1 British Heart Foundation Cardiovascular Medicine Unit, National Heart and Lung Institute and 2 Department of Immunology, Imperial College School of Technology and Medicine, Hammersmith Hospital, London W12 ONN; 3 Ludwig Institute for Cancer Research, University College Medical School, London W1W 7BS; and 4 Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom
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ABSTRACT |
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Although
mouse endothelial cells (EC) may advance our understanding of
endothelial function, primary EC remain difficult to isolate. We have
established a murine cardiac endothelial cell line (MCEC-1) from
transgenic mice harboring a temperature-sensitive simian virus 40 large
TAg gene (tsA58 TAg) under H-2Kb class I promoter control.
MCEC-1 cells were characterized by their ability to form tubes,
Griffonia simplicifolia isolectin B4 binding, and CD31,
intercellular adhesion molecule (ICAM)-2, and endoglin expression.
MCEC-1 cells proliferated rapidly under permissive conditions [33°C
with interferon (IFN)-], where the T antigen is active and
transcription is activated by the presence of IFN-
, whereas under
nonpermissive conditions (38°C without IFN-
) proliferation was
reduced by 30-fold and the EC showed enhanced proliferation in response
to growth factors. Expression of E- and P-selectin, ICAM-1, and
vascular cell adhesion molecule-1 was upregulated by tumor necrosis
factor-
and interleukin-1
, and MCEC-1 cells, in contrast to
primary EC, were amenable to transfection by lipofection. This novel
line will allow further study of the role of the endothelium in
cardiovascular disease. Moreover, this technique will allow EC to be
readily obtained from genetically modified mice backcrossed with
H-2Kb-tsA58 mice.
endothelium; proliferation; adhesion; Immortomouse
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INTRODUCTION |
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VASCULAR ENDOTHELIUM is a vital component of the vascular system, playing a central role in vasculogenesis, angiogenesis, hemostasis, vascular permeability, and the regulation of inflammation. The development of techniques for the isolation and propagation of endothelial cells (EC) in vitro (13) has resulted in a dramatic increase in our understanding of endothelial function. In addition, the development of genetically modified mice has allowed the precise definition of the function of specific genes in vivo (5, 12). To define in more detail the functional consequences of gene targeting, it is desirable to establish in culture EC from both transgenic and knockout mice. EC have been isolated from the murine lung (4, 8, 21), lymph nodes (24,), and brain (15). However, the isolation of primary EC from mouse organs is both time consuming and costly, and the EC of some organs, including the heart, can only be passaged two or three times before significant senescence occurs (E. A. Lidington, unpublished data). Although these problems can be overcome by the use of immortalized cell lines, the process of immortalization may result in phenotypic changes such as the loss of surface antigens (10) and reduced responsiveness to cytokines (20). Furthermore, immortalized cell lines are unsuitable for studying the regulation of EC proliferation.
The generation of H-2Kb-tsA58 transgenic mice, which
express a thermolabile strain (tsA58) of the simian virus (SV)40 large T antigen (tsA58 TAg) linked to an inducible major histocompatibility complex H-2K promoter, provides the opportunity for overcoming many of
the intrinsic problems of immortalized cell lines (14). In
these mice, T antigen expression is only functionally evident at the
reduced temperature of 33°C and promoter activity can be upregulated
by interferon (IFN)- treatment (14). Furthermore, these
mice have been used to produce cell lines that are conditionally immortal, including a murine brain EC line (15).
In this paper we describe a method for the routine isolation and propagation of murine cardiac EC from H-2Kb-tsA58 transgenic mice. We demonstrate that these EC can be rapidly expanded, retain responsiveness to EC growth factors, and compare favorably with primary murine EC in terms of adhesion molecule expression and support of leukocyte adhesion. We propose that intercrossing H-2Kb-tsA58-transgenic mice with genetically modified mice of interest will provide a ready source of EC from a variety of different vascular beds for further study, thus enhancing our understanding of the role of the endothelium in the pathophysiology of cardiovascular disease.
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METHODS |
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Antibodies and reagents.
Monoclonal antibodies (MAbs) against murine antigens used include YN1
(IgG2a) against intercellular adhesion molecule (ICAM)-1 and MK2.7
(IgG1) against vascular cell adhesion molecule (VCAM)-1 [American Type
Culture Collection (ATCC), Rockville, MD], MES-1 (IgG2a) against
E-selectin (a kind gift from Dr. D. Brown, Celltech Chiroscience,
Slough, UK), RB40 (IgG1) against P-selectin (a kind gift from Dr. D. Vestweber, University of Munster, Munster, Germany), EA3 (IgG1) against
platelet endothelial cell adhesion molecule (PECAM)-1, H202.106.7.4
against junctional adhesion molecule (JAM)-1 (both kind gifts from
Prof. B. Imhof, University of Geneva, Geneva, Switzerland), F-8 (IgG1)
(Santa Cruz Biotechnology, CA) and BV13 (a kind gift from Prof. E. Dejana, Mario Negri Institute for Pharmacological Research, Milan,
Italy), both against VE-cadherin, and OKT8 (IgG2a) against human CD8
(ATCC). The hybridoma for MAb MJ7/18 anti-endoglin (IgG2a) was
obtained from the Developmental Studies Hybridoma Bank, University of
Iowa (Iowa City, IA). MAbs 3C4 (IgG2a) anti-ICAM-2 and PAb101 (IgG2a)
anti-SV40 large T antigen were purchased from Pharmingen (San Diego,
CA). The FITC-conjugated affinity-purified goat anti-rat
F(ab')2 was from Jackson Immunoresearch Laboratories (West
Grove, PA). Biotinylated Griffonia simplicifolia isolectin B4 (GSL I-B4) and phycoerythrin-conjugated streptavidin were from Vector Laboratories (Peterborough, UK), Dil-Ac-low-density lipoprotein (LDL) from Biogenesis (Poole, UK), murine vascular endothelial growth
factor (VEGF), human basic fibroblast growth factor (bFGF), and murine
IFN- from PeproTech (London, UK), and murine tumor necrosis factor
(TNF)-
and murine interleukin (IL)-1
from R and D Systems
(Abingdon, UK).
Animals. Female C57BL/6 and CBA/Ca mice were purchased from Harlan Olac (Bicester, UK). The H-2Kb-tsA58 transgenic mice (Immortomouse; Ref. 14) were bred in house [H-2Kb-tsA58 transgenic mice (Immortomouse) can also be obtained commercially from Charles River Laboratories]. All mice were kept in the same controlled climatic conditions in filter-topped microisolator cages with autoclaved bedding. Irradiated food and drinking water were made readily available. All animals were housed and studied according to United Kingdom Home Office guidelines. Sentinel mice were housed alongside test animals and screened regularly for a standard panel of murine pathogens.
Isolation and transfection of murine EC.
Hearts were aseptically removed, after incision at the base of the
aorta, from five 6-wk-old H-2Kb-tsA58 transgenic or
wild-type mice (C57BL/6 and CBA/Ca). After being washed, hearts were
examined to ensure that all of the aorta, including the aortic root and
the large pulmonary vessels, was removed before the remaining tissue
was diced and incubated with collagenase A (Boehringer Mannheim, Lewes,
UK) at 1 mg/ml for 30 min at 37°C. The cell suspension was washed in
Hanks' balanced salt solution (HBSS) and centrifuged at 250 g, and the pellet was resuspended in trypsin-EDTA (ICN,
Basingstoke, UK) for 10 min at 37°C before passing through a 100-µm
filter. The cell suspension was incubated with OKT8 MAb at 4°C to
block Fc receptors on macrophages and then washed and incubated with
anti-endoglin MAb MJ7/18 (6). After incubation with goat
anti-rat IgG microbeads (Miltenyi Biotec, Bisley, UK), EC bound to the
beads were isolated using MS + miniMAC columns and a miniMAC
magnet (Miltenyi Biotec) and were then collected into complete medium
consisting of DMEM (GIBCO BRL Life Technologies, Paisley, UK)
supplemented with 10% fetal bovine serum (FBS) (Helena Biosciences,
Sunderland, UK), 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 2 mM
L-glutamine (all from GIBCO), 10 U/ml heparin (Leo
Laboratories, Prince Risborough, UK), and 30 µg/ml EC growth factor (ECGF; Sigma-Aldrich, Poole, UK) and cultured in 1%
gelatin-coated tissue culture flasks. The EC were initially expanded by
culture at 33°C in the presence of recombinant mouse IFN- (20 U/ml; PeproTech). Murine cardiac endothelial cells (MCEC-1) were
routinely used up to passage 20. For transfection, MCEC-1
plated at subconfluence in 24-well plates (4 × 104
cells/well) were cultured overnight at 33°C in normal growth medium.
Plasmid DNA (0.75 µg; pCMV.SPORT-
gal) was incubated with 5 µl of
Lipofectamine (2 mg/ml) or Cellfectin (1 mg/ml) in 0.3 ml of Opti-MEM
(all from GIBCO) for 30 min to allow complexes to form. The growth
medium was replaced by the lipid-DNA complexes and incubated with the
MCEC-1 cells at 33°C for 5 h before returning to normal growth
medium. After 48 h, wells were washed with PBS, fixed (2%
formaldehyde, 0.05% glutaraldehyde), and incubated with 1 mg/ml X-gal
staining solution (50 mM potassium ferricyanide, 50 mM potassium
ferrocyanide), resulting in transfected cells staining blue.
Flow cytometry. EC were stained with the appropriate primary MAb followed by FITC-conjugated affinity purified goat anti-rat F(ab')2, both for 30 min at 4°C, followed by washing and fixation in 1% paraformaldehyde. Samples were analyzed on an Epics XL-MCL flow cytometer (Coulter, High Wycombe, UK) by counting 10,000 cells per sample. Results are expressed as the relative fluorescent intensity, which represents the mean fluorescent intensity (MFI) with test MAb divided by the MFI with an isotype-matched irrelevant MAb.
Proliferation assay. EC from one 75-cm2 flask were split into four and cultured at 33° or 38°C for 48 h before being passaged into 96-well plates at 1,000 cells/well in 200 µl of growth medium. Monolayers were cultured for up to 96 h before pulsing with 0.2 µCi of [3H]thymidine/well (Amersham Pharmacia Biotech, Little Chalfont, UK) 18 h before the end of the assay. EC were harvested and counts per minute were determined with a Betaplate 96-well harvester (Wallac Oy, Turku, Finland).
Tube formation assay. MCEC-1 cells (3 × 105) in 1 ml of growth medium were plated on 600 µl of polymerized Matrigel (Collaborative Biomedical Products, Bedford, MA) in 35-mm petri dishes and cultured for 6 h at 37°C. Tube formation was imaged by phase-contrast microscopy, and representative fields were photographed.
Staining for tsA58 TAg expression. MCEC-1 cells (1 × 105) were transferred onto glass slides using a Shandon cytospin (Runcorn, UK). The slides were fixed in ice-cold acetone and incubated for 30 min with anti-SV40 large T antigen MAb PAb101 in 1% mouse serum. After washing in PBS, biotinylated rabbit anti-murine Ig (DAKO, Ely, UK) in 1% rabbit serum was added for 30 min. After further washing, phycoerythrin-conjugated streptavidin was added for 30 min before washing, mounting in Aquamount, and examination under an Olympus Bx4 fluorescence microscope.
Immunofluorescent staining of junctional components. MCEC-1 cells (at 2 × 105/well) were cultured on glass two-well chamber slides (Becton Dickinson, Bedford, MA) for 3 days. Monolayers were fixed in 3% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 min at room temperature, and then blocked with 5% BSA for 10 min. They were then incubated with antibodies (20 µg/ml) against JAM-1, PECAM-1, or VE-cadherin for 20 min, followed by three washes in HBSS and incubation with the appropriate FITC-conjugated secondary antibody for 20 min. After a further three washes, the wells were removed and the slides were mounted in Perma Fluor aqueous mountant (Shandon) before visualization by fluorescence microscopy.
Apoptosis assays. To measure condensed nuclei undergoing apoptosis the nuclear stain acridine orange (Sigma) was used. MCEC-1 cells were plated at 38°C at 1 × 105/well in six-well plates in DMEM containing 0.5% FBS and no ECGF. After 24 h, the medium was replaced with complete medium, medium deficient in ECGF, or ECGF and FBS, and the EC were then incubated for a further 48 h. Monolayers were fixed in ice-cold 70% ethanol for 10 min at 4°C and then washed three times in PBS before incubation with 1 µg/ml acridine orange for 5 min. After a final wash in PBS, the nuclei were visualized by immunofluorescence microscopy and condensed nuclei were counted and expressed as a percentage of total cells.
To assess dependence on anchorage for survival, MCEC-1 cells were plated into three 75-cm2 tissue culture flasks (1 × 106/flask). One flask was precoated with 1% gelatin, and cells were cultured under static conditions in complete medium at 38°C. In the remaining two flasks, EC were incubated at 38°C on an orbital shaker (at 2 rpm), to prevent cell adhesion, in complete medium or in complete medium deficient in ECGF. After 24 h the cells were harvested, centrifuged, and counted after addition of 1% trypan blue. The number of cells excluding trypan blue were counted and expressed as a proportion of the total number of cells plated (because of their loss of form and smearing, the trypan blue-positive cells could not be accurately quantified).Statistics. Differences between the results of experimental treatments were evaluated by the unpaired Student's t-test. Differences were considered significant at P values of <0.05.
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RESULTS |
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Generation of MCEC-1 cells.
Primary EC isolated from hearts of wild-type C57BL/6 or CBA/Ca mice
formed typical cobblestone monolayers but were slow to reach confluence
and could only be maintained for two or three passages before reaching
senescence. This occurred consistently in a variety of culture
conditions and was in contrast to our experience with primary EC
isolated from murine lung, which can be maintained for up to 11 passages (21). EC isolated from the hearts of
H-2Kb-tsA58 mice and cultured initially at 33°C with
IFN- reached confluence by day 7. These cells were
passaged repeatedly, giving rise to the MCEC-1 cell line, which has not
been cloned. MCEC-1 cells, although largely derived from the
microvasculature of the heart, are likely to be heterogeneous and may
include EC from the endocardium and coronary vessels. MCEC-1 cells had
a typical cobblestone morphology when grown on gelatin-coated tissue
culture plastic at 38°C (Fig.
1A) and
rapidly realigned to form characteristic microtubules when cultured on
Matrigel (Fig. 1B). The morphology of MCEC-1 cells was found
to differ somewhat between permissive and nonpermissive
culture conditions, with cells grown at 33°C in growth medium
containing IFN-
having more active nuclei with increased mitotic
figures (Fig. 1C). Monolayers cultured on glass chamber
slides showed strong staining at the cell junctions with an antibody to
JAM-1 (Fig. 1D) and a similar, but weaker, pattern of
staining with an antibody against VE-cadherin (data not shown). No
signal was visible in the negative control samples (Fig.
1E).
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MCEC-1 cells show increased adhesion molecule expression in
response to IL-1 and TNF-
.
MCEC-1 cells were found to efficiently incorporate Dil-labeled Ac-LDL
(data not shown). Further characterization was performed using flow
cytometric analysis to measure surface expression of endoglin (CD105),
ICAM-2 (CD102), PECAM-1 (CD31), and binding of the lectin GSL I-B4. As
seen in Fig. 2, PECAM-1 and ICAM-2, which
are found on EC, platelets, and some subtypes of leukocytes, were both
detectable on the surface of MCEC-1 cells. Although PECAM-1 expression
was relatively weak (Fig. 2A), this is consistent with
previous studies of murine EC (4, 21). Endoglin was highly
constitutively expressed on >99% of EC, with the same percentage of
cells also binding GSL I-B4.
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Effect of different culture conditions on MCEC-1 cell proliferation
and tsA58 TAg expression.
Indirect immunofluorescence was used to demonstrate the expression of
tsA58 TAg antigen under permissive conditions. As seen in Fig.
3B, MCEC-1 cells cultured at
33°C with IFN- demonstrated abundant nuclear staining with
anti-SV40 large T antigen MAb PAb101. However, after 48 h at
38°C in the absence of IFN-
, tsA58 TAg expression was undetectable
(Fig. 3A). Comparison of EC proliferation using either cell
counting or the uptake of [3H]thymidine demonstrated a
dramatic difference between cells cultured at 33°C with IFN-
and
those cultured at 38°C without IFN-
. A minimum of 24 h was
required for EC to adapt to the change in temperature, and therefore EC
derived from a single flask were cultured under the required conditions
for 48 h before experiments comparing culture conditions. As shown
in Fig. 4, MCEC-1 cells maintained under
permissive conditions entered the log phase of growth and proliferated
rapidly. MCEC-1 cell proliferation was increased by up to 16-fold by
reducing the culture temperature from 38°C to 33°C and by 32-fold
by reducing the temperature and adding IFN-
(20 U/ml; Fig.
4A). In contrast, the same cells cultured at 38°C in the
absence of IFN-
showed a markedly reduced proliferation rate (Fig.
4B). MCEC-1 cells have been cultured under permissive conditions for >90 passages over 12 mo with no signs of senescence and
have retained their temperature and IFN-
responsiveness. Moreover,
early-passage MCEC-1 cells have been frozen in liquid nitrogen and
subsequently thawed as required, with no change in phenotype (data not
shown).
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MCEC-1 cells proliferate in response to growth factor stimulation.
To assess the responsiveness of MCEC-1 cells to growth factors, we
initially stimulated cells grown at 38°C in the absence of IFN-
with ECGF (30 µg/ml) and measured DNA synthesis by the uptake of
[3H]thymidine for up to 4 days after stimulation. As
shown in Fig. 5A, there was
very little proliferation of cells in the absence of ECGF, whereas
addition of the growth factor led to a significant increase in
proliferation over 4 days. Further experiments were performed using the
proangiogenic growth factors bFGF and VEGF. As shown in Fig.
5B, MCEC-1 cells were also responsive to bFGF and VEGF and
both of these growth factors enhanced the proliferative effect of ECGF.
The results of these experiments were also confirmed by cell counting
(data not shown).
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DISCUSSION |
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The development of transgenic mice, or those with null mutations in specific genes of interest, continues to provide valuable insights into endothelial function and represents an important approach in the study of atherosclerosis and other vascular diseases (5, 12). Previous studies, in both human and murine models, demonstrated the presence of functional heterogeneity between EC derived from different organs (7, 9). Hence, the availability of EC from different vascular beds of genetically modified mice would facilitate further definition of the role of the target gene at specific sites and would allow questions to be addressed in vitro that are difficult or impossible to address in vivo. However, the isolation and propagation of EC from mice is technically difficult and often yields small numbers of cells.
We have circumvented these problems through the generation of a conditionally immortalized cardiac EC line, MCEC-1, from the H-2Kb-tsA58 temperature-sensitive Immortomouse (14), using positive selection with magnetic microbeads coated with MAb MJ7/18 against murine endoglin. This novel method of targeting endoglin for murine EC isolation proved to be easy to perform and highly reproducible and was also used to generate a lung EC line (not shown). In view of the fact that endoglin may be expressed on cells of monocytic lineage and smooth muscle cells in addition to EC (2, 19, 22), careful characterization of the MCEC-1 cell line was performed. The cells were confirmed as being EC in origin by their distinct morphology and ability to form microtubules on Matrigel, expression of CD31, binding of GSL I-B4 (18), and uptake of Dil-Ac-LDL. In addition, both JAM-1 and VE-cadherin were expressed at intercellular junctions. However, CD31 was not detectable at cell borders, possibly reflecting loss of expression with serial passage and/or trypsin sensitivity as previously reported (21). Primary cardiac EC were also isolated from wild-type C57BL/6 and CBA/Ca mice for comparison with MCEC-1 cells in these assays, and they behaved similarly (data not shown). However, although primary cardiac EC formed typical cobblestone monolayers, they took longer to reach confluence and could only be maintained for two or three passages before reaching senescence, thus limiting the number of experiments that could be performed. This occurred consistently in a variety of culture conditions and was in contrast to our experience with primary EC isolated from murine lung (21).
The presence of the inducible tsA58 TAg allows MCEC-1 cells to be
rapidly expanded under permissive conditions (33°C + IFN-), so as to obtain sufficient EC for analysis, before switching to the
untransformed state for experimentation by culture at 38°C in the
absence of IFN-
. Previous studies suggested that the immortalization of EC may alter their constitutive and activated phenotype compared with primary EC (3, 10, 20). Thus MCEC-1 cells, compared with conventional SV40-transformed EC lines, have the advantage of an
inducible promoter that is inactive when the cells are cultured at
38°C in the absence of IFN-
. This was confirmed by the lack of
detectable tsA 58 TAg and by a 30-fold reduction in proliferation in
MCEC-1 maintained under nonpermissive conditions compared with MCEC-1
cells cultured at 33°C with IFN-
.
Of particular interest was the finding that when maintained at 38°C MCEC-1 cells were growth factor dependent and proliferated at a rate similar to that of primary EC. Moreover, MCEC-1 cells cultured at 38°C in the presence of ECGF can be maintained for up to 20 passages. As with human EC, ECGF and bFGF were the most potent mitogens, with VEGF stimulation resulting in a less marked but significant increase in MCEC-1 cell proliferation. Furthermore, the growth factor-dependent state of MCEC-1 cells under these conditions will allow the study of genes involved in EC proliferation and differentiation. In contrast, in MCEC-1 cells maintained at 33°C the effect of the tsA58 TAg was dominant, with no additional proliferation being observed after the addition of growth factors (data not shown). This demonstration of the conditional nature of the promoter in MCEC-1 was used to facilitate transfection of the cells. Under permissive conditions, 40% transfection efficiency was achieved using a lipofection method, a level that is not readily achieved in primary EC. Thus it should be possible to generate stably transfected EC lines using this approach, with subsequent study of the transfected cells in the differentiated state under nonpermissive conditions.
In light of the fact that the MCEC-1 cells are immortalized we also investigated their susceptibility to undergo apoptosis under nonpermissive conditions. These experiments demonstrated that the MCEC-1 cells require both adhesion and exogenous growth factors for optimum survival in culture (17, 23). Thus, when cultured in suspension, EC undergo apoptosis that is only partially reversed by the presence of ECGF. In addition, adherent EC will undergo apoptosis after withdrawal of FBS and ECGF from the culture medium, and this is reduced by 50% after replacement of the growth factor.
MCEC-1 cells will also be useful in the further study of the role of
leukocyte-EC interactions in cardiovascular pathology. To this end, the
expression of cellular adhesion molecules on resting and
cytokine-stimulated MCEC-1 cells was studied. Because the
H-2Kb-tsA58 transgenic mice are on a mixed background,
MCEC-1 cells were compared with primary EC from both C57BL/6 and CBA/Ca
mice (11). Flow cytometric analysis of resting
MCEC-1 cells and primary EC confirmed comparable constitutive
expression of CD31, endoglin, ICAM-1, ICAM-2, and VCAM-1. In contrast
to human EC, murine lung and lymph node-derived EC have been reported
to constitutively express significant amounts of VCAM-1 (4, 8,
21, 24) and similar levels of VCAM-1 were found on resting
cardiac EC in this study. MCEC-1 cells and primary cardiac EC retained
their sensitivity to TNF-, IL-1
, and lipopolysaccharide (not
shown), as evidenced by adhesion molecule upregulation. A combination of TNF-
and IL-1
was more effective than either cytokine alone, resulting in the induction of E- and P-selectin expression and the
upregulation of both ICAM-1 and VCAM-1. Cytokine-stimulated MCEC-1
cells were able to support the rolling and firm adhesion of human
neutrophils and the murine monocytic cell line WEHI-3 (1)
under physiological flow (data not shown).
In conclusion, we have described a simple method for the isolation of conditionally immortalized murine cardiac EC that can be rapidly expanded under permissive conditions before switching to the differentiated state for experimentation. The EC obtained retain the phenotypic characteristics of primary EC, are growth factor responsive, and can be readily transfected by lipofection. This approach will facilitate the isolation of EC lines from genetically modified mice within two generations, by crossing gene-targeted mice with the commercially available H-2Kb-tsA58 transgenic mice and then intercrossing the offspring. This in turn will further enhance our understanding of the role of the endothelium in the pathogenesis of cardiovascular diseases.
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ACKNOWLEDGEMENTS |
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We thank R. C. Landis, S. Alexander, S. Ahmad, and D. Marshall for help with this study.
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FOOTNOTES |
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This study was funded by the British Heart Foundation (Grant PG/98115) and by the Arthritis Research Campaign (Grant M0620).
Address for reprint requests and other correspondence: J. C. Mason, BHF Cardiovascular Medicine Unit, National Heart and Lung Inst., Imperial College School of Technology and Medicine, Hammersmith Hosp., Du Cane Rd., London W12 ONN, UK (E-mail: justin.mason{at}ic.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 February 2001; accepted in final form 30 August 2001.
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