VCAM-1 In Human Umbilical Vein Endothelial Cells Research Articles

Anti-Atherosclerotic Properties of Aronia melanocarpa Extracts Influenced by Their Chemical Composition Associated with the Ripening Stage of the Berries.

The high content of bioactive compounds in Aronia melanocarpa fruit offers health benefits. In this study, the anti-atherosclerotic effect of Aronia extracts was assessed. The impact on the level of adhesion molecules and the inflammatory response of human umbilical vein endothelial cells (HUVECs) was shown in relation to the chemical composition and the stage of ripening of the fruits. Samples were collected between May (green, unripe) and October (red, overripe) on two farms in Poland, which differed in climate. The content of chlorogenic acids, anthocyanins, and carbohydrates in the extracts was determined using HPLC-DAD/RI. The surface expression of ICAM-1 and VCAM-1 in HUVECs was determined by flow cytometry. The mRNA levels of VCAM-1, ICAM-1, IL-6, and MCP-1 were assessed using the quantitative real-time PCR method. The farms' geographical location was associated with the quantity of active compounds in berries and their anti-atherosclerotic properties. Confirmed activity for green fruits was linked to their high chlorogenic acid content.

Inhibitory Effect of Punicalagin on Inflammatory and Angiogenic Activation of Human Umbilical Vein Endothelial Cells.

Punicalagin, a major ellagitannin isolated from pomegranate, is proved to have various pharmacological activities with an undefined therapy mechanism. The objective of this research was to demonstrate the effect of punicalagin on anti-inflammatory and angiogenic activation in human umbilical vein endothelial cells (HUVECs) and their potential mechanisms. Endothelial-leukocyte adhesion assay was applied to evaluate primary cultures of HUVECs activation following tumor necrosis factor alpha (TNF-α) treatment. The endothelial cell proliferation, migration, permeability and tube formation were assessed by EdU assay, wound migration assay, trans-endothelial electrical resistances (TEER) assay, and capillary-like tube formation assay, respectively. In addition, the expression of relevant proteins was assessed using Western blot analysis. We confirmed that punicalagin could reduce the adhesion of human monocyte cells to HUVECs in vitro and in vivo. Further, punicalagin decreased the expression of mRNA and proteins of ICAM-1 and VCAM-1 in HUVECs. Moreover, punicalagin inhibited permeability, proliferation, migration, and tube formation in VEGF-induced HUVECs, suppressed IKK-mediated activation of NF-κB signaling in TNF-α-induced endothelial cells, and inhibited vascular endothelial growth factor receptor 2 (VEGFR2) activation and downstream p-PAK1. Our findings indicated that punicalagin might have a protective effect on HUVECs activation, which suggested that punicalagin functions through an endothelial mediated mechanism for treating various disorders such as, cancer, rheumatoid arthritis, and cardiovascular disease.

Hyperoside Protects Human Umbilical Vein Endothelial Cells Against Anticardiolipin Antibody-Induced Injury by Activating Autophagy.

Anticardiolipin antibody (aCL), an important characterization of antiphospholipid syndrome, shows an intense association with vascular endothelial injury. Hyperoside is a flavonoid extracted from medicinal plants traditionally used in Chinese medicines, displaying anti-inflammatory, anti-cancer, and anti-oxidative properties in various diseases. Recent studies have shifted the focus on the protective effects of hyperoside on vascular endothelial injury. However, little is known about the mechanisms involved. In the present study, we investigated the effect of hyperoside on aCL-induced injury of human umbilical vein endothelial cells (HUVECs) in vitro. Our data illustrated that aCL induced HUVEC injury via inhibiting autophagy. Hyperoside reduced aCL-induced secretion of proinflammatory cytokines IL-1β and IL-8 and endothelial adhesion cytokines TF, ICAM1, and VCAM1 in HUVECs. Additionally, hyperoside activated autophagy and suppressed the mTOR/S6K and TLR4/Myd88/NF-κB signaling transduction pathways in aCL-induced HUVECs. To the best of our knowledge, this is the first study to investigate the effect of hyperoside on aCL-induced injury, as well as offer insights into the involved mechanisms, which is of great significance for the treatment of antiphospholipid syndrome.

Role of activin A in the pathogenesis of endothelial cell dysfunction in preeclampsia.

Circulating markers for endothelial activation such as endothelin-1 (ET-1), ICAM-1 and VCAM-1 are elevated in women with preeclampsia. Using human umbilical vein endothelial cells (HUVECs) as an in vitro model of the maternal vasculature, we show that activin A and preeclamptic serum upregulate ET-1, ICAM-1, and VCAM-1 in HUVECs. Further, we show that follistatin, a specific binding protein for activin, mitigates the upregulation of ET-1, ICAM-1 and VCAM-1 in HUVECs exposed to either activin A or preeclamptic serum. These data are consistent with activin A contributing to the pathophysiology of preeclampsia and suggest that therapies targeting activin signalling are worth exploring.

Abstract 10859: Preventive Effects of Neopterin on Atherosclerosis

Introduction: Neopterin, a GTP metabolite produced by activated macrophages after stimulation by interferon-γ released by T cells, is expressed at high levels in the carotid atherosclerotic lesions in patients with angina pectoris. However, the modulatory effect of neopterin on atherogenesis has not yet been reported. Objective and Methods: We aimed to clarify the effects of neopterin on atherogenesis. We investigated the atheroprotective effects of neopterin on human umbilical vein endothelial cells (HUVECs), human monocyte-derived macrophages, human aortic smooth muscle cells (HASMCs) in vitro , and aortic lesions in ApoE -/- mice in vivo . Plasma neopterin levels were compared between 25 patients with acute coronary syndrome (ACS) and 15 healthy volunteers. Results: Neopterin attenuated TNF-α-mediated monocyte adhesion to HUVECs. Neopterin suppressed TNF-α-induced upregulation of MCP-1, ICAM-1, and VCAM-1 in HUVECs. Neopterin shifted the phenotype overwhelmingly to anti-inflammatory M2 rather than inflammatory M1 via NF-κB downregulation during differentiation of human monocytes into macrophages. Neoptein significantly suppressed oxidized LDL-induced foam cell formation associated with reduced CD36 expression and increased both expressions and activities of ABCA1 and ABCG1 in human monocyte-derived macrophages. Neopterin significantly suppressed angiotensin II-induced migration of HASMCs, but increased fibronectin and MMP2 expressions accompanied with enhanced MMP2 activity in HASMCs. Four-week-infusion of neopterin into ApoE -/- mice significantly retarded the development of aortic atherosclerotic lesions with reduced monocyte/macrophage infiltration and lowered plasma levels of total cholesterol. In addition, plasma neopterin levels were markedly increased in ACS patients than healthy volunteers, suggesting that neopterin increased to counteract the formation of atherothrombosis. Conclusions: Our results indicate that neopterin prevents atherogenesis by suppressing monocyte adhesion to endothelial cells, macrophage foam cell formation, and VSMC migration. Thus, neopterin could serve as a novel therapeutic target and potential biomarker for atherosclerotic cardiovascular diseases.

Chemerin plays a protective role by regulating human umbilical vein endothelial cell-induced nitric oxide signaling in preeclampsia.

The aim of this study was to determine chemerin levels in preeclampsia and to assess the effects of this anti-inflammatory factor on endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-κB, and vascular cell adhesion molecule (VCAM) expression in human umbilical vein endothelial cells (HUVECs). Serum chemerin and eNOS levels were measured by enzyme-linked immunosorbent assays, while chemerin mRNA and protein levels were measured by fluorescent quantitative polymerase chain reaction and Western blotting, respectively. Nitric oxide (NO) concentrations were determined with a colorimetric method. Akt and eNOS phosphorylation were assessed by Western blotting. We also tested the effects of the phosphoinositide 3-kinase inhibitor LY294002 and the eNOS inhibitor L-NAME. NF-κB p65 and VCAM-1 phosphorylation were assessed by Western blotting to investigate the role of chemerin in tumor necrosis factor (TNF)-α-induced HUVEC injury. Serum chemerin levels were increased in preeclampsia, while eNOS was decreased. Chemerin mRNA and protein were both increased in placentae from patients with preeclampsia. Furthermore, chemerin serum level positively correlated with blood pressure, body mass index, and serum insulin and was negatively correlated with serum eNOS. Chemerin dose-dependently increased NO concentrations in supernatants. Chemerin can increase eNOS and Akt levels in HUVECs, and these results could be partly blocked by LY294002 and L-NAME. Chemerin significantly decreased TNF-α-induced NF-κB and VCAM-1 in HUVECs, and these changes were partly inhibited by LY294002 and L-NAME. Chemerin may play a protective role by regulating NO signaling. Future studies should assess the role of chemerin in preeclampsia and other vascular diseases.

Apelin-13 Promotes Monocyte Adhesion to Human Umbilical Vein Endothelial Cell Mediated by Phosphatidylinositol 3-Kinase Signaling Pathway*

Previously we reported that G protein-coupled receptor APJ endogenous ligand apelin-13 induced adhesion of monocytes to human umbilical vein endothelial cells(HUVECs).Now we investigated whether phosphatidylinositol 3-kinase(PI3K) signaling pathway mediated monocytes(MCs) adhesion to HUVECs induced by apelin-13.Human umbilical vein endothelial cell line ECV304 was cultured in DMEM medium and the monocyte cell line THP-1 was cultured in 1640 medium.Myeloperoxidase(MPO) assay was used to identify effects of monocytes adhesion to HUVECs.Expression of vascular cell adhesion molecule(VCAM)-1,phospho-PI3K and PI3K were detected by Western blotting.Apelin-13 promoted PI3K phosphorylation in concentration-dependent and time-dependent manner,which reached the peak at 1 μmol/L and 30 min respectively.The PI3K inhibitor LY294002 inhibited PI3K phosphorylation and the expression of VCAM-1 in HUVECs induced by apelin-13.And the PI3K inhibitor LY294002 inhibited the MCs adhesion to HUVECs induced by apelin-13.It can be concluded that apelin-13 promoted monocytes adhesion to HUVECs through VCAM-1 mediated by PI3K signaling pathway.

In vitro biocompatibility evaluation of surface‐modified titanium alloys

The present work is aimed to evaluate the effects of a surface modification process on the biocompatibility of three vanadium-free titanium alloys with biomedical applications interest. Chemical composition of alloys investigated, in weight %, were Ti-7Nb-6Al, Ti-13Nb-13Zr, and Ti-15Zr-4Nb. An easy and economic method intended to improve the biocompatibiblity of these materials consists in a simple thermal treatment at high temperature, 750 degrees C, in air for different times. The significance of modification of the surface properties to the biological response was studied putting in contact both untreated and thermally treated alloys with human cells in culture, Human Umbilical Vein Endothelial Cells (HUVEC) and Human Peripheral Blood Mononuclear Cells (PBMC). The TNF-alpha release data indicate that thermal treatment improves the biological response of the alloys. The notable enhancement of the surface roughness upon oxidation could be related with the observed reduction of the TNF-alpha levels for treated alloys. A different behavior of the two cell lines may be observed, when adhesion molecules (ICAM-1 and VCAM-1 in HUVEC, ICAM-1, and LFA-1 in PBMC) were determined, PBMC being more sensitive than HUVEC to the contact with the samples. The data also distinguish surface composition and corrosion resistance as significant parameters for the biological response.

P.233 Transgingival lag-screw (TLS) osteosynthesis of alveolar process fractures

Certain infectious pathogens contribute to atherogenesis. Indeed, the strong relationship between periodontal pathogens, such as Porphyromonas gingivalis (P.g.) and coronary heart disease has been demonstrated. We investigated the potential role of P.g. in monocyte–endothelial interaction. Lipopolysaccharide (LPS) fraction was extracted from P.g. cultured under anaerobic conditions and compared to that obtained from an Escherichia coli (E. coli) strain (JM109). Human umbilical vein endothelial cells (HUVECs) were incubated in the presence of P.g.-LPS fraction or E. coli-LPS fraction for various periods and mononuclear cell adhesion assays were conducted under flow. The adhesion of mononuclear cells to HUVECs treated with P.g.-LPS fraction peaked after 24 h of incubation, whereas those treated with E. coli-LPS fraction maximized after 4 h of incubation. A fluorescent immunobinding assay revealed that P.g.-LPS fraction significantly upregulated ICAM-1 and VCAM-1 in HUVECs. Antibodies against ICAM-1 and Toll-like receptor (TLR)-2, but not TLR-4, attenuated P.g.-LPS fraction-facilitated mononuclear cell adhesion to HUVECs.In conclusion, these results suggest that chronic P.g. infection may facilitate monocyte recruitment to vascular endothelium through sustained upregulation of ICAM-1 and VCAM-1. Our findings provide new evidence that the TLR-2 pathway may contribute to atherogenesis by mediating P.g.-LPS signal transduction.

Extended exposure of lipopolysaccharide fraction from Porphyromonas gingivalis facilitates mononuclear cell adhesion to vascular endothelium via Toll-like receptor-2 dependent mechanism

Certain infectious pathogens contribute to atherogenesis. Indeed, the strong relationship between periodontal pathogens, such as Porphyromonas gingivalis (P.g.) and coronary heart disease has been demonstrated. We investigated the potential role of P.g. in monocyte–endothelial interaction. Lipopolysaccharide (LPS) fraction was extracted from P.g. cultured under anaerobic conditions and compared to that obtained from an Escherichia coli (E. coli) strain (JM109). Human umbilical vein endothelial cells (HUVECs) were incubated in the presence of P.g.-LPS fraction or E. coli-LPS fraction for various periods and mononuclear cell adhesion assays were conducted under flow. The adhesion of mononuclear cells to HUVECs treated with P.g.-LPS fraction peaked after 24h of incubation, whereas those treated with E. coli-LPS fraction maximized after 4h of incubation. A fluorescent immunobinding assay revealed that P.g.-LPS fraction significantly upregulated ICAM-1 and VCAM-1 in HUVECs. Antibodies against ICAM-1 and Toll-like receptor (TLR)-2, but not TLR-4, attenuated P.g.-LPS fraction-facilitated mononuclear cell adhesion to HUVECs.In conclusion, these results suggest that chronic P.g. infection may facilitate monocyte recruitment to vascular endothelium through sustained upregulation of ICAM-1 and VCAM-1. Our findings provide new evidence that the TLR-2 pathway may contribute to atherogenesis by mediating P.g.-LPS signal transduction.

Thrombin-induced Autoinhibitory Factor, Down Syndrome Critical Region-1, Attenuates NFAT-dependent Vascular Cell Adhesion Molecule-1 Expression and Inflammation in the Endothelium

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. We recently reported that thrombin and vascular endothelial growth factor, but not tumor necrosis factor-alpha, results in dramatic up-regulation of Down syndrome critical region (DSCR)-1 gene in endothelial cells, a negative feedback regulator of calcineurin-NFAT signaling. Constitutive expression of DSCR-1 in activated endothelial cells markedly impaired NFAT nuclear localization, proliferation, tube formation, and tumor growth. The goal of the present study was to elucidate the relative roles of NFAT/DSCR-1 and NF-kappaB/I-kappaB in mediating thrombin-responsive gene expression in endothelial cells. DNA microarrays of thrombin-treated human umbilical vein endothelial cells overexpressing DSCR-1 or constitutive active IkappaBalpha revealed genes that were dependent on NFAT and/or NF-kappaB activity. Vascular cell adhesion molecule-1 was inhibited both by DSCR-1 and I-kappaB at the level of mRNA, protein, promoter activity, and function (monocyte adhesion). Using a combination of transient transfections, electrophoretic mobility shift assays, and chromatin immunoprecipitation, thrombin was shown to induce time-dependent coordinate binding of RelA and NFATc to a tandem NF-kappaB element in the upstream promoter region of vascular cell adhesion molecule-1. Together, these findings suggest that thrombin-mediated activation of endothelial cells involves an interplay between NFAT and NF-kappaB signaling pathways and their negative feedback inhibitors, DSCR-1 and I-kappaB, respectively. As natural brakes in the inflammatory process, DSCR-1 and I-kappaB may lend themselves to therapeutic manipulation in vasculopathic disease states.

Inhibition of Lipopolysaccharide Activity by a Bacterial Cyclic Lipopeptide Surfactin

Compounds that inactivate lipopolysaccharide (LPS) activity have the potential of being new anti-inflammatory agents. Therefore, we searched among microbial secondary metabolites for compounds that inhibited LPS-stimulated adhesion between human umbilical vein endothelial cells (HUVEC) and HL-60 cells. By this screening, we found a cyclic lipopeptide surfactin from the culture broth of Bacillus sp. BML752-121F2 to be inhibitory. The addition of the surfactin prior to the LPS stimulation decreased HL-60 cell-HUVEC adhesion without showing any cytotoxicity. We confirmed that surfactin inhibited LPS-induced expression of ICAM-1 and VCAM-1 in HUVEC. It also inhibited the cellular adhesion induced by lipid A, the active component of LPS; but it did not inhibit TNF-alpha or IL-1 beta-induced cell adhesion. Then, surfactin was shown to suppress the interaction of lipid A with LPS-binding protein (LBP) that mediates the transport of LPS to its receptors. Finally, surface plasmon resonance (SPR) analysis revealed the surfactin to interact reversibly with lipid A. Thus, this Bacillus surfactin was shown to be an inhibitor of LPS-induced signal transduction, directly interacting with LPS.

Time sequence of the inhibition of endothelial adhesion molecule expression by reconstituted high density lipoproteins

We have used discoidal reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I and dimyristoyl phosphatidylcholine (DMPC) as a tool to investigate the time sequence of the HDL-mediated inhibition of vascular cell adhesion molecule (VCAM)-1 and E-selectin expression in cytokine-activated human umbilical vein endothelial cells (HUVECs). Specifically, we have asked a few questions — (i) how long do the cells need to be exposed to the rHDL before adhesion molecule expression is inhibited and (ii) how long does the inhibition persist after removing the rHDL from the cells. When the cells were not pre-incubated with the rHDL, there was no inhibition. The magnitude of the inhibition increased progressively with increasing duration of pre-incubation up to 16 h. Inhibition did not require the rHDL to be physically present during the activation of adhesion molecule expression by tumour necrosis factor(TNF)-α, excluding the possibility that the rHDL was merely interfering with the interaction between TNF-α and the cells. When HUVECs were pre-incubated for 16 h with rHDL, the inhibition remained substantial even if the rHDL were removed from the medium up to 8 h prior to addition of TNF-α. The HDL-mediated inhibition of VCAM-1 in HUVECs was unaffected by the presence of puromycin, an inhibitor of protein synthesis, excluding the possibility that HDL may have acted by stimulating the synthesis of a cell protein that itself inhibits adhesion molecule expression. These results have important implications in terms of understanding the mechanism(s) of the HDL-mediated inhibition of endothelial adhesion molecule expression.

Cobalt chloride-induced signaling in endothelium leading to the augmented adherence of sickle red blood cells and transendothelial migration of monocyte-like HL-60 cells is blocked by PAF-receptor antagonist.

In response to hypoxia, sickle red blood cells (SS RBC) and leukocytes exhibit increased adherence to the vascular endothelium, while diapedesis of leukocytes through the blood vessel increases. However, the cellular signaling pathway(s) caused by hypoxia is poorly understood. We utilized CoCl2 as a mimetic molecule for hypoxia to study cellular signaling pathways. We found that in human umbilical vein endothelial cells (HUVEC), CoCl2 at 2 mM concentration induced the surface expression of a subset of CAMs (VCAM-1) and activation of transcription factor NF-kappaB in the nuclear extracts of HUVEC. Furthermore, CoCl2 also caused time-dependent tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoform ERK2 without significantly affecting ERK1, indicating ERK2 is the preferred substrate for upstream kinase of the MAPK pathway. Inhibitors of MAP kinase (PD98059) or platelet-activating factor (PAF)- receptor antagonist (CV3988) inhibited the CoCl2-induced NF-kappaB activation and VCAM-1 expression. Augmented expression of VCAM-1 led to increased SS RBC adhesion, inhibitable by a VCAM-1 antibody. Additionally, CoCl2 caused a two- to threefold increase in the rate of transendothelial migration of monocyte-like HL-60 cells and a twentyfold increase in phosphorylation of platelet endothelial cell adhesion molecules (PECAM-1). The transendothelial migration of monocytes was inhibited by an antibody to PECAM-1. Both phosphorylation of PECAM-1 and transendothelial migration of monocytes in response to CoCl2 were inhibited by protein kinase inhibitor (GF109203X) and augmented by protein phosphatase inhibitor (Calyculin A). Our data suggests that CoCl2-induced cellular signals directing increased expression of VCAM-1 in HUVEC involve downstream activation of MAP kinase and NF-kappaB, while the phosphorylation of PECAM-1 occurs as a result of activation of PKC. We conclude that PAF-receptor antagonist inhibits the CoCl2- or hypoxia-induced increase in the adhesion of SS RBC, PECAM-1 phosphorylation, and the concomitant transendothelial migration of monocytes.

Interaction of Sickle Erythrocytes With Endothelial Cells in the Presence of Endothelial Cell Conditioned Medium Induces Oxidant Stress Leading to Transendothelial Migration of Monocytes

The abnormal adherence of sickle red blood cells (SS RBC) to endothelial cells has been thought to contribute to vascular occlusion, a major cause of morbidity in sickle cell disease (SCD). We determined whether the interaction of SS RBC with cultured endothelial cells induced cellular oxidant stress that would culminate in expression of cell adhesion molecules (CAMs) involved in the adhesion and diapedesis of monocytes and the adherence of SS reticulocytes. We showed that the interaction of SS RBC at 2% concentration in the presence of multimers of von Willebrand factor (vWf), derived from endothelial cell-derived conditioned medium (E-CM) with cultured human umbilical vein endothelial cells (HUVEC), resulted in a fivefold increased formation of thiobarbituric acid-reactive substances (TBARS) and activation of the transcription factor NF-kB, both indicators of cellular oxidant stress. Normal RBC show none of these phenomena. The oxidant stress-induced signaling resulted in an increased surface expression of a subset of CAMs, ICAM-1, E-selectin, and VCAM-1 in HUVEC. The addition of oxygen radical scavenger enzymes (catalase, superoxide dismutase) and antioxidant (probucol) inhibited these events. Additionally, preincubation of HUVEC with a synthetic peptide Arg-Gly-Asp (RGD) that prevents vWf-mediated adhesion of SS RBC reduced the surface expression of VCAM-1 and NF-kB activation. Furthermore, SS RBC-induced oxidant stress resulted in a twofold increase in the transendothelial migration of both monocyte-like HL-60 cells and human peripheral blood monocytes, and approximately a sixfold increase in platelet-endothelial cell adhesion molecule-1 (PECAM-1) phosphorylation, each of which was blocked by protein kinase C inhibitor and antioxidants. These results suggest that the adherence/contact of SS RBC to endothelial cells in large vessel can generate enhanced oxidant stress leading to increased adhesion and diapedesis of monocytes, as well as heightened adherence of SS reticulocytes, indicating that injury/activation of endothelium can contribute to vaso-occlusion in SCD.

Interaction of Sickle Erythrocytes With Endothelial Cells in the Presence of Endothelial Cell Conditioned Medium Induces Oxidant Stress Leading to Transendothelial Migration of Monocytes

The abnormal adherence of sickle red blood cells (SS RBC) to endothelial cells has been thought to contribute to vascular occlusion, a major cause of morbidity in sickle cell disease (SCD). We determined whether the interaction of SS RBC with cultured endothelial cells induced cellular oxidant stress that would culminate in expression of cell adhesion molecules (CAMs) involved in the adhesion and diapedesis of monocytes and the adherence of SS reticulocytes. We showed that the interaction of SS RBC at 2% concentration in the presence of multimers of von Willebrand factor (vWf), derived from endothelial cell-derived conditioned medium (E-CM) with cultured human umbilical vein endothelial cells (HUVEC), resulted in a fivefold increased formation of thiobarbituric acid-reactive substances (TBARS) and activation of the transcription factor NF-kB, both indicators of cellular oxidant stress. Normal RBC show none of these phenomena. The oxidant stress-induced signaling resulted in an increased surface expression of a subset of CAMs, ICAM-1, E-selectin, and VCAM-1 in HUVEC. The addition of oxygen radical scavenger enzymes (catalase, superoxide dismutase) and antioxidant (probucol) inhibited these events. Additionally, preincubation of HUVEC with a synthetic peptide Arg-Gly-Asp (RGD) that prevents vWf-mediated adhesion of SS RBC reduced the surface expression of VCAM-1 and NF-kB activation. Furthermore, SS RBC-induced oxidant stress resulted in a twofold increase in the transendothelial migration of both monocyte-like HL-60 cells and human peripheral blood monocytes, and approximately a sixfold increase in platelet-endothelial cell adhesion molecule-1 (PECAM-1) phosphorylation, each of which was blocked by protein kinase C inhibitor and antioxidants. These results suggest that the adherence/contact of SS RBC to endothelial cells in large vessel can generate enhanced oxidant stress leading to increased adhesion and diapedesis of monocytes, as well as heightened adherence of SS reticulocytes, indicating that injury/activation of endothelium can contribute to vaso-occlusion in SCD.