Platelet-endothelial Cell Interactions Research Articles

Platelet and endothelial cell responses under concurrent shear stress and tensile strain

Thrombosis can lead to significant mortality and morbidity. Both platelets and vascular endothelial cells play significant roles in thrombosis. Platelets' response to blood flow-induced shear stress can vary greatly depending on shear stress magnitude, pattern and shear exposure time. Endothelial cells are also sensitive to the biomechanical environment. Endothelial cell activation and dysfunction can occur under low oscillatory shear stress and low tensile strain. Platelet and endothelial cell interaction can also be affected by mechanical conditions. The goal of this study was to investigate how blood flow-induced shear stress, vascular wall tensile strain, platelet-endothelial cell stress history, and platelet-endothelial cell interaction affect platelet thrombogenicity. Platelets and human coronary artery endothelial cells were pretreated with physiological and pathological shear stress and/or tensile strain separately. The pretreated cells were then put together and exposed to pulsatile shear stress and cyclic tensile strain simultaneously in a shearing-stretching device. Following treatment, platelet thrombin generation rate, platelet and endothelial cell activation, and platelet adhesion to endothelial cells was measured. The results demonstrated that shear stress pretreatment of endothelial cells and platelets caused a significant increase in platelet thrombin generation rate, cell surface phosphatidylserine expression, and adhesion to endothelial cells. Shear stress pretreatment of platelets and endothelial cells attenuated endothelial cell ICAM-1 expression under stenosis conditions, as well as vWF expression under recirculation conditions. These results indicate that platelets are sensitized by prior shearing, while in comparison, the interaction with shear stress-pretreated platelets may reduce endothelial cell sensitivity to pathological shear stress and tensile strain.

The efficacy of therapeutic plasma exchange in COVID-19 patients on endothelial tightness in vitro is hindered by platelet activation.

Coronavirus disease (COVID)-19 is characterised in particular by vascular inflammation with platelet activation and endothelial dysfunction. During the pandemic, therapeutic plasma exchange (TPE) was used to reduce the cytokine storm in the circulation and delay or prevent ICU admissions. This procedure consists in replacing the inflammatory plasma by fresh frozen plasma from healthy donors and is often used to remove pathogenic molecules from plasma (autoantibodies, immune complexes, toxins, etc.). This study uses an in vitro model of platelet-endothelial cell interactions to assess changes in these interactions by plasma from COVID-19 patients and to determine the extent to which TPE reduces such changes. We noted that exposure of an endothelial monolayer to plasmas from COVID-19 patients post-TPE induced less endothelial permeability compared to COVID-19 control plasmas. Yet, when endothelial cells were co-cultured with healthy platelets and exposed to the plasma, the beneficial effect of TPE on endothelial permeability was somewhat reduced. This was linked to platelet and endothelial phenotypical activation but not with inflammatory molecule secretion. Our work shows that, in parallel to the beneficial removal of inflammatory factors from the circulation, TPE triggers cellular activation which may partly explain the reduction in efficacy in terms of endothelial dysfunction. These findings provide new insights for improving the efficacy of TPE using supporting treatments targeting platelet activation, for instance.

Platelet Interactions with Liver Sinusoidal Endothelial Cells and Hepatic Stellate Cells Lead to Hepatocyte Proliferation

(1) Background: Platelets were postulated to constitute the trigger of liver regeneration. The aim of this study was to dissect the cellular interactions between the various liver cells involved in liver regeneration and to clarify the role of platelets. (2) Methods: Primary mouse liver sinusoidal endothelial cells (LSECs) were co-incubated with increasing numbers of resting platelets, activated platelets, or platelet releasates. Alterations in the secretion of growth factors were measured. The active fractions of platelet releasates were characterized and their effects on hepatocyte proliferation assessed. Finally, conditioned media of LSECs exposed to platelets were added to primary hepatic stellate cells (HSCs). Secretion of hepatocyte growth factor (HGF) and hepatocyte proliferation were measured. After partial hepatectomy in mice, platelet and liver sinusoidal endothelial cell (LSEC) interactions were analyzed in vivo by confocal microscopy, and interleukin-6 (IL-6) and HGF levels were determined. (3) Results: Co-incubation of increasing numbers of platelets with LSECs resulted in enhanced IL-6 secretion by LSECs. The effect was mediated by the platelet releasate, notably a thermolabile soluble factor with a molecular weight over 100 kDa. The conditioned medium of LSECs exposed to platelets did not increase proliferation of primary hepatocytes when compared to LSECs alone but stimulated hepatocyte growth factor (HGF) secretion by HSCs, which led to hepatocyte proliferation. Following partial hepatectomy, in vivo adhesion of platelets to LSECs was significantly increased when compared to sham-operated mice. Clopidogrel inhibited HGF secretion after partial hepatectomy. (4) Conclusion: Our findings indicate that platelets interact with LSECs after partial hepatectomy and activate them to release a large molecule of protein nature, which constitutes the initial trigger for liver regeneration.

Emergence of AnnexinVpos CD31neg CD42blow/neg extracellular vesicles in plasma of humans at extreme altitude

BackgroundHypobaric hypoxia has been reported to cause endothelial cell and platelet dysfunction implicated in the formation of microvascular lesions, and in its extremes may contribute to vascular leakage in high altitude pulmonary edema or blood brain barrier disruption leading to cerebral micro-hemorrhage (MH). Platelet function in the development of microvascular lesions remained ill defined, and is still incompletely understood. In this study platelet- and endothelial cell-derived extracellular vesicles (PEV and EEV, respectively) and cell adhesion molecules were characterized in plasma samples of members of a high altitude expedition to delineate the contribution of platelets and endothelial cells to hypobaric hypoxia-induced vascular dysfunction.Methods and findingsIn this observational study, platelet and endothelial cell-derived extracellular vesicles were analysed by flow-cytometry in plasma samples from 39 mountaineers participating in a medical research climbing expedition to Himlung Himal, Nepal, 7,050m asl. Megakaryocyte/platelet-derived AnnexinVpos, PECAM-1 (CD31) and glycoprotein-1b (GP1b, CD42b) positive extracellular vesicles (PEV) constituted the predominant fraction of EV in plasma samples up to 6,050m asl. Exposure to an altitude of 7,050m led to a marked decline of CD31pos CD42neg EEV as well as of CD31pos CD42bpos PEV at the same time giving rise to a quantitatively prevailing CD31neg CD42blow/neg subpopulation of AnnexinVpos EV. An almost hundredfold increase in the numbers of this previously unrecognized population of CD31neg CD42blow/neg EV was observed in all participants reaching 7,050m asl.ConclusionsThe emergence of CD31neg CD42blow/neg EV was observed in all participants and thus represents an early hypoxic marker at extreme altitude. Since CD31 and CD42b are required for platelet-endothelial cell interactions, these hypobaric hypoxia-dependent quantitative and phenotypic changes of AnnexinVpos EV subpopulations may serve as early and sensitive indicators of compromised vascular homeostasis.

Aspirin-Dependent Platelet Inflammatory Inhibition in Healthy Subjects Decreases NLRP-1 Inflammasome

Inflammation and endothelial dysfunction are implicated in the onset of atherosclerosis. Inflammasome activation takes part in the pathogenesis of the atherosclerotic disease. This study investigated the influence of platelet inflammatory inhibition on the transcription of intracitosolic nucleotide-binding oligomerization domain-like receptor protein 1 (NLRP-1) inflammasome in endothelial cells. This experimental study enrolled 10 healthy volunteers with no cardiovascular risk factors and normal results on vascular examination. They received low doses of aspirin (100mg/day) for seven days. A venous blood sample was collected in all subjects before aspirin intake and after the experimental week. Human aortic endothelial cell (HAEC) cultures were exposed to baseline plasma and plasma from subjects after aspirin intake. NLRP-1 gene expression was analyzed in these cultures. HAEC cultures that were exposed to plasma from subjects at baseline showed higher expression of NLRP-1 than HAECs exposed to plasma of healthy volunteers after one week on salicilate intake (relative quantification, 1.077±0.05 vs. 1.002±0.06; odds ratio, 1.8; 95 confidence interval, 1.1-2.9; P<0.01). Data observed in the our study indicate that in HAECs, the intracytosolic NLRP-1 expression is attenuated by the auto/paracrine platelet inhibition by aspirin, without direct platelet-endothelial cell interaction.

Platelet-endothelial cell interactions modulate smooth muscle cell phenotype in an in vitro model of type 2 diabetes mellitus.

Platelet (PLT)-endothelial cell (EC) interaction appears to contribute to phenotypic transition of vascular smooth muscle cells (VSMCs), which play an important role in the physiological and pathological process of vascular complications in type 2 diabetes mellitus (DM2). However, the precise mechanisms by which interactions between PLTs and ECs affect VSMC phenotype have largely remained unclear. We determined the effect of diabetic PLT-EC interaction to influence VSMC migration, proliferation, and phenotypic transformation in triple-cell coculture models using the quantitative real-time PCR, Western blot, fluorescence microscopy, wound scratch assays, CCK-8 assays, and gelatin zymography assays. Our results revealed DM2 PLT-EC interaction to be associated with a significant downregulation of VSMC-specific contractile phenotypic genes and proteins, including SM22α, smooth muscle actin, Smoothelin-B, and smooth muscle-myosin heavy chain. Inversely, VSMC-specific proliferative phenotype gene and protein levels, including cyclin D1 and 2, nonmuscle myosin heavy chain B, and PCNA were in upregulation. Furthermore, the DM2-originated PLT-EC interaction promoted the expression level of transforming growth factor-β1, and the PI3K/Akt and matrix metalloproteinase 9 signaling pathway was activated subsequently. Finally, these reactions contributed to a synthetic phenotype of VSMCs, including the proliferation, migration, and gelatinolytic activities. These findings suggest that PLT-EC interaction modulates the phenotypic transition of VSMCs between a contractile and proliferative/synthetic phenotype under diabetic conditions, conceivably providing important implications regarding the mechanisms controlling the VSMC phenotypic transition and the development of cardiovascular complications.

New Interfaces of Thyroid Hormone Actions With Blood Coagulation and Thrombosis.

Substantial clinical evidence indicates hyperthyroidism enhances coagulation and increases the risk of thrombosis. In vitro and clinical evidence implicate multiple mechanisms for this risk. Genomic actions of thyroid hormone as 3,5,3′-triiodo-L-thyronine (T3) via a nuclear thyroid hormone receptor have been implicated, but recent evidence shows that nongenomic mechanisms initiated at the receptor for L-thyroxine (T4) on platelet integrin αvβ3 are prothrombotic. The T4-initiated mechanisms involve platelet activation and, in addition, cellular production of cytokines and chemokines such as CX3CL1 with procoagulatory activities. These procoagulant actions of T4 are particulary of note because within cells T4 is not seen to be functional, but to be only a prohormone for T3. Finally, it is also possible that thyroid hormone stimulates platelet-endothelial cell interaction involved in local thrombus generation. In this brief review, we survey mechanisms by which thyroid hormone is involved in coagulation and platelet functions. It is suggested that the threshold should be lowered for considering the possibility that clinically significant clotting may complicate hyperthyroidism. The value of routine measurement of partial thromboplastin time or circulating D-dimer in patients with hyperthyroid or in patients treated with thyrotropin-suppressing dosage of T4 requires clinical testing.

Sequence alignment analysis of proteins involved in platelet-endothelial cell interaction identifies molecular incompatibilities between Homo sapiens and Sus scrofa

Background: Platelets play a vital role in acute humoral xenograft rejection (AHXR) in pig-to-primate xenotransplantation, presenting as microvascular thrombosis in the graft and/or consumptive coagulopathy in the recipient. Adhesion and aggregation of primate platelets to the activated vascular endothelial cells through sequential binding of ligands on endothelial cells and subendothelial matrix ultimately trigger a complex biological process of prothrombotic signaling cascades. Increasing evidence suggests that the molecular incompatibilities in effector molecules across species may partially contribute to dysregulated microvascular thrombosis in xenografts. Method: We selected candidate genes from the NCBI database with keywords: platelet-endothelium interaction，platelet adhension, platelet aggregation, and subendothelial matrix ligands. Pair-wise amino acid alignments were made using the Emboss Needle method. Emboss needle created optimal global alignment of the amino acid sequences of human genes and pig genes using ClustalW2.Results: Most of the proteins involved in platelet-EC interaction in Homo sapiens share high sequence similarity with their homologues in Sus scrofa. Cytokines that potentially induce endothelial damage (such as CD40L, TNF-α) were highly conserved between Homo sapiens and Sus scrofa. Some endothelium-derived cytokines (such as IL-8, CCL2, CCL5) that can induce platelet activation or enhance aggregation share high sequence similarity between Homo sapiens and sus scrofa. Some regulators that potentially transduce inhibitory signaling to control platelet activation or complement activation have relatively poor sequence identity between Homo sapiens and Sus scrofa, and some even lack their homologues in Sus scrofa.Conclusion: The characteristic of sequence similarity of proteins involved in platelet-EC interaction indicated the molecular incompatibilities between humans (Homo sapiens) and pigs (Sus scrofa). This study provides a clue for explanation of excessive platelet activity in pig-to-primate xenotransplantation.

Platelets modulate endothelial cell response to dynamic shear stress through PECAM-1

IntroductionBoth vascular endothelial cells and platelets are sensitive to blood flow induced shear stress. We have recently reported that platelet-endothelial cell interaction could greatly affect platelet activation under flow. In the present study, we aimed to investigate how platelet-endothelial cell interaction affected endothelial cell inflammatory responses under flow. Materials and methodsHuman coronary artery endothelial cells were exposed to normal or low pulsatile shear stress with or without the presence of platelets. Following shear exposure, endothelial cell ICAM-1 expression was measured using ELISA, Western blot and PCR; cell surface PECAM-1 expression/phosphorylation was measured using ELISA. Platelet adhesion to endothelial cells was quantified using immunofluorescence microscopy. To determine the role of PECAM-1 in platelet-endothelial cell interaction, endothelial cell PECAM-1 expression was suppressed using siRNA. ResultsPathological low shear stress induced a significant increase in endothelial cell ICAM-1 expression, at both protein and mRNA levels. Platelet adhesion to endothelial cells increased significantly under low shear stress, co-localizing with PECAM-1 at endothelial cell junctions. The presence of platelets inhibited low shear stress-induced ICAM-1 upregulation. When endothelial cell PECAM-1 expression was suppressed, platelet adhesion to endothelial cells under low shear stress decreased significantly; endothelial cell ICAM-1 expression was not affected by shear stress, with or without platelets. ConclusionsThese results suggested that PECAM-1 could mediate platelet adhesion to endothelial cells under shear stress. Platelets binding to endothelial cells interfered with endothelial cell mechanotransduction through PECAM-1, affecting endothelial cell inflammatory responses towards pathological shear flow.

Antiplatelet activity of flavonoid and coumarin drugs

Polyphenols are used as phlebotonic drugs, but their mechanism of action remains unknown. Since platelet activity and platelet-endothelial cell interactions are involved in the pathogenesis of cardiovascular disease, this work examines whether different flavonoid and coumarin drugs are able to inhibit platelet aggregation. This specific case of coumarins, the antiplatelet effect is not linked with a possible interaction over blood coagulation since this effect only dicoumarols have it.The antiplatelet capacity of polyphenols was assayed using peripheral blood platelets from healthy controls. The distribution of the different platelets subsets was quantified by flow cytometry, using the calcium ionophore as a pro-aggregant. The number of GPIIb/IIIa receptors occupied by the drugs was assayed by flow cytometry using two CD61 surface fluorescein antibodies.All the polyphenols tested inhibited platelet aggregation. A percentage antiplatelet activity of 88.91±7.98% was recorded for naringin, 48.43±8.84% for naringenin, 53.83±7.87% for esculetin, 54.65±6.91% for fraxetin, and 25.75±4.12% for coumarin. Naringin showed significantly greater percentage occupation of GPIIb/IIIa receptors than did naringenin (14.82±0.81% vs. 3.90±0.55%), and esculetin returned significantly higher values than fraxetin and coumarin (12.47±0.97 vs. 7.53±0.49 and 7.90±0.69 respectively).All drugs show important antiplatelet activity. Naringin was the best antiplatelet compound, showing the greatest antiplatelet activity and the highest percentage binding of GPIIb/IIIa receptors. However, any of the compounds used could be used in the prevention of cardiovascular disease.

The Coronary Microcirculation in Hamster-to-Rat Cardiac Xenografts

Background: The aim of this study was to establish a new experimental model to directly analyse the coronary microcirculation in cardiac xenografts. Methods: Intravital fluorescence microscopy (IVM) of the subepicardial microcirculation in heterotopically transplanted hamster-to-rat cardiac xenografts was performed at 30 and 90 min of reperfusion. We quantitatively assessed the microcirculatory perfusion characteristics as well as the interactions of leukocytes and platelets with the endothelium of postcapillary coronary venules in non-sensitised as well as sensitised recipients. Results: In this first experimental IVM study of cardiac xenografts, we successfully visualised the subepicardial microcirculation, i.e. feeding arterioles, nutritive capillaries and draining postcapillary venules, during reperfusion. Leukocyte-endothelial and platelet-endothelial cell interactions could be quantified. In the non-sensitised group, the myocardial microcirculation remained stable during the observation period of 90 min, whereas in the sensitised group, xenografts were rejected immediately. Conclusions: We established a model for the assessment of the microcirculatory dysfunction and inflammation during ischaemia/reperfusion injury in hamster-to-rat cardiac xenografts.

Platelet-endothelial cell interaction in brain microvessels of angiotensin II type 1 receptor knockout mice following transient bilateral common carotid artery occlusion

Platelet-endothelial cell interaction in brain microvessels of angiotensin II type 1 receptor knockout mice following transient bilateral common carotid artery occlusion

CD100 and plexins B2 and B1 mediate monocyte-endothelial cell adhesion and might take part in atherogenesis.

Leukocyte migration is essential for the function of the immune system. Their recruitment from the vessels to the tissues involves sequential molecular interactions between leukocytes and endothelial cells (ECs). Many adhesion molecules involved in this process have already been described. However, additional molecules may be important in this interaction, and here we explore the potential role for CD100 and plexins in monocyte-EC binding. CD100 was shown to be involved in platelet-endothelial cell interaction, an important step in atherogenesis and thrombus formation. In a recent work we have described CD100 expression in monocytes and in macrophages and foam cells of human atherosclerotic plaques. In the present work, we have identified plexin B2 as a putative CD100 receptor in these cells. We have detected CD100 expression in the endothelium as well as in in vitro cultured endothelial cells. Blocking of CD100, plexin B1 and/or B2 in adhesion experiments have shown that both CD100 and plexins act as adhesion molecules involved in monocyte-endothelial cell binding. This effect may be mediated by CD100 expressed in both cell types, probably coupled to the receptors endothelial plexin B1 and monocytic plexin B2. These results can bring new insights about a possible biological activity of CD100 in monocyte adhesion and atherosclerosis, as well as a future candidate for targeting therapeutics.

Therapeutic effect of methotrexate encapsulated in cationic liposomes (EndoMTX) in comparison to free methotrexate in an antigen-induced arthritis study in vivo

Objectives: Cationic lipid complexes bind to angiogenic endothelial cells of solid tumours and microvessels of chronic inflammatory tissue. Methotrexate (MTX) is one of the drugs used in the therapy of rheumatoid arthritis (RA); it is applied systemically but can have serious side-effects. The aim of this study was to investigate the impact of MTX encapsulated in cationic liposomes (EndoMTX) in comparison to treatment with free MTX.Method: We used an antigen-induced arthritis (AiA) model and investigated the leucocyte– and platelet–endothelial cell interaction in arthritic female C57/Bl6 mice and in healthy controls. The arthritic animals were divided into four different groups receiving either trehalose, free MTX, EndoMTX placebo, or EndoMTX. These parameters and functional capillary density (FCD) were measured and assessed by intravital microscopy (IVM). We controlled clinical parameters such as the knee joint diameter (KJD) throughout the observation period.Results: Animals treated with EndoMTX showed a significant and superior reduction in leucocyte– and platelet–endothelial cell interaction, FCD, and KJD. Free MTX or empty liposomes also showed a reduction in these parameters but not to a significant level. FCD decreased in the EndoMTX group in comparison to using free drugs or empty carrier-like liposomes.Conclusions: This study demonstrates the advantage of using MTX encapsulated in cationic liposomes in contrast to free and generic MTX, with a higher efficacy in anti-inflammatory and anti-angiogenic abilities. Targeting with cationic liposomes may be a promising treatment option and should be elucidated in further experiments regarding dose reduction and side-effects due to MTX usage.

Platelet PI3Kγ Contributes to Carotid Intima-Media Thickening under Severely Reduced Flow Conditions.

Studies have begun to focus on the emerging function of platelets as immune and inflammatory cells that initiate and accelerate vascular inflammation. Phosphoinositide 3-kinase gamma (PI3Kγ) is critically involved in a number of inflammatory and autoimmune diseases. This study aims to investigate the contribution of platelet PI3Kγ to vascular remodeling under flow severely reduced conditions. Mouse partial left carotid artery ligation with adoptive transfer of activated, washed wild-type or PI3Kγ-/- platelets was used as the model. Intima-media area, leukocyte recruitment, and proinflammatory mediator expression were assessed. In vitro PI3Kγ-/- platelets were used to verify the effect of PI3Kγ on platelet activation, interaction with leukocytes, and endothelial cells. Mice injected with activated platelets showed a significant increase in intima-media thickening, recruitment of neutrophils (at 3 d) and macrophages (at 21 d), and intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor alpha, and interleukin-6 expression (at 3 d) in the flow-reduced area. These effects were abrogated by platelet PI3Kγ deficiency. Circulating platelet-leukocyte aggregates were reduced in PI3Kγ-/- mice after partial ligation. In vivo data confirmed that PI3Kγ mediated Adenine di-Phosphate -induced platelet activation through the Akt and p38 MAP kinase signaling pathways. Moreover, platelet PI3Kγ deficiency reduced platelet-leukocyte aggregation and platelet-endothelial cell (EC) interaction. These findings indicate that platelet PI3Kγ contributes to platelet-mediated vascular inflammation and carotid intima-media thickening after flow severely reduced. Platelet PI3Kγ may be a new target in the treatment of vascular diseases.

Investigating Platelet Interactions in Sickle Cell Disease Using a Novel Multi-Shear "Endothelialized" Microfluidic System

▪Sickle Cell Disease (SCD) is an inherited disorder of the β-globin chain of hemoglobin, in which a single point mutation leads to decreased deformability of red blood cells (RBCs) and increased cellular adhesion to endothelium. The effect of this mutation on RBCs has been well characterized, and the interplay of endothelial cells, RBCs, and white blood cells (WBCs) have also been well characterized. However, few studies have specifically investigated how platelets interact with endothelial cells and other blood cells in the context of SCD and the role these cell fragments may have in vaso-occlusion.To that end, we utilized microfluidic technology previously developed in our lab to perform a “real time” in vitro analyses of platelet-endothelial cell interactions in SCD patient samples. This “microvasculature-on-a-chip” enables the visualization of blood cell-endothelial cell interactions under a controlled hemodynamic environment (Tsai et al, JCI, 2012).As shear stress can trigger platelet activation, we further modified and optimized our standard microfluidic devices to encompass 3 different physiologic shear rates. Our device features microchannels 50µm in diameter with human umbilical vein endothelial cells (HUVEC) confluently lining the channels; there are 12 channels in each device, grouped in 3 sets of 4 channels with graduating shear rates spanning 3 orders of magnitude (Figure 1). Our initial experiments were performed under normoxic conditions allowing characterization of platelet-endothelial interactions in an “arterial” in vitro environment. Whole blood samples were obtained from 3 patient populations: patients with HgbSS SCD on hydroxyurea (HgbSS+HU), patients with HgbSS SCD not on hydroxyurea (HgbSS-no HU), and normal healthy controls. Over 30 minutes, whole blood stained with fluorescently labeled CD41 to identify platelets and Hoeschst to identify HUVEC nuclei was perfused at a rate of 1.5µl/minute under videomicroscopy. Accumulation of platelets on the endothelialized channels and platelet aggregates were quantified based on anti-CD41 fluorescence. Within 1 minute of perfusion, HgbSS-no HU whole blood samples exhibited extensive platelet aggregates at 1 and 10 dyne/cm2 (Figure 2); this phenomenon did not occur under any of the shear conditions in blood samples from Hgb SS+HU or healthy control samples. In HgbSS-no HU blood samples, some of these “thrombi”-like aggregates were stable under flow, increased in size, and persisted for the remainder of the 30 minute experiments. In contrast, mild, uniform, platelet adhesion slowly developed at high shear conditions in Hgb SS+HU with fewer platelet aggregates forming as compared to patients with HgbSS- no HU. Healthy control samples did not exhibit this platelet aggregation. There appears to be an attenuating effect of hydroxyurea on platelets that prevents platelet clumping from occuring as frequently under various shear conditions that is not present in the Hgb SS-no HU samples (Figure 3).In conclusion, using our novel in vitro system, we have demonstrated the platelets from Hgb SS-no HU patients have a significantly increased propensity to adhere, aggregate, and accumulate in endothelialized microvasculature-sized microchannels. Interestingly, this effect appears to be attenuated in blood samples from Hgb SS+HU patients and not present in healthy controls, demonstrating that hydroxyurea appears to be an important modifier of this phenomenon. Experiments investigating the underlying mechanisms of this phenomenon, the effects of deoxygenation and the potential role of platelets in vaso-occlusion, the effects of sickle cell platelet adhesion/aggregation on endothelial function, and how hydroxyurea may or may not affect any or all of these parameters, are all currently ongoing. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells.

The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 μg/ml, and up to 33 % for 15 μg/ml, p < 0.05 or less) in a concentration-dependent manner, and enhanced endothelial nitric oxide synthase (eNOS) activation (T495 phosphorylation decreased by 31 ± 6 % for 2.5 μg/ml and 48 ± 6 % for 15 μg/ml; S1177 phosphorylation increased by 13 ± 3 % for 2.5 μg/ml and 18 ± 7 % for 15 μg/ml, compared to untreated cells, p < 0.05 or less). Additionally, incubation for 24 or 48 h with BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 μg/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p < 0.0001). The increased CD39 expression and the increased eNOS activation in HUVEC can be regarded as the beneficial markers of the improvement of antiplatelet action of endothelial cells. Unexpectedly, these assumptions were not confirmed in the experimental model of platelet-endothelial cell interactions. These observations lead to the conclusion that BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium.

Simvastatin reduces leucocyte– and platelet–endothelial cell interaction in murine antigen-induced arthritis in vivo

Objectives: The use of statins in the prevention and treatment of cardiovascular diseases is well established. Their use as anti-inflammatory and immunomodulatory agents in the treatment of rheumatoid arthritis (RA) has also been investigated, with several clinical and experimental studies indicating an anti-inflammatory effect of statins for RA, but other studies showing no effect or even the opposite. The current study was designed to examine the effect of simvastatin in an in vivo murine model of arthritis using intravital microscopy.Method: We assigned four groups (n = 7, female C57Bl6 mice), two with and two without antigen-induced arthritis (AiA), from which one of the non-AiA groups and one of the AiA groups were treated with simvastatin 40 mg/kg i.p. daily for 14 consecutive days after induction of arthritis. Platelet– and leucocyte–endothelial cell interaction was assessed by measurement of rolling and adherent fluorescence-labelled platelets and leucocytes, functional capillary density (FCD) was evaluated, and knee joint diameter was determined as a clinical parameter.Results: In arthritic mice treated with simvastatin, a significant reduction in platelet– and leucocyte–endothelial cell interaction was observed in comparison to arthritic mice treated with vehicle. In addition, a significant reduction in FCD was seen in arthritic mice treated with simvastatin, along with a reduction in knee joint swelling of the AiA mice.Conclusions: Treatment of AiA mice with simvastatin showed significant reductions in platelet– and leucocyte–endothelial cell interactions, in FCD, and in the swelling of the knee joint. These results support the hypothesis of the anti-inflammatory effects of statins in the treatment of RA.

Abstract 646: The Paradoxical Role of TREM-Like 1 in Atherosclerosis

Atherosclerosis is a chronic inflammatory process of the vessel wall driven by an inflammatory state caused by the involvement of different cell populations, including platelets. The receptor Triggering Receptor Expressed on Myeloid Cells (TREM)-like transcript (TLT)-1 is prepacked in platelet α-granules and brought to the surface upon activation. TLT-1 has both membrane-bound and soluble forms (sTLT-1) and the latter has been found to enhance platelet activation as well as platelet-endothelial cell interactions. TLT-1 null mice (treml1-/-) have reduced platelet aggregation, are significantly more susceptible to lipopolysaccharide challenge compared to their wild type counterparts, and accordingly, hemorrhage in response to an acute inflammatory challenge. These results suggest that TLT-1 is a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms and in light of this, we hypothesized that TLT-1 plays an important role in atherosclerosis progression. To address this, we generated apoE-/-/treml1-/- double knockout mice [DKO]. We found that DKO mice fed an atherogenic diet (HFD) showed significantly increased weight gain when compared to apoE-/-. Accordingly, DKO mice showed increased total cholesterol and triglycerides compared to controls. Surprisingly, assessment of lesion size revealed that DKO mice have significantly smaller lesions in the aortic sinus at four and 12 weeks after HFD compared to apoE-/-. At 20 weeks, lesion differences are no longer significant, but compositional analysis revealed that DKO atherosclerotic lesions are less vulnerable as seen by decreased lesion calcification and increased smooth muscle cell content. Furthermore, q-rtPCR analysis of atherosclerosis-related genes revealed that TLT-1 differentially affects genes related to lipid metabolism (apo AI and B) and vascular inflammation (thrombospondin 4, PAI-1 and VGFR-2). While our data suggests that the smaller lesion size may be explained by lower platelet reactivity, differences in cholesterol levels suggest altered lipid metabolism which is supported by our metabolomics studies (González, M. et.al). Taken together TLT-1 plays a dual role in the progression of atherosclerosis.

Lactobacillus reuteri Maintains a Functional Mucosal Barrier during DSS Treatment Despite Mucus Layer Dysfunction

Treatment with the probiotic bacterium Lactobacillus reuteri has been shown to prevent dextran sodium sulfate (DSS)-induced colitis in rats. This is partly due to reduced P-selectin-dependent leukocyte- and platelet-endothelial cell interactions, however, the mechanism behind this protective effect is still unknown. In the present study a combination of culture dependent and molecular based T-RFLP profiling was used to investigate the influence of L. reuteri on the colonic mucosal barrier of DSS treated rats. It was first demonstrated that the two colonic mucus layers of control animals had different bacterial community composition and that fewer bacteria resided in the firmly adherent layer. During DSS induced colitis, the number of bacteria in the inner firmly adherent mucus layer increased and bacterial composition of the two layers no longer differed. In addition, induction of colitis dramatically altered the microbial composition in both firmly and loosely adherent mucus layers. Despite protecting against colitis, treatment with L. reuteri did not improve the integrity of the mucus layer or prevent distortion of the mucus microbiota caused by DSS. However, L. reuteri decreased the bacterial translocation from the intestine to mesenteric lymph nodes during DSS treatment, which might be an important part of the mechanisms by which L. reuteri ameliorates DSS induced colitis.