Activity of sap from Croton lechleri on rat vascular and gastric smooth muscles

The effects of red sap from Croton lechleri (SdD), Euphorbiaceae, on vascular and gastric smooth muscles were investigated. SdD, from 10 to 1000 μg/ml, induced concentration-dependent vasoconstriction in rat caudal arteries, which was endothelium-independent. In arterial preparations pre-constricted by phenylephrine (0.1 μM) or KCl (30 mM), SdD also produced concentration-dependent vasoconstriction. To study the mechanisms implicated in this effect we used selective inhibitors such as prazosin (0.1 μM), an antagonist of α₁-adrenoceptors, atropine (0.1 μM), an antagonist of muscarinic receptors, and ritanserin (50 nM), a 5-HT_2A antagonist; none of these influenced vasoconstriction caused by SdD. Likewise, nifedipine (50 nM), an inhibitor of L-type calcium channels, did not modify the action of SdD. Capsaicin (100 nM), an agonist of vanilloid receptors, also did not affect vasoconstriction by SdD.We also investigated the action of SdD (10–1000 μg/ml) on rat gastric fundus; per se the sap slightly increased contractile tension. When the gastric fundus was pre-treated with SdD (100 μg/ml) the contraction induced by carbachol (1 μM) was increased, whereas that by KCl (60 mM) or capsaicin (100 nM) were unchanged.The data shows that SdD increased contractile tension in a concentration-dependent way, both on vascular and gastric smooth muscles. The vasoconstriction is unrelated to α₁, M, 5-HT_2A and vanilloid receptors as well as L-type calcium channels. SdD increased also contraction by carbachol on rat gastric fundus. Thus for the first time, experimental data provides evidence that sap from C. lechleri owns constricting activity on smooth muscles.     

Endothelin induces functional hypertrophy of peritubular smooth muscle cells

When chronically stimulated with agonists of contraction, smooth muscle cells (SMCs) undergo cell hypertrophy, a process defined as increase in size and potentiation of the contractile phenotype in the absence of proliferation. Hypertrophic response has long been associated to a number of pathologies of the cardiovascular and respiratory systems. We have investigated the phenotypic and functional response of SMCs to long-term treatment with endothelin. Our model was primary cultures of peritubular smooth muscle cells (PSMC) a testicular cell type target of locally produced endothelin and characterized by an unusual phenotypic stability when cultured in simple medium in complete absence of serum. We report the following responses of PSMC to 4-day exposure to ET-1: (i) increased protein synthesis without induction of cell proliferation; (ii) increase in cell size (evaluated by means of flow cytometry) and increased expression of SM-alpha-actin, desmin, caldesmon and calponin, markers of the contractile phenotype. In experiments of selective stimulation of either ETA or ETB receptor subtypes, both proved to be involved in inducing the observed hypertrophic responses. The hypertrophic cells exhibit the ultrastructural features of differentiated SMCs and are capable of calcium mediated contractile response when acutely stimulated with ET-1 specifically through ETA and/or ETB receptors, as evaluated by calcium imaging and scanning electron microscopy. These observations demonstrate that engagement of ET receptors is capable of inducing potentiation of the contractile phenotype and functional hypertrophy of PSMC.     

Cadherins in vascular smooth muscle cell (patho)biology: Quid nos scimus?

Vascular smooth muscle cells (SMCs) phenotypes span a reversible continuum from quiescent/contractile (differentiated) to proliferative/synthetic (dedifferentiated) enabling them to perform a diversity of functions that are context-dependent and important for vascular tone-diameter homeostasis, vasculogenesis, angiogenesis or vessel reparation after injury. Dysregulated phenotype modulation and failure to maintain/regain the mature differentiated and contractile phenotypic state is pivotal in the development of vascular diseases such as atherosclerosis and restenosis after angioplasty and coronary bypass grafting. Many functions of SMCs such as adhesion, migration, proliferation, contraction, differentiation and apoptosis are regulated by a broad spectrum of cell-cell and cell-matrix adhesion molecules. Cadherins represent a superfamily of cell surface homophilic adhesion molecules with fundamental roles in morphogenetic and differentiation processes during development and in the maintenance of tissue integrity and homeostasis in adults. The cadherins have major inputs on signalling pathways and cytoskeletal assemblies that participate in regulating processes such as cell polarity, migration, proliferation, survival, phenotype and differentiation. Abnormalities in these processes have long been recognized to underlie pathological SMC-driven reparation, but knowledge on the involvement of cadherins is remarkably limited. This article presents a comprehensive review of cadherin family members currently identified on vascular SMCs in relation to their functions, molecular mechanisms of action and relevance for vascular pathology.     

Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.     

Mechanical effects of ET-1 in cardiomyocytes isolated from normal and heart-failed rabbits

Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca²⁺ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca²⁺ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca²⁺ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC₅₀ obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca²⁺ channels were similar with a peak amplitude at 1 nM ET-1 of –3.26 ± 0.8 in control cardiomyocytes and –3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca²⁺ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.Recipient of Servier Investigator Award     

O2 Level Controls Hematopoietic Circulating Progenitor Cells Differentiation into Endothelial or Smooth Muscle Cells

Background

Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium.

Methodology/Principal Findings

Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O₂) or normoxic (21% O₂) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3^rd passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability.

Conclusion/Significance

We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3^rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells.     

Regulation of parietal cell calcium signaling in gastric glands

The ligands interacting with enterochromaffin-like (ECL) and parietal cells and the signaling interactions between these cells were investigated in rabbit gastric glands using confocal microscopy. Intracellular calcium concentration ([Ca(2+)](i)) changes were used to monitor cellular responses. Histamine and carbachol increased [Ca(2+)](i) in parietal cells. Gastrin (1 nM) increased [Ca(2+)](i) in ECL cells and adjacent parietal cells. Only the increase of [Ca(2+)](i) in parietal cells was inhibited by H(2) receptor antagonists (H(2)RA). Gastrin (10 nM) evoked an H(2)RA-insensitive [Ca(2+)](i) increase in parietal cells. Carbachol produced large H(2)RA- and somatostatin-insensitive signals in parietal cells. Pituitary adenylate cyclase-activating peptide (PACAP, 100 nM) elevated [Ca(2+)](i) in ECL cells and adjacent parietal cells. H(2)RAs abolished the PACAP-stimulated [Ca(2+)](i) increase in adjacent parietal cells. Somatostatin did not inhibit the increase of [Ca(2+)](i) in parietal cells stimulated with histamine, high gastrin concentrations, or carbachol but abolished ECL cell calcium responses to gastrin or PACAP. Hence, rabbit parietal cells express histaminergic, muscarinic, and CCK-B receptors coupled to calcium signaling but insensitive to somatostatin, whereas rabbit and rat ECL cells express PACAP and CCK-B calcium coupled receptors sensitive to somatostatin.     

Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility

Heterogeneity of embryological origins is a hallmark of vascular smooth muscle cells (SMCs) and may influence the development of vascular disease. Differentiation of human pluripotent stem cells (hPSCs) into developmental origin-specific SMC subtypes remains elusive. Here we describe a chemically defined protocol in which hPSCs were initially induced to form neuroectoderm, lateral plate mesoderm or paraxial mesoderm. These intermediate populations were further differentiated toward SMCs (>80% MYH11(+) and ACTA2(+)), which displayed contractile ability in response to vasoconstrictors and invested perivascular regions in vivo. Derived SMC subtypes recapitulated the unique proliferative and secretory responses to cytokines previously documented in studies using aortic SMCs of distinct origins. Notably, this system predicted increased extracellular matrix degradation by SMCs derived from lateral plate mesoderm, which was confirmed using rat aortic SMCs from corresponding origins. This differentiation approach will have broad applications in modeling origin-dependent disease susceptibility and in developing bioengineered vascular grafts for regenerative medicine.     

Contraction of vascular smooth muscle in cell culture

The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells. This limitation has been overcome by the cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane). Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist-induced contraction. Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2-dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture.     

MicroRNA-145-based differentiation of human mesenchymal stem cells to smooth muscle cells

Objectives

To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs.

Results

Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation.

Conclusion

Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease.

     

Aceclidine and pilocarpine interact differently with muscarinic receptor in isolated rabbit iris muscle

The relationship between muscarinic receptor affinity states and the contractile response to the muscarinic agonists carbachol, aceclidine, and pilocarpine, has been examined in the isolated rabbit iris muscle. Contraction of the iris muscle by carbachol and aceclidine was more potent and/or more efficacious than the response to pilocarpine. Analysis of [³H]-Quinuclidinyl benzilate (QNB) binding showed that while both carbachol and aceclidine bound to high- and low-affinity forms of the muscarinic receptor, pilocarpine bound to one affinity state. The efficacy of carbachol and aceclidine to stimulate contraction of the iris muscle was consistent with receptor occupancy theory only when considering the low-affinity state of the muscarinic receptor, and activation of the low-affinity rather than high-affinity binding state of the receptor is likely to mediate the contraction of iris muscle. Therefore, the typical anti-glaucoma muscarinic agonists aceclidine and pilocarpine may interact differently with their target receptors in isolated rabbit iris muscle.     

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains.

We have previously demonstrated that alpha-smooth muscle (alpha-SM) actin is predominantly distributed in the central region and beta-non-muscle (beta-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and alpha-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha-SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with alpha-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.     

Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells

Proliferative or synthetic vascular smooth muscle cells (VSMCs) are widely accepted to be mainly derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs, i.e., a phenotype switch from a normally quiescent and contractile type into a proliferative or synthetic form. However, this theory has been challenged by recent evidence that synthetic VSMCs predominantly originate instead from media-derived multipotent vascular stem cells (MVSCs). To test these hypotheses further, we re-examine whether the conventional rat aortic SMC (RASMC) culture involves the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs and the potential mechanism for controlling the synthetic phenotype of RASMCs. We enzymatically isolated RASMCs and cultured the cells in both a regular growth medium (RGM) and a stem cell growth medium (SCGM). Regardless of culture conditions, only a small portion of freshly isolated RASMCs attaches, survives and grows slowly during the first 7 days of primary culture, while expressing both SMC- and MVSC-specific markers. RGM-cultured cells undergo a process of synthetic SMC differentiation, whereas SCGM-cultured cells can be differentiated into not only synthetic SMCs but also other somatic cells. Notably, compared with the RGM-cultured differentiated RASMCs, the SCGM-cultured undifferentiated cells exhibit the phenotype of MVSCs and generate greater amounts of reactive oxygen species (ROS) that act as a negative regulator of differentiation into synthetic VSMCs. Knockdown of phospholipase A2, group 7 (Pla2g7) suppresses ROS formation in the MVSCs while enhancing SMC differentiation of MVSCs. These results suggest that cultured synthetic VSMCs can be derived from the SMC differentiation of MVSCs with ROS as a negative regulator.     

Changes in three-dimensional architecture of microfilaments in cultured vascular smooth muscle cells during phenotypic modulation

To investigate changes in the three-dimensional microfilament architecture of vascular smooth muscle cells (SMC) during the process of phenotypic modulation, rabbit aortic SMCs cultured under different conditions and at different time points were either labelled with fluorescein-conjugated probes to cytoskeletal and contractile proteins for observation by confocal laser scanning microscopy, or extracted with Triton X-100 for scanning electron microscopy. Densely seeded SMCs in primary culture, which maintain a contractile phenotype, display prominent linear myofilament bundles (stress fibres) that are present throughout the cytoplasm with alpha-actin filaments predominant in the central part and beta-actin filaments in the periphery of the cell. Intermediate filaments form a meshed network interconnecting the stress fibres and linking directly to the nucleus. Moderately and sparsely seeded SMCs, which modulate toward the synthetic phenotype during the first 5 days of culture, undergo a gradual redistribution of intermediate filaments from the perinuclear region toward the peripheral cytoplasm and a partial disassembly of stress fibres in the central part of the upper cortex of the cytoplasm, with an obvious decrease in alpha-actin and myosin staining. These changes are reversed in moderately seeded SMCs by day 8 of culture when they have reached confluence. The results reveal two changes in microfilament architecture in SMCs as they undergo a change in phenotype: the redistribution of intermediate filaments probably due to an increase in synthetic organelles in the perinuclear area, and the partial disassembly of stress fibres which may reflect a degradation of contractile components.     

Gene expression profiling of resting and activated vascular smooth muscle cells by serial analysis of gene expression and clustering analysis

Migration and proliferation of vascular smooth muscle cells (SMCs) are key events in atherosclerosis. However, little is known about alterations in gene expression upon transition of the quiescent, contractile SMC to the proliferative SMC. We performed serial analysis of gene expression (SAGE) of cultured, human SMCs, either grown under resting circumstances or activated with an atherogenic stimulus. Analysis of tags, representing 47,209 and 47,259 mRNAs from a library of resting and activated SMCs, respectively, identified 105 tags induced and 52 tags repressed greater than fivefold. To evaluate the relevance in SMC biology of unmatched, regulated tags, we performed hierarchical clustering analysis, based on their expression profiles in public SAGE databases, and clustered these novel genes in distinct groups. The regulation in SMCs was confirmed by Northern blotting for representative genes of these groups. Plasminogen activator inhibitor-2 has not been associated with atherosclerosis before and was localized to atherosclerotic lesions.     

Effect of changes of pHi on intracellular calcium in a smooth muscle-like cell line

The effect of changes of pH_i on Ca_i were studied using fluorescent dyes in cells of the cultured smooth muscle-like line, BC3H-1. Resting Ca_i in these cells was 182 ± 12 nM (n = 74) at pH_o of 7.4. Upon exposure to NH₄Cl, which rapidly alkalinized cells, a transient increase of Cai to 349 ± 55 nM (n = 29) was observed. The peak of the transient occurred within 30 s of exposure to NH₄Cl and returned to baseline within 1 minute. Two other procedures which resulted in rapid cellular alkalinization also caused a transient rise in Ca_i: exposure to and then removal of CO₂ (Ca_i increased from 182 ± 22 to 248 ± 28 nM; n = 8); and exposure to and then removal of Na propionate (Ca_i increased from 242 ± 32 to 456 ± 71 nM; n = 9). The NH4Cl-induced Ca_i transient was eliminated by exposure to 0.2 mM TMB8 and to Ca-free solutions, but not by exposure to 0.5 mM LaCl₃. Sustained changes of pH_i can be induced by varying pH_o. When pH_o was lowered to 6.9, Ca_i fell by 49 ± 11 nM but increased by 203 ± 51 nM (n = 6) when pH_o was raised to 7.9. These data indicate that rapid alkalinization of BC3H-1 cells results in a rapid transient rise of Ca_i. This transient is most likely due to the release of Ca from intracellular stores but may also involve an increase of Ca influx. Steady state values of Cai are positively correlated with steady state pH_i. These data may have implications for the contractile state of smooth muscle during periods of acid/base disturbances and relate to the role of elevated pH_i in cells from hypertensive animals.     

Plasma fibronectin promotes modulation of arterial smooth-muscle cells from contractile to synthetic phenotype

Isolated arterial smooth-muscle cells (SMCs) cultured in medium containing whole blood serum or plasma-derived serum undergo modulation from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and cessation of the ability to contract. Instead, an extensive rough endoplasmic reticulum and a large Golgi complex are formed and, if properly stimulated, the cells start to proliferate actively and to produce extracellular-matrix components. In vivo, a similar change in the differentiated properties of SMCs appears to be an early key event in atherogenesis. The purpose of the present investigation was to try to identify plasma components that promote the modulation of the smooth-muscle phenotype. SMCs were enzymatically isolated from rat aorta and cultured in a defined, serum-free medium. The phenotypic state of the cells was determined by transmission electron microscopy, and their growth status was followed by 3H-thymidine autoradiography and cell counting. Under these conditions, Cohn fractions I (fibrinogen) and V (albumin) were found to partially support cell attachment and transition from the contractile to the synthetic phenotype, whereas fractions II-III and IV (globulins) were inactive in this respect. Analysis on adsorptive columns of gelatin Sepharose 4B indicated that Cohn fraction I, but not fraction V, contained fibronectin, an adhesive protein that is present in plasma and binds to fibrinogen. When seeded on a substrate of plasma fibronectin, the cells attached with high efficiency and modulated into the synthetic phenotype at a rate similar to that observed in serum-containing medium. In the absence of exogenous mitogens, the structural transformation of the cells was not accompanied by a proliferative response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-mediated contraction of collagen lattices in serum-free medium: Effect of serum and nonserum factors

Summary This study was conducted to identify a defined, serum-free culture medium that supports cell dependent contraction of a collagen lattice. Collagen lattices were found to contract in cultures containing human foreskin fibroblasts (HFF) or rabbit aortic smooth muscle (RASM) cells incubated in serum-free medium. HFF and RASM cells required different supplements to contract the collagen gels. HFF cultured in Dulbecco’s modified Eagle’s (DME) medium supplemented with bovine serum albumin (BSA) and either endothelial cell growth supplement (EnGS), insulin (In), or platelet derived growth factor (PDGF) supported collagen lattice contraction. Replacement of BSA with casein without the addition of other supplements improved contraction. In contrast, RASM cells supplemented with BSA, EnGS, In, and PDGF were able to contract collagen gels only minimally. Similar to HFF, RASM cells cultured in DME medium supplemented with casein, but without the addition of other supplements, contracted collagen lattices. HFF-mediated collagen contraction was inhibited by prostaglandins E₁ or E₂, fibronectin, or ascorbic acid. The reported serum-free model provides a useful in vitro method to investigate the role of serum and nonserum factors regulating cell mediated-contraction of insoluble collagen fibrils. Presented in part as abstract 1963 in the 1985 Federation of American Societies for Experimental Biology, April 21–26, Anaheim, California, and published in Fed. Proc. (44):747; 1985.