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1
* Experimental Cell Research Program, Methodist Research Institute, Clarian Health Partners, Inc., Indianapolis, Indiana 46202, USA;
Department of Pulmonary and Critical Care Medicine, John Hopkins University School of Medicine, Baltimore, Maryland 21224, USA; and
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA
1Correspondence: Experimental Cell Research Program, The Methodist Research Institute, 1701 N. Senate Ave., Indianapolis, IN 46219, USA. E-mail: denglish{at}msn.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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dimer of heterotrimeric G-proteins. SPP-induced
chemotaxis in sorted cells coexpressing ßARKct was inhibited by 80%,
demonstrating that chemotaxis was driven by a ß-dependent pathway.
However, no significant inhibition was observed in cells transfected
with ßARKct but not enriched by sorting. Thus, we have developed a
method for enriching transfected cells that allows the elucidation of
crucial mechanisms of endothelial cell activation and function. This
method should find wide applicability in studies designed to define
pathways responsible for regulation of motility and other functions in
these dynamic cells.—Kovala, A. T., Harvey, K. A., McGlynn,
P., Boguslawski, G., Garcia, J. G. N., English, D.
High-efficiency transient transfection of endothelial cells for
functional analysis.
Key Words: FACS sorting • chemotaxis • heterotrimeric G-protein ß subunits
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
). The regulation of
angiogenesis, either by enhancement or inhibition, has important
clinical applications and has therefore been the focus of intense
research. A vast array of both natural mediators and pharmacological
agents that influence angiogenesis have been identified and studied
(4)
. Unfortunately, examination of the molecular and
biochemical events by which these mediators influence endothelial cells
in vitro has largely been limited to pharmacological
approaches. Endothelial cells have not been amenable to molecular
genetic investigations because of the difficulty of effectively
transfecting primary cells with mutated signal transduction mediators.
Transfection efficiencies in endothelial cells using standard calcium
phosphate, DEAE-dextran, or cationic liposome techniques have not
exceeded 20–30% (5
, 6)
. Efficiencies exceeding 90% have
been observed with adenovirus vectors (7
, 8)
, but the
utility of viral vectors is limited by the investment of time,
facilities, overall expense, and safety considerations. Therefore, the
ability to transiently transfect endothelial cells with high
efficiencies is essential for the development of a thorough
understanding of the signal transduction pathways involved in
endothelial cell angiogenic responses in vitro. We undertook
the present investigation to develop a high-efficiency technique for
reliable and reproducible transient transfection of primary endothelial
cells for functional analyses.
Maximization of transfection efficiency and protein expression is
critical to many transient transfection experiments. A convenient
reporter gene for the transfection of mammalian cell is the green
fluorescent protein (GFP). Derived from the jellyfish Aequorea
victoria, GFP is widely used as a reporter for studies involving
protein expression, subcellular localization, or as a marker for
transfection because of its easily detected fluorescence (reviewed in
refs 9
,10
). Enhanced GFP contains two amino acid
substitutions that red-shift its excitation spectra and increase its
fluorescence intensity (11)
. GFP fluorescence does not
require additional cofactors, substrates, or gene products, making it
an ideal reporter gene for transfection. Exposure of the protein to
blue or UV light produces a strong green fluorescence. The more
recently available red fluorescent protein (RFP) was isolated from the
sea anemone relative Discosoma and is unrelated to GFP. The
coding sequences of both GFP and RFP have been modified for human
codon-usage preferences to increase translational efficiency
(12)
. An important advantage of both of the fluorescent
proteins is that they can be detected in living cells without fixation,
staining, or processing and can emit fluorescence at wavelengths
sufficiently different as to allow simultaneous detection.
Fluorescence-activated cell sorting (FACS) allows rapid analysis and
purification of intact cells expressing the fluorescent proteins.
However, very few studies have used FACS-isolated cells for subsequent
functional studies (13)
.
In this report, we describe a highly efficient method for the transient
transfection of endothelial cells for functional analysis. As a
starting point, transfection efficiencies of 60–70% were achieved
when measuring GFP expression in endothelial cells as by the highly
sensitive flow cytometry analysis. We then developed strategies for the
efficient cotransfection and simultaneous expression of GFP and RFP,
and used fluorescence-activated cell sorting to isolate a population of
cells expressing both proteins. These strategies provided the
opportunity to apply molecular genetic techniques to the study of
signal transduction pathways involved in the regulation of endothelial
cell chemotaxis. The value of these procedures was demonstrated by
cotransfection, FACS sorting, and functional analysis of cells
simultaneously expressing GFP and the carboxyl-terminal domain of ß
adrenergic receptor kinase (ßARKct), an inhibitor of signaling
through the ß dimer of the heterotrimeric G-proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue culture
Primary BPAECs were grown in DMEM supplemented with 20% FBS,
1% nonessential minimal amino acids, 15 µg/ml ECGS, 100 U/ml
penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphtericin B
(complete medium). BPAECs were grown to confluence in 75
cm2 flasks under 5%
CO2-95% air at 37°C. Only cells from passage 6
to passage 15 were used for reported experiments.
Transfection
The expression vectors pEGFP-N1, and, pDsRed1-N1 were purchased
from CLONTECH Laboratories. The plasmid encoding the ßARK1
carboxyl-terminal (ßARKct) ß-binding domain and the
corresponding empty vector (pRK) are described elsewhere (Garcia et
al., unpublished results). Fugene 6 reagent, in the amounts indicated
in the figure legends, was diluted directly into 1 ml of OPTI-MEM and
incubated at room temperature for 5–10 min before the diluted reagent
was added to the plasmid DNA in polystyrene tubes. The DNA-Fugene
mixture was then incubated at room temperature for an additional 30 min
to allow complex formation. Confluent BPAE cells were washed twice with
Dulbecco’s phosphate-buffered saline, trypsinized, washed, and
resuspended (2x106 cells/ml) in complete media.
One milliliter of the resuspended cells was added to 10 ml of complete
medium in 100 mm plates immediately prior to transfection. Cells were
incubated for 40–46 h in the presence of the transfection mixture
before analysis.
Flow cytometry
Cells were harvested by trypsinization, washed, and resuspended
in DMEM without phenol red for cytometric analysis. Cytometric analysis
and sorting were performed using a FACStarPLUS
flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose,
Calif.) equipped with a water-cooled argon laser emitting at 488 nm.
Analysis was performed using CELLQuest Version 3.1f software
(Becton-Dickinson Immunocytometry Systems). Green fluorescence (FL1)
was measured using a 530 + 30 nm band pass filter and red fluorescence
(FL3) was determined with a 630 + 22 band pass filter. Gates were set
to exclude necrotic cells and cellular debris and the fluorescence
intensity of events within the gated regions was quantified. Data were
collected from 10,000–20,000 events for each sample. Signal
amplification was decreased to normalize the dot plot for analysis, and
compensation was used to exclude overlap between the two signals. Two
distinct populations of cells were visible on the flow histograms;
therefore, determination of the percentage of transfected cells was
based on the inclusion of only cells exhibiting high levels of
fluorescence and exclusion of cells adjacent to autofluorescent,
nontransfected cells.
FACS
BPAECs that had been cotransfected with pEGFP-N1 and either
pRed1-N1, pßARKct, or, pRK, were harvested as described above.
Fluorescence-activated cell sorting was performed with a low forward
scatter threshold to detect transfected cells while ensuring that
debris and electronic noise were not captured as legitimate events.
Cells were sorted at a rate of 2500–3000 events/s. Control sorts were
performed to ensure a greater than 98.5% sorting efficiency. The green
fluorescent cell population of interest was gated based on light
scatter and fluorescence. In some of the experiments gates were
established to allow the isolation of cells that were either GFP(+) and
RFP(+) or GFP(+) and GFP(-). Cells that were diluted during the
sorting process into a 0.85% w/v sodium chloride solution (Sigma
Diagnostics) were collected in 12 x 75 mm glass tubes at
0.1–0.5 x 106 cells/ml. For chemotaxis
analysis, the cells were concentrated and resuspended in DMEM.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and Western blotting
Cell lysates were prepared by pelleting 5 x
105 cells and resuspending the pellets in 100
µl 1X lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.01%
Nonidet P-40, 5 mg/ml Na Deoxycholate, 0.1% SDS, 100 mM NaF, 1%
glycerol, 1 mM Na orthovanadate, one Complete protease inhibitor
mixture tablet (Boehringer Mannheim, Indianapolis, Ind.)/25 ml, pH
7.4). Protein concentrations were determined using the BCA protein
assay (Pierce, Rockford, Ill.) with bovine serum albumin as a standard.
For each sample, 1.5 or 3 µg total protein/lane were separated on
10–20% SDS-PAGE and transferred to Immobilon-P membranes (Millipore
Corporation, Bedford, Mass.). Duplicate membranes were probed with
either 1 µg/ml Living Colors Peptide Antibody for GFP expression or
0.5 µg/ml Living Colors D.s. Peptide Antibody for RFP. Signals were
detected by ECL Western blotting analysis system and Hyperfilm ECL
(Amersham Pharmacia Biotech, Buckinghamshire, U.K.). The
autoradiographs were scanned on a Hewlett Packard ScanJet 6200C and
analyzed using Un-Scan-It version 4.1 software (Silk Scientific
Corporation, Orem, Utah).
Chemotaxis assay
Both GFP(+) and GFP(-) populations of cells isolated by
fluorescence-activated sorting were tested for SPP-induced chemotaxis
on gelatin coated Transwell filters (Corning Inc., N.Y.). The GFP(-)
population served as a control for the effects of the transfection
process and for the sorting process. Cells cotransfected with pEGFP-N1
and the empty vector pRK were also used as a control to identify any
effect due to the GFP expression. Transfection and preparation of
control cells for assays were performed in parallel to that of the
GFP/pßARKct. Confluent BPAE cells grown in 100 mm dishes were
harvested 40–46 h after transfection, washed, and resuspended in DMEM
without phenol red for fluorescence-activated sorting. Chemotaxis
assays were carried out in Boyden chambers with 8 µm pore filters
that had been coated with 0.1% gelatin, as described previously
(14)
. Cells (105) were added to the
top chamber and preincubated for 2 h to allow monolayer formation
before stimulation. Chemotaxis was induced by the addition of SPP at a
final concentration of 500 nM to 300 µl DMEM in the bottom chamber.
After 2 h of incubation at 37°C, migration was stopped by
removal of the top chamber and the nonmigrating cells on the upper
surface of the filter were carefully removed with a cotton swab. Cells
that had migrated across the filter were fixed with formaldehyde,
stained with hematoxylin, and counted (three fields per filter under
200x magnification). Results are the average of two experiments (±
SE), using three separate chambers per experiment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, both untransfected cells and mock-transfected cells exhibited
identical patterns of autofluorescence. GFP transfected cells exhibited
a strong increase in fluorescence measured in the FL1 channel. The
percentage of transfected cells in the population was determined by
establishing a gate well above the level of autofluorescence seen in
untransfected cells. Cells within the gate were scored as GFP(+) and,
therefore, were considered successfully transfected. While this high
cut-off eliminates some of the cells expressing lower levels of GFP and
results in an underestimate of the true transfection
efficiency, it ensures that only strongly GFP-expressing cells are
scored as transfected and that no cells are included due to
autofluorescence. This was important for subsequent sorting
experiments.
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To optimize transfection conditions, we measured the effects of
increasing amounts of plasmid DNA while maintaining a constant volume
of Fugene 6 (Fig. 2A
). Maximum efficiency was found to occur between 5 and 10
µg of plasmid per 100 mm dish; 60–70% of cells were typically
GFP(+). Using more than 10 µg DNA per dish lowered the transfection
efficiency. As both the total amount of DNA and the DNA:Fugene 6 ratio
are important factors in transfection efficiency, we tested various
DNA:Fugene 6 (µg:µl) ratios using 5, 10, or 20 µg of plasmid DNA.
The efficiency of transfection increased with increasing DNA:Fugene 6
ratios for both the 5 and 10 µg DNA per samples (Fig. 2B
).
Up to 60 µl of Fugene 6 could be used without affecting cell
viability or morphology. At 20 µg DNA, the percentage of GFP(+) cells
was slightly reduced at all DNA:Fugene 6 ratios tested. Therefore, a
combination of 5–10 µg DNA and the highest possible DNA:Fugene 6
ratio (60 µl or less of Fugene 6) was used in subsequent experiments.
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Cotransfection of GFP and RFP
The cotransfection and expression of GFP and RFP in endothelial
cells was examined using the conditions established above. The dot
plots for samples transfected with each plasmid individually or
together were analyzed by flow cytometry. GFP fluorescence was analyzed
in the FL1 channel and RFP fluorescence was analyzed in the FL3
channel. Quadrants were established that allowed the discrimination of
endothelial cell autofluorescence (lower left), RFP-associated
fluorescence (upper left), GFP-associated fluorescence (lower right),
and fluorescence for cells expressing both RFP and GFP (upper right),
as shown in Fig. 3
. Under identical conditions, the transfection efficiency, as measured
by GFP fluorescence, was found to be at 
2.5-fold higher than the
transfection efficiency as measured by RFP fluorescence (Fig. 4B
). When cells cotransfected with both RFP and GFP were
analyzed, there was an almost complete shift of the RFP-expressing
cells into the upper right quadrant, indicating that these cells were
expressing both proteins. In contrast, only 50% of the GFP
fluorescent cells also exhibited RFP fluorescence. This suggested that
either pRed1-N1 had an inherently lower ability to transfect cells,
that RFP was expressed at lower levels, or that the detection of RFP
fluorescence was less sensitive than the detection of GFP fluorescence.
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Given the apparent difference between the transfection efficiencies of
GFP and RFP as determined by fluorescence, the effect of differences in
the ratio of pDsRed1-N1 to pEGFP-N1 DNA cotransfected into endothelial
cells was examined. Ratios of 4:1 or 9:1 (pDsRed1N1:pEGFP-N1) were
found to result in the highest percentage of cells expressing both RFP
and GFP (Fig. 4A
). Approximately 20% of the total cell
population at these ratios express both RFP and GFP, whereas only
10–15% of the cells expressed GFP alone and 2% or fewer cells
expressed RFP alone.
We then examined the effect of changing the total amount of DNA while
maintaining a 4:1 pDsRed1-N1:pEGFP-N1 ratio on the percentage of
cotransfected cells. The percentage of GFP(+) cells that were also
RFP(+) consistently remained at 50% over a range of 5 to 40 µg of
plasmid DNA (Fig. 4C
).
To increase the proportion of RFP(+) cell in the population, we used
fluorescence-activated cell sorting to isolate GFP(+) cells. This
approach eliminated all untransfected cells and those expressing only
RFP, thereby enriching cotransfected population by 2.5-fold from 20%
of the total population to 50% of the GFP+ population. Cells
cotransfected at a 4:1 ratio of plasmids were sorted for GFP
fluorescence. FACS analysis of the sorted GFP(+) population for RFP
fluorescence confirmed that the RFP(+) population had been increased to
over 50% (data not shown). Western blotting confirmed an increase in
the GFP protein levels in the GFP(+) cells compared to the unsorted
controls and the complete absence of GFP protein in the GFP(-) cells
(Fig. 5A
). When parallel blots were probed for RFP
expression, a nearly identical pattern of distribution was observed,
with only trace levels of RFP expression detectable in the GFP(-)
cells. Therefore, sorting for GFP(+) cells enhanced the concentration
of RFP+ cells within the population.
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The relatively low transfection efficiency based on RFP fluorescence
prompted an examination of RFP expression. Two populations of cells
were sorted from cotransfected cells expressing both RFP and GFP. The
cell population exhibiting dual fluorescence [GFP(+), RFP(+)] and the
population with only GFP fluorescence [GFP(+), RFP(-)] were isolated
and lysates were examined for the expression of both fluorescent
proteins by Western blotting (Fig. 5B
). Expression of RFP
was manifest in both populations of cells, demonstrating that although
RFP was expressed, not all of the protein had undergone fluorophore
formation and was therefore not detectable by fluorescence. The
densitometric quantitation of the Western blots indicated that the
GFP(+), RFP(-) cell population expressed 30% of both the GFP (29%
± 9, n=3) and the RFP (30% ± 4, n=3) protein
detected in the GFP(+) population as a whole. This indicated that the
efficiency of both transfection and expression with pDsRed1-N1 was
equal to that of pEGFP-N1, and that the apparent difference was due to
inherent limitations in the detection of RFP by fluorescence alone.
Functional assay of endothelial cells
To demonstrate the applicability of the transfection procedures to
the study of biological functions of endothelial cells, the procedures
developed above were applied to the cotransfection of plasmids encoding
GFP and the ßARKct inhibitor of signal transduction. The ßARKct
domain binds the ß subunit of the heterotrimeric G-proteins, thus
depleting the pool of active ß protein (15)
. It had
previously been demonstrated that SPP induces endothelial cell
chemotaxis through activation of the G-protein-coupled receptor Edg-1
(14)
. Therefore, BPAE cells were transfected with either
pEGFP-N1 and pßARKct or pEGFP-N1 and pRK, the empty vector. The
SPP-induced chemotactic response of cells cotransfected with pEGFP-N1
and either the vector or pßARKct were not significantly different
when the total, unsorted cell populations was assayed for migration
(Fig. 6A
). In contrast when fluorescence-activated sorting for GFP
expression was used to isolate GFP(+) and GFP(-) populations of cells,
chemotaxis in the GFP(+) cells cotransfected with pßARKct
transfections were inhibited by 82% (± 3%) relative to the GFP(-)
cells (Fig. 6B
). The combination of pEGFP-N1 and pRK reduced
migration by 28% (± 10%). Thus, the inhibition of chemotaxis in
the pEGFP-N1/pßARKct transfected cells was only evident after FACS
isolation of transfected cells, confirming the essential utility of
enriching the transfected cell population prior to functional assay.
Inhibition of chemotaxis by ßARKct expression demonstrated that
SPP-induced chemotaxis relies on the generation of free ß
heterodimers after agonist binding to the receptor.
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The cotransfection efficiency of the ßARKct could not be directly determined because of the lack of an appropriate antibody. The available antibodies to ßARK are directed against the amino-terminal region of the kinase, the very region that has been deleted in the creation of the dominant negative ßARKct. Preliminary experiments in which different dominant negative isoforms were used and identical patterns of expression in sorted cells have been observed (unpublished data) support our conclusion that high levels of cotransfection were consistently achieved with our procedure. The cotransfection efficiency is independent of the plasmid used. This, however, does not apply to expression levels of the proteins expressed from different plasmids, which can vary depending on the promoters and the inherent properties of the proteins involved. Most important, the marked functional inhibition of SPP-induced chemotaxis in the GFP(+) cells on cotransfection with ßARKct strongly supports our contention that high levels of cotransfection have been achieved in these experiments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Techniques
commonly used in other cells, such as the introduction of dominant
negative variants of signal transduction intermediates, have proved
difficult in endothelial cells. Endothelial cells are generally studied
as primary cells rather than immortalized cell lines; thus low
transfection efficiencies make it difficult to study the activities of
exogenously supplied genes. Adenovirus vectors have been used to
successfully transfect over 90% of cultured endothelial cells
(7
, 8)
. Adenovirus vectors have also been tested as
in vivo for possible application in gene therapy (7
, 17)
. The use of viral transfection vectors, though effective, is
labor intensive and expensive, and requires dedicated facilities. The
complexing of DNA to replication deficient adenovirus particles has
been reported to allow transfection of up to 20% of the cells
(18)
. Transient transfection with plasmids has been used
successfully when high-efficiency transfection is not required, as in
cases where transcriptional regulation of promoter constructs is linked
to reporter genes (19)
. However, transfection levels of
20–30%, which are the highest achieved with endothelial cells using a
variety of methods (5
, 6)
, are lower than those required
for many other types of experiments, such as those involving the
expression of dominant negative isoforms of signaling molecules.
Therefore the development of the effective, inexpensive and simple
method of transiently transfecting endothelial cells with plasmids
encoding molecules of interest is essential for developing a clear
understanding of the signal transduction events that transpire after
the activation of membrane receptors by extracellular ligands.
Among several lipid transfection reagents tested, Fugene 6 provided the
highest transfection efficiency (data not shown). We have also found
that transfecting endothelial cells in suspension immediately after
trypsinization markedly enhanced transfection efficiency. Several
factors may combine to produce this enhancement. The use of cell
suspensions rather than adherent monolayers may provide greater
transfection efficiency due to the increased number of cells that would
enter the growth cycle after initial plating. Transfection efficiencies
are generally higher in cells that are undergoing division than in
resting cells (20)
. The increased transfection rate may
also be due to the increased surface area the suspended cells present
for interaction with the lipid-DNA complexes.
We established the optimal transfection conditions using 5–10 µg DNA and 60 µl Fugene 6. The observed high transfection rate of 60–70% represents an efficiency far superior to that typically achieved previously with endothelial cells. Transfection of endothelial cells at these levels is in itself sufficient for many experimental situations. Nevertheless, to further increase the percentage of transfected cells in the population for vigorous analysis of signaling events, a cotransfection procedure, followed by fluorescence-activated sorting, was developed.
The conditions for cotransfection of endothelial cells with two
plasmids were established with pEGFP-N1 and pDsRed1-N1. The different
transfection efficiencies observed with pEGPF-N1 and pDsRed1-N1 under
identical conditions were found to be due to differential detection
efficiency of the fluorescence proteins (Fig. 3)
. The inserted cDNAs
for both proteins are of a similar size and the same vector was used
for each construct; both are expressed from the same human
cytomegalovirus promoter. The pEGFP-N1 plasmid encodes a red-shifted
variant of GFP where two amino acid substitutions, a Phe-64 to Leu and
a Ser-65 to Thr, result in a 35-fold increase in fluorescence over
wild-type GFP (11)
. The increased fluorescence allows
detection of cells that weakly express the GFP; this is not be the case
in cells expressing similar quantities of RFP. The rate of fluorophore
formation by the RFP protein is much slower that of the GFP protein,
resulting in a larger proportion of the cells that are not yet
fluorescent at any given time after transfection. These two
factors—lower inherent fluorescence and a slow rate for fluorophore
formation—explain the presence of RFP protein in cells that do not
exhibit RFP fluorescence (Fig. 5B
), and indicated that the
actual transfection efficiency for RFP is higher than that measured by
fluorescence. In fact, the identical ratios of GFP and RFP proteins
found in the sorted GFP(+), RFP(+) and GFP(+), RFP(-) cells indicate
that the RFP transfection efficiency was nearly identical to the GFP
transfection efficiency. Therefore, cotransfection of cells with GFP
and a second plasmid expressing a protein of interest, and sorting for
GFP, result in population of cells that express the second protein at
levels nearly equal to that of GFP.
We also examined the possibility of using fluorescence-activated cell
sorting to produce a cell population enriched in cotransfected cells.
By sorting for GFP after cotransfection, a 2.5-fold increase in the
percentage of cells that were RFP(+) as measured by fluorescence was
achieved in the GFP(+) population. Given that the GFP(+), RFP(-)
population was expressing RFP protein at levels similar to GFP (Fig. 5B
), essentially the entire GFP(+) population should also be
RFP(+), despite the difficulties in fluorescent detection. Replacement
of pDsRed1-N1 with a vector carrying a gene of interest would similarly
permit enrichment for cells transfected with that gene after sorting
for GFP. A possible alternative to cotransfecting cells with two
separate plasmids would be to have both cDNAs expressed from the same
plasmid, ensuring that cells expressing the fluorescent protein also
expressed the second protein. However, this approach often results in
diminished expression of both proteins. Cotransfection of a second
plasmid with pEGFP-N1 provides for greater versatility, allowing
various cDNAs to be rapidly tested without subcloning.
Several recent publications have identified sphingosine 1-phosphate as
a powerful chemoattractant for endothelial cells (14
, 21
, 22)
. Extracellular SPP-initiated signaling involves the binding
of SPP to, and subsequent activation of, members of the Edg family of
G-protein-coupled receptors (GPCR) (23)
. In particular,
the Edg1 receptor has been implicated to have a major role in
SPP-induced chemotaxis in endothelial cells (14)
. The
receptor-driven activation of the heterotrimeric G-proteins results in
the dissociation of the 
subunit from the ß heterodimer; both
sets of subunits in turn become responsible for the activation of
divergent signaling pathways (24)
. The ß heterodimer
has been implicated in the regulation of GPCR-activated chemotaxis in
Dictyostelium discoideum (25
, 26)
and in
leukocytes (27
, 28)
. After receptor activation, the ß
subunit mediates targeting of the ß-adrenergic receptor kinase
(ßARK) to the membrane through binding to a specific domain located
in the carboxyl-terminal 125 residues of ßARK. The expression of the
truncated ßARK carboxyl-terminal (ßARKct) domain has been
demonstrated to bind and sequester free ß subunits, thus acting in
a dominant negative fashion specific for the ß heterodimer
signaling pathway (15
, 29)
. Expression of the ßARKct in
endothelial cells results in a substantial inhibition of SPP-induced
chemotaxis, demonstrating a major role for the ß heterodimer in
endothelial cell chemotaxis (Fig. 6)
. Chemotaxis induced through
agonist binding to GPCRs has been found to involve activation of
phosphoinositide (PI) 3-kinase in neutrophils (30)
and
macrophages (31
, 32)
. The activation of PI 3-kinase
occurs downstream of free ß subunit generation in transfection
studies of COS-7 cells (33)
. However, it has recently been
demonstrated that SPP-induced chemotaxis of BPAE cells is not inhibited
by the PI 3-kinase inhibitor LY-294002 (14)
, suggesting
that activation of a different signaling pathway is required in
endothelial cells. The identity of the pathways involved is currently
under investigation, using the techniques described here.
In conclusion, we have developed a procedure that permits the highly efficient transient transfection and subsequent enrichment of functional endothelial cell populations expressing exogenous molecules of interest. The method will facilitate the elucidation of the molecular mechanisms of endothelial cell activation and function and should be applicable to studies of pathways responsible for angiogenesis, migration, permeability, adhesion, and the secretion in these dynamic cells.
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ACKNOWLEDGMENTS |
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Received for publication March 30, 2000.
Revision received May 24, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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complex in living cells during chemotaxis. Science 287,1034-1036 subunit of Gi, not by activating Gq or Gs. Proc. Natl. Acad. Sci. USA 94,14489-11494 dimers released by activation of G-coupled receptors. Proc. Natl. Acad. Sci. USA 94,14495-11499-mediated signaling. J. Biol. Chem. 269,6193-6197 in thymocyte development. T cell activation, and neutrophil migration. Science 287,1040-1046 in inflammation. Science 287,1049-1053. Science 275,394-397This article has been cited by other articles:
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