The Evaluation of Hypoxia-Inducible Factor 1 in N-Nitro-L-Arginine Methyl Ester Preeclampsia Model of Pregnant Rats

Abstract

Objective

The objective of the study was to evaluate hypoxia-inducible factor 1 (HIF-1), which plays a major role in the stimulation of angiogenesis in placental tissues, by using immunohistochemical staining in preeclampsia model of rats, developed by N-nitro-L-arginine methyl ester (L-NAME)

Methods

Thirty pregnant rats were randomized into 2 groups (n = 15 in each group) on day 10 of gestation. L-NAME was given to rats in the study group by gavage. On days 0, 10, and 20 of gestation, rats were weighted, and urine protein values and blood pressures were measured. Hypoxia-inducible factor 1 expressions were assessed with immunohistochemical staining by using avidin-biotin peroxidase via selecting preparation.

Results

Systolic and diastolic blood pressures and urine protein value of L-NAME group on day 20 of gestation were found to be significantly higher than those obtained on days 0 and 10 of gestation in the same group and those obtained on day 20 of gestation in the sham group (P < 0.05). Maternal weight, number of fetuses, and mean fetal weight of rats in L-NAME group on day 20 of gestation were found to be significantly lower than those obtained from rats in the sham group (P < 0.05). Regarding HIF-1 expression of placental tissues, mild immunohistochemical staining was found in 2 rats (13.4%) and moderate in 13 rats (86.6%) in the L-NAME group. A significant difference was found in terms of HIF-1 positivity in the maternal placentas of both groups (P < 0.05).

Conclusions

L-NAME preeclampsia model of pregnant rats is consistent with human preeclampsia in terms of hypertension, proteinuria, and intrauterine growth retardation; in addition, it also shows evidence of placental hypoxia findings.

Keywords

hypoxia-inducible factor 1 preeclampsia rat

Each year, many women die of pregnancy-related complications worldwide. Preeclampsia is the most common hypertensive disorder associated with pregnancy. Preeclampsia is characterized by increased blood pressure and proteinuria in the mother, accompanied by growth retardation in the fetus. The pathophysiology of preeclampsia in the second half of gestation and early postpartum period has not been fully elucidated. However, there is strong evidence that underlying mechanisms merge at endothelial dysfunction. It has been also reported that endothelial dysfunction is related to placental ischemia.¹

Nitric oxide (NO), produced from L-arginine in the presence of oxygen via NO synthase (NOS) in the vascular endothelium, elevates in maternal serum during a normal pregnancy and plays an important role in the control of vascular tone and regulation of vascular functions.^2,3 In hypertensive disorders of pregnancy, lack of NO or its decreased concentration is a part of endothelial dysfunction. It was shown that NO degradation products of NO are increased in preeclamptic women and is related to decreased blood flow in uteroplacental unit.⁴ Several laboratory studies have shown that hypertension—as well as peripheral or renal vasoconstriction, glomerular proteinuria, intrauterine growth retardation (IUGR), and fetal morbidity—was shown in models developed by applying N-nitro-L-arginine methyl ester (L-NAME) or other NOS inhibitors.^5–7 Both animal studies by NOS inhibitors and decreased NO levels and subsequent hypertension in preeclamptic women only partially explain decreased placental perfusion and intrauterine growth retardation. On the other hand, it has been reported that the expression of vascular endothelial growth factor, which has recently gained importance in the pathophysiology of preeclampsia, is decreased in L-NAME models.^8,9 However, preeclampsia remains as a serious problem with multiple questions and options, despite current knowledge.

Vascular development during implantation and placentation is essential for successful gestation. Vascular deficiency and hypoxia during placentation cause obstetric complications such as preeclampsia, but we know little about regulation of placental angiogenesis and placental hypoxia.¹⁰ Accordingly, it is interesting to demonstrate the relationship between NO, which is closely associated with placental perfusion and angiogenesis, and hypoxia-inducible factor 1 (HIF-1), which has increased expression by hypoxia.

The aim of this study was to evaluate HIF-1 in placental tissues of rats in a preeclampsia model that is formed by L-NAME.

Materials and Methods

Obtaining Pregnant Rats

Thirty Wistar albino rats weighing between 200 and 250 g (12-16 weeks old) were obtained from Test Animal Laboratory of Cumhuriyet University, Medicine Faculty (Sivas, Turkey) for use in this study. Consent was obtained from Institutional Test Animal Ethic Committee for Use and Investigation of Animals. Care was taken to select animals of similar age and weight. Animals were maintained with 8-mm standard rat pellets in special cages at standard conditions (12-hour light-dark cycle, air conditioned, stable temperature). Each female rat was mated with male rats between 5:00 PM and 9:00 AM at 22°C. The next day, vaginal smear tests were performed to seek sperm. Day 0 of gestation was defined as the day when spermatozoa were found in a vaginal smear. On day 10 of gestation, rats were randomized into 2 groups (sham and L-NAME groups), with equal number of rats in each group.

Study Groups

sham group: 15 pregnant rats, which received no treatment

L-NAME group: 15 pregnant rats, which received daily L-NAME (50 mg/kg per day) by gavage for 7 days beginning on day 11 of gestation.

Experimental Procedure

Rats were killed using 40 mg/kg intraperitoneal ketamine HCl and 1 mg/kg xylazine hydrochloride on day 20 of gestation after performing a cesarean section under combined general anesthesia (ketamine HCl 75 mg/kg and xylazine hydrochloride 1 mg/kg).

Placentas and pups were removed by cesarean section. The number and weight of pups and placentas were recorded and put into formalin for histopathologic examination after being washed with saline.

Measurement of Weight and Blood Pressure and Collection of Urine Sample

Weight and blood pressure of rats were measured on days 0 (D₀), 10 (D₁₀), and 20 (D₂₀) of gestation. On the same days, urine samples were collected. Blood pressure was measured by using the tail-cuff blood pressure monitoring system (BPHR 9610 Blood Pressure System; Commat, Ankara, Turkey).¹¹ Tails of unanesthetized rats were heated to dilate tail vessels in a closed environment with a constant temperature of 39°C to 40°C.

Protein Measurement in Urine Samples of Rats

Protein quantity was measured by dipstick method (006T250; Ulti Med Products GmbH, Ahrensburg, Germany) in urine samples, which were obtained on the days 0 (D₀), 10 (D₁₀), and 20 (D₂₀) of gestation.¹² Proteinuria was identified by dropping urine samples onto colorimetric strips. Proteinuria was graded as “−,” “1+,” “2+,” “3+,” or “4+” by comparing color reaction to color scale: “−” to “2+” reflects mild proteinuria (30-100 mg/dL), whereas “3+” and “4+,” severe proteinuria (300-2000 mg/dL).

Light Microscopy and Immunohistochemical Evaluation

Preparations include abundant cells that best reflect the histological grade of the cases selected among placental specimens of the groups. Three-micrometer sections were obtained from paraffin blocks of these specimens and put on the poly-L-lysine-coated slides. Hypoxia-inducible factor 1 kit antibodies (alpha antibody [H₁ alpha 67] GTX20001, lot: 12782 GeneTex concentrate; GeneTex, Irvine, CA) were used. Antibody was diluted by 1:100. Immunohistochemical staining was achieved by using the avidin-biotin peroxidase method.

Staining procedure was performed at a humidified condition, heated up to 26°C to 27°C with wet floor. Several 3-μm-thickness tissue sections were fixed by formalin, embedded in paraffin, and placed in an oven at 65°C overnight. They were then deparaffinized in xylene for 30 minutes at 50°C. After this process, tissues were dehydrated for 5 minutes by 80°C, 90°C, and 96°C alcohol. Then, to remove endogen peroxidase activity, they were incubated with hydrogen peroxide for 10 minutes, followed by washout with distilled water for 5 minutes and PBS (phosphate-buffered saline) solution for 10 minutes. For better antibody exposition, they were boiled in water for 40 minutes after being washed with PBS solution for 10 minutes. EDTA buffer (pH 8.7) was used as a boiling solution. They were then placed in the same solution for 20 minutes at room temperature. Sections were incubated for 20 minutes by UV block after being washed for 10 minutes in PBS. Then, sections were incubated with HIF for 90 minutes at room temperature and placed in PBS for 10 minutes. They were then incubated with binding solution (Link) for 20 minutes at room temperature and placed in PBS for 10 minutes. Next, they were incubated with streptavidin peroxidase (label) for 20 minutes at room temperature and placed in PBS for 10 minutes. Finally, colorization was performed by using a substrate/chromogen mixture. They were incubated with a solution prepared from a dilution consisting of a mixture of substrate and 1 drop AEC chromogen and 2 mL H₂O₂. Mayer hematoxylin was used for counterstaining for 30 seconds. The sections were then washed with distilled water. Tissues were dried and closed with cover glass using immune mount.

Slides were assessed by using light microscope at ×4 magnification to detect areas of HIF-1 staining. Staining grade was scored at 5 random areas, and the area with the highest score was identified. Within the 2 groups, 100 cells were labeled in each ×40 magnification field. Scoring was achieved by a semiquantitative method, which uses percentage of stained cells and staining grade in sections. Staining grades were classified as follows: 0 (no staining), +1 (weak staining), +2 (moderate), and +3 (strong). Scoring of immunohistochemical staining for each section was done by using a score algorithm, H-SCORE, which uses the following formula: I × PC, where I is staining grade; PC, percentage of stained cells in each grade.

Statistical Analyses

Results were evaluated by SPSS software version 14.0. Mann-Whitney U test; Fisher exact test, and χ² test were used in the assessment of data. Data were expressed as mean (SD), number, and percentage of subject in tables. P < 0.05 was considered to be statistically significant.

Results

Indirect systolic and diastolic blood pressures were measured at D₀, D₁₀, and D₂₀. No difference was found in maternal systolic and diastolic blood pressure between D₀, D₁₀, and D₂₀ in the sham group (P > 0.05). Systolic and diastolic blood pressures of rats in the L-NAME group at D₂₀ were found to be significantly higher than those obtained at D₀ and D₁₀ in the same group and those obtained at the D₂₀ in the sham group (P < 0.05; Table 1).

Table 1

Maternal Systolic and Diastolic Blood Pressures, Weight, and Urine Protein Levels Parameters

Maternal weights at D₀ were similar in both groups (P > 0.05). Maternal weights at D₂₀ were significantly higher than those obtained at D₀ and D₁₀ in both groups (P < 0.05). Again, maternal weights at D₁₀ were significantly higher than those obtained at D₀ in both groups (P < 0.05). However, maternal weights at D₂₀ in L-NAME groups were significantly lower than those measured at D₂₀ in the sham group (P < 0.05; Table 1).

No protein (−/+) was detected in urine samples of rats in the sham group on D₀, D₁₀, and D₂₀. In the L-NAME group, no protein (−/+) was found in urine samples collected at D₀ and D₁₀, whereas protein (++/+++) was found in those at D₂₀. Protein values of urine samples collected at D₂₀ in the L-NAME group were significantly higher than those collected at D₀ and D₁₀ in the same group and those collected at D₂₀ in the sham group (P < 0.05; Table 1).

Both the number of fetuses and mean (SD) fetal weight were significantly lower than those of the sham group (respectively, 8.4 [5.2], 1.9 [0.5], 12.4 [4.2], 3.3 [0.5]) (P < 0.05; Table 2).

Table 2

Number of Fetuses and Fetal Weight

Regarding HIF-1 expression of placental tissues, mild immunohistochemical staining was found in 2 rats (13.4%) and moderate in 13 rats (86.6%) in the L-NAME group, whereas no staining was detected in the sham group (Fig. 1). Hypoxia-inducible factor 1 expression status of placental tissues was assessed as negativity or positivity. When placental HIF-1 positivity of rats was compared between the 2 groups, a significant difference was observed (P < 0.05; Table 3).

Figure 1

A, HIF-1 positive trophoblast, IHC ×20. B, HIF-1 negative trophoblast, IHC ×20.

Table 3

HIF-1 Expression Scores in Placental Cytotrophoblast

Discussion

We observed the increased maternal blood pressure, increased urine protein, IUGR, and significantly increased HIF-1 expression in placental tissue in our study that was based on the preeclamptic rat model induced by L-NAME administration.

Preeclampsia is a syndrome characterized by high blood pressure and proteinuria. Two blood pressure measurements greater than 140/90 mm Hg (at least 6 hours between the 2 measurements) after week 20 of gestation are diagnostic for hypertension in patients who had had no hypertension at the beginning of pregnancy. Proteinuria is the presence of protein 300 mg or more in 24-hour urine collection or at least 30 mg/dL protein (1+ dipstick) in a spot urine sample.¹³ In preeclampsia, IUGR, preterm birth, placental abruption, and oligohydramnios can develop as a result of placental insufficiency. Perinatal mortality or fetal asphyxia can occur because of placental abruption.¹⁴

In our study, systolic and diastolic blood pressures of the L-NAME rats at D₂₀ were found higher than those of the sham rats. Blood pressure of the L-NAME rats at D₂₀ was also higher than those at D₀ and at D₁₀. However, no change occurred in systolic and diastolic blood pressure between D₀, D₁₀, and D₂₀ in the sham groups. This shows that giving L-NAME to pregnant rats causes an increase in blood pressure. In a study, continuous L-NAME was given to pregnant rats from day 18 of gestation to postpartum hour 24 via venous catheter; these rats developed hypertension and proteinuria.⁶ The infusion of L-NAME to rats from day 11 to day 20 of gestation developed hypertension in these rats.⁵ In another study, administration of L-NAME to rats from day 17 of pregnancy caused hypertension and proteinuria in these rats.⁷ Our findings were consistent with these studies. These results shows that NO plays an important role in the control of vascular tone and in the regulation of vascular functions during pregnancy.

Preeclampsia is characterized by generalized endothelial dysfunction. To date, data have suggested that endothelial dysfunction plays a role in the pathogenesis of preeclampsia by altering both vascular response and intravascular coagulation.¹⁵ Because of injury to the endothelium, the integrity of cellular membrane is disrupted, a protein leakage occurs, and then proteinuria develops.¹⁶ In our study, no protein was found in urine samples collected at D₀, D₁₀, and D₂₀ in the rats of the sham group, whereas proteinuria was found in urine samples collected at D₂₀ in rats of the L-NAME group. Additional studies support our results in terms of proteinuria.^5–7 Considering these findings, the preeclampsia model, which was developed using L-NAME, increased urinary protein excretion. Thus, giving L-NAME to pregnant rats reflects the clinical features of preeclampsia by resulting in proteinuria as well as hypertension.

In preeclampsia, the PGI2/TxA2 ratio is changed in the fetoplacental area, and NO release decreases. Therefore, IUGR, chronic hypoxia, and perinatal mortality can occur, because fetoplacental perfusion is deteriorated because of the previously mentioned changes.¹⁷ By taking the concept that IUGR in preeclampsia is caused by the decrease in uteroplacental blood flow in consideration, Morris et al performed a study in which NOS activity was measured in the placental bed, placenta, and umbilical cords of pregnancies with IUGR and those of healthy pregnancies with normal fetal development.¹⁷ They found that NOS activity was low in preeclamptic pregnancies. In our study, the maternal weight of rats in the L-NAME groups at D₂₀ was found to be significantly lower than those in the sham group at D₂₀. This is significant in terms of IUGR. The studies performed with L-NAME also demonstrated IUGR in fetuses of pregnant rats with hypertension.^5,7 In our study, the number and weight of fetuses in rats in the L-NAME group were found to be significantly lower than in the sham group. In light of these findings, it should be suggested that there is a relationship between L-NAME application and IUGR, one of known criteria for severe preeclampsia.

In a preeclampsia model, developed by giving L-NAME to rats on day 5 of pregnancy, Biswas et al¹⁸ demonstrated that implantation failure occurred because of NO insufficiency, secondary pre-embryo development was weakened, and embryonic growth retardation occurred. In our study, the mean number of fetuses in rats of the L-NAME group was lower than that in the sham group. Although L-NAME was initiated on day 10 of gestation in our study, results support those of Biswas et al.¹⁸

Hypoxia-inducible factor 1 is an α/β heterodimeric protein complex and regulates the expression of many genes such as angiogenesis, vascular tone, cell growth, and survival in response to changes in oxygen availability.¹⁹ Hypoxia-inducible factor 1 activity is regulated by prolyl hydroxylase (PHD1, PHD2, PHD3) and factor-inhibiting HIF 1. At the same time, other signaling mechanisms such as kinases and NO levels modulate overall HIF activity.¹⁹ Many pathways in the inflammatory microenvironment (hypoxia, NO, or several cytokines) affected HIF-1α levels.²⁰ Relation among NO and HIF-1, which is an important regulator of cellular homeostasis, has been shown in some recent studies.^21,22 Bahtiyar et al²³ were the first to hypothesize that NO inhibition had synergistic effects with chronic hypoxia in pregnant rats, but they showed that their effect was not synergistic, suggesting independent pathways.

Among the animal models of gestational hypertension, an increase was shown in the expressions of placental HIF-1α and endoglin in the reduced uterine perfusion pressure model of pregnant rats.²⁴ In the pregnant rat, elevations of circulating soluble endoglin produced a preeclampsia-like syndrome, including the development of hemolysis, elevated liver enzymes, and low platelets, reduced fetal growth, severe hypertension, and nephritic range proteinuria.²⁵ A priori study showed that HIF-α proteins were increased in placenta tissues obtained from preeclamptic women.²⁶

Placental hypoxia plays an important role in placental disorders. Recent studies have reported that the endoglin gene is regulated by HIF-1 and that both are increased in the hypoxic and preeclamptic placenta.^27,28 So we hypothesized that NO inhibition by L-NAME produces similar effects like reduced uterine perfusion pressure model and human studies by causing experimental preeclampsia, thus increasing HIF-1 expression. Our study showed increased HIF-1 expression in placental tissues in preeclamptic rats, similar to the previous studies.

Conclusions

There are no other studies of placental HIF-1 expression by L-NAME administration in pregnant rats, so our study is the first on this subject. We showed increased HIF-1 expression after L-NAME administration in pregnant rats, similar to preeclamptic placentas. This leads us to think that the HIF-α protein level may be increased in hypertensive pregnant rats because of fetoplacental perfusion defect and hypoxia.

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