A Blocking Antibody to the Hyaluronan Receptor for Endocytosis (HARE) Inhibits Hyaluronan Clearance by Perfused Liver

Abstract

Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125 I-HA at 37°C by rat liver sinusoidal endothelial cells.125 I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 g/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues.125 I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.After Meyer and Palmer (1) discovered hyaluronan (HA), 1 it was found to be a component of essentially all vertebrate extracellular matrices (ECMs). Fibroblasts, keratinocytes, chondrocytes, and other cells continuously synthesize and secrete HA, which is a linear polymer with a native molecular mass that may exceed 10 7 Da and is composed of the repeating disaccharide 2-deoxy,2-acetamido-D-glucopyranosyl-␤ (1, 4)-D-glucuronopyranosyl-␤ (1, 3). Despite its simple structure, HA is involved in many cell functions including migration, differentiation, and phagocytosis (2-6). HA is important in development (4, 7), wound healing (8, 9), angiogenesis (10, 11), and tumor growth and metastasis (12, 13). Although previously believed to be only a structural component in the ECM, HA is now also recognized as an active cell-signaling molecule. Some cell types show distinct physiological responses to HA of different sizes. In particular, some cell types respond physiologically to very small, but not large, HA. Small HA oligosaccharides containing 14 -20 sugars stimulate angiogenesis by endothelial cells (10,11,14), induce gene expression in activated macrophages (15), and induce NO synthase expression in sinusoidal LECs and Kupffer cells, but not hepatocytes or stellate cells (16). Cell surface HA receptors identified to date include CD44, RHAMM (CD168), ICAM-1 (CD54), LYVE-1 (5), and an endocytic receptor that is specific for HA and chondroitin sulfate. This latte...

The Hyaluronan Receptor for Endocytosis (HARE) Activates NF-κB-mediated Gene Expression in Response to 40–400-kDa, but Not Smaller or Larger, Hyaluronans

“…The results confirm that HA binding to 190-hHARE is required for HARE-mediated NF-B-activated gene expression. Although we do not have a similar stable cell line expressing rHARE(⌬Link), previous studies showed that mAb-174, which was raised against rHARE, completely blocks HA uptake in stable cells expressing rHARE (29), in primary rat liver sinusoidal endothelial cells, and in intact perfused rat liver (30,49). mAb-174 does not recognize hHARE.…”

Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

“…This is in contrast to other ASGPr ligand-targeted hepatocyte delivery systems, in which significant uptake by other organs was also observed (30,45,50). Although HA receptors are present in spleen and lymph nodes (22), differences in receptor characteristics and a significantly greater avidity of LSEC HARE receptors may account for the efficient uptake of the s50 HA nanocapsules by liver (51). Similarly, while receptors capable of recognizing the galactose moiety are present in other tissues (30,45,50), the greater avidity of the ASGPr for it natural ligand ASOR versus galactose (by 3 orders of magnitude; ref.…”

“…We targeted the hepatocyte asialoglycoprotein receptor (ASGPr) using its natural ligand, asialoorosomucoid (ASOR) (21), while LSECs were targeted using hyaluronan (HA), the endogenous ligand for the HA receptor for endocytosis (HARE) (22). We developed an atomization method to prepare nanocapsules of less than 50 in diameter for delivery of plasmids ranging in size from 5.2 to 12.8 kb and coated with targeting ligand, e.g.…”

Hyaluronan Endocytosis: Mechanisms of Uptake and Biological Functions

“…Indeed, blocking HARE results in an inhibition of HA clearance in the liver [35]. HARE also plays a role in chondroitin sulfate proteoglycan endocytosis [36].…”