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Circulation Research. 1996;78:388-394

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(Circulation Research. 1996;78:388-394.)
© 1996 American Heart Association, Inc.

Articles

Tropoelastin Gene Expression in Individual Vascular Smooth Muscle Cells

Relationship to DNA Synthesis During Vascular Development and After Arterial Injury

James K. Belknap, Nicole A. Grieshaber, Phillip E. Schwartz, E. Christopher Orton, Michael A. Reidy, Richard A. Majack1

From the Departments of Pediatrics and Cell and Structural Biology (J.K.B., N.A.G., P.E.S., R.A.M.), University of Colorado Health Sciences Center, Denver, Colo; the Departments of Physiology and Clinical Sciences (J.K.B., E.C.O.), Colorado State University, Ft Collins; and the Department of Pathology (M.A.R.), University of Washington, Seattle.

Correspondence to Dr James K. Belknap, Department of Pediatrics, Campus Box B131, University of Colorado Health Sciences Center, Denver, CO 80262.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Abstract After vascular injury, quiescent adult smooth muscle cells (SMCs) revert to a more immature synthetic-state phenotype concomitant with the onset of cell replication. The relationship between SMC proliferation and the reexpression of genes characteristic of immature SMCs (eg, tropoelastin [TE]), on an individual cell basis, has not been determined. Using a combined bromodeoxyuridine (BrdU) immunocytochemistry–TE in situ hybridization technique, we determined the relationship between DNA synthesis and TE gene expression in the rat vascular wall during development and after experimental injury. During the early development of the aortic media (embryonic days 13 to 18), low but detectable levels of TE expression occurred equally in both replicating and nonreplicating SMCs. TE message levels dramatically increased in the late fetal and early postnatal periods (fetal day 19 to 1 month postpartum), after a precipitous drop in SMC replication, and then decreased to undetectable levels by postpartum day 60. After balloon catheter injury in the adult, a developmental sequence of SMC replication followed by TE gene expression was reiterated in both the media and in the developing neointima. On an individual cell basis, adult SMCs replicating after injury expressed little or no TE message; detectable TE gene expression occurred only in nonreplicating SMCs. The most important implications of these data are that (1) adult SMCs replicating after injury appear to revert to a preelastogenic embryonic phenotype; (2) maximal TE expression occurs in SMCs only after the cessation of cell replication; and (3) in both the media and the neointima, adult SMCs responding to injury undergo temporally sequential changes in phenotype reflective of SMC development.


Key Words: vascular smooth muscle • cell replication • aorta • development • tropoelastin


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
During blood vessel development, vascular SMCs undergo extensive replication and synthesize an ECM, which, in part, establishes the structural integrity of the vessel wall. SMCs in the adult vasculature, in contrast, are remarkably quiescent but can be stimulated by injury to again replicate and to express certain genes (such as those encoding structural matrix genes) characteristic of earlier developmental states.1 2 3 4 5 6 This "dedifferentiation" process is believed to be an important event in the pathogeneses of atherosclerosis and hypertension,7 8 in the restenosis of vessels after angioplasty and atherectomy,9 10 11 12 13 14 and in the adaptation of SMCs to culture conditions. A variety of poorly defined terms have been proposed to describe certain phenotypic states of vascular SMCs. Developing SMCs, cultured SMCs, and adult SMCs responding to injury have been described as being in a "synthetic" state due to specific morphological characteristics of the cells,13 15 whereas normal adult SMCs in vivo have been considered to be in a "contractile" state.13 14 15 The process by which contractile-state adult SMCs become synthetic in nature has been described as phenotypic "modulation."13

The temporal relationship between SMC replication and the expression of specific genes (such as those for TE or other markers of the immature or dedifferentiated state) has not been examined in individual SMCs. This relationship is of considerable importance, given the major contribution of ECM production to lesion formation16 and the positive correlation between numbers of synthetic-state SMCs in arterial lesions with the incidence and severity of subsequent restenotic events.10 12 In an effort to clarify some of the complex issues concerning SMC "phenotype" and its relationship to cell replication, we used a combined BrdU immunohistochemistry–TE in situ hybridization protocol to determine the relationship, on an individual cell basis, of SMC replication and TE gene expression during development and after injury. Specifically, we sought to address the following questions: (1) What is the relationship between the onset of SMC replication and changes in mRNA phenotype after injury (ie, do postinjury SMCs replicate and express synthetic-state markers simultaneously)? (2) What importance does anatomic position play in the determination of SMC phenotype (ie, do medial SMCs replicating after injury express immature or synthetic-state markers, or is a change in mRNA phenotype dependent on migration into the neointimal compartment)? (3) Does a stable "neointimal" SMC phenotype exist, or do the phenotypic markers expressed by neointimal SMCs reflect sequential and transitory stages in neointimal maturation?

The results reported herein reinforce previous observations suggesting that sequential temporally distinct phases of cellular replication and TE gene expression occur during vascular development and clearly demonstrate that a similarly timed sequence is reiterated in the tunica media, and subsequently in the forming neointima, of adult blood vessels after injury. Although the relationship between DNA synthesis and the expression of an immature program of gene expression in SMCs appears complex, it seems clear that high levels of structural matrix gene expression are largely limited to quiescent SMCs after a period of rapid replication and that the various phenotypes expressed by adult SMCs after vascular injury3 6 17 may represent successive stages along a developmental continuum of SMC maturation.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Aortic Developmental Time Course, Animal Surgery, and Tissue Preparation
For the aortic developmental series, pregnant female Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, Indianapolis, Ind. At gestational days 12 to 20, pregnant females were injected intraperitoneally with BrdU (100 mg/kg body wt, Sigma Chemical Co) at 17, 9, and 1 hour before euthanasia.14 Pregnant females were killed at increasing stages of gestation (days e13 to e21), and embryos or fetuses were removed and placed in 10% phosphate-buffered formaldehyde for 16 hours at 4°C. Tissues were embedded in paraffin and sectioned. Aortic tissues from postnatal animals (day 1, day 16, 1 month, 2 months, and 3 months) were obtained using similar protocols. At least three animals were used for each developmental time point.

For balloon injury, adult (>3 months) male Sprague-Dawley rats, weighing {approx}400 g, were purchased from either Simonsen Laboratories (Gilroy, Calif) or Tyler Laboratories, Inc (Bellevue, Wash). Rats were anesthetized with an intraperitoneal injection of xylazine (2.2 mg/kg Anased, Lloyd Laboratories) and ketamine (50 mg/kg body wt Ketaset, Aveco Co, Inc). Balloon injury of the carotid artery was performed as described previously.14 Briefly, a 2F balloon embolectomy catheter was introduced through the left external carotid artery, advanced caudally through the common carotid artery to the aortic arch, inflated, and withdrawn. The sequence was repeated three times to attain complete removal of endothelium from the common carotid artery. The contralateral uninjured carotid artery was used as control tissue. Surgically treated animals were housed and fed ad libitum after surgery. Rats were injected intraperitoneally with BrdU as described above. At 2, 4, 7, 14, and 42 days after surgery, the rats were euthanatized, and both common carotid arteries were extracted and fixed in 4% paraformaldehyde before embedding in paraffin. Three to five rats were used for each time point.

Immunohistochemistry
Immunoperoxidase staining for BrdU was used to visualize replicating SMCs in the developing aortic wall, as previously described and quantified by Cook et al.18 Briefly, paraffin-embedded sections were deparaffinized, treated with proteinase K, denatured with 2N HCl, blocked by incubation with normal horse serum, and then exposed to a monoclonal antibody against BrdU (Becton-Dickinson).19 20 21 An avidin-biotin immunoperoxidase system (Pierce) and 3,3'-diaminobenzidine substrate kit (Vector Laboratories) were used to detect the antigen-antibody complexes. Immunohistochemistry was performed on at least three rats from each time point in both the developmental and balloon injury time courses.

In Situ Hybridization
Sections of the tissues described above were deparaffinized, rehydrated, and then serially incubated in 0.2% Triton X-100/PBS, 1 mg/mL proteinase K, and acetic anhydride/0.1 mol/L tetraethylammonium. The slides were sequentially dehydrated in a graded ethanol series, air-dried, prehybridized for 2 hours, and then hybridized to a 35S-labeled riboprobe for TE overnight (1x106 cpm per section; the cDNA for rat TE was kindly provided by Dr W. Parks, Washington University, St Louis, Mo). Duplicate sections were used for both sense and antisense probe hybridizations. After hybridization, tissues were washed in 2x SSC, incubated with RNase A (Sigma Chemical Co), and washed several times with 2x SSC both at room temperature and at 55°C and with 0.1x SSC at 55°C. The sections were dehydrated through a graded ethanol series, air-dried, and dipped in NTB-2 emulsion (Eastman Kodak Co). The slides were developed after 5 to 7 days and counterstained with hematoxylin and eosin. In situ hybridizations were performed on three to five animals per time point in both the developmental and carotid balloon injury time courses. For slides in the developmental time course, the relative amount of aortic TE expression at different time points was assessed by counting exposed silver grains per square micrometer in the aortic tunica media of rats (ages e15 to 90 days postpartum) using a Macintosh computer and IMAGE (National Institutes of Health) software. Counts were performed on three animals for each developmental time point.

Combined Immunohistochemistry/In Situ Hybridization
The two above techniques were combined by sequentially performing immunohistochemistry for BrdU, followed by in situ hybridization for TE. Sections were deparaffinized, rehydrated in a graded ethanol series, and incubated in 100 µg/mL proteinase K for 10 minutes. The sections were then incubated in 2N HCl for 20 minutes, followed by incubation in horse serum for 20 minutes. The remaining immunocytochemistry protocol was performed as described above. After exposure to 3,3'-diaminobenzidine, samples were washed in 0.1x PBS, incubated in acetic anhydride, dehydrated in a graded ethanol series, and air-dried. The slides were prehybridized for 2 hours, and the remaining in situ protocol was performed as described above. The technique was performed on three to five animals for each time point in both the developmental and carotid balloon injury time courses. Quantification of BrdU-positive and BrdU-negative (hematoxylin-stained nuclei) SMCs expressing TE mRNA was performed in the carotid artery wall 7 and 14 days after injury to establish the pattern of TE expression in replicating and nonreplicating SMCs. A minimum of 10 grains over and immediately surrounding the nucleus was used to establish a cell as positive for TE message (the threshold of 10 grains was used because 2 to 9 grains were present over nuclei in sections hybridized with sense probe; most positive cells had 25 to 30 grains). A minimum of 400 carotid artery SMCs from four or five different rats was counted for each time point.

Statistical Analysis
Data are presented as mean±SEM. For mean comparison of BrdU-positive and BrdU-negative SMCs expressing TE message, a one-tailed unpaired Student's t test was used. A value of P<.05 was considered significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
SMC Replication and TE Gene Expression in the Developing Aortic Wall
A majority of SMCs in the developing rat aorta actively replicate during embryonic and early fetal life, exhibiting a 24-hour BrdU labeling index of >80% from days e13 to e17.18 As reported by Cook et al,18 the number of BrdU-positive SMCs in the aortic wall drops precipitously between days e17 and e19 and continues to decrease until replication rates reach <0.5% by 60 days postpartum and <0.06% by 90 to 120 days postpartum.18 22 Fig 1Down, left (a through c, g through i), illustrates the temporal and spatial distribution of replicating SMCs in the developing aortic wall.




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  		Figure 1. Peak TE gene expression follows peak cell replication in SMCs during development. Left, BrdU immunohistochemistry (a through c, g through i) and TE in situ hybridization (d through f, j through l) analyses of the rat aorta during development from day e19 (a and d), day e21 (b and e), and postpartum day 1 (c and f), day 16 (g and j), 1 month (h and k), and 2 months (i and l). Luminal surface of vascular wall faces top of pictures. Bar=50 µm. Right, Quantification of silver grains per 100 µm2 aortic tunica media at the developmental time points (ppd indicates postpartum day) presented vs BrdU labeling in the developing rat aorta. Aortas from three different animals were counted for each time point. Note that TE message increases in fetal (days e19 and e21) and early postnatal (days 1 and 16, 1 month) time points after a developmental period of high replication. (Aortic BrdU labeling values represented in the bar graph were taken from Cook et al.18 )

TE gene expression in the developing rat aorta was assessed by in situ hybridization with a cRNA probe (Fig 1Up, left) and subsequent quantification of exposed silver grains over the tunica media using IMAGE (National Institutes of Health) software (Fig 1Up, right). At the earliest time point examined (day e13), TE expression was minimal but clearly detectable throughout the tunica media. Aortic TE expression remained low throughout embryonic and early fetal life and increased dramatically during late fetal and early neonatal life (Fig 1Up, left, d through f, and j; Fig 1Up, right); this increase correlated with the marked decrease in replication occurring during this period. TE expression remained high through 1 month postpartum (Fig 1Up, left, k; Fig 1Up, right). By 2 months postpartum, TE expression was decreased in the media, but small nests of positive cells remained visible in the adventitia (Fig 1Up, left, l; Fig 1Up, right). No TE expression was detectable in any compartment of the 3-month vascular wall. The developing aortic wall therefore undergoes a period of intense replication in the embryonic stage (through day e18),18 followed by a period of active TE gene expression in fetal (days e19 to e21) and early postnatal life.

BrdU immunohistochemistry was combined with TE in situ hybridization to determine the relationship between SMC replication and structural matrix gene expression in the individual SMCs. In the embryonic and early fetal aorta (through day e18), most SMCs were labeled with BrdU, and all SMCs expressed TE mRNA at extremely low levels. In the late fetal and early postnatal aorta, all SMCs (replicating and nonreplicating) expressed markedly increased levels of TE mRNA, although there was some variability in TE expression among individual cells (data not shown). Therefore, while no clear relationship between TE gene expression and DNA replication was apparent when assessing individual SMCs in the developing aorta, a temporal relationship of expression of the two phenotypic markers was strikingly apparent in the vessel as a whole (Fig 1Up), with the embryonic period of peak replication being followed by a fetal/neonatal period of intense TE gene expression.

SMC Replication and TE Gene Expression in the Injured Adult Vascular Wall
After experimental balloon catheter injury of the rat carotid artery, SMC replication proceeds in a well-characterized fashion.14 23 Using this model system and the combined TE in situ hybridization–BrdU immunocytochemistry technique described above, we sought to determine the relationship between DNA synthesis and TE gene expression in adult SMCs after injury. Two and 4 days after balloon injury, replicating SMCs were present in the tunica media, but no TE message was detectable at that time (Fig 2aDown and 2eDown). At 7 days after injury, the tunica media contained few replicating cells, but many medial SMCs expressed TE mRNA (Fig 2bDown and 2fDown). By 14 days after injury, no SMC replication and no TE gene expression were apparent in the tunica media. SMCs in the forming neointima were not visible until 7 days, at which time the majority of neointimal cells were BrdU positive (Fig 2bDown). Minimal TE expression was evident in neointimal SMCs at 7 days. By 14 days after injury, the majority of neointimal SMCs were replicatively quiescent, and TE expression was present in most cells (Fig 2gDown). At 6 weeks after injury, only a few luminal SMCs were replicating, and no TE mRNA expression was detectable (Fig 2dDown and 2hDown).



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  		Figure 2. TE gene expression follows peak replication in both the neointima and tunica media in the balloon-injured carotid artery. BrdU immunohistochemistry (a through d) and TE in situ hybridization (e through h) analyses of balloon-injured rat carotid arteries were obtained 2 days (a and e), 7 days (b and f), 14 days (c and g), and 42 days (d and h) after injury. Note that the highest SMC replication rates are observed in the tunica media and neointima at 2 and 7 days, respectively, whereas the greatest TE gene expression is observed at 7 days in the tunica media and 14 days in the neointima. By 6 weeks, both the neointima and tunica media undergo minimal replication and no TE gene expression. Arrows indicate the internal elastic lamina. Bar=50 µm.

Results from double-labeled samples (Fig 3Down) confirmed the results obtained with the two individual protocols, demonstrating that SMCs in the injured tunica media and neointima rarely undergo concomitant DNA synthesis and TE gene expression and that TE mRNA accumulation primarily occurs in nonreplicative SMCs. At 7 days after injury, nonreplicating medial SMCs expressed significant amounts of TE message, whereas essentially no TE message was present in replicating neointimal SMCs (Fig 3aDown and 3bDown). In sections in which both replicating and nonreplicating SMCs could be visualized in the 7-day neointima (Fig 3cDown and 3dDown), only the nonreplicating neointimal SMCs appeared to express TE message. By 14 days after injury, most neointimal cells actively expressed TE mRNA but were replicatively quiescent; the few replicating neointimal SMCs were essentially devoid of TE mRNA (Fig 3eDown and 3fDown). Quantification of BrdU-positive and BrdU-negative cells expressing TE mRNA revealed that in the carotid artery wall 7 days after injury, 86±3% of the cells that were BrdU negative were positive for TE, whereas only 4±1% of BrdU-positive SMCs expressed TE message (Fig 4Down). At 14 days after injury, 94±3% of BrdU-negative cells were TE positive, whereas 4±1% of BrdU-positive cells expressed TE mRNA. The differences in percentage of BrdU-positive and BrdU-negative SMCs expressing TE were significant (P<.05) in both the 7- and 14-day postinjury vessels. Therefore, a distinct sequence of SMC replication followed by TE gene expression occurred initially in the tunica media, and subsequently in the neointima, of injured carotid arteries.



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  		Figure 3. Individual SMCs replicating after injury do not appear to express TE mRNA. Light-field (a, c, and e) and dark-field (b, d, and f) micrographs of 7-day (a through d) and 14-day (e and f) postinjury carotid arteries after the simultaneous detection of BrdU and TE mRNA. Note that BrdU-positive SMCs (long arrows, a, b, e, and f) in both the 7-day and 14-day neointima express minimal or no TE message compared with BrdU-negative SMCs in both the tunica media (short arrows, a through d) and neointima (short arrows, e and f). Bar=50 µm.



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  		Figure 4. The majority of SMCs replicating in the injured vascular wall do not express detectable TE message. Bar graph shows quantification of replicating (BrdU+) and nonreplicating (BrdU-) SMCs undergoing TE mRNA expression in the rat carotid artery 7 and 14 days after balloon injury. TE message expression by BrdU+ and BrdU- SMCs was determined (as described in text) in tissue sections on which a combined in situ hybridization (TE)/immunohistochemistry (BrdU) technique was performed (see Fig 3Up).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
Little is known about the in vivo relationship between cell replication and the expression of genes (such as those encoding structural ECM molecules), which in part define the "synthetic" state in vascular SMCs.24 Such information is crucial to our overall understanding of the molecular events accompanying vascular injury, given the importance ascribed to the acquisition of the synthetic state as an essential step in SMC replication,8 13 15 the role of synthetic-state SMCs in determining the incidence and severity of restenosis in human lesions,10 12 and the considerable ECM accumulation found in the mature neointima.16 In the present study, we have compared the patterns of expression of TE, a developmentally regulated structural matrix gene and a marker of synthetic-state SMCs, and cell replication in the blood vessel wall during development and after injury. Using a double-labeling technique, we have determined the relationship between TE expression and DNA synthesis in the vascular wall as a whole and in individual SMCs.

SMC Replication and TE mRNA Expression in the Developing Vascular Wall
During embryonic life, SMCs in the developing rat aorta exhibit a very high index of replication ({approx}80% per day from days e13 to e17),18 resulting in the establishment of the adult component of SMC layers in the tunica media by the end of the embryonic period.25 At the transition from embryonic to fetal life, SMC replication decreases dramatically to {approx}40% and remains at that level throughout the fetal period.9 A further gradual decrease in vascular SMC replication occurs postpartum, with the replication index dropping to <0.5% by 1 month and <0.06% by 3 to 4 months of age.18 22 The pattern of TE mRNA expression has not been described, to date, in the developing rat aorta. Morphologically, recognizable elastic fibers first occur in the rat aorta at day e13 but do not show a large increase in accumulation until the late embryonic/early fetal stages (days e17 to e19),25 coincident with a dramatic decrease in SMC replication.18 In the postpartum rat aorta, elastin and collagen content continue to increase for the first few weeks of life,26 27 28 29 while DNA synthesis continues to decline.18 26 28 This combination of processes results in a remarkable change in the makeup of the tunica media, from the newborn vessel wall (in which 65% of the media is composed of SMCs) to the 12-week vessel wall (in which 70% to 75% of the tunica media is matrix).27

By in situ hybridization, TE message expression in the developing rat aorta showed a pattern similar to that described morphologically and biochemically for elastin protein accumulation.25 28 Although TE mRNA expression was present at low levels at the earliest time point examined (day e13), the highest levels of TE mRNA expression were observed during late fetal and early postnatal life. This time course of expression of TE mRNA in the developing rat aorta is similar to that described for the rat pulmonary artery30 and for the aortas of other species.31 32 Thus, in the developing rat aorta, the highest TE mRNA expression occurs in the late fetal and early postnatal periods, following an embryonic period of intense SMC replication.

SMC Replication and TE mRNA Expression in the Injured Vessel Wall: Neointimal Formation as a Reiteration of Arterial Development
SMC replication and structural ECM synthesis are important components of the pathogenesis of atherosclerosis and restenosis after balloon angioplasty. The importance of matrix production in restenosis is underscored by the fact that ECM occupies {approx}70% to 80% of the "mature" neointima, with the remainder occupied by SMCs.1 33 The kinetics of SMC replication in the adult rat carotid artery after experimental balloon catheter injury have been extensively described,14 23 whereas the kinetics of matrix production have received little attention.16 Although ECM synthesis has been reported to increase when SMCs are stimulated to divide in vitro,24 34 35 36 the exact correlation between SMC replication and structural matrix gene expression in vivo has not previously been investigated.

In the present studies, TE gene expression followed replication in both the tunica media and the developing neointima, with maximal TE mRNA levels occurring at 7 days in the media and at 14 days in the neointima. Using a BrdU/TE double-labeling technique, we demonstrated that the earliest replicating cells in both the tunica media and neointima are not elastogenic but rather appear to pass through an elastogenic phase subsequent to replication.

The hypothesis that neointimal SMCs reexpress a more immature phenotype has been supported by a great deal of experimental work using a number of phenotypic markers.8 37 38 Increased expression of several developmentally regulated ECM components, including TE, {alpha}1(I) procollagen, extradomain-A fibronectin, and osteopontin, has been extensively documented in neointimal SMCs.3 6 17 39 40 A similar pattern of expression has been described for cytoskeletal and contractile proteins (vimentin, desmin, actin isoforms, tropomyosin, and myosin),2 5 41 42 43 44 45 46 H19,4 cytochrome p-450IA1,47 and platelet-derived growth factor-B chain.6 Cumulatively, the available data suggest that vascular injury causes adult SMCs to dedifferentiate into cells expressing characteristics of those composing the fetal or neonatal aorta. The data are incomplete, however, given that a majority of the investigations cited above typically assessed the phenotypic characteristics of neointimal SMCs at a single time point (eg, 2 weeks after balloon injury). Our studies show that both medial and neointimal SMCs undergo a timed sequence of DNA synthesis and TE gene expression subsequent to injury similar to that observed during normal aortic development. The data suggest several important points concerning the expression of immature synthetic-state characteristics in SMCs replicating after injury: (1) The expression of immature markers such as TE may not necessarily occur concomitantly with the onset of cell replication in adult SMCs. (2) SMCs in the injured vascular wall appear to revert to a "preelastogenic" phenotype rather than to a "fetal" or "neonatal" phenotype, as has been previously suggested. (3) Expression of immature characteristics by adult SMCs in injured vessels does not appear to be dependent on movement of the SMCs into the neointimal space. (4) Expression of synthetic-state markers such as TE in neointimal SMC does not appear to represent a stable or static expression of a more immature phenotype but, rather, varies according to the time after injury. Importantly, the data suggest that a single neointimal cell phenotype may not exist per se; the phenotypic characteristics expressed by neointimal SMCs at any given time point may only reflect the developmental stage through which the cells are passing at any given time.

In summary, the relationships among DNA synthesis, cellular phenotype, and matrix gene expression in vascular SMCs appear complex, given the plasticity of SMC gene expression and the lack of specific molecular definitions for specific SMC phenotypes. In the present study, we described the pattern of expression of TE, a structural matrix gene that may serve as a marker for the synthetic phenotype, in developing SMCs and in adult SMCs after vascular injury and have correlated its expression with DNA synthesis in individual cells. Further detailed studies using a variety of molecular probes will clearly be required before we can achieve an integrated understanding of how mature fully differentiated SMCs alter their phenotypic properties after vascular injury, how these changes relate to SMC replication, and how these alterations contribute to restenosis and vascular lesion formation.


*    Selected Abbreviations and Acronyms
 
BrdU = bromodeoxyuridine
e (associated = embryonic or fetal day
with number)
ECM = extracellular matrix
SMC = smooth muscle cell
TE = tropoelastin


*    Acknowledgments
 
This study was supported by grants HL-46841 and HL-47685 from the National Institutes of Health. Dr Majack was a Genentech–American Heart Association Established Investigator. Dr Belknap was supported by a postdoctoral National Research Service Award.


*    Footnotes
 
1 Deceased. Back

Received May 3, 1995; accepted November 20, 1995.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 

  1. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest. 1983;49:208-215. [Medline] [Order article via Infotrieve]
  2. Gabbiani G, Kocher O, Bloom WS, Vandekerckove J, Weber K. Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media. J Clin Invest. 1984;73:148-152.
  3. Glukhova MA, Frid MG, Shekhonin BV, Vasilevsdaya TD, Grunwald J, Saginati M, Koteliansky VE. Expression of extra domain A fibronectin in vascular smooth muscle cells is phenotype dependent. J Cell Biol. 1989;109:357-366. [Abstract/Free Full Text]
  4. Kim D-K, Zhang L, Dzau V, Pratt RE. H19, a developmentally regulated gene is reexpressed in rat vascular smooth muscle cells after injury. J Clin Invest. 1994;93:355-360.
  5. Kocher O, Skalli O, Bloom WS, Gabbiani G. Cytoskeleton of rat aortic smooth muscle cells: normal conditions and experimental intimal thickening. Lab Invest. 1984;50:645-652. [Medline] [Order article via Infotrieve]
  6. Majesky MW, Giachelli CM, Reidy MA, Schwartz SM. Rat carotid neointimal smooth muscle cells reexpress a developmentally regulated mRNA phenotype during repair of arterial injury. Circ Res. 1992;71:759-768. [Abstract/Free Full Text]
  7. Ross R, Glomset JA. The pathogenesis of atherosclerosis (first of two parts). N Engl J Med. 1976;295:369-377. [Medline] [Order article via Infotrieve]
  8. Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular disease. Circ Res. 1986;58:427-444. [Abstract/Free Full Text]
  9. Johnson DE, Hinohara T, Selmon MR, Braden LJ, Simpson JB. Primary peripheral arterial stenoses and restenoses excised by transluminal atherectomy: a histopathologic study. J Am Coll Cardiol. 1990;15:419-425. [Abstract]
  10. Leclerc G, Isner JM, Kearney M, Simons M, Safian RD, Baim DS, Weir L. Evidence implicating nonmuscle myosin in restenosis: use of in situ hybridization to analyze human vascular lesions obtained by directional atherectomy. Circulation. 1992;85:543-553. [Abstract/Free Full Text]
  11. McBride W, Lange RA, Hillis LC. Restenosis after successful coronary angioplasty. N Engl J Med. 1988;318:1734-1737. [Medline] [Order article via Infotrieve]
  12. Simons M, Leclerc G, Safian RD, Isner JM, Weir L, Baim DS. Relation between activated smooth-muscle cells in coronary-artery lesions and restenosis after atherectomy. N Engl J Med. 1993;328:608-613. [Abstract/Free Full Text]
  13. Chamley-Campbell JH, Campbell GR. What controls smooth muscle phenotype? Atherosclerosis. 1981;40:347-357. [Medline] [Order article via Infotrieve]
  14. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury: I, smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327-333. [Medline] [Order article via Infotrieve]
  15. Campbell GR, Campbell JH, Manderson JA, Horrigan S, Rennick RE. Arterial smooth muscle: a multifunctional mesenchymal cell. Arch Pathol Lab Med. 1988;112:977-986. [Medline] [Order article via Infotrieve]
  16. Nikkari ST, Jarvelainen HT, Wight TN, Ferguson M, Clowes AW. Smooth muscle cell expression of extracellular matrix genes after arterial injury. Am J Pathol. 1994;144:1348-1355. [Abstract]
  17. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993;92:1686-1696.
  18. Cook CL, Weiser MCM, Schwartz PE, Jones CL, Majack RA. Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells. Circ Res. 1994;74:189-196. [Abstract/Free Full Text]
  19. Hayashi Y, Koike M, Matautani M, Hoshino T. Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue. J Histochem Cytochem. 1988;36:511-514. [Abstract]
  20. Orton EC, LaRue SM, Ensley B, Stenmark K. Bromodeoxyuridine labeling and DNA content of pulmonary arterial medial cells from hypoxia-exposed and nonexposed healthy calves. Am J Vet Res. 1992;53:1925-1930. [Medline] [Order article via Infotrieve]
  21. Sugihara H, Hattori T, Fukuda M. Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues. Histochemistry. 1986;85:193-195. [Medline] [Order article via Infotrieve]
  22. Lombardi DM, Reidy MA, Schwartz SM. Methodologic considerations important in the accurate quantitation of aortic smooth muscle cell replication in the normal rat. Am J Pathol. 1991;138:441-446. [Abstract]
  23. Clowes AW, Clowes MM. Kinetics of cellular proliferation after arterial injury, II: inhibition of smooth muscle growth by heparin. Lab Invest. 1985;52:611-616. [Medline] [Order article via Infotrieve]
  24. Sjolund M, Madsen K, von der Mark D, Thyberg J. Phenotype modulation in primary cultures of smooth muscle cells from rat aorta: synthesis of collagen and elastin. Differentiation. 1986;32:173-80. [Medline] [Order article via Infotrieve]
  25. Nakamura H. Electron microscopic study of the prenatal development of the thoracic aorta in the rat. Am J Anat. 1988;181:406-418. [Medline] [Order article via Infotrieve]
  26. Berry CL, Looker T, Germain J. The growth and development of the rat aorta. J Anat. 1972;113:1-16. [Medline] [Order article via Infotrieve]
  27. Gerrity RG, Cliff WJ. The aortic tunica media of the developing rat. Lab Invest. 1975;32:585-600. [Medline] [Order article via Infotrieve]
  28. Looker T, Berry CL. The growth and development of the rat aorta: II, changes in nucleic acid and scleroprotein content. J Anat. 1972;113:17-34. [Medline] [Order article via Infotrieve]
  29. Olivetti G, Anversa P, Melissari M, Loud AV. Morphometric study of early postnatal development of the thoracic aorta in the rat. Circ Res. 1980;47:417-424. [Abstract/Free Full Text]
  30. Noguchi A, Samaha H. Developmental changes in tropoelastin gene expression in the rat lung studied by in situ hybridization. Am J Respir Cell Mol Biol. 1991;5:571-578.
  31. Bendeck MP, Langille BL. Rapid accumulation of elastin and collagen in the aortas of sheep in the immediate perinatal period. Circ Res. 1991;69:1165-1169. [Abstract/Free Full Text]
  32. Davidson JM, Hill KE, Alford JL. Developmental changes in collagen and elastin biosynthesis in the porcine aorta. Dev Biol. 1986;118:103-111. [Medline] [Order article via Infotrieve]
  33. Snow AD, Bolender RP, With TN, Clowes AW. Heparin modulates the composition of the extracellular matrix domain surrounding arterial smooth muscle cells. Am J Pathol. 1990;137:313-330. [Abstract]
  34. Breton M, Berrow E, Brahimi-Horn MC, Deudon E, Picard J. Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta. Exp Cell Res. 1986;66:416-426.
  35. Holderbaum D, Ehrhart K. Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro. Exp Cell Res. 1984;153:16-24. [Medline] [Order article via Infotrieve]
  36. Wight TN, Potter-Perigo S, Aulinskas T. Proteoglycans and cell proliferation. Am J Respir Dis. 1989;140:1132-1135.
  37. Majesky MW, Schwartz SM. Smooth muscle diversity in arterial wound repair. Toxicol Pathol. 1990;18:554-559. [Medline] [Order article via Infotrieve]
  38. Thyberg J, Hedin U, Sjolund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis. 1990;10:966-990. [Free Full Text]
  39. Giachelli C, Bae N, Lombardi D, Majesky M, Schwartz S. Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR). Biochem Biophys Res Commun. 1991;177:867-873. [Medline] [Order article via Infotrieve]
  40. Glukhova MA, Frid MG, Shekhonin BV, Galabanov YV, Koteliansky VE. Expression of fibronectin variants in vascular and visceral smooth muscle cells in development. Dev Biol. 1990;141:193-202. [Medline] [Order article via Infotrieve]
  41. Eddinger TJ, Murphy RA. Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle. Arch Biochem Biophys. 1991;284:232-237. [Medline] [Order article via Infotrieve]
  42. Glukhova MA, Frid MG, Koteliansky VE. Developmental changes in expression of contractile and cytoskeletal proteins in human aortic smooth muscle. J Biol Chem. 1990;265:13042-13046. [Abstract/Free Full Text]
  43. Kocher O, Gabbiani G. Cytoskeletal features of normal and atheromatous human arterial smooth muscle cells. Hum Pathol. 1986;17:875-880. [Medline] [Order article via Infotrieve]
  44. Kocher O, Skalli O, Cerutti D, Gabbiani F, Gabbiani G. Cytoskeletal features of rat aortic cells during development: an electron microscopic, immunohistochemical, and biochemical study. Circ Res. 1985;56:829-838. [Abstract/Free Full Text]
  45. Osborn M, Caselitz J, Puschel K, Weber K. Intermediate filament expression in human vascular smooth muscle and in arteriosclerotic plaques. Virchows Arch A Pathol Anat Histopathol. 1987;411:449-458. [Medline] [Order article via Infotrieve]
  46. Owens GK, Thompson MM. Developmental changes in isoactin expression in rat aortic smooth muscle cells in vivo. J Biol Chem. 1986;261:13373-13380. [Abstract/Free Full Text]
  47. Giachelli CM, Majesky MW, Schwartz SM. Developmentally regulated cytochrome P-450IA1 expression in cultured rat vascular smooth muscle cells. J Biol Chem. 1991;266:3981-3986. [Abstract/Free Full Text]
  48. Rosenquist TH, Beall AC. Elastogenic cells in the developing cardiovascular system. Ann N Y Acad Sci. 1990;588:106-119.[Medline] [Order article via Infotrieve]



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