Dexamethasone and mifepristone increase retroviral infectivity through different mechanisms

Using adapted retroviruses for gene delivery is a modern and powerful tool in biological research as well as a promising approach for gene therapy. An important limitation for the extensive use of retroviral vectors is the low infection rate in target cells such as pulmonary vascular endothelial cells due to the insufficient infectivity of standard retrovirus supernatants that can only be overcome by complicated methods of virus concentration. This paper describes two easy methods to augment target cell infectivity, first by increasing the retroviral titer in the medium collected from packaging cells by stimulation of viral propagation with dexamethasone, and second, by increasing target cell sensitivity to retroviral infection by the glucocorticoid receptor antagonist, mifepristone. Using this method, we increased the infectivity of pulmonary microvascular endothelial cells from 16% to 85%. We demonstrate that mifepristone increased the susceptibility of target cells to retroviruses without increasing the viral titer of the supernatant. Dexamethasone, but not mifepristone, increased expression of delivered proteins such as GFP that are important for early identification of infected cells. Each, or both step(s), may be included in a standard protocol for retrovirus propagation and infection of target cells

Serum can overcome contact inhibition in confluent human pulmonary artery smooth muscle cells.

Pulmonary artery endothelial cells (PAEC) in an intact vessel are continually exposed to serum, but unless injured, do not proliferate, constrained by confluence. In contrast, pulmonary artery smooth muscle cells (PASMC) attain, and maintain, confluence in the presence of minimal serum, protected from serum's stimulatory effects except when the endothelial barrier becomes more permeable. We hypothesized therefore, that confluent PASMC may be less constrained by contact inhibition in the presence of serum than PAEC and tested this idea by exposing confluent non-transformed human PAEC and PASMC to media containing increasing concentrations of fetal bovine serum (FBS) and determining cell growth over 7 days. PAEC that had attained confluence in low serum did not proliferate even when exposed to 5% serum, the highest concentration tested. In contrast, PASMC that attained confluence in low serum did proliferate once serum levels were increased, an effect that was dose dependent. Consistent with this observation, PASMC had more BrdU incorporation and a greater percentage of cells in S phase in 5% compared to 0.2% FBS, whereas no such difference was seen in PAEC. These results suggest that confluent human PAEC are resistant to the stimulatory effects of serum, whereas confluent PASMC can proliferate when serum levels are increased, an effect mediated in part by differences in phosphoinositide 3-kinase activation. This observation may be relevant to understanding the PASMC hyperplasia observed in humans and animals with pulmonary hypertension in which changes in endothelial permeability due to hypoxia or injury expose the underlying smooth muscle to serum

Pulmonary Microvascular Endothelial Cells Form a Tighter Monolayer when Grown in Chronic Hypoxia

Unique among the vascular beds, loss of endothelial integrity in the pulmonary microcirculation due to injury can lead to rapidly fatal hypoxemia. The ability to regain confluence and re-establish barrier function is central to restoring proper gas exchange. The adult respiratory distress syndrome (ARDS) is a heterogeneous disease, however, meaning that endothelial cells within different regions of the lung do not likely see the same oxygen tension as they attempt to proliferate and re-establish an intact endothelial monolayer; the effect of hypoxia on the integrity of this newly formed endothelial monolayer is not clear. Immortalized human pulmonary microvascular endothelial cells (PMVEC) (ST1.6R cells) were sparsely plated and grown to confluence over 4 days in either normoxia (21% oxygen) or hypoxia (5% oxygen). Confluence attained in a hypoxic environment resulted in a tighter, less permeable endothelial monolayer (as determined by an increase in transendothelial electrical resistance, decreased permeability to fluorescently labeled macromolecules, and decreased hydraulic conductance). PMVEC grown to confluence under hypoxia had decreased RhoA activity; consistent with this finding, inhibition of Rho kinase, a well-described downstream target of RhoA, markedly increased electrical resistance in normoxic, but not hypoxic, PMVEC. These results were confirmed in primary human and rat PMVEC. These data suggest that PMVEC grown to confluence under hypoxia form a tighter monolayer than similar cells grown under normoxia. This tighter barrier appears to be due, in part, to the inhibition of RhoA activity in hypoxic cells

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo

The PI3-kinase inhibitors, LY294002 and wortmannin, block serum-induced proliferation in confluent PASMC.

<p>Human PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum in the presence or absence of the PI3-kinase inhibitors LY294002 (15 µmol) or wortmannin (50 nmol) and cell cycle profile determined 24 hours later. A) Representative plot of single experiment; B) Aggregate results from 3 separate experiments (n = 3 experiments, * indicates p<.05).</p

Exposure to increased serum stimulates growth in confluent PASMC, but not in confluent PAEC.

<p>Human PAEC (A) and PASMC (B) attained confluence in low (0.2%) serum as determined by light microscopy and the absence of proliferation. Cells were then exposed to increasing concentrations of serum and cell number determined in triplicate seven days later. (n = 3 experiments; * indicates p<.05 compared with starting cell number).</p

Increased serum stimulates cell cycle progression/DNA synthesis in confluent PASMC, but not PAEC.

<p>Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and cell cycle profile (A and C) and BrdU incorporation (B and D) determined 24 hours later. (n = 3 experiments; * indicates p<.05).</p

PASMC that attain confluence at high serum cannot maintain that cell density in low serum.

<p>Human PASMC were grown to confluence in 10% FBS and maintained in 10% serum for 7 days. Confluence was determined by light microscopy and by the demonstration of stable cell number over 7 days. Cells were then exposed to various concentrations of serum ranging from 0.1% to 10%: A) Cell counts done 7 days later. B) Cells were stained with the membrane impermeable dye ethidium homodimer-1 at different time points to determine cell viability. Ethidium homodimer-1 can only stain apoptotic or necrotic cells and is a marker of cell death. Cell death is increased by day 2 and returns to baseline by day 7. (n = 3 separate experiments, * indicates p<.05 compared with starting cell number for (A) or compared to day 0 at 10% serum for (B).</p

PI3-kinase inhibition prevents retinoblastoma hyperphosphorylation in confluent PASMC exposed to high serum concentrations.

<p>Human PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.1% or 5% serum in the presence or absence of LY294002 or wortmannin and cell lysates harvested 24 hours later for protein analysis. Representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma].</p