JOURNAL TRANSCRIPT
1113
Growth-Promoting Effects of Substance P Endothelial Cells In Vitro
on
Synergism With Calcitonin Gene-Related Peptide, Insulin, and Plasma Factors Amparo C. Villablanca, Christopher J. Murphy, Ted W. Reid
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Abstract The purpose of this study was to determine the effects of the vasoactive perivascular neuropeptide substance P (SP) on the growth and function of vascular endothelial cells in serum-free culture conditions with cells quiescent in the GO-G, phase of the cell cycle and to characterize the response. In addition, interactions between SP and other growth factors and neuropeptides including insulin, platelet factors, neurokinin A, neurokinin B, and calcitonin gene-related peptide (CGRP) were studied on endothelial cell growth and compared. Growth effects were determined by stimulation of tritiated thymidine incorporation into DNA and cell proliferation. SP exhibited differential effects on cell growth that were a function of concentration, incubation time, interaction with other growth factors, and cell culture conditions. DNA synthesis in response to SP showed a bell-shaped distribution with a maximal effect that was 10.5-fold over control at 500 ,ug/mL of SP after 48 hours of incubation. The effect showed marked synergism with insulin (10 ,ug/mL) and with CGRP (0.01 to 10
,ug/mL), which is colocalized with SP in vivo. Insulin and CGRP alone had no significant effect on endothelial cell growth. Furthermore, no synergism was observed between SP and platelet-derived growth factor or platelet-derived endothelial cell growth factor. Endothelial cell proliferation increased in response to SP to 2.6-fold over control at 48 hours, was maximal at 10 ,tg/mL SP, and also demonstrated synergism with insulin (10 ,ug/mL). Our studies indicate that neuropeptides play a significant role in regulating endothelial cell growth and proliferation. The demonstrated synergism between SP and other known growth factors and neuropeptides implies that synergism between differing signal transduction pathways may be significant in the total observed response. These findings suggest that SP may have an important role in the normal and pathophysiological states of the vasculature. (Circ Res. 1994;75:1113-1120.) Key Words * substance P * neuropeptides * cell growth * endothelial cell * tachykinins
T he vascular endothelium is strategically poised between blood and the subendothelial layers of the vascular wall. The endothelium consists of a quiescent contact-inhibited cell monolayer seemingly structurally simple but functionally complex and participates in a variety of cellular events, including the elaboration of growth factors and regulation of vascular cell growth.' In addition, the endothelium is important in pathophysiological events, including atherogenesis and wound healing.' Substance P (SP) is a ubiquitous vasoactive perivascular member of the tachykinin family of neuropeptides, which share a common carboxy-terminal sequence and include the structural analogues neurokinin A (NK-A) and neurokinin B (NK-B). SP is present in capsaicin-sensitive unmyelinated primary afferent nerve fibers in peripheral organs and envelops the vasculature, with the highest density of SP-containing networks concentrated in the aorta and venae cavae close to the heart.2 SP has also been localized in the adventitia and the medial-adventitial border of the vessel wall and is synthesized and released from arterial endothelial cells (ECs),3-6 where it has been localized to the cytoplasm.5 SP is colocalized in vascular
afferent nerve endings with calcitonin gene-related peptide (CGRP), where it has a role as a mediator of neurotransmission, neurotropism, and neurogenic inflammation.5'7-10 SP is also present in the blood of humans and other mammals and can be detected in plasma by radioimmunoassay,1" with increased plasma levels reported in conditions characterized by uncontrolled cellular growth, including malignancies.12"13 A variety of circulatory and vascular effects of SP have been elucidated or postulated. These effects include its role in stimulating immune function,14 tissue repair,15 increased vascular permeability,16"17 response to hypoxia3 and its role as a mediator of inflammation,18 potent peripheral and coronary vasodilator, and modulator of coronary blood flow.19-22 The two latter effects are associated with SP-mediated endothelial prostacyclin (PGI2) production23'24 and endothelium-dependent production of endothelium-derived relaxing factor (EDRF).25,26 Evidence from recent studies27-29 suggests that sensory neuropeptides may also play an important role in regulating in vitro cellular proliferation. However, the role of SP on arterial endothelial cell growth is unknown. We hypothesized that SP is a stimulus for arterial EC growth. The purpose of the present study was to use a serum-free culture environment with cells in growth quiescence to determine the role of SP on EC growth, to compare the response with that of other neuropeptides to include NK-A, NK-B, and CGRP, and to characterize the growth response by evaluating the
Received May 27, 1994; accepted September 12, 1994. From the Departments of Internal Medicine and Ophthalmology, School of Medicine, University of California-Davis. Correspondence to Amparo C. Villablanca, University of California-Davis, Division of Cardiovascular Medicine, TB 172, Bioletti Way, Davis, CA 95616. C) 1994 American Heart Association, Inc.
1114
Circulation Research Vol 75, No 6 December 1994
modulating effects of known growth factors and blood elements, including insulin, serum, and platelet-poor plasma. Growth-promoting cellular responses were studied by determining incorporation of [3H]thymidine into DNA and by cell counts.
Materials and Methods Materials Chemicals SP was obtained from Sigma Chemical Co. Aqueous solutions of SP were found to be unstable with repeated freezethawing but could be kept at 5°C for up to 1 week with loss of half of the maximal activity. All solutions were made up fresh before their use. SP was determined to be >99% pure by high-performance liquid chromatography (HPLC) done in our laboratory on the purchased peptide. Norleucine SP (NL-SP) was obtained from Multiple Peptide Systems. Human CGRP, NK-B, SP fragments 1-9, 5-11, and 6-11, bovine insulin, and hydroxyurea were purchased from Sigma; NK-A was from Peptide Institute Inc; [3H]thymidine was obtained from Amersham; and the metalloendopeptidase enkephalinase (EK) was acquired from Genentech Inc and was stable in 0.15 mol/L NaCl in 0.01 mol/L HEPES (pH 7.2) at 5°C for over 1 year. Platelet-derived growth factor (PDGF, human AB) was purchased from JR Scientific. Platelet-derived EC growth factor (PD-ECGF) was prepared from the plasma fraction of human platelets30 and partially purified (=50%) by CM and QAESephadex chromatography and ammonium sulfate precipitation as described previously.31 This material stimulated EC DNA synthesis threefold to fourfold over control at dilutions of up to 1:125.
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Medium 199 was purchased from Fisher; fetal bovine and iron-supplemented calf serum were from Hyclone; penicillin/ streptomycin/fungizone, trypsin, thymidine, and EDTA were obtained from Sigma; tissue culture plates were from Falcon; tissue culture flasks were from Corning Glass Works; and cell scrapers were from Costar.
Cell Culture Secondary cultures of calf pulmonary artery ECs were obtained from the American Type Culture Collection (CRL 1733) and as a generous gift from Una Ryan, St Louis, Mo. Cells were routinely cultured in medium 199 supplemented with 5% fetal bovine serum, 5% iron-supplemented calf serum, and 10` mol/L thymidine, hereafter referred to as growth medium, plus antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 ,ug/mL amphotericin B) on plastic tissue-culture flasks in a humidified atmosphere of 5% CO2 and air at 37°C. Cells were passaged weekly nonenzymatically at a split ratio of 1:2 by using a cell scraper and were subcultured an average of three or four times (range, 1 to 10 times) after they were received before being used for studies. No differences were seen in the cellular responses to neuropeptides and growth factors for the range of subcultures
in the wells. This thymidine depletion procedure was sufficient to bring the cells to a synchronized quiescent phase of the cell cycle (GO/G1), as determined by low levels of [3H]thymidine incorporation into DNA and flow cytometric analysis. We chose these study conditions because ECs in vivo are in growth quiescence and experience a very low turnover rate32 and because we wished to study the ability of the various neuropeptides to stimulate cells to enter the DNA synthesis (S) phase of the cell cycle. At this point, the culture medium was changed, and the desired growth factors or peptides were then added in serum-free medium 199. [3H]thymidine (0.1 ,uCi) was inoculated into each well immediately after the addition of the desired growth stimulus. Serum-free medium 199 alone was used for the control condition. Initially, cells (three to six wells per experimental point) were incubated with the added growth factors for 48 hours to 6 days. For incubations longer than 3 days, on the third day of incubation the conditioned medium was discarded, and [3H]thymidine and growth factors were readded. Various time-response curves were constructed, and because DNA synthesis was maximal after 2 days of incubation, all further assays were performed for 2 days. Therefore, after 48 hours of sparse
incubation with the added growth factors, cells were washed with 0.15 mol/L NaCl, DNA-precipitated with 0.3 mol/L HCI
for 20 minutes at 5°C, washed again with 0.3 mol/L HCI, rinsed with 95% ethanol, allowed to air-dry, and then solubilized with 0.5 mol/L NaOH. An aliquot from each well was taken for liquid scintillation counting (LKB), and data were expressed as counts per minute of [3H]thymidine incorporated into DNA. Incubation of cells with HCI during the assay procedure precipitated the DNA and permeabilized the cells so that any low levels of thymidine associated with intracellular pools could be released and removed during the subsequent wash step. In this manner, measurements of [3H]thymidine incorporated into DNA correspond to de novo DNA synthesis and not to shifts in levels of the cold thymidine precursor pool.
Proliferation Studies To determine that an increase in [3H]thymidine incorporated into DNA was accompanied by cellular proliferation, ECs were incubated in the presence and absence of SP and then counted. Cells to be counted were plated and treated in a manner identical to that used in the DNA synthesis studies, except [3H]thymidine was not added. Growth factor(s) was incubated with the cells (5 to 10 wells per experimental point) for 48 hours to 6 days, with a maximal effect noted by 2 days of incubation. Therefore, after 48 hours the wells were emptied and washed with phosphate-buffered saline, pH 7.4. Cells were detached from the wells by incubating them with trypsin in EDTA for 2 minutes at 37°C as described above. Growth medium was added to stop the reaction, and the contents of each well were then mixed with a P-200 micropipette. This procedure was repeated once more to ensure that all cells were
removed from the wells. The cells for each experimental point then pooled and centrifuged and resuspended in medium 199 containing 50% (vol/vol) trypan blue. Cell counts were performed by use of a Neubauer counting chamber (C.A. Hauser and Son). Nonviable cells (approximately one to three per high-powered field) were excluded from counting.
were
used.
Data Analysis
DNA Synthesis Assay
DNA synthesis experiments were performed in three to six wells per point, and cell proliferation assay experiments were performed in 5 to 10 wells per point. Results are expressed as
To perform accurate counts for plating, ECs were removed from the tissue culture flasks by incubating them for 2 minutes at 37°C with 0.25% trypsin in EDTA (25 mmol/L in 50 mmol/L NaCl, pH 7.4) and then plated in the inner 60 wells of 96-well plastic plates at a density of 6 x103 cells per well in serumcontaining growth medium. The outer 36 wells were rimmed with sterile water. Two days later the cells were fed with fresh growth medium devoid of thymidine and allowed to deplete this medium for an additional 2 days, at which point the cells were preconfluent (40% to 60% confluent) and still relatively
mean
+±SEM of data from experiments performed
in triplicate
(n=3). Data for all studies were analyzed by two-tailed Student's t test, and significance was determined at P