Upregulation Of Endothelial Adhesion Molecules Research Articles

Shenfu injection ameliorates endotoxemia-associated endothelial dysfunction and organ injury via inhibiting PI3K/Akt-mediated glycolysis

Ethnopharmacological relevanceMicrocirculatory dysfunction is one of the main characteristics of sepsis. Shenfu Injection (SFI) as a traditional Chinese medicine is widely applied in clinical severe conditions. Recent studies have shown that SFI has the ability to ameliorate sepsis-induced inflammation and to improve microcirculation perfusion. Aim of the studyThis study aims to investigate the underlying mechanism of SFI for ameliorating sepsis-associated endothelial dysfunction and organ injury. Materials and methodsSide-stream dark-field (SDF) imaging was used to monitor the sublingual microcirculation of septic patients treated with or without SFI. Septic mouse model was used to evaluate the effects of SFI in vivo. Metabolomics and transcriptomics were performed on endothelial cells to identify the underlying mechanism for SFI-related protective effect on endothelial cells. ResultsSFI effectively abolished the disturbance and loss of sublingual microcirculation in septic patients. Twenty septic shock patients with or without SFI administration were enrolled and the data showed that SFI significantly improved the levels of total vessel density (TVD), perfused vessel density (PVD), microvascular flow index (MFI), and the proportion of perfused vessels (PPV). The administration of SFI significantly decreased the elevated plasma levels of Angiopoietin-2 (Ang2) and Syndecan-1, which are biomarkers indicative of endothelial damage in sepsis patients. In the mouse septic model in vivo, SFI inhibited the upregulation of endothelial adhesion molecules and Ly6G + neutrophil infiltration while restored the expression of VE-Cadherin in the vasculature of the lung, kidney, and liver tissue. Additionally, SFI reduced the plasma levels of Ang2, Monocyte Chemoattractant Protein-1(MCP1), and Interleukin-6 (IL6), and alleviated liver and kidney injury in septic mice. Moreover, SFI significantly inhibited the inflammatory activation and increased permeability of endothelial cells induced by endotoxins in vitro. By performing metabolomics and transcriptomics, we identified the activation of PI3K/Akt-mediated glycolysis as the underlying mechanism for SFI-related protective effect on endothelial cells. ConclusionsOur findings revealed that SFI may improve microcirculation perfusion and endothelial function in sepsis via inhibiting PI3K/Akt-mediated glycolysis, providing theoretical evidence for the clinical application of SFI.

Endothelial Cell-Selective Adhesion Molecule Enhances Tumor Growth in a HFpEF Mouse Model

Accumulating epidemiological evidence shows that the incidence of multiple types of cancer, such as melanoma and GI cancers, is significantly higher in patients with heart failure with preserved ejection fraction (HFpEF). However, the mechanistic relationships between HFpEF and cancer development are poorly understood. Impaired angiogenesis, upregulation of endothelial adhesion molecules and chronic low-grade inflammation are common in HFpEF. Here we examined the role of endothelial cell-selective adhesion molecule (ESAM) in the progression of malignant melanoma in a HFpEF mouse model. Orthotopic melanoma xenografts (B16F10-Luc2) were used and implanted subcutaneously in mice underwent uninephrectomy and aldosterone infusion (UNX-Aldo with 1% high salt drinking water for 4 weeks) and tumor growth was monitored with bioluminescent imaging. We found that ESAM expression and serum levels were elevated in the UNX-Aldo mice, which was accompanied by enhanced melanoma growth and metastasis rate. ESAM promoted tube formation and increased phospho-Akt, phospho-PKCα and phospho-ERK1/2 in cultured endothelial cells. Flow cytometry analysis showed elevated number of tumor-associated neutrophils (CD11b+Ly6G+Ly6C-) and myeloid-derived suppressor cells (MDSCs, CD11b+F4/80-Gr-1+) in the UNX-Aldo mice. UNX-Aldo mice with genetic deletion of ESAM (ESAM KO) showed a reduced tumor growth and reduced number of tumor-associated neutrophils and MDSCs. Collectively, our results indicate that in HFpEF, a proinflammatory ESAM activation may act to promote tumor angiogenesis, neutrophil and MDSCs tumor infiltration, thereby mitigating a proper immune response and facilitate tumor growth. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Type 2 Diabetes Mellitus Provokes Rat Immune Cells Recruitment into the Pulmonary Niche by Up-regulation of Endothelial Adhesion Molecules.

Purpose: Diabetes mellitus, especially type 2, is conceived as a devastating chronic metabolic disease globally. Due to the existence of an extensive vascular network in the pulmonary tissue, it is suggested that lungs are sensitive to the diabetic condition like other tissues. This study was designed to address the possible effect of type 2 diabetes mellitus on the promotion of pathological changes via vascular injury. Methods: Sixteen male Wistar rats were randomly allocated to the two of control and T2D groups. To induce type 2 diabetes (T2D), rats were received high-fat and a single dose of streptozotocin (STZ). On week 12, rats were euthanized and lungs samples were taken. Using hematoxylin and eosin (H&E) staining, the pathological changes were monitored. The expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and interleukin 10 (IL-10) was monitored using real-time PCR assay. The level of tumor necrosis factor-α (TNF-α) was detected using ELISA assay. Nitrosative stress was monitored using the Griess assay. Results: Pathological examination in bronchoalveolar discharge revealed the existence of mild to moderate interstitial bronchopneumonia and increased neutrophilic leukocytosis compared to the control. Enhanced ICAM-1 and VCAM-1 expression and suppression of IL-10 was found using real-time PCR analysis (P < 0.05). The levels of TNF-α and NO were increased with diabetic changes compared to the control rats (P < 0.05). Conclusion: T2D could promote pulmonary tissue injury via the production of TNF-α and up-regulation of vascular ICAM-1 and VCAM-1. The inflammatory status and vascular ICAM-1 and VCAM-1 increase immune cell recruitment into the pulmonary niche.

Anti-inflammatory therapies in myocardial infarction: failures, hopes and challenges.

In the infarcted heart, the damage-associated molecular pattern proteins released by necrotic cells trigger both myocardial and systemic inflammatory responses. Induction of chemokines and cytokines and up-regulation of endothelial adhesion molecules mediate leukocyte recruitment in the infarcted myocardium. Inflammatory cells clear the infarct of dead cells and matrix debris and activate repair by myofibroblasts and vascular cells, but may also contribute to adverse fibrotic remodelling of viable segments, accentuate cardiomyocyte apoptosis and exert arrhythmogenic actions. Excessive, prolonged and dysregulated inflammation has been implicated in the pathogenesis of complications and may be involved in the development of heart failure following infarction. Studies in animal models of myocardial infarction (MI) have suggested the effectiveness of pharmacological interventions targeting the inflammatory response. This article provides a brief overview of the cell biology of the post-infarction inflammatory response and discusses the use of pharmacological interventions targeting inflammation following infarction. Therapy with broad anti-inflammatory and immunomodulatory agents may also inhibit important repair pathways, thus exerting detrimental actions in patients with MI. Extensive experimental evidence suggests that targeting specific inflammatory signals, such as the complement cascade, chemokines, cytokines, proteases, selectins and leukocyte integrins, may hold promise. However, clinical translation has proved challenging. Targeting IL-1 may benefit patients with exaggerated post-MI inflammatory responses following infarction, not only by attenuating adverse remodelling but also by stabilizing the atherosclerotic plaque and by inhibiting arrhythmia generation. Identification of the therapeutic window for specific interventions and pathophysiological stratification of MI patients using inflammatory biomarkers and imaging strategies are critical for optimal therapeutic design.

Exercise-induced upregulation of endothelial adhesion molecules in human skeletal muscle and number of circulating cells with remodeling properties.

Multipotent cells have received great interest because of their potential capacity to repair and remodel peripheral tissues. We examined the effect of an acute exercise bout on the number of circulating cells with known remodeling properties and the level of factors in plasma and skeletal muscle tissue with potential to recruit these cells. Twenty healthy male subjects performed a 60-min cycling exercise. Blood samples for flow cytometry were drawn from 10 subjects (group 1) before and up to 2 h after exercise, and absolute cell counts of the classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocyte (MO) subpopulations and of CD45dimCD34+VEGFR2+ endothelial progenitor cells (EPCs) were measured by bead-based determination. Plasma samples and vastus lateralis muscle biopsies were obtained from the other 10 subjects (group 2). In group 1, all MO subsets were increased directly after exercise, with CD14+CD16++ MOs showing the greatest fold increase. After 2 h, only CD14++CD16- MOs were increased compared with resting levels. The number of EPCs showed a trend toward increasing with exercise (P = 0.08). In group 2, the mRNA levels of the endothelial adhesion molecules ICAM-1, VCAM-1, and E-selectin increased in the skeletal muscle tissue. VEGF-A increased in exercised skeletal muscle and stimulated the expression of VCAM-1 and E-selectin in human umbilical vein endothelial cells. In conclusion, exercise increases MO subsets with different temporal patterns and enhances the capacity of skeletal muscle tissue to recruit circulating cells as shown by increased expression of endothelial adhesion molecules.NEW & NOTEWORTHY In the present study we showed for the first time that the adhesion molecules ICAM-1, VCAM-1, and E-selectin, known to be able to recruit circulating cells to the peripheral tissue, increased in exercised human skeletal muscle concurrently with increased circulating levels of cells shown to have importance for skeletal muscle remodeling. These findings support the concept of cell recruitment from the circulation playing a role in skeletal muscle adaptation to exercise.

Neuroprotectant androst-3β, 5α, 6β-triol suppresses TNF-α-induced endothelial adhesion molecules expression and neutrophil adhesion to endothelial cells by attenuation of CYLD-NF-κB pathway

Neuroinflammation is one of key pathologic element in neurological diseases including stroke, traumatic brain injury, Alzheimer’ s Disease, Parkinson's Disease, and multiple sclerosis as well. Up-regulation of endothelial adhesion molecules, which facilitate leukocyte adhesion to the endothelium, is the vital process of endothelial cells mediated neuroinflammation. Androst-3β, 5α, 6β-triol (Triol) is a synthetic steroid which has been reported to have neuroprotective effects in hypoxia/re-oxygenation-induced neuronal injury model. In the present study, we firstly investigated whether Triol inhibited the TNF-α-induced inflammatory response in rat brain microvascular endothelial cells (RBMECs). Our data showed that Triol decreased TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and the adhesion of neutrophil to RBMECs. We also found that Triol inhibited TNF-α-induced degradation of IκBα and phosphorylation of NF-κBp65 that are required for NF-κB activation. Furthermore, Triol significantly reversed TNF-α-induced down-expression of CYLD, which is a deubiquitinase that negatively regulates activation of NF-κB. These results suggest that Triol displays an anti-inflammatory effect on TNF-α-induced RBMECs via downregulating of CYLD-NF-κB signaling pathways and might have a potential benefit in therapeutic neuroinflammation related diseases.

Propofol ameliorates endothelial inflammation induced by hypoxia/reoxygenation in human umbilical vein endothelial cells: Role of phosphatase A2

Hypoxia/reoxygenation (H/R) induces endothelial inflammation with augmentation of endothelial adhesion molecules over-expression. Propofol was reported to attenuate endothelial adhesion molecule expression in some situations. Here, we examined the molecular mechanism for how propofol restored H/R-mediated up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs). Compared with the control group, H/R up-regulated expression of Pin-1 and PP2A, increased p66Shc–Ser36 phosphorylation, induced p66Shc mitochondrial translocation, O2− accumulation and NF-κB activation, and decreased eNOS–Ser1177 phosphorylation and nitric oxide (NO) production, thus up-regulating expression of endothelial adhesion molecules and increasing mononuclear-endothelial interaction. More importantly, except that propofol had no effect on H/R-induced p66Shc–Ser36 phosphorylation, most of H/R-mediated changes were alleviated by propofol, resulting in the reduction of endothelial adhesion molecules expression and mononuclear-endothelial adhesion. Moreover, we demonstrated the protective effect of propofol on H/R-induced endothelial inflammation was similar to that of calyculin A, an inhibitor of PP2A. In contrast, FTY720, an activator of PP2A, antagonized the effect of propofol. Our data indicated that propofol down-regulated PP2A expression, leading to reduced dephosphorylation of p66Shc–Ser36 and eNOS–Ser1177, which is associated with ROS accumulation and NO reduction, resulting in inhibition of endothelial adhesion molecule expression and mononuclear-endothelial interaction.

Noninvasive Molecular Imaging of Mouse Atherosclerosis.

Molecular imaging offers great potential for noninvasive visualization and quantitation of the cellular and molecular components involved in atherosclerotic plaque stability. In this chapter, we review emerging molecular imaging modalities and approaches for quantitative, noninvasive detection of early biological processes in atherogenesis, including vascular endothelial permeability, endothelial adhesion molecule up-regulation, and macrophage accumulation, with special emphasis on mouse models. We also highlight a number of targeted imaging nanomaterials for assessment of advanced atherosclerotic plaques, including extracellular matrix degradation, proteolytic enzyme activity, and activated platelets using mouse models of atherosclerosis. The potential for clinical translation of molecular imaging nanomaterials for assessment of atherosclerotic plaque biology, together with multimodal approaches is also discussed.

Neuregulin1-β decreases IL-1β-induced neutrophil adhesion to human brain microvascular endothelial cells.

Neuroinflammation contributes to the pathophysiology of diverse diseases including stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, and multiple sclerosis, resulting in neurodegeneration and loss of neurological function. The response of the microvascular endothelium often contributes to neuroinflammation. One such response is the upregulation of endothelial adhesion molecules which facilitate neutrophil adhesion to the endothelium and their migration from blood to tissue. Neuregulin-1 (NRG1) is an endogenous growth factor which has been reported to have anti-inflammatory effects in experimental stroke models. We hypothesized that NRG1 would decrease the endothelial response to inflammation and result in a decrease in neutrophil adhesion to endothelial cells. We tested this hypothesis in an in vitro model of cytokine-induced endothelial injury, in which human brain microvascular endothelial cells (BMECs) were treated with IL-1β, along with co-incubation with vehicle or NRG1-β. Outcome measures included protein levels of endothelial ICAM-1, VCAM-1, and E-selectin, as well as the number of neutrophils that adhere to the endothelial monolayer. Our data show that NRG1-β decreased the levels of VCAM-1, E-selectin, and neutrophil adhesion to brain microvascular endothelial cells activated by IL1-β. These findings open new possibilities for investigating NRG1 in neuroprotective strategies in brain injury.

Propofol protects human umbilical vein endothelial cells from cisplatin-induced injury

The anticancer drug cisplatin can up-regulate endothelial adhesion molecule expression, and trigger vascular endothelial injury. Propofol, an intravenous anesthetic, can inhibit endothelial adhesion molecule expression in some situations. Here, we explored whether and how propofol improved cisplatin-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells. Compared with control group, cisplatin reduced endothelial nitric oxide synthase dimer/monomer ratio, activated protein kinase C and enhanced endothelial nitric oxide synthase-Thr495 phosphorylation, decreased nitric oxide production, augmented intercellular adhesion molecule 1 expression and monocyte-endothelial adhesion. These cisplatin-mediated effects were attenuated by propofol treatment. Nω-Nitro-L-arginine methyl ester hydrochloride, a nitric oxide synthase inhibitor, inhibited the effect of propofol on cisplatin-induced intercellular adhesion molecule 1 expression. Propofol improved cisplatin-mediated tetrahydrobiopterin reduction and nitrotyrosine overexpression. Compared with control group, cisplatin and PMA, a protein kinase C activator, both increased endothelial nitric oxide synthase-Thr495 phosphorylation, while propofol and GFX, a protein kinase C inhibitor, both decreased cisplatin-induced endothelial nitric oxide synthase-Thr495 phosphorylation. Our data indicated that propofol, via reducing cisplatin-induced endothelial nitric oxide synthase uncoupling and endothelial nitric oxide synthase-Thr495 phosphorylation, restored nitric oxide production, intercellular adhesion molecule 1 expression and monocyte-endothelial interaction.

Attenuation of Inflammatory Response and Reduction in Infarct Size by Postconditioning Are Associated With Downregulation of Early Growth Response 1 During Reperfusion in Rat Heart

Early growth response 1 (EGR-1) works as a master regulator that plays a key role in triggering inflammation-induced tissue injury after ischemia and reperfusion. This study tested the hypothesis that postconditioning (Postcon) or anti-inflammatory compound, curcumin, ameliorates inflammatory responses and further reduces infarct size by normalizing EGR-1 expression during reperfusion. In the control group, male Sprague-Dawley rats were subjected to 30-min ischemia and 180-min reperfusion. Postcon with four cycles of 10-s/10-s reperfusion/ischemia was applied at the onset of reperfusion. Curcumin (150 mg/kg per day) was fed 5 days before ischemia. Relative to the control, Postcon reduced expression of EGR-1 mRNA and protein, as further identified by less EGR-1 immunoreactivity in myocardial nuclei and microvessels during reperfusion. Along with EGR-1 downregulation, levels of plasma and myocardial tumor necrosis factor α and interleukin 6 (IL-6) were significantly decreased. Upregulated P-selectin and intercellular adhesion molecule 1 mRNA and protein as well as their immunoreactivity at area at risk myocardium were significantly attenuated. Neutrophil extravasation identified by myeloperoxidase immunohistochemical staining was inhibited. Infarct size, determined with triphenyltetrazolium chloride staining, was smaller in the Postcon group than that in the control. The protection achieved with pretreatment with curcumin was comparable to the benefits gained by Postcon in all end points measured. In H9C2 rat cardiomyoblast cell line, EGR-1 siRNA downregulated hydrogen peroxide-induced EGR-1 mRNA expression and subsequently reduced tumor necrosis factor α mRNA level. These results suggest that EGR-1 seems to play a critical role in myocardial reperfusion injury because downregulation of EGR-1 either by Postcon or the use of pharmacological intervention reduces infarct size, most likely through an inhibition of inflammation-mediated processes.

Anti-Donor HLA Class I Antibodies

The development of donor-specific human leukocyte antigen (HLA) class I antibodies after organ transplantation is associated with subsequent acute and chronic rejection. The aim of this study was to examine the role of anti-HLA class I antibody in modulating endothelium-leukocyte interaction. Human microvascular endothelial cells (HMEC-1) stimulated with HLA class I antibody (W6/32) or allospecific antibodies from sensitized patients (n=6) were examined for activation of transcription factor CREB by Western blotting. Up-regulation of endothelial adhesion molecules and chemokines was measured by flow cytometry and quantitative polymerase chain reaction, respectively. Leukocyte adhesion was evaluated by chemotaxis and in vitro flow-based assays. Treatment of HMEC-1 cells with HLA class I antibody resulted in the phosphorylation of CREB in protein kinase A-dependent pathway. Furthermore, there was a significant increase in the expression of cell surface VCAM-1 (Akt-dependent) and ICAM-1 in Akt-dependent and extracellular signal-regulated kinase-dependent manner (P<0.001). Additionally, exposure to W6/32 antibody induced significant expression of interleukin-6, CXCL8, CXCL10, and CCL5. Knockdown of CREB produced a reduction in W6/32-induced CXCL8 expression (P<0.001). Media from W6/32-treated endothelial cells induced a significant monocyte chemotaxis (P<0.001) and flow-based adhesion assay demonstrated an increase in monocyte adhesion to endothelial cells compared with the control group (P<0.001). Importantly, allospecific antibodies from sensitized patients also activated endothelial CREB and significantly up-regulated VCAM-1, ICAM-1, and CXCL8. These findings suggest that donor-specific HLA class I antibodies directly activate endothelial cells leading to an increase in their potential to recruit and bind recipient leukocytes, thereby increasing the potential for allograft inflammation.

Propofol protects against high glucose-induced endothelial adhesion molecules expression in human umbilical vein endothelial cells

BackgroundHyperglycemia could induce oxidative stress, activate transcription factor nuclear factor kappa B (NF-κB), up-regulate expression of endothelial adhesion molecules, and lead to endothelial injury. Studies have indicated that propofol could attenuate oxidative stress and suppress NF-κB activation in some situations. In the present study, we examined whether and how propofol improved high glucose-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs).MethodsProtein expression of endothelial adhesion molecules, NF-κB, inhibitory subunit of NF-κBα (IκBα), protein kinase Cβ2 (PKCβ2), and phosphorylation of PKCβ2 (Ser660) were measured by Western blot. NF-κB activity was measured by electrophoretic mobility shift assay. PKC activity was measured with SignaTECT PKC assay system. Superoxide anion (O2.-) accumulation was measured with the reduction of ferricytochrome c assay. Human peripheral mononuclear cells were prepared with Histopaque-1077 solution.ResultsHigh glucose induced the expression of endothelial selectin (E-selectin), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and increased mononuclear-endothelial adhesion. High glucose induced O2.- accumulation, PKCβ2 phosphorylation and PKC activation. Further, high glucose decreased IκBα expression in cytoplasm, increased the translocation of NF-κB from cytoplasm to nuclear, and induced NF-κB activation. Importantly, we found these high glucose-mediated effects were attenuated by propofol pretreatment. Moreover, CGP53353, a selective PKCβ2 inhibitor, decreased high glucose-induced NF-κB activation, adhesion molecules expression, and mononuclear-endothelial adhesion.ConclusionPropofol, via decreasing O2.- accumulation, down-regulating PKCβ2 Ser660 phosphorylation and PKC as well as NF-κB activity, attenuated high glucose-induced endothelial adhesion molecules expression and mononuclear-endothelial adhesion.

Murine Kupffer Cells Are Protective in Total Hepatic Ischemia/Reperfusion Injury with Bowel Congestion through IL-10

Kupffer cells (KCs) are thought to mediate hepatocyte injury via their production of proinflammatory cytokines and reactive oxygen species in response to stress. In this study, we depleted KCs from the liver to examine their role in total warm hepatic ischemia/reperfusion (I/R) injury with bowel congestion. We injected 8-wk-old C57BL/10J mice with liposome-encapsulated clodronate 48 h before 35 min of hepatic ischemia with bowel congestion, followed by 6 or 24 h of reperfusion. KC-depleted animals had a higher mortality rate than diluent-treated animals and a 10-fold elevation in transaminase levels that correlated with increases in centrilobular necrosis. There was extensive LPS binding to the endothelial cells, which correlated with an upregulation of endothelial adhesion molecules in the KC-depleted animals versus diluent-treated animals. There was an increase in the levels of proinflammatory cytokines in KC-depleted animals, and a concomitant decrease in IL-10 levels. When KC-depleted mice were treated with recombinant IL-10, their liver damage profile in response to I/R was similar to diluent-treated animals, and endothelial cell adhesion molecules and proinflammatory cytokine levels decreased. KCs are protective in the liver subjected to total I/R with associated bowel congestion and are not deleterious as previously thought. This protection appears to be due to KC secretion of the potent anti-inflammatory cytokine IL-10.

Effects of G-CSF treatment on neutrophil mobilization and neurological outcome after transient focal ischemia

Several recent studies demonstrated beneficial effects of G-CSF treatment (granulocyte colony-stimulating factor) in various CNS disease. Possible mechanisms underlying this activity are neuroprotection, anti-apoptosis, angiogenesis and anti-inflammation. Hence, we investigated the efficacy of G-CSF administration in experimental stroke by determining infarct volume and neurological score in wildtype, G-CSF-deficient and G-CSF-treated G-CSF-deficient mice. Besides, cerebral ischemia is followed by an upregulation of endothelial adhesion molecules which promote leukocyte recruitment to the injured area. In combination with G-CSF-induced leukocytosis, increased peripheral neutrophils could aggregate within microvasculature and additionally impair blood perfusion of the ischemic tissue. Therefore, we analyzed the neutrophil counts in both vessel and tissue compartment 2 and 5 days post-stroke by immunohistochemistry. Here we show that G-CSF deficiency leads to increased infarct volumes, whereas G-CSF substitution revokes detrimental effects by reducing lesion size and enhancing neurological outcome compared to untreated animals. Administration of G-CSF is accompanied by significant increase of circulating neutrophils 2 days post-ischemia but leukocytosis is restricted to the vessel compartment and has no deleterious effect on lesion formation and functional recovery. These observations are likely to be important for therapeutic targeting of G-CSF-mediated neuroprotection in stroke.

Endothelial Activation by Sickle Mouse Red Cells and Their Endothelial Adhesion In Vivo is Abolished by Catalase Treatment of Sickle Cells, but Not Quiescent Endothelium, Implying a Role of Heme-Mediated Peroxide Generation in Sickle Cell Adhesion.

Abstract 902Multiple adhesion molecules, expressed on sickle red blood cells (SS RBCs) and activated endothelium, have been implicated in SS RBC adhesion to vascular endothelium. Moreover, intrinsic differences among heterogeneous SS RBC subpopulations, involving differences in red cell adhesion molecules and cell deformability, may contribute to their adhesive and obstructive properties and lead to postcapillary obstruction. However, the role of SS RBCs in endothelium activation and adhesion has not been evaluated despite the insightful studies of Hebbel and coworkers (JCI, 1982) demonstrating that SS RBCs generate excessive amounts of reactive oxygen species due to the presence of unstable hemoglobin S (HbS) and autoxidation of iron in heme. RBCs from transgenic-knockout sickle (BERK) mice similarly show a pronounced increase in heme degradation (Nagababu et. al. Blood Cells Mol Dis, 2008). We hypothesize that hypoxic conditions in venules (oxygen tension,∼30 mm Hg) will accelerate autoxidation of RBC membrane-bound HbS and release H2O2that will be transferred to adjoining endothelium resulting in its activation (i.e., up-regulation of endothelial adhesion molecules) and SS RBC adhesion. To test the hypothesis that HbS-containing red cells from BERK mice will result in activation of quiescent endothelium in normal mice, we infused FITC (fluorescein isothiocynate)-labeled BERK red cells into congenic C57BL mice. BERK mice, expressing exclusively human βS- and α-globins, have been extensively backcrossed onto C57BL background. Intravital observations were made in the cremaster muscle microcirculatory bed. A single bolus of 150 μl of FITC-labeled BERK RBCs (Hct 30%) was infused into the recipient C57BL mouse via the jugular vein over a period of 5 min to avoid any shear related platelet aggregation. Infusion of FITC-labeled control (C57BL) mouse RBCs into C57BL recipient mice resulted in rare or no RBC adhesion, suggesting that there was no activating effect on endothelium. In contrast, infusion of BERK mouse RBCs into C57BL mice resulted in time-dependent increase in adhesion to venular endothelium. Adhesion became discernable after 3 minutes and showed a 3-5 fold increase after 5-min compared with the number of adherent RBCs at 3 min (P<0.01). Next, we investigated if the infusion of BERK mouse RBCs would induce increased endothelial oxidants. To this end, the cremaster preparation was suffused for 15 min with 123 dihydrorhodamine (DHR), a H2O2-sensitive probe (10 μl/L), followed by a bolus infusion of BERK mouse RBCs, and time-dependent changes in DHR fluorescence intensity were monitored in venules, the sites of adhesion. Infusion of BERK mouse RBCs, but not C57BL RBCs, resulted in time-dependent increase in the fluorescence intensity (ΔI) in venular endothelium, with almost 5-fold increase in DHR intensity after 5 min of BERK RBC infusion (P<0.001) compared with ΔI at 1 min. When infusion of catalase (900 U/mouse) into recipient C57BL mice was followed 30 min later by a bolus of FITC-labeled BERK mouse RBCs, BERK RBC adhesion and pronounced DHR fluorescence in endothelium were observed, demonstrating that intravascular infusion of catalase had little effect on oxidant generation by BERK mouse RBCs. In contrast, infusion of BERK RBCs pre-treated with catalase (100 U in 0.2 ml RBC suspension, 9-fold less catalase per mouse) to quench RBC generated H2O2 inhibited endothelial DHR fluorescence and BERK RBC adhesion. These results strongly suggest an obligatory role of heme-mediated peroxide generation by SS RBC in endothelial activation and SS RBC adhesion, and support the notion that heme-mediated oxidant generation may play a vital role in endothelial dysfunction in sickle cell disease. Disclosures:No relevant conflicts of interest to declare.

Tumor Necrosis Factor-α in Airway Secretions from Cystic Fibrosis Patients Upregulate Endothelial Adhesion Molecules and Induce Airway Epithelial Cell Apoptosis: Implications for Cystic Fibrosis Lung Disease

Airway inflammation plays a crucial role in lung damage in cystic fibrosis (CF) and is characterized by a persistent influx of neutrophils into the airways. We hypothesized that the high levels of inflammatory products that accumulate in the microenvironment of the CF lung contribute to induce the persistent neutrophil recruitment and the airway epithelial damage. Thus, we evaluated the in vitro effect of sputum sol phase (SSP) from CF patients on a) adhesion molecule expression by human microvascular endothelial cells (HMECs) and b) apoptosis of human bronchial epithelial cells (HBECs), both wild-type and CFTR-defective. SSP was obtained from 7 clinically stable adult CF patients and 8 patients with an acute exacerbation. HMECs and HBECs were cultured in the absence or presence of SSP. Cell adhesion molecule expression was assessed by flow cytometry and cell death by the detection of histone-associated DNA fragments, caspase activation, and cytochrome c release. SSP obtained from CF patients, especially at the time of an acute exacerbation, induced a) an upregulation of endothelial adhesion molecules on cultured HMECs that was associated with an increase of neutrophil adhesion to these cells, and was mediated at least in part by TNF-alpha and IL-1 and b) apoptosis of airway epithelial cells, mainly activated by TNF- alpha pathway. These results suggest that the high concentrations of inflammatory mediators in CF airways contribute both to the chronic neutrophil influx and the airway damage, and support the crucial role of early anti-inflammatory treatment in the disease.

BMP-7 protects mesangial cells from injury by polymeric IgA

Bone morphogenetic protein-7 (BMP-7) is a potential therapeutic agent for acute and chronic renal diseases. Here we found that addition of polymeric IgA, isolated from patients with IgA nephropathy, increased the synthesis of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and fibronectin in cultured human mesangial cells, effects blunted by BMP-7. When mesangial cells were cultured with both polymeric IgA and BMP-7 there was an increase in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The activation of NF kappaB and TNF-alpha synthesis induced by polymeric IgA or TNF-alpha were downregulated by BMP-7 or rosiglitazone. BMP-7 inhibited TNF-alpha release from polymeric IgA-stimulated mesangial cells by activation of PPAR-gamma but suppressed TGF-beta release by mechanisms independent of PPAR-gamma. The expression of inhibitory Smad6 and 7 was increased whereas the expression of active Smad2 and 3 was reduced in these mesangial cells by BMP-7. Our study shows that BMP-7 ameliorates IgA nephropathy-derived polymeric IgA-induced TNF-alpha and TGF-beta synthesis in human mesangial cells through multiple mechanisms involving inhibitory Smads and PPAR-gamma.

IL-1 Cytokines in Cardiovascular Disease: Diagnostic, Prognostic and Therapeutic Implications

Interleukins (ILs) are key mediators in the chronic vascular inflammatory response underlying several aspects of cardiovascular disease. Due to their powerful pro-inflammatory potential, and the fact that they are highly expressed by almost all cell types actively implicated in atherosclerosis, members of the IL-1 cytokine family were the first to be investigated in the field of vessel wall inflammation. The IL-1 family is comprised of five proteins that share considerable sequence homology: IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1Ra), IL-18 (also known as IFNgamma-inducing factor), and the newly discovered ligand of the ST2L receptor, IL-33. Expression of IL-1s and their receptors has been demonstrated in atheromatous tissue, and serum levels of IL-1-cytokines have been correlated with various aspects of cardiovascular disease and their outcome. In vitro studies have confirmed pro-atherogenic properties of IL-1alpha, IL-1beta and IL-18 such as, up-regulation of endothelial adhesion molecules, the activation of macrophages and smooth muscle cell proliferation. In contrast with this, IL-1Ra, a natural antagonist of IL-1, possesses anti-inflammatory properties, mainly through the endogenous inhibition of IL-1 signaling. IL-33 was identified as a functional ligand of the, till recently, orphan receptor, ST2L. IL-33/ST2L signaling has been reported as a mechanically activated, cardioprotective paracrine system triggered by myocardial overload. As the roles of individual members of the IL-1 family are being revealed, novel therapies aimed at the modulation of interleukin function in several aspects of cardiovascular disease, are being proposed. Several approaches have produced promising results. However, none of these approaches has yet been applied in clinical practice.

Perplexity of monocyte responses to C-reactive protein (CRP)

Thromb Haemost 2008; 99: 461–462 C-reactive protein (CRP), a member of the pentraxin family, is one of the prototypic plasma biomarkers of inflammation (1, 2). The physiological roles of CRP in the innate immune response have, however, remained elusive as it possesses both proand anti-inflammatory actions (1, 2). Inflammation is pivotal in all phases of atherosclerosis from the nascent lesion through progression to the culmination in acute coronary syndromes (3). To date, over two dozen large-scale prospective clinical studies have reported that moderate elevations in base-line CRP levels predict, albeit to varying degrees, future cardiovascular disease and death across all levels of lowdensity lipoprotein (LDL) cholesterol and of the Framingham Risk Score (4, 5). CRP has been implicated inmultiple aspects of atherogenesis and plaque vulnerability; however, whether CRP plays a direct causal role in these events remains controversial (reviewed in [6]). For example, transgenic expression of human CRP in apolipoprotein E-deficient mice has been reported to accelerate atherogenesis (7, 8), to show no effect (9) or even to slow atherosclerosis development (10). In vitro, CRP induces release of proinflammatory interleukins IL-1, IL-6, IL-8 and tumor necrosis factor-α NF (11, 12) from human monocytes; promotes monocye adhesion to endothelial cells through upregulation of endothelial adhesion molecules (13, 14) and enhances phagocytosis of oxidized LDL (6). These observations are consistent with the role of monocytes in the initiation and progression of atherosclerosis (3). However, monocytes may have a more complex role in atherogenesis than previously thought. In this issue of Thrombosis and Haemostasis, Hanriot et al. (15) show that CRP modulates expression of genes known to mediate both proand anti-inflammatory responses. These novel observations raise the intriguing possibility that CRP could activate yet unidentified pathways in human monocytes. Using a custom-made 250 oligonucleotide microarray for screening CRP regulated genes in monocytes isolated from apparently healthy men and women (aged 25 to 35 years), Hanriot et al. detected changes in approximately 10% of the genes represented on the microarray. The authors confirm previous studies on CRP induction of pro-inflammatory cytokine gene expression with the exception of TNF, which was unaffected by CRP, and show increased mRNA expression of monocyte chemotactic protein-1 (MCP-1), Gro-β, the chemokine receptors CCR-8 and CXCR-4, and plasminogen activator inhibitor type 2 (PAI-2). In concert with IL-8,MCP-1 andGro-β facilitatemonocyte recruitment and adhesion to endothelial cells, whereas CCR-8 and CXCR-4 mediate monocyte (and macrophage) chemotaxis. The function of PAI-2 appears to be more complex. PAI-2 promotes fibrin deposition in the arterial wall, thereby contributing to progression of the initial lesion, whereas it may stabilize more advanced plaques by inhibiting activation of matrix metalloproteinases, enzymes overexpressed in human atheromata (3). While the concept of CRP’s anti-inflammatory role is not new (reviewed in [1, 2]), Hanriot et al. also report CRP-induced changes in monocyte gene expression that are consistent with suppression of inflammation. Thus, the authors show that CRP attenuates expression of the monocyte/lymphocyte chemotactic proteins MIP-1α and MIP-1β and α2-macroglobulin, which antagonize the actions of the anti-inflammatory cytokine transforming growth factor-β. Protection of mice overexpressing human CRP from allergic encephalomyelitis is mediated through reduction of MIP-1 (16). The most striking finding is that CRP increases expression of the nuclear receptor liver X receptor-α (LXR-α) and one of its target genes the ATP-binding cassette transporter A1 (ABCA1). ABCA1 promotes reverse cholesterol transport from macrophages in atherosclerotic plaques to high density lipoprotein (HDL) and suppresses genes involved in macrophage inflammatory signaling and apoptosis, thereby inhibiting atherosclerosis (17). CRP induction of LXR-α expression and activation is mediated through the high affinity IgG receptor FcγR1 (CD64) and, to lesser a degree, the low affinity IgG receptor FcγRII (CD32), and involves activation of both p42/44MAPK and phosphatidylinositol 3-kinase signaling pathways. A critical issue whether CRP could effectively facilitate reverse cholesterol transport in monocytes (and macrophages) remains to be elucidated. The complexity of LXR-α responses makes it difficult to extrapolate the beneficial or deleterious effects of a target gene in isolation to human pathology when target genes are likely beingmodulated in concert. For example, an-