Massimo Conese

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes

The GPI‐anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the α2‐macroglobulin receptor/low density lipoprotein receptor‐related protein (α2MR‐LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol‐specific phospholipase C (PI‐PLC). In this paper we show that during internalization of uPA:serpins at 37°C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface‐biotinylated, uPA: PAI‐1‐exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37°C. In fact, uPAR was resistant to PI‐PLC after the 4°C binding of uPA:PAI‐1 to biotinylated cells, but upon incubation at 37°C PI‐PLC‐sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI‐1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.

Gene Transfer by Means of Lipo- and Polyplexes: Role of Clathrin and Caveolae-Mediated Endocytosis

In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway. The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.

Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs.

Airway inflammation frequently found in congenital and acquired lung diseases may interfere with gene delivery by direct administration through either instillation or aerosol. Systemic delivery by the intravenous administration represents an alternative route of delivery that might bypass this barrier. A nonviral approach for transfecting various airway-derived cell lines in vitro showed that cationic polymers (PEI 22K and 25K) and lipids (DOTAP, GL-67/DOPE) are able to transfect with high efficiency the reporter genes firefly luciferase and E. coli lacZ. Notably, two properties predicted that cationic vectors would be useful for a systemic gene delivery approach to the lung: (1) transfection was not inhibited or increased when cells were incubated with cationic lipids or polymers in the presence of serum; and (2) cationic vectors protected plasmid DNA from DNase degradation. A single injection of DNA complexed to the cationic polymer PEI 22K into the tail vein of adult mice efficiently transfected primarily the lungs and to a lesser extent, heart, spleen, kidney and liver. The other vectors mediated lower to undetectable levels of luciferase expression in the lungs, with DOTAP > GL67/DOPE > PEI 25K > DOTMA/DOPE. A double injection protocol with a 15-min interval between the two doses of DOTAP/DNA complexes was investigated and showed a relevant role of the first injection in transfecting the lungs. A two log increase in luciferase expression was obtained either when the two doses were comprised of luciferase plasmid or when an irrelevant plasmid was used in the first injection. The double injection of luciferase/PEI 22K complexes determined higher transgene levels than a single dose, but a clear difference using an irrelevant plasmid as first dose was not observed. Using lacZ as a reporter gene, it was shown that only cells in the alveolar region, including type II penumocytes, stained positively for the transgene product.

Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs.

Biodistribution of nonviral cationic vector/DNA complexes was studied after systemic or intratracheal administration to the lungs and correlated with transgene expression. Intravenous injection in C57Bl/6 mice gave maximal and significant luciferase expression in the lungs with the cationic polymer PEI 22K/DNA complexes at the highest ratios of positive/negative charges versus DNA alone. While DOTAP/DNA complexes with high charge ratio determined lower but still significant luciferase activity versus uncomplexed DNA, GL-67A and PEI 25K mediated negligible luciferase expression. Labelled PEI 22K and DOTAP complexes were evenly distributed in the alveolar region, where GFP expression was revealed, while PEI 25K and GL-67A complexes were not detected, suggesting a different interaction of these complexes with the plasma membrane of endothelial cells. Following an intratracheal injection, the highest and significant levels of transfection were obtained with slightly positive PEI complexes as compared with DNA alone, whereas cationic lipid-based vectors, DOTAP and GL-67A, gave not significant luciferase activity. Both types of polyplexes gave similar levels of lung luciferase expression by targeting different airway cell populations. PEI 25K complexes determined high levels of GFP in the bronchial cells, confirming confocal data on fluorescent complexes internalization. PEI 22K complexes gave mainly high GFP signal in the distal tract of the bronchial tree, where tagged complexes were recovered. Fluorescent lipid complexes were found in aggregates in the lumen of bronchi totally (DOTAP) or partially (GL-67A) co-localizing with surfactant protein A. Results indicated that cationic polymers could overcome the surfactant barrier which inhibited airway cell transfection mediated by cationic lipids.

Nonmucoid Pseudomonas aeruginosa Expresses Alginate in the Lungs of Patients with Cystic Fibrosis and in a Mouse Model

BACKGROUND In patients with cystic fibrosis (CF), lung infection with mucoid Pseudomonas aeruginosa strains overexpressing the exopolysaccaride alginate is preceded by colonization with nonmucoid strains. We investigated the kinetics, impact of environmental signals, and genetics of P. aeruginosa alginate expression in a mouse model and in patients with CF. METHODS Using indirect immunofluorescence, microarray technology and real-time reverse-transcription polymerase chain reaction, we assessed alginate gene expression during aerobic and anaerobic growth of the nonmucoid strain PAO1 in vitro, in a mouse lung-infection model and in sputum specimens from patients with CF infected with nonmucoid or mucoid P. aeruginosa strains. RESULTS Anaerobic conditions increased the transcription of alginate genes in vitro and in murine lungs within 24 h. Alginate production by PAO1 in murine lungs and by nonmucoid P. aeruginosa strains in patients with CF was reversible after in vitro culture under aerobic conditions. A subpopulation of P. aeruginosa clones revealing stable alginate production was detected in murine lungs 2 weeks after infection. CONCLUSIONS Anaerobiosis and lung infection rapidly induce alginate production by gene regulation in nonmucoid P. aeruginosa. This trait may contribute to early persistence, leading to chronic P. aeruginosa infection once stable mucoid strains are generated.

Na+/H+ Exchanger Regulatory Factor Isoform 1 Overexpression Modulates Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Expression and Activity in Human Airway 16HBE14o- Cells and Rescues ΔF508 CFTR Functional Expression in Cystic Fibrosis Cells

There is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) interacting proteins play critical roles in the proper expression and function of CFTR. The Na+/H+ exchanger regulatory factor isoform 1 (NHERF1) was the first identified CFTR-binding protein. Here we further clarify the role of NHERF1 in the regulation of CFTR activity in two human bronchial epithelial cell lines: the normal, 16HBE14o-, and the homozygous ΔF508 CFTR, CFBE41o-. Confocal analysis in polarized cell monolayers demonstrated that NHERF1 distribution was associated with the apical membrane in 16HBE14o- cells while being primarily cytoplasmic in CFBE41o- cells. Transfection of 16HBE14o- monolayers with vectors encoding for wild-type (wt) NHERF1 increased both apical CFTR expression and apical protein kinase A (PKA)-dependent CFTR-mediated chloride efflux, whereas transfection with NHERF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent chloride efflux. These data led us to hypothesize an important role for NHERF1 in regulating CFTR localization and stability on the apical membrane of 16HBE14o- cell monolayers. Importantly, wt NHERF1 overexpression in confluent ΔF508 CFBE41o- and ΔF508 CFT1-C2 cell monolayers induced both a significant redistribution of CFTR from the cytoplasm to the apical membrane and a PKA-dependent activation of CFTR-dependent chloride secretion.

Neutrophil recruitment and airway epithelial cell involvement in chronic cystic fibrosis lung disease

The pathological hallmark of cystic fibrosis (CF) chronic inflammatory response is the massive neutrophil influx into the airways. This dysregulated neutrophil emigration may be caused by the abnormal secretion of chemoattractants by respiratory epithelial cells and polarised lymphocyte T-helper response. Neutrophils from CF patients have a different response to inflammatory mediators than neutrophils from normal subjects, indicating that they are primed in vivo before entering the CF airways. CF neutrophils secrete more myeloperoxidase and elastase, mobilise less opsonin receptors and release less L-selectin than non-CF neutrophils. Moreover, they show altered cytokine production and a dysregulated chemotaxis response. Laboratory studies now suggest that CFTR is involved in regulating some neutrophil functions and indicate that altered properties of CF neutrophils may depend on genetic factors. Current gene therapy approaches are targeted to the respiratory epithelium, but many hurdles oppose an efficient and efficacious CFTR gene transfer. The possibility of CFTR gene therapy-based approach targeting CF neutrophils at the hematopoietic stem cell level is discussed.

6 The urokinase/urokinase-receptor system and cancer invasion

u-PA binds with high affinity to its specific GPI-anchored receptor on the cell surface. The binding has at least two important consequences: (1) it enhances the rate of plasminogen activation on the cell surface; and (2) it focuses the u-PA proteolytic activity at the leading front of migrating cells. Several recent findings suggest that surface-bound u-PA is essential for the invasive ability of tumour cells, even if a picture is emerging indicating a concerted action with other proteases, like collagenases and cathepsin B (Kobayashi et al, 1992; Ossowski, 1992; Schmitt et al, 1992; (Danø et al, 1994). In some tumours, e.g. colon, breast and skin cancer, in situ hybridization studies have given an insight into the u-PA/u-PAR tumour biology showing a complex interplay between stromal and cancer cells Danø et al, 1994). u-PA, u-PAR, and PAI-1 tumour content are now well established prognostic factor in breast cancer. This body of knowledge could be used for theurapeutic purposes. For example, a large study with 671 patients has allowed the identification of node-negative patients which, according to their u-PA levels, would need adjuvant therapy (Foekens et al, 1992). Many other tumours, especially colorectal cancer, expect a direct clinical evaluation of u-PA, u-PAR and serpins as prognostic factors.

Gene Therapy Progress and Prospects: Episomally maintained self-replicating systems

The use of nonviral gene therapy vectors has been hampered by low level of transfection efficiency and lack of sustained gene expression. Episomal self-replicating systems may overcome these hurdles through their large packaging capacity, stability and reduced toxicity. This article reviews three classes of episomal molecules that have been tested with possible therapeutic genes: (1) self-replicating circular vectors, containing the Epstein–Barr virus (EBV) elements oriP and EBNA1; (2) small circular vectors containing scaffold/matrix attachment regions (S/MARs) as cis-acting elements to maintain the episomal status of the vector; (3) chromosomal vectors, based on the functional elements of the natural chromosomes. The studies reported validate the use of episomal vectors to obtain stable and prolonged gene expression, although reveal some limitations that necessitate additional work.

Gene and cell therapy for cystic fibrosis: From bench to bedside

Clinical trials in cystic fibrosis (CF) patients established proof-of-principle for transfer of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells. However, the limited efficacy of gene transfer vectors as well as extra- and intracellular barriers have prevented the development of a gene therapy-based treatment for CF. Here, we review the use of new viral and nonviral gene therapy vectors, as well as human artificial chromosomes, to overcome barriers to successful CFTR expression. Pre-clinical studies will surely benefit from novel animal models, such as CF pigs and ferrets. Prenatal gene therapy is a potential alternative to gene transfer to fully developed lungs. However, unresolved issues, including the possibility of adverse effects on pre- and postnatal development, the risk of initiating oncogenic or degenerative processes and germ line transmission require further investigation. Finally, we discuss the therapeutic potential of stem cells for CF lung disease.