Ligand Binding and Signaling of HARE/Stabilin-2

The Stabilin receptors are a two-member family in the type H class of scavenger receptors. These dynamic receptors bind and internalize multiple ligands from the cell surface for the purpose of clearing extracellular material including some synthetic drugs and for sensing the external environment of the cell. Stabilin-1 was the first receptor to be cloned, though the biological activity of Hyaluronic Acid Receptor for Endocytosis (HARE)/Stabilin-2 was observed about 10 years prior to the cloning of Stabilin-1. Stabilin-1 has a more diverse expression profile among the tissues than HARE/Stabilin-2. This review will focus on HARE/Stabilin-2 and its interactions with hyaluronan, heparin, and phosphorothioate antisense oligonucleotides and what is known about how this receptor participates in signaling upon ligand binding

Role of the Hyaluronan Receptor, Stabilin-2/HARE, in Health and Disease

Stabilin-2/HARE is the primary clearance receptor for circulating hyaluronan (HA), a polysaccharide found in the extracellular matrix (ECM) of metazoans. HA has many biological functions including joint lubrication, ocular turgor pressure, skin elasticity and hydration, cell motility, and intercellular signaling, among many others. The regulatory system for HA content in the tissues, lymphatics, and circulatory systems is due, in part, to Stabilin-2/HARE. The activity of this receptor was discovered about 40 years ago (early 1980s), cloned in the mid-1990s, and has been characterized since then. Here, we discuss the overall domain organization of this receptor and how it correlates to ligand binding, cellular signaling, and its role in known physiological disorders such as cancer

\u3ci\u3eIn vivo\u3c/i\u3e Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2. SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7. With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11. In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SECspecific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis

Antisense oligonucleotides: treatment strategies and cellular internalization

The clinical application of antisense oligonucleotides (ASOs) is becoming more of a reality as several drugs have been approved for the treatment of human disorders and many others are in various phases in development and clinical trials. ASOs are short DNA/RNA oligos which are heavily modified to increase their stability in biological fluids and retain the properties of creating RNA-RNA and DNA-RNA duplexes that knock-down or correct genetic expression. This review outlines several strategies that ASOs utilize for the treatment of various congenital diseases and syndromes that develop with aging. In addition, we discuss some of the mechanisms for specific non-targeted ASO internalization within cells

Tissue-specific Splice Variants of HARE/Stabilin-2 are Expressed in Bone Marrow, Lymph Node, and Spleen

The hyaluronan receptor for endocytosis (HARE), or Stabilin-2, is the mammalian endocytic clearance receptor for HA, heparin, advanced glycation end-products, acetylated and oxidized low-density lipoproteins and collagen N-terminal propeptides. This large 2551 amino acid receptor is encoded by a gene that covers over 180 kbp on human chromosome 12 and is predicted to be composed of 69 exons. Due to the expression profile of this gene and the number of exons it contains, we hypothesized that splice variants of stab2 are encoded in these tissues. In addition, a correlation between alternative splice variants and cancer progression has been shown in other HA receptors such as RHAMM and CD42. In this study, two methods were utilized in identifying and/or isolating the HARE splice variants. The first method used primer sets to amplify the 190-HARE encoding region that could contain splice junctions; therefore, they could be removed from the gel, purified, and sequenced. Five splice variants were detected in that manner. In the second approach, the entire open reading frame of HARE was amplified. This allowed four splice variants with extensive exon splicing to be isolated. After the splice variants were sequenced, three were cloned into a mammalian expression vector. Next, stable cell lines expressing the variants were created in order to determine stable protein expression. In this study, the splice variants were found to be tissue specific in most cases. This means that tissue specific regulatory splicing mechanisms may lead to differences in functionality between the splice variants

Antisense oligonucleotides: treatment strategies and cellular internalization

The clinical application of antisense oligonucleotides (ASOs) is becoming more of a reality as several drugs have been approved for the treatment of human disorders and many others are in various phases in development and clinical trials. ASOs are short DNA/RNA oligos which are heavily modified to increase their stability in biological fluids and retain the properties of creating RNA-RNA and DNA-RNA duplexes that knock-down or correct genetic expression. This review outlines several strategies that ASOs utilize for the treatment of various congenital diseases and syndromes that develop with aging. In addition, we discuss some of the mechanisms for specific non-targeted ASO internalization within cells

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2

SECs (Sinusoidal Endothelial Cells), Liver Microenvironment, and Fibrosis

Liver fibrosis is awound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs phenotype including lack of LSEC fenestration, capillarization, and formation of an organized basement membrane have been shown to precede fibrosis and promote hepatic stellate cell activation. Here, we review the interplay of LSECs with the dynamic changes in the fibrotic liver microenvironment such as matrix rigidity, altered ECM protein profile, and cell-cell interactions to provide insight into the pivotal changes in LSEC physiology and the extent towhich it mediates the progression of liver fibrosis. Establishing the molecular aspects of LSECs in the light of fibrotic microenvironment is valuable towards development of novel therapeutic and diagnostic targets of liver fibrosis

Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin‑2 Clearance Receptor

The Stabilin receptors are systemic clearance receptors for some classes of chemically modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarizationbased assay. The tested ASOs varied in their backbone composition, modification of the ribose 2′ position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5−10−5 pattern of flanking 2′-O-methoxyethyl modifications was then used to test the effects of pH and salt concentration on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissociation and fit within the biological parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors