ARTICLE |
Correspondence to: Rumiko Matsuoka, Dept. of Pediatric Cardiology, The Heart Institute of Japan, Tokyo Women's Medical U., 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
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Summary |
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To determine which part of the smooth muscle cells (SMCs) of the ductus arteriosus (DA) contribute to duct closure after birth, we looked for areas in which SM2 myosin heavy chain (MHC) mRNA expression, which is associated with contraction of smooth muscle, and apoptosis could be detected in the DA during development. In situ hybridization revealed that the SM2 MHC mRNA was strongly positive in the longitudinally oriented SMCs and inner layer of the circularly oriented SMCs just before birth. Apoptotic cells were detected in the SMCs of the DA from 1 day after birth. Histochemical analysis using terminal deoxynucleotidyl transferase-mediated dUTPdigoxigenin nick end-labeling (TUNEL) revealed significant numbers of TUNEL-positive nuclei in the longitudinally oriented SMCs and the inner layer of the circularly oriented SMCs. Masson-stained sections showed that the TUNEL-positive area in the DA was replaced by connective tissue from 1 day after birth. These results suggest that the increase in the SM2 MHC mRNA expression and the induction of apoptosis are present at the same site in the media of the DA. Therefore, the SMCs in this area may play an important role in duct constriction and remodeling of the vessel wall after birth. (J Histochem Cytochem 48:3544, 2000)
Key Words: ductus arteriosus, duct closure, smooth muscle, myosin heavy chain isoform, apoptotic cell death, rabbit
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Introduction |
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The ductus arteriosus (DA) is a unique blood vessel that changes dramatically, from patency of the vessel during fetal life to closure at birth. In addition, a dramatic change in its shape occurs during the fetal and neonatal periods (
In the blood vessel smooth muscle, at least two smooth muscle-type myosin heavy chain (MHC) isoforms, SM1 and SM2 (
Recent studies have shown that apoptosis, originally described by
To determine which part of the SMCs of the DA contribute to its closure after birth, we looked for areas in which SM2 MHC mRNA expression and apoptosis could be detected in the DA during development, using fetal and neonatal rabbits. Our results indicate that the longitudinally oriented SMCs and the inner layer of the circularly oriented SMCs play an important role in duct constriction and remodeling of the vessel wall after birth.
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Materials and Methods |
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Sample Preparation
Japanese White rabbits on gestational Day 29 and at 1, 3, 5, 7, and 9 days after birth were used. Briefly, the DA was excised from the animal with or without the pulmonary artery (PA) and aortic artery (Ao). A few samples were embedded in OCT compound (Miles; Elkhart, IN), quickly frozen in normal hexaneliquid nitrogen, and stored at -80C for in situ hybridization to detect smooth muscle MHC mRNAs. For the terminal deoxynucleotidyl transferase-mediated dUTPdigoxigenin nick end-labeling (TUNEL) analysis, samples were fixed in 10% neutral buffered formalin at room temperature (RT) overnight. For electron microscopic analysis, samples were fixed in 3% glutaraldehyde buffered in 0.1 M sodium cacodylate (pH 7.3) at 4C for 1.5 hr and in 1% osmium oxide buffered in 0.1 M sodium cacodylate (pH 7.3) at 4C for 1.5 hr. For electrophoretic analysis of DNA ladder formation, samples were frozen in liquid nitrogen and stored at -80C until assayed.
DNA Probes
The probe (antisense oligo-DNA with auxiliary ATT repeats at the 3' and 5' ends; antisense SM2 probe) was designed from the 39 nucleotides from rabbit SM2 smooth muscle cDNA which were inserted in SM1, and is therefore not homologous with SM1 (
In Situ Hybridization
The procedures, sensitivity, and reliability of in situ hybridization using the T-T dimerized oligo-DNA probe have been described previously in detail (
Densitometry
Densitometric analysis was performed on a Macintosh computer using the public domain NIH Image (ver. 1.61) program (developed at the U.S. National Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh.nih.gov, or on floppy disk from the National Technical Information Service, Springfield, Virginia, part number PB95-500195GEI).
In Situ Detection of DNA Strand Breaks
To identify nuclei with DNA strand breaks at the cellular level, TUNEL was performed using an ApopTag Plus kit (Oncor; Gaithersburg, MD). Briefly, paraffin-embedded 3-µm-thick longitudinal sections through the DA were cut onto silane-coated glass slides and dewaxed in a routine manner. After washing with PBS, the sections were treated with 50 µg/ml proteinase K in PBS at RT for 20 min. Endogenous peroxidase activity was quenched with 2% hydrogen peroxide in PBS at RT for 5 min. Then the ApopTag Plus kit was used. Counterstaining was performed with methyl green. Hematoxylineosin (H-E) and Masson trichrome staining was also performed for control and detection of connective tissue, respectively.
Transmission Electron Microscopy
Transmission electron microscopic analysis was performed to confirm the existence of nuclear shrinkage and chromatin condensation. Fixed samples were embedded in epoxy resin (Poly/Bed 812 embedding kit; PolySciences, Warrington, PA) and ultrathin sections were double stained with saturated uranyl acetate (Merck; Darmstadt, Germany) and 0.4% lead citrate (Nacalai Tesque) and examined in a JEM 1200EX (JEOL, Japan) electron microscope.
Electrophoretic Analysis of Extracted DNA
Frozen samples were minced and 510 µl of the samples were used. Forty microliters of lysis buffer [50 mM Tris-HCl (pH 7.8), 10 mM EDTA, 0.5% w/v sodium-N-lauroylsarcosinate (Nacalai Tesque)] was added and mixed well. Then 4 µl of proteinase K (10 mg/ml) was added and mixed well, and the samples were incubated at 50C for 90 min. Next, 2 µl of RNase A (10 mg/ml; Sigma) was added and the samples were incubated at 50C for 30 min. These samples were then run on 2% agarose gel (1 g of agarose/50 ml, 40 mM Trizma base, 5 mM glacial acetic acid, 2 mM EDTA) and stained with ethidium bromide.
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Results |
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In Situ Hybridization of SM2 MHC mRNA
On gestational Day 29 (just before birth, when the DA is still patent), strong SM2 MHC mRNA expression was detected in the longitudinally oriented SMCs and in the inner layer of the circularly oriented SMCs of the DA (Figure 1A, Figure 1B, and Figure 2). However, in the outer layer of the circularly oriented SMCs of the DA, and in the PA and Ao, SM2 MHC mRNA expression was weak (Figure 1A and Figure 1B). It has been shown that the SM2 MHC protein also appears strongly in the same area of the DA (
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To verify the specificity of signals, we used an irradiated complementary 28S rRNA probe as a positive control, and sense probes and a nonirradiated complementary 28S rRNA probe as negative controls. Strong positive signals were detected with the irradiated complementary 28S rRNA probe (Figure 3A and Figure 3B), but no signals were detected with the sense probes (gray squares in Figure 1A and Figure 1C) and the nonirradiated complementary 28S rRNA probe (Figure 3C). Therefore, the signals of the SM2 MHC mRNA with the SM2 antisense probe confirmed its specificity.
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Apoptotic Features by TUNEL
On gestational Day 29, the TUNEL-positive nuclei could not be seen in the cells of the DA, PA, and Ao (Figure 4D). However, TUNEL analysis revealed stained nuclei in the SMCs of the DA (Figure 4H) which was completely closed (Figure 4E) at 1 day after birth. In the PA and Ao, TUNEL-positive nuclei were not detected. Interestingly, a significant number (>90%) of TUNEL-positive nuclei were detected in the longitudinally oriented SMCs and the inner layer of the circularly oriented SMCs of the DA (Figure 4H). Very few nuclei of the circularly oriented SMCs in the media were stained by TUNEL. Furthermore, the Masson-stained section showed that the TUNEL-positive area in the DA was replaced by connective tissue from 1 day after birth (Figure 4G). A significant number (almost 100%) of TUNEL-positive nuclei were also observed in the longitudinally oriented SMCs and the inner layer of the circularly oriented SMCs of the DA, and this area was also replaced by connective tissue at 3 days after birth (Figure 5C and Figure 5D). At 9 days after birth, the DA had become thin (Figure 5E). The number of TUNEL-positive nuclei had decreased and the amount of connective tissue had increased. However, a few SMCs in the media (outer layer) were still alive (Figure 5F5H).
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Apoptotic Features by Transmission Electron Microscopy
The transmission electron micrograph of the SMCs of the DA obtained on gestational Day 29 is shown as a control (Figure 6A). At 1 and 3 days after birth, the SMCs of the DA had become smaller and nuclear chromatin condensation and nuclear shrinkage could be seen, but the integrity of the plasma membrane was retained (Figure 6B and Figure 6C). These observations are characteristic features of the apoptotic process (
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Apoptotic Features by Electrophoretic Analysis
As shown in Figure 7, a faint but visible ladder, which is an early characteristic feature of apoptosis (
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Discussion |
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In this study we confirmed the localization of SM2 MHC mRNA expression in the DA just before birth. It is especially important to note that strong SM2 MHC mRNA expression was detected in the longitudinally oriented SMCs and in the inner layer of the circularly oriented SMCs (Figure 1A, Figure 1B, and Figure 2). We have already confirmed that the ratio of SM2/(SM1 + SM2) MHC mRNA expression is higher in the DA than in the PA and Ao at the fetal stage, and this ratio conversely becomes low compared with the PA and Ao at the neonatal stage (
At 1 day after birth, a significant number (>90%) of TUNEL-positive nuclei were detected in the longitudinally oriented SMCs and the inner layer of the circularly oriented SMCs of the DA (Figure 4H). Furthermore, the Masson-stained section revealed that the TUNEL-positive area in the DA was quickly replaced by connective tissue (Figure 4G). At 3 days after birth, the number of TUNEL-positive nuclei observed in the inner layer of the circularly oriented SMCs increased compared with 1 day after birth (Figure 5D). These findings indicate that the remodeling of the DA after birth is rapidly induced from the media of the constricted DA, especially from the longitudinally oriented and the inner layer of the circularly oriented SMCs.
Interestingly, increases in SM2 MHC mRNA expression and induction of apoptosis were observed at the same site in the media of the DA. This strongly suggests that the SMCs located in the longitudinally oriented and the inner layer of the circularly oriented area play an important role in duct constriction and remodeling of the vessel wall. Several investigators (
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Acknowledgments |
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Supported by a Grant-in-Aid for Encouragement of Young Scientists (1995, 1996) from the Ministry of Education, Science and Culture, by an open research grant (1997, 1998) from the Japan Research Promotion Society for Cardiovascular Diseases, and by the Japan Shipbuilding Industry Foundation (1997, 1998).
We thank Ms Barbara Levene for reading the manuscript. We greatly appreciate the technical advice of Dr Takehiko Koji at Nagasaki University School of Medicine in establishing in situ hybridization using a nonradioactive T-T dimerized oligo-DNA probe. We also thank Yoshiyuki Furutani, BSc, at Tokyo Women's Medical University for technical help with densitometric analysis.
Received for publication July 19, 1999; accepted September 15, 1999.
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