Isolation of two morphologically distinct cell lines from rat arterial smooth muscle expressing high tumorigenic potentials

Summary Smooth muscle cell proliferation is an important feature of atherogenesis. Some works have hypothesized that a transformation of smooth muscle cells could arise during this pathological process. The present paper describes two spontaneously transformed cell lines of arterial smooth muscle cells (SMC) established from aortic media of adult rat. The cell lines have been designated V6 and V8; some of their morphologic, growth, and metabolic characteristics are described and compared to their parent cells. The two cell lines appeared distinct by their morphology and by their degree of transformation. V6 cells appeared as elongated spindle-shaped cells whereas V8 cells were spread cells with a cobblestone pattern. Karyotypes of both cell lines showed a high polyploidy level. V6 and V8 cell lines were immortalized and showed growth characteristics of transformed cells: low requirement of serum to grow, ability to form colonies in soft agar and tumorigenicity in nude mice; V8 cells presented a higher malignancy than V6 cells. Both V6 and V8 cells exhibited characteristics of cultured arterial SMC: ultrastructure, alpha actin expression at the protein and mRNA level, prostacyclin production. The remarkably different morphologies of the V6 and V8 lines and their transformed phenotype suggest that these cell lines could be useful models to study SMC differentiation and proliferation with respect to atherosclerotic or hypertensive vascular diseases. Electron microscopy was performed in the Center of Electron Microscopy Applied to Biology and Geology (CEMABG), Claude Bernard University, Lyon I. Flow cytofluorometry was performed in the Center of Fluorometry, Department of Human Biology, Claude Bernard University, Lyon I and funded by ARC N^o 6055-80. This work was supported by INSERM, by MRT grant 86-C-0301 and by ARC grant 415-87.     

Isolation and characterization of vasoformative endothelial cells from the rat aorta

Summary We describe here a modified nonenzymatic method for the isolation of rat aortic endothelial cells with vasoformative properties. Aortic rings placed on plastic or gelatin-coated surfaces generated outgrowths primarily composed of endothelial cells. Prompt removal of aortic explants after endothelial migration minimized fibroblast contamination. However, fibroblasts, because of their high proliferative rate tended to overgrow the endothelial cells even when present in small numbers. This potential pitfall was avoided by weeding out fibroblasts with the rounded tip of a bent glass pipette. Primary endothelial colonies free of fibroblasts were segregated in cloning rings, trypsin-treated, and transferred to gelatin-coated dishes. Endothelial cells were cultured in MCDB 131 growth medium containing 10% fetal bovine serum, endothelial cell growth supplement, and heparin. Using this technique, pure endothelial cell strains were obtained from single aortic rings. Confluent endothelial cells formed a contact-inhibited monolayer with typical cobblestone pattern. The endothelial cells were positive for Factor VIII-related antigen, took up DiI-Ac-LDL, and bound the Griffonia Simplicifolia-isolectin-B4. Endothelial cells cultured on collagen gel formed a polarized monolayer, produced basement membrane, displayed Weibel-Palade bodies and caveolae, and were connected by tight junctions. In addition, they reorganized into a network of microvascular cords and tubes when overlaid with a second layer of collagen and formed microvascular sprouts in response to fibroblast-conditioned medium. This isolation procedure yields stable strains of vasoformative endothelial cells, which can be used to study aortic endothelium-related angiogenesis and its mechanisms.     

Enhanced proliferation and altered intracellular zinc levels in early- and late-passage mouse aorta smooth muscle cells

Cell growth and DNA synthesis were studied from a cultured early- and late- passage mouse aorta smooth muscle cell (MASMC) because the proliferation of vascular smooth muscle cell (VSMC) is a key factor in development of atherosclerosis. In this study, the cells were cultured in fetal bovine serum (FBS) and stimulated by growth factors such as thrombin and platelet-derived growth factor-BB (PDGF-BB). Compared to the number of early-passage MASMC (passage 3 to 9) the number of late-passage MASMC (passage 30 to 40) in a normal serum state was increased 2 fold at Day 1,3 and 6 in culture, respectively. Incorporation of [³H] thymidine into DNA induced by serum, PDGF and thrombin in late-passage MASMC was greater than those in early-passage MASMC. We also examined whether intracellular zinc levels would be an aging factor or not. The intracellular zinc level in, early- and late-passage MASMC was monitored by using the zinc probe dyeN-(6-methoxy-8-quinolyl)-p-toluenesulfonamide. It is interested that late-passage MASMC increased the intracellular fluorescence level of zinc, more than the early passage MASMC did. The alterations of intracellular zinc level occur concurrently with changes in MASMC proliferation rate during aging. This data suggest that the age-associated changes in zinc concentrations may provide a newin vitro model for the study of smooth muscle cell differentiation.     

Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas

Summary Vascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar¹,Ile⁸-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTPγS. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.     

Collagen synthesis and accumulation in long-term rabbit aortic smooth muscle cell cultures

Summary This study describes the ability of aortic smooth muscle cells to synthesize and accumulate collagen with time in culture. Inasmuch as smooth muscle cell cultures multilayer and continue to divide, albeit slowly, and can be maintained in the same vessels where seeded for extended periods of time, a long-term aging study from a single subcultivated population of cells was carried out. This is different from the usual cell-culture aging achieved by an increase in cell population doublings obtained by repeated subcultivations. The latter process, which is trypsin induced, involves a changing cellular environment including the extracellular matrix that is produced by the cells in culture. Second subcultures of weanling rabbit, aortic media, smooth muscle cells maintained for different periods of time up to 14 wk displayed decreasing hydroxyproline formation with time. Proline hydroxylation was determined by pulsing these second-passage cells with [¹⁴C]proline for 24 h at various times during the 14 wk period. The cell layer and medium were evaluated separately for radioactive proline and hydroxyproline and the medium for bacterial collagenase-susceptible protein as well. The percent of hydroxylation in the medium decreased from >31% within 1 wk after plating to 15.2% after 14 wk in culture. The percent of collagenase-susceptible protein in the medium decreased in a comparable manner. The DNA levels increased during the entire period although initially somewhat more rapidly. Accumulation of protein in the extracellular matrix continued during the 14-wk span. The accumulation of hydroxyproline in the extracellular matrix also continued to increase throughout the culture period, but it did slow down significantly. Yet the cells appear not to have lost their ability to accumulate connective tissue and protein in the insoluble cell layer. The data suggest clearly that the percent collagen synthesis relative to total protein synthesis decreases in the older cultures; total protein synthesis also decreases as expected. This study was supported by NIH Program Projects AG00001 and HL 13262.     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.     

普罗托品对大鼠胸主动脉血管平滑肌细胞内游离钙浓度和蛋白激酶C活性的影响

本实验室先前的研究已证实,普罗托品(protopine,Pro)舒张家兔主动脉的作用,可能与其增加血管平滑肌细胞内cAMP和cGMP水平有关.为了深入探讨Pro的扩血管作用机制,实验采用等张收缩记录大鼠离体血管条张力,利用Fura-2/AM负载的大鼠胸主动脉培养细胞直接测定细胞内游离Ca2+浓度([Ca2+]i),并应用同位素γ-32p-ATP催化活性法测定蛋白激酶C(PKC)活性等方法,分别观察了Pro的相关效应.结果表明,Pro(30和100 μmol/L)明显降低去甲肾上腺素(NA)和高钾所致的动脉条收缩幅度,使二者的量效曲线呈非平行右移,最大反应压低;pD2'值分别为3.7±0.25和3.97±0.15;Pro(50和100μmol/L)对静息状态下[Ca2+]i没有任何影响,但对NA和高钾引起的[Ca2+]i升高均有明显抑制作用;Pro(30和100 μmol/L)对未经NA处理血管条的胞浆和胞膜PKC活性均无明显影响;但在NA预处理的血管条,Pro使NA所升高的胞浆内PKC的活性趋于降低,而明显升高胞膜PKC的活性,对PKC的总活性无明显影响.结果提示,在有NA存在的情况下,Pro似能促使PKC从胞浆向细胞膜转移,其扩血管效应似为其降Ca2+作用、升高cAMP和cGMP的作用及其对PKC影响等几方面的综合结果.     

Compartmentalized coculture of porcine arterial endothelial and smooth muscle cells on a microporous membrane

Summary Endothelial and smooth muscle cells were harvested from porcine pulmonary arteries and grown to two passages from primary culture in serum-containing medium. Thereafter, the cells were plated on the opposite sides of a microporous poly-(ethylene terephthalate) membrane and cultivated in a chemically defined, serum-free medium. The membrane with pores of 1 μm diameter allowed the passage of molecules and the extension of cell processes, while maintaining separate homogeneous cell populations. Pores of 3 μm diameter permitted the crossing of smooth muscle cells through the membrane. The coating of the polymer with constituents of the extracellular matrix optimized cell adhesion. Morphological analysis of the model showed typical cobblestone pattern and ultrastructure of endothelial cells, which lost rapidly the expression of von Willebrand factor but kept that of angiotensin-converting enzyme. Smooth muscle cells were spindle shaped and specific α-actin was revealed by immunochemistry and quantitated by enzyme-linked immunosorbent assay (ELISA). Their ultrastructure featured an intermediate contractile-synthetic phenotype. Permeability studies to different molecules showed a marked reduction of the albumin clearance. Finally, in coculture in the presence of endothelial cells, the smooth muscle cells proliferation was increased, whereas it was not the case in autologous cocultures. In conclusion, such a coculture model may help to a better understanding of the interactions between endothelial and smooth muscle cells that may be important in the pathogenesis of vascular diseases.     

Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta

Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan. Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory effect on either class of [³⁵S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation of [³⁵S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by promoting the core protein synthesis. Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA 37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291).     

Hypertriglyceridemic serum from magnesium-deficient rats induces proliferation and lipid accumulation in cultured vascular smooth muscle cells

An important characteristic of hyperlipemia associated with magnesium deficiency in rats is the postprandial accumulation of triglyceride-rich lipoproteins. The present investigation was performed to determine the effect of serum from magnesium-deficient animals on cultured vascular smooth muscle cells (VSMC). Sera were obtained from control and magnesium-deficient rats fed adequate or deficient diets for 8 days. Magnesium-deficient animals were hypertriglyceridemic compared with control rats, but their total cholesterolemia was not significantly modified. Pooled sera from control and magnesium-deficient animals were added to the culture medium at various concentrations. The maximum of proliferation for both control and magnesium-deficient sera was reached when they were added at 6% to the culture medium and on day 4 after the begining of incubation. Medium containing serum from magnesium-deficient rats stimulated the cell proliferation as monitored by cell count and [³H]thymidine incorporation. Staining of VSMC with Oil red O and measuring lipids have shown a marked lipid accumulation (triglycerides) in cells incubated with serum obtained from magnesium-deficient animals compared with serum from control rats. These results indicate that serum from magnesium-deficient rats contains factors that stimulate proliferation of arterial medial cells and that hyperlipemia associated with magnesium-deficiency may cause lipid accumulation in vascular cells.     

Biological characteristics of foam cell formation in smooth muscle cells derived from bone marrow stem cells

Bone marrow mesenchymal stem cells (BMSC) can differentiate into diverse cell types, including adipogenic, osteogenic, chondrogenic and myogenic lineages. There are lots of BMSC accumulated in atherosclerosis vessels and differentiate into VSMC. However, it is unclear whether VSMC originated from BMSC (BMSC-SMC) could remodel the vessel in new tunica intima or promote the pathogenesis of atherosclerosis. In this study, BMSC were differentiated into VSMC in response to the transforming growth factor β (TGF-β) and shown to express a number of VSMC markers, such as α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain1 (SM-MHC1). BMSC-SMC became foam cells after treatment with 80 mg/L ox-LDL for 72 hours. Ox-LDL could upregulate scavenger receptor class A (SR-A) but downregulate the ATP-binding cassette transporter A1 (ABCA1) and caveolin-1 protein expression, suggesting that modulating relative protein activity contributes to smooth muscle foam cell formation in BMSC-SMC. Furthermore, we found that BMSC-SMC have some biological characteristics that are similar to VSMC, such as the ability of proliferation and secretion of extracellular matrix, but, at the same time, retain some biological characteristics of BMSC, such as a high level of migration. These results suggest that BMSC-SMC could be induced to foam cells and be involved in the development of atherosclerosis.     

Nitroalkenes induce rat aortic smooth muscle cell apoptosis via activation of caspase-dependent pathways

Nitroalkene derivatives of nitro-linoleic acid (LNO₂) and nitro-oleic acid (OA-NO₂) are nitrated unsaturated fatty acids that can be detected in healthy human plasma, red blood cells and urine. It has been shown that nitroalkenes have potent anti-inflammatory properties in multiple disease models. In the present study, we are the first to investigate the apoptotic effects of nitroalkenes in rat aortic smooth muscle cells (RASMCs). We observed that nitroalkenes induce RASMCs apoptosis in a dose-dependent manner. In addition, nitroalkenes stimulate extrinsic caspase-8 and intrinsic caspase-9 activity to trigger the caspase-3 apoptotic signaling cascade, resulting in RASMCs death. Furthermore, the pro-apoptotic protein, Bad was upregulated and antiapoptotic protein, Bcl-xl was downregulated during nitroalkene-induced apoptosis. These results demonstrate that nitroalkenes can induce RASMCs apoptosis via stimulation of caspase activity and the regulation of apoptotic protein expression levels.     

Silencing CCL8 inhibited the proliferation and migration of PDGF-BB-stimulated human aortic smooth muscle cells

ABSTRACT

C-C motif Chemokine ligand 8 (CCL8) has been found in diseases’ pathogenesis. But its molecular mechanism in atherosclerosis (AS) remains to be elucidated. Human aortic smooth muscle cells (HASMCs) were stimulated by PDGF-BB to establish cell model. α-SMA in PDGF-BB-stimulated HASMCs was measured by immunofluorescence staining. Relative gene expressions in PDGF-BB-stimulated HASMCs were detected by quantitative real-time polymerase chain reaction and western blot. HASMCs proliferation, migration, and cell cycle were assessed by cell counting kit-8, wound-healing assay, and flow cytometry. HASMCs viability was increased after PDGF-BB stimulation, with α-SMA downregulation yet CCL8 upregulation. Silencing CCL8 inhibited PDGF-BB-stimulated HASMCs proliferation and migration, and increased cells percentage in G1 phases but decreased those in S phase. Also, silencing CCL8 decreased OPN and cyclinD1 expressions and AKT and ERK1/2 phosphorylation while increased those of α-SMA and Sm22α. However, upregulating CCL8 led to opposite effects, suggesting CCL8 could be an atherosclerosis therapeutic target.     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. II. The platelet-derived growth factor A-chain contains a sequence that specifically binds heparin

Summary Synthetic oligopeptides were used to study the specificity of the interaction between heparin and platelet-derived growth factor (PDGF) in competition experiments. DNA synthesis in PDGF-dependent human arterial smooth muscle cell (hASMC) cultures was used as a biological tracer of PDGF activity. Oligo-108-124 (corresponding to amino acid residues 108-124 of the long PDGF A-chain isoform) had no effect on DNA synthesis in itself but competed at 10⁻¹⁰ M concentration effectively with PDGF for binding to heparin and released the block on thymidine incorporation induced by heparin. Poly-lysine-serine (lysine:serine ratio 3:1) was also effective but at a considerably higher concentration (10⁻⁶ M). Poly-arginine-serine did not compete with PDGF for heparin as deduced from the cell assay. This suggested that among basic amino acids, lysine was more important than arginine for heparin binding. Deletion of lysine residues 115 and 116 in Oligo-108-124 abolished its effect on the interaction between PDGF and heparin in the cell assay. Likewise, Oligo-69-84 (corresponding to the PDGF A-chain residues 69–84), with three lysine residues interrupted by a proline, was ineffective. In Oligo-108-124, the lysine residues are interrupted by an arginine. Our results suggested that the binding between PDGF and heparin is specific and that the amino acid sequence [-Lys¹¹⁵-Lys¹¹⁶-Arg¹¹⁷-Lys¹¹⁸-Arg¹¹⁹-] is of major importance. They do not however, exclude other domains of the PDGF A or B chains as additional binding sites for heparin nor do they exclude the possibility that heparin and the PDGF receptor share a common binding site.     

大鼠细小肺动脉平滑肌细胞原代培养和鉴定方法的研究

目的：建立一种重复性好、培养周期短及传代次数多的大鼠细小肺动脉平滑肌细胞（PASMCs）培养方法。方法：在无菌条件下,分离雄性SD大鼠肺细小动脉,剥离外膜和剔除内皮细胞,经胶原酶I消化,培养PASMCs。0.4%台盼蓝染色测定细胞活力;倒置相差显微镜观察;免疫细胞化学法和免疫荧光染色法,进行平滑肌α-肌动蛋白（α-SMactin）鉴定。结果：形态学观察、免疫细胞化学法及免疫荧光染色法鉴定表明培养细胞为PASMCs;细胞存活率在96.5%以上;原代培养后4～7d即可传代,并且生长特点、细胞形态不易发生改变。结论：采用胶原酶I消化法培养PASMCs,方法简单、酶消化时间易控制、培养周期短、重复性好,培养的原代PASMCs具有数量多和生长迅速的特点。     

Tissue inhibitor of metalloproteinases-4 suppresses vascular smooth muscle cell migration and induces cell apoptosis

In a previous study, we have demonstrated that overexpression of the tissue inhibitors of metalloproteinases-4 (TIMP-4) can inhibit the neointima formation in the rat carotid model. To define the functions of tissue inhibitor of metalloproteinases-4 (TIMP-4) in SMCs, we transduced human TIMP-4 cDNA into rat aortic SMCs by using adenoviral vector. Overexpression of TIMP-4 blocked the conversion of pro-MMP-2 to the active form and inhibited basic fibroblast growth factor-induced migration by 56.7% (p < 0.01). Overexpression of TIMP-4 markedly increased apoptotic cell death without changing their proliferation. Importantly, overexpression of human TIMP-4 in the wall of balloon-injured rat carotid artery also increased SMC apoptosis. The percentages of TUNEL-positive cells of total cells increased significantly in AdTIMP-4 infected group compared with AdGFP infected group. These findings demonstrate that TIMP-4 can inhibit SMCs migration and induce apoptosis in vitro and in vivo, which may generate new targets for prevention and treatment of vascular diseases.     

Comparative endocrinology-paracrinology-autocrinology of human adult large vessel endothelial and smooth muscle cells

Summary Endothelial and smooth muscle cells were isolated from human adult large blood vessels to compare their proliferative response to hormones and growth factors. Neural extracts and the medium from differentiated hepatoma cells were used as concentrated sources of required hormones and growth factors that supported both cell types. Active hormones and growth factors were identified from the neural extracts and hepatoma medium by substitution or direct isolation and biochemical characterization. Epidermal growth factor, lipoproteins, and heparin-binding growth factors elicited growth-stimulatory effects on both endothelial and smooth muscle cells. Both types of human vascular cells displayed 7600 to 8600 specific heparin-binding growth factor receptors per cell with a similar apparent dissociation constant (Kd) of 200 to 250 pM. Heparin modified the response of both endothelial and smooth muscle cells to heparin-binding growth factors dependent on the type of heparin-binding growth factor and amount of heparinlike material present. In addition, heparin exerted a growth factor-independent inhibition of smooth muscle cell proliferation. Platelet-derived growth factor, insulinlike growth factors, and glucocorticoid specifically supported proliferation of smooth muscle cells with no apparent effect on endothelial cell proliferation. Growth-factorlike proteinase inhibitors had an impact specifically on endothelial cell proliferation. Transforming growth factor beta was a specific inhibitor of endothelial cells, but had a positive effect on smooth muscle cell proliferation. The results provide a framework for differential control of the two vascular cell types at normal and atherosclerotic blood vessel sites by the balance among positive and negative effectors of endocrine, paracrine and autocrine origin. This research was supported by NIH grants CA37589, HL33847, and AM35310 from the National Institutes of Health, Bethesda, MD; grant 1718 from the Council for Tobacco Research; and a grant from RJR/Nabisco, Inc.