Expression In Smooth Muscle Cells Research Articles

Pathogenesis of abdominal pain in bowel obstruction: role of mechanical stress-induced upregulation of nerve growth factor in gut smooth muscle cells.

Abdominal pain is one of the major symptoms in bowel obstruction (BO); its cellular mechanisms remain incompletely understood. We tested the hypothesis that mechanical stress in obstruction upregulates expression of nociception mediator nerve growth factor (NGF) in gut smooth muscle cells (SMCs), and NGF sensitizes primary sensory nerve to contribute to pain in BO. Partial colon obstruction was induced with a silicon band implanted in the distal bowel of Sprague-Dawley rats. Colon-projecting sensory neurons in the dorsal root ganglia (T13 to L2) were identified for patch-clamp and gene expression studies. Referred visceral sensitivity was assessed by measuring withdrawal response to stimulation by von Frey filaments in the lower abdomen. Membrane excitability of colon-projecting dorsal root ganglia neurons was significantly enhanced, and the withdrawal response to von Frey filament stimulation markedly increased in BO rats. The expression of NGF mRNA and protein was increased in a time-dependent manner (day 1-day 7) in colonic SMC but not in mucosa/submucosa of the obstructed colon. Mechanical stretch in vitro caused robust NGF mRNA and protein expression in colonic SMC. Treatment with anti-NGF antibody attenuated colon neuron hyperexcitability and referred hypersensitivity in BO rats. Obstruction led to significant increases of tetrodotoxin-resistant Na currents and mRNA expression of Nav1.8 but not Nav1.6 and Nav1.7 in colon neurons; these changes were abolished by anti-NGF treatment. In conclusion, mechanical stress-induced upregulation of NGF in colon SMC underlies the visceral hypersensitivity in BO through increased gene expression and activity of tetrodotoxin-resistant Na channels in sensory neurons.

Olfactomedin 2 Regulates Smooth Muscle Phenotypic Modulation and Vascular Remodeling Through Mediating Runt-Related Transcription Factor 2 Binding to Serum Response Factor.

The objective of this study is to investigate the role and underlying mechanism of Olfactomedin 2 (Olfm2) in smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Platelet-derived growth factor-BB induces Olfm2 expression in primary SMCs while modulating SMC phenotype as shown by the downregulation of SMC marker proteins. Knockdown of Olfm2 blocks platelet-derived growth factor-BB-induced SMC phenotypic modulation, proliferation, and migration. Conversely, Olfm2 overexpression inhibits SMC marker expression. Mechanistically, Olfm2 promotes the interaction of serum response factor with the runt-related transcription factor 2 that is induced by platelet-derived growth factor-BB, leading to a decreased interaction between serum response factor and myocardin, causing a repression of SMC marker gene transcription and consequently SMC phenotypic modulation. Animal studies show that Olfm2 is upregulated in balloon-injured rat carotid arteries. Knockdown of Olfm2 effectively inhibits balloon injury-induced neointima formation. Importantly, knockout of Olfm2 in mice profoundly suppresses wire injury-induced neointimal hyperplasia while restoring SMC contractile protein expression, suggesting that Olfm2 plays a critical role in SMC phenotypic modulation in vivo. Olfm2 is a novel factor mediating SMC phenotypic modulation. Thus, Olfm2 may be a potential target for treating injury-induced proliferative vascular diseases.

CRE and SRE mediate LPA-induced CCN1 transcription in mouse aortic smooth muscle cells

Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein (ox-LDL), is a potent bioactive phospholipid. Our recent data reveal that LPA induces matricellular protein CCN1 (also known as Cyr61) expression in aortic smooth muscle cells (SMCs) and that CCN1 bridges LPA and integrin signaling pathways leading to SMC migration. Whether and how LPA regulates the transcriptional machinery of the CCN1 gene are unknown. In this study, we found that LPA markedly induces CCN1 mRNA expression in SMCs. Using deleting mutation and reporter gene strategies, we demonstrated regions from -2038 to -1787 and from -101 to +63 of the CCN1 promoter contain the essential regulatory elements. The serum response element (SRE) and cyclic AMP-response element (CRE) are located in these regions. LPA induced time-dependent phosphorylation of serum response factor (SRF) and CRE-binding protein (CREB) in mouse SMCs. Luciferase assays of a series of deleted, mutated CCN1 promoter-reporter gene constructs and dominant negative construct revealed the distal SRE and the proximal CRE in the CCN1 promoter are required for LPA-induced CCN1 gene expression. Our results imply that elevated LPA levels may trigger SMC migration and exacerbate restenosis and atherosclerotic lesions through the induced CCN1, which communicates with a set of plasma membrane proteins and intracellular kinases.

Telomerase Reverse Transcriptase Deficiency Prevents Neointima Formation Through Chromatin Silencing of E2F1 Target Genes.

Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC.

Abstract 13838: Smooth Muscle Cells Are the Origin of the Majority of Foam Cells in Atherosclerotic Lesions of Apolipoprotein E-deficient Mice

Background: Apolipoprotein E-deficient (ApoE -/- ) mice are the most frequently used model of human atherosclerosis. Foam cells in mouse lesions have been believed to be primarily macrophage-derived using traditional staining with macrophage markers. Recent studies indicated that smooth muscle cells (SMCs) comprise at least 50% of foam cells in human coronary atherosclerosis, and express reduced levels of ATP-binding cassette transporter A1 (ABCA1) when compared to intimal leucocytes. Identification of SMC-derived foam cells is challenging due to the absence of classic SMC markers and expression of macrophage markers by a high percentage of intimal SMCs in mouse and human lesions. Hypothesis: Unlike human atherosclerosis, foam cells in ApoE -/- mice are primarily macrophage- rather than SMC-derived. Methods: 8-week-old male and female ApoE -/- mice were fed a Western diet for 6 weeks to promote lesion development. Flow cytometry was used to estimate the proportion of leukocyte- and SMC-derived foam cells in cell suspensions from digested ascending aortas. Foam cells were identified by the neutral lipid dye BODIPY, and leukocyte-derived were distinguished from SMC-derived foam cells by expression of CD45 (a pan-leukocyte marker), and I-A/I-E (antigen presenting cell markers), which have not been reported to be expressed by SMCs. SMC origin was confirmed using a SMC-specific epigenetic lineage marker. ABCA1 level was also measured in the two foam cell populations. Results: 69.47±3.20 % (male) and 73.33±2.35 % (female) (mean ± SEM, n=9 each) of foam cells in ApoE -/- mouse lesions were CD45 and I-A/I-E negative, and were confirmed by SMC lineage marker to be predominantly SMC-derived. SMC-derived foam cells were thus significantly greater than leukocyte-derived foam cells in ApoE -/- mice ( P <0.01), and exhibited three times lower ABCA1 protein than leukocyte-derived foam cells in both sexes. Conclusions: Contrary to our hypothesis, SMCs contribute the majority of foam cells in ApoE -/- mouse atherosclerosis. Impaired ABCA1 expression in SMC foam cells, as seen in human intimal SMC foam cells, suggests a likely mechanism for preferential SMC foam cell development in both species, and a potential target for atherosclerosis treatment and prevention.

Dicer generates a regulatory microRNA network in smooth muscle cells that limits neointima formation during vascular repair.

MicroRNAs (miRNAs) coordinate vascular repair by regulating injury-induced gene expression in vascular smooth muscle cells (SMCs) and promote the transition of SMCs from a contractile to a proliferating phenotype. However, the effect of miRNA expression in SMCs on neointima formation is unclear. Therefore, we studied the role of miRNA biogenesis by Dicer in SMCs in vascular repair. Following wire-induced injury to carotid arteries of Apolipoprotein E knockout (Apoe -/-) mice, miRNA microarray analysis revealed that the most significantly regulated miRNAs, such as miR-222 and miR-21-3p, were upregulated. Conditional deletion of Dicer in SMCs increased neointima formation by reducing SMC proliferation in Apoe -/- mice, and decreased mainly the expression of miRNAs, such as miR-147 and miR-100, which were not upregulated following vascular injury. SMC-specific deletion of Dicer promoted growth factor and inflammatory signaling and regulated a miRNA-target interaction network in injured arteries that was enriched in anti-proliferative miRNAs. The most connected miRNA in this network was miR-27a-3p [e.g., with Rho guanine nucleotide exchange factor 26 (ARHGEF26)], which was expressed in medial and neointimal SMCs in a Dicer-dependent manner. In vitro, miR-27a-3p suppresses ARHGEF26 expression and inhibits SMC proliferation by interacting with a conserved binding site in the 3' untranslated region of ARHGEF26 mRNA. We propose that Dicer expression in SMCs plays an essential role in vascular repair by generating anti-proliferative miRNAs, such as miR-27a-3p, to prevent vessel stenosis due to exaggerated neointima formation.

Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells

Substrate-mediated delivery of functional plasmid DNA (pDNA) has been proven to be a promising strategy to promote competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs), which is beneficial to inducing fast endothelialization of implanted vascular devices. Thus, it is of great importance to develop universal approaches with simplicity and easiness to immobilize DNA complex nanoparticles on substrates. In this study, the bioinspired polydopamine (PDA) coating was employed in immobilization of DNA complex nanoparticles, which were composed of protamine (PrS) and plasmid DNA encoding with hepatocyte growth factor (HGF-pDNA) gene. We demonstrated that the DNA complex nanoparticles can be successfully immobilized onto the PDA surface. Consequently, the HGF expression of both ECs and SMCs were significantly improved when they cultured on the DNA complex nanoparticles-immobilized substrates. Furthermore, EC proliferation was specifically promoted due to bioactivity of HGF, leading to an enhancement of EC competitiveness over SMCs. Our findings demonstrated the substrate-mediated functional gene nanoparticle delivery through PDA coating as a simple and efficient approach. It may hold great potential in the field of interventional cardiovascular implants.

Role of macrophage colony-stimulating factor in the development of neointimal thickening following arterial injury.

Evidence suggests that macrophage colony-stimulating factor (M-CSF) participates critically in atherosclerosis; little is known about the role of M-CSF in the development of neointimal hyperplasia following mechanical vascular injury. We examined the expression of M-CSF and its receptor, c-fms, in rodent and rabbit models of arterial injury. Injured rat carotid arteries expressed 3- to 10-fold higher levels of M-CSF and c-fms mRNA and protein following balloon injury as compared to uninjured arteries. In the rabbit, M-CSF protein expression was greatest in neointimal smooth muscle cells (SMCs) postinjury, with some expression in medial SMCs. M-CSF-positive SMCs exhibited markers of proliferation. At 30days postinjury, neointimal SMCs in the adjacent healed area near the border between injured and uninjured zone lost both proliferative activity and overexpression of M-CSF. The presence of induced M-CSF and c-fms expression correlated with the initiation of SMCs proliferation. M-CSF stimulated incorporation of [(3)H] thymidine in human aortic smooth muscle cells in a concentration-dependent manner. Serum-free conditioned medium from aortic SMCs also promoted DNA synthesis, and this effect was blocked by M-CSF specific antibody. To test further the role of M-CSF in vivo, we induced arterial injury by placing a periadventitial collar around the carotid arteries in compound mutant mice lacking apolipoprotein apoE (apoE(-/-)) and M-CSF. Loss of M-CSF abolished the neointimal hyperplastic response to arterial injury in apoE(-/-) mice. Local delivery of M-CSF to the injured artery restored neointimal proliferation, suggesting a critical role of M-CSF for the development of neointimal thickening following arterial injury.

Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis

Coronary atherosclerosis complicated by plaque disruption and thrombosis is a critical event in myocardial infarction and stroke, the major causes of cardiovascular death. In atherogenesis, macrophages (MAC) and smooth muscle cells (SMC) are key actors; they synthesize matrix components and numerous factors involved in the process. Here, we design experiments to investigate whether SMC–MAC communication induces changes in ECM protein composition and/or neo-angiogenesis. Cell to cell communication was achieved using trans-well chambers, where SMCs were grown in the upper chamber and differentiated MAC in the bottom chamber for 24 or 72h. We found that cross-talk between MAC and SMC during co-culture: (i) significantly decreased the expression of ECM proteins (collagen I, III, elastin) in SMC; (ii) increased the expression and activity of metalloprotease MMP-9 and expression of collagenase MMP-1, in both MAC and SMC; (iii) augmented the secretion of soluble VEGF in the conditioned media of cell co-culture and VEGF gene expression in both cell types, compared with control cells. Moreover, the conditioned media collected from MAC–SMC co-culture promoted endothelial cell tube formation in Matrigel, signifying an increased angiogenic effect. In addition, the MAC–SMC communication led to an increase in inflammatory IL-1β and TLR-2, which could be responsible for cellular signaling. In conclusion, MAC–SMC communication affects factors and molecules that could alter ECM composition and neo-angiogenesis, features that could directly dictate the progression of atheroma towards the vulnerable plaque. Targeting the MAC–SMC cross-talk may represent a novel therapeutic strategy to slow-down or retard the plaque progression.

Free fatty acid activates MMP‐1 and MMP‐3 through PPAR‐γ mitigation

Obesity is positively related with the prevalence of coronary arterial disease (CAD). It is well known about the plasma concentrations of free fatty acid (FFA) are up‐regulated in cases with obesity. Oleic acid (OA) is known as the most abundant monounsaturated fatty acid in human circulatory system. Several pro‐atherosclerotic responses of OA had been established. Sirtuin 1 (SIRT1) acts a key role in regulation of the normal physical function in smooth muscle cells (SMCs). SIRT1 activation is thought as a novel approach to delay the progression of atherosclerotic injuries. However, the mechanism is still unclear as to whether OA affects SIRT1 expression and activity in SMCs. This study was designed to validate the hypothesis that OA treatment mitigates the expression level of SITR1 and its down‐stream genes, which is a negative regulator of NF‐κB pathway. We confirmed that OA treatment represses SIRT1 and PPAR‐γ levels in SMCs. Moreover, OA enhances transforming growth factor‐β1 (TGF‐b1) release by activation of NF‐κB. OA causes NO production by inducible nitric oxide synthase (iNOS) overexpression, thereby promoting the secretion of matrix metalloproteinases‐1 (MMP‐1) and matrix metalloproteinases‐3 (MMP‐3). Taken together, we suggested OA enhances MMPs activation by SIRT1 down‐regulation. MMPs activation had been shown to positively link with the atherosclerotic plaque generation, therefore, our findings might provide a novel route for developing new therapeutic treatments for FFAs‐related CAD.Support or Funding InformationMOST‐103‐2320‐B‐006‐050

Endothelial Cells Can Regulate Smooth Muscle Cells in Contractile Phenotype through the miR-206/ARF6&NCX1/Exosome Axis.

Active interactions between endothelial cells and smooth muscle cells (SMCs) are critical to maintaining the SMC phenotype. Exosomes play an important role in intercellular communication. However, little is known about the mechanisms that regulate endothelial cells and SMCs crosstalk. We aimed to determine the mechanisms underlying the regulation of the SMC phenotype by human umbilical vein endothelial cells (HUVECs) through exosomes. We found that HUVECs overexpressing miR-206 upregulated contractile marker (α-SMA, Smoothelin and Calponin) mRNA expression in SMCs. We also found that the expression of miR-206 by HUVECs reduced exosome production by regulating ADP-Ribosylation Factor 6 (ARF6) and sodium/calcium exchanger 1 (NCX1). Using real-time PCR and western blot analysis, we showed that HUVEC-derived exosomes decreased the expression of contractile phenotype marker genes (α-SMA, Smoothelin and Calponin) in SMCs. Furthermore, a reduction of the miR-26a-containing exosomes secreted from HUVECs affects the SMC phenotype. We propose a novel mechanism in which miR-206 expression in HUVECs maintains the contractile phenotype of SMCs by suppressing exosome secretion from HUVECs, particularly miR-26a in exosomes, through targeting ARF6 and NCX1.

Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-Mediated Contractility via Activation of Focal Adhesion Kinase and Extracellular Regulated Kinase 1/2 in Cerebral Arteries from Rat.

Cerebral ischaemia results in enhanced endothelin B (ETB ) receptor-mediated contraction and receptor protein expression in the affected cerebrovascular smooth muscle cells (SMC). Organ culture of cerebral arteries is a method to induce similar alterations in ETB receptor expression. We suggest that rapid and sustained reduction in wall tension/stretch is a possible trigger mechanism for this vascular remodelling. Isolated rat middle cerebral artery (MCA) segments were incubated in a wire myograph with or without mechanical stretch, prior to assessment of their contractile response to the selective ETB receptor agonist sarafotoxin 6c. The involvement of extracellular regulated kinase (ERK) 1/2 and focal adhesion kinase (FAK) was studied by their specific inhibitors U0126 and PF-228, respectively. Compared with their stretched counterparts, unstretched MCA segments showed a significantly increased ETB receptor-mediated contractile response after 12 hr of incubation, which was attenuated by either U0126 or PF-228. The functionally increased ETB -mediated contractility could be attributed to two different mechanisms: (i) a difference in ETB receptor localization from primarily endothelial expression to SMC expression and (ii) an increased calcium sensitivity of the SMCs due to an increased expression of the calcium channel transient receptor potential canonical 1. Collectively, our results present a possible mechanism linking lack of vessel wall stretch/tension to changes in ETB receptor-mediated contractility via triggering of an early mechanosensitive signalling pathway involving ERK1/2 and FAK signalling. A mechanism likely to be an initiating factor for the increased ETB receptor-mediated contractility found after cerebral ischaemia.

Prevention of vascular smooth muscle cell proliferation and injury-induced neointimal hyperplasia by CREB-mediated p21 induction: An insight from a plant polyphenol

Cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element (CRE)-binding protein (CREB) signaling cascade negatively regulates platelet-derived growth factor BB (PDGF-BB)-induced smooth muscle cell (SMC) proliferation, which is a critical event in the initiation and development of restenosis and atherosclerotic lesions. Salvianolic acid A (SAA) is one of the most abundant polyphenols extracted from salvia. The aim of this study is to investigate whether SAA exerts an action on PDGF-BB-induced proliferation via cAMP/PKA/CREB mechanism. SAA blunts PDGF-BB-induced human umbilical artery smooth muscle cell (hUASMC) proliferation via p21 induction, as evidenced by its increased mRNA and protein expression levels. The SAA-induced upregulation of p21 involves the cAMP/PKA signaling pathway; a cAMP analog mimicked the effects of SAA and a specific cAMP/PKA inhibitor opposed these effects. SAA also activated CREB, including phosphorylation at Ser133, and induced its nuclear translocation. Deletion and mutational analysis of p21 promoters, co-immunoprecipitation, and western blot analysis showed that CRE is essential for SAA-induced p21 protein expression. Transfection of dominant-negative CREB (mutated Ser133) plasmids into hUASMCs attenuated SAA-stimulated p21 expression. SAA upregulated p21 expression and activated CREB in the neointima of balloon-injured arteries in vivo. Our results indicate that SAA promotes p21 expression in SMCs through the cAMP/PKA/CREB signaling cascade in vitro and prevents injury-induced neointimal hyperplasia.

Telomerase Inhibition by Everolimus Suppresses Smooth Muscle Cell Proliferation and Neointima Formation Through Epigenetic Gene Silencing

SummaryProliferation of smooth muscle cells (SMCs) during neointima formation is prevented by drug-eluting stents. The replicative capacity of mammalian cells is enhanced by telomerase expression; however, the contribution of telomerase to the proliferative response underlying neointima formation and its potential role as a pharmacological target are unknown. The present study investigated the mechanisms underlying the mitogenic function of telomerase, and tested the hypothesis that everolimus, which is commonly used on drug-eluting stents, suppresses SMC proliferation by targeting telomerase. Inhibition of neointima formation by everolimus was lost in mice overexpressing telomerase reverse transcriptase (TERT), indicating that repression of telomerase confers the anti-proliferative efficacy of everolimus. Everolimus reduced TERT expression in SMC through an Ets-1-dependent inhibition of promoter activation. The inhibition of TERT-dependent SMC proliferation by everolimus occurred in the absence of telomere shortening but rather as a result of a G1→S-phase arrest. Although everolimus failed to inhibit phosphorylation of the retinoblastoma protein as the gatekeeper of S-phase entry, it potently repressed downstream target genes. Chromatin immunoprecipitation assays demonstrated that TERT induced E2F binding to S-phase gene promoters and supported histone acetylation. These effects were sensitive to inhibition by everolimus. These results characterize telomerase as a previously unrecognized target for the antiproliferative activity of everolimus, and further identify a novel mitogenic pathway in SMC that depends on the epigenetic activation of S-phase gene promoters by TERT.

Dynamic polymer substrates with increasing stiffness for regulating smooth muscle cells

Event Abstract Back to Event Dynamic polymer substrates with increasing stiffness for regulating smooth muscle cells Xifeng Liu1, Lei Cai1 and Shanfeng Wang1, 2 1 The University of Tennessee, Knoxville, Department of Materials Science and Engineering, United States 2 The University of Tennessee, Knoxville, Institute of Biomedical Engineering, United States Cardiovascular tissues bear constant blood shear and dynamic hardening under diseased conditions, which causes the tissue stiffness varies all the time. To mimic the dynamic changing environment in the vessel tissues and investigate the influence of dynamically changing substrate mechanical properties on the cell behaviors, we fabricated a model polymer network from poly(e-caprolactone) triacrylate that can gradually stiffen in 24 h through impeded crystallization at body temperature (37ºC). Rat primary smooth muscle cells (SMCs) were cultured on both static and dynamic substrates and distinct SMC attachment, proliferation and spreading were found. Quantification of contractile gene expression and protein content showed that the dynamic substrates could facilitate the contractile conversion process of SMCs. The analysis of focal adhesions and integrin expression indicated that the cellular abilities to sensing and adhering to the substrate surface were enhanced by the dynamic stiffening stimulation. These results extend the knowledge about SMC mechanosensing to dynamic substrates with increasing stiffness, and demonstrate a new method of regulating SMC adhesion, growth, and functional conversion on substrates. We have developed biodegradable and photo-crosslinkable poly(ε-caprolactone) triacrylates (PCLTAs) that can be photo-crosslinked into networks with high gel fractions and controllable thermal and mechanical properties[1]-[3]. Elastic modulus ranging from 1 to 200 MPa was achieved by modulating the crosslinking density and crystallinity through the nominal molecular weight (Mn) of the polymer[2],[3]. When the molecular weight increases from 2000 to 20000 g/mol, the PCL network changes from an amorphous, compliant elastomer to a semi-crystalline, stiff material with a crystallinity of 42%. For the semi-crystalline PCLTA networks, the melting temperature (Tm) of the substrate increased from 13.8ºC for Mn of 5000 g/mol to 50.4ºC for Mn of 20000 g/mol. Because of the constraints of the crosslinks, polymer crystallization in a network is suppressed and impeded, as demonstrated by reduced crystallinity and slower crystallization. By controlling the Tm of the PCLTA network to be slightly above 37ºC, PCL segments in the network crystallized very slowly at 37ºC and the substrate stiffness increased gradually with the increase in crystallinity over a time period of 24 h. Based on this unique mechanism of impeded polymer crystallization in PCLTA networks, we explore to apply this spontaneously increasing substrate stiffness to regulate SMCs when they are seeded on the substrates and cell attachment occurs at the same time. To study the effect of the gradually increasing substrate stiffness on SMC adhesion, spreading, proliferation, and phenotypic conversion, we prepared a series of PCLTA networks, melted and crystallized at 37ºC for 0, 4, 8, or 24 h. The PCLTA synthesized in this study had Mn of 7985 g/mol, weight-average molecular weight of 9626 g/mol, and polymer molecular weight distribution of 1.206. Figure 1. Scheme of PCLTA photo-crosslinking and crystallization at 37°C. Figure 2. (a) The elastic modulus (G') of the PCLTA networks as monitored using a rheometer at 37ºC. (b) The strain-stress curves for four melted amorphous PCLTA networks pre-treated at 37ºC for 0, 4, 8, and 24 h, respectively. (c) Tensile modulus of these networks at 37ºC. Figure 3. (a) SMC attachment rate on the dynamic and static networks at 4 h post-seeding, as normalized to that on TCPS positive control. (b) SMC spread area calculated at 12 h and day 1 post-seeding on these substrates. (c) SMC numbers at 12 h, days 1, 2, and 4 post-seeding on the dynamic and static samples using TCPS as positive control. *: p < 0.5 to the 24 h static sample. $: p < 0.5 to the 0 h dynamic sample. Conclusions: SMCs receiving dynamic stiffening stimulation had stronger focal adhesions and higher expression of environmental sensing integrin subunits. SMC attachment, spreading, proliferation, and expression of contractile gene markers and calponin protein were all promoted upon receiving the dynamic mechanical stimulation. National Science Foundation (DMR-11-06142)

Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

Background. Both endothelin receptor type B ([ETBR], a G protein-coupled receptor that mediates the vascular effects of the potent vasoconstrictor endothelin-1) and human cytomegalovirus ([HCMV], a ubiquitous herpesvirus) have been implicated in the pathogenesis of cardiovascular disease (CVD). The effects of HCMV infection on ETBR expression are unknown. We hypothesized that HCMV may contribute to the pathogenesis of CVD via ETBR modulation.Methods. Human CMV effects on ETBR were studied in vitro in endothelial cells (ECs) and smooth muscle cells (SMCs) and ex vivo in human carotid plaque tissue specimens. Expression of ETBR and viral immediate-early were quantified using quantitative polymerase chain reaction. Functional consequences after ETBR blockade in ECs were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation, wound healing, tube formation, and flow adhesion assays.Results. Human CMV is capable of upregulating both ETBR mRNA and protein expression in ECs and SMCs. The ETBR was also abundantly expressed in ECs, foam cells, and SMCs, and, more importantly, in HCMV-positive cells in human carotid plaques. Endothelin receptor type B blockade led to decreased proliferation and reduced tumor necrosis factor α-mediated leukocyte recruitment in both uninfected and HCMV-infected ECs. Direct HCMV infection was antimigratory and antiangiogenic in ECs.Conclusions. Human CMV may contribute to CVD via ETBR induction.

Role of smooth muscle Nox4-based NADPH oxidase in neointimal hyperplasia

Elevated levels of reactive oxygen species (ROS) in the vascular wall play a key role in the development of neointimal hyperplasia. Nox4-based NADPH oxidase is a major ROS generating enzyme in the vasculature, but its roles in neointimal hyperplasia remain unclear. ObjectiveOur purpose was to investigate the role of smooth muscle cell (SMC) Nox4 in neointimal hyperplasia. Approach and resultsMice overexpressing a human Nox4 mutant form, carrying a P437H dominant negative mutation (Nox4DN) and driven by SM22α promoter, to achieve specific expression in SMC, were generated in a FVB/N genetic background. After wire injury-induced endothelial denudation, Nox4DN had significantly decreased neointima formation compared with non-transgenic littermate controls (NTg). ROS production, serum-induced proliferation and migration, were significantly decreased in aortic SMCs isolated from Nox4DN compared with NTg. Both mRNA and protein levels of thrombospondin 1 (TSP1) were significantly downregulated in Nox4DN SMCs. Downregulation of TSP1 by siRNA decreased cell proliferation and migration in SMCs. Similar to Nox4DN, downregulation of Nox4 by siRNA significantly decreased TSP1 expression level, cell proliferation and migration in SMCs. ConclusionsDownregulation of smooth muscle Nox4 inhibits neointimal hyperplasia by suppressing TSP1, which in part can account for inhibition of SMC proliferation and migration.

The role of demineralized bone particle in a PLGA scaffold designed to create a media equivalent for a tissue engineered blood vessel

Poly(lactic-co-glycolic acid) (PLGA) is a copolymer used for scaffolds in tissue engineering due to its biodegradable and biocompatible properties. Demineralized bone particle (DBP) is a natural material containing bone morphogenic proteins (BMPs). The objective of this study was to determine if DBP induced differentiation of mesenchymal stem cells (MSCs) into a smooth muscle cells (SMCs) phenotype and act in a similar manner when treated with TGF-β1 as SMCs under the same conditions. PLGA scaffolds were fabricated with or without DBP using the solvent casting/salt leaching technique. Proliferation of MSCs and SMCs was measured using the MTT assay, while the expression of the smooth muscle-specific markers α-smooth muscle actin (α-SMA), smooth muscle 22 alpha (SM22α), and calponin was measured using RT-PCR and western blotting. Results showed PLGA with DBP scaffolds increased cell proliferation of SMCs (1 and 3 days) and MSCs (3 and 7 days) compared to the PLGA only scaffolds. Smooth muscle-specific genes were significantly increased in SMCs cultured for 7 days on PLGA with DBP scaffolds compared to PLGA only scaffolds and were also significantly increased when used SMC medium containing TGF-β1. Interestingly, the smooth muscle-specific genes were not altered in MSCs by the addition of TGF-β1 to the PLGA with DBP. However, in the 6-well and PLGA only groups, TGF-β1 addition caused increased levels of SM22α and calponin mRNA. Western blotting results were similar to the mRNA levels. These results suggest that the cell growth and gene expression of SMCs were up-regulated by DBP and TGF-β1 in PLGA scaffold; however, they were inadequate for the shift of MSCs into a SMCs phenotype.

Serum Response Factor Is Essential for Prenatal Gastrointestinal Smooth Muscle Development and Maintenance of Differentiated Phenotype.

Background/AimsSmooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized.MethodsWe generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence.ResultsCongenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRαlow) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRαlow cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression.ConclusionsSRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRαlow cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.

THORACIC AORTIC DILATIONS IN PATIENTS WITH BICUSPID AORTIC VALVE DISEASE IS MARKED BY ACCELERATED SMOOTH MUSCLE CELL AGING

Individuals with a bicuspid aortic valve (BAV) are at increased risk for ascending aortic dilation and dissection. Loss of aortic medial smooth muscle cells (SMCs) and disruption of the extracellular matrix are well-recognized pathologies, but the underlying cellular mechanisms remain elusive. We tested the hypothesis that the dilated aorta in patients with BAV was marked by accelerated cellular aging. Samples of human ascending aorta were obtained from individuals with BAV undergoing thoracic aorta replacement (n=37, age 54.7±2.2, aortic diameter 4.8±0.9 cm) or patients with a tricuspid aortic valve and non-dilated aorta undergoing heart transplantation or coronary bypass procedures (n=8, age 55.3±8.1, aortic diameter 3.1±0.3 cm). Assessment of fresh aortic samples for senescence-associated β-galactosidase revealed evidence for rare medial cell senescence that was 4.2-fold more prevalent in dilated aortas (0.83±0.10%) than in non-dilated aortas (0.20±0.10%, p=0.048). Expression of p16 was abundantly detected in medial SMCs within dilated aortas (24.0±1.5%) and 3-fold more abundant than in non-dilated aortas (8.2±1.5%, p<0.0001). Interestingly, medial p16 expression did not correlate with telomere length of medial cells, as determined by quantitative and standardized PCR (p=0.35). Immunostaining for γH2A.X (phosphorylated Ser139) interestingly revealed discrete nuclear DNA double-strand breakage signals in 25.7±3.8% of medial cells in dilated aortas from patients with BAV, which was 2.3-fold higher than that found in non-dilated aortas (11.0±4.9, p=0.03). Medial cell outgrowths from BAV patients showed increased senescence-associated ß-galactosidase activity compared to control patient SMC outgrowths (38.9±2.9 vs. 19.5±1.9, p=0.0005). Furthermore, incubation of early passage SMCs with 50nM of angiotensin II for 3 days caused a 1.4-fold increase in senescence-associated ß-galactosidase expressing SMCs from BAV patients (p=0.0006), but elicited no change in senescence-associated ß-galactosidase expression in SMCs from control patients (p=0.83). These findings identify a previously unrecognized phenomenon of accelerated SMC aging in the aortas of patients with BAV, with cellular senescence and unresolved DNA breaks. Accelerated cell aging could thus be a driver of aortic wall degeneration in these patients and a potential therapeutic target.