Endothelial Cell P-selectin Expression Research Articles

Molecular mechanisms underlying synergistic adhesion of sickle red blood cells by hypoxia and low nitric oxide bioavailability

AbstractThe molecular mechanisms by which nitric oxide (NO) bioavailability modulates the clinical expression of sickle cell disease (SCD) remain elusive. We investigated the effect of hypoxia and NO bioavailability on sickle red blood cell (sRBC) adhesion using mice deficient for endothelial NO synthase (eNOS) because their NO metabolite levels are similar to those of SCD mice but without hypoxemia. Whereas sRBC adhesion to endothelial cells in eNOS-deficient mice was synergistically upregulated at the onset of hypoxia, leukocyte adhesion was unaffected. Restoring NO metabolite levels to physiological levels markedly reduced sRBC adhesion to levels seen under normoxia. These results indicate that sRBC adherence to endothelial cells increases in response to hypoxia prior to leukocyte adherence, and that low NO bioavailability synergistically upregulates sRBC adhesion under hypoxia. Although multiple adhesion molecules mediate sRBC adhesion, we found a central role for P-selectin in sRBC adhesion. Hypoxia and low NO bioavailability upregulated P-selectin expression in endothelial cells in an additive manner through p38 kinase pathways. These results demonstrate novel cellular and signaling mechanisms that regulate sRBC adhesion under hypoxia and low NO bioavailability. Importantly, these findings point us toward new molecular targets to inhibit cell adhesion in SCD.

A biohybrid artificial lung prototype with active mixing of endothelialized microporous hollow fibers

Acute respiratory distress syndrome (ARDS) affects nearly 150,000 patients per year in the US, and is associated with high mortality ( approximately 40%) and suboptimal options for patient care. Mechanical ventilation and extracorporeal membrane oxygenation are limited to short-term use due to ventilator-induced lung injury and poor biocompatibility, respectively. In this report, we describe the development of a biohybrid lung prototype, employing a rotating endothelialized microporous hollow fiber (MHF) bundle to improve blood biocompatibility while MHF mixing could contribute to gas transfer efficiency. MHFs were surface modified with radio frequency glow discharge (RFGD) and protein adsorption to promote endothelial cell (EC) attachment and growth. The MHF bundles were placed in the biohybrid lung prototype and rotated up to 1,500 revolutions per minute (rpm) using speed ramping protocols to condition ECs to remain adherent on the fibers. Oxygen transfer, thrombotic deposition, and EC p-selectin expression were evaluated as indicators of biohybrid lung functionality and biocompatibility. A fixed aliquot of blood in contact with MHF bundles rotated at either 250 or 750 rpm reached saturating pO(2) levels more quickly with increased rpm, supporting the concept that fiber rotation would positively contribute to oxygen transfer. The presence of ECs had no effect on the rate of oxygen transfer at lower fiber rpm, but did provide some resistance with increased rpm when the overall rate of mass transfer was higher due to active mixing. RFGD followed by fibronectin adsorption on MHFs facilitated near confluent EC coverage with minimal p-selectin expression under both normoxic and hyperoxic conditions. Indeed, even subconfluent EC coverage on MHFs significantly reduced thrombotic deposition adding further support that endothelialization enhances, blood biocompatibility. Overall these findings demonstrate a proof-of-concept that a rotating endothelialized MHF bundle enhances gas transfer and biocompatibility, potentially producing safer, more efficient artificial lungs.

Simvastatin Attenuates Hepatic Sensitization to Lipopolysaccharide After Partial Hepatectomy

Hepatic resection may be curative in patients with hepatobiliary malignancies but excessive loss of liver volume may cause hepatic dysfunction and increase susceptibility to subsequent postoperative infections and sepsis. Herein, we hypothesized that pretreatment with simvastatin may protect against lipopolysaccharide (LPS)-induced liver damage after partial hepatectomy. Male C57Bl/6 mice underwent 68% hepatectomy and exposed to LPS after 24h. Animals were pretreated with simvastatin (0.02 and 0.2mg/kg). Serum alanine aminotransferase (ALT) and tumor necrosis factor-alpha (TNF-alpha) as well as leukocyte infiltration and hepatocyte apoptosis were examined 6h after LPS challenge. An in vitro endothelial cell adhesion assay was used to study the mechanisms of simvastatin on leukocyte adhesion and the role of P-selectin and lymphocyte function antigen-1 (LFA-1). Partial hepatectomy increased the sensitivity of the remnant liver tissue to LPS-provoked tissue injury. Simvastatin pretreatment reduced the LPS-induced increase in serum levels of ALT by 65% in hepatectomized animals. Moreover, simvastatin decreased leukocyte infiltration and hepatocyte apoptosis in the liver remnant of endotoxemic mice. LPS-provoked serum levels of TNF-alpha were decreased by 83% in hepatectomized animals treated with simvastatin. TNF-alpha-induced leukocyte adhesion as well as P-selectin expression in endothelial cells and LFA-1 function were inhibited by simvastatin in vitro. These novels findings demonstrate that simvastatin protects the remnant liver against endotoxemic injury following major hepatectomy. Thus, simvastatin reduced LPS-induced leukocyte recruitment and hepatocyte apoptosis in the liver remnant. One key mechanism appears to be related to the inhibition of TNF-alpha formation and function (P-selectin expression) as well as to direct actions on LFA-1 function. Thus, simvastatin may represent a novel therapeutic approach to prevent septic liver damage after partial liver resection.

A Radical Scavenger, Edaravone, Protects Canine Kidneys from Ischemia-Reperfusion Injury after 72 Hours of Cold Preservation and Autotransplantation

Cold ischemia-reperfusion (I/R) injury is a prominent cause of delayed graft function after kidney transplantation. Reactive oxygen species play a crucial role in I/R injury. Edaravone is a synthetic radical scavenger that has been used in acute stroke. Some animal experiments have revealed its beneficial effects against I/R injury, but its effects after cold preservation and transplantation of canine kidneys are unknown. Female hybrid dogs weighing 11 to 13 kg were used. Under anesthesia, the left kidney was harvested. After 72 hr of preservation in cold histidine-tryptophan-ketoglutarate solution, autotransplantation was performed in the right iliac fossa, with contralateral nephrectomy. Animals were divided into control and treatment groups (n=6 per group). In the treatment group, edaravone was administered intravenously at harvest and at reperfusion (3 mg/kg) and in addition was added to the preservation solution (50 microM). Animal survival at 2 weeks was four of six in the control group and six of six in the treatment group. Compared with controls, treated animals had higher mean urine output, higher mean glomerular filtration rate, improved tubular cell function, lower mean serum creatinine, and lower renal vascular resistance. Biopsy specimens from treated animals showed less tubular cell damage and decreased P-selectin expression in endothelial cells. Lipid peroxidation of renal tissue and urinary excretion of 8-hydroxy-2'-deoxyguanosine were suppressed by the treatment. Edaravone reduced cold I/R injury in canine renal transplantation. The agent has the potential to ameliorate preservation injury in clinical transplantation.

Cytokines in Sickle Cell Disease

Sickle red cells express adhesion molecules including integrin 4β1, CD36, band 3 protein, sulfated glycolipid, Lutheran protein, phosphatidylserine and integrin-associated protein. The proadhesive sickle cells may bind to endothelial cell P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD36 and integrins leading to its activation. Monocytes also activate endothelium by releasing proinflammatory cytokines like tumor necrosis factor alpha (TNF-) and interleukin 1β (IL-1β). Sickle monocytes also express increased surface CD11b and cytoplasmic cytokines TNF and IL-1β indicating activated state. Polymorphonuclear leukocytes (PMNs) are also activated with reduced L-selectin expression, enhanced CD64 expression and elevated levels of sL-selectin, sCD16 and elastase resulting in increased adhesiveness to the endothelium. Platelets are also activated and secrete thrombospondin (TSP) and cytokine IL-1. They also form platelet- monocytes aggregates causing endothelial cell P-selectin expression. Endothelial cell activation by these multiple mechanisms leads to a loss of vascular integrity, expression of leukocyte adhesion molecules, change in the surface phenotype from antithrombotic to prothrombotic, excessive cytokine production and upregulation of HLA molecules. Furthermore, contraction of these activated endothelial cells leads to exposure of extracellular matrix proteins, such as TSP, laminin, and fibronectin and their participation in adhesive interactions with bridging molecules from the plasma such as von Willebrand factor (vWf) released from endothelial cells, ultimately culminating in vasoocclusion and local tissue ischemia, the pathognomonic basis of vasoocclusive crisis.

Mitochondrial reactive oxygen species regulate spatial profile of proinflammatory responses in lung venular capillaries.

Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor- vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-alpha and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca(2+), both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca(2+) increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-alpha, but not arachidonate, released Ca(2+) from endoplasmic stores and increased mitochondrial Ca(2+). Furthermore, Ca(2+) depletion abrogated TNF-alpha responses partially, but arachidonate responses completely. These differences in Ca(2+) mobilization by TNF-alpha and arachidonate were reflected in spatial patterning in the capillary in that the TNF-alpha effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-alpha- but not the arachidonate-induced responses. These findings indicate that the different modes of Ca(2+) mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-alpha revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.

Effect of Prematurity on the Presence of Weibel-Palade Bodies in Human Umbilical Vein Endothelial Cell In Situ

Abstract Newborn, premature infants have greater susceptibility to infection compared to older newborn infants. Although the reason for greater susceptibility to bacterial infection in premature infants is unknown, there are many reports that focus on polymorphonuclear leukocytes (neutrophils) as the defective cell type in the immature neonatal system. However, we have recently reported that in an rat model of inflammation, neutrophils from adult rats failed to migrate efficiently in neonatal rats. Furthermore, we found that this defect was associated with decreased expression of P-selectin on the surface of neonatal endothelial cells in situ. P-selectin is an adherence molecule that is expressed by activated endothelial cells and serves as the counterligand for CD11/CD18, the β2- integrin that is expressed by activated neutrophils. We hypothesized that endothelial cell P-selectin expression would be less in human umbilical cord veins from prematurely born (preterm) infants compared to normal term infants. To test this hypothesis, we collected umbilical cords from preterm infants (<27 weeks gestation) and term infants (>38 weeks gestation).

Inhibition of gastric secretion relieves post-prandial urgency, diarrhea, and cramps in a variety of GI disorders

While several endothelial cell adhesion molecules (CAM) have been implicated as mediators of the leukocyte-endothelial cell adhesion observed in inflamed microvessels, P-selectin appears to be the major CAM that accounts for the very rapid (minutes) leukocyte rolling observed in these vessels. P-selectin can be rapidly mobilized from a preformed storage pool (WeibelPalade bodies) within endothelial cells by different stimuli, including histamine, thrombin, hydrogen peroxide, and leukotrienes. The objective of this study was to determine whether bradykinin, a well-known participant in inflammatory reactions can elicit P-selectin expression in the gastrointestinal microvasculature. The dual radiolabelled antibody technique was used to quantify endothelial cell P-selectin expression in rat metentery, small intestine, large intestine, pancreas and stomach. Intraperitoneal administration of bradykinin (25-200 lag) induced a dose-dependent up-regulation of P-selectin in most of the gastrointestinal organs. The increased P-selectin in these vascular beds occured within 15 min after bradykinin administration; no increases were noted 3 hrs after bradykinin. In order to determine whether the bradykinin B 2 receptor mediates the increased P-selectin expression elicited by bradykinin, some animals were pretreated with a Bz-receptor antagonist (HOE). In all gastrointestinal organs, HOE completely prevented the bradykinin-induced increase in P-selectin expression. These findings indicate that bradykinin can mobilize the preformed pool of P-selectin in vascular endothelial ceils and that bradykinin-mediated P-selectin involves the engagement of B~-receptors. Supported by P01 DK43785