Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure

Abstract

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC-MS/MS, suggesting that ACR modification of Lys¹³⁹ (6-kDa linker), Lys¹⁰⁷, and Lys¹⁶¹, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

Keywords: acrolein; chondroitin sulfate; glycocalyx; heparan sulfate; heparanase; hyaluronan; hyaluronidase; ischemic stroke; stroke.

Figure 1

Degradation of glycosaminoglycans after stroke induction.A, levels of HS, CS, and HA in infarct region or normal area (in the contralateral hemisphere) of mouse brain tissues at 24 h after stroke induction. Extraction of GAGs and analysis of their unsaturated disaccharides were performed as described under “Experimental procedures.” Compositions of unsaturated disaccharides of HS and CS are shown in Fig. S1. B, plasma contents of HS and CS after onset of ischemic stroke. Data are expressed as the mean ± S.D. (error bars). C, expression levels of HYAL, HPSE, and MMP9 in infarct region or normal area of mouse brain tissues after onset of ischemic stroke. Western blotting of each protein was performed using 20 µg of protein extracted from tissues. Experiments were repeated three times and the same results were obtained. N, normal, I, infarct region. D, HPSE activity in the infarct region or normal area after onset of stroke. HPSE activity in tissue extracts (150 µg of protein) was measured using a heparan-degrading enzyme assay kit. Horizontal line indicates median. *p < 0.05; **p < 0.01; ns, not significant.

Figure 2

Induction of HPSE expression in microvascular endothelial cells 24 h after stroke onset. Mouse brain was removed 24 h after the onset of ischemic stroke. The nuclei, HPSE, and endothelial cells were stained using DAPI, anti-HPSE antibody (H-80), and isolectin-B4, respectively (Table S2). Bar = 50 µm. Note that the primary HPSE (H-80) antibody can recognize 101–180 amino acids of HPSE and proHPSE. Experiments were repeated three times, and reproducible results were obtained.

Figure 3

Effect of coadministration of NAC, LMWH, and pLMWCS on the size of infarct volume. Preparation of PIT mouse model, triphenyltetrazolium chloride staining of brain tissue, and analysis of infarct volume were carried out as described in “Experimental procedures.” Intraperitoneal administration of NAC (250 mg/kg), enoxaparin (2.5 mg/kg), pLMWHS (100 mg/kg), and pLMWCS (100 mg/kg) in PBS was performed immediately after induction of infarction. The therapeutic range (0.3–0.7 IU/ml) for anti-FXa activity was observed at 0.5–4 h after injection of enoxaparin (2.5 mg/kg) (data not shown). Data are expressed as the means ± S.D. (error bars). *p < 0.05, ***p < 0.001, ****p < 0.0001 against vehicle. #p < 0.05 against NAC. ns, not significant. Bar = 5 mm. Enox., enoxaparin.

Figure 4

Acrolein induces HS degradation in HBMEC/ciβ. Cell growth (A), expression levels of HS and CS (B), expression levels of HPSE, proHPSE, and MMP9 (C), and expression level of HPSE mRNA (D) of HBMEC/ciβ exposed to acrolein (ACR) or hydrogen peroxide (H₂O₂). A, to examine the toxicity of ACR and H₂O₂, 1.0 × 10⁵ cells of HBMEC/ciβ were inoculated into 35-mm dishes and cultured for 72 h. B, GAGs were extracted using 1.0 × 10⁷ cells of HBMEC/ciβ and analyzed using HPLC. C, Western blotting of each protein was performed using 20 µg of protein extracted from the cell lysate. Experiments were repeated three times and the same results were obtained. D, data were calculated using the 2^−ΔΔCt method. Transcription of the housekeeping gene GAPDH was used to normalize data. A, B, and D, data are expressed as the mean ± S.D. (error bars). *p < 0.05 against none.

Figure 5

Effect of acrolein or hydrogen peroxide on HPSE activity of HEK293 cells transfected with hHPSE cDNA.A, HPSE activity can be measured using GPC with post-column derivatization. Aldehyde groups at the reducing end of polysaccharides react with 2-cyanoacetoamide as a fluorogenic post-labeling reagent. In general, scission activity (HPSE activity) is evaluated by GPC through the monitoring of the prelabeled heparin degradation or by a heparan-degrading enzyme assay kit with prelabeled heparin (Fig. S2A). However, it was difficult to prepare the free form of proHPSE from conditioned medium, and measurement of scission activity of proHPSE by heparan-degrading enzyme assay kit was also disturbed by unfractionated heparin (data not shown). Therefore, unfractionated heparin which is supplemented to conditioned medium was directly used as a substrate of proHPSE, and post-label derivatization was employed to evaluate the scission activity. B, HEK293 cells transfected with hHPSE1 cDNA was lysed in lysis buffer (pH 6.0 or pH 7.0), and the resulting cell lysates (2.5 or 25 µg) were incubated with unfractionated heparin (2.0 µg). Extraction and GPC were performed as described under “Experimental procedures.” C, effect of ACR on HPSE activity. D, effect of H₂O₂ on HPSE activity. Experiments were repeated in triplicate with reproducible results.

Figure 6

Activated scission activity of proHPSE by acrolein exposure. Effect of ACR (A) or H₂O₂ (B) on the scission activity of proHPSE in conditioned medium containing 50 µg of unfractionated heparin. Preparation of conditioned medium of HEK293 cells transfected with hHPSE1 cDNA and measurement of scission activity of proHPSE by GPC were carried out as described under “Experimental procedures.” Experiments were repeated in triplicate with reproducible results.

Figure 7

Identification of acrolein-modified amino acid residues in proHPSE.A, SDS-PAGE of proHPSE-rich fraction from conditioned medium of HEK293 cells transfected with hHPSE1 cDNA. The 65-kDa protein was recognized by Coomassie Brilliant Blue staining. B, amino acid sequences of proHPSE were determined using nano LC–MS/MS. Identified amino acid sequences are shown in green, based on the results of peptide analysis by nano LC–MS/MS. The acrolein-modified amino acids of proHPSE are shown in yellow (incubated without acrolein for 24 h) or red (exposed to 500 µm acrolein for 24 h). The 6-kDa linker is underlined. C, location of acrolein-modified amino acid residues on proHPSE (PDB ID: 5LA4). Cyan, 8-kDa subunit; green, 6-kDa linker; light blue, 50-kDa subunit.