WBC Adhesion Research Articles

Caspase-1 As a Therapeutic Target in Intravascular Hemolysis-Induced Microvascular Inflammation and Occlusive Processes

Hemolytic anemias, such as sickle cell disease (SCD), are a group of acute and acquired conditions characterized by the accelerated destruction of red blood cells (RBC). Intravascular hemolysis (IVH), or RBC lysis within the bloodstream, incurs the release of damage-associated molecular patterns (DAMPs), trigging cell activation and immune responses. Caspase-1 (Casp-1), a protease enzyme, plays a critical role during inflammatory processes by initiating programed cell death and promoting the cleavage of pro-interleukin (IL)-1β into its active form, IL-1β, through inflammasome assembly. Here, we aimed to elucidate the complex mechanisms involved in IVH-induced inflammation, leukocyte-endothelium interactions, and microvascular dynamics. Acute IVH was induced in C57BL/6 mice (HEM) by the injection of sterile water (i.v., 150 µl), and control mice received i.v. saline (CON) (n=3-8 per group). Intravital microscopy was performed at 15 min post IVH induction on the cremaster muscle of mice to quantify venular leukocyte (WBC) rolling and adhesion, and non-invasive laser Doppler flowmetry (LDF) measured blood perfusion in the murine pelvis microvasculature. Blood samples were collected at 1-hour post-IVH for ELISA and flow cytometry analyses. Immortalized human endothelial cells (HUVEC; n=7 per group) were stimulated with heme or S100A8 for 3 hours. HEM mice demonstrated significantly increased plasma heme (P<0.0001) and cell-free hemoglobin (P<0.001), in association with significantly decreased plasma haptoglobin (P<0.05), compared to CON mice, thereby confirming the occurrence of IVH. Furthermore, HEM mice exhibited significantly increased plasma concentrations of the S100A8 alarmin (P<0.001) and IL1-β (P<0.001), as well as augmentations (P<0.001) in plasma TNF-ɑ and IL-6 levels. Flow cytometry was used to measure active Casp-1 in neutrophils and monocytes of mice using the FAM-FLICA probe. FLICA staining was significantly higher in the neutrophils (P<0.05) and monocytes (P<0.05) of HEM mice, compared to CON mice, implying Casp-1 activation in these cell subsets. WBC-endothelium interactions are a hallmark of vaso-occlusion in SCD. HEM mice exhibited elevated venular WBC rolling and adhesion to the endothelium, compared to CON mice (P<0.001), as previously shown (Almeida et al., 2015; PMID: 26019278). In addition, IVH significantly reduced the velocity of microvascular blood flow in mice (P<0.001), and impaired perfusion (P<0.001). To address the role of IVH-induced Casp-1 activation in microvascular dynamics, mice received i.p. administrations of the Casp-1 inhibitor, YVAD, at 1 hour prior to induction of IVH, or not, and were subjected to intravital microscopy or LDF. Interestingly, the inhibition of Casp-1 significantly abrogated IVH-induced WBC rolling (P<0.01) and adhesion to vessel walls (P<0.05) in HEM mice and significantly improved microvascular blood perfusion and flow velocity (P<0.05), suggesting a role for Casp-1 activation in the WBC-endothelium and occlusive interactions observed (See Figure). We then evaluated how heme and S100A8 may induce endothelial cell activation, as these DAMPs were found elevated in the plasma of HEM mice. As expected, compared to unstimulated HUVEC, heme induced endothelial cell activation in vitro, as shown by higher expressions of ICAM-1 (P<0.05), VCAM-1 (P<0.01) and E-selectin (P<0.01), and increased Casp-1 activity (P<0.01). In turn, S100A8 alone significantly elevated ICAM-1 (P<0.05) and E-selectin (P<0.01) expressions on HUVEC, as well as IL-1β release (P<0.001), however no significant increase in Casp-1 activity was observed. HUVEC, when pre-treated with YVAD, displayed a significant reduction in S100A8-induced E-selectin expression (P<0.05), but neither ICAM-1 nor VCAM-1 expression were significantly decreased. In contrast, no changes in the expressions of these adhesion molecules were observed in heme-stimulated cells after YVAD treatment. Taken together, these findings suggest that targeting caspase-1 activation and its associated pathways may represent a potential therapeutic intervention to mitigate the adverse inflammatory and microvascular effects of IVH, a disease mechanism that occurs in severe hemolytic anemias such as SCD.

P-Selectin Blockage Potentiates the Inhibiting Effects of Inflammasome Cytokine Neutralization on Leukocyte Recruitment in an In Vivo Model of Acute Sickle Cell Vaso-Occlusion

The vaso-occlusive (VO) processes that characterize sickle cell disease (SCD) pathophysiology result from a complex interplay between altered blood rheology, inflammatory mechanisms, pan-cellular activation and adhesive interactions between red blood cells, leukocytes (WBCs), platelets and the endothelium. Inhibition of the binding of the P-selectin (CD62P) adhesion molecule, expressed on activated endothelium and platelets, in the form of crizanlizumab monoclonal antibody (mAb) therapy, has demonstrated documented success for preventing VO crisis (VOC) in SCD patients (N Engl J Med (2017) 376(5):429-39), leading to marketing authorization by numerous agencies. In turn, blockade of interleukin (IL)-1β activity, in the form of canakinumab administration, was well tolerated in SCD patients and associated with inflammatory marker reduction in a multicenter phase 2a study (Blood (2022)139(17):2642-52), with in vivo studies suggesting a role for the inflammasome-processed cytokines, IL-1β and IL-18, in amplifying vaso-occlusion in SCD mice (Blood Abstract: (2021) 138 (S1):856). As targeting multiple mechanisms may enhance VOC prevention in SCD, we evaluated the combined effects of P-selectin blockage, together with IL-1β and/or IL-18 neutralization, on WBC recruitment during tumor necrosis factor-α (TNF)-induced VO processes in SCD mice. Townes mice (5-mo old) received (i.p.) either saline, anti-murine IL-1β mAb (01BSUR IgG2a; 200 µg/mouse), and/or anti-murine IL-18 (SK113AE-4 IgG1; 500 µg/mouse), and/or anti-murine CD62P (RB40.34 IgG1, BD; 30 µg/mouse), or an IgG1 antibody (iProt105125 or A110-1, BD). Previous dosing intravital microscopy (IM) protocols determined the optimal concentrations of antibodies in TNF-stimulated Townes mice (doses tested in µg/mouse; 100, 200 anti-IL-1β [N=3; 4]; 250, 500 anti-IL-18, [N=3; 4]; 10-60 anti-CD62P [N=4-7 each]; data not shown). VO-like processes were induced in mice at 30 min after anti-CD62P treatment or 21h post anti-IL1β / anti-IL-18 administrations by TNF injection (0.5μg, i.p.). At 3h after TNF, the cremaster muscles of the anesthetized mice were surgically exposed, and the rolling, adhesion and extravasation of WBCs in 6-10 venules each were observed using IM. As expected, anti-CD62P administration significantly inhibited the rolling of leukocytes along venule walls of TNF-induced SCD mice, but did not significantly affect WBC adhesion (Fig. 1A). In contrast, the administration of anti-IL-1β mAb significantly inhibited both WBC rolling and adhesion, when compared to saline (Fig 1B); likewise, IL-18 neutralization significantly reduced WBC rolling and adhesion at the venule walls, when compared to IgG1 (Fig. 1C). As previously demonstrated, the combined administration of anti-IL-1β and anti-IL-18 did not further reduce TNF-induced WBC rolling or adhesion in the microvasculature, compared to anti-IL-18 alone (Fig. 1D). Importantly, the co-administration of anti-CD62P, together with either anti-IL1β or anti-IL-18, further inhibited WBC rolling (Fig. 1B and C) and amplified the effect of IL-18 neutralization on WBC adhesion (Fig. 1C). Finally, CD62P inhibition in association with the neutralization of both IL1β and IL-18 further reduced WBC rolling in a non-significant manner and incurred a further significant decrease in the adhesion of WBCs (Fig. 1D). As regards WBC extravasation from cremaster venules, when evaluating the effect of CD62P inhibition together with either anti-IL1β or anti-IL-18, the additive effect observed was not significant (P>0.05; N=4-5); however, CD62P inhibition further reduced the extravasation of WBC in mice subjected to both IL1β and IL-18 neutralization (reduced by 71.1±8.3%, P=0.002; N=4-5). In conclusion, P-selectin blockage potentiates the inhibiting effects of IL-1β and IL-18 neutralization on TNF-induced WBC adhesion to the microvasculature of a murine model of SCD. This potentiating effect may be mediated by further inhibition of WBC rolling and tethering to the endothelium, but concurrent inhibition of platelet-mediated cellular interactions by P-selectin inhibition should not be ruled out. Given that WBC recruitment to blood vessel walls is associated with the initiation of VO processes, we suggest that the combined use of biological therapies may boost these approaches’ individual beneficial effects on SCD VOC. Financial support: Novartis Pharma AG. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Relative Shedding of Glycosaminoglycans from the Endothelial Glycocalyx During Inflammation

The endothelial (EC) surface layer (glycocalyx) has been shown to act as a barrier to transvascular exchange of solutes, and adhesion of leukocytes (WBCs) during the inflammatory process. It is a labile structure whose components are readily shed by the action of proteases and endoglycosidases. Evidence to date suggests that during the inflammatory process, activation of matrix metalloproteases on the EC surface may cleave the principal proteoglycans (syndecans and glypicans), and EC derived lyases may result in shedding of the associated glycosaminoglycans (GAGs) heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA). Shedding of these components of the glycocalyx serves to increase leukocyte (WBC) access to adhesion receptors on the EC membrane. The objective of the present study was to elucidate the relative role of HS (the dominant GAG) and HA as the major barrier to WBC‐EC adhesion.Shedding of glycans from the EC in post‐capillary venules of the mesentery (rat) in response to topical application of the chemoattractant f‐met‐leu‐phe (fMLP, 10−6 M) was observed by intravital microscopy. The presence and loss of HS, CS and HA was assessed by labeling all components with fluorescently labelled lectin (BS‐1) or HS antibodies, and HA with fluorescently labelled hyaluronan binding protein (HBP). During exposure to fMLP, GAG concentration exhibited an exponential decay. Following a 30 min period about 50% of HBP was lost in contrast to a previously shown loss of 20% of lectin labelled GAGs, and 25% loss of Mab labelled HS. The exponential time‐constant for HBP shedding was 5.8 min, which was one‐third the 14.3 min for BS‐1 labelled GAGs.The relatively rapid shedding of HA compared to HS was consistent with the hypothesis that HA may form the dominant barrier to WBC‐EC adhesion. In situ observations (in rat mesentery) of fMLP induced transient adhesion of circulating beads coated with antibody to ICAM‐1 (1A29) revealed a time constant for adhesion of 10.9 min, which was less than that for BS‐1 labelled GAGs. Prior observations by Fan et al., (Biorheology, 2019, DOI: 10.3233/BIR‐180204) using stochastic optical reconstruction microscopy suggest that HA lies closer to and parallel to the endothelial membrane, compared to HS which lies further away and normal to the EC surface. Thus, a compact layer of HA near the EC membrane may surround WBC adhesion receptors that are much shorter in length than the total thickness of the glycocalyx. Its more rapid shedding, compared to other GAGs, may be the major factor affecting exposure of WBC adhesion receptors on the EC.Support or Funding InformationSupported in part by NIH R01 HL39286

Relative shedding of glycosaminoglycans from the endothelial glycocalyx during inflammation and their contribution to stiffness of the glycocalyx.

The endothelial (EC) surface layer (glycocalyx) has been shown to act as a barrier to transvascular exchange of solutes, and adhesion of leukocytes (WBCs) during the inflammatory process. It is a labile structure whose components are readily shed by the action of proteases and endoglycosidases. Details of shedding of specific constituents of the glycocalyx remain to be determined. To review the contributions of the primary glycosaminoglycans that comprise the glycocalyx, heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA), as barrier to WBC-EC adhesion, and elucidate the rates of shedding of each component in response to an inflammatory stimulus. Assess the potential role that stiffness of the glycocalyx plays in resisting infiltration by WBCs during the adhesion process. Quantitate shedding of the glycocalyx in post-capillary venules of rat mesentery in response to superfusion of the tissue with 10-6M fMLP. The presence and loss of HS, CS and HA was assessed by labeling all components with fluorescently labelled lectin (BS-1) or HS antibodies, and HA with fluorescently labelled hyaluronan binding protein (HBP). Following a 30min exposure of the mesentery to fMLP about 50% of HBP was lost in contrast to a previously shown loss of 20% of lectin labelled GAGs, and 25% loss of Mab labelled HS. The time constant for HBP shedding (5.8min) was one-third that for BS-1 labelled GAGs (14.3min). An attempt was made to assess stiffness of the glycocalyx by observing the motion of adhered lectin coated fluorescently labelled microspheres (FLM) under oscillatory flow conditions. Estimates of the elastic modulus of the glycocalyx revealed a value of 26mPa, which was orders of magnitude below published data obtained by atomic force microscopy. The relatively rapid shedding of HA compared to HS was consistent with the hypothesis that HA may form the dominant barrier to WBC-EC adhesion. Prior observations that HA lies closer to and parallel to the endothelial membrane, compared to HS suggests that the compact layer of HA near the EC membrane surrounds WBC adhesion receptors that are much shorter in length than the total thickness of the glycocalyx. The relatively low elastic modulus of the glycocalyx under shear is consistent with the hypothesis that the FLMs adhered to strands of HS normal to the EC surface that extended above the relatively more compact and stiffer HA layer below. Gradients of stiffness within the glycocalyx may not be detected by compressive indentation tests published to date.

The Ribonucleotide Reductase Inhibitor, Didox, Reduces the In Vivo Vascular Inflammation and Oxidative Stress Induced By Acute Hemolysis

Introduction: Several diseases and disorders, including hereditary and acquired hemolytic anemia, blood transfusion reactions, preeclampsia and some infections, incur intravascular hemolysis to varying degrees. The destruction of red blood cells and the release of hemoglobin (Hb) and heme into the circulation results in vascular inflammation, characterized by the recruitment of leukocytes to the vascular endothelium, reduced nitric oxide bioavailability and oxidative stress, all of which may contribute to complications seen in hemolytic diseases, such as pulmonary hypertension, cutaneous leg ulcers and priapism. Didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor, has been shown to have anti-inflammatory and anti-oxidative effects. This molecule has potential as a cancer therapy and has demonstrated beneficial effects in numerous pathologies, diminishing vaso-occlusive processes in mice with sickle cell disease, and suppressing mast cell activation and degranulation, amongst other effects. Aims: The aim of this study was to evaluate the effects of didox on the vascular inflammatory effects of in vivo acute hemolysis. Methods: C57BL/6 mice received i.v. didox (10 mg/animal) or the same volume of saline vehicle immediately before receiving the hemolytic stimulus. In some mice, didox (10 mg) was given intraperitonally (i.p.) 30 min before hemolysis. Acute hemolysis (HEM) was induced by injecting mice (i.v.) with 100 µl sterile water; for non-hemolysis controls (CON), the same quantity of saline was administered i.v.. At 15 min post-hemolysis, blood was obtained by cardiac puncture for biochemical and flow cytometry analyses; other animals were submitted to cremaster muscle exposure followed by intravital microscopy. Results: Intravascular H2O administration successfully induced hemolysis within 15 min, as demonstrated by elevated plasma cell-free Hb and heme levels (plasma Hb: 0.20±0.05 and 0.66±0.16 mg/ml, P<0.01; total plasma heme: 26.3±4.9 and 87.1±18.4 µM, for CON and HEM, respectively, N=≥6, P<0.001). Interestingly, pre-administration of mice with didox slightly, but significantly, decreased plasma Hb and Heme (plasma Hb: 0.43±0.07 and 0.54±0.07 mg/ml, P<0.05; total plasma heme: 62.7.3±9.5 and 71.5±6.7 µM for i.v. and i.p. didox, respectively, N=6, P<0.05). Concomitantly, acute hemolysis significantly augmented leukocyte (WBC) recruitment and extravasation in the cremaster microcirculation (WBC adhesion: 3.07±0.28 and 13.41±1.02 WBC µm-1, P<0.001; WBC extravasation: 0.72±0.16 and 2.93±0.45 WBC (100x50 µm-2) for CON and HEM, respectively, N=≥5, P<0.001). Didox significantly abrogated this effect of hemolysis, decreasing WBC adhesion to 6.12±0.62 and 2.10±0.30 WBC µm-1 and WBC extravasation to 1.32±0.24 and 1.14±0.21 WBC (100x50 µm-2) (for i.v. and i.p. didox, respectively, N≥5, P<0.001). Flow cytometry demonstrated that hemolysis elevated both the generation of reactive oxygen species (ROS) in the granulocytes of mice and their expression of the Mac-1 integrin subunit, CD11b (ROS: 590±58 and 1140±187 MFI, P<0.01; CD11b: 3028±387 and 4534±416 MFI, for CON and HEM, respect. N=3-6, P<0.01). Importantly, the inhibition of vascular inflammation by didox was associated with a significant decrease in both ROS generation and CD11b expression by granulocytes (ROS: 591±53 and 699±120 MFI, P<0.01; CD11b 2399±126 and 2379±207 MFI, for i.v. and i.p. didox, respectively. N=3-6, P<0.01). Conclusion: Didox, when administrated both intravascularly and intraperitoneally in mice, can inhibit the significant elevations in leukocyte integrin expression and cellular recruitment in the microcirculation that are induced by acute hemolysis. This improvement in vascular inflammation is accompanied by the prevention of oxidative stress in the leukocytes. While didox was found to slightly reduce hemolysis in response to osmotic shock, an interesting observation in itself, it is probable that the beneficial effects of this molecule on vascular inflammation are mediated largely by its effect on oxidative stress parameters. As such, this molecule may provide a novel therapeutic approach to reduce the oxidative stress and vascular inflammation caused by hemolytic events. This includes potential clinical use to treat sickle cell disease and other disorders in which hemolysis is a contributing factor to the pathophysiology. DisclosuresElford:Molecules for Health, Inc.: Equity Ownership, Patents & Royalties: Didox.

Recombinant human vimentin binds preferentially to P‐selectin through the rod domain to block leukocyte adhesion to platelets

BackgroundPlatelet‐leukocyte interactions play an important role in inflammation. Leukocyte adhesion to vascular endothelium is a step‐wise process that is initiated by leukocyte (WBC) P‐selectin glycoprotein ligand‐1 (PSGL‐1) to P‐selectin on adhered platelets and endothelium. These interactions then start a cascade of WBC firm adhesion and transmigration, which could lead to organ injury. Finding novel therapeutics to block specific aspects of inflammation may be useful in controlling unwanted inflammation. Recent reports suggest that vimentin may bind to PSGL‐1, although the function of this had not been previously described. We hypothesize that rhVim may block P‐selectin‐PSGL‐1 interactions and that this may be through the rod domain.MethodsAll research was approved by the Institutional Review Boards of the Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine. Recombinant human vimentin (rhVim) and its rod domain (Rod) were expressed in E. coli and His‐purified using affinity chromatography. Blood was collected from healthy adult donors and mepacrine‐labelled whole blood or isolated neutrophils (PMN) were perfused in microfluidic channels at 2 dyn/cm2 over fibrinogen or isolated platelets in the presence of rhVim, Rod, or vehicle control. Blocking antibodies to P‐selectin (AK4) and PSGL‐1 (KPL‐1) were used in conjunction with rhVim to assess for additional decreases in PMN adhesion to platelets. Protein binding was assessed using ELISA and the equilibrium dissociation constant, KD, was measured using surface plasmon resonance (BIACore).ResultsrhVim significantly decreased WBC adhesion to fibrinogen coated surfaces by ~50% (p&lt;0.05) and PMN adhesion to platelets in a dose dependent fashion (up to 40 ug/mL; p&lt;0.05). In the presence of AK4 (10 ug/mL) and KPL‐1 (2.5 ug/mL) blocking antibodies, the addition of a submaximal dose of rhVim (5 ug/mL) did not further decrease PMN adhesion to platelets. Similar to rhVim, the Rod decreased WBC adhesion to fibrinogen‐coated channels by ~50% as compared to vehicle control (42 ± 13 vs 79 ± 20 WBC per field; p&lt;0.05). There was no difference in WBC adhesion when comparing equimolar concentrations of rhVim and Rod. Likewise, the binding of rhVim and Rod to P‐sel/Fc were also similar (67 ± 1.2 and 125 ± 1.2 nM, respectively) and there was no binding of either to IgG or PSGL‐1.DiscussionOur data suggests that rhVim blocks adhesion of WBC and PMN to platelets via binding to P‐selectin, and not PSGL‐1. This effect may be through binding within the rod domain. Further studies are warranted to determine the binding epitope of rhVim to P‐selectin as a potential therapy for unwanted inflammation.Support or Funding InformationThis work is supported by NIH/NHLBI HL‐116524, NIH/NIGMS GM‐112806 and GM‐123261, NIH/NINDS NS‐094280, and a Merit Review Award Number I01 BX002551 from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of inflammation induced shedding of the endothelial glycocalyx with low molecular weight heparin

The endothelial surface layer (ESL) consists of the endothelial cell (EC) glycocalyx and adsorbed proteins, and forms a barrier between blood and the EC. Enzymatic shedding of the ESL in response to cytokines may expose receptors for leukocyte (WBC) adhesion and increase vascular permeability. Thus, intravital microscopy was used to explore stabilization of the ESL with low molecular weight heparin (LMWH) to mitigate structural changes with inflammation. Following bolus infusions (i.v.) of LMWH (0.12–1.6mg/kg), shedding of glycans in response to 10−7M fMLP was measured by loss of fluorescently labeled lectins bound to the EC and WBC-EC adhesion was monitored in post-capillary venules of rat mesentery. During a 30min exposure to fMLP, a 50% reduction in fluorescence (indicative of glycan shedding) occurred at the lowest dose of LMWH whereas a 50% increase occurred (indicative of ESL compaction) at the highest dose. Shedding was reduced by LMWH in a dose dependent manner with an EC50 of 0.6mg/kg. Concomitant WBC-EC adhesion increased over 3-fold for all doses of LMWH. However, at a dose of 1.6mg/kg, WBC-EC adhesion did not rise significantly during the initial 10min exposure to fMLP. Correlation of WBC adhesion with intensity of the lectin stain for all measurements revealed a significant 40% reduction in adhesion as intensity increased 50%. This relationship was attributed to LMWH inhibition of heparanase and/or binding to components of the glycocalyx that resulted in mitigation of glycan shedding, compaction of the lectin stain and stabilization of the glycocalyx.

Endothelin a Receptor (ETRA) Blockade Inhibits Leukocyte Adhesion to the Vascular Endothelium in Sickle Cell Disease

▪Objective:Recurrent acute episodes of vaso-occlusion (VOC) are one of the hallmarks of sickle cell disease (SCD). Leukocytes are in an activated state in SCD and their recruitment to the vascular endothelium marks a critical event in the pathophysiology of the vaso-occlusive process. Endothelin-1 (ET-1), a potent vasoconstrictive and pro-inflammatory endogenous peptide, is elevated in steady state SCD, increases during acute VOC crisis, and remains elevated post crisis. This study asked whether the ET-1-ETRA signaling pathway is involved in promoting leukocyte adhesion in a hypoxia-reoxygenation challenged mouse model of SCD. Secondly, we wondered whether ET-1 is able to directly activate and increase the proadhesive phenotype of the peripheral leukocyte population.Methods: Animals:We used the UAB knockout-transgenic mouse model of SCD for our studies (Ryan, 1997). Experimental groups consisted of homozygous (SCD-/-), heterozygous (SCD+/-), and C57BL/6 control mice. Intravital Microscopy:Bosentan (BOS; dual ETRA/B antagonist) or Ambrisentan (AMB; specific ETRA antagonist) was administered by medicated chow for 8 weeks. Prior to imaging, animals were exposed to 5 hrs of 8% O2 followed by 2 hrs of reoxygenation in room air. Leukocytes were stained in vivo with a PE-conjugated anti-CD45 antibody. Mice were anesthetized and the calvarial bone marrow microvascular network was observed using an intravital microscope with water-immersion objectives. Images were analyzed off-line for leukocyte velocity, adhesion, and flow dynamics (Image Pro-Plus 5.0). Superoxide/Cytokine/Integrin measurements: Leukocytes were isolated from freshly harvested peripheral blood (SCD-/- and controls) and exposed to ET-1 or vehicle. ROS production was measured by luminescence (Lumistar Galaxy Luminometer). Intracellular cytokine and surface integrin expression was analyzed by flow cytometry. Neutrophil Adhesion Assay: Peripheral neutrophils were purified by a Ficoll gradient from SCD-/- mice and controls. Following ex vivo incubation with ET-1 or vehicle, the purified neutrophils were added to pre-activated (thrombin) rat PMVECs. After washing with PBS, the cells were fixed and stained with MPO to identify adherent neutrophils.Results: Intravital microscopy revealed that ETR blockade with BOS, but not AMB, enhanced WBC rolling and adhesion at steady state without altering flow velocity, suggesting an important role for ETRB signaling at baseline in SCD-/- animals. Following a hypoxia/reoxygenation protocol, both antagonists were equally effective in decreasing WBC rolling and adhesion in SCD-/- animals. Both ETR antagonists were able to correct H/R-induced decreases in blood flow velocity to near baseline values. Next we asked if ET-1 is capable of activating WBCs by first measuring levels of receptor mRNA in sorted peripheral neutrophils and monocytes. Next we measured intracellular cytokine, surface integrins and ROS production following ex vivo exposure to ET-1. Results indicated a very low amount of ETRA mRNA in neutrophils of SCD-/- mice and controls. ETRB mRNA was predominantly expressed in monocytes of SCD-/- mice. ET-1 ex vivo stimulation failed to increase cytokine, integrin and ROS levels beyond baseline in sickle neutrophils and monocytes. Functionally, more neutrophils from SCD-/- mice adhered to pre-activated endothelial cells than controls; however, ex vivo ET-1 stimulation failed to increase adhesion.Summary and Conclusions: Our results demonstrate that selective ETRA blockade is sufficient to abrogate H/R-induced leukocyte-endothelial interactions in a preclinical model of SCD. While the dual ETRA/B antagonist produced similar results after H/R, its proadhesive effect at steady state suggests an important role for the endothelin B receptor under baseline conditions. Although the long-term significance of these findings remains to be elucidated, at this early stage our results suggest a potential therapeutic advantage for a selective ETRA treatment strategy in SCD. The lack of response of the leukocyte population to direct ET-1 stimulation suggests that ET-1 targets primarily the vascular endothelium to promote leukocyte adhesion. In conclusion, this study established a potentially important role for clinically available ETRA antagonists in the treatment portfolio for patients with SCD. DisclosuresGutsaeva:Grifols: Research Funding.

Soluble vimentin decreases neutrophil adhesion to platelets by inhibiting P‐selectin glycoprotein ligand‐1‐P‐selectin interactions

BackgroundVimentin is an intermediate filament protein mainly thought to be involved in maintaining the cytoskeleton. Recently, there has been increasing evidence of vimentin's role during inflammation, although its precise role is unclear. In a model of murine peritonitis, vimentin‐deficient mice have an increased ability to clear bacteria, suggesting that vimentin somehow inhibits inflammation. This effect may be through inhibiting the interaction between platelets and leukocytes (WBC), since platelets are known to play an important role in the capture and activation of WBC at sites of injury and infection. We and others have described the important role of P‐selectin glycoprotein ligand‐1 (PSGL‐1)‐P‐selectin (P‐sel) interactions on WBC capture by platelets and neutrophil (PMN) transmigration. We hypothesize that soluble vimentin (sVim) decreases PMN‐platelet interactions via interrupting PSGL‐1‐P‐selectin interactions.MethodsAll research was approved by the Institutional Review Board and Institutional Animal Care and Use Committee at Baylor College of Medicine. Recombinant sVim was expressed in E. coli and His‐purified using affinity chromatography. Blood was collected from healthy adult donors and mepacrine‐labelled whole blood or isolated PMN were perfused over fibrinogen, washed platelet monolayers, or P‐selectin/Fc chimeric protein at 2 dyn/cm2 in the presence of varying concentrations of sVim using a microfluidic parallel‐plate flow system (Bioflux). Finally, to determine the role of vimentin on WBC rolling in vivo, mean WBC rolling velocities in both wild type (C57Bl/6) and vimentin−/−mice were measured in cremaster muscle venules (46 ± 0.5 μm) using intravital video microscopy. In vitro data were analyzed as paired t‐test or repeated‐measured analysis of variance, where appropriate, whereas in vivo data were compared using t‐test.ResultsPerfusion of whole blood over fibrinogen resulted in platelet adherence to the fibrinogen coating. WBC adhesion to platelets was reduced in the presence of sVim (50 μg/mL; 29±15% of vehicle control; p&lt;0.05, mean±SEM, n=3). To determine whether this effect was directly related to PMN‐platelet interactions, isolated PMN in the presence of varying concentrations of sVim was perfused over washed platelet monolayers. sVim decreased the number of rolling and adhered PMN on washed platelets in a dose‐dependent fashion (88±22, 35±6, 11±2, 4±2, &amp; 0±0% of vehicle control values for 2.5, 5, 10, 20, &amp; 40 μg/mL, respectively; p&lt;0.05, n=5). To determine whether these observations were due to PSGL‐1‐P‐sel interactions, isolated PMN in the presence varying concentrations of sVim were perfused through P‐sel/Fc chimeric protein (10 μg/mL)‐coated channels. Similar to their effects on PMN‐platelet interactions, sVim significantly decreased PMN adhesion to P‐sel/Fc in a dose‐dependent manner (53±15, 21±3, 16±2, 10±2, 2±1% of vehicle control values for 2.5, 5, 10, 20, &amp; 40 μg/mL, respectively; p&lt;0.05, n=4). In vivo, mean WBC rolling velocities were lower in venules of mice lacking vimentin as compared to wild type mice (42.5±8.8 vs 122.5±23.3 μm/sec; p&lt;0.05, mean±SEM, n=3–4).ConclusionSoluble vimentin decreases neutrophil adhesion to platelets by inhibiting PSGL‐1‐P‐selectin interactions. Further studies need to take place to determine how vimentin disrupts WBC capture and rolling and whether vimentin can be used to modulate inflammation caused by platelet‐leukocyte interactions.Support or Funding InformationNIH grants HL116524, GM0112806, &amp; EY018239 and a Merit Review Grant from the Department of Veterans Affairs

SPH simulations of WBC adhesion to the endothelium: the role of haemodynamics and endothelial binding kinetics.

A multiscale Lagrangian particle solver introduced in our previous work is extended to model physiologically realistic near-wall cell dynamics. Three-dimensional simulation of particle trajectories is combined with realistic receptor-ligand adhesion behaviour to cover full cell interactions in the vicinity of the endothelium. The selected stochastic adhesion model, which is based on a Monte Carlo acceptance-rejection method, fits in our Lagrangian framework and does not compromise performance. Additionally, appropriate inflow/outflow boundary conditions are implemented for our SPH solver to enable realistic pulsatile flow simulation. The model is tested against in-vitro data from a 3D geometry with a stenosis and sudden expansion. In both steady and pulsatile flow conditions, results show close agreement with the experimental ones. Furthermore we demonstrate, in agreement with experimental observations, that haemodynamics alone does not account for adhesion of white blood cells, in this case U937 monocytic human cells. Our findings suggest that the current framework is fully capable of modelling cell dynamics in large arteries in a realistic and efficient manner.

Acute Inflammatory Processes Are Induced By Hemolysis and Reversed By Hydroxyurea

BackgroundPlasma free hemoglobin (PFHb) can be elevated by hemolysis in several diseases, including malaria, sepsis and hemolytic anemias such as sickle cell disease (SCD). When PFHb is not neutralized, it can trigger vascular and organ dysfunction, leading to adverse clinical effects. As an example, increased PFHb in SCD is proposed to reduce nitric oxide (NO) bioavailability and induce vascular oxidative stress and inflammation. Vaso-occlusive (VO) processes in SCD may be triggered by the adhesion of leukocytes (WBC), and subsequently red blood cells, to the vessel wall. Hydroxyurea (HU), used as a fetal Hb-inducing drug in SCD, may also exert some of its effects by acting as an NO donor. We induced hemolytic processes in mice to observe WBC recruitment and inflammatory processes; the ability of HU to affect such processes was also investigated. MethodsHemolysis was induced in wild-type C57BL/6 mice (2-4 months old) by injecting water (H2O). For intravital microscopy (IVM), cremaster muscles were prepared before or after administration of H2O and/or other agents, and WBC adhesion and extravasation were quantified (7-8 microvenules/mouse). Inflammation status after TNF-α (i.p., 210 min) or H2O (15 min) administration was evaluated by IVIS (Caliper) using a chemiluminescent probe to detect WBC myeloperoxidase. PFHb was measured with Drabkin's solution. ResultsMice that received H2O presented marked vascular hemolysis within 15 min; levels of PFHb were doubled in H2O mice, compared to mice receiving PBS (2.57±0.43g/dL, 1.08±0.08g/dL Hb, respect., p<0.01, N=3-5) and were similar to PFHb levels observed in a SCD mouse model (data not shown). In contrast, mice that received a TNF-α-inflammatory stimulus showed no alteration in PFHb (1.29±0.43 g/dL, 1.23±0.09 g/dL; PBS and TNF-α, respect., N=3, P>0.05). IVIS demonstrated induction of significant systemic inflammation by H2O, compared to PBS control, particularly in the abdominal area (27.8x105±9.7x105p/s, 6.6x105±1.0x105p/s, respect., P<0.05, N=3), almost reaching levels observed for TNF-α-treated mice (41.0x105±1.7x105p/s, p<0.01 comp. to PBS, N=3). IVM showed that these same TNF-α and H2O stimuli induced WBC adhesion to vessel walls and WBC extravasation (See Table). When an NO donor (DEANO) was given together with H2O, this drug was able to prevent WBC recruitment, reducing both WBC adhesion and extravasation (See Table), similar to effects that were observed when DEANO was given post TNF-α to inflammatory mice (data not shown). Surprisingly, when HU was given following H2O-induced hemolysis, both increased WBC adhesion and extravasation were reversed in a similar manner to that observed for DEANO. Importantly, neither the administration of DEANO nor HU altered H2O-induced elevations in PFHb (p>0.05, data not shown). Finally, using ODQ, a soluble guanylyl cyclase inhibitor, together with HU, the beneficial effects of HU on H2O-induced WBC recruitment were abolished (See Table). ConclusionThis H2O-mouse model displays substantial hemolysis, culminating in rapid-systemic inflammation and significant alterations in WBC recruitment to the vessel wall, comparable to those of a major inflammatory stimulus. Acute HU administration abolished H2O-induced WBC recruitment in a manner similar to that of an established NO donor. The anti-inflammatory effects of HU in this model were not mediated by the inhibition of hemolysis, rather they were reversed by inhibition of cGMP production. Data indicate that hemolytic events alone may be sufficient to induce major inflammatory events. As WBC recruitment and inflammation are thought to trigger SCD VO processes, results indicate that hemolysis could contribute to this phenomenon. Furthermore, HU may have important NO/cGMP-mediated anti-inflammatory effects, possibly indicating potential for use in other diseases that exhibit hemolysis or inflammation. Disclosures:No relevant conflicts of interest to declare.

Shear‐Dependent Adhesion of Leukocytes and Lectins to the Endothelium and Concurrent Changes in Thickness of the Glycocalyx of Post‐Capillary Venules in the Low‐Flow State

To elucidate shear-dependent effects of deformation of the endothelial glycocalyx on adhesion of circulating ligands in post-capillary venules, and delineate effect of MMPs. Adhesion of WBCs and lectin-coated FLMs (0.1μm diameter) to EC of post-capillary venules in mesentery was examined during acute reductions in shear rates (γ·, hemorrhagic hypotension). Adhesion was examined with or without superfusion with 0.5μm doxycycline to inhibit MMPs. Thickness of the glycocalyx was measured by exclusion of fluorescent 70kDa dextran from the EC surface. During superfusion with Ringers, rapid reductions in γ· resulted in a significant rise in WBC adhesion and a twofold rise in microsphere adhesion. With addition of doxycycline WBC and FLM adhesion increased twofold under high- and low-flow conditions. FLM adhesion was invariant with γ· throughout the network in the normal (high)-flow state. With reductions in γ·, thickness of the glycocalyx increased significantly, with or without doxycycline. The concurrent increase in WBC and FLM adhesion with increased thickness of the glycocalyx during reductions in shear suggests that glycocalyx core proteins recoil from their deformed steady-state configuration, which increases exposure of binding sites for circulating ligands.

The Endothelial Glycocalyx as a Barrier to Leukocyte Adhesion and Its Mediation by Extracellular Proteases

The endothelial cell (EC) surface is coated with a layer of polysaccharides linked to membrane-bound and trans-membrane proteoglycans that comprise the glycocalyx, which is augmented by adsorbed proteins derived from the blood stream. This surface layer has been shown to affect hemodynamics in small blood vessels of the microcirculation, the resistance to flow, and leukocyte (WBC) to EC adhesion. Parallel studies of WBC-EC adhesion in response to chemoattractants and cytokines, and shedding of constituents of the glycocalyx, have suggested a role for activation of extracellular proteases in mediating the dynamics of WBC adhesion in response to inflammatory and ischemic stimuli. Likely candidates among the many proteases present are the matrix metalloproteases (MMPs). Inhibition of MMP activation with sub-antimicrobial doses of doxycycline, or zinc chelators, has also inhibited WBC adhesion and shedding of glycans from the EC surface in response to the chemoattractant fMLP. Taken together, these studies suggest that shedding of the EC glycocalyx exposes adhesion receptors and thus enhances WBC-EC adhesion. Future therapeutic strategies for treating pathologies such as the low flow state and inflammation may benefit by further exploration of the mechanics of the glycocalyx in light of protease activation and shear-dependent effects.

Inhibition of Glycan Shedding from the Endothelial Glycocalyx and Adhesion of Leukocytes by Doxycycline in Post‐capillary Venules

Previous studies have demonstrated that shedding of glycans from the endothelial cell (EC) glycocalyx and leukocyte (WBC)‐EC adhesion in response to the chemoattractant fMLP could be attenuated by doxycycline (Doxy) through the inhibition of matrixmetalloproteases (MMPs). Given that Doxy is a strong calcium chelator, the present studies were undertaken to determine if the calcium chelator EDTA could produce a similar effect. To this end, WBC‐EC adhesion and adhesion of lectin (BS‐1) coated microspheres (0.1 um diameter) in post‐capillary venules of mesentery (rat) were studied during superfusion of the tissue with Ringers solution plus EDTA in the presence or absence of fMLP. In the absence of fMLP, with 1 mM EDTA sequestration of microspheres steadily increased with time, and with 3 mM sequestration reached levels 50–100% greater than without EDTA. In contrast, with 0.5 uM Doxy alone, microsphere adhesion remained constant under control conditions. Calcium chelation at levels (1 mM EDTA) that inhibited fMLP induced WBC adhesion to the same extent as Doxy (0.5 uM) caused a disruption of the venular permeability barrier that resulted in increased microsphere sequestration in the venular wall. Thus, the inhibition of microsphere shedding and WBC adhesion in response to fMLP by doxycycline appear to be caused by inhibition of MMPs in contrast to calcium chelation. Supported by R01 HL39286.

Neutrophils, lymphocytes, and monocytes exhibit diverse behaviors in transendothelial and subendothelial migrations under coculture with smooth muscle cells in disturbed flow

Atherosclerosis develops at regions of the arterial tree exposed to disturbed flow. The early stage of atherogenesis involves the adhesion of leukocytes (white blood cells [WBCs]) to and their transmigration across endothelial cells (ECs), which are located in close proximity to smooth muscle cells (SMCs). We investigated the effects of EC/SMC coculture and disturbed flow on the adhesion and transmigration of 3 types of WBCs (neutrophils, peripheral blood lymphocytes [PBLs], and monocytes) using our vertical-step flow (VSF) chamber, in which ECs were cocultured with SMCs in collagen gels. Such coculture significantly increased the adhesion and transmigration of neutrophils, PBLs, and monocytes under VSF, particularly in the reattachment area, where the rolling velocity of WBCs and their transmigration time were decreased, as compared with the other areas. Neutrophils, PBLs, and monocytes showed different subendothelial migration patterns under VSF. Their movements were more random and shorter in distance in the reattachment area. Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines, which contributed to the increased WBC adhesion and transmigration. Our findings provide insights into the mechanisms of WBC interaction with the vessel wall (composed of ECs and SMCs) under the complex flow environments found in regions of prevalence for atherogenesis.

Antithrombin ameliorates endotoxin-induced organ dysfunction more efficiently when combined with danaparoid sodium than with unfractionated heparin

This study investigated the potential benefits of combination therapy using antithrombin (AT) with danaparoid sodium (DA) compared with the use of AT with unfractionated heparin (UFH) in the treatment of sepsis. Rats infused with lipopolysaccharide were treated with either DA alone, AT alone, AT plus DA, AT plus UFH, or human serum albumin as controls. AT (125 U/kg) was injected into the AT group immediately after lipopolysaccharide infusion. The AT/DA and AT/UFH groups received the same dose of AT in conjunction with either DA (400 U/kg) or UFH (400 U/kg). The status of the mesenteric microcirculation was examined by intra-vital microscopy and the laboratory indices of coagulation, inflammation, and organ dysfunction were measured. The coagulation markers were improved following the administration of DA or UFH. The decreases in the WBC counts were significantly suppressed in the AT/DA group. The elevation of IL-6 decreased in the AT, DA, and AT/DA groups (all p<0.01) but not in the AT/UFH group. The prostaglandin I2 levels were significantly elevated only in the AT/DA group (p<0.05). The WBC adhesion was significantly suppressed in the DA, AT/UFH, and AT/DA groups (p<0.05), and the RBC velocity was best maintained in the AT/DA group with no associated increase in capillary hemorrhage. The elevation of ALT and BUN significantly improved only in the AT/DA group. ONCLUSION: Organ dysfunction can thus be alleviated by even moderate doses of AT replacement when co-administered with DA.

Role of glycocalyx in leukocyte-endothelial cell adhesion.

The binding of fluorescently labeled microspheres (FLMs, 0.1-microm diameter) coated with antibody (1a29) to ICAM-1 was studied in postcapillary venules during topical application of the chemoattractant N-formylmethionyl-leucyl-phenylalanine (fMLP). FLM adhesion to endothelial cells (ECs) increased dramatically from 50 to 150 spheres per 100-microm length of venule after superfusion of the mesentery with fMLP and equaled or exceeded levels of leukocyte (WBC) adhesion. Removal of the EC glycocalyx by micropipette infusion of the venule with heparinase increased FLM-EC adhesion to levels attained with fMLP. Subsequent application of fMLP did not increase FLM adhesion further, suggesting that the FLMs saturated all ICAM-1 binding sites. Perfusion with heparinase after suffusion with fMLP significantly increased FLM-EC adhesion above levels attained with fMLP. However, WBC adhesion fell because of possible removal of selectins necessary to maintain WBC rolling at the wall. It is concluded that the glycocalyx serves as a barrier to adhesion and that its shedding during natural activation of ECs may be an essential part of the inflammatory response.

Human promyelocytic cell line: a convenient tool for studying the molecular basis of WBC filtration.

Blood filtration is a technique widely used to reduce the levels of WBCs in blood components. Several studies have been conducted to define the factors that are involved in WBC reduction, but the various mechanisms are not clearly delineated. This study explored the role of WBC adhesion molecules in WBC reduction during filtration. A minifilter has been developed that has properties similar to those of the standard filter (Sepacell, Asahi Medical) but that allows a smaller volume of blood to be used (15 mL). WBC reduction was achieved to a similar extent in the standard filter and the minifilter (4.15 log and 4.18 log, respectively). Samples of human promyelocytic cell line (HL60) were filtered before and after differentiation induced by vitamin D3 (D3-HL60). Flow cytometry was used to characterize the D3-HL60 filtrates and to count the WBCs after filtration. HL60 was retained in the filter to the same extent as all other WBCs. A higher level of integrin receptors (CD11b/CD18; CD11c/CD18) was expressed by D3-HL60 than by HL60. When the blood was incubated with anti-CD11b, anti-CD11c, or anti-CD18, fewer D3-HL60 cells were trapped by the filter, while only anti-CD11b alters HL60 retention in the filter. The receptors CD11b/CD18 and CD11c/CD18 appear to bind to the filter fibers and to be one of the mechanisms responsible for WBC retention.

Leukocyte sequestration in the microvasculature in normal and low flow states.

Techniques of indicator dilution were applied to determine the relative transit times (TTs) of fluorescently labeled leukocytes (TTWBC), red blood cells (TTRBC), and plasma (TTpl) between functionally paired arterioles and venules in hamster cremaster muscle in normal and low flow states. In the normal flow state, paired measurements of TTWBC/TTpl in arteriovenous (A-V) pairs bounding the true capillaries averaged 0.75 +/- 0.08 (SE) s and were not significantly different from an average TTRBC/TTpl of 0.78 +/- 0.06 (SE) s as WBCs appeared to traverse the capillary segment through more centralized preferential pathways. In larger-diameter A-V pairs, significantly larger (10%) values of TTWBC/TTpl were found compared with TTRBC/TTpl due to margination of WBCs in postcapillary venules. To assess the relative effects of WBC-capillary plugging and WBC adhesion in venules on flow resistance, TTWBC was measured in normal and low flow states, with the latter induced by systemic administration of sodium nitroprusside, which resulted in an increase in TTpl from a norm of 1.08 +/- 0.16 to 2.62 +/- 0.44 (SE) s (P < 0.05). With onset of the low flow state, TTWBC/TTpl, the number of plugged capillaries, and the duration of capillary plugs did not change significantly from the norm. In contrast, the rate at which WBC-endothelium (-EC) adhesion increased with successive bolus injections increased approximately eight-fold during hypoperfusion. Estimates of the percentage increase in segmental resistance at the capillary level (due to plugging) and in postcapillary venules (due to EC adhesion) revealed that venous resistance may increase at a threefold greater rate due to WBC sequestration with each successive bolus infusion. Inasmuch as hemodynamic resistance in capillary and venular segments is of the same order of magnitude in the normal flow state, it appears that WBC adhesion in venules may have a far greater deleterious effect on microvascular blood flow in the low flow state.

Molecular Understanding of Cellular Adhesion on Artificial Surfaces

This study was conducted to clarify cellular adhesion mechanisms of blood cells (platelets [PLT] and white blood cells [WBC]) and vascular endothelial cells at the molecular level. This study indicated that the adhesion of three cellular systems to proteins such as fibronectin and fibrinogen proceeds via the RGD (Arg-Gly-Asp) ligand-receptor interaction, in which the RGD tripeptidyl sequence is the minimal amino acid sequence common to adhesive proteins. This was evident from the dose-dependent inhibitory effect of RGD-containing peptide on cellular adhesion. Additional supporting evidence was the presence of PLT and WBC receptors, which molecularly recognize RGD, verified by fluorescein-labelled RGD-containing peptide. The adhesion of vascular endothelial cells was also predominantly controlled by the ligand-receptor mechanism, and participation of complement activation on WBC adhesion was demonstrated as well. The adhesion of WBCs on surface hydroxyl group-bearing polymers proceeded via the CR3 receptor-C3b ligand interaction, in which activated complement factor C3b is chemically fixed upon complement activation. Thus, the molecular understanding of cellular adhesion mechanisms provide the basis of biocompatibility for implantation and extracorporeal circulation, as well as molecular design of artificial and bioartificial organs.