Novel embryonic genes are preferentially expressed by autonomously replicating rat embryonic and neointimal smooth muscle cells.

We sought to identify and characterize the expression pattern of genes expressed by smooth muscle cells (SMCs) during periods of self-driven replication during vascular development and after vascular injury. Primary screening of a rat embryonic aortic SMC-specific cDNA library was accomplished with an autonomous embryonic SMC-enriched, nonautonomous adult SMC-subtracted cDNA probe. Positive clones were rescreened in parallel with embryonic SMC-specific and adult SMC-specific cDNA probes. We identified 14 clones that hybridized only with the embryonic cDNA ("emb" clones), 11 of which did not share significant homology with sequences in any of the databases. Five of these novel emb genes (emb7, emb8, emb20, emb37, and emb41) were selectively and only transiently reexpressed in vivo by neointimal SMCs during periods of rapid replication. The emb8:embryonic growth-associated protein (EGAP), which was studied the most extensively, was expressed at high levels by cultured, autonomously replicating embryonic and neointimal SMCs but was detected only at low levels even in mitogenically stimulated adult SMCs. Finally, the administration of antisense EGAP oligonucleotides markedly attenuated embryonic and neointimal SMC replication rates. We suggest that autonomous replication of SMCs may be essential for normal vascular morphogenesis and for the vascular response to injury and that these newly identified "embryonic" genes may be part of the molecular machinery that drives this unique growth phenotype.

Relationship between perlecan and tropoelastin gene expression and cell replication in the developing rat pulmonary vasculature.

Smooth-muscle-cell (SMC) replication and extracellular matrix protein expression are two vital and interrelated processes necessary for normal development of the vasculature. To understand better the nature of this relationship in the developing rat lung, we investigated the relationship between SMC proliferation and the expression of perlecan, a basement membrane (BM) heparan sulfate proteoglycan implicated in the control of SMC growth and differentiation, and tropoelastin (TE), a structural matrix protein not known to influence directly the replicative state of SMCs. Using bromodeoxyuridine (BrdU) incorporation to assess DNA synthesis, we first established the time course of SMC proliferation in the hilar pulmonary artery (PA) from embryonic to adult life. We found a labeling index of > 80% during the embryonic period (embryonic Day 13 [e13] to fetal Day 18 [f18]), a dramatic decline to approximately 40% during the fetal period of development, and a steady decrease in proliferation rates following birth such that, by 30 d of age, a labeling index of < 2% was noted. Using in situ hybridization, we found that although peak expression of both perlecan and TE messenger RNA (mRNA) occurred in the fetal and early postnatal periods following the major decrease in cell replication, TE mRNA expression was clearly observed in the PA as early as embryonic Day 14, whereas perlecan transcripts were virtually undetectable until fetal Day 19. Therefore, to evaluate further the relationship between cell replication and perlecan and/or TE gene expression, we used a combined in situ hybridization/BrdU immunohistochemistry technique and demonstrated that, on an individual cell basis, perlecan message was predominantly expressed by nonreplicating (BrdU-negative) PA, whereas TE mRNA was equally expressed in replicating and nonreplicating PA SMCs. Interestingly, a very similar pattern of replication and relationship to perlecan and TE mRNA expression was noted in airway SMCs and epithelial cells. Thus, in the lung as a whole, maximal expression of both the BM protein perlecan and the interstitial matrix protein TE occurs coordinately and follows the period of maximal SMC proliferation. However, in individual SMCs, perlecan mRNA expression varies inversely with DNA synthesis, whereas TE mRNA expression appears independent of the proliferative state of the cell.

Perlecan regulates Oct-1 gene expression in vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) are very quiescent in the mature vessel and exhibit a remarkable phenotype-dependent diversity in gene expression that may reflect the growth responsiveness of these cells under a variety of normal and pathological conditions. In this report, we describe the expression pattern of Oct-1, a member of a family of transcription factors involved in cell growth processes, in cultured and in in vivo SMCs. Oct-1 mRNA was undetectable in the contractile-state in vivo SMCs; was induced upon disruption of in vivo SMC-extracellular matrix interactions; and was constitutively expressed by cultured SMCs. Oct-1 transcripts were repressed when cultured SMCs were plated on Engelbreth-Holm-Swarm tumor-derived basement membranes (EHS-BM) but were rapidly induced after disruption of SMC-EHS-BM contacts; reexpression was regulated at the transcriptional level. To identify the EHS-BM component involved in the active repression of Oct-1 mRNA expression, SMCs were plated on laminin, type IV collagen, fibronectin, or perlecan matrices. Oct-1 mRNA levels were readily detectable when SMCs were cultured on matrices composed of laminin, type IV collagen, or fibronectin but were repressed when SMCs were cultured on perlecan matrices. Finally, the Oct-1-suppressing activity of EHS-BM was sensitive to heparinase digestion but not to chondroitinase ABC or hyaluronidase digestion, suggesting that the heparan sulfate side chains of perlecan play a biologically important role in negatively regulating the expression of Oct-1 transcripts.

Developmental regulation of perlecan gene expression in aortic smooth muscle cells.

Heparan sulfate proteoglycans (HSPGs) are believed to act as potent endogenous regulators of vascular smooth muscle cell (SMC) replication, migration, gene expression and differentiation. Here we describe the pattern of expression of perlecan, the predominant basement membrane HSPG, during aortic development in the rat. Expression of perlecan mRNA and protein in the aortic SMC was first significantly observed at day e19 (day 19 of embryonic development), a time which marks a dramatic switch in SMC replication rate and growth phenotype. Expression of perlecan message and protein was high throughout fetal and early neonatal life, and it remained readily detectable in the adult aorta. Using a double-labeling technique (in situ hybridization for perlecan message coupled with bromodeoxyuridine immunohistochemistry), we determined the relationship between DNA synthesis and perlecan mRNA expression in individual SMC at days e17-e21; we found that perlecan gene expression was largely limited to non-replicating cells. Consistent with the in vivo data, perlecan mRNA was undetectable in cultured e17 SMC by Northern or RT-PCR analysis, while in cultured adult SMC, perlecan mRNA was significantly higher in non-replicating (serum-starved) cultures compared to replicating cultures. Treatment of growth-arrested adult SMC cultures with heparin caused a further accumulation in perlecan mRNA levels. The data suggest that the expression of perlecan by vascular SMC is regulated by apparent developmental age as well as by cellular growth state. The developmentally times expression of perlecan in the aortic wall may contribute to the establishment and/or maintenance of vascular SMC differentiation and quiescence.

Developmental regulation of angiotensin converting enzyme and angiotensin type 1 receptor in the rat pulmonary circulation.

Factors that influence the development of the normal pulmonary vasculature are poorly understood. Since increased local production of angiotensin II (AII) by angiotensin converting enzyme (ACE) has been implicated in the medial hypertrophy of systemic and pulmonary hypertension, we questioned whether ACE and angiotensin receptor expression may influence the muscularization of the normal pulmonary vasculature during development. The approach employed measurement of lung ACE activity, assessment of local ACE expression by immunohistochemistry, and angiotensin type 1 receptor (AT1) expression by in situ hybridization in rat lungs ranging from 15 days of intrauterine life (term = 21 d) to adulthood. The temporal and spatial pattern of ACE expression was compared with that of the endothelial marker, von Willebrand factor (vWF), and the smooth muscle cell markers, alpha smooth muscle actin and smooth muscle myosin. ACE activity was first detected in lung homogenates on day 17 of gestation (1 +/- 0.2 mU/mg) and increased progressively to term (27.7 +/- 3.2 mU/mg). However, the greatest increase in lung ACE activity to adult levels (379 +/- 25.2 mU/mg) occurred between 2 and 4 wk of postnatal life. Immunohistochemistry demonstrated vWF expression by vascular endothelium throughout the lung as early as day 15 of gestation. In contrast, ACE expression was observed in the endothelium of only hilar pulmonary arteries on day 15 of gestation, and thereafter was noted to be expressed in endothelial cells of progressively more distal arteries, such that by term, endothelial cells of all muscularized arteries expressed ACE. Alveolar capillary ACE expression was not detected until day 20 of gestation, and increased dramatically after birth. Smooth muscle actin expression in lung arteries closely paralleled the expression of endothelial ACE. AT1 receptor mRNA was first expressed in the peripheral lung on day 17 of gestation by non-epithelial undifferentiated mesenchyme. In contrast, AT1 mRNA signal was much reduced in differentiated smooth muscle. We speculate that ACE expression in the fetal lung circulation may influence the muscularization of fetal pulmonary arteries by the interaction of locally produced angiotensin II with the AT1 receptor.

Smooth muscle cells isolated from the neointima after vascular injury exhibit altered responses to platelet-derived growth factor and other stimuli.

A variety of evidence suggests that vascular smooth muscle cells (SMC) exhibit a more immature phenotype when stimulated by injury to replicate in the adult. One growth characteristic common to immature (embryonic, fetal, and neonatal) SMC is a markedly reduced responsiveness to platelet-derived growth factor (PDGF) and other mitogenic stimuli. Here we demonstrate that SMC isolated from the 14-day neointima of experimentally injured carotid arteries exhibit a similar growth phenotype. The proliferative responses of neointimal cells to the BB homodimer of PDGF, which interacts with both forms of the PDGF receptor, were up to twenty-fold less (as assessed by BrdU immunocytochemistry) than that of adult control tunica media cells over a wide range of PDGF concentrations. Paradoxically, these cells expressed abundant mRNA for the alpha- and beta-subunits of the PDGF receptor (by RT-PCR) and expressed abundant PDGF receptor protein (by Western blotting). Addition of PDGF-BB to neointimal SMC induced significant autophosphorylation of the PDGF receptor, suggesting that the PDGF receptors were fully functional. The chemotactic responses of neointimal SMC to PDGF, in in vitro migration assays, were identical to that of control medial cells. The data further establish the existence of vascular SMC phenotypes characterized by a refractoriness to growth stimulation by specific mitogens, and provide further evidence for the reiteration of developmentally regulated programs following vascular injury in vivo.

Tropoelastin gene expression in individual vascular smooth muscle cells. Relationship to DNA synthesis during vascular development and after arterial injury.

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 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 a 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 pre-elastogenic 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 temporarily sequential changes in phenotype reflective of SMC development.

Multiple growth factors are released from mechanically injured vascular smooth muscle cells.

Local release of mitogenic and chemotactic signals during angioplasty-induced vascular injury may initiate restenosis. We investigated whether mechanical injury to vascular smooth muscle cells (VSMC) results in the release of biologically active peptide growth factors. Monolayers of bovine SMC cultures were mechanically injured by cell scraping. Conditioned medium (CM) from control and injured SMC cultures was collected, and the mitogenic activity was measured by [3H]thymidine incorporation in recipient SMC cultures. Mitogenic activity from injured CM was detected within 15 min after injury. When the CM from injured cells was removed 15 min after injury and replaced with serum-free media, there was no detectable mitogenic activity in the replacement CM assessed 1-6 days postinjury. Suramin, a nonspecific peptide growth factor antagonist, significantly inhibited the mitogenic activity of injured CM. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF A chain), and epidermal growth factor (EGF) were detected in CM from injured cells by immunoblot analysis. The mitogenic activity of injured CM was significantly inhibited with neutralizing antibodies to bFGF (34%), PDGF-AA (32%), PDGF-BB (25%), and EGF (25%). A neutralizing antibody to transforming growth factor (TGF)-beta had no effect. In conclusion, bFGF, PDGF, and EGF are immediately released from mechanically injured VSMC. VSMC likely contain preformed, biologically active growth factors that are efficiently released from the cell cytoplasm following mechanical injury. Conditioned medium from injured VSMC is highly mitogenic, and this activity is probably due to multiple growth factors interacting synergistically.

Quantitative export of FGF-2 occurs through an alternative, energy-dependent, non-ER/Golgi pathway.

Although basic fibroblast growth factor (bFGF/FGF-2) is found outside cells, it lacks a conventional signal peptide sequence; the mechanism underlying its export from cells is therefore unknown. Using a transient COS-1 cell expression system, we have identified a novel membrane-associated transport pathway that mediates export of FGF-2. This export pathway is specific for the 18-kD isoform of FGF-2, is resistant to the anti-Golgi effects of Brefeldin A, and is energy-dependent. In FGF-2-transfected COS-1 cells, this ER/Golgi-independent pathway appears to be constitutively active and functions to quantitatively export metabolically-labeled 18-kD FGF-2. Co-transfection and co-immunoprecipitation experiments, using a vector encoding the cytoplasmic protein neomycin phosphotransferase, further demonstrated the selectivity of this export pathway for FGF-2. When neomycin phosphotransferase was appended to the COOH-terminus of 18-kD FGF-2, the chimera was exported. However, the transmembrane anchor sequence of the integral membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) blocked export. The chimeric protein localized to the plasma membrane with its FGF-2 domain extracellular and remained cell-associated following alkaline carbonate extraction. Taken together, the data suggest that FGF-2 is "exported" from cells via a unique cellular pathway, which is clearly distinct from classical signal peptide-mediated secretion. This model system provides a basis for the development and testing of therapeutic agents which may block FGF-2 export. Such an intervention may be of considerable use for the treatment of angiogenesis-dependent diseases involving FGF-2.

Static tension is associated with increased smooth muscle cell DNA synthesis in rat pulmonary arteries.

During the development of pulmonary hypertension, vascular cell proliferation closely parallels the rise in pulmonary intravascular pressure. The possible direct physical effect that elevated pressures may have on inducing vascular cell proliferation in pulmonary hypertension is unclear. To address this question, static force (0, 1, 5, and 10 g) was applied to hilar pulmonary arterial rings cultured in a serum-free medium. Incorporation of the thymidine analogue, bromodeoxyuridine (BrdU), into medial and adventitial cells was analyzed by immunohistochemistry. Medial cell BrdU incorporation (%positive cells) was increased (P < 0.0001) at all levels of force compared with 0-g controls (unmounted and mounted, but without applied force) (unmounted: 0.65 +/- 0.08; mounted: 0 g, 1.8 +/- 0.39; 1 g, 3.7 +/- 0.35; 5 g, 5.2 +/- 0.43; 10 g, 2.8 +/- 0.17). Hypoxia exposure and endothelial denudation of arteries attenuated (P < 0.05) tension-induced medial cell BrdU labeling (2.5 +/- 0.96 and 3.3 +/- 0.63, respectively) compared with control arteries (6.0 +/- 0.54). Nifedipine reduced tension-induced medial cell BrdU incorporation (P < 0.05). There was no difference in DNA synthesis in adventitial cells at the various levels of force, although hypoxia decreased adventitial cell BrdU incorporation overall (P < 0.05). We conclude that static wall tension may be an important direct stimulus for medial cell DNA synthesis.

Extinction of autonomous growth potential in embryonic: adult vascular smooth muscle cell heterokaryons.

Vascular smooth muscle cells (SMC) isolated from embyronic and early fetal (e13-e18) rat aortas exhibit an "embryonic growth phenotype" in culture (Cook, C. L., M. C. M. Weiser, P. E. Schwartz, C. L. Jones, and R. A. Majack. 1994. Circ. Res. 74:189-196). Cells in this growth phenotype exhibit autonomous, serum-independent replication, in contrast to SMC in the "adult" growth phenotype, whose proliferation in culture is dependent on exogenous mitogens. To determine which of these two phenotypes is genetically dominant, heterokaryons were constructed between adult and embryonic (day e17) rat aortic SMC. The fused cells were maintained in serum-free medium for 3 d, then were labeled with bromodeoxyuridine (BrdU) for an additional 24 h. Under these conditions, parental e17 SMC exhibited a high rate of self-driven DNA synthesis (73-85% BrdU-positive cells), while parental adult SMC showed minimal replication (13-21% BrdU-positive cells). Homokaryons of parental cells exhibited parental growth phenotypes and showed the expected mitogenic response when stimulated with serum. Heterokaryons between e17 and adult SMC exhibited a nonautonomous growth phenotype; the "adult" growth phenotype was calculated to be dominant in > 89% of all true heterokaryons. The data suggest that adult SMC express molecules capable of genetically extinguishing or otherwise inhibiting the autonomous replication of embryonic SMC.

Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells.

Little is known about the phenotypic changes that occur in vascular smooth muscle cells (SMCs) as the developing aorta undergoes the transition from a loosely organized, highly replicative tissue to a morphologically mature, quiescent tissue. In the present study, we have characterized the in vivo pattern of SMC replication during intrauterine and neonatal aortic development in the rat and have cultured and assessed the in vitro growth properties of embryonic, fetal, and neonatal vascular SMCs. Embryonic SMCs, which exhibited a very high in vivo replication rate (75% to 80% per day), demonstrated a significant potential for self-driven replication, as assessed by the ability to proliferate under serum-deprived conditions. Several lines of evidence suggest that the autonomous growth of SMCs in the "embryonic growth phenotype" may be driven by a unique mechanism independent of known adult SMC mitogens: embryonic SMC replication was not associated with the detectable secretion of mitogenic activity capable of stimulating adult SMCs, and embryonic SMCs were mitogenically unresponsive to a variety of known adult SMC growth factors. The capacity for self-driven growth was lost by embryonic day 20, suggesting that important changes in gene expression and phenotype occur in developing SMCs between embryonic days 18 and 20. Taken together, the data describe a unique embryonic growth phenotype of vascular SMCs and suggest that the replication of aortic SMCs during intrauterine development is self driven, self regulated, and controlled by a developmental timing mechanism. The conversion of SMCs from the embryonic to the late fetal/adult growth phenotype will likely be found to be an important component of a developmental system controlling vascular morphogenesis.

Role of basic fibroblast growth factor in vascular lesion formation.

In the present study we investigated whether basic fibroblast growth factor (bFGF) plays a role in the proliferative response of smooth muscle cells (SMCs) to denuding injury. Rat carotid smooth muscle was found to express the mRNA for bFGF, and bFGF protein was found to be present in rat aorta by immunoblot analysis. Systemically administered bFGF was a potent mitogen for vascular SMCs in arteries denuded with a balloon catheter, increasing replication from 11.5% in controls to 54.8%. Denudation with a device (filament loop), which causes only minimal damage to medial SMCs, showed a similar increase in replication (1.3% versus 43.3%) after bFGF infusion. In unmanipulated vessels, however, SMCs were unresponsive to infused bFGF. Infusion of a "mitotoxin" (bFGF conjugated to saporin) caused a greater than 50% decrease in the number of viable SMCs in the arterial wall after balloon injury. Prolonged administration of bFGF (12 micrograms/day for 2 weeks) after balloon injury caused an approximately twofold increase in intimal thickening. These results show that bFGF, which is synthesized by the arterial wall, could be a potent mitogen for SMCs in vivo and suggest that any release of endogenous bFGF may be capable of stimulating SMC proliferation, which may subsequently lead to intimal lesion formation.

Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory peptide that can inhibit or promote the proliferation of cultured vascular smooth muscle cells (SMCs), depending on cell density (Majack, R. A. 1987. J. Cell Biol. 105:465-471). In this study, we have examined the mechanisms underlying the growth-promoting effects of TGF-beta in confluent SMC cultures. In mitogenesis assays using confluent cells, TGF-beta was found to potentiate the stimulatory effects of serum, PDGF, and basic fibroblast growth factor (bFGF), and was shown to act individually as a mitogen for SMC. In gene and protein expression experiments, TGF-beta was found to regulate the expression of PDGF-A and thrombospondin, two potential mediators of SMC proliferative events. The induction of thrombospondin protein and mRNA was density-dependent, delayed relative to its induction by PDGF and, based on cycloheximide experiments, appeared to depend on the de novo synthesis of an intermediary protein (probably PDGF-A). The relationship between PDGF-A expression and TGF-beta-mediated mitogenesis was investigated, and it was determined that a PDGF-like activity (probably PDGF-A) was the biological mediator of the growth-stimulatory effects of TGF-beta on confluent SMC. The effects of purified homodimers of PDGF-A on SMC replication were investigated, and it was determined that PDGF-AA was mitogenic for cultured SMC, particularly when used in combination with other growth factors such as bFGF and PDGF-BB. The data suggest several molecular mechanisms that may account for the ability of TGF-beta to promote the growth of confluent SMC in culture.

Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries.

A large percentage of vascular reconstructions, endarterectomies, and angioplasties fail postoperatively due to thrombosis and restenosis. Many of these failures are thought to result from an inability of the vascular endothelium to adequately regenerate and cover the denuded area. After balloon catheter denudation of the rat carotid artery, regrowth of endothelium ceases after approximately 6 wk, leaving a large area devoid of endothelium. Here we show that this cessation of reendothelialization can be overcome by the systemic administration of basic fibroblast growth factor (bFGF). Administration of 120 micrograms bFGF over an 8-h period caused a highly significant increase in the replication rate of endothelial cells at the leading edge of 38.5 vs. 2.1% in controls, and, when given over a longer period of time (12 micrograms daily for 12 d), resulted in a significant increase in the extent of endothelial outgrowth onto the denuded surface. Furthermore, total regrowth could be achieved within 10 wk after balloon catheter denudation when 12 micrograms bFGF was injected twice per week for a period of 8 wk. Endothelium in unmanipulated arteries responded to bFGF with a significant increase in replication, but no increase in endothelial cell density was observed in these arteries. These data demonstrate that bFGF can act as a potent mitogen for vascular endothelial cells in vivo, and add considerably to our understanding of the mechanism underlying endothelial repair after in vivo vascular injuries.

Vascular smooth muscle cells express distinct transforming growth factor-beta receptor phenotypes as a function of cell density in culture.

Transforming growth factor-beta (TGF-beta) is a bifunctional, density-dependent regulator of vascular smooth muscle cell (SMC) proliferation in vitro (at sparse densities SMC are growth-inhibited by the peptide, whereas at confluent densities TGF-beta potentiates their growth). We have used affinity labeling and ligand binding techniques to characterize cell surface receptors for TGF-beta under sparse and confluent culture conditions. Confluent SMC, whose growth are promoted by TGF-beta, exhibited a single class of high affinity TGF-beta binding sites (Kd = 6 pM, 3,000 sites/cell). In contrast, sparse SMC (whose growth are inhibited by TGF-beta) expressed two distinct classes of high affinity binding sites with binding constants of 6 pM (3,000 sites/cell) and 88 pM (11,000 sites/cell). By affinity labeling using 125I-TGF-beta and disuccinimidyl suberate cross-linking, confluent cells were found to express a major Mr = 280,000 TGF-beta receptor as well as trace amounts of low molecular weight (Mr = 85,000 and 65,000) receptor subtypes. All three of these receptors were determined, by ligand competition, to show similar affinity for TGF-beta. The predominant receptor subtypes expressed by sparse SMC exhibited apparent Mr = 75,000 and 65,000. In ligand competition experiments, the Mr = 75,000 receptor subtype (never present in confluent cultures) exhibited lower relative affinity for TGF-beta than did the Mr = 65,000 form. The ability of TGF-beta to inhibit SMC proliferation, therefore, correlates with the expression of a unique TGF-beta-binding protein on the SMC surface. The data suggest that TGF-beta may exert opposite biological effects on the same cell type via an interaction with distinct, selectively expressed receptor subtypes.

Expression of apolipoprotein E by cultured vascular smooth muscle cells is controlled by growth state.

Rat vascular smooth muscle cells (SMC) in culture synthesize and secrete a approximately 38,000-Mr protein doublet or triplet that, as previously described (Majack and Bornstein. 1984. J. Cell Biol. 99:1688-1695), rapidly and reversibly accumulates in the SMC culture medium upon addition of heparin. In the present study, we show that this approximately 38,000-Mr heparin-regulated protein is electrophoretically and immunologically identical to apolipoprotein E (apo-E), a major plasma apolipoprotein involved in cholesterol transport. In addition, we show that expression of apo-E by cultured SMC varies according to growth state: while proliferating SMC produced little apo-E and expressed low levels of apo-E mRNA, quiescent SMC produced significantly more apo-E (relative to other proteins) and expressed markedly increased levels of apo-E mRNA. Northern analysis of RNA extracted from aortic tissue revealed that fully differentiated, quiescent SMC contain significant quantities of apo-E mRNA. These data establish aortic SMC as a vascular source for apo-E and suggest new functional roles for this apolipoprotein, possibly unrelated to traditional concepts of lipid metabolism.

Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation.

Thrombospondin (TS) is an extracellular glycoprotein whose synthesis and secretion by vascular smooth muscle cells (SMC) is regulated by platelet-derived growth factor. We have used a panel of five monoclonal antibodies against TS to determine an essential role for thrombospondin in the proliferation of cultured rat aortic SMC. All five monoclonal antibodies inhibited SMC growth in 3-d and extended cell number assays; the growth inhibition was specific for anti-TS IgG. The effects of one antibody (D4.6) were examined in detail and were found to be reversable and dose dependent. Cells treated with D4.6 at 50 micrograms/ml (which resulted in a greater than 60% reduction in cell number at day 8) were morphologically identical to control cells. D4.6-treated SMC were analyzed by flow cytofluorimetry and were found to be arrested in the G1 phase of the cell cycle. To determine a possible cellular site of action of TS in cell growth, SMC were examined by immunofluorescence using a polyclonal antibody against TS. TS was observed diffusely bound to the cell surface of serum- or platelet-derived growth factor-treated cells. The binding of TS to SMC was abolished in the presence of heparin, which prevents the binding of TS to cell surfaces and inhibits the growth of SMC. Monoclonal antibody D4.6, like heparin, largely abolished cell surface staining of TS but had no detectable effect on the cellular distribution of fibronectin. These results were corroborated by metabolic labeling experiments. We conclude that cell surface-associated TS is functionally essential for the proliferation of vascular SMC, and that this requirement is temporally located in the G1 phase of the cell cycle. Agents that perturb the interaction of TS with the SMC surface, such as heparin, may inhibit SMC proliferation in this manner.

Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures.

In culture, vascular smooth muscle cells (SMC) grow in a "hill-and-valley" (multilayered) pattern of organization. We have studied the growth, behavioral organization, and biosynthetic phenotype of rat aortic SMC exposed to purified platelet-derived growth regulatory molecules. We show that multilayered growth is not a constitutive feature of cultured SMC, and that beta-type transforming growth factor (TGF-beta) is the primary determinant of multilayered growth and the hill-and-valley pattern of organization diagnostic for SMC in culture. TGF-beta inhibited, in a dose-dependent manner, the serum- or platelet-derived growth factor-mediated proliferation of these cells in two-dimensional culture, but only when cells were plated at subconfluent densities. The ability of TGF-beta to inhibit SMC growth was inversely correlated to plating cell density. When SMC were plated at monolayer density (5 X 10(4) cells/cm2) to allow maximal cell-to-cell contact, TGF-beta potentiated cell growth. This differential response of SMC to TGF-beta may contribute to the hill-and-valley pattern of organization. Unlike its effect on other cell types, TGF-beta did not enhance the synthesis of fibronectin or its incorporation into the extracellular matrix. However, the synthesis of a number of other secreted proteins was altered by TGF-beta treatment. SMC treated with TGF-beta for 4 or 8 h secreted markedly enhanced amounts of an Mr 38,000-D protein doublet whose synthesis is known to be increased by heparin (another inhibitor of SMC growth), suggesting metabolic similarities between heparin- and TGF-beta-mediated SMC growth inhibition. The data suggest that TGF-beta may play an important and complex regulatory role in SMC proliferation and organization during development and after vascular injury.

Induction of thrombospondin messenger RNA levels occurs as an immediate primary response to platelet-derived growth factor.

We have investigated the regulation of mRNA levels for thrombospondin, a platelet-derived growth factor (PDGF)-regulated secreted glycoprotein, in cultures of rat vascular smooth muscle cells (SMC). A thrombospondin cDNA hybridizes to a single 5.5-kilobase SMC message which is greatly induced by serum or PDGF. When quiescent SMC are treated with PDGF, thrombospondin levels are induced rapidly (within 15 min) and in a dose-dependent manner. The induction of thrombospondin message levels parallels PDGF-mediated mitogenesis. PDGF-mediated increases in thrombospondin mRNA can be blocked in the presence of actinomycin D, suggesting that PDGF regulates the thrombospondin message at the level of gene transcription. In the presence of cycloheximide, PDGF "super-induces" the thrombospondin message. The data establish that mRNA levels for thrombospondin are regulated by PDGF in a manner similar to that of c-myc, c-fos, and other growth-regulatory gene products and extend our previous findings that thrombospondin secretion by SMC is dependent on exposure to PDGF. These observations are strongly suggestive of an important role for thrombospondin in the growth response of mesenchymal cells.