N-oleoyl glycine and N-oleoyl alanine attenuate alcohol self-administration and preference in mice.

The endocannabinoid system (ECS) plays a key modulatory role during synaptic plasticity and homeostatic processes in the brain and has an important role in the neurobiological processes underlying drug addiction. We have previously shown that an elevated ECS response to psychostimulant (cocaine) is involved in regulating the development and expression of cocaine-conditioned reward and sensitization. We therefore hypothesized that drug-induced elevation in endocannabinoids (eCBs) and/or eCB-like molecules (eCB-Ls) may represent a protective mechanism against drug insult, and boosting their levels exogenously may strengthen their neuroprotective effects. Here, we determine the involvement of ECS in alcohol addiction. We first measured the eCBs and eCB-Ls levels in different brain reward system regions following chronic alcohol self-administration using LC-MS. We have found that following chronic intermittent alcohol consumption, N-oleoyl glycine (OlGly) levels were significantly elevated in the prefrontal cortex (PFC), and N-oleoyl alanine (OlAla) was significantly elevated in the PFC, nucleus accumbens (NAc) and ventral tegmental area (VTA) in a region-specific manner. We next tested whether exogenous administration of OlGly or OlAla would attenuate alcohol consumption and preference. We found that systemic administration of OlGly or OlAla (60 mg/kg, intraperitoneal) during intermittent alcohol consumption significantly reduced alcohol intake and preference without affecting the hedonic state. These findings suggest that the ECS negatively regulates alcohol consumption and boosting selective eCBs exogenously has beneficial effects against alcohol consumption and potentially in preventing relapse.

Nanoparticles of VAV1 siRNA combined with LL37 peptide for the treatment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer-related death, and it is highly resistant to therapy owing to its unique extracellular matrix. VAV1 protein, overexpressed in several cancer diseases including pancreatic cancer (PC), increases tumor proliferation and enhances metastases formation, which are associated with decreased survival. We hypothesized that an additive anti-tumor effect could be obtained by co-encapsulating in PLGA nanoparticles (NPs), the negatively charged siRNA against VAV1 (siVAV1) with the positively charged anti-tumor LL37 peptide, as a counter-ion. Several types of NPs were formulated and were characterized for their physicochemical properties, cellular internalization, and bioactivity in vitro. NPs' biodistribution, toxicity, and bioactivity were examined in a mice PDAC model. An optimal siVAV1 formulation (siVAV1-LL37 NPs) was characterized with desirable physicochemical properties in terms of nano-size, low polydispersity index (PDI), neutral surface charge, high siVAV1 encapsulation efficiency, spherical shape, and long-term shelf-life stability. Cell assays demonstrated rapid engulfment by PC cells, a specific and significant dose-dependent proliferation inhibition, as well as knockdown of VAV1 mRNA levels and migration inhibition in VAV1+ cells. Treatment with siVAV1-LL37 NPs in the mice PDAC model revealed marked accumulation of NPs in the liver and in the tumor, resulting in an increased survival rate following suppression of tumor growth and metastases, mediated via the knockdown of both VAV1 mRNA and protein levels. This proof-of-concept study validates our hypothesis of an additive effect in the treatment of PC facilitated by co-encapsulating siVAV1 in NPs with LL37 serving a dual role as a counter ion as well as an anti-tumor agent.

A functional eEF2K-eEF2 pathway in the NAc is critical for the expression of cocaine-induced psychomotor sensitisation and conditioned place preference.

Recent evidence links synaptic plasticity and mRNA translation, via the eukaryotic elongation factor 2 kinase (eEF2K) and its only known substrate, eEF2. However, the involvement of the eEF2 pathway in cocaine-induced neuroadaptations and cocaine-induced behaviours is not known. Knock-in (KI) mice and shRNA were used to globally and specifically reduce eEF2K expression. Cocaine psychomotor sensitization and conditioned place preference were used to evaluate behavioural outcome. Changes in eEF2 phosphorylation were determined by western blot analyses. No effect was observed on the AMPA/NMDA receptor current ratio in the ventral tegmental area, 24 h after cocaine injection in eEF2K-KI mice compared with WT. However, development and expression of cocaine psychomotor sensitization were decreased in KI mice. Phosphorylated eEF2 was decreased one day after psychomotor sensitization and returned to baseline at seven days in the nucleus accumbens (NAc) of WT mice, but not in eEF2K-KI mice. However, one day following cocaine challenge, phosphorylated eEF2 decreased in WT but not KI mice. Importantly, specific targeting of eEF2K expression by shRNA in the NAc decreased cocaine condition place preference. These results suggest that the eEF2 pathway play a role in cocaine-induced locomotor sensitization and conditioned place preference.

Spatial, temporal, and cell-type-specific expression of NADPH Oxidase isoforms following seizure models in rats.

The NADPH Oxidase (NOX) enzymes are key producers of reactive oxygen species (ROS) and consist of seven different isoforms, distributed across the tissues and cell types. The increasing level of ROS induces oxidative stress playing a crucial role in neuronal death and the development of epilepsy. Recently, NOX2 was reported as a primary source of ROS production, activated by NMDA receptor, a crucial marker of epilepsy development. Here, we demonstrate spatial, temporal, and cellular expression of NOX2 and NOX4 complexes in in-vitro and in-vivo seizure models. We showed that the expression of NOX2 and NOX4 was increased in the initial 24 h following a brief seizure induced by pentylenetetrazol. Interestingly, while this elevated level returns to baseline 48 h following seizure in the cortex, in the hippocampus these levels remain elevated up to one week following the seizure. Moreover, we showed that 1- and 2- weeks following status epilepticus (SE), expression of NOX2 and NOX4 remains significantly elevated both in the cortex and the hippocampus. Furthermore, in in-vitro seizure model, NOX2 and NOX4 isoforms were overexpressed in neurons and astrocytes following seizures. These results suggest that NOX2 and NOX4 in the brain have a transient response to seizures, and these responses temporally vary depending on, seizure duration, brain region (cortex or hippocampus), and cell types.

The Prominin-1-Derived Peptide Improves Cardiac Function Following Ischemia.

Myocardial infarction (MI) remains the leading cause of death in the western world. Despite advancements in interventional revascularization technologies, many patients are not candidates for them due to comorbidities or lack of local resources. Non-invasive approaches to accelerate revascularization within ischemic tissues through angiogenesis by providing Vascular Endothelial Growth Factor (VEGF) in protein or gene form has been effective in animal models but not in humans likely due to its short half-life and systemic toxicity. Here, we tested the hypothesis that PR1P, a small VEGF binding peptide that we developed, which stabilizes and upregulates endogenous VEGF, could be used to improve outcome from MI in rodents. To test this hypothesis, we induced MI in mice and rats via left coronary artery ligation and then treated animals with every other day intraperitoneal PR1P or scrambled peptide for 14 days. Hemodynamic monitoring and echocardiography in mice and echocardiography in rats at 14 days showed PR1P significantly improved multiple functional markers of heart function, including stroke volume and cardiac output. Furthermore, molecular biology and histological analyses of tissue samples showed that systemic PR1P targeted, stabilized and upregulated endogenous VEGF within ischemic myocardium. We conclude that PR1P is a potential non-invasive candidate therapeutic for MI.

The role of mitochondria in cocaine addiction.

The incidence of cocaine abuse is increasing especially in the U.K. where the rates are among the highest in Europe. In addition to its role as a psychostimulant, cocaine has profound effect on brain metabolism, impacting glycolysis and impairing oxidative phosphorylation. Cocaine exposure alters metabolic gene expression and protein networks in brain regions including the prefrontal cortex, the ventral tegmental area and the nucleus accumbens, the principal nuclei of the brain reward system. Here, we focus on how cocaine impacts mitochondrial function, in particular through alterations in electron transport chain function, reactive oxygen species (ROS) production and oxidative stress (OS), mitochondrial dynamics and mitophagy. Finally, we describe the impact of cocaine on brain energy metabolism in the developing brain following prenatal exposure. The plethora of mitochondrial functions altered following cocaine exposure suggest that therapies maintaining mitochondrial functional integrity may hold promise in mitigating cocaine pathology and addiction.

Targeted siRNA Nanoparticles for Mammary Carcinoma Therapy.

Non-viral, polymeric-based, siRNA nanoparticles (NPs) have been proposed as promising gene delivery systems. Encapsulating siRNA in targeted NPs could confer improved biological stability, extended half-life, enhanced permeability, effective tumor accumulation, and therapy. In this work, a peptide derived from apolipoprotein B100 (ApoB-P), the protein moiety of low-density lipoprotein, was used to target siRNA-loaded PEGylated NPs to the extracellular matrix/proteoglycans (ECM/PGs) of a mammary carcinoma tumor. siRNA against osteopontin (siOPN), a protein involved in breast cancer development and progression, was encapsulated into PEGylated poly(d,l-lactic-co-glycolic acid) (PLGA) NPs using the double emulsion solvent diffusion technique. The NPs obtained possessed desired physicochemical properties including ~200 nm size, a neutral surface charge, and high siOPN loading of ~5 µg/mg. ApoB-P-targeted NPs exhibited both enhanced binding to isolated ECM and internalization by MDA-MB-231 human mammary carcinoma cells, in comparison to non-targeted NPs. Increased accumulation of the targeted NPs was achieved in the primary mammary tumor of mice xenografted with MDA-MB-231 mammary carcinoma cells as well as in the lungs, one of the main sites affected by metastases. siOPN NPs treatment resulted in significant inhibition of tumor growth (similar bioactivity of both formulations), accompanied with significant reduction of OPN mRNA levels (~40% knockdown of mRNA levels). We demonstrated that targeted NPs possessed enhanced tumor accumulation with increased therapeutic potential in mice models of mammary carcinoma.

The role of monocyte subpopulations in vascular injury following partial and transient depletion.

The innate immunity system plays a critical role in vascular repair and restenosis development. Liposomes encapsulating bisphosphonates (LipBPs), but not free BPs, suppress neointima formation following vascular injury mediated in part by monocytes. The objective of this study was to elucidate the role of monocyte subpopulations on vascular healing following LipBP treatment. The potency- and dose-dependent treatment effect of clodronate (CLOD) and alendronate (ALN) liposomes on restenosis inhibition, total monocyte depletion, and monocytes subpopulation was studied. Rats subjected to carotid injury were treated by a single IV injection of LipBPs at the time of injury. Low- and high-dose LipALN treatment (3 and 10 mg/kg, respectively) resulted in a dose-dependent effect on restenosis development after 30 days. Both doses of LipALN resulted in a dose-dependent inhibition of restenosis, but only high dose of LipALN depleted monocytes (-60.1 ± 4.4%, 48 h post injury). Although LipCLOD treatment (at an equivalent potency to 3 mg/kg alendronate) significantly reduced monocyte levels (72.1 ± 6%), no restenosis inhibition was observed. The major finding of this study is the correlation found between monocyte subclasses and restenosis inhibition. Non-classical monocyte (NCM) levels were found higher in LipALN-treated rats, but lower in LipCLOD-treated rats, 24 h after injury and treatment. We suggest that the inhibition of circulating monocyte subpopulations is the predominant mechanism by which LipBPs prevent restenosis. The effect of LipBP treatment on the monocyte subpopulation correlates with the dose and potency of LipBPs.

Monocyte-mediated drug delivery systems for the treatment of cardiovascular diseases.

Major advances have been achieved in understanding the mechanisms and risk factors leading to cardiovascular disorders and consequently developing new therapies. A strong inflammatory response occurs with a substantial recruitment of innate immunity cells in atherosclerosis, myocardial infarction, and restenosis. Monocytes and macrophages are key players in the healing process that ensues following injury. In the inflamed arterial wall, monocytes, and monocyte-derived macrophages have specific functions in the initiation and resolution of inflammation, principally through phagocytosis, and the release of inflammatory cytokines and reactive oxygen species. In this review, we will focus on delivery systems, mainly nanoparticles, for modulating circulating monocytes/monocyte-derived macrophages. We review the different strategies of depletion or modulation of circulating monocytes and monocyte subtypes, using polymeric nanoparticles and liposomes for the therapy of myocardial infarction and restenosis. We will further discuss the strategies of exploiting circulating monocytes for biological targeting of nanocarrier-based drug delivery systems for therapeutic and diagnostic applications.

Polymeric nanoparticles of siRNA prepared by a double-emulsion solvent-diffusion technique: Physicochemical properties, toxicity, biodistribution and efficacy in a mammary carcinoma mice model.

siRNA-loaded nanoparticles (NPs) administered systemically can overcome the poor stability and rapid elimination of free double-stranded RNA in circulation, resulting in increased tumor accumulation and efficacy. siRNA against osteopontin (siOPN), a protein involved in breast cancer development, was encapsulated in poly(D,L-lactic-co-glycolic acid) NPs by a double emulsion solvent diffusion (DESD) technique. We also compared the effect of polyethylenimine (PEI) molecular weight (800 Da and 25 kDa), used as the counter-ion for siRNA complexation, on the physicochemical properties of the NPs, cytotoxicity, and cellular uptake. NPs prepared by the DESD technique were obtained at the desired size (∼170 nm) using both types of PEIs, and were characterized with a neutral surface charge, high encapsulation yield (up to ∼60%), siOPN concentration of 5.6-8.4 µg/mg, stability in physiologic conditions in vitro and in vivo, and long-term shelf-life stability (> 3 years). The NPs prepared using both PEIs exhibited no cytotoxicity in primary smooth muscle culture, and no detrimental effect on mice liver enzymes following their IV administration. Following cellular uptake and biodistribution studies, the therapeutic potential of the NPs was demonstrated by a significant decrease of tumor progression and size in an ectopic xenograft model of mammary carcinoma in mice.

Liposomal temozolomide drug delivery using convection enhanced delivery.

Even though some progress in diagnosis and treatment has been made over the years, there is still no definitive treatment available for Glioblastoma multiforme (GBM). Convection-enhanced delivery (CED), a continuous infusion-mediated pressure gradient via intracranial catheters, studied in clinical trials, enables in situ drug concentrations several orders of magnitude greater than those achieved by systemic administration. We hypothesized that the currently limited efficacy of CED could be enhanced by a liposomal formulation, thus achieving enhanced drug localization to the tumor site with minimal toxicity. We hereby describe a novel approach for treating GBM by CED of liposomes containing the known chemotherapeutic agent, temozolomide (TMZ). A new technique for encapsulating TMZ in hydrophilic (PEGylated) liposomes, characterized by nano-size (121nm), low polydispersity index (<0.13) and with near-neutral charge (-ʒ,0.2mV), has been developed. Co-infusion of PEGylated Gd-DTPA liposomes and TMZ-liposomes by CED in GBM bearing rats, resulted in enhanced tumor detection with longer residence time than free Gd-DTPA. Treatment of GBM-bearing rats with either TMZ solution or TMZ-liposomes resulted in greater tumor inhibition and significantly higher survival. However, the longer survival and smaller tumor volumes exhibited by TMZ liposomal treatment in comparison to TMZ in solution were insignificant (p<0.053); and only significantly lower edema volumes were observed. Thus, there are no clear-cut advantages to use a liposomal delivery system of TMZ via CED over a drug solution.

C-reactive protein and atherothrombosis: Cause or effect?

The complex relationship between the inflammatory response and vascular injury and repair is of major importance to the pathogenesis of cardiovascular disease. CRP is not only a strong marker for cardiovascular morbidity but a modulator that suppresses local and systemic thromboregulatory pathways. In the present review we address the question of whether CRP is involved in atherogenesis, in thrombosis, or in both components of the atherothrombotic process. While CRP is present in the atherosclerotic lesion, it is probably not pro-atherogenic and correlates only minimally with atherogenesis. Alas, CRP promotes thrombus formation and vascular occlusion. Thus, CRP is most likely not affecting atheroma build-up but rather the deleterious process of plaque vulnerability and thrombus formation. Dwelling into CRP mechanism of action may lead to the design of new diagnostic modalities that will add to the predictive value of CRP in identifying those patients at high cardiovascular risk. Furthermore, defining the mechanistic domain is the foundation to the cause-effect detection of possible therapeutic interventions to counter CRP morbid effects.

Role of thromboxane receptor in C-reactive protein-induced thrombosis.

OBJECTIVE: Thromboxane A(2) and prostacyclin are thromboregulatory prostaglandins. The inflammatory C-reactive protein (CRP) promotes thrombosis after vascular injury, presumably via potentiation of thromboxane activity. Using a genetic approach, we investigated the role of thromboxane receptor (TP) pathway in CRP-induced thrombosis. METHODS AND RESULTS: Four genetically engineered mice strains were used: C57BL/6 wild-type, human CRP transgenic (CRPtg), thromboxane receptor-deficient (Tp(-/-)), and CRPtgTp(-/-) mice. CRP and TP expression were correlated, and suppression of CRP expression using small interfering RNA/CRP led to reduction in TP expression. Platelet-endothelial adherence was increased in CRPtg and suppressed in CRPtgTP(-/-)and CRPtg cells that were suppressed with TP small interfering RNA. TP deficiency in both platelets and endothelial cells was synergistic in affecting platelet-endothelial interactions. Time until arterial occlusion, measured after photochemical injury, was significantly shorter in CRPtg and prolonged in CRPtgTp(-/-) compared with controls (n=10-15, 35±3.4, 136±13.8, and 67±8.9 minutes, respectively; P<0.05). CONCLUSIONS: TP pathway is of major importance in CRP-induced thrombosis. The expression of TP is increased in CRPtg endothelial cells, and its blockade significantly suppresses the prothrombotic effect of CRP.

Endothelial C-reactive protein increases platelet adhesion under flow conditions.

While data regarding the pathogenetic role of C-reactive protein (CRP) in atherothrombosis are accumulating, it is still controversial whether local CRP secretion is of any pathobiological significance. The present study examined whether endothelial-derived CRP modulates autocrine prothrombotic activity. Endothelial cells were isolated from hearts of mice transgenic to human CRP and grown in primary cultures. Human CRP expression was confirmed in these cells compared with no expression in cultures derived from wild-type congenes. Adhesion of human platelets to endothelial cells was studied in the "cone and plate" flow system. Platelet adhesion to cells expressing CRP was significantly increased compared with that in controls (n = 6, P < 0.01). The proadhesive effect of CRP was significantly suppressed in mouse heart endothelial cells and in human umbilical vein endothelial cells following treatment with small interfering RNA for human CRP. Adhesion was modulated by an increase in P-selectin. P-selectin expression correlated with a proadhesive phenotype, and blocking P-selectin with neutralizing antibody significantly decreased the adhesion of platelets to CRP-expressing cells (40.4 ± 10.5 to 9.4 ± 6.9 platelets/high-power field, n = 5 to 6, P < 0.01). In conclusion, human CRP that is locally produced in endothelial cells increases platelet adhesion to endothelial cells under normal shear flow conditions. These findings indicate that CRP exerts a local effect on endothelial cells via P-selectin expression, which promotes platelet adhesion and subsequent thrombus formation.

Suppression of FoxO1 activity by long-chain fatty acyl analogs.

OBJECTIVE: Overactivity of the Forkhead transcription factor FoxO1 promotes diabetic hyperglycemia, dyslipidemia, and acute-phase response, whereas suppression of FoxO1 activity by insulin may alleviate diabetes. The reported efficacy of long-chain fatty acyl (LCFA) analogs of the MEDICA series in activating AMP-activated protein kinase (AMPK) and in treating animal models of diabesity may indicate suppression of FoxO1 activity. RESEARCH DESIGN AND METHODS: The insulin-sensitizing and anti-inflammatory efficacy of a MEDICA analog has been verified in guinea pig and in human C-reactive protein (hCRP) transgenic mice, respectively. Suppression of FoxO1 transcriptional activity has been verified in the context of FoxO1- and STAT3-responsive genes and compared with suppression of FoxO1 activity by insulin and metformin. RESULTS: Treatment with MEDICA analog resulted in total body sensitization to insulin, suppression of lipopolysaccharide-induced hCRP and interleukin-6-induced acute phase reactants and robust decrease in FoxO1 transcriptional activity and in coactivation of STAT3. Suppression of FoxO1 activity was accounted for by its nuclear export by MEDICA-activated AMPK, complemented by inhibition of nuclear FoxO1 transcriptional activity by MEDICA-induced C/EBPß isoforms. Similarly, insulin treatment resulted in nuclear exclusion of FoxO1 and further suppression of its nuclear activity by insulin-induced C/EBPß isoforms. In contrast, FoxO1 suppression by metformin was essentially accounted for by its nuclear export by metformin-activated AMPK. CONCLUSIONS: Suppression of FoxO1 activity by MEDICA analogs may partly account for their antidiabetic anti-inflammatory efficacy. FoxO1 suppression by LCFA analogs may provide a molecular rational for the beneficial efficacy of carbohydrate-restricted ketogenic diets in treating diabetes.

Neointimal formation is reduced after arterial injury in human crp transgenic mice.

OBJECTIVES/METHODS: Elevated CRP levels predict increased incidence of cardiovascular events and poor outcomes following interventions. There is the suggestion that CRP is also a mediator of vascular injury. Transgenic mice carrying the human CRP gene (CRPtg) are predisposed to arterial thrombosis post-injury. We examined whether CRP similarly modulates the proliferative and hyperplastic phases of vascular repair in CRPtg when thrombosis is controlled with daily aspirin and heparin at the time of trans-femoral arterial wire-injury. RESULTS: Complete thrombotic arterial occlusion at 28 days was comparable for wild-type and CRPtg mice (14 and 19%, respectively). Neointimal area at 28d was 2.5 fold lower in CRPtg (4190+/-3134 microm(2), n=12) compared to wild-types (10,157+/-8890 microm(2), n=11, p<0.05). Likewise, neointimal/media area ratio was 1.10+/-0.87 in wild-types and 0.45+/-0.24 in CRPtg (p<0.05). Seven days post-injury, cellular proliferation and apoptotic cell number in the intima were both less pronounced in CRPtg than wild-type. No differences were seen in leukocyte infiltration or endothelial coverage. CRPtg mice had significantly reduced p38 MAPK signaling pathway activation following injury. CONCLUSIONS: The pro-thrombotic phenotype of CRPtg mice was suppressed by aspirin/heparin, revealing CRP's influence on neointimal growth after trans-femoral arterial wire-injury. Signaling pathway activation, cellular proliferation, and neointimal formation were all reduced in CRPtg following vascular injury. Increasingly we are aware of CRP multipotent effects. Once considered only a risk factor, and recently a harmful agent, CRP is a far more complex regulator of vascular biology.

Effects of estradiol and raloxifene on arterial thrombosis in ovariectomized mice.

OBJECTIVE: The effects of estrogen and selective estrogen receptor modulators (eg, raloxifene) on arterial thrombosis are not well defined. This study assessed the manner and mechanism by which estrogen and raloxifene affect homeostatic pathways in ovariectomized mice after acute arterial injury. DESIGN: Female mice (3 weeks old) underwent ovariectomy or sham operation. Five days after surgery, mice were assigned to treatment with estradiol (5.3 nmol/kg), raloxifene (2.7 micromol/kg), or placebo (n = 10-12/group). The biological effects of both treatments were assessed by measurements of bone mass and the degree of uterine atrophy. After 4 months of therapy, carotid artery thrombosis was induced by photochemical injury, and the time to vascular occlusion was measured. RESULTS: Both treatments increased bone mineral density (4.1%-7.85%). Reversal of macroscopic uterine atrophy was observed only in estrogen-treated mice. Ovariectomized mice had a shorter time to occlusion compared with sham-operated mice (70.8 +/- 7.4 vs 103 +/- 11.3 min), suggesting accelerated thrombosis. Both estradiol and raloxifene significantly inhibited intra-arterial thrombosis in ovariectomized mice, prolonging the time to occlusion to 136.33 +/- 13.5 and 141.43 +/- 9.26 min, respectively. Cyclooxygenase-2 levels in the lung tissue were significantly increased by both raloxifene and estradiol with endothelial nitric oxide synthase expression being unaltered. Platelet adhesion (measured by surface coverage under a shear rate of 1,800 s for 2 min) was significantly reduced in ovariectomized animals, being 4.63% +/- 1.47%, 5.78% +/- 1.58%, and 10.04% +/- 1.33% for raloxifene, estradiol, and placebo, respectively. CONCLUSIONS: Ovariectomy amplifies thrombosis. We found that 4 months of treatment with both estradiol and raloxifene attenuates intravascular thrombosis. The antithrombotic effect was accompanied by increased expression of cyclooxygenase-2 and suppression of platelet surface adhesion.

Cannabidiol, a nonpsychoactive Cannabis constituent, protects against myocardial ischemic reperfusion injury.

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.

Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury.

C-reactive protein (CRP) is a risk marker and a potential modulator of vascular disease. Whether CRP modulates nitric oxide (NO) synthase (NOS) activity and NO metabolism remains unclear. We studied the effect of CRP on NO metabolism in transgenic mice that express human CRP (CRPtg). CRPtg and wild-type mice were subjected to controlled femoral artery wire injury. CRP serum levels at baseline and 6 and 24 h after injury were 12.4 +/- 9, 18.6 +/- 6.9, and 58.4 +/- 13 mg/l, respectively, in CRPtg mice but were undetectable at all time points in wild-type mice. Endothelial NOS protein and mRNA expression were significantly suppressed in the injured arteries of CRPtg mice (n = 5, P < 0.05). A similar reduction in eNOS expression was observed in the distant lung and heart. NO release after injury was significantly lower in CRPtg mice, as measured by nitrate and nitrite breakdown products, with a concomitant suppression of cGMP NO signaling after injury. Endothelial NOS and NO expression after vascular injury are locally and systemically suppressed in mice that express human CRP. These in vivo observations support the hypothesis that CRP modulates NO metabolism and may have implications regarding the mechanisms by which CRP modulates vascular disease.

C-reactive protein promotes monocyte-platelet aggregation: an additional link to the inflammatory-thrombotic intricacy.

INTRODUCTION: C-reactive protein (CRP) is a risk marker for cardiovascular events in humans with pro-thrombotic activities in men and mice. Formation of monocyte-platelet aggregates (MPAs) in the blood correlates with acute cardiovascular disease and provides a possible inflammatory-thrombotic link. We investigated the effect of CRP on MPA ex vivo and in vivo. METHODS AND RESULTS: Monocyte-platelet aggregation was examined by flow cytometry with dual-labeling for monocytes and platelets. Incubation of human blood with rhCRP doubled MPA formation. CRP-induced MPA formation is calcium and P-selectin dependent. Blocking antibodies to the Fc gamma receptor II had no significant effect on MPA formation. Similar effects were noted in transgenic mice, which express the human CRP gene (CRPtg). Constitutive monocyte counts and MPA levels were similar in wild-type and CRPtg mice. Lipopolysaccharide injection more than fourfold increased monocyte levels in wildtype and CRPtg mice, and preferentially increased MPA in CRPtg compared with wildtype mice. CONCLUSIONS: CRP promotes MPAtion ex vivo and in vivo. CRP-induced aggregation is calcium-dependent and mediated via P-selectin glycoprotein ligand-1 binding. Our results suggest an inflammatory-thrombotic link that is regulated by high levels of CRP. This relationship provides a potential mechanism for CRP's thrombogenic effects and a potential therapeutic target for future intervention.