Carmen Barnes

siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.

Maintenance of normal endothelial function is critical to various aspects of blood vessel function, but its regulation is poorly understood. In this study, we show that disruption of baseline fibroblast growth factor (FGF) signaling to the endothelium leads to a dramatic reduction in let-7 miRNA levels that, in turn, increases expression of transforming growth factor (TGF)-β ligands and receptors and activation of TGF-β signaling, leading to endothelial-to-mesenchymal transition (Endo-MT). We also find that Endo-MT is an important driver of neointima formation in a murine transplant arteriopathy model and in rejection of human transplant lesions. The decline in endothelial FGF signaling input is due to the appearance of an FGF resistance state that is characterized by inflammation-dependent reduction in expression and activation of key components of the FGF signaling cascade. These results establish FGF signaling as a critical factor in maintenance of endothelial homeostasis and point to an unexpected role of Endo-MT in vascular pathology.

A mechanism of ion transport across membranes is reported. Microbial transport of Fe(3+) generally delivers iron, a growth-limiting nutrient, to cells via highly specific siderophore-mediated transport systems. In contrast, iron transport in the fresh water bacterium Aeromonas hydrophila is found to occur by means of an indiscriminant siderophore transport system composed of a single multifunctional receptor. It is shown that (i) the siderophore and Fe(3+) enter the bacterium together, (ii) a ligand exchange step occurs in the course of the transport, and (iii) a redox process is not involved in iron exchange. To the best of our knowledge, there have been no other reports of a ligand exchange mechanism in bacterial iron transport. The ligand exchange step occurs at the cell surface and involves the exchange of iron from a ferric siderophore to an iron-free siderophore already bound to the receptor. This ligand exchange mechanism is also found in Escherichia coli and seems likely to be widely distributed among microorganisms.

Thyroid hormone is a critical mediator of cellular metabolism and differentiation. Precise tissue-specific regulation of the concentration of the active ligand, T(3), is achieved by iodothyronine monodeiodination. Type 3 iodothyronine deiodinase (D3) is the major inactivating pathway, preventing activation of the prohormone T(4) and terminating the action of T(3). Using nontransformed human cells, we show that TGF-beta stimulates transcription of the hDio3 gene via a Smad-dependent pathway. Combinations of Smad2 or Smad3 with Smad4 stimulate hDio3 gene transcription only in cells that express endogenous D3 activity, indicating that Smads are necessary but not sufficient for D3 induction. TGF-beta induces endogenous D3 in diverse human cell types, including fetal and adult fibroblasts from several tissues, hemangioma cells, fetal epithelia, and skeletal muscle myoblasts. Maximum stimulation of D3 by TGF-beta also requires MAPK and is synergistic with phorbol ester and several mitogens known to signal through transmembrane receptor tyrosine kinases but not with estradiol. These data reveal a previously unrecognized interaction between two pluripotent systems, TGF-beta and thyroid hormone, both of which have major roles in the regulation of cell growth and differentiation.