Heme Oxygenase Expression in Selected Regions of Term Human Placenta

Abstract

Carbon monoxide (CO), formed during heme oxygenase (HO)-catalyzed oxidation of heme, has been proposed to play a complementary role with nitric oxide in the regulation of placental hemodynamics. The objective of this study was to elucidate HO enzymatic activity and HO-1 (inducible) and HO-2 (constitutive) protein content in the microsomal subcellular fraction of homogenate of selected regions of placenta from normotensive and mild pre-eclamptic pregnancies. HO enzymatic activity was measured under optimized conditions by gas chromatography using CO formation as an index of activity, and HO-1 and HO-2 protein content were determined by Western immunoblot analysis. Microsomal HO activity in each of the four placental regions was not different between normotensive and mild pre-eclamptic pregnancies. Microsomal HO-2 protein content was not different between normotensive and mild pre-eclamptic pregnancies, whereas there was increased expression of microsomal HO-1 protein in chorionic villi and fetal membranes from pre-eclamptic pregnancy compared with normotensive pregnancy. Microsomal HO enzymatic activity correlated with HO-2, but not HO-1, protein content.

It has been proposed that carbon monoxide (CO), produced during the heme oxygenase (HO)-catalyzed oxidation of heme, plays a complementary role with nitric oxide in the regulation of placental hemodynamics (1–5). In support of this concept, HO has been shown to be present in human placenta using in vitro CO formation as an index of HO activity (4) and by immunohistochemistry (3, 5). Furthermore, inhibition of the HO enzymatic system has been reported to increase perfusion pressure of human placenta in vitro (3), indicating a role for CO as a vasorelaxant molecule in this organ system. Recently, we have demonstrated that CO in concentrations that have been measured in human umbilical cord blood decreases fetoplacental perfusion pressure in a term human placental cotyledon preparation (6). This CO-induced decrease in fetoplacental perfusion pressure appears to be mediated primarily by cGMP.

There are three isoforms of HO protein: HO-1 (inducible), HO-2 (constitutive) (7), and HO-3 (function unknown) (8). In a recent study, Western immunoblot analysis showed no overall difference in HO-2 protein content of human placental homogenate isolated from pregnancies complicated by pre-eclampsia or fetal growth restriction compared with normotensive controls (9). Immunohistochemical analysis, however, showed decreased HO-2 protein expression in endothelial cells of placenta from pre-eclampsia or fetal growth restriction (9). It has been reported that the human placenta contains relatively little HO-1 protein, based on Western immunoblot and immunohistochemical analysis (3, 9).

Currently, there are no data on the relationship between HO enzymatic activity and HO-1 and HO-2 protein content in the human placenta. The objective of this study was to elucidate HO enzymatic activity and HO-1 and HO-2 protein content in the microsomal subcellular fraction of homogenate of selected regions of the placenta from term normotensive pregnancy and mild pre-eclampsia, a disease of pregnancy involving maternal hypertension, proteinuria and/or edema.

Materials and Methods

Drugs and Solutions.

Hemin, ethanolamine, bovine serum albumin, ethylenediaminetetraacetic acid, and NADPH were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Chromium mesoporphyrin was purchased from Porphyrin Products Inc. (Logan, UT). All other chemicals were obtained from VWR Canlab (Toronto, ON) and were at least reagent grade. Working solutions of methemalbumin and chromium mesoporphyrin were prepared as described previously (2).

Tissue Preparation.

Human placentas were obtained from elective cesarean section delivery of normal term pregnancies and mild pre-eclamptic pregnancies. Mild pre-eclampsia was defined by the development of blood pressure of at least 140 mmHg systolic (but lower than 160 mmHg) and 90 mmHg diastolic on two or more separate occasions after the 20th week of pregnancy, having previously been normotensive and without a history of renal disease. The definition also included proteinuria (either >300 mg per 24-hr collection or 2+ on a voided random urine sample) in the absence of urinary tract infection (10). Gestational ranges were 38 to 42 weeks.

Within 1 hr of delivery, placentas were transported from the delivery room to the laboratory and selected regions of the placenta were dissected, viz., chorionic villi, chorionic plate, basal plate, and fetal membranes (amnion and chorion); frozen with liquid nitrogen; and stored at −70°C. A microsomal fraction of 10% (w/v) homogenate of each placental region was prepared and protein content was determined, as described previously (2), with spectrophotometric analysis using a microplate reader (EL 800, Bio Tek Instruments, Inc., Winooski, VT). The microsomal samples were stored at −70°C, until analyzed.

HO Enzymatic Activity.

In the individual regions of the placenta, HO enzymatic activity was determined in vitro by measuring the rate of CO formation during the NADPH-dependent oxidative biotransformation of heme (11), using optimized experimental conditions (4). HO enzymatic activity in the microsomal fraction of homogenate of each placental region was expressed as nmol CO formed/g tissue wet weight/hr.

HO-1 and HO-2 Protein Content.

For each sample, HO-1 and HO-2 protein content was determined by Western immunoblot analysis. Thirty micrograms (for HO-2 analysis) or 50 μg (for HO-1 analysis) of microsomal protein from each placental region was loaded onto 12.5% (w/v) polyacrylamide gels, separated by electrophoresis under reducing conditions, and then transferred onto Immobilon-P™ membranes (Millipore Corp., Bedford, MA). Membranes were blocked overnight at 4°C in a phosphate-buffered saline solution containing 0.01% Tween 20 and 5% skim milk powder. The immunoblots were then incubated with a 1:2000 dilution of the polyclonal anti-HO-1 (SPA-896; StressGen, Victoria, BC) or anti-HO-2 (SPA-897; StressGen) antibody, followed by incubation with a peroxidase labeled goat anti-rabbit IgG secondary antibody (Vector Lab. Inc., Burlingame, CA). The specificity of the antibodies was initially determined by Western blot analysis of HO-1 and HO-2 protein in extracts of cultured first trimester human cytotrophoblasts (12). In that study, the anti-HO-1 and anti-HO-2 antibodies detected protein bands corresponding in mass to HO-1 and HO-2, respectively. Although additional bands at approximately 90 kDa, 70 kDa, 50 kDa, and 20 kDa were also detected, only detection of the bands corresponding to HO-1 (32 kDa) or HO-2 (36 kDa) were abolished by preincubation of the antibodies with the HO-1 and HO-2 immunogen peptides used to raise the antibodies. Evidence that the antibodies do not exhibit crossreactivity was provided by the fact that preincubations of the HO-1 peptide with the anti-HO-2 antibody and the HO-2 peptide with the anti-HO-1 antibody did not inhibit detection of the corresponding HO proteins. Whereas both the anti-HO-1 and HO-2 antibodies detected additional nonspecific proteins by Western blot analysis, these proteins were not detected by immunohistochemistry, as preincubation with their corresponding peptides completely abolished all immunostaining. In the present study, antigen was detected by enhanced chemiluminescence (Amersham Canada Inc., Toronto, ON) and exposure of the membrane onto Dupont Reflection NEF film (Dupont Canada Inc., Toronto, ON). Each immunoblot was quantified by optical densitometry (Un-Scan-It, Silk Scientific, Orem, Utah).

Data Analysis.

The data for microsomal HO enzymatic activity and HO-1 and HO-2 protein content of the four placental regions from term placentas of normotensive and mild pre-eclamptic pregnancies are presented as group means ± SD (n = 4). Parametric statistical analysis of the data was conducted by two-way, randomized-design analysis of variance, after confirming homogeneity of variance of the data by Bartlett’s test. For a statistically significant F-statistic (P < 0.05), a one-way, randomized-design analysis of variance followed by Newman-Keuls test or unpaired Student’s t test was conducted, depending on which test was statistically appropriate. Correlational analysis of microsomal HO enzymatic activity and HO-1 and HO-2 protein content was conducted in which the correlation coefficient was determined and statistical significance was set at P < 0.05.

Results

HO Enzymatic Activity.

CO formation in the microsomal fraction of homogenate of chorionic villi from term placenta of normotensive pregnancy and mild pre-eclamptic pregnancy was NADPH dependent and primarily due to HO enzymatic activity as demonstrated by 72 ± 4% and 71 ± 6% inhibition, respectively, of heme-derived CO formation by chromium mesoporphyrin, a competitive inhibitor of HO (13, 14). Similar results were obtained for the chorionic plate, basal plate and fetal membranes regions (data not shown). In placenta from term normotensive pregnancy, HO enzymatic activity in the microsomal fraction of homogenate of chorionic villi and basal plate was greater (P < 0.05) than that of the microsomal fraction of homogenate of chorionic plate and fetal membranes (Fig. 1). Regional differences in HO activity were the same for term placenta from mild pre-eclamptic pregnancy (Fig. 1 ). HO activity for each of the four selected placental regions was not different for mild pre-eclamptic pregnancy compared with normotensive pregnancy.

HO-1 and HO-2 Protein Content.

HO-2 (constitutive) protein and HO-1 (inducible) protein were measurable by Western immunoblot analysis of the microsomal fraction of homogenate from the four selected regions of placenta from normotensive and pre-eclamptic pregnancies (Fig. 2 ). For the HO-1 immunoblots, a band at 32 kDa corresponding to HO-1 was detected. There were additional non-specific bands detected at 110 and 52 kDa, which were of similar intensity in all tissue regions examined. The anti-HO-2 antibody only detected a major band at 36 kDa corresponding in mass to HO-2. Based on the intensity of the bands of the Western immunoblots, there was an apparent greater expression of HO-2 protein compared with HO-1 protein in each of the four selected regions of term human placenta from both normotensive and mild pre-eclamptic pregnancies. There was no apparent difference in HO-2 protein expression between pre-eclamptic pregnancy and normotensive pregnancy for any of the four regions. There was, however, an apparent increase in the expression of HO-1 protein in chorionic villi and fetal membranes from pre-eclamptic pregnancy compared with normotensive pregnancy (chorionic villi, 75.7 ± 21.8 and 38.0 ± 5.6 arbitrary units, respectively, P < 0.05; fetal membranes, 38.9 ± 10.1 and 24.5 ± 1.5 arbitrary units, respectively, P < 0.05).

Correlation of Microsomal HO Activity with HO-1 and HO-2 Protein Content.

For the four selected regions of term placenta from normotensive pregnancy and mild pre-eclamptic pregnancy, there was a statistically significant (P < 0.05) positive correlation between microsomal HO enzymatic activity and HO-2 protein content (Fig. 3 ). There was no correlation between microsomal HO enzymatic activity and HO-1 protein content (data not presented).

Discussion

In this study, we determined that microsomal HO activity in each of the four placental regions was not statistically different between normotensive and mild pre-eclamptic pregnancies. These data indicate that the capacity of these placental regions for heme-derived CO formation is not compromised by mild pre-eclampsia, at least for optimized enzyme assay conditions. Microsomal HO-2 protein content was not different between normotensive and mild pre-eclamptic pregnancies, whereas there was apparent greater expression of microsomal HO-1 protein in chorionic villi and fetal membranes from mild pre-eclamptic pregnancy compared with normotensive pregnancy. Microsomal HO enzymatic activity correlated with HO-2, but not HO-1, protein content. This completed investigation has clarified our initial preliminary study (15), which indicated an apparent difference in microsomal enzymatic HO activity of term placenta between normotensive and mild pre-eclamptic pregnancies.

These new findings extend our previous observations that microsomal HO activity is not altered in morphologically normal chorionic villi from mild pre-eclamptic pregnancy compared with normotensive pregnancy (12, 16), to other key regions of the placenta. In the present study, regional differences in HO enzymatic activity observed in placenta from mild pre-eclamptic pregnancy—with greater activity in chorionic villi and basal plate compared with chorionic plate and fetal membranes—were similar to those found in placenta from gestational-age-matched normotensive pregnancies of this study and our previous investigation (4).

In our study, the apparent greater expression of HO-2 protein than HO-1 protein in the microsomal fraction of homogenate from each of the four selected placental regions from normotensive and mild pre-eclamptic pregnancies is consistent with previously reported data for normotensive pregnancy and pregnancies complicated by pre-eclampsia and fetal growth restriction (3, 9). Furthermore, HO-2 protein expression was not different between mild pre-eclamptic pregnancy compared with normotensive pregnancy for each of chorionic villi, basal plate, chorionic plate and fetal membranes, which is also consistent with a previous study of HO-2 protein content in placenta from pre-eclamptic and fetal growth restricted pregnancies compared to normotensive pregnancy (9). However, in our study, there was increased expression of HO-1 protein in chorionic villi and fetal membranes from mild pre-eclamptic pregnancy compared with normotensive pregnancy. This increased microsomal HO-1 protein expression occurred without an increase in HO enzymatic activity. The current study shows, for the first time, that there is a positive relationship between microsomal HO enzymatic activity and HO-2, but not HO-1, protein content in the selected regions of placenta from mild pre-eclamptic and normotensive pregnancies. Therefore, it appears that microsomal HO-2 protein plays a more important role in determining microsomal HO enzymatic function and activity in these regions of the placenta.

Figure 1.

HO enzymatic activity in the microsomal fraction of homogenate of chorionic villi (CV), chorionic plate (CP), basal plate (BP), and fetal membranes (FM) of term human placenta from normotensive (open bars) and mild pre-eclamptic (hatched bars) pregnancies. The data are presented as group means ± SD of four placentas. Group means with different letters are statistically different from each other (P < 0.05), for comparison of the four placental regions for normotensive pregnancy (regular font) and for comparison of the four placental regions for mild pre-eclamptic pregnancy (italics).

Figure 2.

Western immunoblot analysis of HO-2 and HO-1 proteins in the microsomal fraction of homogenate of chorionic villi (CV), chorionic plate (CP), basal plate (BP), and fetal membranes (FM) of term human placenta from normotensive (n = 4) and mild pre-eclamptic (n = 4) pregnancies. The individual subject data are presented for the four selected placental regions.

Figure 3.

Linear-linear plot of mean HO enzymatic activity and mean HO-2 protein content of the four selected regions of placenta from normotensive pregnancy (n = 4) and mild pre-eclamptic pregnancy (n = 4). There was a statistically significant positive correlation between these two variables: Pearson correlation coefficient (r), 0.708; P < 0.05.

Footnotes

This research was supported by operating grants from the Heart and Stroke Foundation of Ontario Grants NA-4438 and T-3361. Dr. Charles Graham was the recipient of a Heart and Stroke Foundation of Canada Research Scholarship. Dr. Gendie Lash is the recipient of a Post-Doctoral Fellowship from the Canadian Hypertension Society.

1

To whom requests for reprints should be addressed at Department of Pharmacology and Toxicology, Queen’s University Kingston, Ontario, Canada K7L 3N6. E-mail:

Acknowledgements

The authors wish to thank Drs. Hendrik J. Vreman and David K. Stevenson, Department of Pediatrics, Stanford University School of Medicine, for the use of their gas chromatograph to quantitate CO formation and for their continuing interest in our research.

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