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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1203-1209.)
© 1997 American Heart Association, Inc.

Articles

Smooth Muscle Cell Heterogeneity in Pulmonary and Systemic Vessels

Importance in Vascular Disease

Maria G. Frid; Edward C. Dempsey; Anthony G. Durmowicz; ; Kurt R. Stenmark

From the Cardiovascular Pulmonary and Developmental Biology Research Laboratories, University of Colorado Health Sciences Center (M.G.F., E.C.D., A.G.D., K.R.S.), and the VA Medical Center (E.C.D.), Denver, Colo.

Correspondence to Kurt R. Stenmark, MD, Developmental Lung Biology Laboratory, University of Colorado Health Sciences Center, 4200 E 9th Ave, Box B131, Denver, CO 80262. E-mail kurt.stenmark{at}uchsc.edu

	Abstract

Top Abstract Introduction SMC Heterogeneity in the... Unique Responses of Specific... In Vitro Analysis of... Summary References

 
Abstract Experimental evidence is rapidly accumulating which demonstrates that the arterial media in both pulmonary and systemic vessels is not composed of a phenotypically homogeneous population of smooth muscle cells (SMCs) but rather of heterogeneous subpopulations of cells with unique developmental lineages. In vivo and in vitro observations strongly suggest that marked differences in the phenotype, growth, and matrix-producing capabilities of phenotypically distinct SMC subpopulations exist and that these differences are intrinsic to the cell type. These data also suggest that differential proliferative and matrix-producing capabilities of distinct SMC subpopulations govern, at least in part, the pattern of abnormal cell proliferation and matrix protein synthesis observed in the pathogenesis of vascular disease. Within the pulmonary circulation, the observation that the isolated medial SMC subpopulations exhibit differential proliferative responses to hypoxic exposure is important, since this in vitro cell-model system can now be used to better understand the mechanisms that regulate increased responsiveness of specific medial cell subpopulations to low oxygen concentrations. Our data also support the idea that protein kinase C is likely to be one important determinant of differential cell growth responses to hypoxia. The data also suggest differential involvement of specific arterial SMC subpopulations in the elastogenic responses of the vessel wall to injury. We believe that a better understanding of the mechanisms contributing to the unique behavior of specific arterial cell subpopulations will provide important future directions for therapies aimed at preventing abnormal cell replication and matrix protein synthesis in vascular disease.

Key Words: smooth muscle cell heterogeneity • protein kinase C • pulmonary hypertension • vascular remodeling • tropoelastin • proliferation • hypoxia

	Introduction

Top Abstract Introduction SMC Heterogeneity in the... Unique Responses of Specific... In Vitro Analysis of... Summary References

 
Experimental evidence is rapidly accumulating which demonstrates that the arterial media in both pulmonary and systemic vessels is not composed of a phenotypically homogeneous population of SMCs but rather of heterogeneous subpopulations of cells with unique developmental lineages. Further, it appears that specific subpopulations of SMCs respond in unique ways to injury and thus make selective contributions to the vascular remodeling process. These findings are not surprising if one considers the fact that SMCs within the media must perform numerous and varied functions to maintain homeostasis of the vessel wall. For example, the MPA has been shown to stretch both longitudinally and circumferentially by 5% to 15% with each systole, a function requiring variable orientation of SM fibers within the wall.¹ The MPA has also been shown to vary in thickness around its circumference, suggesting nonuniformity in pulmonary artery wall properties.² In response to sympathetic neural stimulation, conduit arteries have been shown to stiffen, demonstrating a significant contractile capability of the SMCs within these vessels.³ In addition, in response to chronic pressure elevation, conduit pulmonary artery SMCs proliferate and increase production of extracellular matrix proteins.^{4 5} It seems likely, then, that the existence of SMCs of different phenotypes within the arterial media would be the best way for a vessel to perform the various functions required. If the vessel is injured, some medial cells would be recruited to repair the injury, while at the same time, other cells would perform the functions required of the vessel to maintain cardiovascular homeostasis. The following paragraphs briefly summarize data from our laboratory demonstrating SMC heterogeneity in the bovine pulmonary and systemic vascular arterial media and the diverse functional responses unique cell populations of the pulmonary circulation exhibit in normal development and disease.

	SMC Heterogeneity in the Pulmonary and Systemic Circulations

Top Abstract Introduction SMC Heterogeneity in the... Unique Responses of Specific... In Vitro Analysis of... Summary References

 
In Vivo Analysis
Experimental evidence supporting the concept of phenotypic heterogeneity of vascular SMCs initially emerged from studies analyzing expression of muscle-specific contractile and cytoskeletal proteins in the systemic circulation. These studies demonstrated unique differences in muscle-specific protein expression by SMCs at different locations along the longitudinal axis of the arterial tree, as well as in distinct arterial wall layers (ie, intima versus media) of normal or injured animal or atherosclerotic human vessels.^{6 7 8 9} More limited, however, is information regarding site-specific heterogeneity of SMCs within the vascular media. A few studies, analyzing the expression of a limited number of contractile proteins, suggested that site-specific heterogeneity of SMC phenotype in the systemic vascular media does exist.^{10 11 12 13} However, no systematic evaluation of SMC heterogeneity within the pulmonary circulation had been performed.

In previous studies in our laboratory using routine staining methods, we had observed SMCs in the outer media of bovine pulmonary arteries that varied in arrangement pattern, orientation, TE mRNA expression, and proliferative indices, observations suggestive of vascular SMC heterogeneity.^{5 4 14} We thus performed experiments to systematically analyze SMC phenotype in the bovine pulmonary and systemic arterial media by using a broad panel of antibodies against muscle-specific proteins that are believed to be reliable markers of SM phenotype (-SM-actin, SM MHC isoforms, calponin, desmin, SM [high-molecular-weight] caldesmon, and meta-vinculin). We demonstrated the existence of a very complex, site-specific heterogeneity in the structure and cell phenotype of the bovine pulmonary and aortic arterial media (Figure).¹⁵ As shown in the Table, at least four phenotypically distinct SMC subpopulations were found to exist within the mature bovine arterial media based on differential expression of muscle-specific contractile and cytoskeletal proteins. Importantly, immunobiochemical studies analyzing cell phenotype alone would have suggested that only three cell phenotypes could be distinguished within the mature vascular media (see "Immunobiochemical characteristics" in the Table). However, when cell morphology, arrangement pattern, orientation, elastic lamellae pattern, and developmental differentiation pathways (see below) were also considered, the existence of four distinct cell subpopulations became obvious.

View larger version (70K): [in this window] [in a new window]   Figure 1. Structural and cellular heterogeneity of the mature bovine MPA. A, Indirect immunofluorescence staining with antibodies against total actin reveals a heterogeneous pattern of cell arrangement within the arterial media. B, Schematic diagram demonstrates the structural and cellular heterogeneity within the bovine arterial media. Differences in the mechanical properties of the vessel wall, based on differential arrangement of elastic lamellae, enabled mechanical separation of the arterial media into three layers. Li indicates preluminal media; L2, middle media; L3, outer media.

View this table: [in this window] [in a new window]   Table 1. Four Distinct Cell Subpopulations Existing Within the Mature Bovine Arterial Media

Analysis of both cross and longitudinal tissue sections, schematically represented in the Figure, panel B, showed that the preluminal media (L1) was composed of small, irregularly shaped cells interspersed among fragmented particles of elastin. As shown in the Table, these cells (L1 cells) were found to be negative for all the SM markers analyzed, and therefore their phenotype could be defined as nonmuscle. The inner-middle media (L2 in the Figure and Table) was composed of traditional-appearing, elongated, spindle-shaped cells oriented circumferentially between well-developed continuous elastic lamellae. Immunobiochemical analysis, including both immunostaining and Western blotting techniques, demonstrated that L2 cells expressed -SM-actin, SM-1 MHC isoform, calponin, and desmin (Table). However, these cells did not express any of the alternatively spliced muscle-specific proteins such as SM-2 MHC isoform, meta-vinculin, or SM (high-molecular-weight) caldesmon (Table). The outer media (L3 in the Figure) was composed of two cell subpopulations, each with distinct morphological appearance and unique patterns of cell arrangement. As schematically presented in the Figure, panel B, large spindle-shaped cells, forming compact clusters (termed L3-C), were observed in areas devoid of elastic lamellae and were oriented longitudinally within the vessel wall. A population of thin spindle-shaped cells oriented circumferentially (termed L3-I) was observed in interstitial areas between the compact cell clusters. These cells were interspersed between well-developed, continuous elastic lamellae. Dramatic differences in the expression of muscle-specific markers were observed between these two cell types (Table). L3-Cells expressed all the muscle-specific proteins tested, including the alternatively spliced variants of vinculin, MHC, and caldesmon, and therefore represented SMCs of a differentiated phenotype. L3-I cells, on the other hand, did not express any of the muscle-specific markers analyzed in this study and therefore their phenotype could be defined as nonmuscle.

The differential expression of contractile and cytoskeletal proteins in vascular SMCs demonstrated that multiple cell phenotypes were expressed within the arterial media at a specific location of the vascular tree. However, analysis performed at a single time point (the mature vessel in this case) did not necessarily establish the existence of distinct SMC subtypes, because it could not rule out the possibility that the observed differences in cell phenotype were simply the result of a temporal "phenotypic modulation" of a single SMC type. Therefore, we performed experiments in which we sought to "follow" the developmental fate of each phenotypically unique cell subpopulation identified in the mature media.¹⁵ In contrast to arteries of small rodents, in which cells of unique phenotype cannot be localized to a specific or distinct region of the media, in large bovine arteries, cells with unique phenotypes could be compared at different developmental stages, because they were either localized to a specific medial layer (as L1 and L2 cells residing in preluminal and middle media, respectively) or exhibited a specific pattern of cell arrangement (as L3-C cells forming compact cell clusters). We analyzed the differentiation pattern of the distinct cell subpopulations within the arterial media by assessing the temporal and spatial pattern of their muscle-specific protein expression at various developmental stages. Our data demonstrated that phenotypically distinct medial SMC subpopulations progressed along distinct differentiation pathways during development, therefore suggesting the existence of unique cell lineages.¹⁵

Thus, it appears that the cell composition of the vascular media is complex, with multiple subsets of both SM and "nonmuscle-like" cells existing in close proximity within the arterial media. The cellular complexity of the vascular media raises questions as to why diversity in cell phenotype is necessary for maintaining homeostasis of the vessel wall and what functions various arterial cell subpopulations might serve under normal conditions and in response to injury.

	Unique Responses of Specific SMC Subpopulations to Injury

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In Vivo Studies
The existence of phenotypically distinct SMC subpopulations within the arterial media raises questions as to the roles these subpopulations play in vascular disease. There is a substantial body of experimental evidence demonstrating that pathological lesions in atherosclerosis, restenosis, and hypertension in humans, as well as neointimal thickening in injured vessels of experimental animals, are composed mainly of cells with nonmuscle-like characteristics.^{7 16 17 18 19 20 21 22} Absence or paucity of muscle-specific markers in these cells was in the past usually attributed to the process of dedifferentiation of a single medial SMC type during its migration into the intimal space. However, identification of nonmuscle-like cells in the normal mature vascular media in our studies as well as in studies by other investigators suggests that the nonmuscle-like cells observed in pathological lesions could also originate via expansion of a subset of medial cells lacking expression of SM-specific markers. For example, recent studies by Holifield et al²³ suggested that the intimal thickening seen after balloon injury in canine arteries is the result of selective proliferation of a subset of nonmuscle-like cells in the arterial media rather than dedifferentiation (or so-called "phenotypic modulation") of fully differentiated SMCs. It is important to note, however, that the existence of SMC heterogeneity and the concept of selective "expansion" or selective response of a unique subset of SMCs do not necessarily preclude previous hypotheses regarding the role of phenotypic modulation of SMCs in vascular disease. Rather, it raises multiple possibilities for cellular responses to stress, which may be injury specific, such as phenotypic modulation of differentiated SMCs, selective expansion of a preexisting subset of nonmuscle-like cells, or initial selective activation and/or expansion of a unique subset of nonmuscle-like cells that subsequently modulate the phenotype of other cells within the vessel wall.

In an effort to test the hypothesis of selective cellular responses to injury in the pulmonary circulation, we evaluated the proliferative behavior of the unique cell subpopulations we had identified to hypoxia-induced pulmonary hypertension. We had previously reported marked increases in medial SMC proliferation in hypoxia-induced neonatal pulmonary hypertension.^{4 24} Further, we had shown that two unique SMC subpopulations, residing in close proximity to each other in the outer media, could be differentiated on the basis of selective expression of alternatively spliced variants of three muscle-specific cytoskeletal proteins (the alternatively spliced forms are meta-vinculin, 150-kD caldesmon, and the SM-2 MHC isoform).¹⁵ To determine whether these distinct SMC subpopulations in the neonate exhibited selective proliferative responses to hypoxia-induced pulmonary hypertension, we performed double-label immunofluorescence staining, with polyclonal antibodies against meta-vinculin to differentiate distinct SMC subpopulations and monoclonal antibodies against the nuclear antigen Ki-67 to assess proliferation. Quantitative analysis showed that at every postinjury time point studied, >95% of the overall proliferation occurred within one, the meta-vinculin–negative SMC subpopulation, while the other, meta-vinculin–positive SMC subpopulation remained quiescent.²⁵ These data strongly suggested that distinct SMC subpopulations within neonatal pulmonary arterial media exhibited markedly different growth responses to the same pathophysiological stimuli.

We then sought to determine whether the remarkable differences in proliferative behavior observed in these two SMC subpopulations of the hypertensive neonate were simply due to the fact that one SMC subpopulation exhibited a more "differentiated" phenotype (ie, cells expressing meta-vinculin and other markers of differentiated SM) than the other, thus rendering the differentiated SMCs incapable of proliferation. To address this possibility, we examined the differentiation and proliferative phenotypes of SMC subpopulations in the late period of fetal development, when high rates of SMC proliferation have been reported²⁶ and when SMC subpopulations identical to those described in the neonatal vessel wall exist.¹⁵ We found that although the pattern of cytodifferentiation (based on expression of meta-vinculin and other muscle-specific proteins) and overall cell replication were similar to those of the hypertensive neonate, proliferation in the fetal arterial wall occurred equally in meta-vinculin–expressing and –nonexpressing SMC subpopulations.²⁵ Thus, marked changes in the proliferative capability of specific SMC subpopulations can occur over a very short period of time. This discovery suggests that although the phenotype of SMCs in pathological lesions in various vascular diseases has often been described as "fetal-like," the mechanisms that control SMC proliferation in vascular disease processes, at least in some SMC subpopulations, differ from those operating in normal fetal development.²⁷ Identification of the genes or gene products suppressing proliferation in specific neonatal or adult arterial SMC subpopulations even under injury conditions will be an important goal for future studies and could aid in developing more effective interventions to inhibit abnormal SMC replication.

Because the pathogenesis of neonatal pulmonary hypertension is also characterized by increased production and deposition of extracellular matrix by medial SMCs,^{14 28} we sought to determine whether the distinct pulmonary arterial SMC subpopulations exhibited differential synthetic behavior in normal development and in response to hypoxic hypertension. We focused on studying the expression pattern of TE, because its mature form, elastin, plays a crucial role in determining the structure and function of large outflow arteries and because elastin expression is tightly regulated throughout development. We evaluated expression of TE mRNA at various time points in normal fetal and neonatal development, as well as in response to hypoxic hypertension.²⁹ Using in situ hybridization and immunohistochemical techniques to simultaneously evaluate TE mRNA expression and the cytodifferentiation state of the cell, respectively, we found that TE mRNA exhibited a biphasic pattern of expression during fetal development that seemed to be influenced by two major factors: the differentiation state of the SMCs and the arterial pressure. In early fetal development (<100 days of gestation), TE mRNA was expressed throughout the entire main pulmonary arterial wall. This is a time when pressure in the pulmonary circulation is low and SMCs are poorly differentiated, based on expression of various muscle-specific proteins. In midgestation (180 days), TE mRNA expression decreased, starting in the outer media, which coincided with the appearance of immunoreactivity to cytodifferentiation-related markers, specifically desmin, SM myosin, and calponin in cells of the outermost media. During late gestation (250 to 270 days), coincident with the rise in fetal pulmonary arterial pressure, TE mRNA was reexpressed throughout the entire vessel wall; however, this expression was now in a heterogenic pattern, which correlated with the existence of distinct SMC phenotypes within the arterial media. TE mRNA was now expressed in all SMC populations except those in the outer media, which were expressing meta-vinculin (L3-C cells, see the Figure and Table). After birth, TE mRNA expression in the vessel wall rapidly declined, such that by 1 month, no TE mRNA signal was detected.¹⁴ However, if newborn calves were exposed to hypoxia on the first day of life and developed severe pulmonary hypertension, TE mRNA persisted at high levels in all SMC subpopulations except those marked by expression of meta-vinculin.^{14 29} These data demonstrated the existence of specific SMC subpopulations within the vascular media that differed dramatically in their matrix-producing capabilities. These observations further support the idea that unique SMC subpopulations exist in the arterial media and vary in the roles they play in vascular elastogenesis during normal development and in pathogenesis of vascular disease.

	In Vitro Analysis of SMC Heterogeneity

Top Abstract Introduction SMC Heterogeneity in the... Unique Responses of Specific... In Vitro Analysis of... Summary References

 
Study of the mechanisms conferring unique proliferative and synthetic responses to specific medial SMC subpopulations requires reliable and reproducible in vitro cell models. Several laboratories have demonstrated that arterial SMC subpopulations with unique biochemical and growth properties can be isolated from normal arterial media.^{30 31 32 33} Since the isolated cell subpopulations maintained these characteristics over multiple passages in culture and did not converge to a common phenotype, the observed in vitro cellular diversity has been attributed to the existence of intrinsic heterogeneity of arterial SMCs rather than to the process of phenotypic modulation.

Our goal was to isolate and maintain in culture arterial SMCs with unique characteristics that correlated with the diverse cell subpopulations observed in vivo. We succeeded in isolating four phenotypically unique SMC subpopulations, each exhibiting distinct morphological and biochemical characteristics³⁴ (Table), which could be broadly categorized into nonmuscle-like cells and SM-like cells on the basis of the pattern of expression of -SM-actin and SM myosin. (Note that recent studies^{12 35 36} have demonstrated that SM myosin can serve as a better marker of the SMC differentiation state than -SM-actin.) In general, the nonmuscle and SM-like cell types exhibited very different morphological appearance under culture conditions (Table). Nonmuscle-like cells (expressing little -SM-actin and no SM MHC) appeared rounded in shape and at confluence formed a monolayer. SMCs (expressing high levels of both -SM-actin and SM myosin) appeared spindle shaped and at confluence exhibited the "hill-and-valley" pattern traditionally described for SMCs. The observed morphological and biochemical differences were maintained by the distinct cell types over multiple passages in culture.

We then attempted to investigate whether these phenotypically distinct cell subpopulations would exhibit unique proliferative and synthetic responses to growth-promoting stimuli. We found that the nonmuscle-like cell subpopulations exhibited markedly enhanced growth under serum-stimulated (10% calf serum) conditions compared with SM cell subpopulations. Moreover, nonmuscle-like cells had the potential to grow in plasma-based (10% plasma-derived serum) media, whereas SM-like cells remained quiescent in 10% plasma. Since the nonmuscle-like cells had increased responsiveness to serum stimulation and because PKC activation can contribute to this general pattern of overall enhanced growth capacity,³⁷ we tested whether the nonmuscle-like cells would also be stimulated to a greater extent than SM-like cells by phorbol 12-myristate 13-acetate, a cell-permeable direct activator of PKC. We found that the nonmuscle-like cells had increased DNA synthesis in response to phorbol 12-myristate 13-acetate treatment. Since we had previously found that activation of PKC was a requisite step for SMCs to proliferate under hypoxic conditions, we questioned whether the two cell types would differ in their responses to hypoxic exposure (3% O₂). The nonmuscle-like L3-I cells had significantly augmented DNA synthesis in response to hypoxia compared with SM L2 cells. Specific isozymes of PKC have been linked to specific cell functions, and we had recently shown that the calcium-dependent isozyme of PKC is an important determinant of hypoxic growth capacity.³⁷ We therefore compared the level of expression of the PKC- isoform in the two cell types and found that nonmuscle-like L3-I cells had increased levels of immunodetectable PKC- compared with the SM L2 cells²⁷ (Table). This pattern of isozyme expression was paralleled by increased whole cellular PKC catalytic activity in nonmuscle L3-I cells compared with SM L2 cells (Table). Thus, distinct arterial cell subpopulations with markedly different growth potential, similar to those observed in vivo, can be isolated and maintained in culture. Study of these cell subpopulations in vitro should provide further insight into the signaling pathways that confer unique proliferative properties to specific cell subpopulations within the vascular media in vivo.

The fact that phenotypically distinct SMC populations can differ in their extracellular matrix protein production in culture has been reported by Lemire et al,³⁸ who found that marked differences in TE and versican mRNA expression exist between rat pup and adult aortic SMC phenotypes. In vitro experiments in our laboratory have shown that SMC subpopulations isolated from neonatal bovine MPA markedly differ in TE mRNA expression and maintain these differences over their lifespan in culture. In situ hybridization experiments on aggregate cell populations derived from the whole bovine neonatal pulmonary arterial media demonstrated the existence of distinct and stable cell types with both high and low levels (sometimes none) of TE mRNA expression.³⁹ Since both transcriptional and posttranscriptional mechanisms may contribute to steady state TE mRNA expression, we used a reverse-transcribed polymerase chain reaction assay developed by Swee and colleagues⁴⁰ to determine transcriptional versus posttranscriptional control of TE mRNA in these cells. In contrast to the posttranscriptional mechanisms reported to control TE mRNA steady state levels in rat fetal versus adult lung cells,⁴⁰ we found that the differences in steady state TE mRNA levels of high- versus low-TE–producing cell populations isolated from neonatal bovine pulmonary arterial media were regulated at the transcriptional level.³⁹ This finding provides further evidence for genetic differences between phenotypically distinct cell subpopulations in the vascular media. It further supports the idea that only specific pulmonary arterial SMC subpopulations contribute to the elastogenic response to vascular injury.

	Summary

Top Abstract Introduction SMC Heterogeneity in the... Unique Responses of Specific... In Vitro Analysis of... Summary References

 
Collectively, our in vivo and in vitro findings and those of others support the concept that heterogeneity in the phenotype, growth, and matrix-producing capabilities of different SMC subpopulations exists and that these differences are intrinsic to the cell type. These data also suggest that differential proliferative and matrix-producing capabilities of distinct SMC subpopulations govern, at least in part, the pattern of abnormal cell proliferation and matrix protein synthesis observed in the pathogenesis of vascular disease. Within the pulmonary circulation, the observation that the isolated medial SMC subpopulations exhibit differential proliferative responses to hypoxic exposure is important, since this in vitro cell model system can now be used to better understand the mechanisms that regulate increased responsiveness of specific medial cell subpopulations to low oxygen concentrations. Our data also support the idea that PKC is likely to be one important determinant of differential cell growth responses to hypoxia. The data also suggest differential involvement of specific arterial SMC subpopulations in the elastogenic responses of the vessel wall to injury. We believe that a better understanding of the mechanisms contributing to unique behavior of specific arterial cell subpopulations will provide important future directions for therapies aimed at preventing abnormal cell replication and matrix protein synthesis in vascular disease.

	Selected Abbreviations and Acronyms

 

-SM-actin = -smooth muscle actin MHC = myosin heavy chain MPA = main pulmonary artery PKC = protein kinase C SM = smooth muscle SMC = SM cell TE = tropoelastin

	Acknowledgments

 
This study was supported by NIH SCOR No. HL46481 and PPG No. HL14985. E.C. Dempsey was supported by a Roerig/ACCP Physician Scientist Award, a Veterans Administration grant, and the Giles Filley Research Award from the American Physiological Society. A.G. Durmowicz was supported by a Career Development Award from the American Heart Association.

Received March 3, 1997; accepted March 18, 1997.

	References

Top Abstract Introduction SMC Heterogeneity in the... Unique Responses of Specific... In Vitro Analysis of... Summary References

 

1. Zuckerman BD, Orton E, Stenmark KR, Trapp JA, Murphy JR, Coffeen PR, Reeves JT. Alteration of the pulsatile load in the high altitude calf model of pulmonary hypertension. J Appl Physiol. 1991;70:859-868.[Abstract/Free Full Text]
2. Reeves JT, Durmowicz AG, Weiser MCM, Orton EC, Stenmark KR. Diversity in the pulmonary circulation. Eur Respir Rev. 1993;3:629-637.
3. Ingram RH, Szidon JP, Fishman AP. Response of the main pulmonary artery of dogs to neuronally released versus blood-borne norepinephrine. Circ Res. 1970;26:249-262.[Abstract/Free Full Text]
4. Orton EC, LaRue SM, Ensley B, Stenmark KR. Bromodeoxyuridine labeling and DNA content of pulmonary arterial medial cells from normal and hypoxia exposed calves. Am J Vet Res. 1992;53:1925-1930.[Medline] [Order article via Infotrieve]
5. Mecham RP, Whitehouse LA, Wrenn DS, Parks WC, Griffin GL, Senior RW, Crouch EC, Voelkel NF, Stenmark KR. Smooth muscle–mediated connective tissue remodeling in pulmonary hypertension. Science. 1987;237:423-426.[Abstract/Free Full Text]
6. Majesky MW, Schwartz SM. Smooth muscle diversity in arterial wound repair. Toxicol Pathol. 1990;18:554-559.[Medline] [Order article via Infotrieve]
7. Glukhova MA, Kabakov AE, Frid MG, Ornatsky OI, Belkin AM, Mukhin DN, Orekhov AN, Koteliansky VE, Smirnov VN. Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression. Proc Natl Acad Sci U S A. 1988;85:9542-9546.[Abstract/Free Full Text]
8. Okamoto E, Imataka K, Fujii J, Kuro-o M, Nakahara K, Nishimura H, Yazaki Y, Nagai R. Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of rabbit carotid artery. Biochem Biophys Res Commun. 1992;185:459-464.[Medline] [Order article via Infotrieve]
9. Babaev VR, Bobryshev YV, Stenina OV, Tararak EM, Gabbiani G. Heterogeneity of smooth muscle cells in atheromatous plaque of human aorta. Am J Pathol. 1990;136:1031-1042.[Abstract]
10. Zanellato AM, Borrione AC, Giuriato L, Tonello M, Scannapieco G, Pauletto P, Sartore S. Myosin isoforms and cell heterogeneity in vascular smooth muscle, I: developing and adult bovine aorta. Dev Biol. 1990;141:431-446.[Medline] [Order article via Infotrieve]
11. Osborn M, Caselitz J, Weber K. Heterogeneity of intermediate filament expression in vascular smooth muscle: a gradient in desmin positive cells from the rat aortic arch to the level of the arteria iliaca communis. Differentiation. 1981;20:196-202.[Medline] [Order article via Infotrieve]
12. Frid MG, Printseva OY, Chiavegato A, Faggin E, Scatena M, Koteliansky VE, Pauletto P, Glukhova MA, Sartore S. Myosin heavy chain isoform composition and distribution in developing and adult human aortic smooth muscle. J Vasc Res. 1993;30:279-292.[Medline] [Order article via Infotrieve]
13. Yablonka-Reuveni Z, Schwartz SM, Crist B. Development of chicken aortic smooth muscle: expression of cytoskeletal and basement membrane proteins defines two distinct cell phenotypes emerging from a common lineage. Cell Mol Biol Res. 1995;41:241-249.[Medline] [Order article via Infotrieve]
14. Stenmark KR, Durmowicz AG, Roby JD, Mecham RP, Parks WC. Persistence of the fetal pattern of tropoelastin gene expression in severe neonatal bovine pulmonary hypertension. J Clin Invest. 1994;93:1234-1242.
15. Frid MG, Moiseeva EP, Stenmark KR. Multiple phenotypically distinct smooth muscle cell populations exist in the adult and developing bovine pulmonary arterial media in vivo. Circ Res. 1994;75:669-681.[Abstract/Free Full Text]
16. Pauletto P, Chiavegato A, Giuriato L, Scatena M, Faggin E, Grisenti A, Sarzani R, Paci MV, Fulgeri PD, Rappelli A, Pessina AC, Sartore S. Hyperplastic growth of aortic smooth muscle cells in renovascular hypertensive rabbits is characterized by the expansion of an immature cell phenotype. Circ Res. 1994;74:774-788.[Abstract/Free Full Text]
17. Campbell GR, Campbell JH. The phenotypes of smooth muscle expressed in human atheroma. Ann N Y Acad Sci. 1990;598:143-158.[Medline] [Order article via Infotrieve]
18. Schwartz SM, deBlois D, O'Brien ERM. The intima: soil for atherosclerosis and restenosis. Circ Res. 1995;77:445-465.[Free Full Text]
19. Desmouliere A, Gabbiani G. The cytoskeleton of arterial smooth muscle cells during human and experimental atheromatosis. Kidney Int. 1992;37:S87-S89.
20. Leclerc G, Isner JM, Kearney M, Simons M, Safiani RD, Baim DS, Weir I. 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]
21. Frid MG, Shekhonin BV, Koteliansky VE, Glukhova MA. Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin. Dev Biol. 1992;153:185-193.[Medline] [Order article via Infotrieve]
22. Kuro-o M, Nagai R, Nakahara K-i, Katoh H, Tsai R-C, Tsuchimochi H, Yazaki Y, Ohkubos A, Takaku F. cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in atherosclerosis. J Biol Chem. 1991;266:3768-3773.[Abstract/Free Full Text]
23. Holifield B, Helgason T, Jemelka S, Taylor A, Navran S, Allen J, Seidel C. Differentiated vascular myocytes: are they involved in neointimal formation? J Clin Invest. 1996;97:814-825.[Medline] [Order article via Infotrieve]
24. Belknap KJ, Orton EC, Ensley B, Stenmark KR. Hypoxia increases bromodeoxyuridine labeling indices in bovine neonatal pulmonary arteries. Am J Respir Cell Mol Biol. 1997;16:366-371.[Abstract]
25. Wohrley JD, Frid MG, Orton EC, Belknap JK, Stenmark KR. Hypoxia selectively induces proliferation in a specific subpopulation of smooth muscle cells in the bovine neonatal pulmonary arterial media. J Clin Invest. 1995;96:273-281.
26. 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]
27. Xu Y, Stenmark KR, Walchak SJ, Ruff LJ, Dempsey EC. Pulmonary artery smooth muscle cells from chronically hypoxic neonatal calves retain fetal-like and acquire new growth properties: importance of the hypoxia-induced proliferative phenotype and calcium-dependent isozymes of protein kinase C-alpha(). Am J Physiol. 1997. In press.
28. Stenmark KR, Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol. 1997;59:89-144.[Medline] [Order article via Infotrieve]
29. Durmowicz AG, Frid MG, Wohrley JD, Stenmark KR. Expression and localization of tropoelastin mRNA in the developing bovine pulmonary artery is dependent on vascular cell phenotype. Am J Respir Cell Mol Biol. 1996;14:569-576.[Abstract]
30. Schwartz SM, Foy L, Bowen-Pope DF, Ross R. Derivation and properties of platelet-derived growth factor–independent rat smooth muscle cells. Am J Pathol. 1990;136:1417-1428.[Abstract]
31. Fujita H, Shimokado K, Yutani C, Takaichi S, Masuda J, Ogata J. Human neonatal and adult vascular smooth muscle cells in culture. Exp Mol Pathol. 1993;58:25-39.[Medline] [Order article via Infotrieve]
32. Bochaton-Piallat M-L, Ropraz P, Gabbiani F, Gabbiani G. Phenotypic heterogeneity of rat arterial smooth muscle cell clones: implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol. 1996;16:815-820.[Abstract/Free Full Text]
33. Topouzis S, Majesky MW. Smooth muscle lineage diversity in the chick embryo. Dev Biol. 1996;178:430-445.
34. Frid MG, Stenmark KR. Distinct pulmonary arterial smooth muscle cell populations exhibit unique growth characteristics in vitro. Am J Respir Crit Care Med. 1995;141:A526. Abstract.
35. Miano J, Cserjesi P, Ligon K, Perisamy M, Olson EN. Smooth muscle myosin heavy chain marks exclusively the smooth muscle lineage during mouse embryogenesis. Circ Res. 1994;75:803-812.[Abstract/Free Full Text]
36. Aikawa M, Sivam PN, Kuro-o M, Kimura K, Nakahara K, Takewaki S, Ueda M, Yamaguchi H, Yazaki Y, Periasamy M, Nagai R. Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis. Circ Res. 1993;73:1000-1012.[Abstract/Free Full Text]
37. Dempsey EC, Badesch DB, Dobyns EL, Stenmark KR. Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: dependence on cell size, time from birth, insulin-like growth factor 1, and auto-activation of protein kinase C. J Cell Physiol. 1994;160:469-481.[Medline] [Order article via Infotrieve]
38. Lemire JM, Potter-Perigo S, Hall KL, Wight TN, Schwartz SM. Distinct rat aortic smooth muscle cells differ in versican/Pg-M expression. Arterioscler Thromb Vasc Biol. 1996;16:821-829.[Abstract/Free Full Text]
39. Durmowicz AG, Swee MH, Frid MG, Stenmark KR, Parks WC. The differential expression of tropoelastin mRNA by pulmonary artery smooth muscle cells is transcriptionally regulated. FASEB J. 1995;9:A294. Abstract.
40. Swee MH, Parks WC, Pierce RA. Developmental regulation of elastin production: expression of TE pre-mRNA persists after downregulation of steady-state mRNA levels. J Biol Chem. 1995;270:14899-14906.[Abstract/Free Full Text]

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