Flow Chamber Assay Research Articles

Abstract TMP25: Targeting Myeloid Cell-Specific Pyruvate Kinase M2 Improves Stroke Outcome following Transient Cerebral Ischemia in the Comorbid Condition of Hyperlipidemia

Background: Activated leukocytes exhibit a high level of aerobic glycolysis compared to quiescent leukocytes. Dimeric form of pyruvate kinase M2 (PKM2) regulates aerobic glycolysis and transcription of pro-inflammatory mediators. By following the stroke therapy academic industry roundtable (STAIR) guidelines, we tested a novel hypothesis that manipulating metabolic plasticity of myeloid cells, by targeting PKM2, will improve stroke outcome. Methods: Using a well-characterized PKM2 activator (ML-265, inhibits PKM2 dimerization and stabilizes tetramer) in standardized neutrophil in vitro assays, we evaluated the mechanistic role of PKM2 in modulating post-stroke neutrophil function. Transient cerebral ischemia was induced using the intraluminal filament model of MCAO. Quantitative assessment of stroke outcome was evaluated (up to day 7) by measuring infarct volume, cerebral blood flow, survival rate, neurological/sensorimotor deficits, and thrombo-inflammation. Results: Treatment with ML-265 resulted in significantly reduced PKM2 nuclear translocation, IL-1 beta secretion and NETs formation in post-stroke peripheral neutrophils. Myeloid specific PKM2 -/- mice (male/female) on wild-type and hyperlipidemic background (PKM2 fl/fl LysM Cre and PKM2 fl/fl LysM Cre Apoe -/- ) exhibited significantly smaller infarcts and improved sensorimotor/neurological outcomes concomitant with enhanced survival rate and reduced post ischemic thrombo-inflammation. Using laser speckle imaging, we found significantly improved post-reperfusion cerebral blood flow in myeloid specific PKM2 -/- mice. In microfluidics flow chamber assay, human and mouse whole blood pretreated with ML-265 formed smaller thrombi when perfused over collagen at an arterial shear rate. Conclusions: Our data suggest that PKM2 regulates post-stroke neutrophil function and thrombo-inflammation. Targeting PKM2 may be explored as a novel strategy to improve stroke outcome.

Correction of Defective T-Regulatory Cells From Patients With Crohn’s Disease by Ex Vivo Ligation of Retinoic Acid Receptor-α

Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by exvivo expansion in the presence of factors that activate their regulatory activities. We measured levels of the integrin α4β7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded exvivo and incubated with rapamycin with or without agonists of the retinoic acid receptor-α (RARA), and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. We found that Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. The pathway that regulates the expression of integrin subunit α is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4β7, as well as CD62L and FOXP3, compared with cells incubated with rapamycin or rapamycin and all-trans retinoic acid. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines, and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labeled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. Incubation of patients' exvivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4β7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. ClinicalTrials.gov, Number: NCT03185000.

Manipulating Metabolic Plasticity By Targeting Pyruvate Kinase M2 in Platelets Inhibits Arterial Thrombosis

Background: Most of the cellular responses initiated upon platelet activation are energy consuming. Like normal cells, resting platelets rely primarily on oxidative phosphorylation (OXPHOS) to generate ATP, whereas activated platelets exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen, a phenomenon referred to as the Warburg effect in tumor cells) suggesting that metabolic plasticity exists in activated platelets. Although aerobic glycolysis yields less total ATP when compared to OXPHOS, the rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which is well suited for high-energy demands during platelet activation. Pyruvate kinases (PKs) catalyzes the final step of glycolysis, the formation of pyruvate and ATP from phosphoenolpyruvate and ADP. Four PK isoforms exist in mammals: L and R isoforms are expressed in the liver and red blood cells; the M1 isoform is expressed in most adult tissues that have high catabolic demands including muscle and brain; M2 is expressed in cells including activated platelets and leukocytes. While PKM1 and tetrameric PKM2 favor ATP production from OXPHOS through the TCA cycle, dimeric PKM2 drives aerobic glycolysis.Objective: We tested an innovative concept that by manipulating the energy demand of activated platelets (metabolic plasticity), by targeting PKM2, will inhibit platelet function and thrombosis.Methods: Using a specific inhibitor of PKM2 (inhibits PKM2 dimerization and stabilizes tetramers) and standardized platelet in vitro assays, we determined the mechanistic role of PKM2 in modulating platelet function in human and mice. To provide definitive evidence, we generated a megakaryocyte or platelet-specific PKM2-/- mouse (PKM2fl/flPF4Cre). Lactate assay was performed in WT and PKM2 null platelets. Susceptibility to thrombosis was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid artery thrombosis and laser injury models) by utilizing intravital microscopy.Results: We found that PKM2 is relatively highly expressed compared to PKM1 in human and murine platelets. Transmission electron microscopy (immunogold staining) revealed that PKM2 is found in the cytoplasm and a- granule in resting platelets, whereas most of PKM2 translocated to cytoplasm upon activation. Human and mouse platelets pretreated with PKM2 inhibitor exhibited decreased platelet aggregation to sub-optimal doses of collagen and convulxin but not to thrombin. In microfluidics flow chamber assay, human and whole mouse blood pretreated with PKM2 inhibitor formed small thrombi when perfused over collagen for 5 min at an arterial shear rate of 1500s-1 (P<0.05 vs. vehicle control). Platelets from PKM2fl/flPF4Cre mice exhibited decreased platelet aggregation to sub-optimal doses of collagen and convulxin, but not to thrombin, compared to PKM2fl/fl mice concomitant with decrease lactate production. In microfluidics flow chamber assay, whole blood from PKM2fl/flPF4Cre mice formed smaller thrombi when perfused over collagen for 5 min at an arterial shear rate of 1500s-1, compared to PKM2fl/fl mice. PKM2fl/flPF4Cre mice were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models (P<0.05 vs. vehicle control, N=10 mice/group), without altering hemostasis. PKM2 regulates the phosphorylation signal transducer and activator of transcription 3 (STAT3) and p-STAT3 act as a protein scaffold that facilitates the catalytic process of activating PLCg by kinase Syk in response to low-doses of collagen and CRP, but not TRAP or ADP in human and murine platelets. Interestingly, we found that PKM2 and STAT3 colocalized in the convulixn- stimulated control platelets and less phosphorylation of STAT-3 was observed in activated PKM2 null platelets (P<0.05 vs. WT), suggesting a non-glycolytic role of the PKM2 in regulating collagen signaling.Conclusions: Our results suggest that dimeric PKM2 regulates platelet function and arterial thrombosis most likely via GPVI signaling pathway. We suggest that manipulating metabolic plasticity by targeting dimeric PKM2 may be explored as a novel strategy to inhibit platelet function and arterial thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Interactions between rheumatoid arthritis synovial fibroblast migration and endothelial cells.

Leukocytes travel within the circulation and enter connective tissues by interactions with endothelium of postcapillary venules mediated by cell adhesion molecules, summarized as the leukocyte adhesion cascade. In the severe combined immunodeficient (SCID) mouse model, rheumatoid arthritis (RA) synovial fibroblasts (SF) migrated to distant cartilage through the vasculature. Therefore, RASF adhesion toward endothelial cells (EC) and E- and P-selectins were analyzed. Cell-to-cell binding assays between SF and EC were performed. Interactions of SF with tumor necrosis factor α (TNFα)-activated EC or selectins were analyzed in flow adhesion assays. Immunohistochemistry for E-selectin ligand CD15s was performed. CD15s induction in RASF by human serum or media was evaluated. Wild-type and E-/-/ P-/- Selectin-SCID mice were used for inverse-wrap surgery. After laser-mediated microdissection, real-time PCR for E-/P-selectin/vascular cell adhesion molecule 1 was performed. Adhesion between SF/EC under static conditions was highest in Roswell Park Memorial Institute-cultured RASF to TNFαα-activated human umbilical vein endothelial cells (2.25-fold) and RASF adhesion was higher toward venous than arterial EC (Dulbecco's modified eagle medium P=0.0419, RPMI P=0.0119). In flow chamber assays, RASF adhesion to E-selectin was higher than to P-selectin (e.g. 0.9dyncm-2 P=0.0001). Osteoarthritis synovial fibroblasts showed lower rolling/adhesion properties (e.g. 0.5dyncm-2 , P=0.0010). RASF adhesion to TNFαα-activated EC was increased (e.g. 0.9dyncm-2 , P=0.0061). CD15s induction in RASF was strongest in RA serum. Vimentin/CD15s double-positive cells were detectable. In E-/P-selectin-deficient mice, contralateral invasion was reduced (P=0.023). E- and P-selectin, and vascular cell adhesion molecule 1 expression in EC of implants was confirmed. Our data indicate that the milieu within vessels induces CD15s which enables RASF to interact with E-selectin/EC under flow. Therefore, RASF may migrate to distant sites and leave the vasculature similarly to leukocytes.

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in invitro and invivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

Uric acid enhances alteplase-mediated thrombolysis as an antioxidant

Uric acid (UA) therapy may prevent early ischemic worsening after acute stroke in thrombolysis patients. The aim of this study was to examine the influence of UA on the thrombolytic efficacy of alteplase in human blood samples by measuring thrombolysis under flow conditions using a newly developed microchip-based flow-chamber assay. Human blood samples from healthy volunteers were exposed to UA, alteplase, or a combination of UA and alteplase. Whole blood and platelet-rich plasma were perfused over a collagen- and thromboplastin-coated microchip, and capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 92% lower in the UA–alteplase-treated group compared with the alteplase-treated group. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, UA significantly inhibited the effect of hydrogen peroxide. Meanwhile, rat models of thromboembolic cerebral ischemia were treated with either alteplase or UA–alteplase combination therapy. Compared with alteplase alone, the combination therapy reduced the infarct volume and inhibited haemorrhagic transformation. UA enhances alteplase-mediated thrombolysis, potentially by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

Dichloroacetate, an inhibitor of pyruvate dehydrogenase kinases, inhibits platelet aggregation and arterial thrombosis

AbstractResting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) to generate adenosine triphosphate, whereas activated platelets exhibit a high level of aerobic glycolysis, suggesting the existence of metabolic flexibility in platelets. Mitochondrial pyruvate dehydrogenase kinases (PDK 1-4) play a pivotal role in metabolic flexibility by inhibiting pyruvate dehydrogenase complex. We determined whether metabolic reprogramming, diverting metabolism from aerobic glycolysis back to OXPHOS, would inhibit platelet function. PDKs activity in human and mouse platelets was inhibited with dichloroacetic acid (DCA), a potent inhibitor of all 4 forms of PDK. Human and mouse platelets pretreated with DCA exhibited decreased platelet aggregation to suboptimal doses of collagen, convulxin, thrombin, and adenosine diphosphate concomitant with decreased glucose uptake. Bioenergetics profile revealed that platelets pretreated with DCA exhibited decreased aerobic glycolysis in response to convulxin only. Furthermore, DCA inhibited ATP secretion, thromboxane A2 generation, and tyrosine phosphorylation of Syk and PLCγ2 in response to collagen or convulxin in human and mouse platelets (P < .05 vs vehicle treated). In the flow chamber assay, human and mouse blood pretreated with DCA formed smaller thrombi when perfused over collagen for 10 minutes at an arterial shear rate of 1500 s−1 (P < .05 vs control). Wild-type mice pretreated with DCA were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury–induced mesenteric artery thrombosis models (P < .05 vs vehicle control), without altering hemostasis. Targeting metabolic plasticity with DCA may be explored as a novel strategy to inhibit platelet function.

Inorganic Polyphosphate Amplifies High Mobility Group Box 1-Mediated Von Willebrand Factor Release and Platelet String Formation on Endothelial Cells.

Objective- Inorganic polyphosphate (polyP) is known to modulate coagulation, inflammation, and metabolic pathways. It also amplifies inflammatory responses of HMGB1 (high mobility group box 1) in endothelial cells. The objective of this study was to evaluate the effect of polyP on von Willebrand factor (VWF) release from endothelial cells with or without HMGB1. Approach and Results- EA.hy926 endothelial cells were treated with different concentrations of polyP70 alone or in combination with different concentrations of HMGB1. VWF release was measured by an ELISA assay in the absence or presence of pharmacological inhibitors of the receptor for advanced glycation end products, P2Y1, and Ca2+. A flow chamber assay was used to monitor polyP70-mediated platelet recruitment and VWF-platelet string formation. PolyP70 and HMGB1 induced VWF release from endothelial cells by a concentration-dependent manner. PolyP70 amplified HMGB1-mediated VWF release from endothelial cells. This was also true if boiled platelet releasate was used as the source of polyP. Gene silencing or pharmacological inhibitors of receptor for advanced glycation end products, P2Y1, and Ca2+ significantly inhibited VWF release. PolyP70 and HMGB1 synergistically promoted VWF-platelet string formation in the flow chamber assay, which was inhibited by the anti-GPIbα (glycoprotein Ib alpha) antibody. VWF release by polyP70-HMGB1 complex required phosphorylation of Src and phospholipase C because inhibitors of Src, phospholipase C, and Ca2+ signaling significantly decreased VWF secretion. The polyP70-HMGB1 complex also increased angiopoietin-2 release, indicating that Weibel-Palade body exocytosis is involved in the VWF release. Conclusions- PolyP70 can promote thrombotic and inflammatory pathways by inducing VWF release and platelet string formation on endothelial cells.

CCR2 mediates transendothelial migration of human pathogenic Th17 cells

Abstract Leukocyte extravasation is a multistep process characterized by rolling and arrest on the endothelium followed by transendothelial migration (TEM). Arrest and TEM by T cells typically requires the activities of chemokines and their receptors. Previously, we showed that CCR2 identifies highly differentiated, readily activated CD4+ memory T cells in human blood with diverse effector capabilities, and we showed that CCR6 is the signature chemokine receptor for Th17 cells. Here we describe studies of the migratory capabilities and effector profiles of CD4+ CCR6+ CCR2+ as compared with CD4+ CCR6+ CCR2− and CD4+ CCR6− CCR2− T cells from human blood. Using flow chamber assays with TNFa-activated human umbilical vein endothelial cells, we found that among the CCR6+ cells, only the CCR2+ subset was able to cross the endothelial monolayer efficiently. Using antibodies against the CCR6 ligand, CCL20, and a specific inhibitor of CCR2, we found that CCR6 was important for arrest and that CCR2 was critical for subsequent TEM. Using intracellular staining of cells activated ex vivo, we found that although IL-17-expressing cells were found within all CCR6+ subsets, the CCR6+CCR2+ cells were distinguished by their production of IFNg, GM-CSF, and TNFa, thereby demonstrating the cytokine profile of pathogenic Th17 cells as identified in mouse models of autoimmune disease. Taken together, our studies suggest that chemokine receptors on memory Th cells play non-redundant roles in the egress from blood, that providing T cells with CCR6 and CCR2 could enhance their trafficking into tissue, and that, on the other hand, blocking CCR2 alone could prevent the extravasation of pathogenic Th17 cells and ameliorate tissue damage in Th17-cell driven disease.

Platelet Munc13-4 regulates hemostasis, thrombosis and airway inflammation

Platelet degranulation is crucial for hemostasis and may participate in inflammation. Exocytosis in platelets is mediated by SNARE proteins and should be controlled by Munc13 proteins. We found that platelets express Munc13-2 and -4. We assessed platelet granule exocytosis in Munc13-2 and -4 global and conditional knockout (KO) mice, and observed that deletion of Munc13-4 ablates dense granule release and indirectly impairs alpha granule exocytosis. We found no exocytic role for Munc13-2 in platelets, not even in the absence of Munc13-4. In vitro, Munc13-4-deficient platelets exhibited defective aggregation at low doses of collagen. In a flow chamber assay, we observed that Munc13-4 acted as a rate-limiting factor in the formation of thrombi. In vivo, we observed a dose-dependency between Munc13-4 expression in platelets and both venous bleeding time and time to arterial thrombosis. Finally, in a model of allergic airway inflammation, we found that platelet-specific Munc13-4 KO mice had a reduction in airway hyper-responsiveness and eosinophilic inflammation. Taken together, our results indicate that Munc13-4-dependent platelet dense granule release plays essential roles in hemostasis, thrombosis and allergic inflammation.

CX3CR1/CX3CL1 Axis Mediates Platelet-Leukocyte Adhesion to Arterial Endothelium in Younger Patients with a History of Idiopathic Deep Vein Thrombosis.

Mechanisms linking deep vein thrombosis (DVT) and subclinical atherosclerosis and risk of cardiovascular events are poorly understood. The aim of this study was to investigate the potential impact of CX3CR1/CX3CL1 axis in DVT-associated endothelial dysfunction. The study included 22 patients (age: 37.5 ± 8.2 years) with a history of idiopathic DVT and without known cardiovascular risk factors and 23 aged-matched control subjects (age: 34 ± 7.8 years). Flow cytometry was used to evaluate peripheral markers of platelet activation, leukocyte immunophenotypes and CX3CR1/CX3CL1 expression in both groups. A flow chamber assay was employed to measure leukocyte arrest under dynamic conditions. Platelet activation and the percentage of circulating CX3CR1-expressing platelets, CX3CR1-expressing platelet-bound monocytes and CD8+ lymphocytes were higher in patients with DVT than in controls. Additionally, patients with DVT had increased plasma levels of CX3CL1, soluble P-selectin and platelet factor 4/CXCL4. Interestingly, this correlated with enhanced platelet-leukocyte interaction and leukocyte adhesion to TNFα-stimulated arterial endothelial cells, which was partly dependent on endothelial CX3CL1 upregulation and increased CX3CR1 expression on platelets, monocytes and lymphocytes. In conclusion, increased CX3CR1 expression on circulating platelets may constitute a prognostic marker for long-term adverse cardiovascular events in patients with DVT. Blockade of CX3CL1/CX3CR1 axis may represent a new therapeutic strategy for the prevention of cardiovascular comorbidities associated with DVT.

Ultrasound molecular imaging of the secreted tumor marker Netrin-1 in multiple breast cancer models

With regard to biomarker expression, tumors are heterogeneous media displaying intra- and interpatient variability. Ultrasound molecular imaging (USMI) is a newly developed tool that can characterize heterogeneous tumors. Here, we focus on Netrin-1-overexpressing breast cancer. A newly developed targeted therapy interfering with Netrin-1 requires patient stratification. We developed Netrin-1-targeted microbubbles (MBs) and assessed USMI for identification of Netrin-1-positive breast cancer. In vitro flow chamber assays showed specific binding of anti-Netrin-1-MBs to human and murine breast cancer cell lines overexpressing Netrin-1. In vivo, in Netrin-1-positive transgenic or implanted mouse breast cancer models, USMI showed significantly increased signals with Netrin-1-targeted MBs, compared with isotype control MBs and targeted MBs after blocking of Netrin-1:—44.8%, 32.0%, 33.7% intensity for MBsNetrin-1, MBsIsotype, or after blocking, resp., in MMTV tumors;—28.2%, 13.7%, 13.4%, resp., in SKBR7 tumors;—60.9%, 47.8%, 47.9%, resp., in 4T1 tumors. In Netrin-1-negative tumors and in normal glands, targeted and control MBs showed no significant differences. Immunohistochemistry confirmed the expression of Netrin-1 in endothelial cells. In conclusion, USMI allowed the characterization of tumor heterogeneity and the differentiation between Netrin-1-positive and -negative tumors, and has the potential to become a companion diagnostic for breast cancer patient stratification.

First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis.

Adequate platelet reactivity is required for maintaining hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis. ML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl3-induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding after treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly support 12-LOX as a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy.

Galectin-3: A Positive Regulator of Leukocyte Recruitment in the Inflamed Microcirculation.

In vivo and ex vivo imaging were used to investigate the function of galectin-3 (Gal-3) during the process of leukocyte recruitment to the inflamed microcirculation. The cremasteric microcirculation of wild-type (C57BL/6), Gal-3-/-, and CX3CR1gfp/+ mice were assessed by intravital microscopy after PBS, IL-1β, TNF-α, or recombinant Gal-3 treatment. These cellular responses were investigated further using flow-chamber assays, confocal microscopy, flow cytometry, PCR analysis, and proteome array. We show that mechanisms mediating leukocyte slow rolling and emigration are impaired in Gal-3-/- mice, which could be because of impaired expression of cell adhesion molecules and an altered cell surface glycoproteome. Local (intrascrotal) administration of recombinant Gal-3 to wild-type mice resulted in a dose-dependent reduction in rolling velocity associated with increased numbers of adherent and emigrated leukocytes, ∼50% of which were Ly6G+ neutrophils. Intrascrotal administration of Gal-3 to CX3CR1gfp/+ mice confirmed that approximately equal numbers of monocytes are also recruited in response to this lectin. Exogenous Gal-3 treatment was accompanied by increased proinflammatory cytokines and chemokines within the local tissue. In conclusion, this study unveils novel biology for both exogenous and endogenous Gal-3 in promoting leukocyte recruitment during acute inflammation.

Indoxyl sulfate induces platelet hyperactivity and contributes to chronic kidney disease–associated thrombosis in mice

AbstractThrombosis is a common complication of chronic kidney disease (CKD), but the causes and mechanisms of CKD-associated thrombosis are not well clarified. Here, we show that platelet activity is remarkably enhanced in CKD mice, with increase of serum indoxyl sulfate (IS), a typical uremic toxin, which cannot be effectively cleared by routine dialysis. Ex vivo and in vitro experiments reveal that IS displays a distinct ability to enhance platelet activities, including elevated response to collagen and thrombin, increases in platelet-derived microparticles, and platelet-monocyte aggregates. The flow chamber assay and carotid artery thrombosis model demonstrate that IS-induced platelet hyperactivity contributes to thrombus formation. Further investigations disclose that reactive oxygen species (ROS)-mediated p38MAPK signaling plays a key role in IS-induced platelet hyperactivity. Moreover, we show that Klotho, which is expressed dominantly in the kidneys, has the capacity to counteract IS-induced platelet hyperactivity by inhibiting ROS/p38MAPK signaling, whereas Klotho reduction may aggravate the effect of IS on platelet activation in CKD and klotho+/− mice. Finally, we demonstrate that Klotho protein treatment can protect against IS-induced thrombosis and atherosclerosis in apoE−/− mice. Our findings uncover the mechanism of platelet hyperactivity induced by IS and provide new insights into the pathogenesis and treatment of CKD-associated thrombosis.

C/EBPd enables the efficient extravasation of human CCR2+ MAIT cells

Abstract Early responses to pathogen challenge depend in part on recruitment of effector-capable T cells from blood. Human mucosal-associated invariant T (MAIT) cells are abundant, mostly CD8+, MR1-restricted, anti-bacterial T cells that could contribute to this response. However, little is known about these cells’ ability to traffic in inflammation. We found that, as compared with non-MAIT CD8+ T cells, human MAIT cells excelled at entering inflamed skin in mice. In flow chamber assays, MAIT cells were efficient at trafficking across activated human umbilical vein endothelial cells (HUVEC) due to high levels of selectin ligands, and chemokine receptors CCR6, and CCR2, which we showed were important for rolling, arrest, and transendothelial migration, respectively. We found that MAIT cells express high levels of the mRNAs for FUT7 and ST3GAL4, which are critical for synthesizing selectin ligands. We also found that expression of FUT7, ST3GAL4, and CCR6 depends on C/EBPd, a bZIP transcription factor that we discovered is expressed preferentially in MAIT cells and that binds to the FUT7, ST3GAL4, and CCR6 promoters. For MAIT cells, knockdown of C/EBPd decreased rolling and arrest on HUVEC and extravasation into inflamed mouse skin. Thus, CCR2+ MAIT cells display trafficking abilities that depend on chemokine receptors with non-redundant roles and the cooperative activities of C/EBPd-regulated genes - and exemplify effector-ready T cells that express a coordinated program enabling rapid entry into sites of inflammation.

Edaravone, a Synthetic Free Radical Scavenger, Enhances Alteplase-Mediated Thrombolysis.

The combination of alteplase, a recombinant tissue plasminogen activator, and edaravone, an antioxidant, reportedly enhances recanalization after acute ischemic stroke. We examined the influence of edaravone on the thrombolytic efficacy of alteplase by measuring thrombolysis using a newly developed microchip-based flow-chamber assay. Rat models of embolic cerebral ischemia were treated with either alteplase or alteplase-edaravone combination therapy. The combination therapy significantly reduced the infarct volume and improved neurological deficits. Human blood samples from healthy volunteers were exposed to edaravone, alteplase, or a combination of alteplase and edaravone or hydrogen peroxide. Whole blood was perfused over a collagen- and thromboplastin-coated microchip; capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 69.9% lower in the edaravone-alteplase- than alteplase-treated group. The thrombolytic effect of alteplase was significantly attenuated in the presence of hydrogen peroxide, suggesting that oxidative stress might hinder thrombolysis. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, edaravone significantly inhibited the decrease. Edaravone enhances alteplase-mediated thrombolysis, likely by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

Endothelial cell-derived CD95 ligand serves as a chemokine in induction of neutrophil slow rolling and adhesion.

Integrin activation is crucial for the regulation of leukocyte rolling, adhesion and trans-vessel migration during inflammation and occurs by engagement of myeloid cells through factors presented by inflamed vessels. However, endothelial-dependent mechanisms of myeloid cell recruitment are not fully understood. Here we show using an autoperfused flow chamber assay of whole blood neutrophils and intravital microscopy of the inflamed cremaster muscle that CD95 mediates leukocyte slow rolling, adhesion and transmigration upon binding of CD95-ligand (CD95L) that is presented by endothelial cells. In myeloid cells, CD95 triggers activation of Syk-Btk/PLCγ2/Rap1 signaling that ultimately leads to integrin activation. Excitingly, CD95-deficient myeloid cells exhibit impaired bacterial clearance in an animal model of sepsis induced by cecal ligation and puncture (CLP). Our data identify the cellular and molecular mechanisms underlying the chemoattractant effect of endothelial cell-derived CD95L in induction of neutrophil recruitment and support the use of therapeutic inhibition of CD95's activity in inflammatory diseases.

Abstract 41: Platelet Dream Plays a Critical Role During Thrombogenesis in Mice

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain. Using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that hematopoietic and endothelial cell DREAM are required for platelet thrombus formation following arteriolar injury. DREAM deletion also prolonged tail bleeding times. In vitro flow chamber assays and in vivo adoptive transfer experiments indicated the importance of platelet DREAM in thrombogenesis. We found that deletion of platelet DREAM does not alter ultrastructural features but significantly impaired aggregation and ATP secretion induced by collagen-related peptide (CRP), ADP, or A23187, but not thrombin or U46619. Biochemical studies showed that platelet DREAM is required for phosphoinositide 3-kinase (PI3K) activation induced by GPVI-, A23187-, or integrin-mediated signaling. Studies using DREAM-null platelets and isoform-specific PI3K inhibitors revealed that platelet DREAM positively regulates granule secretion, Ca2+ mobilization, and aggregation induced by CRP or A23187 through PI3K class Iβ (PI3K-Iβ) activity. Genetic and pharmacological studies in megakaryoblastic MEG-01 cells showed that DREAM regulates A23187-induced Ca2+ mobilization and that the regulatory function of DREAM requires Ca2+ binding and PI3K-Iβ activity. Taken together, we have identified platelet DREAM as a novel regulator of PI3K-Iβ activity during thrombus formation.

Targeting the PDE8A-Raf-1 kinase signaling complex to treat autoimmune inflammation

Abstract The levels of cAMP are regulated by phosphodiesterases (PDE), which are targets for the treatment of inflammatory disorders. We previously showed that PDE8A regulates T cell motility. We report here that PDE8A exerts its control of T cell function through the Raf-1 kinase signaling pathway. To mimic T cell migration under physiologic conditions, we performed flow chamber assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 significantly suppresses rolling and adhesion of in vivo MOG35-55 activated inflammatory CD4+ T effector (Teff) cells while interacting with inflamed brain endothelial cells under shear flow conditions. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP around Raf-1 thereby protecting it from protein kinase A mediated inhibitory phosphorylation. To test this in Teff cells, we used a cell permeable peptide (DP) that disrupts the PDE8A-Raf-1 signaling complex. Disruption of the PDE8A-Raf-1 complex by DP significantly reduces adhesion of Teff cells to endothelial cells. We further observed that disrupting PDE8A-Raf-1 through DP specifically reduces adhesion, spreading and locomotion of Teff cells while interacting with the vascular adhesion molecule ICAM-1. Our investigation of the effect of PDE8 inhibitor on chronic and relapsing-remitting experimental autoimmune encephalomyelitis (EAE) in vivo indicate suppression of clinical and histopathological signs of disease. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase signaling complex disruptors significantly decreases Teff cell adhesion and migration on endothelial cells, and represents a novel approach to treat autoimmune inflammation in vivo.