Endothelial and epithelial expression of sialyl Lewisx and sialyl Lewisa in lesions of breast carcinoma

Tumor cells can invade and generate metastasis via either lymphatics or blood vessels. When tumor cells are circulating in the blood, they must adhere to the vessel wall, which is lined by endothelium, before they can extravasate and form new metastases. Several families of adhesion molecules have been identified to play a role in the extravasation cascade. Selectins and their sialyl Lewis^x and/or sialyl Lewis^a (sLe^x and sLe^a, respectively) containing ligands play an initiating role in this cascade; we have now analyzed their role in the generation of metastatic breast carcinoma lesions. We examined expression of endothelial E- and P-selectin, expression of epithelial and endothelial sLe^x and sLe^a normal tissues compared with primary and metastatic breast in carcinoma lesions within individual patients. While normal breast epithelial cells do not express sLe^x or sLe^a, epithelial expression of these oligosaccharide epitopes was enhanced in primary breast carcinoma lesions. Furthermore, epithelial expression levels of sLe^x and/or sLe^a were even higher in most patients (9 of 12) who had metastatic compared with primary lesions. We show that endothelia in primary lesions express more sLe^x than in normal tissue and that metastatic lesions express even higher amounts of sLe^x compared with primary lesions. The expression of P- and E-selectin was also greatly enhanced in tumor-bearing tissue compared with normal tissue. Our data support the hypothesis that while they are circulating in the blood, sLe^x- and/or sLe^a-expressing carcinoma cells have a higher probability for extravasation at sites where the endothelium expresses E- and P-selectin and for generation of new metastases. Int. J. Cancer 74:296-300, 1997. © 1997 Wiley-Liss, Inc.     

The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 β-1,6- N -acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

Background  

The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLe^x) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 β1,6 N -acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLe^x (C2-O-sLe^x). Our goal was to determine the expression profiles of C2-O-sLe^x in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLe^x present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLe^x was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 β1,6- N -acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).     

Attachment of human colon cancer cells to vascular endothelium is enhanced by N-acetylglucosaminyltransferase V

Expression of N-acetylglucosaminyltransferase V (GnT-V) in colon cancer has been shown to be related to hematogenous metastasis and poor prognosis. To investigate the mechanism by which cancer cells expressing GnT-V metastasize to distant organs, we established GnT-V-overexpressing DLD-1 and WiDr cells (human colon cancer cell lines) by transfecting them with a GnT-V expression vector. Attachment to endothelial cells expressing E-selectin was studied, and expression of the E-selectin ligand, sialyl Lewis x, in colon cancer cells was investigated. Both of the cell lines showed reduced adhesion to fibronectin as compared with mock transfectants. In contrast, attachment to human umbilical vein endothelial cells expressing E-selectin was significantly enhanced by GnT-V expression (p < 0.01). Sialyl Lewis x is a ligand for E-selectin and a marker for poor prognosis of colon cancer. Its synthesis in cells has been shown to involve GnT-V. We demonstrated that expression of sialyl Lewis x in colon cancer cells was induced by GnT-V expression. These results suggest that GnT-V induces sialyl Lewis x expression and leads colon cancer cells to metastasize by enhancing their ability to attach to vascular endothelium in distant organs, such as liver or lung. Inhibition of GnT-V activity may prevent metastasis in colon cancer patients with high sialyl Lewis x expression.     

Increased level of circulating adhesion molecules in the sera of breast cancer patients with distant metastases

The adhesion of circulating cancer cells to the vascular endothelium is an important at step in the hematogenous metastasis of cancer. E-selectin expressed on endothelial cells and carbohydrate ligands expressed on cancer cells mediate this adhesion. We investigated the clinical significance of such cell adhesion molecules in breast cancer. The cytosol concentration of sialyl Lewis(x) was found more elevated in cancerous tissue than that in adjacent non-cancerous tissue. In the serum, sialyl Lewis(x) and soluble E-selectin were seen elevated in patients with advanced and recurrent breast cancer, especially in those with distant metastases. From the above, we have concluded that sialyl Lewis(x) and soluble E-selectin could be used as tumor markers with a close relationship to the metastasis of breast cancer.      

E-selectin binding by pancreatic tumor cells is inhibited by cancer sera

Tumor cells interact with endothelial cells during both intraand extravasation. Understanding how these interactions are modulated could lead to the development of ways to alter the metastatic potential of tumor cell. Three pancreatic cancer cell lines, SW1990, CAPAN-2 and PANC-1, were examined for their ability to bind to the endothelial cell adhesion molecule E-selectin (ELAM-1). SW1990 cells exhibited highest binding, highest surface expression of the carbohydrate antigens sialylated Lewis² (sLe²) and sialylated Lewis^x (sLe^x) and released the most high m. w. sLe² and sLe^x antigens. Expression of sLe² and sLe^x antigens and binding to E-selectin were reduced by pre-treatment of SW1990 cells with the O-linked glycosylation inhibitor benzyl-α-GaINAc but not with the N-linked glycosylation inhibitor tunicamycin. Expression of peptide epitopes associated with MUCI apomucins was increased by benzyl-α-GaINAc. Cell binding was greatly reduced by mucins purified from SW1990 xenografts and by an antibody against sLe². An antibody against sLe^x had a much less marked effect. Sera from pancreatic cancer patients reduced SW1990 cell binding to E-selectin but sera from normals did not. The degree of inhibition was related to the sLe^x level in the sample. When cancer serum was separated by column chromatography on Sephacryl S-400, the void volume fractions contained most of the sLe² and sLe^x antigens and most of the inhibitory activity to E-selectin binding. Differences in the relative availability of sLe² and sLe^x ligands on serum molecules and on the SW1990 cell surface may account for the differences between antibody and serum inhibition results. Thus SW1990 cell adhesion to E-selectin is mediated by ligands on mucinous glycoproteins, and adhesion can be inhibited by mucins, high blood levels of sLe^x and reduction of cellular O-linked glycosylation. © 1994 Wiley-Liss, Inc.     

Tumor-microenvironment interactions: the fucose-generating FX enzyme controls adhesive properties of colorectal cancer cells

Extravasation of tumor cells is a pivotal step in metastasis formation. This step is initiated by an interaction of extravasating tumor cells with endothelial cells. Among the molecules mediating tumor-endothelium interactions are selectins and their fucosylated ligands. In a previous study, we demonstrated that the fucose-generating FX enzyme regulates the expression of selectin ligands by B and T lymphocytes and by head and neck squamous cell carcinoma cells. It was also shown that the FX enzyme regulated important interaction parameters between these cancer cells and endothelial cells. The present study was aimed to determine whether the FX enzyme controls adhesive interactions between colorectal cancer cells and endothelial cells. The results clearly indicate that this is indeed the case. Overexpressing the FX enzyme by the transfer of FX cDNA to low FX-expressing colorectal cancer cells resulted in an increased adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. Down-regulating FX levels in colorectal cancer cells expressing high levels of endogenous FX by transfection with small-interfering RNA resulted in a down-regulated expression of the selectin ligand sialyl Lewis-a and a decrease in the adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. These transfection experiments also indicated that manipulating the levels of the FX enzyme affected global cellular fucosylation and altered the interaction of colorectal cancer cells with some extracellular matrix components such as fibronectin. We also found that highly metastatic colorectal cancer variants express higher levels of FX and of sialyl Lewis-a than low metastatic variants originating in the same tumors. These results lead us to hypothesize that the FX enzyme controls the capacity of colorectal cancer to extravasate and form metastasis. If this hypothesis will be confirmed the FX enzyme could become a target molecule for metastasis prevention.     

Liver metastasis and adhesion to the sinusoidal endothelium by human colon cancer cells is related to mucin carbohydrate chain length

Mucin production by human colon cancer cells correlates with liver metastasis in animal models, but it is not known which steps in metastasis depend on specific alterations in mucin synthesis. Clonal variants of cell line LS174T selected for differences in mucin core carbohydrate expression have been further characterized biochemically, and tested for their ability to participate in metastasis-related events. LS-C mucin contains truncated carbohydrates enriched for sialyl Tn and these cells bind to basement membrane matrix to a greater extent than LS-B cells. This binding is partially inhibitable by antibody to sialyl Tn. LS-B produces more fully glycosylated mucin and preferentially binds to hepatic sinusoidal endothelial cells and E-selectin through sialylated peripheral mucin-associated carbohydrate structures. Adhesion of LS-B to endothelial cells is inhibited by neutralizing antibody to E-selectin, and inhibition of glycosylation or desialylation of LS-B mucin abrogates binding to E-selectin in vitro<0R>. LS-B cells spontaneously metastasized from cecum to liver and colonized the liver of athymic mice after splenic-portal injection to a significantly greater extent than LS-C, suggesting that expression of peripheral mucin carbohydrate structures is most important for metastasis of human colon cancer cells. Int. J. Cancer 76:556–562, 1998.© 1998 Wiley-Liss, Inc.     

Cyclooxygenase-2 activity altered the cell-surface carbohydrate antigens on colon cancer cells and enhanced liver metastasis

Cyclooxygenase-2 (COX-2) was recently reported (M. Tsujii and R. N. DuBois, Cell, 83: 493-501, 1995) to affect the metastatic potential of cells. Previous studies (M. Fukuda, Cancer Res., 56: 2237-2244, 1996) indicated that sialyl Lewis antigen expression is correlated with hematogenous metastasis of colon cancer. In the present study, we investigated the interaction between COX-2 activity, expression of sialyl Lewis antigens, in vitro cancer cell adhesion to endothelial cells, and in vivo metastatic potential. Effects of COX-2 activity and prostaglandin E(2) on cell adhesion, expression of sialyl Lewis antigens, and glycosyltransferase genes were determined in Caco-2-m (COX-2 low level), Caco-2-COX-2 (programmed to overexpress COX-2), and HT-29 (COX-2 high level) cells. Metastatic spread of these cells to the liver was also investigated. Caco-2-COX-2 cells had increased SPan-1 levels and increased adherence to endothelial cells via SPan-1 compared with Caco-2-m cells. HT-29 cells expressed sialyl Lewis a and adhered to endothelial cells via sialyl Lewis a. Treatment with a COX-2 inhibitor, celecoxib, decreased SPan-1 and sialyl Lewis a expression and adherence to endothelial cells. beta 3Gal-T5 and ST3Gal III and IV expression was inhibited by celecoxib and was enhanced by prostaglandin E(2) treatment. Caco-2-COX-2 and HT-29 cells metastasized to the liver, whereas Caco-2-m cells did not. Pretreatment with celecoxib reduced the metastatic potential as well as anti-sialyl Lewis antibodies. Our results indicate a direct link between COX-2 and enhanced adhesion of carcinoma cells to endothelial cells, and enhanced liver metastatic potential via accelerated production of sialyl Lewis antigens. COX-2 inhibitors may suppress metastasis.     

SA-Lea and tumor metastasis: the old prediction and recent findings

Several in vivo studies demonstrated that tumor metastasis depend on the expression of carbohydrate Lewis structures. Lewis antigens and their derivatives such as Lewis b (Leb), Lewis X (LeX), sialyl Lewis X (SA-LeX), sialyl Lewis a (SA-Lea), and Lewis Y (LeY) were identified as tumor-associated structures approximately 20 years ago by Koprowski et al. using hybridoma technology and showed that upregulation and/or de novo expression of these determinants on the tumor cell surface is associated with a poor prognosis. LeX and SA-LeX are ligands for selectin adhesion molecules; E- and P-selectins are vascular receptors expressed on activated endothelial cells (ECs) and L-selectin is expressed on leukocytes. Leukocytes also express on their surface LeX and SA-LeX determinants, which are involved in the initial steps of extravasation, that is, rolling, which is alpha step mediated by interaction with E-selectin on ECs. We hypothesized that the tumor cells transmigration from the bloodstream to metastatic sites is similar to lymphocyte extravasation and that adhesion of cancer cells in analogy with the lymphocyte rolling is mediated by interaction of carbohydrate determinants on tumor cells with selectins on ECs. To assess the role of interaction of carbohydrate structures with E-selectin in metastatic process in vivo, we demonstrated that the peptides mimicking SA-Lea blocked colonization of tumor cells in experimental model of lung metastasis in vivo. Furthermore, the metastases formation was completely attenuated in E-selectin-knock out (KO) mice demonstrating the importance of selectin-mediated interaction in this process. We also showed that a peptide mimicking SA-Lea E-selectin ligand has an ability to significantly reduce neutrophil recruitment into peritoneal cavity in acute inflammatory conditions. These studies support the hypothesis that the interaction of tumor cells via the carbohydrate SA-Lea determinant and E-selectin constitutes the important step in the metastatic process in analogy with lymphocyte extravasation and that carbohydrate antigen mimics have a potential as anti-inflammatories and anti-adhesive tumor therapeutics.     

Sialyl-lewis-x antigen immunoreaction of colorectal-cancer and its relationship to hematogenous metastasis

The adhesion of cancer cells to endothelial cells of the target organ is one of the most important steps of hematogenous metastasis. Especially, sialyl Le(X) plays an important role in defining the metastasis. The expression of sialyl Le(X) antigen in colorectal cancer and its usefulness not only as an indicator of metastatic potential but also as a prognostic factor was studied immunohistochemically. Fifty-five (32.4%) sialyl Le(X) antigen-positive tumors were found in 170 colorectal cancers. There was a significant correlation between the expression of sialyl Le(X) antigen and the histological tumor type, venous invasion, lymph node metastasis, as well as liver metastasis. Hematogenous metastases were significantly more frequent in patients with sialyl Le(X)-positive tumor than in those with sialyl Le(X)-negative tumor; and prognosis was significantly poorer in the former. The results suggest that sialyl Le(X) antigen plays a role in hematogenous metastasis of colorectal cancer, and that the expression of sialyl Le(X) is associated with poor prognosis.     

Adhesion of human breast cancer cells to vascular endothelium mediated by Sialyl Lewisx/E-selectin

The adhesion molecules involved in the attachment of breast cancer cells to endothelial cells were investigated in vitro. All six human breast cancer cell lines expressed sialyl Lewis &supx; antigen(s-Le &supx;). Only two cell lines expressed sialyl Lewis&supa; antigen. A correlation was found between the degree of s-Le&supx; expression and the attachment of cancer cells to human umbilical vein endothelial cells (HUVECs) activated by IL-1beta. Monoclonal antibodies against s-Le &supx; or E-selectin inhibited this adhesion. These findings suggest that s-Le &supx; on the surface of cancer cells and E-selectin on the surface of endothelial cells play roles in adheision of breast cancer cells to vascular endothelium.     

Human colon cancer cells express multiple glycoprotein ligands for E-selectin

The interaction between the colon tumor cell surface and the endothelial cell layer is an important component of tumor intravasation, extravasation, and metastasis. Multiple studies suggest that tumor cells may bind to E-selectin expressed on endothelial cells during these processes. To identify possible E-selectin ligands on tumor cells that may participate in this mechanism, we used E-selectin-Ig chimera affinity chromatography to isolate glycoproteins from the human colon cancer cell line Colo-205. Binding of these cells to E-selectin was specific, required the presence of calcium, and could be blocked by antibodies against E-selectin. We identified LAMP-1 (lysosomal membrane glycoprotein-1), LAMP-2, and two high molecular weight glycoproteins (>400 kDa and 300 kDa) as the main E-selectin ligands on Colo-205 cells. Treatment of the cells with N-glycanase and O-sialoglycoprotease abolished their binding to E-selectin. The high MW glycoproteins contained sialyl Lewis X and/or sialyl Lewis A glycoconjugates, and appeared to be either alternatively spliced or alternatively glycosylated forms of MUC-1 (mucin-1).     

Dimethyl sulfoxide (DMSO) increases expression of sialyl lewis X antigen and enhances adhesion of human gastric carcinoma (NUGC4) cells to activated endothelial cells

Dimethyl sulfoxide (DMSO) exerts a number of biological effects including the promotion of cell differentiation in cultured cells. In this study, we examined the effect of DMSO on the adhesion of tumor cells to endothelial cells. In vitro treatment of human gastric adenocarcinoma (NUGC4) cells with DMSO resulted in increased adhesion to interleukin-1 (IL-l)-activated human endothelial cells compared with DMSO-untreated NUGC4 cells. In flow cytometry, treating NUGC4 cells with DMSO enhanced the expression of sialyl Lewis x (sialyl Le^x) and sialyl dimeric Le^x antigens on their surface. Also, the binding of Limulus polyphemus agglutinin (LPA), which specifically binds to cell-surface sialic acids, was increased by DMSO. The adhesion of DMSO-treated NUGC4 cells to activated endothelial cells was blocked by neuraminidase pre-treatment of tumor cells or by antibody against either endothelial leukocyte adhesion molecule-1 (ELAM-I) or sialyl Le^x. Thus, it is suggested that enhanced adhesion following DMSO treatment is mediated by the interaction of sialyl Le^x expressed on NUGC4 cells with ELAM-I of endothelial cells. The modulation of sialyl Le^x antigen by DMSO provides a useful system for studying the regulatory mechanism of Lewis-related carbohydrate antigens and also for understanding the metastatic properties of cancer cells.     

Inhibition of hepatic endothelial E-selectin expression by C-raf antisense oligonucleotides blocks colorectal carcinoma liver metastasis

Cytokine-dependent induction of E-selectin expression is mediated through cooperative signaling involving the Ras/Raf/mitogen-activated protein kinase pathway. We previously reported that metastatic tumor cells entering the hepaticcirculation rapidly induce a cytokine cascade leading to E-selectin induction (A-M. Khatib, et al., Cancer Res., 59:1356-1361, 1999).Here, we investigated the effect of a blockade of E-selectin induction on colorectal carcinoma metastasis using rodent (host)-specific C-raf antisense oligonucleotides and human colorectal carcinoma CX-1 cells. Pretreatment of hepatic endothelial cells in vitro with the antisense oligonucleotides abrogated E-selectin-dependent CX-1 adhesion. In vivo, pretreatment of nude mice with these oligonucleotides abrogated E-selectin induction in response to intrasplenic/portal inoculation of CX-1 cells, and this reduced the number of liver metastases by 86% relative to controls. The results suggest that the inhibition of tumor-induced, hepatic microvessel E-selectin expression may provide a useful strategy for the prevention of hepatic metastasis.     

Introduction of Sd(a) carbohydrate antigen in gastrointestinal cancer cells eliminates selectin ligands and inhibits metastasis

The Sd(a) blood group carbohydrate structure is expressed in the normal gastrointestinal mucosa. We reported previously that the expression of Sd(a) carbohydrate structures and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT) activity responsible for Sd(a) synthesis were remarkably decreased in cancer lesions of the gastrointestinal tract. In this study, we found that Sd(a) antigen was expressed mainly in chief cells of normal stomach but not in cancer tissue by immunohistologic staining. In separated gastric mucosal cells, the Sd(a) glycolipids and beta1,4GalNAcT activity were concentrated in a fraction that contained chief cells as a major population. We cloned the cDNA encoding the glycosyltransferase that catalyzes the synthesis of Sd(a) (Sd(a)-beta1,4GalNAcT). Introduction of this cloned cDNA into KATO III gastric or HT29 colonic cancer cell lines, which originally expressed the E-selectin ligands, sialyl Lewis(x) and sialyl Lewis(a), resulted in a marked increase in cell-surface expression of Sd(a) along with the concomitant total loss of both sialyl Lewis(x) and sialyl Lewis(a). Both KATO III and HT29 cells transfected with the Sd(a)-beta1,4GalNAcT gene showed significantly decreased adhesion to activated human umbilical vein endothelial cells when compared with mock-transfected cells. Sd(a) determinants showed no direct binding to Siglec-3, -5, -7, and -9. These Sd(a)-beta1,4GalNAcT-transfected cells showed strikingly reduced metastatic potential in vivo when compared with mock-transfected cells. In summary, forced expression of Sd(a) carbohydrate determinant caused remarkable elimination of carbohydrate ligands for selectin and reduced metastasis of human gastrointestinal tract cancer cells.     

Altered mRNA expression of specific molecular species of fucosyl- and sialyl-transferases in human colorectal cancer tissues

Human colorectal cancers express various cancer-associated carbohydrate determinants such as Lewis Y or sialyl Lewis A, suggesting a considerable alteration in glycosyltransferase activities occurring upon malignant transformation. We investigated the mRNA amounts of fucosyltransferase (Fuc-T) and sialyltransferase (ST) isoenzymes, including Fuc-T III, IV, V, VI and VII and ST-3N, ST-30 and ST-4, in human colorectal cancer tissues by Northern blotting and RT-PCR. Regarding fucosyltransferases, mRNA of Fuc-T III and VI was not significantly altered, and only Fuc-T IV mRNA showed a moderate increase in cancer tissues when compared with adjacent non-malignant colonic epithelia taken from the same patient (273 ± 96%; p < 0.001). The moderate increase of Fuc-T IV message may be related to an enhanced expression of Lewis Y in colon cancer tissues. In the ST isoenzymes, mRNA for ST-3N remained unchanged, whereas that for ST-4 decreased significantly in cancer tissues, to 32 ± 29%, (p < 0.005). The most remarkable finding was that the message of ST-30 was prominently increased in cancer tissues compared with non-malignant colorectal mucosa. When further investigated by quantitative RT-PCR assays on a larger series of patients with colorectal cancers, the average increase in mRNA for ST-30 was 459 ± 200% compared with that in adjacent non-malignant epithelium (significant at p < 0.0001). The increase of ST-30 message was more prominent in the cancer tissues strongly expressing sialyl Lewis A than in the cancer tissues expressing sialyl Lewis A only weakly or moderately (significant at p < 0.05). The marked increase in the message of ST-30 is suggested to be related to an enhanced expression of sialylated carbohydrate determinants in colon cancer tissues including sialyl Lewis A, since the enzyme exhibited a significant activity against the type I chain carbohydrate substrate and produced the precursors for sialyl Lewis A synthesis, when its cDNA was expressed in Cos-7 cells. Int. J. Cancer 71:556-564, 1997 © 1997 Wiley-Liss, Inc.     

E-selectin regulates gene expression in metastatic colorectal carcinoma cells and enhances HMGB1 release

Extravasation of cancer cells is a pivotal step in the formation of hematogenous metastasis. Extravasation is initiated by the loose adhesion of cancer cells to endothelial cells via an interaction between endothelial selectins and selectin ligands expressed by the tumor cells. The present study shows that the interaction between recombinant E-selectin (rE-selectin) and colorectal cancer (CRC) cells alters the gene expression profile of the cancer cells. A DNA microarry analysis indicated that E-selectin-mediated alterations were significantly more pronounced in the metastatic CRC variants SW620 and KM12SM than in the corresponding non-metastatic local SW480 and KM12C variants. The number of genes altered by E-selectin in the metastatic variants was about 10-fold higher than the number of genes altered in the corresponding local variants. Aiming to identify genes involved in CRC metastasis, we focused, by using a DNA microarry analysis, on genes that were altered by E-selectin in a similar fashion exclusively in both metastatic variants. This analysis indicated that E-selectin down regulated (at least by 1.6-folds) the expression of 7 genes in a similar fashion, in both metastatic cells. The DNA microarry analysis was validated by real time PCR or by RT-PCR. HMGB1 was among these genes. Confocal microscopy indicated that E-selectin down regulated the cellular expression of the HMGB1 protein and enhanced the release of HMGB1 into the culture medium. The released HMGB1 in turn, activated endothelial cells to express E-selectin.