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Background.Inflammatory bowel diseases (IBD) comprise two chronic relapsing intestinal disorders: Crohn's disease (CD) and ulcerative colitis (UC). IBD are the result of an abnormal inflammatory response to intestinal microbes in a genetically susceptible host. Clinically, rectal bleeding, severe diarrhea, weight loss and abdominal pain characterize IBD. Moreover, IBD are associated with a variety of extraintestinal manifestations (EIM). Increased risk of arterial and venous thrombosis in IBD patients, with a 3-fold higher risk for development venous thromboembolism is one of the most important EIM in terms of morbidity and mortality. Although the mechanisms underlying the increased thrombotic risk in IBD are not completely understood, there is evidence of abnormalities in coagulation, fibrinolysis and platelet function. However, in this context, the contribution of the vessel wall has been less explored. Endothelial dysfunction (ED) and abnormal activation of RhoA/Rho kinase (ROCK) pathway have been shown to participate in multiple pathological processes associated with thrombotic complications. We hypothesize that in IBD, the chronic inflammatory process settles the conditions for a chronic endothelial stimulation and increased ROCK activation contributing to the pathogenesis of thrombotic complications in IBD.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Background:Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We showed that chronic cocaine use is associated with markers of endothelial dysfunction (ED) (Sáez et al. Thromb Res 2011; 128:18). The hallmark of ED is a change to a prothrombotic and proadhesive phenotype associated with a decreased bioavailability of nitric oxide (NO). Several experimental studies have shown that cocaine induces ED in vitro and in vivo; however, the receptor and the signaling pathways involved in the interaction of cocaine with endothelial cells (EC) are unknown. Sigma-1 receptor (sigma-1R) belongs to the family of nonopioid sigma receptors that regulate a variety of cellular functions in neuronal, glial and peripheral cells and are involved in pathological conditions such as neurodegenerative diseases, drug addiction, smooth muscle cell contraction and ischemia. Cocaine binding to sigma-1R has been demonstrated in microglial cell lines, where is located in the lipid raft microdomains of the endoplasmic reticulum being translocated into the plasma membrane upon activation. The effect of cocaine on sigma-1R expression and function in systemic endothelial cells EC are largely unknown. We hypothesize that activation of sigma-1R in EC and its downstream signaling pathway may be involved in the pathogenesis of cocaine-associated endothelial dysfunction, a key phenomenon in the onset and progression of atherosclerosis.

Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We have recently demonstrated that chronic cocaine use is associated with endothelial dysfunction (Sáez et al. Thromb Res 2011; 128: 18), a key event in the onset and progression of atherosclerosis. There is growing evidence that the RhoA/Rho kinase (ROCK) pathway has an important pathophysiological role in vascular endothelial dysfunction. Accordingly, we hypothesized that cocaine use induces activation of RhoA/ROCK pathway.The main aim of this work was to investigate the activation of RhoA/ROCK pathway through ex vivo, in vivo and in vitro studies.Ex vivo studies. We studied 13 cocaine dependent individuals (aged 19–52 years mean age 37 years) who met DSM-IV criteria for cocaine dependence, seeking treatment for cocaine abuse and age, sex-matched healthy controls (aged 20–49 years, mean age 35 years). Samples were obtained at admission, within 72 hours of drug exposure. Endothelial cell damage was determined by enumerating circulating endothelial cells (CECs). Rho-kinase activity was assessed by the levels of phosphorylated to total myosin light chain phosphatase 1 (MYPT1-P/T) in circulating leukocytes. In vivo studies. Adult male Sprague-Dawley rats were randomly assigned to receive either cocaine (30mg/kg, provided by NIDA, USA) or saline intraperitoneally once daily for 21 days. The levels of aortic phosphorylated MYPT1 (phospho-MYPT1) were assessed by western blot in aorta extracts. In vitro experiments. Human umbilical vein endothelial cells (HUVECs) were cultured under standard conditions and supplemented for 5 hours with plasma from chronic cocaine users, normal plasma, cocaine (10μM) or vehicle. After media removal, HUVECs were either lysed for determination of ROCK activity or co-cultured with resting platelets and immunostained for von Willebrand factor (FVW). Platelet adhesion was evaluated by immunofluorescence microsocopy. Experiments were conducted in the presence or absence of ROCK inhibitors, Y-27632 (10 μM) or atorvastatin (10μM).Cocaine users showed significantly elevated number of CECs compared to the controls (65 ± 6.6 vs 14 ± 3.4 cells/mL, p: 0.0002). In the control subjects, leukocyte mean MYPT1-P/T ratio was 2.2 ± 0.8 whereas in cocaine addicts were significantly increased (9.8 ± 2.8; p 0.015). ROCK activity was higher by 100% (p: 0.019) in the aortic wall of the cocaine-treated rats compared to sham animals. HUVECs supplemented with plasma from cocaine users showed an increase in ROCK activity by 25% (p: 0.039), released significantly higher amount of FVW (p<0.05) and adhered a larger number of platelets (22.6±5 vs 7.9±3 platelets/cell, respectively; p: 0.006) compared with control plasma. Cocaine exposure induced a dramatically higher number of platelets adhered to HUVEC than in vehicle-treated cells (220±73 vs 10.2±1.1 platelets/cell, respectively; p>0.001).ROCK inhibitors, atorvastatin and Y-27632 reduced the release of FVW by HUVECs exposed to plasma from cocaine users by 65% (p: 0.004) and strongly inhibited platelet adhesion (by 75% in plasma-treated cells and by 90% in HUVECs exposed to cocaine, p:< 0.006).We found an increase in Rho kinase activity in peripheral leukocytes of cocaine abusers, in the vessel wall of rats exposed to cocaine and a marked positive effect of ROCK inhibitors on the cellular injury induced by cocaine or plasma from cocaine consumers on endothelial cells. Collectively, these data suggest that activation of RhoA/ROCK pathway plays a key role in cocaine-induced endothelial dysfunction. Inhibition of ROCK may provide therapeutic benefits in a comprehensive treatment for cocaine addiction.No relevant conflicts of interest to declare.

Abstract 2177

Washed human platelets, isolated from ACD anticoagulated whole blood, were resuspended in Tyrode's saline solution-HEPES to a final concentration of 2.5 × 108/mL, stimulated with collagen and collagen related peptide (CRP), and their activation and secretion measured by whole-blood lumi-aggregometry in the presence or absence of Panx1-specific channel blockers: carbenoxolone (Cbnx) 100 μM; probenecid (Pbn) 100 μM and mefloquine (Mfq) 10 μM. Expression of Panx1 was determined by western blot, flow cytometry and fluorescence microscopy using a specific rabbit polyclonal anti-Panx 1 antibody. Panx hemichannel function was assessed by sulforhodamine B dye uptake taken up by ADP or collagen-stimulated platelet in the presence or absence of known Panx1 channel blockers.Western blots of platelet lysates with rabbit anti-Panx1 antibody revealed a 47 kDa band corresponding to the known molecular weight of the Panx1 monomer. Flow cytometry performed on permeabilized platelets showed a significant shift in fluorescence intensity in platelets incubated with the anti-Panx1 antibody. Strong, specific staining was also observed by fluorescent microscopy of permeabilized platelets incubated with the anti-Panx1 antibody. Both platelet shape change and ATP release induced by CRP (0.5μg/ml) or collagen (1.0 μg/ml) were inhibited more than 50 % by Cbnx and Pbn; however, the most significant effect was obtained with platelets exposed to mefloquine 10 μM (see table):Platelet stimulation with Collagen or ADP induced an increase in sulforhodamine B uptake which could be significantly inhibited by preincubation with the Panx1 channel blocker probenecid.Our results demonstrated that human platelets express Panx1 Taken together, these data demonstrate that Panx1 is expressed by human platelets, evidenced by the presence of the protein by western blot, flow cytometry and immunofluorescence microscopy. From a functional standpoint, the significant effect of carbenoxolone, probenecid and especially mefloquine on collagen and CRP-induced shape change and ATP release, suggest that in human platelets Panx1 is involved in ATP release. In other cell types Panx1 channels can be opened by activation of P2 receptors; however, further studies are required to elucidate a possible association and functional interaction of Panx1 with P2X1 in platelets. Since purinergic signaling is a fundamental mechanism in platelet activation, inhibition of Panx1 hemichannels could have therapeutic potential when dampening platelet activation is desired.No relevant conflicts of interest to declare.

Abstract 1132

Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We have previously shown (Pereira et al. J Thromb Haemost 2009; 7[suppl 2]: 232a) that chronic cocaine use is associated with markers of endothelial dysfunction and platelet activation in subjects studied after recent consumption. It has been suggested that many of the adverse effects of cocaine on the vessel wall are due to its acute effects related to the sympathomimetic properties of the drug. However, from a pathogenic standpoint, we hypothesized that important pathologic consequences, such as early onset atherosclerosis and regional brain perfusion, defects, are not solely explained by the acute vasomotor actions of cocaine.The main aim of this work was to investigate the effect of short-term abstinence on markers of endothelial injury and platelet activation in chronic cocaine consumers.. We studied 23 cocaine dependent individuals (aged 19–52years mean age 30 years) who met DSM-IV criteria for cocaine dependence, seeking treatment for cocaine abuse and 25 healthy controls (aged 20–49 years, mean age 31 years). Samples were obtained at admission within 72 hours of drug exposure and after 4 weeks of strict, controlled abstinence in a rehabilitation clinic.Endothelial cell damage was assessed by enumerating circulating endothelial cells (CECs) and plasma levels of soluble markers: soluble intercellular adhesion molecule (sICAM); monocyte chemoattractant protein (MCP-1), von Willebrand factor (VWF) and high-sensitivity C reactive protein (hs-CRP). Plasma levels of soluble CD40L (sCD40L), NAP-2 and RANTES were determined to demonstrate platelet activation in vivo.Markers of endothelial cell damage/activation and platelet activation, were significantly higher in cocaine dependents individuals after recent consumption (baseline) as compared with the controls. The variations observed after 4 weeks drug withdrawal (post-abstinence) are shown in Table 1:Our results demonstrate that cocaine use is associated to endothelial dysfunction and platelet activation which are prominent findings after recent consumption. Evidence of endothelial cell activation/damage is still present after 4 weeks of strict and controlled abstinence when markers of platelet activation returned to their baseline levels. Taken together, these observations suggest that cocaine induced-endothelial cell damage is maintained independent of the acute effect of the drug on the blood vessels. The persistence of this condition may play a role in long-term ischemic complications associated with cocaine abuse such as early onset atherosclerosis and regional brain perfusion defects. Further studies on the mechanisms underlying cocaine-induced endothelial dysfunction might provide novel strategies to improve endothelial function as part of the treatment in recently abstinent cocaine addicts.No relevant conflicts of interest to declare.

Abstract 4205