Paul Declerck

Effectiveness of the electronic cigarette: An eight-week Flemish study with six-month follow-up on smoking reduction, craving and experienced benefits and complaints.

Background: Smoking reduction remains a pivotal issue in public health policy, but quit rates obtained with traditional quit-smoking therapies remain disappointingly low. Tobacco Harm Reduction (THR), aiming at less harmful ways of consuming nicotine, may provide a more effective alternative. One promising candidate for THR are electronic cigarettes (e-cigs). The aim of this study was to investigate the efficacy of second-generation e-cigs both in terms of acute craving-reduction in the lab and in terms of smoking reduction and experienced benefits/complaints in an eight-month Randomized Controlled Trial (RCT). Design: RCT with three arms. Methods: Participants (N = 48) unwilling to quit smoking were randomized into two e-cig groups and one control group. During three lab sessions (over two months) participants, who had been abstinent for four hours, vaped/smoked for five minutes, after which we monitored the effect on craving and withdrawal symptoms. eCO and saliva cotinine levels were also measured. In between lab sessions, participants in the e-cig groups could use e-cigs or smoke ad libitum, whereas the control group could only smoke. After the lab sessions, the control group also received an e-cig. The RCT included several questionnaires, which repeatedly monitored the effect of ad libitum e-cig use on the use of tobacco cigarettes and the experienced benefits/complaints up to six months after the last lab session. Results: From the first lab session on, e-cig use after four hours of abstinence resulted in a reduction in cigarette craving which was of the same magnitude as when a cigarette was smoked, while eCO was unaffected. After two months, we observed that 34% of the e-cig groups had stopped smoking tobacco cigarettes, versus 0% of the control group (difference p < 0.01). After five months, the e-cig groups demonstrated a total quit-rate of 37%, whereas the control group showed a quit rate of 38% three months after initiating e-cig use. At the end of the eight-month study, 19% of the e-cig groups and 25% of the control group were totally abstinent from smoking, while an overall reduction of 60% in the number of cigarettes smoked per day was observed (compared to intake). eCO levels decreased, whereas cotinine levels were the same in all groups at each moment of measurement. Reported benefits far outweighed the reported complaints. Conclusion: In a series of controlled lab sessions with e-cig naïve tobacco smokers, second generation e-cigs were shown to be immediately and highly effective in reducing abstinence induced cigarette craving and withdrawal symptoms, while not resulting in increases in eCO. Remarkable (>50 pc) eight-month reductions in, or complete abstinence from tobacco smoking was achieved with the e-cig in almost half (44%) of the participants.

The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent

Plasminogen activator inhibitor 1 (PAI‐1) is believed to control proteolytic activity and cell migration during angiogenesis. We previously demonstrated in vivo that this inhibitor is necessary for optimal tumor invasion and vascularization. We also showed that PAI‐1 angiogenic activity is associated with its control of plasminogen activation but not with the regulation of cell‐matrix interaction. To dissect the role of the various components of the plasminogen activation system during angiogenesis, we have adapted the aortic ring assay to use vessels from gene‐inactivated mice. The single deficiency of tPA, uPA, or uPAR, as well as combined deficiencies of uPA and tPA, did not dramatically affect microvessel formation. Deficiency of plasminogen delayed microves‐sel outgrowth. Lack of PAI‐1 completely abolished angio‐genesis, demonstrating its importance in the control of plasmin‐mediated proteolysis. Microvessel outgrowth from PAI‐1‐/‐ aortic rings could be restored by adding exogenous PAI‐1 (wild‐type serum or purified recombi‐nant PAI‐1). Addition of recombinant PAI‐1 led to a bell‐shaped angiogenic response clearly showing that PAI‐1 is proangiogenic at physiological concentrations and antiangiogenic at higher levels. Using specific PAI‐1 mutants, we could demonstrate that PAI‐1 promotes an‐giogenesis at physiological (nanomolar) concentrations through its antiproteolytic activity rather than by interacting with vitronectin.—Devy, L., Blacher, S., Grignet‐Debrus, C., Bajou, K., Masson, V., Gerard, R. D., Gils, A., Carmeliet, G., Carmeliet, P., Declerck, P. J., Noèl, A., Foidart, J. M. The pro‐ or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 16, 147–154 (2002)

Plasma plasminogen activator inhibitor-1 in angina pectoris. Influence of plasma insulin and acute-phase response.

Plasminogen activator Inhibitor-1 (PAI-1) is an Important physiological Inhibitor of fibrlnolysls. It circulates in blood both In free active form and In Inactive form comptexed with tissue type plasminogen activator (t-PA). Control mechanisms for Its synthesis and release from hepatocytes and endothellal cells are important In the pathogenesis of thrombosis. Possible risk factors for myocardlal Infarction Include high insulin and PAH levels, which correlate with one another In healthy subjects, and fibrlnogen, which together with PAI-1, Is an acute-phase reactant We therefore studied the Interrelationships between PAI-1, plasma Insulin, and acute-phase proteins In 67 patients with angina pectoris. Plasma Insulin correlated strongly (r=0.59, p<0.001) with PAI activity, free PAI-1 antigen (r=0.60, p<0.001), and total PAI-1 antigen (r=0.58, p<0.001). The acute-phase proteins, fibrlnogen and C-reactive protein, correlated significantly with t-PA antigen, total PAI-1 antigen, and PAH/t-PA complexes but not with PAI activity or free PAI-1. The results suggest that Insulin stimulates synthesis and release of free PAI-1 (probably via hepatocytes as previously shown with cell culture) and that endothellal cell synthesis and release of t-PA, together with PAI-1, reflects a nonspecific acute-phase response to chronic vascular disease. Hyperlnsullnemia found In patients with angina pectoris could play a role In the development of myocardlal Infarction via the Induction of high plasma PAI-1 activity.

Age-Dependent Spontaneous Coronary Arterial Thrombosis in Transgenic Mice That Express a Stable Form of Human Plasminogen Activator Inhibitor-1

Background—Plasminogen activator inhibitor-1 (PAI-1) regulates fibrinolysis and has been reported to be an independent risk factor for ischemic cardiovascular events. This study describes the age-dependent development of spontaneous coronary arterial thrombi that are associated with evidence of subendocardial myocardial infarction in mice transgenic for human PAI-1. Methods and Results—We generated two independent transgenic mice founder lines that express a stable variant of active human PAI-1 under control of the murine preproendothelin-1 (mPPET-1) promoter. Backcrossed homozygous transgenic animals from founder line I had plasma PAI-1 levels of 23±12 ng/mL. PAI-1 transgenic animals younger than 4 months do not exhibit any evidence of arterial or venous thrombosis. Ninety percent of transgenic animals (n=10) older than 6 months developed spontaneous occlusions of typically multiple, penetrating coronary arteries, with histological evidence of subendocardial infarction identified in 50% of animals. Conclusions—This study shows that chronically elevated levels of PAI-1 are associated with age-dependent coronary arterial thrombosis in mice transgenic for human PAI-1. This is the first study of a murine model of coronary thrombosis that occurs in the absence of severe hypercholesterolemia or multiple genetic manipulations. These findings provide new evidence to support the hypothesis that PAI-1 excess contributes to the development of coronary arterial thrombosis.

Host-derived plasminogen activator inhibitor-1 (PAI-1) concentration is critical for in vivo tumoral angiogenesis and growth

Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in tumor progression and is believed to control proteolytic activity and cell migration during angiogenesis. We report here that host PAI-1, at physiological concentration, promotes in vivo tumor invasion and angiogenesis. In sharp contrast, inhibition of tumor vascularization was observed when PAI-1 was produced at supraphysiologic levels, either by host cells (transgenic mice overexpressing PAI-1) or by tumor cells (after transfection with murine PAI-1 cDNA). This study provides for the first time in vivo evidence for a dose-dependent effect of PAI-1 on tumor angiogenesis. Of great interest is the finding that PAI-1 produced by tumor cells, even at high concentration, did not overcome the absence of PAI-1 in the host, emphasizing the importance of the cellular source of PAI-1.

Plasminogen activator inhibitor 1 may promote tumour growth through inhibition of apoptosis

Plasminogen activator inhibitor 1 (PAI-1) has been found to be a bad prognostic factor in a number of tumours but the reason has not been fully explained. The human prostate cancer cell line PC-3 and the human promyelocytic leukaemia cell line HL-60 were used in this study to determine the effect of PAI-1 on spontaneous and induced apoptosis in culture. Apoptosis was induced with camptothecin or etoposide. Addition of a stable variant of PAI-1 or wild-type PAI-1 to these cells resulted in a significant inhibition of apoptosis. In contrast, both the latent form of PAI-1 and the stable variant of PAI-1 inactivated by a specific neutralizing monoclonal antibody, or the stable variant of PAI-1 in a complex with recombinant urokinase did not inhibit apoptosis. This indicated that the inhibitory activity of PAI-1 was required for its anti-apoptotic effect but the urokinase-type plasminogen activator receptor was not involved. These findings provide an explanation for the bad prognostic correlation of high PAI-1 levels in tumours. The anti-apoptotic effect was also found in non-tumoural cells including human umbilical vein endothelial cells and the benign human breast epithelial cell line MCF-10A, suggesting that this is a novel physiologic function of PAI-1.

Plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is an important component of the plasminogen/plasmin system as it is the main inhibitor of tissue-type and urokinase-type plasminogen activator. Consequently, PAI-1 plays an important role in cardiovascular diseases (mainly through inhibition of t-PA) and in cell migration and tumor development (mainly through inhibition of u-PA). As a member of the serpin superfamily, PAI-1 shares important structural properties with other serpins. However, PAI-1 also exhibits unique conformational and functional properties. The current paper provides an overview of the knowledge on PAI-1 gathered since its discovery two decades ago. We are discussing (a) its structural properties and their subsequent association with the functional properties, (b) its role in a wide variety of (patho)physiological processes and (c) a number of strategies to interfere with its functional properties eventually aiming at pharmacological modulation of this risk factor.

Development of a Genotype 325–Specific proCPU/TAFI ELISA

Objective—A Thr/Ile polymorphism at position 325 in the coding region of proCPU has been reported. Immunological assays, fully characterized (including genotype dependency), are required for the quantitation of proCPU levels. Methods and Results—We have generated a panel of monoclonal antibodies against human, plasma-derived proCPU. Two combinations exhibiting distinct reactivities were selected for measurement of proCPU in plasma. T12D11/T28G6-HRP yielded values of 10.1±3.1 &mgr;g/mL (mean±SD, n=86; normal donors), and T32F6/T9G12-HRP yielded values of 5.4±3.0 &mgr;g/mL. Grouping according to the 325 genotype demonstrated that T12D11/T28G6-HRP was independent to this polymorphism whereas T32F6/T9G12-HRP revealed a complete lack of reactivity with the Ile/Ile genotype (ie, 0.0±0.0, 4.2±1.7, and 7.3±2.9 &mgr;g/mL for the Ile/Ile, Ile/Thr, and Thr/Thr isoforms, respectively). Commercially available antigen assays appeared to be partially dependent on the 325 genotype (eg, 44±8.9% and 100±30% for the Ile/Ile and Thr/Thr isoforms, respectively). Conclusions—Our data demonstrate that great care should be taken when evaluating proCPU antigen values as a putative causative agent or as a diagnostic risk marker for cardiovascular events.

Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two different mechanisms

Plasminogen activator inhibitor-1 (PAI-1), a unique member of the serpin superfamily, plays an important role in fibrinolysis and is an established risk factor for cardiovascular diseases. PAI-1 can occur in three interconvertible conformations: an active, a latent and a substrate form. To study conformational and functional relationships in PAI-1, a wide variety of monoclonal antibodies were evaluated for their influence on PAI-1 activity. Out of 77 monoclonal antibodies, directed against human PAI-1, six were selected for their strong inhibitory effect towards PAI-1 activity, i.e., 80 to 100% inhibition in the presence of a 1- to 16-fold molar excess of monoclonal antibody. Detailed analysis of the reaction products formed during the interaction between PAI-1 and its target proteinases tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA), in the presence of these monoclonal antibodies, revealed two distinct mechanisms of PAI-1 inactivation. Incubation of PAI-1 with one series of monoclonal antibodies resulted in the absence of any reaction indicative for direct interaction with the reactive-site loop or a facilitated conversion to the latent conformation. The loss of PAI-1 activity in the presence of the other group of monoclonal antibodies was associated with the concomitant formation of a 41 kDa cleavage product after interaction with the target proteinase. The latter observation demonstrates that binding of these antibodies induced a conformational change thereby converting the inhibitory, active conformation to the non-inhibitory substrate conformation. No conformational changes could be observed in latent PAI-1 under these conditions. Analysis of cross-reactivity revealed that some of these functionally important epitopes were conserved throughout PAI-1 obtained from various species including rabbit mouse and/or pig, resulting in similar functional and conformational effects induced by these antibodies. Thus, we have demonstrated the occurrence of two distinct mechanisms by which the inhibitory activity of PAI-1 can be neutralized. This may have implications for the design of therapeutic or preventive strategies to interfere with PAI-1 activity. Cross-reactivity of these inhibitory antibodies with PAI-1 from various species may also allow their application in experimental animal models studying the in vivo role of PAI-1 in various diseases (e.g. atherosclerosis, thrombosis, angiogenesis,...).

The structural basis for the pathophysiological relevance of PAI-1 in cardiovascular diseases and the development of potential PAI-1 inhibitors

Plasminogen activator inhibitor-I (PAI-I) is an important component of the plasminogen/plasmin system as it is the main inhibitor of tissue-type and urokinase-type plasminogen activator. Consequently, PAI-I plays an important role in cardiovascular diseases (mainly through inhibition of t-PA), and in cell migration and tumor development (mainly through inhibition of u-PA and interaction with vitronectin). As a member of the serpin superfamily, PAI-I shares important structural properties with other serpins. However, PAI-I also exhibits unique conformational and functional properties. The current review provides an overview of the knowledge on PAI-I gathered since its discovery two decades ago. We discuss (a) its structural properties of the protein and their subsequent relation to functional activities, (b) its role in a wide variety of (patho)physiological processes and (c) a number of strategies to interfere with its functional properties eventually aiming at pharmacological modulation of this risk factor.