The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-α and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-α, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-α injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-α in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-α. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

The endocannabinoid N-arachidonoyl ethanolamine (anandamide), found both in the CNS and in the periphery, plays a role in numerous physiological systems. One might expect that the chemically related N-arachidonoyl-L-serine (ARA-S) could also be formed alongside anandamide. We have now isolated ARA-S from bovine brain and elucidated its structure by comparison with synthetic ARA-S. Contrary to anandamide, ARA-S binds very weakly to cannabinoid CB 1 and CB2 or vanilloid TRPV1 (transient receptor potential vanilloid 1) receptors. However, it produces endothelium-dependent vasodilation of rat isolated mesenteric arteries and abdominal aorta and stimulates phosphorylation of p44͞42 mitogen-activated protein (MAP) kinase and protein kinase B͞Akt in cultured endothelial cells. ARA-S also suppresses LPS-induced formation of TNF-␣ in a murine macrophage cell line and in wild-type mice, as well as in mice deficient in CB 1 or CB2 receptors. Many of these effects parallel those reported for abnormal cannabidiol (Abn-CBD), a synthetic agonist of a putative novel cannabinoidtype receptor. Hence, ARA-S may represent an endogenous agonist for this receptor.abnormal cannabidiol ͉ anandamide ͉ cannabinoids ͉ endothelium ͉ reactive oxygen intermediates T he identification, structural elucidation, and syntheses of the plant cannabinoids in the early 1960s led to thorough investigations of the chemistry, metabolism, and pharmacology of these compounds, in particular of the psychoactive constituent ⌬ 9 -tetrahydrocannabinol (1, 2). However, until the late 1980s and early 1990s, when specific receptors were identified and shortly thereafter cloned, the mechanism of the numerous cannabinoid actions remained an enigma (3-5). Two main receptors are now known: the CB 1 receptor, found in the CNS, as well as in some peripheral tissues, and the CB 2 receptor, found predominantly in the immune system (6-8). Additional, not yet fully identified receptors are present both in the CNS and in the periphery (6, 9, 10).Because receptors in mammals are not formed to encounter a plant constituent, research was initiated to discover endogenous ligands. In the 1990s two endogenous cannabinoids (endocannabinoids) were identified, N-arachidonoyl ethanolamine (anandamide) (11) and 2-arachidonoyl-glycerol (12, 13). Additional endocannabinoids have been reported, but their biological roles are yet obscure (6, 14). Anandamide and 2-arachidonoyl-glycerol have large spectrum of physiological actions, most of which are associated with the neural and immune systems. However, cardiovascular effects, which are in part CB 1 -mediated (15), are also well established (9,14,16).Anandamide is a product of phosphatidylethanolamine (17). Because phosphatidylserine is found alongside phosphatidylethanolamine in body tissues, one might expect that arachidonoyl-Lserine (ARA-S) is also an endogenous constituent (see Fig. 1A for the structures of anandamide and ARA-S). We report that we have isolated ARA-S from bovine brain and have evaluated some of its biological propertie...

Cannabidiol (CBD) is a major, nonpsychoactive Cannabis constituent with anti-inflammatory activity mediated by enhancing adenosine signaling. Inasmuch as adenosine receptors are promising pharmaceutical targets for ischemic heart diseases, we tested the effect of CBD on ischemic rat hearts. For the in vivo studies, the left anterior descending coronary artery was transiently ligated for 30 min, and the rats were treated for 7 days with CBD (5 mg/kg ip) or vehicle. Cardiac function was studied by echocardiography. Infarcts were examined morphometrically and histologically. For ex vivo evaluation, CBD was administered 24 and 1 h before the animals were killed, and hearts were harvested for physiological measurements. In vivo studies showed preservation of shortening fraction in CBD-treated animals: from 48 +/- 8 to 39 +/- 8% and from 44 +/- 5 to 32 +/- 9% in CBD-treated and control rats, respectively (n = 14, P < 0.05). Infarct size was reduced by 66% in CBD-treated animals, despite nearly identical areas at risk (9.6 +/- 3.9 and 28.2 +/- 7.0% in CBD and controls, respectively, P < 0.001) and granulation tissue proportion as assessed qualitatively. Infarcts in CBD-treated animals were associated with reduced myocardial inflammation and reduced IL-6 levels (254 +/- 22 and 2,812 +/- 500 pg/ml in CBD and control rats, respectively, P < 0.01). In isolated hearts, no significant difference in infarct size, left ventricular developed pressures during ischemia and reperfusion, or coronary flow could be detected between CBD-treated and control hearts. Our study shows that CBD induces a substantial in vivo cardioprotective effect from ischemia that is not observed ex vivo. Inasmuch as CBD has previously been administered to humans without causing side effects, it may represent a promising novel treatment for myocardial ischemia.

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Streptococcus mutans (S. mutans) is a gram-positive facultatively anaerobic bacterium and the most common pathogen associated with tooth caries. The organism is acid tolerant and can undergo physiological adaptation to function effectively in acid environments such as carious dental plaque. Some cannabinoids have been found to have potent anti-microbial activity against gram-positive bacteria. One of these is the non-psychoactive, minor phytocannabinoid Cannabigerol (CBG). Here we show that CBG exhibits anti-bacterial activities against S. mutans. CBG halts the proliferation of planktonic growing S. mutans, which is affected by the initial cell density. High-resolution scanning electron microscopy showed that the CBG-treated bacteria become swollen with altered membrane structures. Transmission electron microscopy provided data showing that CBG treatment leads to intracellular accumulation of membrane structures. Nile red, DiOC2(3) and laurdan staining demonstrated that CBG alters the membrane properties, induces membrane hyperpolarization, and decreases the membrane fluidity. CBG-treated bacteria showed increased propidium iodide uptake and reduced calcein AM staining, suggesting that CBG increases the membrane permeability and reduces the metabolic activity. Furthermore, CBG prevented the drop in pH caused by the bacteria. In summary, we present here data showing the mechanisms by which CBG exerts its anti-bacterial effect against S. mutans.

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Streptococcus mutans is a common cariogenic bacterium in the oral cavity involved in plaque formation. Previous studies showed that Cannabigerol (CBG) has bacteriostatic and bacteriocidic activity against S. mutans. The aim of the present study was to study its effect on S. mutans biofilm formation and dispersion. S. mutans was cultivated in the presence of CBG, and the resulting biofilms were examined by CV staining, MTT assay, qPCR, biofilm tracer, optical profilometry, and SEM. Gene expression was determined by real-time qPCR, extracellular polysaccharide (EPS) production was determined by Congo Red, and reactive oxygen species (ROS) were determined using DCFH-DA. CBG prevented the biofilm formation of S. mutans shown by reduced biofilm biomass, decreased biofilm thickness, less EPS production, reduced DNA content, diminished metabolic activity, and increased ROS levels. CBG altered the biofilm roughness profile, resulting in a smoother biofilm surface. When treating preformed biofilms, CBG reduced the metabolic activity of S. mutans with a transient effect on the biomass. CBG reduced the expression of various genes involved in essential metabolic pathways related to the cariogenic properties of S. mutans biofilms. Our data show that CBG has anti-biofilm activities against S. mutans and might be a potential drug for preventive treatment of dental caries.

Recent findings on the inhibition of angiogenesis and vascular endothelial cell proliferation by anthracycline antibiotics, which contain a quinone moiety, make this type of compound a very promising lead in cancer research/therapy. We have reported that a new cannabinoid anticancer quinone, cannabidiol hydroxyquinone (HU-331), is highly effective against tumor xenografts in nude mice. For evaluation of the antiangiogenic action of cannabinoid quinones, collagen-embedded rat aortic ring assay was used. The ability of cannabinoids to cause endothelial cell apoptosis was assayed by TUNEL staining and flow cytometry analysis. To examine the genes and pathways targeted by HU-331 in vascular endothelial cells, human cDNA microarrays and polymerase chain reaction were used. Immunostaining with anti-CD31 of tumors grown in nude mice served to indicate inhibition of tumor angiogenesis. HU-331 was found to be strongly antiangiogenic, significantly inhibiting angiogenesis at concentrations as low as 300 nM. HU-331 inhibited angiogenesis by directly inducing apoptosis of vascular endothelial cells without changing the expression of pro-and antiangiogenic cytokines and their receptors. A significant decrease in the total area occupied by vessels in HU-331-treated tumors was also observed. These data lead us to consider HU-331 to have high potential as a new antiangiogenic and anticancer drug.