Am J Physiol Cell Physiol 306: C1184–C1190, 2014. First published April 16, 2014; doi:10.1152/ajpcell.00269.2013.
Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium Rebecca Lever,1 Mark J. Rose,2 Edward A. McKenzie,3 and Clive P. Page2 1
Department of Pharmacology, University College London School of Pharmacy, London, United Kingdom; 2Sackler Institute of Pulmonary Pharmacology, King’s College London, London, United Kingdom; and 3Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Submitted 3 September 2013; accepted in final form 15 April 2014
Lever R, Rose MJ, McKenzie EA, Page CP. Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium. Am J Physiol Cell Physiol 306: C1184 –C1190, 2014. First published April 16, 2014; doi:10.1152/ajpcell.00269.2013.—Heparanase (HPSE1) is known to be involved in mechanisms of metastatic tumor cell migration. This enzyme selectively cleaves heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed in mammals and are known to be involved in regulating the activity of an array of inflammatory mediators. In the present study, we have investigated the effects of human recombinant heparanase, the inactive precursor of this enzyme (proheparanase) and enzymatically inactivated heparanase, on inflammatory cell recruitment in the rat and on human leukocyte-endothelial adhesion in vitro. Intraperitoneal injection of heparanase (500 g) induced a significant inflammatory cell infiltrate in the rat, as assessed by peritoneal lavage 4 h later. Intravital microscopy of the mesenteric microcirculation of anesthetized rats showed an increase in rolling and adherent cells in postcapillary venules that was sensitive to heparin, a nonselective inhibitor of heparanase activity. In vitro, heparanase augmented the adhesion of human neutrophils and mononuclear cells to human umbilical vein endothelial cells in a concentration-dependent manner. Proheparanase had similar effects to the active enzyme both with respect to leukocyte accumulation in the peritoneal cavity and adhesion in vitro. However, heat-inactivated heparanase induced cell adhesion in vitro but was without effect in vivo. Together, these data indicate a role for heparanase in inflammatory cell trafficking in vivo that appears to require enzymatic activity. cell trafficking; heparan sulfate; inflammation; mononuclear leukocyte; neutrophil THE HUMAN ENDO- GLUCURONIDASE, heparanase (HPSE1), cloned in 1999 (13, 35), is known to be involved in mechanisms of metastatic tumor cell migration into tissues. The substrates of this enzyme are heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed throughout the body (4, 36) and form a major component of the subendothelial matrix, as well as being strongly expressed as part of the glycocalyx on the surface of endothelial cells and circulating blood elements, affording negative charge density to cell surfaces. Loss of this charge is known to be associated with leukocyte adhesion (29). It has also been suggested that, at least during sepsis, HPSE1 may well contribute to the observed loss in endothelial HSPG (29). Inhibition of heparanase using antisense strategies (7, 34), heparin (26, 28), or relatively selective inhibitors such as phosphomannopentaose (PI-88) (3, 14) is effective in reducing tumor cell adhesion, migration, and subsequent colonization in
Address for reprint requests and other correspondence: R. Lever, Dept. of Pharmacology, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK (e-mail: [email protected]). C1184
tissues, and this enzyme is an established therapeutic target for the development of anticancer drugs (23). However, fewer studies have investigated the role of this enzyme in leukocyte emigration during inflammatory responses, which shares many similarities with tumor cell metastasis (37). Indeed, the wellestablished role of HSPGs in the physiological functioning of a range of inflammatory mediators, including chemokines (20, 21, 27, 38), growth factors (10, 27), and certain adhesion molecules (38), suggests that enzymatic disruption of these proteoglycans should impact upon inflammatory responses. Furthermore, it is known that heparan-degrading enzymes are released by certain leukocytes when they diapedese (19, 22) and by endothelial cells in response to proinflammatory stimuli (5, 8). It is also known that these enzymes are inhibited by heparin (2), accounting for some of the described anti-inflammatory effects of this structural relative of heparan sulfate (16). For example, when heparin is used at low doses in models of inflammatory disease, especially in lymphocyte-driven processes such as allergic encephalomyelitis (19), delayed-type hypersensitivity (33), and graft vs. host reactions (26), it significantly reduces leukocyte infiltration into tissues. Moreover, the development of delayed-type hypersensitivity reactions in mice has been found to correlate with endothelial heparanase production and to be sensitive to inhibition of this enzyme (8). The HPSE1 enzyme is initially synthesized as a 65-kDa inactive precursor, proheparanase (pro-HPSE1) (9), which is then processed into an active form in lysosomes (30) in a pH-dependent manner (6). The active form of the enzyme is comprised of and secreted as a heterodimer, with subunits of 50 and 8 kDa, respectively (9). It has been shown that that both pro-HPSE1 (32) and active HPSE1 (12) can mediate cell adhesion via mechanisms unrelated to enzymatic activity. This appears to be a 1-integrin-dependent process and also utilizes cell surface HSPG (32), presenting a further mechanism by which heparanase may promote inflammation in vivo, in addition to those mechanisms likely to be dependent on cleavage of cell surface and matrix HSPG. In the present study, we examined the effects of pro-HPSE1, HPSE1, and enzymatically inactivated HPSE1 [heat inactivated (HI-HPSE)] on human neutrophil and mononuclear cell adhesion in vitro, as well as the possible proinflammatory effects of these molecules in vivo in a rodent model of peritoneal inflammation. MATERIALS AND METHODS
Expression and purification of heparanase enzymes and preparation of HI-HPSE1. HPSE1 was expressed, purified, and analyzed as described previously (24). HI-HPSE1 was prepared by heating
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The pro-HPSE1 cDNA was PCR amplified from a mammary gland cDNA library (Clontech) using pfu turbo polymerase (Stratagene) and the following cycling conditions [94°C 1’, (94°C 30 s, 60°C 30 s, 72°C 2’) 30 cycles, 72°C 10’, 4°C]. Primers used were forward: 5=-ccc ggg cag gac gtc gtg gac ctg gac ttc ttc acc-3= and reverse: 5=-gaa ttc tca gat gca agc agc aac ttt ggc att tct-3=. PCR product was separated using standard agarose gel electrophoresis techniques, gel purified (Qiaquick-Qiagen), and then cloned into pZero blunt vector (Invitrogen). Correct sequence was verified by sequencing with dideoxy dye terminators (ABI big dye version 1.1). The product was excised from pZero blunt by double digestion with Sma1/EcoR1 enzymes (Promega) and ligated into the baculovirus transfer vector pAcGP67A (Pharmingen) cut with the same enzymes. pAcGP67A:pro-HPSE1 transfer vector was cotransfected, along with baculovirus DNA into sf9 cells exactly as described previously (24). Secreted protein was collected and purified by heparin sepharose chromatography as described previously. Average yields were ⬃6 mg/l of purified protein at ⬎90% purity as visualized by SDS-PAGE Coomassie blue staining. All recombinant proteins were concentrated to 1 mg/ml in a final buffer containing 25 mM Tris·HCl pH 7.5 and 150 mM NaCl and kept at 4°C for immediate use or ⫺80°C for long-term storage. In vivo studies. All experiments on animals were performed under UK Home Office and local ethical approval from King’s College London. Male Sprague-Dawley rats (150 –200 g; Harlan), housed under a 12:12-h light/dark cycle for 7 days with food and water provided ad libitum, were injected intraperitoenally with IL-1 (R&D
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Fig. 1. Heparanase-induced cell recruitment to the peritoneal cavity of the rat. Total cell numbers (open bars) in peritoneal lavage 4 h after intraperitoneal administration of saline, IL-1 (20 ng), or active HPSE1 (500 g). The neutrophil component of lavages is indicated by the filled bars. Data are expressed as means ⫾ SE and were analyzed by ANOVA, followed by Dunnett’s test. *P ⬍ 0.05, compared with saline control; n ⫽ 6 per group.
HPSE1 protein to 80°C for 45 min, followed by cooling to room temperature and ultracentrifugation. The protein content in the resultant solution was confirmed by Bradford assay, and the loss of enzymatic activity was confirmed by lack of ability to cleave FITC-heparan sulphate (24).
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Fig. 2. Heparanase-induced cell rolling and adhesion in the rat mesenteric microcirculation. A: rolling flux in 100-m sections of mesenteric postcapillary venule 4 h after intraperitoneal administration of saline (hatched bar) or IL-1 (20 ng; open bars) with and without cotreatment with unfractionated heparin. B: number of cells stationary in 100-m sections of mesenteric postcapillary venule 4 h after the treatments described in A. C: number of cells rolling in 100-m sections of mesenteric postcapillary venule 4 h after intraperitoneal administration of saline (hatched bar) or active HPSE1 (500 g; dotted bars) with and without cotreatment with unfractionated heparin. D: number of cells stationary in 100-m sections of mesenteric postcapillary venule 4 h after the treatments described in C. Data are expressed as means ⫾ SE and were analyzed by ANOVA, followed by Dunnett’s test. #P ⬍ 0.05, compared with saline control. *P ⬍ 0.05, compared with IL-1 or active HPSE1 control; n ⫽ 6 per group.
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Systems) or the study compounds in a total volume of 500 l. Four hours later, animals were either euthanized and the peritoneal cavity was lavaged with 20 ml saline, or they were anesthetized [thiobutabarbital sodium (Inactin RBC); 100 mg/kg ip; Sigma-Aldrich] for intravital microscopy. Total cells in lavage fluids were counted, and differential cell counts were obtained from cytospin preparations, stained using the DiffQuick system (Gamidor). Anesthetized animals were maintained at 37°C on a heated microscope stage. Single loops of terminal ileum were carefully exteriorized, following midline incision. Sections of mesentery were placed over an optical window within the stage and superfused with bicarbonate buffer. Unbranched, postcapillary venules were viewed under a Zeiss Axioskop 2 FS microscope, fitted with a ⫻40 water-immersion lens and a ⫻10 eyepiece. Digital images were captured using a Sony XC-003P color vision digital camera, and images were viewed and recorded for subsequent offline analysis using Axiovision Software (Image Associates). Animals were euthanized at the end of experiments. Leukocyte rolling flux was quantified as the number of rolling cells passing a fixed point on the venular wall per minute. Adherent leukocytes were considered those cells that were stationary for at least 30 s within a given 100-m vessel wall segment. In vitro adhesion assays. Leukocyte-endothelial adhesion was examined using an assay described previously (15, 31). Peripheral venous blood was drawn from healthy volunteers into citrated tubes. Healthy blood donors were recruited, written informed consent was provided, and samples were obtained under the approval of the Research Ethics Committee of King’s College London. Neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated by standard density-dependent centrifugation on Histopaque-1077 and were radiolabeled with sodium 51 chromate (37 kBq per 106 cells; Amersham). Human umbilical vein endothelial cells (HUVECs; Cambrex), at passage five or below, were cultured to confluency in supplemented MCDB 131 medium (Cambrex) in the central 60 wells of flatbottomed 96-well plates (200 l culture medium per well; 5% CO2; 37°C). Some wells were stimulated for 6 h with human rIL-1 (100 U/ml; Sigma-Aldrich) before use in adhesion assays. Monolayers were washed with warmed HBSS before addition of 200 l radiolabeled leukocyte suspension per well (106 cells/ml) in the absence and presence of test compounds. Plates were incubated for 30 min (37°C), and nonadherent cells were then removed by gentle aspiration and washed with warmed HBSS. Following assessment of monolayer integrity by microscopy, adherent cells were lysed with 1% Igepal (Sigma) and samples were transferred to scintillation vials and ␥-counted, alongside samples of the original radiolabeled leukocyte suspension (input). The number of adherent cells was calculated as the percentage of input counts present in sample counts, corrected for background radioactivity. RESULTS
Heparanase induces inflammatory cell recruitment in vivo. Administration of recombinant HPSE1 to the peritoneal cavity of the rat induced a robust infiltration of leukocytes to this site (Fig. 1), which was similar in magnitude to that elicited by IL-1; this cytokine was used as a positive control in all experiments with heparanase enzymes, due to its well-characterized proinflammatory effects in the models used. We went on to examine the effect of HPSE1 on leukocyte rolling and adhesion in the mesenteric microcirculation of the rat using intravital microscopy. HPSE1 increased the number of rolling and adherent cells in postcapillary venules, again, to a similar extent as IL-1 (Fig. 2, A–D). In these experiments, we sought to examine the effect of inhibiting the enzymatic activity of HPSE1 on inflammation. Heparin is a nonselective inhibitor of heparanase and is known to affect leukocyte adhesion in its own right. However, it is notable that, in our experiments,
heparin inhibited the response to HPSE1 (Fig. 2, C and D), in particular with respect to adhesion, at lower concentrations than those required to modulate the cytokine-induced response (Fig. 2, A and B). This is commensurate with the heparanase inhibitor activity of heparin, which occurs at lower concentrations than those required for other potentially anti-inflammatory activities of this molecule (16). Proheparanase induces cell recruitment in vivo but heatinactivated heparanase does not. An important question lies in whether heparanase needs to be catalytically active to induce inflammation in vivo, given reports that this molecule possesses proadhesive effects in vitro that are independent of HSPG cleavage (12, 32). Therefore, we examined the effects of both the inactive precursor of HPSE1 and heat-inactivated HPSE1 enzyme on cell recruitment to the peritoneal cavity. Human recombinant pro-HPSE1 was expressed and purified (summarized in Fig. 3, A and B) and was found to induce an inflammatory response that again was comparable to that elicited by IL-1. By contrast, heat-inactivated heparanase (HI-HPSE1) had no effect in this model (Fig. 3C).
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Fig. 3. Proheparanase- and heat-inactivated heparanase-induced cell recruitment to the peritoneal cavity of the rat. A: pro-form of HPSE1 (amino acids 36 –543) was cloned into the baculovirus secretory transfer vector pACGP67 to direct secretion into the culture media. B: recombinant pro-HPSE1 virus was used to infect Hi5 insect cells. Culture medium containing protein was purified by heparin sepharose chromatography and eluted fractions analyzed by SDS PAGE. C: total cell numbers (open bars) in peritoneal lavage 4 h after intraperitoneal administration of saline, IL-1 (20 ng), pro-HPSE1 (500 g) or HI-HPSE1 (500 g). The neutrophil component of lavages is indicated by the filled bars. Data are expressed as means ⫾ SE and were analyzed by ANOVA, followed by Dunnett’s test. *P ⬍ 0.05 compared with saline control; n ⫽ 6 per group.
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Heparanase induces and enhances leukocyte-endothelial interactions in vitro in a manner that is not dependent on enzymatic activity. Having found differential effects of proHPSE1 and enzymatically inactive, HI-HPSE1 in vivo, with respect to inflammatory cell accumulation, and in light of previous reports that cell adhesion can be enhanced by proHPSE1 in vitro in a manner independent of enzymatic activity (12, 32), we were interested to apply these proteins to an in vitro model of leukocyte-endothelial adhesion. Heparanase concentration dependently increased the adhesion of both neutrophils (Fig. 4A, closed squares) and PBMCs (Fig. 5A, closed symbols) to cultured HUVEC monolayers, compared with the basal adhesion seen in unstimulated control wells (Figs. 4D and 5D, closed bars). Interestingly, when endothelial cells were prestimulated with IL-1, which enhances subsequent leukocyte adhesion to these cells (Figs. 4D and 5D; open bars show the IL-1-induced adhesion seen in stimulated control wells), the presence of HPSE1 further augmented adhesion, again in a concentration-dependent manner (Fig. 4A and 5A, open squares). Therefore, we investigated the effects of pro-HPSE1 in the same model and found that adhesion was similarly promoted (Figs. 4B and 5B). These data suggest that either endothelial cells or leukocytes, or both, possess the necessary machinery for processing of pro-HPSE1 to its active form or
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The results of this study show that heparanase proteins are capable of modulating inflammatory cell adhesion in vitro and trafficking in vivo. In vitro, HPSE1, pro-HPSE1, and HIHPSE1 all increased the adhesion of both neutrophils and PBMCs to cultured endothelial monolayers. By contrast, in vivo, HPSE1 and pro-HPSE1 induced the infiltration of inflammatory cells to the peritoneal cavity, whereas HI-HPSE1 was without effect. It is most likely that the effects of HPSE1 in vivo depend at least in part on enzymatic activity, given that the heat inactivated form of the enzyme, shown to lack the ability to cleave HSPG, was without effect despite being able
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simply that the protein does not require enzymatic activity for its proadhesive effects in this in vitro system. To address this point further, we assessed the effects of HI-HPSE1 and found that, despite a lack of HSPG-degrading activity, adhesion was still enhanced (Figs. 4C and 5C). Finally, we heat-inactivated pro-HPSE1 in the same manner and found that this material, by contrast, was entirely without effect under any of the experimental conditions tested (data not shown), suggesting that any proadhesive effects that are retained after heat inactivation are restricted, nonetheless, to actively processed heparanase.
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Fig. 4. Heparanase-induced neutrophil adhesion to cultured endothelial cells. The effect of active HPSE1 (A), pro-HPSE1 (B), or HIHPSE1 (C) on adhesion of human neutrophils to unstimulated human umbilical vein endothelial cells (HUVECs; ) or to HUVECs prestimulated with IL-1 (100 U/ml) for 6 h (䊐). D: basal and IL-1-induced control adhesion. Data (A–C) are presented as %relevant control (D) and represent the means ⫾ SE of 6 independent experiments, each carried out in triplicate. Data were analyzed by ANOVA, followed by Dunnett’s test. *P ⬍ 0.05, compared with saline or IL-1 control (A–C) or to saline (D).
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Fig. 5. Heparanase-induced mononuclear cell adhesion to cultured endothelial cells. The effect of HPSE1 (A), pro-HPSE1 (B), or HI-HPSE1 (C) on adhesion of human peripheral blood mononuclear cells (MNC) to unstimulated HUVECs () or to HUVECs prestimulated with IL-1 (100 U/ml) for 6 h (䊐). D: basal and IL-1-induced control adhesion. Data (A–C) are presented as %relevant control (D) and represent the means ⫾ SE of 6 independent experiments, each carried out in triplicate. Data were analyzed by ANOVA, followed by Dunnett’s test. *P ⬍ 0.05, compared with saline or IL-1 control (A–C) or to saline (D).
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to induce adhesion in vitro, and the effects of native HPSE1 in vivo were sensitive to inhibition by heparin. It is logical that cell recruitment in vivo, in the presence of shear force at the vessel wall and a matrix-bound chemotactic gradient, would be sensitive to HSPG degradation, and our results indeed suggest the enzymatic activity of HPSE1 to be involved in the proinflammatory effects seen in vivo. However, and in accordance with previous studies, our data suggest that breakdown of extracellular matrix or cell surface HSPGs is unlikely to be required for the firm adhesion of immune cells to endothelium per se, as HI-HPSE1 was found to induce adhesion to the same extent as the untreated molecule in vitro. As described previously, with respect to lymphocytes (32), the enzymatically inactive proenzyme pro-HPSE1 also induced the adhesion of both PBMCs and neutrophils in our assay. Clearly, it is possible that the moiety required for these proadhesive effects is present on all three forms of the protein and would appear not to be destroyed by heat inactivation, given the effect of HI-HPSE. However, we also found that heat treatment of pro-HPSE1 removed its proadhesive activity. It has previously been shown that pro-HPSE1 and pointmutated, enzymatically inactive HPSE enhanced adhesion in vitro by promoting the clustering of cell surface HSPGs, an
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effect that is shared by a heparin/heparan-binding peptide sequence of HPSE1, Lys158-Asp171 (18, 27), which maps to the NH2 terminus of the 50-kDa subunit of active HPSE1 (17). It is possible, therefore, that destruction of the tertiary structure of pro-HPSE1 by heat treatment serves to make this important sequence, which also corresponds to a potential cleavage site on pro-HPSE1 at Gln157-Lys158 (1, 9), functionally inaccessible. Furthermore, pro-HPSE1 has been shown to induce Akt phosphorylation in endothelial cells in a manner that is independent of both enzymatic activity and the presence of HSPG and that is thought to contribute to the proangiogenic role of the protein. (11). This property may additionally be involved in regulating the trafficking of leukocytes in vivo, given that phosphatidylinositol 3-kinase activity within endothelial cells has been shown to be required for the diapedesis stage of lymphocyte extravasation, following their firm adhesion (25). Importantly, activation of endothelial phosphatidylinositol 3-kinase signaling by pro-HPSE1 was associated with its uptake and preceded processing to active HPSE1 (11), which may in part explain why heat inactivated, “processed” HPSE1 lacks activity in vivo; enzymatic activity is lost and at least one alternative, nonenzymatic mode of action is non-applicable.
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Interestingly, the cellular infiltrate seen in response to HPSE1 or pro-HPSE1 comprised a greater proportion of mononuclear cells than the neutrophil-dominated IL-1-induced infiltrate. While it is not possible to compare directly the responses to IL-1 and heparanase enzymes, given the large difference in total protein administered, we believe that the IL-1 controls serve as a useful comparator in these experiments and were included on account of the robust and welldescribed effects of the cytokine in these models. We also found that coadministration of HPSE1 or pro-HPSE1 with IL-1, at the same doses used in the single stimulus experiments, led to no additional increase in cell numbers and also showed a neutrophil dominant lavage (data not shown). A plausible explanation is that HPSE1 has differential effects on leukocyte subtypes, possibly via 1-integrin-mediated effects (32), thus selectively promoting mononuclear cell, over neutrophil, adhesion in the mesenteric microcirculation. The pulmonary endothelial glycocalyx has recently been observed to regulate neutrophil adhesion and lung injury during sepsis (29), and inhibition of HPSE1 prevented the glycocalyx loss and neutrophil adhesion associated with endotoxemia (29). These results are consistent with our data presented here that implicate heparanase as a proinflammatory substance. In conclusion, these data provide further evidence of a role for heparanase proteins in the context of inflammation. They also suggest that the enzymatic activity of heparanase, or at least the ability to be processed to an enzymatically active form, is essential for its proinflammatory activity in vivo. ACKNOWLEDGMENTS We thank Dr. Robert Felix, James Bennett, and Maina Bhaman for assaying the enzymatic activity of the HPSE1 and HI-HPSE1 used in our experiments. DISCLOSURES This work was funded by Oxford Glycosciences (now UCB/Celltech). AUTHOR CONTRIBUTIONS Author contributions: R.L., M.J.R., E.A.M., and C.P.P. conception and design of research; R.L., M.J.R., and E.A.M. performed experiments; R.L. and M.J.R. analyzed data; R.L., M.J.R., E.A.M., and C.P.P. interpreted results of experiments; R.L. and M.J.R. prepared figures; R.L. drafted manuscript; R.L., E.A.M., and C.P.P. edited and revised manuscript; R.L., E.A.M., and C.P.P. approved final version of manuscript. REFERENCES 1. Abboud-Jarrous G, Rangini-Guetta Z, Aingorn H, Atzmon R, Elgavish S, Peretz T, Vlodavsky I. Site-directed mutagenesis, proteolytic cleavage, and activation of human proheparanase. J Biol Chem 280: 13568 –13575, 2005. 2. Bar-Ner M, Eldor A, Wasserman L, Matzner Y, Cohen IR, Fuks Z, Vlodavsky I. Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species. Blood 70: 551–557, 1987. 3. Basche M, Gustafson DL, Holden SN, O’Bryant CL, Gore L, Witta S, Schultz MK, Morrow M, Levin A, Creese BR, Kangas M, Roberts K, Nguyen T, Davis K, Addison RS, Moore JC, Eckhardt SG. A phase I biological and pharmacologic study of the heparanase inhibitor PI-88 in patients with advanced solid tumors. Clin Cancer Res 12: 5471–5480, 2006. 4. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68: 729 –777, 1999. 5. Chen G, Wang D, Vikramadithyan R, Yagyu H, Saxena U, Pillarisetti S, Goldberg IJ. Inflammatory cytokines and fatty acids regulate endothelial cell heparanase expression. Biochemistry 43: 4971–4977, 2004.
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6. Cohen E, Atzmon R, Vlodavsky I, Ilan N. Heparanase processing by lysosomal/endosomal protein preparation. FEBS Lett 579: 2334 –2338, 2005. 7. Edovitsky E, Elkin M, Zcharia E, Peretz T, Vlodavsky I. Heparanase gene silencing, tumor invasiveness, angiogenesis, and metastasis. J Natl Cancer Inst 96: 1194 –1195, 2004. 8. Edovitsky E, Lerner I, Zcharia E, Peretz T, Vlodavsky I, Elkin M. Role of endothelial heparanase in delayed-type hypersensitivity. Blood 107: 3609 –3616, 2006. 9. Fairbanks MB, Mildner AM, Leone JW, Cavey GS, Mathews WR, Drong RF, Slightom JL, Bienkowski MJ, Smith CW, Bannow CA, Heinrikson RL. Processing of the human heparanase precursor and evidence that the active enzyme is a heterodimer. J Biol Chem 274: 29587–29590, 1999. 10. Gallagher JT, Turnbull JE. Heparan sulphate in the binding and activation of basic fibroblast growth factor. Glycobiology 2: 523–528, 1992. 11. Gingis-Velitski S, Zetser A, Flugelman MY, Vlodavsky I, Ilan N. Heparanase induces endothelial cell migration via protein kinase B/Akt activation. J Biol Chem 279: 23536 –23541, 2004. 12. Goldshmidt O, Zcharia E, Cohen M, Aingorn H, Cohen I, Nadav L, Katz BZ, Geiger B, Vlodavsky I. Heparanase mediates cell adhesion independent of its enzymatic activity. FASEB J 17: 1015–1025, 2003. 13. Hulett MD, Freeman C, Hamdorf BJ, Baker RT, Harris MJ, Parish CR. Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis. Nat Med 5: 803–809, 1999. 14. Joyce JA, Freeman C, Meyer-Morse N, Parish CR, Hanahan D. A functional heparan sulfate mimetic implicates both heparanase and heparan sulfate in tumor angiogenesis and invasion in a mouse model of multistage cancer. Oncogene 24: 4037–4051, 2005. 15. Lever R, Hoult JR, Page CP. The effects of heparin and related molecules upon the adhesion of human polymorphonuclear leukocytes to vascular endothelium in vitro. Br J Pharmacol 129: 533–540, 2000. 16. Lever R, Page CP. Novel drug development opportunities for heparin. Nat Rev Drug Discov 1: 140 –148, 2002. 17. Levy-Adam F, Abboud-Jarrous G, Guerrini M, Beccati D, Vlodavsky I, Ilan N. Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase. J Biol Chem 280: 20457–20466, 2005. 18. Levy-Adam F, Feld S, Suss-Toby E, Vlodavsky I, Ilan N. Heparanase facilitates cell adhesion and spreading by clustering of cell surface heparan sulfate proteoglycans. PLoS One 3: e2319, 2008. 19. Lider O, Mekori YA, Miller T, Bar-Tana R, Vlodavsky I, Baharav E, Cohen IR, Naparstek Y. Inhibition of T lymphocyte heparanase by heparin prevents T cell migration and T cell-mediated immunity. Eur J Immunol 20: 493–439, 1990. 20. Luster AD. Chemokines– chemotactic cytokines that mediate inflammation. N Engl J Med 338: 436 –445, 1998. 21. Massena S, Christoffersson G, Hjertström E, Zcharia E, Vlodavsky I, Ausmees N, Rolny C, Li JP, Phillipson M. A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils. Blood 116: 1924 –1931, 2010. 22. Matzner Y, Vlodavsky I, Bar-Ner M, Ishai-Michaeli R, Tauber AI. Subcellular localization of heparanase in human neutrophils. J Leukoc Biol 51: 519 –524, 1992. 23. McKenzie EA. Heparanase: a target for drug discovery in cancer and inflammation. Br J Pharmacol 151: 1–14, 2007. 24. McKenzie E, Young K, Hircock M, Bennett J, Bhaman M, Felix R, Turner P, Stamps A, McMillan D, Saville G, Ng S, Mason S, Snell D, Schofield D, Gong H, Townsend R, Gallagher J, Page M, Parekh R, Stubberfield C. Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells. Biochem J 373: 423–435, 2003. 25. Nakhaei-Nejad M, Hussain AM, Zhang QX, Murray AG. Endothelial PI3-kinase activity regulates lymphocyte diapedesis. Am J Physiol Heart Circ Physiol 293: H3608 –H3616, 2007. 26. Naparstek E, Slavin S, Weiss L, Sidi H, Ohana M, Reich S, Vlodavsky I, Cohen IR, Naparstek Y. Low-dose heparin inhibits acute graft versus host disease in mice. Bone Marrow Transplant 12: 185–189, 1993. 27. Parish CR. The role of heparan sulphate in inflammation. Nat Rev Immunol 6: 633–643, 2006. 28. Sciumbata T, Caretto P, Pirovano P, Pozzi P, Cremonesi P, Galimberti G, Leoni F, Marcucci F. Treatment with modified heparins inhibits experimental metastasis formation and leads, in some animals, to longterm survival. Invasion Metastasis 16: 132–143, 1996.
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29. Schmidt EP, Yang Y, Janssen WJ, Gandjeva A, Perez MJ, Barthel L, Zemans RL, Bowman JC, Koyanagi DE, Yunt ZX, Smith LP, Cheng SS, Overdier KH, Thompson KR, Geraci MW, Douglas IS, Pearse DB, Tuder RM. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis. Nat Med 18: 1217–1223, 2012. 30. Shafat I, Vlodavsky I, Ilan N. Characterization of mechanisms involved in secretion of active heparanase. J Biol Chem 281: 23804 –23811, 2006. 31. Smailbegovic A, Lever R, Page CP. The effects of heparin on the adhesion of human peripheral blood mononuclear cells to human stimulated umbilical vein endothelial cells. Br J Pharmacol 134: 827–386, 2001. 32. Sotnikov I, Hershkoviz R, Grabovsky VN, Ilan N, Cahalon L, Vlodavsky I, Alon R, Lider O. Enzymatically quiescent heparanase augments T cell interactions with VCAM-1 and extracellular matrix components under versatile dynamic contexts. J Immunol 172: 5185–5193, 2004. 33. Sy MS, Schneeberger E, McCluskey R, Greene MI, Rosenberg RD, Benacerraf B. Inhibition of delayed-type hypersensitivity by heparin depleted of anticoagulant activity. Cell Immunol 82: 23–32, 1983. 34. Uno F, Fujiwara T, Takata Y, Ohtani S, Katsuda K, Takaoka M, Ohkawa T, Naomoto Y, Nakajima M, Tanaka N. Antisense-
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mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells. Cancer Res 61: 7855– 7860, 2001. Vlodavsky I, Friedmann Y, Elkin M, Aingorn H, Atzmon R, IshaiMichaeli R, Bitan M, Pappo O, Peretz T, Michal I, Spector L, Pecker I. Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nat Med 5: 793–802, 1999. Vlodavsky I, Friedmann Y. Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis. J Clin Invest 108: 341–347, 2001. Vlodavsky I, Eldor A, Haimovitz-Friedman A, Matzner Y, IshaiMichaeli R, Lider O, Naparstek Y, Cohen IR, Fuks Z. Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion Metastasis 12, 112–127, 1992. Wang L, Fuster M, Sriramarao P, Esko JD. Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. Nat Immunol 6: 902– 910, 2005.
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Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium Rebecca Lever,1 Mark J. Rose,2 Edward A. McKenzie,3 and Clive P. Page2 1
Department of Pharmacology, University College London School of Pharmacy, London, United Kingdom; 2Sackler Institute of Pulmonary Pharmacology, King’s College London, London, United Kingdom; and 3Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Submitted 3 September 2013; accepted in final form 15 April 2014
Lever R, Rose MJ, McKenzie EA, Page CP. Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium. Am J Physiol Cell Physiol 306: C1184 –C1190, 2014. First published April 16, 2014; doi:10.1152/ajpcell.00269.2013.—Heparanase (HPSE1) is known to be involved in mechanisms of metastatic tumor cell migration. This enzyme selectively cleaves heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed in mammals and are known to be involved in regulating the activity of an array of inflammatory mediators. In the present study, we have investigated the effects of human recombinant heparanase, the inactive precursor of this enzyme (proheparanase) and enzymatically inactivated heparanase, on inflammatory cell recruitment in the rat and on human leukocyte-endothelial adhesion in vitro. Intraperitoneal injection of heparanase (500 g) induced a significant inflammatory cell infiltrate in the rat, as assessed by peritoneal lavage 4 h later. Intravital microscopy of the mesenteric microcirculation of anesthetized rats showed an increase in rolling and adherent cells in postcapillary venules that was sensitive to heparin, a nonselective inhibitor of heparanase activity. In vitro, heparanase augmented the adhesion of human neutrophils and mononuclear cells to human umbilical vein endothelial cells in a concentration-dependent manner. Proheparanase had similar effects to the active enzyme both with respect to leukocyte accumulation in the peritoneal cavity and adhesion in vitro. However, heat-inactivated heparanase induced cell adhesion in vitro but was without effect in vivo. Together, these data indicate a role for heparanase in inflammatory cell trafficking in vivo that appears to require enzymatic activity. cell trafficking; heparan sulfate; inflammation; mononuclear leukocyte; neutrophil THE HUMAN ENDO- GLUCURONIDASE, heparanase (HPSE1), cloned in 1999 (13, 35), is known to be involved in mechanisms of metastatic tumor cell migration into tissues. The substrates of this enzyme are heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed throughout the body (4, 36) and form a major component of the subendothelial matrix, as well as being strongly expressed as part of the glycocalyx on the surface of endothelial cells and circulating blood elements, affording negative charge density to cell surfaces. Loss of this charge is known to be associated with leukocyte adhesion (29). It has also been suggested that, at least during sepsis, HPSE1 may well contribute to the observed loss in endothelial HSPG (29). Inhibition of heparanase using antisense strategies (7, 34), heparin (26, 28), or relatively selective inhibitors such as phosphomannopentaose (PI-88) (3, 14) is effective in reducing tumor cell adhesion, migration, and subsequent colonization in
Address for reprint requests and other correspondence: R. Lever, Dept. of Pharmacology, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK (e-mail: [email protected]). C1184
tissues, and this enzyme is an established therapeutic target for the development of anticancer drugs (23). However, fewer studies have investigated the role of this enzyme in leukocyte emigration during inflammatory responses, which shares many similarities with tumor cell metastasis (37). Indeed, the wellestablished role of HSPGs in the physiological functioning of a range of inflammatory mediators, including chemokines (20, 21, 27, 38), growth factors (10, 27), and certain adhesion molecules (38), suggests that enzymatic disruption of these proteoglycans should impact upon inflammatory responses. Furthermore, it is known that heparan-degrading enzymes are released by certain leukocytes when they diapedese (19, 22) and by endothelial cells in response to proinflammatory stimuli (5, 8). It is also known that these enzymes are inhibited by heparin (2), accounting for some of the described anti-inflammatory effects of this structural relative of heparan sulfate (16). For example, when heparin is used at low doses in models of inflammatory disease, especially in lymphocyte-driven processes such as allergic encephalomyelitis (19), delayed-type hypersensitivity (33), and graft vs. host reactions (26), it significantly reduces leukocyte infiltration into tissues. Moreover, the development of delayed-type hypersensitivity reactions in mice has been found to correlate with endothelial heparanase production and to be sensitive to inhibition of this enzyme (8). The HPSE1 enzyme is initially synthesized as a 65-kDa inactive precursor, proheparanase (pro-HPSE1) (9), which is then processed into an active form in lysosomes (30) in a pH-dependent manner (6). The active form of the enzyme is comprised of and secreted as a heterodimer, with subunits of 50 and 8 kDa, respectively (9). It has been shown that that both pro-HPSE1 (32) and active HPSE1 (12) can mediate cell adhesion via mechanisms unrelated to enzymatic activity. This appears to be a 1-integrin-dependent process and also utilizes cell surface HSPG (32), presenting a further mechanism by which heparanase may promote inflammation in vivo, in addition to those mechanisms likely to be dependent on cleavage of cell surface and matrix HSPG. In the present study, we examined the effects of pro-HPSE1, HPSE1, and enzymatically inactivated HPSE1 [heat inactivated (HI-HPSE)] on human neutrophil and mononuclear cell adhesion in vitro, as well as the possible proinflammatory effects of these molecules in vivo in a rodent model of peritoneal inflammation. MATERIALS AND METHODS
Expression and purification of heparanase enzymes and preparation of HI-HPSE1. HPSE1 was expressed, purified, and analyzed as described previously (24). HI-HPSE1 was prepared by heating
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The pro-HPSE1 cDNA was PCR amplified from a mammary gland cDNA library (Clontech) using pfu turbo polymerase (Stratagene) and the following cycling conditions [94°C 1’, (94°C 30 s, 60°C 30 s, 72°C 2’) 30 cycles, 72°C 10’, 4°C]. Primers used were forward: 5=-ccc ggg cag gac gtc gtg gac ctg gac ttc ttc acc-3= and reverse: 5=-gaa ttc tca gat gca agc agc aac ttt ggc att tct-3=. PCR product was separated using standard agarose gel electrophoresis techniques, gel purified (Qiaquick-Qiagen), and then cloned into pZero blunt vector (Invitrogen). Correct sequence was verified by sequencing with dideoxy dye terminators (ABI big dye version 1.1). The product was excised from pZero blunt by double digestion with Sma1/EcoR1 enzymes (Promega) and ligated into the baculovirus transfer vector pAcGP67A (Pharmingen) cut with the same enzymes. pAcGP67A:pro-HPSE1 transfer vector was cotransfected, along with baculovirus DNA into sf9 cells exactly as described previously (24). Secreted protein was collected and purified by heparin sepharose chromatography as described previously. Average yields were ⬃6 mg/l of purified protein at ⬎90% purity as visualized by SDS-PAGE Coomassie blue staining. All recombinant proteins were concentrated to 1 mg/ml in a final buffer containing 25 mM Tris·HCl pH 7.5 and 150 mM NaCl and kept at 4°C for immediate use or ⫺80°C for long-term storage. In vivo studies. All experiments on animals were performed under UK Home Office and local ethical approval from King’s College London. Male Sprague-Dawley rats (150 –200 g; Harlan), housed under a 12:12-h light/dark cycle for 7 days with food and water provided ad libitum, were injected intraperitoenally with IL-1 (R&D
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Fig. 1. Heparanase-induced cell recruitment to the peritoneal cavity of the rat. Total cell numbers (open bars) in peritoneal lavage 4 h after intraperitoneal administration of saline, IL-1 (20 ng), or active HPSE1 (500 g). The neutrophil component of lavages is indicated by the filled bars. Data are expressed as means ⫾ SE and were analyzed by ANOVA, followed by Dunnett’s test. *P ⬍ 0.05, compared with saline control; n ⫽ 6 per group.
HPSE1 protein to 80°C for 45 min, followed by cooling to room temperature and ultracentrifugation. The protein content in the resultant solution was confirmed by Bradford assay, and the loss of enzymatic activity was confirmed by lack of ability to cleave FITC-heparan sulphate (24).
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Fig. 2. Heparanase-induced cell rolling and adhesion in the rat mesenteric microcirculation. A: rolling flux in 100-m sections of mesenteric postcapillary venule 4 h after intraperitoneal administration of saline (hatched bar) or IL-1 (20 ng; open bars) with and without cotreatment with unfractionated heparin. B: number of cells stationary in 100-m sections of mesenteric postcapillary venule 4 h after the treatments described in A. C: number of cells rolling in 100-m sections of mesenteric postcapillary venule 4 h after intraperitoneal administration of saline (hatched bar) or active HPSE1 (500 g; dotted bars) with and without cotreatment with unfractionated heparin. D: number of cells stationary in 100-m sections of mesenteric postcapillary venule 4 h after the treatments described in C. Data are expressed as means ⫾ SE and were analyzed by ANOVA, followed by Dunnett’s test. #P ⬍ 0.05, compared with saline control. *P ⬍ 0.05, compared with IL-1 or active HPSE1 control; n ⫽ 6 per group.
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Systems) or the study compounds in a total volume of 500 l. Four hours later, animals were either euthanized and the peritoneal cavity was lavaged with 20 ml saline, or they were anesthetized [thiobutabarbital sodium (Inactin RBC); 100 mg/kg ip; Sigma-Aldrich] for intravital microscopy. Total cells in lavage fluids were counted, and differential cell counts were obtained from cytospin preparations, stained using the DiffQuick system (Gamidor). Anesthetized animals were maintained at 37°C on a heated microscope stage. Single loops of terminal ileum were carefully exteriorized, following midline incision. Sections of mesentery were placed over an optical window within the stage and superfused with bicarbonate buffer. Unbranched, postcapillary venules were viewed under a Zeiss Axioskop 2 FS microscope, fitted with a ⫻40 water-immersion lens and a ⫻10 eyepiece. Digital images were captured using a Sony XC-003P color vision digital camera, and images were viewed and recorded for subsequent offline analysis using Axiovision Software (Image Associates). Animals were euthanized at the end of experiments. Leukocyte rolling flux was quantified as the number of rolling cells passing a fixed point on the venular wall per minute. Adherent leukocytes were considered those cells that were stationary for at least 30 s within a given 100-m vessel wall segment. In vitro adhesion assays. Leukocyte-endothelial adhesion was examined using an assay described previously (15, 31). Peripheral venous blood was drawn from healthy volunteers into citrated tubes. Healthy blood donors were recruited, written informed consent was provided, and samples were obtained under the approval of the Research Ethics Committee of King’s College London. Neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated by standard density-dependent centrifugation on Histopaque-1077 and were radiolabeled with sodium 51 chromate (37 kBq per 106 cells; Amersham). Human umbilical vein endothelial cells (HUVECs; Cambrex), at passage five or below, were cultured to confluency in supplemented MCDB 131 medium (Cambrex) in the central 60 wells of flatbottomed 96-well plates (200 l culture medium per well; 5% CO2; 37°C). Some wells were stimulated for 6 h with human rIL-1 (100 U/ml; Sigma-Aldrich) before use in adhesion assays. Monolayers were washed with warmed HBSS before addition of 200 l radiolabeled leukocyte suspension per well (106 cells/ml) in the absence and presence of test compounds. Plates were incubated for 30 min (37°C), and nonadherent cells were then removed by gentle aspiration and washed with warmed HBSS. Following assessment of monolayer integrity by microscopy, adherent cells were lysed with 1% Igepal (Sigma) and samples were transferred to scintillation vials and ␥-counted, alongside samples of the original radiolabeled leukocyte suspension (input). The number of adherent cells was calculated as the percentage of input counts present in sample counts, corrected for background radioactivity. RESULTS
Heparanase induces inflammatory cell recruitment in vivo. Administration of recombinant HPSE1 to the peritoneal cavity of the rat induced a robust infiltration of leukocytes to this site (Fig. 1), which was similar in magnitude to that elicited by IL-1; this cytokine was used as a positive control in all experiments with heparanase enzymes, due to its well-characterized proinflammatory effects in the models used. We went on to examine the effect of HPSE1 on leukocyte rolling and adhesion in the mesenteric microcirculation of the rat using intravital microscopy. HPSE1 increased the number of rolling and adherent cells in postcapillary venules, again, to a similar extent as IL-1 (Fig. 2, A–D). In these experiments, we sought to examine the effect of inhibiting the enzymatic activity of HPSE1 on inflammation. Heparin is a nonselective inhibitor of heparanase and is known to affect leukocyte adhesion in its own right. However, it is notable that, in our experiments,
heparin inhibited the response to HPSE1 (Fig. 2, C and D), in particular with respect to adhesion, at lower concentrations than those required to modulate the cytokine-induced response (Fig. 2, A and B). This is commensurate with the heparanase inhibitor activity of heparin, which occurs at lower concentrations than those required for other potentially anti-inflammatory activities of this molecule (16). Proheparanase induces cell recruitment in vivo but heatinactivated heparanase does not. An important question lies in whether heparanase needs to be catalytically active to induce inflammation in vivo, given reports that this molecule possesses proadhesive effects in vitro that are independent of HSPG cleavage (12, 32). Therefore, we examined the effects of both the inactive precursor of HPSE1 and heat-inactivated HPSE1 enzyme on cell recruitment to the peritoneal cavity. Human recombinant pro-HPSE1 was expressed and purified (summarized in Fig. 3, A and B) and was found to induce an inflammatory response that again was comparable to that elicited by IL-1. By contrast, heat-inactivated heparanase (HI-HPSE1) had no effect in this model (Fig. 3C).
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Fig. 3. Proheparanase- and heat-inactivated heparanase-induced cell recruitment to the peritoneal cavity of the rat. A: pro-form of HPSE1 (amino acids 36 –543) was cloned into the baculovirus secretory transfer vector pACGP67 to direct secretion into the culture media. B: recombinant pro-HPSE1 virus was used to infect Hi5 insect cells. Culture medium containing protein was purified by heparin sepharose chromatography and eluted fractions analyzed by SDS PAGE. C: total cell numbers (open bars) in peritoneal lavage 4 h after intraperitoneal administration of saline, IL-1 (20 ng), pro-HPSE1 (500 g) or HI-HPSE1 (500 g). The neutrophil component of lavages is indicated by the filled bars. Data are expressed as means ⫾ SE and were analyzed by ANOVA, followed by Dunnett’s test. *P ⬍ 0.05 compared with saline control; n ⫽ 6 per group.
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Heparanase induces and enhances leukocyte-endothelial interactions in vitro in a manner that is not dependent on enzymatic activity. Having found differential effects of proHPSE1 and enzymatically inactive, HI-HPSE1 in vivo, with respect to inflammatory cell accumulation, and in light of previous reports that cell adhesion can be enhanced by proHPSE1 in vitro in a manner independent of enzymatic activity (12, 32), we were interested to apply these proteins to an in vitro model of leukocyte-endothelial adhesion. Heparanase concentration dependently increased the adhesion of both neutrophils (Fig. 4A, closed squares) and PBMCs (Fig. 5A, closed symbols) to cultured HUVEC monolayers, compared with the basal adhesion seen in unstimulated control wells (Figs. 4D and 5D, closed bars). Interestingly, when endothelial cells were prestimulated with IL-1, which enhances subsequent leukocyte adhesion to these cells (Figs. 4D and 5D; open bars show the IL-1-induced adhesion seen in stimulated control wells), the presence of HPSE1 further augmented adhesion, again in a concentration-dependent manner (Fig. 4A and 5A, open squares). Therefore, we investigated the effects of pro-HPSE1 in the same model and found that adhesion was similarly promoted (Figs. 4B and 5B). These data suggest that either endothelial cells or leukocytes, or both, possess the necessary machinery for processing of pro-HPSE1 to its active form or
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The results of this study show that heparanase proteins are capable of modulating inflammatory cell adhesion in vitro and trafficking in vivo. In vitro, HPSE1, pro-HPSE1, and HIHPSE1 all increased the adhesion of both neutrophils and PBMCs to cultured endothelial monolayers. By contrast, in vivo, HPSE1 and pro-HPSE1 induced the infiltration of inflammatory cells to the peritoneal cavity, whereas HI-HPSE1 was without effect. It is most likely that the effects of HPSE1 in vivo depend at least in part on enzymatic activity, given that the heat inactivated form of the enzyme, shown to lack the ability to cleave HSPG, was without effect despite being able
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simply that the protein does not require enzymatic activity for its proadhesive effects in this in vitro system. To address this point further, we assessed the effects of HI-HPSE1 and found that, despite a lack of HSPG-degrading activity, adhesion was still enhanced (Figs. 4C and 5C). Finally, we heat-inactivated pro-HPSE1 in the same manner and found that this material, by contrast, was entirely without effect under any of the experimental conditions tested (data not shown), suggesting that any proadhesive effects that are retained after heat inactivation are restricted, nonetheless, to actively processed heparanase.
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Fig. 4. Heparanase-induced neutrophil adhesion to cultured endothelial cells. The effect of active HPSE1 (A), pro-HPSE1 (B), or HIHPSE1 (C) on adhesion of human neutrophils to unstimulated human umbilical vein endothelial cells (HUVECs; ) or to HUVECs prestimulated with IL-1 (100 U/ml) for 6 h (䊐). D: basal and IL-1-induced control adhesion. Data (A–C) are presented as %relevant control (D) and represent the means ⫾ SE of 6 independent experiments, each carried out in triplicate. Data were analyzed by ANOVA, followed by Dunnett’s test. *P ⬍ 0.05, compared with saline or IL-1 control (A–C) or to saline (D).
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Fig. 5. Heparanase-induced mononuclear cell adhesion to cultured endothelial cells. The effect of HPSE1 (A), pro-HPSE1 (B), or HI-HPSE1 (C) on adhesion of human peripheral blood mononuclear cells (MNC) to unstimulated HUVECs () or to HUVECs prestimulated with IL-1 (100 U/ml) for 6 h (䊐). D: basal and IL-1-induced control adhesion. Data (A–C) are presented as %relevant control (D) and represent the means ⫾ SE of 6 independent experiments, each carried out in triplicate. Data were analyzed by ANOVA, followed by Dunnett’s test. *P ⬍ 0.05, compared with saline or IL-1 control (A–C) or to saline (D).
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to induce adhesion in vitro, and the effects of native HPSE1 in vivo were sensitive to inhibition by heparin. It is logical that cell recruitment in vivo, in the presence of shear force at the vessel wall and a matrix-bound chemotactic gradient, would be sensitive to HSPG degradation, and our results indeed suggest the enzymatic activity of HPSE1 to be involved in the proinflammatory effects seen in vivo. However, and in accordance with previous studies, our data suggest that breakdown of extracellular matrix or cell surface HSPGs is unlikely to be required for the firm adhesion of immune cells to endothelium per se, as HI-HPSE1 was found to induce adhesion to the same extent as the untreated molecule in vitro. As described previously, with respect to lymphocytes (32), the enzymatically inactive proenzyme pro-HPSE1 also induced the adhesion of both PBMCs and neutrophils in our assay. Clearly, it is possible that the moiety required for these proadhesive effects is present on all three forms of the protein and would appear not to be destroyed by heat inactivation, given the effect of HI-HPSE. However, we also found that heat treatment of pro-HPSE1 removed its proadhesive activity. It has previously been shown that pro-HPSE1 and pointmutated, enzymatically inactive HPSE enhanced adhesion in vitro by promoting the clustering of cell surface HSPGs, an
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effect that is shared by a heparin/heparan-binding peptide sequence of HPSE1, Lys158-Asp171 (18, 27), which maps to the NH2 terminus of the 50-kDa subunit of active HPSE1 (17). It is possible, therefore, that destruction of the tertiary structure of pro-HPSE1 by heat treatment serves to make this important sequence, which also corresponds to a potential cleavage site on pro-HPSE1 at Gln157-Lys158 (1, 9), functionally inaccessible. Furthermore, pro-HPSE1 has been shown to induce Akt phosphorylation in endothelial cells in a manner that is independent of both enzymatic activity and the presence of HSPG and that is thought to contribute to the proangiogenic role of the protein. (11). This property may additionally be involved in regulating the trafficking of leukocytes in vivo, given that phosphatidylinositol 3-kinase activity within endothelial cells has been shown to be required for the diapedesis stage of lymphocyte extravasation, following their firm adhesion (25). Importantly, activation of endothelial phosphatidylinositol 3-kinase signaling by pro-HPSE1 was associated with its uptake and preceded processing to active HPSE1 (11), which may in part explain why heat inactivated, “processed” HPSE1 lacks activity in vivo; enzymatic activity is lost and at least one alternative, nonenzymatic mode of action is non-applicable.
AJP-Cell Physiol • doi:10.1152/ajpcell.00269.2013 • www.ajpcell.org
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Interestingly, the cellular infiltrate seen in response to HPSE1 or pro-HPSE1 comprised a greater proportion of mononuclear cells than the neutrophil-dominated IL-1-induced infiltrate. While it is not possible to compare directly the responses to IL-1 and heparanase enzymes, given the large difference in total protein administered, we believe that the IL-1 controls serve as a useful comparator in these experiments and were included on account of the robust and welldescribed effects of the cytokine in these models. We also found that coadministration of HPSE1 or pro-HPSE1 with IL-1, at the same doses used in the single stimulus experiments, led to no additional increase in cell numbers and also showed a neutrophil dominant lavage (data not shown). A plausible explanation is that HPSE1 has differential effects on leukocyte subtypes, possibly via 1-integrin-mediated effects (32), thus selectively promoting mononuclear cell, over neutrophil, adhesion in the mesenteric microcirculation. The pulmonary endothelial glycocalyx has recently been observed to regulate neutrophil adhesion and lung injury during sepsis (29), and inhibition of HPSE1 prevented the glycocalyx loss and neutrophil adhesion associated with endotoxemia (29). These results are consistent with our data presented here that implicate heparanase as a proinflammatory substance. In conclusion, these data provide further evidence of a role for heparanase proteins in the context of inflammation. They also suggest that the enzymatic activity of heparanase, or at least the ability to be processed to an enzymatically active form, is essential for its proinflammatory activity in vivo. ACKNOWLEDGMENTS We thank Dr. Robert Felix, James Bennett, and Maina Bhaman for assaying the enzymatic activity of the HPSE1 and HI-HPSE1 used in our experiments. DISCLOSURES This work was funded by Oxford Glycosciences (now UCB/Celltech). AUTHOR CONTRIBUTIONS Author contributions: R.L., M.J.R., E.A.M., and C.P.P. conception and design of research; R.L., M.J.R., and E.A.M. performed experiments; R.L. and M.J.R. analyzed data; R.L., M.J.R., E.A.M., and C.P.P. interpreted results of experiments; R.L. and M.J.R. prepared figures; R.L. drafted manuscript; R.L., E.A.M., and C.P.P. edited and revised manuscript; R.L., E.A.M., and C.P.P. approved final version of manuscript. REFERENCES 1. Abboud-Jarrous G, Rangini-Guetta Z, Aingorn H, Atzmon R, Elgavish S, Peretz T, Vlodavsky I. Site-directed mutagenesis, proteolytic cleavage, and activation of human proheparanase. J Biol Chem 280: 13568 –13575, 2005. 2. Bar-Ner M, Eldor A, Wasserman L, Matzner Y, Cohen IR, Fuks Z, Vlodavsky I. Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species. Blood 70: 551–557, 1987. 3. Basche M, Gustafson DL, Holden SN, O’Bryant CL, Gore L, Witta S, Schultz MK, Morrow M, Levin A, Creese BR, Kangas M, Roberts K, Nguyen T, Davis K, Addison RS, Moore JC, Eckhardt SG. A phase I biological and pharmacologic study of the heparanase inhibitor PI-88 in patients with advanced solid tumors. Clin Cancer Res 12: 5471–5480, 2006. 4. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68: 729 –777, 1999. 5. Chen G, Wang D, Vikramadithyan R, Yagyu H, Saxena U, Pillarisetti S, Goldberg IJ. Inflammatory cytokines and fatty acids regulate endothelial cell heparanase expression. Biochemistry 43: 4971–4977, 2004.
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