Matrix Vol. 12/1992, pp. 321-332 © 1992 by Gustav Fischer Verlag, Stuttgart
In-Situ Hybridization of Tropoelastin mRNA during the Development of the Multilayered Neonatal Rat Aortic Smooth Muscle Cell Culture PAUL TOSElLl, BARBARA FARIS, DAVIDSASSOON, BRUCE A. JACKSON and CARL FRANZBlAU Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, USA.
Abstract
Cultured neonatal rat aortic smooth muscle cells are active in synthesizing and depositing large amounts of elastin in their extracellular matrix, making this an ideal system for studying elastogenesis. In this study, the ability of individual cells to synthesize tropoelastin was examined by in-situ hybridization methods. One-micron semi-thin epoxy resin-embedded transverse sections of cells cultured 1, 2, 3 and 4 weeks showed an increase with time in both the number of cells with hybridization signal and the signal intensity; tropoelastin mRNA hybridization signal intensity decreased thereafter up to 8 weeks in culture. In longitudinal sections through the early cultures (I-week), we observed mitotic cells with no detectable hybridization signal, and nonmitotic cells with either no, little or high signal intensity. These data suggest that mitotic cells do not synthesize tropoelastin, and that there is a strong correlation between the hybridization signal intensity and the rate of tropoelastin synthesis. These data also suggest in-situ hybridization methods can detect which cell(s) contain tropoelastin mRNA, their location in the multilayer, and variations in signal intensity. We conclude it is possible to correlate hybridization signal intensity with varitions of tropoelastin mRNA levels within individual cells of the cultured smooth muscle cell multilayer. Key words: in-situ hybridization, plastic sections, rat aorta, vascular smooth muscle cell culture, tropoelastin.
Introduction
The elasticity of the wall of blood vessels permits considerable expansion. The vessel thus acts as a reservoir and converts the intermittent flow of blood from the heart into a continuous, but pulsatile stream. Its elastic recoil is responsible for the diastolic pressure which propels the blood during diastole. The media of the large vessels, which consist of an orderly array of several layers of smooth muscle cells (SMC) embedded in an extracellular matrix, is responsible for its elastic recoil. The chief components of the vascular wall matrix are elastic fibers, collagen and proteoglycans.
Neonatal rat aortic smooth muscle cell (NRSMC) morphology in primary cell cultures was initially described by Hinek and Thyberg (1977) and later by Oakes et al. (1982). These cultures demonstrate rapid growth in the first two weeks and develop 3- to 5-celllayers (Gundrum, 1986). Growth continues each week so that 5-week cultures consist of 12- to 14-cell layers. The cultures are active in synthesizing and depositing large amounts of elastin in their extracellular matrix, making this an ideal system for studying elastogenesis. In a previous study, we provided electron microscopic immunohistochemical evidence for insoluble elastin localization in 8-day NRSMC cultures (Faris et al., 1986).
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The present study was undertaken to further define by insitu hybridization methods elastogenesis during the development of the multilayered NRSMC culture. Our results indicate that tropoelastin mRNA is highly expressed in some, but not all, SMC grown in culture for only 7 days. At various stages throughout development, variations in hybridization signal are detected; and, there is a strong correlation between the hybridization signal intensity and the rate of elastin synthesis.
Methods Rat aortic smooth muscle cell cultures
Neonatal rat aortic smooth muscle cells (NRSMC) were isolated and grown from the aortas of 2- to 3-day old Sprague-Dawley rats (Charles River Breeding Laboratories, Boston, MA) as described previously by Oakes et a1. (1982) and modified by Barone et a1. (1988). Briefly, cells in primary culture were seeded at 5 x 105 cells/ 25 cm 2 tissue culture flask and maintained for 7 days. The cells were subcultivated by trypsination for 5 to 8 min at 3rc. They were subsequently seeded at 5 X 105 cells/ 25 cm 2 flask and maintained for various periods of time. At specified times of harvest, the medium was removed from two flasks and each cell layer was washed two times with Puck's saline, harvested in water and then homogenized in a glass-glass homogenizer. Duplicate aliquots of the homogenate were removed for DNA analyses and another aliquot was lyophilized and used for elastin and total protein determinations. Also, at these times, separate flasks were removed for tropoelastin mRNA determinations and for insitu hybridization analyses as described below. Insoluble elastin preparation and quantification
Each lyophilized cell layer preparation was suspended in 0.1 N NaOH and placed at 98 °C for 45 min with occasional shaking as described by Lansing et a1. (1952). After centrifugation, the supernatents and residues (elastin) were hydrolyzed separately. Samples were hydrolyzed, in vacuo, at 110°C for 20h in 6N HCI and analyzed on a Beckman Model 6300 amino acid analyzer. Total protein content was calculated by adding the fiM of amino acids in both the Lansing supernatant and residue fractions from each cell preparation. The percent elastin per flask is calculated by dividing the elastin content by the total protein. DNA Determination
DNA content of the cell layers was routinely determined by the diphenylamine method of Burton (1956).
RNA extraction and purification
Total RNA from the NRSMC cultures was isolated as described by Chomczynski and Sacchi (1987) with some modifications using RNAzoI B (Biotecx Laboratories; Houston, TX). For each time point 2 flasks were harvested. Northern blot hybridization
Northern blot analysis of tropoelastin mRNA was performed essentially according to the method of Lehrach et a1. (1979). Total RNA from first passage NRSMC grown for 1, 3, 4, 5 and 6 weeks were subjected to electrophoresis on 1% agarose gels. Rat (1.5 kb) tropoelastin cDNA probes (Rich and Foster, 1989) were radiolabeled by the random priming method (Boehringer-Mannheim). The specific activity of the resulting cDNAs ranged from 107 to 108 cpm/ fig of purified insert. In-Situ hybridization Preparation of NRSMC cultures. For each time point, two 25 cm 2 flasks were used. The cultured NRSMC were fixed in 4% formaldehyde (freshly prepared from paraformaldehyde) for 30 min in PBS, pH 7.4, chilled to 4°C. The cultures were rinsed in PBS (30 min), 0.85% saline (30 min), and dehydrated in a graded series of ethanol. An embedding mixture consisting of 1:1 mixture Araldite 502:dodecenyl succinic anhydride and 2% benzyldimethyl amine catalyst was thoroughly mixed and then deareated in a 4°C vacuum oven for 30 min. After complete dehydration, ethanol was removed from the flasks and replaced with the embedding mixture. The flasks were placed in the 40°C vacuum oven and deareated for 15 min. Following one or two changes of embedding mixture, a final thin layer of the mixture was added to each flask. The flasks were again deareated for 15 min at 40°C and then placed in a 60 °C oven for 18 h for polymerization to occur. Each flask was quickly cracked open while still warm after removal from the oven. The embedded cell layer was then easily pulled away from the flask. Using a LKB Ultramicrotome IV, sections (1.0 fim thick) were cut with a diamond knife. The cell culture blocks were sectioned in two planes: a transverse plane in which the cells were sectioned along a plane oriented perpendicular to the cell layer, and a horizontal plane oriented parallel to the cell layer. Preparation of samples for the determination of tropoelastin riboprobe specificity. For these control studies, 6day neonatal rats were sacrificed and the thoracic organs removed as a tissue block. Then, the samples were rinsed, fixed with 4% paraformaldehyde in PBS, dehydrated and embedded in Paraplast as described by Sassoon et a1. (1988). Sections 6 fim thick were cut and stored at 4 °C until used for hybridization. In addition, rat pulmonary fibroblasts (Foster et aI., 1990) were grown on Tissue-Tek
Tropoelastin mRNA Expression chamber glass slides. The cultures were fixed with 4% paraformaldehyde in PBS, dehydrated and stored at 4°C until used for hybridization. For positive control studies, we employed the mouse beta-actin coding sequence (pAL 41.20) cloned into the PstI site of pBluescript plus (Alonso et ai., 1986). To synthesize anti-sense [3s S]-labeled probe, DNA template linearized with EcoR I was transcribed by T 3 RNA polymerase. Preparation of tropoelastin riboprobe. An EcoR I fragment (1.5 kb) rat tropoelastin cDNA (Rich and Foster, 1989) was inserted into pBluescript plus (Stratagene). To synthesize anti-sense eSS]-labeled RNA probes (complementary riboprobe), DNA templates linearized with Sad were transcribed by T 7 RNA polymerase. Sense RNA probes (homologous riboprobe) were obtained with DNA templates linearized by Hind III digestion and T 3 RNA polymerase. Three-hundred nanograms of linearized DNA templates were transcribed in 36 mM Tris, pH 7,5,5 mM MgCh, 2 mM Spermidine, 9 mM NaCl, 9 mM dithiothreitol, 1 unit human placenta ribonuclease inhibitor, 0.5 mM each of CTP, ATP, GTP, 2.3 mM eSS]-alpha UTP (250 Ci/mM; NEN) and either T 3 or T 7 RNA polymerase. The transcription reaction mixture was incubated 1-2 h at 37°C. This procedure yielded RNA transcripts with a specific activity greater than 5 x 10 8 cpm/Jlg. For removal of DNA template, RQl was added and the mixture incubated at 3rc for 15 min. Total yeast RNA was added to obtain a concentration of 250 Jlg/ml of final mixture concentration. Probe length was reduced to an approximate mean size of 60 nucleotides by alkali hydrolysis, and unincorporated nucleotides were removed by gel filtration on G-50 Sephadex. The radiolabeled riboprobe transcript fragments were concentrated by cold ethanol precipitation and suspended in hybridization buffer (see below for hybridization buffer composition). In-Situ hybridization procedure. The procedures used for probe preparation (see above), section treatment, hybridization and washing were based upon those used by Sassoon et ai. (1988) (unless otherwise indicated). Araldite embedded sections were deplasticized as described by Lisca et ai. (1988) and Paraplast-embedded sections were deparaffinized in xylene. Deplasticized sections or deparaffinized sections were rehydrated through a graded series of ethanols (100% to 30%), rinsed in 0.85% saline solution (5 min), PBS (5 min), and post-fixed in a prepared and filtered solution of 4% formaldehyde (freshly prepared from paraformaldehyde) in PBS buffer (30 min). Slides carrying sections were then rinsed in PBS (2 x 10 min) and treated with a fresh solution of proteinase K (20 Jlg/ml, 7.5 min) in Tris-HCl, EDTA (50 mM, 5 mM, pH 7.2). They were then rinsed in PBS (5 min), refixed in 4% formaldehyde solution in PBS, dipped in distilled water and acetylated in a 0.09 M solution of triethanolamine with acetic anhydride (1 :400 v/v) for 10 min. The slides were subsequently rinsed in PBS and 0.85% saline (5 min each),
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quickly dehydrated through a series of ethanols (30% to 100%), and allowed to dry at least 2h prior to hybridization. Probe was applied directly to tissue sections (20 JlI) at a final adjusted concentration of 1 x 106 cpm in hybridization buffer (50% deionized formamide, 0.3 M NaCl, 20mM Tris-HCI (pH7.4), 5mM EDTA, 10mM NaP0 4 (pH 8.0): 10% dextran sulfate, Denhardt's, 50 Jlg/ml total yeast RNA} and tissue and probe were covered with a siliconized cover-glass (22 x 22 mm). Hybridization was carried out at 50°C for approximately 16 h in a humid chamber. Coverslips were gently floated off in a 5X SSC (IX SSC, 0.15 M NaCl, 0.015 M Sodium Citrate), 10 mM dithiothreitol (DIT) at 50°C and subsequently the sections were subjected to a stringent washing at 60°C in 50% formamide, 2X SSC, 0.1 M DIT. Slides were then rinsed in STE (0.1 M NaCl, 0.01 M Tris-HCI, 0.001 M EDTA) washing buffer and treated with RNase A (20 Jlg/ml) in washing buffer for 45 min at 37°C. Following washes at room temperature in 2X SSC (15 min) and O.IX SSC (15 min), respectively, the slides were rapidly placed in a series of ethanols containing 0.3 M ammonium acetate. Autoradiography. The samples were processed for standard autoradiography using Kodak NTB-2 nuclear track emulsion. They were exposed for either 1 day (cultured rat pulmonary fibroblasts), or 3 days (cultured NRSMC), or 7 days (neonatal rat heart and lung) in light-tight boxes with desiccant at 4 0C. Electron microscopy and electron- and light-microscopic immunocytochemistry
Cultured NRSMC were examined by conventional electron microscopic staining (Toselli and Pepe, 1968) and by light and electron microscopic antibody staining (Faris et ai., 1986). Rabbit polyclonal antiserum raised against alpha-elastin (Faris et ai., 1986) was used for immunocytochemicallocalization studies.
Results The purpose of this study was to characterize elastin gene expression as a function of cell growth in culture. NRSMC in first passage (25 cm 2 flasks) were cultured for 6 weeks, and the cell layers were analyzed at various times for DNA, insoluble elastin, and total protein (Table I). Both the elastinlDNA content and the percent of the total protein attributed to elastin increase at a slow rate, if at all, after 4 weeks in culture. Northern blot analyses were done to measure the steady state levels of tropoelastin mRNA over the time course. Equal amounts of total mRNA (15 Jlg) extracted from cells collected for 1, 3, 4, 5 and 6 weeks in first passage were hybridized to [32 P]-labeled elastin cDNA. Analysis of the autoradiogram from the Northern blot of the tropoelastin
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is always consistent. As seen in Figure 2 (ll-week old culture), each cell layer is usually formed by slightly overlapping cells. With increase time in culture, the separation between cells increases, and the spaces become occupied with deposits of extracellular matrix. Examination of the cell culture revealed differences in accumulation, composition and arrangement of the formed elements of the extracellular matrix at varous depths in culture. By dividing the full depth of the culture into thirds, distinct differences were observed. For example, after culturing cells three weeks in first passage, the bottom of the culture contained large organized deposits of elastic material. The middle third of the culture consisted of smaller coalescing elastic fibers, and the top one third of the culture showed only small accumulations of elastic material. The top 3 to 5 layers in all cultures are tightly packed (see upper portion, Fig.2) and there is relatively less extracellular material when compared to the lower portion of the culture. Examination of the bottom third of the culture (Fig. 3) reveals a nearly continuous lamella of elastic fibers.
uIture
Fig. 1. Northern blot hybridization analysis of tropoelastin mRNA as a function of time in culture. Preparations of total mRNA (15 !!g lane) extracted from NRSMC were analyzed by Northern blots with a tropoelastin-specific eDNA clone. The arrow indicates probe hybridizing to 3.9-kb tropoelastin mRNA. mRNA (Fig. 1) shows small amounts present at 1- and 6week time periods. At 3 weeks, the steady state level of tropoelastin mRNA appears most intense and is seen to decease each week thereafter.
Culture morphology - development ofthe multilayered culture The NRSMC cultures demonstrate intense growth in the first two weeks. By one week in first passage, the cell layer is 7 to 9 !lm thick and consistes of 3- to 5-celilayers. At two weeks the cultures' thickness roughly doubled to become 16 to 18 !lm, and the number of cell layers is increased from 5 to 8. Over the next three weeks, growth increases only by approximately 20% each week, so that by 5 weeks, the cultures is 25 to 31!lm thick consisting of 12- to 14-cell layers (Gundrum, 1986). While absolute number of layers may vary slightly from one experiment to another, the trend
Determination oftropoelastin riboprobe specificity During recent years an alternative and most useful application of the in-situ hybridization technique has evolved to directly localize cellular mRNA on semi-thin, epoxy resinembedded tissue sections (Liscia et aI., 1988). We extended this technique to visualize tropoelastin mRNA on semithin, epoxy resin-embedded cell-culture sections. Since in-situ hybridization of tissue sections provide a greater opportunity for non-specific interaction than Northern blot analysis, we conducted the following controls to assess tropoelastin riboprobe specificity. First we demonstrated tropoelastin riboprobe hybridized to appropriate sites on tissue sections. For this, we used sections derived from 6-day neonatal rat heart/lung tissue blocks. Then we hybridized the sections with either: (1) complementary (anti-sense) tropoelastin riboprobe (positive control), anti-sense beta-actin riboprobe (positive control), or (3) homologous (sense) tropoelastin (negative control). Figure 4 A illustrates the hybridization of tropoelastin anti-sense probe to neonatal rat heart. Note the strong hybridization signal in the vascular NRSMC (medium arrow) and epicardial cells (small arrow). The strong signal present in some endocardial cells (large arrows) is best observed at
Fig. 2. Electron microscopy of transverse section through the top portion of NRSMC cultured for 11 weeks. Note the tightly packed cells in the upper portion of the figure. E - Extracellular elastin. Bar = 0.5 microns. Fig. 3. Electron microscopy of horizontal section rhrough the bottom portion of NRSMC cultured for 3 weeks. Note extracellular elastic fiber lamella stained with anti-alpha-elastin antiserum and secondary antibody linked with 10-nm diameter colloidal gold particles. Insert: Elastic fiber lamella as visualized by light r:-:icroscopy immunocytochemistry at low magnification. E - Extracellular elastin; C Collagen. Arrow - lamella stained using the light microscopical immunogold silver staining method Uanssen Life Sciences). Bar = 0.5 microns.
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4A
B
Tropoelastin mRNA Expression high magnification (not shown). Figure4B shows neonatal rat heart hybridized with anti-sense beta-actin. Note the distinct difference in the vascular SMC and epicardial cells hybridization signal pattern when compared with Figure4A. Also, there is a noteworthy difference in hybridization signal pattern observed in the pulmonary tissue portion of the heart/lung tissue block sample (data not shown). In the case of the anti-sense tropoelastin probe, tropoelastin mRNA hybridization signal is present in pulmonary vascular and airway smooth muscle, spetal interstitial, alveolar wall and pleural cells. In the case of the anti-sense beta-actin probe, beta-actin mRNA signal is present chiefly in pulmonary alveolar wall and pleural cells. We did not detect hybridization signal in neonatal rat heart or lung rissue hybridized with sense tropoelastin riboprobe. Second, we demonstrated tropoelastin riboprobe hybridized to appropriate sites on pulmonary fibroblasts cultured for 10 days on Tissue-Tek chamber slides. Pulmonary fibroblasts express tropoelastin in vitro (Foster and Rich, 1990), and we detected mature elastic fibers in these cultures when stained with Verhoeff's elastic tissue stain (data not shown). For this control study, all hybridization and signal detection procedures were conducted on un-sectioned, "whole mounted" fibroblasts. Figure4C shows fibroblasts, hybridized with anti-sense tropoelastin riboprobe, lying near the edge of the growing surface. Note the srrong hybridization signal present in most cells; also note some cells have a weak signal. We did not detect hybridization signal in fibroblasts hybridized with sense tropoelastin riboprobe. Third, we fixed, embedded, sectioned and then hybridized I-week cultured NRSMC with either anti-sense or sense tropoelastin riboprobe. Horizontal sections hybridized with anti-sense tropoelastin probe (Fig. 4 D) show most cells display strong hybridization signal intensity; also, we frequenrly observed easily identified pairs of cells with litrle or no detectable hybridization signal. As shown in Figure 4 E, we did not detect hybridization signal in NRSMC hybridized with sense tropoelastin (compare Figure4E with Figure4D). Finally, Figure 4 F shows a control photomicrograph of horizontally sectioned I-week cultured NRSMC hybrid-
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ized with anti-sense beta-actin riboprobe. Note the distinct difference in the hybridization signal pattern when compared to Figure 4 D.
Expression oftropoelastin mRNA during development ofthe multilayered culture Throughout cell growth the NRSMC display variations in tropoelastin mRNA hybridization signal intensity. When I-week cultured cells are sectioned parallel to the plane of the cell culture flask substratum, cells with litrle or no detectable signal lie adjacent to cells with strong signal (Fig. 5) (also, see Fig. 4 D). In addition, Figure 5 shows a cell in mitosis (curved arrow). This cell is in close contact with and almost completely surrounded by cells. We did not detect levels of tropoelastin mRNA in the mitotic cell, and adjacent cells exhibit weak signal at best (arrowhead). In contrast, distant NRSMC have a strong signal (arrow). At higher magnification (Fig. 6), the 4-week culture (Fig.6A) and the 7-week culture (Fig. 6B) are cut perpendicular to the plane of the cell culture flask substratum. All NRSMC seen in Figure 6 A exhibit a high level of tropoelastin mRNA. Figure6B shows the 7-week SMC with either no detectable tropoelastin mRNA, or a reduced level of mRNA when compared to the 4-week cells. Low magnification survey of SMC grown in culture for 1,2,3,4,6 and 8 weeks (Fig. 7), sectioned transversely and hybridized indicate they contain sparse amounts of tropoelastin mRNA at 1 week in culture. The mRNA signal progressively increases in intensity during the time course, peaking at 4-weeks; then, the mRNA level appears to decrease in intensity each week thereafter.
Phenotype, tropoelastin mRNA expression and elastin distribution An attempt was made to determine differences, if any, in the tropoelastin mRNA in-situ hybridization signal pattern in the NRSMC cultures. As stated earlier, the bottom third of the culture multilayer reveals a nearly continuous lamella of elastic fibers (see Fig. 3), the middle third consists of smaller coalescing fibers, and the upper third has a minimal
.... Fig. 4. Control hybridization for the determination of tropoelastin riboprobe specificity. Figure A and B: Paraffin-embedded 6-micron thick section through right ventricular wall of six-day neonatal rat heart hybridized with either anti-sense tropoelastin riboprobe (Figure A) or anti-sense beta-actin riboprobe (Figure B). Figure C: Whole-mount rat pulmonary fibroblasts cultured on Tissue-Tek chamber slides and hybridized with anti-sense tropoelastin riboprobe. FiguresO-F: Plastic-embedded I-micron semi-thin horizontal sections of I-week NRSMC cultures hybridized with either anti-sense tropoelastin (Figure 0), sense tropoelastin (Figure E) or anti-sense beta-actin riboprobe (Figure F). In Figure A, note the strong signal present in areas where there is a high cell-density of positive cells such as the coronary artery (medium arrow) and the pericardium (small arrow). The strong signal present in areas containing a low cell-density of positive cells in the endocardium (large arrows) is less apparent and is best demonstrated at high magnification (not shown). Figure C shows signal variation in the individual, whole-mounted cultured fibroblasts. Figure 0 also shows signal variation in cultured NRSMC. Note the easily identified pair of cells with little or no detectable hybridization signal. L - Coronary artery lumen; Small arrow Pericardium; Medium arrow - Coronary artery; Large arrow - Endocardium; n - Nucleus. Bar (Figures A-B) = SO microns; (Figure C- F) = 20 microns.
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amount of elastin. Fig. 8 shows the variation in tropoelastin mRNA hybridization signal intensity in a transverse section through the 5-week culture. There is abundant signal in both the bottom third and middle third of the culture multilayer. Note the cells located in the lower two-thirds of the culture (Fig.8) have a flat shape, which is best demonstrated at a higher magnification (see Fig. 6). In con-
trast, there is little or no signal intensity in the upper third of the multilayer where most cells are more spherical (left top portion of Fig. 8). This result suggests that there is a quantitative difference in the amount of tropoelastin mRNA between the upper third and the lower two-thirds of the culture. Also, the data suggest that is a change in elastogenic phenotype from spherical (little or no tropoelastin mRNA)
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Fig. 8. Plastic-embedded I-micron semi-thin transverse section of 5-week NRSMC cultures hybridized with anti-sense tropoelastin riboprobe. (~) - delineates bottom of culture; (~) - delineates top of culture. Bar = 10 microns. to a flat profile (abundant tropoelastin mRNA) during the development of the culture multilayer.
Discussion NRSMC in culture generate an extracellular matrix which contains many, if not all, of the key components identified in vitro blood vessels including elastin, collagen, proteoglycans and fibronectin. The synthesis of the subunit of microfibrillar protein was demonstrated by Muir et al. (1976). Using transmission electron microscopy, Toselli et al. (1981) studied the long-term accumulation of elastin in rabbit aortic SMC cultures and found microfibrils in these interstices of the amorphous component of mature elastic fibers, a finding similar to that described for in vivo systems. The NRSMC cultures are extremely active in synthesizing and deposing large amounts of insoluble elastin in their extracellular matrix, making this an ideal system for studying insults affecting the elastic fibers. Previous work from our laboratory (Faris et aI., 1986) described the effect of elastases on the extracellular matrix of these cultures. Elastase effect was monitored both ultrastructurally and chemically. An interesting observation in the present report was the differences in tropoelastin in-situ hybridization signal pattern throughout the time course studied. We detect at least three patterns in NRSMC grown in culture for 1 week (Fig. 5). The mitotic cell (curved arrow) shows no detectable signal. Nearby non-dividing SMC (arrowhead) exhibit little or no signal, and more distant non-dividing SMC (arrow) have abundant signal. Such signal differences may reflect underlying variations in tropoelastin mRNA steady state levels. We conclude from our observations that mitotic cells do not contain tropoelastin mRNA resulting in no detectable hybridization signal. Also, daughter cells may delay in tropoelastin mRNA biosynthesis resulting in little
or no detectable hybridization signal (see the pair of cells identified in Fig.4D); and, with time, the daughter cells may initiate elastogenesis resulting in intense hybridization signal. It is possible that such events are not limited to tropoelastin mRNA and that, for example, mitotic cells may not contain the mRNAs of other extracellular matrix components such as fibronectin and the procollagens. Charnley-Campbell et al. (1981) discussed the concept of cellular modulation in enzymatically dissociated SMC from intact mammalian aortae. They postulated that SMC reversibly modulate over the first several days in primary cell culture from a phenotypic state in which they are contracted, contain thick myofilaments, and do not undergo migration or mitosis to a synthetic state in which they become capable of mitosis and migration, lose their thick myofilaments and ability to contract and their cytoplasm are filled with organelles usually associated with synthesis of secretory protein. NRSMC tropoeiastin mRNA hybridization signal pattern variations we observe are compatible with the SMC synthetic state described by ChamleyCampbell et al. (1981). Initial evaluation of elastin biosynthesis pattern of the NRSMC cultures indicate a continuous increase in elastin accumulation over time in culture. In the early cultures (TableI), there is an approximately 30-fold increase in elastin content in cells cultured for 1 and 4 weeks. Concomitant with this is an approximately 3.5 increase in DNA content in cells cultured 1- 2 and 4 weeks. These finding are consistent with our earlier report (Barone et aI., 1988). Our results using in-situ hybrization confirm and extend these findings. Transverse sections of NRSMC grown in culture for 1, 2, 3 and 4 weeks (Fig. 7) show an increase in both the number of cells with signal and the signal intensity.
Tropoelastin mRNA Expression Table 1. Insoluble elastin accumulation in neonatal rat aortic smooth muscle cell culture. Time in Culture
mg Elastin
mgDNA
mg Elastin! % Elastin mgDNA
1 week 1 2
0.036 0.034
0.037 0.033
0.83 1.04
3.4 3.9
2 weeks 1 2
0.259 0.162
0.049 0.034
5.34 4.77
12.4 8.5
3 weeks 1 2
0.416 0.428
N.D. N.D
N.D. N.D.
16.8 19.1
4 weeks 1 2
1.250 1.326
0.117 0.139
10.73 9.55
27.2 24.1
5 weeks 1 2
1.945 2.314
0.204 0.197
9.54 11.74
28.6 28.7
6 weeks 1 2
2.844 2.332
0.204 0.167
13.95 13.96
35.4 30.7
N.D. = Not Determined Additional signal patterns are apparent during continued cell growth. These patterns are clearly demonstrated in transversely sectioned cultures photographed at high magnification (Fig. 6). For example, Figure6A (4-week material) shows that all NRSMC have high signal intensity. In the 7-week material (Fig. 6B), there is a decrease in signal intensity per cell when compared to the 4-week cells. High magnification examination of transversely sectioned 8week material show a further decrease in signal intensity, and many cells have no signal (data not shown). There is approximately a 2-fold increase in mgs of DNA from 4weeks to 6-weeks in culture (Table I). Concomitant with this is an approximately 2-fold increase in elastin accumulation. We conclude there is a decrease in the steady-state tropoelastin mRNA expression in cells cultured from 4weeks to 8-weeks. Survey of Northern blot analyses (Fig. 1) of the older cultures also depict a decrease in tropoelastin mRNA expression. In Northern blot analysis, the decrease in signal relates to the average number of cells presnt in the flask. In-situ hybridization can detect which cells contain tropoelastin mRNA, their location in the multilayer, and differences in signal intensity. From the data, we conclude there is a relationship between: (1) signal intensity associated with an individual cell, (2) the number of cells with signal (either high or low) intensity, and (3) time in culture. These conclusions suggest a correlation between the amount of functional tropoelastin mRNA present within an individual cell and the overall biosynthetic rate of elastin by the multilayer. The un-sectioned, whole-mounted neonatal rat pulmo-
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nary fibroblasts hybridized with tropoelastin riboprobe for use in control studies also displayed individual cells with easily identifiable differences in signal intensity. Cells lying most proximal to the Tissue-Tek culture substratum edge were sparsely populated (i. e. low cellular density areas), and these cells displayed little or no signal intensity (see Fig. 4 C). More distally located cells were observed in other regions of the chamber; here, there was a high cellular density and the cells generally exhibited a high signal intensity (data not shown). In addition, cells lying adjacent to the edge cells were observed showing a signal intensity intermediate in strength to that observed in cells lying more proximal or distal to the edge. Such correlations between hybridization signal intensity and cell location are also consistent with variations of tropoelastin mRNA levels within individual cells. Also, more meaningful quantitative analysis of cellular mRNA hybridization signal is likely when un-sectioned, sparsely populated cells are measured. In this case, one does not have to take into account either (1) the percent of cell present in the plane of the section, or (2) section-thickness variations. In summary, we demonstrate it is possible to gain insight into elastin gene expression as a function of cell growth in culture. Tropoelastin mRNA data derived from both in-situ hybridization studies employing plastic-embedded cultures and Northern blot analyses can be correlated with insoluble elastin accumulation. We found that elastogenesis continues with time in culture, and that tropoelastin mRNA steady state levels peaked in the cells cultured for 3-4 weeks. Finally, we show in-situ hybridization signal degree is compatible with variations of tropoelastin mRNA steady state levels within individual cells of the cultured NRSMC multilayer. Acknowledgements We thank Judith A. Foster and Celeste B. Rich for the gifts of tropoelastin specific cDNA probe RE-2 and the pulmonary fibroblasts cultures and Margaret Buckingham for the gift of mouse beta-actin coding sequence pAL 41.20. We also thank Alfie Tsay and Jag Bhawan for their helpful comments. Valerie Verbitski and Rosemarie Moscaritolo kindly helped uy by providing cultured cells. One of the authors, P.T., thanks Anna Kadar for encouraging him to pursue molecular biology skills, and special thanks for Xun Weng for help in acquiring the skills. This study was supported by United States Public Health Sciences grants HL 19 717 and HL 13 262. References Alonso, A., Minty, A., Bourlet, Y. and Buckingham, M.: Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates. J. Mol. Evol. 23: 11-22,1986. Barone, L. M., Wolfe, B. L., Faris, B. and Franzblau, c.: Elastin mRNA levels and insoluble elastin accumulation in neonatal rat smooth muscle cell cultures. Biochemistry 27: 3175 - 3182, 1988.
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Burton, K.: A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: 315 - 323, 1956. Chamley-Campbell,]. H., Campbell, G. R. and Ross, R.: Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. J. Cell Bioi. 89: 379-383, 1981. Chomczynski, P. and Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. Faris, B., Toselli, P., Kispert,J., Wolfe, B. L., Pratt, C. A., Mogayzel, P.]., Jr. and Franzblau, c.: Elastase effect on the extracellular matrix of rat aortic smooth muscle cells in culture. Exp. Mol. Path. 45: 105-117,1986. Foster,]. A., Rich, C. B. and Miller, M. F.: Pulmonary fibroblasts: An in vitro model of emphysema. Regulation of elastin gene expression. J. BioI. Chem. 265: 15544-15549,1990. Gundrum,].].: An ultrastructural study of cultured neonatal rat aortic smooth muscle and its response to elastase. Ph. D. Dissertation: Boston University, Boston, Massachusetts, 1986. Hinek,A. and Thyberg,].: Electron microscopy observations on the formation of elastic fibers in primary cultures of aortic smooth muscle cells. J. Ultrastruct. Res. 60: 12-20,1977. Lansing, A.J., Rosenthal, T.B., Alex, M. and Dempsey, E. W.: The structure and chemical characterization of elastic fibers as revealed by elastase and by electron microscopy. Anat. Rec. 114: 555 - 575, 1952. Lehrach, H., Frischauf, A. M., Hanahan, D., Wozney,]., Fuller, F. and Boedtker, H.: Construction and characterization of proalpha-l collagen. Complementary deoxyribonucleic acid clones. Biochemistry 18: 3146-52, 1979.
Liscia, D. S., Doherty, P.]. and Smith, G. H.: Localization of alphacasein gene transcription in sections of epoxy resin-embedded mouse mammary tissue by in-situ hybridization. J. Histochem. Cytochem. 36: 1503-1510,1988. Muir, L. W., Bornstein, P. and Ross, R.: A presumptive subunit of elastic fiber microfibrils secreted by arterial smooth-muscle cells in culture. Eur. J. Biochem. 64: 105 -14, 1976. Oakes,B. W., Batty, A. c., Handler, c.]. and Sandberg,L.B.: The synthesis of elastin, collagen and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle: An ultrastructural and biochemical study. Eur. J. Cell BioI. 27: 34-46, 1982. Rich, C. B. and Foster,]' A.: Characterization of rat heart tropoelastin. Arch. Biochem. Biophys. 268: 551-558,1989. Sassoon, D. A., Garner,1. and Buckingham, M.: Transcripts of alpha-cardiac and alpha-skeletal actins are early markers for myogenesis in the mouse embryo. Development 104: 155 -164, 1988. Toselli,P.A. and Pepe,F.A.: The fine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolixus during the molting cycle. 1. Muscle strucrure at molting. J. Cell Bioi. 37: 445 -461,1968. Toselli, P., Salcedo, L. L., Oliver, P. and Franzblau, c.: Formation of elastic fibers and elastin in rabbit aortic smooth muscle cell cultures. Conn. Tiss. Res. 8: 231-239, 1981. Dr. Paul Toselli, Department of Biochemistry, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, USA.
In-Situ Hybridization of Tropoelastin mRNA during the Development of the Multilayered Neonatal Rat Aortic Smooth Muscle Cell Culture PAUL TOSElLl, BARBARA FARIS, DAVIDSASSOON, BRUCE A. JACKSON and CARL FRANZBlAU Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, USA.
Abstract
Cultured neonatal rat aortic smooth muscle cells are active in synthesizing and depositing large amounts of elastin in their extracellular matrix, making this an ideal system for studying elastogenesis. In this study, the ability of individual cells to synthesize tropoelastin was examined by in-situ hybridization methods. One-micron semi-thin epoxy resin-embedded transverse sections of cells cultured 1, 2, 3 and 4 weeks showed an increase with time in both the number of cells with hybridization signal and the signal intensity; tropoelastin mRNA hybridization signal intensity decreased thereafter up to 8 weeks in culture. In longitudinal sections through the early cultures (I-week), we observed mitotic cells with no detectable hybridization signal, and nonmitotic cells with either no, little or high signal intensity. These data suggest that mitotic cells do not synthesize tropoelastin, and that there is a strong correlation between the hybridization signal intensity and the rate of tropoelastin synthesis. These data also suggest in-situ hybridization methods can detect which cell(s) contain tropoelastin mRNA, their location in the multilayer, and variations in signal intensity. We conclude it is possible to correlate hybridization signal intensity with varitions of tropoelastin mRNA levels within individual cells of the cultured smooth muscle cell multilayer. Key words: in-situ hybridization, plastic sections, rat aorta, vascular smooth muscle cell culture, tropoelastin.
Introduction
The elasticity of the wall of blood vessels permits considerable expansion. The vessel thus acts as a reservoir and converts the intermittent flow of blood from the heart into a continuous, but pulsatile stream. Its elastic recoil is responsible for the diastolic pressure which propels the blood during diastole. The media of the large vessels, which consist of an orderly array of several layers of smooth muscle cells (SMC) embedded in an extracellular matrix, is responsible for its elastic recoil. The chief components of the vascular wall matrix are elastic fibers, collagen and proteoglycans.
Neonatal rat aortic smooth muscle cell (NRSMC) morphology in primary cell cultures was initially described by Hinek and Thyberg (1977) and later by Oakes et al. (1982). These cultures demonstrate rapid growth in the first two weeks and develop 3- to 5-celllayers (Gundrum, 1986). Growth continues each week so that 5-week cultures consist of 12- to 14-cell layers. The cultures are active in synthesizing and depositing large amounts of elastin in their extracellular matrix, making this an ideal system for studying elastogenesis. In a previous study, we provided electron microscopic immunohistochemical evidence for insoluble elastin localization in 8-day NRSMC cultures (Faris et al., 1986).
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The present study was undertaken to further define by insitu hybridization methods elastogenesis during the development of the multilayered NRSMC culture. Our results indicate that tropoelastin mRNA is highly expressed in some, but not all, SMC grown in culture for only 7 days. At various stages throughout development, variations in hybridization signal are detected; and, there is a strong correlation between the hybridization signal intensity and the rate of elastin synthesis.
Methods Rat aortic smooth muscle cell cultures
Neonatal rat aortic smooth muscle cells (NRSMC) were isolated and grown from the aortas of 2- to 3-day old Sprague-Dawley rats (Charles River Breeding Laboratories, Boston, MA) as described previously by Oakes et a1. (1982) and modified by Barone et a1. (1988). Briefly, cells in primary culture were seeded at 5 x 105 cells/ 25 cm 2 tissue culture flask and maintained for 7 days. The cells were subcultivated by trypsination for 5 to 8 min at 3rc. They were subsequently seeded at 5 X 105 cells/ 25 cm 2 flask and maintained for various periods of time. At specified times of harvest, the medium was removed from two flasks and each cell layer was washed two times with Puck's saline, harvested in water and then homogenized in a glass-glass homogenizer. Duplicate aliquots of the homogenate were removed for DNA analyses and another aliquot was lyophilized and used for elastin and total protein determinations. Also, at these times, separate flasks were removed for tropoelastin mRNA determinations and for insitu hybridization analyses as described below. Insoluble elastin preparation and quantification
Each lyophilized cell layer preparation was suspended in 0.1 N NaOH and placed at 98 °C for 45 min with occasional shaking as described by Lansing et a1. (1952). After centrifugation, the supernatents and residues (elastin) were hydrolyzed separately. Samples were hydrolyzed, in vacuo, at 110°C for 20h in 6N HCI and analyzed on a Beckman Model 6300 amino acid analyzer. Total protein content was calculated by adding the fiM of amino acids in both the Lansing supernatant and residue fractions from each cell preparation. The percent elastin per flask is calculated by dividing the elastin content by the total protein. DNA Determination
DNA content of the cell layers was routinely determined by the diphenylamine method of Burton (1956).
RNA extraction and purification
Total RNA from the NRSMC cultures was isolated as described by Chomczynski and Sacchi (1987) with some modifications using RNAzoI B (Biotecx Laboratories; Houston, TX). For each time point 2 flasks were harvested. Northern blot hybridization
Northern blot analysis of tropoelastin mRNA was performed essentially according to the method of Lehrach et a1. (1979). Total RNA from first passage NRSMC grown for 1, 3, 4, 5 and 6 weeks were subjected to electrophoresis on 1% agarose gels. Rat (1.5 kb) tropoelastin cDNA probes (Rich and Foster, 1989) were radiolabeled by the random priming method (Boehringer-Mannheim). The specific activity of the resulting cDNAs ranged from 107 to 108 cpm/ fig of purified insert. In-Situ hybridization Preparation of NRSMC cultures. For each time point, two 25 cm 2 flasks were used. The cultured NRSMC were fixed in 4% formaldehyde (freshly prepared from paraformaldehyde) for 30 min in PBS, pH 7.4, chilled to 4°C. The cultures were rinsed in PBS (30 min), 0.85% saline (30 min), and dehydrated in a graded series of ethanol. An embedding mixture consisting of 1:1 mixture Araldite 502:dodecenyl succinic anhydride and 2% benzyldimethyl amine catalyst was thoroughly mixed and then deareated in a 4°C vacuum oven for 30 min. After complete dehydration, ethanol was removed from the flasks and replaced with the embedding mixture. The flasks were placed in the 40°C vacuum oven and deareated for 15 min. Following one or two changes of embedding mixture, a final thin layer of the mixture was added to each flask. The flasks were again deareated for 15 min at 40°C and then placed in a 60 °C oven for 18 h for polymerization to occur. Each flask was quickly cracked open while still warm after removal from the oven. The embedded cell layer was then easily pulled away from the flask. Using a LKB Ultramicrotome IV, sections (1.0 fim thick) were cut with a diamond knife. The cell culture blocks were sectioned in two planes: a transverse plane in which the cells were sectioned along a plane oriented perpendicular to the cell layer, and a horizontal plane oriented parallel to the cell layer. Preparation of samples for the determination of tropoelastin riboprobe specificity. For these control studies, 6day neonatal rats were sacrificed and the thoracic organs removed as a tissue block. Then, the samples were rinsed, fixed with 4% paraformaldehyde in PBS, dehydrated and embedded in Paraplast as described by Sassoon et a1. (1988). Sections 6 fim thick were cut and stored at 4 °C until used for hybridization. In addition, rat pulmonary fibroblasts (Foster et aI., 1990) were grown on Tissue-Tek
Tropoelastin mRNA Expression chamber glass slides. The cultures were fixed with 4% paraformaldehyde in PBS, dehydrated and stored at 4°C until used for hybridization. For positive control studies, we employed the mouse beta-actin coding sequence (pAL 41.20) cloned into the PstI site of pBluescript plus (Alonso et ai., 1986). To synthesize anti-sense [3s S]-labeled probe, DNA template linearized with EcoR I was transcribed by T 3 RNA polymerase. Preparation of tropoelastin riboprobe. An EcoR I fragment (1.5 kb) rat tropoelastin cDNA (Rich and Foster, 1989) was inserted into pBluescript plus (Stratagene). To synthesize anti-sense eSS]-labeled RNA probes (complementary riboprobe), DNA templates linearized with Sad were transcribed by T 7 RNA polymerase. Sense RNA probes (homologous riboprobe) were obtained with DNA templates linearized by Hind III digestion and T 3 RNA polymerase. Three-hundred nanograms of linearized DNA templates were transcribed in 36 mM Tris, pH 7,5,5 mM MgCh, 2 mM Spermidine, 9 mM NaCl, 9 mM dithiothreitol, 1 unit human placenta ribonuclease inhibitor, 0.5 mM each of CTP, ATP, GTP, 2.3 mM eSS]-alpha UTP (250 Ci/mM; NEN) and either T 3 or T 7 RNA polymerase. The transcription reaction mixture was incubated 1-2 h at 37°C. This procedure yielded RNA transcripts with a specific activity greater than 5 x 10 8 cpm/Jlg. For removal of DNA template, RQl was added and the mixture incubated at 3rc for 15 min. Total yeast RNA was added to obtain a concentration of 250 Jlg/ml of final mixture concentration. Probe length was reduced to an approximate mean size of 60 nucleotides by alkali hydrolysis, and unincorporated nucleotides were removed by gel filtration on G-50 Sephadex. The radiolabeled riboprobe transcript fragments were concentrated by cold ethanol precipitation and suspended in hybridization buffer (see below for hybridization buffer composition). In-Situ hybridization procedure. The procedures used for probe preparation (see above), section treatment, hybridization and washing were based upon those used by Sassoon et ai. (1988) (unless otherwise indicated). Araldite embedded sections were deplasticized as described by Lisca et ai. (1988) and Paraplast-embedded sections were deparaffinized in xylene. Deplasticized sections or deparaffinized sections were rehydrated through a graded series of ethanols (100% to 30%), rinsed in 0.85% saline solution (5 min), PBS (5 min), and post-fixed in a prepared and filtered solution of 4% formaldehyde (freshly prepared from paraformaldehyde) in PBS buffer (30 min). Slides carrying sections were then rinsed in PBS (2 x 10 min) and treated with a fresh solution of proteinase K (20 Jlg/ml, 7.5 min) in Tris-HCl, EDTA (50 mM, 5 mM, pH 7.2). They were then rinsed in PBS (5 min), refixed in 4% formaldehyde solution in PBS, dipped in distilled water and acetylated in a 0.09 M solution of triethanolamine with acetic anhydride (1 :400 v/v) for 10 min. The slides were subsequently rinsed in PBS and 0.85% saline (5 min each),
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quickly dehydrated through a series of ethanols (30% to 100%), and allowed to dry at least 2h prior to hybridization. Probe was applied directly to tissue sections (20 JlI) at a final adjusted concentration of 1 x 106 cpm in hybridization buffer (50% deionized formamide, 0.3 M NaCl, 20mM Tris-HCI (pH7.4), 5mM EDTA, 10mM NaP0 4 (pH 8.0): 10% dextran sulfate, Denhardt's, 50 Jlg/ml total yeast RNA} and tissue and probe were covered with a siliconized cover-glass (22 x 22 mm). Hybridization was carried out at 50°C for approximately 16 h in a humid chamber. Coverslips were gently floated off in a 5X SSC (IX SSC, 0.15 M NaCl, 0.015 M Sodium Citrate), 10 mM dithiothreitol (DIT) at 50°C and subsequently the sections were subjected to a stringent washing at 60°C in 50% formamide, 2X SSC, 0.1 M DIT. Slides were then rinsed in STE (0.1 M NaCl, 0.01 M Tris-HCI, 0.001 M EDTA) washing buffer and treated with RNase A (20 Jlg/ml) in washing buffer for 45 min at 37°C. Following washes at room temperature in 2X SSC (15 min) and O.IX SSC (15 min), respectively, the slides were rapidly placed in a series of ethanols containing 0.3 M ammonium acetate. Autoradiography. The samples were processed for standard autoradiography using Kodak NTB-2 nuclear track emulsion. They were exposed for either 1 day (cultured rat pulmonary fibroblasts), or 3 days (cultured NRSMC), or 7 days (neonatal rat heart and lung) in light-tight boxes with desiccant at 4 0C. Electron microscopy and electron- and light-microscopic immunocytochemistry
Cultured NRSMC were examined by conventional electron microscopic staining (Toselli and Pepe, 1968) and by light and electron microscopic antibody staining (Faris et ai., 1986). Rabbit polyclonal antiserum raised against alpha-elastin (Faris et ai., 1986) was used for immunocytochemicallocalization studies.
Results The purpose of this study was to characterize elastin gene expression as a function of cell growth in culture. NRSMC in first passage (25 cm 2 flasks) were cultured for 6 weeks, and the cell layers were analyzed at various times for DNA, insoluble elastin, and total protein (Table I). Both the elastinlDNA content and the percent of the total protein attributed to elastin increase at a slow rate, if at all, after 4 weeks in culture. Northern blot analyses were done to measure the steady state levels of tropoelastin mRNA over the time course. Equal amounts of total mRNA (15 Jlg) extracted from cells collected for 1, 3, 4, 5 and 6 weeks in first passage were hybridized to [32 P]-labeled elastin cDNA. Analysis of the autoradiogram from the Northern blot of the tropoelastin
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is always consistent. As seen in Figure 2 (ll-week old culture), each cell layer is usually formed by slightly overlapping cells. With increase time in culture, the separation between cells increases, and the spaces become occupied with deposits of extracellular matrix. Examination of the cell culture revealed differences in accumulation, composition and arrangement of the formed elements of the extracellular matrix at varous depths in culture. By dividing the full depth of the culture into thirds, distinct differences were observed. For example, after culturing cells three weeks in first passage, the bottom of the culture contained large organized deposits of elastic material. The middle third of the culture consisted of smaller coalescing elastic fibers, and the top one third of the culture showed only small accumulations of elastic material. The top 3 to 5 layers in all cultures are tightly packed (see upper portion, Fig.2) and there is relatively less extracellular material when compared to the lower portion of the culture. Examination of the bottom third of the culture (Fig. 3) reveals a nearly continuous lamella of elastic fibers.
uIture
Fig. 1. Northern blot hybridization analysis of tropoelastin mRNA as a function of time in culture. Preparations of total mRNA (15 !!g lane) extracted from NRSMC were analyzed by Northern blots with a tropoelastin-specific eDNA clone. The arrow indicates probe hybridizing to 3.9-kb tropoelastin mRNA. mRNA (Fig. 1) shows small amounts present at 1- and 6week time periods. At 3 weeks, the steady state level of tropoelastin mRNA appears most intense and is seen to decease each week thereafter.
Culture morphology - development ofthe multilayered culture The NRSMC cultures demonstrate intense growth in the first two weeks. By one week in first passage, the cell layer is 7 to 9 !lm thick and consistes of 3- to 5-celilayers. At two weeks the cultures' thickness roughly doubled to become 16 to 18 !lm, and the number of cell layers is increased from 5 to 8. Over the next three weeks, growth increases only by approximately 20% each week, so that by 5 weeks, the cultures is 25 to 31!lm thick consisting of 12- to 14-cell layers (Gundrum, 1986). While absolute number of layers may vary slightly from one experiment to another, the trend
Determination oftropoelastin riboprobe specificity During recent years an alternative and most useful application of the in-situ hybridization technique has evolved to directly localize cellular mRNA on semi-thin, epoxy resinembedded tissue sections (Liscia et aI., 1988). We extended this technique to visualize tropoelastin mRNA on semithin, epoxy resin-embedded cell-culture sections. Since in-situ hybridization of tissue sections provide a greater opportunity for non-specific interaction than Northern blot analysis, we conducted the following controls to assess tropoelastin riboprobe specificity. First we demonstrated tropoelastin riboprobe hybridized to appropriate sites on tissue sections. For this, we used sections derived from 6-day neonatal rat heart/lung tissue blocks. Then we hybridized the sections with either: (1) complementary (anti-sense) tropoelastin riboprobe (positive control), anti-sense beta-actin riboprobe (positive control), or (3) homologous (sense) tropoelastin (negative control). Figure 4 A illustrates the hybridization of tropoelastin anti-sense probe to neonatal rat heart. Note the strong hybridization signal in the vascular NRSMC (medium arrow) and epicardial cells (small arrow). The strong signal present in some endocardial cells (large arrows) is best observed at
Fig. 2. Electron microscopy of transverse section through the top portion of NRSMC cultured for 11 weeks. Note the tightly packed cells in the upper portion of the figure. E - Extracellular elastin. Bar = 0.5 microns. Fig. 3. Electron microscopy of horizontal section rhrough the bottom portion of NRSMC cultured for 3 weeks. Note extracellular elastic fiber lamella stained with anti-alpha-elastin antiserum and secondary antibody linked with 10-nm diameter colloidal gold particles. Insert: Elastic fiber lamella as visualized by light r:-:icroscopy immunocytochemistry at low magnification. E - Extracellular elastin; C Collagen. Arrow - lamella stained using the light microscopical immunogold silver staining method Uanssen Life Sciences). Bar = 0.5 microns.
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4A
B
Tropoelastin mRNA Expression high magnification (not shown). Figure4B shows neonatal rat heart hybridized with anti-sense beta-actin. Note the distinct difference in the vascular SMC and epicardial cells hybridization signal pattern when compared with Figure4A. Also, there is a noteworthy difference in hybridization signal pattern observed in the pulmonary tissue portion of the heart/lung tissue block sample (data not shown). In the case of the anti-sense tropoelastin probe, tropoelastin mRNA hybridization signal is present in pulmonary vascular and airway smooth muscle, spetal interstitial, alveolar wall and pleural cells. In the case of the anti-sense beta-actin probe, beta-actin mRNA signal is present chiefly in pulmonary alveolar wall and pleural cells. We did not detect hybridization signal in neonatal rat heart or lung rissue hybridized with sense tropoelastin riboprobe. Second, we demonstrated tropoelastin riboprobe hybridized to appropriate sites on pulmonary fibroblasts cultured for 10 days on Tissue-Tek chamber slides. Pulmonary fibroblasts express tropoelastin in vitro (Foster and Rich, 1990), and we detected mature elastic fibers in these cultures when stained with Verhoeff's elastic tissue stain (data not shown). For this control study, all hybridization and signal detection procedures were conducted on un-sectioned, "whole mounted" fibroblasts. Figure4C shows fibroblasts, hybridized with anti-sense tropoelastin riboprobe, lying near the edge of the growing surface. Note the srrong hybridization signal present in most cells; also note some cells have a weak signal. We did not detect hybridization signal in fibroblasts hybridized with sense tropoelastin riboprobe. Third, we fixed, embedded, sectioned and then hybridized I-week cultured NRSMC with either anti-sense or sense tropoelastin riboprobe. Horizontal sections hybridized with anti-sense tropoelastin probe (Fig. 4 D) show most cells display strong hybridization signal intensity; also, we frequenrly observed easily identified pairs of cells with litrle or no detectable hybridization signal. As shown in Figure 4 E, we did not detect hybridization signal in NRSMC hybridized with sense tropoelastin (compare Figure4E with Figure4D). Finally, Figure 4 F shows a control photomicrograph of horizontally sectioned I-week cultured NRSMC hybrid-
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ized with anti-sense beta-actin riboprobe. Note the distinct difference in the hybridization signal pattern when compared to Figure 4 D.
Expression oftropoelastin mRNA during development ofthe multilayered culture Throughout cell growth the NRSMC display variations in tropoelastin mRNA hybridization signal intensity. When I-week cultured cells are sectioned parallel to the plane of the cell culture flask substratum, cells with litrle or no detectable signal lie adjacent to cells with strong signal (Fig. 5) (also, see Fig. 4 D). In addition, Figure 5 shows a cell in mitosis (curved arrow). This cell is in close contact with and almost completely surrounded by cells. We did not detect levels of tropoelastin mRNA in the mitotic cell, and adjacent cells exhibit weak signal at best (arrowhead). In contrast, distant NRSMC have a strong signal (arrow). At higher magnification (Fig. 6), the 4-week culture (Fig.6A) and the 7-week culture (Fig. 6B) are cut perpendicular to the plane of the cell culture flask substratum. All NRSMC seen in Figure 6 A exhibit a high level of tropoelastin mRNA. Figure6B shows the 7-week SMC with either no detectable tropoelastin mRNA, or a reduced level of mRNA when compared to the 4-week cells. Low magnification survey of SMC grown in culture for 1,2,3,4,6 and 8 weeks (Fig. 7), sectioned transversely and hybridized indicate they contain sparse amounts of tropoelastin mRNA at 1 week in culture. The mRNA signal progressively increases in intensity during the time course, peaking at 4-weeks; then, the mRNA level appears to decrease in intensity each week thereafter.
Phenotype, tropoelastin mRNA expression and elastin distribution An attempt was made to determine differences, if any, in the tropoelastin mRNA in-situ hybridization signal pattern in the NRSMC cultures. As stated earlier, the bottom third of the culture multilayer reveals a nearly continuous lamella of elastic fibers (see Fig. 3), the middle third consists of smaller coalescing fibers, and the upper third has a minimal
.... Fig. 4. Control hybridization for the determination of tropoelastin riboprobe specificity. Figure A and B: Paraffin-embedded 6-micron thick section through right ventricular wall of six-day neonatal rat heart hybridized with either anti-sense tropoelastin riboprobe (Figure A) or anti-sense beta-actin riboprobe (Figure B). Figure C: Whole-mount rat pulmonary fibroblasts cultured on Tissue-Tek chamber slides and hybridized with anti-sense tropoelastin riboprobe. FiguresO-F: Plastic-embedded I-micron semi-thin horizontal sections of I-week NRSMC cultures hybridized with either anti-sense tropoelastin (Figure 0), sense tropoelastin (Figure E) or anti-sense beta-actin riboprobe (Figure F). In Figure A, note the strong signal present in areas where there is a high cell-density of positive cells such as the coronary artery (medium arrow) and the pericardium (small arrow). The strong signal present in areas containing a low cell-density of positive cells in the endocardium (large arrows) is less apparent and is best demonstrated at high magnification (not shown). Figure C shows signal variation in the individual, whole-mounted cultured fibroblasts. Figure 0 also shows signal variation in cultured NRSMC. Note the easily identified pair of cells with little or no detectable hybridization signal. L - Coronary artery lumen; Small arrow Pericardium; Medium arrow - Coronary artery; Large arrow - Endocardium; n - Nucleus. Bar (Figures A-B) = SO microns; (Figure C- F) = 20 microns.
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amount of elastin. Fig. 8 shows the variation in tropoelastin mRNA hybridization signal intensity in a transverse section through the 5-week culture. There is abundant signal in both the bottom third and middle third of the culture multilayer. Note the cells located in the lower two-thirds of the culture (Fig.8) have a flat shape, which is best demonstrated at a higher magnification (see Fig. 6). In con-
trast, there is little or no signal intensity in the upper third of the multilayer where most cells are more spherical (left top portion of Fig. 8). This result suggests that there is a quantitative difference in the amount of tropoelastin mRNA between the upper third and the lower two-thirds of the culture. Also, the data suggest that is a change in elastogenic phenotype from spherical (little or no tropoelastin mRNA)
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Dark-field
Fig. 8. Plastic-embedded I-micron semi-thin transverse section of 5-week NRSMC cultures hybridized with anti-sense tropoelastin riboprobe. (~) - delineates bottom of culture; (~) - delineates top of culture. Bar = 10 microns. to a flat profile (abundant tropoelastin mRNA) during the development of the culture multilayer.
Discussion NRSMC in culture generate an extracellular matrix which contains many, if not all, of the key components identified in vitro blood vessels including elastin, collagen, proteoglycans and fibronectin. The synthesis of the subunit of microfibrillar protein was demonstrated by Muir et al. (1976). Using transmission electron microscopy, Toselli et al. (1981) studied the long-term accumulation of elastin in rabbit aortic SMC cultures and found microfibrils in these interstices of the amorphous component of mature elastic fibers, a finding similar to that described for in vivo systems. The NRSMC cultures are extremely active in synthesizing and deposing large amounts of insoluble elastin in their extracellular matrix, making this an ideal system for studying insults affecting the elastic fibers. Previous work from our laboratory (Faris et aI., 1986) described the effect of elastases on the extracellular matrix of these cultures. Elastase effect was monitored both ultrastructurally and chemically. An interesting observation in the present report was the differences in tropoelastin in-situ hybridization signal pattern throughout the time course studied. We detect at least three patterns in NRSMC grown in culture for 1 week (Fig. 5). The mitotic cell (curved arrow) shows no detectable signal. Nearby non-dividing SMC (arrowhead) exhibit little or no signal, and more distant non-dividing SMC (arrow) have abundant signal. Such signal differences may reflect underlying variations in tropoelastin mRNA steady state levels. We conclude from our observations that mitotic cells do not contain tropoelastin mRNA resulting in no detectable hybridization signal. Also, daughter cells may delay in tropoelastin mRNA biosynthesis resulting in little
or no detectable hybridization signal (see the pair of cells identified in Fig.4D); and, with time, the daughter cells may initiate elastogenesis resulting in intense hybridization signal. It is possible that such events are not limited to tropoelastin mRNA and that, for example, mitotic cells may not contain the mRNAs of other extracellular matrix components such as fibronectin and the procollagens. Charnley-Campbell et al. (1981) discussed the concept of cellular modulation in enzymatically dissociated SMC from intact mammalian aortae. They postulated that SMC reversibly modulate over the first several days in primary cell culture from a phenotypic state in which they are contracted, contain thick myofilaments, and do not undergo migration or mitosis to a synthetic state in which they become capable of mitosis and migration, lose their thick myofilaments and ability to contract and their cytoplasm are filled with organelles usually associated with synthesis of secretory protein. NRSMC tropoeiastin mRNA hybridization signal pattern variations we observe are compatible with the SMC synthetic state described by ChamleyCampbell et al. (1981). Initial evaluation of elastin biosynthesis pattern of the NRSMC cultures indicate a continuous increase in elastin accumulation over time in culture. In the early cultures (TableI), there is an approximately 30-fold increase in elastin content in cells cultured for 1 and 4 weeks. Concomitant with this is an approximately 3.5 increase in DNA content in cells cultured 1- 2 and 4 weeks. These finding are consistent with our earlier report (Barone et aI., 1988). Our results using in-situ hybrization confirm and extend these findings. Transverse sections of NRSMC grown in culture for 1, 2, 3 and 4 weeks (Fig. 7) show an increase in both the number of cells with signal and the signal intensity.
Tropoelastin mRNA Expression Table 1. Insoluble elastin accumulation in neonatal rat aortic smooth muscle cell culture. Time in Culture
mg Elastin
mgDNA
mg Elastin! % Elastin mgDNA
1 week 1 2
0.036 0.034
0.037 0.033
0.83 1.04
3.4 3.9
2 weeks 1 2
0.259 0.162
0.049 0.034
5.34 4.77
12.4 8.5
3 weeks 1 2
0.416 0.428
N.D. N.D
N.D. N.D.
16.8 19.1
4 weeks 1 2
1.250 1.326
0.117 0.139
10.73 9.55
27.2 24.1
5 weeks 1 2
1.945 2.314
0.204 0.197
9.54 11.74
28.6 28.7
6 weeks 1 2
2.844 2.332
0.204 0.167
13.95 13.96
35.4 30.7
N.D. = Not Determined Additional signal patterns are apparent during continued cell growth. These patterns are clearly demonstrated in transversely sectioned cultures photographed at high magnification (Fig. 6). For example, Figure6A (4-week material) shows that all NRSMC have high signal intensity. In the 7-week material (Fig. 6B), there is a decrease in signal intensity per cell when compared to the 4-week cells. High magnification examination of transversely sectioned 8week material show a further decrease in signal intensity, and many cells have no signal (data not shown). There is approximately a 2-fold increase in mgs of DNA from 4weeks to 6-weeks in culture (Table I). Concomitant with this is an approximately 2-fold increase in elastin accumulation. We conclude there is a decrease in the steady-state tropoelastin mRNA expression in cells cultured from 4weeks to 8-weeks. Survey of Northern blot analyses (Fig. 1) of the older cultures also depict a decrease in tropoelastin mRNA expression. In Northern blot analysis, the decrease in signal relates to the average number of cells presnt in the flask. In-situ hybridization can detect which cells contain tropoelastin mRNA, their location in the multilayer, and differences in signal intensity. From the data, we conclude there is a relationship between: (1) signal intensity associated with an individual cell, (2) the number of cells with signal (either high or low) intensity, and (3) time in culture. These conclusions suggest a correlation between the amount of functional tropoelastin mRNA present within an individual cell and the overall biosynthetic rate of elastin by the multilayer. The un-sectioned, whole-mounted neonatal rat pulmo-
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nary fibroblasts hybridized with tropoelastin riboprobe for use in control studies also displayed individual cells with easily identifiable differences in signal intensity. Cells lying most proximal to the Tissue-Tek culture substratum edge were sparsely populated (i. e. low cellular density areas), and these cells displayed little or no signal intensity (see Fig. 4 C). More distally located cells were observed in other regions of the chamber; here, there was a high cellular density and the cells generally exhibited a high signal intensity (data not shown). In addition, cells lying adjacent to the edge cells were observed showing a signal intensity intermediate in strength to that observed in cells lying more proximal or distal to the edge. Such correlations between hybridization signal intensity and cell location are also consistent with variations of tropoelastin mRNA levels within individual cells. Also, more meaningful quantitative analysis of cellular mRNA hybridization signal is likely when un-sectioned, sparsely populated cells are measured. In this case, one does not have to take into account either (1) the percent of cell present in the plane of the section, or (2) section-thickness variations. In summary, we demonstrate it is possible to gain insight into elastin gene expression as a function of cell growth in culture. Tropoelastin mRNA data derived from both in-situ hybridization studies employing plastic-embedded cultures and Northern blot analyses can be correlated with insoluble elastin accumulation. We found that elastogenesis continues with time in culture, and that tropoelastin mRNA steady state levels peaked in the cells cultured for 3-4 weeks. Finally, we show in-situ hybridization signal degree is compatible with variations of tropoelastin mRNA steady state levels within individual cells of the cultured NRSMC multilayer. Acknowledgements We thank Judith A. Foster and Celeste B. Rich for the gifts of tropoelastin specific cDNA probe RE-2 and the pulmonary fibroblasts cultures and Margaret Buckingham for the gift of mouse beta-actin coding sequence pAL 41.20. We also thank Alfie Tsay and Jag Bhawan for their helpful comments. Valerie Verbitski and Rosemarie Moscaritolo kindly helped uy by providing cultured cells. One of the authors, P.T., thanks Anna Kadar for encouraging him to pursue molecular biology skills, and special thanks for Xun Weng for help in acquiring the skills. This study was supported by United States Public Health Sciences grants HL 19 717 and HL 13 262. References Alonso, A., Minty, A., Bourlet, Y. and Buckingham, M.: Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates. J. Mol. Evol. 23: 11-22,1986. Barone, L. M., Wolfe, B. L., Faris, B. and Franzblau, c.: Elastin mRNA levels and insoluble elastin accumulation in neonatal rat smooth muscle cell cultures. Biochemistry 27: 3175 - 3182, 1988.
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