The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo.

The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.

Galectin-9 mediates neutrophil capture and adhesion in a CD44 and ß2 integrin-dependent manner.

Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin-9 (Gal-9) on neutrophil recruitment. Our data indicate that Gal-9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal-9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal-9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal-9 knockout mice in a model of zymosan-induced peritonitis. We also provide evidence for Gal-9 binding sites on human neutrophils; Gal-9 binding induced neutrophil activation (increased expression of ß2 integrins and reduced expression of CD62L). Intra-vital microscopy confirmed a pro-recruitment role for Gal-9, with increased numbers of transmigrated neutrophils following Gal-9 administration. We studied the role of both soluble and immobilized Gal-9 on human neutrophil recruitment. Soluble Gal-9 significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM-1. When immobilized, Gal-9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal-9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro-adhesive effects of Gal-9. Our data indicate that Gal-9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal-9 in leukocyte recruitment in different inflammatory settings.

Podoplanin regulates the migration of mesenchymal stromal cells and their interaction with platelets.

Mesenchymal stromal cells (MSCs) upregulate podoplanin at sites of infection, chronic inflammation and cancer. Here, we investigated the functional consequences of podoplanin expression on the migratory potential of MSCs and their interactions with circulating platelets. Expression of podoplanin significantly enhanced the migration of MSCs compared to MSCs lacking podoplanin. Rac-1 inhibition altered the membrane localisation of podoplanin and in turn significantly reduced MSC migration. Blocking Rac-1 activity had no effect on the migration of MSCs lacking podoplanin, indicating that it was responsible for regulation of migration through podoplanin. When podoplanin-expressing MSCs were seeded on the basal surface of a porous filter, they were able to capture platelets perfused over the uncoated apical surface and induce platelet aggregation. Similar microthrombi were observed when endothelial cells (ECs) were co-cultured on the apical surface. Confocal imaging shows podoplanin-expressing MSCs extending processes into the EC layer, and these processes could interact with circulating platelets. In both models, platelet aggregation induced by podoplanin-expressing MSCs was inhibited by treatment with recombinant soluble C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b). Thus, podoplanin may enhance the migratory capacity of tissue-resident MSCs and enable novel interactions with cells expressing CLEC-2.

Appropriation of GPIbα from platelet-derived extracellular vesicles supports monocyte recruitment in systemic inflammation.

Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-ß1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-ß1-stimulated cremaster muscle, while in the ApoE-/- model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic.

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062-1074.

Monocyte Subsets Coregulate Inflammatory Responses by Integrated Signaling through TNF and IL-6 at the Endothelial Cell Interface.

Two major monocyte subsets, CD14+CD16- (classical) and CD14+/dimCD16+ (nonclassical/intermediate), have been described. Each has different functions ascribed in its interactions with vascular endothelial cells (EC), including migration and promoting inflammation. Although monocyte subpopulations have been studied in isolated systems, their influence on EC and on the course of inflammation has been ignored. In this study, using unstimulated or cytokine-activated EC, we observed significant differences in the recruitment, migration, and reverse migration of human monocyte subsets. Associated with this, and based on their patterns of cytokine secretion, there was a difference in their capacity to activate EC and support the secondary recruitment of flowing neutrophils. High levels of TNF were detected in cocultures with nonclassical/intermediate monocytes, the blockade of which significantly reduced neutrophil recruitment. In contrast, classical monocytes secreted high levels of IL-6, the blockade of which resulted in increased neutrophil recruitment. When cocultures contained both monocyte subsets, or when conditioned supernatant from classical monocytes cocultures (IL-6hi) was added to nonclassical/intermediate monocyte cocultures (TNFhi), the activating effects of TNF were dramatically reduced, implying that when present, the anti-inflammatory activities of IL-6 were dominant over the proinflammatory activities of TNF. These changes in neutrophil recruitment could be explained by regulation of E-selectin on the cocultured EC. This study suggests that recruited human monocyte subsets trigger a regulatory pathway of cytokine-mediated signaling at the EC interface, and we propose that this is a mechanism for limiting the phlogistic activity of newly recruited monocytes.

Signalling through Src family kinase isoforms is not redundant in models of thrombo-inflammatory vascular disease.

The Src family kinases (SFK) are a group of signalling molecules with important regulatory functions in inflammation and haemostasis. Leucocytes and platelets express multiple isoforms of the SFKs. Previous studies used broad-spectrum pharmacological inhibitors, or murine models deficient in multiple SFK isoforms, to demonstrate the functional consequences of deficiencies in SFK signalling. Here, we hypothesized that individual SFK operate in a non-redundant fashion in the thrombo-inflammatory recruitment of monocyte during atherosclerosis. Using in vitro adhesion assays and single SFK knockout mice crossed with the ApoE-/- model of atherosclerosis, we find that SFK signalling regulates platelet-dependent recruitment of monocytes. However, loss of a single SFK, Fgr or Lyn, reduced platelet-mediated monocyte recruitment in vitro. This translated into a significant reduction in the burden of atherosclerotic disease in Fgr-/- /ApoE-/- or Lyn-/- /ApoE-/- animals. SFK signalling is not redundant in thrombo-inflammatory vascular disease and individual SFK may represent targets for therapeutic intervention.

Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2.

Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte-endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling.

Adipogenic Differentiation of Mesenchymal Stem Cells Alters Their Immunomodulatory Properties in a Tissue-Specific Manner.

Chronic inflammation is associated with formation of ectopic fat deposits that might represent damage-induced aberrant mesenchymal stem cell (MSC) differentiation. Such deposits are associated with increased levels of inflammatory infiltrate and poor prognosis. Here we tested the hypothesis that differentiation from MSC to adipocytes in inflamed tissue might contribute to chronicity through loss of immunomodulatory function. We assessed the effects of adipogenic differentiation of MSC isolated from bone marrow or adipose tissue on their capacity to regulate neutrophil recruitment by endothelial cells and compared the differentiated cells to primary adipocytes from adipose tissue. Bone marrow derived MSC were immunosuppressive, inhibiting neutrophil recruitment to TNFα-treated endothelial cells (EC), but MSC-derived adipocytes were no longer able to suppress neutrophil adhesion. Changes in IL-6 and TGFß1 signalling appeared critical for the loss of the immunosuppressive phenotype. In contrast, native stromal cells, adipocytes derived from them, and mature adipocytes from adipose tissue were all immunoprotective. Thus disruption of normal tissue stroma homeostasis, as occurs in chronic inflammatory diseases, might drive "abnormal" adipogenesis which adversely influences the behavior of MSC and contributes to pathogenic recruitment of leukocytes. Interestingly, stromal cells programmed in native fat tissue retain an immunoprotective phenotype. Stem Cells 2017;35:1636-1646.

The Relationship Between Serum Interleukin-1α and Asymptomatic Infrarenal Abdominal Aortic Aneurysm Size, Morphology, and Growth Rates.

OBJECTIVE/BACKGROUND: In a pilot study, a relationship between abdominal aortic aneurysm (AAA) diameter and serum interleukin (IL)-1α levels was reported, and that endothelial cell (EC) activation in vitro in response to serum from patients with AAA was blocked by anti-IL-1α antibodies. The aim of the present study was to further investigate the relationship between serum IL-1α and asymptomatic infrarenal AAA size, morphology, and growth rates. METHODS: Serum IL-1α was measured using enzyme linked immunosorbent assay in 101 patients with asymptomatic, infrarenal AAA and related to aneurysm size, morphology, and growth rates. RESULTS: IL-1α was measured in 101 patients. There was no statistically significant difference in mean age between men and women. IL-1α was detectable in 62.4% of patients; median IL-1α titre was 3.26 pg/mL. There was no statistically significant relationship between IL-1α and maximum AAA antero-posterior diameter as measured by ultrasound (p = .649), AAA morphology (aortic length [p = .394], sac [p = .369], and thrombus volume [p = .629]) as measured on computed tomography, absolute increase in AAA diameter (p = .214), or AAA growth rate (p = .230). CONCLUSION: IL-1α is detectable in the majority of patients with infrarenal AAA, but the cause and clinical significance of this novel observation remains unknown.

Bone Morphogenetic Protein 9 Enhances Lipopolysaccharide-Induced Leukocyte Recruitment to the Vascular Endothelium.

Bone morphogenetic protein (BMP)9 is a circulating growth factor that is part of the TGF-ß superfamily and is an essential regulator of vascular endothelial homeostasis. Previous studies have suggested a role for BMP9 signaling in leukocyte recruitment to the endothelium, but the directionality of this effect and underlying mechanisms have not been elucidated. In this study, we report that BMP9 upregulates TLR4 expression in human endothelial cells and that BMP9 pretreatment synergistically increases human neutrophil recruitment to LPS-stimulated human endothelial monolayers in an in vitro flow adhesion assay. BMP9 alone did not induce neutrophil recruitment to the endothelium. We also show that E-selectin and VCAM-1, but not ICAM-1, are upregulated in response to BMP9 in LPS-stimulated human endothelial cells. Small interfering RNA knockdown of activin receptor-like kinase 1 inhibited the BMP9-induced expression of TLR4 and VCAM-1 and inhibited BMP9-induced human neutrophil recruitment to LPS-stimulated human endothelial cells. BMP9 treatment also increased leukocyte recruitment within the pulmonary circulation in a mouse acute endotoxemia model. These results demonstrate that although BMP9 alone does not influence leukocyte recruitment, it primes the vascular endothelium to mount a more intense response when challenged with LPS through an increase in TLR4, E-selectin, and VCAM-1 and ultimately through enhanced leukocyte recruitment.

Modulation of VEGF-induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

Identification of a transitional fibroblast function in very early rheumatoid arthritis.

OBJECTIVES: Synovial fibroblasts actively regulate the inflammatory infiltrate by communicating with neighbouring endothelial cells (EC). Surprisingly, little is known about how the development of rheumatoid arthritis (RA) alters these immunomodulatory properties. We examined the effects of phase of RA and disease outcome (resolving vs persistence) on fibroblast crosstalk with EC and regulation of lymphocyte recruitment. METHODS: Fibroblasts were isolated from patients without synovitis, with resolving arthritis, very early RA (VeRA; symptom ≤12 weeks) and established RA undergoing joint replacement (JRep) surgery. Endothelial-fibroblast cocultures were formed on opposite sides of porous filters. Lymphocyte adhesion from flow, secretion of soluble mediators and interleukin 6 (IL-6) signalling were assessed. RESULTS: Fibroblasts from non-inflamed and resolving arthritis were immunosuppressive, inhibiting lymphocyte recruitment to cytokine-treated endothelium. This effect was lost very early in the development of RA, such that fibroblasts no longer suppressed recruitment. Changes in IL-6 and transforming growth factor beta 1 (TGF-ß1) signalling appeared critical for the loss of the immunosuppressive phenotype. In the absence of exogenous cytokines, JRep, but not VeRA, fibroblasts activated endothelium to support lymphocyte. CONCLUSIONS: In RA, fibroblasts undergo two distinct changes in function: first a loss of immunosuppressive responses early in disease development, followed by the later acquisition of a stimulatory phenotype. Fibroblasts exhibit a transitional functional phenotype during the first 3 months of symptoms that contributes to the accumulation of persistent infiltrates. Finally, the role of IL-6 and TGF-ß1 changes from immunosuppressive in resolving arthritis to stimulatory very early in the development of RA. Early interventions targeting 'pathogenic' fibroblasts may be required in order to restore protective regulatory processes.

CLEC-2 expression is maintained on activated platelets and on platelet microparticles.

The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in FcγRIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.

The role of platelets in the recruitment of leukocytes during vascular disease.

Besides their role in the formation of thrombus during haemostasis, it is becoming clear that platelets contribute to a number of other processes within the vasculature. Indeed, the integrated function of the thrombotic and inflammatory systems, which results in platelet-mediated recruitment of leukocytes, is now considered to be of great importance in the propagation, progression and pathogenesis of atherosclerotic disease of the arteries. There are three scenarios by which platelets can interact with leukocytes: (1) during haemostasis, when platelets adhere to and are activated on sub-endothelial matrix proteins exposed by vascular damage and then recruit leukocytes to a growing thrombus. (2) Platelets adhere to and are activated on stimulated endothelial cells and then bridge blood borne leukocytes to the vessel wall and. (3) Adhesion between platelets and leukocytes occurs in the blood leading to formation of heterotypic aggregates prior to contact with endothelial cells. In the following review we will not discuss leukocyte recruitment during haemostasis, as this represents a physiological response to tissue trauma that can progress, at least in its early stages, in the absence of inflammation. Rather we will deal with scenarios 2 and 3, as these pathways of platelet-leukocyte interactions are important during inflammation and in chronic inflammatory diseases such as atherosclerosis. Indeed, these interactions mean that leukocytes possess means of adhesion to the vessel wall under conditions that may not normally be permissive of leukocyte-endothelial cell adhesion, meaning that the disease process may be able to bypass the regulatory pathways which would ordinarily moderate the inflammatory response.

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development.

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.

Crosstalk between mesenchymal stem cells and endothelial cells leads to downregulation of cytokine-induced leukocyte recruitment.

Mesenchymal stem cells (MSC) have immunomodulatory properties, but their effects on endothelial cells (EC) and recruitment of leukocytes are unknown. We cocultured human bone marrow-derived MSC with EC and found that MSC could downregulate adhesion of flowing neutrophils or lymphocytes and their subsequent transendothelial migration. This applied for EC treated with tumor necrosis factor-α (TNF), interleukin-1ß (IL-1), or TNF and interferon-γ combined. Supernatant from cocultures also inhibited endothelial responses. This supernatant had much higher levels of IL-6 than supernatant from cultures of the individual cells, which also lacked inhibitory functions. Addition of neutralizing antibody against IL-6 removed the bioactivity of the supernatant and also the immunomodulatory effects of coculture. Studies using siRNA showed that IL-6 came mainly from the MSC in coculture, and reduction in production in MSC alone was sufficient to impair the protective effects of coculture. Interestingly, siRNA knockdown of IL-6-receptor expression in MSC as well as EC inhibited anti-inflammatory effects. This was explained when we detected soluble IL-6R receptor in supernatants and showed that receptor removal reduced the potency of supernatant. Neutralization of transforming growth factor-ß indicated that activation of this factor in coculture contributed to IL-6 production. Thus, crosstalk between MSC and EC caused upregulation of production of IL-6 by MSC which in turn downregulated the response of EC to inflammatory cytokines, an effect potentiated by MSC release of soluble IL-6R. These studies establish a novel mechanism by which MSC might have protective effects against inflammatory pathology and cardiovascular disease.

The role of platelet-endothelial cell adhesion molecule-1 in atheroma formation varies depending on the site-specific hemodynamic environment.

OBJECTIVE: Polymorphisms in the platelet-endothelial cell adhesion molecule (PECAM-1)-1 gene are linked to increased risk of coronary artery disease. Because PECAM-1 has been demonstrated to form a mechanosensory complex that can modulate inflammatory responses in murine arterial endothelial cells, we hypothesized that PECAM-1 contributes to atherogenesis in a shear-dependent and site-specific manner. APPROACH AND RESULTS: ApoE(-/-) mice that were wild-type, heterozygous, or deficient in PECAM-1 were placed on a high-fat diet. Detailed analysis of the aorta at sites with differing hemodynamics revealed that PECAM-1-deficient mice had reduced disease in areas of disturbed flow, whereas plaque burden was increased in areas of steady, laminar flow. In concordance with these observations, bone marrow chimera experiments revealed that hematopoietic PECAM-1 resulted in accelerated atheroma formation in areas of laminar and disturbed flow, however endothelial PECAM-1 moderated disease progression in areas of high sheer stress. Moreover, using shear stress-modifying carotid cuffs, PECAM-1 was shown to promote macrophage recruitment into lesions developing in areas of low shear stress. CONCLUSIONS: PECAM-1 on bone marrow cells is proatherogenic irrespective of the hemodynamic environment, however endothelial cell PECAM-1 is antiatherogenic in high shear environments. Thus, targeting this pathway therapeutically would require a cell-type and context-specific strategy.

The rheology of interactions between leukocytes, platelets and the vessel wall in thrombo-inflammation.

Leukocytes and platelets must adhere to the wall of blood vessels to carry out their protective functions in inflammation and haemostasis. Recruitment is critically dependent on rheological variables (wall shear rate and stress, red cell aggregation and haematocrit) which affect delivery to the vessel wall as well as velocities and forces experienced there. Leukocyte recruitment is efficient only up to wall shear rates of about 300 s-1 and usually restricted to low-shear post-capillary venules in inflammation. Being smaller, platelets experience lower velocities and shear forces adjacent to the wall and can adhere at much higher shear rates for haemostasis in arteries. In addition, we found quite different effects of variations in haematocrit or red cell aggregation on attachment of neutrophils or platelets, which also assist their separate recruitment in venules or arteries. However, it has become increasingly evident that inflammatory and thrombotic responses may occur together, with platelets promoting the adhesion and activation of neutrophils and monocytes. Indeed, it is 30 years since we demonstrated that platelets could cause neutrophils to aggregate in suspension and, when attached to a surface, could support selectin-mediated rolling of all leukocytes. Thrombin-activated platelets could further induce neutrophil activation and immobilisation. In some conditions, platelets could bind to intact endothelial monolayers and capture neutrophils or monocytes. Subsequently, we found that extracellular vesicles released by activated platelets (PEV) fulfilled similar functions when deposited on surfaces or bound to endothelial cells. In murine models, platelets or PEV could act as bridges for monocytes in inflamed vessels. Thus, leukocytes and platelets are rheologically adapted for their separate functions, while novel thrombo-inflammatory pathways using platelets or PEV may underlie pathogenic leukocyte recruitment.

Comparative rheology of the adhesion of platelets and leukocytes from flowing blood: why are platelets so small?

We investigated rheological adaptation of leukocytes and platelets for their adhesive functions in inflammation and hemostasis, respectively. Adhesion and margination of leukocytes or platelets were quantified for blood perfused through capillaries coated with P-selectin or collagen, when flow rate, suspending phase viscosity, red cell aggregation, or rigidity was modified. Independent variation of shear rate and shear stress indicated that the ability of platelets to attach at higher levels than leukocytes was largely attributable to their smaller size, reducing their velocity before attachment, and, especially, drag after attachment. Increasing red cell aggregation increased the number of marginated and adhering leukocytes but inhibited platelet adhesion without effect on the number marginated. Increasing red cell rigidity tended to inhibit leukocyte adhesion but promote platelet adhesion. The effects on platelets may be explained by changes in the depth of the near-wall, red cell-depleted layer; broadening (or narrowing) this layer to greater (or less) than the platelet diameter would decrease (or increase) the normal force applied by red blood cells and make attachment less (or more) efficient. Thus different adhesive capabilities of leukocytes and platelets may arise from their differences in size, both directly because of influence on cell velocity and force experienced at the wall and indirectly through effects of size on margination in the bloodstream and interaction with the cell-free layer. In addition, red cell aggregation (of hitherto uncertain physiological significance) may be useful in promoting leukocyte adhesion in inflamed venules but inhibiting unwanted platelet deposition in veins.