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LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.

LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.

The distribution of lamprey gonadotropin-releasing hormone (GnRH)-I and -III has been extensively characterized by immunocytochemistry in the forebrain of the sea lamprey, Petromyzon marinus. However, the cellular location of lamprey GnRH-III mRNA expression by in situ hybridization in the lamprey brain has not been determined. We show for the first time the location of expression of lamprey GnRH-III, as well as provide a more comprehensive in situ study of lamprey GnRH-I and glutamic acid decarboxylase (GAD; GABA-synthesizing enzyme) mRNA expression in the brain of the lamprey in different reproductive life stages. Colorimetric and dual-label fluorescent amplification methods of in situ hybridization were used on brain tissue sections of adult, juvenile, and larval sea lamprey. In each life stage of the lamprey, expression of lamprey GnRH-I was shown in the preoptic area (POA) and the hypothalamus forming the characteristic arc-like cell population extending from the preoptic nucleus (NPO) to the neurohypophysis. Lamprey GnRH-III expression was also seen in the POA of each life stage in close proximity to lamprey GnRH-I mRNA containing neurons. GAD expression was shown in distinct cell clusters in and around the POA, in the olfactory bulb, in the dorsal thalamus beneath the habenular region, and also in the ventral-medial hypothalamus stretching from the periventricular region to the anterior portion of the rhombencephalon. Using dual-label in situ hybridization, we have shown that lamprey GnRH-I and -III mRNA are colocalized in the same cells in the POA in adult lampreys. Dual-label in situ hybridization also showed close proximity of GAD mRNA containing neurons and GnRH containing neurons in the POA. These data suggest that γ-aminobutyric acid (GABA) may directly affect GnRH release in the brain of the sea lamprey.