Euglycemic Hyperinsulinemia Increases Blood Pressure in Pregnant Rats Independent of Placental Antiangiogenic and Inflammatory Factors

Ana C Palei; Frank T Spradley; Joey P Granger

doi:10.1093/ajh/hpt137

. 2013 Aug 16;26(12):1445–1451. doi: 10.1093/ajh/hpt137

Euglycemic Hyperinsulinemia Increases Blood Pressure in Pregnant Rats Independent of Placental Antiangiogenic and Inflammatory Factors

Ana C Palei ^1,², Frank T Spradley ^1,², Joey P Granger ^1,^2,^✉

PMCID: PMC3816628 PMID: 23955606

Abstract

BACKGROUND

Although pregnancies associated with hyperinsulinemia and altered placental angiogenic and inflammatory factors are at increased risk for developing preeclampsia, the effects of euglycemic hyperinsulinemia on placental factors and blood pressure regulation during pregnancy are unclear. We hypothesized that chronic hyperinsulinemia results in increased placental soluble fms-like tyrosine kinase 1(sFlt-1) and tumor necrosis factor α (TNF- α) levels and hypertension in pregnant rats.

METHODS

On gestational day (GD) 14, Sprague-Dawley rats were assigned as normal pregnant or pregnant + insulin. Insulin was infused subcutaneously by osmotic minipump for 5 days at a dose of 1.5 mU/kg/min. Those rats receiving insulin were supplemented with 20% glucose in drinking water to maintain euglycemia. On GD 19, mean arterial pressure (MAP) and heart rate (HR) were assessed in conscious rats by indwelling carotid catheters, followed by collections of blood, placentas, and fetuses. In addition to pl acental sFlt-1 and TNF-α levels, circulating insulin, glucose, leptin, cholesterol, triglyceride, and free fatty acid concentrations were measured.

RESULTS

MAP was higher in pregnant + insulin vs. normal pregnant rats; however, HR was similar between groups. Although litter size and placental weight were comparable, fetuses from pregnant + insulin rats were heavier. Importantly, circulating insulin concentration was elevated in the pregnant + insulin group, with no change in glucose level. Moreover, circulating leptin, cholesterol, triglyceride, and free fatty acid concentrations were increased in the pregnant + insulin group. There were no differences in placental sFlt-1 and TNF-α concentrations between groups.

CONCLUSIONS

In summary, sustained euglycemic hyperinsulinemia, comparable with insulin levels in preeclamptic women, can raise blood pressure in pregnancy independent of recognized placental factors associated with preeclampsia.

Keywords: hyperinsulinemia, blood pressure, pregnancy, hypertension, preeclampsia.

Preeclampsia is a relatively common obstetric complication that contributes significantly to high maternal and fetal morbidity and mortality rates.^1,2 Although its etiology remains unclear, impaired cytotrophoblast invasion followed by placental ischemia is thought to cause the placental release of antiangiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt-1) and inflammatory cytokines such as tumor necrosis factor α (TNF-α) that ultimately lead to maternal hypertension.^3,4 In addition, there are several recognized risk factors for the development of preeclampsia, including preexisting metabolic diseases such as obesity and diabetes.^5,6 Such disturbances in metabolic factors, including insulin, may affect multiple steps in the cascade that links placental ischemia with maternal hypertension—for instance, enhancing the placental secretion of vasoactive factors that promotes hypertension.

Major metabolic adaptations arise in pregnancy to ensure the nutrient supply of the growing fetus. Studies have shown that insulin sensitivity is decreased by approximately 50% by 34–36 weeks of gestation in normal pregnant women.⁷ There is also a gradual increase in insulin response to the oral glucose tolerance test during the course of pregnancy, suggesting a progressive increase in insulin secretion throughout gestation.⁸ Insulin resistance also occurs during normal rat pregnancy.⁹ Intriguingly, several studies have reported that insulin resistance and hyperinsulinemia are markedly exacerbated during preeclampsia.^10,11

Abnormal circulating levels of insulin have been implicated in endothelial dysfunction¹² by many of the pathways associated with preeclampsia, such as nitric oxide, endothelin 1, and reactive oxygen species. Interestingly, insulin can also augment the inflammatory effects of TNF-α in vascular endothelial cells¹³ and regulate the gene expression of vascular endothelial growth factor in fibroblasts.¹⁴ Although pregnancies associated with hyperinsulinemia and altered placental angiogenic and inflammatory factors are at increased risk for developing preeclampsia, the effects of hyperinsulinemia on placental factors and blood pressure regulation during pregnancy are unclear. Although previous findings suggest that chronic treatment of pregnant rats with insulin increases systolic blood pressure and decreases fetal body weight, these changes were confounded by significant reductions in circulating glucose levels.^15,16 Because most preeclamptic patients are hyperinsulinemic and euglycemic or hyperglycemic (not hypoglycemic), we used an euglycemic hyperinsulinemic pregnant rat model in an attempt to recapitulate more closely the preeclamptic scenario in humans. Therefore, the purpose of this study was to test the hypothesis that sustained euglycemic hyperinsulinemia, comparable with insulin levels observed in preeclamptic women,^17,18 increases blood pressure and placental sFlt-1 and TNF-α levels in pregnant rats.

METHODS

Animals

All protocols were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Age-matched, timed-pregnant Sprague-Dawley rats (Harlan, Indianapolis, IN) were received on gestational day (GD) 11. They were maintained on a 12:12 hour light/dark cycle at 23 °C and were provided food (Harlan 8604) and water ad libitum.

Insulin treatment

On GD 14, rats were randomly assigned to the normal pregnant group (n = 8) or to the pregnant + insulin group (n = 10). Under isoflurane anesthesia (Butler Schein Animal Health, Dublin, OH), which was delivered by an anesthetic vaporizer (Ohmeda, BOC Health Care, Steeton, England), an osmotic minipump (model 1007D; Alzet, Cupertino, CA) was placed subcutaneously in the scapular region of pregnant + insulin rats to deliver insulin (Lantus, insulin glargine (recombinant DNA origin) injection; Sanofi-Aventis, Bridgewater, NJ) at a dose of 1.5 mU/kg/min.^19,20 To prevent changes in blood glucose levels during insulin infusion, 20% glucose (Fisher Chemical, Pittsburgh, PA) was added to the drinking water of those rats treated with insulin. From GD 13 to GD 19, body weight, food intake, and water intake were recorded daily. Average caloric intake (combined calories from food and glucose in drinking water) per day was calculated on GD 16–18. We excluded caloric intake on GD 15 and 19 from the average caloric intake per group because animals were still recovering from the surgeries performed on GD 14 (minipump placement) and 18 (carotid catheter placement), respectively.

Blood pressure measurement

On GD 18, rats were anesthetized with isoflurane as described above and implanted with in-dwelling carotid catheters consisting of V-1 tubing attached to V-3 tubing (Scientific Commodities, Lake Havasu City, AZ). Catheters were tunneled under the skin and externalized at the scapular region. The next day, rats were placed in individual restraining cages, and catheters were connected to pressure transducers (MLT0699; ADInstruments, Colorado Springs, CO) coupled to a computerized data acquisition system (PowerLab and Lab Chart Pro V7 software; ADInstruments). After an acclimation period, mean arterial blood pressure and heart rate were measured in conscious rats.

Tissue harvest

On GD 19, rats were anesthetized with isoflurane, and a ventral midline incision was made to externalize the uterus. Blood was collected into Corvac separator tubes (Tyco Healthcare Kendall, Mansfield, MA) and Vacutainer K₂EDTA tubes (BD, Franklin Lakes, NJ) by punching the abdominal aorta. The number of viable and reabsorbed fetuses in each animal was recorded, and individual fetuses and placentas were weighed. Representative placentas from each horn were flash-frozen in liquid nitrogen and stored at −80 °C until used. Serum and plasma samples were obtained by centrifugation of whole blood at 2,000g for 12 minutes at 4 °C and stored at −20 °C until assayed.

Measurements in serum and plasma

Blood glucose before insulin infusion was measured using a glucometer (OneTouch UltraMini; LifeScan, Milpitas, CA). Plasma glucose concentration at GD 19 was determined by the glucose oxidation method (Analox Instruments, Lunenburg, MA). Serum insulin and leptin were quantified by enzyme-linked immunosorbent assay (all R&D Systems, Minneapolis, MN) following the manufacturer’s instructions. Plasma total cholesterol (Cayman Chemical, Ann Arbor, MI) was quantified by fluorimetric assay, whereas triglycerides (Cayman Chemical) and free fatty acids (Zen-bio, Durham, NC) were quantified by colorimetric assays.

Measurements in placenta

Frozen placentas were crunched with mortar and pestle in liquid nitrogen, and tissue fragments were suspended in radioimmunoprecipitation lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) with a protease inhibitor cocktail containing phenylmethyl-sulfonyl fluoride and sodium orthovanadate (all Santa Cruz Biotechnology). After homogenization with a glass dounce tissue grinder, homogenates were centrifuged at 14,000g for 25 minutes, and supernatants were used to quantify sFlt-1 and TNF-α by enzyme-linked immunosorbent assay (both R&D Systems). Total protein concentration in samples was measured using the bicinchoninic acid method (Thermo Scientific, Rockford, IL).

Statistical analysis

All statistical analyses and graphs were prepared using GraphPad Prism 5.0 software (San Diego, CA). Comparisons between groups were performed by unpaired Student t test or Mann–Whitney U test as appropriate. Values are shown as mean ± SEM. A value of P < 0.05 was considered statistically significant.

RESULTS

Circulating metabolic factors

On GD 14, blood glucose levels were comparable in the normal pregnant group and the pregnant + insulin group (70.6±8.3mg/dl vs. 69.7±4.1mg/dl). At GD 19, insulin treatment caused an approximately 48% increase in serum insulin concentrations over normal pregnant rats (Figure 1a) (P < 0.05). Importantly, plasma glucose levels were not different between groups (Figure 1b).

Figure 1. — Effects of chronic insulin infusion during euglycemia in pregnant rats on circulating insulin (a) and glucose (b) levels at gestational day 19. Normal pregnant group (n = 8); pregnant + insulin (1.5 mU/kg/min) group supplemented with 20% glucose in drinking water (n = 10). *P < 0.05.

Table 1 shows that serum leptin and plasma cholesterol, triglyceride, and free fatty acid concentrations were elevated at GD 19 in insulin-treated pregnant rats compared with their normal pregnant counterparts (all P < 0.05).

Table 1.

Effects of chronic euglycemic hyperinsulinemia during pregnancy on placental, fetal, and circulating measurements at gestational day 19

Parameter	Pregnant	Pregnant + insulin
Litter size	13.8±0.6	13.9±0.7
Fetal body weight, g	2.32±0.03	2.41±0.02*
Placental weight, g	0.55±0.01	0.57±0.01
Serum leptin concentration, ng/ml	1.09±0.16	1.86±0.22*
Plasma cholesterol concentration, mg/dl	183.2±13.9	238.6±17.1*
Plasma triglyceride concentration, mg/dl	135.2±23.7	437.9±46.7*
Plasma FFA concentration, mg/dl	102.9±18.9	198.0±32.7*

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Values are mean ± SEM. Normal pregnant rats (n = 8); pregnant + insulin (1.5 mU/kg/min) rats supplemented with 20% glucose in drinking water (n = 10).

Abbreviation: FFA, free fatty acids.

*P < 0.05.

Blood pressure

On GD 19, mean arterial pressure was significantly increased by about 10mm Hg in pregnant + insulin rats compared with normal pregnant rats (Figure 2a) (P < 0.05). Although there was a trend to increased heart rate in pregnant + insulin rats, heart rate was not statistically different between groups (Figure 2b).

Figure 2. — Effects of chronic insulin infusion during euglycemia in pregnant rats on mean arterial pressure (a) and heart rate (b) at gestational day 19. Normal pregnant group (n = 8); pregnant + insulin (1.5 mU/kg/min) group supplemented with 20% glucose in drinking water (n = 10). *P < 0.05.

Fetal weights

As detailed in Table 1, fetal weight from the pregnant + insulin group was significantly greater than those from the normal pregnant group (P < 0.05). However, no differences in litter size or placental weight were seen in response to the insulin treatment in pregnant rats.

Placental antiangiogenic and inflammatory factors

There were no differences in placental sFlt-1 (Figure 3a) or TNF-α (Figure 3b) concentrations between pregnant + insulin rats and normal pregnant rats at GD 19.

Body weights and intakes

Initial body weight at GD 14 was similar for the rats designated for the normal pregnant group (274.0±8.8g) and for the pregnant + insulin group (274.4±3.6g). Baseline food intake and water intake were assessed from GD 13 to 14, and no differences in food intake (16.3±1.1 vs. 15.2±1.6g/day) or water intake (37.4±2.2 vs. 37.6±1.9ml/day) were found between the normal pregnant group and the pregnant + insulin group, respectively.

At the end of the study on GD 19, there was a trend to increased body weight in pregnant + insulin rats (344.4±4.7g) compared with normal pregnant rats (328.9±10.2g), although not statistically significant. From GD 16 to 18, pregnant + insulin rats had lower food intake than normal pregnant rats (GD 16: 17.7±0.7 vs. 22.8 ±0.6g/day; GD 17: 19.9±0.9vs. 24.6±0.7g/day; GD 18: 17.7±0.5vs. 22.2±1.0g/day; all P < 0.05). Water intake was similar in pregnant + insulin and normal pregnant groups on GD 16 and 17 (GD 16: 46.5±1.6 vs. 49.6±1.9ml/day; GD 17: 50.9± 2.1vs. 54.1±2.1ml/day); however, water intake was significantly decreased in pregnant + insulin rats compared with normal pregnant rats on GD 18 (48.9±1.9 vs. 57.7±2.3ml/day; P < 0.05). Because glucose was added in the drinking water of animals treated with insulin, this group presented higher average caloric intake on GD 16–18 than their normal pregnant counterparts (94.3±1.9 vs. 70.0 ±1.4 kcal/day; P < 0.05), which explains partially the increase in body weight of pregnant + insulin rats.

DISCUSSION

Our main finding is that sustained hyperinsulinemia increases mean arterial pressure in pregnant rats, which occurred independently of changes in glycemia. In addition, fetal weight was increased by chronic insulin treatment during euglycemia in pregnant rats. These alterations after insulin infusion were not dependent on changes in typical placental factors linked to preeclampsia, namely sFlt-1 and TNF-α. Intriguingly, circulating leptin, cholesterol, triglyceride, and free fatty acid levels were increased. These data highlight that hyperinsulinemia is an important risk factor for the development of hypertension in pregnancy.

In favor of a role for insulin in preeclampsia, Hamasaki and colleagues¹⁷ showed that hyperinsulinemic pregnant women have increased systolic and diastolic blood pressure levels compared with normal pregnant women. Moreover, these patients presented higher incidence of pregnancy-induced hypertension.¹⁷ In addition, association studies have found that insulin resistance is present in pregnant women who subsequently developed preeclampsia,^18–21,22 and in nonpregnant women who have previously experienced this syndrome.^23–25 Collectively these reports suggest that hyperinsulinemia and insulin resistance are involved in the pathophysiology of preeclampsia and thus provide the impetus for mechanistic studies in rodents examining how hyperinsulinemia alters blood pressure in pregnancy.

Bursztyn’s group performed a series of studies to evaluate the effects of chronic hyperinsulinemia during gestation.^{15,16,26–28} As expected, they observed that pregnant rats treated with insulin 1 week before and throughout pregnancy by subcutaneously implanted sustained-release insulin pellets had elevated serum insulin levels (approximately 78%) compared with normal pregnant rats.¹⁵ They showed that systolic blood pressure measured by tail cuff was increased in insulin-treated pregnant rats on GD 19 and 20.^15,16 Moreover, these blood pressure values returned to pregestational values 30 days after delivery.¹⁶ However, it is important to note that the insulin-treated pregnant rats presented a significant reduction in plasma glucose levels.¹⁵ Hence, these previous studies actually investigated the effects of hyperinsulinemia-induced hypoglycemia on blood pressure regulation in pregnant rats. Additionally, it has been demonstrated that hypoglycemia per se induced by various stimuli, including hyperinsulinemia, activates the autonomic nervous system,²⁹ which can then contribute to the effects of hyperinsulinemia on increasing blood pressure. Therefore, we believe that the hypertensive actions of hyperinsulinemia in pregnant rats reported previously were confounded by hypoglycemia.

Importantly, Hall’s group performed several studies to evaluate the effects of chronic euglycemic hyperinsulinemia in male rats.^{19,20,30–33} They showed that 5 days of moderate hyperinsulinemia during euglycemia produced a modest raise in mean arterial pressure.¹⁹ However, there is large body of evidence indicating that blood pressure regulation is different in males and females, especially pregnant females.^34,35 Moreover, sex steroids can affect insulin sensitivity in different tissues.³⁶ ^, ³⁷ Additionally, it is though that the high circulating levels of estrogen and progesterone associated with pregnancy contributes for the development of gestational insulin resistance.^36,37 In light of these considerations, although earlier studies in males had evaluated the effects of euglycemic hyperinsulinemia on blood pressure, it was not clear whether chronic euglycemic hyperinsulinemia could elicit hypertension and alterations in sFLT-1 and TNF-α during pregnancy. Indeed, we demonstrated here that sustained euglycemic hyperinsulinemia increases mean arterial pressure in pregnant rats. In addition, the increased blood pressure was not associated with increases in the placental factors sFlt-1 and TNF-α. These findings highlight that other mechanisms are at play.

Several avenues that mediate insulin’s hypertensive effects have been examined before. Increased sympathetic activity and muscle blood flow have been implicated as possible mechanisms whereby elevated circulating insulin raises blood pressure.³⁸ However, because we did not observe significant heart rate differences in response to the insulin infusion in pregnant rats, our data suggest that enhanced adrenergic activity was not required for the hypertensive actions of hyperinsulinemia. Indeed, Keen and colleagues³¹ showed that pharmacological blockade of α1- and β-adrenergic receptors did not prevent the increases in blood pressure induced by chronic euglycemic insulin treatment in male rats. Moreover, Brands and colleagues²⁰ found that hyperinsulinemia-induced hypertension was not mediated by increased cardiac output but by increased total peripheral resistance and decreased glomerular filtration rate. These hemodynamic alterations were dependent on a normal renal ability to synthesize angiotensin II and thromboxane.^30,32,33 Although these factors have been reported to play a role in insulin-induced hypertension in male rats, the role of angiotensin II and thromboxane in mediating the blood pressure response to insulin during pregnancy remains to be determined.

Although we previously reported that fasting insulin, glucose tolerance, and triglycerides were not altered in our rodent model of placental ischemia-induced hypertension,³⁹ these observations do not rule out the possibility that metabolic dysfunction may contribute to the pathogenesis of preeclampsia. Actually, our new findings that chronic insulin infusion during euglycemia leads to increased blood pressure in pregnant rats imply that hyperinsulinemia contributes to the development of hypertension in preeclampsia rather than results of placental ischemia. Nevertheless, hyperinsulinemia could also affect vascular function through alterations in other metabolic factors.¹² Podjarny et al. ¹⁵ described that circulating triglycerides were elevated in insulin-treated pregnant rats compared with normal pregnant rats. We also observed dyslipidemia and hyperleptinemia in euglycemic hyperinsulinemic pregnant rats. Indeed, human studies have demonstrated that increased circulating cholesterol, triglyceride, free fatty acid, and leptin levels can precede the appearance of the clinical symptoms of preeclampsia.^18,21,40,41 Therefore, we cannot rule out a role for hyperlipidemia and hyperleptinemia in mediating the blood pressure response to euglycemic hyperinsulinemia in pregnant rats. Whether hyperinsulinemia directly elicited high blood pressure in our pregnant rats or whether this response was mediated through combined action with hyperleptinemia and dyslipidemia remains to be determined.

Insulin is also thought to play a key role in feto-placental development and growth.⁴² An inverse correlation between maternal insulin sensitivity and neonatal weight has been noted in humans,^43,44 suggesting that insulin resistance and the associated hyperinsulinemia can lead to increased birth weight. In addition, patients with fetal growth retardation have been associated with higher insulin sensitivity and lower plasma insulin levels after glucose load compared with pregnant women with normal fetal growth.⁴⁵ In regard to fetal outcome, we found that chronic insulin infusion during euglycemia promoted increased fetal weight. However, Podjarny et al. ¹⁶ observed that fetal weight from insulin-treated pregnant rats was reduced compared with normal pregnant rats. Our contrasting findings to those of Podjarny may result from decreased circulating glucose and therefore decreased glucose delivery to the fetuses in their model, whereas glucose levels were maintained in our insulin-infused pregnant rats by supplementing their drinking water with 20% glucose. We speculate that the presence of hyperinsulinemia in our pregnant rats without hypoglycemia promoted glucose shunting to the placenta-fetal compartment, resulting in increased fetal growth. Although infants born macrosomic or large for gestational age have increased risk of developing cardiovascular diseases later in life,⁴⁶ future studies should address whether in utero exposure to euglycemic hyperinsulinemia affects the long-term health of the offspring.

In conclusion, we found evidence that sustained hyperinsulinemia during euglycemia leads to increased mean arterial blood pressure and fetal weight in pregnant rats, representing a mechanism that contributes to the development of hypertension during pregnancy. However, these changes were independent of recognized placental factors (sFlt-1 and TNF-α) linked to the pathophysiology of preeclampsia. Furthermore, these data highlight mechanisms that may link hyperinsulinemia and insulin resistance with a heightened risk for preeclampsia.

Preeclampsia is estimated to affect 5%–7% of all pregnancies in the United States and approaches rates of 15% in blacks. Despite its position as a leading cause of maternal death and major contributor to maternal and perinatal morbidity, the pathophysiology of preeclampsia has yet to be fully elucidated. Moreover, the incidence of preeclampsia has increased by 40% over the last several decades as a result of a significant increase in the incidence of metabolic diseases such as obesity.

An important initiating event in preeclampsia is thought to involve reduced placental perfusion that leads to the placental release of antiangiogenic factors such as sFlt-1, which antagonizes endogenous vascular endothelial growth factor, and release of inflammatory cytokines such as TNF-α. Although recent studies have demonstrated an important effect of obesity on placental function, the effects of obesity-related metabolic factors such as insulin on the pathways that link placental ischemia and maternal blood pressure are unknown. Although several studies have reported that insulin resistance and hyperinsulinemia are markedly exacerbated during preeclampsia, the effects of euglycemic hyperinsulemia on placental factors and blood pressure regulation during pregnancy are unclear. In this study we tested the hypothesis that chronic euglycemic hyperinsulinemia, comparable with insulin levels observed in preeclamptic women, increases blood pressure and placental antiangiogenic and inflammatory factors in pregnant rats. Our data suggest that sustained euglycemic hyperinsulinemia can raise blood pressure independent of changes in classic placental factors associated with preeclampsia. These data highlight a potential mechanism contributing to the development of hypertension in obese pregnant women.

DISCLOSURE

The authors declared no conflict of interest.

ACKNOWLEDGMENTS

We would like to thank Marietta Arany, Kathy Cockrell, and Haiyan Zhang for their technical expertise. This work was supported by National Institutes of Health grants HL051971 and 1T32HL105324.

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41. Lorentzen B, Endresen MJ, Clausen T, Henriksen T. Fasting serum free fatty acids and triglycerides are increased before 20 weeks of gestation in women who later develop preeclampsia. Hypertens Pregnancy 1994; 13: 103–109 [Google Scholar]
42. Ciampelli M, Lanzone A, Caruso A. Insulin in obstetrics: a main parameter in the management of pregnancy. Hum Reprod Update 1998; 4: 904–914 [DOI] [PubMed] [Google Scholar]
43. Bernstein IM, Goran MI, Copeland KC. Maternal insulin sensitivity and cord blood peptides: relationships to neonatal size at birth. Obstet Gynecol 1997; 90: 780–783 [DOI] [PubMed] [Google Scholar]
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45. Caruso A, Paradisi G, Ferrazzani S, Lucchese A, Moretti S, Fulghesu AM. Effect of maternal carbohydrate metabolism on fetal growth. Obstet Gynecol 1998; 92: 8–12 [DOI] [PubMed] [Google Scholar]
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PERMALINK

Euglycemic Hyperinsulinemia Increases Blood Pressure in Pregnant Rats Independent of Placental Antiangiogenic and Inflammatory Factors

Ana C Palei

Frank T Spradley

Joey P Granger