Endothelial P-selectin Expression Research Articles

Erianin alleviates LPS-induced acute lung injury via antagonizing P-selectin-mediated neutrophil adhesion function

Ethnopharmacological relevanceDendrobium officinale Kimura et Migo, known as “Tiepi Shihu” in traditional Chinese medicine, boasts an extensive history of medicinal use documented in the Chinese Pharmacopoeia. “Shen Nong Ben Cao Jing” records D. officinale as a superior herbal medicine for fortifying “Yin” and invigorating the five viscera. Erianin, a benzidine compound, emerges as a prominent active constituent derived from D. officinale, with the pharmacological efficacy of D. officinale closely linked to the anti-inflammatory properties of erianin. Aim of the studyAcute lung injury (ALI) is a substantial threat to global public health, while P-selectin stands out as a promising novel target for treating acute inflammatory conditions. This investigation aims to explore the therapeutic potential of erianin in ALI treatment and elucidate the underlying mechanisms. Experimental designThe effectiveness of erianin in conferring protection against ALI was investigated through comprehensive histopathological and biochemical analyses of lung tissues and bronchoalveolar lavage fluid (BALF) in an in vivo model of LPS-induced ALI in mice. The impact of erianin on fMLP-induced neutrophil chemotaxis was quantitatively assessed using the Transwell and Zigmond chamber, respectively. To determine the therapeutic target of erianin and elucidate their binding capability, a series of sophisticated assays were employed, including drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and molecular docking analyses. ResultsErianin demonstrated a significant alleviation of LPS-induced acute lung injury, characterized by reduced total cell and neutrophil counts and diminished total protein contents in BALF. Moreover, erianin exhibited a capacity to decrease proinflammatory cytokine production in both lung tissues and BALF. Notably, erianin effectively suppressed the activation of NF-κB signaling in the lung tissues of LPS- challenged mice; however, it did not exhibit in vitro inhibitory effects on inflammation in LPS-induced human pulmonary microvascular endothelial cells (HPMECs). Additionally, erianin blocked the adhesion and rolling of neutrophils on HPMECs. While erianin did not influence endothelial P-selectin expression or cytomembrane translocation, it significantly reduced the ligand affinity between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). ConclusionsErianin inhibits P-selectin-mediated neutrophil adhesion to activated endothelium, thereby alleviating ALI. The present study highlights the potential of erianin as a promising lead for ALI treatment.

Oral famotidine reduces the plasma level of soluble P-selectin in children with sickle cell disease.

Plasma histamine levels are increased in patients with sickle cell disease (SCD), potentially promoting endothelial P-selectin expression and vaso-occlusion via histamine type 2 (H2) receptors. We conducted a prospective, non-comparative, single-centre study to determine whether famotidine, a H2 receptor antagonist, reduces P-selectin expression in SCD children. The median plasma P-selectin level was significantly reduced after 29 days of oral famotidine (53.2 ng/mL [IQR: 46.7-63.4] vs. 69.9 ng/mL [IQR: 53.6-84.2], median difference -10.2 ng/mL [IQR: -21.8 to -2.7], p = 0.005) in 28 patients. No effect was observed on other adhesion molecules, inflammation or haemolysis markers, except decreased reticulocyte count. No adverse events deemed related to famotidine were observed. Randomized controlled trials are now needed to assess the efficacy of famotidine in preventing vaso-occlusion in SCD.

A novel galactoxylan derived from Viola diffusa alleviates LPS-induced acute lung injury via antagonizing P-selectin-mediated adhesion function

Acute lung injury (ALI) greatly threatens human health worldwide. P-selectin is a potential target for the treatment of acute inflammatory diseases, and natural polysaccharides exhibit high-affinity for P-selectin. Viola diffusa, a traditional Chinese herbal, shows strong anti-inflammatory effects, but pharmacodynamic substances and underlying mechanisms are still unclear. In this study, a galactoxylan polysaccharide (VDPS) derived from Viola diffusa was isolated and characterized, evaluated the protective effect on LPS induced ALI and underlying mechanism. VDPS significantly alleviated LPS-induced pathological lung injury, and decreased the numbers of total cells and neutrophils as well as the total protein contents in the bronchoalveolar lavage fluid (BALF). Moreover, VDPS reduced proinflammatory cytokine production both in BALF and lung. Interestingly, VDPS significantly restrained the activation of NF-κB signaling in the lung of LPS-exposed mice, but it cannot inhibit LPS-induced inflammation in human pulmonary microvascular endothelial cells (HPMECs) in vitro. Additionally, VDPS disrupted neutrophil adhesion and rolling on the activated HPMECs. VDPS cannot impact the expression or cytomembrane translocation of endothelial P-selectin, but remarkably interrupt the binding of P-selectin and PSGL-1. Overall, this study demonstrated that VDPS can alleviate LPS-induced ALI via inhibiting P-selectin-dependent adhesion and recruitment of neutrophils on the activated endothelium, providing a potential treatment strategy for ALI.

NLRP3 inflammasome activation in neutrophils directs early inflammatory response in murine peritonitis.

NLR family pyrin domain containing 3 (NLRP3) inflammasome mediates caspase-1-dependent processing of inflammatory cytokines such as IL-1β, an essential endothelial activator, and contributes to the pathology of inflammatory diseases. To evaluate the role of NLRP3 in neutrophils in endothelial activation, which is still elusive, we used the thioglycollate-induced peritonitis model characterized by an early neutrophil influx, on Nlrp3-/- and Nlrp3+/+ mice. Nlrp3-/- mice recruited fewer neutrophils than Nlrp3+/+ into the peritoneum and showed lower IL-1β in peritoneal lavage fluid. The higher production of IL-1β in Nlrp3+/+ was neutrophil-dependent as neutrophil depletion prevented the IL-1β production. The Nlrp3+/+ neutrophils collected from the peritoneal fluid formed significantly more filaments (specks) than Nlrp3-/- neutrophils of ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain), a readout for inflammasome activation. Intravital microscopy revealed that leukocytes rolled significantly slower in Nlrp3+/+ venules than in Nlrp3-/-. Nlrp3-/- endothelial cells isolated from mesenteric vessels demonstrated a lower percentage of P-selectin-positive cells with lower intensity of surface P-selectin expression than the Nlrp3+/+ endothelial cells evaluated by flow cytometry. We conclude that neutrophils orchestrate acute thioglycollate-induced peritonitis by producing IL-1β in an NLRP3-dependent manner. This increases endothelial P-selectin expression and leukocyte transmigration.

Endothelial Cell Activation Enhances Thromboinflammation in Vitt

Aim: Vaccine induced thrombosis thrombocytopenia (VITT) syndrome is a rare complication of ChAdOx1 vaccination for COVID19. VITT shares similarities with heparin induced thrombocytopenia (HIT). In both VITT and HIT, patients develop antibodies against platelet factor 4 (PF4) which promote platelet activation and thrombosis. Involvement of the endothelium in the pathogenic process has been described for HIT. Binding of HIT antibodies to PF4/glycosaminoglycan complexes on the surface of endothelial cells leads to their activation and increased procoagulant activity (Madeeva, J Thromb Haemost, 2016). However, the contribution of the endothelium to VITT has not been described. Here, we evaluate the effect of VITT plasma or serum on endothelial cell activation and thromboinflammation using an endothelialized biochip. Method: Serum and plasma samples were received from patients fulfilling clinical criteria of VITT including venous and/or arterial thrombosis, thrombocytopenia <150 x 10^3/uL, D-dimer >5-fold of upper limit of normal within 4-42 days after 1st dose of ChAdOx1 vaccine (termed "Clinical VITT") and from healthy, age and sex matched, ChAdOx1 vaccinated individuals within the same timeframe from vaccination (termed "Vaccine controls"). PF4 ELISA and VITT functional assays were performed on all samples (Lee, Blood Adv, 2022). Human umbilical vein endothelial cells (HUVECs) were treated with heat inactivated serum or plasma in the presence of peptide GPRP. Polydimethylsiloxane microfluidic straight channels were seeded with HUVECs (Endochip) and treated with VITT or control serum and compared with TNF alpha treatment. The endothelial surface expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), P-selectin, tissue factor (TF) and von Willebrand factor (vWF) were measured by immunostaining in the presence or absence of native platelet factor 4 (PF4). In some experiments HUVECs were treated with ChAdOx1 vaccine or vehicle for 48-120h. Thromboinflammation was measured by fluorescence area of antibody-stained platelets, neutrophils and fibrin on the Endochip after perfusion of recalcified blood from HIT antibody-reactive healthy volunteers (Morel-Kopp, J Thromb Haemost, 2016) at a shear rate of 100s-1. Results: Samples from 10 patients presenting with clinically confirmed VITT were paired with samples from 10 vaccinated individuals. Clinical VITT samples were separated into negative and positive for both PF4 ELISA and VITT functional assay (defined as VITT ELISA positive and VITT ELISA negative, Panel A). Endothelial cell surface expression of P-selectin was increased after 12 h incubation with 25% v/v VITT serum or plasma compared with samples from AZ vaccinated non-VITT individuals (median integrated intensity/cell 8065 vs 3515 a.u, p=0.03). The expression of TF was significantly increased by addition of PF4 to the VITT plasma (median integrated intensity/cell 42038 vs 14881 a.u., p=0.01 by Mann Whitney test). ChAdOx1 treatment of HUVECs at 1:1000 dilution induced the expression of spike protein but did not significantly enhance thromboinflammation compared with media, as assessed by the perfusion of whole blood through the Endochip. Treatment of the Endochip with VITT serum, without the addition of PF4, significantly increased the adhesion of platelets compared with control serum (median 2.2-fold, p=0.002), neutrophils (2.5-fold, p=0.003) and fibrin (5.4-fold, p=0.002 by Wilcoxon paired samples test (Panel B). There was no difference in the thromboinflammatory response between ELISA negative and positive group (Panel B). Conclusion: Endothelial inflammation contributes to the prothrombotic tendency in VITT in an in vitro model of thromboinflammation. VITT serum induces the endothelial expression of P-selectin and TF. Endothelial inflammation may explain thrombotic events in cerebral or splanchnic veins of vaccinated individuals without detectable anti-PF4 antibodies by ELISA or platelet-based VITT functional assays. Endothelialised biochips have diagnostic potential for detecting immune prothrombotic tendencies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mast Cells Induce Blood Brain Barrier Damage in SCD by Causing Endoplasmic Reticulum Stress in the Endothelium.

Endothelial dysfunction underlies the pathobiology of cerebrovascular disease. Mast cells are located in close proximity to the vasculature, and vasoactive mediators released upon their activation can promote endothelial activation leading to blood brain barrier (BBB) dysfunction. We examined the mechanism of mast cell-induced endothelial activation via endoplasmic reticulum (ER) stress mediated P-selectin expression in a transgenic mouse model of sickle cell disease (SCD), which shows BBB dysfunction. We used mouse brain endothelial cells (mBECs) and mast cells-derived from skin of control and sickle mice to examine the mechanisms involved. Compared to control mouse mast cell conditioned medium (MCCM), mBECs incubated with sickle mouse MCCM showed increased, structural disorganization and swelling of the ER and Golgi, aggregation of ribosomes, ER stress marker proteins, accumulation of galactose-1-phosphate uridyl transferase, mitochondrial dysfunction, reactive oxygen species (ROS) production, P-selectin expression and mBEC permeability. These effects of sickle-MCCM on mBEC were inhibited by Salubrinal, a reducer of ER stress. Histamine levels in the plasma, skin releasate and in mast cells of sickle mice were higher compared to control mice. Compared to control BBB permeability was increased in sickle mice. Treatment of mice with imatinib, Salubrinal, or P-selectin blocking antibody reduced BBB permeability in sickle mice. Mast cells induce endothelial dysfunction via ER stress-mediated P-selectin expression. Mast cell activation contributes to ER stress mediated endothelial P-selectin expression leading to increased endothelial permeability and impairment of BBB. Targeting mast cells and/or ER stress has the potential to ameliorate endothelial dysfunction in SCD and other pathobiologies.

Thrombin-Mediated Activation of PAR-1 Contributes to Microvascular Stasis in Mouse Models of Sickle Cell Disease Via Increased Endothelial Expression of P-Selectin and VWF

Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene, resulting in an altered red cell physiology that causes vascular complications such as hemolytic anemia, chronic inflammation, activation of coagulation, and vaso-occlusion. We have shown that infusion of hemin results in tissue factor (TF)-dependent activation of coagulation in mice. Furthermore, TF-dependent activation of coagulation contributes to inflammation in a mouse model of SCD. Interestingly, thrombin-dependent inflammation in sickle cell mice was not attenuated by deficiency of the main thrombin receptor protease activated receptor-1 (PAR-1). However, others have shown that the activation of endothelial cell PAR-1 with agonist peptide enhances interactions of these cells with sickle RBCs in a P-selectin-dependent manner. Importantly, P-selectin inhibition reduces microvascular stasis in mouse models of SCD and prevents vaso-occlusive crisis in sickle cell patients.We propose that thrombin-mediated PAR-1 activation promotes microvascular stasis in mouse models of SCD via increased expression of P-selectin and VWF on the endothelium, triggered by exocytosis of Weibel-Palade bodies.To investigate if the TF/thrombin/PAR-1 pathway contributes to microvascular stasis, dorsal skinfold chambers were implanted in NY1DD sickle mice 3 days before the experiment. Microvascular stasis was determined in 20-25 preselected micro-vessels in response to intravenous infusion of stroma-free hemoglobin (SFH, 1.6 µmol/kg) and was expressed as % non-flowing vessels (mean ± SEM). We previously demonstrated that infusion of SFH results in microvascular stasis that is inhibited by hemopexin in NY1DD mice, indicating that hemoglobin releases heme into the circulation. In NY1DD mice treated with control IgG antibody, SFH infusion caused stasis in 28.3 ± 1.7% and 36.7 ± 1.7% of preselected vessels at 1 and 4 hrs after infusion, respectively. Treatment with an inhibitory anti-TF antibody 1H1 (25 mg/kg; IP) 30 minutes before SFH infusion significantly reduced stasis to only 3.3 ± 1.5% and 2.8 ± 1.6% of vessels at 1 and 4 hrs, respectively (p<0.01). To investigate if TF contributes to SFH-induced microvascular stasis via generation of downstream coagulation proteases, NY1DD mice were fed with control chow or chow containing either Factor Xa (FXa) inhibitor rivaroxaban (0.4 mg/g chow) or thrombin inhibitor dabigatran (10 mg/g chow) ad libitum for 4 days prior to stasis experiments. We previously showed that these doses efficiently anticoagulated sickle mice without bleeding complications. FXa inhibition significantly reduced stasis at 1 hr (4.9 ± 0.1% versus 22.4 ± 3.8%, p<0.001) and 4 hrs (1.7 ± 1.6% versus 12.8 ± 1.9%, p<0.001) after SFH injection. Similarly, dabigatran also significantly attenuated SFH-induced stasis (3.1 ± 1.5% non-flowing vessels at 1 hour and 1.6 ± 1.6% non-flowing vessels at 4 hours). Next, to investigate if thrombin contributes to SFH-induced stasis through activation of PAR-1, we used bone marrow transplantation to generate sickle mice lacking PAR-1 expression in all non-hematopoietic cells. PAR-1+/+ and PAR-1-/- mice were lethally irradiated and transplanted with bone marrow from Townes sickle (SS) mice. Efficient reconstitution of bone marrow was confirmed by hemoglobin electrophoresis. We found that PAR-1-/- SS mice were significantly protected from SFH-induced stasis compared to PAR-1+/+ SS mice at both 1 hr (13.2 ± 3.4% versus 37.6 ± 3.9% stasis; P<0.001) and 4 hrs (6.6 ± 1.7% versus 18.0 ± 1.5% stasis; p<0.05) after hemoglobin challenge. We have previously shown that a monoclonal antibody to murine P-selectin or a polyclonal antibody to VWF markedly inhibit stasis in sickle mice. After the 4 hr stasis measurement, lungs were harvested from PAR-1+/+ SS and PAR-1-/- SS the mice and stained for P-selectin, VWF, and CD31 (a marker for endothelial cells). We found that intensity of P-selectin and VWF staining was markedly reduced in the lungs of PAR-1-/- SS mice compared to the staining observed in lungs of PAR-1+/+ SS mice.Our data indicates that SFH-induced microvascular stasis is significantly diminished by inhibition of TF, FXa, or thrombin activity or knockout of endothelial PAR-1, which leads to reduced expression of P-selectin and VWF on endothelium in a mouse model of SCD. We speculate that inhibiting the TF/thrombin/PAR-1 axis may reduce vaso-occlusive crises in SCD patients. DisclosuresVercellotti:CSL Behring: Research Funding. Belcher:CSL Behring: Research Funding.

Mast Cells Contribute to Brain Microvascular Permeability in Sickle Cell Disease

Mast cells are in close proximity to the vasculature and cause endothelial activation, plasma extravasation, and vascular dysfunction (Gupta & Harvima, Immunol Rev 2018). Vascular dysfunction in sickle cell disease (SCD) is accompanied by increased expression of P-selectin. Treatment with Crizanlizumab, an antibody against P-selectin, led to significantly less sickle cell-related pain crises (Ataga et al., NEJM 2017) highlighting the role of endothelial P-selectin in vasoocclusive crises (VOC). Earlier studies demonstrated that mast cell activation with morphine or ischemia/reperfusion stimulates endothelial E- and/or P-selectin expression. However, it is unknown how mast cells stimulate endothelial selectin expression in SCD. Endothelial dysfunction contributes significantly to the pathobiology of SCD including VOC and may play a critical role in increased blood-brain barrier (BBB) permeability, which may contribute to stroke, another major comorbidity of SCD. One of the known triggers of endothelial dysfunction, inflammation and oxidative stress is endoplasmic reticulum (ER) stress. We hypothesize that in a sickle microenvironment, mediators derived from activated mast cells stimulate endothelial P-selectin expression via ER stress leading to increased BBB permeability. We examined the ability of mast cells to stimulate P-selectin expression and BBB permeability via ER stress in a sickle microenvironment.We isolated MCs from HbAA-BERK and HbSS-BERK, control and sickle mice, respectively; incubated them in vitro and collected mast cell conditioned media (MCCM) from HbAA MCs and HbSS MCs. Normal mouse brain microvascular endothelial cells (mBMECs) were treated with unconditioned MCCM, HbAA MCCM, or HbSS MCCM to examine the effect of mast cell activation on endothelium. We observed increased mast cell activity in HbSS mice evinced by significantlyhigher plasma and skin histamine levels, compared to HbAA mice (p< 0.02 for both). Mast cells from HbSS mouse skin showed significantly increased expression of histamine compared to HbAA skin mast cells (p< 0.04). mBMECs incubated with HbAA and HBSS MCCM exhibited about 3- and 6-fold fold increases in P-selectin expression, compared to unconditioned culture medium, respectively (p< 0.0001 for both). Therefore, mast cells in culture release substances that stimulate P-selectin expression which is further increased by mast cells from sickle (HbSS) microenvironment. Preincubation of mBMEC with 5 microM salubrinal, an inhibitor of dephosphorylation of elongation initiation factor-a, which reduces ER stress, significantly inhibited HbSS MCCM-induced P-selectin expression on mBMEC (p< 0.0001) to the level induced by HbAA-MCCM. In contrast, salubrinal did not inhibit HbAA-MCCM-induced P-selectin expression on mBMEC, suggesting that in a sickle microenvironment mast cells contribute to P-selectin expression via ER stress.We next examined mast cell activity on endothelial permeability in vitro on mBMEC monolayers and in vivo in the brain of HbSS mice. mBMECs incubated with HbSS MCCM showed a significant increase in Evans blue leakage compared to unconditioned or HbAA MCCM (p<0.0001 for both), which was inhibited by preincubation of mBMEC with 5 microM salubrinal prior to incubation with HbSS MCCM (p< 0.0001). In vivo female HbSS mice showed a significantly increased leakage in the brain of FITC-dextran injected through tail vein compared to HbAA mice (p< 0.01). HbSS mice treated with 1 mg/kg salubrinal demonstrated inhibition of FITC-dextran leakage in the brain compared to vehicle (p< 0.05). Thus, ER stress contributes to increased BBB permeability in HbSS mice. We observed activated degranulating mast cells in the brain parenchyma of HbSS mice. In HbAA mice, quiescent mast cells were confined to the meninges of the brain but not seen in the parenchyma. Together, these data suggest that mast cell activation contributes to BBB permeability in a sickle microenvironment via ER stress-mediated P-selectin expression. In turn, this mast cell-initiated activity in the brain may underlie the pathobiology of stroke in SCD. Inhibitors of mast cells and P-selectin have been tested clinically leading to reduced VOC in SCD without known adverse events. Therefore, mast cell activation-induced P-selectin via ER stress may serve as a treatable target for reducing the risk of stroke in SCD. DisclosuresGupta:Novartis: Honoraria; Tau tona: Consultancy.

Vascular endothelial cell expression of JAK2V617F is sufficient to promote a pro-thrombotic state due to increased P-selectin expression.

Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F-expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F-expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F-expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F-positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses.

Phosphodiesterase III inhibitor attenuates rat sinusoidal obstruction syndrome through inhibition of platelet aggregation in Disse's space.

Sinusoidal obstruction syndrome (SOS) is a serious drug-induced liver injury. However, the pathophysiology of the disease remains unclear. This study investigated the effects of cilostazol (CZ), a phosphodiesterase III inhibitor, in a monocrotaline (MCT)-induced rat model of SOS. Male Wistar rats were administrated MCT to induce SOS. Rats were divided into control, MCT, and MCT+CZ groups. In the MCT+CZ group, CZ was administered at 48h, 24h, and 30min prior to and 8h and 24h after MCT administration. The MCT group was treated with water instead of CZ. At 48h after MCT administration, blood and liver samples were collected to assess biochemistry and liver histology. Expression of rat endothelial cell antigen, CD34, CD41, P-selectin, and caspase-3 in the liver were analyzed. Plasminogen activator inhibitor-1 (PAI-1) in hepatocytes was analyzed using western blotting and polymerase chain reaction. In the MCT group, macroscopic findings showed a dark-red liver surface. Histological findings showed sinusoidal dilatation, coagulative necrosis of hepatocytes, and endothelial damage of the central vein. These changes were attenuated in the MCT+CZ group. Elevated serum transaminase and decreased platelet counts were observed in the MCT+CZ group compared with those in the MCT group. Treatment with CZ reduced MCT-induced damage to the liver sinusoidal endothelial cells, inhibited extravasated platelet aggregation, and suppressed hepatocyte apoptosis around the central vein. CZ attenuated hepatic PAI-1 protein and mRNA levels. Cilostazol attenuated MCT-induced SOS by preventing damage to liver sinusoidal endothelial cells and extravasated platelet aggregation. Hepatic PAI-1 levels were suppressed with CZ treatment.

Vitamin C and Microvascular Dysfunction in Systemic Inflammation

Sepsis, life-threatening organ dysfunction caused by a dysfunctional host response to infection, is associated with high mortality. A promising strategy to improve the outcome is to inject patients intravenously with ascorbate (vitamin C). In animal models of sepsis, this injection improves survival and, among others, the microvascular function. This review examines our recent work addressing ascorbate’s ability to inhibit arteriolar dysfunction and capillary plugging in sepsis. Arteriolar dysfunction includes impaired vasoconstriction/dilation (previously reviewed) and impaired conduction of vasoconstriction/dilation along the arteriole. We showed that ascorbate injected into septic mice prevents impaired conducted vasoconstriction by inhibiting neuronal nitric oxide synthase-derived NO, leading to restored inter-endothelial electrical coupling through connexin 37-containing gap junctions. Hypoxia/reoxygenation (confounding factor in sepsis) also impairs electrical coupling by protein kinase A (PKA)-dependent connexin 40 dephosphorylation; ascorbate restores PKA activation required for this coupling. Both effects of ascorbate could explain its ability to protect against hypotension in sepsis. Capillary plugging in sepsis involves P-selectin mediated platelet-endothelial adhesion and microthrombi formation. Early injection of ascorbate prevents capillary plugging by inhibiting platelet-endothelial adhesion and endothelial surface P-selectin expression. Ascorbate also prevents thrombin-induced platelet aggregation and platelet surface P-selectin expression, thus preventing microthrombi formation. Delayed ascorbate injection reverses capillary plugging and platelet-endothelial adhesion; it also attenuates sepsis-induced drop in platelet count in systemic blood. Thrombin-induced release of plasminogen-activator-inhibitor-1 from platelets (anti-fibrinolytic event in sepsis) is inhibited by ascorbate pH-dependently. Thus, under acidotic conditions in sepsis, ascorbate promotes dissolving of microthrombi in capillaries. We propose that protected/restored arteriolar conduction and capillary bed perfusion by ascorbate contributes to reduced organ injury and improved survival in sepsis.

Fluid-induced lung injury-role of TRPV4 channels.

Administration of bolus intravenous fluid is associated with respiratory dysfunction and increased mortality, findings with no clear mechanistic explanation. The objective of this study was to examine whether bolus intravenous (i.v.) fluid administration results in acute lung injury in a rat model and further, to examine whether this injury is associated with transient receptor potential vallinoid (TRPV)4 channel function and endothelial inflammatory response. Healthy male Sprague-Dawley rats were administered 60ml/kg 0.9% saline i.v. over 30min. Manifestation of acute lung injury was assessed by lung physiology, morphology, and markers of inflammation. The role of TRPV4 channels in fluid-induced lung injury was subsequently examined by the administration of ruthenium red (RR) in this established rat model and again in TRPV4 KO mice. In endothelial cell culture, permeability and P-selectin expression were measured following TRPV4 agonist with and without antagonist; 0.9% saline resulted in an increase in lung water, lavage protein and phospholipase A2, and plasma angiopoietin-2, with worsening in arterial blood oxygen (PaO2), lung elastance, surfactant activity, and lung histological injury score. These effects were ameliorated following i.v. fluid in rats receiving RR. TRPV4 KO mice did not develop lung edema. Expression of P-selectin increased in endothelial cells following administration of a TRPV4 agonist, which was ameliorated by simultaneous addition of RR. Bolus i.v. 0.9% saline resulted in permeability pulmonary edema. Data from ruthenium red, TRPV4 KO mice, and endothelial cell culture suggest activation of TRPV4 and release of angiopoietin 2 and P-selectin as the central mechanism.

Ascorbate inhibits platelet-endothelial adhesion in an in-vitro model of sepsis via reduced endothelial surface P-selectin expression.

Plugging of the capillary bed can lead to organ failure and mortality in sepsis. We have reported that intravenous ascorbate injection reduces platelet adhesion to the capillary wall and capillary plugging in septic mice. Both platelet adhesion and capillary plugging require P-selectin, a key adhesion molecule. To elucidate the beneficial effect of ascorbate, we hypothesized that ascorbate reduces platelet-endothelial adhesion by reducing P-selectin surface expression in endothelial cells. We used mouse platelets, and monolayers of cultured microvascular endothelial cells (mouse skeletal muscle origin) stimulated with lipopolysaccharide, to examine platelet-endothelial adhesion. P-selectin mRNA expression in endothelial cells was determined by real-time PCR and P-selectin protein expression at the surface of these cells by immunofluorescence. Secretion of von Willebrand factor from cells into the supernatant (a measure of P-selectin-containing granule exocytosis) was determined by ELISA. Lipopolysaccharide (10 μg/ml, 1 h) increased platelet-endothelial adhesion. P-selectin-blocking antibody inhibited this adhesion. Lipopolysaccharide also increased P-selectin mRNA in endothelial cells, P-selectin expression at the endothelial surface, and von Willebrand factor secretion. Ascorbate pretreatment (100 μmol/l, 4 h) inhibited the increased platelet adhesion, surface expression of P-selectin, and von Willebrand factor secretion, but not the increase in P-selectin mRNA. The lipopolysaccharide-induced increase in platelet-endothelial adhesion requires P-selectin presence at the endothelial surface. Ascorbate's ability to reduce this presence could be important in reducing both platelet adhesion to the capillary wall and capillary plugging in sepsis.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003.

BackgroundAntibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling.MethodsPrimary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003).ResultsTreatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement.ConclusionsDespite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.

Upregulation of endothelial cell adhesion molecules characterizes veins close to granulomatous infiltrates in the renal cortex of cats with feline infectious peritonitis and is indirectly triggered by feline infectious peritonitis virus-infected monocytes in vitro.

One of the most characteristic pathological changes in cats that have succumbed to feline infectious peritonitis (FIP) is a multifocal granulomatous phlebitis. Although it is now well established that leukocyte extravasation elicits the inflammation typically associated with FIP lesions, relatively few studies have aimed at elucidating this key pathogenic event. The upregulation of adhesion molecules on the endothelium is a prerequisite for stable leukocyte-endothelial cell (EC) adhesion that necessarily precedes leukocyte diapedesis. Therefore, the present work focused on the expression of the EC adhesion molecules and possible triggers of EC activation during the development of FIP. Immunofluorescence analysis revealed that the endothelial expression of P-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) was elevated in veins close to granulomatous infiltrates in the renal cortex of FIP patients compared to non-infiltrated regions and specimens from healthy cats. Next, we showed that feline venous ECs become activated when exposed to supernatant from feline infectious peritonitis virus (FIPV)-infected monocytes, as indicated by increased adhesion molecule expression. Active viral replication seemed to be required to induce the EC-stimulating activity in monocytes. Finally, adhesion assays revealed an increased adhesion of naive monocytes to ECs treated with supernatant from FIPV-infected monocytes. Taken together, our results strongly indicate that FIPV activates ECs to increase monocyte adhesion by an indirect route, in which proinflammatory factors released from virus-infected monocytes act as key intermediates.

Platelet CD40 Exacerbates Atherosclerosis by Transcellular Activation of Endothelial Cells and Leukocytes

Beyond their eminent role in hemostasis and thrombosis, platelets are recognized as mediators of inflammation. Platelet cluster of differentiation 40 (CD40) ligand (CD40L and CD154) plays a key role in mediating platelet-induced inflammation in atherosclerosis. CD40, the receptor for CD40L, is present on platelets; however, the role of CD40 on this cell type is until now undefined. We found that in both mice and humans, platelet CD40 mediates the formation of platelet-leukocyte aggregates and the release of chemokine (C-X-C motif) ligand 4. Leukocytes were also less prone to adhere to CD40-deficient thrombi. However, platelet CD40 was not involved in platelet aggregation. Activated platelets isolated from Cd40(-/-)Apoe(-/-) mice adhered less to the endothelium upon injection into Apoe(-/-) mice when compared with CD40-sufficient platelets. Furthermore, lack of CD40 on injected platelets led to reduced leukocyte recruitment to the carotid artery as assayed by intravital microscopy. This was accompanied by a decrease in endothelial vascular cell adhesion molecule-1, platelet endothelial cell adhesion molecule, VE-cadherin, and P-selectin expression. To investigate the effect of platelet CD40 in atherosclerosis, Apoe(-/-) mice received thrombin-activated Apoe(-/-) or Cd40(-/-)Apoe(-/-) platelets every 5 days for 12 weeks, starting at the age of 17 weeks, when atherosclerotic plaques had already formed. When compared with mice that received Apoe(-/-) platelets, those receiving Cd40(-/-)Apoe(-/-) platelets exhibited a >2-fold reduction in atherosclerosis. Plaques of mice receiving CD40-deficient platelets were less advanced, contained less macrophages, neutrophils, and collagen, and displayed smaller lipid cores. Platelet CD40 plays a crucial role in inflammation by stimulating leukocyte activation and recruitment and activation of endothelial cells, thereby promoting atherosclerosis.

Therapeutic Potential of Cerium Oxide Nanoparticles for the Treatment of Peritonitis Induced by Polymicrobial Insult in Sprague-Dawley Rats

Peritonitis is a life-threatening disease that is associated with high mortality. The purpose of this study was to determine if cerium oxide nanoparticles can be used to diminish intra-abdominal infection-induced mortality and systemic inflammatory response syndrome in the laboratory rat. Randomized, controlled animal study and cell culture study. University research laboratory. Male Sprague-Dawley rats aged 12 weeks, RAW 246.7 macrophage cell line. Intra-abdominal infection or peritonitis was induced by intraperitoneal injection of cecal material (600 mg/kg in 5% sterile dextrose water at a dosage of 5 mL/kg) obtained from healthy donors. Rats in control and peritonitis groups received 200 μL of sterile deionized water IV via the tail vein, whereas rats in cerium oxide-only group and peritonitis+cerium oxide group received cerium oxide nanoparticles (0.5 mg/kg) IV at the time of polymicrobial injection. Survival rate was monitored for 14 days, while in other experiments, animals were killed at 3 and 18 hours after induction of peritonitis for biochemical analysis. Administration of a single dose (0.5 mg/kg) of cerium oxide nanoparticles IV to rats in the peritonitis group significantly improved survival rates and functioned to restore core body temperature toward baseline. Treatment-induced increases in animal survivability were associated with reduced systemic and hepatic oxidative stress, diminished serum cytokines, and chemokine levels. Changes in serum inflammatory markers with treatment were accompanied by decreased monocyte and lymphocyte extravasation into the peritoneal cavity along with decreased infiltration of macrophages into liver. In the heart, treatment diminished extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase-Stat-3 signaling and attenuated endothelial expression of P-selectin and vascular cell adhesion molecule-1. Cerium oxide nanoparticles attenuate the systemic inflammatory response associated with peritonitis, suggesting potential use as a novel therapeutic agent for the treatment of severe intra-abdominal infection.

Pentoxifylline attenuates leukocyte–endothelial interactions in a two-hit model of shock and sepsis

BackgroundThis study investigated the effects of pentoxifylline (PTX) combined with resuscitation fluids on microcirculatory dysfunctions in a two-hit model of shock and sepsis. Materials and methodsMale Wistar rats (250 g) were submitted to hemorrhagic shock and reperfusion followed by sepsis induced by cecal ligation and puncture. For the initial treatment of shock, rats were randomly divided into: sham, no injury, no treatment; hypertonic saline solution (HS) (7.5%, 4 mL/kg); lactated Ringer's solution (LR, 3 × shed blood volume); HS + PTX (4 mL/Kg + 25 mg/kg PTX); and LR + PTX (3 × shed blood volume + 25 mg/kg PTX). After 48 h of being exposed to the double injury, leukocyte–endothelial interactions were assessed by intravital microscopy of the mesentery. Endothelial expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) was evaluated by immunohistochemistry, as well as lung neutrophil infiltration by histology. ResultsLactated Ringer's solution induced marked increases (P < 0.001) in the number of rolling leukocytes per 10 min (two-fold), adherent leukocytes per 100 μm venule length (six-fold), migrated leukocytes per 5000 μm2 (eight-fold), P-selectin and ICAM-1 expression (four-fold), and lung neutrophil infiltration (three-fold) compared with sham. In contrast, PTX attenuated leukocyte–endothelial interactions, P-selectin and ICAM-1 expression at the mesentery when associated with either LR (P < 0.001) or HS (P < 0.05). Neutrophil migration into the lungs was similarly reduced by PTX (P < 0.05). ConclusionsData presented showed thatpentoxifylline attenuates microcirculatory disturbances at the mesenteric bed with significant minimization of lung inflammation after a double-injury model of hemorrhagicshock and reperfusion followed by sepsis.

Skin Repair Properties of d-Limonene and Perillyl Alcohol in Murine Models

The orange-peel derived terpene d-Limonene, probably through its metabolite, perillyl alcohol (POH), has been reported to have tissue-repair properties. Two murine models of respectively 12-O-Tetradecanoylphorbol-13-Acetate (TPA)-induced dermatitis and mechanical skin lesion were used here to assess the efficacy of d-Limonene or POH applied topically. Macroscopic and microscopic evaluation of skin lesions was performed as well as that of P-selectin expression, together with measurements of serum concentrations of IL-1β, IL-6 and TNF-α in the first model. Healing and angiogenesis around the scar were examined in the second model. Because differences in angiogenesis were noted, the effect of both d-Limonene and POH was further tested on an in vitro model of endothelial microtubules formation. Both d-Limonene and POH reduced the severity and extension of TPA-induced skin lesions with significantly lowered macroscopic and microscopic scores (p<0.04 in both cases). Moreover, the expression of P-selectin induced by TPA was abrogated by POH and significantly lower serum concentrations of IL-6 and TNF-α were observed in d-Limonene- and POH-treated mice (p<0.04 and 0.03). In the second model, tissue regeneration was improved, especially by POH, and was clearly associated with reduced neovascularization. This surprising anti-angiogenic effect was confirmed in the matrigel model of endothelial microtubules formation. These studies show that d-Limonene and POH demonstrate significant anti-inflammatory effects in murine dermal inflammation and wound-healing. The decreased systemic cytokine production as well as a consistent inhibition of endothelial P-selectin expression and neo-vascularization induced by these terpenic compounds contribute to their healing effects on the epidermal barrier.

No signs of inflammation during knee surgery with ischemia: a study involving inhaled nitric oxide.

Nitric oxide donors and inhaled nitric oxide (iNO) may decrease ischemia/reperfusion injury as reported in animal and human models. We investigated whether the attenuation of reperfusion injury, seen by others, in patients undergoing knee arthroplasty could be reproduced when patients had spinal anesthesia. 45 consecutive patients were randomized into three groups (n = 15). Groups 1 and 3 were receiving iNO 80 ppm or placebo (nitrogen, N2) throughout the entire operation, and group 2 only received iNO in the beginning and at the end of the operation. Blood samples were collected before surgery, at the end of the surgery, and 2 hours postoperatively. Muscle biopsies were taken from quadriceps femoris muscle before and after ischemia. There were no increases in plasma levels of soluble adhesion molecules: ICAM, VCAM, P-selectin, E-selectin, or of HMGB1, in any of the groups. There were low numbers of CD68+ macrophages and of endothelial cells expression of ICAM, VCAM, and P-selectin in the muscle analyzed by immunohistochemistry, prior to and after ischemia. No signs of endothelial cell activation or inflammatory response neither systemically nor locally could be detected. The absence of inflammatory response questions this model of ischemia/reperfusion, but may also be related to the choice of anesthetic method EudraCTnr.