Differential release of prostaglandin E-like and F-like substances by endothelial cells cultured from human umbilical arteries and veins

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Differential Substances

16,119-131(1978)

Release of Prostaglandin E-Like and F-Like by Endothelial Cells Cultured from Human Umbilical Arteries and Veins’ WILLIAML.JOYNERANDJAMESC.STRAND

Department of Physiology and Biophysics, University of Nebraska College of Medicine, Omaha, Nebraska 68105 Received June 13,1977 Endotheliai cells, isolated from human umbilical vessels and grown in culture, displayed morphological characteristics similar to those described for endothelial cells in vivo. After 7-10 days in culture, endothelial cells from human umbilical arteries and veins were found to release prostaglandm F-like substances in excess of prostaglandm E-like substances into the media bathing the cells. Quantitative differences in concentration of prostaglandin E-like and F-like substances as well as alterations in the rate of prostaglandin release were determined by the presence of some unidentified component(s) of fetal calf serum. Implications of prostaglandin release by cellular components of human umbilical vessels are discussed.

INTRODUCTION Recent studies have clearly demonstrated that the function of the endothelial cell (EC) in viuo is much more complex than previously described. ECs synthesize vasoactive (Alexander and Gimbrone, 1976), clotting (Jaffe, 1975), and fibrinolytic substances (McDonald et al., 1973); metabolize certain compounds (Shepro et al., 1975), contain numerous types of neurochemical receptors (Venter et al., 1975); are electrically coupled (Buonassisi and Venter, 1976); and contain contractile filaments (Becker and Nachman, 1973). Evidence is accumulating rapidly to demonstrate that the EC not only functions as a passive barrier to the movement of solutes but also plays a dramatic role in controlling and modifying the internal environment of the vasculature. Thus, the EC may be pivotal in physiological as well as pathological processes which alter the transport of nutrients to tissues. In the last several years, the structure and function of ECs in culture have been the subject of intense research due largely to the success of isolating and growing these cells. Although Maruyama (1963) and Pomerat and Slick (1963) were the first investigators to isolate and culture these cells, a decade elapsed before other reports appeared demonstrating successful isolation, growth, and replication of homogeneous populations of ECs in culture (Lewis et al., 1973; Jaffe et al., 1973; McDonald et al., 1973). Likely reasons for more recent successes are related to improved techniques for isolating, growing, and characterizing cells in vitro. With such improved techniques, it is now possible to differentiate ECs in culture from other vascular cell types, viz., smooth ‘This study was supported, in part, by National Institutes of Health Grant No. HL-19455 and University of Nebraska Medical Center Seed Grant. 119 0026-2862/78/0161-43119%02.00/0 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

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muscle cells and fibroblasts, by morphological and immunological criteria (Jaffe et al., 1973). Consequently, various groups of researchers have confirmed that homogeneous populations of ECs from human and bovine arteries and veins can be isolated and cultured (Booyse et al., 1975; Jatfe et al., 1973; Gimbrone et al., 1974). The identification of prostaglandins (PG) E,, E,, Fi,, and F,, in extracts of human umbilical vessels was reported by Karim (1967). Subsequently, Tuvemo and Wide (1973) identified the release of PGF,, into media bathing isolated human umbilical arteries. However, cellular sources of the PGs were unexplored until immunoreactive PGE (iPGE) was measured in media bathing ECs (Gimbrone and Alexander, 1975) and smooth muscle cells (Alexander and Gimbrone, 1976) cultured from human umbilical veins. About the same time, Terragno et al. (1975) reported that sections of bovine mesenteric arteries and veins possessed PG biosynthetic capacity. Furthermore, these investigators found that bradykinin stimulated increases in PGE and PGF production from arterial and venous sections, respectively. These observations suggested to us that ECs cultured from human umbilical arteries might have a different prostaglandin biosynthetic capacity than EC cultured from human umbilical veins. Using light and electron microscopy, we document the successful culture of ECs isolated from human umbilical arteries and veins. Furthermore, we describe the synthesis and release of prostaglandin Elike (“PGE”) and F-like (“PGF”) substances from ECs in culture. Finally, we report preliminary findings regarding the effect of fetal calf serum (FCS) on the release rate of “PGE” and “PGF” by ECs cultured from a human umbilical vein. MATERIAL

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Preparation of endothelial cell cultures. Human umbilical cords obtained from normal, term births at local hospitals were immediately placed in cold phosphatebuffered saline (PBS), pH 7.2, containing penicillin-streptomycin. Within 24 hr, EC cultures were prepared according to the following procedure. Using sterile techniques, portions of the cord which were clamped during delivery or which contained large clots were removed and discarded. Both ends of either the umbilical artery and/or vein were cannulated, and the vessel was rinsed with PBS and filled with collagenase (O.l%, in PBS, Worthington). After 10-15 min the solution was removed and centrifuged, and the cell pellet was resuspended in PBS. The vessel was rinsed again with PBS, the solution was centrifuged, and the cell pellet was resuspended in PBS and combined with the previous cell suspension. Following centrifugation of the total cell suspension, the cells were then resuspended in Lewis medium (Medium 199, GIBCO), pH 7.2, containing 20% FCS (GIBCO). Using a hemocytometer, the total number of cells in the suspension was determined. Dye exclusion using erythrosin B was employed to assess the number of viable cells. Subsequently, culture flasks (Corning or Falcon) were seeded at concentrations of lo5 cells/cm2, additional culture medium (2-4 ml) was added, and incubation at 37O was begun. After 3, 6, and 24 hr the medium was changed. Subsequently, fresh medium was added to the culture flasks every 48 hr. Growth of the EC cultures was evaluated by determining the number of cells in each flask at 2-day intervals for a 2- to 3-week period. Total cell population in each flask was ascertained

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by counting the number of cells in five separate areas (0.28 mmz), determining the average number of cells per unit area, and extrapolating to the total area of the flask. Morphological characterization of the EC cultures was performed at the light (LM) and electron (EM) microscopic level. For LM observation, 35mm photographs of EC cultures were taken on an inverted microscope (Nikon) at various periods of time after seeding. For EM observation, the ECs were fixed and embedded in situ using Chang’s technique (1972). Sections were viewed under a Philips 201 electron microscope. Prostaglandin assay. To determine prostaglandin (PG) activity in EC cultures, the medium was removed from the incubation flasks and deposited in 5-10 vol of cold 95% ethanol. After filtration of the mixture, the filtrate was evaporated to a smaller volume (50 ml) and extracted three times each with ethyl acetate, 0.1 M sodium phosphate buffer (pH 8.0), and chlorform to remove neutral lipids and inorganic material. The chloroform extract was evaporated to dryness with nitrogen and weighed. The acidic lipid extract was reconstituted in 0.5 ml of chloroform : methanol (4 : 1) and applied to silica gel layers (0.5 mm thick) for thin-layer chromatography (tic) in a chloroform :methanol : acetic acid (18 :2 : 1) solvent system. The tic zones, tentatively identified as “PGF” and “PGE” zones by comparison to the migration of simultaneously chromatographed standards of PGF,, and PGE,, were eluted from the silica gel and reconstituted in 0.5 ml of Krebs-bicarbonate solution. Aliquots were bioassayed at least three times. To determine losses of PGs due to the extraction and separation procedures, four 30ml volumes of heparinized arterial whole blood from a dog were rapidly withdrawn, and each was deposited separately in 5 vol of 95% ethanol at O”. Known quantities of PGE, and PGF,, were added to three blood-ethanol mixtures either together or separately to achieve blood concentrations of either 0.1, 1.0, or 10.0 &ml; no prostaglandiis were added to the fourth mixture. Following filtration, acidic lipid extraction, and thin-layer chromatographic separation, bioassay of aliquots of eluates from “PGE” and “PGF” zones indicated that 63% (range = 53-79%) of the added PGE, and 64% (range = 5273%) of the added PGF,, were recovered. No detectable PG activity was present in the mixture to which PGs were not added. Values which we report for “PGE” and “PGF” have not been corrected for losses due to extraction and separation procedures. To bioassay for PGs, we use a modification of the blood-bathed organ technique of Vane (1969) which allows us to determine both “PGE” and “PGF” activity in acidic lipid extracts of biological fluids (McGiff et al., 1970b). In brief, three assay tissues (rat stomach strip, RSS; rat colon, RC; chick rectum, CR), selected for their sensitivity and specificity to PGs, were superfused in series with Krebs-bicarbonate solution at the rate of 1 ml/min. Activity (either contraction or relaxation) of each tissue was transduced via an auxotonic lever, and all changes in activity were recorded simultaneously on a multichannel polygraph. Standards and eluates from tic zones (Fig. 1) were alternately injected (0.01-0.1 ml) in the superfusate to assay for biological activity using the principle of bracket assay. Since RSS displays the greatest sensitivity and stability, maximum contractions of this tissue were used to quantitate unknowns by comparison to standards. Typically, thresholds of measurable activity to injected standards were produced by 0.02 ng of PGE, and 0.2 ng of PGF,,. Precision of this assay procedure, expressed as the coefficient of variation, was 11.8%.

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FIG. 1. Comparative responses of rat stomach strip (RSS), rat colon (RC) and chick rectum (CR) to doses of PGE, and a “PGE” zone eluate (top) and to doses of PGF,, and a “PGF” zone eluate (bottom). In addition to chromatographic separation of PGs, further certainty of identification is supported by the observation that PGE, and an aliquot of a “PGE” zone eluatc produced strong contractions of RSS and CR, but weaker contraction of RC (top). In contrast, PGF,, and an sliquot of a “PGE” zone eluate strongly contracted RSS and RC, but weakly contracted CR (bottom). However, since the responses of RSS to doses of PGE, and PGF,, were not comparable in this figure, the differential responses of RC were not evident, whereas the differential responses of CR were obvious. Time scale indicates 2 min; vertical scale of 2 cm indicates perspective of assay tissue activity.

PGs released by ECs culturedfrom human umbilical arteries and veins. After 7-10 days in culture, ECs from human umbilical arteries and/or veins were washed three times with PBS, and 5.0 ml of fresh Lewis medium containing 20% FCS was added to each flask. Cultures and blank flasks containing Lewis medium with 20% FCS were incubated for 24 hr at 37O. Following the incubation period, the medium was removed from the ECs and the blank flasks. Each sample was prepared for PG assay as previously described. Distilled water (2.0 ml) was added to each flask and the cells were removed from the inside surface by scraping with a rubber policeman. After this procedure was repeated, the cell suspensions were combined, frozen and thawed five times, and assayed for total protein by the method of Lowry et al. (195 1). We expressed PG concentrations per unit of total protein to standardize between samples. Rate of PG release by ECs culturedfrom a human umbilical vein. After 7-10 days in culture, four flasks containing ECs from a single human umbilical vein were washed three times with PBS, and 5.0 ml of fresh Lewis medium containing 20% FCS was added to each flask and the blank flask. All flasks were placed simultaneously in the incubator at 37O. Medium was removed successively from each flask after 30,60, 120, and 240 min of incubation (medium was removed from the blank flask at 240 mm), and each sample was prepared for PG assay as previously described. Fresh Lewis medium

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containing 20% FCS (5.0 ml) was added to each culture, and the flasks were placed in the incubator at 37O for 24 hr. To determine the effect of FCS on the rate of PG release from ECs, the previously described procedure was repeated using the same cultures, but in the absence of FCS. At the end of the respective time periods, each sample was prepared for PG assay as previously described. Finally, the ECs were removed from each culture flask, and total protein was determined. RESULTS Characterization of Cultured Endothelial Cells ECs isolated from human umbilical veins and arteries were inoculated into culture flasks at concentrations of 1.5 x lo5 cells/cm2 with a range of 0.5-2.6 x 105. Using erythrosin B, N-90% of these cells were determined to be viable. Within 24 hr, the total number of cells isolated from umbilical veins (Fig. 2, O-) had increased three- to fourfold, and during the next 4 days the total number of cells doubled. Thereafter, the number of cells remained relatively constant (6.0 x 10’ cells/cm9 ECs cultured from human umbilical arteries (Fig. 2, O- - -) increased in number by five- to six-fold within 48 hr after inoculation. Over the next 4- to 6-day interval the growth rate was moderate (1.3- to 1.5fold), and on Day 10 the total number of arterial ECs was not significantly different from that of venous ECs. At this time, both of the EC cultures were confluent and were maintained in monolayers (Figs. 3A and B). In the logarithmic phase of growth, population doubling times were 24-30 and 12-24 hr for venous and arterial cell cultures, respectively. These cells could be maintained in culture with 20% FCS for 46 weeks before noticeable slutJing was observed. -

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FIG. 2. Growth pattern for ECs isolated from human umbilical cords. By visually counting the cells in culture (number of cells per square centimeter x 103, the pattern of growth for five cultures of venous ECs (a-) and four cultures of arterial ECs (@ - -) was followed for 2 weeks (time). Mean + SEM.

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FIG. 3. Light micrographs of ECs isolated from human umbilical veins (A) and arteries (B) after 10 days in culture, stained with silver nitrate and methylene blue. x 1036.

In a separate series of experiments, ECs which had been in culture for 4-8 days were placed in culture medium containing either 5, 10, 20, or 30% FCS. These cells were observed for confluency over the following 2-3 weeks. If the concentration of FCS was decreased to 10% or less, cell confluency decreased substantially within 4-6 days; however, all other concentrations of FCS were adequate to sustain the ECs for 4-6 weeks. Light microscopic observations of ECs cultured from veins and arteries are depicted in Figs. 3A and B. Like other characterizations of ECs in culture, these micrographs show that both venous and arterial ECs are polygonal (30 x 19 pm) in shape, and they grow in single layers with no discernable pattern. In contrast, fibroblasts and smooth muscle cells are spindleshaped and grow in parallel arrays or whorls. Electron micrographs show that normal cytoplasmic constituents were present in these ECs (Fig. 4). In addition, rod-shaped structures, identified as Weibel-Palade bodies (Weibel and Palade, 1964) which are unique to ECs, were present in the cytoplasm of these ECs.

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FIG. 4. Etectron micrographs of ECs isolated from human umbilical veins after 10 days in culture. Weibel-Palade bodies are demonstrated (T) in upper and lower panels. Small (70-100 A) intracellular filaments (t) can be seen in the upper panel. Magnification: x 42,000 and 48,000 for upper and lower panels respectively.

“PGE” and “PGF’ Activity Released by ECs Culturedfrom Human Umbilical Arteries and Veins PG activity was present in media obtained from EC cultures after a 24-hr incubation period. Figure 5 shows “PGF” and “PGE” activity (ordinate), expressed as nanograms of PG per microgram of cell protein, generated by ECs cultured from human umbilical veins (left) and from human umbilical arteries (right). After a 24-hr incubation, Lewis medium with 20% FCS obtained from five blank flasks was found to contain 0.45 + 0.16 rig/ml of “PGF” and 0.04 + 0.01 rig/ml of “PGE.” To determine PG activity released from ECs, blank PG values were substracted from each individual PG value obtained from flasks containing ECs. No detectable PG activity was found in Lewis medium without FCS after a 24-hr incubation period. In four experiments, medium from ECs cultured from human umbilical veins was found to contain 156.74 ? 36.09 ng of “PGF,” and cell protein measured 282 f 103 pg. “PGF” activity was 0.74 &- 0.21 ng/pg of cell protein. In addition, this medium was found to contain 14.99 + 5.53 ng of

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FIG. 5. Prostaglandin activity, expressed as nanograms per microgram of cell protein, released from cultured endothelial cells isolated from human umbilical veins (left) and arteries (right). Each bar represents the mean f SEM for prostaglandin activity from venous cells (N = 4) and arterial cells (IV = 3). Unshaded areas denote “PGF” activity; shaded areas denote “PGE” activity.

“PGE;” therefore, “PGE” activity was 0.07 + 0.02 ng/,ug of cell protein. “PGF” activity was significantly (Student t test) greater than “PGE” activity (p < 0.05), and the ratio of “PGF” : “ PGE” activity was 11.4 : 1. In three experiments, medium from ECs cultured from human umbilical arteries was found to contain 148.97 + 72.33 ng of “PGF” and cell protein measured 503 & 190 ,ug. “PGF” activity was 0.35 + 0.12 rig/lug of cell protein. This same medium was found to contain 43.44 + 17.08 ng of “PGE;” therefore, “PGE” activity was 0.14 f 0.08 ng/pg of cell protein. “PGF” and “PGE” activities in medium from ECs cultured from human umbilical arteries were not significantly (Student t test) different, although the ratio of “PGF” : “PGE” activity was 2.5 : 1. Rate of “PGF’ and “PGF’ Release by ECs Culturedfrom Human Umbilical Vein The rate of PG release by ECs cultured from a human umbilical vein was determined from measurements of “PGF” and “PGE” activity in medium from ECs incubated for varying periods in either the presence or absence of 20% FCS. Figure 6 shows “PGF” and “PGE” activity ordinate), expressed as nanograms of PG per milligram of cell protein, generated by ECs cultured from a human umbilical vein with respect to time (abscissa). Thus, “PGF” (left) and “PGE” (right) activity increased linearly with respect to time in both the presence and absence of FCS. The difference in scales of the ordinate for the left and right sides of Fig. 6 supports an observation made in the 24-hr study, viz., EC-generated “PGF” activity was approximately IO-fold greater than “PGE” activity in the presence of FCS. In contrast, in the absence of FCS, “PGF” activity was reduced significantly to twice the “PGE” activity. The rate of “PGF” release was 11.54 ng/mg of cell protein/hr in the presence of FCS, but was decreased by two-thirds to 3.64 ng/mg of cell protein/hr in the absence of FCS. The rate of “PGE” release, which was 1.22 ng/mg of cell protein/hr in the presence of FCS, was unaffected by the absence of FCS (1.30 ng/mg of cell protein/hr).

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-WFCS o---MFCS

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FIG. 6. Effect of the presence and absence of fetal calf serum (FCS) in medium on the rate of release of “PGF” (left) and “PGE” (right) activity from endothelial cells cultured from a human umbilical vein. Endothelial cells were incubated for various intervals (20-240 min) in the presence (e) and absence (O- - -) of FCS. Each regression equation and correlation coefficient (r) appear adjacent to the line of best fit. In all cases, r was sign&ant (P < 0.05). Abscissa = time in minutes; ordinate = prostaglandin activity in nanograms per milligram of cell protein.

DISCUSSION ECs were cultured from human umbilical arteries and veins using collagenase for dissociation. Lewis et al. (1973) seeded at lower concentrations, but in our study lower seeding concentrations of either arterial or venous cells were insufficient to establish confluency. Confluency for both venous and arterial ECs was reached within 4-6 days, and during the logarithmic phase of growth doubling times were 24-30 hr. These growth characteristics are comparable to other studies which used ECs cultured from either bovine aortas (McDonald et al., 1973; Shepro et al., 1975) or human umbilical veins (Gimborne et al., 1974). Others using ECs cultured from human umbilical veins (Jaffe et al., 1973) or bovine aortas (Booyse et al., 1975) found that doubling times were longer (48-92 and 32-34 hr, respectively). Monolayers of venous and arterial ECs reach confluency within 5-7 days after inoculation, and the density at confluency was 104-IO5 cells/cm* (Gimbrone et al., 1974; Haudenschild et al., 1976; Booyse et al., 1975). Our data is in accord with the above observations but, undoubtedly, the confluency of cell cultures as well as growth patterns can be varied by the initial seeding concentration (Fenselau and Mello, 1976). One of the major questions concerning the isolation and culture of ECs is the identification of the cell population. Earlier studies describing the culture of ECs (Maruyama, 1963; Pomerat and Slick, 1963) were criticized because the cells could not be described accurately as ECs. Many investigators have carefully compared the morphological and immunological characteristics of ECs in culture with fibroblasts and

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smooth muscle cells (McDonald et al., 1973; Jaffe et al., 1973; Booyse et aZ., 1975). At the light microscopic level, ECs are polygonal and grow in single layers with no discernible patterns. In contrast, fibroblasts and smooth muscle cells are spindle shaped and grow in multilayers which exhibit parallel arrays or whorls. Electron micrographs showed that these ECs possessed characteristic Weibel-Palade bodies and other cytoplasmic constituents, e.g., vacuoles, vesicles, and filaments. Thus, using an established technique for the culture of ECs, the cells cultured from human umbilical arteries or veins in our study maintained all of the morphological characteristics of ECs. We report for the first time the differential release of “PGF” and “PGE” by confluent monolayers of ECs in primary cultures derived from human umbilical arteries and veins. Although Gimbrone and Alexander (1975) showed that iPGE could be extracted from medium bathing ECs cultured from human umbilical veins, they did not measure other PGs present in the medium, nor did they explore the capacity of ECs cultured from human umbilical arteries to release PGs. In our study, ECs cultured from human umbilical arteries and veins released 2.5- and 11.4-fold more “PGF,” respectively, than “PGE.” Furthermore, ECs cultured from human umbilical arteries demonstrated biosynthetic capacity of approximately two-thirds that of ECs cultured from human umbilical veins. These disparities suggested to us that some unknown substance(s) present in the medium could be responsible for the observed differences. In addition, two separate pieces of evidence support the notion that substances present in the incubation medium could stimulate selectively PGF and/or PGE release. First, Gimbrone and Alexander (1975) demonstrated that the addition of angiotensin II to medium bathing ECs cultured from human umbilical veins stimulated an increase in iPGE release. Second, Terragno et al. (1975) selectively increased PGE synthesis in homogenates of bovine mesenteric arteries and PGF synthesis in homogenates of bovine mesenteric veins by the addition of bradykinin to the incubation mixture. Determination of “PGF” and “PGE” in medium bathing ECs cultured from a single human umbilical vein showed that the rate of PG release, and presumably synthesis, were linear up to 4 hr of incubation in either the presence or absence of FCS. However, in the absence of FCS, “PGF” release was reduced from ll- to 2.5-fold more than “PGE” release, whereas the rate of “PGE” release was unaffected. Two separate steps in PG metabolism working either alone or in concert could be responsible for the observed differences. First, the presence of a substance(s) in FCS could selectively stimulate “PGF” synthesis. The nature of this substance remains a mystery. Second, there could be increased interconversion of PGE to PGF compounds by intracellular PGE-9-ketoreductase in the presence of FCS. If the rate of “PGE” synthesis was increased in the presence of FCS, the augmentation in biosynthetic rate might activate PGE-9-ketoreductase, thereby converting PGE compounds to PGF compounds. Since we are measuring two products of PG synthetase activity and not enzymic activity itself, this interpretation would be consistent with our results, viz., no apparent increased rate of PGE release, but an increased rate of PGF release. Since all known components required for PG synthesis are provided either by the cells themselves or by exogenous administration via the medium we believe the ratio of “PGF” : ‘6PGE” (2.5 : 1) released into the media in the absenceof FCS represents the basal state of PG release by ECs cultured from umbilical vessels. This is consistent with the study by Karim (1967), who first identified the presence of PGE,, PGE,, PGF,,,

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and PGF,, in extracts of human umbilical vessels and reported values that indicated PGF compounds to be present in concentrations exceeding those of PGE compounds by 2.6-fold. Subsequently, using ECs cultured from human umbilical veins, Gimbrone and Alexander (1975) found the rate of iPGE release into the media in the absence of FCS to be 0.009 + 0.002 ng/$g of cell protein/2hr, a value similar to that observed by us (0.005 & 0.002 rig/a of cell protein/2 hr). Furthermore, under the same conditions smooth muscle cells cultured from the same source have been shown to release iPGE at a basal rate 20-fold greater (per milligram of protein) than that released by ECs (Alexander and Gimbrone, 1976). Thus, we believe the basal eflux of PGs from umbilical vessels to be substantial, and we agree with other investigators (Tuvemo and Wide, 1973; Strandberg and Tuvemo, 1975) who have suggested that PGs contribute importantly to umbilical vascular tone and, possibly, to the regulation of umbilical vessel blood flow. In our study, the addition of FCS to the medium resulted in a threefold increase in “PGF” production by ECs cultured from human umbilical vein, although the component(s) responsible remain unidentified. Hong et al. (1976) previously reported that FCS prolonged biosynthesis of PGE, from methylcholanthrene-transformed mouse BALB/3T3 cells, and suggested that FCS may provide additional substrate (arachidonic acid) for PG biosynthesis and/or phospholipases capable of releasing PG precursors from membrane phospholipids. Further, they suggested that FCS may contain a variety of vasoactive compounds capable of stimulating PG biosynthesis. In this regard, Gimbrone and Alexander (1975) found that angiotensin II could stimulate a IO-fold increase in iPGE secretion from human umbilical vein ECs in culture. In addition, bradykinin, angiotensin II, and histamine were shown to increase secretion of iPGE from smooth muscle cells cultured from the same source by lo-, 3.5-, and 2.3-fold basal, respectively (Alexander and Gimbrone, 1976). This is not surprising, since several vasoactive substances including angiotensin II (McGiff et al., 1970a), norepinephrine (McGiff et al., 1972a), and bradykinin (McGiff et al., 1972b) have been previously reported to stimulate PG synthesis. The effects of PGs of the E and F series have been shown to be potent constrictors of longitudinally and helically cut strips of human umbilical arteries and veins (Park et al., 1972), although Hillier and Karim (1968) have indicated that PGE, was a vasodilator when applied to human umbilical vessels. Recently, Tuvemo et al. (1976) have demonstrated the formation and release of PG endoperoxides by human umbilical arteries; when applied to strips of human umbilical arteries, PGG, and PGH, were lOO- and 200-fold more potent than PGE, in producing vascular contractions. Therefore, given the appropriate hormonal stimulation during parturition, PGEs, PGFs, and their endoperoxide precursors may contribute significantly to cessation of umbilical vessel blood flow by inducing potent, sustained contractions of vascular smooth muscle. Finally, closure of the ductus arteriosus has been related to the synergistic effect of a vasoconstrictor substance (PGF,) and increased oxygen tension in the blood perfusing that vessel (Starling and Elliott, 1974). Since ECs cultured from human umbilical veins preferentially synthesize “PGF” compounds, a potent vasoconstrictor effect in vivo on the ductus arteriosus by PGFs, distal to the site of synthesis and release, is not unlikely. On the other hand, Coceani et al. (1976) believe that endogenous synthesis of PGE,, which dilates the ductus arteriosus, regulates the patency of that vessel. This

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concept is supported by the observation that inhibition of PG synthesis with indomethacin (Vane, 1971) can result in closure of a patent ductus arteriosus in the premature infant (Friedman et al., 1976). Based on these observations, we believe that a reasonable and unifying hypothesis regarding the closure of the ductus arteriosus might include the following sequence of events: (1) Patency of the ductus arteeosus in utero during gestation may be sustained by preferential PGE synthesis by that vessel; (2) with the onset of parturition, preferential PGF synthesis and release into the circulation by umbilical ECs, and possibly by smooth muscle cells, could result in high blood concentrations of PGF compounds being delivered to the ductus arteriosus; and (3) the vasoconstrictor activity of PGF compounds may overcome the vasodilator activity of PGE compounds resulting in closure of a patent ductus arteriosus. ACKNOWLEDGMENTS We thank P. A. De&o, S. W. May, and P. A. Bestmann for their excellent technical assistance. Dr. C. M. Moriarty provided helpful suggestions during all phases of the study. Dr. John E. Pike of the Upjohn Company generously supplied prostaglandins. REFERENCES ALEXANDER, R. W., AND GIMBRONE, M. A. (1976). Stimulation of prostaglandin E synthesis in cultured human umbilical vein smooth muscle cells. Proc. Nat. Acud. Sci. USA 73,1617-1620. BECKER, C. G., AND NACHMAN, R. L. (1973). Contractile proteins of endothelial cells, platelets and smooth muscle. Amer. J. Pathof. 71, 1-18. BOOYSE, F. M., SEDLAK, B. J., AND RAFELSON, M. E. (1975). Culture of arterial endothelial cells: Characterization and growth of bovine aortic cells. Thromb. Diath. Haemorrh. 34,825-839. BUONASSISI, V., AND VENTER, J. C. (1976). Hormone and neurotransmitter receptors in an established vascular endothelial cell line. Proc. Nat. Acad. Sci. USA 73, 1612-1616. CALD~ELL, P. R. B., SEEGAL, B. C., Hsu, K. C., DAS, M., AND SOWER, R. L. (1976). Angiotensinconverting enzyme: Vascular endothelial localization. Science 191,105~1051. CHANG, J. H. T. (1972). Fixation and embedment, in situ, of tissue culture cells for electron microscope. Tissue Cell 4,56 l-574. COCEANI, F., OLLEY, P. M., AND BODACH, E. (1976). Prostaglandins: A possible regulator of muscle tone in the ductus arteriosus. In “Advances in Prostaglandin and Thromboxane Research” (B. Samuelsson and R. Paoletti, eds.), Vol. 1, pp. 417-424. Raven Press, New York. FENSELAU, A., AND MELLO, R. J. (1976). Growth stimulation of cultured endothelial cells by tumor cell homogenates. Cancer Res. 36, 3269-3273. FRIEDMAN, W. F., HIRSCHKLAU, M. J., PRINTZ, M. P., PITLICK, P. T., AND KIRKPATRICK, S. E. (1976). Pharmacological closure of patent ductus arteriosus in the premature infant. N. Engl. J. Med. 295,526529. GIMBRONE, M. A., AND ALEXANDER, R. W. (1975). Angiotensin II stimulation of prostaglandin production in cultured human vascular endothelium. Science 189,219-220. GWBRONE, M. A., COTRAN, R. S., AND FOLKMAN, J. (1974). Human vascular endothelial cells in culture; Growth and DNA synthesis. J. Ceil Biol. 60,673-684. HAUDENSCHILD, C. C., ZAHNISER, D., FOLKMAN, J., AND KLAGSBRUN, M. (1976). Human vascular endothelial cells in culture: Lack of response to serum growth factors. Exp. Cell Res. 98,175-183. HILLIER, K., AND KAIUM, S. M. M. (1968). Effects of prostaglandins E,, E,, F,,; F, on isolated human umbilical and placental blood vessels.J. Obstet. Gynaecol. Brit. Commonw. 75,667-673. HONG, S.?. L., POLSKY-CYNKIN, R., AND LEVINE, L. (1976). Stimulation of prostaglandin biosynthesis by vasoactive substances in methylcholanthrene-transformed mouse BALB/3T3. J. Biol. Chem. 25 1, 776780. JAFFE, E. A. (1975). Synthesis of factor VIII antigen by cultured human endothelial cells. Ann. N.Y. Acad. Sci. 240,62-69.

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