Preferential binding of polyethylene glycol-coated liposomes containing a novel cationic lipid, TRX-20, to human subendthelial cells via chondroitin sulfate.

PURPOSE: To design novel cationic liposomes, polyethylene glycol (PEG)-coated cationic liposomes containing a newly synthesized cationic lipid, 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20) were formulated and their cellular binding and uptake investigated in vitro in the following cells: human subendothelial cells (aortic smooth muscle cells and mesangial cells) and human endothelial cells. METHODS: Three different PEG-coated cationic liposomes were prepared by the extrusion method, and their mean particle size and zeta potential were determined. Rhodamine-labeled PEG-coated cationic liposomes were incubated with smooth muscle cells, mesangial cells, and endothelial cells at 37 degrees C for 24 h. The amounts of cellular binding and uptake of liposomes were estimated by measuring the cell-associated fluorescence intensity of rhodamine. To investigate the binding property of the liposomes, the changes of the binding to the cells pretreated by various kinds of glycosaminoglycan lyases were examined. Fluorescence microscopy-is used to seek localization of liposomes in the cells. RESULTS: The cellular binding and uptake of PEG-coated cationic liposomes to smooth muscle cells was depended strongly on the chemical species of cationic lipids in these liposomes. Smooth muscle cells bound higher amount of PEG-coated TRX-20 liposomes than other cationic liposomes containing N-(1-(2.3-dioleoyloxy) propyl)-N, N, N-trimethylammonium salts or N-(alpha-(trimethylammonio)acetyl)-D-glutamate chloride. Despite of the higher affinity of PEG-coated TRX-20 liposomes for subendothelial cells, their binding to endothelial cells was very small. The binding to subendothelial cells was inhibited when cells were pretreated by certain kinds of chondroitinase, but not by heparitinase. These results suggest that PEG-coated TRX-20 liposomes have strong and selective binding property to subendothelial cells by interacting with certain kinds of chondroitin sulfate proteoglycans (not with heparan sulfate proteoglycans) on the cell surface and in the extracellular matrix of the cells. This binding feature was different from that reported for other cationic liposomes. CONCLUSIONS: PEG-coated TRX-20 liposomes can strongly and selectively bind to subendothelial cells via certain kinds of chondroitin sulfate proteoglycans and would have an advantage to use as a specific drug delivery system.

The regulatory region and transcription factor required for the expression of rat and salmon pituitary hormone-encoding genes show cell-type and species specificity.

The promoter regions of the genes encoding the rat and chum salmon growth hormones (GH) and rat prolactin (PRL) were combined with a reporter gene and introduced into GH- and/or PRL-producing cells from rat. The rat GH and PRL promoters (pGH and pPRL, respectively) were most active in cells producing GH and PRL, respectively. The activity of the salmon pGH was much less than that of the rat pGH in rat GH-producing cells. The regulatory region required for cell-type-specific gene expression of pituitary hormones thus contains information, not only for cell-type specificity, but possibly for species specificity as well. A reporter plasmid containing the GH or somatolactin (SL) promoter and an effector plasmid having a gene encoding transcription factor Pit-1 (rat or salmon) were cotransfected into HeLa (human) or EPC (carp) cells. Rat and salmon Pit-1 were more active in HeLa and EPC cells, respectively, indicating that Pit-1 appears to interact species specifically with the transcription machinery.

Pituitary-specific transcription factor Pit-1 in the rdw rat with growth hormone- and prolactin-deficient dwarfism.

The pituitary gland of the rdw rat (gene symbol: rdw) with hereditary dwarfism expresses 30-100 times less GH and prolactin (PRL) mRNA than normal controls. To clarify the features of rdw rats, TSH and the pituitary-specific transcription factor Pit-1, which is involved not only in the gene expression of GH and PRL but in somatotroph, lactotroph and thyrotroph development as well, were examined. The rdw pituitary contained about seven times more TSH beta mRNA than the normal control, whereas Pit-1 mRNA expression in rdw and control was the same. Nucleotide sequencing of PCR-amplified Pit-1 cDNA indicated that the deduced amino acid sequence of rdw Pit-1 was identical with that of the normal rat. Using an antibody against rat Pit-1 protein produced in E. coli, Western blotting analysis demonstrated the presence of the same amount of Pit-1 protein in rdw and normal rat pituitaries. The distribution of Pit-1-positive cells in the anterior pituitary was essentially the same in rdw and normal rats. It follows from these findings that the defective gene in the rdw rat is unrelated to the Pit-1 gene and the normal quantity of Pit-1 protein is insufficient to produce normal amounts of GH and PRL in the rdw pituitary. These and previous results suggest that the reduction in GH and PRL production in the rdw pituitary might be due to that in thyroid hormone production.

Pit-1/GH factor-1 involvement in the gene expression of somatolactin.

A presumed pituitary hormone, somatolactin (SL), belonging to the GH/PRL family, is produced in the intermediate lobe of teleost pituitary. The pituitary-specific transcription factor, Pit-1, is indispensable not only for the expression of mammalian GH and PRL genes, but for the development of GH- and PRL-producing cells present in the anterior lobe of the pituitary as well. Thus, in this study, examination was made of the possible involvement of Pit-1 in the intermediate lobe-specific expression of the SL gene. Using antibodies against chum salmon Pit-1, the presence of the 40-kilodalton Pit-1 protein in anterior and neurointermediate lobes of rainbow trout pituitary was demonstrated. By immunohistochemical examination, Pit-1 protein was shown to be located in the nuclei of SL-producing cells in the intermediate lobe. In experiments of cotransfection into HeLa cells, rat Pit-1 enhanced the promoter activity of SL gene, and a 0.5-kilobase upstream region from the transcriptional start site was sufficient for this enhancement. It follows from these results that Pit-1 protein may possibly be involved in SL gene expression as well as the development of SL-producing cells.