Etty Grad

C-reactive protein promotes platelet adhesion to endothelial cells: a potential pathway in atherothrombosis.

C‐reactive protein (CRP) is a strong predictor for acute cardiovascular events. Several endothelial prothrombotic effects of CRP have been recently reported. This study examined the effect of CRP on bovine aortic endothelial cell (EC) activation and capacity to recruit human platelets under flow conditions using the cone and plate(let) analyser method. Human recombinant CRP promoted platelet adhesion in a dose‐ and time‐dependent manner, with a maximal effect at 20 μg/ml (increase of 174% over baseline, P < 0·01). Similar effects were observed following incubation of EC with sera of transgenic mice that express human CRP (10 μg/ml). Anti‐intercellular adhesion molecule‐1 neutralising monoclonal antibody and nitric oxide donor, sodium nitroprusside, blocked the effect of CRP, reducing adhesion from 202% to 128% (P < 0·05) and 114% (P = 0·02) respectively. The pro‐adhesive effect of CRP was abolished by calphostin C (a protein kinase C inhibitor), whereas the extracellular signal‐regulated kinase antagonist, PD98059, did not have any effect. CRP promoted P‐selectin expression on the EC surface and blockade of P‐selectin reversed CRP‐induced platelet adhesion. In conclusion, CRP promoted platelet adhesion to EC. Our results emphasise the possible role of CRP in linking inflammation and thrombosis and provide a potential mechanism for the high incidence of vascular events associated with high CRP levels.

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 CRPs thrombogenic effects and a potential therapeutic target for future intervention.

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.

Aspirin reduces the prothrombotic activity of C‐reactive protein

Summary.  Aim: C‐reactive protein (CRP) is a risk marker and a potential modulator of vascular disease. Previous studies support a prothrombotic activity of CRP, with impaired thromboregulation. The present study examined the antithrombotic effect of aspirin in mice transgenic for human CRP (CRPtg mice). Mechanistic investigations further elucidated the effect of CRP on prostanoid metabolism in vivo and in vitro. Methods and Results: Administration of aspirin (30 mg kg−1 day−1) to CRPtg mice slowed the accelerated thrombosis after photochemical injury to the carotid (99 ± 32 vs. 45 ± 24 min and 75 ± 23 vs. 82 ± 26 min in wild‐type mice vs. CRPtg mice, without and following aspirin treatment, respectively). Vascular injury modulated the expression of key pathways in prostanoid metabolism differently in CRPtg mice and wild‐type mice. Suppression of cyclo‐oxygenase 2 (COX‐2)‐derived metabolism with suppression of prostaglandin I2 (PGI2) synthase and PGI2 metabolism was recorded in the injured artery with increased thromboxane receptor expression. Aspirin therapy reduced the difference in PGI2 biosynthesis between CRPtg mice and wild‐type mice. In vitro studies in human‐derived cells further supported these findings. Incubation of human umbilical vein endothelial cells (HUVECs) with human recombinant CRP (5 μg mL−1) suppressed PGI2 synthase expression and significantly increased thromboxane receptor levels. Incubation of smooth muscle cells with CRP did not affect prostanoid expression. Conclusions: CRP modulates prostanoid metabolism to favor vascular occlusion. Elevated CRP levels might predispose to the cardiovascular hazard conferred by selective COX‐2 inhibitors, and the risk mediated by CRP may be limited by aspirin.

Role of Thromboxane Receptor in C-Reactive Protein–Induced Thrombosis

Objective—Thromboxane A2 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). Conclusion—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.

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 CRPs 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 &mgr;mol/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−1 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.

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/EBPβ isoforms. Similarly, insulin treatment resulted in nuclear exclusion of FoxO1 and further suppression of its nuclear activity by insulin-induced C/EBPβ 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.

liposomal temozolomide drug delivery using convection enhanced delivery

Abstract 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 (121 nm), low polydispersity index (<0.13) and with near‐neutral charge (−3,0.2 mV), 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. Graphical abstract Figure. No Caption available.

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.