Population Of Smooth Muscle Cells Research Articles

Arterial territory-specific phosphorylated retinoblastoma protein species and CDK2 promote differences in the vascular smooth muscle cell response to mitogens

Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries.

Myocardin

There is a general consensus that perturbations to the vessel wall provoke a population of mature vascular smooth muscle cells (VSMCs) to either dedifferentiate into proliferating/migrating cells or transdifferentiate into new cell types.1 Such phenotypic plasticity has stimulated much research on the signaling pathways and transcriptional mediators, coordinating the expression of a growing number of genes (both coding and noncoding) that constitute a unique transcriptome for the differentiated state of VSMCs. The majority of such genes harbor 1 or more 10-bp codes known as CArG boxes to which the serum response factor (SRF) binds.2 More than 60 proteins interact with SRF to drive cofactor-dependent programs of gene expression. One prominent SRF cofactor is myocardin (MYOCD), which was first described as a cardiac muscle–enriched transcription factor that powerfully stimulates CArG-dependent promoters driving expression of cardiac-specific genes and contributes to normal cardiac muscle gene expression in vivo.3 Soon after its discovery in 2001, MYOCD was shown to similarly enhance activity of VSMC-restricted promoters in a CArG-dependent manner and induce expression of several of the corresponding endogenous transcripts in a cell line that does not express VSMC-restricted genes. Furthermore, Myocd mRNA was discovered to be abundantly expressed in aortic SMC, only to be reduced when such cells are placed in culture, suggesting MYOCD is important in maintaining the normal VSMC contractile phenotype.4 Subsequent studies confirmed and extended many of these findings, thus validating the role of MYOCD as a key component …

Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells.

ObjectiveTo obtain a pure population of smooth muscle cells (SMC) derived from mouse embryonic stem cells (ESC) and further assess their functions.MethodsA vector, expressing both puromycin resistance gene (puror) and enhanced green fluorescent protein (EGFP) gene driven by smooth muscle 22α (SM22α) promoter, named pSM22α-puror-IRES2-EGFP was constructed and used to transfect ESC. Transgenic ESC (Tg-ESC) clones were selected by G418 and identified by PCR amplification of puror gene. The characteristics of Tg-ESC were detected by alkaline phosphatase (ALP) staining, SSEA-1 immunofluorescence and teratoma formation test in vivo. After induction of SMC differentiation by all-trans retinoic acid, differentiated Tg-ESC were treated with 10 µg/mL puromycin for three days to obtain purified SMC (P-SMC). Percentage of EGFP+ cells in P-SMC was assessed by flow cytometer. Expressions of smooth muscle specific markers were detected by immunostaining and Western blotting. Proliferation, migration and contractility of P-SMC were analyzed by growth curve, trans-well migration assay, and carbachol treatment, respectively. Finally, both P-SMC and unpurified SMC (unP-SMC) were injected into syngeneic mouse to see teratoma development.ResultsTg-ESC clone was successfully established and confirmed by PCR detection of puror gene in its genomic DNA. The Tg-ESC was positive for ALP staining, SSEA-1 staining and formed teratoma containing tissues derived from three germ layers. After retinoic acid induction, large amount of EGFP positive cells outgrew from differentiated Tg-ESC. Three days of puromycin treatment produced a population of P-SMC with an EGFP+ percentage as high as 98.2% in contrast to 29.47% of unP-SMC. Compared with primary mouse vascular smooth muscle cells (VSMC), P-SMC displayed positive, but lowered expression of SMC-specific markers including SM α-actin and myosin heavy chain (SM-MHC) detected either, by immunostaining, or immunoblotting, accelerated proliferation, improved migration (99.33 ± 2.04 vs. 44.00 ± 2.08 migrated cells/field, P < 0.05), and decreased contractility in response to carbachol (7.75 ± 1.19 % vs. 16.50 ± 3.76 % in cell area reduction, P < 0.05). In vivo injection of unP-SMC developed apparent teratoma while P-SMC did not.ConclusionsWe obtained a pure population of ESC derived SMC with less mature (differentiated) phenotypes, which will be of great use in research of vascular diseases and in bio-engineered vascular grafts for regenerative medicine.

Abstract 186: Aortic IL-17C Producing Cells in Atherosclerosis.

The progression of atherosclerosis from fatty acid streaks to complex lesions involves regular communication between hematopoetic cells within the arterial wall and vascular cells via cytokine and chemokine networks. Recent evidence has demonstrated the presence of Interleukin-17A (IL-17A)-producing T cells within atherosclerotic Apolipoprotein E-deficient (Apoe-/-) aortas. IL-17A is a member of the IL-17 cytokine family, which consists of six related isoforms (IL-17A-IL-17F). While the functions of IL-17A, IL-17E, and IL-17F are the subject of ongoing studies, the roles and producers of IL-17B and IL-17C remain elusive. Recent studies have demonstrated a pro-inflammatory role for epithelial cell and keratinocyte-derived IL-17C in the context of experimental colitis, and psoriasis. In the present study, we sought to examine the expression of IL-17C within the aortas of atherosclerotic Apoe-/- and Il17a-/-Apoe-/- mice. We detected Il17c mRNA within the aortas of both 12-week western diet-fed Apoe-/- and Il17a-/-Apoe-/- mice. To further identify and characterize the cellular sources of aortic IL-17C, we examined aortic leukocytes and vascular cells by flow cytometry. We identified several major cellular producers of IL-17C in vivo, including a major population of smooth muscle cells (5±0.8% of Apoe-/- CD45-αSMA+ SMCs) and a population of CD45-αSMA- cells (5.8±0.8% of Apoe-/- CD45-αSMA- cells). Interestingly, the percentage of aortic IL-17C producing cells was elevated within 12 week western diet-fed Il17a-/-Apoe-/- mice in comparison to Apoe-/- littermates (6.16±0.24% of the Il17a-/-Apoe-/- aorta vs 3.6±0.1% of the Apoe-/- aorta), suggesting an inverse relationship between T cell derived-IL-17A and vascular-IL-17C. To examine the effects of IL-17C and the possibility of synergy between TNFα and IL-17C on aortic chemokine and cytokine production, we explanted and treated whole aortas with IL-17C or IL-17C and TNFα in vitro. IL-17C with and without TNFα broadly supported the expression of Ccl2, Ccl7, Cxcl1, Cxcl2, Cxcl5, and Il6 in explanted WD Apoe-/- aortas. Together, these results suggest that vascular smooth muscle cell-derived IL-17C may play a pro-inflammatory role in atherosclerosis via the production of chemokines and cytokines.

Abstract 4282: Crenolanib inhibition of PDGFRA+ fibroblast-like cells in visceral smooth muscle.

Abstract Platelet derived growth factors (PDGFs) and their receptors (PDGFRs) play important roles in cell proliferation, survival, and migration . The PDGF/PDGFR signaling pathway plays a crucial role in embryonic development and for interstitial and mesenchymal cell proliferation . Primary cancer cells in the GI with abnormal expression of receptor tyrosine kinases like c-Kit and PDGFRs have been implicated in the formation of gastro-intestinal stromal tumors (GIST). Fibroblast-like cells (FLCs) exist in the smooth muscle of all visceral organs yet their functional role throughout the viscera is unclear . It has recently been demonstrated by studies within our group that these FLCs express PDGFRα . These studies demonstrated for the first time that PDGFRα+ cells are a novel class of excitable cells with the molecular and ionic apparatus to mediate enteric inhibitory responses to purines in GI muscles. Using Crenolanib, a novel potent inhibitor of PDGFRα, we have disrupted PDGFRα expression and signaling in normal BALB/c mice. Crenolanib is being developed to treat cancers with populations of primary cells over-expressing PDGFRα including GIST . To track the specific changes in GI visceral smooth muscle cell populations and changes in post-junctional neural responses, we examined changes in protein abundance, gene expression, and used intracellular microelectrodes to examine electrophysiological properties and responses to stimuli. Given the intimate anatomical relationship between Kit+ ICCs and PDGFRα+ FLCs, their structural similarities, and their defining expression of receptor tyrosine kinases, changes in the dynamics of both of these cell populations during Crenolanib inhibition was monitored within this model. Age and species matched pairs of mice injected with either vehicle or Crenolanib were collected from postpartum days 10 through 25 and analyzed for differences in gross anatomy and weight, electrophysiology, PDGFRα protein distribution by western blot and immunofluorescence, and gene expression by both end-point PCR and qPCR. These analyses show a distinct decrease in the protein abundance and gene expression of PDGFRα in Crenolanib injected mice; inhibitory neurological signaling is also affected, with a highly significant decrease in purinergic inhibitory responses to stimulation, which indicates this as one of the functional roles of FLCs in the Tunica muscularis. Monitoring FLCs and ICCs in this inhibition model has provided valuable information on the endogenous cellular dynamics that exist between ICCs and FLCs; it also sheds light on their relationship during pathophysiolgical disorders, including the development of GISTs . Citation Format: Evan Colletti, Abhijit Ramachandran, Monique Carter, Sean Ward. Crenolanib inhibition of PDGFRA+ fibroblast-like cells in visceral smooth muscle. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4282. doi:10.1158/1538-7445.AM2013-4282

Modeling and Rescue of the Vascular Phenotype of Williams-Beuren Syndrome in Patient Induced Pluripotent Stem Cells

Elastin haploinsufficiency in Williams-Beuren syndrome (WBS) leads to increased vascular smooth muscle cell (SMC) proliferation and stenoses. Our objective was to generate a human induced pluripotent stem (hiPS) cell model for in vitro assessment of the WBS phenotype and to test the ability of candidate agents to rescue the phenotype. hiPS cells were reprogrammed from skin fibroblasts of a WBS patient with aortic and pulmonary stenosis and healthy control BJ fibroblasts using four-factor retrovirus reprogramming and were differentiated into SMCs. Differentiated SMCs were treated with synthetic elastin-binding protein ligand 2 (EBPL2) (20 μg/ml) or the antiproliferative drug rapamycin (100 nM) for 5 days. We generated four WBS induced pluripotent stem (iPS) cell lines that expressed pluripotency genes and differentiated into all three germ layers. Directed differentiation of BJ iPS cells yielded an 85%-92% pure SMC population that expressed differentiated SMC markers, were functionally contractile, and formed tube-like structures on three-dimensional gel assay. Unlike BJ iPS cells, WBS iPS cells generated immature SMCs that were highly proliferative, showed lower expression of differentiated SMC markers, reduced response to the vasoactive agonists, carbachol and endothelin-1, impaired vascular tube formation, and reduced calcium flux. EBPL2 partially rescued and rapamycin fully rescued the abnormal SMC phenotype by decreasing the smooth muscle proliferation rate and enhancing differentiation and tube formation. WBS iPS cell-derived SMCs demonstrate an immature proliferative phenotype with reduced functional and contractile properties, thereby recapitulating the human disease phenotype. The ability of rapamycin to rescue the phenotype provides an attractive therapeutic candidate for patients with WBS and vascular stenoses.

Percutaneous Tissue-Engineered Pulmonary Valved Stent Implantation: Comparison of Bone Marrow-Derived CD133+-Cells and Cells Obtained from Carotid Artery

The aim of tissue engineering is, for instance, to create a new valve or organ that is similar to the native one. This new construct may have the capability of growth, repair, and remodeling. One instance where tissue engineering offers an enormous advantage is in the treatment of Tetralogy of Fallot. After repair of this defect, pulmonary valve regurgitation with late ventricular dysfunction is not uncommonly observed. In this study, we compared the use of endothelial cells (ECs) and smooth muscle cells (SMCs) derived from the carotid artery (group 1/n=5) with CD133+-cells derived from the bone-marrow (group 2/n=5) of juvenile sheep. Western blot analysis and histology were done before and after percutaneous pulmonary valved stent implantation. After seeding of the pulmonary valved stent with one of the cell groups, the construct was placed in a dynamic bioreactor for 16 days. After incubation, the valved stent was seeded again for 3 days. Finally, the valved stent was implanted percutaneously under angiographic control. In group 1, α-actin and calponin were found in the EC and SMC population by using Western blot analysis. In contrast, CD31 was found in EC, but not in the SMC population. Cells from group 2 demonstrated a pure population. CD133+-cells were detected in the CD133+-cell population and no CD133+-cells were found in the negative fraction. Three months after implantation, the transvalvular gradient was significantly higher in group 1 compared to group 2. CD31-staining demonstrated a confluent monolayer in both groups. Immunohistochemistry revealed strong expression of α-smooth-muscle-actin and an in-growth into the leaflets of both groups (p=ns). CD3-, CD20-, CD45-, and CD68-staining confirmed no signs of inflammation in group 2, whereas in group 1 small amounts of inflammation were detected in all analyzed animals. Von Kossa staining revealed mild to moderate calcifications in the annular region of group 1. In contrast, less calcification was detected in group 2. Autologous CD133+-cells derived from bone marrow had a better outcome with regard to calcification, inflammation, and transvalvular gradient compared to autologous cells derived from the carotid artery. Therefore, CD133+-cells are more advantageous for tissue engineering of heart valves.

Premature differentiation of vascular smooth muscle cells in human congenital diaphragmatic hernia

BackgroundCongenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterized by the herniation of abdominal organs into the chest cavity. The high mortality and morbidity of CDH patients are primarily caused by the associated pulmonary hypertension (PH), characterized by the thickening of the vascular media and adventitia. The media consist of heterogeneous populations of vascular smooth muscle cells (VSMC), ranging from synthetic to the characteristic contractile cells. VSMCs are influenced by developmental and environmental cues and may play a role in the development of the structural changes observed in CDH patients. Therefore, we hypothesized that the distribution of the VSMC populations may already be different at the origin of CDH development. MethodologyWe analyzed the protein expression of specific markers associated with synthetic and contractile VSMC phenotypes in human lungs at different developmental stages. Next, we compared lungs of premature and term CDH patients, as well as patients with lung hypoplasia due to renal agenesis or PROM, with age-matched controls. ResultsSynthetic and contractile VSMCs are distributed in a temporal and spatial specific pattern along the proximodistal axis of the lung. CDH patients have more abundant contractile VSMCs which are also more distally distributed. This different distribution pattern is already observed from 19weeks of gestation onwards. ConclusionOur data suggest that the more extensive distribution of contractile VSMCs is associated with an early maturation of the pulmonary vasculature, contrasting the concept that CDH might be the result of delayed maturation of the epithelium.

Differential susceptibility of human primary aortic and coronary artery vascular cells to RNA interference

BackgroundRNAi technology is a promising tool for gene therapy of vascular disease. However, the biological heterogeneity between endothelial (EC) and vascular smooth muscle cells (SMC) and within different vascular beds make them differentially susceptible to siRNA. This is further complicated by the task of choosing the right transfection reagent that leads to consistent gene silencing across all cell types with minimal toxicity. The goal of this study was to investigate the intrinsic RNAi susceptibility of primary human aortic and coronary artery endothelial and vascular smooth muscle cells (AoEC, CoEC, AoSMC and CoSMC) using adherent cell cytometry. MethodsCells were seeded at a density of 5000cells/well of a 96well plate. Twenty four hours later cells were transfected with either non-targeting unlabeled control siRNA (50nM), or non-targeting red fluorescence labeled siRNA (siGLO Red, 5 or 50nM) using no transfection reagent, HiPerFect or Lipofectamine RNAiMAX. Hoechst nuclei stain was used to label cells for counting. For data analysis an adherent cell cytometer, Celigo was used. ResultsRed fluorescence counts were normalized to the cell count. EC displayed a higher susceptibility towards siRNA delivery than SMC from the corresponding artery. CoSMC were more susceptible than AoSMC. In all cell types RNAiMAX was more potent compared to HiPerFect or no transfection reagent. However, after 24h, RNAiMAX led to a significant cell loss in both AoEC and CoEC. None of the other transfection conditions led to a significant cell loss. ConclusionThis study confirms our prior observation that EC are more susceptible to siRNA than SMC based on intracellular siRNA delivery. RNAiMax treatment led to significant cell loss in AoEC and CoEC, but not in the SMC populations. Additionally, this study is the first to demonstrate that coronary SMC are more susceptible to siRNA than aortic SMC.

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.

Abstract 176: Extracellular S100A4 Is a Key Modulator of Arterial Smooth Muscle Cell Phenotypic Transition

Background: It has been proposed that smooth muscle cells (SMCs) from the arterial wall are heterogeneous and that only a subset of medial SMCs are prone to accumulate into the intima leading to atheromatous plaque formation. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as a marker of R-SMCs in vitro and of intimal SMCs both in pig and human. S100A4 is an intracellular Ca2+ binding protein that can also be secreted; it has extracellular functions probably via the receptor for advanced glycation end products (RAGE). Objective: Investigate the role of S100A4 in SMC phenotypic change, a process characteristic of atherosclerotic plaque formation. Methods and results: Transfection of a human S100A4-containing plasmid in S-SMCs (practically devoid of S100A4) led to overexpression of S100A4 in approximately 10% of SMCs, release of S100A4, and a transition towards a R-phenotype of the whole SMC population. Moreover, treatment of S-SMCs with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition towards a R-phenotype, which was associated with increased proliferative and migratory activities as well as downregulation of SMC differentiation markers (i.e. alpha-smooth muscle actin and smooth muscle myosin heavy chains). It yielded activation of NF-kappa B without changing RAGE mRNA baseline level. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulated SMC differentiation markers. In contrast, silencing of S100A4 mRNA in R-SMCs and during platelet-derived growth factor-BB-induced S-to-R-phenotypic change did not affect SMC morphology in spite of decreased proliferative activity. Conclusions: Our results show that extracellular S100A4 modulates SMC phenotypic changes and is essential for the establishment of the R-phenotype. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosis.

Extension of bladder-based organ regeneration platform for tissue engineering of esophagus

Recent successes in regenerative medicine and tissue engineering of bladder and bladder-like neo-organs have leveraged regenerative constructs composed of a biodegradable scaffold seeded with a population of smooth muscle cells. We have shown that such smooth muscle cells are isolatable from adipose and other sources alternate to the primary organ. We hypothesize that this regenerative platform is not limited to regeneration of bladder and bladder-like neo-organs, but rather represents a foundational technology platform broadly applicable for regeneration of laminarly organized hollow organs. Using esophagus as an illustrative example in support of this hypothesis, we demonstrate that patch constructs composed of adipose-derived smooth muscle cells seeded on a biodegradable matrix catalyze complete regeneration of the esophageal wall in a rodent model of esophageal injury. By implication, such regenerative constructs may potentially be used to mediate the regeneration of any laminarly organized tubular organ.

Cell Reorganization in Gastric Transplant during Esophagoplasty

We studied peculiarities of cell reorganization of the gastric transplant during the delayed period after esophagoplasty for benign pathologies of the esophagus. Polymorphism of adaptive and pathological structural rearrangements in the mucosa of the gastric tube was observed. Degenerative and atrophic changes in epithelial structures, hypertrophy of smooth muscular components, and sclerotic reorganization of the stroma were predominating morphological phenomena. Heterogeneity of ultrastructural modifications of the epithelium reflected high secretory activity of foveal mucocytes in combination with degenerative changes in cytoplasmic organelles and impaired secretory function of fundic gland cells. Hypertrophied cells with minor ultrastructural changes, leiomyocytes with signs of biosynthesis, and degenerated smooth muscle fibers were revealed in the population of smooth muscle cells.

Microtubule Dynamics Regulate Cyclic Stretch-Induced Cell Alignment in Human Airway Smooth Muscle Cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.

Effects of Fluid Shear Stress on a Distinct Population of Vascular Smooth Muscle Cells

Vascular smooth muscle cells (SMCs) are a major cell type involved in vascular remodeling. The various developmental origins of SMCs such as neural crest and mesoderm result in heterogeneity of SMCs, which plays an important role in the development of vascular remodeling and diseases. Upon vascular injury, SMCs are exposed to blood flow and subjected to fluid shear stress. Previous studies have shown that fluid shear stress inhibits SMC proliferation. However, the effect of shear stress on the subpopulation of SMCs from specific developmental origin and vascular bed is not well understood. Here we investigated how shear stress regulates human aortic SMCs positive for neural crest markers. DNA microarray analysis showed that shear stress modulates the expression of genes involved in cell proliferation, matrix synthesis, cell signaling, transcription and cytoskeleton organization. Further studies demonstrated that shear stress induced SMC proliferation and cyclin D1, downregulated cell cycle inhibitor p21, and activated Akt pathway. Inhibition of PI-3 kinase blocked these shear stress-induced changes. These results suggest that SMCs with neural crest characteristics may respond to shear stress in a different manner. This finding has significant implications in the remodeling and diseases of blood vessels.

Vascular Wall Engineering Via Femtosecond Laser Ablation: Scaffolds with Self-Containing Smooth Muscle Cell Populations

For tissue-engineered vascular grafts to reach their full potential, three-dimensional (3D) cellular micro-integration will be necessary. In this study, we utilize femtosecond laser ablation to produce microchannels inside electrospun polycaprolactone (PCL) scaffolds. These microchannels potentially provide spatially controlled cell distributions approaching those observed in vivo. The ability of such laser-ablated microchannels to direct cell seeding was evaluated. The dimensions chosen were 100 μm wide, 100 μm deep and 10 mm long. Femtosecond laser ablation successfully produced these microchannels in the scaffolds without substantially altering the ~900 nm diameter fibers. Flow within these microchannels was studied by injecting fluorescent polystyrene bead solutions. Direct measurement of bead motion yielded an inlet velocity of 2.78 cm s(-1). This was used for modeling two-dimensional (2D) flow using computational fluid dynamics to estimate flow profiles within the microchannel. Successful demonstrations of bead flow were followed by seeding of 500,000 human coronary artery smooth muscle cells (HCASMCs) in proliferative medium at a rate of ~500 μL min(-1). Confocal microscopy and scanning electron microscopy confirmed that the HCASMCs were seeded down the full 10-mm length of the microchannel and stayed within its boundaries. Both nuclei and F-actin were observed within the seeded cells. The presence of F-actin filaments shows that the cells were adhered strongly to the scaffold and remained viable throughout the culture. The concept of "vascular wall engineering" producing intricate cell seeding through microchannels produced via femtosecond laser ablation was validated.

Phenotypic changes in cultured smooth muscle cells: limitation or opportunity for tissue engineering of hollow organs?

Smooth muscle cells (SMCs) are typically used as a cell source for the reconstruction of hollow organs by conventional tissue engineering techniques. However, the necessity for and advantage of the use of tissue-specific SMCs are unknown. The present study investigated the phenotypic changes that occur following isolation and in vitro expansion of rat SMC populations isolated from three different tissues: the aorta, oesophagus and urinary bladder. rSMCs were isolated by enzymatic dispersion and expanded by conventional cell culture techniques, yielding microscopically homogeneous populations. SMC phenotypes were monitored according to their expression of marker proteins during the first two passages. Two of the three SMC populations (rSMC-a and rSMC-e) showed a marked change in their marker protein profiles during the first two passages, which resulted in a homogeneous phenotype that was neither fully contractile nor fully synthetic. SMCs from the urinary bladder did not show such a shift. Differences between the three rSMC populations were observed with regard to proliferative activity and gene expression patterns, suggesting the retention of some tissue-specific cell characteristics. In summary, phenotypic changes in SMCs occur as a result of conventional cell isolation and expansion techniques, thus questioning the necessity for a tissue-specific cell source for regenerative medicine applications.

Functional Modeling of the Shift in Cellular Calcium Dynamics at the Onset of Synchronization in Smooth Muscle Cells

In the present paper we address the nature of synchronization properties found in populations of mesenteric artery smooth muscle cells. We present a minimal model of the onset of synchronization in the individual smooth muscle cell that is manifested as a transition from calcium waves to whole-cell calcium oscillations. We discuss how different types of ion currents may influence both amplitude and frequency in the regime of whole-cell oscillations. The model may also explain the occurrence of mixed-mode oscillations and chaotic oscillations frequently observed in the experimental system.

Surgical Management of Endobronchial Inflammatory Myofibroblastic Tumors

Endobronchial myofibroblastic tumors are neoplasms composed of clonal populations of smooth muscle cells and a variable lymphocytic inflammatory component. They represent a challenge with respect to diagnosis, classification, and surgical resection due to their infrequent occurrence. We retrospectively reviewed our experience with patients who had myofibroblastic tumors in the major airways over a 15-year period, in order to understand the incidence, natural biology, treatment, and long-term outcome of individuals with this type of neoplasm in an endobronchial location. Between 1995 and 2010, 11 patients (9 female, 2 male) underwent surgical resection of a myofibroblastic tumor arising within the tracheobronchial tree. The mean age was 39.6 years (range, 22.3 to 53.6 years). All patients were symptomatic, with cough and dyspnea as the most common presenting complaints. Rigid bronchoscopy with endobronchial biopsy was utilized to establish the diagnosis in 9 of 11 patients. Laser-mechanical debulking was performed to relieve airway obstruction prior to operation in 10 of 11 patients. Because of wide submucosal infiltration of the neoplasms, surgical resection for complete removal was required for all individuals. Tracheal resection was performed in 3 patients, carinal resection in 1 patient, mainstem bronchial resection in 2 patients, sleeve resection in 3 patients, bilobectomy in 1 patient, and right lower lobectomy in 1 patient. Resection with tumor-free margins was accomplished in all patients. Mean tumor size was 2.3 cm (range, 1.5 to 3.5 cm). There were no operative deaths, with all patients alive and disease-free at a mean of 6.1 ± 3.7 years. Complete surgical resection of inflammatory myofibroblastic tumors presenting in a major airway is safe and leads to excellent survival for patients with this uncommon disease.

MicroRNA-1 Regulates Smooth Muscle Cell Differentiation by Repressing Kruppel-Like Factor 4

The role of microRNA-1 (miR-1) has been studied in cardiac and skeletal muscle differentiation. However, it remains unexplored in vascular smooth muscle cells (SMCs) differentiation. The aim of this study was to uncover novel targets of and shed light on the function of miR-1 in the context of embryonic stem cell (ESC) differentiation of SMCs in vitro. miR-1 expression is steadily increased during differentiation of mouse ESC to SMCs. Loss-of-function approaches using miR-1 inhibitors uncovered that miR-1 is required for SMC lineage differentiation in ESC-derived SMC cultures, as evidenced by downregulation of SMC-specific markers and decrease of derived SMC population. In addition, bioinformatics analysis unveiled a miR-1 binding site on the Kruppel-like factor 4 (KLF4) 3' untranslated region (3'UTR), in a region that is highly conserved across species. Consistently, miR-1 mimic reduced KLF4 3'UTR luciferase activity, which can be rescued by mutating the miR-1 binding site on the KLF4 3'UTR in the reporter construct. Additionally, repression of the miR-1 expression by miR-1 inhibitor can reverse KLF4 downregulation during ESC-SMC differentiation, which subsequently inhibits SMC differentiation. We conclude that miR-1 plays a critical role in the determination of SMC fate during retinoid acid-induced ESC/SMC differentiation, which may indicate that miR-1 has a role to promote SMC differentiation.