INTRODUCTION

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VASCULAR ENDOTHELIAL CELLS play an important role in a variety of physiological processes, including vasoregulation, repair of the vascular intima, blood clotting, and development of new blood vessels (angiogenesis). Vascular tone and blood flow can be regulated by endothelial release of compounds such as nitric oxide (NO), prostacyclin (PGI₂), ATP, and endothelin, whereas endothelial proliferation and migration are important components of a continual process of endothelial repair in established blood vessels (15). Additionally, during the formation of new blood vessels, endothelial cells release enzymes to degrade the basement membrane, migrate through the vascular wall, and proliferate to create capillaries that extend perpendicularly to the original blood vessel (8). Angiogenesis occurs in many physiological and pathophysiological conditions, including development, wound healing, ovarian and menstrual cycling, rheumatoid arthritis, and tumor growth.

Many compounds control vascular endothelial cell function. Release of vasoactive compounds such as PGI₂ and NO is regulated by acetylcholine, bradykinin, and ATP (23). Many factors have been reported to either promote or interfere with chemotaxis, mitogenesis, and/or angiogenesis in vascular endothelial cells; these include basic fibroblast growth factor (28), vascular endothelial growth factor (VEGF) (7), placental growth factor (PIGF) (36), and NO (27). The suggestion has been made that purines or purine nucleotides might be capable of acting as angiogenic agents (29). ATP and adenosine have been shown to promote proliferation (32) and migration (21) of endothelial cells in culture. A definitive role for purines in the regulation of nonvasoactive functions in the endothelium has yet, however, to be demonstrated.

A number of studies have now shown significant effects of exogenous UTP on a variety of vascular processes. UTP has been shown to stimulate release of vasodilatory compounds such as NO and PGI₂ from endothelial cells (26, 33) and also to produce changes in tension in vascular smooth muscle in an endothelium-independent manner (31, 33). Several different receptors for pyrimidine nucleotides have now been cloned and expressed; among these are the P2Y2 receptor, which exhibits equal responsiveness to ATP and UTP (20); P2Y4, a UTP-selective receptor with no significant affinity for UDP (5, 24); and P2Y6, a UDP-selective receptor with very low affinity for ATP (4, 25). Using functional assays, we previously reported (35) that single endothelial cells isolated from the guinea pig cardiac vasculature can express at least three different nucleotide receptors, including two that respond to UTP. The question of why a single vascular endothelial cell would express three different types of nucleotide receptors prompted our present hypothesis that UTP receptors in vascular endothelial cells are not associated with moment-to-moment regulation of blood flow and pressure but rather with processes such as endothelial cell migration and proliferation. We now report that UTP is a potent chemotactic and mitogenic agent in endothelial cells isolated from the guinea pig cardiac vasculature and is angiogenic in the chorioallantoic membrane of the chicken embryo (CAM). To our knowledge, these findings constitute the first report of a chemotactic or angiogenic effect of any pyrimidine and also the first description of direct chemotactic or mitogenic effects of a pyrimidine in endothelial cells; our results also may be used to assign a role for pyrimidine nucleotide receptors in the mediation of endothelial cell functions that are distinct from those subserved by purine nucleotide receptors.

	METHODS

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Cardiac endothelial cell isolation and culture. Unless otherwise stated, reagents were obtained from Sigma Chemical (St. Louis, MO). VEGF (165-amino acid form) and PIGF-1 were human recombinant products expressed in Sf21 insect cells and Escherichia coli, respectively, and were obtained from Sigma Chemical. Endothelial cells from the cardiac vasculature (CEC) of 250-g guinea pigs were isolated and cultured as described previously (35). Cells were allowed to grow to confluency in a culture medium of DMEM supplemented with penicillin (763 U/ml), streptomycin (100 U/ml), gentamycin (150 µg/ml), Fungizone (5 µg/ml), and amikacin (1 µg/ml) in 0.1% (vol/vol) fetal bovine serum (FBS) in a water-saturated atmosphere of 95% O₂-5% CO₂; confluency was usually achieved within 1 wk. The endothelial origin of cultured cells was routinely confirmed by positive staining in immunofluorescent assays using FITC-labeled antibodies directed against factor VIII and by intracellular calcium responses to agonists such as bradykinin; these characteristics were present in cells through the fifth passage, although cells were used for experiments between passages 0 and 2. Subculturing was accomplished by a 5-min trypsin (0.05% wt/vol, 1:250, GIBCO BRL, Gaithersburg, MD) digest in calcium- and magnesium-free buffer, centrifugation, and resuspension in culture medium before passage seeding.

Mitogenesis assay. CEC were harvested by trypsin digest and resuspended in culture medium containing 0.1% FBS and antibiotics. Approximately 45,000 cells were added to 1 ml of culture medium in each well of 12-well tissue culture plates (Corning Costar, Cambridge, MA); test reagents or appropriate volumes of PBS were then added, and plates were cultured for 72 h. Cells were harvested by trypsin digest, resuspended in 1 ml of culture medium, and counted by either hemocytometry or Coulter counter. Each test (or control) condition was present in triplicate for each experiment, and each sample was counted at least in triplicate.

Chemotaxis assay. Chemotaxis experiments were performed using a modified, open-well Boyden chamber with a polycarbonate membrane (Transwell, 12-well plate format, Corning Costar). Membranes were 6.5 mm in diameter, had 8-µm pores, and had a porosity (% of total filter area occupied by pores) of 5%. Each membrane was coated for 24 h at 4°C with 0.01% (wt/vol) collagen type I, dried, and then irradiated with ultraviolet light for 24 h in a laminar-flow cell culture hood; membranes were subsequently handled under aseptic conditions. Test agents were added to an experimental buffer consisting of culture medium (with antibiotics) plus 1% (wt/vol) BSA (fraction V, Calbiochem, La Jolla, CA); these solutions were then added to culture wells so that fluid levels in the bottom chambers were equal to those in the upper chambers to eliminate hydrostatic pressures. CEC in standard culture vessels were harvested by 2-min trypsin (0.01% wt/vol) digest; after cells were counted by hemocytometry, 90,000 cells were added to the upper chamber of each culture well. Pilot time course experiments determined that the maximum chemotaxis response in these cells occurs between 4 and 6 h; cell culture plates were therefore incubated for 5 h at 37°C, after which membranes were removed and immediately scraped free of cells on the top (upper chamber) side using a rubber policeman fashioned from a hypodermic needle tipped with a length of 2-mm-diameter rubber tubing. Membranes were fixed and stained using the Diff-Quick staining system (Dade, Dudingen, Switzerland). Cells that had moved through membranes were quantified by counting 10 random fields of the bottom (lower chamber) side of each membrane at ×250; numerous pilot experiments detected no significant number of cells in either the upper or lower chamber media (or adherent to the bottom or sides of the lower chamber) at the conclusion of the 5-h experiments. In all experiments, chemotaxis controls, in which test agents were added at equal concentrations to both upper and lower chamber media, were used to control for the possibility that test compounds might affect overall cell motility and/or division in a nonspecific (nonchemotactic) manner.

Angiogenesis assay. Angiogenesis experiments were performed on the chicken CAM in vitro. Chicken eggs were obtained on day 1 after fertilization (California Golden Eggs, Sacramento, CA) and incubated at 39°C in room atmosphere with 85% humidity for 3 days before explantation. Embryos were removed from shells by placing the large air sac end onto a Delta bench-top belt sander to create a window ~2 cm in diameter; the air sac membranes were then torn, and a small hole was created in the opposite end of the shell by cracking. Internal membranes now accessible through the original hole were ruptured, which gently released the shell contents into a 177-ml Styrofoam cup into which had been placed 55 ml of water followed by a plastic wrap liner (17). Glass petri dish bottoms were used to cover the cups, and embryos were returned to incubators until day 8, at which point experiments were begun. Explantation success rates using these techniques averaged 98%, and ~90% of embryos survived through the end of the experiments.

Data analysis. Cell counts, whether by microscopy or hemocytometry, were evaluated statistically by means of one-way ANOVA followed by the Newman-Keuls multiple-comparison test. Effects of treatment in angiogenesis experiments were evaluated by paired Student's t-tests performed on means obtained from 10 random fields from each sample. Differences were considered to be significant when P values were <0.05. All experiments performed on the products of a single cell preparation (to which 4 different animals contributed) were considered to be a single sample of the population (n); in all, cells from ~60 guinea pigs were used in this study; in CAM assays, each embryo was considered to be a single n. Results are presented as means ± SE.

	RESULTS

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Cardiac endothelial cells harvested from confluent cultures in the first or second passage were cultured for 72 h in the presence of 0.1% FBS and either PBS or the test agents ATP, UTP, or VEGF at concentrations determined in preliminary experiments to be maximal; the time course was determined as sufficient for the fastest- and slowest-growing cultures to become ~90 and 40% confluent, respectively. The number of cells present at the conclusion of experiments is shown in Fig. 1. Compared with cells cultured without test reagents (controls), ATP, VEGF, and UTP all produced statistically significant increases in the number of cells present at the end of the 72-h period; although UTP appeared to stimulate the greatest proliferation of cells under these conditions, there was no significant difference among the ATP, VEGF, and UTP means obtained.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Mitogenesis in cultured guinea pig cardiac endothelial cells (CEC). CEC were cultured in 0.1% fetal bovine serum (FBS) in absence (control) or presence of ATP (100 µM), vascular endothelial growth factor (VEGF; 100 ng/ml), or UTP (100 µM) for 72 h, after which cell counts were obtained by hemocytometry. Values presented are means ± SE from n = 5 experiments in which each sample was assayed in triplicate. * P < 0.01, ** P < 0.001 compared with control.

Concentration-response relationships were established for UTP, ATP, VEGF, and PIGF with respect to the ability of each compound to promote directional movement of CEC through permeable supports (chemotaxis); these data are presented in Fig. 2. Although statistically significant slopes were obtained for the relationship between chemotaxis and concentration for UTP, VEGF, and PIGF, no such relationship was observed for ATP, suggesting that this purine nucleotide is not a significant chemotactic agent toward CEC. Apparent EC₅₀ values for chemotaxis by UTP, VEGF, and PIGF were determined to be 6 µM, 0.01 ng/ml, and 85 ng/ml, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent chemotaxis in CEC. For migration assays, 12-well Transwells were used as described in METHODS. Each test agent was diluted to the appropriate concentration in DMEM containing 0.1% FBS and added to bottom compartment of each chamber; CEC harvested from subconfluent cultures were seeded into top chamber and allowed 5 h to migrate to underside of membrane before counting. Responses are expressed as percentage of maximum achieved with each agent for each experiment and are results of n = 3 experiments performed in triplicate; bars indicating SE averaged 20% of each mean value and were omitted for clarity. PIGF, placental growth factor.

A direct comparison of chemotactic effects among all test compounds at their maximum effective concentrations is presented in Fig. 3. In these end-point assays, UTP and VEGF were both found to be highly chemotactic toward CEC (P < 0.001), whereas neither ATP nor PIGF demonstrated any significant chemotactic effects. That PIGF would fail to elicit a significant end-point chemotaxis response while demonstrating a positive slope in its concentration-response curve probably is best explained by the very small increase in chemotaxis observed in the concentration-response curves (Fig. 2), i.e., PIGF elicits at best a small increase in chemotaxis in Fig. 2 compared with the nonspecific movement of cells across the membranes. Chemotaxis in response to UTP in end-point assays was significantly greater than that seen in response to either ATP or PIGF (P < 0.001), and VEGF was also significantly more chemotactic than either ATP or PIGF (P < 0.01 and P < 0.05, respectively). Chemokinesis controls included in each experiment were used to rule out the possibility that agents demonstrating chemotactic activity were not simply increasing overall random cell motility and proliferation; in no case did the chemokinesis control for a given treatment ever produce chemotaxis results that were significantly different from the normal control (no test agent added) values (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   UTP is chemotactic toward CEC. End-point cell migration assays were performed in 0.1% FBS and maximal concentrations of ATP (100 µM), PIGF (100 ng/ml), VEGF (10 ng/ml), or UTP (100 µM); control wells contained only 0.1% FBS, and experiments lasted for 5 h. Values presented are percentage of total number of cells that migrated through membrane (as calculated from 10 random fields quantified from membrane with 33-mm2 area) and are means ± SE from triplicate assays of n = 3-7 preparations of 4 animals each. ** Mean significantly (P < 0.001) different from control mean.

Angiogenesis in response to test agents was evaluated in the CAM assay, followed by microscopic examination of both intact CAM and tissue sections. On examination with a dissecting microscope, implanted sponges that had been soaked in UTP or VEGF were surrounded by a large number of very small, radially arranged blood vessels, a characteristic commonly seen in angiogenesis experiments using sponge implants soaked with other angiogenic agents (data not shown; see, e.g., Ref. 36). Quantitative measurements of the angiogenic effects of UTP and VEGF were obtained by light microscopic analysis of tissue sections of control and treated sponges, and the summary of these data is presented in Fig. 4; neither ATP nor PIGF was used in these experiments, because neither demonstrated chemotactic activity in CEC assays (Fig. 3). Treatment of CAM with either UTP or VEGF resulted in statistically significant increases in the blood vessel density of the CAM compared with paired control samples obtained from the same embryos, a finding that controls for the possibility that the observed increases in vascularity were due simply to inflammatory responses to the sponges used to deliver test compounds.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   UTP is angiogenic in chick chorioallantoic membranes (CAM). Angiogenesis in CAM tissues induced by UTP or VEGF was evaluated by paired analyses as described in METHODS. Microscopic analyses of tissue sections from both control (PBS treatment) and UTP- or VEGF-treated samples revealed a statistically significant increase in number of blood vessels present within tissues treated with either UTP or VEGF compared with controls. All microscopic fields (10 random fields/sample) were normalized for tissue section area. Number of blood vessels in control samples averaged 1.33/mm2. * P < 0.05, n = 5 for VEGF vs. paired controls and n = 5 for UTP vs. paired controls.

	DISCUSSION

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Extracellular UTP has now been shown to stimulate responses in a number of different tissue types, including vascular (31) and nonvascular (34) smooth muscle, liver (14), neutrophils (10), mesangial cells (16), and salivary gland (30). In the vasculature, UTP elicits an increase in endothelial release of PGI₂ (19), and there is evidence that endothelium-dependent UTP relaxation of vascular smooth muscle is mediated by NO (33). UTP is also capable of directly stimulating vascular smooth muscle when the endothelium is removed (13, 22).

The main question addressed in the present study arose from our earlier determination (35) of multiple nucleotide receptors expressed in vascular endothelial cells in culture and centers on why a single endothelial cell would express two receptors capable of responding to ATP (P2Y1 and P2Y2) as well as two receptors capable of responding to UTP (P2Y2 and another, UTP-selective, P2Y receptor). We therefore undertook experiments designed to address the hypothesis that pyrimidine nucleotides play a more important role in the regulation of endothelial cell movement and proliferation than do purine nucleotides such as ATP. Our findings suggest that UTP acts as both a chemotactic and a mitogenic factor in vascular endothelial cells in culture; by way of contrast, ATP demonstrates less mitogenic activity and no significant chemotactic activity in these same assays. Additionally, given our finding of a significant effect of UTP on blood vessel development in an in vitro CAM assay, we now propose that UTP can act as an angiogenic factor.

Concentration-response relationships were established in chemotaxis experiments for UTP, ATP, VEGF, and PIGF (Fig. 2). Although direct comparison of potencies between the growth factors and the nucleotides was not possible because of differences in units used, VEGF was clearly more potent than PIGF in eliciting chemotaxis in endothelial cells (1 ng/ml vs. 85 ng/ml for PIGF); UTP exhibited a potency of ~6 µM, whereas no true concentration-response relationship existed for ATP in chemotaxis experiments. Although the data are not shown, when concentration-response curves were carried out to higher-than-maximal concentrations, the response to UTP, PIGF, and VEGF quickly fell off, a finding noted in a number of chemotaxis experiments performed by others as being consistent with the relative inability of cells to directionally and specifically respond to high concentrations of chemotactic agonists. A direct comparison of relative efficacy in chemotaxis was accomplished in Fig. 3, which shows that maximal concentrations of UTP or VEGF produced statistically significant chemotaxis during the course of the 5-h experiment; neither ATP nor PIGF produced any significant chemotaxis under these same conditions. One explanation for the failure of PIGF to elicit chemotactic responses may be its origin, because previous studies have suggested that bacterially produced PIGF may be devoid of biological activity in mammalian cells because of improper folding of the PIGF homodimer (36). As a result of these concerns, no attempt was made to evaluate the effect of PIGF on endothelial proliferation (Fig. 1). Although maximal concentrations of ATP produced demonstrable mitogenic activity in cultured endothelial cells, the same could not be said for its effects on chemotaxis in CEC, in which the response to ATP was not significantly different from that seen in control cells (Fig. 3). It is possible that the relatively low efficacy of ATP in the latter experiments is caused by an increased tendency for purine nucleotides to be broken down to the inactive inosine by extracellular nucleotidases and phosphatases during the experiment; however, repeated addition of ATP at maximal concentrations during the course of some of the experiments did nothing to increase the chemotactic response of cells treated with this agent (not shown).

Given the lack of selective ligands for receptors that bind UTP, we are unable to definitively identify the receptors mediating mitogenic or chemotactic responses to UTP in our cultured endothelial cells or the receptor(s) that are responding to UTP as it promotes angiogenesis in the chick CAM. Since we previously reported (35) the existence in CEC of two receptors capable of responding to UTP (P2Y2 and another, UTP-selective, receptor), we also are unable to assign relative contributions by these two receptors to the responses to UTP seen in the CEC studies reported here. However, we have obtained indirect evidence that permits us to suggest P2Y4 as a likely candidate for the UTP-selective receptor expressed in CEC. Repeated attempts to use UDP as either a chemotactic or mitogenic agent failed to demonstrate responses in either type of experiment (data not shown); furthermore, attempts to elicit a change in intracellular calcium concentrations in these cells with UDP were also unsuccessful (M. E. Bradley, unpublished observations). Given that the P2Y6 receptor exhibits strong reactivity to UDP, whereas the P2Y4 exhibits much less reactivity (see Ref. 1 for summary of receptor characteristics), our observations suggest P2Y4 as the UTP-selective receptor that is expressed in cultured cardiac endothelial cells. Furthermore, the fact that ATP exhibits no significant chemotactic activity in our experiments would tend to rule out a contribution by P2Y2 to this response, a finding that further supports our hypothesis that UTP, through interactions with UTP-selective (rather than UTP/ATP responsive) receptors, plays a relatively larger role in endothelial cell growth and proliferation than do purine nucleotides.

Purines such as ATP and adenosine have been demonstrated to possess either mitogenic or angiogenic properties in cells cultured from endothelium (21, 29) or vascular smooth muscle (6). Although no such findings have heretofore been reported from experiments on the effects of pyrimidines, the stimulation by UTP of mitogen-activated protein in the endothelial cell line EAhy 926 (12) provides one possible mechanism by which UTP might signal endothelial cells to proliferate, at least in culture. The possibility that this mechanism is involved in UTP signaling of mitogenesis in cardiac endothelial cells, as well as the mechanisms associated with UTP-stimulated chemotaxis in CEC or UTP-stimulated angiogenesis in the chick CAM, remains to be determined.

The significance of our work lies in our finding that UTP promotes mitogenesis and chemotaxis in vascular endothelial cells and is angiogenic in the chick CAM. Although UTP has been shown to stimulate the release of compounds such as NO and PGI₂ from endothelial cells from a variety of vascular sources, it is not clear how the effects of UTP might differ from those mediated by extracellular purine nucleotides. As such, the role of pyrimidine nucleotide receptors in endothelial function has not been well understood. The present findings, namely, that UTP may be more important in the regulation of endothelial cell proliferation and migration than purine nucleotides, may help clarify this question.

Angiogenesis occurs in development, reproductive cycling in the ovary and endometrium, wound healing, and tumor development; in addition, there are a number of known diseases that result from improper or inappropriate angiogenesis, including rheumatoid arthritis and diabetic retinopathy. Angiogenesis plays an important role in the increased vascularity associated with developing tumors (9). Because endothelial cell proliferation and migration are key steps in the development of new blood vessels in both normal and tumor-supporting angiogenesis (2), our finding of a possible role for UTP receptors in the mediation of mitogenesis, chemotaxis, and angiogenesis in vascular endothelial cells makes it conceivable that a defect in the expression of UTP receptors on the surface of endothelial cells could be involved in the pathogenesis of angiogenic disorders or even that UTP could be involved in the development of new blood vessels necessary to support rapidly developing tumors.

UTP has been shown to be released from platelets during degranulation reactions (11), and UTP has now been shown to be released from 1321N1 cells as a consequence of mechanical stimulation (18). The fact that UTP can be released at millimolar concentrations at the luminal surface of endothelial cells permits a number of suggestions of roles for UTP in the regulation of endothelial cell function. Foremost perhaps among these is the possibility that UTP is a primary signal in endothelial repair; because degranulation reactions in platelets are stimulated by (among other things) the presence of a damaged endothelium (3), it is conceivable that the UTP released during this reaction is destined not so much for nucleotide receptors mediating blood flow in the region but rather UTP-selective receptors on the undamaged, adjacent endothelial cells that mediate cell division and migration, with the end result being a repopulation of the damaged endothelium. The answers to these questions should warrant further study in both endothelial and other vascular cell types.