Cannabinoid receptor 2 signaling does not modulate atherogenesis in mice

Background

Strong evidence supports a protective role of the cannabinoid receptor 2 (CB₂) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB₂ receptor in Murine atherogenesis.

Methods and Findings

Low density lipoprotein receptor-deficient (LDLR^−/−) mice subjected to intraperitoneal injections of the selective CB₂ receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB₂ activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB₂ ^−/−/LDLR^−/− mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB₂ ^+/+/LDLR^−/− controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-illicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB₂ receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro.

Conclusion

Our study demonstrates that both activation and deletion of the CB₂ receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB₂ in other inflammatory processes. However, in the context of atherosclerosis, CB₂ does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque.     

Tumour Cannabinoid CB(1) receptor and phosphorylated epidermal growth factor receptor expression are additive prognostic markers for prostate cancer

Background

In cultured prostate cancer cells, down-regulation of epidermal growth factor receptor (EGFR) has been implicated in mediating the antiproliferative effect of the endogenous cannabinoid (CB) ligand anandamide. Using a well-characterised cohort of prostate cancer patients, we have previously reported that expression levels of phosphorylated EGFR (pEGFR-IR) and CB₁ receptor (CB₁IR) in tumour tissue at diagnosis are markers of disease-specific survival, but it is not known whether the two markers interact in terms of their influence on disease severity at diagnosis and disease outcome.

Methodology/Principal Findings

Data from a cohort of 419 patients who were diagnosed with prostate cancer at transurethral resection for voiding difficulties was used. Scores for both tumour CB₁IR and pEGFR-IR were available in the database. Of these, 235 had been followed by expectancy until the appearance of metastases. For patients scored for both parameters, Cox proportional-hazards regression analyses using optimal cut-off scores indicated that the two measures provided additional diagnostic information not only to each other, but to that provided by the tumour stage and the Gleason score. When the cases were divided into subgroups on the basis of these cut-off scores, the patients with both CB₁IR and pEGFR-IR scores above their cut-off had a poorer disease-specific survival and showed a more severe pathology at diagnosis than patients with high pEGFR-IR scores but with CB₁IR scores below the cut-off.

Conclusions/Significance

These data indicate that a high tumour CB₁ receptor expression at diagnosis augments the deleterious effects of a high pEGFR expression upon disease-specific survival.     

High tumour cannabinoid CB1 receptor immunoreactivity negatively impacts disease-specific survival in stage II microsatellite stable colorectal cancer

Background

There is good evidence in the literature that the cannabinoid system is disturbed in colorectal cancer. In the present study, we have investigated whether CB₁ receptor immunoreactive intensity (CB₁IR intensity) is associated with disease severity and outcome.

Methodology/Principal Findings

CB₁IR was assessed in formalin-fixed, paraffin-embedded specimens collected with a consecutive intent during primary tumour surgical resection from a series of cases diagnosed with colorectal cancer. Tumour centre (n = 483) and invasive front (n = 486) CB₁IR was scored from 0 (absent) to 3 (intense staining) and the data was analysed as a median split i.e. CB₁IR <2 and ≥2. In microsatellite stable, but not microsatellite instable tumours (as adjudged on the basis of immunohistochemical determination of four mismatch repair proteins), there was a significant positive association of the tumour grade with the CB₁IR intensity. The difference between the microsatellite stable and instable tumours for this association of CB₁IR was related to the CpG island methylation status of the cases. Cox proportional hazards regression analyses indicated a significant contribution of CB₁IR to disease-specific survival in the microsatellite stable tumours when adjusting for tumour stage. For the cases with stage II microsatellite stable tumours, there was a significant effect of both tumour centre and front CB₁IR upon disease specific survival. The 5 year probabilities of event-free survival were: 85±5 and 66±8%; tumour interior, 86±4% and 63±8% for the CB₁IR<2 and CB₁IR≥2 groups, respectively.

Conclusions/Significance

The level of CB₁ receptor expression in colorectal cancer is associated with the tumour grade in a manner dependent upon the degree of CpG hypermethylation. A high CB₁IR is indicative of a poorer prognosis in stage II microsatellite stable tumour patients.     

Increased expression of cannabinoid CB₁ receptors in Achilles tendinosis

Background

The endogenous cannabinoid system is involved in the control of pain. However, little is known as to the integrity of the cannabinoid system in human pain syndromes. Here we investigate the expression of the cannabinoid receptor 1 (CB₁) in human Achilles tendons from healthy volunteers and from patients with Achilles tendinosis.

Methodology

Cannabinoid CB₁ receptor immunoreactivity (CB₁IR) was evaluated in formalin-fixed biopsies from individuals suffering from painful Achilles tendinosis in comparison with healthy human Achilles tendons.

Principal Findings

CB₁IR was seen as a granular pattern in the tenocytes. CB₁IR was also observed in the blood vessel wall and in the perineurium of the nerve. Quantification of the immunoreactivity in tenocytes showed an increase of CB₁ receptor expression in tendinosis tissue compared to control tissue.

Conclusion

Expression of cannabinoid receptor 1 is increased in human Achilles tendinosis suggesting that the cannabinoid system may be dysregulated in this disorder.     

Fatty Acid Amide Hydrolase in Prostate Cancer: Association with Disease Severity and Outcome,CB1 Receptor Expression and Regulation by IL-4

Background

Recent data have indicated that there may be a dysregulation of endocannabinoid metabolism in cancer. Here we have investigated the expression of the endocannabinoid metabolising enzyme fatty acid amide hydrolase (FAAH) in a well characterised tissue microarray from patients diagnosed with prostate cancer at transurethral resection for voiding problems.

Methodology/Principal Findings

FAAH immunoreactivity (FAAH-IR) was assessed in formalin-fixed paraffin-embedded non-malignant and tumour cores from 412 patients with prostate cancer. CB₁ receptor immunoreactivity (CB₁IR) scores were available for this dataset. FAAH-IR was seen in epithelial cells and blood vessel walls but not in the stroma. Tumour epithelial FAAH-IR was positively correlated with the disease severity at diagnosis (Gleason score, tumour stage, % of the specimen that contained tumour) for cases with mid-range CB₁IR scores, but not for those with high CB₁IR scores. For the 281 cases who only received palliative therapy at the end stages of the disease, a high tumour epithelial FAAH-IR was associated with a poor disease-specific survival. Multivariate Cox proportional-hazards regression analyses indicated that FAAH-IR gave additional prognostic information to that provided by CB₁IR when a midrange, but not a high CB₁IR cutoff value was used. Interleukin-4 (IL-4) receptor IR was found on tumour epithelial cells and incubation of prostate cancer PC-3 and R3327 AT1 cells with IL-4 increased their FAAH activity.

Conclusions/Significance

Tumour epithelial FAAH-IR is associated with prostate cancer severity and outcome at mid-range, but not high, CB₁IR scores. The correlation with CB₁IR in the tumour tissue may be related to a common local dysregulation by a component of the tumour microenvironment.     

The Expression Level of CB1 and CB2 Receptors Determines Their Efficacy at Inducing Apoptosis in Astrocytomas

Background

Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB₁ and CB₂ receptors mediate this therapeutic effect is unclear.

Principal Findings

We generated astrocytoma subclones that express set levels of CB₁ and CB₂, and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB₁, CB₂ and AKT, but still through a mechanism involving ERK1/2.

Significance

The high expression level of CB₁ and CB₂ receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB₁ and CB₂ receptors, yet still activate ERK1/2.     

Anandamide Suppresses Proliferation and Cytokine Release from Primary Human T-Lymphocytes Mainly via CB2 Receptors

Background

Anandamide (AEA) is an endogenous lipid mediator that exerts several effects in the brain as well as in peripheral tissues. These effects are mediated mainly by two types of cannabinoid receptors, named CB₁R and CB₂R, making AEA a prominent member of the “endocannabinoid” family. Also immune cells express CB₁ and CB₂ receptors, and possess the whole machinery responsible for endocannabinoid metabolism. Not surprisingly, evidence has been accumulated showing manifold roles of endocannabinoids in the modulation of the immune system. However, details of such a modulation have not yet been disclosed in primary human T-cells.

Methodology/Significance

In this investigation we used flow cytometry and ELISA tests, in order to show that AEA suppresses proliferation and release of cytokines like IL-2, TNF-α and INF-γ from activated human peripheral T-lymphocytes. However, AEA did not exert any cytotoxic effect on T-cells. The immunosuppression induced by AEA was mainly dependent on CB₂R, since it could be mimicked by the CB₂R selective agonist JWH-015, and could be blocked by the specific CB₂R antagonist SR144528. Instead the selective CB₁R agonist ACEA, or the selective CB₁R antagonist SR141716, were ineffective. Furthermore, we demonstrated an unprecedented immunosuppressive effect of AEA on IL-17 production, a typical cytokine that is released from the unique CD4+ T-cell subset T-helper 17.

Conclusions/Significance

Overall, our study investigates for the first time the effects of the endocannabinoid AEA on primary human T-lymphocytes, demonstrating that it is a powerful modulator of immune cell functions. In particular, not only we clarify that CB₂R mediates the immunosuppressive activity of AEA, but we are the first to describe such an immunosuppressive effect on the newly identified Th-17 cells. These findings might be of crucial importance for the rational design of new endocannabinoid-based immunotherapeutic approaches.     

Association between Cannabinoid CB1 Receptor Expression and Akt Signalling in Prostate Cancer

Background

In prostate cancer, tumour expression of cannabinoid CB₁ receptors is associated with a poor prognosis. One explanation for this association comes from experiments with transfected astrocytoma cells, where a high CB receptor expression recruits the Akt signalling survival pathway. In the present study, we have investigated the association between CB₁ receptor expression and the Akt pathway in a well-characterised prostate cancer tissue microarray.

Methodology/Principal Findings

Phosphorylated Akt immunoreactivity (pAkt-IR) scores were available in the database. CB₁ receptor immunoreactivity (CB₁IR) was rescored from previously published data using the same scale as pAkt-IR. There was a highly significant correlation between CB₁IR and pAkt-IR. Further, cases with high expression levels of both biomarkers were much more likely to have a more severe form of the disease at diagnosis than those with low expression levels. The two biomarkers had additive effects, rather than an interaction, upon disease-specific survival.

Conclusions/Significance

The present study provides data that is consistent with the hypothesis that at a high CB₁ receptor expression, the Akt signalling pathway becomes operative.     

Hepatitis C Virus Induces the Cannabinoid Receptor 1

Background

Activation of hepatic CB₁ receptors (CB₁) is associated with steatosis and fibrosis in experimental forms of liver disease. However, CB₁ expression has not been assessed in patients with chronic hepatitis C (CHC), a disease associated with insulin resistance, steatosis and metabolic disturbance. We aimed to determine the importance and explore the associations of CB₁ expression in CHC.

Methods

CB₁ receptor mRNA was measured by real time quantitative PCR on extracted liver tissue from 88 patients with CHC (genotypes 1 and 3), 12 controls and 10 patients with chronic hepatitis B (CHB). The Huh7/JFH1 Hepatitis C virus (HCV) cell culture model was used to validate results.

Principal Findings

CB₁ was expressed in all patients with CHC and levels were 6-fold higher than in controls (P<0.001). CB₁ expression increased with fibrosis stage, with cirrhotics having up to a 2 fold up-regulation compared to those with low fibrosis stage (p<0.05). Even in mild CHC with no steatosis (F0-1), CB₁ levels remained substantially greater than in controls (p<0.001) and in those with mild CHB (F0-1; p<0.001). Huh7 cells infected with JFH-1 HCV showed an 8-fold upregulation of CB₁, and CB₁ expression directly correlated with the percentage of cells infected over time, suggesting that CB₁ is an HCV inducible gene. While HCV structural proteins appear essential for CB₁ induction, there was no core genotype specific difference in CB₁ expression. CB₁ significantly increased with steatosis grade, primarily driven by patients with genotype 3 CHC. In genotype 3 patients, CB₁ correlated with SREBP-1c and its downstream target FASN (SREBP-1c; R = 0.37, FASN; R = 0.39, p<0.05 for both).

Conclusions/Significance

CB₁ is up-regulated in CHC and is associated with increased steatosis in genotype 3. It is induced by the hepatitis C virus.     

Crosstalk between chemokine receptor CXCR4 and cannabinoid receptor CB2 in modulating breast cancer growth and invasion

Background

Cannabinoids bind to cannabinoid receptors CB₁ and CB₂ and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB₂ may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis.

Methodology/Principal Findings

We observed high expression of both CB₂ and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB₂-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems.

Conclusions/Significance

This study provides novel insights into the crosstalk between CB₂ and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB₂ receptors could be used for developing innovative therapeutic strategies against breast cancer.     

Functional characterization of the octenol receptor neuron on the maxillary palps of the yellow fever mosquito, Aedes aegypti

Background

1-Octen-3-ol (octenol) is a common attractant released by vertebrates which in combination with carbon dioxide (CO₂) attracts hematophagous arthropods including mosquitoes. A receptor neuron contained within basiconic sensilla on the maxillary palps of adult mosquitoes responds selectively to 1-octen-3-ol. Recently, an odorant receptor (AaegOR8) known to occur on the maxillary palps was expressed in a heterologous system and demonstrated to be selectively sensitive to (R)-(−)-1-octen-3-ol, one of two enantiomeric forms. Lesser responses were elicited by stimulation with the (S)-enantiomer and various structural analogs.

Methodology/Principal Findings

Here we characterize the specificity of the octenol receptor neuron in the yellow fever mosquito, Aedes aegypti (L.), in vivo using single cell recordings. The octenol neuron is exquisitely sensitive to (R)-(−)-1-octen-3-ol; comparable responses to (S)-(+)-1-octen-3-ol were elicited only at stimulus doses over 100× that required for the (R)-enantiomer. An intermediate response closer to that elicited by the (R)-(−)-enantiomer was elicited by racemic 1-octen-3-ol. Small structural changes in (R)-(−)-1-octen-3-ol resulted in large decreases in responses. Increases in spike activity were also elicited in the octenol neuron by 2-undecanone, a known repellent; other repellents (DEET, IR3535 and picaridin) were inactive.

Conclusions/Significance

The results of our electrophysiological studies of the octenol receptor neuron in vivo approximates results of a previous study of the octenol receptor (AaegOR8 with its obligate partner Aaeg\ORco) expressed heterologously in Xenopus oocytes. By comparison of our current results with those of the heterologous expression study, we conclude that specificity of the octenol receptor neuron can be explained largely by characteristics of the OR alone without other associated proteins present in vivo. Our findings show that repellents may have specific stimulatory effects on receptor neurons and support the notion of repellents as modulators of mosquito odorant receptor activity.     

Antibodies to the α(1)-Adrenergic Receptor Cause Vascular Impairments in Rat Brain as Demonstrated by Magnetic Resonance Angiography

Background

Circulating agonistic autoantibodies acting at G protein-coupled receptors have been associated with numerous sever pathologies in humans. Antibodies directed predominantly against the α₁-adrenergig receptor were detected in patients suffering from widespread diseases such as hypertension and type 2 diabetes. Their deleterious action has been demonstrated for peripheral organs. We postulate that antibodies to the α₁-adrenergig receptor are relevant pathomolecules in diseases of the central nervous system associated with vascular impairments.

Methodology/Principal Findings

Using a rat model we studied the long-term action of antibodies against the α₁-adrenergig receptor either induced by immunization with a receptor peptide or applied by intravenous injection. The vasculature in the rat brains was investigated by time-of-flight magnetic resonance angiography using a 9.4 Tesla small animal MR imaging system. Visual examination of maximum-intensity-projections (MIPs) of brain angiographs revealed the development of vascular defects in antibody- exposed animals between three and eight months of treatment. Relative vascular areas were derived from representative MIP image sections by grayscale analysis and used to form an index of vascular circulation. Animals exposed to the action of α₁-adrenergig receptor antibodies showed significantly reduced vascular areas (p<0.05). Calculated index values indicated attenuated blood flow in both antibody-treated cohorts compared to their respective controls reaching with (relative units ± standard error, n = 10) 0.839±0.026 versus 0.919±0.026 statistical significance (p<0.05) for peptide-immunized rats.

Conclusion/Significance

We present evidence that antibodies to the α₁-adrenergig receptor cause cerebrovascular impairments in the rat. Our findings suggest the pathological significance of these antibodies in pathologies of the human central nervous system linked to impairments of brain vasculature such as stroke and dementia.     

Restoration of Corticosteroid Sensitivity by p38 Mitogen Activated Protein Kinase Inhibition in Peripheral Blood Mononuclear Cells from Severe Asthma

Background

Severe asthma accounts for a small number of asthmatics but represents a disproportionate cost to health care systems. The underlying mechanism in severe asthma remains unknown but several mechanisms are likely to be involved because of a very heterogeneous profile. We investigated the effects of a p38MAPK inhibitor in corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) from severe asthmatics and the profile of its responders.

Methodology/Principal Findings

Corticosteroid sensitivity was determined by measuring dexamethasone inhibition of CD3/28 and TNF-α induced IL-8 production in PBMCs by using ELISA. PBMCs from severe asthmatics were relatively less sensitive to dexamethasone (Dex) as compared to those of non-severe asthmatics and healthy volunteers. The IC₅₀ values of Dex negatively correlated with decreased glucocorticoid receptor (GR) nuclear translocation assessed using immunocytochemistry (r = −0.65; p<0.0005) and with decreased FEV₁ (% predicted) (r = 0.6; p<0.0005). A p38α/β inhibitor (SB203580) restored Dex-sensitivity in a subpopulation of severe asthma that was characterized by a defective GR nuclear translocation, clinically by lower FEV₁ and higher use of oral prednisolone. We also found that SB203580 partially inhibited GR phosphorylation at serine 226, resulting in increased GR nuclear translocation in IL-2/IL-4 treated corticosteroid insensitive U937s.

Conclusions/Significance

p38MAPKα/β is involved in defective GR nuclear translocation due to phosphorylation at Ser226 and this will be a useful biomarker to identify responders to p38MAPKα/β inhibitor in the future.     

Hyperbaric oxygen prevents early death caused by experimental cerebral malaria

Background

Cerebral malaria (CM) is a syndrome characterized by neurological signs, seizures and coma. Despite the fact that CM presents similarities with cerebral stroke, few studies have focused on new supportive therapies for the disease. Hyperbaric oxygen (HBO) therapy has been successfully used in patients with numerous brain disorders such as stroke, migraine and atherosclerosis.

Methodology/Principal Findings

C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were exposed to daily doses of HBO (100% O₂, 3.0 ATA, 1–2 h per day) in conditions well-tolerated by humans and animals, before or after parasite establishment. Cumulative survival analyses demonstrated that HBO therapy protected 50% of PbA-infected mice and delayed CM-specific neurological signs when administrated after patent parasitemia. Pressurized oxygen therapy reduced peripheral parasitemia, expression of TNF-α, IFN-γ and IL-10 mRNA levels and percentage of γδ and αβ CD4⁺ and CD8⁺ T lymphocytes sequestered in mice brains, thus resulting in a reduction of blood-brain barrier (BBB) dysfunction and hypothermia.

Conclusions/Significance

The data presented here is the first indication that HBO treatment could be used as supportive therapy, perhaps in association with neuroprotective drugs, to prevent CM clinical outcomes, including death.     

Ulcerative Colitis Induces Changes on the Expression of the Endocannabinoid System in the Human Colonic Tissue

Background

Recent studies suggest potential roles of the endocannabinoid system in gastrointestinal inflammation. Although cannabinoid CB₂ receptor expression is increased in inflammatory disorders, the presence and function of the remaining proteins of the endocannabinoid system in the colonic tissue is not well characterized.

Methodology

Cannabinoid CB₁ and CB₂ receptors, the enzymes for endocannabinoid biosynthesis DAGLα, DAGLβ and NAPE-PLD, and the endocannabinoid-degradating enzymes FAAH and MAGL were analysed in both acute untreated active ulcerative pancolitis and treated quiescent patients in comparison with healthy human colonic tissue by immunocytochemistry. Analyses were carried out according to clinical criteria, taking into account the severity at onset and treatment received.

Principal Findings

Western blot and immunocytochemistry indicated that the endocannabinoid system is present in the colonic tissue, but it shows a differential distribution in epithelium, lamina propria, smooth muscle and enteric plexi. Quantification of epithelial immunoreactivity showed an increase of CB₂ receptor, DAGLα and MAGL expression, mainly in mild and moderate pancolitis patients. In contrast, NAPE-PLD expression decreased in moderate and severe pancolitis patients. During quiescent pancolitis, CB₁, CB₂ and DAGLα expression dropped, while NAPE-PLD expression rose, mainly in patients treated with 5-ASA or 5-ASA+corticosteroids. The number of immune cells containing MAGL and FAAH in the lamina propria increased in acute pancolitis patients, but dropped after treatment.

Conclusions

Endocannabinoids signaling pathway, through CB₂ receptor, may reduce colitis-associated inflammation suggesting a potential drugable target for the treatment of inflammatory bowel diseases.     

Warfarin anticoagulation exacerbates the risk of hemorrhagic transformation after rt-PA treatment in experimental stroke: therapeutic potential of PCC

Background

Oral anticoagulant therapy (OAT) with warfarin is the standard of stroke prevention in patients with atrial fibrillation. Approximately 30% of patients with cardioembolic strokes are on OAT at the time of symptom onset. We investigated whether warfarin exacerbates the risk of thrombolysis-associated hemorrhagic transformation (HT) in a mouse model of ischemic stroke.

Methods

62 C57BL/6 mice were used for this study. To achieve effective anticoagulation, warfarin was administered orally. We performed right middle cerebral artery occlusion (MCAO) for 3 h and assessed functional deficit and HT blood volume after 24 h.

Results

In non-anticoagulated mice, treatment with rt-PA (10 mg/kg i.v.) after 3 h MCAO led to a 5-fold higher degree of HT compared to vehicle-treated controls (4.0±0.5 µl vs. 0.8±0.1, p<0.001). Mice on warfarin revealed larger amounts of HT after rt-PA treatment in comparison to non-anticoagulated mice (9.2±3.2 µl vs. 2.8±1.0, p<0.05). The rapid reversal of anticoagulation by means of prothrombin complex concentrates (PCC, 100 IU/kg) at the end of the 3 h MCAO period, but prior to rt-PA administration, neutralized the exacerbated risk of HT as compared to sham-treated controls (3.8±0.7 µl vs. 15.0±3.8, p<0.001).

Conclusion

In view of the vastly increased risk of HT, it seems to be justified to withhold tPA therapy in effectively anticoagulated patients with acute ischemic stroke. The rapid reversal of anticoagulation with PCC prior to tPA application reduces the risk attributed to warfarin pretreatment and may constitute an interesting therapeutic option.     

Expression and Functional Relevance of Cannabinoid Receptor 1 in Hodgkin Lymphoma

Background

Cannabinoid receptor 1 (CB₁) is expressed in certain types of malignancies. An analysis of CB₁ expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.

Design and Methods

We examined the distribution of CB₁ protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB₁ signaling on cell survival was investigated.

Results

A predominant expression of CB₁ was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB₁-abundance and displayed a dose-dependent decline of viability under CB₁ inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.

Conclusions

The present study identifies CB₁ as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.     

δ-Opioid Receptor Activation Rescues the Functional TrkB Receptor and Protects the Brain from Ischemia-Reperfusion Injury in the Rat

Objectives

δ-opioid receptor (DOR) activation reduced brain ischemic infarction and attenuated neurological deficits, while DOR inhibition aggravated the ischemic damage. The underlying mechanisms are, however, not well understood yet. In this work, we asked if DOR activation protects the brain against ischemic injury through a brain-derived neurotrophic factor (BDNF) -TrkB pathway.

Methods

We exposed adult male Sprague-Dawley rats to focal cerebral ischemia, which was induced by middle cerebral artery occlusion (MCAO). DOR agonist TAN-67 (60 nmol), antagonist Naltrindole (100 nmol) or artificial cerebral spinal fluid was injected into the lateral cerebroventricle 30 min before MCAO. Besides the detection of ischemic injury, the expression of BDNF, full-length and truncated TrkB, total CREB, p-CREB, p-ATF and CD11b was detected by Western blot and fluorescence immunostaining.

Results

DOR activation with TAN-67 significantly reduced the ischemic volume and largely reversed the decrease in full-length TrkB protein expression in the ischemic cortex and striatum without any appreciable change in cerebral blood flow, while the DOR antagonist Naltrindole aggregated the ischemic injury. However, the level of BDNF remained unchanged in the cortex, striatum and hippocampus at 24 hours after MCAO and did not change in response to DOR activation or inhibition. MCAO decreased both total CREB and pCREB in the striatum, but not in the cortex, while DOR inhibition promoted a further decrease in total and phosphorylated CREB in the striatum and decreased pATF-1 expression in the cortex. In addition, MCAO increased C11b expression in the cortex, striatum and hippocampus, and DOR activation specifically attenuated the ischemic increase in the cortex but not in the striatum and hippocampus.

Conclusions

DOR activation rescues TrkB signaling by reversing ischemia/reperfusion induced decrease in the full-length TrkB receptor and reduces brain injury in ischemia/reperfusion     

p-Cresol Affects Reactive Oxygen Species Generation,Cell Cycle Arrest,Cytotoxicity and Inflammation/Atherosclerosis-Related Modulators Production in Endothelial Cells and Mononuclear Cells

Aims

Cresols are present in antiseptics, coal tar, some resins, pesticides, and industrial solvents. Cresol intoxication leads to hepatic injury due to coagulopathy as well as disturbance of hepatic circulation in fatal cases. Patients with uremia suffer from cardiovascular complications, such as atherosclerosis, thrombosis, hemolysis, and bleeding, which may be partly due to p-cresol toxicity and its effects on vascular endothelial and mononuclear cells. Given the role of reactive oxygen species (ROS) and inflammation in vascular thrombosis, the objective of this study was to evaluate the effect of p-cresol on endothelial and mononuclear cells.

Methods

EA.hy926 (EAHY) endothelial cells and U937 cells were exposed to different concentrations of p-cresol. Cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion technique, respectively. Cell cycle distribution was analyzed by propidium iodide flow cytometry. Endothelial cell migration was studied by wound closure assay. ROS level was measured by 2′,7′-dichlorofluorescein diacetate (DCF) fluorescence flow cytometry. Prostaglandin F_2α (PGF_2α), plasminogen activator inhibitor-1 (PAI-1), soluble urokinase plasminogen activator receptor (suPAR), and uPA production were determined by Enzyme-linked immunosorbant assay (ELISA).

Results

Exposure to 100–500 µM p-cresol decreased EAHY cell number by 30–61%. P-cresol also decreased the viability of U937 mononuclear cells. The inhibition of EAHY and U937 cell growth by p-cresol was related to induction of S-phase cell cycle arrest. Closure of endothelial wounds was inhibited by p-cresol (>100 µM). P-cresol (>50 µM) also stimulated ROS production in U937 cells and EAHY cells but to a lesser extent. Moreover, p-cresol markedly stimulated PAI-1 and suPAR, but not PGF_2α, and uPA production in EAHY cells.

Conclusions

p-Cresol may contribute to atherosclerosis and thrombosis in patients with uremia and cresol intoxication possibly due to induction of ROS, endothelial/mononuclear cell damage and production of inflammation/atherosclerosis-related molecules.