Sulfatase 2 (SULF2) Monoclonal Antibody 5D5 Suppresses Human Cholangiocarcinoma Xenograft Growth Through Regulation of a SULF2-Platelet-Derived Growth Factor Receptor Beta-Yes-Associated Protein Signaling Axis.

BACKGROUND AND AIMS: Existing therapeutic approaches to treat cholangiocarcinoma (CCA) have limited effectiveness, prompting further study to develop therapies for CCA. We report a mechanistic role for the heparan sulfate editing enzyme sulfatase 2 (SULF2) in CCA pathogenesis. APPROACH AND RESULTS: In silico analysis revealed elevated SULF2 expression in human CCA samples, occurring partly through gain of SULF2 copy number. We examined the effects of knockdown or overexpression of SULF2 on tumor growth, chemoresistance, and signaling pathway activity in human CCA cell lines in vitro. Up-regulation of SULF2 in CCA leads to increased platelet-derived growth factor receptor beta (PDGFRß)-Yes-associated protein (YAP) signaling activity, promoting tumor growth and chemotherapy resistance. To explore the utility of targeting SULF2 in the tumor microenvironment for CCA treatment, we tested an anti-SULF2 mouse monoclonal antibody, 5D5, in a mouse CCA xenograft model. Targeting SULF2 by monoclonal antibody 5D5 inhibited PDGFRß-YAP signaling and tumor growth in the mouse xenograft model. CONCLUSIONS: These results suggest that SULF2 monoclonal antibody 5D5 or related agents may be potentially promising therapeutic agents in CCA.

Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.

The extracellular lysophospholipase D autotaxin (ATX) and its product, lysophosphatidic acid, have diverse functions in development and cancer, but little is known about their functions in the immune system. Here we found that ATX had high expression in the high endothelial venules of lymphoid organs and was secreted. Chemokine-activated lymphocytes expressed receptors with enhanced affinity for ATX, which provides a mechanism for targeting the secreted ATX to lymphocytes undergoing recruitment. Lysophosphatidic acid induced chemokinesis in T cells. Intravenous injection of enzymatically inactive ATX attenuated the homing of T cells to lymphoid tissues, probably through competition with endogenous ATX and exertion of a dominant negative effect. Our results support the idea of a new and general step in the homing cascade in which the ectoenzyme ATX facilitates the entry of lymphocytes into lymphoid organs.

Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue.

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.

Autotaxin through lysophosphatidic acid stimulates polarization, motility, and transendothelial migration of naive T cells.

Blood-borne lymphocytes home to lymph nodes by interacting with and crossing high endothelial venules (HEVs). The transendothelial migration (TEM) step is poorly understood. Autotaxin (ATX) is an ectoenzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a bioactive lipid and a close relative of sphingosine 1-phosphate. HEVs produce and secrete ATX into the blood. A prior study implicated ATX in the overall homing process, but the step in which it functions and its mechanism of action have not been defined. In this article, we show that HA130, an inhibitor of the enzymatic activity of ATX, slows T cell migration across lymph node HEVs in vivo. Ex vivo, ATX plus LPC or LPA itself induces the polarization of mouse naive T cells and stimulates their motility on an ICAM-1 substratum. Under physiologic shear conditions in a flow chamber, LPA or ATX/LPC strongly enhances TEM of integrin-arrested T cells across an endothelial monolayer. HA130 blunts the TEM-promoting activity of ATX, paralleling its in vivo effects. T cells possess Mn(+2)-activatable receptors for ATX, which are localized at the leading edge of polarized cells. ATX must bind to these receptors to elicit a maximal TEM response, providing a mechanism to focus the action of LPA onto arrested lymphocytes in flowing blood. Our results indicate that LPA produced via ATX facilitates T cell entry into lymph nodes by stimulating TEM, substantiating an additional step in the homing cascade. This entry role for LPA complements the efflux function of sphingosine 1-phosphate.

KSGal6ST generates galactose-6-O-sulfate in high endothelial venules but does not contribute to L-selectin-dependent lymphocyte homing.

The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. Although sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression implicate Gal6S as a binding determinant for the lymphocyte homing receptor, L-selectin. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). In the present study, we use mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin in vivo. First, we establish that KSGal6ST is selectively expressed in high endothelial venules (HEVs) in lymph nodes and Peyer's patches. We also determine by mass spectrometry that KSGal6ST generates Gal6S on several classes of O-glycans in peripheral lymph nodes. Furthermore, KSGal6ST, but not C6ST-1, is required for the generation of the Gal6S-containing glycan, 6,6'-disulfo-3'sLN (Siaα2→3[6S]Galß1→4[6S]GlcNAc) or a closely related structure in lymph node HEVs. Nevertheless, L-selectin-dependent short-term homing of lymphocytes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures we detected do not contribute to L-selectin ligand recognition in this setting. These results refine our understanding of the biological ligands for L-selectin and introduce a mouse model for investigating the functions of Gal6S in other contexts.

Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs.

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs.

"Home sweet home" for lymphocytes.

In this issue of Blood, Hernandez Mir and colleagues provide the most detailed analysis to date of the glycans on an HEV-expressed ligand (CD34) isolated from a human lymphoid organ, (tonsils), adding to our understanding of how L-selectin mediates lymphocyte homing.

Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88.

Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O-sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293, and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O, and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes.

Lymphocyte-HEV interactions in lymph nodes of a sulfotransferase-deficient mouse.

The interaction of L-selectin expressed on lymphocytes with sulfated sialomucin ligands such as CD34 and GlyCAM-1 on high endothelial venules (HEV) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte recruitment. HEC-GlcNAc6ST-deficient mice lack an HEV-restricted sulfotransferase with selectivity for the C-6 position of N-acetylglucosamine (GlcNAc). HEC-GlcNAc6ST-/- animals exhibit faster lymphocyte rolling and reduced lymphocyte sticking in HEV, accounting for the diminished lymphocyte homing. Isolated CD34 and GlyCAM-1 from HEC-GlcNAc6ST-/- animals incorporate approximately 70% less sulfate than ligands from wild-type animals. Furthermore, these ligands exhibit a comparable reduction of the epitope recognized by MECA79, a function-blocking antibody that reacts with L-selectin ligands in a GlcNAc-6-sulfate-dependent manner. Whereas MECA79 dramatically inhibits lymphocyte rolling and homing to lymph nodes in wild-type mice, it has no effect on HEC-GlcNAc6ST-/- mice. In contrast, in vitro rolling on purified GlyCAM-1 from HEC-GlcNAc6ST-/- mice, although greatly diminished compared with that on the wild-type ligand, is inhibited by MECA79. Our results demonstrate that HEC-GlcNAc6ST contributes predominantly, but not exclusively, to the sulfation of HEV ligands for L-selectin and that alternative, non-MECA79-reactive ligands are present in the absence of HEC-GlcNAc6ST.

Gene trap disruption of the mouse heparan sulfate 6-O-endosulfatase gene, Sulf2.

Heparan sulfate (HS) chains are found in the extracellular matrix, covalently linked to core proteins collectively termed heparan sulfate proteoglycans (HSPGs). A wealth of data has demonstrated roles for HSPGs in the regulation of many cell surface signaling pathways that are crucial during development. Variations in the sulfation pattern along the HS chains influence their ability to interact with molecules such as growth factors, chemokines, morphogens, and adhesion molecules. Sulf1 and Sulf2 are members of a class of recently identified genes that encode heparan sulfate 6-O-endosulfatases (Sulf genes). The removal of 6-O-sulfate from HS via SULF activity influences the function of many factors, including Wnt, fibroblast growth factor, hepatocyte growth factor, heparin-binding epidermal growth factor, and bone morphogenetic protein. Given their possible developmental roles, we have examined Sulf gene expression during mouse embryogenesis. The two Sulf genes are expressed in a broad range of tissues throughout development with largely nonoverlapping expression patterns. Sulf2 transcripts are expressed in the lung, heart, placenta, and ribs. We generated a mouse line possessing a gene trap disruption of the Sulf2 gene. Mice homozygous for the Sulf2 gene trap allele are viable and fertile and have no major developmental defects on several genetic backgrounds. However, we observed strain-specific, nonpenetrant defects affecting viability, lung development, and growth in Sulf2 homozygous animals. These data suggest that Sulf2 may have roles in several tissues but that there is compensation by and/or redundancy with Sulf1.

Endoglycan, a member of the CD34 family of sialomucins, is a ligand for the vascular selectins.

The interactions of the selectin family of adhesion molecules with their ligands are essential for the initial rolling stage of leukocyte trafficking. Under inflammatory conditions, the vascular selectins, E- and P-selectin, are expressed on activated vessels and interact with carbohydrate-based ligands on the leukocyte surface. While several ligands have been characterized on human T cells, monocytes and neutrophils, there is limited information concerning ligands on B cells. Endoglycan (EG) together with CD34 and podocalyxin comprise the CD34 family of sialomucins. We found that EG, previously implicated as an L-selectin ligand on endothelial cells, was present on human B cells, T cells and peripheral blood monocytes. Upon activation of B cells, EG increased with a concurrent decrease in PSGL-1. Expression of EG on T cells remained constant under the same conditions. We further found that native EG from several sources (a B cell line, a monocyte line and human tonsils) was reactive with HECA-452, a mAb that recognizes sialyl Lewis X and related structures. Moreover, immunopurified EG from these sources was able to bind to P-selectin and where tested E-selectin. This interaction was divalent cation-dependent and required sialylation of EG. Finally, an EG construct supported slow rolling of E- and P-selectin bearing cells in a sialic acid and fucose dependent manner, and the introduction of intact EG into a B cell line facilitated rolling interactions on a P-selectin substratum. These in vitro findings indicate that EG can function as a ligand for the vascular selectins.

Induction of PNAd and N-acetylglucosamine 6-O-sulfotransferases 1 and 2 in mouse collagen-induced arthritis.

BACKGROUND: Leukocyte recruitment across blood vessels is fundamental to immune surveillance and inflammation. Lymphocyte homing to peripheral lymph nodes is mediated by the adhesion molecule, L-selectin, which binds to sulfated carbohydrate ligands on high endothelial venules (HEV). These glycoprotein ligands are collectively known as peripheral node addressin (PNAd), as defined by the function-blocking monoclonal antibody known as MECA-79. The sulfation of these ligands depends on the action of two HEV-expressed N-acetylglucosamine 6-O-sulfotransferases: GlcNAc6ST-2 and to a lesser degree GlcNAc6ST-1. Induction of PNAd has also been shown to occur in a number of human inflammatory diseases including rheumatoid arthritis (RA). RESULTS: In order to identify an animal model suitable for investigating the role of PNAd in chronic inflammation, we examined the expression of PNAd as well as GlcNAc6ST-1 and -2 in collagen-induced arthritis in mice. Here we show that PNAd is expressed in the vasculature of arthritic synovium in mice immunized with collagen but not in the normal synovium of control animals. This de novo expression of PNAd correlates strongly with induction of transcripts for both GlcNAc6ST-1 and GlcNAc6ST-2, as well as the expression of GlcNAc6ST-2 protein. CONCLUSION: Our results demonstrate that PNAd and the sulfotransferases GlcNAc6ST-1 and 2 are induced in mouse collagen-induced arthritis and suggest that PNAd antagonists or inhibitors of the enzymes may have therapeutic benefit in this widely-used mouse model of RA.

Measuring the activities of the Sulfs: two novel heparin/heparan sulfate endosulfatases.

Sulfatases hydrolyze sulfate esters on a variety of molecules including glycosaminoglycans, sulfoglycolipids, and cytosolic steroids. These enzymes are found in a wide range of organisms with their basic enzymatic mechanisms broadly conserved. In mammals, many of the sulfatases localize in the lysosome and exhibit enzymatic activity on a small aryl substrate such as 4-methylumbelliferyl sulfate (4-MUS). They are known as arylsulfatases. Sulf-1 and Sulf-2 have been cloned and identified as sulfatases that release sulfate groups on the C-6 position of GlcNAc residue from an internal subdomain in intact heparin. Hence, these enzymes are endosulfatases. The Sulfs are secreted in an active form into conditioned medium of transfected Chinese hamster ovary (CHO) cells. In this chapter, arylsulfatase and endoglucosamine-6-sulfatase assays for the Sulfs are described. A solid-phase binding assay is also detailed, which allows investigation of the ability of the Sulfs to modulate the interaction of heparin-binding proteins with immobilized heparin. The example illustrated is vascular endothelial growth factor (VEGF). This assay is projected to be very useful in the investigation of the biological functions of the Sulfs.

HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1.

BACKGROUND: Heparin/heparan sulfate (HS) proteoglycans are found in the extracellular matrix (ECM) and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs) interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF), cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin. RESULTS: Our results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC) to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities. CONCLUSION: Our results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.

Expression of the extracellular sulfatase SULF2 is associated with squamous cell carcinoma of the head and neck.

Sulfatase 2 (SULF2), an extracellular sulfatase that alters sulfation on heparan sulfate proteoglycans, is involved in the tumorigenesis and progression of several carcinomas. SULF2 expression has not been evaluated in squamous cell carcinoma of the head and neck (HNSCC). Here we report results of IHC of SULF2 expression in HNSCC tissue. SULF2 was detected in 57% of tumors (n = 40) with a significant increase in intensity and number of stained cells compared to adjacent cancer-free tissue (p-value < 0.01), increasing with cancer stage when comparing stages 1 and 2 to stages 3 and 4 (p-value 0.01). SULF2 was not detected in epithelial cells of cancer-free controls, and expression was independent of patient demographics, tumor location and etiological factors, smoking and HPV infection by p16 IHC analysis. Sandwich ELISA was performed on serum of HNSCC patients (n = 28) and controls (n = 35), and although SULF2 was detectable, no change was observed in HNSCC. Saliva, collected by mouthwash, from HNSCC patients (n = 8) and controls (n = 8) was also tested by ELISA in a preliminary investigation and an increase in SULF2 was observed in HNSCC (p-value 0.041). Overall, this study shows that SULF2 is increased in HNSCC independent of tissue location (oral cavity, oropharynx, larynx and hypopharynx), patient demographics and etiology. Although no change in SULF2 was detected in HNSCC serum, its detection in saliva makes it worthy of further investigation as a potential HNSCC biomarker.

Sulf-2, a proangiogenic heparan sulfate endosulfatase, is upregulated in breast cancer.

Sulf-2 is an endosulfatase with activity against glucosamine-6-sulfate modifications within subregions of intact heparin. The enzyme has the potential to modify the sulfation status of extracellular heparan sulfate proteoglycan (HSPG) glycosaminoglycan chains and thereby to regulate interactions with HSPG-binding proteins. In the present investigation, data mining from published studies was employed to establish Sulf-2 mRNA upregulation in human breast cancer. We further found that cultured breast carcinoma cells expressed Sulf-2 mRNA and released enzymatically active proteins into conditioned medium. In two mouse models of mammary carcinoma, Sulf-2 mRNA was upregulated in comparison to its expression in normal mammary gland. Although mRNA was present in normal tissues, Sulf-2 protein was undetectable; it was, however, detected in some premalignant lesions and in tumors. The protein was localized to the epithelial cells of the tumors. In support of the possible mechanistic relevance of Sulf-2 upregulation in tumors, purified recombinant Sulf-2 promoted angiogenesis in the chick chorioallantoic membrane assay.

A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-alpha/beta and TNF-alpha in cultured endothelial cells.

BACKGROUND: The recruitment of lymphocytes to secondary lymphoid organs relies on interactions of circulating cells with high endothelial venules (HEV). HEV are exclusive to these organs under physiological conditions, but they can develop in chronically-inflamed tissues. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV results in lymphocyte rolling, which represents the initial step in lymphocyte homing. HEV expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, GST-3, LSST or CHST4), an HEV-restricted sulfotransferase, is essential for the elaboration of L-selectin functional ligands as well as a critical epitope recognized by MECA-79 mAb. RESULTS: We examined the expression of GlcNAc6ST-2 in relationship to the MECA-79 epitope in rheumatoid arthritis (RA) synovial vessels. Expression of GlcNAc6ST-2 was specific to RA synovial tissues as compared to osteoarthritis synovial tissues and localized to endothelial cells of HEV-like vessels and small flat-walled vessels. Double MECA-79 and GlcNAc6ST-2 staining showed colocalization of the MECA-79 epitope and GlcNAc6ST-2. We further found that both TNF-alpha and lymphotoxin-alphabeta induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells. CONCLUSION: These observations demonstrate that GlcNAc6ST-2 is induced in RA vessels and provide potential cytokine pathways for its induction. GlcNAc6ST-2 is a novel marker of activated vessels within RA ectopic lymphoid aggregates. This enzyme represents a potential therapeutic target for RA.

Differential gene expression profile of human tonsil high endothelial cells: implications for lymphocyte trafficking.

Lymphocyte recirculation is dependent on the interactions of adhesion and signaling molecules expressed on lymphocytes and their partners on high endothelial cells (HEC). Many of the events in this process have yet to be molecularly characterized. To identify novel HEC-specific proteins with potential function in the recruitment cascade, we sequenced a normalized human tonsil HEC cDNA library (generated from an inflamed tonsil) from which lymphocyte and human umbilical vein endothelial cell cDNAs had been subtracted. One-thousand forty-nine sequences were analyzed. All but three mapped to known cDNAs or genomic DNAs. The two most abundant transcripts encoded alpha2-macroglobulin and hevin. The next-abundant transcripts encoded several other protease inhibitors, making this protein class the most prominent in HEC. Several endothelial-specific transcripts were also identified, including those encoding E-selectin, vascular cell adhesion molecule-1, vascular endothelial-junctional adhesion molecule, and platelet-endothelial cell adhesion molecule-1. The library contains a great diversity of transcripts, and studies of the encoded proteins will provide further insight into the complex biology of these specialized endothelial cells.

SULF2, a heparan sulfate endosulfatase, is present in the blood of healthy individuals and increases in cirrhosis.

BACKGROUND: SULF2 is an extracellular sulfatase that acts on heparan sulfate proteoglycans and modulates multiple signaling pathways. It is normally bound to the cell surface but can be released into the medium of cultured cells. SULF2 is known to be increased in cirrhotic liver compared to healthy liver. We asked whether SULF2 protein was present in the blood of healthy controls and increased in patients with liver cirrhosis. METHODS: We devised a sandwich ELISA for SULF2 using 2 novel monoclonal antibodies (mAbs) and measured its levels in sera of normal individuals and cirrhosis patients. RESULTS: SULF2 was higher in cirrhosis patients (1460 ± 1160 pg/ml, N=34) than in healthy individuals (728 ± 400 pg/ml, N=37). SULF2 levels increased with age in both healthy and patient groups. CONCLUSIONS: SULF2 may be a useful serologic biomarker for liver cirrhosis.