properties and may be relevant to endothelial dysfunction in COPD.

Objectives: To determine if PGI2 expression is altered in smoking-related lung disease and if it may be protective in COPD-associated endothelial dysfunction.

Methods: We evaluated, by immunohistochemistry, Western blotting, and polymerase chain reaction, human emphysema tissue compared with normal tissue for expression of prostacyclin synthase (PGI2S). We examined the effects of cigarette smoke extract (CSE) and aldehyde components on eicosanoid expression in primary human pulmonary microvascular endothelial cells. Finally, we used a murine model of lung-specific PGI2S overexpression and in vitro studies to determine if PGI2 expression has protective effects on cigarette smoke–induced endothelial apoptosis.

Measurements and Main Results: Human emphysema lung tissue exhibited lower PGI2S expression within the pulmonary endothelium than in normal lung. In vitro studies demonstrated that CSE, and in particular the α,β unsaturated aldehyde acrolein, suppressed PGI2S gene expression, whereas CSE significantly induced the upstream mediators COX-2 and cytosolic phospholipase A2 in human pulmonary microvascular endothelial cells. Mice with lung-specific PGI2S overexpression exhibited less endothelial apoptosis after chronic smoke exposure. In vitro, iloprost exhibited protective effects on CSE-induced apoptosis.

Conclusions: PGI2 has protective effects in the pulmonary vasculature after acute and chronic cigarette smoke exposure. An imbalance in eicosanoid expression may be important to COPD-associated endothelial dysfunction.