0021-972X/90/7005-1285$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright© 1990 by The Endocrine Society
Vol. 70, No. 5 Printed in U.S.A.
Partial Characterization of a Novel Growth Factor from the Blood of Women with Preeclampsia* ROBERT N. TAYLOR, THOMAS J. MUSCI, ROBERT W. KUHN, AND JAMES M. ROBERTS Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0132
ABSTRACT. Sera obtained before delivery from women with preeclampsia contain greater mitogenic activity than sera drawn from the same women 24-48 h after parturition or sera from normal parturients. These studies describe the initial characterization of the blood-borne mitogenic factor(s) from preeclamptic women which we have named ELMER (Endogenous Ligand conferring MitogEnic Response). ELMER appears to be a unique mitogen with characteristics that are not identical to those of other known growth factors. ELMER is present in
serum as an acid- and heat-labile protein, approximately 160,000 daltons in size, which is a potent mitogen for human fibroblasts but not for human endothelial cells. Its presence in plasma suggests that it is a circulating factor rather than a product of blood coagulation ex vivo. We believe that ELMER represents a potential serum marker of preeclampsia and that it may play roles in the vasospasm and proliferative vascular lesion, termed atherosis, frequently associated with the preeclamptic syndrome. {J Clin Endocrinol Metab 70: 1285-1291,1990)
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REECLAMPSIA is a pregnancy-specific disease manifest by vasospasm, coagulopathy, and impaired tissue perfusion. Its etiology and pathophysiology remain enigmatic. Our laboratory has recently presented testable hypotheses which invoke a central role of endothelial cell
injury in the pathogenesis of this disorder (1). We have attempted to elucidate the molecular factors involved in the pathogenic state by the isolation and identification of circulating products that are likely to translate an abnormality of placentation into a systemic disease. These efforts have provided direct evidence that predelivery sera from preeclamptic women sublethally injure human endothelial cells in vitro (2). Moreover, we have demonstrated that the prepartum sera of women with preeclampsia contain greater mitogenic activity than matched postpartum sera or those of normal parturients (3). Since endothelial cell injury stimulates the secretion of potent mitogenic growth factors (4), our findings support the concept that preeclampsia is associated with endothelial injury in vivo. The rapid postpartum disappearance of these in vitro markers is consistent with the Received February 21, 1989. Address all correspondence and requests for reprints to: Dr. Robert N. Taylor, W. M. Keck Laboratory of Developmental and Molecular Genetics, Department of Obstetrics, Gynecology, and Reproductive Sciences, M-1489, University of California, San Francisco, California 94143-0132. * This work was supported by NIH Grants HD-24180, -22873 and the Analytical Separation Core (Grant HD-11979). This is publication 5 from the University of California-San Francisco Preeclampsia Project.
clinical resolution of preeclampsia after delivery. Moreover, the vasospasm and vascular histopathology of preeclampsia bear significant resemblance to those of allograft rejection and atherosclerosis (5), and recent investigations of the latter disease suggest an important link between endothelial injury, vasospasm, and vascular mesenchymal cell hyperplasia (4, 6). \ The present study focused on characterization of the mitogenic factor(s) observed in women with active preeclampsia. By comparing this activity with the biochemical qualities of other well characterized growth factors we have initiated attempts to identify and purify this factor (s). Of the known mitogenic factors present in blood, platelet-derived growth factor (PDGF), transforming growth factors-a and -0 (TGFa and TGF/3), and epidermal growth factor appear to have their greatest concentrations in platelets (7-11). Of all of the growth factors, insulin-like growth factors (IGFs) circulate in blood at the highest concentration, due to their association with high mol wt carrier proteins which stabilize these factors (12). While they are not potent mitogens on their own, IGFs can act in concert with PDGF and epidermal growth factor to promote human fibroblast DNA synthesis (13). The level of fibroblast growth factor (FGF) is very low in the circulation, and FGF appears to be bound to plasma proteins that inhibit its biological activity (Gospodarowicz, D. J., personal communication). The studies presented in this paper suggest that the
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mitogenic activity present in the sera of preeclamptic women is primarily due to a growth factor(s) with biochemical characteristics that differentiate it from other established mitogens. We have named this factor ELMER (Endogenous Ligand conferring MitogEnic Response). Experimental Subjects Pregnant women admitted to the Obstetrical Service of the Medical Center at the University of California-San Francisco (UCSF) with term singleton gestations were recruited to participate in a protocol approved by the UCSF Committee on Human Research. These patients were assigned as preeclamptic or normal by well established criteria, which included elevated blood pressure, proteinuria, and elevated serum uric acid levels, as detailed by us previously (3).
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[125I]PDGF. The displacement of [125I]PDGF binding was determined as described by Escobedo et al. (15). Nonspecific binding accounted for 25% of the bound [125I]PDGF. PDGF neutralization Purified immunoglobulin G (IgG) from rabbits immunized with highly purified native human platelet PDGF was obtained from R&D Systems, Inc. (Minneapolis, MN). A final IgG concentration of 50 ^g/mL was added to quiescent fibroblast cultures according to the manufacturer's instructions before stimulation with pre- and postdelivery preeclamptic serum. This concentration of IgG has been shown to neutralize 90% of the mitogenic activity of 5 ng/mL pure PDGF. From the RRA quantification of PDGF in pregnancy serum (see Results below), we estimated that the concentration of PDGF in 2% serum-supplemented media ranged from 0.5-2 ng/mL. Protease digestion of ELMER
Materials and Methods Serum and plasma specimens were rapidly prepared from whole blood obtained before and within 48 h after delivery. The specimens were aliquoted and stored at — 70 C until assayed. In some experiments, sera were subjected to thermal inactivation at 60 C for 120 min or acidified by the addition of small volumes of 1 N HC1 to achieve a final pH below 3. The acidified sera were maintained at 4 C for 60 min and neutralized to pH 7 by the addition of 1 N NaOH. Serum samples were adjusted to a final concentration of 2% (1.5 ± 0.1 mg protein/mL) in Dulbecco's Modified Eagle's Medium before their addition to the bioassay, as previously described (3).
Insoluble S. griseus protease, covalently derivatized to agarose beads (P4531), was purchased from Sigma (St. Louis, MO) and used according to the manufacturer's specifications. Patients' serum samples were diluted to a concentration of 50% in Dulbecco's Modified Eagle's Medium, and agarose-protease beads were added at a final concentration of 2 mg/mL agarose. The protease digestion was performed at 25 C for 60 min, and the beads were sedimented from the serum-supplemented media by centrifugation at 5000 x g for 15 min at 4 C. The supernatant fraction was collected, diluted further in medium to yield a final serum concentration of 2%, and tested in the fibroblast culture bioassay as described for untreated serum. Undigested samples of the same sera were subjected to identical manipulations, excluding addition of the protease resin.
Cell cultures
Molecular sizing
Human foreskin fibroblasts and human umbilical vein endothelial cells were obtained and maintained in culture as described previously (2, 3). The cells were grown to confluence in 24-well Falcon dishes and maintained in serum-free medium for 48 h. A final concentration of 2% pregnancy serum or plasma was added to the quiescent cells for 20 h, and labeling with [3H]thymidine (New England Nuclear, Boston, MA; 0.5 jttCi/well) was carried out for 4 additional h. [3H]Thymidine incorporation was determined by trichloroacetic acid precipitation and scintillation counting, as previously described (3).
Centricon ultrafiltration units with a nominal mol wt cutoff of 10,000 daltons were purchased from Amicon (Danvers, MA). Patient sera were diluted to 2% in culture medium, and 2-mL aliquots were centrifuged at 5,000 X g for 25 min at 4 C in the ultrafiltration unit. The retained fraction (retentate) was added to the fibroblast culture bioassay. Control samples of the same diluted sera were subjected to centrifugation in polycarbonate tubes without filters.
Blood specimens
Quantification of pregnancy serum PDGF concentration by RRA Human foreskin fibroblasts were grown to confluence for 5 days and allowed to deplete their media of growth factors. Subsequently, the cells were cultured in the presence of serumfree medium for 2 additional days. The cells were harvested, and 100,000 cells were aliquoted to borosilicate glass tubes containing 250 nL of 10% platelet-poor plasma in phosphatebuffered saline. Human PDGF was purified from outdated platelets, as described by Williams et al. (14), to generate a standard competition curve for PDGF binding. Increasing amounts of human PDGF or pregnancy sera were added to the reaction tubes, followed by the addition of 20,000 cpm (~1 ng)
High pressure liquid chromatographic (HPLC) sizing To more accurately ascertain the mol wt of ELMER, gel permeation chromatography of preeclamptic sera was performed using a TSK-125 column (Bio-Rad Laboratories, Richmond, CA). Samples (100 /JL, each) of neat pre- and postpartum preeclamptic sera were separated isocratically using phosphatebuffered saline (pH 7.4) as the mobile phase. Fractions (500 ^L each) were collected and added to replicate wells of quiescent fibroblasts in serum-free media. The incorporation of [3H] thymidine was determined as described above. The counts incorporated by cells exposed to the postdelivery fractions were subtracted from those of the identical predelivery fractions, and these arithmetic differences were used to assess ELMER activity.
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Statistical analyses Experimental data reported in this paper were analyzed using the Statview 512+ microcomputer program (Abacus Concepts, Inc., Calabasas, CA). Most aspects of the characterization of this mitogenic factor(s) necessitated the comparison of serum fractions obtained pre- and postdelivery from the same patients. Unless indicated otherwise, the results were expressed as the mean ± SE of replicate experiments using three or more patients' blood specimens. Comparisons were made using Student's t test, generally with paired data, although in some cases it was appropriate to use the unpaired t test, as noted. Our hypothesis was that physicochemical perturbations would increase the ratio of prepartum/postpartum mitogenic activities. We have chosen to present the data both as raw counts per min of [3H]thymidine incorporated as well as by normalizing the percent mitogenic stimulation [(cpm incorporated predelivery)/(cpm incorporated postdelivery) x 100] to correct for variation among individuals. Tests with P < 0.05 were considered to reflect significant differences between patient and treatment groups. In the case of the HPLC separation, analysis of variance with repeated measures was used to compare the activity chromatograms of matched pre- and postdelivery samples.
Results Lack of mitogenic response in human endothelial cells The cellular specificity of various mitogens was used to initiate the characterization of ELMER. We have previously shown that this factor(s) was mitogenic for human foreskin fibroblasts, resulting in a stimulation of
cellular [3H]thymidine incorporation (expressed as predelivery/postdelivery cpm X 100) by preeclamptic (137 ± 6%) us. normal parturients (107 ± 6%; P < 0.01; n = 14) (3). The effects of paired sera from preeclamptic and normal pregnancies were similarly studied using human umbilical vein endothelial cells as the bioassay for mitogenic response. Under the same conditions used for the fibroblast experiments, endothelial cells failed to show a significant difference in [3H]thymidine incorporation when stimulated with preeclamptic (109 ± 4%) vs. normal pregnant (106 ± 4%) sera (P > 0.3; n = 11; see Fig. 1). These results revealed that ELMER was mitogenic for mesenchymal, but not endothelial, cells, indicating that it was not likely to be FGF (16) or one of the two major colony-stimulating factors (17). Conversely, TGF/3, which has been shown to be growth inhibitory for human umbilical vein endothelial cells (18), did not seem to account for ELMER activity.
fibroblast
endothelial
FIG. 1. Cellular specificity of ELMER's mitogenic activity. Paired serum samples from preeclamptic (•) and normal patients {M) were assayed as described in the text, with the results expressed as mean percent mitogenic stimulation ± SE (see Materials and Methods). The n value represents the number of patients in each group analyzed. The results demonstrated that fibroblasts, but not endothelial cells, were sensitive to mitogenic stimulation by predelivery preeclamptic sera.
in trace amounts and is cleared rapidly from primate plasma, with a tA of less than 2 min (19). PDGF is primarily released from platelets during blood clotting. However, under conditions of endothelial cell injury, PDGF is also released from these cells (4). The concentration of PDGF in normal human serum has been reported to range from 15-50 ng/mL (19-21). Using a RRA based on the competitive inhibition of [125I]PDGF binding to quiescent human fibroblasts, we determined that the mean concentration (±SE) of PDGF in the predelivery serum of normal parturients was 52 ± 22 ng/ mL, while the PDGF concentration in predelivery sera from preeclamptic women was 44 ± 15 ng/mL (n = 6; P > 0.3; see Fig. 2). Thus, PDGF did not appear to account for the increased mitogenic activity in predelivery preeclamptic serum over that of normal parturients. Finally, coincubation of quiescent cells with preeclamptic sera and PDGF-neutralizing antibody caused a small decrease, but failed to significantly diminish their mitogenic activity ratio (pre-/postdelivery cpm X 100). The mean results of two experiments, each performed in triplicate, yielded mitogenic stimulation values of 131% in the absence and 119% in the presence of the neutralizing antibody.
Serum concentrations of PDGF in normal and preeclamptic women
Protease sensitivity of mitogenic activity
Of the peptide growth factors that stimulate mesenchymal, but not endothelial, cell mitosis, PDGF is the best characterized. Human PDGF is present in the blood
Conditions to establish the relative protease sensitivity of ELMER were developed using agarose beads to which proteinases were stably derivatized. This approach ob-
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100000 1 3000-
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[PDGF] ng/ml FIG. 2. Quantification of serum PDGF concentrations by RRA. A RRA using human foreskin fibroblasts in suspension was carried out as described in Materials and Methods. Unbound labeled PDGF was removed by centrifugation on a Ficoll gradient. The concentrations of PDGF in prepartum sera from preeclamptic and normal (arrows) parturients were calculated from the displacement of specifically bound [125I]PDGF by known concentrations of pure PDGF (•). The values are shown as the mean (±SE) of six samples from each group.
viated contamination of the test sera with soluble proteases, which per se may possess mitogenic activity. Preand postdelivery preeclamptic sera were incubated with agarose-protease beads at 25 C for 1 h, while untreated serum samples were subjected to the incubation conditions in the absence of protease. The results revealed that gentle protease exposure eliminated the typically observed pre-/postpartum increase in mitogenic activity (Fig. 3). Effects of heat and acid treatments on ELMER The heat stability of ELMER was assessed by incubating aliquots of serum specimens at 60 C for 120 min before addition to the fibroblast cultures. The mitogenic effects of heat-treated sera were directly compared to those of untreated paired sera from nine preeclamptic patients. The results of these experiments are presented in Table 1 and demonstrated the thermal lability of ELMER. To correct for individual variation in [3H] thymidine incorporation by different serum specimens, the raw data were normalized to the postdelivery serum activity of each patient, as described in Materials and Methods. The incremental mitogenic stimulation of predelivery preeclamptic sera (155 ± 19%) was destroyed after heat treatment (95 ± 17%; P < 0.01, by paired t test; n = 9) compared to its postpartum activity (100%). As noted in Table 1, the predominant loss of mitogenic effect resulted from a decrease in the prepartum activity. The heat-stable components of these sera accounted for 63 ± 3% of the total mitogenic activity. This contribution
untreated
protease
FIG. 3. Protease sensitivity of ELMER. Paired t tests of mitogenesis assay data from five preeclamptic patients' sera before (untreated) and after protease digestion. These results showed that the mitogenic factor(s) responsible for the statistical increase in pre-/postpartum activity was lost after mild protease digestion (see Materials and Methods). D, Prepartum samples; H, postpartum samples. TABLE 1. Thermal sensitivity of ELMER [3H]Thymidine cpm incorporated/well Predelivery untreated Postdelivery untreated
58,620 ± 9,040 37,210 ± 5,340°
Predelivery heated Postdelivery heated
34,600 ± 10,330 29,450 ± 9,560"
Paired t tests of raw data comparing untreated and heat-treated sera were calculated from nine preeclamptic patients. The mean values (± SE) are presented. Despite the large standard errors resulting from individual variation within the bioassay, the results demonstrated that heating preeclamptic sera to 60 C for 120 min destroyed the relative mitogenic activity of pre-/postpartum samples. " P < 0.02 vs. predelivery untreated. b P > 0.3 us. predelivery heated.
is in agreement with the proportion of serum mitogenic activity (~60%) previously attributed to heat-stable factors such as PDGF and TGF/3 (4). Using a similar design, preeclamptic sera were acidified to pH values below 3 for 60 min at 4 C and neutralized to pH 7 before their addition to the fibroblast cultures. This treatment decreased the mitogenic activity of predelivery sera by 45 ± 8%, but had no effect on the postpartum serum samples (Table 2), indicating that ELMER was acid sensitive. The findings of heat and acid lability essentially excluded PDGF, IGFs, and TGF/3 as possible candidates for ELMER. ELMER activity in preeclamptic plasma Plasma samples, obtained pre- and postdelivery from preeclamptic women, were also studied using the [3H]
MITOGENIC GROWTH FACTOR IN PREECLAMPSIA TABLE 2. Acid sensitivity of ELMER 3
H]Thymidine cpm incorporated/well
Predelivery untreated Postdelivery untreated
78,260 ± 8,330 54,530 ± 7,990"
Predelivery acidified Postdelivery acidified
43,750 ± 8,080 51,790 ± 7,250*
Paired t tests of raw data from three preeclamptic patients' sera were examined by the mitogenic assay. Mean values (±SE) are presented. These results showed that after the acidification treatment described in Materials and Methods, predelivery sera lost their mitogenic advantage compared to their corresponding postpartum samples. " P < 0.05 us. predelivery untreated. * P > 0.3 us. predelivery acidified.
thymidine incorporation bioassay. In the initial experiments, matched plasma and serum samples, obtained pre- and postpartum, were analyzed for mitogenic activity. In our assay, 2% plasma effected only 47 ± 7% the mitogenic stimulation of 2% serum from the same patients (n = 9). However, when pre- and postpartum plasma samples were compared, the results were, in fact, more dramatic than those obtained with serum. In normal patients, pre-/postpartum plasma effected a 113 ± 16% stimulation of [3H]thymidine incorporation. In contrast, predelivery preeclamptic plasma promoted 239 ± 31% mitogenic stimulation of quiescent fibroblasts compared to postdelivery plasma from the same women (P < 0.01, by unpaired t test; n = 15). The relative ratio of serum mitogenic activity in the same preeclamptic patients was 141 ± 11%, a value very similar to our previously reported figure of 137 ± 6% for preeclamptic women (3). These data suggested that ELMER was not an artifact induced by serum clotting ex vivo, but indeed reflected a circulating factor(s) in preeclampsia. The observation of a similar activity in plasma mitigates against a role for a-thrombin, which is a known mitogen for fibroblasts (22). Further evidence against a-thrombin as the protein responsible for ELMER activity is that hirudin, a potent and specific inhibitor of a-thrombin activity, had no effect on ELMER activity (data not shown).
191%. In contrast, a normal parturient's serum had a mitogenic stimulation of 89% before and 111% after ultrafiltration. An experiment representative of a preeclamptic patient is shown in Fig. 4. The results suggested that ELMER was more than 10,000 daltons in size. A more precise estimation of the molecular mass of ELMER was afforded by HPLC gel permeation chromatography. Using this technique, matched sera from preeclamptic patients were fractionated and assayed for mitogenic activity. Significant differences between preand postdelivery serum fraction activities were demonstrated by repeated measures analysis of variance (P < 0.01). The serum fraction (fraction 4) that possessed the greatest relative pre-/postpartum mitogenic activity corresponded to a molecular mass of about 160,000 (Fig. 5). In addition to providing a more accurate estimate of the apparent mol wt of ELMER, HPLC afforded a significant purification of this activity. The specific activity of fraction 4 of the HPLC eluate of predelivery preeclamptic serum (1865 ± 62 cpm incorporated/well • /xg serum protein) was approximately 50-fold greater than that of the same unfractionated predelivery preeclamptic serum (38 ± 2 cpm incorporated/well • fig serum protein). Discussion The experiments described in this report served to initiate the characterization of a blood-borne factor(s) and potential diagnostic marker of preeclampsia. These studies, like our preliminary report (3), relied on the 80000
• 0
Ultrafiltration of small aliquots of diluted preeclamptic sera was performed to estimate the mol wt of the factor(s) responsible for increased mitogenic activity in predelivery preeclamptic sera. Using Centricon microconcentators with a mol wt cut-off of 10,000 daltons, we found that ELMER activity was dramatically concentrated in the retentate. Unfiltered serum samples from two preeclamptic women had a mean mitogenic stimulation ratio of 142% (pre-/postpartum activity x 100), but after ultrafiltration of the same sera this ratio increased to
prepartum postpartum
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retentate untreated FIG. 4. Molecular sieving of ELMER. Paired prepartum (D) and postpartum (H) sera from preeclamptic patients were assayed for mitogenic activity (see Materials and Methods). In a representative patient, the untreated samples yielded a typical pre-/post ratio (124% in this example). After ultrafiltration over a 10,000-dalton molecular sieve, the pre-/post activity ratio in the retentate fraction increased to 220%. Raw [3H]thymidine counts per min incorporated for each sample are represented as the mean ± SE of triplicate determinations.
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FIG. 5. Gel permeation chromatography of ELMER by HPLC. Paired pre- and postdelivery sera from preeclamptic patients were separated by HPLC, as described in Materials and Methods. The column eluate fractions were incubated for 24 h with serum-free media in replicate
wells of quiescent human fibroblasts and assayed for mitogenic activity. Differences between the 3H counts incorporated by identical fractions from matched pre- and postpartum samples are plotted in histogram above. These are the mean (±SE) results from HPLC analyses of four preeclamptic sera. Molecular mass (Mr) standards (thyroglobulin, IgG, ovalbumin, and myoglobin) are plotted against their corresponding elution fractions.
incorporation of [3H]thymidine by fibroblasts exposed to a low fixed concentration of pregnancy serum or plasma. The relatively increased potency of pre- vs. postdelivery serum in preeclamptic, but not normal, parturients served as the bioassay for the characterization studies. These experiments demonstrated that ELMER is mitogenic for fibroblasts, but not endothelial cells, and is likely to be a protein, as gentle protease digestion destroyed its activity. This factor(s) is also sensitive to thermal and acid denaturation. These characteristics mitigate against its identification as TGF0, IGF, or PDGF. While FGF is heat and acid labile, it, unlike ELMER, is a potent mitogen for human umbilical endothelial cells (16). Acid treatment, which destroyed ELMER, does not appear to diminish PDGF activity (21) and has been reported to enhance TGF0 (23) and IGF (12) activities by dissociating these peptides from their serum binding proteins. Furthermore, quantification of PDGF concentrations by RRAs failed to reveal significant differences between preeclamptic and normal sera, and neutralizing PDGF antibodies did not significantly alter their relative ratio of mitogenic activity. Interleukin-1, a cytokine primarily involved in the differentiation of B lymphocytes, is also a potent mitogen for dermal fibroblasts (24). Interleukin-1 is protease sensitive and heat labile, although it has been reported to be active after mild acidification (25). a-Thrombin and other proteases (22) have been shown to stimulate DNA synthesis in cultured human fibroblasts; however, the presence of mitogenic activity in plasma and the failure of hirudin to inhibit ELMER activity suggest that a-thrombin is
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not a likely candidate. j82-Microglobulin, which is reported to be elevated in the serum of preeclamptic women (26), has been proposed as mesenchymal cell growth factor (27), although this conclusion has been refuted recently (28). Finally, Sen-Majumdar et al. (29) have characterized a unique heat-labile placental growth factor of 34,000 daltons whose potential role in preeclampsia has not been investigated to date. The apparent mol wt of ELMER is about 160,000 daltons, which exceeds the sizes of the classic growth factors and the other potential mitogens described above. It is possible that the HPLC fraction that contains the most ELMER activity represents an aggregate of smaller proteins comprised of previously identified mitogens. Alternatively, the approximately 160-kDa fraction may contain a serum carrier protein to which a smaller heatand acid-labile growth factor is complexed. Fibronectin, a high mol wt cell matrix protein known to be elevated in plasma of preeclamptic women (30), is mitogenic for human fibroblasts (31), but has been reported to be stable to heat and acid exposure (32). We are currently applying the information obtained from this characterization to purify ELMER, which will ultimately allow its precise identification. Of primary importance in this study is the observation that ELMER activity is present in predelivery preeclamptic plasma as well as serum, supporting a potential in vivo role, rather than an artifact of platelet release during blood clotting. These findings are compatible with our hypothesis that endothelial cell injury underlies the pathophysiology of the preeclamptic syndrome (1), as a variety of growth factors are known to be released from endothelial cells into the local circulation at sites of injury (4). In summary, a mitogenic factor(s) has been characterized and partially purified from pregnancy serum. At the present time we cannot be certain whether it represents a unique growth factor, an activity comprised of multiple mitogenic effectors, or a circulating mitogen-binding protein complex. This activity, which we have called ELMER, is a marker of preeclampsia and might contribute to the pathological changes manifest by vasospasm and vascular wall proliferation in the atherosis lesions associated with this disorder (5, 33). Acknowledgments We thank Mitra Jazayeri, Erin Newman, Jean Perry, R.N., M.S., and the members of the Clinical Data Core for their expert assistance, and Louvina Alfaro Forkin for her help in the manuscript and figure preparation.
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MITOGENIC GROWTH FACTOR IN PREECLAMPSIA Obstet Gynecol. 1989;161:1200-4. 2. Rodgers GM, Taylor RN, Roberts JM. Preeclampsia is associated with a serum factor cytotoxic to human endothelial cells. Am J Obstet Gynecol. 1988;159:908-14. 3. Musci TJ, Roberts JM, Rodgers GM, Taylor RN. Mitogenic activity is increased in the sera of preeclamptic women prior to delivery. Am J Obstet Gynecol. 1988;159:1446-51. 4. Ross R, Raines EW, Bowen-Pope DF. The biology of plateletderived growth factor. Cell. 1986;46:155-69. 5. Labarrere CA. Acute atherosis: a histopathological hallmark of immune aggression. Placenta. 1988;9:95-108. 6. Barrett TB, Benditt EP. Platelet-derived growth factor gene expression in human atherosclerotic plaques and in normal artery wall. Proc Natl Acad Sci USA. 1988;85:2810-4. 7. Ross R, Glomset J, Kariya B, Harker L. A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc Natl Acad Sci USA. 1974;71:1207-10. 8. Childs CB, Proper JA, Tucker RF, Moses HL. Serum contains a platelet-derived transforming growth factor. Proc Natl Acad Sci USA. 1982;79:5312-6. 9. Assoian RK, Komoriya A, Meyers CA, Miller DM, Sporn MB. Transforming growth factor-/3 in human platelets. J Biol Chem. 1983;258:7155-50. 10. Oka Y, Orth DN. Human plasma epidermal growth factor/jSurogastrone is associated with blood platelets. J Clin Invest. 1983;72:249-59. 11. Roberts AB, Anzano MA, Lamb LC, Smith JM, Sporn MB. New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues. Proc Natl Acad Sci USA. 1981;78:5339-43. 12. Hintz RL, Liu F. Demonstration of specific plasma protein binding sites for somatomedin. J Clin Endocrinol Metab. 1977;45:988-95. 13. Conover CA, Rosenfeld RG, Hintz RL. Hormonal control of the replication of human fetal fibroblasts: role of somatomedin C/insulin-like growth factor I. J Cell Physiol. 1986;128:47-54. 14. Williams LT, Tremble P, Antoniades HN. Platelet-derived growth factor binds specifically to receptors on vascular smooth muscle cells and the binding becomes nondissociable. Proc Natl Acad Sci USA. 1982;79:5867-70. 15. Escobedo JA, Navankasatussas S, Cousens LS, Coughlin SR, Bell GI, Williams LT. A common PDGF receptor is activated by homodimeric A and B forms of PDGF. Science. 1988;240:1532-4. 16. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev. 1987;8:95-114. 17. Bussolino F, Wang JM, Defilippi P, et al. Granulocyte and granulocyte-macrophage-colony stimulating factors induce human endo-
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thelial cells to migrate and proliferate. Nature. 1989;337:471-3. 18. Baird A, Durkin T. Inhibition of endothelial cell proliferation by type beta transforming growth factor: interactions with acidic and basic fibroblast growth factors. Biochem Biophys Res Commun. 1986;138:476-482. 19. Bowen-Pope DF, Malpass TW, Foster DM, Ross R. Plateletderived growth factor in vivo: levels, activity and rate of clearance. Blood. 1984;64:458-69. 20. Singh JP, Chaikin MA, Stiles CD. Phylogenetic analysis of plateletderived growth factor by radio-receptor assay. J Cell Biol. 1982;95:667-71. 21. Huang SJ, Huang SS, Deuel TF. Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein. J Cell Biol. 1983;97:383-8. 22. Carney DH, Cunningham DD. Cell surface action of thrombin is sufficient to initiate division of chick cells. Cell. 1978;14:811-23. 23. Lawrence DA, Pircher R, Jullien P. Conversion of a high molecular weight latent /3-TGF from chicken embryo fibroblasts into a low molecular weight active /3-TGF under acidic conditions. Biochem Biophys Res Commun. 1985;133:1026-34. 24. Dinarello CA. Interleukin-1. Rev Infect Dis. 1984;6:51-95. 25. Mizel SB. Physicochemical characterization of lymphocyte activating factor (LAF). J Immunol. 1979;122:2167-72. 26. Oian P, Monrad-Hansen H-P, Maltau JM. Serum uric acid correlates with /32-microglobulin in pre-eclampsia. Acta Obstet Gynecol Scand. 1986;65:103-6. 27. Canalis E, McCarthy T, Centrella M. A bone-derived growth factor isolated from rat calvariae is beta2 microglobulin. Endocrinology. 1987;121:1198-200. 28. Jennings JC, Mohan S, Baylink DJ. /32-Microglobulin in not a bone cell mitogen. Endocrinology. 1989;125:404-9. 29. Sen-Majumdar A, Murthy U, Das M. A new trophoblast-derived growth factor from human placenta: purification and receptor identification. Biochemistry. 1986;25:627-34. 30. Lazarchick J, Stubbs TM, Romein L, Van Dorsten JP, Loadholt CB. Predictive value of fibronectin levels in normotensive gravid women destined to become preeclamptic. Am J Obstet Gynecol. 1986;154:1050-2. 31. Bitterman PB, Rennard SI, Adelberg S, Crystal RG. Role of fibronectin as a growth factor for fibroblasts. J Cell Biol. 1983;97:1925-32. 32. Greenburg G, Gospodarowicz D. Inactivation of a basement membrane component responsible for cell proliferation but not cell attachment. Exp Cell Res. 1982;140:l-14. 33. DeWolf F, Robertson WB, Brosens I. The ultrastructure of acute atherosis in hypertensive pregnancy. Am J Obstet Gynecol. 1975;123:164-74.
Vol. 70, No. 5 Printed in U.S.A.
Partial Characterization of a Novel Growth Factor from the Blood of Women with Preeclampsia* ROBERT N. TAYLOR, THOMAS J. MUSCI, ROBERT W. KUHN, AND JAMES M. ROBERTS Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0132
ABSTRACT. Sera obtained before delivery from women with preeclampsia contain greater mitogenic activity than sera drawn from the same women 24-48 h after parturition or sera from normal parturients. These studies describe the initial characterization of the blood-borne mitogenic factor(s) from preeclamptic women which we have named ELMER (Endogenous Ligand conferring MitogEnic Response). ELMER appears to be a unique mitogen with characteristics that are not identical to those of other known growth factors. ELMER is present in
serum as an acid- and heat-labile protein, approximately 160,000 daltons in size, which is a potent mitogen for human fibroblasts but not for human endothelial cells. Its presence in plasma suggests that it is a circulating factor rather than a product of blood coagulation ex vivo. We believe that ELMER represents a potential serum marker of preeclampsia and that it may play roles in the vasospasm and proliferative vascular lesion, termed atherosis, frequently associated with the preeclamptic syndrome. {J Clin Endocrinol Metab 70: 1285-1291,1990)
P
REECLAMPSIA is a pregnancy-specific disease manifest by vasospasm, coagulopathy, and impaired tissue perfusion. Its etiology and pathophysiology remain enigmatic. Our laboratory has recently presented testable hypotheses which invoke a central role of endothelial cell
injury in the pathogenesis of this disorder (1). We have attempted to elucidate the molecular factors involved in the pathogenic state by the isolation and identification of circulating products that are likely to translate an abnormality of placentation into a systemic disease. These efforts have provided direct evidence that predelivery sera from preeclamptic women sublethally injure human endothelial cells in vitro (2). Moreover, we have demonstrated that the prepartum sera of women with preeclampsia contain greater mitogenic activity than matched postpartum sera or those of normal parturients (3). Since endothelial cell injury stimulates the secretion of potent mitogenic growth factors (4), our findings support the concept that preeclampsia is associated with endothelial injury in vivo. The rapid postpartum disappearance of these in vitro markers is consistent with the Received February 21, 1989. Address all correspondence and requests for reprints to: Dr. Robert N. Taylor, W. M. Keck Laboratory of Developmental and Molecular Genetics, Department of Obstetrics, Gynecology, and Reproductive Sciences, M-1489, University of California, San Francisco, California 94143-0132. * This work was supported by NIH Grants HD-24180, -22873 and the Analytical Separation Core (Grant HD-11979). This is publication 5 from the University of California-San Francisco Preeclampsia Project.
clinical resolution of preeclampsia after delivery. Moreover, the vasospasm and vascular histopathology of preeclampsia bear significant resemblance to those of allograft rejection and atherosclerosis (5), and recent investigations of the latter disease suggest an important link between endothelial injury, vasospasm, and vascular mesenchymal cell hyperplasia (4, 6). \ The present study focused on characterization of the mitogenic factor(s) observed in women with active preeclampsia. By comparing this activity with the biochemical qualities of other well characterized growth factors we have initiated attempts to identify and purify this factor (s). Of the known mitogenic factors present in blood, platelet-derived growth factor (PDGF), transforming growth factors-a and -0 (TGFa and TGF/3), and epidermal growth factor appear to have their greatest concentrations in platelets (7-11). Of all of the growth factors, insulin-like growth factors (IGFs) circulate in blood at the highest concentration, due to their association with high mol wt carrier proteins which stabilize these factors (12). While they are not potent mitogens on their own, IGFs can act in concert with PDGF and epidermal growth factor to promote human fibroblast DNA synthesis (13). The level of fibroblast growth factor (FGF) is very low in the circulation, and FGF appears to be bound to plasma proteins that inhibit its biological activity (Gospodarowicz, D. J., personal communication). The studies presented in this paper suggest that the
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mitogenic activity present in the sera of preeclamptic women is primarily due to a growth factor(s) with biochemical characteristics that differentiate it from other established mitogens. We have named this factor ELMER (Endogenous Ligand conferring MitogEnic Response). Experimental Subjects Pregnant women admitted to the Obstetrical Service of the Medical Center at the University of California-San Francisco (UCSF) with term singleton gestations were recruited to participate in a protocol approved by the UCSF Committee on Human Research. These patients were assigned as preeclamptic or normal by well established criteria, which included elevated blood pressure, proteinuria, and elevated serum uric acid levels, as detailed by us previously (3).
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[125I]PDGF. The displacement of [125I]PDGF binding was determined as described by Escobedo et al. (15). Nonspecific binding accounted for 25% of the bound [125I]PDGF. PDGF neutralization Purified immunoglobulin G (IgG) from rabbits immunized with highly purified native human platelet PDGF was obtained from R&D Systems, Inc. (Minneapolis, MN). A final IgG concentration of 50 ^g/mL was added to quiescent fibroblast cultures according to the manufacturer's instructions before stimulation with pre- and postdelivery preeclamptic serum. This concentration of IgG has been shown to neutralize 90% of the mitogenic activity of 5 ng/mL pure PDGF. From the RRA quantification of PDGF in pregnancy serum (see Results below), we estimated that the concentration of PDGF in 2% serum-supplemented media ranged from 0.5-2 ng/mL. Protease digestion of ELMER
Materials and Methods Serum and plasma specimens were rapidly prepared from whole blood obtained before and within 48 h after delivery. The specimens were aliquoted and stored at — 70 C until assayed. In some experiments, sera were subjected to thermal inactivation at 60 C for 120 min or acidified by the addition of small volumes of 1 N HC1 to achieve a final pH below 3. The acidified sera were maintained at 4 C for 60 min and neutralized to pH 7 by the addition of 1 N NaOH. Serum samples were adjusted to a final concentration of 2% (1.5 ± 0.1 mg protein/mL) in Dulbecco's Modified Eagle's Medium before their addition to the bioassay, as previously described (3).
Insoluble S. griseus protease, covalently derivatized to agarose beads (P4531), was purchased from Sigma (St. Louis, MO) and used according to the manufacturer's specifications. Patients' serum samples were diluted to a concentration of 50% in Dulbecco's Modified Eagle's Medium, and agarose-protease beads were added at a final concentration of 2 mg/mL agarose. The protease digestion was performed at 25 C for 60 min, and the beads were sedimented from the serum-supplemented media by centrifugation at 5000 x g for 15 min at 4 C. The supernatant fraction was collected, diluted further in medium to yield a final serum concentration of 2%, and tested in the fibroblast culture bioassay as described for untreated serum. Undigested samples of the same sera were subjected to identical manipulations, excluding addition of the protease resin.
Cell cultures
Molecular sizing
Human foreskin fibroblasts and human umbilical vein endothelial cells were obtained and maintained in culture as described previously (2, 3). The cells were grown to confluence in 24-well Falcon dishes and maintained in serum-free medium for 48 h. A final concentration of 2% pregnancy serum or plasma was added to the quiescent cells for 20 h, and labeling with [3H]thymidine (New England Nuclear, Boston, MA; 0.5 jttCi/well) was carried out for 4 additional h. [3H]Thymidine incorporation was determined by trichloroacetic acid precipitation and scintillation counting, as previously described (3).
Centricon ultrafiltration units with a nominal mol wt cutoff of 10,000 daltons were purchased from Amicon (Danvers, MA). Patient sera were diluted to 2% in culture medium, and 2-mL aliquots were centrifuged at 5,000 X g for 25 min at 4 C in the ultrafiltration unit. The retained fraction (retentate) was added to the fibroblast culture bioassay. Control samples of the same diluted sera were subjected to centrifugation in polycarbonate tubes without filters.
Blood specimens
Quantification of pregnancy serum PDGF concentration by RRA Human foreskin fibroblasts were grown to confluence for 5 days and allowed to deplete their media of growth factors. Subsequently, the cells were cultured in the presence of serumfree medium for 2 additional days. The cells were harvested, and 100,000 cells were aliquoted to borosilicate glass tubes containing 250 nL of 10% platelet-poor plasma in phosphatebuffered saline. Human PDGF was purified from outdated platelets, as described by Williams et al. (14), to generate a standard competition curve for PDGF binding. Increasing amounts of human PDGF or pregnancy sera were added to the reaction tubes, followed by the addition of 20,000 cpm (~1 ng)
High pressure liquid chromatographic (HPLC) sizing To more accurately ascertain the mol wt of ELMER, gel permeation chromatography of preeclamptic sera was performed using a TSK-125 column (Bio-Rad Laboratories, Richmond, CA). Samples (100 /JL, each) of neat pre- and postpartum preeclamptic sera were separated isocratically using phosphatebuffered saline (pH 7.4) as the mobile phase. Fractions (500 ^L each) were collected and added to replicate wells of quiescent fibroblasts in serum-free media. The incorporation of [3H] thymidine was determined as described above. The counts incorporated by cells exposed to the postdelivery fractions were subtracted from those of the identical predelivery fractions, and these arithmetic differences were used to assess ELMER activity.
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Statistical analyses Experimental data reported in this paper were analyzed using the Statview 512+ microcomputer program (Abacus Concepts, Inc., Calabasas, CA). Most aspects of the characterization of this mitogenic factor(s) necessitated the comparison of serum fractions obtained pre- and postdelivery from the same patients. Unless indicated otherwise, the results were expressed as the mean ± SE of replicate experiments using three or more patients' blood specimens. Comparisons were made using Student's t test, generally with paired data, although in some cases it was appropriate to use the unpaired t test, as noted. Our hypothesis was that physicochemical perturbations would increase the ratio of prepartum/postpartum mitogenic activities. We have chosen to present the data both as raw counts per min of [3H]thymidine incorporated as well as by normalizing the percent mitogenic stimulation [(cpm incorporated predelivery)/(cpm incorporated postdelivery) x 100] to correct for variation among individuals. Tests with P < 0.05 were considered to reflect significant differences between patient and treatment groups. In the case of the HPLC separation, analysis of variance with repeated measures was used to compare the activity chromatograms of matched pre- and postdelivery samples.
Results Lack of mitogenic response in human endothelial cells The cellular specificity of various mitogens was used to initiate the characterization of ELMER. We have previously shown that this factor(s) was mitogenic for human foreskin fibroblasts, resulting in a stimulation of
cellular [3H]thymidine incorporation (expressed as predelivery/postdelivery cpm X 100) by preeclamptic (137 ± 6%) us. normal parturients (107 ± 6%; P < 0.01; n = 14) (3). The effects of paired sera from preeclamptic and normal pregnancies were similarly studied using human umbilical vein endothelial cells as the bioassay for mitogenic response. Under the same conditions used for the fibroblast experiments, endothelial cells failed to show a significant difference in [3H]thymidine incorporation when stimulated with preeclamptic (109 ± 4%) vs. normal pregnant (106 ± 4%) sera (P > 0.3; n = 11; see Fig. 1). These results revealed that ELMER was mitogenic for mesenchymal, but not endothelial, cells, indicating that it was not likely to be FGF (16) or one of the two major colony-stimulating factors (17). Conversely, TGF/3, which has been shown to be growth inhibitory for human umbilical vein endothelial cells (18), did not seem to account for ELMER activity.
fibroblast
endothelial
FIG. 1. Cellular specificity of ELMER's mitogenic activity. Paired serum samples from preeclamptic (•) and normal patients {M) were assayed as described in the text, with the results expressed as mean percent mitogenic stimulation ± SE (see Materials and Methods). The n value represents the number of patients in each group analyzed. The results demonstrated that fibroblasts, but not endothelial cells, were sensitive to mitogenic stimulation by predelivery preeclamptic sera.
in trace amounts and is cleared rapidly from primate plasma, with a tA of less than 2 min (19). PDGF is primarily released from platelets during blood clotting. However, under conditions of endothelial cell injury, PDGF is also released from these cells (4). The concentration of PDGF in normal human serum has been reported to range from 15-50 ng/mL (19-21). Using a RRA based on the competitive inhibition of [125I]PDGF binding to quiescent human fibroblasts, we determined that the mean concentration (±SE) of PDGF in the predelivery serum of normal parturients was 52 ± 22 ng/ mL, while the PDGF concentration in predelivery sera from preeclamptic women was 44 ± 15 ng/mL (n = 6; P > 0.3; see Fig. 2). Thus, PDGF did not appear to account for the increased mitogenic activity in predelivery preeclamptic serum over that of normal parturients. Finally, coincubation of quiescent cells with preeclamptic sera and PDGF-neutralizing antibody caused a small decrease, but failed to significantly diminish their mitogenic activity ratio (pre-/postdelivery cpm X 100). The mean results of two experiments, each performed in triplicate, yielded mitogenic stimulation values of 131% in the absence and 119% in the presence of the neutralizing antibody.
Serum concentrations of PDGF in normal and preeclamptic women
Protease sensitivity of mitogenic activity
Of the peptide growth factors that stimulate mesenchymal, but not endothelial, cell mitosis, PDGF is the best characterized. Human PDGF is present in the blood
Conditions to establish the relative protease sensitivity of ELMER were developed using agarose beads to which proteinases were stably derivatized. This approach ob-
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[PDGF] ng/ml FIG. 2. Quantification of serum PDGF concentrations by RRA. A RRA using human foreskin fibroblasts in suspension was carried out as described in Materials and Methods. Unbound labeled PDGF was removed by centrifugation on a Ficoll gradient. The concentrations of PDGF in prepartum sera from preeclamptic and normal (arrows) parturients were calculated from the displacement of specifically bound [125I]PDGF by known concentrations of pure PDGF (•). The values are shown as the mean (±SE) of six samples from each group.
viated contamination of the test sera with soluble proteases, which per se may possess mitogenic activity. Preand postdelivery preeclamptic sera were incubated with agarose-protease beads at 25 C for 1 h, while untreated serum samples were subjected to the incubation conditions in the absence of protease. The results revealed that gentle protease exposure eliminated the typically observed pre-/postpartum increase in mitogenic activity (Fig. 3). Effects of heat and acid treatments on ELMER The heat stability of ELMER was assessed by incubating aliquots of serum specimens at 60 C for 120 min before addition to the fibroblast cultures. The mitogenic effects of heat-treated sera were directly compared to those of untreated paired sera from nine preeclamptic patients. The results of these experiments are presented in Table 1 and demonstrated the thermal lability of ELMER. To correct for individual variation in [3H] thymidine incorporation by different serum specimens, the raw data were normalized to the postdelivery serum activity of each patient, as described in Materials and Methods. The incremental mitogenic stimulation of predelivery preeclamptic sera (155 ± 19%) was destroyed after heat treatment (95 ± 17%; P < 0.01, by paired t test; n = 9) compared to its postpartum activity (100%). As noted in Table 1, the predominant loss of mitogenic effect resulted from a decrease in the prepartum activity. The heat-stable components of these sera accounted for 63 ± 3% of the total mitogenic activity. This contribution
untreated
protease
FIG. 3. Protease sensitivity of ELMER. Paired t tests of mitogenesis assay data from five preeclamptic patients' sera before (untreated) and after protease digestion. These results showed that the mitogenic factor(s) responsible for the statistical increase in pre-/postpartum activity was lost after mild protease digestion (see Materials and Methods). D, Prepartum samples; H, postpartum samples. TABLE 1. Thermal sensitivity of ELMER [3H]Thymidine cpm incorporated/well Predelivery untreated Postdelivery untreated
58,620 ± 9,040 37,210 ± 5,340°
Predelivery heated Postdelivery heated
34,600 ± 10,330 29,450 ± 9,560"
Paired t tests of raw data comparing untreated and heat-treated sera were calculated from nine preeclamptic patients. The mean values (± SE) are presented. Despite the large standard errors resulting from individual variation within the bioassay, the results demonstrated that heating preeclamptic sera to 60 C for 120 min destroyed the relative mitogenic activity of pre-/postpartum samples. " P < 0.02 vs. predelivery untreated. b P > 0.3 us. predelivery heated.
is in agreement with the proportion of serum mitogenic activity (~60%) previously attributed to heat-stable factors such as PDGF and TGF/3 (4). Using a similar design, preeclamptic sera were acidified to pH values below 3 for 60 min at 4 C and neutralized to pH 7 before their addition to the fibroblast cultures. This treatment decreased the mitogenic activity of predelivery sera by 45 ± 8%, but had no effect on the postpartum serum samples (Table 2), indicating that ELMER was acid sensitive. The findings of heat and acid lability essentially excluded PDGF, IGFs, and TGF/3 as possible candidates for ELMER. ELMER activity in preeclamptic plasma Plasma samples, obtained pre- and postdelivery from preeclamptic women, were also studied using the [3H]
MITOGENIC GROWTH FACTOR IN PREECLAMPSIA TABLE 2. Acid sensitivity of ELMER 3
H]Thymidine cpm incorporated/well
Predelivery untreated Postdelivery untreated
78,260 ± 8,330 54,530 ± 7,990"
Predelivery acidified Postdelivery acidified
43,750 ± 8,080 51,790 ± 7,250*
Paired t tests of raw data from three preeclamptic patients' sera were examined by the mitogenic assay. Mean values (±SE) are presented. These results showed that after the acidification treatment described in Materials and Methods, predelivery sera lost their mitogenic advantage compared to their corresponding postpartum samples. " P < 0.05 us. predelivery untreated. * P > 0.3 us. predelivery acidified.
thymidine incorporation bioassay. In the initial experiments, matched plasma and serum samples, obtained pre- and postpartum, were analyzed for mitogenic activity. In our assay, 2% plasma effected only 47 ± 7% the mitogenic stimulation of 2% serum from the same patients (n = 9). However, when pre- and postpartum plasma samples were compared, the results were, in fact, more dramatic than those obtained with serum. In normal patients, pre-/postpartum plasma effected a 113 ± 16% stimulation of [3H]thymidine incorporation. In contrast, predelivery preeclamptic plasma promoted 239 ± 31% mitogenic stimulation of quiescent fibroblasts compared to postdelivery plasma from the same women (P < 0.01, by unpaired t test; n = 15). The relative ratio of serum mitogenic activity in the same preeclamptic patients was 141 ± 11%, a value very similar to our previously reported figure of 137 ± 6% for preeclamptic women (3). These data suggested that ELMER was not an artifact induced by serum clotting ex vivo, but indeed reflected a circulating factor(s) in preeclampsia. The observation of a similar activity in plasma mitigates against a role for a-thrombin, which is a known mitogen for fibroblasts (22). Further evidence against a-thrombin as the protein responsible for ELMER activity is that hirudin, a potent and specific inhibitor of a-thrombin activity, had no effect on ELMER activity (data not shown).
191%. In contrast, a normal parturient's serum had a mitogenic stimulation of 89% before and 111% after ultrafiltration. An experiment representative of a preeclamptic patient is shown in Fig. 4. The results suggested that ELMER was more than 10,000 daltons in size. A more precise estimation of the molecular mass of ELMER was afforded by HPLC gel permeation chromatography. Using this technique, matched sera from preeclamptic patients were fractionated and assayed for mitogenic activity. Significant differences between preand postdelivery serum fraction activities were demonstrated by repeated measures analysis of variance (P < 0.01). The serum fraction (fraction 4) that possessed the greatest relative pre-/postpartum mitogenic activity corresponded to a molecular mass of about 160,000 (Fig. 5). In addition to providing a more accurate estimate of the apparent mol wt of ELMER, HPLC afforded a significant purification of this activity. The specific activity of fraction 4 of the HPLC eluate of predelivery preeclamptic serum (1865 ± 62 cpm incorporated/well • /xg serum protein) was approximately 50-fold greater than that of the same unfractionated predelivery preeclamptic serum (38 ± 2 cpm incorporated/well • fig serum protein). Discussion The experiments described in this report served to initiate the characterization of a blood-borne factor(s) and potential diagnostic marker of preeclampsia. These studies, like our preliminary report (3), relied on the 80000
• 0
Ultrafiltration of small aliquots of diluted preeclamptic sera was performed to estimate the mol wt of the factor(s) responsible for increased mitogenic activity in predelivery preeclamptic sera. Using Centricon microconcentators with a mol wt cut-off of 10,000 daltons, we found that ELMER activity was dramatically concentrated in the retentate. Unfiltered serum samples from two preeclamptic women had a mean mitogenic stimulation ratio of 142% (pre-/postpartum activity x 100), but after ultrafiltration of the same sera this ratio increased to
prepartum postpartum
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retentate untreated FIG. 4. Molecular sieving of ELMER. Paired prepartum (D) and postpartum (H) sera from preeclamptic patients were assayed for mitogenic activity (see Materials and Methods). In a representative patient, the untreated samples yielded a typical pre-/post ratio (124% in this example). After ultrafiltration over a 10,000-dalton molecular sieve, the pre-/post activity ratio in the retentate fraction increased to 220%. Raw [3H]thymidine counts per min incorporated for each sample are represented as the mean ± SE of triplicate determinations.
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FIG. 5. Gel permeation chromatography of ELMER by HPLC. Paired pre- and postdelivery sera from preeclamptic patients were separated by HPLC, as described in Materials and Methods. The column eluate fractions were incubated for 24 h with serum-free media in replicate
wells of quiescent human fibroblasts and assayed for mitogenic activity. Differences between the 3H counts incorporated by identical fractions from matched pre- and postpartum samples are plotted in histogram above. These are the mean (±SE) results from HPLC analyses of four preeclamptic sera. Molecular mass (Mr) standards (thyroglobulin, IgG, ovalbumin, and myoglobin) are plotted against their corresponding elution fractions.
incorporation of [3H]thymidine by fibroblasts exposed to a low fixed concentration of pregnancy serum or plasma. The relatively increased potency of pre- vs. postdelivery serum in preeclamptic, but not normal, parturients served as the bioassay for the characterization studies. These experiments demonstrated that ELMER is mitogenic for fibroblasts, but not endothelial cells, and is likely to be a protein, as gentle protease digestion destroyed its activity. This factor(s) is also sensitive to thermal and acid denaturation. These characteristics mitigate against its identification as TGF0, IGF, or PDGF. While FGF is heat and acid labile, it, unlike ELMER, is a potent mitogen for human umbilical endothelial cells (16). Acid treatment, which destroyed ELMER, does not appear to diminish PDGF activity (21) and has been reported to enhance TGF0 (23) and IGF (12) activities by dissociating these peptides from their serum binding proteins. Furthermore, quantification of PDGF concentrations by RRAs failed to reveal significant differences between preeclamptic and normal sera, and neutralizing PDGF antibodies did not significantly alter their relative ratio of mitogenic activity. Interleukin-1, a cytokine primarily involved in the differentiation of B lymphocytes, is also a potent mitogen for dermal fibroblasts (24). Interleukin-1 is protease sensitive and heat labile, although it has been reported to be active after mild acidification (25). a-Thrombin and other proteases (22) have been shown to stimulate DNA synthesis in cultured human fibroblasts; however, the presence of mitogenic activity in plasma and the failure of hirudin to inhibit ELMER activity suggest that a-thrombin is
JCE & M • 1990 Vol 70 • No 5
not a likely candidate. j82-Microglobulin, which is reported to be elevated in the serum of preeclamptic women (26), has been proposed as mesenchymal cell growth factor (27), although this conclusion has been refuted recently (28). Finally, Sen-Majumdar et al. (29) have characterized a unique heat-labile placental growth factor of 34,000 daltons whose potential role in preeclampsia has not been investigated to date. The apparent mol wt of ELMER is about 160,000 daltons, which exceeds the sizes of the classic growth factors and the other potential mitogens described above. It is possible that the HPLC fraction that contains the most ELMER activity represents an aggregate of smaller proteins comprised of previously identified mitogens. Alternatively, the approximately 160-kDa fraction may contain a serum carrier protein to which a smaller heatand acid-labile growth factor is complexed. Fibronectin, a high mol wt cell matrix protein known to be elevated in plasma of preeclamptic women (30), is mitogenic for human fibroblasts (31), but has been reported to be stable to heat and acid exposure (32). We are currently applying the information obtained from this characterization to purify ELMER, which will ultimately allow its precise identification. Of primary importance in this study is the observation that ELMER activity is present in predelivery preeclamptic plasma as well as serum, supporting a potential in vivo role, rather than an artifact of platelet release during blood clotting. These findings are compatible with our hypothesis that endothelial cell injury underlies the pathophysiology of the preeclamptic syndrome (1), as a variety of growth factors are known to be released from endothelial cells into the local circulation at sites of injury (4). In summary, a mitogenic factor(s) has been characterized and partially purified from pregnancy serum. At the present time we cannot be certain whether it represents a unique growth factor, an activity comprised of multiple mitogenic effectors, or a circulating mitogen-binding protein complex. This activity, which we have called ELMER, is a marker of preeclampsia and might contribute to the pathological changes manifest by vasospasm and vascular wall proliferation in the atherosis lesions associated with this disorder (5, 33). Acknowledgments We thank Mitra Jazayeri, Erin Newman, Jean Perry, R.N., M.S., and the members of the Clinical Data Core for their expert assistance, and Louvina Alfaro Forkin for her help in the manuscript and figure preparation.
References 1. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J
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