Change In Smooth Muscle Phenotype Research Articles

Novel Inflammatory Signaling via Interleukin‐13 Decreases Potassium Channel Expression and Increases Vascular Smooth Muscle Excitability

The transcriptional repressor element‐1 (RE1) silencing transcription factor (REST), also known as neuron‐restrictive silencer factor (NRSF) is a gene silencing transcription factor, which has been characterized as a master regulator of the neuronal phenotype. REST plays a key role in the development of the nervous system and is a critical player in the etiology of neurodegenerative diseases such as Huntington's Disease, though little is known about the role of REST in non‐neuronal cells. We are exploring a potential role of REST as a mediator between inflammation and changes in smooth muscle phenotype. Kv7.5 voltage‐dependent potassium channels, encoded by the KCNQ5 gene, are expressed in smooth muscle cells where they play an important role in determining resting membrane potential. Interleukin‐13 (IL‐13) is an inflammatory cytokine, which is implicated as a primary mediator of the phenotypic changes induced by allergic inflammation in many tissues. In the present study, we hypothesized that IL‐13 can increase nuclear REST and decrease KCNQ5 mRNA expression and Kv7.5 protein in vascular smooth muscle cells, providing a potential link between inflammation, REST and potassium channel‐mediated vascular hyper‐responsiveness. In support of this hypothesis, using A7r5 rat aortic smooth muscle cells, we found that 1) IL‐13 treatment significantly increased nuclear REST, and 2) significantly suppressed the expression of KCNQ5 and Kv7.5 currents, resulting in increased sensitivity of A7r5 cells to arginine vasopressin (AVP). 3) REST overexpression also significantly decreased Kv7.5 currents, and 4) significantly increased the sensitivity of A7r5 cells to AVP. We conclude that inflammation that induces IL‐13 may increase nuclear REST levels and thereby decrease KCNQ5 expression and Kv7.5 currents, ultimately increasing vascular smooth muscle cell excitability and contributing to vascular hyper‐responsiveness.Support or Funding InformationThe project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contribution of nuclear factor E2‐related factor 2 to the atherogenic phenotype transition in coronary arterial smooth muscle cells lacking CD38 gene (1065.16)

Recent studies have reported that CD38 gene deficiency results in dedifferentiation or transdifferentiation of arterial smooth muscle (SMCs) upon atherogenic stimulations. However, the molecular mechanisms mediating this SMC phenotypic change remain unknown. In the present study, we first characterized the phenotypic change in the primary cultures of coronary arterial myocytes (CAMs) from CD38‐/‐ mice. It was shown that CD38 deficiency decreased the expression of contractile marker calponin and α‐SMA and increased the expression of SMC dedifferentiation marker vimentin, which was accompanied by collagen I accumulation and cell proliferation. This phenotypic change was enhanced by 7‐ketocholesterol (7‐Ket), an atherogenic stimulus in CAMs from CD38‐/‐ mice. We further found that the CD38 deficiency decreased the protein level of nuclear factor E2‐related factor 2 (Nrf2) and inhibited its activity. Given that Nrf2 is a transcription factor that responds to oxidative stress, we determined the role of redox signaling in the regulation of Nrf2 expression and activity. It was demonstrated that in CD38‐/‐ CAMs 7‐Ket failed to produce O2‐., while in CD38+/+ CAMs 7‐Ket‐induced O2‐. production was substantially attenuated by Nox4 gene silencing. In addition, 7‐Ket‐induced increase in Nrf2 activity in CD38+/+ CAMs was significantly inhibited by silencing Nox4 gene. Finally, we demonstrated that both Nrf2 gene silencing and CD38 deletion had similar effect to increase CAM dedifferentiation upon 7‐Ket stimulation. In contrast, the overexpression of Nrf2 gene markedly abolished 7‐Ket‐induced dedifferentiation in CD38‐/‐ CAMs. These results suggest that CD38 activity is required for 7‐Ket‐induced O2‐. production, which increases Nrf2 activity maintaining CAMs in a differentiated status. When CD38 gene expression and function are deficient, the Nrf2 activity is suppressed thereby leading to dedifferentiation of CAMs.Grant Funding Source: HL057244, HL091464 and HL075316

Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries

Chronic hypoxia increases vascular endothelial growth factor (VEGF) and thereby promotes angiogenesis. The present study explores the hypothesis that hypoxic increases in VEGF also remodel artery wall structure and contractility through phenotypic transformation of smooth muscle. Pregnant and nonpregnant ewes were maintained at sea level (normoxia) or 3,820 m (hypoxia) for the final 110 days of gestation. Common carotid arteries harvested from term fetal lambs and nonpregnant adults were denuded of endothelium and studied in vitro. Stretch-dependent contractile stresses were 32 and 77% of normoxic values in hypoxic fetal and adult arteries. Hypoxic hypocontractility was coupled with increased abundance of nonmuscle myosin heavy chain (NM-MHC) in fetal (+37%) and adult (+119%) arteries. Conversely, hypoxia decreased smooth muscle MHC (SM-MHC) abundance by 40% in fetal arteries but increased it 123% in adult arteries. Hypoxia decreased colocalization of NM-MHC with smooth muscle α-actin (SM-αA) in fetal arteries and decreased colocalization of SM-MHC with SM-αA in adult arteries. Organ culture with physiological concentrations (3 ng/ml) of VEGF-A(165) similarly depressed stretch-dependent stresses to 37 and 49% of control fetal and adult values. The VEGF receptor antagonist vatalanib ablated VEGF's effects in adult but not fetal arteries, suggesting age-dependent VEGF receptor signaling. VEGF replicated hypoxic decreases in colocalization of NM-MHC with SM-αA in fetal arteries and decreases in colocalization of SM-MHC with SM-αA in adult arteries. These results suggest that hypoxic increases in VEGF not only promote angiogenesis but may also help mediate hypoxic arterial remodeling through age-dependent changes in smooth muscle phenotype and contractility.

Counterpoint: Alterations in airway smooth muscle phenotype do not cause airway hyperresponsiveness in asthma

We have been assigned the task of showing that “alterations in airway smooth muscle phenotype DO NOT cause the airways hyperresponsiveness of asthma.” In the following paragraphs we will briefly review the overwhelming lack of evidence that any fundamental change in the intrinsic properties of

Airway Structural Components Drive Airway Smooth Muscle Remodeling in Asthma

Chronic asthma is an inflammatory airways disease characterized by pathological changes in the airway smooth muscle (ASM) bundle that contribute to airway obstruction and hyperresponsiveness. Remodeling of the ASM is associated with an increased smooth muscle mass, involving components of cellular hypertrophy and hyperplasia, and changes in the phenotype of the muscle that facilitate proliferative, synthetic and contractile functions. These changes are considered major contributing factors to the pathophysiology of asthma, because of their role in exaggerated airway narrowing. The mechanisms that regulate changes in ASM mass and phenotype are incompletely understood, but likely involve the regulatory role of mediators and growth factors secreted from inflammatory cells on ASM cell proliferation and phenotype. An alternative hypothesis is that cellular and structural components that together constitute the airway wall, such as the airway epithelium, airway nerves, and the extracellular matrix, interact with the ASM bundle to facilitate changes in smooth muscle phenotype and function that drive remodeling under inflammatory conditions. This review discusses the mechanisms by which structural components of the airway wall communicate with the ASM bundle to regulate remodeling and discusses these mechanisms in the context of the pathophysiology of asthma.

AT1 receptor antagonist inhibits aortic smooth muscle phenotypic change through no and AKT signaling in hypertension in vivo

The precise mechanisms of angiotensin II type 1 (AT1) receptors on the vascular smooth muscle cell (VSMCs) phenotype in vivo remain unclear, as studies using passaged VSMCs under conventional culture conditions have been unable to adequately investigate the molecular mechanisms underlying the phenotypic modulation of VSMCs. To clarify the mechanisms of smooth muscle (SM) cell phenotype and AT1 receptor-mediated signal transductions in hypertension, we compared the effects of an angiotensin-converting enzyme (ACE) inhibitor and AT1 receptor antagonist on vascular remodeling, oxidative stress and phenotypic changes of VSMCs. Fourteen-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were divided into 3 groups and treated for 6 weeks with either a vehicle, an ACE inhibitor (cilazapril; 10 mg/kg/day), and AT1 receptor antagonist (E4177; 30 mg/kg/day). Compared to the vehicle group, blood pressure, wall-to-lumen ratio, collagen content and the total cell number in aortic media were significantly reduced in the 2 drug-treated groups; with no significant differences between the 2 drug-treated groups. Both drugs significantly up-regulated the contractile-type SM myosin heavy chain (MHC); SM2. Whereas, they significantly down-regulated the synthetic-type SM-MHC; NMHC-B/SMemb. Total aortic reactive oxygen species (ROS) assessed by 8-iso-PGF2α, the expression of NAD(P)H oxidase essential subunit, p22phox, and p38 mitogen-activated protein kinase (p38 MAPK) were significantly reduced in the 2 drug-treated groups, and superoxide content of the aortic media assessed by hydroxyetidine staining did not differ between the 2 drug-treated groups. However, eNOS, phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) and Cu/Zn SOD were significantly up-regulated in the 2 drug-treated groups. Additionally, E4177 induced greater increases in SM2, eNOS and p-Akt expression than did cilazapril. AT1 receptor antagonists inhibited aortic VSMC phenotypic changes by the up-regulation of eNOS expression and activation of the Akt pathway, but not p38-MAPK signaling, than did ACE inhibitors in SHRSP; suggesting that AT1 receptor-mediated NO and Akt signaling might be an important determinant pathway for the aortic VSMC phenotype in hypertension in vivo.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation.

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

Molecular Basis by Which Garlic Suppresses Atherosclerosis

The aim of this study was to determine the mechanism by which the aged garlic extract "Kyolic" has a protective effect against atherosclerosis. Plasma cholesterol of rabbits fed a 1% cholesterol-enriched diet for 6 wk was not reduced by supplementation with 800 microL Kyolic/(kg body. d). In spite of this, Kyolic reduced by 64% (P < 0.05) the surface area of the thoracic aorta covered by fatty streaks and significantly reduced aortic arch cholesterol. Kyolic also significantly inhibited by approximately 50% the development of thickened, lipid-filled lesions in preformed neointimas produced by Fogarty 2F balloon catheter injury of the right carotid artery in cholesterol-fed rabbits. In vitro studies found that Kyolic completely prevented vascular smooth muscle phenotypic change from the contractile, high volume fraction of filament (V(v)myo) state, and inhibited proliferation of smooth muscle cells in the synthetic state with a 50% effective dose (ED(50)) of 0.2%. Kyolic also slightly inhibited the accumulation of lipid in cultured macrophages but not smooth muscle, and had no effect on the expression of adhesion molecules on the surface of the endothelium or the adherence of leukocytes. It is concluded that Kyolic exerts antiatherogenic effects through inhibition of smooth muscle phenotypic change and proliferation, and by another (unclarified) effect on lipid accumulation in the artery wall.

Maturation of ovine uterine smooth muscle during development and the effects of parity

To characterize changes in myometrial contractile proteins and myosin heavy chain (MHC) isoforms during ovine fetal and neonatal development and after pregnancy. We hypothesized that ovine myometrium demonstrates progressive cellular differentiation and maturation which begins in utero and extends into the postnatal period, and that pregnancy causes further cellular alterations. Myometrium was obtained from female fetal (72- to 140-days of gestation, n = 19; term = approximately 145 days), postnatal (1 day to 3 months, n = 25), and parous noncycling nonpregnant (n = 9) sheep to measure total and soluble proteins, actin, MHC, and MHC isoforms. Contractile proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and expression of 200-kD MHC isoforms were determined with Western immunoblots. The contents of total and soluble proteins and actin and total myosin gradually increase (P <.003) during ovine development. Although the contribution of smooth-muscle 204-kD MHC increased (P <.001) from 23 +/- 8% of total MHC at <100 days of gestation to 75 +/- 2% 3 to 4 months postnatally, the 200-kD species fell proportionately. Before birth, MHC-B, a fetal isoform, is the predominant 200-kD protein; postnatally, it is replaced by SM2, demonstrating a switch from a synthetic to a mature contractile smooth-muscle phenotype. Pregnancy is associated with further increases in actin contents and redistribution of the contents of the 204-kD and SM2 MHC isoforms. Although the fetal and postnatal uterus has no known functional demand, ovine myometrial differentiation and maturation begin in the midtrimester and continue throughout the postnatal period. Thus, changes in smooth-muscle phenotype occur prenatally, as evidenced by a switch from MHC-B to SM2, which may signal completion of organ development and preparation for adult function. Pregnancy results in further modifications in myometrial proteins.

The extracellular matrix dynamically regulates smooth muscle cell responsiveness to PDGF.

Focal accumulation of smooth muscle (SMC) within the arterial intima contributes to the formation of lesions of atherosclerosis. Platelet-derived growth factor (PDGF) is a potent stimulant of SMC migration and proliferation in culture that may play a role in the accumulation of SMC in atherogenesis. SMCs normally reside in the media of the artery wall surrounded by extracellular matrix (ECM), including type I collagen. In atherogenesis, the ECM is degraded, new ECM components, such as fibronectin, are synthesized and assembled, and these alterations in ECM components are associated with changes in SMC phenotype. To model the changes in ECM in normal and diseased arteries, we have analyzed SMCs cultured on different forms of type I collagen. Our studies demonstrate that integrin-mediated signals from various forms of type I collagen lead to specific and rapid modulation of the integrin signaling complex, including cytoskeletal connections, and of the responsiveness of SMC to PDGF stimulation.

Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype

Macrophages cocultured with rabbit aortic smooth muscle cells at a ratio of 1:3 degraded all the 35S-labeled heparan sulfate proteoglycan from the smooth muscle surface into free sulfate (Kav of 0.84 on Sepharose 6B). Concomitantly, the same macrophages induced a decrease in the volume fraction of myofilaments (Vvmyo) of the smooth muscle cells and a decrease in alpha-actin mRNA as a percentage of total actin mRNA. Both macrophage lysosomal lysate at neutral pH and heparinase degraded cell-free 35S-labeled matrix deposited by smooth muscle cells into fragments which eluted at a Kav of 0.63 and which were identified as heparan sulfate chains by their complete degradation in the presence of low pH nitrous acid. At acid pH the macrophage lysosomal lysate completely degraded the heparan sulfate to free sulfate (Kav 0.84). Both macrophage lysosomal lysate and commercial heparinase at neutral pH induced smooth muscle phenotypic change while other enzymes such as trypsin and chondroitin ABC lyase had no effect. It was therefore suggested that the active factor present in the macrophages is a lysosomal heparan sulfate-degrading endoglycosidase (heparinase). Only a small amount of heparan sulfate-degrading activity was released into the incubation medium by living macrophages, and there was no heparinase activity on their isolated plasma membranes, although proteolytic enzymes were evident in both instances. In pulse-chase studies, high Vvmyo smooth muscle cells were seen to constantly internalize and degrade 35S-labeled heparan sulfate proteoglycan from their own pericellular compartment, suggesting that this may be the mechanism by which smooth muscle phenotype is maintained under normal circumstances and that removal of heparan sulfate from the surface of smooth muscle cells and its degradation by macrophages temporarily interrupts this process, inducing smooth muscle phenotypic change.

Cytoskeletal and extracellular matrix proteins in cerebral arteries following subarachnoid hemorrhage in monkeys

It is unclear if vasospasm after subarachnoid hemorrhage (SAH) is predominantly due to smooth-muscle contraction, proliferative vasculopathy, or other changes within the arterial wall such as fibrosis or change in smooth-muscle phenotype. In this study, immunohistochemistry was used to examine changes in extracellular and cytoskeletal proteins in cerebral arteries after SAH that might support one of these mechanisms. Following baseline cerebral angiography, bilateral SAH was created in nine monkeys. Three animals each were killed 7, 14, or 28 days after SAH. Cerebral angiography was repeated on Day 7 in all animals and immediately prior to sacrifice in animals killed on Days 14 and 28. Both middle cerebral arteries and four control basilar arteries were examined using fluorescent antibody techniques with antisera to alpha-actin, myosin, fibronectin, fibrinogen, vimentin, desmin, laminin, and collagens (types I, III, IV, and V). Angiography showed that vasospasm was most severe on Day 7, present but resolving on Day 14, and completely resolved by Day 28. Microscopic study of arterial sections and blinded review of microphotographs of arterial sections by five independent observers did not reveal changes in intensity of density of staining for collagens, desmin, myosin, laminin, or alpha-actin in the tunica media of tunica adventitia. Fibronectin immunoreactivity increased 14 days after SAH. Seven days after SAH, occasional areas of tunica media showed immunoreactivity to fibrinogen. On Day 28, intimal thickening was observed in four of six middle cerebral arteries and this tissue demonstrated immunoreactivity to alpha-actin, myosin, vimentin, desmin, fibronectin, laminin, and each type of collagen. No significant increases in the number of intimal cells showing immunoreactivity to alpha-actin were seen and no significant changes in the hydroxyproline content of cerebral arteries developed at any time after SAH. These results suggest that rigidity and lumen narrowing of vasospasm are not due to increased arterial collagen, although other proteins in the arterial wall or an alteration in cross-linking of existing proteins could produce these changes. There is no indication that smooth-muscle contractile proteins change during vasospasm or that increases in the number of alpha-actin-containing myointimal cells contribute to vasospasm. The occurrence of intimal thickening and increased tunica media fibronectin after vasospasm suggests that vasospasm damages smooth muscle, possibly as a result of intense prolonged smooth-muscle contraction.

THE MACROPHAGE AS AN INITIATOR OF ATHEROSCLEROSIS

1. Heparan sulfate proteoglycan in the basal lamina of smooth muscle cells is important in the maintenance of the 'contractile', high volume fraction of myofilaments (Vvmyo) phenotype. The mechanism by which this occurs may involve the continuous internalization of heparan sulfate by the smooth muscle cells themselves. 2. One macrophage can degrade all the heparan sulfate from three smooth muscle cells by the action of heparan sulfate-degrading enzymes in their lysosomes, thus leaving none available for internalization by the smooth muscle cell until it has synthesized more, and leading to the induction of smooth muscle phenotypic change from a high Vvmyo to a low Vvmyo. 3. In this altered phenotype the smooth muscle cells proliferate in response to mitogens, synthesize large amounts of extracellular matrix and accumulate lipid, all characteristics of the smooth muscle cell in developing atheroma.

Balloon catheter injury to rabbit carotid artery. I. Changes in smooth muscle phenotype.

Stereology was used to investigate the changes in ultrastructure of smooth muscle cells during the formation of an experimental intimal thickening induced by injury with an inflated balloon catheter. The volume density of myofilaments in the cell cytoplasm was measured in smooth muscle cell-lined areas (which are freely permeable to Evans blue dye and, hence, stain blue) and in re-endothelialized areas (which remain white after injection of Evans blue) of the rabbit carotid artery. Two weeks after injury, the volume densities of myofilaments in the intimal smooth muscle cells in both white and blue areas were significantly less than that for control medial smooth muscle (67.9% +/- 3.6%; mean +/- SE), being 38.8% +/- 1.0% and 35.9 +/- 3.3%, respectively. By 6 weeks after injury, the volume density had increased significantly in both white (55.1% +/- 3.4%) and blue areas (53.5% +/- 3.0%), and these values did not change significantly by 18 weeks. The volume density of myofilaments in the luminal (lining) smooth muscle cells in the blue areas was significantly less than that of control medial cells and remained low (26.7% +/- 2.1%) up to 18 weeks after injury. The initial balloon-induced injury caused considerable damage to the smooth muscle cells in the media, and the remaining medial cells underwent similar changes in ultrastructure to the cells in the neointima. At 2 weeks, the cells had a low volume density of myofilaments (44.9% +/- 2.4%), which returned to a level not significantly different from the control artery by 6 weeks after injury. There were no differences in the estimates of the volume density of myofilaments between the inner and outer media of the injured arteries. These findings suggest that, after injury produced by a balloon catheter, the smooth muscle cells in both the media and the resultant intimal thickening undergo a reversible change in ultrastructure.

Potential Role of Heparanase in Atherosclerosis

The mechanism by which a change in smooth muscle phenotype is effected in the artery wall during atherogenesis may be via release of extracellular matrix-degrading enzymes, particularly heparanase, from activated macrophages and T lymphocytes.

Endothelial cell influences on vascular smooth muscle phenotype.

In the normal arterial wall the smooth muscle cells and abluminal surface of the endothelium are surrounded by a basal lamina consisting of (among other substances) laminin, heparan sulfate proteoglycan, and type IV collagen (59, 75). In turn the basal laminae are closely associated with the continuum of collagen, elastin, and proteoglycans of the extracellular matrix. This associa­ tion maintains spatial arrangements of components within the vessel wall (93), but may also regulate the proper homeostatic balance of the cellular con­ stituents. In this article, the relationship between the endothelium, smooth muscle, and an extracellular matrix molecule they produce is explored. In particular, we will discuss the proposal that a heparin-like glycosaminoglycan present in the basal lamina of both smooth muscle and endothelium plays an important role in controlling the phenotype the smooth muscle expresses, and its response to serum mitogens. Change in smooth muscle phenotype leads to many changes in the biology of the cell, and these will also be reviewed.

Lipid accumulation in arterial smooth muscle cells: Influence of phenotype

Isolated smooth muscle cells from the adult pig and rabbit aorta in primary culture undergo a spontaneous change in phenotype from a contractile to a synthetic state over 6–8 days, losing their capacity to contract and gaining the capacity to divide. The change in smooth muscle phenotype to the synthetic state is accompanied by distinct changes in the cells' ability to metabolize LDL, with the rate of degradation of 125I-labelled LDL decreasing to about one fifth of the level in contractile state cells. This does not appear to be due to changes in the number or affinity of LDL receptors since saturable binding of LDL is unaltered. The specific activities of the lysosomal enzymes acid phosphatase and N-acetyl-β-glucosamini-dase increase with change to the synthetic state as do cytochrome c oxidase (mitochondria) and NADPH-dependent cytochrome c reductase (endoplasmic reticulum). In contrast there is a slight but not significant decrease in the specific activity of the lysosomal enzyme acid cholesteryl esterase of rabbit smooth muscle cells and a significant decrease in the activity of pig cells with change in phenotype to the synthetic state. Significantly more [ 3H]cholesteryl oleate is recovered in synthetic state than in contractile state cells following incubation with 20 tug/ml unlabelled LDL and [ 3H]sodium oleate. Morphologically there is no difference in the number of lipid droplets in contractile and synthetic state cells after incubation in 5% normolipemic serum, but in cells grown in 10% hyperlipemic serum for 4 days synthetic state cells become almost completely filled with lipid droplets while contractile state cells are unaffected. Lipid accumulation also occurs selectively in vivo in synthetic is compared with contractile state smooth muscle cells within intimal fibromuscular thickening induced by de-endothelialization of the carotid artery of cholesterol-fed rabbits. We suggest that accumulation of lipid in smooth muscles cells of atherosclerotic plaques is related to reduced catabolism of LDL following smooth muscle phenotypic change from the contractile to synthetic state.