Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36.

Transmembrane protein CD36 binds multiple ligands, including oxidized low-density lipoproteins (oxLDLs) and long-chain fatty acids (LCFAs). Our aim was to determine whether LCFAs compete with oxLDLs for binding to CD36. We addressed this issue by examining the inhibitory effect of LCFAs against the binding of Alexa-fluor-labeled oxLDLs (AFL-oxLDL) to a synthetic peptide representing the oxLDL-binding site on CD36 (3S-CD36150₋168). All of the unsaturated LCFAs tested, inhibited the binding of AFL-oxLDL to 3S-CD36150₋168, albeit to varying degrees. For instance, the concentrations required for 50% inhibition of binding for oleic, linoleic, and α-linolenic acids were 0.25, 0.97, and 1.2 mM, respectively. None of the saturated LCFAs tested (e.g. stearic acid) exhibited inhibitory effects. These results suggest that at least unsaturated LCFAs can compete with oxLDLs for binding to CD36. The study also provides information on the structural requirements of LCFAs for inhibition of oxLDLs-CD36 binding.

A synthetic peptide-based assay system for detecting binding between CD36 and an oxidized low-density lipoprotein.

CD36 is an integral membrane protein that mediates the cellular uptake of oxidized low-density lipoprotein (oxLDL) through recognition of the oxidized glycerophospholipids (oxPLs) formed during LDL oxidation. We aimed to devise an assay system to detect binding between CD36 and oxLDL/oxPL without using recombinant proteins. A peptide corresponding to amino-acid residues 149-168 of mouse CD36 with biotin at its N-terminus (named biotin-CD36(149-168)) and variants of it were synthesized and immobilized onto streptavidin-coated plates. oxLDL labeled with Alexa-Fluor-488 bound specifically and saturably to immobilized biotin-CD36(149-168), but poorly or not at all to the variants, such as that with a scrambled amino-acid sequence. The binding of fluorescence-labeled oxLDL to biotin-CD36(149-168) was inhibited efficiently by an oxPL species, but not by a nonoxidized glycerophospholipid. This assay system using biotin-CD36(149-168) provides a convenient means not only of characterizing binding profiles between CD36 and oxLDL/oxPL but also of finding competitors for the binding.

Wrapping tissues with a pre-established cage-like layer composed of living cells.

We report a method for wrapping tissues with a pre-established cage-like layer composed of living cells. We encapsulated multicellular aggregates of human hepatoma HepG2 cells as a model of tissues such as pancreatic islets and hepatocyte spheroids in alginate-based hydrogel microcapsules and subsequently coated the microcapsule surface with a gelatin derivative through a horseradish peroxidase-catalyzed reaction. Human aorta endothelial (HAE) cells grew on the surface and formed a cell layer within 24 h of incubating the microcapsules in a medium containing the cells. Subsequent degradation of the hydrogel microcapsule using a non-proteolytic enzyme, alginate lyase, resulted in a cage-like structure of HAE cells formed around the microcapsule. The HAE cell layer shrank without fragmenting and wrapped the inner spherical tissue. This method was also effective for wrapping multiple cellular aggregates within a single cage of HAE cells. In addition, it was possible to wrap tissue grown from individual cells in spherical cavities within the microcapsules.

Rapidly serum-degradable hydrogel templating fabrication of spherical tissues and curved tubular structures.

Development of the techniques for fabricating three-dimensional tissues still poses significant challenges for tissue engineering. We used hydrogels obtained from phenol-substituted amylopectin (AP-Ph) as templates for preparing multicellular spherical tissues (MSTs) and endothelialized curved tubular structures in type I collagen gel. AP-Ph hydrogel microparticles of diameter 200 µm and fibers of diameter 500 µm disappeared within hours of soaking in a serum-containing medium. HeLa cells and human endothelial cells were enclosed in the microparticles and hydrogel fibers, respectively, and then embedded in Ca-alginate microcapsules or the collagen gel. The enclosed cells were released in cavities formed by hydrogel degradation in the serum-containing medium. The released HeLa cells in the spherical cavities grew and formed MSTs, eventually filling the cavities. The spherical tissues were easily harvested by liquefying the Ca-alginate hydrogel microcapsule membrane by chelation using sodium citrate. The released endothelial cells grew on the tubular cavity surfaces and formed tubular structures. An endothelial cell network was formed by cell migration into the collagen gel. These results demonstrate the potential of serum-degradable AP-Ph hydrogels in constructing three-dimensional tissues.

Cell-enclosing gelatin-based microcapsule production for tissue engineering using a microfluidic flow-focusing system.

Gelatin-based microcapsule production using a microfluidic system and the feasibility of the resultant microcapsules for constructing spherical tissues surrounded by heterogeneous cells were studied. The first cell-encapsulation and subsequent cell-enclosing microparticle encapsulation were achieved using a microfluidic flow-focusing droplet production system. A hollow-core structure of about 150 µm in diameter was developed by incubating the resultant microparticles at 37 °C, which induced thermal melting of the enclosed unmodified gelatin microparticles. Mammalian cells filled the hollow-cores after 4 days of incubation. A cell layer on the cell-enclosing microcapsules was developed by simply suspending the microcapsules in medium containing adherent fibroblast cells. This method may prove useful for the generation of gelatin microcapsules using a microfluidic system for formation of artificial tissue constructs.

Development of an in vitro system for screening the ligands of a membrane glycoprotein CD36.

It has well been known that human and rodents exhibit a preference for fats. This suggests the existence of an orosensory system responsible for the detection of dietary fats. A plasma membrane glycoprotein CD36, besides the role in the uptake of long-chain fatty acids (LCFAs) as well as oxidized low-density lipoprotein (OxLDL) in a variety of cells, has been postulated to be a candidate fat taste receptor on the tongue. Therefore, molecules that bind with CD36 to cause intracellular signaling but have fewer calories could be substitutes for dietary fats. In the present study, we developed an in vitro system for the screening of CD36 ligands using Chinese hamster ovary-K1 cells (CHO-K1) stably transfected with human or mouse CD36. When incubated with OxLDL labeled with fluorescence dye, the fluorescence was much higher in the transfected CHO-K1 cells than in non-transfected CHO-K1 cells. Incubation of the transfected cells with OxLDL caused a rapid phosphorylation of extracellular signal regulated kinase, and the degree was significantly higher compared with that in non-transfected CHO-K1 cells. The expression system using CHO-K1 cells could be a convenient tool to screen the novel ligands of CD36.