Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis.

Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.

Human ATP-binding cassette transporter-2 (ABCA2) positively regulates low-density lipoprotein receptor expression and negatively regulates cholesterol esterification in Chinese hamster ovary cells.

We present evidence that the ATP binding-cassette transporter-2 (ABCA2) is a sterol-responsive gene that has a role in the trafficking of low-density lipoprotein-derived free cholesterol (LDL-FC). In HepG2 cells ABCA2 was coordinately expressed with other sterol-responsive genes. Stable constitutive expression of ABCA2 in Chinese hamster ovary cells (CHOA2) was accompanied by an increase the expression of the low-density lipoprotein receptor (LDLR) and other genes involved in the regulation of cholesterol homeostasis. LDLR mRNA was elevated greater than ninefold and 3-hydroxy-3-methylglutaryl CoA synthase (HMGCoA S) expression was elevated sevenfold in CHOA2 cells. The increase in LDLR expression was regulated at the level of transcription; however, culture of CHO and CHOA2 cells in medium containing lipoprotein-deficient serum (LPDS) results in similar levels of LDLR promoter expression. No differences were measured in the dose-dependent uptake of fluorescently labeled 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchorate-LDL (DiI-LDL) between CHO and CHOA2 cells cultured in medium containing LPDS. Ultraviolet microscopy revealed a similar distribution of the DiI-LDL label in cytoplasmic vesicles. We measured an LDL dose-dependent reduction in esterification of LDL-FC in intact CHOA2 cells cultured in medium containing LPDS, however, no significant difference was measured in acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in cell-free extracts of CHO and CHOA2 cells. CHO cells or CHOA2 cells treated with the hydrophobic amine, U18666A, showed similar filipin staining of unesterified cholesterol in cytoplasmic vesicles. Addition of progesterone or U18666A to CHO cells elevated ABCA2 expression. Finally, we found that ABCA2 expression was elevated in Niemann-Pick type C1 (NPC1) fibroblasts and in Familial Hypercholesterolemia (FHC) fibroblasts.

Identification of a novel first exon of the human ABCA2 transporter gene encoding a unique N-terminus.

The human ABCA2 transporter is a member of a large family of ATP-binding proteins that transport a variety of molecules across biological membranes. Using RNA ligation-mediated PCR (RLM-PCR), we have identified a novel first exon, which we designate 1B that is located 699 bp upstream of the previously characterized first exon, which we designate 1A. These first exons are alternatively spliced to the second exon of the ABCA2 transcript resulting in a protein that has a unique amino terminus. For exon 1B, the new amino terminus encoded by the first exon is 52 amino acids and for exon 1A, 22 amino acids. We observed that among adult tissues examined, the highest expression of the 1B isoform was in peripheral blood leukocytes (PBL). Laser scanning confocal microscopy revealed that the 1A isoform and the 1B isoform co-localize with lysosome-associated membrane proteins-1 and -2 (LAMP-1 and -2). Cytotoxicity assays suggested a role for ABCA2 in estramustine and estradiol resistance, and overexpression of ABCA2 is seen in an estramustine-resistant prostate carcinoma line. Since both isoforms of the ABCA2 transporter have identical subcellular localization and both are overexpressed in a resistant cell line, we propose that they are also functionally redundant. It is likely that expression of ABCA2 by two independent promoters constitutes locus of regulation controlling expression of the protein to meet requirements in different tissues.

Cellular Senescence: Ex Vivo p53-Dependent Asymmetric Cell Kinetics.

Although senescence is a defining property of euploid mammalian cells, its physiologic basis remains obscure. Previously, cell kinetics properties of normal tissue cells have not been considered in models for senescence. We now provide evidence that senescence is in fact the natural consequence of normal in vivo somatic stem cell kinetics extended in culture. This concept of senescence is based on our discovery that cells engineered to conditionally express the well-recognized tumor suppressor protein and senescence factor, p53, exhibit asymmetric cell kinetics. In vivo, asymmetric cell kinetics are essential for maintenance of somatic stem cells; ex vivo, the same cell kinetics yield senescence as a simple kinetic endpoint. This new "asymmetric cell kinetics model" for senescence suggests novel strategies for the isolation and propagation of somatic tissue stem cells in culture.