Normal Smooth Muscle Cells Research Articles

Correlation between antizyme 1 and differentiation of vascular smooth muscle cells cultured in honeycomb-like type-I collagen matrix

Vascular smooth muscle cells (SMC) are able to proliferate when cultured on plates, but become differentiated when maintained in three-dimensional type I collagen matrices (honeycombs). SMC grown in honeycombs contained a low level of polyamines due to the presence of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase (ODC) and of polyamine uptake. To clarify the role of AZ1 in differentiation of SMC in honeycombs, an ODC gene was stably transfected into SMC (ODC-SMC). Although proliferation of ODC-SMC on plates was accelerated together with an increase in phosphorylated focal adhesion kinase (FAK) and a decrease in α-actin and myosin, maker proteins of differentiation, growth of ODC-SMC ceased in honeycombs similarly to normal SMC with a low level of phosphorylated FAK and a high level of α-actin and myosin. AZ1 expression in ODC-SMC on plates was low, but that in honeycombs was high. Antizyme in ODC-SMC in honeycombs not only decreased the level of ODC but also inhibited polyamine uptake activity. These results taken together suggest that low levels of polyamines caused by AZ1 in SMC in honeycombs inhibit phosphorylation of FAK and enhance expression of α-actin and myosin, resulting in differentiation through inhibition of focal adhesions.

Antitumor effects and cell selectivity of temporin-1CEa, an antimicrobial peptide from the skin secretions of the Chinese brown frog (Rana chensinensis)

Many antimicrobial peptides from amphibian exhibit additional anticancer properties due to a similar mechanism of action at both bacterial and cancer cells. We have previously reported the cDNA sequence of the antimicrobial peptide temporin-1CEa precursor cloned from the Chinese brown frog Rana chensinensis. In this study, we purified, synthesized and structurally characterized temporin-1CEa from the skin secretions of R. chensinensis. The cytotoxicity and cell selectivity of temporin-1CEa were further examined on twelve human carcinoma cell lines and on normal human umbilical vein smooth muscle cells (HUVSMCs). Our results indicated that temporin-1CEa has the amino acid sequence of FVDLKKIANIINSIF-NH2, and exhibits 50–56% identity with temporin family peptides from other frog species. The CD spectra for temporin-1CEa adopted a well-defined α-helical structure in 50% TFE/water solution. The results of MTT assay showed that temporin-1CEa exhibits cytotoxicity to all tested cancer cell lines in a concentration-dependent manner, being MCF-7 cells the most sensitive. Moreover, temporin-1CEa had lower hemolytic effect to human erythrocytes and had no significant cytotoxicity to normal HUVSMCs at concentrations showed potent antitumor activity. In summary, temporin-1CEa, an amphiphilic α-helical cationic peptide, may represent a novel anticancer agent for breast cancer therapy, considering its cancer cell selectivity and relatively lower cytotoxicity to normal cells.

In This Issue

HomeCirculation ResearchVol. 109, No. 5In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published19 Aug 2011https://doi.org/10.1161/RES.0b013e31822e8eaaCirculation Research. 2011;109:475Arginase 1 Is Regulated by LXRα (p 492)Pourcet et al identify a new mechanism that promotes the regression of atherosclerotic plaques.Due to its slow life-long progression, the development of atherosclerotic plaques is difficult to control, even with optimal therapy. Therefore, attempts to promote the regression of preformed plaques are particularly attractive. Even though plaque regression is difficult to study, it has been reported that transplantation of atherosclerotic aorta from atherosclerosis-prone apoE-null mice to wild-type nonatherosclerotic mice results in plaque reduction. Using this ingenious model, Pourcet et al now show that plaque regression is accompanied by an increase in the expression of the anti-inflammatory enzyme arginase1 (Arg1). They report that that this increase is regulated by the liver X receptor alpha (LXRα) and that deletion of LXRα prevents the upregulation of Arg1 in regressive lesions. Nevertheless, LXRα did not directly bind to the Arg-1 gene; instead, it increased the expression of IRF8, which in concert with PU.1, stimulated Arg1 gene expression in macrophages. Although previous studies have shown that LXRs prevent inflammation, the work of Pourcet and colleagues reveals a new molecular pathway linking LXRs to plaque regression, raising the possibility that the pathway could be gainfully targeted to reduce inflammation in atherosclerotic lesions and to promote plaque regression.Orai1 and Neointima Formation (p 534)Zhang et al find a new way to prevent abnormal smooth muscle cell growth.Download figureDownload PowerPointAlthough smooth muscle cells in normal blood vessels remain quiescent and contractile, disease or injury can cause these cells to change their phenotype and proliferate, thereby contributing to the formation of atherosclerotic lesions, hypertension, or restenosis after angioplasty. The processes that regulate smooth muscle cell proliferation are not fully understood; however, calcium signaling has been shown to play an essential role. Zhang et al report that Orail1, a protein involved in the generation of calcium release–activated calcium current, is required for the proliferation of smooth muscle cells in rat carotid arteries after balloon injury. They found that Orail1 levels were increased in proliferating smooth muscle cells of the injured artery and that knockdown of Orail1 decreased neointima formation. Because in cultured smooth muscle cells the knockdown of Orail prevented the nuclear translocation of the transcription factor NFAT, the authors suggest that calcium influx via Orail1 promotes cell growth by stimulating the nuclear translocation of NFAT and that Orail1 may be a new drug target for preventing abnormal smooth muscle cell growth.Cell Senescence in Pulmonary Hypertension (p 543)Noureddine et al say that pulmonary hypertension may be due to accelerated aging of smooth muscle cells.Download figureDownload PowerPointBecause chronic obstructive pulmonary disease (COPD) is usually diagnosed in the middle-aged or elderly, it has been long suspected to be a symptom of accelerated aging. Clarifying this relationship, Noureddine et al report that in pulmonary artery smooth muscle cells of COPD patients, the extent of telomere shortening, which is indicative of replicative senescence, is directly related to the severity of pulmonary hypertension. They found that the senescent cells were located near actively dividing cells, and, when the senescent cells were put in culture, they excreted factors that stimulated the growth of normal smooth muscle cells. Thus, not only do some cells in the lung age quickly, they also coax neighboring cells to grow. This causes thickening of the blood vessels and thereby an abnormal increase in blood pressure (pulmonary hypertension). Further understanding of the causes and consequences of pulmonary smooth muscle cell aging could lead to the development of new treatments for COPD, which, despite its status as the 4th leading cause of death in the United States, has no known cure.Written by Aruni Bhatnagar Previous Back to top Next FiguresReferencesRelatedDetails August 19, 2011Vol 109, Issue 5 Advertisement Article InformationMetrics © 2011 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31822e8eaa Originally publishedAugust 19, 2011 PDF download Advertisement

Low-intensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca2+-sensitive K+ current in miniature swine with LV hypertrophy

Coronary vascular dysfunction has been observed in several models of heart failure (HF). Recent evidence indicates that exercise training is beneficial for patients with HF, but the precise intensity and underlying mechanisms are unknown. Left ventricular (LV) hypertrophy can play a significant role in the development of HF; therefore, the purpose of this study was to assess the effects of low-intensity interval exercise training on coronary vascular function in sedentary (HF) and exercise trained (HF-TR) aortic-banded miniature swine displaying LV hypertrophy. Six months postsurgery, in vivo coronary vascular responses to endothelin-1 (ET-1) and adenosine were measured in the left anterior descending coronary artery. Baseline and maximal coronary vascular conductance were similar between all groups. ET-1-induced reductions in coronary vascular conductance (P < 0.05) were greater in HF vs. sedentary control and HF-TR groups. Pretreatment with the ET type A (ET(A)) receptor blocker BQ-123 prevented ET-1 hypersensitivity in HF animals. Whole cell voltage clamp was used to characterize composite K(+) currents (I(K(+))) in coronary smooth muscle cells. Raising internal Ca(2+) from 200 to 500 nM increased Ca(2+)-sensitive K(+) current in HF-TR and control, but not HF animals. In conclusion, an ET(A)-receptor-mediated hypersensitivity to ET-1, elevated resting LV wall tension, and decreased coronary smooth muscle cell Ca(2+)-sensitive I(K(+)) was found in sedentary animals with LV hypertrophy. Low-intensity interval exercise training preserved normal coronary vascular function and smooth muscle cell Ca(2+)-sensitive I(K(+)), illustrating a potential mechanism underlying coronary vascular dysfunction in a large-animal model of LV hypertrophy. Our results demonstrate the potential clinical impact of exercise on coronary vascular function in HF patients displaying pathological LV hypertrophy.

Glutathione peroxidase-deficient smooth muscle cells cause paracrine activation of normal smooth muscle cells via cyclophilin A

Background/aimsReduced activity of the antioxidant glutathione peroxidase-1 (GPx1) correlates with increased risk of cardiovascular events in patients with coronary artery disease. However, it remains unclear whether this imbalance in antioxidant capacity directly contributes to activation of vascular cells. In response to oxidative stress, smooth muscle cells (SMCs) secrete the pro-inflammatory immunomodulator cyclophilin A (CyPA). We hypothesized that reduction in vascular cell GPx1 activity causes secretion of CyPA and paracrine-mediated activation of NF-κB and proliferation of SMCs. Methods/resultsUsing a murine model of GPx1 deficiency (GPx1+/−), we found elevated levels of hydrogen peroxide levels and increased secretion of CyPA in both arterial segments and cultured SMCs as compared to wild type (WT). Conditioned media from GPx1+/− SMCs caused increased NF-κB activation of quiescent WT SMCs, and this was inhibited by the antioxidant N-acetyl-l-cysteine or by cyclosporine A (CsA). In co-culture experiments, SMCs derived from GPx1+/− aorta caused increased proliferation of WT SMCs, which was also inhibited by CsA. ConclusionsReduction in vascular cell GPx1 activity and the associated increase in oxidative stress cause CyPA-mediated paracrine activation of SMCs. These findings identify a novel mechanism by which an imbalance in antioxidant capacity may contribute to vascular disease.

Oxygen- and glucose-deprived culture promotes cell proliferation and invasion of vascular smooth muscle cells

Excessive proliferation of vascular smooth muscle cells (VSMCs) is an important reason for the formation and development of many vascular remodeling diseases. In pathological conditions, necrosis of VSMCs may result in the release of inflammatory cytokines, which can lead to stimulation of other normal smooth muscle cells, and promote the proliferation of VSMCs. The purpose of this study was to investigate the effect of oxygen- and glucose-deprived (OGD) conditioned medium on VSMC cell proliferation and invasion. Following culture of VSMCs in OGD-conditioned medium, the cell cycle distributions were remarkably altered. The number of cells in the G0/G1 phase decreased, while the number of cells in G2/M and S phase increased. The expression of cell cycle proteins D1 (Cyclin D1) in VSMCs increased correspondingly. These results suggested that after being cultured in OGD medium, VSMCs can pass through the G0/G1 phase by up-regulation of Cyclin D1 expression, and promote cell proliferation. In addition, we found that the expression of matrix metalloproteinase (MMP)-2 and MMP-9 was increased in OGD medium cultured VSMCs. Using a Transwell invasion assay, we showed that the OGD medium enhanced VSMC cell invasion. These results suggest that MMP-2 and MMP-9 degraded the basement membrane and promoted VSMC invasion. Taken together, our data demonstrate that OGD-conditioned medium can promote VSMC proliferation and invasion by up-regulating Cyclin D1 and MMP-2 and MMP-9 expression, which may contribute to the formation and development of vascular remodeling diseases.

Mechanism related to docosahexaenoic acid induced large conductance calcium-activated potassium channel currents increase in coronary smooth muscle cells

To investigate the mechanism of enhanced large conductance calcium-activated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16, 17-epoxydocosapentaenoic acid (16, 17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2 ± 2.7)% of total potassium currents (n = 20). DHA could activate BK channels, and its 50% effective concentration (EC(50)) was (0.23 ± 0.03) µmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16, 17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC(50) was (19.7 ± 2.8) nmol/L. DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.

Adherence Junctions and Cadherin-11 in Normal and Overactive Human Detrusor Smooth Muscle Cells

We investigated whether analysis of adherence junctions in human detrusor could be used as a diagnostic tool to determine detrusor overactivity. We characterized the protein composition of adherence junctions in the human bladder using cadherin-11 since our group previously found that cadherin-11 could be an integral structural protein of adherence junctions. We obtained a total of 46 biopsies from 23 patients categorized into 4 groups, including 5 who were normal, and 6 each with neurogenic disease with detrusor overactivity, bladder outlet obstruction with detrusor overactivity and idiopathic detrusor overactivity. Specimens were processed to study cadherin-11 expression using combined immunohistochemical and immunogold electron microscopy techniques. Cadherin-11 expression was semiquantitatively analyzed and correlated to muscle fascicle structure and collagen in the extracellular spaces. Immunogold labeling showed highly specific cadherin-11 expression at adherence junctions in detrusor smooth muscle cells. During immunohistochemical staining a wide variety of cadherin-11 expression and fascicle structure was found in the same specimen. No correlation was noted between detrusor overactivity and cadherin-11 expression. However, cadherin-11 seemed to be down-regulated with intercellular space widening and collagenosis. Cadherin-11 is an integral structural protein of the adherence junction. Defects in the overactive detrusor are highly punctate. Quantitative analysis of adherence junctions using biopsy cannot replace urodynamic evaluation as a predictor of detrusor overactivity in the human bladder.

GLI3 repressor controls functional development of the mouse ureter

Obstructive and nonobstructive forms of hydronephrosis (increased diameter of the renal pelvis and calyces) and hydroureter (dilatation of the ureter) are the most frequently detected antenatal abnormalities, yet the underlying molecular mechanisms are largely undefined. Hedgehog (Hh) proteins control tissue patterning and cell differentiation by promoting GLI-dependent transcriptional activation and by inhibiting the processing of GLI3 to a transcriptional repressor. Genetic mutations that generate a truncated GLI3 protein similar in size to the repressor in humans with Pallister-Hall syndrome (PHS; a disorder whose characteristics include renal abnormalities) and hydroureter implicate Hh-dependent signaling in ureter morphogenesis and function. Here, we determined that Hh signaling controls 2 cell populations required for the initiation and transmission of coordinated ureter contractions. Tissue-specific inactivation of the Hh cell surface effector Smoothened (Smo) in the renal pelvic and upper ureteric mesenchyme resulted in nonobstructive hydronephrosis and hydroureter characterized by ureter dyskinesia. Mutant mice had reduced expression of markers of cell populations implicated in the coordination of unidirectional ureter peristalsis (specifically, Kit and hyperpolarization-activation cation-3 channel [Hcn3]), but exhibited normal epithelial and smooth muscle cell differentiation. Kit deficiency in a mouse model of PHS suggested a pathogenic role for GLI3 repressor in Smo-deficient embryos; indeed, genetic inactivation of Gli3 in Smo-deficient mice rescued their hydronephrosis, hydroureter, Kit and Hcn3 expression, and ureter peristalsis. Together, these data demonstrate that Hh signaling controls Kit and Hcn3 expression and ureter peristalsis.

Cigarette Smoke Extract-induced Reduction in Migration and Contraction in Normal Human Bronchial Smooth Muscle Cells

The proliferation, migration, cytokine release, and contraction of airway smooth muscle cells are key events in the airway remodeling process that occur in lung disease such as asthma, chronic obstruction pulmonary disease, and cancer. These events can be modulated by a number of factors, including cigarette smoke extract (CSE). CSE-induced alterations in the viability, migration, and contractile abilities of normal human airway cells remain unclear. This study investigated the effect of CSE on cell viability, migration, tumor necrosis factor (TNF)-α secretion, and contraction in normal human bronchial smooth muscle cells (HBSMCs). Treatment of HBSMCs with 10% CSE induced cell death, and the death was accompanied by the generation of reactive oxygen species (ROS). CSE-induced cell death was reduced by N-acetyl-l-cysteine (NAC), an ROS scavenger. In addition, CSE reduced the migration ability of HBSMCs by 75%. The combination of NAC with CSE blocked the CSE-induced reduction of cell migration. However, CSE had no effect on TNF-α secretion and NF-κB activation. CSE induced an increase in intracellular Ca2+ concentration in 64% of HBSMCs. CSE reduced the contractile ability of HBSMCs, and the ability was enhanced by NAC treatment. These results demonstrate that CSE treatment induces cell death and reduces migration and contraction by increasing ROS generation in normal HBSMCs. These results suggest that CSE may induce airway change through cell death and reduction in migration and contraction of normal HBSMCs.

Dedifferentiation of prostate smooth muscle cells in response to bacterial LPS

Prostate smooth muscle cells (SMCs) are strongly involved in the development and progression of benign prostatic hyperplasia and prostate cancer. However, their participation in prostatitis has not been completely elucidated. Thus, we aimed to characterize the response of normal SMC to bacterial lipopolysaccharide (LPS). Primary prostate SMCs from normal rats were stimulated with LPS (0.1, 1, or 10 µg/ml) for 24 or 48 hr. The phenotype was evaluated by electron microscopy, immunofluorescence, and Western blot of SMCα-actin (ACTA2), calponin, vimentin, and tenascin-C, while the innate immune response was assessed by immunodetection of TLR4, CD14, and nuclear NF-κB. The secretion of TNFα and IL6 was determined using ELISA. Bacterial LPS induces SMCs to develop a secretory phenotype including dilated rough endoplasmic reticulum cisternae with well-developed Golgi complexes. Furthermore, SMCs displayed a decrease in ACTA2 and calponin, and an increase in vimentin levels after LPS challenge. The co-expression of ACTA2 and vimentin, together with the induction of tenascin-C expression indicate that a myofibroblastic-like phenotype was induced by the endotoxin. Moreover, LPS elicited a TLR4 increase, with a peak in NF-κB activation occurring after 10 min of treatment. Finally, LPS stimulated the secretion of IL6 and TNFα. Prostate SMCs are capable of responding to LPS in vitro by dedifferentiating from a contractile to a miofibroblastic-like phenotype and secreting cytokines, with the TLR4 signaling pathway being involved in this response. In this way, prostate SMCs may contribute to the pathophysiology of inflammatory diseases by modifying the epithelial-stromal interactions.

Endogenous IGFBP-3 regulates excess collagen expression in intestinal smooth muscle cells of Crohnʼs disease strictures

Stricture formation occurs in ≈30% of patients with Crohn's disease (CD) and is a significant cause of morbidity. Strictures are characterized by intestinal smooth muscle cell hyperplasia, smooth muscle cell hypertrophy, and fibrosis due to excess net extracellular matrix production, including collagen. Transforming growth factor-β1 (TGF-β1) has profibrotic effects in many tissues due to its ability to regulate collagen expression and extracellular matrix dynamics. We previously showed that both insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) and TGF-β1 are expressed by normal human intestinal smooth muscle cells, bind to, and activate TGF-βRII/I receptors in these cells. Smooth muscle cells isolated from the muscularis propria of patients were used to prepare RNA, protein lysates, or placed into primary culture. IGFBP-3, TGF-β1, and collagen IαI expression was measured with quantitative reverse-transcription polymerase chain reaction (RT-PCR) and protein levels by enzyme-linked immunosorbent assay (ELISA) or immunoblot. Expression and production of IGFBP-3, TGF-β1, and collagen IαI were significantly increased specifically in smooth muscle cells isolated from regions of strictured intestine in CD compared to nonstrictured histologically normal resection margin. IGFBP-3 and TGF-β1 regulated collagen IαI expression and production via a TGF-βRII/I-dependent and Smad2/3-dependent mechanism. Upregulated (excess) collagen IαI expression and production in smooth muscle cells of strictures and basal collagen IαI in smooth muscle cells of normal margin were inhibited by immunoneutralization of IGFBP-3 or TGF-β1. The findings indicate that upregulated endogenous IGFBP-3 and TGF-β1 expression regulates excess collagen IαI production and contributes to fibrosis and stricture formation in CD.

A novel anti‐apoptotic role for apolipoprotein L2 in IFN‐γ‐induced cytotoxicity in human bronchial epithelial cells

Airway epithelium functions not only as a physical barrier, but also a regulator of lung inflammation. IFN-γ plays a critical role in airway inflammation associated with respiratory viral infection. We investigated differential protein profiling in IFN-γ-stimulated normal human bronchial epithelial cells (HBEC) using a 2-dimensional gel electrophoresis followed by MALDI-TOF-MS/MS. IFN-γ markedly stimulated apolipoprotein L2 (ApoL2) protein expression in normal HBEC. ApoL2 mRNA expression was also elevated in normal human lung fibroblasts and smooth muscle cells stimulated with IFN-γ, in lung tissues from an IFN-γ-predominant influenza A virus-infected mouse lung injury model, and in cancer lung tissues from human patients. Normal HBEC showed strong resistance to IFN-γ-induced cytotoxicity. ApoL2 knockdown by siRNA promoted IFN-γ-induced cytotoxicity as revealed by a significant drop in cell viability using MTT and CyQUANT NF cell proliferation assays, and a marked increase in hypodiploid sub-G1 cell population in cell cycle analysis. Furthermore, depletion of ApoL2 facilitated IFN-γ-induced membrane damage and chromatin condensation as observed in Hoechst and propidium iodide-double staining and in transmission electron microscopy, and DNA fragmentation using a DNA laddering assay, in a caspase-dependent manner. Our results reveal a novel function for ApoL2 in conferring anti-apoptotic ability of human bronchial epithelium to the cytotoxic effects of IFN-γ, in maintaining airway epithelial layer integrity.

A family of cell-adhering peptides homologous to fibrinogen C-termini

A family of cell-adhesive peptides homologous to sequences on different chains of fibrinogen was investigated. These homologous peptides, termed Haptides, include the peptides Cβ, preCγ, and CαE, corresponding to sequences on the C-termini of fibrinogen chains β, γ, and αE, respectively. Haptides do not affect cell survival and rate of proliferation of the normal cell types tested. The use of new sensitive assays of cell adhesion clearly demonstrated the ability of Haptides, bound to inert matrices, to mediate attachment of different matrix-dependent cell types including normal fibroblasts, endothelial, and smooth muscle cells. Here we present new active Haptides bearing homologous sequences derived from the C-termini of other proteins, such as angiopoietin 1&2, tenascins C&X, and microfibril-associated glycoprotein-4. The cell adhesion properties of all the Haptides were found to be associated mainly with their 11 N-terminal residues. Mutated preCγ peptides revealed that positively charged residues account for their attachment effect. These results suggest a mechanism of direct electrostatic interaction of Haptides with the cell membrane. The extended Haptides family may be applied in modulating adhesion of cells to scaffolds for tissue regeneration and for enhancement of nanoparticulate transfection into cells.

Signalling pathway of isophorone diisocyanate-responsive interleukin-8 in airway smooth muscle cells

This study is the first to analyse the soluble factors secreted by the bronchial epithelium after exposure to isophorone diisocyanate (IPDI) that are responsible for increasing migration and proliferation of primary normal human bronchial smooth muscle cells (BSMCs). We treated immortalised, nontumorigenic human bronchial epithelial cells (cell line BEAS-2B) and primary normal human bronchial epithelial cells (HBEC) with IPDI, and then collected the conditioned culture media (IPDI-BEAS-2B-CM and IPDI-HBEC-CM, respectively), which was added to BSMCs. Exposure of BEAS-2B cells and HBECs to IPDI increased interleukin (IL)-8 production. Culture of BSMCs with IPDI-BEAS-2B-CM and IPDI-HBEC-CM increased BSMC proliferation and migration, which are major features in asthma-related airway remodelling. Induction of BSMC proliferation and migration by IPDI-BEAS-2B-CM and IPDI-HBEC-CM was associated with increased focal adhesion kinase (FAK), Src, extracellular signal-regulated kinase (ERK)1/2 and AKT activation. Blocking FAK with a specific inhibitor significantly decreased BSMC migration and proliferation by inhibiting ERK1/2 activation. FAK and ERK1/2 inhibitor also decreased IPDI-BEAS-2B-CM-, IPDI-HBEC-CM- and recombinant human IL-8-mediated BSMC proliferation and migration, whereas blocking Rnd3 using small interfering RNA failed to affect BSMC proliferation, suggesting that Rnd3 was only involved in the regulation of BSMC migration. Our study suggests that inhibition of IL-8 or IL-8-mediated FAK/ERK/Rnd3 signalling is an attractive therapeutic target for IPDI-mediated asthma.

Dicing Up MicroRNA Gene Expression Profiles in Normal and Neoplastic Smooth Muscle Cells

This Commentary discusses the role of miRNA expression profiles in identifying malignancy.

Urinary bladder smooth muscle regeneration utilizing bone marrow derived mesenchymal stem cell seeded elastomeric poly(1,8-octanediol-co-citrate) based thin films

Bladder regeneration studies have yielded inconclusive results possibly due to the use of unfavorable cells and primitive scaffold design. We hypothesized that human mesenchymal stem cells seeded onto poly(1,8-octanediol-co-citrate) elastomeric thin films would provide a suitable milieu for partial bladder regeneration. POCfs were created by reacting citric acid with 1,8-octanediol and seeded on opposing faces with human MSCs and urothelial cells; normal bladder smooth muscle cells and UCs, or unseeded POCfs. Partial cystectomized nude rats were augmented with the aforementioned POCfs, enveloped with omentum and sacrificed at 4 and 10 weeks. Isolated bladders were subjected to Trichrome and anti-human γ-tubulin, calponin, caldesmon, smooth muscle γ-actin, and elastin stainings. Mechanical testing of POCfs revealed a Young’s modulus of 138 kPa with elongation 137% its initial length without permanent deformation demonstrating its high uniaxial elastic potential. Trichrome and immunofluorescent staining of MSC/UC POCf augmented bladders exhibited typical bladder architecture with muscle bundle formation and the expression and retention of bladder smooth muscle contractile proteins of human derivation. Quantitative morphometry of MSC/UC samples revealed muscle/collagen ratios approximately 1.75× greater than SMC/UC controls at 10 weeks. Data demonstrate MSC seeded POCfs support partial regeneration of bladder tissue in vivo.

An endocrine-disrupting chemical, fenvalerate, induces cell cycle progression and collagen type I expression in human uterine leiomyoma and myometrial cells

Fenvalerate (Fen), widely used for its high insecticidal potency and low mammalian toxicity, is classified as an endocrine-disrupting chemical. Recently, Fen has received great attention for its adverse effects on human reproductive health. In this study, we found that Fen (10 μM) had a stimulatory effect on the growth of both cell lines at 24 h compared with controls by MTS ( p < 0.01) and BrdU ( p < 0.01) assays in hormonally responsive uterine leiomyoma (UtLM) cells and normal uterine smooth muscle cells (UtSMC). Flow cytometry results showed that Fen enhanced the escape of cells from the G 0–G 1 checkpoint and promoted progression of both cell types into the S phase. An Annexin V assay showed that Fen had an anti-apoptotic effect on both cell types. By Real-time PCR, we found that collagen I mRNA expression increased ( p < 0.05) in Fen-treated cells compared to controls, although it was greater in UtLM tumor cells. Accordingly, Fen increased ( p < 0.05) collagen I protein levels in both cell lysate and supernatant when compared to controls. To further test the mechanism of Fen's effects, transactivation and competitive binding assays were done. The results showed Fen did not significantly stimulate luciferase activity at concentrations of 0.1 μM, 1.0 μM or 10.0 μM in either of the cell types. Competitive binding assays revealed that the affinity of Fen binding to estrogen receptors (ERs) was non-detectable compared to E 2. Our data show that Fen can stimulate the growth of both UtLM cells and UtSMC, which involves a combination of enhanced cell cycle progression and inhibition of apoptosis. Also this compound can increase collagen I expression, at both mRNA and protein levels. Interestingly, the ER is less likely involved in either the hyperplasia or extracellular matrix (ECM) overproduction induced by Fen. Our results indicate that Fen exposure could be considered a novel risk factor for uterine fibroids through molecular mechanisms that do not directly involve the ERs.

SIX1 acts synergistically with TBX18 in mediating ureteral smooth muscle formation

Dysfunction of the ureter often leads to urine flow impairment from the kidney to the bladder, causing dilation of the ureter and/or renal pelvis. Six1 is a crucial regulator of renal development: mutations in human SIX1 cause branchio-oto-renal (BOR) syndrome and Six1(-/-) mice exhibit renal agenesis, although the ureter is present. It remains unclear whether Six1 plays a role in regulating ureter morphogenesis. We demonstrate here that Six1 is differentially expressed during ureter morphogenesis. It was expressed in undifferentiated smooth muscle (SM) progenitors, but was downregulated in differentiating SM cells (SMCs) and had disappeared by E18.5. In Six1(-/-) mice, the ureteral mesenchymal precursors failed to condense and differentiate into normal SMCs and showed increased cell death, indicating that Six1 is required for the maintenance and normal differentiation of SM progenitors. A delay in SMC differentiation was observed in Six1(-/-) ureters. A lack of Six1 in the ureter led to hydroureter and hydronephrosis without anatomical obstruction when kidney formation was rescued in Six1(-/-) embryos by specifically expressing Six1 in the metanephric mesenchyme, but not the ureter, under control of the Eya1 promoter. We show that Six1 and Tbx18 genetically interact to synergistically regulate SMC development and ureter function and that their gene products form a complex in cultured cells and in the developing ureter. Two missense mutations in SIX1 from BOR patients reduced or abolished SIX1-TBX18 complex formation. These findings uncover an essential role for Six1 in establishing a functionally normal ureter and provide new insights into the molecular basis of urinary tract malformations in BOR patients.

Normal Shear Stress and Vascular Smooth Muscle Cells Modulate Migration of Endothelial Cells Through Histone Deacetylase 6 Activation and Tubulin Acetylation

Endothelial cells (ECs) line the innermost of the blood vessel wall and are constantly subjected to shear stress imposed by blood flow. ECs were also influenced by the neighboring vascular smooth muscle cells (VSMCs). The bidirectional communication between ECs and VSMCs modulates vascular homeostasis. In this study, the involvement of histone deacetylase 6 (HDAC6) in modulating migration of ECs co-cultured with VSMCs by the normal level of laminar shear stress (NSS) was investigated. ECs was either cultured alone or co-cultured with VSMCs under static conditions or subjected to NSS of 15 dyne/cm2 by using a parallel-plate co-culture flow chamber system. It was demonstrated that both NSS and VSMCs could increase EC migration. The migration level of ECs co-cultured with VSMCs under NSS was not higher than that under the static condition. The process of EC migration regulated by VSMCs and NSS was associated with the increased expression of HDAC6 and low level of acetylated tubulin. The increase in HDAC6 expression was accompanied by a time-dependent decrease in the acetylation of tubulin in ECs co-cultured with VSMCs. Inhibition of the HDAC6 by siRNA or tributyrin, an inhibitor of HDACs, induced a parallel alteration in the migration and the acetylated tubulin of ECs co-cultured with VSMCs. It was observed by immunofluorescence staining that the acetylated tubulin was distributed mostly around the cell nucleus in ECs co-cultured with VSMCs. The results suggest that the NSS may display a protective function on the vascular homeostasis by modulating EC migration to a normal level in a VSMC-dependent manner. This modulation process involves the down-regulation of acetylated tubulin which results from increased HDAC6 activity in ECs.