Sickle-trait hemoglobin reduces adhesion to both CD36 and EPCR by Plasmodium falciparum-infected erythrocytes

Sickle-trait hemoglobin protects against severe Plasmodium falciparum malaria. Severe malaria is governed in part by the expression of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that are encoded by var genes, specifically those variants that bind Endothelial Protein C Receptor (EPCR). In this study, we investigate the effect of sickle-trait on parasite var gene expression and function in vitro and in field-collected parasites. We mapped var gene reads generated from RNA sequencing in parasite cultures in normal and sickle-cell trait blood throughout the asexual lifecycle. We investigated sickle-trait effect on PfEMP1 interactions with host receptors CD36 and EPCR using static adhesion assays and flow cytometry. Var expression in vivo was compared by assembling var domains sequenced from total RNA in parasites infecting Malian children with HbAA and HbAS. Sickle-trait did not alter the abundance or type of var gene transcripts in vitro, nor the abundance of overall transcripts or of var functional domains in vivo. In adhesion assays using recombinant host receptors, sickle-trait reduced adhesion by 73–86% to CD36 and 83% to EPCR. Similarly, sickle-trait reduced the surface expression of EPCR-binding PfEMP1. In conclusion, Sickle-cell trait does not directly affect var gene transcription but does reduce the surface expression and function of PfEMP1. This provides a direct mechanism for protection against severe malaria conferred by sickle-trait hemoglobin. Trial Registration: ClinicalTrials.gov Identifier: NCT02645604.

Inflammatory Smooth Muscle Cells Induce Endothelial Cell Alterations to Influence Cerebral Aneurysm Progression via Regulation of Integrin and VEGF Expression

Cerebral aneurysm growth is characterized by vessel wall frailness, although the underlying cellular mechanisms are unclear. Here, we examined the relationship between inflammatory smooth muscle cells (SMCs) and endothelial cells (ECs) in cerebral aneurysms, including the mechanisms underlying inflammatory SMC-induced changes in ECs. Five saccular cerebral aneurysms were collected and five temporal artery samples were used as controls. Cells and cytokines were detected by immunohistochemistry and TUNEL (transferase dUTP nick end labeling) assays performed to evaluate apoptosis. Human umbilical vein endothelial cells (HUVECs) were seeded on collagen I, IV, and VI-coated plates for cell adhesion assays and inflammatory SMCs (iSMCs) were established by culture in flexible silicone chambers subjected to cyclic mechanical stretch. HUVECs were cultured in iSMC-conditioned medium, followed by evaluation of their viability, apoptosis, and function, and determination of VEGF (vascular endothelial growth factor) -A and integrin levels by western blotting. Aneurysm tissue contained fewer SMCs and lacked ECs. In aneurysm walls, more matrix metalloproteinase (MMP) -1, MMP-3, and apoptotic cells were detected, accompanied by decreased collagen IV and VI levels. Cell adhesion assays revealed that more HUVECs were attached in collagen IV and VI-coated plates compared with controls. iSMC-conditioned medium significantly reduced HUVEC viability and apoptosis showed an increased trend; however, the difference was not significant. iSMC medium also reduced tube formation and migration of HUVECs. Moreover, iSMC medium reduced HUVEC expression of VEGF-A, integrin α1, integrin α2, and integrin β. Our data demonstrate a lack of SMCs and ECs in aneurysm walls, accompanied by elevated MMP and decreased collagen levels. In vitro assays showed that iSMCs induced reduction in EC adhesion, and caused EC dysfunction. Understanding of the relationships among SMC, EC, and collagens during aneurysm progression provides an additional therapeutic option for prevention of cerebral aneurysm progression.

A Comparative Medicine Study of Platelet Biophysics Among Hemostasis Models of Different Species

Background: Animal models of hemostasis play a crucial role in understanding hemostasis and developing novel therapeutic treatments. Hemostasis is a mechanical phenomenon and recent studies have demonstrated that biophysical parameters, such as platelet margination (Walton et al. Blood 2017), thrombus porosity (Welsh et al. Blood 2014), shear forces (Nesbitt et al. Nat Med 2009), compression forces (Ju et al. Nat Comm 2018), and single platelet forces, as measured by platelet contraction cytometry, developed by our laboratory, (Myers et al. Nat Mat. 2017) affect hemostatic processes and may be pathologically altered in disease states. However, little is known about the most basic biophysical interspecies differences between platelets of various animal hemostasis models, which could provide critical insight into interpreting data and results. Here, we report the results of our initial study on platelet biophysics in humans (n=5), mice (n=8), pigs (n=5), and dogs (n=6), which are commonly used for hemostasis models due to anatomical similarities to human vessels, organs, and cell function (Siller-Matula et al. Thromb & Haem 2008) (Jagadeeswaran et al. Circ Res 2015). We focus on understanding differences in platelet adhesion on collagen and fibrinogen, spreading area, singe cell contraction forces, and bulk contraction, which all may influence the initiation and stability of blood clots (Fig. 1). Our data demonstrates striking differences between each species, especially concerning adhesion, spreading area, and single platelet force. Surprisingly, measurements of volumetric bulk contraction, the most commonly used biophysical assay in hemostasis research, appear to be quite consistent between species. Methods: We performed a comprehensive study of platelet biophysics using adhesion assays, bulk contraction, and single platelet contractile forces on humans, mice, pigs, and dogs' platelets. For the adhesion assays, spreading area, and platelet contractile forces, platelets were gel-filtered as described previously (Myers et al. 2017). Cell Adhesion & Spreading Assay: Gel filtered platelets were diluted to 20M/mL, incubated on coverslips that had been incubated with type 1 collagen (100 µg/mL) or fibrinogen (100 µg/mL), allowed to adhere for 2 hours, counted, and measured. Platelet Contraction Cytometry: Polyacrylamide hydrogels with pairs of fibrinogen microdots are fabricated at physiologically relevant stiffnesses. Gel-filtered thrombin-activated platelets (1 U/mL) are then plated to attach, spread, and contract the microdots together. Individual platelet force is directly proportional to the microdot displacement measured with fluorescent imaging. Bulk Clot Contraction Assay: Platelets are washed as described previously (Lam et al. Nat Mat 2011) and then diluted to 200M/mL in a solution of fibrinogen (2 mg/mL), CaCl2 (5 mM), and thrombin (1 U/mL). Clots are left to form in cuvettes lined with gridded paper and measured every 30 mins for 2.5 hours. All assays were performed on at least 3 subjects from each species and in triplicate when possible. Results: We found both significant biophysical differences and similarities between platelets of different species (Fig. 2). Overall, murine platelets have the highest biophysical similarity to human platelets regarding adhesion, volumetric contraction, and single platelet contractile force despite substantial size differences. Porcine platelets exhibited enhanced adhesion on collagen with little adhesion to fibrinogen; while canine platelets showed the opposite. Such differences could have significant implications on the biophysical initiation of blood hemostasis and thrombosis, as well as the thrombogenicity tests of new materials. In bulk and single platelet contraction, we noted that similarities between most species except for canine platelets which produced forces nearly triple that of humans, the highest contractile forces of any platelets measured to date. As recent work has shown that forces are linked to clot porosity and therefore activation (Mirramezani et al. JTH 2018), such high forces could influence the clot hierarchical structure and subsequent lysis. Taken together, these results demonstrate how the biophysical properties of platelets differ across species and may be another reason why animal studies of hemostasis do not always translate to humans. Disclosures No relevant conflicts of interest to declare.

Synthesis and determination of absolute configuration of a non-peptidic αvβ6 integrin antagonist for the treatment of idiopathic pulmonary fibrosis

Integrin inhibitor (cell adhesion assays) pIC50 αvβ6 = 8.4, αvβ3 = 6.0, αvβ5 = 5.9 and αvβ8 = 7.7.

Sevuparin Reduces Adhesion Of Both Sickle Red Cells and Leukocytes To Endothelial Cells In Vitro and Inhibits Vaso-Occlusion In Vivo

Introduction Sevuparin is a new chemically modified heparin with low anticoagulant activity currently being studied as an adjuvant therapy for severe malaria. Heparins generally have multiple biological properties, including antithrombin III-dependent inhibition of thrombin as well as blockade of P-selectin-mediated adhesion. Selectins have been shown to contribute to both sickle red cell (SS RBC) and neutrophil (PMN) adhesion in vitro and in mice with sickle cell disease (SCD). Sickle mice lacking both P- and E-selectins are relatively resistant to tumor necrosis factor-α (TNFα)-induced vaso-occlusion. We therefore theorized that sevuparin would show activity in inhibiting the selectin-dependent adhesion of red cells and leukocytes seen in the context of SCD, with the potential to decrease vaso-occlusion at a low level of anti-coagulation. Methods We studied both RBC and PMN adhesion to human umbilical vein endothelial cells (ECs) in an in vitro flow chamber. For studies of SS RBC adhesion, we exposed confluent cultures of ECs on gelatin-coated glass slides to interleukin (IL)-13 (50 ng/ml) for 48 h, followed by histamine (100 μM) for 10 min at 37°C immediately prior to performance of adhesion assays. Upregulation of P-selectin expression was confirmed by FACS of mechanically dislodged ECs. RBC adhesion was quantified during continuous flow. For assays of neutrophil adhesion, ECs were treated with human TNFα (10 μg/ml) overnight, followed by incubation for 10 min at 37°C with 100 µM histamine. All slides bearing ECs were washed after incubation with histamine and immediately mounted in the flow chamber for adhesion assays. Inhibition by sevuparin was tested by pre-incubating ECs with different concentrations of sevuparin for 20 min at 37°C prior to adhesion assays. PMNs from SCD patients were tested for adhesion to treated ECs with and without preceding exposure of ECs to sevuparin. In addition, PMNs from normal donors were first exposed to SS RBCs, as previously described, and then tested for adhesion to ECs with and without pre-exposure to sevuparin. PMN adhesion was quantitated after 10 min of no flow, followed by wash out at various shear stresses. The ability of sevuparin to prevent vaso-occlusion in vivo was studied by infusing red fluorescent SS RBCs into nude mice to effect in vivo adhesion and vaso-occlusion visible in implanted window chambers (Zennadi et al. 2007); 500 ng of murine TNFα in 100 μl was injected intraperitoneally (IP) 4 h before infusion of SS RBCs to increase endothelial expression of selectins. Sevuparin or saline control solution was injected SQ at various dose levels prior to infusion of SS RBCs into nude mice. Results SS RBC adhesion to ECs stimulated with IL13 and histamine was greater than adhesion to similarly stimulated ECs pretreated with sevuparin at 100, 200, 400 and 600 µg/ml prior to exposure to flowing SS RBCs (p = 0.047, 0.031, 0.094, 0.065, respectively, using a paired t test, in which each patient sample was only compared to itself), with a robust dose-response (p < 0.001) (Fig 1A). In a similar analysis, 7.5 µg/ml of function blocking monoclonal antibody to CD62P (9E10) also significantly reduced RBC adhesion (p = 0.038). Sevuparin significantly inhibited adhesion of SCD PMNs to ECs treated with TNFα and histamine, and this inhibition exhibited a modest dose-response relationship (Fig 1B). When normal PMNs stimulated by SS RBCs were studied, the differences between adhesion in the absence of sevuparin and in the presence of either 600 or 800 µg/mL sevuparin were also highly significant at both 1 and 2 dyne/cm2 (p = 0.019 and p = 0.011 at 1 dyne/cm2 and p = 0.013 and p = 0.008 at 2 dynes/cm2, respectively) (Fig 1C). In vivo, injection of sevuparin prior to infusion of SS RBCs significantly decreased SS RBC adhesion to vessel walls, as measured by the percent of vessel lengths occupied by adherent cells (Fig 2A). Sevuparin treatment also significantly increased the percent of venules that maintained normal blood flow (Fig 2B). Conclusions Sevuparin is an effective inhibitor of SS RBC adhesion and both SCD and normal PMN adhesion to endothelial cells in vitro. In vivo, sevuparin effectively decreased vaso-occlusion and improved blood flow after TNFα treatment. Therefore, we consider sevuparin a promising anti-adhesion agent with potential to reduce vaso-occlusion in SCD, via reducing RBC adhesion and leukocyte adhesion, possibly through its effect on selectins. Disclosures: Batchvarova: Dilaforette, AB: Research Funding. Shan:Dilaforette, AB: Research Funding. Zennadi:Dilaforette, AB: Research Funding. Lindgren:Dilaforette, AB: Employment. Leitgeb:Dilaforette, AB: Employment. Sulila Tamsen:Dilaforette, AB: Employment. Telen:GlycoMimetics, Inc.: Research Funding; Dilaforette, NA: Research Funding; Pfizer, Inc.: Consultancy.