Behavior Of Smooth Muscle Cells Research Articles

Phytoncide, Nanochemicals from Chamaecyparis obtusa, Inhibits Proliferation and Migration of Vascular Smooth Muscle Cells.

Phytoncide, nanochemicals extracted from Chamaecyparis obtusa (C. obtusa), is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, antioxidant, and antiinflammatory activities. However, the effect of phytoncide in vascuar diseases, especially on the behavior of vascular smooth muscle cells, has not yet been clearly elucidated. Therefore, in the present study, we investigated the effects of 15 kinds of phytoncide by various extraction conditions from C. obtusa on the proliferation and migration in rat aortic smooth muscle cells (RAoSMCs). First of all, we determined the concentration of each extracts not having cytotoxicity by MTT assay. We observed that the proliferation rate measured using BrdU assay was significantly reduced by supercritical fluid, steam distillation, Me-OH, and hexane extraction fraction in order with higher extent, respectively. Moreover, the treatment of above nanofractions inhibit the migration of RAoSMCs by 40%, 60%, and 30%, respectively, both in 2-D wound healing assay and 3-D boyden chamber assay. Immunoblot revealed that the phosphorylated levels of Akt and ERK were significantly reduced in nanofractions treated RAoSMCs. Taken together, these data suggest that phytoncide extracted from C. obtusa inhibits proliferation and migration in RAoSMCs via the modulation of phosphorylated levels of Akt and ERK. Therefore, phytoncide nanomolecules might be a potential therapeutic approach to prevent or treat atheroscrelosis and restenosis.

Fabrication of micro‐patterned titanium dioxide nanotubes thin film and its biocompatibility

In this study, in order to obtain a novel biomaterials surface which possesses the functions of an anticoagulant, inhibiting smooth muscle proliferation and pro-endothelialisation simultaneously, a micro-patterned titanium dioxide (TiO2)-nanotubes (P-TiO2-nanotubes) thin film was prepared on titanium (Ti) surface by photolithography combining with anodising method. The authors found that the P-TiO2-nanotubes could reduce platelet attachment and conformational change of fibrinogen (FGN) as well as promoting endothelial cells (ECs) growth compared with the TiO2-nanotubes. Notably, P-TiO2-nanotubes could significantly reduce smooth muscle cells (SMCs) proliferation compared with TiO2-nanotubes and TiO2, and the ECs and SMCs cultured on the P-TiO2-nanotubes thin film were elongated, which was suggested to be beneficial for maintaining the cell function. Therefore it is suggested that the P-TiO2-nanotubes thin film can contribute more to biocompatible functions of regulating and coordinating the behaviour of platelets, ECs and SMCs.

Divergent effects of Wnts on vascular smooth muscle cell (VSMC) behaviour

Divergent effects of Wnts on vascular smooth muscle cell (VSMC) behaviour

Adsorption of plasma proteins and fibronectin on poly(hydroxylethyl methacrylate) brushes of different thickness and their relationship with adhesion and migration of vascular smooth muscle cells.

The surface-grafted poly(hydroxylethyl methacrylate) (PHEMA) molecules were demonstrated to show a brush state regardless of their molecular length (molecular weight). Adsorption of proteins from 10% fetal bovine serum (FBS), fibronectin (Fn) and bovine serum albumin (BSA) was quantified by ellipsometry, revealing that the amounts of FBS and Fn decreased monotonously along with the increase of PHEMA thickness, whereas not detectable for BSA when the PHEMA thickness was larger than 6 nm. Radio immunoassay found that the adsorption of Fn from 10% FBS had no significant difference regardless of the PHEMA thickness. However, ELISA results showed that the Arg-Gly-Asp (RGD) activity of adsorbed Fn decreased with the increase of PHEMA thickness. By comparison of cellular behaviors of vascular smooth muscle cells (VSMCs) being cultured in vitro in the normal serum-containing medium and the Fn-depleted serum-containing medium, the significant role of Fn on modulating the adhesion and migration of VSMCs was verified. Taking account all the results, the Fn adsorption model and its role on linking the biomaterials surface to the VSMCs behaviors are proposed.

A correlation study of protein adsorption and cell behaviors on substrates with different densities of PEG chains

The adsorption of proteins, in particular fibronectin (Fn), was studied on poly(ethylene glycol) (PEG, 5kDa)-grafted surfaces, and was correlated with the adhesion behaviors of smooth muscle cells (SMCs). The PEG molecules were covalently grafted on aldehyde-activated substrates with different densities of amino groups. The thickness of PEG layer increased nearly 10 fold in a hydrated state, reaching to 27nm on the surface of highest PEG chain density with a brush configuration. On the lower PEG-grafted surfaces, however, the PEG molecules adopted a mushroom configuration. The adsorption of Fn without and with the competition of bovine serum albumin (BSA) and serum was studied by using ellipsometry, fluorescence microscopy and radio-labeling techniques. The adsorption amount of Fn in serum decreased initially with increased PEG chain density until 0.12chains/nm2 PEG, and then slightly increased on the 0.29chains/nm2 PEG. A series of protein preadsorption experiments were carried out under different conditions before SMCs culture in vitro. Compared with those substrates without Fn preadsorption, the cell adhesion and spreading were significantly enhanced on all the PEG surfaces preadsorbed with Fn and serum, although they overall decreased along with the increase of PEG grafting density. The adhesion force of Fn decreased monotonously with the increase of PEG grafting density, which was in accordance with the cell adhesion force. The correlation between the PEG-grafted surfaces, Fn adsorption, and cellular behaviors is finally suggested.

Abstract 169: Smad3 Drives Cross-Talk Between TGFß and Wnt/ß-Catenin Signaling Pathways in Smooth Muscle Cells

Restenosis (neo-intimal hyperplasia) occurs in approximately 25-50% of patients undergoing arterial interventions, primarily due to the proliferation and migration of arterial smooth muscle cells (SMCs) into the peri-luminal area. Recently, Wnt/β-catenin signaling has been shown to promote SMC proliferation and enhance neo-intimal hyperplasia but its mechanism of activation is unclear. Interestingly, Wnt/β-catenin has been shown to be activated by TGFβ in mesenchymal stem cells and fibroblasts. We have shown that TGFβ and its downstream signaling protein, Smad3, are upregulated following vascular injury and that Smad3 overexpressing SMCs display enhanced proliferation, migration, and neo-intimal hyperplasia. These results led us to hypothesize that TGFβ, through Smad3, activates Wnt/β-catenin to regulate SMC behavior following arterial injury . In primary rat SMCs, TGFβ (5ng/mL) led to β-catenin activation and relocalization from the plasma membrane to the cytoplasm / nucleus within 24 hours. Furthermore, qRT-PCR results demonstrated that expression of Wnt11 (22 fold) and Wnt9a (3.9 fold) were significantly upregulated after 24 hours of TGFβ stimulation (p<0.05, n=3). In addition, 24 hours of TGFβ stimulation in SMCs overexpressing Smad3 (TGFβ/Smad3) further enhanced the gene expression of Wnt11 (>300 fold) and Wnt9a (14 fold) and also stimulated significant increases in Wnt2b (41 fold), Wnt5a (2.9 fold), and Wnt4 (3.2 fold) (p<0.05, n=3) as measured by qRT-PCR. Western blot results demonstrated that the combined TGFβ/Smad3 stimulation increased β-catenin protein levels, suggesting that TGFβ activates canonical Wnt signaling leading to stabilization of β-catenin protein. In normal rat carotid arteries, β-catenin protein was undetectable via immunohistochemistry but could be seen in SMCs of the vessel media at 3 days post-balloon angioplasty and in neo-intimal cells at 7 and 14 days. Smad3 was also expressed in neo-intimal cells at 7 and 14 days post-angioplasty suggesting that TGFβ, through Smad3, is responsible for Wnt/β-Catenin activation during vascular injury. In conclusion, this work describes a novel cross-talk in SMCs between TGFβ and Wnt signaling which may provide a viable target for future anti-restenotic treatments.

Phenotypic Modulation of Smooth Muscle Cells by Chemical and Mechanical Cues of Electrospun Tecophilic/Gelatin Nanofibers

The ability of mature smooth muscle cells (SMCs) to modulate their phenotype in response to environmental cues is a critical issue related to vascular diseases. A tissue engineered vascular graft shall promote the contractile phenotype of vascular SMCs. To this aim, Tecophilic/gelatin (TP/gel) was electrospun at different weight ratios of TP/gelatin (100:0, 70:30, 50:50, 30:70), leading to differences in biochemical and mechanical properties of the nanofibers which in turn influenced the phenotype of SMCs. Results indicated that both the substrate with higher ligand density and lower stiffness could enhance SMC contractility and reduce cell proliferation. However, observing the highest SMCs contractility on electrospun TP(70)/gel(30) among the composite scaffolds demonstrated stiffness as the most critical parameter. Due to conflicting effects of softness versus minor fraction of gelatin (reduced ligand density) within TP(70)/gel(30) fibers, a relatively high proliferation of SMCs was still observed on TP(70)/gel(30) scaffold. The surface of TP(70)/gel(30) scaffold was further modified through physical adsorption of gelatin molecules so as to increase the ligand density on its surface, whereby a functional vascular construct that promotes the contractile behavior of SMCs with low cell proliferation was developed.

Effect of 20-hydroxyeicosatetraenoic acid on biological behavior of human villous trophoblasts and uterine vascular smooth muscle cells

During pregnancy, disorders in uterine spiral artery remodeling (USAR) cause preeclampsia and other diseases. The aim of this study was to investigate the effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on the biological behavior of human villous trophoblasts (HVTs) and human uterine vascular smooth muscle cells (HUVSMCs), and explore the role of 20-HETE in USAR. 20-HETE and its inhibitor HET0016 were used to study migration, invasion and apoptosis in the HVT and HVT-HUVSMC models. 20-HETE inhibited the migration and invasion of HVTs, and inhibited apoptosis in HUVSMCs and HUVSMCs co-cultured with HVTs. 20-HETE had thus obvious effects on the biological behavior of HVTs and HUVSMCs. These effects may cause USAR disorders and vascular dysfunction, leading to preeclampsia.

Effects of polydopamine functionalized titanium dioxide nanotubes on endothelial cell and smooth muscle cell

Previous investigations have demonstrated that TiO2 nanotubes (NTs) with particular structure cues could control the behavior of different types of cells, including endothelial cells (ECs) and smooth muscle cells (SMCs). Besides, polydopamine (PDA) modified surfaces were reported to be beneficial to increase the proliferation and viability of ECs and meanwhile could inhibit the proliferation of SMCs. The TiO2 nanotubes (NTs) were functionalized with polydopamine (PDA) (PDA/NTs) to study the synergetic effect of both nanotopography (NTs) and chemical cues (PDA) of TiO2 nanotubes on the regulation of cellular behavior of ECs and SMCs. The PDA-modified TiO2 nanotubes were subjected to field-emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and water contact angle (WCA) analysis. In vitro cell culture tests confirmed that, comparing with flat titanium (Ti) and TiO2 nanotubes, PDA/NTs surface synergistically promoted ECs attachment, proliferation, migration and release of nitric oxide (NO). Meanwhile, the PDA/NTs performed well in reducing SMCs adhesion and proliferation. This novel approach might provide a new platform to investigate the synergistic effect of local chemistry and topography, as well as the applications for the development of titanium-based implants for enhanced endothelialization.

Bisulfite and sulfite as derivatives of sulfur dioxide alters biomechanical behaviors of airway smooth muscle cells in culture

Sulfur dioxide (SO2) is a common air pollutant that triggers asthmatic symptoms, but its toxicological mechanisms are not fully understood. Specifically, it is unclear how SO2 in vivo affects airway smooth muscle (ASM) cells of which the mechanics is known to ultimately mediate airway hyperresponsiveness (AHR) – a hallmark feature of asthma. To this end, we investigated the effects of bisulfite/sulfite (1:3 M/M in neutral fluid to simulate the in vivo derivatives of inhaled SO2 in the airways), on the viability, migration, stiffness and contractility of ASM cells cultured in vitro. The results showed that bisulfite/sulfite consistently increased viability, migration, F-actin intensity and stiffness of ASM cells in similar fashion as concentration increasing from 10−4 to 10−1 mmol/L. However, bisulfite/sulfite increased the ASM cell contractility induced by KCl only at the concentration between 10−4 and 10−3 mmol/L (p < 0.05), while having no consistent effect on that induced by histamine. At the concentration of 100 mmol/L, bisulfite/sulfite became acutely toxic to the ASM cells. Taken together, the data suggest that SO2 derivatives at low levels in vivo may directly increase the mass, stiffness and contractility of ASM cells, which may help understand the mechanism in which specific air pollutants contribute in vivo to the pathogenesis of asthma.

Directional migration of vascular smooth muscle cells guided by synergetic surface gradient and chemical pattern of poly(ethylene glycol) brushes

Directional migration of cells in vitro can mimic corresponding biological events in vivo, which provides a way to determine the cascade responses in the tissue regeneration process and develop novel criteria for the design of tissue-inductive biomaterials. In this work, a density gradient of methoxy poly(ethylene glycol) brushes (from 0.37 to 0.95 chains/nm2) on plain and striped pattern surfaces was fabricated, using a dynamically controlled reaction process, and characterized by X-ray photoelectron spectroscopy and quartz crystal microbalance with dissipation. Adhesion and migration behavior of vascular smooth muscle cells were studied on the gradient and gradient-patterned surfaces. The vascular smooth muscle cells exhibited preferential orientation and enhanced directional migration on the gradient surface toward the lower end of the methoxy poly(ethylene glycol) density. By introducing methoxy poly(ethylene glycol) chemical striped patterns in parallel with the gradient direction on the surface, the extent of cell orientation and directional migration were significantly improved. Due to the synergetic effects of surface methoxy poly(ethylene glycol) striped patterns and gradient cues, almost all cells were oriented, and 67% of the cells were observed to move unidirectionally.

A Critical Role for Proinflammatory Behavior of Smooth Muscle Cells in Hemodynamic Initiation of Intracranial Aneurysm

BackgroundIntracranial aneurysm initiation is poorly understood, although hemodynamic insult is believed to play an important role in triggering the pathology. It has recently been found in a rabbit model that while macrophages are absent during hemodynamic aneurysm initiation, matrix metalloproteinases (MMPs) are elevated and co-localize with smooth muscle cells (SMCs). This study investigates whether SMCs play a mechanistic role in aneurysm initiation triggered by hemodynamics.MethodsAneurysmal damage was induced at the basilar terminus via bilateral common carotid artery ligation in rabbits (n = 45, plus 7 sham controls). 16 ligated rabbits were treated with doxycycline to inhibit MMPs, 7 received clodronate liposomes to deplete circulating monocytes, and the rest received no drug. Effects of the treatments on aneurysm development were assessed histologically 5 days and 6 months after ligation. MMP production and expression of inflammatory markers by SMCs was monitored by immunohistochemistry and in situ hybridization.ResultsTreatment with doxycycline attenuated aneurysmal development examined at 5 days and 6 months, suggesting that MMPs contribute to aneurysm initiation. However, systemic depletion of macrophages did not decrease MMPs or suppress aneurysmal development. Immunofluorescence showed that during aneurysm initiation MMP-2 and MMP-9 were distributed in SMCs, and in situ hybridization indicated that they were transcribed by SMCs. In regions of early aneurysmal lesion, SMCs exhibited decreased expression of smooth muscle actin and increased NF-κB and MCP-1 expressions.ConclusionsDuring aneurysm initiation triggered by hemodynamics, SMCs rather than macrophages are responsible for MMP production that is critical for aneurysmal lesion development. These SMCs exhibit proinflammatory behavior.

An adipokine omentin prevents pathological vascular remodeling

Background: Obese states are associated with the development of atherosclerosis and vascular restenosis after angioplasty. Omentin is an adipocytokine whose concentrations are reduced in obese individuals. Recently, we have reported that circulating omentin levels are down-regulated in association with obesity-linked disorders including carotid atherosclerosis and coronary artery disease. Here, we investigated the role of omentin on neointimal hyperplasia and Vascular Smooth Muscle Cell (VSMC) behavior in vivo and in vitro. Methods and results: An adenoviral vector expressing human Omentin (Ad-OMT) or β-galactosidase as a control was injected into the jugular vein of Wild-Type (WT) mice 3 d prior to vascular injury. Left femoral arteries of mice were injured by a stainless-steel wire inserted from the lumen. Although human omentin protein could not be detected in plasma in control WT mice, plasma human omentin levels were increased to 1353.9±169.6 ng/ml in Ad-OMT-treated WT mice on the 5th day after injection. At 21 days after vascular injury, systemic delivery of Ad-OMT significantly attenuated neointimal thickening compared with control (p<0.01, n=8). Ad-OMT decreased the number of bromodeoxyuridine (BrdU) positive proliferating cells in the neointima at day 7 after vascular injury (p<0.01, n=5). In cultured VSMCs, treatment with recombinant omentin protein attenuated DNA synthesis and chemotactic activity induced by various growth factors as assessed by BrdU incorporation and Boyden chamber assays, respectively. Treatment with omentin suppressed growth factor-induced phosphorylation of ERK in VSMCs. Treatment of VSMCs with omentin stimulated the phosphorylation of AMPK in a time-dependent manner. Furthermore, transduction with dominant-negative AMPK prevented the inhibitory effect of omentin on growth factor-stimulated VSMC proliferation and ERK phosphorylation. Conclusion: Omentin attenuates neointimal hyperplasia after vascular injury and suppresses VSMC growth through its ability to activate AMPK, suggesting that omentin can represent a novel target molecule for the prevention of pathological remodeling.

Cell Durotaxis on Polyelectrolyte Multilayers with Photogenerated Gradients of Modulus

Behaviors of rat aortic smooth muscle (A7r5) and human osteosarcoma (U2OS) cells on photo-cross-linked polyelectrolyte multilayers (PEMUs) with uniform, or gradients of, moduli were investigated. The PEMUs were built layer-by-layer with the polycation poly(allylamine hydrochloride) (PAH) and a polyanion poly(acrylic acid) (PAA) that was modified with photoreactive 4-(2-hydroxyethoxy) benzophenone (PAABp). PEMUs with different uniform and gradients of modulus were generated by varying the time of uniform ultraviolet light exposure and by exposure through optical density gradient filters. Analysis of adhesion, morphology, cytoskeletal organization, and motility of the cells on the PEMUs revealed that A7r5 cells established a polarized orientation toward increasing modulus on shallow modulus gradients (approximately 4.7 MPa mm(-1)) and durotaxed toward stiffer regions on steeper gradients (approximately 55 MPa mm(-1)). In contrast, U2OS cells exhibited little orientation or durotaxis on modulus gradients. These results demonstrate the utility of photo-cross-linked PEMUs to direct vascular and osteoblast cell behavior, a potential application for PEMU coatings on biomedical implants.

Non‐canonical MMP1‐PAR1 signaling drives vascular smooth muscle cell de‐differentiation and restenosis

Cardiovascular disease remains the leading cause of death in the United States. Aberrant vascular repair following plaque rupture or iatrogenic intervention is mediated by smooth muscle cells (SMCs). SMC proliferation, migration, and de‐differentiation from a contractile to a synthetic phenotype are crucial contributors to the pathology associated with maladaptive remodeling. The GPCR, protease activated receptor‐1 (PAR1), has been implicated in both vascular remodeling and atherothrombosis. Despite the well documented contribution of PAR1 to arterial thrombosis, it is unclear whether PAR1 promotes or mitigates the hyperplastic response to arterial injury. We sought to determine whether inhibition of upstream activating proteases of PAR1, matrix metalloprotease‐1 (MMP‐1) and thrombin, would have differing effects on SMC behavior and restenosis. We describe a previously unknown pathway through which MMP‐1 drives intimal hyperplasia and leads to SMC de‐differentiation through biased agonism of PAR1. In contrast, thrombin‐PAR1 signaling promotes a contractile phenotype. Also, pharmacologic blockade of MMP‐1 suppresses intimal hyperplasia following arterial injury. These data suggest a new strategy for targeted PAR1 inhibition that does not rely on receptor antagonism, but instead directly targets the detrimental activating protease. NIH grants F30 HL108590 (to K.M.A.), R01 HL64701 (to A.K.)

Application of a mechanobiological simulation technique to stents used clinically

Many cardiovascular diseases are characterised by the restriction of blood flow through arteries. Stents can be expanded within arteries to remove such restrictions; however, tissue in-growth into the stent can lead to restenosis. In order to predict the long-term efficacy of stenting, a mechanobiological model of the arterial tissue reaction to stress is required. In this study, a computational model of arterial tissue response to stenting is applied to three clinically relevant stent designs. We ask the question whether such a mechanobiological model can differentiate between stents used clinically, and we compare these predictions to a purely mechanical analysis. In doing so, we are testing the hypothesis that a mechanobiological model of arterial tissue response to injury could predict the long-term outcomes of stent design.Finite element analysis of the expansion of three different stent types was performed in an idealised, 3D artery. Injury was calculated in the arterial tissue using a remaining-life damage mechanics approach. The inflammatory response to this initial injury was modelled using equations governing variables which represented tissue-degrading species and growth factors. Three levels of inflammation response were modelled to account for inter-patient variability. A lattice-based model of smooth muscle cell behaviour was implemented, treating cells as discrete agents governed by local rules.The simulations predicted differences between stent designs similar to those found in vivo. It showed that the volume of neointima produced could be quantified, providing a quantitative comparison of stents. In contrast, the differences between stents based on stress alone were highly dependent on the choice of comparison criteria. These results show that the choice of stress criteria for stent comparisons is critical.This study shows that mechanobiological modelling may provide a valuable tool in stent design, allowing predictions of their long-term efficacy. The level of inflammation was shown to affect the sensitivity of the model to stent design. If this finding was verified in patients, this could suggest that high-inflammation patients may require alternative treatments to stenting.

JunB Mediates Basal- and TGFβ1-Induced Smooth Muscle Cell Contractility

Smooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFβ1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFβ1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFβ1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton.

Influence of fibre diameter and orientation of electrospun copolyetheresterurethanes on smooth muscle and endothelial cell behaviour

Polymers exhibiting cell-selective effects represent an extensive research field with high relevance for biomedical applications e.g. in the cardiovascular field supporting re-endothelialization while suppressing smooth muscle cell overgrowth. Such an endothelial cell-selective effect could be recently demonstrated for a copolyetheresterurethane (PDC) containing biodegradable poly(p-dioxanone) and poly(ε-caprolactone) segments, which selectively enhanced the adhesion of human umbilical vein endothelial cells (HUVEC) while suppressing the attachment of smooth muscle cells (SMC). In this study we investigated the influence of the fibre orientation (random and aligned) and fibre diameter (2 μm and 500 nm) of electrospun PDC scaffolds on the adhesion, proliferation and apoptosis of HUVEC and SMC. Adhesion, viability and proliferation of HUVEC was diminished when the fibre diameter was reduced to a submicron scale, while the orientation of the microfibres did only slightly influence the cellular behaviour. In contrast, a submicron fibre diameter improved SMC viability. In conclusion, PDC scaffolds with micron-sized single fibres could be promising candidate materials for cell-selective stent coatings.

Age-related increase of stem marker expression influences vascular smooth muscle cell properties

ObjectiveAging represents a major risk factor for vascular disease development. With aging, changes of the biological properties of vascular smooth muscle cells (SMCs) are observed. Stem marker expression characterizes SMCs during developmental growth and atherosclerosis, but the contribution of SMCs with stem features to the age-related arterial remodeling remains largely unknown. Methods and resultsImmunostaining revealed rare vascular growth factor receptor-1+ (flt-1+) and c-kit+ cells in tunica media of grossly normal human young (17–30 years old) large arteries and 2-month old rat aorta, whereas CD133+ cells were absent. In large arteries of human aged donors (64–77 years), flt-1+ and c-kit+ cell number increased in the intimal thickening and tunica media. Double immunofluorescence revealed that 30.6 ± 3% of flt-1+ intimal cells co-expressed α-smooth muscle actin. Immunostaining, blots and RT-PCR documented the increased expression of flt-1 and c-kit in 20–24-month old rat aortic media. In vitro, old rat aortic SMCs proliferated and migrated more with greater flt-1, c-kit, NF-κB, VCAM-1, IAP-1 and MCP-1 levels and less α-smooth muscle actin and myosin compared to young SMCs. Old SMCs were also more susceptible to all-trans retinoic and NF-κB inhibition-induced apoptosis compared to young SMCs. Anti-flt-1 blocking antibody reduced migration and placental growth factor-induced but not serum and PDGF-BB-stimulated proliferation of old SMCs. ConclusionsThe increase of flt-1+ and c-kit+ SMCs characterizes large arteries of aged donors; the blocking of flt-1 signaling influences the behavior of old SMCs, suggesting that the accumulation of SMCs with a stem phenotype contributes to the age-dependent adverse arterial remodeling.

Abstract 519: Calmodulin Is a Marker of a Distinct Smooth Muscle Cell Population Recruited by Plaque-Derived Macrophages from the Human Carotid Artery Media

Background: Different experimental observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset during atheromatous plaque establishment and restenosis. Objectives: We investigated the presence and features of such SMC subset in cell cultures derived from human carotid endarterectomy (CEA) specimens; moreover, we aimed at finding markers that could play a role in SMC behavior. Methods and Results: CEA specimens comprised a grossly undiseased (UP) and a diseased portion (DP). The UP contained a thin intimal thickening with the underlying media and the DP an atherosclerotic plaque with the underlying media. Cell cultures were initiated by tissue explantation. From plaque tissues only macrophage-derived foam cells were retrieved. From medial tissues 2 distinct SMC phenotypes were identified: 1) large SMCs, flat with a monolayered growth pattern, isolated from the UP media; 2) small SMCs, fusiform and growing in multilayers, isolated from both the UP and DP media only when cocultured with plaque-derived macrophages. Small SMCs displayed higher proliferative and migratory activities and a poor level of differentiation compared with large SMCs. Two-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry showed that calmodulin (CaM), a calcium-binding protein involved in cell-cycle regulation, was mainly expressed in small SMCs. This result was confirmed using a specific CaM antibody. Coculture of large SMCs with plaque-derived macrophages induced a switch to the small phenotype and was associated with increased CaM expression. Consistently, the specific CaM inhibitor W-7 hampered the large-to-small switch and predominantly inhibited the proliferation of small SMCs. In vivo, CaM was markedly expressed in atherosclerotic plaques whereas it was barely detectable in the media. Conclusions: Plaque-derived macrophages promote the selective migration of a distinct SMC subpopulation exhibiting features of an “atheroma-prone” phenotype. CaM is a marker of small SMCs in vitro and of plaque SMCs in vivo, and plays a role in SMC phenotypic modulation. Further studies on these SMC populations can be instrumental in understanding and influencing the evolution of atherosclerosis.