Novel Genes for Mitogen-Independent Smooth Muscle Replication
Vascular smooth muscle cells (SMCs) are generally assumed to require mitogenic stimulation to replicate. The mitogen may originate in the circulation, be derived from nearby cell types in the vessel wall, or be produced by SMCs themselves. Regardless of the source of the mitogen, requirements for activation of cell surface growth factor receptors and consequent downstream signaling pathways have been guiding principles to understand SMC growth control in vivo. This long-held assumption is being challenged by Weiser-Evans et al1 in this issue of Circulation Research. These authors report the cloning of novel genes expressed by SMCs that exhibit a mitogen-independent or autonomous growth phenotype in vitro. One of these cDNAs, emb8:embryonic growth–associated protein (emb8:EGAP), may be at least partially responsible for controlling autonomous growth potential in SMCs. The authors suggest that these newly identified emb genes may represent an entirely novel class of genes that confer autonomous growth potential to SMCs during high rates of replication in vivo (during development, wound repair, and intimal disease).
Autonomous Growth Phenotype
The concept of a self-driven, autonomous growth phenotype for vascular SMCs stems, in part, from work begun by Cook et al2 about 10 years ago. Their initial goal was to define factors produced during normal vascular development that function to shut off SMC replication in the late fetal and early postnatal periods. The assumption was that SMCs produce a growth-inhibitory pericellular matrix and that a better understanding of the composition of this natural growth inhibitory matrix and how it was produced would lead to more effective inhibitors of SMC proliferation in diseased adult vessels. A necessary first step was to precisely determine when SMCs stopped proliferating during development of the rat aorta in utero. Analysis of bromodeoxyuridine (BrdU)-labeling index values showed a sharp transition from very high replication rates (70% to 80% per day) at embryonic days 13 to 17 (e13 to e17) to ≈20% per day at e19 to birth. The first surprise came when e17 aortic SMCs placed in cell culture were found to proliferate at high rates (up to 40% per day) in defined medium entirely devoid of exogenous growth factors. Comparable cultures of adult aortic SMCs became growth arrested and failed to replicate under identical conditions. The ability of embryonic SMCs (eSMCs) to proliferate in the complete absence of whole blood serum or plasma-derived serum supplements was a stable property independent of passage number or population doublings.2 Autonomously replicating eSMCs failed to secrete detectable mitogenic activity into conditioned medium and were mitogenically unresponsive to a variety of known SMC growth factors, including platelet-derived growth factor (PDGF), fibroblast growth factor-2, epidermal growth factor, or 10% calf serum. Taken together, these results raised the possibility of a unique mechanism of SMC growth control, one that seemed to be self-regulated and uncoupled from a dependence on exogenous growth factors. The capacity for self-driven SMC replication was lost by e20, correlating in time with a large drop in BrdU-labeling index values in fetal rat aorta in vivo. These early studies suggested that the high SMC replication rates observed in embryonic rat aorta in vivo are driven by mechanisms intrinsic to eSMCs and that developmentally timed loss of this phenotype results in the acquisition of proliferative quiescence during normal vascular development.
Subsequent work showed that an autonomous growth phenotype is reexpressed in adult rat carotid SMCs during neointimal formation after balloon injury.3 As obtained from the carotid artery wall 7 days after injury, autonomously replicating neo7 SMCs in vitro are epithelioid in shape and proliferate in serum-free medium at rates comparable to those exhibited by e17 SMCs. As observed for eSMCs, adult neo7 SMCs also failed to secrete detectable mitogenic activity into conditioned medium.3 Furthermore, direct coculture of neo7 SMCs with mitogen-dependent adult aortic SMCs failed to stimulate DNA synthesis in the target aortic SMCs.3 The autonomous growth behavior of SMCs from injured carotid arteries was a transient property that could no longer be observed in cells isolated from vessels at 21 and 28 days after balloon injury. These findings were consistent with a large body of literature suggesting that adult rat carotid SMCs reexpress a developmental sequence during neointimal formation after arterial injury.
What is the mechanism for mitogen-independent, autonomous SMC growth? To address this question, Majack4 examined the growth phenotypes of heterokaryons made by fusing autonomously growing e17 SMCs with mitogen-dependent adult SMCs. Heterokaryon analysis had been used previously to study mechanisms involved in tumor suppression, cellular differentiation, postmitotic growth state, and apoptosis.5 The results clearly showed that the adult, mitogen-dependent phenotype was dominant and that heterokaryons required serum or growth factor supplements to replicate.4 This was also found to be true for heterokaryons made by fusing adult carotid neo7 SMCs with mitogen-dependent adult aortic SMCs.3 These results led to the concept that mitogen-dependent adult SMCs produce an intracellular suppressor that extinguishes the expression of genes required to maintain the autonomous growth phenotype.4 Although the molecular nature of this autonomous growth suppressor activity awaits additional characterization, the heterokaryon results failed to provide insight into the identity or functional properties of genes responsible for conferring autonomous growth properties on embryonic and neointimal SMCs. The cloning studies described by Weiser-Evans et al1 were designed with this important objective in mind.
Cloning and Analysis of Emb Genes
A cDNA library constructed from autonomously growing eSMCs was screened with a subtracted eSMC cDNA probe from which sequences common to proliferating mitogen-dependent adult SMCs had been removed. The subtracted probe identified cDNA clones representing 3% of the total eSMC cDNA library. After several iterations and selections, a manageable set of 14 independent clones was obtained and called emb genes.1 Sequence comparison against the GenBank database identified 3 known genes (pendulin, S6 ribosomal protein, and cdc25A) and 11 potentially novel genes. One of these novel genes, emb8:EGAP, was representative and selected for additional study. Emb8:EGAP is widely expressed throughout the early embryo, including aortic SMCs. Although emb8:EGAP mRNA levels declined abruptly around e18 to e19 and were undetectable in postnatal aortic SMCs, this was not true for all tissues. High levels of emb8:EGAP expression were maintained in adult spermatocytes, intestinal crypts, basal epidermis, and interstitial fibroblasts of skeletal muscle. Emb8:EGAP was reexpressed after carotid injury in adult rats in a pattern that closely matched the gain and loss of autonomous growth potential in vitro. Emb8:EGAP encodes a 725-amino acid protein that contains a nuclear localization sequence and lacks a signal sequence for sorting by the secretory pathway. A BLAST search of expressed sequence tag databases showed strongly conserved homologues of emb8:EGAP among human, mouse, Drosophila and Caenorhabditis elegans expressed sequence tags.
Expression data alone are often misinterpreted as evidence for a functional role of a gene product in the tissues and cell types where it is expressed. Loss-of-function experiments are required to critically test for biological activity. Weiser-Evans et al1 added sense and antisense emb8:EGAP oligodeoxynucleotides (ODN) to eSMCs, neo7 SMCs, and mitogen-dependent adult SMC cultures to determine if emb8:EGAP plays a functional role in autonomous SMC growth. The results showed that antisense ODN (5 μmol/L) reduced BrdU-labeling index values in serum-free medium by 57% for neo7 SMCs and by 20% for eSMCs but had no effect on BrdU index values for mitogen-dependent adult SMCs stimulated with 10% whole blood serum. Sense ODN had no effect, and neutralizing antibodies to interferon-γ did not reverse growth inhibition by antisense ODN. Lack of more substantial inhibition of autonomous SMC growth by antisense emb8:EGAP ODN may indicate that strategies to inhibit multiple emb genes at the same time will be required to see greater inhibition. These data offer support, although not proof, that emb8:EGAP may play an important functional role in conferring autonomous growth potential on vascular SMCs in which it is expressed. Critical next steps will be to verify that emb8:EGAP antisense ODN reduces target RNA and emb8:EGAP protein levels in cells whose growth is inhibited; determine if forced expression of emb8:EGAP in mitogen-dependent SMCs converts them to a mitogen-independent growth phenotype; characterize emb gene transcriptional regulation and, in particular, whether emb genes exhibit positive autoregulation; determine what the adult autonomous growth suppressor activity is; and make germ-line mutations in emb8:EGAP in mice to assess their effects on embryonic SMC replication and vascular development in vivo.
Summary and Implications
Although the significance of emb genes for SMC growth control awaits the results of additional studies, it important to ask whether and to what extent the autonomous growth phenotype described by Weiser-Evans et al1 resembles the kinds of SMC diversity previously reported by others. In this regard, the most striking similarity is with a PDGF-independent subset of adult rat aortic SMCs described by Schwartz et al.6 This SMC subtype was identified by its ability to be grown and serially passaged in mitogen-depleted plasma-derived serum. Like the autonomously growing SMCs described above, PDGF-independent SMCs are epithelioid in shape, do not secrete mitogenic activity into conditioned medium, express PDGF receptors, and exhibit only marginal mitogenic responses to exogenous PDGF.6 Similarly, neointimal SMCs isolated 14 days after balloon injury to rat carotid artery were also found to be epithelioid in shape and to grow in the absence of exogenous PDGF.7 However, unlike neo7 SMCs described above, intimal SMCs described by Walker et al7 secreted mitogenic activity into conditioned medium and produced PDGF themselves. In fact, the properties of neointimal SMCs described by Walker et al7 strongly resemble a subset of epithelioid SMCs derived from newborn rat aortas,8 9 adult rat aortas,10 and inner media of bovine aorta and main pulmonary artery.11 Frid et al11 have recently made significant progress toward identification of critical intracellular pathways that maintain autonomous growth potential in bovine medial SMCs.
In summary, it is now apparent that SMC diversity is an important feature of the structure and function of normal arteries. This diversity extends to the level of SMC growth control, but the number of distinct growth phenotypes and the control of their expression in the vessel wall remain unclear. The cloning of SMC emb genes by Weiser-Evans et al1 provides an important new set of tools to reexamine these questions and additionally explore the implications of autonomous growth potential for SMC growth control in the intact artery wall.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- © 2000 American Heart Association, Inc.
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