Effects of maternally administered epidermal growth factor on placental permeability

BASIC SCIENCE SECTION Effects of maternally administered epidermal growth factor on placental permeability Francis Mimouni, MD, Steven B. Hoath, MD, and Gail Hammond, BS Cincinnati, Ohio Epidermal growth factor is a well-studied modulator of epithelial membrane structure and function. Mammalian placentas are a rich source of epidermal growth factor receptors, but the role of epidermal growth factor in placental pathophysiologic conditions is unclear. To determine whether epidermal growth factor could affect mechanisms of placental transfer, we used an in situ rat placental perfusion model. Fourteen Sprague-Dawley rats that were 20 days pregnant were randomized to epidermal growth factor or placebo during placental transport experiments. We chose ethylenediaminetetraacetate tagged with chromium 51 as a marker of placental permeability. Epidermal growth factor treatment led to a dramatic increase of maternofetal clearances of chromium 51-ethylenediaminetetraacetate. We conclude that maternally administered epidermal growth factor has a potent action on placental permeability. (AM J OBSTET GVNECOL 1991 ;165:173-6.)

Key words: Epidermal growth factor, placenta, placental transport Because of the importance of the placenta in maintaining mammalian pregnancy, there is increasing interest in biochemical factors that regulate placental function and allow the placenta to act as a critical interface between mother and fetus. Recently it has been recognized that the placenta is a rich source of growth factors and growth factor receptors, I as well as protooncogenes. 2 These findings have led to the suggestion that these molecular regulators play an important role in the control of placental or fetal growth. One of the best-studied growth factors in this regard is epidermal growth factor. Although the human placenta is one of the richest known sources of epidermal growth factor receptor,3 the roles of epidermal growth factor and that of the structurally homologous protein transforming growth factor-a remain unclear. In the placenta of normal, near-term fetal rats," 5 there is a developmental increase in epidermal growth factor-binding sites; this is also the case for the human placenta during the last half of gestation. 6 . 7 Placental membranes of intrauterine growth-restricted rats have increased binding of epidermal growth factor,' From the Division of Neonatology, Department of Pediatrics, University of Cincinnati College of Medicine. Supported in part by National Institutes of Health grant HD 11725, United States Public Health Service Training in Perinatal Care and Research grant MCH MCT 000174, National Institutes of Health Clinical Research Center grant RR00068, National Institutes of Health grant HD 20748, and the Perinatal Research Institute. Received for publication September 6, 1990; revised January 29, 1991; accepted February 1, 1991. Reprint requests: Francis Mamouni, MD, Department of Pediatrics, Magee-Womens Hospital, Forbes Ave. and Halket St., Pittsburgh, PA 15213. 6/1 /28487

whereas placentas from streptozocin-treated diabetic rats have decreased the binding of epidermal growth factor! Since epidermal growth factor has been demonstrated to influence ionic signal transduction (sodiumhydrogen exchange)8 and to increase transport of aaminoisobutyrate 9 and deoxyglucose lO in isolated cell culture systems, we undertook a study of the effect of maternally administered epidermal growth factor on placental transport. As an index of placental permeability, we selected chromium 51-ethylenediaminetetraacetate (EDTA), a known diffusion marker, believed to travel across the placenta by means of "small pores,"" because of its small molecular size. We used an in situ system of placental perfusion in the rat l2 to test the null hypothesis that maternally administered epidermal growth factor would not affect placental transport of 5lCr-EDTA. Material and methods All chemicals were reagent grade, purchased from Sigma Chemical Co. (St. Louis). Radioisotopes were purchased from New England Nuclear (Boston). Receptor-grade epidermal growth factor was purchased from Bioproducts for Science (Indianapolis). Epidermal growth factor is extracted from mouse salivary glands and is 2::99% pure. Animals and surgical procedure. Fourteen timemated Sprague-Dawley rats were purchased from Zivic Miller Lab Inc. (Allison Park, Pa.) on day 14 of gestation. They were housed in individual cages and allowed free access to water and Purina Rat Chow (RalstonPurina Co., St. Louis). 173

174

Mimouni, Hoath, and Hammond

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clearances of "\Cr-EDTA in epidermal growth factor versus placebo-infused

animals.

Table I. Comparability of epidermal growth factor and control groups Va ria blf Maternal weight (gm) Fetal weight (gm) Placental weight (gill)

Ditfn{'/l(f

443 ± :l:l

NS

4.0 ± 0.5

:l.9 ± 0.2

NS

0.51 ± 0.04

0.49 ± 0.08

;\is

418 ± 41

NS, Not significant.

Placental perfusion. On day 20 of gestation, the dams were anesthetized with 110 mg/kg sodium thiobutabarbital, which was injected intraperitoneally. The maternal rectal temperature was maintained at 37" C through the experiment. A maternal jugular vein was cannulated for injection of tracers; a carotid artery, for blood pressure monitoring and blood sampling; and a femoral vein, for the administration of epidermal growth factor (or placebo). Maternal arterial pressure and perfusion pressure were measured with precalibrated Gould-Statham pressure transducers (Gould Inc., Cleveland) and recorded continuously on a chart recorder. After laparotomy and hysterotomy, one of the fetuses was delivered. Modified Krebs-Ringer solution containing 118 mmoll L sodium chloride, 4.7 mmoll L potassium chloride, 1.25 mmollL calcium chloride, I.IH rnmollL potassium orthophosphate dihydrate, 1.1 H mrnoll L magnesium sulfate, 25 mmoll L sodium bicarbonate, II mmoll L glucose, and 3.5% dextran

(40,000 d) was gassed continuously with a mixture of 95% oxygen and 5% carbon dioxide at 37° C and pumped by a peristaltic pump (Hake-Buchler, Saddle Brook, N.J.) into the fetal circulation of the placenta, through a cannula in the umbilical artery. The perfusion rate varied from 0.28 to 0.39 mllmin between experiments but remained constant during any individual experiment. After a single pass through the placenta, perfusate effluent was collected from a cannula in the umbilical vein. Each fetus whose placenta was perfused was removed after the vessels had been cannulated, blotted with tissue paper, and weighed to an accuracy of 0.1 mg with a Mettler H5I AR balance (Mettler Corp, Cleveland). At the end of the experiment the perfused placenta was detached from the uterine wall, and its wet weight was measured to an accuracy of 0.001 mg after fetal membranes and the umbilical cord had been dissected and discarded. Randomization. Animals were randomized to maternal administration of epidermal growth factor (n = 8) or placebo (n = 6). Epidermal growth factor or placebo was given through the catheter, which was inserted through the maternal femoral vein. Epidermal growth factor was given as a bolus of 5 f..lg, followed by continuous infusion of 0.8 f..lg/min. This infusion was continued until the end of the experiment. The investigators were blinded as to whether the animal received epidermal growth factor in normal saline solution or placebo (saline solution alone). Placental transfer. After 20 minutes of epidermal growth factor or placebo infusion, 40 f..lCi of>iCr-EDTA (500 to 800 mCi/mg) in 0.1 to 0.2 ml 0.9% sodium

Epidermal growth factor and placental permeability

Volume 165 Number I

chloride was injected at time 0 as a bolus into the maternal circulation through a cannula in the jugular vein. At approximately 3, 13, and 25 minutes, 0.5 ml heparinized maternal blood samples were drawn from a cannula in the carotid artery for radioisotope counting. Three 4-minute collections of perfusate effluent were taken in each experiment. slCr activity (in counts per minute) in aliquots of plasma and of perfusate effluent were counted for 10 minutes in a Packard 5330 auto'Y spectrometer (Packard Instruments Co.). Exclusion criteria. A successful perfusion was one in which the perfused placenta was uniformly blanched (because of removal of fetal blood), and placental perfusate effluent recovery was 95% to 100% of the inflow volume. Exclusions were made before counting of plasma and perfusate samples. Calculations. The steady-state clearance of 51Cr_ EDTA from maternal plasma to each perfusate sample (in microliters per minute) was defined as maternofetal clearance of sICr-EDTA (K mr 5ICr-EDTA):

Kmf 3ICr-EDTA = (P x F 1M) x 1000 where P is the 51Cr concentration (counts per minute per milliliter) in perfusate effluent samples, M is the 51Cr concentration (counts per minute per milliliter) in the maternal plasma at the midpoint of each 4-minute collection period, and F is the rate at which the perfusate was pumped through the placenta (milliliters per minute). To normalize for differences in placental size in different experiments, the clearance was related to the wet weight of the perfused placenta (microliters per minute per gram of placenta). Statistics. The data are presented as mean ± SE. Differences between means in each group and over time were analyzed by means of repeated measures analysis of variance; p < 0.05 was considered significant.

Results Epidermal growth factor and control groups were comparable by maternal, fetal, and placental weight at baseline Cfable I). Clearances of 5ICr-EDTA were significantly affected by the infusion of epidermal growth factor; the Kml "Cr-EDTA at baseline (i.e., after 20 minutes of epidermal growth factor or placebo infusion), which was calculated from the first effusate collection, was almost doubled in the epidermal growth factor group (5.45 ± 0.61 vs 2.88 ± 0.15 IJollmin/gm, p < 0.01); it increased further in the epidermal growth factor group, to reach a value of 8.52 ± 1.84 IJollmin/gm (p < 0.001) in the last perfusate, whereas it remained the same in the control group at 3.16 ± 0.14 IJollminl gm (not significant, compared with baseline) (Fig. 1).

175

Comment Experimentally our results demonstrate that maternal-fetaI 5I Cr-EDTA clearance is increased by maternal administration of epidermal growth factor. In adult rats epidermal growth factor is promptly removed from the circulation by hepatic sequestration and biliary secretion. 13 Thus, because of the fact that we could not infuse epidermal growth factor directly to the uterine artery for technical reasons, we used a dose of epidermal growth factor that was clearly pharmacologic both for bolus and for continuous infusion. Whether maternal serum concentrations remained elevated within the physiologic or the pharmacologic range after hepatic degradation was not determined in this pilot study. Regardless of this consideration, the mechanism by which placental permeability was increased by the exogenous growth factor requires explanation. Theoretically, augmented permeability to small molecules could be due either to an increase in "pore" number or to "pore" size. ll Epidermal growth factor has been demonstrated to have potent vasoconstrictor or vasodilator effects in different species, depending on the vascular bed studied. 14 -16 An increase in uterine blood flow induced by epidermal growth factor, however, would have been unlikely to enhance 5ICr-EDTA clearance; indeed 51Cr_ EDTA is a known diffusion marker that crosses the placenta according to a concentration gradient and therefore is unlikely to be affected by an increase in flow. l l We speculate that epidermal growth factor might be one of the regulators of placental transport of nutrients that cross the placenta by simple diffusion. This speculation would be consistent with the observation that intrauterine growth retardation in the rat increases epidermal growth factor binding to the placenta5 (which theoretically could increase non-energy-dependent transfer of nutrients through the placenta). In contrast, in situations of excessive fetal nutrient availability such as the diabetic pregnancy in the rat, epidermal growth factor binding to placenta is reduced 4 (which theoretically could decrease non-energy-dependent transfer of nutrients through the placenta). Whether epidermal growth factor does or does not have a physiologic effect on placental function, its potent action on placental permeability, as demonstrated in this study, might provide a unique model for studying the molecular regulation of nutrient transport across the placenta. All known biologic effects of epidermal growth factor are mediated through epidermal growth factor binding to specific cell-surface receptors. 17 Placebo (normal saline solution) did not have any measurable effect on placental permeability. We cannot, however, exclude the possibility that transforming growth factor-ex, the only other protein known to bind to the epidermal

176 Mimouni, Hoath, and Hammond

growth factor receptor, 18. in placental permeability.

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July 1991 Am J Obstet Gynecol

may elicit similar changes 10.

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