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.

Heparin regulates the collagen phenotype of vascular smooth muscle cells: induced synthesis of an Mr 60,000 collagen.

The effect of heparin on the biosynthetic phenotype of rat vascular smooth muscle cells (SMC) was investigated in vitro. Addition of heparin to the culture medium of early passage rat SMC resulted in a marked (3-15-fold) increase of a cell layer-associated Mr 60,000 protein that was sensitive to digestion by purified bacterial collagenase and contained significant amounts of hydroxyproline. Pulse-chase analysis of heparin-treated SMC revealed that the Mr 60,000 collagen was a primary and abundant product of these cells and was not processed extracellularly to a smaller form. The inductive effect of heparin could be mimicked by iota carrageenan or dextran sulfates but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfates. The induction was concentration dependent with a maximal effect observed at a heparin concentration of 10 micrograms/ml. Synthesis of the Mr 60,000 collagen increased 18-24 h after addition of heparin to the cultures. Following induction and subsequent removal of heparin, synthesis of the protein remained maximal for at least 12 h and required 72 h to return to a basal level. These data demonstrate that the biosynthetic phenotype of vascular SMC in vitro can be controlled, at least in part, by heparin and related polyanions and suggest a role for similar molecules endogenous to the vessel wall in the regulation of SMC function.

Heparin and related glycosaminoglycans modulate the secretory phenotype of vascular smooth muscle cells.

Previous studies have established a role for heparin-like molecules in the regulation of vascular smooth muscle cell growth and migration in vitro. We present data indicating that the secretory phenotype of cultured rat aortic smooth muscle cells can be modulated by exogenous soluble heparin, heparan sulfate, and dermatan sulfate glycosaminoglycans. In the presence of these molecules, smooth muscle cells secrete increased amounts of two noncollagenous proteins (Mr 37,000 and 39,000). This effect can be mimicked by iota carrageenan and dextran sulfate but not by hyaluronic acid, chondroitin-4-sulfate, or chondroitin-6-sulfate. The inductive effect of heparin was dose-dependent and occurred rapidly (within 1 h) with maximal induction (three- to fivefold over controls) occurring after 10-12 h of treatment. The effect was rapidly reversible (within 1 h) and was not altered in the presence of actinomycin D, suggesting regulation at a posttranscriptional level. These data indicate that the biosynthetic expression of specific smooth muscle cell proteins may be determined, at least in part, by components of the smooth muscle cell extracellular matrix.

Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells.

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The induction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 micrograms/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 microgram/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.

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.

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.

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.

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.

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.

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.

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.

Ultrastructural characteristics of endothelial permeability in chronic hypertension.

This study examined characteristics of paracellular and pinocytotic permeability pathways across the middle cerebral artery endothelium of 12- to 16- month-old spontaneously hypertensive rats (SHR). Interendothelial junctions in SHR, like those of age-matched Wistar-Kyoto controls, were impermeable to lanthanum and horseradish peroxidase (HRP) tracers. Freeze-fracture preparations revealed that interendothelial junctions of chronically hypertensive rats are characterized by a twofold increase over controls in the number of tight junctional strands and the mean apical-basal depth. It is believed that this tight junctional hypertrophy may function to increase adhesive forces between neighboring endothelial cells, and may play a role inthe prevention of hypertension-induced paracellular permeability increases. Morphological and tracer studies of pinocytotic pathways indicated that, it the middle cerebral artery, endothelial vesicular transport activity is not increased during chronic hypertension. No evidence was found to indicate the presence of transendothelial permeability channels across control or hypertensive arterial endothelium. Thus, increased transendothelial permeability, commonly observed in acute hypertension, does not appear to occur during chronic hypertension, at least in the middle cerebral artery. Our findings suggest that the arterial endothelium may undergo structural (tight junctional) adaptation in response to prolonged hypertension.

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.

The bicellular and reflexive membrane junctions of renomedullary interstitial cells: functional implications of reflexive gap junctions.

Renomedullary interstitial cells are loosely organized within the interstitial space surrounding collecting ducts, limbs of Henle, and capillaries of the rat renal medulla. These cells possess long processes, which interact with each other and with cell bodies to form bicellular tight, intermediate, and gap junctions. In addition, both cell bodies and cell processes possess "reflexive" gap and intermediate junctions. Possible functions of renomedullary interstitial cell membrane junctions are discussed. Particular attention is given to a consideration of the functional significance of "reflexive" gap junctions.

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.

Inhibition of vascular smooth muscle cell migration by heparin-like glycosaminoglycans.

Previous studies have suggested that heparin-like glycosaminoglycans may be endogenous inhibitors of smooth muscle proliferation in the vessel wall. The purpose of this study was to determine the effects of exogenous glycosaminoglycans on rat vascular (aortic) smooth muscle cell migration following wounding in vitro. Our data indicate that heparin and related molecules (iota carrageenan, dextran sulfate), but not other glycosaminoglycans (hyaluronate, chondroitin, and dermatan sulfates), inhibit smooth muscle cell motility in a cell-specific, dose-dependent, and reversible fashion. The effect of heparin was maximal (60% inhibition) at 10 micrograms/ml; a half-maximal effect was observed at 1 microgram/ml. Heparin did not significantly affect the migration of bovine aortic endothelium or Swiss 3T3 cells. These observations support the concept that heparin-like glycosaminoglycans may be important regulators of vascular smooth muscle cell function.

An ameloblastoma with myofibroblasts and intracellular septate junctions.

This report represents a review of the ultrastructural features of the simple ameloblastoma. Our ultrastructural data indicate that the stromal component of ameloblastoma is composed of myofibroblasts with associated collagen and basal lamina material. A unique feature of these myofibroblasts is the formation of plaque-like structures on extended cell processes which were identified as intracellular septate junctions. The epithelial component of ameloblastoma was shown to be composed of three distinct cell types which appear to represent a continuum for the ameloblast-like basal cell through an intermediate to an acanthomatous cell. The actual relationship between the three cell types is unclear.

The ultrastructural localization of membrane ATPase in rat thin limbs of the loop of Henle.

The cytochemical distribution of nonspecific membrane ATPase activity in the epithelial membranes of the thin limbs of the loops of Henle of rat nephrons was studied at the ultrastructural level. Membrane ATPase activity was localized in the luminal, lateral, and (to a lesser extent) basal membranes of only the outer medullary segment of the thin descending limbs of long nephrons (Type II epithelium). The reaction product was lacking in the thin limb of short nephrons (Type I epithelium) as well as in the inner medullary descending (Type III epithelium) and ascending (Type IV epithelium) segments of the thin limbs of long nephrons. These data reinforce the concept of thin limb heterogeneity and may indicate a specialized role for the outer medullary segment of thin descending limbs of long nephrons in the concentrating mechanism.

Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin.

Addition of platelet-derived growth factor (PDGF) to growth-arrested cultured smooth muscle cells (SMC) induces the synthesis and secretion of thrombospondin (TS), a glycoprotein component of the SMC extracellular matrix in vitro. This induction occurs at PDGF concentrations that are suboptimal for a mitogenic response. In this study we examined the effect of TS on the proliferation of SMC, using a serum-free mitogenesis assay. Addition of either epidermal growth factor (EGF) or purified human platelet TS to quiescent rat vascular SMC did not substantially stimulate mitogenesis; the 30-hr nuclear labeling index increased from a mean of 7% in control cells to 20% for EGF-treated SMC and 17% for cells exposed to TS alone. However, TS and EGF acted synergistically to stimulate DNA synthesis by SMC, increasing the labeling index to 47%. The facilitative effect of TS on EGF-mediated mitogenesis was inhibited by heparin, a known inhibitor of SMC growth and migration that also blocks incorporation of TS into the SMC extracellular matrix. The effect was specific for EGF; TS did not augment the response of cells to insulin or insulin-like growth factor 1. These data establish a functional role for cell-derived TS and provide evidence for the presence of an autocrine, growth-supportive mechanism involving the extracellular matrix. In addition, our experiments support the existence of a novel, heparin-sensitive SMC mitogenic pathway and suggest a mechanism whereby heparin-like molecules may inhibit SMC proliferation.