Establishment of an immortalized fetal intrapulmonary artery endothelial cell line

Margaret C. Pace1, Ken L. Chambliss1, Zohre German1, Ivan S. Yuhanna1, Michael E. Mendelsohn2, and Philip W. Shaul1

1 Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9063; and 2 Molecular Cardiology Research Institute, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. In an effort to establish an immortalized cell line, ovine fetal pulmonary artery endothelial cells (PAECs; passage 5) were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16, and phenotypes related to nitric oxide (NO) production were evaluated up to passage 28. Acetylated low-density lipoprotein uptake, endothelial NO synthase (eNOS) expression, and proliferation rates were unaltered by immortalization. Acetylcholine-stimulated eNOS activity was 218-255% above basal levels in immortalized cells, and this was comparable to the 250% increase seen in primary PAECs (passage 6). eNOS was also acutely activated by estradiol to levels 197-309% above basal, paralleling the stimulation obtained in primary cells. In addition, the expression of estrogen receptor-alpha , which has recently been shown to mediate the acute response in primary PAECs, was conserved. Thus fetal PAECs transfected with E6 and E7 show no signs of senescence with passage, and mechanisms of NO production, including those mediated by estradiol, are conserved. Immortalized PAECs will provide an excellent model for further studies of eNOS gene expression and function in fetal pulmonary endothelium.

cell immortalization; endothelial nitric oxide synthase; estrogen receptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ENDOTHELIUM plays a critical role in the regulation of the growth and function of the developing pulmonary circulation (5, 36). The central importance of this cell type is, at least in part, related to its production of the vasodilators nitric oxide (NO) and prostacyclin (PGI2) and the vasoconstrictors endothelin and thromboxane A2. These endothelium-derived compounds are responsible for mediating pulmonary vasomotor tone in the fetus and newborn (5, 36), and NO and PGI2 are especially important in producing the normal decline in pulmonary vascular resistance that occurs immediately at birth (5, 13, 36). In addition, there is evidence that pulmonary endothelial dysfunction plays a key role in the pathogenesis of a variety of pulmonary vascular diseases in the perinatal period, including persistent pulmonary hypertension of the newborn (24, 28). As such, studies of fetal pulmonary endothelial cell gene expression and function are essential to further our understanding of the fundamental mechanisms underlying these disorders. To date, however, the use of cultured cell models in direct investigations of the fetal pulmonary endothelium have been limited by the requirement for early-passage primary cells.

One approach to overcome the constraints of primary cell culture models is the use of immortalized cell lines. The introduction of viral genes, such as the simian virus large T oncogene, into early-passage, dividing primary cells inactivates cellular proteins that are responsible for limiting cell passage number and for senescence (10). DNA from the human papillomavirus type 16 (HPV-16) has been used in the immortalization of a variety of cell types including bronchiolar and mammary epithelial cells (12, 31, 33, 35), human umbilical vein endothelial cells (6), embryonic fibroblasts (32), smooth muscle cells (20), and keratinocytes (18). The transforming and immortalizing functions of HPV-16 have been localized to the E6/E7 region of the viral genome (8). The oncoprotein products of the E6 and E7 genes bind and degrade the tumor suppressor proteins p53 and pRB, respectively (6, 22, 30, 34). Inactivation of p53 and pRB results in inhibition of the cellular programs that limit the rate and number of cell divisions, thus establishing immortalization (10). Although some studies (18, 33) have found that the E7 gene alone is sufficient for immortalization, most studies (7, 12, 18, 31-33) have demonstrated that both the E6 and E7 genes are necessary for the maintenance of long-term immortalization.

The purpose of the current investigation was to establish an immortalized fetal intrapulmonary artery endothelial cell line from primary cells of a known phenotype. Cell immortalization was accomplished by permanent transfection of two retroviral vectors containing the HPV-16 E6 or E7 gene into primary ovine fetal pulmonary artery endothelial cells (PAECs), which we have employed previously in studies of fetal pulmonary endothelial cell gene expression and function. The availability and use of the latter cell type has been limited due to senescence beyond passages 6-8 (11, 14, 15, 19, 27). Once established, functionally relevant phenotypes of the immortalized cells related to NO production by the endothelial isoform of NO synthase (eNOS) were examined and compared with primary PAECs. This included studies of rapid eNOS activation by acetylcholine and also 17beta -estradiol (E2beta ), which Lantin-Hermoso et al. (14) have recently shown is a potent eNOS agonist at physiological concentrations.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and immortalization. Primary PAECs were obtained from third-generation intrapulmonary arteries of mixed-breed fetal lambs at 125-135 days of gestation (term = 144 ± 4 days) with methods previously described by Shaul and Wells (27). The procedures followed in the care and euthanasia of the study animals were approved by the Institutional Review Board for Animal Research. PAECs were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% iron-supplemented calf serum, 10% lamb serum, 1% L-glutamine, 1% penicillin-streptomycin, 0.5% ampicillin, 0.15% gentamicin, 0.15% nystatin, and 0.1% tylosin in a humidified incubator with 5.0% CO2 in air at 37°C.

To obtain a pure culture of PAECs, primary cells at passage 5 were sorted based on the uptake of 1,1'-dioctadecyl-1,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL; Biomedical Technologies, Stoughton, MA). The cells were plated at a density of 1 × 106 cells/56-cm2 culture dish, grown for 48 h, and incubated with DiI-Ac-LDL diluted aseptically in medium (10 µg/ml) for 4 h at 37°C. After washing, trypsinization, and pelleting, the cells were resuspended in sterile phosphate-buffered saline (PBS) containing 138 mM NaCl, 2.67 mM KCl, 1.47 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4, and were sorted with a Becton Dickinson (Sparks, MD) FACS IV cell sorter. For excitation, the 514-nm line of an argon laser was used, and fluorescence emission > 550 nm was collected. Endothelial cells were collected into the medium and replated onto 56-cm2 culture dishes.

The PAECs were immortalized in collaboration with Eon (Boston, MA). Sorted primary PAECs were grown to 50% confluence and infected with established techniques with two amphotrophic, replication-defective retroviral constructs, each containing the gene for G418 resistance and either the E6 or E7 transforming genes of HPV-16 (17). Surviving cells were selected and propagated up to passage 28. In all experiments, primary cells at passage 6 were compared with immortalized cells at passages 18, 23, and 28, unless otherwise stated.

Reverse transcription-polymerase chain reaction assay for E6 and E7. To provide evidence that HPV-16 E6 and E7 used for cell immortalization continue to be expressed, reverse transcription (RT)-polymerase chain reaction (PCR) assays were performed with primers designed from the published cDNA sequence of human HPV-16 E6 and E7 (23). Total cellular RNA was obtained from immortalized PAECs at passage 28 by a single-extraction method with an acid guanidinium thiocyanate-phenol-chloroform mixture (27). Total RNA was also obtained from primary PAECs to serve as a negative control. cDNA synthesis was carried out with 2 µg of total RNA, 200 U of Moloney murine leukemia virus enzyme, 0.2 µg of each oligo(dT), 1 mM deoxynucleotide triphosphates, 10 mM dithiothreitol, and 1.5 mM Mg2+ in a total volume of 50 µl. In selected tubes, reverse transcriptase was omitted to control for amplification from contaminating cDNA or genomic DNA. The RT primer for E6 was 5'-ACG TGT TCT TGA TGA TCT GC-3' and for E7 was 5'-GCT TGT CCA GCT GGA CCA TC-3'. The reactions were incubated at 42°C for 1 h. After RT, the 50-µl reaction mixture was precipitated with 3 M sodium acetate and 100% ethanol. The resulting pellet was resuspended and subjected to PCR with specific oligonucleotide primers designed from the E6 and E7 coding regions of HPV-16 (22). The sequence of the sense primer for E6 was 5'-TGC ACA GAG CTG CAA ACA AC-3' and for the antisense primer was 5'-ACG TGT TCT TGA TGA TCT GC-3'. The sequence of the sense primer for E7 was 5'-ATG CAT GGA GAT ACA CCT AC-3' and for the antisense primer was 5'-GCT TGT CCA GCT GGA CCA TC-3'. The PCR reactions contained 2.0 mM Mg2+, 1 µM primers, 200 µM deoxynucleotide triphosphates, 20 mM Tris · HCl (pH 8.4)-50 mM KCl, 5 µl of cDNA, and 2.5 U of Taq DNA polymerase in a final volume of 50 µl. The PCR temperature profile consisted of 35 cycles of 94°C for 45 s (denaturing), 52°C for 2 min (annealing), and 72°C for 1 min (extension). The PCR products were size fractionated by agarose gel electrophoresis, and their identities were confirmed by sequencing with standard methods.

Cell morphology and proliferation. The phenotype of the immortalized cells was assessed by examining the morphological characteristics common to endothelial cells, including a cobblestone appearance and contact inhibition of cell growth. The uptake of DiI-Ac-LDL was compared in primary and immortalized PAECs plated onto acid-washed coverslips at a density of 3 × 105 cells/9.6-cm2 culture well with the methods described in Cell culture and immortalization.

Cell proliferation rates in primary and immortalized cells were also evaluated by plating primary cells at passage 6 and immortalized cells at passages 18 and 28 at a density of 1.0 × 106 cells/56 cm2. Cell numbers were determined daily in each group for 4 days during growth in 20% serum. The mean value of four independent cell counts per plate was calculated. Quadruplicate plates were examined in each study group.

NOS enzymatic activity in cell lysates. Primary and immortalized PAECs were grown to 80% confluence, and the cells were rinsed twice in ice-cold PBS, pelleted, and resuspended in ice-cold buffer containing 50 mM Tris · HCl (pH 7.4), 0.1 mM EDTA, 30 µM pepstatin A, 0.4 mM leupeptin, 1.2 mM Nalpha -p-tosyl-L-lysine chloromethyl ketone, 20 µM tetrahydrobiopterin, 3 mM dithiothreitol, and 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The cells were disrupted by sonication (Branson Ultrasonics, Chicago, IL) three times for 10 s each. NOS enzymatic activity in the resulting cell lysates was determined by measuring the conversion of L-[3H]arginine to L-[3H]citrulline (19). Fifty microliters of cell lysate were added to 50 µl of buffer, yielding final concentrations of reagents as follows: 2 mM beta -NADPH, 12 µM tetrahydrobiopterin, 10 µM flavin adenine dinucleotide, 10 µM flavin mononucleotide, 0.5 mM CaCl2 in excess of EDTA, 15 nM calmodulin, 2 µM cold L-arginine, and 2.0 µCi/ml of L-[3H]arginine. After incubation at 37°C for 1 h, the assay was terminated by the addition of 400 µl of 40 mM HEPES buffer, pH 5.5, with 2 mM EDTA and 2 mM EGTA. The terminated reactions were applied to 1-ml columns of Dowex AG50WX-8 (Tris form) and eluted with 1 ml of 40 mM HEPES buffer. The L-[3H]citrulline generated was collected into scintillation vials and quantified by liquid scintillation spectroscopy. NOS activity was fully inhibited by the addition of 2.0 mM nitro-L-arginine methyl ester (L-NAME). The protein content of the cell lysates was determined with the method of Bradford (2), with bovine serum albumin as the standard. Under the conditions employed, the limiting factor is the abundance of NOS enzyme. We have previously found this determination to be a sensitive indicator of differences in eNOS abundance (15, 19).

Immunoblot analysis. Immunoblot analysis was performed to evaluate the levels of expression of eNOS and estrogen receptor-alpha (ER-alpha ) in the primary and immortalized cells. Chen et al. (4) have recently demonstrated that ER-alpha is involved in the rapid activation of eNOS by physiological concentrations of estradiol. The methods used for immunoblot analysis generally followed those previously reported by North et al. (19). Primary and immortalized PAECs were grown to 80% confluence, harvested, pelleted, resuspended in the above-mentioned 50 mM Tris · HCl buffer, and disrupted by sonication. The protein content of the resulting cell lysates was determined with the Bradford (2) method. SDS-PAGE was performed on 50 µg of protein with 10% acrylamide, and the proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked for 1 h in buffer containing 150 mM NaCl and 10 mM Tris (pH 7.5) with 0.5% Tween 20 and 5% dried milk. After being blocked, the membranes were incubated either overnight at 4°C with a 1:2,000 dilution of polyclonal primary antiserum generated to the unique midmolecule peptide PYNSSPRPEQHKSYK of human eNOS (3, 16) or for 1 h at room temperature with a 2.5 µg/ml concentration of monoclonal antibody to human ER-alpha (AER320, NeoMarkers, Fremont, CA). After incubation with primary antisera, the polyvinylidene difluoride membranes were washed with 150 mM NaCl buffer with 0.5% Tween 20 and incubated for 1 h with a 1:2,000 dilution of either goat anti-rabbit Ig antibody-horseradish peroxidase conjugate (Amersham) or a sheep anti-mouse Ig antibody-horseradish peroxidase conjugate (Amersham) for eNOS or ER-alpha detection, respectively. The membranes were washed in 150 mM NaCl buffer with 0.5% Tween 20, and the bands for eNOS or ER-alpha were visualized by chemiluminescence (ECL Western Blotting Analysis System, Amersham). The antiserum to eNOS was the kind gift of Dr. Thomas Michel (Cardiovascular Division, Brigham and Woman's Hospital, Harvard Medical School, Boston, MA).

NOS activation in intact cells. Rapid NOS activation was measured in intact primary and immortalized PAECs by measuring L-[3H]arginine conversion to L-[3H]citrulline with methods previously described by Lantin-Hermoso et al. (14). In contrast to the determinations of NOS enzymatic activity in cell lysates in the presence of excess cofactors and substrates that reveal enzyme abundance, the assessment of NOS stimulation in the intact cells evaluates rapid activation of existing enzyme by functional signal transduction mechanisms. Near-confluent plated cells were placed in L-arginine-deficient, serum-free endothelial-SFM growth medium (Life Technologies, Grand Island, NY) containing 138 mM NaCl, 2.67 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, 1.0% penicillin-streptomycin, 0.5% ampicillin, 0.15% gentamicin, 0.15% nystatin, and 0.1% tylosin for 18 h. The medium was replaced with buffer containing 120 mM NaCl, 4.2 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 7.5 mM glucose, 10 mM HEPES, 1.2 mM Na2HPO4, and 0.37 mM KH2PO4, pH 7.4, for a 15-min preincubation. In selected wells serving as blanks, 500 µl of 1 M TCA were added in lieu of PBS. After the preincubation period, the incubation for NOS activity was initiated by aspirating the PBS from the wells and replacing it with 400 µl of PBS containing 1.5 µCi/ml of L-[3H]arginine (Amersham International). The cells were incubated at 37°C for 15 min in the absence (basal) and presence of acetylcholine (10-6 M) or E2beta (10-12 to 10-8 M) at 37°C. A previous study (14) has demonstrated that maximal stimulation of eNOS is obtained with an acetylcholine concentration of 10-6 M. The NOS reaction was terminated by adding 500 µl of ice-cold 1 M TCA to each well. The cells were freeze fractured in liquid nitrogen for 2 min, thawed at 37°C for 5 min, and scraped with a rubber spatula. The contents of each well were aspirated and transferred to ice-cold sialonized glass test tubes. Ether extraction was performed three times with water-saturated ether. The samples were neutralized with 1.5 ml of 25 mM HEPES, pH 8, and processed further as described above for determination of NOS enzymatic activity in cell lysates. Basal and stimulated NOS activities in the intact cells were fully inhibited by the addition of 2.0 mM L-NAME. In individual experiments, 4 wells of a 24-well plate were used to evaluate basal NOS activity and 6 wells were used for each treatment group. Results are expressed as the percentage of basal NOS activity in the same plate. Findings were replicated in at least three independent experiments.

Statistical analyses. Analysis of variance with Newman-Keuls post hoc testing was employed to compare mean values between groups. Results are presented as means ± SE. Significance was accepted at the 0.05 level of probability.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

E6 and E7 expression. RT-PCR studies were performed to determine whether the expression of HPV-16 E6 and E7 persisted after cell immortalization. Typical negative images of ethidium bromide-stained agarose gels of the RT-PCR products for E6 and E7 are shown in Fig. 1. A single major PCR product was obtained for both E6 (Fig. 1A) and E7 (Fig. 1B) at the predicted sizes of 287 and 76 bp, respectively. RNA from primary PAECs, which were used as a negative control, did not yield PCR products. PCR products were also not obtained when the reverse transcriptase enzyme was omitted from the RT step. The PCR products were confirmed to be the E6 and E7 regions of HPV-16 by direct sequencing.


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Fig. 1.   Ethidium bromide-stained gels of RT-PCR products from E6 (A) and E7 (B) mRNAs in immortalized pulmonary artery endothelial cells (PAECs; right 2 lanes) at passage 28. Left 2 lanes, RNA from primary PAECs serving as negative controls. RT step was performed in presence (RT+) and absence (RT-) of RT enzyme. Negative images are shown. Nos. at right, band sizes.

Cell morphology and proliferation. After transfection with E6 and E7, the immortalized PAECs appeared similar to primary cells, retaining common morphological features indicative of an endothelial cell phenotype, including a cobblestone appearance and contact inhibition of growth. At all passages examined, the uptake of DiI-Ac-LDL by immortalized PAECs was comparable to that of primary cells (data not shown), and uptake was observed in the immortalized cells up to at least passage 28.

Figure 2 compares the proliferation of primary and immortalized PAECs. Before reaching confluence, primary PAECs had a doubling time of ~26 h. Similarly, immortalized PAECs at passages 18 and 28 had approximate doubling times of 30 and 33 h, respectively. The immortalized cells were noted to be slightly smaller in size, resulting in the achievement of confluence after 4 days compared with 3 days for primary cells.


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Fig. 2.   Growth characteristics of primary and immortalized PAECs at passages 18 (T18) and 28 (T28). Primary PAECs at passage 6 or immortalized PAECs were plated at a density of 1.0 × 106 cells, and cell number was determined daily for 4 days of growth in 20% serum. Values are means ± SE; n = 4 determinations. For some data points, error bars are smaller than symbols.

NOS expression. The comparison of NOS activity in lysates of primary and immortalized PAECs is shown in Fig. 3A. NOS activity in the primary PAECs was 15.3 ± 3.6 pmol · mg protein-1 · min-1. There was no difference in NOS activity levels in the primary and immortalized cells at passages 18, 23, and 28. To confirm that comparable NOS activity levels reflected equivalent eNOS expression, immunoblot analysis for eNOS protein was performed. A representative immunoblot is shown in Fig. 3B. Primary and immortalized cells at passages 18, 23, and 28 exhibited a signal for eNOS protein at the expected size of 135 kDa, and band intensity was similar in all four groups. These findings were replicated in three independent experiments.


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Fig. 3.   A: nitric oxide synthase (NOS) enzymatic activity in lysates of primary and immortalized PAECs. Studies were performed in primary PAECs at passage 6 and immortalized PAECs at T18, passage 23 (T23), and T28. L-[3H]arginine conversion to L-[3H]citrulline was measured in presence of all required cofactors and substrates. Values are means ± SE; n = 4 determinations. B: immunoblot analysis for endothelial NOS (eNOS) protein in primary (1°) and immortalized cells. Signal for eNOS protein was detected at 135 kDa. There was no difference in eNOS protein abundance between primary and immortalized cells. Similar findings were obtained in 3 independent experiments.

NOS activation in intact cells. The activation of eNOS by acetylcholine in intact primary and immortalized PAECs is shown in Fig. 4. In primary cells, NOS activity increased 2.5-fold over basal levels in response to acetylcholine (10-6 M). Similarly, acetylcholine caused a 2.2- to 2.6-fold stimulation of eNOS in the immortalized cells at passages 18, 23, and 28.


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Fig. 4.   ACh stimulation of eNOS in primary and immortalized PAECs. L-[3H]arginine conversion to L-[3H]citrulline by intact cells was measured over 15 min in absence (basal) and presence of 10-6 M ACh. Values are means ± SE; n = 4 basal and 6 ACh determinations. * P < 0.05 vs. basal value.

The activation of eNOS by E2beta in intact primary and immortalized PAECs is shown in Fig. 5. NOS activity in primary PAECs rose in a dose-dependent manner, increasing to 181-221% of the basal level, with a threshold concentration of 10-10 M E2beta . Similarly, in immortalized cells, NOS activity increased to 197-309% of the basal level, with a threshold concentration of 10-10 M E2beta .


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Fig. 5.   17beta -Estradiol (E2beta ) stimulation of eNOS in primary and immortalized PAECs. Primary PAECs at passage 6 (A) and immortalized PAECs at T18 (B), T23 (C), and T28 (D) were studied. L-[3H]arginine conversion to L-[3H]citrulline by intact cells was measured over 15 min in absence and presence of 10-12 to 10-8 M E2beta . [E2beta ], E2beta concentration. Values are means ± SE; n = 4 basal and 6 E2beta determinations. * P < 0.05 vs. 0 E2beta .

ER-alpha expression. To determine whether ER-alpha expression is conserved in the immortalized PAECs, immunoblot analysis was performed. A representative immunoblot for ER-alpha protein is shown in Fig. 6. Primary cells at passage 6 and immortalized cells at passages 18, 23, and 28 exhibited a signal for ER-alpha protein at the expected size of 67 kDa, and the intensity of the signal was equivalent in all groups. Similar findings to these were obtained in three independent experiments.


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Fig. 6.   Immunoblot analysis for estrogen receptor-alpha (ER-alpha ) protein in primary and immortalized PAECs. Signal for ER-alpha protein was detected at 67 kDa. There was no difference in ER-alpha protein abundance in primary and immortalized cells. Similar findings were obtained in 3 independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Direct investigations of pulmonary endothelial cell gene expression and function to date have been hampered by the need for early-passage primary cells. The number of primary PAECs available for study is limited, and the level of ascertainment of relevant phenotypes may differ between primary cell harvests. In addition, the use of nonpulmonary endothelial cells to evaluate processes in the pulmonary endothelium can be highly misleading. Phenomena such as the decrease in NO production that occurs in response to rapid changes in oxygenation are unique to the pulmonary endothelium compared with endothelium from other vascular beds (25, 27), making it necessary to study endothelial cells of specific origin. The embryological source of the intrapulmonary endothelium is unique (29), and this may explain numerous specific aspects of pulmonary endothelial cell phenotype. The present report describes the establishment of an immortalized ovine fetal intrapulmonary artery endothelial cell line that overcomes many of the limitations of primary cells.

After cell sorting, primary PAECs at early passage were immortalized by permanent transfection with the E6 and E7 open reading frames of HPV-16. When employed simultaneously, these proteins have been shown to successfully immortalize a variety of cell types, including human umbilical vein endothelial cells (7, 8, 20), by downregulating cell cycle control mechanisms. However, to our knowledge, this is the first report of immortalization of pulmonary endothelial cells. The immortalized PAECs retained common morphological characteristics of endothelial cells, including a cobblestone appearance, contact inhibition of cell growth, and uptake of LDL. The immortalized cells also had similar growth rates to primary cells but were slightly smaller in size and therefore required a longer time to reach confluence. Interestingly, the decrease in cell size observed in the immortalized PAECs has been noted in other cell lines permanently transfected with E6 and E7, including umbilical vein endothelial cells (7), smooth muscle cells (20), and fibroblasts (32). The basis for the modest change in cell volume after E6 and E7 transfection has yet to be determined.

To evaluate whether critical phenotypic features are conserved in the immortalized PAECs, the expression and function of eNOS were assessed and compared with those in primary, early-passage cells. The level of eNOS expression was determined in studies of NOS enzymatic activity in cell lysates and immunoblot analyses for the protein. It was found that NOS enzymatic activity was comparable in the primary PAECs and in immortalized cells at passages 18, 23, and 28. In addition, the abundance of eNOS protein was similar. The consistent expression of eNOS in the immortalized cells, which remain viable after multiple passages, is in marked contrast with the senescence that is observed after passage 8 in primary PAECs (14, 15, 19, 27). As a result, the immortalized cells will serve as an excellent model for further studies of the regulation of eNOS expression in the developing lung. This would include future investigations of the basis for eNOS upregulation in fetal pulmonary endothelium by estrogen and increased oxygenation (15, 19). In more general terms, the immortalized PAECs will also be invaluable in further studies of eNOS trafficking to plasmalemmal caveolae, which was first delineated in primary PAECs (26). Large volumes of cells are necessary for investigations involving isolated caveolae membrane preparations, and this is now feasible with the immortalized cell line.

In addition to studies of eNOS expression, the capacity to activate the enzyme was also assessed by measuring L-[3H]arginine conversion to L-[3H]citrulline in intact cells. In contrast to the determinations of NOS enzymatic activity in lysates in the presence of excess cofactors and substrates that reveal enzyme abundance, the assessment of NOS stimulation in the intact cell evaluates rapid activation of existing enzyme by functional signal transduction mechanisms. The primary PAECs from ovine fetal lambs have been previously employed in studies of acetylcholine-induced eNOS activity (14), taking advantage of the conservation of a classic response that is typically studied in intact arteries and that has been difficult to reproduce in other endothelial cell lines (25). As previously observed, NOS activity increased more than twofold over basal levels in response to acetylcholine in the primary cells. More importantly, the response to acetylcholine was readily apparent in the immortalized PAECs as well. As a result, the immortalized cell line will provide an excellent model for additional investigations, specifically of acetylcholine-mediated responses in fetal pulmonary endothelium and more generally of rapid signal transduction events in endothelial cells.

Lantin-Hermoso et al. (14) have previously demonstrated that eNOS is also rapidly activated by physiological concentrations of 17-estradiol, with a threshold concentration that is 100-fold lower than that for acetylcholine. This process may play a critical role in the atheroprotective properties of the hormone because estrogen causes a rapid vasodilation that is at least partially related to its ability to enhance the bioavailability of NO (4). In addition, this mechanism may be involved in NO-mediated pulmonary vasodilation in the perinatal period because fetal plasma estrogen levels rise markedly with the onset of parturition due to enhanced placental production of the hormone (21). The initial studies of this process were done in early-passage PAECs, and conservation of this phenotype in the immortalized cells would greatly enhance the ability to delineate the mechanisms underlying this nongenomic response to estrogen. In the present investigation, the rapid activation of eNOS in primary PAECs by E2beta was confirmed, with a threshold concentration of 10-10 M. Comparable findings were also obtained in the immortalized cells up to at least passage 28. As such, it will be possible to continue to study the rapid effects of estrogen on endothelial cell function in the immortalized cells and to be able to do so at levels of the hormone that are well below those found in normal cycling women (4).

Chen et al. (4) have recently demonstrated that the acute activation of eNOS by estradiol is mediated by ER-alpha , which was previously known to function solely as a transcription factor. This was accomplished by demonstrating that the response to estradiol in early-passage PAECs is fully inhibited by concomitant acute treatment with specific ER-alpha antagonists and by overexpressing ER-alpha in the primary PAECs and demonstrating that the response is augmented (4). Studies of ER-alpha function have been possible in the primary PAECs because the expression of the receptor is conserved in culture up to passage 8 (11, 14, 15). To determine whether this characteristic remains faithful in the immortalized cells, immunoblot analysis for ER-alpha was performed in cells up to passage 28. It was found that ER-alpha expression is conserved and that receptor abundance is comparable in the immortalized and primary cells. These findings contrast with the observation that ER-alpha expression is lost after repeated passage of other endothelial cell types (9). The immortalized cells will therefore make it possible to investigate both the nongenomic and genomic functions of ER-alpha and also the mechanisms determining the levels of ER-alpha expression in endothelium.

Thus we have established an immortalized fetal intrapulmonary artery endothelial cell line by permanent transfection of the E6 and E7 transforming proteins of HPV-16 into early-passage primary cells. The immortalized cells retain the morphological and growth characteristics of primary cells, and functionally relevant phenotypes related to the regulation of NO production by eNOS are also conserved. However, numerous characteristics have not yet been compared in primary versus immortalized PAECs. This includes processes regulating endothelin production, which is coordinated with eNOS expression during the development of fetal pulmonary hypertension in the lamb model (1). It is anticipated that this new cell line will simplify studies focused on determining the cellular and molecular bases of diseases such as persistent pulmonary hypertension of the newborn. In addition, the immortalized PAECs will be invaluable in future investigations of a variety of issues in vascular biology, including those related to the effects of estrogen on the vascular wall.


    ACKNOWLEDGEMENTS

We are indebted to Wendy Baur for technical assistance and to Marilyn Dixon for preparing this manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-58888 and HL-53546 and National Institute of Child Health and Human Development Grant HD-30276 (all to P. W. Shaul) and NHLBI Grants HL-56069 and HL-59953 (to M. E. Mendelsohn).

The project was done during the tenure of Established Investigatorships of the American Heart Association (P. W. Shaul and M. E. Mendelsohn).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. W. Shaul, Dept. of Pediatrics, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9063 (E-mail: PSHAUL{at}MEDNET.SWMED.EDU).

Received 25 January 1999; accepted in final form 10 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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